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1 Microbiology Laboratory, Department of Physiology, University College of Science, Technology and Agriculture, University of Calcutta, Kolkata, India
2 Department of Chemistry, Bose Institute, Kolkata, India
3 Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University College of Science, Technology and Agriculture, University of Calcutta, Kolkata, India
Correspondence
Manjusri Bal
manjusrb{at}vsnl.net
Received 2 January 2007
Accepted 8 August 2007
64 µg ml–1) was obtained from a Kolkata hospital in June 2005. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc gene, which encodes the thermostable nuclease that is highly specific for S. aureus. The VRSA isolate was also resistant to beta-lactams (amoxicillin, ampicillin, cefepime, cefotaxime, cefuroxime, cephalexin and meticillin), chloramphenicol, streptomycin, macrolides (erythromycin and roxithromycin), clindamycin, rifampicin and trimethoprim-sulfamethoxazole. However, the isolate was susceptible to gentamicin (an aminoglycoside) and ciprofloxacin (a fluoroquinolone). The resistance to vancomycin was inducible in vitro, because the MIC of vancomycin increased from 64 µg ml–1 initially to 1024 µg ml–1 during culture of this VRSA strain in the presence of vancomycin. The VRSA isolate contained a large plasmid (
53.4 kb) and four small plasmids of 6, 5.5, 5.1 and 1.5 kb. The large plasmid of 53.4 kb harboured the vancomycin-resistance genes vanHAX, which was confirmed by PCR amplification using the same plasmid as template and, separately, primers specific for the 2.61 kb vanHAX gene cluster, vanH (969 bp), vanA (1032 bp) and vanX (609 bp). The VRSA isolate was also positive for mecA. Vancomycin resistance was successfully transferred from this VRSA donor to a vancomycin-sensitive recipient S. aureus clinical isolate by a broth mating procedure. The MIC of vancomycin for the transconjugant was 32 µg ml–1, as against 2 µg ml–1 for the parent strain. Nucleotide sequencing of the PCR product showed partial homology with van genes of an enterococcal transposon Tn1546-like element. This is believed to be the first Indian S. aureus isolate that has been shown to be phenotypically vancomycin-resistant, presumably due to a vanHAX analogue.
Abbreviations: DAD, disc agar diffusion; PG, peptidoglycan; VRSA, vancomycin-resistant S. aureus; VSSA, vancomycin-sensitive S. aureus.
The GenBank/EMBL/DDBJ accession numbers for the S. aureus sequences determined in this paper are EU019995 and EU016096.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Conjugative transfer of high-level vancomycin resistance from Enterococcus faecalis to S. aureus (Noble et al., 1992), and transfer of glycopeptide- and macrolide-resistance genes by transconjugation among enterococci and from Ent. faecalis to S. aureus (M
ynarczyk et al., 2002), have been reported. Vancomycin-resistance gene acquisition by S. aureus from Ent. faecium in the clinical environment has also been reported by Weigel et al. (2007).
In this study, we have shown the emergence of vancomycin resistance in Kolkata, India, and its conjugative transfer from one clinical strain to another.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Confirmation of species identification. Identification of the clinical isolates of S. aureus was performed by traditional biochemical tests, including catalase, coagulase and mannitol fermentation tests, and Gram staining (Bannerman, 2003; Turk & Porter, 1978). PCR amplification of the nuc gene (Brakstad et al., 1992) was performed only for VRSA STM2. PCR amplification of 16S rDNA was also performed for VRSA STM2, induced STM2, MC48 and transconjugant T48.
Antibiotic susceptibility testing. The antibiotic-resistance profile was determined by the disc agar diffusion (DAD) technique (Acar, 1980; Bauer et al., 1966), using 18 antibiotic discs. Among these antibiotics, some discs were prepared in the Microbiology Laboratory, Department of Physiology, and a few were obtained commercially from Himedia. The antimicrobials, the manufacturers and the amounts (µg) of antimicrobial per disc were as follows: amoxicillin (Rexcel, 30 µg), ampicillin (Biochem Pharmaceutical Industries, 10 µg), cefepime (Unichem Laboratories, 30 µg), cefotaxime (Alkem Laboratories, 30 µg), cefuroxime (Glaxo Smith Kline, 30 µg), cephalexin (Ranbaxy, 30 µg), chloramphenicol (Sigma, 30 µg), ciprofloxacin [Pharma(Ran), 5 µg], clindamycin (Indipharma, 2 µg), erythromycin (Alembic, 15 µg), gentamicin (Nicholas, 10 µg), meticillin (Himedia, 5 µg), oxacillin (Himedia, 1 µg), rifampicin (Lupin, 5 µg), roxithromycin (Alembic, 15 µg), streptomycin (Synbiotics, 10 µg), trimethoprim-sulfamethoxazole (1 : 5) (Piramal Health Care, 5 µg) and vancomycin (Lilly Pharma and Himedia, 30 µg). S. aureus ATCC 25923, an all-sensitive reference strain, was used as a quality control strain for the DAD test.
