Increased biofilm-associated Carbapenem-resistant Acinetobacter- calcoaceticus-baumannii complex infections among the hospitalized patients in Kathmandu Model Hospital, Nepal

Increased biofilm-associated Carbapenem-resistant Acinetobacter- calcoaceticus-baumannii complex infections among the hospitalized patients in Kathmandu Model Hospital, Nepal | 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 Increased biofilm-associated Carbapenem-resistant Acinetobacter- calcoaceticus-baumannii complex infections among the hospitalized patients in Kathmandu Model Hospital, Nepal Shova Bhandari, Milan Kumar Upreti, Khadga Bikram Angbuhang, Basudha Shrestha, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4413953/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Background Acinetobacter calcoaceticus-baumannii complex (ACBC) has emerged as a global burden to various clinical infections. The increasing resistance to the majority of antibiotics adds a huge problem in empirical therapy and control of ACBC infections. In addition, the ability of ACBC to produce biofilm has not only contributed to its antimicrobial resistance but also helped its persistence and survival in the environment. Most tertiary care hospitals in Nepal are facing similar ACBC infections. Methods Hence, this hospital-based cross-sectional study was carried out to associate the biofilm formation with carbapenem-resistant ACBC isolates detecting biofilm-forming genes Bap , csuE , and bla PER1 from February 2020 to August 2020 at Kathmandu Model Hospital, Kathmandu, Nepal. The clinical bacterial isolates were identified by standard Microbiological procedures including Gram staining, and cultural and biochemical characteristics. A modified Kirby-Bauer disk diffusion method was performed to assay the antibiotic susceptibility testing of ACBC isolates to various antibiotic classes. A quantitative adherence assay was used to determine the biofilm assay. A conventional Polymerase Chain Reaction (PCR) method was used to find the targeted biofilm-related genes using specific primers. Results Out of 665 different clinical samples, bacterial growth was observed in 281 (42.3%) clinical samples. Of these, 32 (11.4%) isolates were identified as ACBC. Out of 32 ACBC isolates, 29 (90.6%) of which were carbapenem-resistant. All carbapenem-resistant ACBC isolates were found to be sensitive to polymixin B and colistin. Out of 29 CR-ACBC, 17.2% of isolates were resistant to tigecycline. A total of 31 ACBC isolates were biofilm producers, out of which 2 were strong biofilm producers followed by 8 moderate, and 21 were weak biofilm producers. The occurrence of biofilm-forming genes; Bap , csuE , and bla PER1 genes were found to be 65.6%, 65.6%, and 56.3% respectively among ACBC clinical isolates. A significant association was observed between carbapenem resistance, biofilm formation, and biofilm-related genes. Conclusion Since ACBC isolates are ubiquitous including in the hospital environment and its infections are alarming to clinical settings, the effective sterilization of clinical equipment and hospital environment are utmost. In addition, a strong policy should be made to prescribe the proper antibiotic based on antibiogram profile to fight against an emerging threat of ACBC infections Acinetobacter baumannii Biofilm Carbapenem-resistant Biofilm-related genes Bap csuE and blaPER1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Acinetobacter calcoaceticus-baumannii complex (ACBC) is a Gram-negative commensal bacterium that often infects immunocompromised patients or patients with indwelling devices, especially in the intensive care unit (ICU) [ 1 ]. It causes a wide range of hospital-acquired infections such as respiratory tract infections, urinary tract infections, bacteremia, sepsis, endocarditis, meningitis, skin and soft tissue infections, burns as well as central and nervous system infections [ 2 ]. A. baumannii infections have become a public health threat due to the rapid rise in the incidence of multidrug-resistant (MDR) strains [ 3 ]. Carbapenem are choice of drug to treat severe infections caused by MDR A. baumannii due to its good activity and low toxicity [ 4 ]. However, the prevalence of carbapenem-resistant A. baumannii has increased globally limiting the treatment options and leading to increased mortality of patients [ 5 ]. The pathogen developed resistance to carbapenems due to the acquisition of antibiotic-resistant genes, decreased permeability, altered penicillin-binding protein, overexpression of efflux pump, production of carbapenemase enzyme mainly class B metallo beta-lactamases (MBLs) and class D oxacillinase and biofilm formation [ 5 – 6 ]. World Health Organization (WHO) has listed carbapenem-resistant A. baumannii as a top priority pathogen requiring additional study and development of new antibiotics in 2017 [ 7 ]. A. baumannii is an emerging pathogen with the ability to produce a biofilm that mostly causes device-related infections such as ventilator-associated pneumonia and catheter-related infections [ 8 ]. The bacteria inside the biofilm are shielded by EPS which acts as a barrier so antibiotics cannot penetrate it leading to antibiotic-resistant [ 9 ]. Biofilm-forming bacteria show 1000-fold higher drug resistance than planktonic cells. So, biofilm-related infections are chronic, prone to relapse, and more difficult to treat [ 10 ]. Within biofilm communities, the bacterial cells are in close proximity and have a high chance of horizontal gene transfer (HGT), particularly via conjugation of antibiotic resistance genes, promoting their survival and spread of antibiotic resistance [ 11 ]. Biofilm-related virulence factors involved in A. bauamnnii infections are biofilm-associated protein ( Bap ), extended-spectrum beta-lactamase (ESBL) family bla PER1 gene, and CsuA/BABCDE pilus usher-chaperone assembly system [ 12 ]. The bap gene encodes a biofilm-forming protein that is crucial for bacterial cell accumulation, intracellular adhesion, and biofilm formation in both biotic and abiotic surfaces [ 13 ]. CsuE is a component of the csuA/BABCDE chaperon-usher assembly system that is required to form a pilus-like bundle during the initial attachment phase of biofilm formation and mainly aids in biofilm formation in abiotic surfaces [ 14 ]. The presence and expression of the bla PER1 promote biofilm formation as well as respiratory epithelial cell adhesion in clinical isolates of A. baumannii [ 15 ]. To address the biofilm-associated carbapenem-resistant A. baumannii infections among hospitalized patients, we conducted a hospital-based cross-sectional study at a Tertiary Care Hospital in Kathmandu, Nepal. We primarily determined the rate of A. baumannii in different clinical specimens. We have also evaluated the association between biofilm formation with carbapenem-resistant ACBC isolates detecting biofilm-forming genes Bap , csuE , and bla PER1 . Methods Study design, study site, and study population A hospital-based cross-sectional study was conducted at Kathmandu Model Hospital, Kathmandu, Nepal from February 2020 to August 2020. The study population of this study includes all the in-patient samples sent to the laboratory for Microbiological processing with the requested form. Inclusion and exclusion criteria All age groups of both sexes admitted to Kathmandu Model Hospital, Kathmandu who gave written consent were enrolled in this study. However, the out-patients were excluded. Sample type and sample size The sample includes pus, sputum, tracheal aspirates, blood, endotracheal tips, catheter tips, wound samples, suction tips, and tissue. A total of 665 clinical samples were included in this study. Bacterial identification and antibiotic susceptibility testing (AST) The samples were inoculated on Mac Conkey Agar (MA) and Blood Agar (BA) (Hi-media) and incubated at 37°C for 24hr and 48hr respectively. The ACBC isolates were identified by standard Microbiological procedures including Gram staining, culture, and biochemical tests. A total of 32 clinical isolates of ACBC were obtained. The antibiotic susceptibility pattern of ACBC was determined by a modified Kirby-Bauer disk diffusion method following CLSI guideline 2019 [16]. The antibiotics used in the study were; Amoxicillin (30µg), cefotaxime (30µg), levofloxacin (5µg), nitrofurantoin (300µg) (for urine samples only), cotrimoxazole (25µg), amikacin (30µg), gentamycin (10µg), ofloxacin (5µg), ciprofloxacin (5µg), amoxicillin/calvulant (30µg), ceftazidime (30µg), doxycycline (30µg), piperacillin (100µg) piperacillin tazobactam (100/10µg), chloramphenicol (30µg), meropenem (10µg), imipenem (10µg), ertapenem, cefepime (30µg), cefoperazone sulbactam (75/30µg), ampicillin/sulbactam (10/10µg), polymyxin B(300U), colistin (30µg) and tigecycline (15µg) (Himedia). Theisolates that are resistant to at least 1 agent in ≥ 3 antimicrobial categories are considered MDR [17]. Subsequently, the prevalence of MDR bacteriawas determined. Detection of biofilm by microtitre plate method An isolated colony of ACBC grown overnight on nutrient agar (NA) was inoculated in 2ml of tryptone soy broth (TSB) containing 1% glucose followed by incubation overnight at 37°C. The cell suspension was diluted in the ratio of 1:100 with fresh TSB. Then, 200µl of diluted suspension was inoculated in a 96-well microtitre plate. Un-inoculated wells with TSB with 1% glucose were used as a negative control. Then, the plate was sealed and incubated at 37°C for 24 hours. The wells were gently washed three times with PBS (pH 7.3) to remove planktonic cells and then dried in an inverted position. Bacterial cells attached to the wells were fixed using 2% sodium acetate followed by staining with 0.1% crystal violet. Then, the wells were washed with distilled water three times and dried in the inverted position. The wells were rinsed again in 95% ethanol to solubilize crystal violet. The optical density (OD) at 630nm was measured using a micro-titer reader(Bio-tek). The assay was carried out at least three times to obtain average optical density [18]. The optical density cutoff value (ODc) was calculated by using the given formula and the results were interpreted as ODc = average OD of negative control + (3 × SD of the negative control), Non-biofilm producer: OD≤ ODc; Weak biofilm producer: ODc ˂ OD ≤ 2×ODc; Medium biofilm producer: 2×ODc ˂ OD≤ 4×ODc and Strong biofilm producer: 4× ODc ≤ OD. Inhibition of biofilm by EDTA An isolated colony of ACBC grown overnight on NA was inoculated in 2 ml of tryptone soy broth (TSB) containing 1% glucose followed by incubation overnight at 37°C. Then, 200µl aliquot of ACBC suspension with 125mg/L EDTA was inoculated into 96 well polystyrene plate and was incubated overnight at 37°C. Then, the inhibition of biofilm was measured and interpreted in the same way as described above [18]. Detection of Bap , csuE and bla PER1 The genomic DNA was extracted by using a genomic extraction kit (Qiagen). The primer used for biofilm genes detection were bap -F(5’-TGCTGACAGTGACGTAGAACCACA-3’), bap -R(5’-TGCAACTAGTGGAATAGCAGCCCA-3’) with expected PCR product size 184bp, csuE -F(5’-CATCTTCTATTTCGGTCCC-3’), csuE -R (5’-CGGTCTGAGCATTGGTAA-3’) with expected product size 168bp and bla PER1 -F (5’- GCAACTGCTGCAATACTCGG-3’), bla PER1 -R (5’-ATGTGCGACCACAGTACCAG-3’) with expected product size 900 bp. The PCR was performed using Qiagen (USA) Master mix in ProFlex thermal cycler. The conditions for PCR amplification were initial denaturation at 95°C for 15 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, primer annealing at 57°C for bap , 59°C for csuE for 30 seconds and extension at 72°C for 30 seconds whereas 40 cycles of denaturation at 94°C for 60 seconds, primer annealing at 50°C for 1 minute, an extension at 72°C for 45 seconds for bla PER1 followed by a final extension at 72°C for 5 minutes. PCR end products were analyzed by electrophoresis in 1.5% agarose gel [13]. Data analysis SPSS version 21 was used for data entry and statistical analysis. The correlation between biofilm formation and carbapenem resistance was analyzed by using the Chi-Square Test. P-value < 0.05 was considered significant. Ethical approval Ethical approval for the study was taken by the International Review Committee (IRC) Phect Nepal (003-2020). Results Distribution of bacterial isolates among positive cultures A total of 665 clinical specimens from in-patients were received and processed in the microbiological lab of KMH during the study period. Among 665 samples, growth was observed in 281 (42.3%) clinical samples while 384 (57.7%) showed no growth. Out of 281 isolates, E. coli (28.8%) was the most predominant pathogen followed by S. aureus (20.3%), K. pneumoniae (16.4%), ACBC (11.4%), P. aeruginosa (8.1) and others (Table 1). Gender-wise and age-wise distribution of Acinetobacter calcoaceticus baumannii complex(ACBC) A significantly higher incidence of ACBC infection was observed among the male patients (26/32; 81.3%) as compared to female patients (6/32; 18.7%). Similarly, the highest incidence of ACBC infection was reported in the patient of age group 20-50 years which was more than half percent (59.6%) followed by the patient of age group 60-70 (15.5%), 50-60 (12.5%), 10-20 (6.2%) and 70-80 years (6.2%). Distribution of Acinetobacter calcoaceticus baumannii complex (ACBC) in various clinical specimens Of 665 clinical samples processed, 32 isolates were identified as ACBC. The highest number of ACBC was isolated from pus samples (n=12, 37.5%) followed by tracheal aspirates (n=6, 18.8%), and sputum (n=3, 9.4%) samples (Figure 1). Antibiotic susceptibility pattern of ACBC isolates All ACBC isolates were resistant to amoxicillin, cefotaxime, and ceftazidime whereas 31 isolates were resistant to amikacin and gentamycin antibiotics. The majority of ACBC isolates (93.8%) were MDR. Most of the isolates were susceptible to doxycycline (53.1%) followed by cotrimoxazole (18.7%), levofloxacin (15.6%), and ofloxacin (15.6%). The respiratory ACBC was highly resistant to azithromycin, a common antibiotic used in respiratory tract infections. All isolates were susceptible to colistin and polymyxin B (Table 2). Biofilm producer among ACBC isolates Out of 32 isolates, 2 isolates were strong biofilm producers, 8 isolates were moderate, and 21 were weak biofilm producers. Only one isolate was non- biofilm producer (Figure 2). Association between Carbapenem resistance and Biofilm production A significant association was observed between carbapenem resistance and biofilm formation (p-value < 0.05) indicating the role of biofilm in carbapenem resistance (Table 3). Distribution of biofilm-associated genes Out of 32 clinical isolates of ACBC, 31 were biofilm positive, and biofilm-related virulence genes Bap and csuE were detected in 67.8% of clinical isolates whereas the frequency of bla PER1 was found to be 58%. No genes were detected in the non-biofilm-producing isolate (Table 4, Figure 3). Association between carbapenem resistance and biofilm-related genes ( Bap , csuE and bla PER1 ) Significant associations between carbapenem-resistant and biofilm-related genes Bap , csuE, and bla PER1 were observed (p-value <0.05) which indicates the role of biofilm-related genes in antibiotic resistance to carbapenem (Table 5). Co-existence of Bap , csuE, and bla PER1 genes Among 32 Acinetobacter isolates, 31 were found to be biofilm producers. Out of 31 biofilm-positive isolates, 21 isolates were positive for both Bap and csuE genes and 18 isolates were positive for the bla PER1 gene. High co-existence of all three genes i.e. Bap , csuE, and bla PER 1 (58%) were found which may have increased their biofilm capacity as well as disease pathogenesis. Whereas the co-existence of only Bap and csuE genes was 9.8%. The presence of only single genes in the clinical isolates was not detected (Figure 4). Correlation between biofilm-related genes with biofilm formation All three genes Bap , csuE, and bla PER1 were present in strong and moderate biofilm-producing isolates. A significant correlation between biofilm-forming genes Bap , csuE, and bla PER1 and biofilm intensity was found (Table 6). Discussion Acinetobacter baumannii infections have become a public health threat due to bacteria’s growing resistance to all available antibiotics [19]. Because of its ability to produce biofilm, it is very challenging to treat and control the infection in hospital settings [20]. In our study, out of 665 in-patient clinical specimens, 281 showed positive bacterial growth. Out of 281 positive cultures, 32 were identified as ACBC i.e. 11.4% of positive growth and third most commonly isolated Gram-negative bacilli after E. coli and K. pneumoniae which is in accordance with the studies done by Khanal et al and Raut et al [21-22]. In our previous study at Manipal Teaching Hospital, Pokhara, we observed ACBC infections (n=117) as the primary cause of nosocomial infections followed by P. aeruginosa infections (n=79) [23]. The majority of isolates were identified from pus and respiratory samples including tracheal aspirates, sputum, and ET tube. In the study conducted in TUTH, the majority of ACBC was detected from respiratory samples 47.2%, followed by pus and swabs 27.3% and body fluids 11.1% [24]. In our previous studies, we had reported an increasing antimicrobial resistance among Gram-negative bacteria from various infections including bacterial isolates from immunocompromised patients [25-27]. Among Gram-negative bacteria, Acinetobacter shows higher resistance to commonly used antibiotics. In this study, 100% ACBC isolates were resistant to Amoxicillin and third-generation cephalosporins (ceftazidime, cefotaxime, and ceftriaxone), 97% ACBC isolates were resistant to gentamycin and amikacin and followed by fluoroquinolones, chloramphenicol, cotrimoxazole and β-lactamase inhibitors. Yadav et al presented similar reports from his study conducted in Nepal [24]. Carbapenem is used to treat MDR ACBC infections but a high prevalence of CR-ACBC 91% was observed in this study which is consistent with a previous study done by Joshi et al [28]. In contrast to our result, Mahto and Dhungel reported 61.2% and 50.5% imipenem and meropenem resistance respectively [29]. In another study, 87.9% ACBC isolates were found to be carbapenem-resistant [30]. This indicates the rise of CR ACBC infections in Nepal in recent years. The higher rate of antimicrobial resistance in bacterial pathogens is due to the irrational use of antibiotics, adherence to empirical therapy without proper AST, extensive use of antibiotics in poultry, direct disposal of antimicrobial waste in the environment, etc [31-32]. High antibiotic susceptibility of ACBC isolates towards doxycycline antibiotics was reported so it can be used to treat MDR ACBC infections. Carbapenem resistance in A. baumannii is mainly caused by class B MBL and class D OXA type β-lactamase which can hydrolyze carbapenem antibiotics [33]. CR-AB infections have a high morbidity and death rate in hospital settings due to their low level of antibiotic susceptibility and subsequent failure of therapy [34]. In this study, all ACBC isolates were susceptible to colistin and polymyxin B whereas 15.6% of isolates were resistant to tigecycline by disk diffusion method. Hence, these can be the choice of drugs for the effective treatment of CR-ACBC-associated infections. A. baumannii has a high biofilm-forming ability which poses serious public health problems because of biofilm-associated resistance to available antimicrobial agents leading to a huge challenge in hospital settings [8]. In this study out of 32 clinical isolates of ACBC, 31 were biofilm producers and only one was non biofilm producer. Among these 2 were strong, 8 were moderate and 21 were weak biofilm producers. Similar biofilm-forming rates (99%) among Acinetobacter species were reported [21]. In this study, biofilm-producing ACBC isolates were more resistant to carbapenem than non-biofilm producers indicating the role of biofilm in antimicrobial resistance. This study shows a significant correlation between biofilm formation and carbapenem resistance which is in accordance with previous studies by Anish et al, Pattanaik and Banashankari, and Sunu Kumari et al [6, 35, 36]. The increase in antimicrobial resistance among biofilm producer A. baumannii is due to the slow growth rate inside biofilm as well as its mechanical and biochemical shield such as low O 2 , high CO 2 , high pH, low water, and nutrient availability leading to poor penetration of antibiotics [37]. Inside the biofilm, the bacteria are near each other and there is a high chance of horizontal gene transfer via the conjugation of resistant genes which promotes their survival in the presence of antibiotics [9, 38]. The prevalence of Bap , csuE, and bla PER-1 was found to be 65.6%, 65.6%, and 56.3% respectively. In another study, the prevalence of Bap , csuE, and bla PER-1 were 79.2%, 38.3%, and 91.6% respectively, and significant correlation between antibiotic resistance, biofilm formation, and related genes [13] which is in support of our study. The presence of biofilm-related genes in all types of clinical specimens in our study indicates biofilm-related genes help in biofilm formation, survival in hospital environments and medical devices as well as disease pathogenesis in hospital settings. No biofilm-related genes were found in carbapenem-sensitive ACBC isolates and a significant association between carbapenem resistance and biofilm-forming genes bap , csuE, and bla PER-1 was found. Further, the co-existence of Bap , csuE, and bla PER1 among positive biofilm isolates was found to be 58% which may have boosted biofilm formation. The co-existence of Bap and csuE was 9.8% and no genes were singly present which also indicates the dependence of genes on biofilm formation such as csuE is critical for initial attachment and bap for biofilm maturation. Conclusion The higher rate of carbapenem resistance ACBC from different clinical specimens demonstrated complete resistance to the majority of antibiotics, however, colistin, polymyxin ‘B’ and tigecycline were still found to be effective drugs to treat CR ACBC infections. The increase in biofilm formation significantly associated with carbapenem resistance adds a big challenge to controlling CR ACBC infections. In addition, this capability of ACBC contributed to antibiotic resistance as well as helped them in environmental survival. Hence, proper sterilization of hospital equipment and environment should be of primary concern and a strong policy to prescribe effective antibiotics based on antibiogram profile should be implemented. Abbreviations ACBC Acinetobacter calcoaceticus-baumannii complex AST Antibiotic susceptibility testing BA Blood Agar Bap biofilm-associated protein CLSI Clinical Laboratory Standard Institutes CR-ACBC Carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex EDTA Ethylene diamine tetra acetic acid ESBL Extended spectrum beta-lactamase HGT Horizontal gene transfer ICU Intensive care unit MA Mac Conkey Agar MBLs Metallo beta lactamases MDR Multidrug resistant NA Nutrient agar OD Optical density PCR Polymerase Chain Reaction TSB Tryptone soy broth WHO World Health Organization Declarations Acknowledgements We express our sincere gratitude to laboratory staff members of GoldenGate International College (GGIC), Kathmandu Model Hospital, and the team of CMDN for their support, in completing this study. We are very much thankful to the participants or their legal guardians for providing samples. Authors contributions SB, MKU, and UTS developed the protocol, collected and analyzed data, and wrote the manuscript. KBA and BS supervised laboratory work at the hospital. UTS supervised Molecular work. UTS and BS were the main reviewers of the manuscript. All authors read and approved the final version of the manuscript to submit for publication. SB = Shova Bhandari, MKU = Milan Kumar Upreti, KBA = Khadga Bikram Angbuhang, BS = Basudha Shrestha, UTS = Upendra Thapa Shrestha. Funding The study was not funded by any organization. No funding is available for publication. Data availability The data used in this study will be available from the corresponding author (Email: [email protected] / [email protected] ) upon request. Ethics approval and consent to participate The study was approved by the International Review Committee (IRC) Phect Nepal (003-2020). Written informed consent was obtained from all the participants and/or their legal guardians. All experiments were performed in accordance with relevant guidelines and regulations (such as the Declaration of Helsinki). Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Rosalino V, Georgina S, Andr LAMM, Nabil E, Vega L, Franyuti-kelly G, Abelardo D, Moncaleano V, Ernesto J, Felix M, Antonio J. Acinetobacter baumannii Resistance: A Real Challenge for Clinicians. Antibiotics. 2020;9(205):1–22. Bulens SN, Sarah HY, Walters MS, Jacob JT, Bower C, Reno J, Kallen AJ. Carbapenem-nonsusceptible Acinetobacter baumannii , 8 US metropolitan areas, 2012–2015.Emerging infectious diseases. 2018;24(4):727. Ozer B, Vatansever C, Dogan O, Keske S, Ergonul O, Can F. Biofilm Formation of Acinetobacter baumannii Under in vitro and in vivo Colistin Exposure. Infect Dis Clin Microbiol. 2019;1(1):26–33. Vijayakumar S, Gopi R, Gunasekaran P, Bharathy M, Walia K, Anandan S, Veeraraghavan B. Molecular Characterization of Invasive Carbapenem-Resistant Acinetobacter baumannii from a Tertiary Care Hospital in South India. Infect Dis Therapy. 