Whole-Genome Analyses of Enterococcus faecium Isolates with Diverse Daptomycin MICs

Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a “last-resort” antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120→Ala and Trp73→Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.     

Genotypic and Phenotypic Evaluation of the Evolution of High-Level Daptomycin Nonsusceptibility in Vancomycin-Resistant Enterococcus faecium

Whole-genome sequencing and cell membrane studies of three clonal Enterococcus faecium strains with daptomycin MICs of 4, 32, and 192 μg/ml were performed, revealing nonsynonymous single nucleotide variants in eight open reading frames, including those predicted to encode a phosphoenolpyruvate-dependent, mannose-specific phosphotransferase system, cardiolipin synthetase, and EzrA. Membrane studies revealed a higher net surface charge among the daptomycin-nonsusceptible isolates and increased septum formation in the isolate with a daptomycin MIC of 192 μg/ml.     

Association of Alterations in ParC and GyrA Proteins with Resistance of Clinical Isolates of Enterococcus faecium to Nine Different Fluoroquinolones

The parC and gyrA genes of 73 ciprofloxacin-resistant and 6 ciprofloxacin-susceptible Enterococcus faecium clinical isolates were partly sequenced. Alterations in ParC and GyrA, possibly in combination with other resistance mechanisms, severely restricted the in vitro activities of the nine quinolones tested. For all isolates, clinafloxacin and sitafloxacin showed the best activities.     

High Prevalence of vanM in Vancomycin-Resistant Enterococcus faecium Isolates from Shanghai,China

The vanM gene was first found in a vancomycin-resistant Enterococcus faecium (VREm) isolate in Shanghai in 2006. In this study, we found that, in 70 VREm strains isolated in nine Shanghai hospitals from 2006 to 2014, vanM was more prevalent than the vanA gene (64.3% [45/70] versus 35.7% [25/70]). The vanM-type isolates showed similar antimicrobial susceptibility patterns with the vanA types. The vanM-type VREm emerged and disseminated in Shanghai.     

Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis

OBJECTIVES: The enterococcal surface protein gene, esp, is a major putative pathogenicity marker in clinical isolates of Enterococcus faecium and Enterococcus faecalis. This study demonstrates in vitro conjugative transfer of the esp gene among E. faecium and E. faecalis. MATERIALS AND METHODS: Enterococcal isolates from clinical samples, positive for esp, were mated on filters with enterococcal recipients. Transconjugants were checked for transfer of antibiotic resistance determinants and co-mobilization of the esp gene. They were also characterized by PCR and plasmid profiling/PFGE typing including Southern hybridizations with labelled esp probes. Transfer as triggered by excision was tested using Taqman PCR. RESULTS: Two of five E. faecalis and five of nine E. faecium transferred antibiotic resistance determinants into a recipient. Of the transconjugants analysed by PCR for acquisition of esp, only isolates from two E. faecalis and a single E. faecium mating were positive. In the donor strains, the esp gene was located on the chromosome. Molecular analysis revealed a plasmid localization of esp in the E. faecium transconjugant and chromosome-to-chromosome transfer in E. faecalis. CONCLUSION: The esp gene is transferable by conjugation among enterococcal isolates.     

Fosfomycin Synergy In Vitro with Amoxicillin,Daptomycin, and Linezolid against Vancomycin-Resistant Enterococcus faecium from Renal Transplant Patients with Infected Urinary Stents

Fosfomycin is a potential option for vancomycin-resistant enterococcus (VRE) infections despite limited in vitro and clinical data. In this study, 32 VRE isolates from renal transplant patients with urinary stent infections were susceptible to fosfomycin, daptomycin, and linezolid and resistant to amoxicillin, minocycline, and nitrofurantoin based on their MIC₅₀s and MIC₉₀s. Fosfomycin was bacteriostatic at 0.5 to 16× the MIC (32 to 2,048 μg/ml); synergy occurred when fosfomycin was combined with daptomycin (2.8 to 3.9 log₁₀ CFU/ml kill; P < 0.001) or amoxicillin (2.6 to 3.4; P < 0.05). These combinations may be potent options to treat VRE urinary infections pending investigation of clinical efficacy.     

