Comparison of high-level gentamicin-resistant Enterococcus faecium isolates from different continents.

Eight clinical isolates of Enterococcus faecium highly resistant to gentamicin (MIC, > 1,000 mg/liter) from patients in six hospitals on three continents were investigated for evidence of spread of either a clone of high-level gentamicin-resistant (HLGR) E. faecium or wide dissemination of a gentamicin resistance (Gmr) plasmid. A combination of ribotypes, plasmid profiles, and extended antimicrobial susceptibilities enabled us to distinguish all but two of the isolates and did not suggest clonal dissemination of a single strain. Two isolates from hospitals situated close together appeared identical by these methods. All of the isolates carried Gmr plasmids which appeared to be closely related following digestion with restriction endonucleases. Cross-hybridization studies confirmed extensive DNA homology between these plasmids. The fragments of these plasmids which hybridized with a probe specific for the aac6'aph2" resistance gene did not resemble those seen in the Gmr transposon Tn5281, which was characterized previously in E. faecalis HH22. This study suggests that there has been widespread dissemination of a single Gmr plasmid and its derivatives amongst isolates of HLGR E. faecium, although a Gmr plasmid from an HLGR E. faecium isolated in the United States showed little homology with the other Gmr plasmids studied.     

Diversity of Structures Carrying the High-Level Gentamicin Resistance Gene (aac6-aph2) in Enterococcus faecalis Strains Isolated in France

Of 24 high-level gentamicin-resistant clinical isolates of Enterococcus faecalis, 20 carried gentamicin resistance (Gm^r) plasmids. The plasmids ranged from 65.0 to 80.0 kb in size. Three of these plasmids were nonconjugative, and 17 transferred by conjugation to an E. faecalis recipient at low frequency (10⁻⁵ to 10⁻⁶ transconjugants per donor). The remaining four strains had a nonconjugative chromosomal Gm^r determinant. On the basis of restriction enzyme and DNA-DNA hybridization profiles, Tn4001-like α elements were located on the chromosome and three types of Tn4001-truncated structures, I, II, and III, were found to be carried by the Gm^r plasmids. Structure I lacked IS256 in the right-hand flanking extremity of Tn4001. Structure II was the same as structure I except that it also had a partial deletion of IS256 in the left-hand flanking extremity of Tn4001. Structure III lacked both the right- and left-hand flanking extremities of Tn4001. One of the wild-type strains carried the Gm^r determinant both on the chromosome, as a Tn4001-like α element, and on a conjugative plasmid, as a Tn4001-truncated type I structure.     

Mechanisms of vancomycin resistance in Staphylococcus aureus

Vancomycin is a glycopeptide antibiotic used for the treatment of Gram-positive bacterial infections. Traditionally, it has been used as a drug of last resort; however, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased susceptibility to vancomycin (vancomycin intermediate-resistant S. aureus [VISA]) and more recently with high-level vancomycin resistance (vancomycin-resistant S. aureus [VRSA]) have been described in the clinical literature. The rare VRSA strains carry transposon Tn1546, acquired from vancomycin-resistant Enterococcus faecalis, which is known to alter cell wall structure and metabolism, but the resistance mechanisms in VISA isolates are less well defined. Herein, we review selected mechanistic aspects of resistance in VISA and summarize biochemical studies on cell wall synthesis in a VRSA strain. Finally, we recapitulate a model that integrates common mechanistic features of VRSA and VISA strains and is consistent with the mode of action of vancomycin.     

屎肠球菌高耐庆大霉素转座子Tn5281的检测

目的　研究高耐庆大霉素 (HLGR)屎肠球菌耐药性与转座子的关系。方法　用聚合酶链反应 (PCR)检测 48株HLGR屎肠球菌Tn5 2 81转座子 ,扩增产物用斑点杂交和测序法验证。结果　 48株HLGR屎肠球菌中 ,13株含有完整的Tn5 2 81,2 6株含有缺少右侧插入序列IS2 5 6的Tn5 2 81缺失型 ,9株不含Tn5 2 81,总阳性率为 81. 3 %。结论　Tn5 2 81在屎肠球菌高耐庆大霉素中起着重要作用。     

Complete Sequence of p07-406, a 24,179-base-pair plasmid harboring the blaVIM-7 metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States

