Low-level Glycopeptide Resistance Research Articles

Prevalence of isolates with reduced glycopeptide susceptibility in orthopedic device-related infections due to methicillin-resistant Staphylococcus aureus

We evaluated, by an improved susceptibility testing method, the prevalence and significance of low-level glycopeptide resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolates, which belonged to a previously described, retrospective cohort of patients treated for orthopedic device-related infections (ODRI) at the Geneva University Hospital between 2000 and 2008. Fifty-seven individual or multiple isolates were retrieved from 41 ODRI patients for glycopeptide susceptibility and clonality studies, including 20 patients with prosthetic joint (PJ) and 21 with osteosynthesis (OS) MRSA infections. Low-level glycopeptide resistance was detected by elevated teicoplanin or/and vancomycin minimum inhibitory concentrations (MICs ≥ 4 mg/L), as determined by a previously validated combination of macrodilution and agar dilution assays of improved sensitivity. MRSA isolates with elevated teicoplanin MICs were detected in 20/41 (49 %) ODRI patients at the onset or during the course of glycopeptide therapy, namely, in 10 of 20 patients with PJ and 10 of 21 patients with OS infections. Only one isolate developed a concomitant increase in vancomycin MIC during therapy. 13/20 (65 %) glycopeptide-intermediate S. aureus (GISA)-infected patients, including 7/10 (70 %) with PJ and 6/10 (60 %) with OS, experienced treatment failure. In contrast, therapy failed in only 5/21 (24 %) ODRI patients with non-GISA isolates (p = 0.012), including 2/10 (20 %) with PJ and 3/11 (27 %) with OS infections. The emergence of low-level teicoplanin resistance could not be explained by teicoplanin administration, since only four patients received teicoplanin. The evaluation of low-level teicoplanin resistance may improve the detection of GISA isolates. Further studies are warranted to evaluate the impact of low-level teicoplanin resistance on the outcome of glycopeptide therapy.

Prevalence and antimicrobial susceptibility pattern of Coagulase-negative Staphylococcus

Background: The vast majority of infections assumed to be caused by Coagulase-negative Staphylococcus (CoNS) are a signifi cant consequence of hospitalization. Current antibiotic-prescribing practices, including preoperative antibiotic prophylaxis, have led to the selection of antibiotic-resistant organisms. The purpose of the present study is to provide a current scenario, species distribution in clinical specimens and antimicrobial susceptibility testing, mainly to screen for methicillin resistance, multi-drug resistant strains. Methodology : Total 113 isolates of Coagulase- negative Staphylococci were isolated from clinical specimens over a 6 month period in a non consecutive non random manner. Antimicrobial resisitance pattern to 20 antimicrobial agents were performed Results : Coagulase-negative Staphylococci consisting of 10 different species were isolated. Staphylococcus epidermidis ( 49), Staphylococcus haemolyticus ( 19) , Staphylococcus hominis ( 11) were the most commonly isolated species. Antimicrobial susceptibility pattern revealed that they were all universally sensitive to vancomycin and linezolid. Methicillin resistance was noted among 48% of the isolates. Maximum resistance was seen with penicillin (91%) and least with Teicoplanin(3%) Conclusion : CoNS are a signifi cant cause of nosocomial bacteremia and their susceptibility to beta-lactamase-stable penicillins is unpredictable. Hence the methicillin resistant CoNS which are also multidrug resistant act as a reservoir for drug resistance in hospitals. Low level glycopeptide resistance encountered in recent years limits its use. For all these reasons, accuracy and promptness in the detection of resistance pattern is of key importance to ensure appropriate antibiotic treatment in infected patients as well as control the spread of resistance in hospital environments.

Whole Genome Sequencing and Complete Genetic Analysis Reveals Novel Pathways to Glycopeptide Resistance in Staphylococcus aureus

The precise mechanisms leading to the emergence of low-level glycopeptide resistance in Staphylococcus aureus are poorly understood. In this study, we used whole genome deep sequencing to detect differences between two isogenic strains: a parental strain and a stable derivative selected stepwise for survival on 4 µg/ml teicoplanin, but which grows at higher drug concentrations (MIC 8 µg/ml). We uncovered only three single nucleotide changes in the selected strain. Nonsense mutations occurred in stp1, encoding a serine/threonine phosphatase, and in yjbH, encoding a post-transcriptional negative regulator of the redox/thiol stress sensor and global transcriptional regulator, Spx. A missense mutation (G45R) occurred in the histidine kinase sensor of cell wall stress, VraS. Using genetic methods, all single, pairwise combinations, and a fully reconstructed triple mutant were evaluated for their contribution to low-level glycopeptide resistance. We found a synergistic cooperation between dual phospho-signalling systems and a subtle contribution from YjbH, suggesting the activation of oxidative stress defences via Spx. To our knowledge, this is the first genetic demonstration of multiple sensor and stress pathways contributing simultaneously to glycopeptide resistance development. The multifactorial nature of glycopeptide resistance in this strain suggests a complex reprogramming of cell physiology to survive in the face of drug challenge.

