Occurrence, distribution and virulence factors of clinically important Acinetobacter species recovered from selected freshwater resources in the Eastern Cape Province, South Africa

Occurrence, distribution and virulence factors of clinically important Acinetobacter species recovered from selected freshwater resources in the Eastern Cape Province, South Africa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Occurrence, distribution and virulence factors of clinically important Acinetobacter species recovered from selected freshwater resources in the Eastern Cape Province, South Africa Mary Ayobami Adewoyin, Anthony Ifeanyi Okoh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.14951/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background : Several Acinetobacter species live in different ecosystems such as soil, freshwater, wastewater, and solid wastes. In this study, we assessed the occurrence of A. baumannii and A. nosocomialis , which are the major two clinically important species of the genus Acinetobacter , in three freshwater resources (Great Fish, Keiskemma, and Tyhume rivers) in the Eastern Cape Province, South Africa over a one year sampling regime (April 2017 - March 2018). Presumptive Acinetobacter species were subjected to molecular identification by using Acinetobacter genus-specific primers targeting the recA gene. The confirmed Acinetobacter species were further delineated into A. baumannii and A. nosocomialis using species-specific primer sets. Similarly, virulence genes, namely; afa/draBC, epsA, fimH, OmpA, PAI, sfa/focDE , and traT in the two Acinetobacter species were also determined using molecular techniques. Result : A total of 1107 presumptive Acinetobacter isolates were recovered from the freshwater resources of which 844 was confirmed positive for the Acinetobacter genus. Of the 844 Acinetobacter isolates, 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great Fish, Keiskemma and Tyhume rivers respectively. Our finding revealed that 410 (48.58%) and 23 (2.7%) of the isolates were confirmed to be A. baumannii and A. nosocomalis , respectively. The presence of these clinically-important Acinetobacter species in the freshwater studied suggests possible contamination of the selected rivers and also that A. baumannii and A. nosocomialis can thrive in aquatic environments. Besides, 308 (75.12%) A. baumannii and 3 (13.04%) A. nosocomialis isolates exhibited one or more virulence genes out of the seven tested, whereas 102 (24.88%) and 20 (86.95%) of the A. baumannii and A. nosocomialis isolates did not harbour any virulence gene. Additionally, OmpA was the most prevalent (p<0.05) virulence gene in A. baumannii with 69 (45.10%), 52 (50.98%) and 77 (49.68%) isolates in Great Fish, Keiskamma and Tyhume rivers respectively. Conclusion : The occurrence of these pathogens in rivers which are consumed by humans and livestock, as well as being used for irrigation system constitutes a risk to public health. Keywords: Freshwater resources, Molecular characterisation, Acinetobacter species, virulence gene s. General Microbiology Freshwater resources Molecular characterisation Acinetobacter species virulence genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The majorities of Acinetobacter species are free-living and are ubiquitous in nature, but the clinically-important species, most especially the Acinetobacter calcoaceticus- baumannii (ACB) complex, are frequently isolated from the hospital environs. Among members of this complex, A . baumannii, A. nosocomialis , and A. pittii are well-reported causative agents of Acinetobacter -related infections in hospitals around the globe [1, 2]. The most virulent Acinetobacter species, A. baumannii , is known to cause disease outbreaks in intensive care units (ICUs). Owing to the importance of this disease-causing species in the clinical settings, some scientists have reported that it is a nosocomial pathogen and its occurrence in other environments is likely not possible [2]. However, in recent discoveries, A. baumannii has been reported to thrive in freshwater, soil, healthy human skin just like other non-pathogenic species in the Genus [3, 4]. The occurrence of A. baumannii and other pathogenic species of the genus Acinetobacter in other environment apart from the hospital could be associated with the indiscriminate disposal of wastewater and materials from the hospital into the environment [5]. The isolation of A. baumannii from hospitalized patients is often associated with serious ailment, with corresponding mortality rate of approximately 30% [6, 7]. Additionally, A. baumannii has been implicated in a range of diseases which includes; Acinetobacter pneumonia-bronchiolitis and tracheobronchitis [8, 9], bloodstream infection [2, 10], wound infection [11], urinary tract infection [2, 11] and meningitis [8, 12]. Similarly, A. nosocomialis was responsible for bacteremia [13, 14], pneumonia [15], and induction of epithelial cell death and host inflammatory responses [16]. The pathogenicity of Acinetobacter species like other microorganisms is strongly associated with the virulence factors they harbour. These factors include porins (OmpA), capsular polysaccharides, lipopolysaccharides (LPS), phospholipase, outer membrane vesicles (OMVs), protein secretion systems, metal acquisition system etc. [8, 17, 18, 19]. The OmpA is a protein located on the outer cell membrane of the bacteria, which is responsible for the selective permeation of materials in and out of the cell. The OmpA also binds to the host epithelial cells in order to gain entry into the cell cytoplasmic environment. As such, it causes cell death (apoptosis) by releasing the cytochrome c. OmpA was also noted as one of the factors with which A. nosocomialis initiates its pathogenesis [20]. Pathogenic Gram-negative bacteria are known to secret outer membrane vesicles (OMVs) [17, 21, 22], for interaction between the bacterial pathogens and the host cells [23]. Kim and others [20] showed that A. nosocomialis uses its outer membrane vesicles (OMVs) for secretion of cytotoxic factors with which it elicits an immune response from host epithelial cell. Phospholipase, like other factors, also contributes to the virulence of the pathogenic Acinetobacter species, by hydrolysing phospholipid bilayer of the host cell membrane in order to destabilize the entire cell [17, 24]. As a result, an investigation of the occurrence and distribution of these clinically-important Acinetobacter species in other environments, other than hospital, is very necessary for the understanding of what nature and diversity truly represent [25, 26]. As such, the time has come to redefine the environmental coverage by these organisms, beyond a clinical setting. Therefore, the focus of this study was to assess the occurrence, distribution and the virulence factors of clinically-important Acinetobacter species such as A . baumannii and A. nosocomialis in the selected freshwater resources in the Eastern Cape, South Africa. Results Isolation and distribution of presumptive Acinetobacter species A total of 1107 presumptive Acinetobacter species was recovered from the three rivers studied, of which 370, 309 and 428 presumptive isolates belonged to Great Fish, Keiskemma and Tyhume rivers respectively as summarized in Table 1. PCR amplification of the recA gene To further validate the occurrence and distribution of the bacteria in the genus Acinetobacter in this study, the identification was achieved by using the PCR-based assay to detect internal recA genes that are specific to all Acinetobacter species. Figure S1 presents the PCR product of the gel electrophoresis and staining for the amplification of the 425 bp fragment, which correlates to recA gene. Out of the 1107 presumptive isolates, 844 were confirmed to belong to the genus Acinetobacter of which 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great fish, Keiskemma and Tyhume rivers respectively. The confirmed isolates belonging to the genus Acinetobacter is summarized and presented in Table 1. Delineation of Genus Acinetobacter into species Figures S2A and S2B shows the PCR products of the gel electrophoresis and staining for the delineation of the genus Acinetobacter into A. baumannii and A. nosocomailis accordingly, while A. pittii was not detected. The occurrence of A. baumannii was 153 (53.68%) in Great Fish river, 102 (46.58%) in Keiskamma river, and 155 (45.59%) in Tyhume river, while 16 (5.61%), 3 (1.37%) and 4 (1.18 %) represent A. nosocomialis from the respective rivers. The presence of A. baumannii in the three rivers studied was significantly higher (p<0.05) than the A. nosocomialis isolates. The delineation into A. baumannii and A. nosocomailis are summarized and presented in Table 1. Detection of Virulence Genes The gel electrophoresis of the virulence genes; afa/draBC , epsA , fimH , OmpA , PAI, sfa/focDE , and traT , are presented in Figure S3. The virulence genes haboured by A. baumannii and A. nosocomialis in this study is presented in Figure 2 and summarized in Table 2. Additionally, the virulence gene(s) associated with each of the Acinetobacter species as well as the river source from which they were isolated is described. The statistical significance of the prevalence virulence genes with respect to rivers was also emphasized. Three hundred and eight (75%) of the 410 A. baumannii and 3 (13%) of the 23 A. nosocomialis isolated from the rivers were observed to exhibit one or more virulence genes, out of the seven tested. Respectively, 102 (24.88%) and 20 (86.95%) of the A. baumannii and A. nosocomialis isolates from the three rivers did not harbour any of the virulence genes tested in this study. However, there was a significant difference (p<0.05) in the prevalence of virulence genes harboured by A. baumannii compared to A. nosocomialis , though the population of the former was significantly higher than the latter in all cases. The prevalence of virulence gene afa/draBC was significantly higher (p<0.05) in the isolates from Great Fish river in comparison to Keiskamma and Tyhume rivers. However, there was a significant difference (p<0.05) in prevalence between the afa/draBC genes haboured by the bacteria recovered from the Tyhume river when compared to those from Keiskamma river. Besides, the prevalence of traT gene in Acinetobacter species from Great Fish and Tyhume rivers showed no statistical difference (p<0.05), but both were significantly higher (p<0.05) than those detected in isolates from Keiskamma river. Similarly, the prevalence of fimH gene detected in isolates recovered from Great Fish river was significantly higher (p<0.05) than those detected in isolates from both Keiskamma and Tyhune river. Nonetheless, the proportion of isolates harbouring the gene in Keiskamma was significantly (p<0.05) lower than those from Tyhume river. The prevalence of the PAI virulence gene was not significantly (p<0.05) different in all the rivers studied. In Great Fish and Keiskamma rivers, there was no significant difference (p<0.05) in the prevalence of sfa/focDE in isolates recovered from the rivers, but the proportion of Acinetobacter species in Tyhume harbouring the gene was significantly higher (p<0.05) than in those from both Great Fish and Keiskamma rivers. The number of Acinetobacter species harbouring epsA virulence gene in the Great Fish river was significantly higher (p<0.05) than those recovered from Keiskamma river, but significantly lower (p<0.05) than in Acinetobacter species recovered from Tyhume river. The prevalence of the OmpA virulence gene was significantly higher (p<0.05) in A. baumannii isolates than all other virulence genes in this study. Besides, OmpA was the most prevalent virulence gene in the rivers, which represented 69 (45.10%), 52 (50.98%) and 77 (49.68%) A. baumannii isolates in Great Fish, Keiskamma and Tyhume rivers respectively. The number of Acienotobacter species harbouring OmpA gene in Great fish river were significantly higher (p<0.05) than isolates from Keiskamma river, whereas Tyhume river maintained the highest level of statistical significance (p<0.05) of the total Acinetobacter species exhibiting the virulence gene. Summarily, OmpA was the most prevalent virulence gene detected in Acinetobacter species in all the three rivers studied followed by fimH and epsA genes, whereas PAI and Sfa/focDE genes were the least exhibited respectively. Furthermore, the number of Acinetobacter species habouring the highest percentage of virulence genes was isolated from the Tyhume River followed by the Great Fish River, while Kieskamma River was the least. Discussion Bacteria in the genus Acinetobacter have been known to colonize a wide array of ecological systems of which water, soil, sludge, wastewater, plants' root, and animals have been prominent. In this study, water samples were collected at five different sites from three selected rivers in the Eastern Cape Province, South Africa for the assessment of the oof Acinetobacter species. Several presumptive species belonging to the genus Acinetobacter were recovered from the water samples. Similar study was carried out by [27] on the microbial community in the urban riverine environment in Croatia, where 57 of the isolates belong to the genus Acinetobacter . Likewise, Krizova et al. [28], noted a widespread of A. bohemicus in the water environment in the Czech Republic. A. baumannii was characterized from a surface water resources in South Nation River (SNR) drainage basin in Eastern Ontario, Canada, in 2013 [26]. Besides, the composition of the bacterial community in a freshwater aquaculture environment was investigated in China by [29]. The study reported that the relative abundance of Acinetobacter species was 0.5% of the total bacterial community. All isolates that were positive for the recA (425 bp) (Figure S1) were taken as belonging to the Acinetobacter genus, according to [30]. A PCR amplification assay based on recA gene-specific