Analysis of Clinical Gram-Negative Carbapenem Resistant Acinetobacter baumannii and Klebsiella pneumoniaeIsolates from Wounds of Patients at a Hospital Reveals Complex Resistome and the First Report of blaSPM and blaSMEGenes in Trinidad, West Indies.

BackgroundMultidrug resistance Enterobacteriaceae causing community-acquired pyelonephritis is an emerging threat; most isolates are EBSL, as per Indian data. After the results of the MERINO trial, most guidelines favors Carbapenem for treating community-acquired pyelonephritis. Overuse of carbapenem may lead to Carbapenem resistance Enterobacteriaceae (CRE) in the community. We analyzed the ESBL and CRE genes in the clinically significant urinary isolates with phenotypic ESBL -E from patients with community-acquired pyelonephritis.Table 1Demographic characteristics of patients with community-acquired acute pyelonephritis caused by phenotypic ESBL -E.MethodsPatients with a diagnosis of community-acquired acute pyelonephritis with age ≥ 18 years and urine culture showing the growth of Enterobacteriaceae with phenotypic ESBL-E are included in the study. If urine culture grew more than one organism in Enterobacteriaceae, other GNBs were excluded. The phenotypic ESBL detection was based on resistance to a third-generation cephalosporin (Cefotaxime, Cefpodoxime, Ceftazidime) and a monobactam (Aztreonam). These positive isolates were further processed using the combination disk method. A ≥5mm increase in zone diameter for either antimicrobial agent tested in combination with clavulanate vs the zone diameter of the agent when tested alone was considered ESBL (Clinical and Laboratory Standards Institute (CLSI) Performance standards for antimicrobial susceptibility tests). Simultaneously, multiplexed Real-time PCR (TRUPCR® UTI AST Panel Kit, Europe) was done to detect ESBL genes (CTX-M, TEM, SHV) and CRE genes (OXA-48, KPC, NDM, VIM, IMP) in these isolates.Table 2:Detection of NDM genes with ESBL genes in patients with community-acquired acute pyelonephritis caused by phenotypic ESBL -E.ResultsA total of 38 isolates with phenotypic ESBL in patients diagnosed with community-acquired pyelonephritis were processed for RT-PCR, and 30 were detected with ESBL and CRE genes. The demographic and clinical characteristics are shown in Table 1. The most common ESBL gene was CTX-M 29 (.96.7%) followed by TEM 25 (83.3%). Multiple ESBL genes were detected in 24(80%) isolates. Among CRE genes, the NDM genes were detected in 15 (50%) isolates with other ESBL genes (Table 2).ConclusionThe presence of CRE genes in phenotypic ESBL -E in community-acquired acute pyelonephritis could be due to the injudicious use of carbapenem. This may imply that CRE will be spread in the community in the future, making it difficult to manage this infection.DisclosuresAll Authors: No reported disclosures

