Transconjugation Experiments Research Articles

Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence.

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

Genetic characterization of coliform bacterial isolates from environmental water in Thailand

IntroductionIn contrast to the study in other part of the world, information about characteristics of plasmids carrying antimicrobial resistance genes (ARGs) in Enterobacteriaceae derived from environmental water in tropical Asian countries including Thailand is limited. This study, therefore, aimed to gain insight into genetic information of antimicrobial resistance in environmental water in Thailand. MethodsColiform bacteria were isolated from environmental water collected at 20 locations in Thailand and identified. Then, susceptibility profiles to ampicillin, cefazoline, cefotaxime, kanamycin, ciprofloxacin, sulfamethoxazole, tetracycline, and nalidixic acid were assessed. In addition, antimicrobial resistant genes integrons, and replicon types were analyzed. And furthermore, plasmids carrying blaTEM and tetM were identified by S1-PFGE analysis and confirmed transmissibility by transconjugation experiments. ResultsIn 130 coliform bacteria isolated, 89 were resistant to cefazoline while 41 isolates were susceptible. Cefazoline-resistant coliform bacteria were found to be significantly resistant to cefotaxime and tetracycline as compared to susceptible isolates. Hence, blaTEM and tetM correlating with β-lactam antibiotics and tetracycline, respectively, were analyzed found to co-localize on the IncFrepB plasmids in isolates from pig farms’ wastewater by S1-PFGE analysis. And furthermore, transmissibility of the plasmids was confirmed. ConclusionsResults obtained in this study suggested that ARGs in coliform bacteria may have been spreading on the farm via IncFrepB plasmids. Hence, appropriate use of antimicrobials and good hygiene management on the farm are required to prevent the emergence and spread of resistant bacteria.

Plasmid-Mediated Ampicillin, Quinolone, and Heavy Metal Co-Resistance among ESBL-Producing Isolates from the Yamuna River, New Delhi, India

Antibiotic resistance is one of the major current global health crises. Because of increasing contamination with antimicrobials, pesticides, and heavy metals, the aquatic environment has become a hotspot for emergence, maintenance, and dissemination of antibiotic and heavy metal resistance genes among bacteria. The aim of the present study was to determine the co-resistance to quinolones, ampicillin, and heavy metals among the bacterial isolates harboring extended-spectrum β-lactamases (ESBLs) genes. Among 73 bacterial strains isolated from a highly polluted stretch of the Yamuna River in Delhi, those carrying blaCTX-M, blaTEM, or blaSHV genes were analyzed to detect the genetic determinants of resistance to quinolones, ampicillin, mercury, and arsenic. The plasmid-mediated quinolone resistance (PMQR) gene qnrS was found in 22 isolates; however, the qnrA, B, C, and qnrD genes could not be detected in any of the bacteria. Two variants of CMY, blaCMY-2 and blaCMY-42, were identified among eight and seven strains, respectively. Furthermore, merB, merP, merT, and arsC genes were detected in 40, 40, 44, and 24 bacterial strains, respectively. Co-transfer of different resistance genes was also investigated in a transconjugation experiment. Successful transconjugants had antibiotic and heavy metal resistance genes with similar tolerance toward antibiotics and heavy metals as did their donors. This study indicates that the aquatic environment is a major reservoir of bacteria harboring resistance genes to antibiotics and heavy metals and emphasizes the need to study the genetic basis of resistant microorganisms and their public health implications.

Detection and Genomic Characterization of a Morganella morganii Isolate From China That Produces NDM-5.