primer, identified 844 Acinetobacter species of the 1107 presumptive isolates, while gyrB gene species-specific primers further delineated them into species namely, A. baumannii (410) and A. nosocomialis (23). According to [30], the method used in this study was 98.2% specific and 92.4% sensitive for Comparably, [31] demonstrated a PCR assay targeting recA gene A. baumannii , A. nosocomialis, and A. pittii are known nosocomial pathogens, which could cause multiple antibiotic resistant infections [2, 30, 32] in immune-compromised patients [2]. Park and others [33] investigated the presence of Acinetobacter species in the bloodstream of patients with blood infection in a tertiary-care hospital in Korea between August 2003 and February 2010. Their findings showed that A. baumannii and A. nosocomialis were prevalent in the samples collected. Similarly, [34], recovered 160 A. baumannii isolates from sputum, blood, pus and fluid aspirates of patients in three Hospitals in Vietnam within a period of two years (between 2012 and 2014). These findings showed that the nosocomial pathogens could colonize any part of the human body to cause infections. However, occurrence of A. baumannii and A. nosocomialis in an aquatic environment, is uncommon in the past few years. Nonetheless, in recent times, these nosocomial pathogens are isolated from other ecosystems [3, 4, 35]. The isolation of the pathogen from water sources might be due to contamination coming from hospital wastewater and materials. The virulence traits of A. baumannii and A. nosocomialis have been a major research focus in recent times [17, 36]. This is due to the nature of A. baumannii infections as well as the role virulence genes play in the pathogenicity of the emerging waterborne pathogen. Virulence genes are the mechanisms through which A. baumannii initiates pathogenesis [36, 37], most especially in the clinical settings. In this study, molecular assay for the detection of virulence factors in both pathogens was performed. Figure S2 shows the electrophoretic images of the seven virulence factors evaluated. Although, A. baumannii had been confirmed to harbour several virulence factors (genes), seven of these genes ( afa/draBC , epsA , fimH , OmpA , PAI, sfa/focDE , and traT ) are reported in this study. Reports on the virulence genes in A. baumannii are usually associated with isolates from clinical environment, whereas such reports are rare on A. baumannii from freshwater resources Thus, this study showed that OmpA gene was predominantly exhibited by the A. baumannii in all the rivers sampled, likewise fimH and epsA genes were also detected in many of the isolates, whereas afa/draBC , PAI, Sfa/focDE , and traT genes were detected in a few Acinetobacter isolates. As such, the exhibition of virulence genes varies from one isolate to the other, which was also reported among clinical isolates known for nosocomial infections [38]. The OmpA gene is the main outer membrane protein (OMP) located on the A. baumannii membrane [39, 40], which [36]. These observations corroborate the findings of this current study which reports that the virulence profiles of an individual isolate varied greatly and OmpA gene was mostly detected [36]. Generally, the outer membranes of Gram-negative bacteria are made up of the OMPs, lipopolysaccharides and phospholipids layer [37]. The presence of outer membrane protein A gene ( OmpA ) in the A. baumanni isolated from the freshwater source is of a major concern based on its role in pathogenesis [36]. Besides, studies have shown that A. baumannii uses OmpA for adhesion to the lung epithelial cell by interacting with a cell cytoskeleton such as fibronectin on the cell surface and thereby inducing pneumonia [41, 42]. It also causes cell death through caspase-3 activation [43, 44]. Similarly, A. baumannii could be responsible for apoptosis through the translocation of its OmpA into the mitochondria and the nucleus of host cells [45, 46]. The combination of the roles played by OmpA makes it an important virulence factor in the pathogenesis of A. baumannii infection. Moreover, antibiotic resistance in A. baumannii is also associated with OmpA [47, 48]. It was suggested that OmpA was involved in the removal of antibiotics from the periplasmic space membrane efflux systems [48]. The survival and persistence of A. baumannii in the cell are enhanced by OmpA due to the formation of biofilms and surface motility. Conclusions In this study, three selected freshwater resources in the Eastern Cape Province, South Africa were evaluated for the occurrence, distribution and virulence genes fingerprints of clinically-relevant Acinetobacter species namely; A. baumannii and A. nosocomialis . This study suggests that these pathogenic Acinetobacter species could also inhabit aquatic environment. As a result, aquatic environment is an important reservoir for pathogenic Acinetobacter species as detected in this study. Besides, the OmpA is a major virulence factor associated with A. baumannii and A. nosocomialis in this study. As such, identification of these opportunistic and virulent waterborne pathogens in the freshwater resources requires public awareness and recognition as important to public health risks. Methods Description of study areas Collection of water samples was carried out from three rivers namely; Great Fish, Keiskamma and Tyhume, in the Eastern Cape Province, South Africa between April 2017 and March 2018. The Great Fish River is located in Chris Hani District Municipality in the Eastern Cape Province and it is one of the major rivers used for irrigation and livestock farming in the area. This river is prone to agricultural and municipal runoffs and also serves as the receiving stream of effluents from many wastewater treatment plants (WWTPs), especially those situated in urban communities such as Craddock. Keiskamma and Tyhume rivers are located in the Amathole District Municipality in the Eastern Cape Province and are exposed to different anthropogenic activities from the rural and urban communities along the river courses, such as livestock drinking and irrigation farming. In addition, these rivers receive effluents from wastewater treatment plants (WWTPs) situated close to their banks. Different sampling points on these rivers were selected based on where humans and animals come into direct contact with them, for example, points where they are used for fishing, drinking and swimming purposes, downstream of the WWTPs, points where irrigation water is released to the water bodies and proximity to hospital facilities. Sampling Water samples were collected at five different sites (S1, S2, S3, S4, and S5) from Great Fish, Keiskamma and Tyhume rivers respectively, for a period of one year, which covers the four seasonal patterns in South Africa (autumn, winter, spring, and summer). Water samples were collected aseptically in sterile 1L glass bottles from different sampling points by midstream-dipping of sample bottles at 25–30 cm down the water column, with the mouth tilting against the flow of the river. All water samples were labelled properly and safely taken to the laboratory (in an ice chest) where they were processed within 6 h of collection [27]. Aliquots of water samples were used for isolation of Acinetobacter species based on standard microbiological procedures [49] Isolation and purification of presumptive Acinetobacter species The isolation of the presumptive density of Acinetobacter species in the water samples was determined by membrane filtration technique [50]. Cellulose membrane of pore size 0.45 𝜇m was used to filter three volumes of 100 mL of the water samples under vacuum [50]. These membranes were aseptically placed on plates with Acinetobacter species selective medium-CHROMagar Acinetobacter base plus selective supplement (CHROMagar, Paris, France) which was prepared according to the manufacturer’ instruction. Each sample plate was subjected to incubation at 37 o C for 24 h after inoculation. Each sample was analysed in triplicate. All bacterial colonies with red colouration on the CHROMagar plates were counted as presumptive Acinetobacter species and were expressed as CFU/100ml. All isolates were sub-cultured on nutrient agar using a streak plate method (Oxoid, UK) and purified for further species identification. Fifty percent (50%) glycerol stocks of the pure culture was prepared and stored at -80 ∘ C. Molecular identification of Acinetobacter species by PCR assays Extraction of genomic DNA : Presumptive Acinetobacter spp. in glycerol stocks was first being resuscitated on tryptic soy broth and incubated for 18 to 24h at 37 ∘ C. DNA extraction from the bacterial isolates was carried out using the direct boiling method according to [51]. The broth culture was centrifuged at 15000 rpm for 5 min using a Mini Spin Microcentrifuge (Lasec, RSA), then the supernatant was dispensed out and the pellet rinsed with sterile normal saline. The pellet was re-suspended in sterile distilled water and boiled in a heating block for 10 minutes using an AccuBlock (Digital dry bath, Labnet). Amplification of unique Acinetobacter species DNA Polymerase chain reaction (PCR) assay was used for the amplification of the Acinetobacter species recA gene as previously described [30]. The forward and reverse primers used were P-rA1 (5′-CCTGAATCTTCTGGTAAAAC-3′) and P-rA2 (5′-GTTTCTGGGCTGCCAAACATTAC-3′) respectively. Briefly, an aliquot of 25 µl containing Taq PCR (12.5 µl) Master Mix (Qiagen, Hilden, Germany), each of the primers (1 µl) (Inqaba, SA), nuclease-free water (6.5 µl) and DNA template (5 µl) was used for the PCR amplification assay. The condition for the amplification included initial denaturation step (94 o C, 5 min), followed by 35 cycles (92 o C, 40 s), annealing (58 o C, 40 s), and the final extension step (72 o C, 10 min) was performed using a thermocycler (Bio-Rad Thermal cycler, USA). Five microlitres (5 µl) of the amplicon was subjected to gel (1.5% agarose) electrophoresis at 100 Volts for 45 min in Tris Boric EDTA buffer (pH 8.0) (0.089 M Tris, 0.089 M boric acid, and 0.002 M EDTA). Ethidium bromide (5 µl of 0.5 mg/ml) (Sigma-Aldrich, USA) was used for gel staining and DNA ladder (100 bp) (Thermo Scientific, (EU) Lithuania) was added into the gels as a standard. Finally, DNA bands were visualized under an ultraviolet transilluminator (Alliance 4.7, France). Delineation of Genus Acinetobacter into species The confirmed Acinetobacter isolates were delineated into species accordingly [30, 52, 53] using a PCR assay. Firstly, optimization of conditions for A. baumannii , A. nosocomialis and A. pittii was carried out using species-specific primers (Table S1 under supplementary Section), while reference strains DSM-102929, DSM-102856, and DSM-9341 (DSMZ, Germany) were used as positive controls for A. baumannii , A. nosocomialis and A. pittii respectively. The PCR amplification was performed as stated in the previous section. Detection of virulence genes Polymerase chain reactions were also carried out for the identification of some Acinetobacter virulence genes including afa/draBC, epsA, fimH, OmpA , PAI, sfa/focDE , and traT genes, which have been previously found in clinical samples [54, 55, 56]. The standard strain of A. baumannii DSM-30007 (DSMZ, Germany) was used as positive control. There was no positive control available for A. nosocomialis . The PCR assay and electrophoresis were conducted as earlier described. Statistical Analysis All statistical analyses were performed using the Statistica software v13.4.0.14 (64-bits). A simple factorial ANOVA was performed for the comparison of normally distributed data. The p -values of less than 0.05 were considered statistically significant for all the statistical tests performed. Abbreviations ACB: Acinetobacter calcoaceticus- baumannii complex; ANOVA: Analysis of Variance; APHA: American Public Health Association; CFU: Colony Forming Unit; CHROMagar: Chromogenic agar; DNA: Deoxyribonucleic Acid; EDTA: Ethylenediamine tetraacetic acid; EspA: Exopolysaccharide A; EU: European Union; ICU: Intensive care unit; L: Ladder; LPS: Lipopolysaccharides; N: Negative control; ND: Not determined; NRF: National Research Foundation; ompA: Outer membrane protein A precursor; OMVs: Outer membrane vesicles; P: Positive control; PAI: Pathogenicity Island; PCR: Polymerase Chain Reaction; RSA: Republic of South Africa; SAMRC: South African Medical Research Council; TWAS: The World Academy of Science; UFH: University of Fort Hare; UK: United Kingdom; USA: United States of America; WWTPs: Wastewater treatment plants. Declarations Ethics approval and consent to participate Collection of freshwater samples for research purposes in this region does not require governmental permission since freshwater resources investigated are within the University jurisdiction. Also, the need for ethical approval was waived by the Research Ethics Committee, University of Fort Hare, because samples analysed in this study do not require human or animal subjects. Consent for publication Not applicable Availability of data and materials The data used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding The research was funded by the National Research Foundation, South Africa/The World Academy of Science, Italy (NRF/TWAS) (grant numbers: 99767 and 116387) and the South African Medical Research Council (grant number: SAMRC/UFH/P790). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. Authors’ contributions MAA conceived and designed the experiment, collected the water samples, isolated the Acinetobacter spp., performed the experiments, data and statistical analyses, and writing of the manuscripts. AIO supervised the research, provided financial support, and reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable Authors’ information Not applicable. References De Vos D, Jean-Paul P, Florence B, Serge J, Gilbert V, Thomas R, Elkana K, Petra B, Thierry P, Mony H, Walter H, Frank DP, Patrick S, Maia M, Pieter D, Mario V, Pierre B, Youri G, Bruno P, Tanny JR, Dijkshoorn L. Molecular Epidemiology and Clinical Impact of Acinetobacter calcoaceticus-baumannii complex in a Belgian Burn Wound Center. PLoS ONE. 2016; 11(5): e0156237. