Whole-Genome Sequencing Insights into Porin-Mediated Resistance and Spread of ESBL- Producing Klebsiella pneumoniae in a Ghanaian Teaching Hospital

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Thus, the current study determined the prevalence and clonal relatedness of MDR K. pneumoniae from hospital environments, patients and healthcare workers in a Ghanaian hospital. Methods Patients (rectal and hand, collected on admission and 48h post admission), healthcare workers (hands) and hospital environment samples were sampled for three months. Antimicrobial susceptibility was determined using VITEK-2. Ten MDR ESBL-producing K. pneumoniae isolates were further analysed by whole-genome sequencing. Results All the isolates were ceftazidime-resistant; 90% were resistant to cefepime, amoxicillin/clavulanic, acid piperacillin/tazobactam, and sulphamethoxazole/trimethoprim. The isolates showed varying resistance to the cephalosporins and were susceptible to tigecycline. One environmental isolate isolate was resistant to meropenem but harboured no carbapenemase gene. The β-lactamase gene, bla SHV, was dominant and harboured by three environmental and five carriage isolates. Furthermore, three environmental and three carriage isolates harboured bla CTX−M−15 . All isolates showed ompK36 and ompK37 mutations. Fluoroquinolone ( qnrB ), aminoglycosides ( aadA1 , aadA2, aac(3)-IIa, aac(6')-Ib-cr,aph(3'')-Ib, aph(6)-Id ) and sulphamethoxazole/trimethoprim ( sul1, sul2 , dfrA14, dfrA15 ) resistance-encoding genes were also detected. A diverse range of sequence types were identified, including ST39, ST307, ST815, ST1552, ST636, ST464, and ST1996, with ST39 being the most frequently observed (environmental = 3; carriage = 1). Three environmental and three carriage isolates harboured the Int1l integron. Many virulence genes, including irp1 , irp2 , iutA , gndA , ompA , fes, fep , mrkD and fimH , were detected in environmental and carriage isolates. IncFIB was the most abundant plasmid replicon in five environmental and four carriage isolates. A clonal relationship was identified between a carriage isolate (ST39) and three environmental isolates (ST39) with shared genetic elements, suggesting that environmental reservoirs may play a role in the transmission and persistence of resistant K. pneumoniae . Conclusion This study highlights the prevalence of MDR ESBL-producing K. pneumoniae in both hospital environments and patients, emphasizing the potential for cross-transmission within healthcare settings. These findings reinforce the urgent need for strengthened infection prevention and control measures, enhanced antimicrobial stewardship, and continuous genomic surveillance to mitigate the spread of resistant K. pneumoniae in healthcare settings. Hospital-acquired infections hospital environment multidrug resistance extended-spectrum β-lactamase carbapenem-resistant bacteria whole genome sequencing public health Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The hospital environment constitutes an important component of infection prevention and control (IPC), as it has been demonstrated that many microorganisms can survive on different surfaces in these environments, with Klebsiella pneumoniae surviving for up to 600 days on inanimate objects [ 1 ]. This represents a potential health threat to users of hospital spaces, including patients, healthcare workers and other visitors. Although measures such as improved hygiene [ 2 ] are implemented to curb disease transmission in hospitals, many microorganisms have developed resistance mechanisms that allow them to escape the effect of disinfectants [ 3 ], allowing them to survive longer in these environments with potential transmission. Of particular importance in these spaces are Gram-negative bacteria, especially drug-resistant ones. Multidrug-resistant Gram-negative bacteria threaten human health and may cause life-threatening infections [ 4 ]. Among these, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant K. pneumoniae have been listed by the World Health Organization (WHO) among pathogens of critical priority for the development of new antibiotics [ 5 ]. This resistant pathotype notably causes nosocomial and community-acquired infections such as pneumonia and urinary tract infections [ 6 ]. K. pneumoniae isolates can harbour many resistance genes and develop diverse antibiotic resistance mechanisms [ 7 ]. In addition to its intrinsic β-lactamase gene, bla SHV−1 , mutations resulting in the loss, reducing the number and diameter of outer membrane porins, ompK35 , ompK36 , and the quiescent ompK37 as well as efflux pumps may contribute to the resistance of K. pneumoniae to β-lactams, including carbapenems [ 8 , 9 ]. Antibiotic resistance genes in K. pneumoniae can be spread widely by the dissemination of epidemic clones and mobile genetic elements, predominantly plasmids [ 10 ]. The Inc-groups of plasmids IncF, IncFII(K1), IncR, IncX, IncX3, IncI2, and ColE1 have particularly been associated with the rapid spread of resistance in K. pneumoniae isolates [ 10 ]. Multilocus sequencing typing (MLST) has revealed the circulation of various sequence types (STs) of MDR K. pneumoniae isolates. Notably, ST307 K. pneumoniae has been recognised as a globally emerging clone with genetic characteristics enabling ease of dissemination and persistence in the hospital setting [ 11 ]. It has been detected in Korea, Germany, South Africa, and Nigeria. [ 12 – 15 ]. The ST39 has also been reported in Russia, South Africa, and Ethiopia. [ 16 – 18 ]. In a study of MDR K. pneumoniae isolates recovered from clinical samples in the Komfo Anokye Teaching Hospital, Ghana, 24.32% of the isolates were resistant to second- and third-generation cephalosporins and carried multiple resistant genes. MLST analyses revealed the circulation of multiple K. pneumoniae STs (ST2171, ST2186, ST17, ST152, ST397, ST101, ST1788 and STS789) in the hospital [ 19 ]. The presence of several virulence genes such as the fimbriae synthesis-related gene, lipopolysaccharide-related gene, capsular polysaccharide synthesis and synthesis regulation-related gene, iron uptake system, urease-related gene, tellurite resistance gene hemolysin among others contribute to the pathogenicity of K. pneumoniae . Capsule and lipopolysaccharide (LPS) antigens, K and O, have been reported to contribute significantly to the pathogenicity of K. pneumoniae isolates [ 20 ]. The structural differences of these antigens have been useful in assigning serotypes which describe the extent of virulence among various K. pneumoniae strains [ 21 ]. Hypervirulent K. pneumoniae associated with increased mortalities emerged in Asia and continues to spread globally [ 22 ]. Investigations into virulent Klebsiella pneumoniae have primarily focused on outbreak settings. The hospital environment in spite of its established role as a reservoir and transmission route for many pathogens, including antibiotic-resistant ones is not usually considered during such investigation as the focus is on the patients. It is equally important to investigate the pathogenicity and virulence of K. pneumoniae isolates colonising patients in non-outbreak settings to establish/improve IPC practices. Thus, despite recent global studies on the epidemiology of ESBL-producing and carbapenem-resistant K. pneumoniae , there is a lack of data on patients’ environments, especially in Sub-Saharan African countries such as Ghana. Using whole genome sequencing and bioinformatics analysis, the current study assessed MDR K. pneumoniae isolates from hospital environments and patients in a teaching hospital in Ghana to establish any clonal relationship between the hospital environment and patient isolates. The study further determined the antimicrobial resistance, virulence, and genetic relatedness of ESBL-producing and carbapenem-resistant isolates in the context of IPC. Materials and Methods Ethical approval Ethical approval for the study was obtained from the Institutional Review Board (IRB) of the Komfo Anokye Teaching Hospital (KATH) (Reference: KATH IRB/AP/107/20) and the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (Reference: BREC/00001917/2020 ). Voluntary, informed written consent was obtained from participating patients and staff. Study population and sample collection The study focused on MDR ESBL-producing K. pneumoniae contaminating hospital environments and colonising patients at the Komfo Anokye Teaching Hospital, Kumasi, from April 2021 to July 2021. Rectal and hand swabs were collected from consenting adult patients within 24 hours of admission and after 48 hours in the wards of three hospital directorates: Obstetrics and Gynaecology, Surgery, and the Intensive Care Unit. Hand swabs were collected concurrently from staff present during patient sampling and from the patient’s immediate environment (bedrails, drip-stand, door handles and taps) in the wards. Isolation and Identification of ESBL-producing MDR K. pneumoniae Briefly, swabs collected from the ward environments, 83 patients and from the hands of healthcare workers were processed on MacConkey agar, and 250 Gram-negative bacteria were identified by Gram staining. Using the VITEK 2 ® automated system (BioMérieux-Vitek, Marcy-l’Étoile, France), 31 isolates were identified among the Gram-negative bacteria as K. pneumoniae . Antibiotic susceptibility testing Antibiotic susceptibility testing was conducted using the VITEK 2 ® automated system (BioMérieux-Vitek, Marcy-l’Étoile, France). The antibiotic panel consisted of 16 antibiotics: cefuroxime (CXM), ceftazidime (CAZ), ceftriaxone (CRO), amoxicillin/clavulanic (AMC), cefepime (FEP), piperacillin/tazobactam (TZP), imipenem (IPM), doripenem (DOR), meropenem (MEM), ertapenem (ERT), gentamicin (GEN), tobramycin (TOB), amikacin (AMK), ciprofloxacin (CIP), trimethoprim-sulphamethoxazole (SXT) and tigecycline (TGC). Isolates were categorized into susceptible, intermediate and resistant using Clinical and Laboratory Standards Institute guidelines (CLSI 2020). DNA extraction, Genome sequencing and analysis Genomic DNA (gDNA) from pure colonies of identified overnight cultures of MDR ESBL-producing K. pneumoniae were extracted using the GenElute® bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, United States) according to the manufacturer’s instructions. The concentration and quality of the extracted gDNA were checked using the Nanodrop 8000 (Thermo Scientific, Waltham, MA, USA). The sequencing and analyses (JEKESA pipeline) were conducted at the Sequencing Core Facility of the South African National Institute for Communicable Diseases (NICD). Using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, United States), Multiplexed paired-end libraries (2 x 300 bp) were prepared. Sequences were determined on an Illumina Nextseq 550 (2 x 150 bp) platform (Illumina, San Diego, CA, United States) with 100× coverage. Quality trimming of raw reads was done using Sickle v1.33 (https://github.com/najoshi/sickle). The raw reads were then assembled spontaneously using the SPAdes v3.6.2 assembler (https://cab.spbu.ru/software/spades/). All contiguous sequences were subsequently submitted to GenBank and assigned accession numbers ( Supplementary Table S1) under BioProject PRJNA823741. Molecular typing of K. pneumoniae isolates Multilocus sequence typing (MLST) was performed in-silico using the WGS data online platform tool with the assembled genomes (https://bigsdb.pasteur.fr/klebsiella/). The serotypes (K types, O types, and wzc and wzi allelic types) of the isolates were determined using the reference Klebsiella WGS data online platform tool, Kaptive-web (http://kaptive.holtlab.net/). Identification of the acquired and chromosomal mutations in the isolates Resistance and virulence genes were determined using ResFinder (https://cge.food.dtu.dk/services/ResFinder/) [accessed on July 18, 2024] and VF analyser via Virulence finder database (https://cge.food.dtu.dk/services/VirulenceFinder/) [accessed on July 20, 2024], respectively. Mutations in the ompK35, omp36 and ompK37 genes were determined via ResFinder. Identification of mobile genetic elements (MGEs)/genetic support The presence of mobile genetic elements was determined using PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/) for plasmids, (Carattoli et al., 2014), INTEGRALL (http://integrall.bio.ua.pt/) for integrons, and RAST SEEDVIEWER (https://rast.nmpdr.org/seedviewer.cgi) for transposons and insertion sequences. Insertion sequences (IS) in genomes were predicted via the ISFinder database (https://www-is.biotoul.fr/). The PHASTER ( https:// phaster.ca/ ) server was used to identify and visualise prophage sequences. The synteny and genetic environment of antibiotic resistance genes and associated mobile genetic elements were investigated using the general feature format (GFF3) files from GenBank. Phylogenomic analyses of the K. pneumoniae isolates Phylogenomic analysis was done to determine the genetic relatedness of isolates in this study. Isolates from this study were also compared with 180 K. pneumoniae isolate genomes from sub-Saharan African countries