Study’s Excerpt/Novelty This study presents a comprehensive evaluation of colistin-resistant and extended-spectrum beta-lactamase (ESBL) gene co-production among Gram-negative clinical isolates from Usmanu Danfodiyo University Teaching Hospital in Sokoto. Notably, 13.9% of the isolates exhibited phenotypic co-production of colistin resistance and ESBL, with a significant presence of blaCTX-M and CTX-M 8 genes among ESBL producers, although no colistin resistance genes (mcr-1 and mcr-2) were detected via PCR. These findings highlight the necessity for integrated molecular and phenotypic investigations to fully elucidate resistance mechanisms in Gram-negative bacteria and underscore the need for further research to uncover alternative pathways contributing to observed resistance phenotypes. Full Abstract The emergence of antimicrobial resistance (AMR) is a major threat to global health. Its effects include high mortality and morbidity rates, treatment failure, and increased treatment costs. This study aimed to evaluate the co-production of colistin-resistant and extended-spectrum beta-lactamase (ESBL) genes among Gram-negative clinical isolates from Usmanu Danfodiyo University Teaching Hospital in Sokoto. Gram-negative bacteria were isolated from clinical specimens, including urine, feces, and wound aspirates. The Double-Disk Synergy Test and the Colistin Agar Test, respectively, were used to phenotypically validate the existence of colistin resistance and ESBL. Polymerase chain reaction (PCR) was used for molecular characterization. Primers were used to target genes linked to colistin resistance (mcr-1 and mcr-2) and ESBL genes (blaCTX-M, CTX-M 1, CTX-M 2, and CTX-M 8). The findings indicated that 13.9% of the isolates displayed co-production of Colistin and ESBL, and of these isolates, 60% had blaCTX-M genes, and 20% had CTX-M 8 linked to ESBL production. However, the presence of colistin resistance genes was not detected by PCR. Therefore, molecular analysis did not confirm the existence of the colistin resistance genes (mcr-1 and mcr-2) in these isolates. Consequently, the findings showed no molecular co-production of the ESBL and colistin resistance genes. This work emphasizes how crucial it is to look into molecular and phenotypic traits to completely comprehend how colistin resistance and ESBL genes coexist in Gram-negative isolates. More research is required to investigate other mechanisms behind the resistance phenotypes identified.

Extended Spectrum Beta Lactamase (ESBL) production in gram negative bacteria confers multiple antibiotic resistance, adversely affecting antimicrobial therapy in infected individuals. ESBLs result from mutations in β-lactamases encoded mainly by the blaTEM,blaSHV and blaCTX-M genes. The prevalence of ESBL producing bacteria has been on the increase globally, especially its upsurge among isolates from community-acquired infections has been observed. To determine ESBL prevalence and identify ESBL genes among clinical isolates in Osun State, Nigeria. A cross-sectional study was carried out from August 2016 - July 2017 in Osun State, Nigeria. Three hundred and sixty Gram-negative bacteria recovered from clinical samples obtained from both community and healthcare-associated infections were tested. They included 147 Escherichia coli (40.8%), 116 Klebsiella spp (32.2%), 44 Pseudomonas aeruginosa (12.2%) and 23 Proteus vulgaris (6.4%) isolates. Others were Acinetobacter baumannii, Serratia rubidae, Citrobacter spp, Enterobacter spp and Salmonella typhi. Disk diffusion antibiotic susceptibility testing was carried out, isolates were screened for ESBL production and confirmed using standard laboratory procedures. ESBLs resistance genes were identified by Polymerase Chain Reaction (PCR). All isolates demonstrated multiple antibiotic resistance. Resistance to ampicillin, amoxicillin with clavulanate and erythromycin was 100%, whereas resistance to Imipenem was very low (5.0%). The overall prevalence of ESBL producers was 41.4% with Klebsiella spp as the highest ESBL producing Enterobacteriacaea. ESBL producers were more prevalent among the hospital pathogens than community pathogens, 58% vs. 29.5% (p=0.003). ESBL genes were detected in all ESBL producers with the blaCTX-M gene predominating (47.0%) followed by blaTEM (30.9%) and blaSHV gene was the least, 22.1%. The blaCTX-M gene was also the most prevalent in the healthcare pathogens (62%) but it accounted for only 25% in those of community origin. A high prevalence of ESBL producing gram-negative organisms occurs both in healthcare and in the community in our environment with the CTX-M variant predominating. Efforts to control the spread of these pathogens should be addressed.