2016;5(3):379–87. Hamidian M, Nigro SJ. Emergence, molecular mechanisms and global spread of carbapenem-resistant Acinetobacter baumannii . Microb Genomics. 2019;5(10). Anish C, Abhisek R, Radha M, Chaudhary A. Evaluation of Biofilm Production in Acinetobacter baumanii with Reference to Imipenem Resistance. Int J Sci Res Publications. 2017;7(12):732–7. Hawkey J, Ascher DB, Judd LM, Wick RR, Kostoulias X, Cleland H, Spelman DW, Padiglione A, Peleg AY, Holt KE. Evolution of carbapenem resistance in Acinetobacter baumannii during a prolonged infection. Microb Genomics. 2018;4(3). Gedefie A, Demsis W, Ashagrie M, Kassa Y, Tesfaye M, Tilahun M, Bisetegn H, Sahle Z. Acinetobacter baumannii biofilm formation and its role in disease pathogenesis: A review. Infect Drug Resist. 2021;14:3711–9. Roy S, Chowdhury G, Mukhopadhyay AK, Dutta S, Basu S. Convergence of Biofilm Formation and Antibiotic Resistance in Acinetobacter baumannii Infection. Front Med. 2022;9:793615. Chen L, Li H, Wen H, Zhao B, Niu Y, Mo Q, Wu Y. Biofilm formation in Acinetobacter baumannii was inhibited by PAβN while it had no association with antibiotic resistance. Microbiol Open. 2020;9(9):1–11. Bowler P, Murphy C, Wolcott R. Biofilm exacerbates antibiotic resistance: Is this a current oversight in antimicrobial stewardship? Antimicrob Resist Infect Control. 2020;9(1):1–5. Longo F, Vuotto C, Donelli G. Biofilm formation in Acinetobacter baumannii . New Microbiol. 2014;37(2):119–27. PMID: 24858639. Yang CH, Su PW, Moi SH, Chuang LY. Biofilm formation in Acinetobacter baumannii : Genotype-phenotype correlation. Molecules. 2019;24(10):1–12. Brossard KA, Campagnari AA. The Acinetobacter baumannii biofilm-associated protein plays a role in adherence to human epithelial cells. Infect Immun. 2012;80(1):228–33. Thummeepak R, Kongthai P, Leungtongkam U, Sitthisak S. Distribution of virulence genes involved in biofilm formation in multi-drug resistant Acinetobacter baumannii clinical isolates. Int Microbiol. 2016;19(2):121–9. CLSI. Clinical Laboratory Standard Institute (CLSI): Performance standards for antimicrobial susceptibility testing. 29 ed: Clinical and Laboratory Standrads Institute antimicrobial susceptibility testing standards M02, M07 and M11. 2019. 118 – 28 p. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268 – 81. 10.1111/j.1469-0691.2011.03570.x . PMID: 21793988. Stepanovic S, Vukovic D, Hola V, Bonaventura GD, Djukic S, Circovic I, Ruzicka F. Quantification of biofilm in microtiter plates. Apmis. 2007;115(8):891–9. Clark NM, Zhanel GG, Lynch JP. Emergence of antimicrobial resistance among Acinetobacter species: A global threat. Curr Opin Crit Care. 2016;22(5):491–9. Bowler P, Murphy C, Wolcott R. Biofilm exacerbates antibiotic resistance: Is this a current oversight in antimicrobial stewardship? Antimicrob Resist Infect Control. 2020;9(1):1–5. Khanal BR, Wagle S, Tiwari BR. Biofilm Formation and Colistin Susceptibility of Clinical Isolates of Acinetobacter Species in a Tertiary Care Hospital of Nepal. Natl J Lab Med. 2019;8:1–4. Raut S, Rijal KR, Khatiwada S, Karna S, Khanal R, Adhikari J, Adhikari B. Trend and characteristics of Acinetobacter baumannii infections in patients attending universal college of medical sciences, Bhairahawa, Western Nepal: A longitudinal study of 2018. Infect Drug Resist. 2020;13:1631–41. Metok Y, Subramanya SH, Thapa Shrestha U, Perez LRR, Adhikari N, Nayak N. Biofilm and MBL production among imipenem resistant Pseudomonas aeruginosa and Acinetobacter species. MicroMedicine. 2020;8(2):63–73 ( http://dx.doi.org/10.5281/zenodo.4195479 ). Yadav SK, Bhujel R, Hamal P, Mishra SK, Sharma S, Sherchand JB. Burden of multidrug-resistant Acinetobacter baumannii infection in hospitalized patients in a tertiary care hospital of Nepal. Infect Drug Resist. 2020;13:725–32. Thapa Shrestha U, Adhikari N, Maharajan R, Banjara MR, Rijal KR, Basnet SR, Agrawal VP. Multidrug resistant Vibrio cholerae O1 from clinical and environmental samples in Kathmandu city. BMC Infect Dis. 2015;15:104. http://www.biomedcentral.com/orderreprints/s12879-015-0844-9 . Nayaju T, Upreti MK, Ghimire A, Shrestha B, Maharjan B, Joshi RD, Lekhak B, Thapa Shrestha U. Higher prevalence of extended spectrum β-lactamase producing uropathogenic Escherichia coli among patients with diabetes from a tertiary care hospital of Kathmandu, Nepal. Am J Trop Med Hyg. 2021;105(5):1347–55. 10.4269/ajtmh.21-0691 . Maharjan R, Bastola A, Adhikari N, Rijal KR, Banjara MR, Ghimire P, Thapa Shrestha U. Multidrug-resistant bacteria with ESBL genes: a growing threat among people living with HIC/AIDS in Nepal. BMC Infect Dis. 2022;22:526. https://doi.org/10.1186/s12879-022-07503-2 . Joshi PR, Acharya M, Kakshapati T, Leungtongkam U, Thummeepak R, Sitthisak S. Co-existence of blaOXA-23 and blaNDM-1 genes of Acinetobacter baumannii isolated from Nepal: Antimicrobial resistance and clinical significance. Antimicrob Resist Infect Control. 2017;6(1):1–7. Mahto M, Dhungel BA. Prevalence of Tigecycline resistance in Multidrug-Resistant Acinetobacter species isolates from clinical specimens. Janaki Med Coll J Med Sci. 2019;7(2):42–7. Neupane L, Sah AK, Rayamajhee B, Pokhrel A, Singh A. Detection of blaoxa-23 Gene from Carbapenem-resistant Acinetobacter Baumannii. J Nepal Health Res Counc. 2023; 20;20(4):899–905. 10.33314/jnhrc.v20i4.4257 . PMID: 37489674. Rijal KR, Banjara MR, Dhungel B, Kafle S, Gautam K, Ghimire B, Dhungel S, Adhikari N, Thapa Shrestha U, Sunuwar DR, Adhikari B, Ghimire P. Use of antimicrobials and antimicrobial resistance in Nepal: a nationwide survey. Sci Rep (Nature). 2021;11:11554. https://doi.org/10.1038/s41598-021-90812-4 . Bista S, Thapa Shrestha U, Dhungel B, Koirala P, Gompo TR, Shrestha N, Adhikari N, Joshi DR, Banjara MR, Adhikari B, et al. Detection of Plasmid-Mediated Colistin Resistant mcr-1 Gene in Escherichia coli Isolated from Infected Chicken Livers in Nepal. Animals. 2020;10:2060. https://doi.org/10.3390/ani10112060 . Benmahmod AB, Said HS, Ibrahim RH. Prevalence and mechanisms of carbapenem resistance among Acinetobacter baumannii clinical isolates in Egypt. Microb Drug Resist. 2019;25(4):480–8. Roberts LW, Forde BM, Hurst T, Ling W, Nimmo GR, Bergh H, Harris PN. Genomic surveillance, characterization and intervention of a polymicrobial multidrug-resistant outbreak in critical care. Microb Genomics. 2021;7(3):mgen000530. Pattanaik A, Banashankari GS. Assessment of Biofilm Production in Carbapenem Resistant Acinetobacter Species Isolated from Different Clinical Specimens. J Med Sci Clin Res. 2017;5(10):29103–10. Sunu Kumari AM, Routray A, Yadav D, Madhavan R. Imipenem resistance and biofilm production in Acinetobacter. Drug Invention Today. 2013;5(3):256–8. Santajit S, Indrawattana N. Mechanisms of antimicrobial resistance in Pasteurellaceae. PBioMed Res Int. 2016;2016(1155):1–8. Tanner WD, Atkinson RM, Goel RK, Toleman MA, Benson LS, Porucznik CA, VanDerslice JA. Horizontal transfer of the blaNDM-1 gene to Pseudomonas aeruginosa and Acinetobacter baumannii in biofilms. FEMS microbiology letters. 2017;364(8). Tables Table 1: Distribution of bacterial isolates among positive cultures Bacterial isolates Number (%) E. coli 81 (28.8) S. aureus 57 (20.3) K. pneumoniae 46 (16.4) ACBC 32 (11.4) P. aeruginosa 23 (8.1) E. faecalis 11 (3.9) E. faecium 2 (0.7) C. freundii 11 (3.9) S. pneumoniae 1 (0.4) P. mirabilis 11 (3.9) P. vulgaris 4 (1.4) S. saprophyticus 1 (0.4) E. aerogenes 1 (0.4) Total 281 (100) Table 2: Antibiotic susceptibility testing of ACBC isolates (n=32) Antibiotic category Antibiotics used No. of ACBC isolates (%) Susceptible N (%) Resistant N (%) First Line Drugs Penicillin + β-lactamase inhibitors Amoxicillin 0 32 (100) Ampicillin/sulbactam 3 (9.4) 29 (90.6) Extended-spectrum cephalosporins; 3 rd and 4 th generation cephalosporins Cefixime a 2 (15.4) 11 (84.6) Cefotaxime 0 32 (100) Ceftazidime 0 32 (100) Folate pathway inhibitors Cotrimoxazole 6 (18.7) 26 (81.3) Macrolides Azithromycin b 1 (10) 9 (90) Gentamycin 1 (3.1) 31 (96.9) Fluoroquinolones Ciprofloxacin 4 (12.5) 28 (87.85) Levofloxacin 5 (15.6) 27 (84.4) Ofloxacin 5 (15.6) 27 (84.4) Phenicols Chloramphenicol 2 (6.2) 30 (93.8) Second Line Drugs Aminoglycosides Amikacin 1 (3.1) 31 (96.9) Penicillin + β-lactamase inhibitors Amoxicillin-clavulanic acid 2 (6.2) 30 (93.8) Extended-spectrum cephalosporins; 3 rd and 4 th generation cephalosporins Cefperazone/Sulbactam 2 (6.2) 30 (93.8) Cefepime 2 (6.2) 30 (93.8) Tetracyclines Doxycycline 17 (53.1) 15 (46.9) Carbapenems Imipenem 3 (9.4) 29 (90.6) Meropenem 3 (9.4) 29 (90.6) Etrapenem 3 (9.4) 29 (90.6) β-lactamase inhibitors Piperacillin/Tazobactam 2 (6.2) 30 (93.8) Third/last Line Drugs Polymyxins Colistin 32 (100) 0 Polymyxin B 32 (100) 0 Glycylcyclines Tigecycline 27 (94.4) 5 (15.6) Note: The susceptibility assay for Cefixime a was used for only blood (n=3) and respiratory ACBC isolates (n=10). Likewise, the susceptibility testing for Azithromycin b was used for respiratory ACBC isolates (n=10). Table 3: Association between Carbapenem resistance and Biofilm production Biofilm Production Number of Carbapenem Total p-value Resistant (%) Sensitive (%) Strong 2 (6.2) 0 22 (6.2) 0.013 Moderate 8 (25) 0 8 (25) weak 19 (59.4) 2 (6.2) 21 (65.6) Non 0 1 (3.1) 1 (3.1) *Chi-square test Table 4: Distribution of biofilm-associated genes Bap, csuE and bla PER1 Biofilm No. of ACBC isolates with biofilm-producing genes (%) Bap csuE bla PER1 Producer 21 (65.6) 21 (65.6) 18 (56.2) Non-producer 11 (34.4) 11 (34.4) 14 (43.8) Total 32 32 32 Table 5: Association between Carbapenem resistance and biofilm-related genes Carbapenem Biofilm related gene Total p-value Detected (%) Not detected (%) Sensitive 0 3 (100) 3 0.033 Resistance 21 (72.4) 8 (27.6) 29 Total 21 11 32 Table 6: Association between Biofilm forming genes with biofilm intensity Biofilm Intensity (Number of biofilm-producing isolates) ACBC isolates possessing biofilm-related genes (%) p-value Bap csuE bla PER1 Strong (n=2) 2 (100) 2 (100) 2 (100) 0.032 Moderate (n=8) 8 (100) 8 (100) 8 (100) Weak (n=21) 11 (52.4) 11 (52.4) 8 (38.1) Non (n=1) 0 0 0 Total (n=32) 21 (65.6) 21 (65.6) 18 (56.2) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 16 May, 2024 Submission checks completed at journal 16 May, 2024 First submitted to journal 13 May, 2024 You are reading this latest preprint version 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4413953","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":305410736,"identity":"ff236b0e-240f-4fae-8209-8a0fa084f231","order_by":0,"name":"Shova Bhandari","email":"","orcid":"","institution":"GoldenGate International College","correspondingAuthor":false,"prefix":"","firstName":"Shova","middleName":"","lastName":"Bhandari","suffix":""},{"id":305410737,"identity":"38da956a-e53f-453b-95ab-8a370ceb20d8","order_by":1,"name":"Milan Kumar Upreti","email":"","orcid":"","institution":"GoldenGate International College","correspondingAuthor":false,"prefix":"","firstName":"Milan","middleName":"Kumar","lastName":"Upreti","suffix":""},{"id":305410739,"identity":"2c1678a5-9c8f-40ac-b7a6-d11c322e4366","order_by":2,"name":"Khadga Bikram Angbuhang","email":"","orcid":"","institution":"GoldenGate International College","correspondingAuthor":false,"prefix":"","firstName":"Khadga","middleName":"Bikram","lastName":"Angbuhang","suffix":""},{"id":305410742,"identity":"281053b6-b0a3-4b81-a705-9453afb5e4ac","order_by":3,"name":"Basudha Shrestha","email":"","orcid":"","institution":"Kathmandu Model Hospital","correspondingAuthor":false,"prefix":"","firstName":"Basudha","middleName":"","lastName":"Shrestha","suffix":""},{"id":305410743,"identity":"80f84cff-69e4-442c-bc12-21bdffa4af88","order_by":4,"name":"Upendra Thapa Shrestha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYLACHhDB3gAkDCyI1yLBwHMApEWCFC0SCSAmEVoMbjcfe/B2j12d/MznVzf8KJBg4G/vTsCv5c6xdMM5z5IlDG7nlN3sATpM4szZDfi13Mgxk+Y5wCxhIJ2TdoMHqMVAIpeQlvxvQC31EvIzz6Td/EOclhw2oJbDEgw32I/dJsoWyTvHzCTnHDguueFMDtttGQMJHoJ+4bvd/EzizYFqfvn2489uvvljI8ff3otfC1JE8BiASfzKUbWwPyCsehSMglEwCkYkAACnSEdOlzLj2wAAAABJRU5ErkJggg==","orcid":"","institution":"Tribhuvan University","correspondingAuthor":true,"prefix":"","firstName":"Upendra","middleName":"Thapa","lastName":"Shrestha","suffix":""}],"badges":[],"createdAt":"2024-05-13 14:41:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4413953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4413953/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57449787,"identity":"fef26689-ba72-43f9-b25c-f1bc1461c5d4","added_by":"auto","created_at":"2024-05-30 20:13:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33984,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of