Nancye C. Clark

Vancomycin-Resistant Staphylococcus aureus Isolate from a Patient in Pennsylvania

ABSTRACT 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 μg/ml), aminoglycosides, β-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.

Characterization of glycopeptide-resistant enterococci from U.S. hospitals.

We examined 105 clinical isolates of glycopeptide-resistant enterococci collected from 31 U.S. hospitals in 14 states during May 1988 to July 1992. The isolates included 82 Enterococcus faecium, 8 E. faecalis, 6 Enterococcus spp., 5 E. gallinarum, 3 E. casseliflavus, and 1 E. raffinosus. The isolates were categorized into the following four phenotypes of glycopeptide resistance on the basis of their MIC patterns: (i) 70 VanA (vancomycin [Vm] MIC, > or = 64 micrograms/ml; teicoplanin [Tei] MIC, 16 to > or = 128 micrograms/ml), (ii) 26 VanB (Vm MIC, 16 to 1,024 micrograms/ml; Tei MIC, < or = 2 micrograms/ml), (iii) 5 VanC (Vm MIC, 4 to 16 micrograms/ml; Tei MIC, < or = 2 micrograms/ml) in E. gallinarum, and (iv) 3 E. casseliflavus and 1 E. raffinosus isolates for which Vm MICs were 4 to 16 micrograms/ml and Tei MICs were < or = 1 micrograms/ml were called unclassified. Of the 101 isolates with the VanA, VanB, and VanC phenotypes, 99 were confirmed by production of a specific 1,030-, 433-, or 796-bp polymerase chain reaction product, respectively, and hybridization with the respective gene probe. The vanA gene was also detected in the E. raffinosus isolate for which the Vm MIC was 16 micrograms/ml and the Tei MIC was 1 microgram/ml. The vanA gene was located on either a 34- or a 60-kb plasmid in all of the U.S. isolates examined. Pulsed-field gel electrophoresis demonstrated both intrahospital and interhospital diversity among Vmr enterococci in the United States and was more useful than plasmid analysis for epidemiologic studies. Images

Vancomycin-Resistant Staphylococcus aureus Isolates Associated with Inc18-Like vanA Plasmids in Michigan

ABSTRACT Five of the seven cases of vancomycin-resistant Staphylococcus aureus (VRSA) infection identified to date have occurred in southeastern Michigan. VRSA isolates from the four most recent cases (all from Michigan) were characterized. The vanA gene was localized to a single plasmid in each VRSA isolate. The pulsed-field gel electrophoresis patterns of chromosomal DNA and the restriction profile of the plasmid demonstrated that the four isolates were unique and differed from the first three VRSA isolates. Vancomycin-resistant Enterococcus (VRE) isolates, all of which were Enterococcus faecalis, were recovered from case patients 4 to 6. Each VRE isolate transferred vancomycin resistance to E. faecalis JH2-2 by conjugation. PCRs for vanA and the Inc18-like plasmid genes traA and repR confirmed the presence of an Inc18-like vanA plasmid in all VRE isolates and transconjugants. An Inc18-like vanA plasmid was identified in the VRSA isolate from case patient 7. These findings suggest a role of Inc18-like plasmids as vanA donors.

Comparison of Tn1546-Like Elements in Vancomycin-Resistant Staphylococcus aureus Isolates from Michigan and Pennsylvania

ABSTRACT 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.

Dissemination of an Enterococcus Inc18-Like vanA Plasmid Associated with Vancomycin-Resistant Staphylococcus aureus

