Genomic Epidemiology And Molecular Features Of Extended Spectrum -lactamase-producing Enterobacteriaceae At Jimma Medical Center Ethiopia.

Introduction: Infections caused by Enterobacterales are common causes of morbidity andrnmortality in hospitals and community. Several species of the Enterobacterales emerged withrnresistance property to extended/broad-spectrum β-lactam antimicrobials. Such strains producernβ-lactamase enzymes that can inactivate extended spectrum β-lactam antimicrobials, andrnhence, are called Extended Spectrum β-lactamase (ESBL)-producing strains. ESBL-producing rnstrains are increasingly being reported from different parts of the world revealing increasedrnprevalence and shift of predominant molecular types over last two decades. However, there isrna limited number of phenotypic studies in Ethiopia. The studies are not conclusive enough tornunravel the magnitude, dissemination, and the actual burden of the problem. rnObjective: The study aimed to determine genomic epidemiology and molecular features ofrnESBL-producing Enterobacteriaceae at Jimma Medical Center (JMC), Ethiopia. rnMethods: A cross-sectional study was conducted at JMC from June-October 2016. Studyrnparticipants were identified with the help of clinicians. And specimens relevant to clinicalrndiagnosis of the patients were collected by experienced nurses. Specimens collected included:rnurine, stool, wound-swab/aspirates, and sputum. Then specimens processed and inoculated onrnMacConkey agar, Blood agar and Salmonella Shigella agar at the microbiology laboratory andrnincubated at 37rnornC for 18-24h. Species identification was performed primarily by conventionalrnbiochemical tests. rnPhenotypic screening and confirmation of ESBL-production was determined by Double DiscrnSynergy Test (DDST). Further validation/confirmation of species identification and antibioticrnsusceptibility test was performed by MALDI-TOF, and disk diffusion for ESBL-phenotype atrnKarolinska University Hospital, Department of Clinical microbiology, Sweden. Strains of E.rncoli and K. pneumoniae with ESBL-phenotype were selected for whole genome sequencingrn(WGS) and sequenced on the Illumina (HiSeq 2500) sequencing platform at SciLifeLab,rnStockholm, Sweden.rnSocio-demographic, clinical data and associated variables were collected using pre-designedrnstructured questionnaire. Ethical clearance was obtained from relevant bodies. Study subjects were given information sheet about the study. Moreover, only study participants who providedrnwritten consent were recruited for the study. rnDescriptive data analysis was performed by using SPSS ver.20 statistical software. Sociodemographic,rnrnclinical data, and species related proportions were summarized by descriptivernparameters. Where applicable, statistical association and significance was defined as p-valuern≤ 0.05 as cut-off value for statistical association. Genome assembly and analysis was rnperformed by using different bioinformatics tools, SPAdes version 3.9 was used for genomernassembly. Enterobase, Center for Genomic Epidemiology (CGE), BIGsdb (Bacterial IsolaternGenome sequence database), and Gubbins (Genealogies Unbiased By recomBination InrnNucleotide Sequences), FastTree v.2, iTOL were tools used for genomic data analysis. rnrnResults: A total of 1,087 study participants either admitted to wards or seeking care as outpatientrnrnat medical, surgical, pediatric, and intensive care units at JMC were recruited. Fromrnthese study participants, 58.9%(n= 640) were male and 41.1%(n=447) were female. A total ofrn1,087 specimens were collected, and 642 bacterial strains of different spp. were isolated. Most,rn63.8% (n=255) were either E. coli(n=144) or Klebsiella spp.