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii : emergence of a successful pathogen. Clin Microbiol Rev. 2008; 21, 538–582. Hamouda A, Findlay J, Al Hassan L, Amyes SG. Epidemiology of Acinetobacter baumannii of animal origin. J. Antimicrob. Agents. 2011; 38, 314–318. Huys G, Bartie K, Cnockaert M, Hoang Oanh DT, Phuong NT, Somsiri T, Chinabut S, Yussoff FD, Shariff M, Giacomini M, Teale A, Swings J. Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments. Microbiol. 2007; 158, 228–235. doi:10.1016/j.resmic.2006.12.011. Hrenovic J, Durn G, Goic-Barisic I, Kovacic A. Occurrence of an Environmental Acinetobacter baumannii Strain Similar to a Clinical Isolate in Paleosol from Croatia. Applied and Environmental Microbiology, 2014; 80(6): 2860–2866. Almasaudi SB. Acinetobacter as nosocomial pathogens: Epidemiology and resistance features. Saudi Journal of Biological Sciences. 2018; 25, 586–596. Jung J, Park W. Acinetobacter species as model microorganisms in environmental microbiology: current state and perspectives. Appl. Microbiol. Biotechnol. 2015; 99, 2533–2548. Doughari HJ, Ndakidemi PA, Human IS, Benade S. The ecology, biology and pathogenesis of Acinetobacter spp.: an overview. Microbes Environ . 2011; 26, 101–112. Whitman TJ, Qasba SS, Timpone JG, Babel BS, Kasper MR, English JF, Sanders JW, Hujer KM, Hujer AM, Endimiani A. Occupational transmission of Acinetobacter baumannii from a United States serviceman wounded in Iraq to a health care worker . Infect. Dis. 2008; 47, 439–443. Garnacho-Montero J, Amaya-Villar R, Ferrandiz-Millon C, Dıaz-Martın A, Lopez-Sanchez JM, Gutierrez-Pizarraya A. Optimum treatment strategies for carbapenem-resistant Acinetobacter baumannii Expert Rev. Anti. Infect. Ther. 2015; 13(6): 769-77. Falagas ME, Vardakas KZ, Kapaskelis A, Triarides NA, Roussos NS. Tetracyclines for multidrug-resistant Acinetobacter baumannii International Journal of Antimicrobial Agents. 2015; 45, 455–460. Basri R, Zueter AR, Mohamed Z, Alam MK, Norsa’adah B, Hasan SA, Hasan H, Ahmad F. Burden of bacterial meningitis: a retrospective review on laboratory parameters and factors associated with death in meningitis, Kelantan Malaysia. Nagoya J. Med. Sci. 2015; 77: 59-68. Lai HH, Liou BH, Chang YY, Kuo SC, Lee YT, Chen TL, Fung CP. Risk factors and clinical outcome of sulbactam non-susceptibility in monomicrobial Acinetobacter nosocomialis Journal of Microbiology, Immunology and Infection. 2016; 49, 371-377. Liu YM, Lee YZ, Kuo SC, Chen TL, Liu CP, Liu CE. Comparison between bacteremia caused by Acinetobacter pittii and Acinetobacter nosocomialis . Journal of Microbiology, Immunology and Infection. 2017; 50, 62–67. Lee YT, Kuo SC, Yang SP, Lin YT, Chiang DH, Tseng FC, Chen TL, Fung CP. Bacteremic nosocomial pneumonia caused by Acinetobacter baumannii and Acinetobacter nosocomialis : a single or two distinct clinical entities. European Society of Clinical Microbiology and Infectious Diseases, CMI. 2012; 19, 640–645. Nho JS, Jun SH, Oh MH, Park TI, Choi CW, Kim S, Choi CH, Lee JC. Acinetobacter nosocomialis secretes outer membrane vesicles that induce epithelial cell death and host inflammatory responses. Microbial Pathogenesis. 2015; 81, 39-45. Lee CR, Lee JH, Park M, Park KS, Bae IK, Kim YB, Cha CJ, Jeong BC, Lee SH. Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options. Frontiers in Cellular Infection Microbiology. 2017; 7 (March) https://doi.org/10.3389/fcimb.2017.00055 . Kurcik-Trajkovska B. Acinetobacter – a serious enemy threatening hospitals worldwide. Macedonian Journal of Medical Science. 2009; 2, 157–162. Kanafani AZ, Kanj SS. Ministry of Health, Kingdome of Saudi Arabia. 2014. http://www.uptodate.com/contents/acinetobacterinfection-treatment-and-prevention . Kim SW, Oh MW, Jun SH, Jeon H, Kim S, Kim K, Lee YC, Lee JC. Outer Membrane Protein A plays a role in pathogenesis of Acinetobacter nosocomialis . Virulence. 2016; 7:4, 413-426, DOI: 10.1080/21505594.2016.1140298. Ellis TN, Kuehn MJ. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiology and Molecular Biology Review. 2010; 74 , 81–94. doi: 10.1128/MMBR.00031-09. Kwon SO, Gho YS, Lee JC, Kim SI. Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii FEMS Microbiology Letter. 2009; 297, 150–156. doi: 10.1111/j.1574-6968.2009.01669. Jun SH, Lee JH, Kim BR, Kim SI, Park TI, Lee JC, Lee YC. Acinetobacter baumannii outer membrane vesicles elicit a potent innate immune response via membrane proteins. PLoS ONE. 2013; 8, e71751. doi: 10.1371/journal.pone.0071751. Fiester SE, Arivett BA, Schmidt RE, Beckett AC, Ticak T, Carrier MV, Ghosh R, Ohneck EJ, Metz ML, Jeffries MKS, Actis LA. Iron-regulated phospholipase C activity contributes to the cytolytic activity and virulence of Acinetobacter baumannii . PLoS ONE. 2016; 11:e0167068. doi: 10.1371/journal.pone.0167068. Diancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. The population structure of Acinetobacter baumannii : expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS ONE. 2010; e10034.doi: 10.1371/journal.pone.0010034. Fernando DM, Khan IUH, Patidar R, Lapen DR,Talbo G, Topp E, Kumar A. Isolation and Characterization of Acinetobacter baumannii recovered from Campylobacter Selective Medium. Frontiers in Microbiology; 2016; 7:1871. Maravic A, Skocibusic, M, Fredotovic Z, Samanic I, Cvjetan S, Knezovic M, Puizina J. Urban riverine environment is a source of multidrug-resistant and ESBL-producing clinically important Acinetobacter Environ Sci Pollut Res. 2016; 23, 3525–3535. Krizova L, Maixnerovaa M, Sedob O, Nemec A. Acinetobacter bohemicus nov.wide spread in natural soil and water ecosystems in the Czech Republic. Systematic and Applied Microbiology . 2014; 37, 467–473. Xiong W, Sun Y, Zhang T, Ding X, Li Y, Wang M, Zeng Z. Antibiotics, Antibiotic Resistance Genes, and Bacterial Community Composition in Fresh Water Aquaculture Environment in China. Microb Ecol. 2015; 70:425–432. Chen TL, Lee YT, Kuo SC, Yang SP, Fung CP, Lee SD. Rapid identification of Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii with a multiplex PCR assay. Journal of Medical Microbiology, 2014; 63, 1154–1159. Chiang MC, Kuo SC, Chen YC, Lee YT, Chen TL, Fung CP. Polymerase chain reaction assay for the detection of Acinetobacter baumannii in endotracheal aspirates from patients in the intensive care unit. Journal of Microbiology, Immunology and Infection. 2011; 44, 106–110. Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol. 2007; 5, 939–951. Park YK, Jung SI, Park KH, Kim DH, Choi JY, Kim SH, Ko KS. Changes in antimicrobial susceptibility and major clones of Acinetobacter calcoaceticus – baumannii complex isolates from a single hospital in Korea over 7 years. Journal of Medical Microbiology . 2012; 61(1) 71–79. Anh NT, Thieu Nga TV, Tuan HM, Tuan NS, Dao Y, Chau NVV, Baker S, Duong HHT. Molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam. Journal of Medical Microbiology . 2017; 66:46–53. Berlau J, Aucken HM, Houang E, Pitt TL. Isolation of Acinetobacter spp including baumannii from vegetables: implications for hospital-acquired infections. Journal of Hospital Infection. 1999; 42: 201–204. Ambrosi C, Scribano D, Aleandri M, Zagaglia C, Di Francesco L, Putignani L, Palamara AT. Acinetobacter baumannii Virulence Traits: A Comparative Study of a Novel Sequence Type with Other Italian Endemic International Clones. Front. Microbiol. 2017; 8:1977. doi: 10.3389/fmicb.2017.01977. Sato Y, Unno Y, Kawakami S, Ubagai T, Ono Y. Virulence characteristics of Acinetobacter baumannii clinical isolates vary with the expression levels of omps. Journal of Medical Microbiology. 2017; 66:203–212. Antunes LCS, Imperi F, Carattoli A, Visca P. Deciphering the Multifactorial Nature of Acinetobacter baumannii PLoS ONE. 2011; 6(8): e22674. doi:10.1371/journal.pone.0022674. Smith MG, Gianoulis TA, Pukatzki S, Mekalanos JJ, Ornston LN, Gerstein M, Snyder M. New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes and Development. 2007; 21:601–614. McConnell MJ, Actis L, Pachon J. Acinetobacter baumannii : human infections, factors contributing to pathogenesis and animal models. FEMS Microbiol Rev. 2013; 37: 130–155. Gaddy JA, Tomaras AP, Actis LA. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infection and Immunity. 2009; 77(8), 3150–3160. Smani Y, Dominguez-Herrera J, Pachon J. Association of the outer membrane protein Omp33 with fitness and virulence of Acinetobacter baumannii . Journal of Infectious Disease. 2013; 208, 1561–1570. doi: 10.1093/infdis/jit386. Lee JC, Oh JY, Kim KS, Jeong YW, Park JC, Cho JW. Apoptotic cell death induced by Acinetobacter baumannii in epithelial cells through caspase-3 activation. APMIS . 2001; 109:679–684. Smani Y, Domnguez-Herrera J, Pachon J. Rifampin protects human lung epithelial cells against cytotoxicity induced by clinical multi and pandrug-resistant Acinetobacter baumannii . European Congress of Clinical Microbiology and Infectious Diseases. 2011; Vienna, Austria, April 2010. Choi CH, Lee EY, Lee YC, Park TI, Kim HJ, Hyun SH, Kim SA, Lee SK, Lee JC. Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cellular Microbiology. 2005; 7, 1127–1138. doi: 10.1111/j.1462-5822.2005.00538. Lee JS, Choi CH, Kim JW, Lee JC. Acinetobacter baumannii outer membrane protein A induces dendritic cell death through mitochondrial targeting. Journal of Microbiology, 2010; 48, 387–392. doi: 10.1007/s12275-010-0155-1. Sugawara E, Nikaido H. OmpA is the principal nonspecific slow porin of Acinetobacter baumannii. Bacteriol . 2012; 194, 4089–4096. doi: 10.1128/JB.00435-12. Smani Y, Fabrega A, Roca I, Sanchez-Encinales V, Vila J, Pachon J. Role of OmpA in the multidrug resistance phenotype of Acinetobacter baumannii . Antimicrob. Agents Chemother. 2014; 58, 1806–1808. doi: 10.1128/AAC.02101-13. Sibanda T, Chigor VN, Koba S, Obi CL, Okoh AI. Characterisation of the physicochemical qualities of a typical rural-based river: ecological and public health implications. Int. J. Environ. Sci. Technol. 2014; 11:1771–1780. American Public Health Association, APHA. Standard Methods for the Examination of Water and Wastewater, 20th Ed.; (APHA): Washington, DC, USA. 2005. Maugeri TL, Carbone M, Fera MT, Gugliandolo C. Detection and differentiation of Vibrio vulnificus in seawater and plankton f a coastal zone of the Mediterranean Sea. Research in Microbiology. 2006; 157(2), 194–200. Chen TL, Siu LK, Wu RCC, Shaio MF, Huang LY, Fung CP, Lee CM, Cho WL. Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS) sequencing for rapid identification of Acinetobacter baumannii . Clin Microbiol Infect; 2007; 13: 801–806. Higgins PG, Wisplinghoff H, Krut O, Seifert H. A PCR-based method to differentiate between Acinetobacter baumannii and Acinetobacter genomic species 13TU. Clin Microbiol Infect. 2007; 13:1199–1201. Thummeepak R, Kongthai P, Leungtongkam U, Sitthisak S. Distribution of virulence genes involved in biofilm formation in multi-drug resistant Acinetobacter baumannii clinical isolates. International Microbiology. 2016; 19:121-129 doi:10.2436/20.1501.01.270. Momtaz H, Seifati SM, Tavakol M. Determining the prevalence and detection of the most prevalent virulence genes in Acinetobacter baumannii isolated from hospital infections. Int J Med Lab. 2015; 2(2):87–97. Braun G, Vidotto MC. Evaluation of Adherence, Hemagglutination, and Presence of Genes Codifying for Virulence Factors of Acinetobacter baumannii Causing Urinary Tract Infection. Mem Inst Oswaldo Cruz, Rio de Janeiro, 2004; 99 (8): 839-844. Tables Table 1: Summary of the relative abundance of the genus Acinetobacter and two clinically important species in the freshwater studied Rivers sampled Presumptive isolates Acinetobacter genus Delineated Acinetobacter species Other species (ND) A. baumannii A. nosocomialis Great Fish 370 285(77.01%) 153(53.68%) 16(5.61%) 116(40.70%) Keiskamma 309 219(70.87%) 102(46.58%) 3(1.37%) 114(52.06%) Tyhume 428 340(79.44%) 155(45.59%) 4(1.18%) 181(53.24%) Total 1107 844(76.24%) 410(48.58%) 23(2.73%) 411(48.70%) ND= not determined Table 2: Virulence gene factors of A. baumannii and A. nosocomialis isolates in addition to which river they belong to. Virulence gene Sample sites Great Fish Kieskamma Tyhume A. baumannii (n=153) A. nosocomialis (n=16) A. baumannii (n=102) A. nosocomialis (n=3) A. baumannii (n=155) A. nosocomialis (n=4) Afa/draBC 16(10.46%) - 4 (3.92%) - 8 (5.81) 1 (25%) espA 36 (23.53%) - 15 (14.71%) - 44 (28.39%) 1 (25%) fimH 44 (28.76%) - 26 (25.49%) 1(33.33%) 30 (19.36%) 1 (25%) OmpA 69 (45.10%) - 52 (50.98%) - 77 (49.68%) - PAI 2 (1.31%) 1(6.26%) 3 (2.94%) - - - Sfa/focDE 3 (1.96%) - 4 (3.92%) - 7 (4.52%) - traT 19 (12.42%) - 4 (3.92%) - 22 14.19%) - Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5622","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":317048,"identity":"85ed55bd-b7c2-4986-8465-aa85ea847fb6","order_by":1,"name":"Mary Ayobami Adewoyin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYDADNgbG9s8/KoAsZuYGonRIsDEwH2NmOAPSwkikFqA9acyMbSA2AS267WefSd2ouVfHJ91j9rhwXm00fztQy4+KbTi1mJ1JN5POOVYswSZzxtx45rbjuTMOMzYw9py5jVvLgTQ26Ry2BAk2iRwDCd5tx3IbgFqALsSj5fwzoJZ/MC1zjuXOJ6jlBtCW3DaQlrQ0ad6GmtwNhLU8Y7bO7UuQbJNIPmw449iB3I1ALQfx+uV8GuPtnG8J/PIzEhsffKipy513/vDBBz8qcGsBAhYJJM5hMHkAn3ogYP6AxKkjoHgUjIJRMApGIgAAnktY+Rzswu8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7489-7402","institution":"University of Fort Hare","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mary","middleName":"Ayobami","lastName":"Adewoyin","suffix":""},{"id":317049,"identity":"9e34329d-4da4-4b5d-aa33-14dc22641418","order_by":2,"name":"Anthony Ifeanyi Okoh","email":"","orcid":"","institution":"University of Fort Hare","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"Ifeanyi","lastName":"Okoh","suffix":""}],"badges":[],"createdAt":"2019-09-20 13:39:03","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.2.14951/v2","doiUrl":"https://doi.org/10.21203/rs.2.14951/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":414901,"identity":"9e4f2b22-b5e8-4b07-8807-e5d7de0b522e","added_by":"auto","created_at":"2020-01-27 21:37:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37232,"visible":true,"origin":"","legend":"Isolates of A. baumannii and A. nosocomialis recovered from each of the sampling sites in Great Fish (GF), Keiskamma (KE) and Tyhume (TY) Rivers. The number of isolates presented in this here represent a pool of the five sampling sites.","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/075b6227-bccf-42bb-b972-a6d327e47c39/v2/fig 1.jpg"},{"id":414902,"identity":"6da48f85-53cb-47ed-952b-00638f01a385","added_by":"auto","created_at":"2020-01-27 21:37:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30657,"visible":true,"origin":"","legend":"Virulence genes in both Acinetobacter species recovered from each of the rivers.","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/075b6227-bccf-42bb-b972-a6d327e47c39/v2/Fig 2.jpg"},{"id":414903,"identity":"f348a0f5-30a9-437b-bf39-1146340be99e","added_by":"auto","created_at":"2020-01-27 21:37:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":387321,"visible":true,"origin":"","legend":"PCR analyses resolved by gel electrophoresis showing confirmed A. nosocomialis targeting the gyrBgene at 294 bp. L= DNA Ladder (100bp); P= Positive control (A. nosocomialis, DSM 102856); Lanes 1 to 10 = Selected A. nosocomialis samples.