ABSTRACT Of the 9 vancomycin-resistant Staphylococcus aureus (VRSA) cases reported to date in the literature, 7 occurred in Michigan. In 5 of the 7 Michigan VRSA cases, an Inc18-like vanA plasmid was identified in the VRSA isolate and/or an associated vancomycin-resistant Enterococcus (VRE) isolate from the same patient. This plasmid may play a critical role in the emergence of VRSA. We studied the geographical distribution of the plasmid by testing 1,641 VRE isolates from three separate collections by PCR for plasmid-specific genes traA, repR, and vanA. Isolates from one collection (phase 2) were recovered from surveillance cultures collected in 17 hospitals in 13 states. All VRE isolates from 2 Michigan institutions (n = 386) and between 60 and 70 VRE isolates (n = 883) from the other hospitals were tested. Fifteen VRE isolates (3.9%) from Michigan were positive for an Inc18-like vanA plasmid (9 E. faecalis [12.5%], 3 E. faecium [1.0%], 2 E. avium, and 1 E. raffinosus). Six VRE isolates (0.6%) from outside Michigan were positive (3 E. faecalis [2.7%] and 3 E. faecium [0.4%]). Of all E. faecalis isolates tested, 6.0% were positive for the plasmid, compared to 0.6% for E. faecium and 3.0% for other spp. Fourteen of the 15 plasmid-positive isolates from Michigan had the same Tn1546 insertion site location as the VRSA-associated Inc18-like plasmid, whereas 5 of 6 plasmid-positive isolates from outside Michigan differed in this characteristic. Most plasmid-positive E. faecalis isolates demonstrated diverse patterns by PFGE, with the exception of three pairs with indistinguishable patterns, suggesting that the plasmid is mobile in nature. Although VRE isolates with the VRSA-associated Inc18-like vanA plasmid were more common in Michigan, they remain rare. Periodic surveillance of VRE isolates for the plasmid may be useful in predicting the occurrence of VRSA.

New Delhi metallo-β-lactamase-producing Enterobacteriaceae, United States.

We characterized 9 New Delhi metallo-β-lactamase–producing Enterobacteriaceae (5 Klebsiella pneumoniae, 2 Escherichia coli, 1 Enterobacter cloacae, 1 Salmonella enterica serovar Senftenberg) isolates identified in the United States and cultured from 8 patients in 5 states during April 2009–March 2011. Isolates were resistant to β-lactams, fluoroquinolones, and aminoglycosides, demonstrated MICs ≤1 µg/mL of colistin and polymyxin, and yielded positive metallo-β-lactamase screening results. Eight isolates had blaNDM-1, and 1 isolate had a novel allele (blaNDM-6). All 8 patients had recently been in India or Pakistan, where 6 received inpatient health care. Plasmids carrying blaNDM frequently carried AmpC or extended spectrum β-lactamase genes. Two K. pneumoniae isolates and a K. pneumoniae isolate from Sweden shared incompatibility group A/C plasmids with indistinguishable restriction patterns and a common blaNDM fragment; all 3 were multilocus sequence type 14. Restriction profiles of the remaining New Delhi metallo-β-lactamase plasmids, including 2 from the same patient, were diverse.

A Cluster of VanD Vancomycin-Resistant Enterococcus faecium: Molecular Characterization and Clinical Epidemiology

VanD-mediated glycopeptide resistance has been reported for an isolate of Enterococcus faecium, BM4339. Three clinical isolates of vancomycin-resistant E. faecium collected from 3 patients during a 6-week period in 1993 had agar dilution MICs of vancomycin and teicoplanin of 128 and 4 microg/mL, respectively. Polymerase chain reaction (PCR) using degenerate primers complementary to genes encoding d-Ala-d-X ligases yielded a 630-bp product that was similar to the published partial sequence of vanD. By use of inverse PCR, vanD, vanHD, and two partial flanking open-reading frames were sequenced. The deduced amino acid sequence of VanD showed 67% identity with VanA and VanB. vanD appeared to be located on the chromosome and was not transferable to other enterococci. The 3 isolates were indistinguishable by pulsed-field gel electrophoresis and differed from BM4339. No other isolates carrying vanD were found in a subset of 875 recent US isolates of vancomycin-resistant enterococci.

Patterns and mechanisms of beta-lactam resistance among isolates of Escherichia coli from hospitals in the United States.