ACBC among different clinical specimens\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4413953/v1/f2e2d0212e73fb4778f34cda.jpg"},{"id":57450382,"identity":"e49f36b5-f963-44c1-bb39-75e37d414856","added_by":"auto","created_at":"2024-05-30 20:21:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27213,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm producer among ACBC isolates\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4413953/v1/350973402782d539454d4d29.jpg"},{"id":57449785,"identity":"e786bea9-ad98-4eeb-8feb-97c4a84a31b5","added_by":"auto","created_at":"2024-05-30 20:13:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85317,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of Bap, csuE, and bla\u003csub\u003ePER1 \u003c/sub\u003egenes in ACBC isolates by conventional PCR\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4413953/v1/a01f56742c324dc9404cc66d.jpg"},{"id":57449788,"identity":"672aff6d-9fd6-4b3d-bec4-144c9c1c1cdc","added_by":"auto","created_at":"2024-05-30 20:13:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33535,"visible":true,"origin":"","legend":"\u003cp\u003eCo-existence of Bap, csuE and bla\u003csub\u003ePER1 \u003c/sub\u003egenes\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4413953/v1/b26edee3ba6b0122cee1dc6c.jpg"},{"id":57450555,"identity":"58022405-a3f1-4a7f-8324-e47f9e078098","added_by":"auto","created_at":"2024-05-30 20:29:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1312561,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4413953/v1/4fd379f8-2da0-4560-b516-225cef364a0f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increased biofilm-associated Carbapenem-resistant Acinetobacter- calcoaceticus-baumannii complex infections among the hospitalized patients in Kathmandu Model Hospital, Nepal","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAcinetobacter calcoaceticus-baumannii complex\u003c/em\u003e (ACBC) is a Gram-negative commensal bacterium that often infects immunocompromised patients or patients with indwelling devices, especially in the intensive care unit (ICU) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It causes a wide range of hospital-acquired infections such as respiratory tract infections, urinary tract infections, bacteremia, sepsis, endocarditis, meningitis, skin and soft tissue infections, burns as well as central and nervous system infections [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e infections have become a public health threat due to the rapid rise in the incidence of multidrug-resistant (MDR) strains [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Carbapenem are choice of drug to treat severe infections caused by MDR \u003cem\u003eA. baumannii\u003c/em\u003e due to its good activity and low toxicity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the prevalence of carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e has increased globally limiting the treatment options and leading to increased mortality of patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The pathogen developed resistance to carbapenems due to the acquisition of antibiotic-resistant genes, decreased permeability, altered penicillin-binding protein, overexpression of efflux pump, production of carbapenemase enzyme mainly class B metallo beta-lactamases (MBLs) and class D oxacillinase and biofilm formation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. World Health Organization (WHO) has listed carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e as a top priority pathogen requiring additional study and development of new antibiotics in 2017 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e is an emerging pathogen with the ability to produce a biofilm that mostly causes device-related infections such as ventilator-associated pneumonia and catheter-related infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The bacteria inside the biofilm are shielded by EPS which acts as a barrier so antibiotics cannot penetrate it leading to antibiotic-resistant [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Biofilm-forming bacteria show 1000-fold higher drug resistance than planktonic cells. So, biofilm-related infections are chronic, prone to relapse, and more difficult to treat [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Within biofilm communities, the bacterial cells are in close proximity and have a high chance of horizontal gene transfer (HGT), particularly via conjugation of antibiotic resistance genes, promoting their survival and spread of antibiotic resistance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiofilm-related virulence factors involved in \u003cem\u003eA. bauamnnii\u003c/em\u003e infections are biofilm-associated protein (\u003cem\u003eBap\u003c/em\u003e), extended-spectrum beta-lactamase (ESBL) family \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePER1\u003c/em\u003e\u003c/sub\u003e gene, and CsuA/BABCDE pilus usher-chaperone assembly system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The \u003cem\u003ebap\u003c/em\u003e gene encodes a biofilm-forming protein that is crucial for bacterial cell accumulation, intracellular adhesion, and biofilm formation in both biotic and abiotic surfaces [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eCsuE\u003c/em\u003e is a component of the \u003cem\u003ecsuA/BABCDE\u003c/em\u003e chaperon-usher assembly system that is required to form a pilus-like bundle during the initial attachment phase of biofilm formation and mainly aids in biofilm formation in abiotic surfaces [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The presence and expression of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePER1\u003c/em\u003e\u003c/sub\u003e promote biofilm formation as well as respiratory epithelial cell adhesion in clinical isolates of \u003cem\u003eA. baumannii\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To address the biofilm-associated carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e infections among hospitalized patients, we conducted a hospital-based cross-sectional study at a Tertiary Care Hospital in Kathmandu, Nepal. We primarily determined the rate of \u003cem\u003eA. baumannii\u003c/em\u003e in different clinical specimens. We have also evaluated the association between biofilm formation with carbapenem-resistant ACBC isolates detecting biofilm-forming genes \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE\u003c/em\u003e, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePER1\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy design, study site, and study population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA hospital-based cross-sectional study was conducted at Kathmandu Model Hospital, Kathmandu, Nepal from February 2020 to August 2020. The study population of this study includes all the in-patient samples sent to the laboratory for Microbiological processing with the requested form.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion and exclusion criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll age groups of both sexes admitted to Kathmandu Model Hospital, Kathmandu who gave written consent were enrolled in this study. However, the out-patients were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample type and sample size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sample includes pus, sputum, tracheal aspirates, blood, endotracheal tips, catheter tips, wound samples, suction tips, and tissue. A total of 665 clinical samples were included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial identification and antibiotic susceptibility testing (AST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were inoculated on Mac Conkey Agar (MA) and Blood Agar (BA) (Hi-media) and incubated at 37\u0026deg;C for 24hr and 48hr respectively. The ACBC isolates were identified by standard Microbiological procedures including Gram staining, culture, and biochemical tests. A total of 32 clinical isolates of ACBC were obtained. The antibiotic susceptibility pattern of ACBC was determined by a\u0026nbsp;modified Kirby-Bauer disk diffusion method\u0026nbsp;following CLSI guideline 2019 [16]. The antibiotics used in the study were; Amoxicillin (30\u0026micro;g), cefotaxime (30\u0026micro;g), levofloxacin (5\u0026micro;g), nitrofurantoin (300\u0026micro;g) (for urine samples only), cotrimoxazole (25\u0026micro;g), amikacin (30\u0026micro;g), gentamycin (10\u0026micro;g), ofloxacin (5\u0026micro;g), ciprofloxacin (5\u0026micro;g), amoxicillin/calvulant (30\u0026micro;g), ceftazidime (30\u0026micro;g), doxycycline (30\u0026micro;g), piperacillin (100\u0026micro;g) piperacillin tazobactam (100/10\u0026micro;g), chloramphenicol (30\u0026micro;g), meropenem (10\u0026micro;g), imipenem (10\u0026micro;g), ertapenem, cefepime (30\u0026micro;g), cefoperazone sulbactam (75/30\u0026micro;g), ampicillin/sulbactam (10/10\u0026micro;g), polymyxin B(300U), colistin (30\u0026micro;g) and tigecycline (15\u0026micro;g) (Himedia).\u0026nbsp;Theisolates that are\u0026nbsp;resistant to at least 1 agent in \u0026ge; 3 antimicrobial categories are considered\u0026nbsp;MDR [17].\u0026nbsp;Subsequently, the prevalence of MDR bacteriawas determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of biofilm by microtitre plate method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn isolated colony of ACBC grown overnight on nutrient agar (NA) was inoculated in 2ml of tryptone soy broth (TSB) containing 1% glucose followed by incubation overnight at 37\u0026deg;C. The cell suspension was diluted in the ratio of 1:100 with fresh TSB. Then, 200\u0026micro;l of diluted suspension was inoculated in a 96-well microtitre plate. Un-inoculated wells with TSB with 1% glucose were used as a negative control. Then, the plate was sealed and incubated at 37\u0026deg;C\u0026nbsp;for 24 hours. The wells were gently washed three times with PBS (pH 7.3) to remove planktonic cells and then dried in an inverted position. Bacterial cells attached to the wells were fixed using 2% sodium acetate followed by staining with 0.1% crystal violet. Then, the wells were washed with distilled water three times and dried in the inverted position. The wells were rinsed again in 95% ethanol to solubilize crystal violet. The optical density (OD) at 630nm was measured using a micro-titer reader(Bio-tek). The assay was carried out at least three times to obtain average optical density [18]. The optical density cutoff value (ODc) was calculated by using the given formula and the results were interpreted as ODc = average OD of negative control + (3 \u0026times; SD of the negative control),\u0026nbsp;Non-biofilm producer: OD\u0026le; ODc; Weak biofilm producer: ODc ˂ OD \u0026le; 2\u0026times;ODc;\u0026nbsp;Medium biofilm producer: 2\u0026times;ODc ˂ OD\u0026le; 4\u0026times;ODc and\u0026nbsp;Strong biofilm producer: 4\u0026times; ODc \u0026le; OD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of biofilm by EDTA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn isolated colony of ACBC grown overnight on NA was inoculated in 2 ml of tryptone soy broth (TSB) containing 1% glucose followed by incubation overnight at 37\u0026deg;C. Then, 200\u0026micro;l aliquot of ACBC suspension with 125mg/L EDTA was inoculated into 96 well polystyrene plate and was incubated overnight at 37\u0026deg;C. Then, the inhibition of biofilm was measured and interpreted in the same way as described above [18].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genomic DNA was extracted by using a genomic extraction kit (Qiagen). The primer used for biofilm genes detection were\u0026nbsp;\u003cem\u003ebap\u003c/em\u003e-F(5\u0026rsquo;-TGCTGACAGTGACGTAGAACCACA-3\u0026rsquo;), \u003cem\u003ebap\u003c/em\u003e-R(5\u0026rsquo;-TGCAACTAGTGGAATAGCAGCCCA-3\u0026rsquo;) with expected PCR product size 184bp, \u003cem\u003ecsuE\u003c/em\u003e-F(5\u0026rsquo;-CATCTTCTATTTCGGTCCC-3\u0026rsquo;), \u003cem\u003ecsuE\u003c/em\u003e-R (5\u0026rsquo;-CGGTCTGAGCATTGGTAA-3\u0026rsquo;) with expected product size 168bp and \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e-F\u0026nbsp;(5\u0026rsquo;- GCAACTGCTGCAATACTCGG-3\u0026rsquo;), \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e-R\u0026nbsp;(5\u0026rsquo;-ATGTGCGACCACAGTACCAG-3\u0026rsquo;) with expected product size 900 bp.