The increasing prevalence and transmission of the carbapenem resistance gene blaNDM–5 has led to a severe threat to public health. So far, blaNDM–5 has been widely detected in various species of Enterobacterales and different hosts across various cities. However, there is no report on the blaNDM–5– harboring Morganella morganii. In January 2016, the first NDM-5-producing Morganella morganii L241 was found in a stool sample of a patient diagnosed as recurrence of liver cancer in China. Identification of the species was performed using 16S rRNA gene sequencing. Carbapenemase genes were identified through both PCR and sequencing. To investigate the characteristics and complete genome sequence of the blaNDM–5-harboring clinical isolate, antimicrobial susceptibility testing, S1 nuclease pulsed field gel electrophoresis, Southern blotting, transconjugation experiment, complete genome sequencing, and comparative genomic analysis were performed. M. morganii L241 was found to be resistant to broad-spectrum cephalosporins and carbapenems. The complete genome of L241 is made up from both a 3,850,444 bp circular chromosome and a 46,161 bp self-transmissible IncX3 plasmid encoding blaNDM–5, which shared a conserved genetic context of blaNDM–5 (ΔIS3000-ΔISAba125-IS5-blaNDM–5-ble-trpF-dsbC-IS26). BLASTn analysis showed that IncX3 plasmids harboring blaNDM genes have been found in 15 species among Enterobacterales from 13 different countries around the world thus far. In addition, comparative genomic analysis showed that M. morganii L241 exhibits a close relationship to M. morganii subsp. morganii KT with 107 SNPs. Our research demonstrated that IncX3 is a key element in the worldwide dissemination of blaNDM-5 among various species. Further research will be necessary to control and prevent the spread of such plasmids.

Transconjugation of erm(X) conferring high-level resistance of clindamycin for Cutibacterium acnes.

Cutibacterium acnes (C. acnes) can become an exacerbating factor in acne vulgaris. Clindamycin has been most frequently used for the treatment of inflammatory acne vulgaris. We studied clindamycin susceptibility and resistance determinants of C. acnes isolated from acne patients in Japan. The isolation rate of clindamycin-resistant C. acnes had significantly increased from 20.3 % in 2009-2010 to 44.1 % in 2016-2017. Strains carrying erm(X), which confers high-level resistance to clindamycin, had significantly increased from 1.4 to 11.8 %. Sequence analysis of the resistance determinant showed that erm(X) was coded on transposon Tn5432. A transconjugation experiment showed that erm(X) can be transferred between C. acnes strains with high frequency and the transconjugants harboured transposon Tn5432 encoding erm(X). Our data show the transconjugation of erm(X) in C. acnes and strongly suggest that the transmission of erm(X) between C. acnes contributes to the increase and spread of clindamycin-resistant C. acnes strains in acne patients.

Characteristics and genetic diversity of multi-drug resistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from bovine mastitis.

A characterization of the drug resistance profiles, identification of PCR-based replicon typing, and multilocus sequence typing (MLST) and analysis of 46 ESBL-producing Escherichia coli from cows with mastitis are described. All multidrug-resistant isolates of various phylogenetic groups (A = 31, B1= 3, B2 = 2, D = 10) were ESBL-producers of genotypes CTX-M-15 (29), CTX-M-55 (4), CTX-M-14 (4), CTX-M-3 (1), CTX-M-1 (1), TEM (22) and SHV (8) that were found on conjugative plasmids of diverse incompatibility groups (primarily IncF). Transconjugation experiments indicated successful (100%) trans-conjugation, which was verified phenotypically and genotypically. A total of 28 sequence types (ST) were identified, with 10% of isolates being ST410, and 9 other ST that were assigned arbitrary numbers, reflecting the degree of diversity. Multilocus sequence analysis revealed two lineages, a dominant and a small lineage. Split-decomposition showed intraspecies recombination clearly contributed in genetic recombination generating genotypic diversity among the isolates, and a lack of interspecies recombination. This coherent analysis on genetic structure of multidrug-resistant pathogenic E. coli population isolated from mastitic-milk weaponized with resistance elements from a large, rapidly developing country will be a helpful contribution for epidemiology and surveillance of drug resistance patterns, and understanding their global diversity.

Emergence of co-production of plasmid-mediated AmpC beta-lactamase and ESBL in cefoxitin-resistant uropathogenic Escherichia coli.

