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

We characterized 9 New Delhi metallo-ß-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 ß-lactams, fluoroquinolones, and aminoglycosides, demonstrated MICs ≤1 µg/mL of colistin and polymyxin, and yielded positive metallo-ß-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 ß-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-ß-lactamase plasmids, including 2 from the same patient, were diverse.

Dissemination of an Enterococcus Inc18-Like vanA plasmid associated with vancomycin-resistant Staphylococcus aureus.

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.

Vancomycin-resistant Staphylococcus aureus isolates associated with Inc18-like vanA plasmids in Michigan.

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.

High-level vancomycin-resistant Staphylococcus aureus isolates associated with a polymicrobial biofilm.

Glycopeptides such as vancomycin are the treatment of choice for infections due to methicillin-resistant Staphylococcus aureus. This study describes the identification of high-level vancomycin-resistant S. aureus (VRSA) isolates in a polymicrobial biofilm within an indwelling nephrostomy tube in a patient in New York. S. aureus, Enterococcus faecalis, Enterococcus faecium, Micrococcus species, Morganella morganii, and Pseudomonas aeruginosa were isolated from the biofilm. For VRSA isolates, vancomycin MICs ranged from 32 to >128 microg/ml. VRSA isolates were also resistant to aminoglycosides, fluoroquinolones, macrolides, penicillin, and tetracycline but remained susceptible to chloramphenicol, linezolid, rifampin, and trimethoprim-sulfamethoxazole. The vanA gene was localized to a plasmid of approximately 100 kb in VRSA and E. faecium isolates from the biofilm. Plasmid analysis revealed that the VRSA isolate acquired the 100-kb E. faecium plasmid, which was then maintained without integration into the MRSA plasmid. The tetracycline resistance genes tet(U) and tet(S), not previously detected in S. aureus isolates, were identified in the VRSA isolates. Additional resistance elements in the VRSA isolate included a multiresistance gene cluster, ermB-aadE-sat4-aphA-3, msrA (macrolide efflux), and the bifunctional aminoglycoside resistance gene aac(6')-aph(2")-Ia. Multiple combinations of resistance genes among the various isolates of staphylococci and enterococci, including vanA, tet(S), and tet(U), illustrate the dynamic nature of gene acquisition and loss within and between bacterial species throughout the course of infection. The potential for interspecies transfer of antimicrobial resistance genes, including resistance to vancomycin, may be enhanced by the microenvironment of a biofilm.

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.

Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus.

Vancomycin is usually reserved for treatment of serious infections, including those caused by multidrug-resistant Staphylococcus aureus. A clinical isolate of S. aureus with high-level resistance to vancomycin (minimal inhibitory concentration = 1024 microg/ml) was isolated in June 2002. This isolate harbored a 57.9-kilobase multiresistance conjugative plasmid within which Tn1546 (vanA) was integrated. Additional elements on the plasmid encoded resistance to trimethoprim (dfrA), beta-lactams (blaZ), aminoglycosides (aacA-aphD), and disinfectants (qacC). Genetic analyses suggest that the long-anticipated transfer of vancomycin resistance to a methicillin-resistant S. aureus occurred in vivo by interspecies transfer of Tn1546 from a co-isolate of Enterococcus faecalis.