that were downloaded from the Bacterial and Viral Bioinformatics Resource Center (BV-BRCB) (https://www.bv-brc.org/ ) . A maximum likelihood tree was constructed on BV-BRCB and rooted on the reference genome K. pneumoniae Ecl8 (accession number: HF536482 CANH01000000). FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and Interactive Tree of Life (iTol) v7 (https://itol.embl.de/) were used to visualise and annotate the phylogenetic tree. To gain a more comprehensive insight into the relationships of isolates from this study, Phandango (https://jameshadfield.github.io/phandango/#/main) was used to visualise the phylogenetic tree with corresponding metadata. Results Isolates identification and selection Thirty-one of the 250 Gram-negative bacteria isolated from swabs were identified as putative K. pneumoniae, of which 20/31 (64.5%) were MDR (15 from patients and five from the hospital environment). Ten of the 20 isolates were confirmed as K. pneumoniae, that is, five carriage isolates and five from the environment (door handle, tap, drip-stand and bed and five from patients) after quality assurance and WGS; low quality genomes and those belonging to Klebsiella variicola or Klebsiella quasipneumoniae , biochemically indistinguishable from K. pneumoniae were excluded. Antimicrobial resistance profiles of selected MDR K. pneumoniae isolates from the hospital environments and patients All (100%) of the ten selected MDR K. pneumoniae isolates were resistant to ceftazidime and cefuroxime. Ninety percent of the isolates were resistant to piperacillin/tazobactam, cefepime, amoxicillin/clavulanic acid and sulphamethoxazole/trimethoprim. Eighty percent (8/10) of the isolates showed resistance to ceftriaxone. However, all isolates were susceptible to tigecycline, amikacin, ertapenem and imipenem. Two carriage isolates (P121 and P60-1) and one (E55-1) from the hospital environment showed intermediate resistance to doripenem and resistance to meropenem and presented with elevated minimum inhibitory concentrations (MICs) of 2-4 µg/mL (Supplementary Table S2) . Sixty percent (3/5) of carriage isolates and 60% (3/5) from hospital environments were resistant to gentamicin, tobramycin and ciprofloxacin (Table 1) . The most common antibiogram pattern among the isolates was CXM-FEP-CAZ-CRO-AMC-TZP-GEN-CIP-GEN-SXT, which was common to five isolates; two from patients (P121 and P129) and three from the hospital environment (E4, E34A and E50-2) (Table 2) . WGS-based capsular serotyping and Multilocus sequence typing (MLST) The isolates showed high MLST variation, with seven STs identified, viz., ST39, ST307, ST815, ST1552, ST636, ST464 and ST1996. Four isolates, including three from the hospital environment, i.e., from a bed (E4), door handle (E35) and drip stand (E50), respectively, and one carriage isolate (P60-1), belonged to the same ST 39. The isolates belonged to different capsular serotypes. Two isolates (E4 and E50-2) from the hospital environment (bed and drip stand respectively), however, shared the same ST 39 and capsular serotype, wzi 2 (Table 2) . The O and K capsules were diverse among the isolates. Predominantly, of the O capsules, O1/O2v1 (n = 5/10) and O1/O2v2 (n = 4/10) were common to the isolates. The other O-capsule type for K. pneumoniae was the O3b. The k capsular types were more diverse, with all but two isolates showing different k capsular types. Isolates of the same ST did not necessarily have the same O and K capsules. For instance, for isolates belonging to ST 39, two isolates (E4 and E50-2 from the hospital environment and a patient, respectively) had the O1/O2v1 capsule with KL2 capsules, while the other two (E35 and P60-1 from the hospital environment and a patient respectively) had the same O capsule (O1/2v2) but different K capsules (KL23 and KL149, respectively). Resistome of the isolates Several β-lactamase genes were identified in the isolates, with the dominant genes being the bla SHV (n = 8) in four carriage and four hospital environment isolates. bla CTX-M-15 gene was found in six isolates (three hospital environment and three carriage isolates). Seven different bla SHV genes were identified in the isolates. These included bla SHV-1 , bla SHV-11 , bla SHV-38 , bla SHV-41 , bla SHV-62 , bla SHV-106 , and bla SHV-172 . The bla SCO-1 gene was detected in two carriage isolates (P121 and P136), which also harboured the bla TEM-1, bla CTX-M-15 genes and the bla SHV-172 (P121) and bla SHV-62 (P136) ( Table 2). The bla DHA gene was found in a carriage isolate (P60-1) with ST39 from a patient, though other isolates of similar ST lacked the bla DHA gene. Co-production of bla CTX-M , bla TEM and bla SHV was evident in three carriage isolates (P121 and P136) and two isolates (E34A and E50-2) from the hospital environment. An isolate from the environment (E35) did not harbour any β-lactamase genes, although it was resistant to cefuroxime, ceftazidime, cefepime, amoxicillin/clavulanic and piperacillin/tazobactam ( Table 2) . WGS analysis did not identify any carbapenem resistance genes in the isolate (E55-1) resistant to meropenem. Nine isolates also harboured sulphamethoxazole-trimethoprim resistance genes, sul and dfrA Aminoglycoside resistance was encoded by the aac(aac(3)-IIa , aac(6’)-Ib-cr ), aph (aph(3”)-Ib , aph(6)-Id) , aad(aadA1, aadA2) and ant(2”)-Ia genes and were associated with increased MICs to gentamicin (≥16 µg/mL) and tobramycin (≥16 µg/mL). Other resistance genes were the tet genes in four isolates ( tetA in P60-1 and E34A and tetD in E35 and P136), encoding tetracycline resistance in carriage isolates and hospital environment isolates. The fosA gene for fosfomycin resistance was harboured by all ten isolates (five environmental and five carriage isolates). The QacE genes encoding resistance to quaternary ammonium compounds were observed in three isolates (carriage isolates P121, P136 and the isolate from the hospital environment E35) (Table 2) . Fluoroquinolone resistance, which was observed in five (three hospital environment and two patient) isolates associated with the plasmid-mediated quinolone resistance (PMQR) genes, Qnr , Oqx and aac(6′)-lb-cr (Table 2 ) and less frequently with chromosomal point mutations in the quinolone resistance determining regions (QRDR) of gyrA (S83Y, D87A) which were observed in a carriage isolate, P129 and parC (S80I) in P129 and E34A (Supplementary Table S4) . The oqxA and oqxB genes encoding the multidrug efflux pump, oqxAB , were found in all ten isolates (five environmental and five carriage). The qnr gene occurred in seven isolates (four isolates environmental and three carriage), the most frequent being the qnrB genes. Five (three environmental and two carriage) isolates harboured the aac(6’)-Ib-cr gene and were associated with high MICs to the aminoglycosides gentamicin and tobramycin (Table 2). Chromosomal point analysis indicated mutations in the acrR efflux pump gene in all the K. pneumoniae isolates. One carriage isolate (P129) had the acrR mutation (F204L) encoding tigecycline resistance, though this was not phenotypically expressed (Table S4) . Mutations in outer membrane proteins To determine the contribution of porins to cephalosporin resistance and the reduced susceptibility to carbapenems in the K. pneumoniae isolates (P121, P60-1 and E55-1), all isolates were further assessed for the presence of mutations in porins that could mediate carbapenem resistance in concert with ESBLs. Investigation of major outer membrane porins showed an intact ompK35 gene in all the MDR K. pneumoniae strains from patients and hospital environments. However, numerous mutations associated with resistance to cephalosporins and reduced susceptibility to carbapenems were found in ompK36 (N49S, L59V, T86V, S89T. D91K, A93S, L191Q. F207W, A217S, N218H, Q227N, L229V, E232R, H235D, T254S, T184P, G189T, F198Y, F207Y, A217S, T222L, D223G, N304E) and ompK37 (I70M, I128M, N230G) genes in all isolates (Table 2) . Mobile Genetic elements and environment of ARGs PlasmidFinder revealed the presence of at least two different plasmid replicon types in all ten isolates from patients and hospital environments. The plasmids included IncFIA, IncFIB, IncFII, IncHI1B, IncQ1, Col and Col440II. IncFIB was the most abundant plasmid replicon in nine (five hospital environment and four carriage) isolates. Different insertion sequences were found in the isolates. The transposable element MITEPlu5 , along with the insertion sequences ISCARN29 and ISSph8 , was identified in two ST39 isolates (E4 and P60-1) from the hospital environment and a patient (Supplementary Table S5) . Six isolates had class I integrons. Similar gene cassettes were found in two isolates (E50-2 and E55-1) from the hospital environment bearing the dfrA14 gene. An Intl1 integrase was associated with dfrA15 together with the sulphamethoxazole gene sul1 and the quaternary ammonium compound resistance gene, QacE , on gene cassettes in the carriage isolates P121 and P136 (Table 3 and Figs. 1 and 2) . The bla CTX-M and bla TEM-1 genes were associated with a transposon, recombinase or insertion sequence (Table S3). The bla CTX-M-15 was particularly associated with the transposon IS1380 in the environmental isolates E4, E34A and E50-2 and the carriage isolate P121. The bla TEM-1 was often bracketed by a transposase and recombinase. In E4, E34A, and E50-2, bla TEM-1 was associated with the transposase IS91 and a recombinase (Table S3). The bla SCO-1 gene was also associated with a recombinase in the carriage isolates P121 and P136. The bla SHV gene was, however, chromosomal and not carried on any mobile element. The bla OXA-1 genes were commonly associated with the aminoglycoside hydrolysing gene, aac(6’)-Ib-cr , and the chloramphenicol acetyltransferase, catB3 (Table S3). In the carriage isolate P136, bla OXA-1 was associated with the IS6 transposase with a gene cassette like the K. pneumoniae strain KPH3 plasmid. aph(6)-Id: aph(3'')-Ib were associated with the Tn3 in the carriage isolate P121. Additionally, P121 harboured resistance genes associated with IskrA4 , IS91 and Tn3 transposons and the intI1 integrase (Table S5) . Table 3. Gene cassettes of ESBL K. pneumoniae isolates from patients and the hospital environment Bacterial ID Source Integron Integron name GC1 GC2 GC3 GC4 E34A Environment Int1l In191 dfrA14 E35 Environment Int1l ln388 dfrA15 QacE sul1 P121 Patient Intl1 ln388 dfrA15 aadA1 QacE sul1 P136 Patient Intl1 ln388 dfrA15 aadA1 QacE sul1 E50-2 Environment Intl1 ln191 dfrA14 P129 Patient Intl1 ln388 dfrA15 aadA1 Virulome of ESBL K. pneumoniae isolates A myriad of virulence genes involved in fimbriae synthesis ( fimH , mrkD ), capsular polysaccharide synthesis and synthesis regulation, genes involved in iron uptake system ( iroE , fes , and fur ) aerobactin, ( iutA , irp1 , irp2 and iucA ) yersiniabactin ( ybtA , ybtP , ybtQ ), and enterobactin entBEF ) were present in the isolates (Table 2) . All isolates (both carriage and from the hospital environment) harboured the iutA , gndA , and ompA factors. entC/E, fes and fep were found in four isolates from the hospital environment and five carriage isolates but were absent in an isolate (E35) from the environment which lacked any β-lactamase or ESBL genes and was carbapenem-resistant . iroE was found in nine isolates (four hospital environment and five carriage isolates). fyuA , irp1 and irp2 commonly occurred together in seven isolates (three hospital environment and four carriage) (Table 2) . Though the isolates possessed several virulence genes, hypervirulent K. pneumoniae , which are typically characterised by the presence of roB , iucA , peg-344 , rmpA , and rmpA2 genes [23] were not found. Phylogenomics of the isolates Phylogenomic analyses based on differences in single nucleotide polymorphisms (SNPs) and core genome analyses of the metadata using Phandango revealed that the isolates were diverse. However, isolates of ST39 (E35, E4 and E50-2), from the hospital environment and P121 (from a patient), grouped into one cluster. P144 (ST636) and E34A (ST307) from a patient and the hospital environment, respectively, were also similar despite belonging to two different sequence types (Fig. 3) . The bla CTX-M-15 gene was present in two ST39 isolates, and ST815, ST1552, ST464 and ST307 isolates. The β-lactamases were frequently associated with plasmids, particularly the IncF1B found in all STs, except for an ST39 isolate (E4). The int1integron was found in six isolates. To determine the clonal relatedness of the K. pneumoniae isolates, the isolates in this study were compared to other isolates from sub-Saharan Africa. The K. pneumoniae isolates from the hospital environment and patient carriage were also found to cluster based on sequence types with other genomes from countries in the sub-Saharan Africa region, indicating a wide dissemination of common STs such as ST307 and ST39 in Africa. The isolates clustered with similar isolates previously isolated from clinical sources such as blood, urine, and stool in patients with infections (Fig. 4) . Discussion The dissemination of antimicrobial-resistant