Study On Resistant Mechanism And Molecular Epidemiology Of Two Antimicrobial Resistant Bacteria In Community

ObjectiveESBLs were initially associated with nosocomial outbreaks caused by enzyme-producing strains;recent studies have revealed more complex situations,with a significant increase in community isolates.No comprehensive data are available on the prevalence and risk factors of extended-spectrumβ-lactamases(ESBLs)-production in community residents.Thus,we investigated and report here an insight into the current prevalence,risk factors and molecular type for ESBLs from E.coli in elderly people in a community setting,and to quantify and characterize the emergence and spread of ESBL-producing E.coli in community.We also investigated the nature of the amino acid motifs found in penicillin-binding protein(PBP)2b,2x,and 1a of penicillin nonsusceptible Streptococcus pneumoniae(PNSP)isolates in this region and to obtain preliminary information regarding the prevalence of these alterations,to explore resistant mechanism of PNSP.In addition,multilocus sequence typing(MLST)had been used to determine the genetic relationship of pneumococcal isolates and identify internationally circulating resistant clones.This study will play an important role in tracking the genetic changes to chart the emergence,spread and evolution of PNSP in this region.MethodsA cross-sectional prospective study was conducted in 5 communities in Tie-xi and Dong-ling District,Shenyang,among elderly people and 286 gave informed consent from which rectal swabs were taken.An interview was done to obtain demographic data(age,gender),underlying diseases(diabetes mellitus),antibiotics use and hospital admission or surgery in previous 3 months.Rectal swabs were inoculated directly onto Eosin Methylene Blue(EMB)agar,and incubated at 35℃for 48 h.After the identification of E.coli isolates was determined,18 antibiotics susceptibility was routinely tested by Kirby-Bauer disc diffusion Colonies were screened by double-disk synergy test for ESBL production,and the clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis.The SPSS(version 13.0) software package was used for analysis of risk factors.Then the genotype of ESBLs were characterized by PCR and sequencing.Twenty-four nonduplicate clinical strains of S.pneumoniae were collected between July 2006 and February 2007 from the First Affiliated Hospital,China Medical University(19 isolates),Liaoning Provincial Hospital(2 isolates),and Shenyang Children' Hospital(3 isolates),isolated from sputum(21 isolates),blood(1 isolates), pus(1 isolate),and eye(1 isolate).The organisms were identified by susceptibility to ethylhydrocuprein(optochin,＞14 mm;Oxoid,England)and bile solubility.For each clinical strain,penicillin and cefotaxime MICs were performed by E test strips.Base upon the penicillin MIC values,18 isolates comprising all PNSP isolates(9 PRSP,5 PISP,and 4 PSSP isolates)were selected for further amplification of the pbp2b,pbp2x and pbp1a gene and nucleotide sequencing.Results1.The prevalence of rectal carriage of ESBL-producing E.coli was 7.0%.All 270 isolates were susceptible to imipenem and meropenem;more than 96%of non-ESBL-producing isolates remain susceptible to cephalosporins,74%susceptible to fluoroquinolones,while all confirmed ESBL-producing strains should be reported as resistant to all cephalosporins and aztreonam accroding to CLSI guidelines.19 ESBL-producing strains were 94.7%susceptible to amikacin,84.2%cefoxitin,63.2% amoxicillin/clavulanic acid respectively.These ESBL-producing strains exhibited co-resistance to fluoroquinolone,trimethoprim-sulfamethoxazole,gentamicin and tetracycline.In univariate analysis,only previous exposure to antimicrobial agents was strongly associated with ESBL-production(OR,3.2;95%CI,1.1-9.0,P=0.03).PFGE patterns of 19 ESBL-producing strains showed clonally unrelated.2.19 ESBL-producing isolates harbored CTX-M type ESBL enzyme,of which eleven(57.89%)with CTX-M-14;three with CTX-M-22;one with CTX-M-24 and three with CTX-M-79;one strain produced CTX-M-24 and CTX-M-79 simultaneously. Sequence analysis showed that the gene sequence of CTX-M-79 was G865→A substitution,resulting D289→N in amino acid sequence,which was different from the sequences in