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/075b6227-bccf-42bb-b972-a6d327e47c39/v2/3.png"},{"id":414904,"identity":"d6eb82f7-22db-471b-9b42-d075ed31c8bc","added_by":"auto","created_at":"2020-01-27 21:37:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":817072,"visible":true,"origin":"","legend":"PCR analyses resolved by gel electrophoresis showing confirmed virulence genes OmpA, PAI, fimH, sfa/focDE, espA and traT at 531, 930, 506, 410, 451 and 290 base pairs respectively. L= DNA Ladder (100bp); Lane 1 to 9 (overall) = Selected Acinetobacter isolates; N= Negative control; P=Positive control (A. baumannii, DSM Number: 102929).","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/075b6227-bccf-42bb-b972-a6d327e47c39/v2/4.png"},{"id":414905,"identity":"ec07113c-51f0-42b3-8453-d1f04f35c3ae","added_by":"auto","created_at":"2020-01-27 21:37:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":146092,"visible":true,"origin":"","legend":"Confirmed virulence genes in the Acinetobacter species recovered from each of the rivers.","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/075b6227-bccf-42bb-b972-a6d327e47c39/v2/5.png"},{"id":13487273,"identity":"6ba83599-58e0-4bb8-9f7a-6d7f83c237a9","added_by":"auto","created_at":"2021-09-16 22:09:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1644176,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5622/v2/0bb84720-790e-486f-a67c-39faba00a390.pdf"}],"financialInterests":"","formattedTitle":"Occurrence, distribution and virulence factors of clinically important Acinetobacter species recovered from selected freshwater resources in the Eastern Cape Province, South Africa","fulltext":[{"header":"Background","content":"\u003cp\u003eThe majorities of \u003cem\u003eAcinetobacter\u003c/em\u003e species are free-living and are ubiquitous in nature, but the clinically-important species, most especially the \u003cem\u003eAcinetobacter\u003c/em\u003e \u003cem\u003ecalcoaceticus- baumannii \u003c/em\u003e(ACB) complex, are frequently isolated from the hospital environs. Among members of this complex, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ebaumannii, \u003c/em\u003e\u003cem\u003eA. nosocomialis\u003c/em\u003e, and \u003cem\u003eA. pittii\u003c/em\u003e are well-reported causative agents of \u003cem\u003eAcinetobacter\u003c/em\u003e-related infections in hospitals around the globe [1, 2]. The most virulent \u003cem\u003eAcinetobacter\u003c/em\u003e species, \u003cem\u003eA. baumannii\u003c/em\u003e, is known to cause disease outbreaks in intensive care units (ICUs). Owing to the importance of this disease-causing species in the clinical settings, some scientists have reported that it is a nosocomial pathogen and its occurrence in other environments is likely not possible [2]. However, in recent discoveries, \u003cem\u003eA. baumannii\u003c/em\u003e has been reported to thrive in freshwater, soil, healthy human skin just like other non-pathogenic species in the Genus [3, 4]. The occurrence of \u003cem\u003eA. baumannii \u003c/em\u003eand other pathogenic species of the genus \u003cem\u003eAcinetobacter \u003c/em\u003ein other environment apart from the hospital could be associated with the indiscriminate disposal of wastewater and materials from the hospital into the environment [5].\u003c/p\u003e\n\u003cp\u003eThe isolation of \u003cem\u003eA. baumannii \u003c/em\u003efrom hospitalized patients is often associated with serious ailment, with corresponding mortality rate of approximately 30% [6, 7]. Additionally, \u003cem\u003eA. baumannii\u003c/em\u003e has been implicated in a range of diseases which includes; \u003cem\u003eAcinetobacter\u003c/em\u003e pneumonia-bronchiolitis and tracheobronchitis [8, 9], bloodstream infection [2, 10], wound infection [11], urinary tract infection [2, 11] and meningitis [8, 12]. Similarly, \u003cem\u003eA. nosocomialis \u003c/em\u003ewas responsible for bacteremia [13, 14], pneumonia [15], and induction of epithelial cell death and host inflammatory responses [16].\u003c/p\u003e\n\u003cp\u003eThe pathogenicity of \u003cem\u003eAcinetobacter\u003c/em\u003e species like other microorganisms is strongly associated with the virulence factors they harbour. These factors include porins (OmpA), capsular polysaccharides, lipopolysaccharides (LPS), phospholipase, outer membrane vesicles (OMVs), protein secretion systems, metal acquisition system etc. [8, 17, 18, 19]. The \u003cem\u003eOmpA\u003c/em\u003e is a protein located on the outer cell membrane of the bacteria, which is responsible for the selective permeation of materials in and out of the cell. The \u003cem\u003eOmpA\u003c/em\u003e also binds to the host epithelial cells in order to gain entry into the cell cytoplasmic environment. As such, it causes cell death (apoptosis) by releasing the cytochrome c. \u003cem\u003eOmpA\u003c/em\u003e was also noted as one of the factors with which \u003cem\u003eA. \u003c/em\u003e\u003cem\u003enosocomialis\u003c/em\u003e initiates its pathogenesis [20]. Pathogenic Gram-negative bacteria are known to secret outer membrane vesicles (OMVs) [17, 21, 22], for interaction between the bacterial pathogens and the host cells [23]. Kim and others [20] showed that \u003cem\u003eA. nosocomialis \u003c/em\u003euses its outer membrane vesicles (OMVs) for secretion of cytotoxic factors with which it elicits an immune response from host epithelial cell. Phospholipase, like other factors, also contributes to the virulence of the pathogenic \u003cem\u003eAcinetobacter \u003c/em\u003especies, by hydrolysing phospholipid bilayer of the host cell membrane in order to destabilize the entire cell [17, 24].\u003c/p\u003e\n\u003cp\u003eAs a result, an investigation of the occurrence and distribution of these clinically-important \u003cem\u003eAcinetobacter\u003c/em\u003e species in other environments, other than hospital, is very necessary for the understanding of what nature and diversity truly represent [25, 26]. As such, the time has come to redefine the environmental coverage by these organisms, beyond a clinical setting. Therefore, the focus of this study was to assess the occurrence, distribution and the virulence factors of clinically-important \u003cem\u003eAcinetobacter \u003c/em\u003especies such as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ebaumannii \u003c/em\u003eand \u003cem\u003eA. nosocomialis \u003c/em\u003ein the selected freshwater resources in the Eastern Cape, South Africa.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIsolation and distribution of presumptive \u003cem\u003eAcinetobacter \u003c/em\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 1107 presumptive \u003cem\u003eAcinetobacter \u003c/em\u003especies was recovered from the three rivers studied, of which 370, 309 and 428 presumptive isolates belonged to Great Fish, Keiskemma and Tyhume rivers respectively as summarized in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR amplification of the \u003cem\u003erecA\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the occurrence and distribution of the bacteria in the genus \u003cem\u003eAcinetobacter\u003c/em\u003e in this study, the identification was achieved by using the PCR-based assay to detect internal \u003cem\u003erecA\u003c/em\u003e genes that are specific to all \u003cem\u003eAcinetobacter \u003c/em\u003especies. Figure S1 presents the PCR product of the gel electrophoresis and staining for the amplification of the 425 bp fragment, which correlates to \u003cem\u003erecA \u003c/em\u003egene. Out of the 1107 presumptive isolates, 844 were confirmed to belong to the genus \u003cem\u003eAcinetobacter\u003c/em\u003e of which 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great fish, Keiskemma and Tyhume rivers respectively. The confirmed isolates belonging to the genus \u003cem\u003eAcinetobacter\u003c/em\u003e is summarized and presented in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDelineation of Genus \u003cem\u003eAcinetobacter\u003c/em\u003e into species\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigures S2A and S2B shows the PCR products of the gel electrophoresis and staining for the delineation of the genus \u003cem\u003eAcinetobacter\u003c/em\u003e into \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eA. nosocomailis \u003c/em\u003eaccordingly, while \u003cem\u003eA. pittii\u003c/em\u003e was not detected.\u003c/p\u003e\n\u003cp\u003eThe occurrence of \u003cem\u003eA. baumannii\u003c/em\u003e was 153 (53.68%) in Great Fish river, 102 (46.58%) in Keiskamma river, and 155 (45.59%) in Tyhume river, while 16 (5.61%), 3 (1.37%) and 4 (1.18 %) represent \u003cem\u003eA. nosocomialis\u003c/em\u003e from the respective rivers. The presence of \u003cem\u003eA. baumannii \u003c/em\u003ein the three rivers studied was significantly higher (p\u0026lt;0.05) than the \u003cem\u003eA. nosocomialis \u003c/em\u003eisolates. The delineation into \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eA. nosocomailis\u003c/em\u003e are summarized and presented in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Virulence Genes \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gel electrophoresis of the virulence genes; \u003cem\u003eafa/draBC\u003c/em\u003e, \u003cem\u003eepsA\u003c/em\u003e, \u003cem\u003efimH\u003c/em\u003e, \u003cem\u003eOmpA\u003c/em\u003e, PAI, \u003cem\u003esfa/focDE\u003c/em\u003e, and \u003cem\u003etraT\u003c/em\u003e, are presented in Figure S3. The virulence genes haboured by \u003cem\u003eA. baumannii and A. nosocomialis\u003c/em\u003e in this study is presented in Figure 2 and summarized in Table 2. Additionally, the virulence gene(s) associated with each of the \u003cem\u003eAcinetobacter\u003c/em\u003e species as well as the river source from which they were isolated is described. The statistical significance of the prevalence virulence genes with respect to rivers was also emphasized.\u003c/p\u003e\n\u003cp\u003eThree hundred and eight (75%) of the 410 \u003cem\u003eA. baumannii\u003c/em\u003e and 3 (13%) of the 23 \u003cem\u003eA. nosocomialis\u003c/em\u003e isolated from the rivers were observed to exhibit one or more virulence genes, out of the seven tested. Respectively, 102 (24.88%) and 20 (86.95%) of the \u003cem\u003eA.\u003c/em\u003e \u003cem\u003ebaumannii\u003c/em\u003e and \u003cem\u003eA. nosocomialis\u003c/em\u003e isolates from the three rivers did not harbour any of the virulence genes tested in this study. However, there was a significant difference (p\u0026lt;0.05) in the prevalence of virulence genes harboured by \u003cem\u003eA. baumannii\u003c/em\u003e compared to \u003cem\u003eA. nosocomialis\u003c/em\u003e, though the population of the former was significantly higher than the latter in all cases.\u003c/p\u003e\n\u003cp\u003eThe prevalence of virulence gene \u003cem\u003eafa/draBC \u003c/em\u003ewas significantly higher (p\u0026lt;0.05) in the isolates from Great Fish river in comparison to Keiskamma and Tyhume rivers. However, there was a significant difference (p\u0026lt;0.05) in prevalence between the \u003cem\u003eafa/draBC \u003c/em\u003egenes haboured by the bacteria recovered from the Tyhume river when compared to those from Keiskamma river. Besides, the prevalence of \u003cem\u003etraT \u003c/em\u003egene in \u003cem\u003eAcinetobacter\u003c/em\u003e species from Great Fish and Tyhume rivers showed no statistical difference (p\u0026lt;0.05), but both were significantly higher (p\u0026lt;0.05) than those detected in isolates from Keiskamma river. Similarly, the prevalence of \u003cem\u003efimH\u003c/em\u003e gene detected in isolates recovered from Great Fish river was significantly higher (p\u0026lt;0.05) than those detected in isolates from both Keiskamma and Tyhune river. Nonetheless, the proportion of isolates harbouring the gene in Keiskamma was significantly (p\u0026lt;0.05) lower than those from Tyhume river. The prevalence of the PAI virulence gene was not significantly (p\u0026lt;0.05) different in all the rivers studied. In Great Fish and Keiskamma rivers, there was no significant difference (p\u0026lt;0.05) in the prevalence of \u0026nbsp;\u003cem\u003esfa/focDE \u003c/em\u003ein isolates recovered from the rivers, but the proportion of \u003cem\u003eAcinetobacter\u003c/em\u003e species in Tyhume harbouring the gene was significantly higher (p\u0026lt;0.05) than in those from both Great Fish and Keiskamma rivers. The number of \u003cem\u003eAcinetobacter\u003c/em\u003e species harbouring \u003cem\u003eepsA \u003c/em\u003evirulence gene in the Great Fish river was significantly higher (p\u0026lt;0.05) than those recovered from Keiskamma river, but significantly lower (p\u0026lt;0.05) than in \u003cem\u003eAcinetobacter\u003c/em\u003e species recovered from Tyhume river. The prevalence of the \u003cem\u003eOmpA\u003c/em\u003e virulence gene was significantly higher (p\u0026lt;0.05) in \u003cem\u003eA. baumannii\u003c/em\u003e isolates than all other virulence genes in this study. Besides, \u003cem\u003eOmpA\u003c/em\u003e was the most prevalent virulence gene in the rivers, which represented 69 (45.10%), 52 (50.98%) and 77 (49.68%) \u003cem\u003eA. baumannii\u003c/em\u003e isolates in Great Fish, Keiskamma and Tyhume rivers respectively. The number of \u003cem\u003eAcienotobacter\u003c/em\u003e species harbouring \u003cem\u003eOmpA\u003c/em\u003e gene in Great fish river were significantly higher (p\u0026lt;0.05) than isolates from Keiskamma river, whereas Tyhume river maintained the highest level of statistical significance (p\u0026lt;0.05) of the total \u003cem\u003eAcinetobacter\u003c/em\u003e species exhibiting the virulence gene.