pathogenic bacteria possessing virulence factors within the hospital environment poses a major threat to patient prognosis [24]. It is thus a concern for IPC in hospitals. ESBL-producing and carbapenem-resistant K. pneumoniae have become a serious concern in hospital and community-acquired infections [12, 25]. The current study revealed the hospital environment as a potential source and transmission route for these resistant strains, necessitating effective measures to prevent their spread to users of the hospital environment. Resistance genes were widely distributed among the K. pneumoniae isolates. β-lactamases commonly identified in both carriage isolates and isolates from hospital environments were bla SHV and bla CTX-M , especially the bla CTX-M-15 gene that is widespread among K. pneumoniae isolates [26-28] . These β-lactamases genes have been reported among K. pneumoniae isolates from South Africa and Spain, showing their widespread nature. bla SHV has been reported as commonly harboured by K. pneumoniae among carriage samples, confirming their constitutive chromosomal expression [28, 29]. Carriage isolates and isolates from the hospital environment also harboured the aminoglycoside and quinolone-resistant genes aac(6′)-Ib-cr and the multidrug efflux pumps acrAB and OqxAB . In association with the IncFII plasmid, these resistance mechanisms, reported in other Enterobacterales [30], could also be circulated in the hospital environment and acquired by other bacteria, leaving limited options for treating infections caused by these ESBL-producing K. pneumoniae . The mutations (N49S, L59V, L191S, F207W, D224E, L228V, and E232R) in ompK36 have been previously reported in association with cephalosporin resistance [31]. In this study, these mutations co-occurred with the presence of ESBLs such as bla CTX-M , which may contribute to the high resistance to cephalosporins, as indicated by elevated MICs among both carriage K. pneumoniae isolates and environmental isolates (Supplementary Table S2). However, since no functional assay was performed, the role of these ompK36 mutations in cephalosporin resistance requires further experimental confirmation. The observed β-lactam resistance, including cephalosporin resistance, in the ESBL-producing K. pneumoniae isolates aligns with previous reports by Humphries et al [32] describing associations between K. pneumoniae resistance to cephalosporins (ceftazidime/avibactam) and point mutations in ompK36 . However, while such mutations have been linked to resistance, additional functional analysis is necessary to confirm their role in the observed phenotypic resistance. The mutations A217S, and N218H in ompK36 and I70M, and I128M in ompK37 , reported to be associated with carbapenem resistance among K. pneumoniae isolates [31], were observed in all the isolates, pointing to developing carbapenem resistance in these ESBL-producing K. pneumoniae isolates. Yang et al. also found numerous mutations in ompK36 and ompK37 but not in ompK35 among all K. pneumoniae isolates resistant to Cefoperazone/sulbactam and piperacillin/tazobactam [33]. The emerging carbapenem resistance in these K. pneumoniae isolates from both patients, and the hospital environment is a serious threat to IPC as they may be disseminated in the wards to cause infections that may be severe and difficult to treat. Hence, IPC enhancement is needed to prevent the spread of these ESBL-producing isolates in hospitals. The high number of virulence genes detected in carriage and hospital environment isolates may contribute to the pathogenicity of the K. pneumoniae isolates [34] in colonised patients without adequate IPC practices. In this study, all the ESBL- K. pneumoniae isolates had type 1 or 3 ( fim or mrk ) fimbriae comparable to 90% of clinical K. pneumoniae isolates, which included ESBLs producers possessing fimbriae in a study of K. pneumoniae isolates from Egypt [35]. Coupled with the different capsular serotypes, siderophores, and a high number of other virulence genes in the isolates, these fimbriae, which are major adhesins, may confer a survival advantage in these K. pneumoniae isolates as they are involved in bacterial colonisation of host, biofilm formation and invasion [36, 37]. Though virulence genes associated with hypervirulent K. pneumoniae were not found in the isolates in this study, hypervirulent K. pneumoniae has been identified among isolates with predominantly K1 and K2 capsules with the O1 O-antigen, which have acquired the hvKP virulence plasmid [38], presenting the potential of isolates becoming hypervirulent if they acquire plasmids harbouring the hyper-virulence genes. There was a high variation of STs among the isolates, showing the dissemination of different STs (ST39, ST307, ST815, ST1552, ST636, ST464 and ST1996) in the hospital. ST464 and ST636 were acquired by patients after admission and could have been circulating in the wards before patient admission, while the other STs (ST815 and ST1552) appeared to be circulating in the community. Notably, the high-risk clone ST307 was detected in one isolate from the hospital environment, suggesting this global clone’s presence and possible dissemination in Ghanaian hospitals. The ST307, which harboured the ESBL genes bla SHV-106 and bla CTX-M-15 , and the β-lactamases bla TEM-1B and bla OXA-1 , compares to the ST307 among clinical K. pneumoniae isolates from South Africa, similarly resistant to aminoglycosides and fluoroquinolones however harbouring the carbapenemase gene, bla OXA-181 in addition to other β-lactamases [39]. The detection of this high-risk ST307 K. pneumoniae with β-lactamases in the hospital environment shows that it is disseminated between patients and the environment and calls for heightened disinfection and IPC practices. To the best of the authors’ knowledge, this study is the first to report ESBL-producing K. pneumoniae belonging to ST307 in Ghana and calls for increased surveillance to enable the detection and control of such high-risk clones. Phylogenetic analyses revealed a clustering of three K. pneumoniae isolates from the hospital environment (from a bed, door handle and drip stand), all belonging to ST39 in the Obstetrics and Gynaecology ward. Two of these ST39 isolates from hospital environments (a bed and a drip stand) in the same ward harboured the β-lactamases, bla SHV-11 , bla CTX-M-15 and same virulence genes suggesting an intra-ward circulation of the ESBL-producing K. pneumoniae isolates. Notably, one ST39 isolate, E35 had no ESBLs nor any β-lactamases although phenotypically resistant to β-lactam antibiotics. This difference could be attributed to other mechanisms such as the mutations in the ompK36 porin or the efflux pumps acrR, oqxA and oqxB detected in the isolate, mediating resistance to the β-lactam antibiotics. In clinical settings, it is thus necessary to consider testing for other mechanisms besides the ESBLs, which are routinely tested, therefore highlighting the importance of genomics in AMR surveillance. Though an ST39 isolate (P60-1) from a patient was acquired on admission, the source of acquisition could not be determined. However, the clonal relatedness of these MDR ST39 K. pneumoniae isolates with several ESBLs, ompK36 mutations and virulence genes associated with plasmids and other MGEs could indicate a transfer of resistance and virulence genes among the isolates. The ST39 clone has been previously reported in Ghana and was detected in K. pneumoniae isolates from a study which screened for plasmids in three E. coli and four K. pneumoniae isolates expressing ESBL mediated by the bla CTX-M-15 gene from chronically infected wounds of Ghanaian patients [26]. The presence of the ESBLs in the isolates confirms the global reports of ST39 lineage of K. pneumoniae , which is known to be a carrier of ESBL genes, notably bla CTX-M-15 and bla CTX-M-14 and has been associated with nosocomial infections [16, 40, 41]. The dissemination of the high-risk clone ST39 K. pneumoniae has also been reported in infections and nosocomial outbreaks in sub-Saharan African countries (Congo, Ethiopia and The Gambia) and China [17, 40, 42, 43] and poses a threat to patients in the hospital. Phylogenomic analyses showed a clustering of the isolates from this study with clinical isolates from other countries in sub-Saharan Africa, indicating the potential of these carriage and hospital environment isolates to cause infections. There is a need for enhanced IPC practices, as patients could acquire these high-risk isolates from other patients and further disseminate them in the hospital environments. It should, however, be noted that the current study only analysed a few selected K. pneumoniae isolates using WGS. Furthermore, the study was conducted in a single tertiary hospital and is thus not representative of the epidemiology of K. pneumoniae isolates in Ghanaian hospitals. Despite the perceived limitations, considering that the hospital serves patients from major hospitals in the Ashanti Region of Ghana and beyond, this study provides a snapshot of the state of ESBL-producing K. pneumoniae in hospitals in Ghana and the role of the environment in the potential dissemination of these resistant isolates. Conclusion The current study described the molecular epidemiology of MDR K. pneumoniae isolate, particularly of ompK mutations in combination with ESBLs and several other antibiotic resistance genes and virulence factors from hospital environments and patients. There was a dissemination of ST39 ESBL-producing K. pneumoniae isolates between the hospital environment and patients. These findings re-emphasise the need for measures such as screening of patients to contain ESBL-producing K. pneumoniae isolates since their carriage in patients and dissemination in the Teaching Hospital environment may indicate an increased risk of transmitting these isolates in hospital settings. There is a need to enhance IPC practices, including improved cleaning and disinfection of surfaces in the hospital to prevent further dissemination of these high-risk isolates and their resistant genes. Abbreviations ESBL: Extended spectrum beta-lactamase IPC: Infection prevention and control IRB: Institutional Review Board LPS: Lipopolysaccharide MDR: Multidrug resistant MLST: Multilocus sequencing typing SNP: Single nucleotide polymorphisms ST: Sequence type WHO: World Health Organization Declarations Ethics approval and consent to participate: The study was approved by the Institutional Review Board (IRB) of the KATH (Reference: KATH IRB/AP/107/20) and the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (Reference: BREC/00001917/2020). Voluntary, informed written consent was obtained from participating patients and staff. Clinical Trial: Not applicable Consent for publication : Not applicable Availability of data and material: Sequence data that support the findings of this study has been deposited in GenBank and assigned accession numbers under BioProject PRJNA823741. All other data supporting this study's findings are available within the paper and in the supplementary information. Competing interests: None declared (EEAY, JM, DGE, NA, LKAA, AOO, AI, SE). Funding: This study was supported by the South African Research Chair Initiative of the Department of Science and Technology and the National Research Foundation of South Africa (Grant No. 98342). The funding sources did not influence the study design, data collection, analysis, interpretation of the data, or manuscript writing. Authors' contributions : EEAY, NA, AOO, and SYE co-conceptualized the study. EEAY undertook sample collection, laboratory and statistical analyses and wrote the original manuscript draft. EEAY, JM, DGA, ALKA and AI conducted data analysis. AI sequenced the isolates. SYE, AOO, NA, ALKA, DGA and JM supervised the work, vetted the results and critically reviewed the manuscript. All authors (EEAY, JM, NA, DGA, ALKA, AOO, AI and SYE) read and approved the manuscript. Acknowledgements : The authors are grateful to the Sequencing Core Facility, National Institute for Communicable Diseases, Johannesburg, South Africa. We are also grateful to study participants at the Obstetrics and Gynaecology Directorate, Surgery, and Intensive Care Units of the Komfo Anokye Teaching Hospital. We thank research assistants (nurses, biomedical scientists, and biostatisticians) at the study sites, the Heads of Departments and staff at the various directorates and the microbiology laboratory, Komfo Anokye Teaching Hospital, and staff for their support during the study. References Porter L, Sultan O, Mitchell BG, Jenney A, Kiernan M, Brewster DJ, et al. How long do nosocomial pathogens persist on inanimate surfaces? A scoping review. J Hosp Infect. 2024;147:25-31. Chakma SK, Hossen S, Rakib TM, Hoque S, Islam R, Biswas T, et al. 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Detection of multiple hypervirulent Klebsiella pneumoniae strains in a New York City hospital through screening of virulence genes. Clin Microbiol Infect. 