Increased frequency of integrons and β-lactamase-coding genes among extraintestinal Escherichia coli isolated with a 7-year interval

We analyzed the level of antimicrobial resistance, and the presence of integrons and β-lactamase-coding genes in 69 clinically relevant Escherichia coli strains originating from extraintestinal infections isolated in 1999–2001 and 2008–2010. Comparison of the two groups showed significant differences in drug resistance frequency, and the presence of integron and β-lactamase-coding genes. The frequency of resistance to all antimicrobials beside imipenem, streptomycin, piperacillin/tazobactam, and sulfamethoxazole increased significantly, especially towards aminoglycosides, β-lactams and fluoroquinolones. Similarly, we noticed an increase in the number of strains with integrons from 31.6 to 80.7 %. The presence of integrase genes was associated with elevated frequency of resistance to each antimicrobial tested besides imipenem, piperacillin/tazobactam and ceftazidime. The presence of integrons was also associated with multidrug resistance phenotype. The genetic content of integrons comprised genes determining resistance toward aminoglycosides, sulfonamides and trimethoprim. Moreover, we noticed a significant increase in the frequency of bla _CTX-M β-lactamases, with appearance of bla _CTX-M-15 variant and newer plasmid-encoded β-lactamases like CMY-15 and DHA. The emergence of strains resistant to several classes of antimicrobials and carrying integrons, ESBL and AmpC β-lactamase-coding genes may predict the spread of isolates with limited treatment options.     

Phenotypic and Molecular Characterization of Multidrug Resistant Klebsiella pneumoniae Isolated from a University Teaching Hospital,China

The multidrug-resistant rate of Klebsiella pneumoniae has risen rapidly worldwide. To better understand the multidrug resistance situation and molecular characterization of Klebsiella pneumoniae, a total of 153 Klebsiella pneumoniae isolates were collected, and drug susceptibility test was performed to detect its susceptibility patterns to 13 kinds of antibiotics. Phenotypic tests for carbapenemases ESBLs and AmpC enzyme-producing strains were performed to detect the resistance phenotype of the isolates. Then PCR amplification and sequencing analysis were performed for the drug resistance determinants. The results showed that 63 strains harbored bla _CTX-M gene, and 14 strains harbored bla _DHA gene. Moreover, there were 5 strains carrying bla _KPC gene, among which 4 strains carried bla _CTX-M, bla _DHA and bla _KPC genes, and these 4 strains were also resistant to imipenem. Our data indicated that drug-resistant Klebsiella pneumoniae were highly prevalent in the hospital. Thus it is warranted that surveillance of epidemiology of those resistant isolates should be a cause for concern, and appropriate drugs should be chosen.     

PCR-Based Detection of Extended-Spectrum β-Lactamases (bla CTX-M-1 and bla TEM ) in Escherichia coli, Salmonella spp. and Klebsiella pneumoniae Isolated from Pigs in North Eastern India (Mizoram)

Cephalosporins are major antimicrobials used to treat serious infections. However, their effectiveness is being compromised by the emergence of extended-spectrum β-lactamases (ESBLs). A total of 138 enteric bacteria were isolated from 53 faecal samples of pigs collected from different districts of Mizoram, of which 102 (73.91 %) were Escherichia coli, 26 (18.84 %) were Salmonella spp. and 10 (7.25 %) were Klebsiella pneumoniae. Phenotypic confirmatory test (Double Discs Synergy Test) showed that 8 (5.80 %) E. coli isolates were ESBLs producer. PCR analysis confirmed that out of the eight isolate, 7 (5.07 %) harboured bla _CTX-M-1 gene and/or bla _TEM gene. Of the eight positive isolates, 7 (5.07 %) and 3 (2.17 %) were found to be positive for bla _CTX-M-1 gene and bla _TEM gene, respectively, of which 3 (2.17 %) isolates were positive for both the genes. Only 4 (2.90 %) E. coli isolates carried bla _CTX-M-1 gene alone. Agarose gel electrophoresis showed that all the isolates were carrying plasmids ranging between 0.9 and ~30 kb. Out of the seven isolates positive for bla _CTX-M-1 and/or bla _TEM , 2 (1.84 %) isolates were confirmed for bla _CTX-M-1 gene in their plasmid. Only one E. coli isolate was found to be positive for both the genes in its plasmid. The resistance plasmid could not be transferred to a recipient by in vitro horizontal gene transfer method.     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.     

