Molecular diversity and evolutionary relationships of Tn1546-like elements in enterococci from humans and animals

We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546 derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS, vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element in orf1, of IS1251 in the vanS-vanH intergenic region, and of IS1216V in the vanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in Hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.     

Comparison of Tn1546-like elements in vancomycin-resistant Staphylococcus aureus isolates from Michigan and Pennsylvania

In 2002, the first two clinical isolates of vancomycin-resistant Staphylococcus aureus (VRSA) containing vanA were recovered in Michigan and Pennsylvania. Tn1546, a mobile genetic element that encodes high-level vancomycin resistance in enterococci, was present in both isolates. With PCR and DNA sequence analysis, we compared the Tn1546 elements from each isolate to the prototype Tn1546 element. The Michigan VRSA element was identical to the prototype Tn1546 element. The Pennsylvania VRSA element showed three distinct modifications: a deletion of nucleotides 1 to 3098 at the 5' end, which eliminated the orf1 region; an 809-bp IS1216V-like element inserted before nucleotide 3099 of Tn1546; and an inverted 1,499-bp IS1251-like element inserted into the vanSH intergenic region. These differences in the Tn1546-like elements indicate that the first two VRSA isolates were the result of independent genetic events.     

Molecular analysis of Tn1546-like elements mediating high-level vancomycin resistance in Enterococcus gallinarum

OBJECTIVES: Four Enterococcus gallinarum isolates, all highly resistant to vancomycin, were studied in order to investigate their relationship and to gain insight into the molecular events responsible for their acquired resistance. METHODS: Extensive molecular analysis was performed to compare the four E. gallinarum isolates and their Tn1546-like elements. RESULTS: The four strains had very similar random amplified polymorphic DNA (RAPD) patterns and different but related PFGE profiles. Genotypic analysis demonstrated that all carried both vanC-1 and vanA genes. Using a vanA probe, no hybridization was detected to plasmid DNA, whereas hybridization to different SmaI fragments of the four strains was obtained with total DNA. Amplification and sequencing experiments showed that all four strains carried a Tn1546-like element that contained the orf2, vanR, vanS, vanH, vanA and vanX genes and was flanked at both ends by oppositely oriented IS1216V sequences. On the left side of the vanA cluster, all lacked IRL, and all had, upstream from orf2, 1029 bp of the 3' end of orf1. On the right side, one of the strains lacked vanY, vanZ and IRR, whereas in one of the other three there was an IS1542 element inserted within the vanZ gene. In one strain, an additional IS1216V element was inserted in the intergenic region vanX-vanY. CONCLUSIONS: This is the first study providing a molecular analysis of chromosomal Tn1546-like elements (possibly composite transposons) associated with high-level vancomycin resistance in human and animal strains of E. gallinarum. These molecular findings, together with those from PFGE and RAPD, suggest that the four E. gallinarum isolates are related and might have a common ancestor.     

Diversity of Tn1546 elements in clinical isolates of glycopeptide-resistant enterococci from Scottish hospitals

The Tn1546-related elements of 48 Van glycopetide-resistant enterococci were compared. Ten distinct Tn1546 types were identified with variation primarily due to IS1542 and IS1216V-like insertions. Clonal isolates frequently differed in their Tn1546 type, indicating instability of Tn1546-related elements. A putative hybrid promoter was identified, generated upstream of vanR by the insertion of IS1542. The presence of this hybrid promoter was associated with constitutive expression of the van genes and elevated teicoplanin resistance.     

Reduction in glycopeptide resistance in vancomycin-resistant enterococci as a result of vanA cluster rearrangements

The molecular characterization of five clinical isolates of vanA-containing vancomycin-resistant enterococci with altered resistance to glycopeptides was examined. One strain represented an IS1216V insertion accompanied by partial deletion of the reading frame of vanX following a transposition event. The other four strains represented IS1216V within the vanX-vanY intergenic region associated with deletion of vanY or vanZ.     