An outbreak involving a Pseudomonas aeruginosa strain that was resistant to all tested antimicrobials except polymyxin B occurred in a hospital in Houston, TX. Previous studies on this strain showed that it possesses a novel mobile metallo-β-lactamase (MBL) gene, designated bla_VIM-7, located on a plasmid (p07-406). Here, we report the complete sequence, annotation, and functional characterization of this plasmid. p07-406 is 24,179 bp in length, and 29 open reading frames were identified related to known or putatively recognized proteins. Analysis of this plasmid showed it to be comprised of four distinct regions: (i) a region of 5,200 bp having a Tn501-like mercuric resistance (mer) transposon upstream of the replication region; (ii) a Tn3-like transposon carrying a truncated integron with a bla_VIM-7 gene and an insertion sequence inserted at the other end of this transposon; (iii) a region of four genes, upstream of the Tn3-like transposon, possessing very high similarity to plasmid pXcB from Xanthomonas campestris pv. citri commonly associated with plants; (iv) a backbone sequence similar to the backbone structure of the IncP group plasmid Rms149, pB10, and R751. This is the first plasmid to be sequenced carrying an MBL gene and highlights the amelioration of DNA segments from disparate origins, most noticeably from plant pathogens.     

Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections: First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin,and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac)

Eighteen identical VanB-type Enterococcus faecalis isolates that were obtained from different hospitalized patients were examined for their drug resistance and plasmid DNAs. Of the 18 strains, 12 strains exhibited resistance to erythromycin (Em), gentamicin (Gm), kanamycin (Km), tetracycline (Tc), and vancomycin (Van) and produced cytolysin (Hly/Bac) and a bacteriocin (Bac) active against E. faecalis strains. Another six of the strains exhibited resistance to Gm, Km, Tc, and Van and produced a bacteriocin. Em and Van resistance was transferred individually to E. faecalis FA2-2 strains at a frequency of about 10⁻⁴ per donor cell by broth mating. The Em-resistant transconjugants and the Van-resistant transconjugants harbored a 65.7-kbp plasmid and a 106-kbp plasmid, respectively. The 106-kbp and 65.7-kbp plasmids isolated from the representative E. faecalis NKH15 strains were designated pMG2200 and pMG2201, respectively. pMG2200 conferred vancomycin resistance and bacteriocin activity on the host strain and responded to the synthetic pheromone cCF10 for pCF10, while pMG2201 conferred erythromycin resistance and cytolysin activity on its host strain and responded to the synthetic pheromone cAD1 for pAD1. The complete DNA sequence of pMG2200 (106,527 bp) showed that the plasmid carried a Tn1549-like element encoding vanB2-type resistance and the Bac41-like bacteriocin genes of pheromone-responsive plasmid pYI14. The plasmid contained the regulatory region found in pheromone-responsive plasmids and encoded the genes prgX and prgQ, which are the key negative regulatory elements for plasmid pCF10. pMG2200 also encoded TraE1, a key positive regulator of plasmid pAD1, indicating that pMG2200 is a naturally occurring chimeric plasmid that has a resulting prgX-prgQ-traE1 genetic organization in the regulatory region of the pheromone response. The functional oriT region and the putative relaxase gene of pMG2200 were identified and found to differ from those of pCF10 and pAD1. The putative relaxase of pMG2200 was classified as a member of the MOB_MG family, which is found in pheromone-independent plasmid pHTβ of the pMG1-like plasmids. This is the first report of the isolation and characterization of a pheromone-responsive highly conjugative plasmid encoding vanB resistance.Multiple-drug-resistant enterococci and vancomycin-resistant enterococci (VRE), in particular, are opportunistic pathogens and major causes of nosocomial infections in immunocompromised patients (4, 7, 44). The isolation of VRE (VanA type) was first reported in 1988 in the United Kingdom (67) and France (41), and shortly thereafter it was reported in the United States (55). Since then, VRE have been identified in many countries. VRE have caused an increasing number of treatment-related problems, especially in the United States (4, 43), where they are estimated to account for about 15% of nosocomial enterococcal isolates (15). In Asia, VRE have been isolated from hospitalized patients or food animals in China, Japan, South Korea, Taiwan, and Thailand (49, 74). In particular, they have frequently been isolated in South Korea (72) and Taiwan (40). Since the first report of the isolation of VanA-type VRE from a patient in Japan, VRE have been isolated from both sporadic individual cases and outbreaks of nosocomial infections in several hospitals (29, 49). However, an outbreak of VRE nosocomial infection is still a very rare event in university teaching hospitals in Japan.VRE isolates of the VanA and VanB types are the most commonly identified VRE isolates to be acquired. Their genomes are composed of operon gene clusters, and isolates of the VanA and VanB types have the same basic mechanism of resistance (12). The VanA-type determinant is encoded on the Tn1546 transposon or a Tn1546-like transposon (2), which frequently resides on a conjugative plasmid in VanA-type Enterococcus faecium (41). The vanB gene has been divided into three subtypes, vanB1, vanB2, and vanB3, on the basis of differences in the sequence of the vanB ligase (13, 22, 50). The vanB2 determinant is encoded on conjugative transposon Tn1549 (34 kb) (30) and the closely related transposon Tn5382 (27 kb) (3), which have similarities with the Tn916 family of conjugative transposons (10, 26). The transposable elements can be located on a conjugative or a nonconjugative plasmid or on the chromosome (3, 30, 53, 54). To our knowledge, there has been no report of a vanB determinant located on the pheromone-responsive highly conjugative plasmid.The first outbreak of a VRE nosocomial infection in Japan was caused by a VanB-type Enterococcus faecalis strain in a hospital setting in July 1999. Twenty VanB-type E. faecalis isolates were obtained from three clinical specimens, nine rectal swab specimens from asymptomatic carriers, and eight swab specimens from the hospital environment and were examined for drug resistance by pulsed-field gel electrophoresis (PFGE) (47). Southern blot analysis of the PFGE gel with a vanB probe implied that the VanB-type determinants resided on a 110-kbp plasmid in 19 strains obtained from among the 20 isolates (47). As described in this report, we examined the plasmids carried by the VanB-type VRE and identified two pheromone-responsive plasmids: one plasmid encoding vancomycin resistance and a bacteriocin and the other plasmid encoding erythromycin resistance and cytolysin.     