Plasmid-mediated AmpC (pAmpC) and ESBL co-production was detected in Escherichia coli a major etiologic agent of urinary tract infection. Isolates resistant to cefoxitin by CLSI methodology were tested for pAmpC beta-lactamase using phenylboronic acid and ESBLs by combined disk diffusion method. pAmpC/ESBL genes were characterized by PCR and sequencing. Transconjugation experiments were done to study the transfer of pAmpC and ESBL production from clinical isolates as donor to E. coli J53 AziR as recipient. Incompatibility groups of transmissible plasmids were classified by PCR-based replicon typing (PBRT). Among 148 urine culture positive isolates, E. coli was reported in 39.86% (59/148), with 93.22% (55/59) of cefoxitin resistance. pAmpC production was detected in 25, with varied distribution of blaCMY-2 and blaDHA-1type genes alone (n = 13 and 7 respectively) or in combination (n = 5). ESBL co-production was observed in 88% (22/25) of pAmpC producing isolates with predominance of blaTEM (n = 20). Twenty-three transconjugants showed transmission of pAmpC-and ESBL-resistant genes with co-carriage of blaCMY-2 and blaTEM (n = 15) in plasmids of IncF type (n = 9) being predominant, followed by IncI1 (n = 4) and IncH1 (n = 2) in combination. All clinical isolates were clonally diverse. Resistance against different beta-lactams in uropathogenic E. coli has been an emerging concern in resource- poor countries such as India. Knowledge on the occurrence of AmpC beta-lactamases and ESBL amongst this pathogen and its transmission dynamics may aid in hospital infection control.

Quinolone Resistance Mechanisms Among Salmonella enterica in Malaysia.

The prevalence of quinolone-resistant Salmonella enterica is on the rise worldwide. Salmonella enterica is one of the major foodborne pathogens in Malaysia. Therefore, we aim to investigate the occurrence and mechanisms of quinolone resistance among Salmonella strains isolated in Malaysia. A total of 283 Salmonella strains isolated from food, humans, and animals were studied. The disk diffusion method was used to examine the quinolone susceptibility of the strains, and the minimum inhibitory concentration (MIC) values of nalidixic acid and ciprofloxacin were also determined. DNA sequencing of the quinolone resistance-determining regions (QRDRs) of gyrase and topoisomerase IV genes and the plasmid-borne qnr genes was performed. The transfer of the qnr gene was examined through transconjugation experiment. A total of 101 nalidixic acid-resistant Salmonella strains were identified. In general, all strains were highly resistant to nalidixic acid (average MICNAL, 170 μg/ml). Resistance to ciprofloxacin was observed in 30.7% of the strains (1 ≤ MICCIP ≤ 2 μg/ml). Majority of the strains contained missense mutations in the QRDR of gyrA (69.3%). Silent mutations were frequently detected in gyrB (75.2%), parC (27.7%), and parE (51.5%) within and beyond the QRDRs. Novel mutations were detected in parC and parE. The plasmid-borne qnrS1 variant was found in 36.6% of the strains, and two strains were found to be able to transfer the qnrS1 gene. Overall, mutations in gyrA and the presence of qnrS1 genes might have contributed to the high level of quinolone resistance among the strains. Our study provided a better understanding on the status of quinolone resistance among Salmonella strains circulating in Malaysia.

Identification and Characterization of KPC-Producing Klebsiella Pneumoniae in Clinical Isolates

The dissemination of carbapenemase-producing Klebsiella pneumoniae (KPC) strains is an increasing problem worldwide. KPC ß-lactamases are Ambler class A enzymes mostly plasmid-encoded; their global spread represents a threat to clinical patients care and public health. Between September-December 2012, have been isolated 60 Klebsiella pneumoniae strains that showed a decrease of susceptibility to carbapenems, collected from patients at the Provincial Laboratory of Clinical Microbiology ‘S. Agostino-Estense' Hospital (Modena, Italy). Species identification and antimicrobial susceptibility testing were carried out using the Vitek 2 and studied for carbapenemases production using phenotypic confirmatory tests. The presence of carbapenemase genes were confirmed by PCR and sequencing. The localization of beta-lactamase genes was established by conjugation experiments. We reported the emergence of KPC-2-producing Klebsiella pneumoniae isolates PCR and sequencing experiments revealed the presence of blaKPC-2 gene in 56/60 isolates. The KPC-encoding plasmid was analysed by transconjugation experiments and DNA sequencing. The findings indicate the need to supplement surveillance systems based on susceptibility data with the surveillance of resistance mechanisms.