Enterococci of animal origin and their significance for public health

Enterococci are commensal bacteria in the intestines of humans and animals, but also cause infections in humans. Most often, Enterococcus faecium isolates from clinical outbreaks belong to different types than E. faecium from animals, food, and humans in the community. The same variants of the vanA gene cluster (Tn1546) encoding vancomycin resistance can be detected in enterococci of both human and animal origin. This could indicate horizontal transfer of Tn1546 between enterococci of different origin. E. faecium isolates of animal origin might not constitute a human hazard in themselves, but they could act as donors of antimicrobial resistance genes for other pathogenic enterococci. Enterococcus faecalis of animal origin seems to be a human hazard, as the same types can be detected in E. faecalis from animals, meat, faecal samples from humans in the community, and patients with bloodstream infections.     

Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium.

A clinical isolate of Enterococcus faecium that contains a chromosomally encoded vanA gene cluster, Tn1546::IS1251, transferred vancomycin resistance to the plasmid-free strain Enterococcus faecalis JH2-2 during filter matings. Hybridization of a vanHAXY probe to SmaI restriction-digested genomic DNA separated by pulsed-field gel electrophoresis showed that the vanA gene cluster was located on a 40-kb fragment in the original donor strain and on fragments of different sizes (150 to 450 kb) in the transconjugants. No hybridization to vanA gene cluster probes was obtained with plasmid DNA preparations from the donor or transconjugants. These results suggested that in each case, the van genes had integrated into the recipient chromosome. The transconjugants in turn could act as donors of vancomycin resistance, and resistance was transferable to a Rec- recipient. The results of restriction analyses and DNA hybridizations of genomic DNA from the donor and transconjugants were consistent with the transfer of a mobile element that includes the 12.3-kb Tn1546::IS1251 gene cluster and at least 13 kb of additional DNA. This element has been tentatively designated Tn5482. DNA sequence analysis of a fragment predicted to contain the left end of Tn5482 revealed two insertion sequence-like elements: IS1216V and an apparently truncated IS3-like element. Restriction mapping and DNA hybridization patterns of the van gene clusters of three additional clinical isolates from New York City showed an element similar to Tn5482. Transfer of Tn5482 and related elements may be involved in dissemination of vancomycin resistance.     

Characterization of a vancomycin-resistant Enterococcus faecium outbreak caused by 2 genetically different clones at a neonatal intensive care unit

In July 2010, we identified an outbreak of vancomycin-resistant enterococci (VRE) in our 26-bed neonatal intensive care unit. We performed an epidemiological investigation after clinical cultures of 2 neonates were positive for VRE. Identification, susceptibility testing, and molecular characterization were performed. Cultures of 3 surveillance stool samples of inpatients and 5 environmental samples were positive for VRE. All isolates were identified as Enterococcus faecium containing the vanA gene. Two distinct clones were identified by performing pulsed-field gel electrophoresis. The 2 clones exhibited different pulsotypes, but they represented identical Tn1546 types. Two sequence types, ST18 and ST192, were identified among all of the isolates with multilocus sequence typing. Our investigation determined that the outbreak in the neonatal intensive care unit was caused by 2 genetically different clones. The outbreak may have occurred through clonal spread and horizontal transfer of the van gene.     

Local genetic patterns within a vancomycin-resistant Enterococcus faecalis clone isolated in three hospitals in Portugal

Eight pulsed-field gel electrophoresis subtypes and six Tn1546 variants were identified among Enterococcus faecalis isolates of a single clone recovered in three geographically separate Portuguese hospitals. Some clonal subtypes were found in particular hospitals, and Tn1546 variants were either widespread or confined to some of them. We also report on the first Tn1546 transposon containing an ISEf1 insertion.     