Background:There are scarce reports about the association ofKlebsiella oxytoca(K. oxytoca) with urinary tract infection (UTI) in children. We aimed to evaluate the prevalence offimA, mrkA, matBandpilQadhesins genes and extended-spectrum beta-lactamase (ESBL) genesblaCTX-M, blaTEMandblaSHVby polymerase chain reaction (PCR) and to study biofilm formation and antibiotics resistance inK. oxytocafrom children with UTI.Methods:This study was a retrospective cross-sectional study that included 120 children with UTI due toK. oxytoca. The bacteria were subjected to molecular detection offimA, mrkA, matBandpilQadhesins genes and ESBL genesblaCTX-M, blaTEMandblaSHVby PCR. Biofilm capacity was determined by the microtiter plate method.Results:The isolatedK. oxytocahad positive ESBL activity in 45.8% of isolates. About 40% of isolates were biofilm producers. The frequency of adhesion genes amongK. oxytocawas 91.7%, 83.3%, 48.3% and 37.5% formatB, pilQ, fimAandmrkAgenes, respectively. For ESBL genes, the frequency was 38.3%, 36.7% and 33.3% forblaCTX-M, blaSHVandblaTEMgenes, respectively. The commonest genes among ESBL isolates wereblaCTX-M(83.6%),blaSHV(80%) thenblaTEMgene (72.7%). A significant association (p=0.048) was detected between ESBL activity and biofilm formation byK. oxytoca.Conclusion:Present study highlights the emergence ofK. oxytocaas a pathogen associated with UTI in children. There was a high prevalence of adhesin genes and ESBL genes among these isolates. The capacity ofK. oxytocato form biofilm was associated with ESBL production.

Background: Drug-resistant hospital-acquired infections (HAIs) are a growing concern in modern medicine throughout the world. Klebsiella pneumoniae is one of the most prominent causative agents of multidrug-resistant nosocomial infections. It is also widely recognized for having a high resistance level to many antibiotic classes, particularly beta-lactams. Carbapenemase-producing K. pneumoniae has been identified as a major global cause of HAIs with adverse clinical outcomes. Therefore, it is of the utmost importance to have an in-depth understanding of the antimicrobial resistance (AMR) genetic determinants of this bacterium to stop the spread of highly resistant K. pneumoniae in healthcare facilities and the resulting patient morbidity and mortality. Objectives: This study aimed to investigate the AMR pattern of K. pneumoniae isolates obtained from intensive care units (ICUs), with a focus on extended-spectrum beta-lactamases (ESBLs) genes blaCTX-M, blaGES, and blaIMP. Methods: A total of 105 K. pneumoniae isolates obtained from the sputum samples of ICU patients were identified and confirmed using standard microbiological tests and 16S rRNA polymerase chain reaction (PCR). The antibiotic susceptibility test was performed for all the isolates. The presence of ESBL genes was determined phenotypically and by PCR. Results: The highest level of resistance was observed against ceftazidime (100%), cefotaxime (99%), and imipenem (93.3%). Approximately 87.6% and 39% of the isolates were sensitive to colistin and gentamicin, respectively. Phenotypic ESBL production was observed in 16 isolates, and the prevalence of blaCTX-M was 86.7%. No blaGES and blaIMP genes were detected. Conclusions: Periodic investigation of AMR-mediating genes is essential due to the high prevalence of ESBL genes in HAIs. The presence of other ESBL genes needs to be investigated for a more accurate understanding of the AMR status of K. pneumoniae in healthcare settings.

Coexistence of PMQR and ESBL genes among clinical Escherichia coli isolates from community-acquired UTI in Mexicali, on the US-Mexico border

We investigated the occurrence and distribution of genes encoding for extended-spectrum beta-lactamase (ESBL) production and quinolone resistance among multi-drug resistant (MDR) Escherichia coli and Klebsiella pneumoniae isolated from poultry, domestic pigs, and environment samples in the Msimbazi basin in Tanzania. A total of 130 non-duplicated isolates obtained from the poultry (n = 40), domestic pigs (n = 52) and environment (n = 38) were screened for ESBL genes (blaCTX-M, blaTEM, and blaSHV) and plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS, qnrC, qnrD, qepA, and aac (6)-Ib-cr) using polymerase chain reaction (PCR). The most commonly detected ESBL genes were blaCTX-M (31.5%, n=41) and blaTEM (10%, n=13), while quinolone resistance genes were qnrS (27.7%, n=36), qnrB (6.9%, n=9) and aac (6)-lb-cr (5.4%, n=7). ESBL gene blaSHV and quinolone resistance genes qnrA, qnrC, and qepA were not detected in any of the isolates. Resistance genes were highest in isolates from the environmental samples (86%, n=33), followed by poultry 72.5% (n=29), and domestic pigs 21.4%, (n=9). Overall, the most frequent resistance gene was qnrS, followed by blaCTX-M and blaTEM. About 17.7% (n=23) of the E. coli and K. pneumoniae isolates depicted co-existence of ESBL and quinolone resistance genes. One Health interventional measures, involving different disciplines across human, animal, and environmental sectors, are needed to control the spread of antimicrobial resistance in the Msimbazi River Basin in Tanzania.