\u003c/p\u003e\n\u003cp\u003eSummarily, \u003cem\u003eOmpA \u003c/em\u003ewas the most prevalent virulence gene detected in \u003cem\u003eAcinetobacter \u003c/em\u003especies in all the three rivers studied followed by \u003cem\u003efimH\u003c/em\u003e and \u003cem\u003eepsA\u003c/em\u003e genes, whereas PAI and \u003cem\u003eSfa/focDE \u003c/em\u003egenes were the least exhibited respectively. Furthermore, the number of \u003cem\u003eAcinetobacter\u003c/em\u003e species habouring the highest percentage of virulence genes was isolated from the Tyhume River followed by the Great Fish River, while Kieskamma River was the least.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBacteria in the genus \u003cem\u003eAcinetobacter \u003c/em\u003ehave been known to colonize a wide array of ecological systems of which water, soil, sludge, wastewater, plants' root, and animals have been prominent. In this study, water samples were collected at five different sites from three selected rivers in the Eastern Cape Province, South Africa for the assessment of the oof \u003cem\u003eAcinetobacter\u003c/em\u003e species. \u0026nbsp;Several presumptive species belonging to the genus \u003cem\u003eAcinetobacter \u003c/em\u003ewere recovered from the water samples. Similar study was carried out by [27] on the microbial community in the urban riverine environment in Croatia, where 57 of the isolates belong to the genus \u003cem\u003eAcinetobacter\u003c/em\u003e. Likewise, Krizova et al. [28], noted a widespread of \u003cem\u003eA. bohemicus \u003c/em\u003ein the water environment in the Czech Republic. \u003cem\u003eA. baumannii\u003c/em\u003e was characterized from a surface water resources in South Nation River (SNR) drainage basin in Eastern Ontario, Canada, in 2013 [26]. Besides, the composition of the bacterial community in a freshwater aquaculture environment was investigated in China by [29]. The study reported that the relative abundance of \u003cem\u003eAcinetobacter\u003c/em\u003e species was 0.5% of the total bacterial community.\u003c/p\u003e\n\u003cp\u003eAll isolates that were positive for the \u003cem\u003erecA\u003c/em\u003e (425 bp) (Figure S1) were taken as belonging to the \u003cem\u003eAcinetobacter \u003c/em\u003egenus, according to [30]. A PCR amplification assay based on \u003cem\u003erecA \u003c/em\u003egene-specific primer, identified 844 \u003cem\u003eAcinetobacter \u003c/em\u003especies of the 1107 presumptive isolates, while \u003cem\u003egyrB \u003c/em\u003egene species-specific primers further delineated them into species namely, \u003cem\u003eA. baumannii \u003c/em\u003e(410) and\u003cem\u003e A. nosocomialis \u003c/em\u003e(23). According to [30], the method used in this study was 98.2% specific and 92.4% sensitive for Comparably, [31] demonstrated a PCR assay targeting \u003cem\u003erecA\u003c/em\u003e gene \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eA. nosocomialis, \u003c/em\u003eand \u003cem\u003eA. pittii\u003c/em\u003e are known nosocomial pathogens, which could cause multiple antibiotic resistant infections [2, 30, 32] in immune-compromised patients [2]. Park and others [33] investigated the presence of \u003cem\u003eAcinetobacter \u003c/em\u003especies in the bloodstream of patients with blood infection in a tertiary-care hospital in Korea between August 2003 and February 2010. Their findings showed that \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eA. nosocomialis\u003c/em\u003e were prevalent in the samples collected. Similarly, [34], recovered 160 \u003cem\u003eA. baumannii\u003c/em\u003e isolates from sputum, blood, pus and fluid aspirates of patients in three Hospitals in Vietnam within a period of two years (between 2012 and 2014). These findings showed that the nosocomial pathogens could colonize any part of the human body to cause infections.\u003c/p\u003e\n\u003cp\u003eHowever, occurrence of \u003cem\u003eA. baumannii \u003c/em\u003eand \u003cem\u003eA. nosocomialis \u003c/em\u003ein an aquatic environment, is uncommon in the past few years. Nonetheless, in recent times, these nosocomial pathogens are isolated from other ecosystems [3, 4, 35]. The isolation of the pathogen from water sources might be due to contamination coming from hospital wastewater and materials.\u003c/p\u003e\n\u003cp\u003eThe virulence traits of \u003cem\u003eA. baumannii \u003c/em\u003eand\u003cem\u003e A. nosocomialis\u003c/em\u003e have been a major research focus in recent times [17, 36]. This is due to the nature of \u003cem\u003eA. baumannii \u003c/em\u003einfections as well as the role virulence genes play in the pathogenicity of the emerging waterborne pathogen. Virulence genes are the mechanisms through which \u003cem\u003eA. baumannii \u003c/em\u003einitiates pathogenesis [36, 37], most especially in the clinical settings.\u003c/p\u003e\n\u003cp\u003eIn this study, molecular assay for the detection of virulence factors in both pathogens was performed. Figure S2 shows the electrophoretic images of the seven virulence factors evaluated. Although, \u003cem\u003eA. baumannii \u003c/em\u003ehad been confirmed to harbour several virulence factors (genes), seven of these genes (\u003cem\u003eafa/draBC\u003c/em\u003e, \u003cem\u003eepsA\u003c/em\u003e, \u003cem\u003efimH\u003c/em\u003e, \u003cem\u003eOmpA\u003c/em\u003e, PAI, \u003cem\u003esfa/focDE\u003c/em\u003e, and \u003cem\u003etraT\u003c/em\u003e) are reported in this study.\u0026nbsp; Reports on the virulence genes in \u003cem\u003eA. baumannii\u003c/em\u003e are usually associated with isolates from clinical environment, whereas such reports are rare on \u003cem\u003eA. baumannii \u003c/em\u003efrom freshwater resources\u003c/p\u003e\n\u003cp\u003eThus, this study showed that \u003cem\u003eOmpA\u003c/em\u003e gene was predominantly exhibited by the \u003cem\u003eA. baumannii \u003c/em\u003ein all the rivers sampled, likewise \u003cem\u003efimH\u003c/em\u003e and \u003cem\u003eepsA \u003c/em\u003egenes were also detected in many of the isolates, whereas \u003cem\u003eafa/draBC\u003c/em\u003e, PAI, \u003cem\u003eSfa/focDE\u003c/em\u003e, and \u003cem\u003etraT\u003c/em\u003e genes were detected in a few \u003cem\u003eAcinetobacter\u003c/em\u003e isolates. As such, the exhibition of virulence genes varies from one isolate to the other, which was also reported among clinical isolates known for nosocomial infections [38]. The \u003cem\u003eOmpA\u003c/em\u003e gene is the main outer membrane protein (OMP) located on the \u003cem\u003eA. baumannii \u003c/em\u003emembrane [39, 40], which [36]. These observations corroborate the findings of this current study which reports that the virulence profiles of an individual isolate varied greatly and \u003cem\u003eOmpA\u003c/em\u003e gene was mostly detected [36].\u003c/p\u003e\n\u003cp\u003eGenerally, the outer membranes of Gram-negative bacteria are made up of the OMPs, lipopolysaccharides and phospholipids layer [37]. The presence of outer membrane protein A gene (\u003cem\u003eOmpA\u003c/em\u003e) in the \u003cem\u003eA. baumanni\u003c/em\u003e isolated from the freshwater source is of a major concern based on its role in pathogenesis [36]. Besides, studies have shown that \u003cem\u003eA. baumannii\u003c/em\u003e uses \u003cem\u003eOmpA\u003c/em\u003e for adhesion to the lung epithelial cell by interacting with a cell cytoskeleton such as fibronectin on the cell surface and thereby inducing pneumonia [41, 42]. It also causes cell death through caspase-3 activation [43, 44]. Similarly, \u003cem\u003eA. baumannii\u003c/em\u003e could be responsible for apoptosis through the translocation of its \u003cem\u003eOmpA\u003c/em\u003e into the mitochondria and the nucleus of host cells [45, 46]. The combination of the roles played by \u003cem\u003eOmpA\u003c/em\u003e makes it an important virulence factor in the pathogenesis of \u003cem\u003eA. baumannii\u003c/em\u003e infection. Moreover, antibiotic resistance in \u003cem\u003eA. baumannii\u003c/em\u003e is also associated with \u003cem\u003eOmpA\u003c/em\u003e [47, 48]. It was suggested that \u003cem\u003eOmpA\u003c/em\u003e was involved in the removal of antibiotics from the periplasmic space membrane efflux systems [48]. The survival and persistence of \u003cem\u003eA. baumannii\u003c/em\u003e in the cell are enhanced by \u003cem\u003eOmpA\u003c/em\u003e due to the formation of biofilms and surface motility.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, three selected freshwater resources in the Eastern Cape Province, South Africa were evaluated for the occurrence, distribution and virulence genes fingerprints of clinically-relevant\u0026nbsp; \u003cem\u003eAcinetobacter \u003c/em\u003especies namely; \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eA. nosocomialis\u003c/em\u003e. This study suggests that these pathogenic \u003cem\u003eAcinetobacter \u003c/em\u003especies could also inhabit aquatic environment. As a result, aquatic environment is an important reservoir for pathogenic \u003cem\u003eAcinetobacter\u003c/em\u003e species as detected in this study. Besides, the \u003cem\u003eOmpA\u003c/em\u003e is a major virulence factor associated with \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eA. nosocomialis\u003c/em\u003e in this study. As such, identification of these opportunistic and virulent waterborne pathogens in the freshwater resources requires public awareness and recognition as important to public health risks.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eDescription of study areas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection of water samples was carried out from three rivers namely; Great Fish, Keiskamma and Tyhume, in the Eastern Cape Province, South Africa between April 2017 and March 2018. The Great Fish River is located in Chris Hani District Municipality in the Eastern Cape Province and it is one of the major rivers used for irrigation and livestock farming in the area. This river is prone to agricultural and municipal runoffs and also serves as the receiving stream of effluents from many wastewater treatment plants (WWTPs), especially those situated in urban communities such as Craddock. Keiskamma and Tyhume rivers are located in the Amathole District Municipality in the Eastern Cape Province and are exposed to different anthropogenic activities from the rural and urban communities along the river courses, such as livestock drinking and irrigation farming.\u0026nbsp; In addition, these rivers receive effluents from wastewater treatment plants (WWTPs) situated close to their banks. Different sampling points on these rivers were selected based on where humans and animals come into direct contact with them, for example, points where they are used for fishing, drinking and swimming purposes, downstream of the WWTPs, points where irrigation water is released to the water bodies and proximity to hospital facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWater samples were collected at five different sites (S1, S2, S3, S4, and S5) from Great Fish, Keiskamma and Tyhume rivers respectively, for a period of one year, which covers the four seasonal patterns in South Africa (autumn, winter, spring, and summer). Water samples were collected aseptically in sterile 1L glass bottles from different sampling points by midstream-dipping of sample bottles at 25\u0026ndash;30 cm down the water column, with the mouth tilting against the flow of the river. All water samples were labelled properly and safely taken to the laboratory (in an ice chest) where they were processed within 6 h of collection [27]. Aliquots of water samples were used for isolation of \u003cem\u003eAcinetobacter \u003c/em\u003especies based on standard microbiological procedures [49]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and purification of presumptive \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eAcinetobacter \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolation of the presumptive density of \u003cem\u003eAcinetobacter \u003c/em\u003especies in the water samples was determined by membrane filtration technique [50]. Cellulose membrane of pore size 0.45 𝜇m was used to filter three volumes of 100 mL of the water samples under vacuum [50]. These membranes were aseptically placed on plates with \u003cem\u003eAcinetobacter \u003c/em\u003especies selective medium-CHROMagar \u003cem\u003eAcinetobacter\u003c/em\u003e base plus selective supplement (CHROMagar, Paris, France) which was prepared according to the manufacturer\u0026rsquo; instruction. Each sample plate was subjected to incubation at 37\u003csup\u003eo\u003c/sup\u003eC for 24 h after inoculation. Each sample was analysed in triplicate. All bacterial colonies with red colouration on the CHROMagar plates were counted as presumptive \u003cem\u003eAcinetobacter\u003c/em\u003e species and were expressed as CFU/100ml. All isolates were sub-cultured on nutrient agar using a streak plate method (Oxoid, UK) and purified for further species identification. Fifty percent (50%) glycerol stocks of the pure culture was prepared and stored at -80 \u003csup\u003e∘\u003c/sup\u003eC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular identification of \u003cem\u003eAcinetobacter \u003c/em\u003especies by PCR assays\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of genomic DNA\u003c/strong\u003e: Presumptive \u003cem\u003eAcinetobacter \u003c/em\u003espp. in glycerol stocks was first being resuscitated on tryptic soy broth and incubated for 18 to 24h at 37 \u003csup\u003e∘\u003c/sup\u003eC. DNA extraction from the bacterial isolates was carried out using the direct boiling method according to [51]. The broth culture was centrifuged at 15000 \u003cem\u003erpm\u003c/em\u003e for 5 min using a Mini Spin Microcentrifuge (Lasec, RSA), then the supernatant was dispensed out and the pellet rinsed with sterile normal saline. The pellet was re-suspended in sterile distilled water and boiled in a heating block for 10 minutes using an AccuBlock (Digital dry bath, Labnet).