Determination of MIC. The MIC of vancomycin was determined by a broth microdilution method using Mueller–Hinton broth (MHB; dehydrated, Himedia), as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (NCCLS, 2000).
Isolation of plasmid DNAs. For molecular studies, plasmid DNAs from the clinical isolate S. aureus STM2 and the reference strain E. coli V517, which contains six plasmids of molecular weights 53.4, 5.5, 5.1, 3, 2.7 and 2.1 kb (Macrina et al., 1978), were isolated using the QIAGEN Midi plasmid purification kit, following the manufacturer's instruction of prewarming the elution buffer (Buffer EB) to 50 °C for elution of large plasmids.
Preparation of genomic DNA. The crude lysates of clinical isolates of S. aureus STM2 (VRSA), MC48 (vancomycin-sensitive S. aureus; VSSA) and T48 (transconjugant) were prepared by sucrose-mediated detergent lysis (Saha et al., 1989). Cell lysates were treated with proteinase K (10 µg ml–1, preactivated at 37 °C for 30 min) at room temperature for 30 min. NaCl (1.44 ml, 5 M) was added to this mixture and incubated at 65 °C for 20 min. An equal volume of chloroform : isoamyl alcohol (24 : 1, v/v) mixture was added, and the aqueous layer was extracted with an equal volume of phenol : chloroform : isoamyl alcohol (25 : 24 : 1, v/v). The aqueous phase was again extracted twice with chloroform : isoamyl alcohol (24 : 1, v/v), and DNA was precipitated by the addition of chilled ethanol. The DNA pellet was washed with cold 70 % ethanol and reconstituted in Tris/EDTA buffer. The genomic DNA was treated with RNase and stored at –20 °C.
PCR. PCR amplification was performed with an ABI 9700 thermal cycler in a volume of 50 µl. For amplification of the nuc gene and mecA, the following components were used: 1.5 mM MgCl2, 200 µM each of dATP, dTTP, dGTP and dCTP, 2 µM of each primer, 0.1 µg template DNA, and 1.25 U Taq polymerase (Invitrogen).
(i) PCR amplification of the nuc gene. A partial nuc gene was amplified using nuc gene primers (Table 1
), which were selected on the basis of the published nucleotide sequence of the 966 bp nuc gene derived from the S. aureus Foggi strain (Brakstad et al., 1992). The cycling parameters consisted of 30 cycles of denaturation at 94 °C for 30 s, primer annealing at 50 °C for 1 min, and extension at 72 °C for 1 min 30 s.
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) were used (Dias et al., 2004). The reaction conditions were 30 cycles of denaturation at 94 °C for 40 s, primer annealing at 52 °C for 45 s, and extension at 72 °C for 30 s.
(iii) PCR amplification of vanHAX, vanH, vanA and vanX. The PCR amplification mixture contained the following components: 1x Phusion GC buffer containing 1.5 mM MgCl2, 200 µm each dNTP, 2 µm each primer, 0.1 µg template DNA, 3 % (v/v) DMSO and 1 U Phusion DNA polymerase (Finnzymes). The amplification conditions were initial denaturation at 98 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s; annealing at 50 °C for 1 min; polymerization at 72 °C for 1 min 30 s for van HAX, 60 s for both vanH and vanA, and 30 s for vanX; and final extension at 72 °C for 5 min for all. The primer sequences specific for vanHAX, vanH, vanA and vanX (Donabedian et al., 2000) are given in Table 1.
Transfer of vancomycin resistance by mating procedure.