Bactericidal activities of antibiotics against vancomycin-resistant Enterococcus faecium blood isolates and synergistic activities of combinations.

The effects of teicoplanin (8 micrograms/ml), ampicillin (64 micrograms/ml), imipenem (32 micrograms/ml), and gentamicin (4 micrograms/ml), alone and in combination, against 13 unique blood isolates of vancomycin-resistance (MIC for 90% of isolates tested [MIC90], 512 micrograms/ml), teicoplanin-susceptible (MIC90, 2.0 micrograms/ml), ampicillin-resistant (MIC90, 128 micrograms/ml), and non-beta-lactamase-producing Enterococcus facium (vancomycin-resistant enterococci [VRE] isolates) were evaluated by time-kill studies. All 13 isolates exhibited high-level resistance to streptomycin; 7 isolates exhibited high-level gentamicin resistance (HLGR). After 24 h of incubation, ampicillin (64 micrograms/ml) combined with gentamicin (4 micrograms/ml) was bactericidal against three of the VRE isolates that did not display HLGR. Synergy between ampicillin and gentamicin was not observed against these isolates. Teicoplanin (8 micrograms/ml) alone was bactericidal at 24 h against five of six VRE isolates that lacked HLGR, but was not bactericidal against any HLGR VRE isolate at that time point. The addition of ampicillin (64 micrograms/ml) or imipenem (32 micrograms/ml) to teicoplanin did not significantly enhance the killing of HLGR VRE isolates as a group (P = 0.335). However, there was a trend toward improved killing of some HLGR VRE isolates by teicoplanin plus imipenem. Vancomycin (32 micrograms/ml) combined with ampicillin (64 micrograms/ml) was neither bactericidal nor synergistic against HLGR VRE isolates. Overall, bactericidal activity was attainable against 7 of 13 VRE isolates at 24 h.     

Plasmid heterogeneity and identification of a Tn5281-like element in clinical isolates of high-level gentamicin-resistant Enterococcus faecium isolated in the UK.