To study the national distribution of beta-lactam resistance patterns and mechanisms among Escherichia coli organisms isolated in U.S. hospitals, 652 ampicillin-resistant (Am(r)) or ampicillin-intermediate (Ami) isolates were submitted to the Centers for Disease Control from March 1983 through July 1984 by nine hospitals participating in the National Nosocomial Infections Study. Among the isolates (most of which caused urinary tract infections), 78% were Am(r) and 22% were Ami by the interpretative criteria established by the National Committee for Clinical Laboratory Standards. Resistance to carboxypenicillins ranged from 73 to 74%, and that to acylureidopenicillins ranged from 43 to 66%. A total of 26% of the isolates were resistant to cephalothin, and 4% were resistant to cefazolin. Resistance to cefoxitin was 1%, while resistances to cefuroxime and cefamandole were 2 and 7%, respectively. With the exception of cefsulodin (98% resistant) and cefoperazone (1% resistant), there was no resistance to newer cephalosporins or aztreonam. In general, only minor differences in the incidence of resistance to beta-lactam antibiotics were noted in hospital-acquired versus non-hospital-acquired isolates as well as among isolates from various regions of the United States. TEM beta-lactamases were produced by 87% of the 237 Am(r) isolates examined. By our methods, OXA and chromosomal (type I) beta-lactamases were detected in 2 and 28 isolates, respectively, and plasmid-mediated extended-spectrum cephalosporinases were detected in none of the isolates. Disk substrate and clavulanic acid inhibition assays revealed that TEM beta-lactamase conferred Am(r) and resistance to carboxypenicillins, acylureidopenicillins, cephalothin, cefamandole, cefsulodin, and cefoperazone. A total of 391 isolates were screened for plasmids, and 259 isolates were examined by DNA hybridization with a TEM probe. Among 462 plasmids probed, 129 plasmids, ranging from 4 to 140 megadaltons, harbored TEM sequences. Although beta-lactam resistance in clinical isolates of E. coli is predominantly mediated by TEM beta-lactamase, the diverse spectrum of resistance appears to be related to additional strain=dependent factors.

A gene probe for TEM type beta-lactamases.

A restriction fragment of plasmid pBR322 bearing the TEM-1 beta-lactamase structural gene was electroeluted from agarose gels after digestion with EcoRI and HinfI. The 1-kilobase fragment was 32P-labeled and used to examine genetic relationships with nucleic acids encoding seven other beta-lactamase classes. The probe hybridized only with TEM-2 and OXA-2 class plasmids. Images

pSK41-Like Plasmid Is Necessary for Inc18-Like vanA Plasmid Transfer from Enterococcus faecalis to Staphylococcus aureus In Vitro

ABSTRACT Vancomycin-resistant Staphylococcus aureus (VRSA) is thought to result from the in vivo conjugative transfer of a vanA plasmid from an Enterococcus sp. to S. aureus. We studied bacterial isolates from VRSA cases that occurred in the United States to identify microbiological factors which may contribute to this plasmid transfer. First, vancomycin-susceptible, methicillin-resistant S. aureus (MRSA) isolates from five VRSA cases were tested for their ability to accept foreign DNA by conjugation in mating experiments with Enterococcus faecalis JH2-2 containing pAM378, a pheromone-response conjugative plasmid. All of the MRSA isolates accepted the plasmid DNA with similar transfer efficiencies (∼10−7/donor CFU) except for one isolate, MRSA8, for which conjugation was not successful. The MRSA isolates were also tested as recipients in mating experiments between an E. faecalis isolate with an Inc18-like vanA plasmid that was isolated from a VRSA case patient. Conjugative transfer was successful for 3/5 MRSA isolates. Successful MRSA recipients carried a pSK41-like plasmid, a staphylococcal conjugative plasmid, whereas the two unsuccessful MRSA recipients did not carry pSK41. The transfer of a pSK41-like plasmid from a successful MRSA recipient to the two unsuccessful recipients resulted in conjugal transfer of the Inc18-like vanA plasmid from E. faecalis at a frequency of 10−7/recipient CFU. In addition, conjugal transfer could be achieved for pSK41-negative MRSA in the presence of a cell-free culture filtrate from S. aureus carrying a pSK41-like plasmid at a frequency of 10−8/recipient CFU. These results indicated that a pSK41-like plasmid can facilitate the transfer of an Inc18-like vanA plasmid from E. faecalis to S. aureus, possibly via an extracellular factor produced by pSK41-carrying isolates.