Emergence of OXA-48-Producing Klebsiella pneumoniae in Taiwan

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla _OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla _CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla _OXA-48 gene in all four isolates was identical to pKP_OXA-48N1, a bla _OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla _OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla _OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern.     

Multidrug resistance and high virulence genotype in uropathogenic Escherichia coli due to diffusion of ST131 clonal group producing CTX-M-15: an emerging problem in a Tunisian hospital

A collection of 201 Escherichia coli strains isolated from urine of patients in a Tunisian hospital between January 2006 and July 2008 was studied. Microbial identification was done by conventional methods, and antibiotic susceptibility with disk diffusion method was performed according to the Clinical Laboratory and Standards Institute guidelines. Detection of extended-spectrum beta-lactamase (ESBL) was performed by double-disk synergy test (DDST) and identification was done by PCR and sequencing. ESBL-producing isolates were subjected to molecular typing by random amplified polymorphic DNA (RAPD) and ST131 detection by PCR. Four phylogenetic groups (A, B1, B2 and D), 18 virulence genes and CTX-M group were individualized using PCR. Statistical analysis was done by Pearson χ2 test and Mann–Whitney U test. The strains were recovered primarily from urology (28 %), maternity (19 %) and medicine (16 %) wards. Antibiotic resistance rates were ampicilin (72.1 %), nalidixic acid (41.8 %), ciprofloxacin (38.8 %), gentamicin (23.9 %) and cefotaxime (17.4 %). Thirty-one of cefotaxime-resistant isolates (n?=?35) had a positive DDST and harboured bla _CTX-M-15 gene. Twenty of them (64.5 %) belonged to the ST131 clone and showed the same RAPD DNA profile. Ciprofloxacin- and cotrimoxazole-susceptible isolates were significantly associated with phylogenetic group B2, whereas isolates that were resistant to these molecules were associated with B1 and D phylogenetic groups, respectively. Virulence genes were significantly more frequent among ciprofloxacin- and cotrimoxazole-susceptible strains than those resistant to these antibiotics. However, CXT-M-15-producing isolates were associated with many virulence genes. Isolates concomitantly susceptible to the three antimicrobials agents (ciprofloxacin, cefotaxime and cotrimoxazole) were significantly associated with group B2 and high virulence score, whereas isolates with resistance patterns especially those including resistance to ciprofloxacin belonged predominantly to B1 phylogroup and haboured few virulence genes. The emergence of virulent and multidrug-resistant E. coli is a concerning development that deserves close attention in our institution.     

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.     

Characterization of a Novel Plasmid Type and Various Genetic Contexts of bla OXA-58 in Acinetobacter spp. from Multiple Cities in China

Background/Objective

Several studies have described the epidemiological distribution of bla _OXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of bla _OXA-58-carrying plasmids and the genetic context surrounding bla _OXA-58 in Acinetobacter spp. in China.

Methodology/Principal Findings

Twelve non-duplicated bla _OXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of bla _OXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of bla _OXA-58-harboring plasmids. The genetic structure surrounding bla _OXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both bla _OXA-58 and bla _OXA-23. bla _OXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in bla _OXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of bla _OXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-bla _OXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-bla _OXA-58 were carbapenem susceptible.

Conclusion

The study revealed the unique features of bla _OXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of bla _OXA-58 contributed to various antibiotics resistance profiles.     

Achromobacter xylosoxidans: An Emerging Pathogen Carrying Different Elements Involved in Horizontal Genetic Transfer

In the last few years, numerous cases of multidrug-resistant Achromobacter xylosoxidans infections have been documented in immunocompromised and cystic fibrosis patients. To gain insights into the molecular mechanisms and mobile elements related to multidrug resistance in this bacterium, we studied 24 non-epidemiological A. xylosoxidans clinical isolates from Argentina. Specific primers for plasmids, transposons, insertion sequences, bla _ampC, intI1, and intI2 genes were used in PCR reactions. The obtained results showed the presence of wide host range IncP plasmids in ten isolates and a high dispersion of class 1 integrons (n?=?10) and class 2 integrons (n?=?3). Four arrays in the variable region (vr) of class 1 integrons were identified carrying different gene cassettes as the aminoglycoside resistance aac(6′)-Ib and aadA1, the trimethoprim resistance dfrA1 and dfrA16, and the β-lactamase bla _OXA-2. In only one of the class 2 integrons, a vr was amplified that includes sat2-aadA1. The bla _ampC gene was found in all isolates, confirming its ubiquitous nature. Our results show that A. xylosoxidans clinical isolates contain a rich variety of genetic elements commonly associated with resistance genes and their dissemination. This supports the hypothesis that A. xylosoxidans is becoming a reservoir of horizontal genetic transfer elements commonly involved in spreading antibiotic resistance.     