Ten clinical isolates of high-level gentamicin-resistant (HLGR) Enterococcus faecium, collected from six hospitals throughout the UK, were studied to determine whether HLGR was attributed to widespread dissemination of a single plasmid or whether different plasmid types were implicated in the dissemination of this phenotype. HLGR was attributed to the presence of the AAC6'-APH2" bifunctional aminoglycoside modifying enzyme. The aac6'-aph2" gene was present on a 70 kb plasmid in all ten isolates. Conjugation studies indicated that the HLGR marker could transfer with varying frequency, with or without the associated 70 kb plasmid. Detailed molecular genetic analysis suggested that four of the isolates harboured a transposon similar to Tn5281, originally identified in Enterococcus faecalis strain HH22 isolated in the USA. The UK transposon, however, lacked the two symmetrically located HaeIII sites found in Tn5281. The six remaining isolates appeared to have a Tn5281-truncated structure in which the aac6'-aph2" gene is flanked by an IS256 element at the 5' end. Further studies with nine restriction endonucleases showed that the aac6'-aph2" gene was associated with two different plasmid types in E. faecium. Pulsed-field gel electrophoresis (PFGE) analysis identified three different patterns. The four E. faecium isolates harbouring the Tn5281-like structure were indistinguishable from each other, while the remaining six isolates exhibited two distinct PFGE patterns. This is the first study to indicate that there is heterogeneity among the plasmids that confer the HLGR phenotype in E. faecium isolates in the UK and to report on the presence of a transposon, similar to Tn5281, in E. faecium harbouring the aac6'-aph2" gene.     

Tn6249, a New Tn6162 Transposon Derivative Carrying a Double-Integron Platform and Involved with Acquisition of the blaVIM-1 Metallo-β-Lactamase Gene in Pseudomonas aeruginosa

The In70.2 integron platform appears to be a conserved structure involved in the dissemination of the bla_VIM-1 metallo-β-lactamase gene in Pseudomonas aeruginosa. The genetic context of the In70.2 integron platform from P. aeruginosa VR-143/97, the VIM-1-producing index strain isolated in Italy in 1997, was fully characterized by a next-generation sequencing approach refined by conventional sequencing. The In70.2 integron platform from VR-143/97 was found to be associated with a defective Tn402-like transposon inserted into the urf2 gene of a Tn3 family transposon of an original structure, named Tn6249, which also carried a partially deleted mer operon and an In90 integron platform in a tail-to-tail orientation. Tn6249 was inserted into a PACS171b-like genomic island, which was in turn inserted into the endA gene of the Pseudomonas chromosomal backbone. Tn6249 showed a similar structure and a conserved location with respect to that of Tn6060, a Tn3 family transposon associated with In70.2 and carrying a double-integron platform, which was detected in a VIM-1-producing P. aeruginosa strain isolated in Australia in 2008. Both Tn6249 and Tn6060 are apparently derived from Tn6162, a mercury resistance transposon carrying an integron platform, which was found in P. aeruginosa isolates from different geographic locations. The conservation of the genetic context of Tn6249 and Tn6060 suggests an in situ evolution of these elements after the insertion of a Tn6162-like ancestor into the PACS171b-like genomic island (GI) present in the genome of a successful widespread P. aeruginosa clonal lineage.     

First Report of a Clinical,Multidrug-Resistant Enterobacteriaceae Isolate Coharboring Fosfomycin Resistance Gene fosA3 and Carbapenemase Gene blaKPC-2 on the Same Transposon,Tn1721

In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain {"type":"entrez-nucleotide","attrs":{"text":"HS102707","term_id":"312647496","term_text":"HS102707"}}HS102707 isolate and an Enterobacter aerogenes strain {"type":"entrez-nucleotide","attrs":{"text":"HS112625","term_id":"313253202","term_text":"HS112625"}}HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the bla_KPC-2 and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain {"type":"entrez-nucleotide","attrs":{"text":"HS092839","term_id":"312447864","term_text":"HS092839"}}HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that bla_KPC-2 was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored bla_KPC-2. Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and bla_KPC-2 colocated in the same Tn1721-Tn3–like composite transposon on a novel IncP group plasmid.     