2021;27(4):583-9. El-Domany RA, Awadalla OA, Shabana SA, El-Dardir MA, Emara M. Analysis of the correlation between antibiotic resistance patterns and virulence determinants in pathogenic Klebsiella pneumoniae isolates from Egypt. Microb Drug Resist. 2021;27(6):727-39. Karampatakis T, Tsergouli K, Behzadi P. Carbapenem-resistant Klebsiella pneumoniae : virulence factors, molecular epidemiology and latest updates in treatment options. Antibiotics. 2023;12(2):234. Stahlhut SG, Struve C, Krogfelt KA, Reisner A. Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS Immunol Med Microbiol. 2012;65(2):350-9. Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae . Clin Microbiol Rev. 2019;32(3):e00001-19. Madni O, Amoako DG, Abia ALK, Rout J, Essack SY. Genomic investigation of carbapenem-resistant Klebsiella pneumoniae colonization in an intensive care unit in South Africa. Genes. 2021;12(7):951. Okomo U, Senghore M, Darboe S, Bojang E, Zaman SM, Hossain MJ, et al. Investigation of sequential outbreaks of Burkholderia cepacia and multidrug-resistant extended spectrum β-lactamase producing Klebsiella species in a West African tertiary hospital neonatal unit: a retrospective genomic analysis. Lancet Microbe. 2020;1(3):e119-e29. Villinger D, Schultze TG, Musyoki VM, Inwani I, Aluvaala J, Okutoyi L, et al. Genomic transmission analysis of multidrug-resistant Gram-negative bacteria within a newborn unit of a Kenyan tertiary hospital: A four-month prospective colonization study. Front Cell Infect Microbiol. 2022;12:1240. Gala J-L, Ambroise J, Bearzatto B, Durant J-F, Bonjean M, Irenge L. Genomic characterization of multidrug-resistant extended spectrum β-lactamase-producing Klebsiella pneumoniae from clinical samples of a tertiary hospital in South Kivu Province, eastern Democratic Republic of Congo. MedRxiv. 2023:2023.01. 05.23284226. Du J, Cao J, Shen L, Bi W, Zhang X, Liu H, et al. Molecular epidemiology of extensively drug-resistant Klebsiella pneumoniae outbreak in Wenzhou, Southern China. J Med Microbiol. 2016;65(10):1111-8. Table 1 and 2 Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1and2.docx K.pneumoniaesupplementarytablesEEAYDGAR2.docx Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 27 May, 2025 Editor assigned by journal 27 May, 2025 Reviews received at journal 12 Apr, 2025 Reviewers agreed at journal 12 Apr, 2025 Reviewers invited by journal 11 Apr, 2025 Submission checks completed at journal 04 Apr, 2025 First submitted to journal 04 Apr, 2025 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. 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This visualization highlights the relative positioning and orientation of the integron (\u003cem\u003eintI1\u003c/em\u003e), resistance genes (\u003cem\u003esul1\u003c/em\u003e, \u003cem\u003edfrA15\u003c/em\u003e, \u003cem\u003eaadA1, qacE\u003c/em\u003e), and associated mobile genetic elements, including \u003cstrong\u003eTn3 family transposase, IS6 insertion elements \u003c/strong\u003eand\u003cstrong\u003e recombinase family proteins\u003c/strong\u003e. These elements are labeled and mapped within their genomic context to illustrate their synteny and genetic environment. The \u003cstrong\u003egreen\u003c/strong\u003e color represents genes, while \u003cstrong\u003eyellow\u003c/strong\u003e indicates protein-coding sequences (CDS). Arrows denote the orientation of individual genes, providing insights into their transcriptional direction.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5671518/v1/0e0ffed2d82bc532776bf6b1.png"},{"id":80787099,"identity":"b669cccc-5de6-4797-bdb7-2a0087fe6645","added_by":"auto","created_at":"2025-04-17 06:03:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":707937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical annotation and comparative alignment of the genetic cassette carrying \u003c/strong\u003e\u003cem\u003eintI1\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e \u003cem\u003edfrA15\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e \u003cem\u003esul1\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003eqacE\u003c/em\u003e\u003cstrong\u003e in carriage isolate P136 (\u003c/strong\u003e\u003cem\u003eAccession number: JALJUS010000062\u003c/em\u003e\u003cstrong\u003e) \u003c/strong\u003epresented in \u003cstrong\u003e(a) circular view \u003c/strong\u003eand \u003cstrong\u003e(b) linear view.\u003c/strong\u003eThis visualization highlights the relative positioning and orientation of the integron (\u003cem\u003eintI1\u003c/em\u003e), resistance genes (\u003cem\u003esul1\u003c/em\u003e, \u003cem\u003edfrA15\u003c/em\u003e, \u003cem\u003eaadA1\u003c/em\u003e, \u003cem\u003eqacE\u003c/em\u003e), and associated mobile genetic elements, including \u003cstrong\u003eTn3 family transposase, IS6 insertion elements \u003c/strong\u003eand\u003cstrong\u003e recombinase family proteins\u003c/strong\u003e. These elements are labeled and mapped within their genomic context to illustrate their synteny and genetic environment. The \u003cstrong\u003egreen \u003c/strong\u003ecolor represents genes, while \u003cstrong\u003eyellow\u003c/strong\u003e indicates protein-coding sequences (CDS). Arrows denote the orientation of individual genes, providing insights into their transcriptional direction. This representation facilitates a clearer understanding of the modular nature of these genetic components and their potential roles in \u003cstrong\u003ehorizontal gene transfer, recombination, and dissemination\u003c/strong\u003e within bacterial populations.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5671518/v1/0c1d3902490883ffe7cc6930.png"},{"id":80784731,"identity":"3dd6d942-7d6f-4a97-832d-5ca786c1e5fd","added_by":"auto","created_at":"2025-04-17 05:37:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":277566,"visible":true,"origin":"","legend":"\u003cp\u003eThe core genome phylogenetic branch and metadata (source; WGS in-silico typing; β-lactamases, \u003cem\u003eompK\u003c/em\u003e mutation, plasmid replicons, integrons, insertion sequences, and intact prophages) coupled using Phandango (https://github.com/jameshadfield/phandango/wiki) in MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates (n = 10) from a teaching hospital in Ghana. The colour codes for β-lactamases, plasmid replicons, integrons, insertion sequences and intact prophages (10) showed presence (green; A) and absence (blue; B) in the isolates. Colour codes for sources are patient (purple; A) and environment (orange; B).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5671518/v1/8d8eeec3714b54b0b06046de.png"},{"id":80784743,"identity":"999d6e22-768b-4d82-adcd-5869d42a1239","added_by":"auto","created_at":"2025-04-17 05:37:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1451586,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from humans between 2013 and 2022 in African countries. The core-genome phylogenetic tree was drawn from 181 genomes on BV-BRC and annotated using iTOL. The tree was built with \u003cem\u003eK. pneumoniae\u003c/em\u003e Ecl8 as the reference genome and rooted with the reference strain. The following metadata indicates the year of isolation on the outer-coloured ring, the country of isolation in the middle and the MLST (isolates with unknown STs are indicated U) on the inner ring. The source of the isolates is indicated using shapes. Isolates from this study are labelled with a blue background.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5671518/v1/3a28afb2f18e1eab2461839c.png"},{"id":86179745,"identity":"8d2394d8-a995-4e27-9686-66df3628f156","added_by":"auto","created_at":"2025-07-07 16:19:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4465282,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5671518/v1/41a9e5ca-2db8-45d1-9482-037ca4adc84f.pdf"},{"id":80784727,"identity":"ce935783-5cdd-486a-a98c-5fc67160f9c3","added_by":"auto","created_at":"2025-04-17 05:37:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39982,"visible":true,"origin":"","legend":"","description":"","filename":"Table1and2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5671518/v1/0afd05e3ed9787e0460e486b.docx"},{"id":80786860,"identity":"7b874177-5397-454f-9a49-864c9d2e472e","added_by":"auto","created_at":"2025-04-17 06:01:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":34329,"visible":true,"origin":"","legend":"","description":"","filename":"K.pneumoniaesupplementarytablesEEAYDGAR2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5671518/v1/87b967a6d4c088200920b051.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-Genome Sequencing Insights into Porin-Mediated Resistance and Spread of ESBL- Producing Klebsiella pneumoniae in a Ghanaian Teaching Hospital","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe hospital environment constitutes an important component of infection prevention and control (IPC), as it has been demonstrated that many microorganisms can survive on different surfaces in these environments, with \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e surviving for up to 600 days on inanimate objects [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This represents a potential health threat to users of hospital spaces, including patients, healthcare workers and other visitors. Although measures such as improved hygiene [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] are implemented to curb disease transmission in hospitals, many microorganisms have developed resistance mechanisms that allow them to escape the effect of disinfectants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], allowing them to survive longer in these environments with potential transmission. Of particular importance in these spaces are Gram-negative bacteria, especially drug-resistant ones.\u003c/p\u003e \u003cp\u003eMultidrug-resistant Gram-negative bacteria threaten human health and may cause life-threatening infections [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e have been listed by the World Health Organization (WHO) among pathogens of critical priority for the development of new antibiotics [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This resistant pathotype notably causes nosocomial and community-acquired infections such as pneumonia and urinary tract infections [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates can harbour many resistance genes and develop diverse antibiotic resistance mechanisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to its intrinsic β-lactamase gene, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;1\u003c/sub\u003e, mutations resulting in the loss, reducing the number and diameter of outer membrane porins, \u003cem\u003eompK35\u003c/em\u003e, \u003cem\u003eompK36\u003c/em\u003e, and the quiescent \u003cem\u003eompK37\u003c/em\u003e as well as efflux pumps may contribute to the resistance of \u003cem\u003eK. pneumoniae\u003c/em\u003e to β-lactams, including carbapenems [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntibiotic resistance genes in \u003cem\u003eK. pneumoniae\u003c/em\u003e can be spread widely by the dissemination of epidemic clones and mobile genetic elements, predominantly plasmids [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The Inc-groups of plasmids IncF, IncFII(K1), IncR, IncX, IncX3, IncI2, and ColE1 have particularly been associated with the rapid spread of resistance in \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMultilocus sequencing typing (MLST) has revealed the circulation of various sequence types (STs) of MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. Notably, ST307 \u003cem\u003eK. pneumoniae\u003c/em\u003e has been recognised as a globally emerging clone with genetic characteristics enabling ease of dissemination and persistence in the hospital setting [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It has been detected in Korea, Germany, South Africa, and Nigeria. [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The ST39 has also been reported in Russia, South Africa, and Ethiopia. [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a study of MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates recovered from clinical samples in the Komfo Anokye Teaching Hospital, Ghana, 24.32% of the isolates were resistant to second- and third-generation cephalosporins and carried multiple resistant genes. MLST analyses revealed the circulation of multiple \u003cem\u003eK. pneumoniae\u003c/em\u003e STs (ST2171, ST2186, ST17, ST152, ST397, ST101, ST1788 and STS789) in the hospital [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presence of several virulence genes such as the fimbriae synthesis-related gene, lipopolysaccharide-related gene, capsular polysaccharide synthesis and synthesis regulation-related gene, iron uptake system, urease-related gene, tellurite resistance gene hemolysin among others contribute to the pathogenicity of \u003cem\u003eK. pneumoniae\u003c/em\u003e. Capsule and lipopolysaccharide (LPS) antigens, K and O, have been reported to contribute significantly to the pathogenicity of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The structural differences of these antigens have been useful in assigning serotypes which describe the extent of virulence among various \u003cem\u003eK. pneumoniae\u003c/em\u003e strains [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e associated with increased mortalities emerged in Asia and continues to spread globally [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInvestigations into virulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e have primarily focused on outbreak settings. The hospital environment in spite of its established role as a reservoir and transmission route for many pathogens, including antibiotic-resistant ones is not usually considered during such investigation as the focus is on the patients. It is equally important to investigate the pathogenicity and virulence of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates colonising patients in non-outbreak settings to establish/improve IPC practices. Thus, despite recent global studies on the epidemiology of ESBL-producing and carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e, there is a lack of data on patients\u0026rsquo; environments, especially in Sub-Saharan African countries such as Ghana.