Broth mating (Clewell et al., 1985; Philippon et al., 1983) was performed as follows. Single colonies of donor and recipient cells were inoculated separately in Luria–Bertani (LB) broth and grown overnight at 37 °C with shaking. These overnight cultures were diluted 1 : 100 in fresh medium, and each was grown to early exponential phase (OD600 0.6). Mating mixture was prepared by adding 0.1 ml of donor cells to 0.9 ml of recipient cells, and was swirled gently for a few minutes and then incubated at 37 °C for 6 h (without shaking), followed by plating (0.2 ml per plate) on Luria agar (LA) medium containing 16 µg vancomycin ml–1 and 2.5 µg ciprofloxacin ml–1. Colonies were counted after 48 and 72 h of incubation. Donor and recipient cells were also plated separately to check their disability to grow on the vancomycin plus ciprofloxacin plate, because the donor was ciprofloxacin-sensitive and the recipient was susceptible to vancomycin.
Nucleotide sequencing. Amplified PCR products (vanHAX and vanH) were purified with the Wizard SV Gel and PCR Clean Up System (Promega). The gel-eluted PCR products were sequenced with an ABI Prism automated DNA sequencer (Applied Biosystems) with the single primer (forward primer for vanH, reverse primer for vanHAX). The sequencing reaction was carried out using 5 pmol primer and the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), following the instructions of the manufacturer. The sequencing amplification conditions were as follows: 96 °C for 10 s, 50 °C for 10 s and 60 °C for 4 min for 25 cycles. After the sequencing PCR, the products (10 µl reaction volume) were treated with 2 µl 125 mM EDTA (pH 8.0), and then precipitated using 2 µl 3 M sodium acetate (pH 4.6) and 50 µl absolute ethanol for 20 min at room temperature (in the dark). The DNA was recovered by centrifugation, washed with 70 % alcohol, dried and resuspended in 15 µl Hi-Di formamide (Applied Biosystems). Sequencing was performed in an ABI PRISM 3130 Genetic Analyzer. Raw sequences were edited and assembled using the Auto Assembler program. The resulting sequences were used for identification with the help of the NCBI BLASTN program.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The clinical isolates were identified using standard biochemical tests. All were catalase- and coagulase-positive. The isolates took 72 h to grow in mannitol–salt (10 % NaCl) agar medium.
The thermostable nuclease-encoding nuc gene is highly specific for S. aureus. PCR amplification of the nuc gene of VRSA STM2 using the gene-specific primers (Table 1) and the chromosomal DNA preparation yielded a 270 bp amplicon, as expected (data not shown). This result confirmed VRSA STM2 as an S. aureus strain.
PCR amplification of 16S rDNA of the clinical isolates VRSA STM2 and S. aureus MC48 using primers specific for staphylococcal 16S rRNA (Table 1) produced an identical 1.5 kb amplicon of 16S rDNA in both the strains (Woo et al., 2003). The VRSA STM2 with a vancomycin MIC of 1024 µg ml–1 was named STM2-I (induced STM2). An identical 1.5 kb amplicon was also obtained from STM2-I and the transconjugant T48. These PCR results again confirmed the identity of MC48, STM2, STM2-I and T48 as S. aureus.
Antimicrobial susceptibility tests
On initial testing, the growth of VRSA STM2 on the NA screen plate containing 32 µg vancomycin ml–1 suggested possible vancomycin resistance. Further confirmation of vancomycin resistance was obtained from the DAD test, since no zone of inhibition surrounding the vancomycin disc was noted.
The VRSA STM2 isolate was resistant to many antibiotics, i.e. amoxicillin, ampicillin, cefepime, cefotaxime, cefuroxime, cephalexin, chloramphenicol, clindamycin, erythromycin, meticillin, rifampicin, roxithromycin, streptomycin, trimethoprim-sulfamethoxazole and vancomycin. However, it was susceptible to gentamicin and ciprofloxacin, as determined by the DAD test (Table 2).
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Plasmid analysis
The plasmid profile of VRSA STM2 revealed five plasmids: one large plasmid of 53.4 kb, and four small plasmids of 6, 5.5, 5.1 and 1.5 kb, when compared with the molecular size of plasmid markers of the reference strain E. coli V517 (Fig. 1).