Characterization ofAcinetobacter baumanniiCo-producing Carbapenemases OXA-23 and OXA-66, andarmA16S Ribosomal RNA Methylase at a University Hospital in South Korea

Background: In the present study, the resistance mechanisms against carbapenems and aminoglyco- sides for 23 strains of multi-drug-resistant Acineto- bacter baumannii isolated at a university hospital were investigated. Methods: The minimal inhibitory concentrations (MICs) were determined via broth microdilution or Etest. The genes encoding OXA-type carbapenemases and 16S rRNA methylase were identified using multiplex PCR, and the amplified products were sequenced. Conju- gation experiments were conducted, and an epi- demiologic study was performed using enterobac- terial repetitive intergenic consensus (ERIC)-PCR. Results: In the isolates, the MICs of the tested ami- noglycosides, including arbekacin, were >1024 μg/ mL; the MICs of aztreonam, cefepime, ceftazidime, and ciprofloxacin ranged from 64 to 128 μg/mL; and the MICs of carbapenem ranged from 32 to 64 μg/ mL, as determined through the broth microdilution test. According to the E-test, the MICs of ampicillin/ sulbactam and colistin were 8 and 0.25 to 0.38 μg/ mL, respectively. Sequence analysis confirmed that all of the isolates expressed carbapenemases OXA- 23 and OXA-66, as well as armA 16S rRNA methy- lase. In addition, ISAba1 was identified upstream of the gene encoding OXA-23. OXA-23 and armA were not transferred to Escherichia coli J53 cells in the transconjugation experiments. ERIC-PCR molecular fingerprinting produced a single pattern in all cases. Conclusion: The co-production of OXA-23 and armA 16S rRNA methylase may be attributed to the multi- drug resistance of the A. baumannii isolates in the present study. Stricter surveillance and more rapid detection are necessary to prevent the spread of this type of resistance in the future. (Korean J Clin Microbiol 2011;14:67-73)

First outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in Germany

We report the first outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in Germany. The presence of KPC was confirmed by polymerase chain reaction (PCR). The KPC-encoding plasmid was analysed by transconjugation experiments, DNA sequencing, Southern blotting and isoelectric focussing. Typing was performed by pulsed-field gel electrophoresis (PFGE). An ertapenem-resistant K. pneumoniae with low minimum inhibitory concentrations (MIC) to other cabapenems (tested by the Vitek system) was isolated from the index patient in January 2008. A KPC-2 was identified after K. pneumoniae with identical susceptibility patterns had been isolated from two more patients. Despite the introduction of infection control measures, transmission occurred in five additional patients and three of the patients died from infections. The source of the outbreak strain remained unclear; however, the Tn4401-containing bla (KPC-2) gene was similar to previously described isolates from Greece. Five months after the end of the outbreak, a KPC-K. pneumoniae was isolated from a patient who had been treated in Greece previously. Retrospectively, this patient was treated in November 2007 on the same unit as the index case. Typing revealed that all patients were colonised by the same strain. KPC-K. pneumoniae has been introduced to Germany possibly from Greece and transmission to other institutions is likely.

Emergence and dissemination of Enterobacteriaceae with plasmid-mediated CMY-6 and CTX-M-15 beta-lactamases in community in North-India

A collection of 88 Escherichia coli and 22 Klebsiella pneumoniae strains previously studied for the carriage of bla CTX-M-15 were screened for the presence of bla ampC using multiplex PCR, and analysed for antibiotic resistance and co-carriage of bla ampC and bla CTX-M-15. Cefoxitin resistance was noticed in total of 41.8% isolates. Concomitant resistance to third and fourth generation cephalosporins, aztreonam, gentamicin, trimethoprim and ciprofloxacin was noted. A total of 20% (22/110) isolates (14.5% E. coli and 5.5% K. pneumoniae) carried plasmidic bla ampC of the CIT-family. Sequencing of the representative isolates revealed CMY-6 AmpC-beta-lactamase. Simultaneous occurrence of bla ampC and bla CTX-M was noticed in 77.3% (17/22) isolates. Plasmid analysis of the selected isolates showed the presence of a single plasmid of ca. ~23 kb. Curing and transconjugation experiments provided evidence of carriage of bla CMY and bla CTX-M on same plasmid. RAPD typing revealed the presence of bla CMY and bla CTX-M in a wider range of strains of E. coli and K. pneumoniae.