Bac 32, a novel bacteriocin widely disseminated among clinical isolates of Enterococcus faecium

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates that had been obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were tested for bacteriocin production. Two hundred seventy-seven (44%) of the strains were bacteriocinogenic; and 193 of these exhibited activity against Enterococcus faecium, Enterococcus hirae, and Enterococcus durans. Strain VRE200 harbors the highly efficient conjugative gentamicin resistance plasmid pG200 (70 kb) and bacteriocin plasmid pTI1 (12.5 kb). The bacteriocin encoded on pTI1 was designated bacteriocin 32 (Bac 32). Bacteriocin 32 was active against E. faecium, E. hirae, and E. durans but showed no activity against Listeria monocytogenes. The Bac 32 genetic locus consists of a bacteriocin gene (bacA) and an immunity gene (bacB). Neither of these genes showed significant homology to any known bacteriocin determinants. The deduced bacA product is 89 amino acids in length, with a putative signal peptide of 19 amino acids at the N terminus. The bacB gene encodes a deduced 55-amino-acid protein without a signal sequence. One hundred eighty-nine strains (97.9%) of the 193 strains with activity against the 3 test enterococcal strains gave rise to the expected specific PCR product with a primer specific for bacA, indicating that there is a high incidence of Bac 32 production among VRE clinical isolates. Data from Southern analyses of plasmid DNA from 189 of the Bac 32-producing strains with a plasmid pTI1-specific probe suggested that 137 (72.5%) of the strains harbored a pTI1-type plasmid. Bac 32 or Bac 32-type bacteriocin activity and the determinant genes were also identified in 22 (39.3%) of a total of 56 vancomycin-sensitive E. faecium clinical isolates, which suggests that this bacteriocin is widely disseminated among E. faecium strains.     

Comparison of Glycopeptide-Resistant Enterococcus faecium Isolates and Glycopeptide Resistance Genes of Human and Animal Origins

One hundred thirty-two glycopeptide-resistant Enterococcus faecium (GREF) isolates from different hospitals and pig and poultry farms in Belgium were compared on the basis of (i) their antibiotic susceptibilities, (ii) their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and (iii) the organization of their Tn1546 or related elements in order to detect possible phenotypic and genotypic relationships among both groups of isolates. Human and animal vanA-positive GREF isolates were found to have similar susceptibility patterns; they remained susceptible to gentamicin and were, in general, susceptible to ampicillin. PFGE demonstrated a very high degree of genomic heterogeneity in both groups of isolates. However, indistinguishable isolates were found within different farms or hospitals, and in two instances, epidemiologically unrelated pig and human isolates showed indistinguishable PFGE patterns. In total, eight different transposon types were identified, and all were related to the prototype transposon Tn1546. The two predominant types, Tn1546 and type 2 transposons, which differed at three band positions, were present in both human and animal isolates. Type 2 transposons were significantly associated with pig isolates. The other types were seldom detected. These data suggest a possible exchange of glycopeptide resistance markers between animals and humans.     

Clonal distribution of vancomycin-resistant Enterococcus faecium in Turkey and the new singleton ST733

BackgroundThe aim of this study was to provide information about the spread and characteristics of the vancomycin‐resistant Enterococcus faecium isolates (VREfm) in Turkey.MethodsSeventy‐one nonduplicate consecutive isolates of VREfm were obtained from various clinical specimens of inpatients treated at university or training hospitals in seven regions of Turkey. Further characteristics included antibiotic susceptibility testing, pulsed‐field gel electrophoresis (PFGE) of SmaI‐digested genomic DNA, and multilocus sequence typing (MLST) of selected isolates. The presence of vancomycin resistance and virulence genes (esp and hyl) was investigated by polymerase chain reaction (PCR).ResultsAll VREfm isolates had MICs to vancomycin of ≥32 mg/L and contained the vanA gene. The presence of esp gene was identified in 64 and hyl in eight VREfm isolates. All VREfm showed the multiresistance phenotype, including ampicillin (99%), penicillin (99%), imipenem (99%), ciprofloxacin (87%), moxifloxacin (87%), erythromycin (97%), streptomycin (86%), gentamicin (82%), tetracycline (70%), and teicoplanin (99%). All were susceptible to tigecycline while quinupristin‐dalfopristin (97%) and linezolid (93%) were the most active other agents. Analysis of the PFGE profiles showed that 53 (74.6%) VREfm isolates shared a similar electrophoretic profile, designed as type 1, and were closely related (>85%). The sequence type was identified by MLST in 44 VRE isolates with unrelated or closely related PFGE patterns. MLST revealed that nosocomial spread of VREfm resulted from dissemination of lineage C1 E faecium clones. Sequence types ST78, ST203, and ST117 were the most frequently isolated. This is the first report of ST733 around the world.ConclusionsLineage C1 clones are disseminated among clinical VREfm isolates in seven different regions in Turkey. Regarding VREfm isolates, the worldwide epidemic strains are in circulation in Turkey.     