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). However, no transconjugant colony was found on the above LA medium with VRSA STM2 as the donor and vancomycin-sensitive ciprofloxacin-resistant S. aureus MC50 (Table 2) as the recipient. No growth of recipient S. aureus strains MC48 and MC50, and the donor strain VRSA STM2, was observed on the above LA medium when inoculated separately. A selected transconjugant (named S. aureus T48) was observed to grow even after three successive overnight passages on NA plates containing 32 µg vancomycin ml–1. This indicated successful and stable transfer of vancomycin resistance from the clinical isolate VRSA STM2 donor to another clinical isolate S. aureus MC48 as recipient. While the MIC of vancomycin for the transconjugant T48 was 32 µg ml–1, that of the recipient S. aureus MC48 strain was 2 µg ml–1.
PCR-based detection of vancomycin-resistance genes (vanHAX, vanH, vanA and vanX) and mecA in VRSA STM2
Initially, genomic DNA was used as template for PCR amplification of vanHAX, vanH and vanX, using primers specific for vanHAX (2.6 kb), vanH (969 bp) and vanX (609 bp) (Donabedian et al., 2000), as shown in Table 1. PCR products of 2.6 kb for vanHAX, 1 kb for vanH and 600 bp for vanX were obtained, which suggests the presence of the vanHAX gene cassette in VRSA STM2.
Later, the plasmid preparation obtained using the QIAGEN kit was used as template for PCR amplification of vanHAX (2.61 kb), vanH (969 bp), vanA (1032 bp) and vanX (609 bp) with the appropriate primers. Amplicons of 2.6 kb for vanHAX, 1 kb for vanH, 1.1 kb for vanA and 500 bp for vanX were obtained (Fig. 2). The results confirmed the possession of van genes by the plasmid(s) of VRSA STM2. Most probably, the genomic DNA preparation had plasmid DNA contamination, and that was the reason for obtaining PCR products of vanHAX using genomic DNA as template.
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). Only in the case of the 53.4 kb plasmid were the same amplicons obtained as by PCR using the whole plasmid preparation (containing all the plasmids of different sizes) as template (Fig. 2). This result clearly established that the 53.4 kb plasmid harbours the vancomycin-resistance gene cluster vanHAX in VRSA STM2.
A 2.3 kb amplicon was also obtained from STM2-I and transconjugant T48 using primers specific for the vanHAX gene cassette. A 1 kb amplicon was obtained from VRSA STM2, STM2-I and transconjugant T48 using primers specific for the vanH gene (969 bp) (Fig. 3, Table 1). However, no 2.3 or 1 kb amplicon was observed in the case of VSSA MC48 (Fig. 3). A 600 bp amplicon was also obtained from VRSA STM2, STM2-I and transconjugant T48 using primers specific for the vanX gene (609 bp) (data not shown). However, no such amplicon was observed in the case of VSSA MC48 using these primers. These results suggested the presence of a vanA gene cassette in VRSA STM2 and STM2-I that mediates high-level vancomycin resistance through alterations of the PG layer of the cell wall and cell-wall metabolism.
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) clearly indicated the transfer of the same vancomycin-resistance genes vanHAX from the donor VRSA STM2 to the recipient VSSA MC48. The meticillin resistance of VRSA STM2 was verified by PCR amplification of the mecA gene; an amplicon of 0.3 kb was obtained (data not shown).