\u0026nbsp;The PCR was performed using Qiagen (USA) Master mix in ProFlex thermal cycler. The conditions for PCR amplification were initial denaturation at 95\u0026deg;C for 15 minutes, followed by 35 cycles of denaturation at 94\u0026deg;C for 30 seconds, primer annealing at 57\u0026deg;C for \u003cem\u003ebap\u003c/em\u003e, 59\u0026deg;C for\u0026nbsp;\u003cem\u003ecsuE\u003c/em\u003e for 30 seconds and extension at 72\u0026deg;C for 30 seconds whereas 40 cycles of denaturation at 94\u0026deg;C for 60 seconds, primer annealing at 50\u0026deg;C for 1 minute, an extension at 72\u0026deg;C for 45 seconds for \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e followed by a final extension at 72\u0026deg;C for 5 minutes. PCR end products were analyzed by electrophoresis in 1.5% agarose gel [13].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS version 21 was used for data entry and statistical analysis. The correlation between biofilm formation and carbapenem resistance was analyzed by using the Chi-Square Test. P-value \u0026lt; 0.05 was considered significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for the study was taken by the International Review Committee (IRC) Phect Nepal (003-2020).\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eDistribution of bacterial isolates among positive cultures\u003c/h2\u003e\n\u003cp\u003eA total of 665 clinical specimens from in-patients were received and processed in the microbiological lab of KMH during the study period. Among 665 samples, growth was observed in 281 (42.3%) clinical samples while 384 (57.7%) showed no growth. Out of 281 isolates, \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003e(28.8%) was the most predominant pathogen followed by \u003cem\u003eS. aureus\u003c/em\u003e (20.3%), \u003cem\u003eK. pneumoniae\u003c/em\u003e (16.4%), ACBC (11.4%), \u003cem\u003eP. aeruginosa\u003c/em\u003e (8.1) and others (Table 1).\u003c/p\u003e\n\u003ch2\u003eGender-wise and age-wise distribution of \u003cem\u003eAcinetobacter calcoaceticus baumannii\u0026nbsp;\u003c/em\u003ecomplex(ACBC)\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eA significantly higher incidence of ACBC infection was observed among the male patients (26/32; 81.3%) as compared to female patients (6/32; 18.7%). Similarly, the highest incidence of ACBC infection was reported in the patient of age group 20-50 years which was more than half percent (59.6%) followed by the patient of age group 60-70 (15.5%), 50-60 (12.5%), 10-20 (6.2%) and 70-80 years (6.2%).\u003c/p\u003e\n\u003ch2\u003eDistribution of \u003cem\u003eAcinetobacter calcoaceticus baumannii complex\u0026nbsp;\u003c/em\u003e(ACBC) in various clinical specimens\u003c/h2\u003e\n\u003cp\u003eOf 665 clinical samples processed, 32 isolates were identified as ACBC. The highest number of ACBC was isolated from pus samples (n=12, 37.5%) followed by tracheal aspirates (n=6, 18.8%), and sputum (n=3, 9.4%) samples (Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAntibiotic susceptibility pattern of ACBC isolates\u003c/h2\u003e\n\u003cp\u003eAll ACBC isolates were resistant to amoxicillin, cefotaxime, and ceftazidime whereas 31 isolates were resistant to amikacin and gentamycin antibiotics. The majority of ACBC isolates (93.8%) were MDR. Most of the isolates were susceptible to doxycycline (53.1%) followed by cotrimoxazole (18.7%), levofloxacin (15.6%), and ofloxacin (15.6%). The respiratory ACBC was highly resistant to azithromycin, a common antibiotic used in respiratory tract infections. All isolates were susceptible to colistin and polymyxin B (Table 2).\u003c/p\u003e\n\u003ch2\u003eBiofilm producer among ACBC isolates\u003c/h2\u003e\n\u003cp\u003eOut of 32 isolates, 2 isolates were strong biofilm producers, 8 isolates were moderate, and 21 were weak biofilm producers. Only one isolate was non- biofilm producer (Figure 2).\u003c/p\u003e\n\u003ch2\u003eAssociation between Carbapenem resistance and Biofilm production\u003c/h2\u003e\n\u003cp\u003eA significant association was observed between carbapenem resistance and biofilm formation (p-value \u0026lt; 0.05) indicating the role of biofilm in carbapenem resistance (Table 3).\u003c/p\u003e\n\u003ch2\u003eDistribution of biofilm-associated genes\u003c/h2\u003e\n\u003cp\u003eOut of 32 clinical isolates of ACBC, 31 were biofilm positive, and biofilm-related virulence genes \u003cem\u003eBap\u003c/em\u003e and \u003cem\u003ecsuE\u003c/em\u003e were detected in 67.8% of clinical isolates whereas the frequency of \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e was found to be 58%. No genes were detected in the non-biofilm-producing isolate (Table 4, Figure 3).\u003c/p\u003e\n\u003ch2\u003eAssociation between carbapenem resistance and biofilm-related genes (\u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e)\u003c/h2\u003e\n\u003cp\u003eSignificant associations between carbapenem-resistant and biofilm-related genes \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u0026nbsp;\u003c/em\u003eand \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e were observed (p-value \u0026lt;0.05) which indicates the role of biofilm-related genes in antibiotic resistance to carbapenem (Table 5).\u003c/p\u003e\n\u003ch2\u003eCo-existence\u0026nbsp;of \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER1\u0026nbsp;\u003c/sub\u003e\u003c/em\u003egenes\u003c/h2\u003e\n\u003cp\u003eAmong 32 Acinetobacter isolates, 31 were found to be biofilm producers. Out of 31 biofilm-positive isolates, 21 isolates were positive for both \u003cem\u003eBap\u003c/em\u003e and \u003cem\u003ecsuE\u003c/em\u003e genes and 18 isolates were positive for the \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e gene. High co-existence of all three genes i.e. \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e1\u0026nbsp;\u003c/sub\u003e(58%)\u0026nbsp;were found which may have increased their biofilm capacity as well as disease pathogenesis. Whereas the co-existence of only \u003cem\u003eBap\u003c/em\u003e and \u003cem\u003ecsuE\u003c/em\u003e genes was 9.8%. The presence of only single genes in the clinical isolates was not detected (Figure 4).\u003c/p\u003e\n\u003ch2\u003eCorrelation between biofilm-related genes with biofilm formation\u003c/h2\u003e\n\u003cp\u003eAll three genes \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e were present in strong and moderate biofilm-producing isolates. A significant correlation between biofilm-forming genes \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003eand biofilm intensity was found (Table 6).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e infections have become a public health threat due to bacteria\u0026rsquo;s growing resistance to all available antibiotics [19]. Because of its ability to produce biofilm, it is very challenging to treat and control the infection in hospital settings [20]. In our study, out of 665 in-patient clinical specimens, 281 showed positive bacterial growth. Out of 281 positive cultures, 32 were identified as\u0026nbsp;ACBC i.e. 11.4% of positive growth and third most commonly isolated Gram-negative bacilli after \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e which is in accordance with the studies done by Khanal et al and Raut et al [21-22]. In our previous study at Manipal Teaching Hospital, Pokhara, we observed ACBC infections (n=117) as the primary cause of nosocomial infections followed by \u003cem\u003eP. aeruginosa\u003c/em\u003e infections (n=79) [23]. The majority of isolates were identified from pus and respiratory samples including tracheal aspirates, sputum, and ET tube. In the study conducted in TUTH, the majority of ACBC was detected from respiratory samples 47.2%, followed by pus and swabs 27.3% and body fluids 11.1% [24].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our previous studies, we had reported an increasing antimicrobial resistance among Gram-negative bacteria from various infections including bacterial isolates from immunocompromised patients [25-27]. Among Gram-negative bacteria, \u003cem\u003eAcinetobacter\u003c/em\u003e shows higher resistance to commonly used antibiotics. In this study, 100% ACBC isolates were resistant to Amoxicillin and third-generation cephalosporins (ceftazidime, cefotaxime, and ceftriaxone), 97% ACBC isolates were resistant to gentamycin and amikacin and followed by fluoroquinolones, chloramphenicol, cotrimoxazole and \u0026beta;-lactamase inhibitors. \u0026nbsp;Yadav et al presented similar reports from his study conducted in Nepal [24]. \u0026nbsp;Carbapenem is used to treat MDR ACBC infections but a high prevalence of CR-ACBC 91% was observed in this study which is consistent with a previous study done by Joshi et al [28]. In contrast to our result, Mahto and Dhungel reported 61.2% and 50.5% imipenem and meropenem resistance respectively [29]. In another study, 87.9% ACBC isolates were found to be carbapenem-resistant [30]. This indicates the rise of CR ACBC infections in Nepal in recent years. The higher rate of antimicrobial resistance in bacterial pathogens is due to the irrational use of antibiotics, adherence to empirical therapy without proper AST, extensive use of antibiotics in poultry, direct disposal of antimicrobial waste in the environment, etc [31-32]. High antibiotic susceptibility of ACBC isolates towards doxycycline antibiotics was reported so it can be used to treat MDR ACBC infections. Carbapenem resistance in \u003cem\u003eA. baumannii\u003c/em\u003e is mainly caused by class B MBL and class D OXA type \u0026beta;-lactamase which can hydrolyze carbapenem antibiotics [33]. CR-AB infections have a high morbidity and death rate in hospital settings due to their low level of antibiotic susceptibility and subsequent failure of therapy [34]. In this study, all ACBC isolates were susceptible to colistin and polymyxin B whereas 15.6% of isolates were resistant to tigecycline by disk diffusion method. Hence, these can be the choice of drugs for the effective treatment of CR-ACBC-associated infections.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eA. baumannii\u003c/em\u003e has a high biofilm-forming ability which poses serious public health problems because of biofilm-associated resistance to available antimicrobial agents leading to a huge challenge in hospital settings [8]. In this study out of 32 clinical isolates of ACBC, 31 were biofilm producers and only one was non biofilm producer. Among these 2 were strong, 8 were moderate and 21 were weak biofilm producers. Similar biofilm-forming rates (99%) among \u003cem\u003eAcinetobacter\u003c/em\u003e species were reported [21]. In this study, biofilm-producing ACBC isolates were more resistant to carbapenem than non-biofilm producers indicating the role of biofilm in antimicrobial resistance. This study shows a significant correlation between biofilm formation and carbapenem resistance which is in accordance with previous studies by Anish et al, Pattanaik and Banashankari, and Sunu Kumari et al [6, 35, 36]. The increase in antimicrobial resistance among biofilm producer \u003cem\u003eA. baumannii\u003c/em\u003e is due to the slow growth rate inside biofilm as well as its mechanical and biochemical shield such as low O\u003csub\u003e2\u003c/sub\u003e, high CO\u003csub\u003e2\u003c/sub\u003e, high pH, low water, and nutrient availability leading to poor penetration of antibiotics [37]. Inside the biofilm, the bacteria are near each other and there is a high chance of horizontal gene transfer via the conjugation of resistant genes which promotes their survival in the presence of antibiotics [9, 38].\u003c/p\u003e\n\u003cp\u003eThe prevalence of \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER-1\u003c/sub\u003e\u003c/em\u003e was found to be 65.6%, 65.6%, and 56.3% respectively. In another study, the prevalence of \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER-1\u003c/sub\u003e\u003c/em\u003e were 79.2%, 38.3%, and 91.6% respectively, and significant correlation between antibiotic resistance, biofilm formation, and related genes [13] which is in support of our study. The presence of biofilm-related genes in all types of clinical specimens in our study indicates biofilm-related genes help in biofilm formation, survival in hospital environments and medical devices as well as disease pathogenesis in hospital settings. No biofilm-related genes were found in carbapenem-sensitive ACBC isolates and a significant association between carbapenem resistance and biofilm-forming genes \u003cem\u003ebap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER-1\u003c/sub\u003e\u003c/em\u003ewas found. Further, the co-existence of \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE,\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e among positive biofilm isolates was found to be 58% which may have boosted biofilm formation. The co-existence of \u003cem\u003eBap\u003c/em\u003e and \u003cem\u003ecsuE\u0026nbsp;\u003c/em\u003ewas 9.8% and no genes were singly present which also indicates the dependence of genes on biofilm formation such as \u003cem\u003ecsuE\u003c/em\u003e is critical for initial attachment and \u003cem\u003ebap\u003c/em\u003e for biofilm maturation.