A clonal lineage of VanA-type Enterococcus faecalis predominates in vancomycin-resistant Enterococci isolated in New Zealand

Avoparcin was used as a feed additive in New Zealand broiler production from 1977 until June 2000. We report here on the effects of the usage and discontinuation of avoparcin on the prevalence of vancomycin-resistant enterococci (VRE) in broilers. Eighty-two VRE isolates were recovered from poultry fecal samples between 2000 and mid-2001. VRE isolates were only obtained from broiler farms that were using, or had previously used, avoparcin as a dietary supplement. Of these VRE isolates, 73 (89%) were VanA-type Enterococcus faecalis and nine (11%) were VanA-type Enterococcus faecium. All E. faecalis isolates were found to have an identical or closely related pulsed-field gel electrophoresis (PFGE) pattern of SmaI-digested DNA and were susceptible to both ampicillin and gentamicin. The PFGE patterns of the nine E. faecium isolates were heterogeneous. All VRE contained both the vanA and ermB genes, which, regardless of species or PFGE pattern, resided on the same plasmid. Eighty-seven percent of the VRE isolates also harbored the tet(M) gene, while for 63 and 100%, respectively, of these isolates, the avilamycin and bacitracin MICs were high (>or=256 microg/ml). Five of eight vancomycin-resistant E. faecalis isolates recovered from humans in New Zealand revealed a PFGE pattern identical or closely related to that of the E. faecalis poultry VRE isolates. Molecular characterization of Tn1546-like elements from the VRE showed that identical transposons were present in isolates from poultry and humans. Based on the findings presented here, a clonal lineage of VanA-type E. faecalis dominates in VRE isolated from poultry and humans in New Zealand.     

Transposon characterization of vancomycin-resistant Enterococcus faecium (VREF) and dissemination of resistance associated with transferable plasmids

OBJECTIVES: VanA glycopeptide resistance has persisted on broiler farms in the UK despite the absence of the antimicrobial selective pressure, avoparcin. This study aimed to investigate the contribution of horizontal gene transfer of Tn1546 versus clonal spread in the dissemination of the resistance. METHODS AND RESULTS: One hundred and one vancomycin-resistant Enterococcus faecium isolated from 19 unrelated farms have been investigated. Tn1546 characterization by long PCR and ClaI-digestions of amplicons showed a very low diversity of Tn types (n=4) in comparison to the high genotypic diversity demonstrated by PFGE (n=62). Conjugation experiments were carried out to assess the transfer of vancomycin resistance. Co-transfer of vanA together with erm(B) positioned on the same conjugative plasmid containing a replicon similar to pRE25 was demonstrated and also the presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms. CONCLUSIONS: Horizontal transfer of vancomycin resistance may play a more important role in the persistence of antimicrobial resistance than clonal spread. The presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms, illustrates the ability of these enterococci to acquire and disseminate mobile genetic elements within integrated livestock systems.     

High frequency of vancomycin-resistant Enterococcus faecium isolates with VanB phenotype and vanA genotype in Korean hospitals

A total of 59 vancomycin-resistant Enterococcus faecium (VREF) clinical isolates were collected from 8 Korean hospitals for 2 months in 2004. They were investigated by genotyping for glycopeptide resistance, multilocus sequence typing (MLST), esp repeat profiling, and structural analysis of Tn1546-like element. Nine of 59 VREF isolates (15.3%) from 5 hospitals in Korea showed VanB phenotype, but they contained vanA gene. MLST and esp repeat profiling indicated that E. faecium isolates with VanB phenotype and vanA genotype occurred from independent genetic background except 3 isolates from 1 hospital. Structural analysis of Tn1546 also showed that these isolates were not clonally related. Data showed a relatively high frequency of VREF isolates with incongruence between phenotype and genotype for glycopeptide resistance in Korean hospitals.     