Daptomycin or teicoplanin in combination with gentamicin for treatment of experimental endocarditis due to a highly glycopeptide-resistant isolate of Enterococcus faecium.

Using an experimental endocarditis model, we studied the activity of daptomycin used alone or in combination with gentamicin against an Enterococcus faecium strain that was highly resistant to glycopeptides and susceptible to gentamicin. In vitro, the MIC of daptomycin was 1 micrograms/ml. In vivo, daptomycin appeared to be effective only when it was used in a high-dose regimen, i.e., 12 mg/kg of body weight every 8 h (-2.5 log10 CFU/g versus controls; P < 0.05), particularly when it was combined with gentamicin (-5.0 log10 CFU/g versus controls; P < 0.01). Since the distribution of daptomycin into cardiac vegetations, as evaluated by autoradiography, appeared to be homogeneous, the poor in vivo activity of daptomycin was considered to be related to its high degree of protein binding, as suggested by killing curves studies. Since the MIC of teicoplanin for the vancomycin-resistant E. faecium strain used in the study was only 64 micrograms/ml and since an in vitro synergy between teicoplanin at high dose and gentamicin was observed, a high-dose regimen of teicoplanin, i.e., 40 mg/kg every 12 h, was also assessed in vivo. This treatment provided marginal activity only when it was combined with gentamicin (-2.3 log10 CFU/g versus controls; P < 0.05). These results suggest that the levels of daptomycin or teicoplanin in serum required to cure experimental endocarditis caused by a highly glycopeptide-resistant strain of E. faecium would not be achievable in humans.     

Novel antibiotic regimens against Enterococcus faecium resistant to ampicillin, vancomycin, and gentamicin.

Enterococci have emerged as significant nosocomial pathogens. Enterococci with resistance to commonly used antibiotics are appearing more frequently. We encountered at our institution several infections caused by Enterococcus faecium with high-level resistance to ampicillin, vancomycin, and gentamicin. The optimal antibiotic therapy for serious infections with unusually resistant enterococci has not been established. Using time-kill studies, we tested the effectiveness of various antibiotic combinations against 15 isolates of multidrug-resistant enterococci. No antibiotic was consistently effective when used alone. The combination of ampicillin plus ciprofloxacin was bactericidal for the 12 isolates for which the ciprofloxacin MIC was < or = 8 micrograms/ml. The combination of ciprofloxacin plus novobiocin also demonstrated activity against these isolates. No combination was found to be bactericidal for the remaining three isolates, which were highly ciprofloxacin resistant. These antibiotic combinations may be important for the future treatment of serious infections caused by these resistant pathogens.     

Prevalence of Fosfomycin Resistance among CTX-M-Producing Escherichia coli Clinical Isolates in Japan and Identification of Novel Plasmid-Mediated Fosfomycin-Modifying Enzymes