Prevalence and Characteristics of Extended-Spectrum β-Lactamase and Plasmid-Mediated Fluoroquinolone Resistance Genes in Escherichia coli Isolated from Chickens in Anhui Province,China

The aim of this study was to characterize the prevalence of extended-spectrum β-lactamase (ESBL) genes and plasmid-mediated fluoroquinolone resistance (PMQR) determinants in 202 Escherichia coli isolates from chickens in Anhui Province, China, and to determine whether ESBL and PMQR genes co-localized in the isolates. Antimicrobial susceptibility for 12 antimicrobials was determined by broth microdilution. Polymerase chain reactions (PCRs), DNA sequencing, and pulsed field gel electrophoresis (PFGE) were employed to characterize the molecular basis for β-lactam and fluoroquinolone resistance. High rates of antimicrobial resistance were observed, 147 out of the 202 (72.8%) isolates were resistant to at least 6 antimicrobial agents and 28 (13.9%) of the isolates were resistant to at least 10 antimicrobials. The prevalence of bla _CTX-M, bla _TEM-1 and bla _TEM-206 genes was 19.8%, 24.3% and 11.9%, respectively. Seventy-five out of the 202 (37.1%) isolates possessed a plasmid-mediated quinolone resistance determinant in the form of qnrS (n = 21); this determinant occurred occasionally in combination with aac(6′)-1b-cr (n = 65). Coexistence of ESBL and/or PMQR genes was identified in 31 of the isolates. Two E. coli isolates carried bla _TEM-1, bla _CTX-M and qnrS, while two others carried bla _CTX-M, qnrS and aac(6′)-1b-cr. In addition, bla _TEM-1, qnrS and aac(6′)-1b-cr were co-located in two other E. coli isolates. PFGE analysis showed that these isolates were not clonally related and were genetically diverse. To the best of our knowledge, this study is the first to describe detection of TEM-206-producing E. coli in farmed chickens, and the presence of bla _TEM-206, qnrS and aac(6′)-1b-cr in one of the isolates.     

Prevalence of Plasmid-Mediated Quinolone Resistance and Aminoglycoside Resistance Determinants among Carbapeneme Non-Susceptible Enterobacter cloacae

Background

Simultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS) isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs) and aminoglycoside resistance determinants (ARDs) among the CNS Enterobacter cloacae (E. cloacae) isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics.

Methods

The β-lactamases genes (including class A carbapenemase genes bla_KPC and bla_SME, metallo-β-lactamase genes (MBLs) bla_IMP, bla_VIM and bla_NDM, and extended spectrum β-lactamases (ESBLs),bla_CTX-M, bla_TEM and bla_SHV), QRDs (including qnrA, qnrB, qnrS and aac(6′)-Ib-cr) and ARDs (including aac(6′)-Ib, armA and rmtB) of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE).

Results

Of the 35 isolates, 9 (25.7%) harbored a carbapenemase gene; 23 (65.7%) carried ESBLs; 24 (68.6%) were QRD positive; and 27 (77.1%) were ARD positive. Among the 5 bla_IMP-8 positive strains, 4 (80%) contained both ESBL and QRD genes, and all the 5 (100%) harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1%) were carbapenemase positive, 14 (60.9%) were QRD positive, and 18 (78.3%) were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination.

Conclusion

QRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing bla_NDM-1, bla_IMP-26, qnrA1 and qnrS1 was first reported.     