Aims: The aims of the present study were to investigate the presence of Plasmid-Mediated Quinolone Resistance (PMQR) determinants and the association of these determinants with Extended Spectrum Beta-Lactamases (ESBLs) genes in ESBL-producing Klebsiella pneumoniae isolates from Teaching Hospital of Bouaké, Côte d’Ivoire.
 Study Design: It is a retrospective study.
 Place of Study: Bacteriology-Virology Laboratory of Teaching Hospital, Bouaké, Côte d'Ivoire.
 Methodology: From January 2015 to December 2016, 96 ESBL-producing Klebsiella pneumoniae isolates were collected from several specimens. Antimicrobial susceptibility of isolates was tested using the standard disk-diffusion method on Mueller-Hinton and interpretation according to recommendations of the 2017 EUCAST. These isolates analyzed for the detection of ESBL (blaCTX-M, blaTEM and blaSHV) and PMQR genes (aac(6’)-Ib-cr, qnrB and qnrS) using simplex PCR.
 Results: Of the 96 ESBL-producing strains, 85 (88.55%) harbored at least one of the ESBL genes tested. Out of the 85 strains encoding ESBL genes, 96.47% carried blaCTX-M and 92.94% blaSHV and blaTEM genes. Eighty nine (89.6%) of the 96 ESBL producing-isolates were resistant to ciprofloxacin and 84.4% to norfloxacin. Among the 96 strains, 80 (83.33%) were found harboring at least one PMQR gene consisting of 78 (81.3%) aac(6’)-Ib-cr, 61 (63.5%) qnrB and 15 (15.6%) qnrS. Among the PMQR-positive strains, 68.4% coharbored qnrB+acc(6’)-Ib-cr genes, 10.5% qnrB+qnrS+acc(6’)-Ib-cr and 6.6% qnrS+acc(6’)-Ib-cr. The qnrB gene was always linked to aac(6’)-Ib-cr gene. Aac(6’)-Ib-cr gene showed the highest association with three ESBL genes (87.6%), followed by qnrB gene (70.6%), then qnrS (17.7%).
 Conclusion: The PMQR genes were highly prevalent in ESBL-producing Klebsiella pneumoniae, primarily the aac(6’)-Ib-cr gene. The high associated was observed between ESBL and PMQR genes, notably with the aac(6’)-Ib-cr gene.

Urinary tract infection (UTI) causes significant renal damage and disease severity is compounded by antimicrobial resistance (AMR) and other comorbidities in the patient. Blood group antigens secreted in body fluids (secretor status) are known to play a role in bacterial adhesion and we studied its influence on AMR in UTI. A total of 2758 patients with UTI were studied with urine culture, qualitative and semiquantitative urine microscopy, serum creatinine and secretor status in saliva samples by adsorption-inhibition method. Of these, AMR from 300 patients with E. coli infection were assessed as per CLSI 2019 guidelines and extended-spectrum beta-lactamase (ESBL) genes (bla TEM, bla CTX-M, bla SHV) and NDM1 genes were studied using TaqMan probes in Real-time polymerase chain reaction. Patients with UTI were followed up for two weeks. Female patients had higher predilection (57%) for E. coli infection while patients with diabetes or non-secretors had none. In our study, ESBL producers were seen in 62% of the E. coli isolates and fosfomycin had 100% susceptibility. Non-secretors were significantly associated with acute kidney injury (AKI), AMR and ESBL genes. Multidrug-resistance (MDR) was noted in 127/160 (79.4%) ESBL and 17/18 (94%) NDM1 gene encoding strains. Quantitative urine microscopy scoring predicted AKI both at presentation and at end of follow up. ESBL producers were common in our study population and non-secretors had a significant association with AMR genes. Urine microscopy scoring system may be a useful tool to predict AKI in patients with UTI.