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe higher rate of carbapenem resistance ACBC from different clinical specimens demonstrated complete resistance to the majority of antibiotics, however, colistin, polymyxin \u0026lsquo;B\u0026rsquo; and tigecycline were still found to be effective drugs to treat CR ACBC infections. The increase in biofilm formation significantly associated with carbapenem resistance adds a big challenge to controlling CR ACBC infections. In addition, this capability of ACBC contributed to antibiotic resistance as well as helped them in environmental survival. Hence, proper sterilization of hospital equipment and environment should be of primary concern and a strong policy to prescribe effective antibiotics based on antibiogram profile should be implemented.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACBC\u003cem\u003e\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Acinetobacter calcoaceticus-baumannii\u0026nbsp;\u003c/em\u003ecomplex\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAST\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Antibiotic susceptibility testing\u003c/p\u003e\n\u003cp\u003eBA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Blood Agar\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBap\u003c/em\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;biofilm-associated protein\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCLSI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Clinical Laboratory Standard Institutes\u003c/p\u003e\n\u003cp\u003eCR-ACBC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Carbapenem-resistant\u0026nbsp;\u003cem\u003eAcinetobacter calcoaceticus-baumannii\u0026nbsp;\u003c/em\u003ecomplex\u003c/p\u003e\n\u003cp\u003eEDTA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ethylene diamine tetra acetic acid\u003c/p\u003e\n\u003cp\u003eESBL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Extended spectrum beta-lactamase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHGT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Horizontal gene transfer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eICU \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Intensive care unit\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mac Conkey Agar\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMBLs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Metallo beta lactamases\u003c/p\u003e\n\u003cp\u003eMDR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Multidrug resistant\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Nutrient agar\u003c/p\u003e\n\u003cp\u003eOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Optical density\u003c/p\u003e\n\u003cp\u003ePCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eTSB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tryptone soy broth\u003c/p\u003e\n\u003cp\u003eWHO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;World Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere gratitude to laboratory staff members of GoldenGate International College (GGIC), Kathmandu Model Hospital, and the team of CMDN for their support, in completing this study. We are very much thankful to the participants or their legal guardians for providing samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSB, MKU, and UTS developed the protocol, collected and analyzed data, and wrote the manuscript. KBA and BS supervised laboratory work at the hospital. UTS supervised Molecular work. UTS and BS were the main reviewers of the manuscript. All authors read and approved the final version of the manuscript to submit for publication.\u0026nbsp;SB = Shova Bhandari, MKU = Milan Kumar Upreti, KBA = Khadga Bikram Angbuhang, BS = Basudha Shrestha, UTS = Upendra Thapa Shrestha.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was not funded by any organization. No funding is available for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study will be available from the corresponding author (Email: [email protected]/[email protected]) upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the International Review Committee (IRC) Phect Nepal (003-2020). Written informed consent was obtained from all the participants and/or their legal guardians.\u0026nbsp;All experiments were performed in accordance with relevant guidelines and regulations (such as the Declaration of Helsinki).\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\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRosalino V, Georgina S, Andr LAMM, Nabil E, Vega L, Franyuti-kelly G, Abelardo D, Moncaleano V, Ernesto J, Felix M, Antonio J. \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Resistance: A Real Challenge for Clinicians. Antibiotics. 2020;9(205):1\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulens SN, Sarah HY, Walters MS, Jacob JT, Bower C, Reno J, Kallen AJ. Carbapenem-nonsusceptible \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, 8 US metropolitan areas, 2012\u0026ndash;2015.Emerging infectious diseases. 2018;24(4):727.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzer B, Vatansever C, Dogan O, Keske S, Ergonul O, Can F. Biofilm Formation of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Under in vitro and in vivo Colistin Exposure. Infect Dis Clin Microbiol. 2019;1(1):26\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVijayakumar S, Gopi R, Gunasekaran P, Bharathy M, Walia K, Anandan S, Veeraraghavan B. Molecular Characterization of Invasive Carbapenem-Resistant Acinetobacter baumannii from a Tertiary Care Hospital in South India. Infect Dis Therapy. 2016;5(3):379\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamidian M, Nigro SJ. Emergence, molecular mechanisms and global spread of carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. Microb Genomics. 2019;5(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnish C, Abhisek R, Radha M, Chaudhary A. Evaluation of Biofilm Production in \u003cem\u003eAcinetobacter baumanii\u003c/em\u003e with Reference to Imipenem Resistance. Int J Sci Res Publications. 2017;7(12):732\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHawkey J, Ascher DB, Judd LM, Wick RR, Kostoulias X, Cleland H, Spelman DW, Padiglione A, Peleg AY, Holt KE. Evolution of carbapenem resistance in Acinetobacter baumannii during a prolonged infection. Microb Genomics. 2018;4(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGedefie A, Demsis W, Ashagrie M, Kassa Y, Tesfaye M, Tilahun M, Bisetegn H, Sahle Z. \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e biofilm formation and its role in disease pathogenesis: A review. Infect Drug Resist. 2021;14:3711\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoy S, Chowdhury G, Mukhopadhyay AK, Dutta S, Basu S. Convergence of Biofilm Formation and Antibiotic Resistance in \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Infection. Front Med. 2022;9:793615.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Li H, Wen H, Zhao B, Niu Y, Mo Q, Wu Y. Biofilm formation in \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e was inhibited by PAβN while it had no association with antibiotic resistance. Microbiol Open. 2020;9(9):1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowler P, Murphy C, Wolcott R. Biofilm exacerbates antibiotic resistance: Is this a current oversight in antimicrobial stewardship? Antimicrob Resist Infect Control. 2020;9(1):1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLongo F, Vuotto C, Donelli G. Biofilm formation in \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. New Microbiol. 2014;37(2):119\u0026ndash;27. PMID: 24858639.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang CH, Su PW, Moi SH, Chuang LY. Biofilm formation in \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e: Genotype-phenotype correlation. Molecules. 2019;24(10):1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrossard KA, Campagnari AA. The \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e biofilm-associated protein plays a role in adherence to human epithelial cells. Infect Immun. 2012;80(1):228\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\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. Int Microbiol. 2016;19(2):121\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCLSI. Clinical Laboratory Standard Institute (CLSI): Performance standards for antimicrobial susceptibility testing. 29 ed: Clinical and Laboratory Standrads Institute antimicrobial susceptibility testing standards M02, M07 and M11. 2019. 118\u0026thinsp;\u0026ndash;\u0026thinsp;28 p.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268\u0026thinsp;\u0026ndash;\u0026thinsp;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1469-0691.2011.03570.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1469-0691.2011.03570.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 21793988.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStepanovic S, Vukovic D, Hola V, Bonaventura GD, Djukic S, Circovic I, Ruzicka F. Quantification of biofilm in microtiter plates. Apmis. 2007;115(8):891\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClark NM, Zhanel GG, Lynch JP. Emergence of antimicrobial resistance among \u003cem\u003eAcinetobacter\u003c/em\u003e species: A global threat. Curr Opin Crit Care. 2016;22(5):491\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowler P, Murphy C, Wolcott R. Biofilm exacerbates antibiotic resistance: Is this a current oversight in antimicrobial stewardship? Antimicrob Resist Infect Control. 2020;9(1):1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhanal BR, Wagle S, Tiwari BR. Biofilm Formation and Colistin Susceptibility of Clinical Isolates of \u003cem\u003eAcinetobacter\u003c/em\u003e Species in a Tertiary Care Hospital of Nepal. Natl J Lab Med. 2019;8:1\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaut S, Rijal KR, Khatiwada S, Karna S, Khanal R, Adhikari J, Adhikari B. Trend and characteristics of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e infections in patients attending universal college of medical sciences, Bhairahawa, Western Nepal: A longitudinal study of 2018. Infect Drug Resist. 2020;13:1631\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetok Y, Subramanya SH, Thapa Shrestha U, Perez LRR, Adhikari N, Nayak N. Biofilm and MBL production among imipenem resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e species. MicroMedicine. 2020;8(2):63\u0026ndash;73 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.5281/zenodo.4195479\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.4195479\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav SK, Bhujel R, Hamal P, Mishra SK, Sharma S, Sherchand JB. Burden of multidrug-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e infection in hospitalized patients in a tertiary care hospital of Nepal. Infect Drug Resist. 2020;13:725\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThapa Shrestha U, Adhikari N, Maharajan R, Banjara MR, Rijal KR, Basnet SR, Agrawal VP. Multidrug resistant \u003cem\u003eVibrio cholerae\u003c/em\u003e O1 from clinical and environmental samples in Kathmandu city. BMC Infect Dis. 2015;15:104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.biomedcentral.com/orderreprints/s12879-015-0844-9\u003c/span\u003e\u003cspan address=\"http://www.biomedcentral.com/orderreprints/s12879-015-0844-9\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNayaju T, Upreti MK, Ghimire A, Shrestha B, Maharjan B, Joshi RD, Lekhak B, Thapa Shrestha U. Higher prevalence of extended spectrum β-lactamase producing uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e among patients with diabetes from a tertiary care hospital of Kathmandu, Nepal. Am J Trop Med Hyg. 2021;105(5):1347\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4269/ajtmh.21-0691\u003c/span\u003e\u003cspan address=\"10.4269/ajtmh.21-0691\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaharjan R, Bastola A, Adhikari N, Rijal KR, Banjara MR, Ghimire P, Thapa Shrestha U. Multidrug-resistant bacteria with ESBL genes: a growing threat among people living with HIC/AIDS in Nepal. BMC Infect Dis. 2022;22:526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12879-022-07503-2\u003c/span\u003e\u003cspan address=\"10.1186/s12879-022-07503-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoshi PR, Acharya M, Kakshapati T, Leungtongkam U, Thummeepak R, Sitthisak S. Co-existence of \u003cem\u003eblaOXA-23\u003c/em\u003e and \u003cem\u003eblaNDM-1\u003c/em\u003e genes of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolated from Nepal: Antimicrobial resistance and clinical significance. Antimicrob Resist Infect Control. 