Study On Plasmid-mediated Resistance Mechamism Of Enterobacter Cloacae

Recent studies have found that Enterobacter cloacae (E. cloacae) can cause various infections including respiratory tract, urogenital tract, skin soft tissue, and bloodstream infections. It has become an important pathogen of nosocomial infections. Furthermore, contrasting to the surveillance of bacterial resistance of Shanghai to our hospital has shown that Enterobacter has prominent resistance to antibiotics ofβ-lactams, fluoroquinolones and aminoglycosides, which are the three commonly used clinical drugs. Studies on the resistance mechanisms of E. cloacae to the above three antibiotics have indicated that:1. the mainβ-lactam antibiotics-resistant mechanism for E. cloacae is the production of the extended spectrumβ-lactamases (ESBLs) and AmpCβ-lactamases (AmpC enzymes), which is primarily mediated by plasmids; 2. the aminoglycoside resistance of E. cloacae is mainly mediated by aminoglycoside-modifying enzyme genes including aac (3)-I, II, III and aac (6')-I, II as well as aminoglycoside methylase genes such as rmtB and armA, which are also generally located on plasmids.3. the plasmid-mediated quinolone resistance discovered recently is an important mechanism for E. cloacae to resist quinolone antibiotics, which is mediated by genes including qnr. According to the report, the plasmid-mediated resistance has some characteristic:①Generally, modifying enzymes were plasmid mediated. Plasmid-mediated enzymes were constitutive hyperproduction without inducers. These enzymes can hydrolyze or modifyβ-lactams or aminoglycosides and cause high level resistance;②one plasmid can containβ-lactams, fluoroquinolones, aminoglycosides resistance gene, together with quaternary ammonium disinfectants and sulfonamides resistance gene simultaneously, it will cause one strain can resist many kinds of antibiotics;③The gene for plasmid-mediated resistance enzymes can disseminate between Enterobacter spp. Due to above reason, plasmid-mediated resistance is very important on the study of bacterial resistance. The results of pulsed-field gel electrophoresis (PFGE) of E. cloacae clinical isolates showed that there were no clone transmission but the resistance pattern was the same, it implied that these strains can have the same or similar plasmids. Therefore, it is necessary to conduct deep studies on the plasmid-mediated mechanisms of E. cloacae.Based on the above situations, the present study investigated a total of 101 strains of E. cloacae clinically isolated in 2005 in our hospital. In this study, drug resistance and homology of these strains were studied; plasmid-mediated resistance genes in these strains to the three clinically commonly used antibiotics were detected; plasmid conjugation experiments of these clinical strains were performed, and the resistance genes and structural genes carried by plasmids were tested.Part 1 Drug sensitivity test and homology analysis of Enterobacter cloacaeThe two-fold agar dilution method was used to determine the minimum inhibitory concentration (MIC) of 14 anti-infective drugs on the 101 strains of clinically isolated E. cloacae. Results showed that the resistance rates of the 101 E. cloacae strains in this group to cefotaxime and ceftazidime were up to 53.4%and 51.4%respectively; while the strains were sensitive to cefepime with the resistance rate of only 10.9%; and the resistance rates to cefoperazone/sulbactam and piperacillin/tazobactam were both 16.9%. The strains were highly sensitive to carbapenem antibiotics without resistance. The resistance rates to quinolone antibiotics and ciprofloxacin were 35.6%and 31.7%; to amikacin and gentamicin were 19.8%and 41.6%respectively. Of 101 strais,30 (29.7%,30/101)strains were resistant to the third cephosporins, together with resistance to quinolone or aminoglycosides, 22 (21.8%,22/101) were resistant to the third cephosporins, quinolone and aminoglycosides.Results of pulsed-field gel electrophoresis (PFGE) characterization for the 101 E. cloacae strains showed that there were a very small number of homogenous strains in this group. Only 3 