GenBank,suggesting a new type of ESBLs which was registered successfully in GenBank(accession numbers EF426798).TEM PCR amplification was positive in 15 strains,which were confirmed to be TEM-1.Of 19 ESBL-producing isolates,8 isolates(42.11%)harbored 2 antibiotic resistant gene cassettes which encoding resistance to trimethoprim(dfr17)and aminoglycosides(aadA5).3.Of 24 isolates,10 isolates(41.67%)were classified as penicillin susceptible S. pneumoniae(penicillin=0.06mg/L),5 isolates(20.83%)were penicillin intermediate S. pneumoniae(penicillin MIC,＞0.06mg/L and＜2mg/L),and 9 isolates(37.5%)were penicillin resistant S.pneumoniae(penicillin MIC=2mg/L).The penicillin MIC at which 50%of isolates are inhibited(MIC₅₀)was 0.625mg/L The penicillin MIC at which 90%of isolates are inhibited(MIC₉₀)was 4mg/L(range 0.016-4mg/L).While 15 isolates(62.5%)were classified as cefotaxime susceptible S.pneumoniae (cefotaxime=1mg/L),2 isolates(8.33%)were cefotaxime intermediate S.pneumoniae (cefotaxime MIC,＞1mg/L and＜4mg/L),and 7 isolates(29.17%)were cefotaxime resistant S.pneumoniae(cefotaxime MIC=4mg/L).The cefotaxime MIC at which 50% of isolates are inhibited(MIC₅₀)was 0.75mg/L The cefotaxime MIC at which 90%of isolates are inhibited(MIC₉₀)was 4mg/L(range 0.032-6mg/L).4.After amplification of the pbp2b,pbp2x and pbp1a gene among S.pneumoniae and further sequencing,18 sequences analysis was done by BioEdit software,using the ClustalW alignment.The most remarkable finding in this study was the Met342→Ile substitution just after the first STMK conserved motifs(PBP2x)in one PRSP isolate sp23,with penicillin MIC=4mg/L and cefotaxime MIC=6mg/L.Sequence analysis revealed that most PRSP isolates(penicillin MIC=1.5mg/L and cefotaxime MIC=2mg/L)shared identical PBP2b,2x,and 1a amino acid profiles,while the distribution of PISP amino acids substitutions differs from those of other countries, showing a unique genetic rearrangement in S.pneumoniae isolates in this region.The novel nucleotides sequences varivants determined in this study were submitted to the GenBank nucleotide sequence database under accession numbers EU035969-EU035971, EU044831,EU056919-EU056922,EU089705-EU089709,EU106881-EU106887, EU124672. 5.9 PNSP isolates were genotyped by MLST.Four PRSP isolates(sp02,sp09, sp23 and sp24)had the same allelic profiles(ST320 4-16-19-15-6-20-1),the double-locus variants(DLVs)of the Taiwan^19F-14 clone(ST236 15-16-19-15-6-20-26), which differed at the aroE and ddl loci.Another one PRSP isolate(sp18)was ST2395 (4-4-2-15-4-1-1),only a single-locus variants(SLVs)of the Spain^23F-1 clone(ST81 4-4-2-4-4-1-1)with a different allele of the recP gene.In addition,PISP isolate(sp01) was ST880(15-29-4-16-30-1-14),only a single-locus variants(SLVs)of the Taiwan^19F-15 clone(ST242 15-29-4-21-30-1-14)with a different allele of the recP gene, and sp14 was ST983(15-16-19-15-3-104-63),triple-locus variants(TLVs)of the Taiwan^19F-14 which differed at the spi,xpt and ddl loci.The sequence type of sp15 was not found in MLST database,suggesting that it is novel allelic profile.Spll was non-typable S.pneumoniae.ConclusionsOur results denote the importance of intestinal tract as a reservoir for ESBL-producing isolates in elderly in community setting in this region and that previous exposure to antibiotics is clearly linked to rectal carriage of ESBL-producing E.coli.Our results might help to some extent in antibiotic therapy dealing with E.coli infections from endogenous origin,such as urinary tract and intra-abdominal infection. Programs and policies for rational antibiotic use are urgently needed to minimize more colonization with ESBL-producers.In this study,most PRSP isolates(shared identical pbp2b,pbp2x,pbp1a gene profiles,and were identified as Taiwan^19F-14 clone,suggesting the clonal spread of specific resistant clone could be one of the major reasons for the rapid increases in penicillin resistance in this region.While the distribution of PISP amino acids substitutions shows a unique genetic rearrangement in S.pneumoniae isolates in this region,and were identified as different clonal complex.Our results will serve as a basis for future monitoring of genetic changes associated with the emergence and spread ofβ-lactam resistance in this region.