Studies On Extended-spectrum β-lactamases And Outer Membrane Protein In Enterobacteriaceae

Enterobacteriaceae is widely distributed and closely related to human. Among infection dieases, Enterobacteriaceae is the most common pathogen, and often characterized as multi-drug resistant bacteria. Extended spectrum (3-lactamases (ESBLs) are the enzymes family which are capable of inactivating third-generation or fourth-generation cephalosporins as well as monobactams (aztreonam), and inhibited by clavulanic acid. On the basis of their encoding gene identity, ESBLs can be divided into TEM-type, SHV-type, CTX-M-type, OXA-type and others, including94of TEM-type ESBLs,44of SHV-type ESBLs,121of CTX-M-type ESBLs,18of OXA-type ESBLs and12of others ESBLs.{http://www.lahey.org/studies). Occurance rate and geneotype of ESBLs are quite different among hospitals, areas and countries because of different use of antibiotics.SHV (3-lactamase is one of earliest finding and detected frequently in Enterobacter-iaceae. Nowadays, more than140SHV subtypes have been reported and new subtypes are being detected continuously. According to the difference of substrates, SHV β-lactamase is described as broad-spectrum β-lactamases, extended-spectrum β-lactamases (ESBLs) and inhibitor-resistant β-lactamases. SHV ESBLs are widely distributed in the world and prevalent subtypes are not same in different areas. A variety of SHV subtypes have been found in China in recent years.Owing to the defect of ESBL confirmatory tests, the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and removed the need to perform ESBL screening and confirmatory tests except for epidemiological or infection control purposes in2010. But preliminary reports suggested that the revised breakpoints may not detect all ESBL-producing strains.Outer membrane protein(OMP) locating at outside of all Gram-negative bacterial cells is known as a permeability barrier for resistance to antibiotics. Membrane porin, the major constitutes of outer membrane protein, has a structure of homotrimer and allows hydrophilic solutes including antibiotics to pass through. The different rate of antibiotics passing through porin can lead to obvious change of resistance in Enterobacteriaceae.Carbapenem-resistant organisms have emerged fast in clinic with the widespread and unreasonable use of carbapenems. The production of carbapenemases is the most common mechanism in Enterobacteriaceae. In2009, the CLSI recommended the modified Hodge test (MHT) to screen for the production of carbapenemase in Enterobacteriaceae. Although the sensitivity and specificity of the MHT have been shown to exceed90%, several studies have reported false positive or false negative results when this method was used to screen for carbapenemase in Enterobacteriaceae.Based on the above situation, in the present study,382consecutive and non-duplicate ESBL-producing strains were collected at Huashan Hospital, including158strains of E. coli,164of K. pneumoniae in2008, and60of P. mirabilis between2007and2008. Our aim was to evaluate the susceptibility of ESBL-producing Enterobacteriaceae using new CLSI breakpoints, the characteristics of two new ESBL SHV subtypes: SHV-136and SHV-137, the change of OmpF in ESBL-producing Proteus mirabilis susceptible to aztreonam and The effect of ESBL-producing combing with outer membrane porin loss on the modified Hodge testing in Enterobacteriaceae. It is very important for the choice of antibiotics in clinic, and reducing the incidence and mortality of health-care associated infection. The study included following four parts of contents.Part one:Susceptibility of ESBL-producing Enterobacteriaceae with the new CLSI breakpoints(一) Analysis of genotypes in ESBL-producing Enterobacteriaceae1. Of382strains with ESBL phenotypes by PCR, genotypes could be determined in363strains, but not in3strains of E. coli,14of K. pneumoniae and2of P. mirabilis.2. blaCTX-M genes alone or in combination with blaSHV were present in92.7%of these ESBL-producing strains.98.1%of E. coli and96.7%of P. mirabilis strains harbored CTX-M type ESBLs.3. WaCTX-M-1group ESBL genes were found in48.2%of K. pneumoniae, significantly higher than that of WaCTX-M-9group (P<0.01). WaCTX-M-9group ESBLs were found in72.8%of is. coli and91.7%of P. mirabilis strains, significantly higher than the WaCTX-M-1group (P<0.01). The CTX-M-2and CTX-M-8groups were not detected.4.26.2%strains of K. pneumoniae harbored SHV type ESBLs alone or in combination with CTX-M:SHV-12(n=29), SHV-2(n=10), SHV-31(n=2), SHV-136(n=1) and SHV-137(n=1). Both CTX-M and SHV ESBL genes were detected in K. pneumoniae strains:CTX-M alone in66.5%, SHV alone in4.3%, and in both together in22%. blaSHV ESBL genes were not detected in any of E. coli and P. mirabilis strains.5. A new TEM subtype was found in a K. pneumoniae, named as TEM-183by Jacoby GA. It was designed as broad-spectrum β-lactamase.