Lenore L. Carias

A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin.

An open reading frame (hyl(Efm)) with homologies to previously described hyaluronidase genes has been identified in nonstool isolates of Enterococcus faecium. E. faecium isolates (n=577) from diverse sources were screened for the presence of hyl(Efm) and esp(Efm), a putative virulence gene associated with epidemic E. faecium strains. The presence of esp(Efm) was roughly twice that of hyl(Efm), but both were found primarily in vancomycin-resistant E. faecium isolates in nonstool cultures obtained from patients hospitalized in the United States. These data suggest that specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Differences in the prevalence of these strains may help explain variations in the clinical importance of multiresistant E. faecium across different continents.

High-Level Expression of Chromosomally Encoded SHV-1 β-Lactamase and an Outer Membrane Protein Change Confer Resistance to Ceftazidime and Piperacillin- Tazobactam in a Clinical Isolate of Klebsiella pneumoniae

ABSTRACT We describe Klebsiella pneumoniae 15571, a clinical isolate resistant to ceftazidime MIC = 32 μg/ml) and piperacillin-tazobactam (MICs = 1,024 and 128 μg/ml). K. pneumoniae 15571 expresses a single β-lactamase with a pI of 7.6. However, when cloned in a high-copy-number vector inEscherichia coli, this blaSHV-1gene did not confer resistance to ceftazidime, a spectrum consistent with the nucleotide sequence, which was nearly identical to those of previously described blaSHV-1 genes. Outer membrane protein (OMP) analysis of K. pneumoniae 15571 revealed a decrease in the quantity of a minor 45-kDa OMP in comparison to that in K. pneumoniae 44NR, a low-level ampicillin-resistant strain that also expresses a chromosomally determined blaSHV-1. Crude β-lactamase enzyme extracts from K. pneumoniae 15571 produced roughly 200-fold more β-lactamase activity than K. pneumoniae 44NR. Northern hybridization analysis revealed that this difference was explainable by quantifiable differences in transcription of theblaSHV-1 gene in the two strains. Primer extension analysis of blaSHV-1 mRNA fromK. pneumoniae 15571 and 44NR indicated that the transcriptional start sites were identical in the two strains. DNA sequencing of the promoter regions upstream of the ofblaSHV-1 open reading frames in the twoK. pneumoniae strains revealed an A→C change in the second position of the −10 region in K. pneumoniae 44NR compared to that in 15571. Site-directed mutagenesis of the clonedK. pneumoniae 15571 blaSHV-1, in which the A in the second position of the 15571 −10 region was changed to a C, resulted in a substantial lowering of the MIC of ampicillin. When the levels of β-lactamase enzyme expression inE. coli were compared, the blaSHV-1downstream of the altered −10 region produced 17-fold less β-lactamase enzyme. These results indicate that elevated levels of ceftazidime resistance can result from a combination of increased enzyme production and minor OMP changes and that levels of chromosomally encoded SHV-1 β-lactamase production can vary substantially with a single-base-pair change in promoter sequence.

Presence of Plasmid-Mediated Quinolone Resistance in Klebsiella pneumoniae Isolates Possessing blaKPC in the United States

ABSTRACT The presence of plasmid-mediated quinolone resistance genes [i.e., qnrA, qnrB, qnrS, aac(6′)-Ib-cr, and qepA] was evaluated among 42 blaKPC-containing Klebsiella pneumoniae isolates collected in the eastern United States. One isolate carried the blaKPC-3 and qnrB19 genes on the same conjugative plasmid, whereas another carried the blaKPC-3 and qnrA1 genes on separate plasmids.

Impact of Specific pbp5 Mutations on Expression of β-Lactam Resistance in Enterococcus faecium

ABSTRACT We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all β-lactams. Affinity for penicillin generally correlated with β-lactam MICs for the mutants, but these associations were not strictly proportional.

