Genomic Epidemiology and Molecular Features of Extended Spectrum β-lactamase-producing Enterobacteriaceae at Jimma Medical Center, Ethiopia.
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2020-04
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Addis Ababa University
Abstract
Introduction: Infections caused by Enterobacterales are common causes of morbidity and mortality in hospitals and community. Several species of the Enterobacterales emerged with resistance property to extended/broad spectrum β-lactam antimicrobials. Such strains produce β-lactamase enzymes that can inactivate extended spectrum β-lactam antimicrobials, and hence, are called Extended Spectrum β-lactamase (ESBL)-producing strains. ESBL producing strains are increasingly being reported from different parts of the world revealing increased prevalence and shift of predominant molecular types over last two decades. However, there is a limited number of phenotypic studies in Ethiopia. The studies are not conclusive enough to unravel the magnitude, dissemination, and the actual burden of the problem.
Objective: The study aimed to determine genomic epidemiology and molecular features of ESBL-producing Enterobacteriaceae at Jimma Medical Center (JMC), Ethiopia.
Methods: A cross-sectional study was conducted at JMC from June-October 2016. Study participants were identified with the help of clinicians. And specimens relevant to clinical diagnosis of the patients were collected by experienced nurses. Specimens collected included: urine, stool, wound swab /aspirates, and sputum. Then specimens processed and inoculated on Mac Conkey agar, Blood agar and Salmonella Shigella agar at the microbiology laboratory and incubated at 37 o C for 18-24h. Species identification was performed primarily by conventional biochemical tests. Phenotypic screening and confirmation of ESBL-production was determined by Double Disc Synergy Test (DDST). Further validation/confirmation of species identification and antibiotic susceptibility test was performed by MALDI-TOF, and disk diffusion for ESBL-phenotype at Karolinska University Hospital, Department of Clinical microbiology, Sweden. Strains of E. coli and K. pneumoniae with ESBL phenotype were selected for whole genome sequencing (WGS) and sequenced on the Illumina (HiSeq 2500) sequencing platform at SciLifeLab, Stockholm, Sweden. Socio-demographic, clinical data and associated variables were collected using pre-designed structured questionnaire. Ethical clearance was obtained from relevant bodies. Study subjects were given information sheet about the study. Moreover, only study participants who provided written consent were recruited for the study. Descriptive data analysis was performed by using SPSS ver.20 statistical software. Sociodemographic, clinical data, and species related proportions were summarized by descriptive parameters. Where applicable, statistical association and significance was defined as p-value ≤ 0.05 as cut-off value for statistical association. Genome assembly and analysis was performed by using different bioinformatics tools, SPAdes version 3.9 was used for genome assembly. Enterobase, Center for Genomic Epidemiology (CGE), BIGsdb (Bacterial Isolate Genome sequence database), and Gubbins (Genealogies Unbiased By recomBination In Nucleotide Sequences), FastTree v.2, iTOL were tools used for genomic data analysis.