strains were of the same clone, while 94 strains had different PFGE fingerprint patterns, indicating that no prevalence of the same clone strains of E. cloacae within our hospital. However, the above strains of different clones often showed the same or similar type of drug resistance spectrums, suggesting that these different strains were likely to carry the same or similar drug resistance plasmids.Part 2 molecular biology study of plasmid-mediated resistance genes in E. cloacaeAfter the extraction of bacterial plasmid DNA templates by phenol/chloroform method, PCR was used to detect plasmid-mediated ESBLs and AmpC, aminoglycoside-modifying enzymes and the plasmid-mediated 16S rRNA aminoglycoside methylase, three kinds of quinolone resistance genes and detecting the amino acid mutations of gyrA and parC genes.1. detecting plasmid-mediated ESBLs and AmpC. After the extraction of bacterial plasmid DNA templates by phenol/chloroform method, the PCR method using CTX-M, SHV and TEM-1 primers, the multiplex PCR using MOX, CIT, ACC, EBC, DHA or FOX-type AmpC primers, found 72 (71.3%) of the 101 E. cloacae strains contained plasmid-mediatedβ-lactamases.37 (36.6%, 37/101) had TEM-1 typeβ-lactamase genes,54 strains (53.5%,54/101) contained SHV-12 type ESBLs genes,31 strains (30.7%,31/101) contained CTX-M-type ESBLs genes.18 strains (17.8%,18/101) were found to contain DHA-1 type plasmid-mediated AmpC enzyme. In 72 containingβ-lactamases strains,1 contained only DHA-1 type AmpC enzymes and 1 contained only TEM-1 type 0-lactamase gene, the other 70 strains (69.3%,70/101) were all ESBLs stains. Among these 70 strains,46 strains (45.5%,46/101) contained two or more than two kinds ofβ-lactamase.2. detecting plasmid-mediated quinolone resistant genes. In this study, with bacterial plasmid DNA as template, using qnrA, qnrB, qnrS, qnrC, qnrD, aac (6')-Ib-cr and qepA primers, plasmid-mediated quinolone resistant genes of the 101 E. cloacae strains were detected by PCR. The qnr genes were sequenced for typing, and the amino acid mutations of gyrA and parC genes encoding DNA gyrase and topoisomerase IV were detected.Of the 101 E. cloacae strains,39 had positive qnr genes (positive rate 38.6%).19 strains (18.8%) were positive for qnrA gene,18 (17.8%) positive for qnrB, and 3 (3.0%) positive for qnrS; 1 of them carried both qnrB and qnrS genes; qnrC and qnrD genes were not found. Through gene sequencing and alignment on GenBank for all qnr positive strains, all qnr A gene positive strains contained qnrAl subtype genes. Among qnrB gene positive strains,16 contained qnrB4 subtype,1 contained qnrB6 subtype, and 1 contained qnrB10 subtype. Among qnrS gene positive strains,1 contained qnrSl subtype,1 contained qnrS2 subtype, and 1 had both qnrB4 and qnrSl genes.44 of the 101 strains were positive for aac (6')-Ib gene by PCR detection. After the PCR products were digested by FokI enzyme, the products of 7 strains could not be cut due to the lack of GGATG site. The 7 strains were determined by sequencing and found to be positive for aac (6')-Ib-cr gene with the positive rate of 6.9%, of which 4 strains were also carried qnrB4 gene. No qepA gene was found through amplification.GyrA and ParC genes of the 39 qnr gene positive strains were tested by PCR, and they were sequenced and aligned on Genbank.23 strains that were positive for qnr gene, having no amino acid mutations at the quinolone resistance determining region (QRDR) of the GyrA and ParC genes, were found sensitive to ciprofloxacin (according to 2006 standard). Another strain with Ser 83 mutating to Tyr at the QRDR of GyrA, was a ciprofloxacin sensitive strain. In the other 15 qnr gene positive strains which 1-3 point mutations of each were found at the QRDR, including Ser 83 mutated to Ile, Phe or Leu, Asp 87 mutated to Ala at the GyrA QRDR, and Ser 80 mutated to Ile at the ParC QRDR, were ciprofloxacin resistant strais.Of the 36 ciprofloxacin-resistant strains,15 were qnr gene positive with the positive rate of 41.7%; of the 63 ciprofloxacin-sensitive strains, 23 were qnr gene positive with the positive rate of 36.5%. The difference of the qnr