Synergism between beta-lactams and glycopeptides against VanA-type methicillin-resistant Staphylococcus aureus and heterologous expression of the vanA operon

Vancomycin resistance of Staphylococcus aureus NY-VRSA and VRSA-5 is due to acquisition of a vanA operon located in a Tn1546-like element. The vanA gene cluster of NY-VRSA contained one copy of insertion sequences IS1251 and IS1216V relative to that of VRSA-5. As evidenced by the nature of the late peptidoglycan precursors and by quantification of d,d-peptidase activities, the vancomycin resistance genes were efficiently expressed in both strains. Study of the stability and inducibility of glycopeptide resistance suggested that low-level glycopeptide resistance of NY-VRSA was most probably due to plasmid instability combined with a long delay for resistance induction. The activity of combinations of vancomycin or teicoplanin with oxacillin against the four VanA-type S. aureus strains already reported was tested by single and double disk diffusion, E-test on agar alone or supplemented with antibiotics, the checkerboard technique, and by determining time-kill curves. A strong synergism against the four clinical isolates, with fractional inhibitory concentration indexes from 0.008 to 0.024, was reproducibly observed between the two antibiotics by all methods. These observations indicate that cell wall inhibitors of the beta-lactam and glycopeptide classes exert strong and mutual antagonistic effects on resistance to each other against VanA-type methicillin-resistant S. aureus.     

Molecular analysis of Tn1546-like elements in vancomycin-resistant enterococci isolated from patients in Europe shows geographic transposon type clustering

Resistance mechanism relatedness was studied in 18 clinical, European vanA vancomycin-resistant enterococci. Molecular analysis revealed 10 Tn1546-like elements, suggesting two evolutionary lineages. Lineage I dominated the European mainland, and lineage II dominated the United Kingdom and Israel. Geographic clustering reflected different types of meat consumption between countries, since each lineage is associated with colonization of different animals.     

Effects of the movement of insertion sequences on the structure of VanA glycopeptide resistance elements in Enterococcus faecium

A Tn1546-related element with IS1216V at position 8839 underwent a structural change after storage of the host strain of Enterococcus faecium at 4 degrees C. The element acquired IS1542 at position 3932, nucleotides 8732 to 8831 were deleted, and the first 3417 nucleotides were lost and replaced by an inverted copy of the IS1216V-vanY-vanZ-inverted-repeat block from the 3' end. Insertion sequence movement is likely to play a key role in the evolution of VanA resistance elements.     

Molecular study of vancomycin-resistant enterococci isolated from humans and from food in a cattle-rearing area of France

OBJECTIVES: Study possible links between vancomycin-resistant enterococci strains isolated from human stool samples and from pork or poultry food products. METHODS: One hundred and eleven vancomycin-resistant enterococci strains (15 VanA and 96 VanC) were isolated from human stool samples and from pork or poultry food products. Characterization of the Tn1546-like element of the 15 VanA strains was realized by restriction analysis of PCR products and polymorphism study. The 96 strains of VanC phenotype (75 Enterococcus gallinarum and 21 Enterococcus casseliflavus) were analysed by pulsed-field gel electrophoresis (PFGE). RESULTS: In the study described here, polymorphism of the Tn1546-like element enabled the establishment of five groups. Groups III, IV and V were found only in human strains. Groups I and II were found to occur in strains isolated from humans and from food, suggesting a possible transfer of the Tn1546-like element. The isolates from Group I harboured the whole Tn1546 element. In Group II, the five strains possessed a novel Tn1546-like element, characterized by a single-nucleotide difference in the vanX gene and a deletion upstream from the nucleotide 164. Analysis by PFGE of the 75 E. gallinarum strains revealed 20 different patterns. One pattern was shared by isolates from pork food and human samples. None of the 21 E. casseliflavus strains tested was found to share similar PFGE patterns. CONCLUSIONS: Results tend to support the possible transfer of the Tn1546-like element between strains of VanA phenotype. Concerning VanC phenotype strains, the transfer was not demonstrated.     