This study investigated the co-carriage of plasmid mediated quinolone resistance (PMQR) and extended spectrum beta-lactamase (ESBL) producing lactose non-fermenting (LNF) Enterobacteriaceae isolated from poultry birds. This was a descriptive cross-sectional study carried out between September, 2016 and March, 2017. The Kirby-Bauer disk diffusion method was used to determine the antimicrobial susceptibility patterns. ESBL screening disc kit was used to detect ESBL activities. Detection of ESBL and PMQR genes was carried out by means of polymerase chain reaction. In total, 207 LNF Enterobacteriaeae isolates were recovered from the cloacal swabs of poultry birds within the Calabar Metropolis. ESBL-producing isolates were 162 (78.3%) while fluroquinolone resistant isolates were 194 (93.7%). Among the ESBL-producing isolates, resistance to Ciprofloxacin, Norfloxacin, Levofloxacin, Ofloxacin and Nalidixic acid was 55 (34.2%), 26 (16.1%), 35 (21.7%), 50 (31.1%), and 162 (100%), respectively. About 65% of the quinolone resistant isolates were positive for at least one of the PMQR and ESBL genes in this study. Strict antimicrobial screening, surveillance of resistant isolates as well as the judicious practice of antimicrobial administration in the poultry setting with special emphasis on fluoroquinolones is advised given the high prevalence of co-existent ESBL and PMQR genes. Key words: LNF enterobacteriaeae, Extended spectrum beta-lactamases, quinolone resistance

BackgroundMulti-drug resistance (MDR) and extensive-drug resistance (XDR) associated with extended-spectrum beta-lactamases (ESBLs) and carbapenemases in Gram-negative bacteria are global public health concerns. Data on circulating antimicrobial resistance (AMR) genes in Gram-negative bacteria and their correlation with MDR and ESBL phenotypes from Nepal is scarce.MethodsA retrospective study was performed investigating the distribution of ESBL and carbapenemase genes and their potential association with ESBL and MDR phenotypes in E. coli, Klebsiella spp., Enterobacter spp. and Acinetobacter spp. isolated in a major tertiary hospital in Kathmandu, Nepal, between 2012 and 2018.ResultsDuring this period, the hospital isolated 719 E. coli, 532 Klebsiella spp., 520 Enterobacter spp. and 382 Acinetobacter spp.; 1955/2153 (90.1%) of isolates were MDR and half (1080/2153) were ESBL producers. Upon PCR amplification, blaTEM (1281/1771; 72%), blaCTXM-1 (930/1771; 53%) and blaCTXM-8 (419/1771; 24%) were the most prevalent ESBL genes in the enteric bacilli. BlaOXA and blaOXA-51 were the most common blaOXA family genes in the enteric bacilli (918/1771; 25%) and Acinetobacter spp. (218/382; 57%) respectively. Sixteen percent (342/2153) of all isolates and 20% (357/1771) of enteric bacilli harboured blaNDM-1 and blaKPC carbapenemase genes respectively. Of enteric bacilli, Enterobacter spp. was the most frequently positive for blaKPC gene (201/337; 60%). The presence of each blaCTX-M and blaOXA were significantly associated with non-susceptibility to third generation cephalosporins (OR 14.7, p < 0.001 and OR 2.3, p < 0.05, respectively).The presence of each blaTEM, blaCTXM and blaOXA family genes were significantly associated with ESBL positivity (OR 2.96, p < 0.001; OR 14.2, p < 0.001 and OR 1.3, p < 0.05 respectively) and being MDR (OR 1.96, p < 0.001; OR 5.9, p < 0.001 and OR 2.3, p < 0.001 respectively).ConclusionsThis study documents an alarming level of AMR with high prevalence of MDR ESBL- and carbapenemase-positive ESKAPE microorganisms in our clinical setting. These data suggest a scenario where the clinical management of infected patients is increasingly difficult and requires the use of last-resort antimicrobials, which in turn is likely to intensify the magnitude of global AMR crisis.