\u003c/p\u003e \u003cp\u003eUsing whole genome sequencing and bioinformatics analysis, the current study assessed MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from hospital environments and patients in a teaching hospital in Ghana to establish any clonal relationship between the hospital environment and patient isolates. The study further determined the antimicrobial resistance, virulence, and genetic relatedness of ESBL-producing and carbapenem-resistant isolates in the context of IPC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for the study was obtained from the Institutional Review Board (IRB) of the Komfo Anokye Teaching Hospital (KATH) \u003cstrong\u003e(Reference: KATH IRB/AP/107/20)\u003c/strong\u003e and the Biomedical Research Ethics Committee of the University of KwaZulu-Natal \u003cstrong\u003e(Reference: BREC/00001917/2020\u003c/strong\u003e). Voluntary, informed written consent was obtained from participating patients and staff.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy population and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study focused on MDR ESBL-producing \u003cem\u003eK. pneumoniae \u003c/em\u003econtaminating hospital environments and colonising patients at the Komfo Anokye Teaching Hospital, Kumasi, from April 2021 to July 2021. Rectal and hand swabs were collected from consenting adult patients within 24 hours of admission and after 48 hours in the wards of three hospital directorates: Obstetrics and Gynaecology, Surgery, and the Intensive Care Unit. Hand swabs were collected concurrently from staff present during patient sampling and from the patient\u0026rsquo;s immediate environment (bedrails, drip-stand, door handles and taps) in the wards. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Identification of ESBL-producing MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, swabs collected from the ward environments, 83 patients and from the hands of healthcare workers were processed on MacConkey agar, and 250 Gram-negative bacteria were identified by Gram staining. Using the VITEK 2\u003csup\u003e\u0026reg;\u003c/sup\u003e automated system (BioM\u0026eacute;rieux-Vitek, Marcy-l\u0026rsquo;\u0026Eacute;toile, France), 31 isolates were identified among the Gram-negative bacteria as \u003cem\u003eK. pneumoniae\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiotic susceptibility testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibiotic susceptibility testing was conducted using the VITEK 2\u003csup\u003e\u0026reg;\u003c/sup\u003e automated system (BioM\u0026eacute;rieux-Vitek, Marcy-l\u0026rsquo;\u0026Eacute;toile, France). The antibiotic panel consisted of 16 antibiotics: cefuroxime (CXM), ceftazidime (CAZ), ceftriaxone (CRO), amoxicillin/clavulanic (AMC), cefepime (FEP), piperacillin/tazobactam (TZP), imipenem (IPM), doripenem (DOR), meropenem (MEM), ertapenem (ERT), gentamicin (GEN), tobramycin (TOB), amikacin (AMK), ciprofloxacin (CIP), trimethoprim-sulphamethoxazole (SXT) and tigecycline (TGC). Isolates were categorized into susceptible, intermediate and resistant using Clinical and Laboratory Standards Institute guidelines (CLSI 2020). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction, Genome sequencing and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA (gDNA) from pure colonies of identified overnight cultures of MDR ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e were extracted using the GenElute\u0026reg; bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, United States) according to the manufacturer\u0026rsquo;s instructions. The concentration and quality of the extracted gDNA were checked using the Nanodrop 8000 (Thermo Scientific, Waltham, MA, USA). The sequencing and analyses (JEKESA pipeline) were conducted at the Sequencing Core Facility of the South African National Institute for Communicable Diseases (NICD). Using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, United States), Multiplexed paired-end libraries (2 x 300 bp) were prepared. Sequences were determined on an Illumina Nextseq 550 (2 x 150 bp) platform (Illumina, San Diego, CA, United States) with 100\u0026times; coverage. \u003c/p\u003e\n\u003cp\u003eQuality trimming of raw reads was done using Sickle v1.33 (https://github.com/najoshi/sickle). The raw reads were then assembled spontaneously using the SPAdes v3.6.2 assembler (https://cab.spbu.ru/software/spades/). All contiguous sequences were subsequently submitted to GenBank and assigned accession numbers (\u003cstrong\u003eSupplementary Table S1)\u003c/strong\u003e under BioProject\u003cstrong\u003e PRJNA823741.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular typing of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultilocus sequence typing (MLST) was performed \u003cem\u003ein-silico \u003c/em\u003eusing the WGS data online platform tool with the assembled genomes (https://bigsdb.pasteur.fr/klebsiella/). The serotypes (K types, O types, and \u003cem\u003ewzc\u003c/em\u003e and \u003cem\u003ewzi\u003c/em\u003e allelic types) of the isolates were determined using the reference \u003cem\u003eKlebsiella\u003c/em\u003e WGS data online platform tool, Kaptive-web (http://kaptive.holtlab.net/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the acquired and chromosomal mutations in the isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResistance and virulence genes were determined using ResFinder (https://cge.food.dtu.dk/services/ResFinder/) [accessed on July 18, 2024] and VF analyser via Virulence finder database (https://cge.food.dtu.dk/services/VirulenceFinder/) [accessed on July 20, 2024], respectively. Mutations in the \u003cem\u003eompK35,\u003c/em\u003e \u003cem\u003eomp36\u003c/em\u003e and \u003cem\u003eompK37\u003c/em\u003e genes were determined via ResFinder. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of mobile genetic elements (MGEs)/genetic support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of mobile genetic elements was determined using PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/) for plasmids, (Carattoli et al., 2014), INTEGRALL (http://integrall.bio.ua.pt/) for integrons, and RAST SEEDVIEWER (https://rast.nmpdr.org/seedviewer.cgi) for transposons and insertion sequences. Insertion sequences (IS) in genomes were predicted via the ISFinder database (https://www-is.biotoul.fr/). The PHASTER (\u003cu\u003ehttps:// phaster.ca/\u003c/u\u003e) server was used to identify and visualise prophage sequences. \u003c/p\u003e\n\u003cp\u003eThe synteny and genetic environment of antibiotic resistance genes and associated mobile genetic elements were investigated using the general feature format (GFF3) files from GenBank. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenomic analyses of the \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenomic analysis was done to determine the genetic relatedness of isolates in this study. Isolates from this study were also compared with 180 \u003cem\u003eK. pneumoniae\u003c/em\u003e isolate genomes from sub-Saharan African countries that were downloaded from the Bacterial and Viral Bioinformatics Resource Center (BV-BRCB) (https://www.bv-brc.org/\u003cu\u003e)\u003c/u\u003e. A maximum likelihood tree was constructed on BV-BRCB and rooted on the reference genome \u003cem\u003eK. pneumoniae \u003c/em\u003eEcl8 (accession number: HF536482 CANH01000000). FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and Interactive Tree of Life (iTol) v7 (https://itol.embl.de/) were used to visualise and annotate the phylogenetic tree.\u003c/p\u003e\n\u003cp\u003eTo gain a more comprehensive insight into the relationships of isolates from this study, Phandango (https://jameshadfield.github.io/phandango/#/main) was used to visualise the phylogenetic tree with corresponding metadata.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIsolates identification and selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty-one of the 250 Gram-negative bacteria isolated from swabs were identified as putative \u003cem\u003eK. pneumoniae,\u003c/em\u003e of which 20/31 (64.5%) were MDR (15 from patients and five from the hospital environment). Ten of the 20 isolates were confirmed as \u003cem\u003eK. pneumoniae,\u003c/em\u003e that is,\u003cem\u003e\u0026nbsp;\u003c/em\u003efive carriage isolates and five from the environment (door handle, tap, drip-stand and bed and five from patients) after quality assurance and WGS; low quality genomes and those belonging to \u003cem\u003eKlebsiella variicola\u003c/em\u003e or \u003cem\u003eKlebsiella quasipneumoniae\u003c/em\u003e, biochemically indistinguishable from \u003cem\u003eK. pneumoniae\u003c/em\u003e were excluded.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial resistance profiles of selected MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from the hospital environments and patients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll (100%) of the ten selected MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were resistant to ceftazidime and cefuroxime. Ninety percent of the isolates were resistant to piperacillin/tazobactam, cefepime, amoxicillin/clavulanic acid and sulphamethoxazole/trimethoprim. Eighty percent (8/10) of the isolates showed resistance to ceftriaxone. However, all isolates were susceptible to tigecycline, amikacin, ertapenem and imipenem. Two carriage isolates (P121 and P60-1) and one (E55-1) from the hospital environment showed intermediate resistance to doripenem and resistance to meropenem and presented with elevated minimum inhibitory concentrations (MICs) of 2-4 \u0026micro;g/mL (Supplementary Table\u003cstrong\u003e\u0026nbsp;S2)\u003c/strong\u003e. Sixty percent (3/5) of carriage isolates and 60% (3/5) from hospital environments were resistant to gentamicin, tobramycin and ciprofloxacin \u003cstrong\u003e(Table 1)\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/strong\u003eThe most common antibiogram pattern among the isolates was CXM-FEP-CAZ-CRO-AMC-TZP-GEN-CIP-GEN-SXT, which was common to five isolates; two from patients (P121 and P129) and three from the hospital environment (E4, E34A and E50-2) \u003cstrong\u003e(Table 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWGS-based capsular serotyping and Multilocus sequence typing (MLST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolates showed high MLST variation, with seven STs identified, viz., ST39, ST307, ST815, ST1552, ST636, ST464 and ST1996. Four isolates, including three from the hospital environment, i.e., from a bed (E4), door handle (E35) and drip stand (E50), respectively, and one carriage isolate (P60-1), belonged to the same ST 39. The isolates belonged to different capsular serotypes. Two isolates (E4 and E50-2) from the hospital environment (bed and drip stand respectively), however, shared the same ST 39 and capsular serotype, wzi 2 \u003cstrong\u003e(Table 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe O and K capsules were diverse among the isolates. Predominantly, of the O capsules, O1/O2v1 (n = 5/10) and O1/O2v2 (n = 4/10) were common to the isolates. The other O-capsule type for \u003cem\u003eK. pneumoniae\u0026nbsp;\u003c/em\u003ewas the O3b. The k capsular types were more diverse, with all but two isolates showing different k capsular types. Isolates of the same ST did not necessarily have the same O and K capsules. For instance, for isolates belonging to ST 39, two isolates (E4 and E50-2 from the hospital environment and a patient, respectively) had the O1/O2v1 capsule with KL2 capsules, while the other two (E35 and P60-1 from the hospital environment and a patient respectively) had the same O capsule (O1/2v2) but different K capsules (KL23 and KL149, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResistome of the isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral \u0026beta;-lactamase genes were identified in the isolates, with the dominant genes being the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026nbsp;\u003c/sub\u003e(n = 8) in four carriage and four hospital environment isolates. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u0026nbsp;\u003c/sub\u003egene was found in six isolates (three hospital environment and three carriage isolates). Seven different \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e genes were identified in the isolates. These included \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-1\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-11\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-38\u003c/sub\u003e,\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-41\u003c/sub\u003e,\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-62\u003c/sub\u003e,\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-106\u003c/sub\u003e, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-172\u003c/sub\u003e. \u0026nbsp;The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSCO-1\u0026nbsp;\u003c/sub\u003egene was detected in two carriage isolates (P121 and P136), which also harboured the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM-1,\u0026nbsp;\u003c/sub\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u0026nbsp;\u003c/sub\u003egenes\u003csub\u003e\u0026nbsp;\u003c/sub\u003eand\u003csub\u003e\u0026nbsp;\u003c/sub\u003ethe \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-172\u0026nbsp;\u003c/sub\u003e(P121) and\u003cem\u003e\u0026nbsp;bla\u003c/em\u003e\u003csub\u003eSHV-62\u0026nbsp;\u003c/sub\u003e(P136)\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eTable 2).\u003c/strong\u003e The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eDHA\u003c/sub\u003e gene was found in a carriage isolate (P60-1) with ST39 from a patient, though other isolates of similar ST lacked the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eDHA\u003c/sub\u003e gene. Co-production of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e was evident in three carriage isolates (P121 and P136) and two isolates (E34A and E50-2) from the hospital environment. An isolate from the environment (E35) did not harbour any \u0026beta;-lactamase genes, although it was resistant to cefuroxime, ceftazidime, cefepime, amoxicillin/clavulanic and piperacillin/tazobactam (\u003cstrong\u003eTable 2)\u003c/strong\u003e. WGS analysis did not identify any carbapenem resistance genes in the isolate (E55-1) resistant to meropenem.\u003c/p\u003e\n\u003cp\u003eNine isolates also harboured sulphamethoxazole-trimethoprim resistance genes, \u003cem\u003esul\u003c/em\u003e and \u003cem\u003edfrA\u003c/em\u003e Aminoglycoside resistance was encoded by the \u003cem\u003eaac(aac(3)-IIa\u003c/em\u003e, \u003cem\u003eaac(6\u0026rsquo;)-Ib-cr\u003c/em\u003e), \u003cem\u003eaph (aph(3\u0026rdquo;)-Ib\u003c/em\u003e, \u003cem\u003eaph(6)-Id)\u003c/em\u003e, \u003cem\u003eaad(aadA1, aadA2)\u003c/em\u003e and \u003cem\u003eant(2\u0026rdquo;)-Ia\u003c/em\u003e genes and were associated with increased MICs to gentamicin (\u0026ge;16\u0026nbsp;\u0026micro;g/mL) and tobramycin (\u0026ge;16\u0026nbsp;\u0026micro;g/mL). Other resistance genes were the \u003cem\u003etet\u0026nbsp;\u003c/em\u003egenes in four isolates (\u003cem\u003etetA\u0026nbsp;\u003c/em\u003ein P60-1 and E34A\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003etetD\u0026nbsp;\u003c/em\u003ein E35 and P136), encoding tetracycline resistance in carriage isolates and hospital environment isolates. The \u003cem\u003efosA\u003c/em\u003e gene for fosfomycin resistance was harboured by all ten isolates (five environmental and five carriage isolates). The \u003cem\u003eQacE\u003c/em\u003e genes encoding resistance to quaternary ammonium compounds were observed in three isolates (carriage isolates P121, P136 and the isolate from the hospital environment E35) \u003cstrong\u003e(Table 2)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFluoroquinolone resistance, which was observed in five (three hospital environment and two patient) isolates associated with the plasmid-mediated quinolone resistance (PMQR) genes, \u003cem\u003eQnr\u003c/em\u003e, \u003cem\u003eOqx\u003c/em\u003e and \u003cem\u003eaac(6\u0026prime;)-lb-cr\u003c/em\u003e \u003cstrong\u003e(Table 2\u003c/strong\u003e) and less frequently with chromosomal point mutations in the quinolone resistance determining regions (QRDR) of \u003cem\u003egyrA\u0026nbsp;\u003c/em\u003e(S83Y, D87A) which were observed in a carriage isolate, P129 and \u003cem\u003eparC\u0026nbsp;\u003c/em\u003e(S80I)\u003cem\u003e\u0026nbsp;\u003c/em\u003ein P129 and E34A \u003cstrong\u003e(Supplementary Table S4)\u003c/strong\u003e. The \u003cem\u003eoqxA\u003c/em\u003e and \u003cem\u003eoqxB\u0026nbsp;\u003c/em\u003egenes encoding the multidrug efflux pump, \u003cem\u003eoqxAB\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ewere found in all ten isolates (five environmental and five carriage). The \u003cem\u003eqnr\u003c/em\u003e gene occurred in seven isolates (four isolates environmental and three carriage), the most frequent being the \u003cem\u003eqnrB\u003c/em\u003e genes. Five (three environmental and two carriage) isolates harboured the \u003cem\u003eaac(6\u0026rsquo;)-Ib-cr\u003c/em\u003e gene and were associated with high MICs to the aminoglycosides gentamicin and tobramycin \u003cstrong\u003e(Table 2).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromosomal point analysis indicated mutations in the \u003cem\u003eacrR\u003c/em\u003e efflux pump gene in all the \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. One carriage isolate (P129) had the acrR mutation (F204L) encoding tigecycline resistance, though this was not phenotypically expressed \u003cstrong\u003e(Table S4)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eMutations in outer membrane proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the contribution of porins to cephalosporin resistance and the reduced susceptibility to carbapenems in the \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates (P121, P60-1 and E55-1), all isolates were further assessed for the presence of mutations in porins that could mediate carbapenem resistance in concert with ESBLs. Investigation of major outer membrane porins showed an intact \u003cem\u003eompK35\u003c/em\u003e gene in all the MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e strains from patients and hospital environments. However, numerous mutations associated with resistance to cephalosporins and reduced susceptibility to carbapenems were found in \u003cem\u003eompK36\u003c/em\u003e (N49S, L59V, T86V, S89T. D91K, A93S, L191Q. F207W, A217S, N218H, Q227N, L229V, E232R, H235D, T254S, T184P, G189T, F198Y, F207Y, A217S, T222L, D223G, N304E) and \u003cem\u003eompK37\u003c/em\u003e (I70M, I128M, N230G) genes in all isolates \u003cstrong\u003e(Table 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMobile Genetic elements and environment of ARGs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmidFinder revealed the presence of at least two different plasmid replicon types in all ten isolates from patients and hospital environments. The plasmids included IncFIA, IncFIB, IncFII, IncHI1B, IncQ1, Col and Col440II. IncFIB was the most abundant plasmid replicon in nine (five hospital environment and four carriage) isolates. Different insertion sequences were found in the isolates. The transposable element \u003cem\u003eMITEPlu5\u003c/em\u003e, along with the insertion sequences \u003cem\u003eISCARN29\u003c/em\u003e and \u003cem\u003eISSph8\u003c/em\u003e, was identified in two ST39 isolates (E4 and P60-1) from the hospital environment and a patient\u0026nbsp;\u003cstrong\u003e(Supplementary Table S5)\u003c/strong\u003e. Six isolates had class I integrons. Similar gene cassettes were found in two isolates (E50-2 and E55-1) from the hospital environment bearing the \u003cem\u003edfrA14\u003c/em\u003e gene. An Intl1 integrase was associated with \u003cem\u003edfrA15\u003c/em\u003e together with the sulphamethoxazole gene \u003cem\u003esul1\u003c/em\u003e and the quaternary ammonium compound resistance gene, \u003cem\u003eQacE\u003c/em\u003e, on gene cassettes in the carriage isolates P121 and P136 \u003cstrong\u003e(Table 3 and Figs. 1 and 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;bla\u003c/em\u003e\u003csub\u003eCTX-M\u0026nbsp;\u003c/sub\u003eand \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM-1\u0026nbsp;\u003c/sub\u003egenes\u003csub\u003e\u0026nbsp;\u003c/sub\u003ewere associated with a transposon, recombinase or insertion sequence (Table S3). The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u0026nbsp;\u003c/sub\u003ewas particularly associated with the transposon IS1380 in the environmental isolates E4, E34A and E50-2 and the carriage isolate P121. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM-1\u0026nbsp;\u003c/sub\u003ewas often bracketed by a transposase and recombinase. In E4, E34A, and E50-2, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM-1\u0026nbsp;\u003c/sub\u003ewas\u003csub\u003e\u0026nbsp;\u003c/sub\u003eassociated with the transposase IS91 and a recombinase (Table S3). The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSCO-1\u0026nbsp;\u003c/sub\u003egene was also associated with a recombinase in the carriage isolates P121 and P136. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e gene was, however, chromosomal and not carried on any mobile element. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-1\u0026nbsp;\u003c/sub\u003egenes were commonly associated with the aminoglycoside hydrolysing gene, \u003cem\u003eaac(6\u0026rsquo;)-Ib-cr\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand the chloramphenicol acetyltransferase, \u003cem\u003ecatB3\u0026nbsp;\u003c/em\u003e(Table S3). In the carriage isolate P136, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-1\u003c/sub\u003e was\u003csub\u003e\u0026nbsp;\u003c/sub\u003eassociated with the IS6 transposase with a gene cassette like the \u003cem\u003eK. pneumoniae\u003c/em\u003e strain KPH3 plasmid. \u003cem\u003eaph(6)-Id: aph(3\u0026apos;\u0026apos;)-Ib\u003c/em\u003e were associated with the Tn3 in the carriage isolate P121. Additionally, P121 harboured resistance genes associated with IskrA4\u003cem\u003e,\u0026nbsp;\u003c/em\u003eIS91 and Tn3 transposons and the intI1\u0026nbsp;integrase \u003cstrong\u003e(Table S5)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Gene cassettes of ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from patients and the hospital environment\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eBacterial ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eIntegron\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eIntegron name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eGC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eGC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eGC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eGC4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eE34A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eEnvironment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eInt1l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eIn191\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003edfrA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eE35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eEnvironment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eInt1l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eln388\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003edfrA15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eQacE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003esul1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eP121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eIntl1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eln388\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003edfrA15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eaadA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eQacE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003esul1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eP136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eIntl1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eln388\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003edfrA15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eaadA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eQacE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003esul1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eE50-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eEnvironment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eIntl1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eln191\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003edfrA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eP129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003eIntl1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eln388\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003edfrA15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eaadA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eVirulome of ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA myriad of virulence genes involved in fimbriae synthesis (\u003cem\u003efimH\u003c/em\u003e, \u003cem\u003emrkD\u003c/em\u003e), capsular polysaccharide synthesis and synthesis regulation, genes involved in iron uptake system (\u003cem\u003eiroE\u003c/em\u003e, \u003cem\u003efes\u003c/em\u003e, and \u003cem\u003efur\u003c/em\u003e)\u0026nbsp;aerobactin, (\u003cem\u003eiutA\u003c/em\u003e, \u003cem\u003eirp1\u003c/em\u003e, \u003cem\u003eirp2\u003c/em\u003e and \u003cem\u003eiucA\u003c/em\u003e) yersiniabactin\u0026nbsp;(\u003cem\u003eybtA\u003c/em\u003e, \u003cem\u003eybtP\u003c/em\u003e, \u003cem\u003eybtQ\u003c/em\u003e), and enterobactin \u003cem\u003eentBEF\u003c/em\u003e) were present in the isolates \u003cstrong\u003e(Table 2)\u003c/strong\u003e. All isolates (both carriage and from the hospital environment) harboured the \u003cem\u003eiutA\u003c/em\u003e, \u003cem\u003egndA\u003c/em\u003e, and \u003cem\u003eompA\u003c/em\u003e factors. \u003cem\u003eentC/E, fes and fep\u0026nbsp;\u003c/em\u003ewere found in four isolates from the hospital environment and five carriage isolates but were\u003cem\u003e\u0026nbsp;\u003c/em\u003eabsent in an isolate (E35) from the environment which lacked any \u0026beta;-lactamase or ESBL genes and was carbapenem-resistant\u003cem\u003e. iroE\u003c/em\u003e was found in nine isolates (four hospital environment and five carriage isolates). \u003cem\u003efyuA\u003c/em\u003e, \u003cem\u003eirp1\u003c/em\u003e and \u003cem\u003eirp2\u0026nbsp;\u003c/em\u003ecommonly occurred together in seven isolates (three hospital environment and four carriage) \u003cstrong\u003e(Table 2)\u003c/strong\u003e. Though the isolates possessed several virulence genes, hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e, which are typically characterised by the presence of \u003cem\u003eroB\u003c/em\u003e, \u003cem\u003eiucA\u003c/em\u003e, \u003cem\u003epeg-344\u003c/em\u003e, \u003cem\u003ermpA\u003c/em\u003e, and \u003cem\u003ermpA2\u003c/em\u003e genes [23] were not found.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenomics of the isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenomic analyses based on differences in single nucleotide polymorphisms (SNPs) and core genome analyses of the metadata using Phandango revealed that the isolates were diverse. However, isolates of ST39 (E35, E4 and E50-2), from the hospital environment and P121 (from a patient), grouped into one cluster. P144 (ST636) and E34A (ST307) from a patient and the hospital environment, respectively, were also similar despite belonging to two different sequence types \u003cstrong\u003e(Fig. 3)\u003c/strong\u003e. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u0026nbsp;\u003c/sub\u003egene was present in two ST39 isolates, and ST815, ST1552, ST464 and ST307 isolates. The \u0026beta;-lactamases were frequently associated with plasmids, particularly the IncF1B found in all STs, except for an ST39 isolate (E4). The int1integron was found in six isolates.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To determine the clonal relatedness of the \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates, the isolates in this study were compared to other isolates from sub-Saharan Africa. The \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from the hospital environment and patient carriage were also found to cluster based on sequence types with other genomes from countries in the sub-Saharan Africa region, indicating a wide dissemination of common STs such as ST307 and ST39 in Africa. The isolates clustered with similar isolates previously isolated from clinical sources such as blood, urine, and stool in patients with infections \u003cstrong\u003e(Fig. 4)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe dissemination of antimicrobial-resistant pathogenic bacteria possessing virulence factors within the hospital environment poses a major threat to patient prognosis [24]. It is thus a concern for IPC in hospitals. ESBL-producing and carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e have become a serious concern in hospital and community-acquired infections [12, 25]. The current study revealed the hospital environment as a potential source and transmission route for these resistant strains, necessitating effective measures to prevent their spread to users of the hospital environment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResistance genes were widely distributed among the \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. \u0026beta;-lactamases commonly identified in both carriage isolates and isolates from hospital environments were \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e, especially the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u003c/sub\u003e gene that is widespread among \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates [26-28]\u003csub\u003e.\u0026nbsp;\u003c/sub\u003eThese \u0026beta;-lactamases genes have been reported among \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from South Africa and Spain, showing their widespread nature. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e has been reported as commonly harboured by \u003cem\u003eK. pneumoniae\u003c/em\u003e among carriage samples, confirming their constitutive chromosomal expression [28, 29]. Carriage isolates and isolates from the hospital environment also harboured the aminoglycoside and quinolone-resistant genes \u003cem\u003eaac(6\u0026prime;)-Ib-cr\u003c/em\u003e and the multidrug efflux pumps \u003cem\u003eacrAB\u003c/em\u003e and \u003cem\u003eOqxAB\u003c/em\u003e. In association with the IncFII plasmid, these resistance mechanisms, reported in other Enterobacterales [30], could also be circulated in the hospital environment and acquired by other bacteria, leaving limited options for treating infections caused by these ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mutations (N49S, L59V, L191S, F207W, D224E, L228V, and E232R) in \u003cem\u003eompK36\u003c/em\u003e have been previously reported in association with cephalosporin resistance [31]. In this study, these mutations co-occurred with the presence of ESBLs such as \u003cem\u003ebla\u003csub\u003eCTX-M\u003c/sub\u003e\u003c/em\u003e, which may contribute to the high resistance to cephalosporins, as indicated by elevated MICs among both carriage \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates and environmental isolates (Supplementary Table S2). However, since no functional assay was performed, the role of these \u003cem\u003eompK36\u003c/em\u003e mutations in cephalosporin resistance requires further experimental confirmation.\u003c/p\u003e\n\u003cp\u003eThe observed \u0026beta;-lactam resistance, including cephalosporin resistance, in the ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates aligns with previous reports by\u0026nbsp;Humphries \u003cem\u003eet al\u003c/em\u003e [32] describing associations between \u003cem\u003eK. pneumoniae\u003c/em\u003e resistance to cephalosporins (ceftazidime/avibactam) and point mutations in \u003cem\u003eompK36\u003c/em\u003e. However, while such mutations have been linked to resistance, additional functional analysis is necessary to confirm their role in the observed phenotypic resistance.\u0026nbsp;The mutations A217S, and N218H in \u003cem\u003eompK36\u0026nbsp;\u003c/em\u003eand I70M, and I128M in \u003cem\u003eompK37\u003c/em\u003e, reported to be associated with carbapenem resistance among \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates [31], were observed in all the isolates, pointing to developing carbapenem resistance in these ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. Yang et al. also found numerous mutations in \u003cem\u003eompK36\u003c/em\u003e and \u003cem\u003eompK37\u003c/em\u003e but not in \u003cem\u003eompK35\u003c/em\u003e among all \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates resistant to Cefoperazone/sulbactam and piperacillin/tazobactam [33]. The emerging carbapenem resistance in these \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from both patients, and the hospital environment is a serious threat to IPC as they may be disseminated in the wards to cause infections that may be severe and difficult to treat. Hence, IPC enhancement is needed to prevent the spread of these ESBL-producing isolates in hospitals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe high number of virulence genes detected in carriage and hospital environment isolates may contribute to the pathogenicity of the \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates [34] in colonised patients without adequate IPC practices. In this study, all the ESBL-\u003cem\u003eK. pneumoniae\u003c/em\u003e isolates had type 1 or 3 (\u003cem\u003efim\u003c/em\u003e or \u003cem\u003emrk\u003c/em\u003e) fimbriae comparable to 90% of clinical \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates, which included ESBLs producers possessing fimbriae in a study of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from Egypt [35]. Coupled with the different capsular serotypes, siderophores, and a high number of other virulence genes in the isolates, these fimbriae, which are major adhesins, may confer a survival advantage in these \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates as they are involved in bacterial colonisation of host, biofilm formation and invasion [36, 37]. Though virulence genes associated with hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e were not found in the isolates in this study, hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e has been identified among isolates with predominantly K1 and K2 capsules with the O1 O-antigen, which have acquired the hvKP virulence plasmid [38], presenting the potential of isolates becoming hypervirulent if they acquire plasmids harbouring the hyper-virulence genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere was a high variation of STs among the isolates, showing the dissemination of different STs (ST39, ST307, ST815, ST1552, ST636, ST464 and ST1996) in the hospital. ST464 and ST636 were acquired by patients after admission and could have been circulating in the wards before patient admission, while the other STs (ST815 and ST1552) appeared to be circulating in the community. Notably, the high-risk clone ST307 was detected in one isolate from the hospital environment, suggesting this global clone\u0026rsquo;s presence and possible dissemination in Ghanaian hospitals. The ST307, which harboured the ESBL genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-106\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u003c/sub\u003e, and the \u0026beta;-lactamases \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM-1B\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-1\u003c/sub\u003e, compares to the ST307 among clinical \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from South Africa, similarly resistant to aminoglycosides and fluoroquinolones however harbouring the carbapenemase gene, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-181\u0026nbsp;\u003c/sub\u003ein addition to other \u0026beta;-lactamases [39]. The detection of this high-risk ST307 \u003cem\u003eK. pneumoniae\u003c/em\u003e with \u0026beta;-lactamases in the hospital environment shows that it is disseminated between patients and the environment and calls for heightened disinfection and IPC practices. To the best of the authors\u0026rsquo; knowledge, this study is the first to report ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e belonging to ST307 in Ghana and calls for increased surveillance to enable the detection and control of such high-risk clones.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhylogenetic analyses revealed a clustering of three \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from the hospital environment (from a bed, door handle and drip stand), all belonging to ST39 in the Obstetrics and Gynaecology ward. Two of these ST39 isolates from hospital environments (a bed and a drip stand) in the same ward harboured the \u0026beta;-lactamases, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV-11\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u003c/sub\u003e and same virulence genes suggesting an intra-ward circulation of the ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. Notably, one ST39 isolate, E35 had no ESBLs nor any \u0026beta;-lactamases although phenotypically resistant to \u0026beta;-lactam antibiotics. This difference could be attributed to other mechanisms such as the mutations in the ompK36 porin or the efflux pumps acrR, oqxA and oqxB detected in the isolate, mediating resistance to the \u0026beta;-lactam antibiotics. In clinical settings, it is thus necessary to consider testing for other mechanisms besides the ESBLs, which are routinely tested, therefore highlighting the importance of genomics in AMR surveillance. Though an ST39 isolate (P60-1) from a patient was acquired on admission, the source of acquisition could not be determined. However, the clonal relatedness of these MDR ST39 \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates with several ESBLs, \u003cem\u003eompK36\u003c/em\u003e mutations and virulence genes associated with plasmids and other MGEs could indicate a transfer of resistance and virulence genes among the isolates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ST39 clone has been previously reported in Ghana and was detected in \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from a study which screened for plasmids in three \u003cem\u003eE. coli\u003c/em\u003e and four \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates expressing ESBL mediated by the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u0026nbsp;\u003c/sub\u003egene from chronically infected wounds of Ghanaian patients [26]. The presence of the ESBLs in the isolates confirms the global reports of ST39 lineage of \u003cem\u003eK. pneumoniae\u003c/em\u003e, which is known to be a carrier of ESBL genes, notably \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-15\u0026nbsp;\u003c/sub\u003eand \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-14\u003c/sub\u003e and has been associated with nosocomial infections [16, 40, 41].