Prevalent phenotypes and antibiotic resistance in Escherichia coli and Klebsiella pneumoniae at an Indian tertiary care hospital: plasmid-mediated cefoxitin resistance

The beta-lactam antibiotics, in combination with aminoglycosides, are among the most widely prescribed antibiotics. However, because of extensive and unnecessary use, resistance to these drugs continues to increase. In recent years, resistance in the Indian bacterial population has increased markedly, the majority showing complex mechanisms. Due to increased transcontinental movement of the human population, it would be wise to know the prevalence and resistance complexity of these strains, well in advance, in order to formulate a policy for empirical therapy. One hundred and eighty-one isolates of Escherichia coli and 61 isolates of Klebsiella pneumoniae obtained from 2655 non-repeat samples of pus (912) and urine (1743) were studied, and their resistance rates and patterns were noted. The isolates were analyzed for prevalent aminoglycoside and cephalosporin resistance phenotypes and for the presence of extended spectrum beta-lactamase (ESBL) and AmpC enzymes by spot-inoculation and modified three-dimensional tests developed in our laboratory. Fourteen isolates of E. coli and six of K. pneumoniae, resistant to all of the antibiotics tested, were selected for plasmid screening, curing, and transconjugation experiments, and for comparative evaluation of the double disk synergy test (DDST) and modified three-dimensional test (TDT) for detection of beta-lactamases. Urinary E. coli isolates showed maximum susceptibility to amikacin (57.1%), followed by tobramycin (38.5%) and gentamicin (31.9%). Eighteen (19.8%) isolates were susceptible to cefotaxime, whereas 11 (12.1%) were susceptible to ceftriaxone. The K. pneumoniae isolates from urine samples showed maximum susceptibility to tobramycin (63.6%) followed by amikacin (54.5%). Of the K. pneumoniae isolates, 31.8% were susceptible to cefotaxime and 13.6% were susceptible to ceftriaxone. A more or less similar trend of antibiotic susceptibility was noted in E. coli and K. pneumoniae isolates from pus samples. Twenty-six (14.4%) E. coli and 15 (24.6%) K. pneumoniae isolates were found to be ESBL-producers by NCCLS-ESBL phenotypic confirmatory test. Eighteen (9.9%) E. coli and 19 (31.1%) K. pneumoniae isolates were found to be AmpC enzyme-producers by our modified TDT. The simultaneous occurrence of ESBL and AmpC enzymes was noted in 7.7% and 9.8% isolates of E. coli and K. pneumoniae, respectively. The prevalence of multidrug-resistant bacterial isolates is quite high in our bacterial population. On comparative evaluation of DDST and TDT in resistant isolates, TDT was found to be the better method, detecting ESBLs in 80% of isolates compared to 15% with DDST. A 19.9-kb plasmid was consistently present in all the screened isolates of E. coli and K. pneumoniae, and was inferred to encode cefoxitin and tetracycline resistance based on curing and transconjugation experiments.

First Outbreak of Klebsiella pneumoniae Clinical Isolates Producing GES-5 and SHV-12 Extended-Spectrum β-Lactamases in Korea