Nucleotide sequencing
Nucleotide sequencing of 652 bases (GenBank accession no. EU019995) with the forward primer of the 1 kb PCR product of vanH showed 47 % homology with the nucleotide sequence of the VanH dehydrogenase of the Tn1546-like element, and 44 % homology with Tn1549 (Garnier et al., 2000). VanH dehydrogenase reduces pyruvate to lactate, one of the building blocks of the PG layer of the cell wall of S. aureus. VanH dehydrogenase, which is functionally like lactate dehydrogenase, is a common metabolic enzyme required for glycolysis and the tricarboxylic acid (TCA) cycle (carbohydrate metabolism), and is ubiquitous in nature. Accordingly, in the VRSA STM2 strain, a mechanism exists that facilitates the expression of VanH dehydrogenase and VanA ligase to form D-Ala-D-lactate. Nucleotide sequencing of 272 bases with the reverse primer of the 2.3 kb PCR product of vanHAX produced a sequence (GenBank accession no. EU016096) that had 45 % homology with, and a region of 21 bases identical to, the Tn1546-like element that contains the vanHAX gene cluster. This sequence also showed 56 % homology with Tn1549, which is responsible for vanB-type vancomycin resistance in Ent. faecalis (Garnier et al., 2000). However, the PCR product vanA (Fig. 2) was obtained using the 53.4 kb plasmid of VRSA STM2 as template and the primers specific for vanA of the Tn1546-like element. The vanA gene is absent in the vanB operon of Tn1549, which has vanR, -S, -H, -B and -X. Accordingly, we may conclude that the vancomycin resistance exhibited by VRSA STM2 is of the vanA type due to the presence of vanHAX analogous to that of the Tn1546-like element. These facts suggest that the VRSA isolate STM2 could have a modified van gene cassette that confers vancomycin resistance. The occurrence of partial sequence similarity might be due to multiple mutations that occurred during multiple replication (many thousand times) of bacterial genes having species specificity and species diversity, or during inter-species mobilization of resistance genes or gene acquisition. Modification of the van gene complex in the Pennsylvania VRSA isolate (the second documented clinical VRSA isolate) has been reported by Clark et al. (2005); it has a deletion of 3098 bp, and two insertions of 809 and 1499 bp. Modification of the van gene complex in the New York VRSA isolate (the third documented clinical VRSA isolate) has also been reported by Weigel et al. (2007).
Intergeneric transfer of high-level vancomycin resistance from Ent. faecalis to S. aureus has been demonstrated by Noble et al. (1992). Intrageneric transfer of such high-level vancomycin and other antibiotic resistance from S. aureus to S. aureus has also been reported (Severin et al., 2004; Pawa et al., 2000). However, in this study, we have demonstrated intrageneric transfer of vancomycin resistance from a clinical strain of S. aureus to another clinical isolate of S. aureus, which has alarming implications for the global dissemination of such high-level glycopeptide resistance in clinical settings with no known effective antibiotic therapy for some Gram-positive bacterial infections.
In summary, the major new findings of the present study are:
(1) This is believed to be the first report of VRSA containing mecA and the vanA gene complex from Kolkata, India, as well as South Asia.
(2) The vancomycin resistance of the clinical isolate VRSA STM2 is inducible in vitro.
(3) PCR amplification and nucleotide sequencing experiments suggest the presence of a vanHAX-type gene cluster, analogous to that of the Tn1546-like element, in VRSA STM2 and transconjugant T48.
(4) The vancomycin resistance obtained in VRSA STM2 is plasmid-mediated and transferable to a sensitive clinical isolate (as found for VSSA MC48).
The emergence of inducible VRSA in Kolkata, India (as well as in South Asia), and its intrageneric transfer is alarming. This may soon become a global problem, unless antimicrobial agents are used more prudently. Scientists, clinicians and other healthcare professionals should identify and report VRSA promptly for appropriate care and treatment of patients, and start to implement infection-control precautions to prevent the spread of VRSA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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ynarczyk, A., Mynarczyk, G. & 
uczak, M. (2002). Conjugative transfer of glycopeptide and macrolide resistant genes among enterococci and from Enterococcus faecalis to Staphylococcus aureus. Med Dosw Mikrobiol 54, 21–28 (in Polish).[Medline]This article has been cited by other articles:
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  B. Perichon and P. Courvalin VanA-Type Vancomycin-Resistant Staphylococcus aureus Antimicrob. Agents Chemother., November 1, 2009; 53(11): 4580 - 4587. [Abstract] [Full Text] [PDF]
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C. Moubareck, D. Meziane-Cherif, P. Courvalin, and B. Perichon VanA-Type Staphylococcus aureus Strain VRSA-7 Is Partially Dependent on Vancomycin for Growth Antimicrob. Agents Chemother., September 1, 2009; 53(9): 3657 - 3663. [Abstract] [Full Text] [PDF]
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 A. Lulitanond, C. Engchanil, P. Chaimanee, M. Vorachit, T. Ito, and K. Hiramatsu The First Vancomycin-Intermediate Staphylococcus aureus Strains Isolated from Patients in Thailand J. Clin. Microbiol., July 1, 2009; 47(7): 2311 - 2316. [Abstract] [Full Text] [PDF]
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X174 DNA HaeIII digest as size and mass standard; 2, 3 and 4, amplicons of vanHAX of STM2, STM2-I and T48, respectively; 5 and 9, MC48; 6, 7 and 8, amplicons of vanH of STM2, STM2-I and T48, respectively.