Van genotype and molecular typing of clinical isolates of vancomycin-resistant Enterococcus北大核心CSCD

Objective To determine the genotype and molecular typing of vancomycin-resistant Enterococcus (VRE). Methods Seventeen clinical isolates of VRE were collected in 2016. The strains were identified to species and confirmed by 16S rRNA sequencing. The minimum inhibitory concentration of antimicrobial agents was determined by microdilution method and agar dilution method. Multilocus sequence typing (MLST) was used for molecular typing. Results The VRE strains were confirmed as Enterococcus faecium by 16S rRNA sequencing. All strains were resistant to vancomycin, but only 12 strains were resistant to teicoplanin. The vanA gene was identified in 13 of the 17 strains. The vanM gene was detected in 9 strains. Both vanA and vanM genes were identified in five of the 17 strains. Six MLST types were identified in the 17 strains, including ST78 (n=8), ST80 (n=4), ST555 (n=2), and one each for ST117, ST262 and ST341. Conclusions The van genotype was primarily vanA (76.5%) and vanM (52.9%) in clinical isolates of VRE. The VRE strains carrying both vanA and vanM were found for the first time. © 2018, Editorial Department of Chinese Journal of Infection. All rights reserved.     

Antibiotic resistance of faecal enterococci in poultry, poultry farmers and poultry slaughterers

The prevalence of resistance in enterococci to antibiotics, commonly used for therapy in poultry or as antimicrobial growth promoters (AMGPs), was determined in faecal samples of two chicken populations: broilers in which antibiotic and AMGP use is common and laying-hens with a low antibiotic usage. In addition faecal samples were examined from three human populations: broiler farmers, laying-hen farmers and poultry slaughterers. MICs of an extended panel of antibiotics for a randomly chosen gentamicin- or vancomycin-resistant enterococcal isolate from each faecal specimen were also determined. The prevalence of resistance for all antibiotics tested was higher in broilers than in laying-hens. Resistance in faecal enterococci of broiler farmers was for nearly all antibiotics higher than those observed in laying-hen farmers and poultry slaughterers. The overall resistance in broilers was correlated with the resistance in broiler farmers and in poultry slaughterers. No correlation between the results obtained in the laying-hens with any of the other populations was found. The 27 gentamicin-resistant isolates all showed high-level resistance to gentamicin and two of these isolates, both Enterococcus faecium, were resistant to all antibiotics tested, except vancomycin. The 73 vancomycin-resistant enterococci (VRE) isolated from the five populations belonged to four different species and in all isolates the vanA gene cluster was detected by blot hybridization. The pulsed-field gel electrophoresis (PFGE) patterns of these vancomycin-resistant enterococci were quite heterogeneous, but Enterococcus hirae isolates with the same or a closely related PFGE pattern were isolated at two farms from the broiler farmer and from broilers. Molecular characterization of vanA-containing transposons of these isolates showed that similar transposon types, predominantly found in poultry, were present. Moreover, similar vanA elements were not only found in isolates with the same PFGE pattern but also in other VRE isolated from both humans and chickens. The results of this study suggest transmission of resistance in enterococci from animals to man. For VRE this might be clonal transmission of animal strains, but transposon transfer seems to occur more commonly.     

First outbreak of vancomycin-resistant enterococci in a tertiary hospital in Turkey

Twenty multidrug-resistant vancomycin-resistant Enterococcus faecium strains of the VanA phenotype were isolated over a 1 year period from five patients in the intensive care unit at the University Hospital of Antalya, Turkey. Molecular investigation showed that these strains belonged to five different pulsotypes and that the vanA gene was carried by a Tn1546-like transposon inserted onto a self-transferable plasmid of approximately 200 kb. One patient was infected by two different strains, suggesting horizontal gene transfer within that patient. This is the first documented outbreak of VRE in Turkey with concomitant spread of plasmid and strains.     

Molecular characterization of glycopeptide-resistant Enterococcus faecium isolates from Portuguese hospitals

Fifty-one pulsed-field gel electrophoresis types and 17 Tn1546 variants were identified among 101 Enterococcus faecium isolates recovered in three distant Portuguese hospitals. Intra- and interhospital dissemination of specific strains and Tn1546 types was detected, which might largely contribute to the endemicity of vancomycin-resistant E. faecium in Portuguese hospitals, as happened previously in other geographical locations.