Background and Aim:Antimicrobial-resistant microorganisms (ARMs) have been increasing among wild animals. Interactions occurring at the interface between wildlife, humans, and livestock can lead to the transmission of ARMs. Thus, the prevalence of ARMs in wild and domestic animals should be determined to address and prevent this issue. This study aimed to determine the resistance patterns of cefotaxime (CTX)-resistant Escherichia coli and identify the presence of extended-spectrum beta-lactamase (ESBL) genes in ESBL-producing E. coli among a population of wild banteng (Bos javanicus) and domestic cattle kept on farms located close to the Lam Pao non-hunting area, Kalasin province, Thailand.Materials and Methods:Forty-five fecal samples were taken from wild bantengs inhabiting the Lam Pao non-hunting area in Thailand, alongside 15 samples from domestic cattle. Bacterial culture, triple sugar iron, and motile indole lysine tests were conducted to identify E. coli. A polymerase chain reaction (PCR) was conducted for specific confirmation. MacConkey agar supplemented with 2 μg/mL of CTX was used to identify CTX-resistant E. coli, which would be used to identify ESBL production based on a double-disk synergy test. Extended-spectrum beta-lactamase-producing samples were subjected to disk diffusion tests to determine resistant patterns, and the sizes of PCR bands and DNA sequencing were used to differentiate ESBL gene types.Results:All samples tested positive for E. coli. Forty-five isolates from 15 banteng samples and three isolates from one domestic cattle sample displayed CTX-resistant and ESBL-producing traits. The banteng and domestic cattle populations exhibited nine and three distinct resistant patterns, respectively. The PCR results indicated that the banteng isolates harbored the following genes: Cefotaxime-M1 (n = 38), CTX-M9 (n = 5), and the SHV group (n = 2). All three isolates from the domestic cattle sample contained the CTX-M1 gene. Classification of ESBL genes based on the DNA sequences of the banteng isolates showed the characteristics of CTX-M15 (n = 20), CTX-M55 (n = 6), CTX-M14 (n = 5), and CTX-M79 (n = 1). The three domestic cattle isolates exhibited the characteristics of CTX-M15, CTX-M55, and CTX-M79.Conclusion:Despite no previous antibiotic applications, approximately one-third of the banteng samples displayed CTX resistance, indicating ARM contamination within the ecosystem. The similarity in ESBL genes between the banteng and domestic cattle populations suggests potential gene transmissions between these animal groups. However, the initial source of ARMs remains unclear, as the banteng population exhibited more ESBL genes than the domestic cattle, suggesting the possibility of multiple ARM sources. These findings raise concerns because the banteng population inhabits an area that is an important source of freshwater and nourishes the entire north-east region of Thailand and other South-east Asian countries, including Laos, Cambodia, and Southern Vietnam.

