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.     

Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors.

The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.     

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.     

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.     

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.     

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.     

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.     

Diversity of Tn1546 and its role in the dissemination of vancomycin-resistant enterococci in Portugal

We characterized the molecular diversity of vanA vancomycin-resistant enterococci (VRE; 176 isolates/87 pulsed-field gel electrophoresis types) from different sources and cities in Portugal (1996 to 2004): (i) food animals (FA; n = 38 isolates out of 31 samples), hospitalized humans (HH; n = 101/101), healthy human volunteers (HV; n = 7/4), and environmental sources (n = 30/10). Some strains were isolated from different hosts and persistently recovered for years. Twenty-four Tn1546 variants were identified, all located on plasmids (30 to 250 kb). Some Tn1546 variants were associated with specific sources such as FA (3 types), HH (11 types), or HV (1 type), while others were recovered from isolates of different origins (8 types). Polymorphisms in the central vanRSHA region of Tn1546 were scarcely detected, while alterations upstream of vanR and downstream of vanA were frequently identified involving mutations (vanS and vanX), deletions (vanY), insertions (IS1216V, ISEf1, and IS19; sequences with or without homology with others available in GenBank databases), and different genetic rearrangements. Most Tn1546 variants contained IS1216V (14 types) or ISEf1 (6 types). IS1216V was found alone or associated with an IS3-like element at different orientations and positions in Tn1546 from human, animal, and environmental samples. ISEf1 was located within vanX-vanY region at nucleotide 9044 of Tn1546 variants mostly associated with clinical isolates, suggesting a common genetic platform. IS19 was observed within the vanX-vanY region in one Tn1546 variant from poultry. Recent spread of VRE in Portugal reflects a complex epidemiology involving both clonal spread and plasmid dissemination containing a variety of Tn1546 types. Apparent Tn1546 heterogeneity among enterococci from human, animal, and environmental sources might reflect frequent genetic exchange events and evolution of particular widely disseminated genetic elements.     

VanB表型-vanA基因型VRE分子特征及遗传背景研究

目的 探讨VanB表型-vanA基因型VRE耐药转座子结构、分子特征及遗传背景,并与VanA表型-vanA基因型VRE进行比较分析,以确定基因型与表型不一致的形成机制.方法 收集2008年3月至2009年1月卫生部北京医院临床标本中21株VRE菌株,用Etest法对10种抗生紊进行MIC测定,并通过PCR、序列测定、接合试验、耐药转座子结构、PFGE及MLST进行分子特征和遗传背景研究.结果 21株VRE均为vanA基因型,其中3株菌呈现VanB表型(万古霉素耐药,替考拉宁敏感);21株菌属于9个不同PFGE型,6个不同MIST型;多对引物对转座子的不同区域PCR扩增并进行序列拼接、比对,发现Tn1546结构中vanX、vanY的缺失及ISEfa4的插入与VanB表型-vanA基因型VRE菌株形成相关.结论 VanB表型-vanA基因型VRE菌株在国内较为罕见,Tn1546结构的改变与菌株的基因型与表型不一致相关.     

Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the VanA cluster.

The MICs of vancomycin and avoparcin were determined for isolates of Enterococcus faecium and isolates of Enterococcus faecalis recovered from the feces of humans and animals in Denmark. Two hundred twenty-one of 376 (59%) isolates of E. faecium and 2 of 133 (1.5%) isolates of E. faecalis were resistant to vancomycin (MICs, 128 to > or = 256 micrograms/ml), and all vancomycin-resistant isolates were resistant to avoparcin (MICs, 64 to > or = 256 micrograms/ml). All vancomycin-resistant isolates examined carried the vanA, vanX, and vanR genes, suggesting that a gene cluster similar to that of the transposon Tn1546 was responsible for the resistance.     

杭州市五家医院万古霉素耐药肠球菌耐药转座子结构及分子分型

目的 明确万古霉素耐药肠球菌(VRE)耐药转座子结构及分子分型.方法 收集2006年4月至2007年4月杭州市5家医院21株VRE菌株,用Etest法进行抗菌药物的药敏试验,并通过PCR、接合试验、质粒提取、耐药转座子结构、脉冲凝胶电泳(PFGE)、多位点序列分型(MIST)及多位点串联重复序列分型(MLVA)进行研究.结果 21株VRE基因型及表型均符合vanA.属于10个不同的PFGE型,7个不同的MLST分型,4个不同的MLVA分型,其中18株属于克隆复合体CC17,另外3株为ST343与CC17接近.所有VRE菌株均对利奈唑胺及替加环素敏感.多对引物对转座子的不同区域的PCR扩增并进行序列拼接、比对,发现其中2株VRE菌株携带典型的耐药转座子Tn1546,其余19株VRE菌株均携带一种新的与Tn1546相似耐药转座子,均在vanXY之间反向插入IS1485.18株VRE菌株均可通过滤膜接合试验进行万古霉素耐药转移,接合菌均含有约54 000 bp大小的质粒.结论 杭州市5家医院21株VRE菌株均为vanA表型及基因型,发现了一种新的万古霉素耐药转座子结构.21株VRE菌株经MLST分型属于7个不同的序列分型,属于或接近易在医院环境里生存并在近年来迅速造成了全球播散的克隆复合体CC17.     