Antimicrobial Resistance of Shigella flexneri Serotype 1b Isolates in China

Shigella flexneri serotype 1b is among the most prominent serotypes in developing countries, followed by serotype 2a. However, only limited data is available on the global phenotypic and genotypic characteristics of S. flexneri 1b. In the present study, 40 S. flexneri 1b isolates from different regions of China were confirmed by serotyping and biochemical characterization. Antimicrobial susceptibility testing showed that 85% of these isolates were multidrug-resistant strains and antibiotic susceptibility profiles varied between geographical locations. Strains from Yunnan were far more resistant than those from Xinjiang, while only one strain from Shanghai was resistant to ceftazidime and aztreonam. Fifteen cephalosporin resistant isolates were identified in this study. ESBL genes (bla _SHV, bla _TEM, bla _OXA, and bla _CTX-M) and ampC genes (bla _MOX, bla _FOX, bla _MIR(ACT-1), bla _DHA, bla _CIT and bla _ACC) were subsequently detected among the 15 isolates. The results showed that these strains were positive only for bla _TEM, bla _OXA, bla _CTX-M, intI1, and intI2. Furthermore, pulsed-field gel electrophoresis (PFGE) analysis showed that the 40 isolates formed different profiles, and the PFGE patterns of Xinjiang isolates were distinct from Yunnan and Shanghai isolates by one obvious, large, missing band. In summary, similarities in resistance patterns were observed in strains with the same PFGE pattern. Overall, the results supported the need for more prudent selection and use of antibiotics in China. We suggest that antibiotic susceptibility testing should be performed at the start of an outbreak, and antibiotic use should be restricted to severe Shigella cases, based on resistance pattern variations observed in different regions. The data obtained in the current study might help to develop a strategy for the treatment of infections caused by S. flexneri 1b in China.     

Increase in Resistance to Extended-Spectrum Cephalosporins in Salmonella Isolated from Retail Chicken Products in Japan

Extended-spectrum β-lactamase (ESBL)-producing Salmonella are one of the most important public health problems in developed countries. ESBL-producing Salmonella strains have been isolated from humans in Asian countries neighboring Japan, along with strains harboring the plasmid-mediated extended-spectrum cephalosporin (ESC)-resistance gene, ampC (pAmpC). However, only a few studies have investigated the prevalence of ESC-resistant Salmonella in chicken products in Japan, which are the main vehicle of Salmonella transmission. The aim of this study was to investigate the prevalence of ESBL-producing, pAmpC-harboring, or carbapenem-resistant Salmonella in chicken products in Japan. In total, 355 out of 779 (45.6%) chicken product samples collected from 1996–2010 contained Salmonella, resulting in 378 distinct isolates. Of these isolates, 373 were tested for resistance to ESCs, cephamycins, or carbapenems. Isolates that showed resistance to one or more of these antimicrobials were then examined by PCR and DNA sequence analysis for the presence of the bla_CMY, bla_CTX-M, bla_TEM, and bla_SHV resistance genes. Thirty-five resistant isolates were detected, including 26 isolates that contained pAmpC (bla_CMY-2), and nine ESBL-producing isolates harboring bla_CTX-M (n = 4, consisting of two bla_CTX-M-2 and two bla_CTX-M-15 genes), bla_TEM (n = 4, consisting of one bla_TEM-20 and three bla_TEM-52 genes), and bla_SHV (n = 1, bla_SHV-12). All pAmpC-harboring and ESBL-producing Salmonella isolates were obtained from samples collected after 2005, and the percentage of resistant isolates increased significantly from 0% in 2004 to 27.9% in 2010 (P for trend = 0.006). This increase was caused in part by an increase in the number of Salmonella enterica subsp. enterica serovar Infantis strains harboring an approximately 280-kb plasmid containing bla_CMY-2 in proximity to ISEcp1. The dissemination of ESC-resistant Salmonella containing plasmid-mediated bla_CMY-2 in chicken products indicates the need for the development of continuous monitoring strategies in the interests of public health.     

In vitro transference and molecular characterization of bla TEM genes in bacteria isolated from Portuguese ready-to-eat foods

The principal aim of this study was to investigate the possibility of transference to Escherichia coli of ??-lactam resistance genes found in bacteria isolated from ready-to-eat (RTE) Portuguese traditional food. From previous screenings, 128 ??-lactam resistant isolates (from different types of cheese and of delicatessen meats), largely from the Enterobacteriaceae family were selected and 31.3% of them proved to transfer resistance determinants in transconjugation assays. Multiplex PCR in donor and transconjugant isolates did not detect bla _CTX, bla _SHV and bla _OXY, but bla _TEM was present in 85% of them, while two new TEMs (TEM-179 and TEM-180) were identified in two isolates. The sequencing of these amplicons showed identity between donor and transconjugant genes indicating in vitro plasmid DNA transfer. These results suggest that if there is an exchange of genes in natural conditions, the consumption of RTE foods, particularly with high levels of Enterobacteriaceae, can contribute to the spread of antibiotic resistance.     