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dissemination of the high-risk clone ST39 \u003cem\u003eK. pneumoniae\u003c/em\u003e has also been reported in infections and nosocomial outbreaks in sub-Saharan African countries (Congo, Ethiopia and The Gambia) and China [17, 40, 42, 43] and poses a threat to patients in the hospital. Phylogenomic analyses showed a clustering of the isolates from this study with clinical isolates from other countries in sub-Saharan Africa, indicating the potential of these carriage and hospital environment isolates to cause infections. There is a need for enhanced IPC practices, as patients could acquire these high-risk isolates from other patients and further disseminate them in the hospital environments.\u003c/p\u003e\n\u003cp\u003eIt should, however, be noted that the current study only analysed a few selected \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates using WGS. Furthermore, the study was conducted in a single tertiary hospital and is thus not representative of the epidemiology of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates in Ghanaian hospitals. Despite the perceived limitations, considering that the hospital serves patients from major hospitals in the Ashanti Region of Ghana and beyond, this study provides a snapshot of the state of ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e in hospitals in Ghana and the role of the environment in the potential dissemination of these resistant isolates.\u0026nbsp;\u003c/p\u003e\n"},{"header":"Conclusion","content":"\u003cp\u003eThe current study described the molecular epidemiology of MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolate, particularly of ompK mutations in combination with ESBLs and several other antibiotic resistance genes and virulence factors from hospital environments and patients. There was a dissemination of ST39 ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates between the hospital environment and patients. These findings re-emphasise the need for measures such as screening of patients to contain ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates since their carriage in patients and dissemination in the Teaching Hospital environment may indicate an increased risk of transmitting these isolates in hospital settings. There is a need to enhance IPC practices, including improved cleaning and disinfection of surfaces in the hospital to prevent further dissemination of these high-risk isolates and their resistant genes.\u0026nbsp;\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003eESBL: Extended spectrum beta-lactamase\u003c/p\u003e\n\u003cp\u003eIPC: Infection prevention and control\u003c/p\u003e\n\u003cp\u003eIRB: Institutional Review Board\u003c/p\u003e\n\u003cp\u003eLPS: Lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eMDR: Multidrug resistant\u003c/p\u003e\n\u003cp\u003eMLST: Multilocus sequencing typing\u003c/p\u003e\n\u003cp\u003eSNP: Single nucleotide polymorphisms\u003c/p\u003e\n\u003cp\u003eST: Sequence type\u003c/p\u003e\n\u003cp\u003eWHO: World Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e The study was approved by the Institutional Review Board (IRB) of the KATH (Reference: KATH IRB/AP/107/20) and the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (Reference: BREC/00001917/2020). Voluntary, informed written consent was obtained from participating patients and staff.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eSequence data that support the findings of this study has been deposited in GenBank and assigned accession numbers under BioProject\u003cstrong\u003e\u0026nbsp;PRJNA823741.\u003c/strong\u003e All other data supporting this study\u0026apos;s findings are available within the paper and in the supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eNone declared (EEAY, JM, DGE, NA, LKAA, AOO, AI, SE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by the South African Research Chair Initiative of the Department of Science and Technology and the National Research Foundation of South Africa (Grant No. 98342). The funding sources did not influence the study design, data collection, analysis, interpretation of the data, or manuscript writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e: EEAY, NA, AOO, and SYE co-conceptualized the study. EEAY undertook sample collection, laboratory and statistical analyses and wrote the original manuscript draft. EEAY, JM, DGA, ALKA and AI conducted data analysis. AI sequenced the isolates. SYE, AOO, NA, ALKA, DGA and JM supervised the work, vetted the results and critically reviewed the manuscript. All authors (EEAY, JM, NA, DGA, ALKA, AOO, AI and SYE) read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors are grateful to the Sequencing Core Facility, National Institute for Communicable Diseases, Johannesburg, South Africa. We are also grateful to study participants at the Obstetrics and Gynaecology Directorate, Surgery, and Intensive Care Units of the Komfo Anokye Teaching Hospital. We thank research assistants (nurses, biomedical scientists, and biostatisticians) at the study sites, the Heads of Departments and staff at the various directorates and the microbiology laboratory, Komfo Anokye Teaching Hospital, and staff for their support during the study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePorter L, Sultan O, Mitchell BG, Jenney A, Kiernan M, Brewster DJ, et al. How long do nosocomial pathogens persist on inanimate surfaces? A scoping review. J Hosp Infect. 2024;147:25-31.\u003c/li\u003e\n\u003cli\u003eChakma SK, Hossen S, Rakib TM, Hoque S, Islam R, Biswas T, et al. Effectiveness of a hand hygiene training intervention in improving knowledge and compliance rate among healthcare workers in a respiratory disease hospital. 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Front Cell Infect Microbiol. 2022;12:1240.\u003c/li\u003e\n\u003cli\u003eGala J-L, Ambroise J, Bearzatto B, Durant J-F, Bonjean M, Irenge L. Genomic characterization of multidrug-resistant extended spectrum \u0026beta;-lactamase-producing \u003cem\u003eKlebsiella pneumoniae \u003c/em\u003efrom clinical samples of a tertiary hospital in South Kivu Province, eastern Democratic Republic of Congo. MedRxiv. 2023:2023.01. 05.23284226.\u003c/li\u003e\n\u003cli\u003eDu J, Cao J, Shen L, Bi W, Zhang X, Liu H, et al. Molecular epidemiology of extensively drug-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e outbreak in Wenzhou, Southern China. J Med Microbiol. 2016;65(10):1111-8.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1 and 2","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hospital-acquired infections, hospital environment, multidrug resistance, extended-spectrum β-lactamase, carbapenem-resistant bacteria, whole genome sequencing, public health","lastPublishedDoi":"10.21203/rs.3.rs-5671518/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5671518/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMultidrug-resistant (MDR) ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e is widely implicated in community and hospital-acquired infections. Thus, the current study determined the prevalence and clonal relatedness of MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e from hospital environments, patients and healthcare workers in a Ghanaian hospital.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePatients (rectal and hand, collected on admission and 48h post admission), healthcare workers (hands) and hospital environment samples were sampled for three months. Antimicrobial susceptibility was determined using VITEK-2. Ten MDR ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were further analysed by whole-genome sequencing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll the isolates were ceftazidime-resistant; 90% were resistant to cefepime, amoxicillin/clavulanic, acid piperacillin/tazobactam, and sulphamethoxazole/trimethoprim. The isolates showed varying resistance to the cephalosporins and were susceptible to tigecycline. One environmental isolate isolate was resistant to meropenem but harboured no carbapenemase gene. The β-lactamase gene, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV,\u003c/sub\u003e was dominant and harboured by three environmental and five carriage isolates. Furthermore, three environmental and three carriage isolates harboured \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e. All isolates showed \u003cem\u003eompK36\u003c/em\u003e and \u003cem\u003eompK37\u003c/em\u003e mutations. Fluoroquinolone (\u003cem\u003eqnrB\u003c/em\u003e), aminoglycosides (\u003cem\u003eaadA1\u003c/em\u003e, \u003cem\u003eaadA2, aac(3)-IIa, aac(6')-Ib-cr,aph(3'')-Ib, aph(6)-Id\u003c/em\u003e) and sulphamethoxazole/trimethoprim (\u003cem\u003esul1, sul2\u003c/em\u003e, \u003cem\u003edfrA14, dfrA15\u003c/em\u003e) resistance-encoding genes were also detected. A diverse range of sequence types were identified, including ST39, ST307, ST815, ST1552, ST636, ST464, and ST1996, with ST39 being the most frequently observed (environmental\u0026thinsp;=\u0026thinsp;3; carriage\u0026thinsp;=\u0026thinsp;1). Three environmental and three carriage isolates harboured the Int1l integron. Many virulence genes, including \u003cem\u003eirp1\u003c/em\u003e, \u003cem\u003eirp2\u003c/em\u003e, \u003cem\u003eiutA\u003c/em\u003e, \u003cem\u003egndA\u003c/em\u003e, \u003cem\u003eompA\u003c/em\u003e, \u003cem\u003efes, fep\u003c/em\u003e, \u003cem\u003emrkD\u003c/em\u003e and \u003cem\u003efimH\u003c/em\u003e, were detected in environmental and carriage isolates. IncFIB was the most abundant plasmid replicon in five environmental and four carriage isolates. A clonal relationship was identified between a carriage isolate (ST39) and three environmental isolates (ST39) with shared genetic elements, suggesting that environmental reservoirs may play a role in the transmission and persistence of resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study highlights the prevalence of MDR ESBL-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e in both hospital environments and patients, emphasizing the potential for cross-transmission within healthcare settings. These findings reinforce the urgent need for strengthened infection prevention and control measures, enhanced antimicrobial stewardship, and continuous genomic surveillance to mitigate the spread of resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e in healthcare settings.\u003c/p\u003e","manuscriptTitle":"Whole-Genome Sequencing Insights into Porin-Mediated Resistance and Spread of ESBL- Producing Klebsiella pneumoniae in a Ghanaian Teaching Hospital","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 05:37:18","doi":"10.21203/rs.3.rs-5671518/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-27T10:18:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-27T10:16:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-12T16:27:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228304162834971739671271971904017150068","date":"2025-04-12T16:10:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-11T21:46:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-05T02:21:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-04-04T12:04:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"800abf3a-1332-4212-b983-ff38d2509a3b","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:11:03+00:00","versionOfRecord":{"articleIdentity":"rs-5671518","link":"https://doi.org/10.1186/s12866-025-04101-5","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-07-02 15:57:51","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2025-04-17 05:37:18","video":"","vorDoi":"10.1186/s12866-025-04101-5","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04101-5","workflowStages":[]},"version":"v1","identity":"rs-5671518","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5671518","identity":"rs-5671518","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}