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.     

Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil

The sequence and gene organization of the van operons in vancomycin (MIC of >256 microg/ml)- and teicoplanin (MIC of > or =32 microg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanA(PT) operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanA(PA) in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanA(PA) by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in D-Ala-D-Lac, as demonstrated by D,D-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor.     

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.     

Transfer of plasmid and chromosomal glycopeptide resistance determinants occurs more readily in the digestive tract of mice than in vitro and exconjugants can persist stably in vivo in the absence of glycopeptide selection

OBJECTIVES AND METHODS: The transferability of vanA and vanB glycopeptide resistance determinants with a defined plasmid (n = 9) or chromosomal (n = 4) location between Enterococcus faecium strains of human and animal origins was compared using filter mating (in vitro) and germ-free mice (in vivo) as experimental models. Moreover, the stability of exconjugants in vivo in the absence of antibiotic selection was examined. RESULTS: Higher transfer rates were observed in vivo for four of six vanA and five of six vanB donor strains. For plasmid-encoded resistance, several log higher transfer frequencies were observed in vivo for some strains. Moreover, the in vivo model supported transfer of plasmid-encoded vanB (1 x 10(-7) exconjugants/donor) when repeated in vitro experiments were negative (estimated < 1 x 10(-9) exconjugants/donor). Readily detectable transfer of plasmid-located vanA and vanB as well as large chromosomal (>200 kb) vanB elements was observed after 24 h. The number of plasmid-mediated vanA exconjugants generally decreased markedly after 3 days. However, exconjugants containing a plasmid harbouring the vanA transposon Tn1546 linked to the post-segregational killing system omega-epsilon-zeta persisted stably in vivo in the absence of glycopeptides for more than 20 days. CONCLUSIONS: The overall results support the notion that the in vitro model underestimates the transfer potential. Rapid transfer of vanA plasmids from poultry- and pig-derived strains to human faecal E. faecium shows that even transiently colonizing strains may provide a significant reservoir for transfer of resistance genes to the permanent commensal flora. Newly acquired resistance genes may be stabilized and persist in new populations in the absence of antibiotic selection.     

Heterologous expression of glycopeptide resistance vanHAX gene clusters from soil bacteria in Enterococcus faecalis

OBJECTIVES: The aim of the study was to determine whether glycopeptide resistance gene clusters from soil bacteria could be heterologously expressed in Enterococcus faecalis and adapt to the new host following exposure to vancomycin. METHODS: The vanHAX clusters from Paenibacillus thiaminolyticus PT-2B1, Paenibacillus apiarius PA-B2B and Amycolatopsis coloradensis DSM 44225 were separately cloned in an appropriately constructed shuttle vector containing the two-component regulatory system (vanRS) of Tn1546. The complete vanA(PT) operon (vanRSHAXY) from P. thiaminolyticus PT-2B1 was cloned in the same shuttle vector lacking enterococcal vanRS. All plasmid constructs were electroporated into E. faecalis JH2-2 and the MICs of vancomycin and teicoplanin were determined for each recombinant strain before and following exposure to sublethal concentrations of vancomycin. RESULTS: The vanHAX clusters from P. thiaminolyticus and P. apiarius conferred high-level vancomycin resistance (MIC > or = 125 mg/L) in E. faecalis JH2-2. In contrast, cloning of the vanHAX cluster from A. coloradensis did not result in a significant increase of vancomycin resistance (MIC = 0.7 mg/L). Resistance to vancomycin was not observed after cloning the complete vanA(PT) operon from P. thiaminolyticus (MIC = 2 mg/L), but this recombinant rapidly adapted to high concentrations of vancomycin (MIC = 500 mg/L) following exposure to sub-lethal concentrations of this antibiotic. CONCLUSION: The results showed that vanA(PT) in P. thiaminolyticus is a possible ancestor of vanA-mediated glycopeptide resistance in enterococci. Experimental evidence supported the hypothesis that enterococci did not acquire glycopeptide resistance directly from glycopeptide-producing organisms such as A. coloradensis.     