(二) Susceptibility testing1. Using the new breakpoints, all382strains were resistant to cefazolin, cefotaxime and ceftriaxone.2.91.7%,85.0%and96.7%of P. mirabilis strains tested susceptible to ceftazidime, cefepime and aztreonam.45.6%and41.8%of E. coli strains appeared susceptible to ceftazidime and cefepime, and20.1%of K. pneumoniae were susceptible to cefepime.3. In general, beta-lactam resistance rates were much higher in K. pneumoniae strains than in E. coli, but still showed that CTX-M-1group strains were more resistant than CTX-M-9group strains. For290strains carrying CTX-M-1or CTX-M-9alone, the susceptibility rates for aztreonam, cefepime and ceftazidime were41%to68%for CTX-M-9group organisms but only3to14%for CTX-M-1group strains.Of99CTX-M-9group E. coli strains,22strains were susceptible to above three antibiotics and11were susceptible to ceftazidime and cefepime, but resistant to aztreonam, while among40CTX-M-1strains,20were resistant to those three antibiotics and8were resistant to ceftazidime and aztreonam, but intermediate to cefepime.A substantial number of ESBL-producing strains would be reported as susceptible to some cephalosporins and aztreonam, notwithstanding2010CLSI lowered the susceptibility breakpoints of some antibiotics for Enterobacteriaceae. The significant difference to the resistance of antibiotics comes from the distribution of ESBL genotypes and bacterias. In regions where CTX-M ESBLs are predominant, resistance to cefotaxime and ceftriaxone may be a better marker for ESBL presence than resistance to ceftazidime, cefepime and aztreonam.Part two:Discovery and characteristics of two new ESBL SHV subtypes: SHV-136and SHV-137We found two new SHV subtypes, named as SHV-136(GenBank:HQ661362) and SHV-137(GenBank:HQ661363) by by Jacoby GA.(http://www.lahey.org/studies)1. The amino acid sequence of SHV-136and SHV-137:it should be noted that WaSHV-136, the gene most closely related to WaSHV-25, is separated by a single-amino-acid substitution (A22V). The WaSHV-137gene differed from blaSHV-l by two substitutions of G147A and N254H.2. Clone and ESBL confirmatory test:the complete encoding sequence of SHV-136and SHV-137was inserted into plasmid pHSG398respectively, then the resultants recombinant plasmid were introduced into E. coli TOP10and the transformants were selected to evaluate by the ESBL confirmatory test. Both of the transformants were designed as ESBL-producing isolates from3twofold concentration decreasing in an MIC for ceftazidime in combination with clavulanic acid than its MIC when tested alone.3. The meaningful mutation in SHV-137:site-directed mutagenesis experiment was performed in SHV-137and both of the substitutions differed from SHV-1were restored (named as SHV-137-M1and SHV-137-M2) respectively. It was demonstrated that the substitution of G147A differed from SHV-1in SHV-137was associated with ESBL activity, however, the substitution of N254H had nothing to do with ESBL activity.4. IEF analysis of two new enzymes:the pIs of blaSHV-136and blaSHV-137were7.5and7.6, respectively.5. Susceptibility testing and the influence of three inhibitors to cloned strains:the MICs of two transformants carrying SHV-136and SHV-137were similar to the tested antibiotics. They were resistant to penicillin and the first generation cephalosporin, susceptible to the second, third and fourth generation cephalosporin. Howerer, the MICs of them to the second, third and fourth generation cephalosporin were obviously higher than that of E. coli TOP10. We also observed6twofold concentration decrease in the MIC of them for amoxicillin/clavulanic acid than its MIC when amoxicillin tested alone, whereas the MICs of them to piperacillin/tazobactam and ampicillin/sulbactam were more than2566μg/ml.Part three:The change of OmpF in ESBL-producing Proteus mirabilis susceptible to aztreonamWe observed that the MICs of fourteen Proteus mirabilis strains containing CTX-M-9group ESBLs to aztreonam were0.04or0.06μg/ml, however, to cefotaxime and cefepime were both resistant. This situation should be contact with the change of membrane porin. Fourteen Proteus mirabilis strains were selected from39tested isolates randomly by a variety of aztreonam MIC and all of them carried CTX-M-9group ESBL. SDS-PAGE for the outer membrane protein didn’t change obviously among them and there was no significant difference in the expression for fadL, ompA, ompF, omp W and tolC.We detected the nucleotide sequence of ompF and ompR in thirty-nine of different aztreonam MIC Proteus mirabilis strains. Interestingly, the mutation of OmpF was observed in all the isolates with aztreonam MIC0.04or0.06μg/ml. Compared to Proteus mirabilis ATCC29906,7amino acid residues were lost,3were inserted and30were muated and the similarity was88.5%. The nucleotide sequence of ompF in the Proteus mirabilis isolates with aztreonam MIC0.5,1or2pg/ml was the same as ATCC29906. OmpR also didn’t change in all the Proteus mirabilis isolates.Part four:The effect of ESBL-producing combing with outer membrane porin loss on the modified Hodge testing in EnterobacteriaceaeThree hundred and one non-duplicate clinical isolates from part one were analysed including153E. coli isolates and148K. pneumoniae isolates. They were all susceptible to imipenem, meropenem, and ertapenem. None of the isolates was found to be susceptible to cefazolin and cefotaxime, and62.1%,48.2%, and19.9%were resistant to ceftazidime, cefepime, and cefoxitin, respectively..Of the301E. coli and K. pneumoniae isolates, none detected carbapenemase genes by PCR. Genes that encoded CTX-M-type ESBLs were detected in280(93.0%) of the isolates, among which CTX-M-14, CTX-M-15, CTX-M-25, CTX-M-14coupled with CTX-M-15, and CTX-M-14coupled with CTX-M-25type ESBL were found in135(48.2%),112(40.0%),2(0.7%),28(10.0%), and3(1.1%) of the isolates, respectively.11.3%were producing more than two types of ESBLs, such as SHV-type and CTX-M-type ESBLs.Although all the isolates were sensitive to carbapenems, the MHT yielded a positive result for3.3%(10/301) of the isolates and all the MHT-positive isolates (except one isolate was SHV-12type ESBL producer) were CTX-M type ESBL producing. Analysis of the outer membrane porin proteins by SDS-PAGE showed that9isolates exhibited loss of one or two of porin protein compared with the sensitive strains. The results suggested that ESBL-producing isolate combining with outer membrane porin loss should play an important role in resulting to the false-positive result.