Nature of transposon-mediated high-level gentamicin resistance in Enterococcus faecalis isolated in the United Kingdom

Forty-two high-level gentamicin-resistant (MIC > 1000 mg/L) strains of Enterococcus faecalis, isolated from diverse geographical locations throughout the UK between 1993 and 1995, were studied to identify the nature of the high-level gentamicin-resistant determinants and the possibility of these determinants being associated with a transposon. High-level gentamicin resistance was attributed to the synthesis of the bifunctional (AAC6'-APH2") aminoglycoside-modifying enzyme. The aac6'-aph2" gene, which was present on a 70 kb plasmid in all 42 isolates, could be transferred by conjugation in association with the 70 kb plasmid in 39 of the isolates studied. In three E. faecalis isolates, however, the high-level gentamicin resistance was transferable independent of the 70 kb plasmid, suggesting the presence of a conjugative transposon. Long-PCR studies showed that all 42 clinical isolates harboured a transposon similar to Tn5281, originally identified in E. faecalis strain HH22 isolated in the USA. Restriction endonuclease and Southern hybridization analysis of the UK transposon showed that it is closely related to the high-level gentamicin resistance-conferring transposon Tn5281. However, the UK transposon lacks the HaeIII site identified in Tn5281. Pulsed-field gel electrophoresis analysis identified seven different patterns. Further studies with nine restriction endonucleases showed that the aac6'-aph2" gene was associated with nine different plasmid types in E. faecalis.     

肠球菌属细菌高水平庆大霉素耐药及其转座子的检测

目的研究2006—2009年北京地区临床分离高水平庆大霉素耐药(HLGR)肠球菌属细菌的双功能修饰酶基因转座子结构。方法采用脑心肉汤微稀释法测定北京地区169株临床分离粪肠球菌和21株屎肠球菌的高水平庆大霉素耐药性,应用菌落PCR扩增方法检测HLGR菌株中双功能修饰酶基因及两端插入序列(IS256),分析转座子结构。结果 169株粪肠球菌中,111株(65.7%)为HLGR株,HLGR菌中97.3%含有双功能修饰酶基因,其中10.2%含有完整结构的转座子(双功能修饰酶基因两端均含有IS256插入序列),89.8%含有截断结构的转座子(缺失一端或两端的插入序列)。21株屎肠球菌中,15株(71.4%)为HLGR株,所有HLGR菌株均含有双功能修饰酶基因,其中13.3%含有完整结构的转座子,86.7%含有截断结构的转座子。结论 2006—2009年北京地区临床分离肠球菌HLGR率高,并且绝大多数HLGR株含双功能修饰酶基因,通常形成截断结构的转座子。     

Tetracycline Resistance in Salmonella enterica subsp. enterica Serovar Dublin

The 47-kbp plasmid pGFT1 from Salmonella enterica subsp. enterica serovar Dublin mediated tetracycline resistance via a tet(A) gene located on an integrated copy of a Tn1721-analogous transposon. The integration site of the transposon was located within the reading frame of a fip gene. Plasmid pGFT1 was shown to be conjugative and to be able to replicate and express tetracycline resistance in Escherichia coli.     

Genetic relatedness among Enterococcus faecalis with transposon-mediated high-level gentamicin resistance in Swedish intensive care units

We studied 45 isolates of Enterococcus faecalis with high-level gentamicin resistance (HLGR), all but one concomitantly resistant to ciprofloxacin, and 25 ciprofloxacin-resistant isolates without HLGR for genetic relatedness using pulsed-field gel electrophoresis (PFGE). E. faecalis were isolated from patients admitted to intensive care units at eight hospitals in southern Sweden from December 1996 through December 1998. Genomic analysis by PFGE resulted in three clusters of genetically related isolates (designated clusters I, II and III) and 23 unique clones. Cluster I was found predominantly in the eastern and central parts of southern Sweden and clusters II and III in south-western Sweden. Among the 45 isolates with HLGR, 69% belonged to cluster I, 20% to cluster II, and 11% had unique PFGE patterns, which suggests that the majority of isolates with HLGR are closely related. Among the 25 ciprofloxacin-resistant isolates without HLGR, 68% had unique PFGE patterns, 12% belonged to cluster I and 20% to cluster III, which suggests the ciprofloxacin-resistant isolates are not related. All isolates with HLGR contained the aac(6')Ie-aph(2")Ia gene, which was carried on a Tn5281-like transposon in all isolates except one. We conclude that HLGR in E. faecalis was mainly due to dissemination of genetically related clones during the time studied, and that HLGR in these isolates was due to the presence of the aac(6')Ie-aph(2")Ia gene.     