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmplification of unique \u003cem\u003eAcinetobacter\u003c/em\u003e species DNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolymerase chain reaction (PCR) assay was used for the amplification of the \u003cem\u003eAcinetobacter\u003c/em\u003e species\u003cem\u003e recA \u003c/em\u003egene as previously described [30]. The forward and reverse primers used were P-rA1 (5\u0026prime;-CCTGAATCTTCTGGTAAAAC-3\u0026prime;) and P-rA2 (5\u0026prime;-GTTTCTGGGCTGCCAAACATTAC-3\u0026prime;) respectively. Briefly, an aliquot of 25 \u0026micro;l containing Taq PCR (12.5 \u0026micro;l) Master Mix (Qiagen, Hilden, Germany), each of the primers (1 \u0026micro;l) (Inqaba, SA), nuclease-free water (6.5 \u0026micro;l) and DNA template (5 \u0026micro;l) was used for the PCR amplification assay. The condition for the amplification included initial denaturation step (94 \u003csup\u003eo\u003c/sup\u003eC, 5 min), followed by 35 cycles (92 \u003csup\u003eo\u003c/sup\u003eC, 40 s), annealing (58 \u003csup\u003eo\u003c/sup\u003eC, 40 s), and the final extension step (72\u003csup\u003eo\u003c/sup\u003eC, 10 min) was performed using a thermocycler (Bio-Rad Thermal cycler, USA). Five microlitres (5 \u0026micro;l) of the amplicon was subjected to gel (1.5% agarose) electrophoresis at 100 Volts for 45 min in Tris Boric EDTA buffer (pH 8.0) (0.089 M Tris, 0.089 M boric acid, and 0.002 M EDTA). Ethidium bromide (5 \u0026micro;l of 0.5 mg/ml) (Sigma-Aldrich, USA) was used for gel staining and DNA ladder (100 bp) (Thermo Scientific, (EU) Lithuania) was added into the gels as a standard. Finally, DNA bands were visualized under an ultraviolet transilluminator (Alliance 4.7, France).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDelineation of Genus \u003cem\u003eAcinetobacter \u003c/em\u003einto species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe confirmed \u003cem\u003eAcinetobacter\u003c/em\u003e isolates were delineated into species accordingly [30, 52, 53] using a PCR assay. Firstly, optimization of conditions for \u003cem\u003eA. baumannii\u003c/em\u003e,\u003cem\u003e A. nosocomialis\u003c/em\u003e and \u003cem\u003eA. pittii\u003c/em\u003e was carried out using species-specific primers (Table S1 under supplementary Section), while reference strains DSM-102929, DSM-102856, and DSM-9341 (DSMZ, Germany) were used as positive controls for \u003cem\u003eA. baumannii\u003c/em\u003e,\u003cem\u003e A. nosocomialis\u003c/em\u003e and \u003cem\u003eA. pittii\u003c/em\u003e respectively. \u0026nbsp;The PCR amplification was performed as stated in the previous section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of virulence genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolymerase chain reactions were also carried out for the identification of some \u003cem\u003eAcinetobacter\u003c/em\u003e virulence genes including \u003cem\u003eafa/draBC, epsA, fimH, OmpA\u003c/em\u003e, PAI, \u003cem\u003esfa/focDE\u003c/em\u003e, and \u003cem\u003etraT\u003c/em\u003e genes, which have been previously found in clinical samples [54, 55, 56]. The standard strain of \u003cem\u003eA. baumannii\u003c/em\u003e DSM-30007 (DSMZ, Germany) was used as positive control. There was no positive control available for \u003cem\u003eA. nosocomialis\u003c/em\u003e. The PCR assay and electrophoresis were conducted as earlier described.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using the Statistica software v13.4.0.14 (64-bits). A simple factorial ANOVA was performed for the comparison of normally distributed data. The \u003cem\u003ep\u003c/em\u003e-values of less than 0.05 were considered statistically significant for all the statistical tests performed.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACB: \u003cem\u003eAcinetobacter\u003c/em\u003e \u003cem\u003ecalcoaceticus- baumannii \u003c/em\u003ecomplex;\u003c/p\u003e\n\u003cp\u003eANOVA: Analysis of Variance;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAPHA: American Public Health Association;\u003c/p\u003e\n\u003cp\u003eCFU: Colony Forming Unit;\u003c/p\u003e\n\u003cp\u003eCHROMagar: Chromogenic agar;\u003c/p\u003e\n\u003cp\u003eDNA: Deoxyribonucleic Acid;\u003c/p\u003e\n\u003cp\u003eEDTA: Ethylenediamine tetraacetic acid;\u003c/p\u003e\n\u003cp\u003eEspA: Exopolysaccharide A;\u003c/p\u003e\n\u003cp\u003eEU: European Union;\u003c/p\u003e\n\u003cp\u003eICU: Intensive care unit;\u003c/p\u003e\n\u003cp\u003eL: Ladder;\u003c/p\u003e\n\u003cp\u003eLPS: Lipopolysaccharides;\u003c/p\u003e\n\u003cp\u003eN: Negative control;\u003c/p\u003e\n\u003cp\u003eND: Not determined;\u003c/p\u003e\n\u003cp\u003eNRF: National Research Foundation;\u003c/p\u003e\n\u003cp\u003eompA: Outer membrane protein A precursor;\u003c/p\u003e\n\u003cp\u003eOMVs: Outer membrane vesicles;\u003c/p\u003e\n\u003cp\u003eP: Positive control;\u003c/p\u003e\n\u003cp\u003ePAI: \u0026nbsp;Pathogenicity Island;\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction;\u003c/p\u003e\n\u003cp\u003eRSA: Republic of South Africa;\u003c/p\u003e\n\u003cp\u003eSAMRC: South African Medical Research Council;\u003c/p\u003e\n\u003cp\u003eTWAS: The World Academy of Science;\u003c/p\u003e\n\u003cp\u003eUFH: University of Fort Hare;\u003c/p\u003e\n\u003cp\u003eUK: United Kingdom;\u003c/p\u003e\n\u003cp\u003eUSA: United States of America;\u003c/p\u003e\n\u003cp\u003eWWTPs: Wastewater treatment plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection of freshwater samples for research purposes in this region does not require governmental permission since freshwater resources investigated are within the University jurisdiction. Also, the need for ethical approval was waived by the Research Ethics Committee, University of Fort Hare, because samples analysed in this study do not require human or animal subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was funded by the National Research Foundation, South Africa/The World Academy of Science, Italy (NRF/TWAS) (grant numbers:\u0026nbsp; 99767\u0026nbsp;and 116387) and the South African Medical Research Council (grant number: SAMRC/UFH/P790). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAA\u003c/strong\u003e conceived and designed the experiment, collected the water samples, isolated the \u003cem\u003eAcinetobacter\u003c/em\u003e spp., performed the experiments, data and statistical analyses, and writing of the manuscripts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAIO\u003c/strong\u003e supervised the research, provided financial support, and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDe Vos D, Jean-Paul P, Florence B, Serge J, Gilbert V, Thomas R, Elkana K, Petra B, Thierry P, Mony H, Walter H, Frank DP, Patrick S, Maia M, Pieter D, Mario V, Pierre B, Youri G, Bruno P, Tanny JR, Dijkshoorn L. Molecular Epidemiology and Clinical Impact of \u003cem\u003eAcinetobacter calcoaceticus-baumannii \u003c/em\u003ecomplex in a Belgian Burn Wound Center. PLoS ONE. 2016; 11(5): e0156237.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"2\"\u003e\n\u003cli\u003ePeleg AY, Seifert H, Paterson DL. \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e: emergence of a successful pathogen. Clin Microbiol Rev. 2008; 21, 538\u0026ndash;582.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"3\"\u003e\n\u003cli\u003eHamouda A, Findlay J, Al Hassan L, Amyes SG. Epidemiology of \u003cem\u003eAcinetobacter baumannii \u003c/em\u003eof animal origin. J. Antimicrob. Agents. 2011; 38, 314\u0026ndash;318.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"4\"\u003e\n\u003cli\u003eHuys G, Bartie K, Cnockaert M, Hoang Oanh DT, Phuong NT, Somsiri T, Chinabut S, Yussoff FD, Shariff M, Giacomini M, Teale A, Swings J. Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments. Microbiol. 2007; 158, 228\u0026ndash;235. doi:10.1016/j.resmic.2006.12.011.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"5\"\u003e\n\u003cli\u003eHrenovic J, Durn G, Goic-Barisic I, Kovacic A. Occurrence of an Environmental \u003cem\u003eAcinetobacter baumannii \u003c/em\u003eStrain Similar to a Clinical Isolate in Paleosol from Croatia. Applied and Environmental Microbiology, 2014; 80(6): 2860\u0026ndash;2866.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"6\"\u003e\n\u003cli\u003eAlmasaudi SB. \u003cem\u003eAcinetobacter\u003c/em\u003e as nosocomial pathogens: Epidemiology and resistance features. Saudi Journal of Biological Sciences. 2018; 25, 586\u0026ndash;596.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"7\"\u003e\n\u003cli\u003eJung J, Park W. Acinetobacter species as model microorganisms in environmental microbiology: current state and perspectives. Appl. Microbiol. Biotechnol. 2015; 99, 2533\u0026ndash;2548.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"8\"\u003e\n\u003cli\u003eDoughari HJ, Ndakidemi PA, Human IS, Benade S. The ecology, biology and pathogenesis of Acinetobacter spp.: an overview. Microbes Environ\u003cem\u003e.\u003c/em\u003e 2011; 26, 101\u0026ndash;112.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"9\"\u003e\n\u003cli\u003eWhitman TJ, Qasba SS, Timpone JG, Babel BS, Kasper MR, English JF, Sanders JW, Hujer KM, Hujer AM, Endimiani A. Occupational transmission of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e from a United States serviceman wounded in Iraq to a health care worker\u003cem\u003e. \u003c/em\u003e Infect. Dis. 2008; 47, 439\u0026ndash;443.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"10\"\u003e\n\u003cli\u003eGarnacho-Montero J, Amaya-Villar R, Ferrandiz-Millon C, Dıaz-Martın A, Lopez-Sanchez JM, Gutierrez-Pizarraya A. Optimum treatment strategies for carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Expert Rev. Anti. Infect. Ther. 2015; 13(6): 769-77.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"11\"\u003e\n\u003cli\u003eFalagas ME, Vardakas KZ, Kapaskelis A, Triarides NA, Roussos NS. Tetracyclines for multidrug-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e International Journal of Antimicrobial Agents. 2015; 45, 455\u0026ndash;460.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"12\"\u003e\n\u003cli\u003eBasri R, Zueter AR, Mohamed Z, Alam MK, Norsa\u0026rsquo;adah B, Hasan SA, Hasan H, Ahmad F. Burden of bacterial meningitis: a retrospective review on laboratory parameters and factors associated with death in meningitis, Kelantan Malaysia. Nagoya J. Med. Sci. 2015; 77: 59-68.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"13\"\u003e\n\u003cli\u003eLai HH, Liou BH, Chang YY, Kuo SC, Lee YT, Chen TL, Fung CP. Risk factors and clinical outcome of sulbactam non-susceptibility in monomicrobial \u003cem\u003eAcinetobacter nosocomialis\u003c/em\u003e Journal of Microbiology, Immunology and Infection. 2016; 49, 371-377.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"14\"\u003e\n\u003cli\u003eLiu YM, Lee YZ, Kuo SC, Chen TL, Liu CP, Liu CE. Comparison between bacteremia caused by \u003cem\u003eAcinetobacter pittii\u003c/em\u003e and \u003cem\u003eAcinetobacter nosocomialis\u003c/em\u003e. Journal of Microbiology, Immunology and Infection. 2017; 50, 62\u0026ndash;67.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"15\"\u003e\n\u003cli\u003eLee YT, Kuo SC, Yang SP, Lin YT, Chiang DH, Tseng FC, Chen TL, Fung CP. Bacteremic nosocomial pneumonia caused by \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and \u003cem\u003eAcinetobacter nosocomialis\u003c/em\u003e: a single or two distinct clinical entities. European Society of Clinical Microbiology and Infectious Diseases, CMI. 2012; 19, 640\u0026ndash;645.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"16\"\u003e\n\u003cli\u003eNho JS, Jun SH, Oh MH, Park TI, Choi CW, Kim S, Choi CH, Lee JC. \u003cem\u003eAcinetobacter nosocomialis\u003c/em\u003e secretes outer membrane vesicles that induce epithelial cell death and host inflammatory responses. Microbial Pathogenesis. 2015; 81, 39-45.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"17\"\u003e\n\u003cli\u003eLee CR, Lee JH, Park M, Park KS, Bae IK, Kim YB, Cha CJ, Jeong BC, Lee SH. Biology of \u003cem\u003eAcinetobacter baumannii:\u003c/em\u003e Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options. Frontiers in Cellular Infection Microbiology. 2017; \u003cem\u003e7\u003c/em\u003e(March) \u003ca href=\"https://doi.org/10.3389/fcimb.2017.00055\"\u003ehttps://doi.org/10.3389/fcimb.2017.00055\u003c/a\u003e.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"18\"\u003e\n\u003cli\u003eKurcik-Trajkovska B. \u003cem\u003eAcinetobacter \u003c/em\u003e \u0026ndash; a serious enemy threatening hospitals worldwide. Macedonian Journal of Medical Science. 2009; 2, 157\u0026ndash;162.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"19\"\u003e\n\u003cli\u003eKanafani AZ, Kanj SS. Ministry of Health, Kingdome of Saudi Arabia. 