Penicillin-Binding Protein 5 and Expression of Ampicillin Resistance in Enterococcus faecium

ABSTRACT We report a structural and transcriptional analysis of thepbp5 region of Enterococcus faecium C68.pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reportedpsr (penicillin-binding protein synthesis repressor) andftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW(ftsWEfm) alone; (ii) psr andpbp5; (iii) pbp5 alone; and (iv)ftsWEfm, psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from theftsWEfm promoter through the pbp5gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faeciumD344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF(pCWR558) and D344-SRF(pCWR583) were 64 μg/ml. Quantities of pbp5transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor ofpbp5 transcription. However, quantities of psrtranscript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.

The KQ Element, a Complex Genetic Region Conferring Transferable Resistance to Carbapenems, Aminoglycosides, and Fluoroquinolones in Klebsiella pneumoniae

ABSTRACT The blaKPC-3 and qnrB19 determinants of transferable Klebsiella pneumoniae plasmid pLRM24 reside within a complex region consisting of a Tn1331 backbone into which a Tn4401-like element and qnrB19 mobilized by an adjacent ISEcp1 insertion sequence have been inserted. This novel element represents a coalescence of genes conferring multidrug resistance in K. pneumoniae.

Transferable Capacity for Gastrointestinal Colonization in Enterococcus faecium in a Mouse Model

A high level of gastrointestinal colonization frequently precedes invasive infection due to Enterococcus faecium. Factors other than antimicrobial resistance that promote gastrointestinal colonization by E. faecium have not been identified. We tested the ability of a colonization-proficient clinical E. faecium isolate (C68) to transfer colonizing ability to noncolonizing E. faecium recipient strains. Transconjugants derived from matings that used E. faecium D344SRF as a recipient strain colonized mouse gastrointestinal tracts in high numbers under selective pressure from clindamycin or vancomycin, compared with control strains that lacked DNA transferred from C68. We transferred DNA into a second recipient strain (E. faecium GE-1), which also colonized mice in significantly greater numbers under selective pressure from clindamycin, compared with a control strain. These results indicate that E. faecium clinical isolates express transmissible factors other than antimicrobial resistance that promote colonization of the mouse gastrointestinal tract.

Enterococcus faecium Low-Affinity pbp5 Is a Transferable Determinant

ABSTRACT Using 15 unrelated Enterococcus faecium isolates as donors, we demonstrated that ampicillin resistance was transferable to an E. faecium recipient containing a pbp5 deletion for all but four strains. The transfers occurred at low frequencies (generally ca. 10−9 transconjugants/recipient CFU), consistent with chromosome-to-chromosome transfer. pbp5 transfer occurred within large genetic regions, and insertion into the recipient genome occurred most commonly into the recipient SmaI restriction fragment that had been created by the previous pbp5 deletion. Restriction mapping of the region upstream of pbp5 revealed a commonality of fragment sizes among the clinical isolates from the United States which differed significantly from those of three strains that were isolated from turkey feces. These data prove conclusively that E. faecium pbp5 is a transferable determinant, even in the absence of a coresiding vancomycin resistance mobile element. They also suggest that the spread of high-level ampicillin resistance among U.S. E. faecium strains is due in part to the transfer of low-affinity pbp5 between clinical isolates.

In vivo efficacies of beta-lactam-beta-lactamase inhibitor combinations against a TEM-26-producing strain of Klebsiella pneumoniae.

We examined the efficacies of the beta-lactam-beta-lactamase inhibitor combinations ampicillin-sulbactam and piperacillin-tazobactam in the treatment of intra-abdominal abscesses caused by a TEM-26-producing strain of Klebsiella pneumoniae. At lower doses, both combinations reduced abscess colony counts by more than 3 log10 CFU/g from that of untreated controls, but treatment with these drugs was inferior to treatment with imipenem. Increasing the doses of the combinations resulted in a further decrease in abscess CFU to a level where both were similar to imipenem in efficacy. These results suggest that the beta-lactam-beta-lactamase inhibitor combinations ampicillin-sulbactam and piperacillin-tazobactam may be viable alternatives for the treatment of serious infections caused by susceptible extended-spectrum beta-lactamase-producing strains of K. pneumoniae.

Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in Enterococcus faecium

Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of beta-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the DeltapbpF DeltaponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the L,D-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the DeltapbpF DeltaponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different beta-lactam antibiotics differed as a function of its partner glycosyltransferase.