Results: A total of 1,087 study participants either admitted to wards or seeking care as outpatient at medical, surgical, pediatric, and intensive care units at JMC were recruited. From these study participants, 58.9%(n= 640) were male and 41.1%(n=447) were female. A total of 1,087 specimens were collected, and 642 bacterial strains of different spp. were isolated. Most, 63.8% (n=255) were either E. coli(n=144) or Klebsiella spp.(n=111) and among nonfermenters, Pseudomonas aeruginosa and Acinetobacter spp. were predominant and accounted for 13% (n=87) of overall isolates. Within the four main units of the hospital, and among all specimens collected, E. coli and Klebsiella spp. were the most common strains isolated. The prevalence of ESBL-producing strains was (54.9%, 144/262) among E. coli and (76%, 111/146) among K. pneumoniae. A small proportion of E. coli strains (5.4%, 14/262) were non-susceptible to carbapenems. But only two E. coli strain encoded carbapenemaseng gene (bla NDM-1 and bla OXA-66 ). Similarly, a total of (22.6%, 33/146) of K. pneumoniae strains were non-susceptible to carbapenems, however, only one strain encoded bla NDM-1. Moreover, E. coli 96% (136/141) and K. pneumoniae 92%(102/111) were resistant to at least three classes of antimicrobials. Several genetic determinants of antimicrobial resistance were identified, where blaCTX-M-15 was the most prevalent ESBL, E. coli (88.4%, 123/139) and K. pneumoniae (84.4%, 92/109). The other genetic determinants included: bla TEM-1B (53.9%, 75/139) , blaOXA-1 (63.3%, 88/139) , dfrA17 (58.2%, 81/139), mph (67.2%, 94/139), aac(6’)-Ib-cr (62.6%, 87/139), and sul (68.3%,95/139) among E. coli strains, and bla TEM-1B (69.7%, 76/109), bla OXA-1 (34.8%, 38/109), sul (61.5%, 67/109), aac(6’)-Ib-cr (65.1%, 71/109), and dfrA27 (32.1%,35/109) among K.pneumoniae strains. Multi-locus sequesnce typing (MLST) revealed several sequence types (STs) carrying the ESBL genes. The most common sequence types (ST) encoding the blaCTX-M-15 were ST410(20/21), ST648 (15/15), ST131 (8/8), ST38 (6/6), ST44 (4/4) among E.coli. Among K. pneumoniae ST218 (7/7), ST15 (6/6), ST147 (6/6), ST17(6/6) and ST39 (5/5) had highest carriage of ESBL-genes. In both K. pneumoniae and E. coli strains, virulence determinant genes were invesitigated. EPEC, ExPEC/UPEC, EAEC were the prevalent pathotypes identified among E. coli strains. The O15/99:H30 ST38 (100%, n=6), O25:H4-ST131 (100%, n=8), O176:H34-ST130 (100%, n=5) encoded multiple toxins/virulence genes. On the other hand, K. pneumoniae strains were characterized by capsular and O-LPS antigens/types. Both classical and hypervirulent strains were identified as multi-drug resistant (MDR) strains carrying blaCTX-M-15 ESBL-genes. Plasmid replicon typing was performed, IncFII(pRSB107) (24.8%, n=32), IncQ1(20%, n=26)and Col(BS512) (14%, n=18) are the most commonly identified replicons among E. coli strains, and IncFIB( K/Mar/PQil) (32.9%, n=32), IncR (24.7%, n=24), and IncFII (14.4%, n=14) were prevalent replicons among K. pneumoniae strains. Single nucleotide polymorphysim (SNP)-based phylogenetic analysis of population structure showed multiple clusters of related strains. Though blaCTX-M-15 is the predominant ESBL-gene, other genetic determinants of resistance to b-lactams and other classes of antimicrobials were identified with a higher proportion in the same clonal complexes. Furthermore, some of these strains (both in E. coli and K. pneumoniae) were high-risk epidemic clones. The dissemination of bla CTX-M-15 mediated antimicrobial resistance was thus polyclonal. Some of the detected conal clusters suggest that an outbreak might have likely occured in the hospital. Furthermore, some possible risk factors for the increased prevalence of ESBL-strains were studied. Among these factors age less than five years, admission, pediatric unit and presence of underlying chronic illness showed statistically significant association with the higher prevalence of ESBL-strains. Gender and current use of antibiotics (reported use of antibiotics) showed no statistically significant association
Conclusion: very high prevalence of ESBL among E. coli and K. pneumoniae was observed. The blaCTX-M-15 was the predominat ESBL-genetic variant. Most of the strains are MDR, also resistant to several other non-β-lactam antimicrobials. The detection of blaNDM-1 is a clear threat of evolving carbapenemases mediated carbapenem resistance. The spread of ESBLs is through multiple clusters of epidemic clones, and some of them are hypervirulent strains. The findings of this study are exteremly important primarly for the current hospital to look into it’s practice, to look into immediate preventive strategies including implemention of strict antimicrobial stewardship programmes and infection control strategies. The findings also shed light on genomic diversity and transmission dynamics of ESBL strains from Ethioipia and hence serve as a baseline data for researchers, planners and policy makers.
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Keywords
ESBL, antimicrobial resistance, E. coli, K. pneumoniae. Jimma, Ethiopia.