2014. \u003ca href=\"http://www.uptodate.com/contents/acinetobacterinfection-treatment-and-prevention\"\u003ehttp://www.uptodate.com/contents/acinetobacterinfection-treatment-and-prevention\u003c/a\u003e.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"20\"\u003e\n\u003cli\u003eKim SW, Oh MW, Jun SH, Jeon H, Kim S, Kim K, Lee YC, Lee JC. Outer Membrane Protein A plays a role in pathogenesis of \u003cem\u003eAcinetobacter nosocomialis\u003c/em\u003e. Virulence. 2016; 7:4, 413-426, DOI: 10.1080/21505594.2016.1140298.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"21\"\u003e\n\u003cli\u003eEllis TN, Kuehn MJ. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiology and Molecular Biology Review. 2010; \u003cem\u003e74\u003c/em\u003e, 81\u0026ndash;94. doi: 10.1128/MMBR.00031-09.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"22\"\u003e\n\u003cli\u003eKwon SO, Gho YS, Lee JC, Kim SI. Proteome analysis of outer membrane vesicles from a clinical \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e FEMS Microbiology Letter. 2009; 297, 150\u0026ndash;156. doi: 10.1111/j.1574-6968.2009.01669.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"23\"\u003e\n\u003cli\u003eJun SH, Lee JH, Kim BR, Kim SI, Park TI, Lee JC, Lee YC. \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e outer membrane vesicles elicit a potent innate immune response via membrane proteins. PLoS ONE. 2013; 8, e71751. doi: 10.1371/journal.pone.0071751.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"24\"\u003e\n\u003cli\u003eFiester SE, Arivett BA, Schmidt RE, Beckett AC, Ticak T, Carrier MV, Ghosh R, Ohneck EJ, Metz ML, Jeffries MKS, Actis LA. Iron-regulated phospholipase C activity contributes to the cytolytic activity and virulence of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. PLoS ONE. 2016; 11:e0167068. doi: 10.1371/journal.pone.0167068.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"25\"\u003e\n\u003cli\u003eDiancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. The population structure of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS ONE. 2010; e10034.doi: 10.1371/journal.pone.0010034.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"26\"\u003e\n\u003cli\u003eFernando DM, Khan IUH, Patidar R, Lapen DR,Talbo G, Topp E, Kumar A. Isolation and Characterization of \u003cem\u003eAcinetobacter baumannii \u003c/em\u003erecovered from Campylobacter Selective Medium. Frontiers in Microbiology; 2016; 7:1871.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"27\"\u003e\n\u003cli\u003eMaravic A, Skocibusic, M, Fredotovic Z, Samanic I, Cvjetan S, Knezovic M, Puizina J. Urban riverine environment is a source of multidrug-resistant and ESBL-producing clinically important \u003cem\u003eAcinetobacter\u003c/em\u003e Environ Sci Pollut Res. 2016; 23, 3525\u0026ndash;3535.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"28\"\u003e\n\u003cli\u003eKrizova L, Maixnerovaa M, Sedob O, Nemec A. \u003cem\u003eAcinetobacter bohemicus\u003c/em\u003e nov.wide spread in natural soil and water ecosystems in the Czech Republic.\u003cem\u003eSystematic and Applied Microbiology\u003c/em\u003e. 2014; 37, 467\u0026ndash;473.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"29\"\u003e\n\u003cli\u003eXiong W, Sun Y, Zhang T, Ding X, Li Y, Wang M, Zeng Z. Antibiotics, Antibiotic Resistance Genes, and Bacterial Community Composition in Fresh Water Aquaculture Environment in China. Microb Ecol. 2015; 70:425\u0026ndash;432.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"30\"\u003e\n\u003cli\u003eChen TL, Lee YT, Kuo SC, Yang SP, Fung CP, Lee SD. Rapid identification of \u003cem\u003eAcinetobacter baumannii, Acinetobacter nosocomialis\u003c/em\u003e and \u003cem\u003eAcinetobacter pittii\u003c/em\u003e with a multiplex PCR assay. Journal of Medical Microbiology, 2014; 63, 1154\u0026ndash;1159.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"31\"\u003e\n\u003cli\u003eChiang MC, Kuo SC, Chen YC, Lee YT, Chen TL, Fung CP. Polymerase chain reaction assay for the detection of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e in endotracheal aspirates from patients in the intensive care unit. Journal of Microbiology, Immunology and Infection. 2011; 44, 106\u0026ndash;110.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"32\"\u003e\n\u003cli\u003eDijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multidrug-resistant \u003cem\u003eAcinetobacter baumannii. \u003c/em\u003eNat Rev Microbiol. 2007; 5, 939\u0026ndash;951.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"33\"\u003e\n\u003cli\u003ePark YK, Jung SI, Park KH, Kim DH, Choi JY, Kim SH, Ko KS. Changes in antimicrobial susceptibility and major clones of \u003cem\u003eAcinetobacter calcoaceticus\u003c/em\u003e\u0026ndash;\u003cem\u003ebaumannii\u003c/em\u003e complex isolates from a single hospital in Korea over 7 years. \u003cem\u003eJournal of Medical Microbiology\u003c/em\u003e. 2012; 61(1) 71\u0026ndash;79.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"34\"\u003e\n\u003cli\u003eAnh NT, Thieu Nga TV, Tuan HM, Tuan NS, Dao Y, Chau NVV, Baker S, Duong HHT. Molecular epidemiology and antimicrobial resistance phenotypes of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolated from patients in three hospitals in southern Vietnam.\u003cem\u003e Journal of Medical Microbiology\u003c/em\u003e. 2017; 66:46\u0026ndash;53.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"35\"\u003e\n\u003cli\u003eBerlau J, Aucken HM, Houang E, Pitt TL. Isolation of Acinetobacter spp including \u003cem\u003e baumannii\u003c/em\u003e from vegetables: implications for hospital-acquired infections. Journal of Hospital Infection. 1999; 42: 201\u0026ndash;204.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"36\"\u003e\n\u003cli\u003eAmbrosi C, Scribano D, Aleandri M, Zagaglia C, Di Francesco L, Putignani L, Palamara AT. \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Virulence Traits: A Comparative Study of a Novel Sequence Type with Other Italian Endemic International Clones. Front. Microbiol. 2017; 8:1977. doi: 10.3389/fmicb.2017.01977.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"37\"\u003e\n\u003cli\u003eSato Y, Unno Y, Kawakami S, Ubagai T, Ono Y. Virulence characteristics of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e clinical isolates vary with the expression levels of omps. Journal of Medical Microbiology. 2017; 66:203\u0026ndash;212.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"38\"\u003e\n\u003cli\u003eAntunes LCS, Imperi F, Carattoli A, Visca P. Deciphering the Multifactorial Nature of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e PLoS ONE. 2011; 6(8): e22674. doi:10.1371/journal.pone.0022674.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"39\"\u003e\n\u003cli\u003eSmith MG, Gianoulis TA, Pukatzki S, Mekalanos JJ, Ornston LN, Gerstein M, Snyder M. New insights into \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes and Development. 2007; 21:601\u0026ndash;614.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"40\"\u003e\n\u003cli\u003eMcConnell MJ, Actis L, Pachon J. \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e: human infections, factors contributing to pathogenesis and animal models. FEMS Microbiol Rev. 2013; 37: 130\u0026ndash;155.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"41\"\u003e\n\u003cli\u003eGaddy JA, Tomaras AP, Actis LA. The \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e 19606 \u003cem\u003eOmpA \u003c/em\u003eprotein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infection and Immunity. 2009; 77(8), 3150\u0026ndash;3160.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"42\"\u003e\n\u003cli\u003eSmani Y, Dominguez-Herrera J, Pachon J. Association of the outer membrane protein Omp33 with fitness and virulence of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. Journal of Infectious Disease. 2013; 208, 1561\u0026ndash;1570. doi: 10.1093/infdis/jit386.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"43\"\u003e\n\u003cli\u003eLee JC, Oh JY, Kim KS, Jeong YW, Park JC, Cho JW. Apoptotic cell death induced by Acinetobacter baumannii in epithelial cells through caspase-3 activation. APMIS\u003cem\u003e.\u003c/em\u003e 2001; 109:679\u0026ndash;684.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"44\"\u003e\n\u003cli\u003eSmani Y, Domnguez-Herrera J, Pachon J. Rifampin protects human lung epithelial cells against cytotoxicity induced by clinical multi and pandrug-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. European Congress of Clinical Microbiology and Infectious Diseases. 2011; Vienna, Austria, April 2010.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"45\"\u003e\n\u003cli\u003eChoi CH, Lee EY, Lee YC, Park TI, Kim HJ, Hyun SH, Kim SA,\u0026nbsp;Lee SK,\u0026nbsp;Lee JC. Outer membrane protein 38 of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e localizes to the mitochondria and induces apoptosis of epithelial cells. Cellular Microbiology. 2005; 7, 1127\u0026ndash;1138. doi: 10.1111/j.1462-5822.2005.00538.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"46\"\u003e\n\u003cli\u003eLee JS, Choi CH, Kim JW, Lee JC. \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e outer membrane protein A induces dendritic cell death through mitochondrial targeting. Journal of Microbiology, 2010; 48, 387\u0026ndash;392. doi: 10.1007/s12275-010-0155-1.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"47\"\u003e\n\u003cli\u003eSugawara E, Nikaido H. \u003cem\u003eOmpA\u003c/em\u003e is the principal nonspecific slow porin of \u003cem\u003eAcinetobacter baumannii. \u003c/em\u003e Bacteriol\u003cem\u003e.\u003c/em\u003e 2012; 194, 4089\u0026ndash;4096. doi: 10.1128/JB.00435-12.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"48\"\u003e\n\u003cli\u003eSmani Y, Fabrega A, Roca I, Sanchez-Encinales V, Vila J, Pachon J. Role of \u003cem\u003eOmpA\u003c/em\u003e in the multidrug resistance phenotype of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. Antimicrob. Agents Chemother. 2014; 58, 1806\u0026ndash;1808. doi: 10.1128/AAC.02101-13.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"49\"\u003e\n\u003cli\u003eSibanda T, Chigor VN, Koba S, Obi CL, Okoh AI. Characterisation of the physicochemical qualities of a typical rural-based river: ecological and public health implications. Int. J. Environ. Sci. Technol. 2014; 11:1771\u0026ndash;1780.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"50\"\u003e\n\u003cli\u003eAmerican Public Health Association, APHA. Standard Methods for the Examination of Water and Wastewater, 20th Ed.; (APHA): Washington, DC, USA. 2005.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"51\"\u003e\n\u003cli\u003eMaugeri TL, Carbone M, Fera MT, Gugliandolo C. Detection and differentiation of \u003cem\u003eVibrio vulnificus\u003c/em\u003e in seawater and plankton f a coastal zone of the Mediterranean Sea. Research in Microbiology. 2006; 157(2), 194\u0026ndash;200.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"52\"\u003e\n\u003cli\u003eChen TL, Siu LK, Wu RCC, Shaio MF, Huang LY, Fung CP, Lee CM, Cho WL. Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS) sequencing for rapid identification of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. Clin Microbiol Infect; 2007; 13: 801\u0026ndash;806.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"53\"\u003e\n\u003cli\u003eHiggins PG, Wisplinghoff H, Krut O, Seifert H. A PCR-based method to differentiate between \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e genomic species 13TU. Clin Microbiol Infect. 2007; 13:1199\u0026ndash;1201.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"54\"\u003e\n\u003cli\u003eThummeepak R, Kongthai P, Leungtongkam U, Sitthisak S. Distribution of virulence genes involved in biofilm formation in multi-drug resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e clinical isolates. International Microbiology. 2016; 19:121-129 doi:10.2436/20.1501.01.270.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"55\"\u003e\n\u003cli\u003eMomtaz H, Seifati SM, Tavakol M. Determining the prevalence and detection of the most prevalent virulence genes in \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolated from hospital infections.\u0026nbsp;Int J Med Lab. 2015; 2(2):87\u0026ndash;97.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"56\"\u003e\n\u003cli\u003eBraun G, Vidotto MC. Evaluation of Adherence, Hemagglutination, and Presence of Genes Codifying for Virulence Factors of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Causing Urinary Tract Infection. Mem Inst Oswaldo Cruz, Rio de Janeiro, 2004; \u003cem\u003e99\u003c/em\u003e(8): 839-844.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style=\"text-align: justify; line-height: 150%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 150%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 150%; font-family: 'Times New Roman',serif;\"\u003eTable 1: Summary of the relative abundance of the genus \u003cem\u003eAcinetobacter\u003c/em\u003e and two clinically important species in the freshwater studied\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\" width=\"660\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 31.0pt;\"\u003e\n\u003ctd style=\"width: 1.0in; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt; height: 31.0pt;\" rowspan=\"2\" width=\"96\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eRivers sampled\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76.5pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt; height: 31.0pt;\" rowspan=\"2\" width=\"102\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ePresumptive isolates\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt; height: 31.0pt;\" rowspan=\"2\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAcinetobacter\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003egenus\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 184.5pt; border: none; border-top: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 31.0pt;\" colspan=\"2\" width=\"246\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eDelineated \u003cem\u003eAcinetobacter \u003c/em\u003especies\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt; height: 31.0pt;\" rowspan=\"2\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eOther species (ND)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.5pt;\"\u003e\n\u003ctd style=\"width: 94.5pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.5pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA.\u0026nbsp; baumannii\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.25in; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.5pt;\" width=\"120\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA. nosocomialis\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 21.75pt;\"\u003e\n\u003ctd style=\"width: 1.0in; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"96\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eGreat Fish \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"102\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e370\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e285(77.01%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e153(53.68%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.25in; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"120\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e16(5.61%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e116(40.70%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 21.75pt;\"\u003e\n\u003ctd style=\"width: 1.0in; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"96\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eKeiskamma\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"102\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e309\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e219(70.87%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e102(46.58%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.25in; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"120\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e3(1.37%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e114(52.06%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 21.0pt;\"\u003e\n\u003ctd style=\"width: 1.0in; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.0pt;\" width=\"96\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eTyhume\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.0pt;\" width=\"102\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e428\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.0pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e340(79.44%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.0pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e155(45.59%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.25in; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.0pt;\" width=\"120\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e4(1.18%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 21.0pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e181(53.24%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 21.75pt;\"\u003e\n\u003ctd style=\"width: 1.0in; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"96\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eTotal\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76.5pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"102\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1107\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e844(76.24%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.5pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e410(48.58%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.25in; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"120\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e23(2.73%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 81.0pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 21.75pt;\" width=\"108\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e411(48.70%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"text-align: justify; line-height: 150%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 150%; font-family: 'Times New Roman',serif;\"\u003eND= not determined\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 150%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"color: windowtext;\"\u003eTable 2: Virulence gene factors of \u003cem\u003eA.\u003c/em\u003e\u003cem\u003ebaumannii\u003c/em\u003e and \u003cem\u003eA. nosocomialis\u003c/em\u003e isolates in addition to which river they belong to.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"color: windowtext;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\" width=\"942\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" rowspan=\"3\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: justify; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eVirulence gene\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 634.95pt; border: none; border-top: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" colspan=\"6\" width=\"847\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eSample sites\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 211.95pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" colspan=\"2\" width=\"283\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eGreat Fish\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 211.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" colspan=\"2\" width=\"282\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eKieskamma\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 211.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" colspan=\"2\" width=\"282\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eTyhume\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 99.45pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"133\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-indent: -.25in; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA.\u003cspan style=\"font: 7.0pt 'Times New Roman';\"\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ebaumannii\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e(n=153)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; text-indent: -.25in; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA.\u003cspan style=\"font: 7.0pt 'Times New Roman';\"\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003enosocomialis\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e(n=16)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; text-indent: -.25in; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA.\u003cspan style=\"font: 7.0pt 'Times New Roman';\"\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ebaumannii\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e(n=102)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; text-indent: -.25in; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA.\u003cspan style=\"font: 7.0pt 'Times New Roman';\"\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003enosocomialis\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e(n=3)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; text-indent: -.25in; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA.\u003cspan style=\"font: 7.0pt 'Times New Roman';\"\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ebaumannii\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e(n=155)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; text-indent: -.25in; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eA.\u003cspan style=\"font: 7.0pt 'Times New Roman';\"\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003enosocomialis\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e(n=4)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 40.0pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 40.0pt;\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAfa/draBC\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 40.0pt;\" width=\"133\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e16(10.46%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 40.0pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 40.0pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e4 (3.92%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 40.0pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 40.0pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e8 (5.81)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 40.0pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1 (25%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eespA\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"133\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e36 (23.53%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e15 (14.71%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e44 (28.39%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1 (25%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003efimH\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"133\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e44 (28.76%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e26 (25.49%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1(33.33%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e30 (19.36%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1 (25%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eOmpA\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"133\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e69 (45.10%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e52 (50.98%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e77 (49.68%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003ePAI\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"133\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e2 (1.31%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1(6.26%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e3 (2.94%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eSfa/focDE\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"133\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e3 (1.96%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e4 (3.92%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 99.0pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"132\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e7 (4.52%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 112.5pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"150\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 6.75pt;\"\u003e\n\u003ctd style=\"width: 71.55pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 6.75pt;\" width=\"95\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; 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In this study, we assessed the occurrence of A. baumannii and A. nosocomialis , which are the major two clinically important species of the genus Acinetobacter , in three freshwater resources (Great Fish, Keiskemma, and Tyhume rivers) in the Eastern Cape Province, South Africa over a one year sampling regime (April 2017 - March 2018). Presumptive Acinetobacter species were subjected to molecular identification by using Acinetobacter genus-specific primers targeting the recA gene. The confirmed Acinetobacter species were further delineated into A. baumannii and A. nosocomialis using species-specific primer sets. Similarly, virulence genes, namely; afa/draBC, epsA, fimH, OmpA, PAI, sfa/focDE , and traT in the two Acinetobacter species were also determined using molecular techniques. \u003c/p\u003e\u003cp\u003eResult : A total of 1107 presumptive Acinetobacter isolates were recovered from the freshwater resources of which 844 was confirmed positive for the Acinetobacter genus. Of the 844 Acinetobacter isolates, 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great Fish, Keiskemma and Tyhume rivers respectively. Our finding revealed that 410 (48.58%) and 23 (2.7%) of the isolates were confirmed to be A. baumannii and A. nosocomalis , respectively. The presence of these clinically-important Acinetobacter species in the freshwater studied suggests possible contamination of the selected rivers and also that A. baumannii and A. nosocomialis can thrive in aquatic environments. Besides, 308 (75.12%) A. baumannii and 3 (13.04%) A. nosocomialis isolates exhibited one or more virulence genes out of the seven tested, whereas 102 (24.88%) and 20 (86.95%) of the A. baumannii and A. nosocomialis isolates did not harbour any virulence gene. Additionally, OmpA was the most prevalent (p\u0026lt;0.05) virulence gene in A. baumannii with 69 (45.10%), 52 (50.98%) and 77 (49.68%) isolates in Great Fish, Keiskamma and Tyhume rivers respectively. \u003c/p\u003e\u003cp\u003eConclusion : The occurrence of these pathogens in rivers which are consumed by humans and livestock, as well as being used for irrigation system constitutes a risk to public health. Keywords: Freshwater resources, Molecular characterisation, Acinetobacter species, virulence gene s.\u003c/p\u003e","manuscriptTitle":"Occurrence, distribution and virulence factors of clinically important Acinetobacter species recovered from selected freshwater resources in the Eastern Cape Province, South Africa","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-01-27 21:37:52","doi":"10.21203/rs.2.14951/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2019-09-25 00:40:36","doi":"10.21203/rs.2.14951/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3a81f3e-c934-43a5-9caa-37315f4c2f56","owner":[],"postedDate":"January 27th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28226,"name":"General Microbiology"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2020-01-27 21:37:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-5622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-5622","version":["v2"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}