Intra-hospital dissemination of quinupristin/dalfopristin- and vancomycin-resistant Enterococcus faecium in a paediatric ward of a German hospital

OBJECTIVES: To demonstrate nosocomial transmission of Enterococcus faecium resistant to quinupristin/dalfopristin and vancomycin/teicoplanin among paediatric patients in a German hospital ward. MATERIALS AND METHODS: Multiply-resistant E. faecium were isolated from three female patients aged 9 months, 2 and 15 years during a 10 day time span. Antibiotic susceptibilities were determined by microbroth dilution. Clonal relatedness among the isolates was investigated via SmaI-macrorestriction analysis by PFGE, multilocus sequence typing (MLST), and plasmid profiling. Presence of virulence and resistance determinants was tested by polymerase chain reaction (PCR). Selected resistance genes were localized by Southern hybridizations. RESULTS: A single E. faecium isolate per patient was investigated. All exhibited resistances to quinupristin/dalfopristin, vancomycin/teicoplanin, streptomycin (high-level), penicillin/ampicillin, erythromycin, oxytetracycline, chloramphenicol, rifampicin and fusidic acid. The isolates were susceptible to linezolid only and intermediately resistant to fluoroquinolones including moxifloxacin. PFGE revealed identical patterns for all three isolates. PCRs for virulence determinants hyaluronidase and enterococcal surface protein, esp, were negative, whereas PCR for the enterocin A gene was positive. MLST identified clonal type [8-5-1-1-1-1-1] belonging to a clonal subgroup C1 of hospital- and outbreak-related E. faecium. Southern hybridizations located several resistance genes (erm(B), vat(D), vanA) on a large plasmid, which was transferable in mating experiments with an E. faecium recipient. CONCLUSIONS: These data show routes of dissemination of resistance to multiple antibiotics including streptogramins and glycopeptides in E. faecium via vertical and/or horizontal gene transfer. The isolates spread in the absence of a direct selective pressure, as none of the patients had received earlier streptogramin or glycopeptide therapy.     

Derivatives of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey Medical Center

Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 microg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 microg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 microg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.     

Vancomycin-resistant Staphylococcus aureus isolate from a patient in Pennsylvania

A vancomycin-resistant Staphylococcus aureus (VRSA) isolate was obtained from a patient in Pennsylvania in September 2002. Species identification was confirmed by standard biochemical tests and analysis of 16S ribosomal DNA, gyrA, and gyrB sequences; all of the results were consistent with the S. aureus identification. The MICs of a variety of antimicrobial agents were determined by broth microdilution and macrodilution methods following National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The isolate was resistant to vancomycin (MIC = 32 micro g/ml), aminoglycosides, beta-lactams, fluoroquinolones, macrolides, and tetracycline, but it was susceptible to linezolid, minocycline, quinupristin-dalfopristin, rifampin, teicoplanin, and trimethoprim-sulfamethoxazole. The isolate, which was originally detected by using disk diffusion and a vancomycin agar screen plate, was vancomycin susceptible by automated susceptibility testing methods. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA indicated that the isolate belonged to the USA100 lineage (also known as the New York/Japan clone), the most common staphylococcal PFGE type found in hospitals in the United States. The VRSA isolate contained two plasmids of 120 and 4 kb and was positive for mecA and vanA by PCR amplification. The vanA sequence was identical to the vanA sequence present in Tn1546. A DNA probe for vanA hybridized to the 120-kb plasmid. This is the second VRSA isolate reported in the United States.     

Heterologous expression of the enterococcal vanA operon in methicillin-resistant Staphylococcus aureus

Two methicillin- and vancomycin-resistant Staphylococcus aureus strains, MI-VRSA and PA-VRSA, and Enterococcus faecalis DMC83006B, considered to be the potential donor of glycopeptide resistance to MI-VRSA, were studied. MI-VRSA is highly resistant to both glycopeptides, whereas PA-VRSA displays low-level resistance to vancomycin and reduced susceptibility to teicoplanin. We have analyzed the expression of the vanA operon in the three clinical isolates. Determination of the relative amounts of late peptidoglycan precursors and quantification of the d,d-peptidase activities, in the absence or after induction by glycopeptides, revealed that the resistance genes were expressed at similarly high levels in the three strains. Glycopeptide resistance stability in the three strains was studied by replica plating. Resistance was lost at high frequency, ca. 50%, after overnight growth of PA-VRSA in the absence of antibiotics, whereas it was fully stable in MI-VRSA and E. faecalis DMC83006B. Induction of resistance by vancomycin was significantly delayed in PA-VRSA relative to MI-VRSA. Low-level glycopeptide resistance of S. aureus PA-VRSA is thus likely due to instability of the genetic element, plasmid or transposon, carrying the vanA operon associated with a longer lag phase before growth resumes after induction by vancomycin.