Functional Analysis of a Bacitracin Resistance Determinant Located on ICECp1, a Novel Tn916-Like Element from a Conjugative Plasmid in Clostridium perfringens

Bacitracins are mixtures of structurally related cyclic polypeptides with antibiotic properties. They act by interfering with the biosynthesis of the bacterial cell wall. In this study, we analyzed an avian necrotic enteritis strain of Clostridium perfringens that was resistant to bacitracin and produced NetB toxin. We identified a bacitracin resistance locus that resembled a bacitracin resistance determinant from Enterococcus faecalis. It contained the structural genes bcrABD and a putative regulatory gene, bcrR. Mutagenesis studies provided evidence that both bcrA and bcrB are essential for bacitracin resistance, and that evidence was supported by the results of experiments in which the introduction of both the bcrA and bcrB genes into a bacitracin-susceptible C. perfringens strain was required to confer bacitracin resistance. The wild-type strain was shown to contain at least three large, putatively conjugative plasmids, and the bcrRABD locus was localized to an 89.7-kb plasmid, pJIR4150. This plasmid was experimentally shown to be conjugative and was sequenced. The sequence revealed that it also carries a tpeL toxin gene and is related to the pCW3 family of conjugative antibiotic resistance and toxin plasmids from C. perfringens. The bcr genes were located on a genetic element, ICECp1, which is related to the Tn916 family of integrative conjugative elements (ICEs). ICECp1 appears to be the first Tn916-like element shown to confer bacitracin resistance. In summary, we identified in a toxin-producing C. perfringens strain a novel mobile bacitracin resistance element which was experimentally shown to be essential for bacitracin resistance and is carried by a putative ICE located on a conjugative plasmid.     

New Transposon Tn6133 in Methicillin-Resistant Staphylococcus aureus ST398 Contains vga(E), a Novel Streptogramin A, Pleuromutilin, and Lincosamide Resistance Gene

A novel streptogramin A, pleuromutilin, and lincosamide resistance determinant, Vga(E), was identified in porcine methicillin-resistant Staphylococcus aureus (MRSA) ST398. The vga(E) gene encoded a 524-amino-acid protein belonging to the ABC transporter family. It was found on a multidrug resistance-conferring transposon, Tn6133, which was comprised of Tn554 with a stably integrated 4,787-bp DNA sequence harboring vga(E). Detection of Tn6133 in several porcine MRSA ST398 isolates and its ability to circularize suggest a potential for dissemination.     

Characterization of Tn6238 with a New Allele of aac(6′)-Ib-cr

Here, we report that the genetic structure of Tn1331 remained conserved in Argentina from 1989 to 2013 (72 of 73 isolates), with the exception being the plasmid-borne Tn1331-like transposon Tn6238 containing a new aac(6′)-Ib-cr allele recovered from a colistin-resistant Klebsiella pneumoniae clinical isolate. A bioinformatic analysis of aac(6′)-Ib-like gene cassettes suggests that this new aac(6′)-Ib-cr allele emerged through mutation or homologous recombination in the Tn1331 genetic platform. Tn6238 is a novel platform for the dissemination of aminoglycoside and fluoroquinolone resistance determinants.     

High-Level Resistance to Trimethoprim in Clinical Isolates of Campylobacter jejuni by Acquisition of Foreign Genes (dfr1 and dfr9) Expressing Drug-Insensitive Dihydrofolate Reductases