Antimicrobial resistance (AMR) is a global public health problem, which presents a huge threat to the treatment of all forms of bacterial infections. A wide range of bacterial pathogens across the globe are increasingly developing resistance to multiple classes of antimicrobial agents rendering the agents concerned ineffective for the treatment of infections. Bloodstream infection (BSI) and other bacterial infections in sub-Saharan Africa (SSA) and Malawi in particular, are a common cause of morbidity and mortality. Few facilities in SSA however, are able to conduct long-term surveillance and as such the full burden of drug resistant infection (DRI) remain largely unknown across the region. In this thesis, blood cultures routinely taken from adult and paediatric medical patients admitted to Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi between 1998 and 2016 were analysed to describe trends in BSI and AMR. The analysis revealed a significant decline of BSI in all major pathogens except S. Typhi. However, the majority of isolates were resistant to the Malawian first-line antimicrobial agents (ampicillin, cotrimoxazole and chloramphenicol). Resistance to all the first line antimicrobial agents was more common in Gram-negative pathogens than Gram-positive pathogens. Non-Salmonellae Enterobacteriaceae that produced extended spectrum beta-lactamase (ESBL) and were fluoroquinolone-resistant were detected, and the proportions of these isolates rose significantly during the surveillance. In contrast, a majority of common Gram-positive pathogens remain susceptible to either penicillin or chloramphenicol. Methicillin resistant S. aureus was first reported in 1998 but became regularly detected in the later years of the surveillance. The analysis of blood culture isolates identified E. coli as one of the common causes of BSI in Blantyre, and the proportion of these isolates that were ESBL producers increased over time. Globally, efforts to treat E. coli infections are increasingly being compromised by the rapid, global spread of ESBL-producing E. coli. In this thesis, a whole genome sequencing (WGS) study was carried out to investigate the genetic population structure and molecular determinants of AMR in E. coli isolates from Malawi. Whole genomes of clinical E. coli isolates from patients admitted to QECH were sequenced and analysed using phylogenetic methods and comparative genomics. It was revealed that the E. coli population in Malawi is highly diverse with isolates belonging to five phylogroups, corresponding to five isolate sequence clusters (SCs) that contained over forty sequence types (STs). A unique sub-lineage of ST131 was identified that was distinct from previously defined sub-lineages of this globally disseminated ST. The most common ESBL gene was blaCT X-M-15. Unlike in other settings where presence of the blaCT X-M-15 gene was strongly linked to ST131, here the gene was not lineage-specific suggesting a distinct genomic landscape of ESBL-producing E. coli in Malawi. This thesis also identified Klebsiella spp. isolates as a common cause of BSI in Blantyre, and an increasing proportion of ESBL-producing and fluoroquinolone resistant isolates were identified. The molecular mechanisms and clones of K. pneumoniae associated with ESBL production and fluoroquinolone resistance were yet to be explored in Malawi. Here, a number of K. pneumoniae isolates were selected for WGS, and placed in a global context by comparison with previously sequenced K. pneumoniae isolates from multiple locations outside SSA, in order to identify the molecular determinants of AMR and determine their relationship with K. pneumoniae population structure. Genomic analysis revealed three main lineages of K. pneumoniae, which corresponded to the previously defined KpI, KpII and KpIII lineages. All three lineages exhibited high genetic diversity. Further phylogenetic analysis revealed a sub-lineage of KpI to be a major cause of CA infections in Malawi. The sub-lineage included the clonally related ST14 and ST15 of K. pneumoniae which cause hospital acquired infection in multiple settings across the globe, A large pool of AMR genes, was identified in the genomes of the Malawian isolates, including multiple ESBL and qnr genes. Plasmid-encoded CTX-M-15 was the most common type of ESBL that was identified. In common with E. coli from Malawi, AMR was not restricted to a particular clade of K. pneumoniae. These findings suggest that dissemination of AMR in the K. pneumoniae population in Malawi was either due to a combination of horizontal gene transfer and clonal expansion, or horizontal gene transfer alone. In conclusion, the thesis has shown that ESBL production and fluoroquinolone resistance is rapidly spreading in Malawi across multiple E. coli and K. pneumoniae lineages that are causing increasing levels of infection. As cephalosporins and fluoroquinolones remain the last resort antimicrobial agents in this setting, urgent action is needed to curb the spread of Gram-negative AMR pathogens.