Co-Carriage of bla KPC-2 and bla NDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India

Global spread of KPC poses to be a serious threat complicating treatment options in hospital settings. The present study investigates the genetic environment of bla _KPC-2 among clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of India. The study isolates were collected from different wards and clinics of Silchar Medical College and Hospital, India, from 2012–2013. The presence of bla _KPC was confirmed by genotypic characterization followed by sequencing. Cloning of the bla _KPC-2 gene was performed and the genetic environment of this gene was characterized as well. Transferability of the resistance gene was determined by transformation assay and Southern hybridization. Additionally, restriction mapping was also carried out. Two isolates of P. aeruginosa were found to harbor bla _KPC-2, were resistant towards aminoglycosides, quinolone and β-lactam-β-lactamase inhibitor combination. In both the isolates, the resistance determinant was associated with class 1 integron and horizontally transferable. Both the isolates were co-harboring bla _NDM-1. The first detection of this integron mediated bla _KPC-2 coexisting with bla _NDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated bla _KPC-2 in terms of infection control and to prevent the spread of this gene in hospitals as well as in the community.     

Characterization of Piperacillin/Tazobactam-Resistant Klebsiella oxytoca Recovered from a Nosocomial Outbreak

We characterized 12 clinical isolates of Klebsiella oxytoca with the extended-spectrum β-lactamase (ESBL) phenotype (high minimum inhibitory concentration [MIC] values of ceftriaxone) recovered over 9 months at a university hospital in Japan. To determine the clonality of the isolates, we used pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and PCR analyses to detect bla _RBI, which encodes the β-lactamase RbiA, OXY-2-4 with overproduce-type promoter. Moreover, we performed the isoelectric focusing (IEF) of β-lactamases, and the determination of the MICs of β-lactams including piperacillin/tazobactam for 12 clinical isolates and E. coli HB101 with pKOB23, which contains bla _RBI, by the agar dilution method. Finally, we performed the initial screening and phenotypic confirmatory tests for ESBLs. Each of the 12 clinical isolates had an identical PFGE pulsotype and MLST sequence type (ST9). All 12 clinical isolates harbored identical bla _RBI. The IEF revealed that the clinical isolate produced only one β-lactamase. E. coli HB101 (pKOB23) and all 12 isolates demonstrated equally resistance to piperacillin/tazobactam (MICs, >128 μg/ml). The phenotypic confirmatory test after the initial screening test for ESBLs can discriminate β-lactamase RbiA-producing K. oxytoca from β-lactamase CTX-M-producing K. oxytoca. Twelve clinical isolates of K. oxytoca, which were recovered from an outbreak at one university hospital, had identical genotypes and produced β-lactamase RbiA that conferred resistance to piperacillin/tazobactam. In order to detect K. oxytoca isolates that produce RbiA to promote research concerning β-lactamase RbiA-producing K. oxytoca, the phenotypic confirmatory test after the initial screening test for ESBLs would be useful.     

Characterization of IS26‐composite transposons and multidrug resistance in conjugative plasmids from Enterobacter cloacae

SHV‐12 is the most widespread resistance determinant of Enterobacter cloacae in Taiwan; however, bla_SHV‐12 has rarely been mobilized. Six multidrug‐resistant E. cloacae isolates were collected. After conjugal transfer, plasmid profiling and analysis of incompatibility groups was performed to characterize the genetic context of bla_SHV‐12‐containing fragments. The presence of mobile genetic elements was demonstrated by PCR, cloning, sequencing and bioinformatics analyses. Four different β‐lactamase genes (bla_TEM‐1, bla_SHV‐12, bla_CTX‐M‐3 and/or bla_CTX‐M‐14) were observed in the conjugative plasmids belonging to the IncHI2 (n = 4), IncI1 or IncP incompatibility groups. The IS26‐bla_SHV‐12‐IS26 locus was located in five different genetic environments. A novel structural organization of a class 1 integron with the aac(6')‐IIc cassette truncated by IS26 was identified in one isolate. Thus, bla_SHV‐12 was obtained from different plasmids through IS26‐mediated homologous recombination. IS26 plays a vital role in the distribution of mobile resistance elements between different plasmids found in multidrug‐resistant E. cloacae isolates.     

Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq

Background

Gram-negative multidrug-resistant (MDR) bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq.

Methodology/Principal Findings

In this study, all MDR Enterobacteriaceae (n = 38) and randomly selected non-MDR counterparts (n = 41) isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥3) plasmids compared to their non-MDR counterparts, which carried ≤2 plasmids (p<0.01). Various large plasmids (∼52 to 100 kb) from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), β-lactam (bla _TEM1, bla _AMPC, bla _CTX-M-15, bla _OXA-1, bla _VIM-2 and bla _SHV), sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol (cat) resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N) were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates.

Conclusions/Significance

This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary relationship with other parts of world. The large plasmids, carrying resistance genes and transfer-associated genes, may be potential factors for regional dissemination of antibiotic resistance.     

IncA/C Plasmid-Mediated Spread of CMY-2 in Multidrug-Resistant Escherichia coli from Food Animals in China

Objectives

To obtain a broad molecular epidemiological characterization of plasmid-mediated AmpC β-lactamase CMY-2 in Escherichia coli isolates from food animals in China.

Methods

A total of 1083 E. coli isolates from feces, viscera, blood, drinking water, and sub-surface soil were examined for the presence of CMY-2 β-lactamases. CMY-2-producing isolates were characterized as follows: the bla _CMY-2 genotype was determined using PCR and sequencing, characterization of the bla _CMY-2 genetic environment, plasmid sizing using S1 nuclease pulsed-field gel electrophoresis (PFGE), PCR-based replicon typing, phylogenetic grouping, XbaI-PFGE, and multi-locus sequence typing (MLST).

Results

All 31 CMY-2 producers were only detected in feces, and presented with multidrug resistant phenotypes. All CMY-2 strains also co-harbored genes conferring resistance to other antimicrobials, including extended spectrum β-lactamases genes (bla _CTX-M-14 or bla _CTX-M-55), plasmid-mediated quinolone resistance determinants (qnr, oqxA, and aac-(6′)-Ib-cr), floR and rmtB. The co-transferring of bla _CMY-2 with qnrS1 and floR (alone and together) was mainly driven by the Inc A/C type plasmid, with sizes of 160 or 200 kb. Gene cassette arrays inserted in the class 1 or class 2 integron were amplified among 12 CMY-2 producers. CMY-2 producers belonged to avirulent groups B1 (n = 12) and A (n = 11), and virulent group D (n = 8). There was a good correlation between phylogenetic groups and sequence types (ST). Twenty-four STs were identified, of which the ST complexes (STC) 101/B1 (n = 6), STC10/A (n = 5), and STC155/B1 (n = 3) were dominant.

Conclusions

CMY-2 is the dominant AmpC β-lactamase in food animals and is associated with a transferable replicon IncA/C plasmid in the STC101, STC10, and STC155 strains.     

Molecular characterization of extended-spectrum-beta-lactamase-producing Escherichia coli isolates from red foxes in Portugal

The presence of broad-spectrum-cephalosporin-resistant Escherichia coli isolates and the implicated mechanisms of resistance and virulence factor genes were investigated in red fox (Vulpes vulpes) in Portugal. Cefotaxime-resistant E. coli isolates were isolated from two of 52 fecal samples (4 %), being both ESBL producers. The β-lactamase genes found in the two isolates were bla _SHV-12 + bla _TEM-1b. The tet(A) and sul2 genes were also detected in these isolates, together with the non-classical class 1 integron (intI1-dfrA12-orfF-aadA2-cmlA1-aadA1-qacH-IS440-sul3) with the PcH1 promoter. The two isolates belonged to the phylogroup A. Amino acid changes in GyrA (S83L + D87G) and ParC (S80I) proteins were identified in our study. Concerning MLST typing, both isolates were assigned to ST1086, never found before in wild animals, and they presented closely related PFGE patterns. This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment to other niches.