The pathogenic bacterium Campylobacter jejuni has been regarded as endogenously resistant to trimethoprim. The genetic basis of this resistance was characterized in two collections of clinical isolates of C. jejuni obtained from two different parts of Sweden. The majority of these isolates were found to carry foreign dfr genes coding for resistant variants of the dihydrofolate reductase enzyme, the target of trimethoprim. The resistance genes, found on the chromosome, were dfr1 and dfr9. In about 10% of the strains, the dfr1 and dfr9 genes occurred simultaneously. About 10% of the examined isolates were found to be negative for these dfr genes and showed a markedly lower trimethoprim resistance level than the other isolates. The dfr9 and dfr1 genes were located in the context of remnants of a transposon and an integron, respectively. Two different surroundings for the dfr9 gene were characterized. One was identical to the right-hand end of the transposon Tn5393, and in the other, the dfr9 gene was flanked by only a few nucleotides of a Tn5393 sequence. The insertion of the dfr9 gene into the C. jejuni chromosome could have been mediated by Tn5393. The frequent occurrence of high-level trimethoprim resistance in clinical isolates of C. jejuni could be related to the heavy exposure of food animals to antibacterial drugs, which could lead to the acquisition of foreign resistance genes in naturally transformable strains of C. jejuni.     

Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of β-Lactam and Vancomycin Resistance in Enterococcus faecium

The transfer of DNA between Enterococcus faecium strains has been characterized both by the movement of well-defined genetic elements and by the large-scale transfer of genomic DNA fragments. In this work, we report on the whole-genome analysis of transconjugants resulting from mating events between the vancomycin-resistant E. faecium C68 strain and the vancomycin-susceptible D344RRF strain to discern the mechanism by which the transferred regions enter the recipient chromosome. Vancomycin-resistant transconjugants from five independent matings were analyzed by whole-genome sequencing. In all cases but one, the penicillin binding protein 5 (pbp5) gene and the Tn5382 vancomycin resistance transposon were transferred together and replaced the corresponding pbp5 region of D344RRF. In one instance, Tn5382 inserted independently downstream of the D344RRF pbp5 gene. Single nucleotide variant (SNV) analysis suggested that entry of donor DNA into the recipient chromosome occurred by recombination across regions of homology between donor and recipient chromosomes, rather than through insertion sequence-mediated transposition. The transfer of genomic DNA was also associated with the transfer of C68 plasmid pLRM23 and another putative plasmid. Our data are consistent with the initiation of transfer by cointegration of a transferable plasmid with the donor chromosome, with subsequent circularization of the plasmid-chromosome cointegrant in the donor prior to transfer. Entry into the recipient chromosome most commonly occurred across regions of homology between donor and recipient chromosomes.     

Diversity and Evolution of the Tn5801-tet(M)-Like Integrative and Conjugative Elements among Enterococcus,Streptococcus, and Staphylococcus

This work describes the diversity and evolution of Tn5801 among enterococci, staphylococci, and streptococci based on analysis of the 5,073 genomes of these bacterial groups available in gene databases. We also examined 610 isolates of Enterococcus (from 10 countries, 1987 to 2010) for the presence of this and other known CTn-tet(M) elements due to the scarcity of data about Tn5801 among enterococci. Genome location (by ICeu-I–pulsed-field gel electrophoresis [PFGE] hybridization/integration site identification), conjugation and fitness (by standard methods), Tn5801 characterization (by long-PCR mapping/sequencing), and clonality (by PFGE/multilocus sequence typing [MLST]) were studied. Twenty-three Tn5801 variants (17 unpublished) clustered in two groups, designated “A” (25 kb; n = 14; predominant in Staphylococcus aureus) and “B” (20 kb; n = 9; predominant in Streptococcus agalactiae). The percent GC content of the common backbone suggests a streptococcal origin of Tn5801 group B, with further acquisition of a 5-kb fragment that resulted in group A. Deep sequence analysis allowed identification of variants associated with clonal lineages of S. aureus (clonal complex 8 [CC8], sequence type 239 [ST239]), S. agalactiae (CC17), Enterococcus faecium (ST17/ST18), or Enterococcus faecalis (ST8), local variants, or variants located in different species and geographical areas. All Tn5801 elements were chromosomally located upstream of the guaA gene, which serves as an integration hot spot. Transferability was demonstrated only for Tn5801 type B among E. faecalis clonal backgrounds, which eventually harbored another Tn5801 copy. The study documents early acquisition of Tn5801 by Enterococcus, Staphylococcus, and Streptococcus. Clonal waves of these pathogens seem to have contributed to the geographical spread and local evolution of the transposon. Horizontal transfer, also demonstrated, could explain the variability observed, with the isolates often containing sequences of different origins.