gene positive rate between the two groups was not statistically significant by the chi-square test (P-0.23), indicating that the detection rate of qnr genes between the quinolone resistant and sensitive groups was similar. In this study,23 of the 39 qnr positive strains were ciprofloxacin-sensitive, but the MIC values were up to 0.06-1mg/l, suggesting that the decreased sensitivity to quinolones for these bacteria may be caused only by qnr, and qnr may be the first appeared resistance mechanism during the formation of quinolone resistance for bacteria.Among 101 strains,70 contained ESBLs, of the 70 ESBLs strains,35(50%, 35/70) were qnr positive strains. Of the other 31 non-ESBLs strains, only 4(12.9%,4/31) strains were qnr positive strains. The difference of the qnr gene positive rate between the two groups was statistically significant by the chi-square test (P=0.005). Of the 18 AmpC strains,14 (77.8%,14/18) were qnr positive strains, of the other 83 non-AmpC strains, 25 strains (30.1%,25/83) were qnr positive strains. It was also statistically significant by the chi-square test between the two groups (P=0.018). It suggested that qnr was more frequent in ESBLs or AmpC strains.3.Detecting aminoglycoside-modifying enzymes and the plasmid-mediated 16S rRNA aminoglycoside methylase. In this study, eight aminoglycoside-modifying enzyme genes of aac (3)-Ⅰ, aac (3)-Ⅱ, aac (3)-Ⅲ, aac (6')-Ⅰb, aac (6')-Ⅱ, ant (2")-Ⅰ, ant (3")-Ⅰ, and aph (3')-Ⅵ, as well as two methylase genes of armA and rmtB were amplified by PCR. Among the 101 E. cloacae strains,48 (47.5%,48/101) contained aminoglycoside-modifying enzyme genes,13 (12.9%,13/101) contained armA or rmtB, in which 10 (9.9%,10/101) contained both aminoglycoside-modifying enzymes and aminoglycoside methylase genes.In the 48 strains containing aminoglycoside-modifying enzyme genes, the most commonly found gene was aac (6')-Ⅰb, which existed in 44 strains (43.6%,44/101). After digestion,7 strains (6.9%) were found to contain aac (6')-Ⅰb-Cr gene, 22 (21.8%) contained ant (2")-Ⅰgene, 6 (5.9%) had aac (3)-Ⅱ, 1 (1.0%) had aac (3)-Ⅰ, and 2 (2.0%) contained ant (3")-Ⅰ. The aac (3)-Ⅲ, aac (6')-Ⅱ, or aph (3')-Ⅵgenes were not found in the 101 strains of E. cloacae in this group.Through PCR detection,13 strains (12.9%) were found to contain methylase genes armA or rmtB, of which 12 (11.9%) contained armA gene, 2 (2.0%) contained rmtB gene, and 1 contained both armA and rmtB genes. The 13 strains with aminoglycoside methylases all showed a high degree of resistance to amikacin and gentamicin, and the MIC values were all above 256ug/ml. The aminoglycoside resistance mediated by aminoglycoside modifying enzymes showed different resistance levels, for example, strains containing aac (6')-Ib gene had MIC values for gentamicin from 4ug/ml to above 256ug/ml.Among 101 strains,70 contained ESBLs, of the 70 ESBLs strains, 47(67.1%,47/70) strains were aminoglycoside-modifying enzyme genes positive. Of the other 31 non-ESBLs strains, only 1(3.3%,1/31) strain were aminoglycoside-modifying enzyme genes positive. The difference of the aminoglycoside-modifying enzyme genes positive rate between the two groups was statistically significant by the chi-square test (P=0.0005). Of the 70 ESBLs strains,13 (18.6%,13/70)strains were aminoglycoside methylases positive strains. Of the other 31 non-ESBLs strains, none strain were aminoglycoside methylases enzyme genes positive. It was also statistically significant by the chi-square test between the two groups (P=0.0003). It suggested that aminoglycoside-modifying and aminoglycoside methylases enzyme genes were more frequent in ESBLs strains.Part 3 Plasmid conjuction of multi-resistant Enterobacter Cloacae resistance genesTo investigate the transferability of resistant plasmid in 101 strains, we selected①44 strains with ESBLs(SHV-12 and CTX-M) and AmpC (DHA-1) enzyme genes;②12 strains with SHV-12, DHA-1, aac (6')-Ib and qnrB4 as as donor strains, E. Coli J53AzR as receptor to do plasmid conjugation tests.Plasmid conjugation tests were conducted for a total of 44 strains with ESBLs or AmpC enzyme genes and 30 strains got