2017;6(1):1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahto M, Dhungel BA. Prevalence of Tigecycline resistance in Multidrug-Resistant \u003cem\u003eAcinetobacter\u003c/em\u003e species isolates from clinical specimens. Janaki Med Coll J Med Sci. 2019;7(2):42\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeupane L, Sah AK, Rayamajhee B, Pokhrel A, Singh A. Detection of blaoxa-23 Gene from Carbapenem-resistant \u003cem\u003eAcinetobacter Baumannii.\u003c/em\u003e J Nepal Health Res Counc. 2023; 20;20(4):899\u0026ndash;905. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.33314/jnhrc.v20i4.4257\u003c/span\u003e\u003cspan address=\"10.33314/jnhrc.v20i4.4257\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 37489674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRijal KR, Banjara MR, Dhungel B, Kafle S, Gautam K, Ghimire B, Dhungel S, Adhikari N, Thapa Shrestha U, Sunuwar DR, Adhikari B, Ghimire P. Use of antimicrobials and antimicrobial resistance in Nepal: a nationwide survey. Sci Rep (Nature). 2021;11:11554. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-90812-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-90812-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBista S, Thapa Shrestha U, Dhungel B, Koirala P, Gompo TR, Shrestha N, Adhikari N, Joshi DR, Banjara MR, Adhikari B, et al. Detection of Plasmid-Mediated Colistin Resistant \u003cem\u003emcr-1\u003c/em\u003e Gene in \u003cem\u003eEscherichia coli\u003c/em\u003e Isolated from Infected Chicken Livers in Nepal. Animals. 2020;10:2060. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani10112060\u003c/span\u003e\u003cspan address=\"10.3390/ani10112060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenmahmod AB, Said HS, Ibrahim RH. Prevalence and mechanisms of carbapenem resistance among \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e clinical isolates in Egypt. Microb Drug Resist. 2019;25(4):480\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts LW, Forde BM, Hurst T, Ling W, Nimmo GR, Bergh H, Harris PN. Genomic surveillance, characterization and intervention of a polymicrobial multidrug-resistant outbreak in critical care. Microb Genomics. 2021;7(3):mgen000530.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePattanaik A, Banashankari GS. Assessment of Biofilm Production in Carbapenem Resistant \u003cem\u003eAcinetobacter\u003c/em\u003e Species Isolated from Different Clinical Specimens. J Med Sci Clin Res. 2017;5(10):29103\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSunu Kumari AM, Routray A, Yadav D, Madhavan R. Imipenem resistance and biofilm production in Acinetobacter. Drug Invention Today. 2013;5(3):256\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantajit S, Indrawattana N. Mechanisms of antimicrobial resistance in Pasteurellaceae. PBioMed Res Int. 2016;2016(1155):1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanner WD, Atkinson RM, Goel RK, Toleman MA, Benson LS, Porucznik CA, VanDerslice JA. Horizontal transfer of the blaNDM-1 gene to \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e in biofilms. FEMS microbiology letters. 2017;364(8).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e \u003cstrong\u003eDistribution of bacterial isolates among positive cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e81 (28.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e57 (20.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e46 (16.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eACBC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e32 (11.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e23 (8.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e11 (3.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eE. faecium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e2 (0.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eC. freundii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e11 (3.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e1 (0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. mirabilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e11 (3.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e4 (1.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. saprophyticus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e1 (0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eE. aerogenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e1 (0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e281 (100)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Antibiotic susceptibility testing of ACBC isolates (n=32)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibiotic category\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibiotics used\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.943820224719104%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of ACBC isolates (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"58.214285714285715%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSusceptible N (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.785714285714285%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResistant N (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"22\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFirst Line Drugs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003ePenicillin + \u0026beta;-lactamase inhibitors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eAmoxicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e32 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eAmpicillin/sulbactam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e3 (9.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e29 (90.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eExtended-spectrum cephalosporins; 3\u003csup\u003erd\u003c/sup\u003e and 4\u003csup\u003eth\u003c/sup\u003e generation cephalosporins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eCefixime\u003cstrong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e2 (15.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e11 (84.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.59649122807018%\" valign=\"top\"\u003e\n \u003cp\u003eCefotaxime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.74561403508772%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.657894736842106%\" valign=\"top\"\u003e\n \u003cp\u003e32 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.59649122807018%\" valign=\"top\"\u003e\n \u003cp\u003eCeftazidime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.74561403508772%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.657894736842106%\" valign=\"top\"\u003e\n \u003cp\u003e32 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003eFolate pathway inhibitors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eCotrimoxazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e6 (18.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e26 (81.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003eMacrolides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eAzithromycin\u003cstrong\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e1 (10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e9 (90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eGentamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e1 (3.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e31 (96.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" rowspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003eFluoroquinolones\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eCiprofloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e4 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e28 (87.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.59649122807018%\" valign=\"top\"\u003e\n \u003cp\u003eLevofloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.74561403508772%\" valign=\"top\"\u003e\n \u003cp\u003e5 (15.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.657894736842106%\" valign=\"top\"\u003e\n \u003cp\u003e27 (84.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.59649122807018%\" valign=\"top\"\u003e\n \u003cp\u003eOfloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.74561403508772%\" valign=\"top\"\u003e\n \u003cp\u003e5 (15.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.657894736842106%\" valign=\"top\"\u003e\n \u003cp\u003e27 (84.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"33\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003ePhenicols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eChloramphenicol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e2 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e30 (93.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecond Line Drugs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003eAminoglycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eAmikacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e1 (3.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e31 (96.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003ePenicillin + \u0026beta;-lactamase inhibitors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eAmoxicillin-clavulanic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e2 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e30 (93.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eExtended-spectrum cephalosporins; 3\u003csup\u003erd\u003c/sup\u003e and 4\u003csup\u003eth\u003c/sup\u003e generation cephalosporins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eCefperazone/Sulbactam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e2 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e30 (93.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.59649122807018%\" valign=\"top\"\u003e\n \u003cp\u003eCefepime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.74561403508772%\" valign=\"top\"\u003e\n \u003cp\u003e2 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.657894736842106%\" valign=\"top\"\u003e\n \u003cp\u003e30 (93.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003eTetracyclines\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eDoxycycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e17 (53.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e15 (46.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eCarbapenems\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eImipenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e3 (9.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e29 (90.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.59649122807018%\" valign=\"top\"\u003e\n \u003cp\u003eMeropenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.74561403508772%\" valign=\"top\"\u003e\n \u003cp\u003e3 (9.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.657894736842106%\" valign=\"top\"\u003e\n \u003cp\u003e29 (90.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eEtrapenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e3 (9.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e29 (90.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026beta;-lactamase inhibitors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003ePiperacillin/Tazobactam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e2 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e30 (93.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eThird/last Line Drugs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003ePolymyxins\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eColistin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e32 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.59649122807018%\" valign=\"top\"\u003e\n \u003cp\u003ePolymyxin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.74561403508772%\" valign=\"top\"\u003e\n \u003cp\u003e32 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.657894736842106%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.80577849117175%\" valign=\"top\"\u003e\n \u003cp\u003eGlycylcyclines\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.25040128410915%\" valign=\"top\"\u003e\n \u003cp\u003eTigecycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.163723916532906%\" valign=\"top\"\u003e\n \u003cp\u003e27 (94.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.780096308186195%\" valign=\"top\"\u003e\n \u003cp\u003e5 (15.