Multidrug-resistant Enterobacterales isolates carrying extended-spectrum beta-lactamases (ESBLs) and mobile colistin resistance (mcr) genes pose a significant healthcare threat as they can lead to difficult-to-treat infections. This study investigates the prevalence of isolates co-harbouring ESBL and mcr genes and characterizes the plasmids co-harbouring those genes. ESBL-producing Enterobacterales (ESBL-E) isolates identified during point prevalence surveys (PPS) in a Dutch hospital were screened for mcr genes. Plasmids co-harbouring mcr and ESBL genes were identified using long- and short-read sequencing data, while detecting resistance and replicon genes using AMRFinderPlus and PlasmidFinder. The plasmid database PLSDB was searched for plasmids containing the same mcr and ESBL gene(s), and SNP and DCJ-Indel distance analyses were conducted to examine plasmid diversity. The most recent common ancestor (MRCA) was inferred through timed phylogeny analyses in BEAST, and putative composite transposons containing the mcr or ESBL genes were identified. Among 188 screened ESBL-E, 11 harboured mcr genes: 9 with mcr-9 and 2 with mcr-9 and mcr-4.3. All plasmids containing mcr and ESBL genes were IncHI plasmids harbouring mcr-9, blaCTX-M-9 and/or blaSHV-12. The plasmid database search resulted in 128 similar plasmids. SNP analysis showed ≤10 SNPs among the PPS study plasmids and up to 924 SNPs among all plasmids. Structural relatedness and phylogenetic analyses revealed clustering of the PPS study plasmids but no additional taxonomical or geographical clustering. The MRCA of the PPS study plasmids likely emerged between 1986 and 2008. Finally, composite transposon analysis indicated that matching complete insertion sequences rarely flanked mcr-9 genes, whereas blaCTX-M-9 and blaSHV-12 frequently were. The prevalence of mcr genes among ESBL-E in this study is 5.9%, with mcr-9 being the most prevalent. Limited plasmid diversity suggests a single introduction event followed by regional and global dissemination across related bacterial species. Persistent ESBL gene mobility suggests a more recent introduction of these genes compared to the mcr-9 genes.

Cases of antibiotic-resistant Escherichia coli (E. coli) infections are becoming increasingly frequent and represent a major threat to our ability to treat cancer patients. The emergence of antimicrobial resistance threatens the treatment of E. coli infections. In this study, the antimicrobial profiles, virulent genes, and the frequency of extended-spectrum beta-lactamase (ESBL) gene carriage in fecal E. coli isolates from cancer patients at the Laquintinie Hospital in Douala (Cameroon) were determined. 507 participants were recruited from October 2021 to March 2023, of whom 307 (60.55%) had cancer and 200 (39.45%) did not. Two hundred and two E. coli were isolated from fecal samples of one hundred and fifteen cancer patients and 47 (87) noncancer patients using EMB LEVINE agar. The antimicrobial resistance profile of the isolates was determined using the Kirby-Bauer disk diffusion method. Virulence and resistance genes were detected by simplex polymerase chain reaction (PCR). E. coli showed significant rates of resistance to amoxicillin, cefotaxime, ceftazidime, piperacillin, tetracycline, and ciprofloxacin in cancer patients compared to noncancer patients. The rate of multidrug resistance (MDR) was significantly (p < 0.05) higher in cancer patients than in noncancer patients. Fifty-five enterovirulent E. coli were identified, of which 24 (43.63%) were EPEC, 13 (23.63%) were EAEC, 6 (10.90%) were ETEC, 10 (18.18%) were STEC, and 2 (3.63%) were EIEC. The frequency of beta-lactamase genes in the 55 ESBL-producing enterovirulent E. coli isolates was determined, and 94.54% harbored at least one ESBL gene, distributed as follows: 80.00% for bla TEM, 67.27% for bla CTX-M, 24.63 for bla OXA, and 36.36% for bla SHV genes. Several associations were observed between virulence factors, resistance genes, and the antimicrobial resistance phenotype. This study revealed the real existence of fecal carriage of ESBL-producing enterovirulent E. coli isolates from cancer patients with a high rate of MDR in the latter.