Klebsiella pneumoniae clinical isolates producing extended-spectrum β-lactamases (ESBLs; TEM and SHV types) are frequently implicated in hospital outbreaks. There are also reports of K. pneumoniae isolates producing various non-TEM, non-SHV ESBLs: CTX-M and GES/IBC types (1). Presently, the different GES-type ESBLs are designated by identical names (8). To clarify the misleading nomenclature of GES-type ESBLs, we propose maintaining the current denomination concerning the fully characterized GES-3 and GES-4 ESBLs reported by Wachino et al. (12, 13) and renaming the variants (GES-3 and GES-4) reported by Vourli et al. (11) as GES-5 and GES-6, respectively. In 2004, six K. pneumoniae clinical isolates producing ESBLs were collected from different patients and distributed among several wards (intensive care unit, neurosurgery, pulmonary internal medicine, and general medicine) at Bundang CHA Medical Center, Republic of Korea. These isolates were identified from sputum samples (for three isolates), urine (for one isolate), bile (for one isolate), and pus (for one isolate). Antibiotic susceptibility testing by disk diffusion tests that were performed according to the recommendations of the Clinical and Laboratory Standards Institute (2) with BBL (Cockeysville, Md.) disks and by the double-disk synergy test of Jarlier et al. (4) suggested the presence of ESBL(s). Double-disk synergies were observed for amoxicillin-clavulanic acid (CLA), ceftazidime, cefotaxime, and aztreonam. All isolates were resistant to penicillins, ceftazidime, cefotaxime, aztreonam, tobramycin, gentamicin, and trimethoprim-sulfamethoxazole. They were susceptible to cefepime and represented reduced susceptibility to imipenem. The analysis of genomic DNA, digested with SpeI and resolved by pulsed-field gel electrophoresis (PFGE) (5), revealed the same macrorestriction pattern among all isolates, which were therefore classified as indistinguishable. Screening for ESBL gene(s) was performed by PCR using the primers for blaTEM, blaSHV, blaGES, blaPER, blaVEB, blaTOHO, blaSFO, blaBES, blaFEC, blaCME, blaTLA, blaCTX-M, and blaOXA (1, 5, 7, 10). PCR amplification experiments using primers designed to amplify GES- and SHV-type ESBL genes gave only positive results for all isolates. The nucleotide sequence of the respective amplicon for the blaSHV gene revealed 100% identity with blaSHV-12 from K. pneumoniae (6). That for the blaGES gene differs by only one silent mutation (G→A) at position 54 from blaGES-5, described elsewhere for Escherichia coli (11). On the isoelectric focusing gel, two β-lactamase activities with pIs of 5.8 and 8.2 were detected in all isolates. On the basis of DNA sequencing and pI values, the β-lactamase activity of pI 8.2 corresponds to that of SHV-12 β-lactamase, and the pI value of 5.8 represents GES-5 β-lactamase. Six isolates were used as donors in transconjugation experiments. Ceftazidime-resistant transconjugants were obtained in all cases at frequencies ranging from 10−4 to 10−5 per donor cell. All transconjugants produced only GES-5. The SHV-12 gene was not cotransferred, indicating that the bla genes for GES-5 and SHV-12 resided on different genetic elements. All GES-5-producing transconjugants were resistant to penicillins and ceftazidime and susceptible to cefoxitin, cefotaxime, and aztreonam. This phenotype is characteristic of GES/IBC-producing Enterobacteriaceae (9-13). All transconjugants were susceptible to cefepime and showed reduced susceptibilities to imipenem. MICs of ticarcillin, ceftazidime, and cefotaxime were reduced by clavulanic acid (Table ​(Table1).1). This resistance was due to the acquisition of large (>70-kb) plasmids that exhibited identical HindIII restriction patterns and harbored the GES-5 ESBL gene. Due to the presence of SHV-12 in all isolates, the MIC patterns of clinical isolates were different from those of their transconjugants. TABLE 1. MICs of β-lactams for ESBL-producing K. pneumoniae clinical isolates, their transconjugants, and the azide-resistant E. coli J53 recipient The carbapenemase bioassay (the modified cloverleaf test) (5) showed that extracts derived from all transconjugants producing GES-5 supported growth of the indicator, E. coli ATCC 25922, in the presence of an imipenem disk (30 μg), resembling the effect of OXA-23, which was used as a positive control. In contrast, cell extracts of a ceftazidime-sensitive K. pneumoniae clinical isolate collected from the Bundang CHA Medical Center did not cause any visible inhibition of carbapenemase activity. These findings indicated that the reduced susceptibilities of all isolates and their transconjugants to imipenem were due to production of GES-5 ESBL with the detectable carbapenemase activity. Finding the same macrorestriction pattern by PFGE indicated that an endemic K. pneumoniae strain producing GES-5 and SHV-12 ESBLs was present in different wards at the Bundang CHA Medical Center in 2004. Six GES-5-producing transconjugants were also resistant to tobramycin, gentamicin, and trimethoprim-sulfamethoxazole. Although the complete nucleotide sequences of integrons from six clinical isolates were not determined, screening for the presence of class 1 through 4 integrase genes from large plasmids by PCR with primer pairs previously described (3) revealed the presence of class 1 integrons. The complete class 1 integron mapping of large plasmids from six clinical isolates and the possible spread of the GES-5 ESBL gene to other Korean hospitals are currently under investigation.

Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel

The extended-spectrum beta-lactamase (ESBL)-producing phenotype is frequent among Enterobacter isolates at the Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. We examined the clonal relatedness and characterized the ESBLs of a collection of these strains. Clonal relatedness was determined by pulsed-field gel electrophoresis. Isoelectric focusing (IEF) and transconjugation experiments were performed. ESBL gene families were screened by colony hybridization and PCR for bla(TEM), bla(SHV), bla(CTX-M), bla(IBC), bla(PER), bla(OXA), bla(VEB), and bla(SFO); and the PCR products were sequenced. The 17 Enterobacter isolates studied comprised 15 distinct genotypes. All isolates showed at least one IEF band (range, one to five bands) whose appearance was suppressed by addition of clavulanate; pIs ranged from 5.4 to > or = 8.2. Colony hybridization identified at least one family of beta-lactamase genes in 11 isolates: 10 harbored bla(TEM) and 9 harbored bla(SHV). PCR screening and sequence analysis of the PCR products for bla(TEM), bla(SHV), and bla(CTX-M) identified TEM-1 in 11 isolates, SHV-12 in 7 isolates, SHV-1 in 1 isolate, a CTX-M-2-like gene in 2 isolates, and CTX-M-26 in 1 isolate. In transconjugation experiments with four isolates harboring bla(TEM-1) and bla(SHV-12), both genes were simultaneously transferred to the recipient strain Escherichia coli HB101. Plasmid mapping, PCR, and Southern analysis with TEM- and SHV-specific probes demonstrated that a single transferred plasmid carried both the TEM-1 and the SHV-12 genes. The widespread presence of ESBLs among Enterobacter isolates in Tel Aviv is likely due not to clonal spread but, rather, to plasmid-mediated transfer, at times simultaneously, of genes encoding several types of enzymes. The dominant ESBL identified was SHV-12.

Interspecies recombination between enterococci: genetic and phenotypic diversity of vancomycin-resistant transconjugants.

Handwerger and colleagues demonstrated that a particular clinical isolate of Enterococcus faecium, designated GUC, and here redesignated as GUCR, can conjugatively transfer vancomycin resistance. The vancomycin resistance is encoded by a chromosomally born linked set of genes in the donor, designated the vanA cluster, to the chromosome of an E. faecalis recipient, JH2-2. Here it is reported that an earlier isolate of E. faecium from the same patient who later harbored the vancomycin-resistant E. faecium GUCR lacks the vanA gene cluster but is otherwise similar (by SmaI chromosomal fingerprint and metabolic fingerprinting) to the vancomycin-resistant GUCR. Therefore, "GUCS" is a strong suspect as the base strain for the clinical acquisition of the vanA cluster present in GUCR. Thirteen laboratory-generated vanA transconjugants derived from conjugation between GUCR and JH2-2 were subjected to further analysis, allowing a comparison between transfer in the laboratory and transfer that occurred in the clinical setting. Surprisingly, each JH2-2 transconjugant had a unique constellation of abilities to oxidize various members of a panel of potential carbon sources. This pattern was stable for each transconjugant, and it was not changed by growing the strains with or without vancomycin. Transconjugants had pulsed-field gel electrophoretic (PFGE) patterns largely consistent with that of JH2-2, the recipient in conjugation experiments. However, PFGE analysis showed that a large but variable amount of DNA, between 145 kb and 277 kb, was transferred into different transconjugants. The mechanism appears to be conjugative transposition in which new DNA is added to the pre-existing genome rather than substituting for a segment in the recipient. Mapping and hybridization studies of several transconjugants showed that each received similar, but not exactly the same, DNA fragment of at least 30 kb from the donor. Sequencing of 16S ribosomal genes was used to confirm that the recipient and donor strains used in transconjugation experiments were different species. Sequence analysis was also used to consider the possibility that rRNA operons might be mobilized in conjugation, but no evidence for the transfer of rDNA operons was found. An apparent insertion sequence in E. faecium almost identical to IS 1485 and 57% sequence identity to IS 199 of Streptococcus mutans was found in the region of DNA transferred. The results imply new consequences of conjugative transfer and interspecies recombination.