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u0026nbsp;\u003c/strong\u003eThe susceptibility assay for\u0026nbsp;Cefixime\u003cstrong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ewas used for only blood (n=3) and respiratory ACBC isolates (n=10). Likewise, the susceptibility testing for\u0026nbsp;Azithromycin\u003cstrong\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003ewas used for respiratory ACBC isolates (n=10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e \u003cstrong\u003eAssociation between Carbapenem resistance and Biofilm production\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.489795918367346%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm Production\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.714285714285715%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of Carbapenem\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.428571428571427%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.88235294117647%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResistant (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.11764705882353%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitive (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\" valign=\"top\"\u003e\n \u003cp\u003eStrong\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\" valign=\"top\"\u003e\n \u003cp\u003e2 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.463917525773196%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.649484536082475%\" valign=\"top\"\u003e\n \u003cp\u003e22 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" rowspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.379746835443036%\" valign=\"top\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.050632911392405%\" valign=\"top\"\u003e\n \u003cp\u003e8 (25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.9873417721519%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.582278481012658%\" valign=\"top\"\u003e\n \u003cp\u003e8 (25)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.379746835443036%\" valign=\"top\"\u003e\n \u003cp\u003eweak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.050632911392405%\" valign=\"top\"\u003e\n \u003cp\u003e19 (59.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.9873417721519%\" valign=\"top\"\u003e\n \u003cp\u003e2 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.582278481012658%\" valign=\"top\"\u003e\n \u003cp\u003e21 (65.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.379746835443036%\" valign=\"top\"\u003e\n \u003cp\u003eNon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.050632911392405%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.9873417721519%\" valign=\"top\"\u003e\n \u003cp\u003e1 (3.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.582278481012658%\" valign=\"top\"\u003e\n \u003cp\u003e1 (3.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e*Chi-square test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4:\u003c/strong\u003e \u003cstrong\u003eDistribution of biofilm-associated genes\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBap, csuE\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.833333333333336%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"63.166666666666664%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of ACBC isolates with biofilm-producing genes (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.09234828496042%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBap\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.07915567282322%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ecsuE\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.82849604221636%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.833333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eProducer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.166666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e21 (65.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19%\" valign=\"top\"\u003e\n \u003cp\u003e21 (65.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22%\" valign=\"top\"\u003e\n \u003cp\u003e18 (56.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.833333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eNon-producer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.166666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e11 (34.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19%\" valign=\"top\"\u003e\n \u003cp\u003e11 (34.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22%\" valign=\"top\"\u003e\n \u003cp\u003e14 (43.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.833333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.166666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5:\u003c/strong\u003e \u003cstrong\u003eAssociation between Carbapenem resistance and biofilm-related genes\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.916943521594686%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbapenem\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm related gene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.11295681063123%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.11295681063123%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.83720930232558%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetected (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"51.16279069767442%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNot detected (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.916943521594686%\" valign=\"top\"\u003e\n \u003cp\u003eSensitive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.930232558139537%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.92691029900332%\" valign=\"top\"\u003e\n \u003cp\u003e3 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.11295681063123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.11295681063123%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"top\"\u003e\n \u003cp\u003eResistance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.95049504950495%\" valign=\"top\"\u003e\n \u003cp\u003e21 (72.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.138613861386137%\" valign=\"top\"\u003e\n \u003cp\u003e8 (27.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.207920792079207%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.95049504950495%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.138613861386137%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.207920792079207%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6:\u003c/strong\u003e \u003cstrong\u003eAssociation between Biofilm forming genes with biofilm intensity\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.166666666666664%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm Intensity (Number of biofilm-producing isolates)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eACBC isolates possessing biofilm-related genes (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.833333333333332%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBap\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ecsuE\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ebla\u003csub\u003ePER1\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.166666666666664%\" valign=\"top\"\u003e\n \u003cp\u003eStrong (n=2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16%\" valign=\"top\"\u003e\n \u003cp\u003e2 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17%\" valign=\"top\"\u003e\n \u003cp\u003e2 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17%\" valign=\"top\"\u003e\n \u003cp\u003e2 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.833333333333332%\" rowspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.879759519038075%\" valign=\"top\"\u003e\n \u003cp\u003eModerate (n=8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.238476953907817%\" valign=\"top\"\u003e\n \u003cp\u003e8 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e8 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e8 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.879759519038075%\" valign=\"top\"\u003e\n \u003cp\u003eWeak (n=21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.238476953907817%\" valign=\"top\"\u003e\n \u003cp\u003e11 (52.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e11 (52.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e8 (38.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.879759519038075%\" valign=\"top\"\u003e\n \u003cp\u003eNon (n=1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.238476953907817%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.879759519038075%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal (n=32)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.238476953907817%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e21 (65.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e21 (65.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.440881763527056%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e18 (56.2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acinetobacter baumannii, Biofilm, Carbapenem-resistant, Biofilm-related genes; Bap, csuE and blaPER1","lastPublishedDoi":"10.21203/rs.3.rs-4413953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4413953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAcinetobacter calcoaceticus-baumannii complex\u003c/em\u003e (ACBC) has emerged as a global burden to various clinical infections. The increasing resistance to the majority of antibiotics adds a huge problem in empirical therapy and control of ACBC infections. In addition, the ability of ACBC to produce biofilm has not only contributed to its antimicrobial resistance but also helped its persistence and survival in the environment. Most tertiary care hospitals in Nepal are facing similar ACBC infections.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHence, this hospital-based cross-sectional study was carried out to associate the biofilm formation with carbapenem-resistant ACBC isolates detecting biofilm-forming genes \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE\u003c/em\u003e, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePER1\u003c/em\u003e\u003c/sub\u003e from February 2020 to August 2020 at Kathmandu Model Hospital, Kathmandu, Nepal. The clinical bacterial isolates were identified by standard Microbiological procedures including Gram staining, and cultural and biochemical characteristics. A modified Kirby-Bauer disk diffusion method was performed to assay the antibiotic susceptibility testing of ACBC isolates to various antibiotic classes. A quantitative adherence assay was used to determine the biofilm assay. A conventional Polymerase Chain Reaction (PCR) method was used to find the targeted biofilm-related genes using specific primers.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOut of 665 different clinical samples, bacterial growth was observed in 281 (42.3%) clinical samples. Of these, 32 (11.4%) isolates were identified as ACBC. Out of 32 ACBC isolates, 29 (90.6%) of which were carbapenem-resistant. All carbapenem-resistant ACBC isolates were found to be sensitive to polymixin B and colistin. Out of 29 CR-ACBC, 17.2% of isolates were resistant to tigecycline. A total of 31 ACBC isolates were biofilm producers, out of which 2 were strong biofilm producers followed by 8 moderate, and 21 were weak biofilm producers. The occurrence of biofilm-forming genes; \u003cem\u003eBap\u003c/em\u003e, \u003cem\u003ecsuE\u003c/em\u003e, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePER1\u003c/em\u003e\u003c/sub\u003e genes were found to be 65.6%, 65.6%, and 56.3% respectively among ACBC clinical isolates. A significant association was observed between carbapenem resistance, biofilm formation, and biofilm-related genes.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSince ACBC isolates are ubiquitous including in the hospital environment and its infections are alarming to clinical settings, the effective sterilization of clinical equipment and hospital environment are utmost. In addition, a strong policy should be made to prescribe the proper antibiotic based on antibiogram profile to fight against an emerging threat of ACBC infections\u003c/p\u003e","manuscriptTitle":"Increased biofilm-associated Carbapenem-resistant Acinetobacter- calcoaceticus-baumannii complex infections among the hospitalized patients in Kathmandu Model Hospital, Nepal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-30 20:13:45","doi":"10.21203/rs.3.rs-4413953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-05-16T17:06:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-16T17:06:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2024-05-13T14:39:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3dec85c-2a7f-4809-9953-5c7f2fa8d401","owner":[],"postedDate":"May 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-30T20:13:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-30 20:13:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4413953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4413953","identity":"rs-4413953","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}