conjugants, conjugated rate was 68.2%(30/44). Seven conjugants were found to have only one plasmid each, so further study had been done to the seven conjugants, the result showed:①donor strain E-41 X J53AZR→conjugant 41TC (CTX-M-1G gene conjugation);②donor strain E-57 X J53 AzR→conjugant 57TC and donor strain E-70×J53 AZR→conjugant 70TC (CTX-M-9G and aac (6')-Ib genes conjugation);③donor strain E-91 X J53AzR→conjugant 91TC and donor strain E-95×J53 AZR→95TC (qnrB, SHV-12, DHA-1 and aac(6')-Ib genes conjugation);④donor strain E-206×J53AZR→conjugant 206TC and donor strain E-209 X J53AZR→conjugant 209TC (CTX-M-9G and aac(6')-Ib genes conjugation).The result of conjugation suggested①OTX-M-1G, CTX-M-3G, CTX-M-9G, SHV-12 and DHA-1 could be transmitted by plasmid conjugation.②Since 7 conjugated strains containing only one plasmid, it implied that CTX-M-3G together with aac(6')-Ib, CTX-M-9G together with aac(6')-Ib, SHV-12 together with aac(6')-Ib can be cotransmitted by one plasmid. The 7 conjugants MIC of cefotaxime and ceftazidim was 4-32mg/l, like donor strains, but it was 66.7-533 times than receptor J53AzR (MIC≤0.06mg/L) The conjugants MIC of Gentamycin and Amikacin was 4-128mg/l, it was 16-1024 times than receptor J53AZR (MIC 0.125mg/L)Plasmid conjugation tests were conducted for 12 strains harboring qnrB, SHV-12, DHA-1 and aac(6')-Ib and 7 strains got conjugants, conjugated rate was 58.8%(7/12).The genes of 7 donor strains were similar, all of them had qnrB4, SHV-12, DHA-1, aac(6')-Ib and ant (21-I genes. The conjugants contained qnrB4, SHV-12, DHA-1, aac(6')-Ib genes. Ciprofloxacin MIC of 7 conjugants was 0.094 mg/L～0.19mg/L, which was 12-24 times more than receptor J53AZR (MIC 0.008mg/L). It also suggested that the decreased sensitivity to quinolones for these bacteria may be caused only by qnr, and qnr may be the first appeared resistance mechanism during the formation of quinolone resistance for bacteria. The 7 conjugants MIC of cefotaxime and ceftazidim was 4-64mg/l, like donor strains, but it was 66.7-1000 times than receptor J53AZR (MIC≤0.06mg/L) The conjugants MIC of Gentamycin and Amikacin was 4-64mg/l, it was 16-1024 times than receptor J53AZR (MIC 0.125mg/L-0.25mg/L)Part 4 Construction analysis of multi-resistant plasmid of Enterobacter CloacaeThe conjugated plasmids containing qnrB, SHV-12, DHA-1 and aac(6')-Ib was futher investigated in the structural features of the plasmids. The plasmids were digested by HindIII and ligated with PUC18. Then the ligated PUC18 was digested again and was found a 14kb fragment was ligated in the PUC18. The 14kb fragment of conjugants 65TC and 404TC was sequenced. Sequencing results showed that the two plasmids had the same structure at the surrounding DNA regions. The structure was similar to the Plasmid pSH7 containing qnrB4 gene in Klebsiella pneumoniae, which was reported previously by our institute, containing sapA-qnrB4-pspF-pspA-pspB-pspC-orfl-pspD-blaBGA-1-ampR gene sequences. The sapA gene was at the upstream of qnrB4, suggesting that it encoded a peptide transport system enzyme. The pspF-pspA-pspB-pspC-pspD was the operon of psp, encoding a phage shock protein, followed by orfl reading frame, and the blaBHA-1 gene encoding AmpC enzyme, as well as the ampC regulatory gene ampR. Detected by PCR, the seven conjugants also contained qacΔ1-sull disinfectant and sulfonamide resistance genes, integron genetic marker Integrase I (integrase gene), and Tn21/Tn501 transposon genetic marker gene (mercury ion reductase gene), indicating that the seven zygotes were resistant to quaternary ammonium disinfectants and sulfonamides, in addition to the third generation of cephalosporins and aminoglycoside antibiotics. Therefore, they were multiple drug resistance plasmids. The seven zygotes harbor qnrB4, SHV-12, DHA-1, aac(6')-Ib, qacΔ1-sull disinfectant and sulfonamide resistance genes, integron genetic marker Integrase I (integrase gene), and Tn21/Tn501 transposon genetic marker gene. This is the first report a plasmid from E. cloacae contain such kinds of resistance genes. SHV-12, aac(6')-Ib locate in which place in plasmid needs futher research.