We evaluated the in vitro activity of fosfomycin against a total of 192 CTX-M β-lactamase-producing Escherichia coli strains isolated in 70 Japanese clinical settings. Most of the isolates (96.4%) were found to be susceptible to fosfomycin. On the other hand, some of the resistant isolates were confirmed to harbor the novel transferable fosfomycin resistance determinants named FosA3 and FosC2, which efficaciously inactivate fosfomycin through glutathione S-transferase activity.Clinical efficacy of an old antibiotic, fosfomycin, is being reassessed owing to its in vitro high potent activity against multidrug-resistant Gram-negative bacilli belonging to the family Enterobacteriaceae (5, 6, 8). In the present study, we investigated the prevalence of fosfomycin resistance among CTX-M extended-spectrum β-lactamase (ESBL)-producing Escherichia coli clinical isolates in Japan and clarified the molecular mechanisms underlying the fosfomycin resistance, with special focus on exogenous resistance determinants, like the FosA^TN protein (9).A total of 192 CTX-M ESBL-producing E. coli isolates, which were collected from 70 medical facilities throughout Japan between 2002 and 2007, were retrospectively subjected to fosfomycin susceptibility testing with the agar dilution method according to the CLSI guideline (4). The result is shown in Fig. ​Fig.1.1. Most of the strains (96.4%) investigated were susceptible to fosfomycin (MIC, ≤64 μg/ml), while seven isolates (3.6%) showed nonsusceptibility to fosfomycin (MIC, ≥128 μg/ml). It seems likely that CTX-M-producing E. coli isolates that have acquired fosfomycin resistance are infrequent in Japan, and these data suggest the probable clinical efficacy of fosfomycin for the treatment of infectious diseases, like urinary tract infections (UTIs), caused by CTX-M-producing E. coli to some extent.Open in a separate windowFIG. 1.Distribution of fosfomycin MICs for the 192 CTX-M-producing E. coli isolates.We evaluated the fosfomycin resistance mechanism of the 10 isolates and found reduced susceptibility to fosfomycin (MIC, ≥64 μg/ml) (Fig. ​(Fig.11 and Table ​Table1).1). The transmissibility of the fosfomycin resistance determinant in the 10 isolates was investigated, and it was found that the nature of fosfomycin resistance of three strains, 08-642, 06-607, and C316, was successfully transferred to a recipient E. coli strain. The cefotaxime resistance phenotype was cotransferred to a recipient strain with the fosfomycin resistance (Table ​(Table11).

TABLE 1.

Characteristics of E. coli strains used in the study

Isolation of a beta-lactamase-producing, aminoglycoside-resistant strain of Enterococcus faecium.

Beta-Lactamase-producing, aminoglycoside-resistant strains of Enterococcus faecalis have been isolated from different geographic areas and are endemic at our institution. We report the isolation of a beta-lactamase-producing, aminoglycoside-resistant strain of E. faecium. The beta-lactamase was plasmid mediated and transferable with high frequency into a plasmid-free E. faecalis recipient strain. MICs suggested that the E. faecium strain also contained intrinsic (chromosomal) resistance to penicillins.     

Whole-Genome Analysis of a Daptomycin-Susceptible Enterococcus faecium Strain and Its Daptomycin-Resistant Variant Arising during Therapy

Development of daptomycin (DAP) resistance in Enterococcus faecalis has recently been associated with mutations in genes encoding proteins with two main functions: (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase [cls]). However, the genetic bases for DAP resistance in Enterococcus faecium are unclear. We performed whole-genome comparative analysis of a clinical strain pair, DAP-susceptible E. faecium S447 and its DAP-resistant derivative R446, which was recovered from a single patient during DAP therapy. By comparative whole-genome sequencing, DAP resistance in R446 was associated with changes in 8 genes. Two of these genes encoded proteins involved in phospholipid metabolism: (i) an R218Q substitution in Cls and (ii) an A292G reversion in a putative cyclopropane fatty acid synthase enzyme. The DAP-resistant derivative R446 also exhibited an S333L substitution in the putative histidine kinase YycG, a member of the YycFG system, which, similar to LiaFSR, has been involved in cell envelope homeostasis and DAP resistance in other Gram-positive cocci. Additional changes identified in E. faecium R446 (DAP resistant) included two putative proteins involved in transport (one for carbohydrate and one for sulfate) and three enzymes predicted to play a role in general metabolism. Exchange of the “susceptible” cls allele from S447 for the “resistant” one belonging to R446 did not affect DAP susceptibility. Our results suggest that, apart from the LiaFSR system, the essential YycFG system is likely to be an important mediator of DAP resistance in some E. faecium strains.