(n=111) and among nonfermenters,rnPseudomonasrnaeruginosarnandrnAcinetobacterrnspp.rnwerernpredominantrnandrnaccountedrnrnforrn13%rn(n=87)rnofrnoverallrnisolates.rnWithinrnthernfourrnmainrnunitsrnofrnthernhospital,rnandrnamongrnallrnrnspecimensrncollected,rnE.rncolirnandrnKlebsiellarnspp.rnwerernthernmostrncommonrnstrainsrnisolated.rnrnrnThernrnprevalence of ESBL-producing strains was (54.9%, 144/262) among E. coli and (76%,rn111/146) among K. pneumoniae. A small proportion of E. coli strains (5.4%, 14/262) were rnnon-susceptible to carbapenems. But only two E. coli strain encoded carbapenemaseng genern(blarnNDM-1rn and blarnOXA-66rn). Similarly, a total of (22.6%, 33/146) of K. pneumoniae strains werernnon-susceptible to carbapenems, however, only one strain encoded blarnNDM-1rn. Moreover, E. colirn96% (136/141) and K. pneumoniae 92%(102/111) were resistant to at least three classes ofrnantimicrobials. rnSeveral genetic determinants of antimicrobial resistance were identified, where blarnCTX-M-15rn wasrnthe most prevalent ESBL, E. coli (88.4%, 123/139) and K. pneumoniae (84.4%, 92/109). Thernother genetic determinants included: blarnTEM-1B rn(53.9%, 75/139) , blarnOXA-1 rn(63.3%, 88/139) ,rndfrA17 (58.2%, 81/139), mph (67.2%, 94/139), aac(6’)-Ib-cr (62.6%, 87/139), and sul (68.3%,rn95/139) among E. coli strains, and blarnTEM-1Brn (69.7%, 76/109), blarnOXA-1 rn(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.rnpneumoniae strains.rnMulti-locus sequesnce typing (MLST) revealed several sequence types (STs) carrying thernESBL genes. The most common sequence types (ST) encoding the blarnCTX-M-15rn were ST410rn(20/21), ST648 (15/15), ST131 (8/8), ST38 (6/6), ST44 (4/4) among E.coli. Among K.rnpneumoniae ST218 (7/7), ST15 (6/6), ST147 (6/6), ST17(6/6) and ST39 (5/5) had highest rncarriage of ESBL-genes. rnIn both K. pneumoniae and E. coli strains, virulence determinant genes were invesitigated.rnEPEC, ExPEC/UPEC, EAEC were the prevalent pathotypes identified among E. coli strains.rnThe O15/99:H30-ST38 (100%, n=6), O25:H4-ST131 (100%, n=8), O176:H34-ST130 (100%,rnn=5) encoded multiple toxins/virulence genes. On the other hand, K. pneumoniae strains wererncharacterized by capsular and O-LPS antigens/types. Both classical and hypervirulent strainsrnwere identified as multi-drug resistant (MDR) strains carrying blarnCTX-M-15rn ESBL-genes. rn Plasmid replicon typing was performed, IncFII(pRSB107) (24.8%, n=32), IncQ1(20%, n=26)rnand Col(BS512) (14%, n=18) are the most commonly identified replicons among E. coli strains,rnand IncFIB(K/Mar/PQil) (32.9%, n=32), IncR (24.7%, n=24), and IncFII (14.4%, n=14) werernprevalent replicons among K. pneumoniae strains. rnSingle nucleotide polymorphysim (SNP)-based phylogenetic analysis of population structurernshowed multiple clusters of related strains. Though blarnCTX-M-15 rnis the predominant ESBL-gene,rnother genetic determinants of resistance to b-lactams and other classes of antimicrobials werernidentified with a higher proportion in the same clonal complexes. Furthermore, some of these rnstrains (both in E. coli and K. pneumoniae) were high-risk epidemic clones. The disseminationrnof blarnCTX-M-15rn mediated antimicrobial resistance was thus polyclonal. Some of the detectedrnconal clusters suggest that an outbreak might have likely occured in the hospital.rnFurthermore, some possible risk factors for the increased prevalence of ESBL-strains werernstudied. Among these factors age less than five years, admission, pediatric unit and presencernof underlying chronic illness showed statistically significant association with the higherrnprevalence of ESBL-strains. Gender and current use of antibiotics (reported use of antibiotics)rnshowed no statistically significant associationrnConclusion: very high prevalence of ESBL among E. coli and K. pneumoniae was observed.rnThe blarnCTX-M-15rn was the predominat ESBL-genetic variant. Most of the strains are MDR, alsornresistant to several other non-β-lactam antimicrobials. The detection of blarnNDM-1rn is a clear threatrnof evolving carbapenemases mediated carbapenem resistance. The spread of ESBLs is throughrnmultiple clusters of epidemic clones, and some of them are hypervirulent strains. The findingsrnof this study are exteremly important primarly for the current hospital to look into it’s practice, rnto look into immediate preventive strategies including implemention of strict antimicrobialrnstewardship programmes and infection control strategies. The findings also shed light onrngenomic diversity and transmission dynamics of ESBL strains from Ethioipia and hence servernas a baseline data for researchers, planners and policy makers.

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