Site-Specific Mutation of Staphylococcus aureus VraS Reveals a Crucial Role for the VraR-VraS Sensor in the Emergence of Glycopeptide Resistance

An initial response of Staphylococcus aureus to encounter with cell wall-active antibiotics occurs by transmembrane signaling systems that orchestrate changes in gene expression to promote survival. Histidine kinase two-component sensor-response regulators such as VraRS contribute to this response. In this study, we examined VraS membrane sensor phosphotransfer signal transduction and explored the genetic consequences of disrupting signaling by engineering a site-specific vraS chromosomal mutation. We have used in vitro autophosphorylation assay with purified VraS[64-347] lacking its transmembrane anchor region and tested site-specific kinase domain histidine mutants. We identified VraS H156 as the probable site of autophosphorylation and show phosphotransfer in vitro using purified VraR. Genetic studies show that the vraS(H156A) mutation in three strain backgrounds (ISP794, Newman, and COL) fails to generate detectable first-step reduced susceptibility teicoplanin mutants and severely reduces first-step vancomycin mutants. The emergence of low-level glycopeptide resistance in strain ISP794, derived from strain 8325 (ΔrsbU), did not require a functional σ(B), but rsbU restoration could enhance the emergence frequency supporting a role for this alternative sigma factor in promoting glycopeptide resistance. Transcriptional analysis of vraS(H156A) strains revealed a pronounced reduction but not complete abrogation of the vraRS operon after exposure to cell wall-active antibiotics, suggesting that additional factors independent of VraS-driven phosphotransfer, or σ(B), exist for this promoter. Collectively, our results reveal important details of the VraRS signaling system and predict that pharmacologic blockade of the VraS sensor kinase will have profound effects on blocking emergence of cell wall-active antibiotic resistance in S. aureus.

Identification by Genomic and Genetic Analysis of Two New Genes Playing a Key Role in Intermediate Glycopeptide Resistance in Staphylococcus aureus

Endogenous, low-level glycopeptide resistance in Staphylococcus aureus results from multifactorial genetic changes. Comparative genomic hybridization analysis revealed the specific deletion of a 1.8-kb segment encompassing two adjacent open reading frames (ORFs) of unknown function in a teicoplanin-susceptible revertant (strain 14-4rev) compared to the sequence of its isogenic, teicoplanin-resistant parental strain, strain 14-4. This provocative finding prompted us to perform a detailed genetic analysis of the contribution of this genomic segment to glycopeptide resistance. Despite repeated efforts in our laboratory, 14-4 and 14-4rev have proven refractory to most genetic manipulations. To circumvent this difficulty, we evaluated the contribution of both putative ORFs (designated teicoplanin resistance factors trfA and trfB) on teicoplanin resistance in a different, genetically tractable background. Genetic analysis showed that single or double trfA and/or trfB mutations abolished teicoplanin resistance in two independent teicoplanin-resistant derivatives of NCTC8325 strain ISP794 generated by two-step passages with the drug. The frequency of teicoplanin-resistant mutants was markedly decreased by the absence of trfAB in the teicoplanin-susceptible ISP794 background. Nevertheless, a low rate of teicoplanin-resistant mutants was selected from ISP794 trfAB, thus indicating an additional contribution of trfAB-independent pathways in the emergence of low-level glycopeptide resistance. Further experiments performed with clinical glycopeptide-intermediate S. aureus isolate NRS3 indicated that the trfAB mutation could affect not only teicoplanin resistance but also vancomycin and oxacillin resistance. In conclusion, our study demonstrates the key role of two novel loci in endogenous, low-level glycopeptide resistance in S. aureus whose precise molecular functions warrant further investigation.

Synergism between beta-lactams and glycopeptides against VanA-type methicillin-resistant Staphylococcus aureus and heterologous expression of the vanA operon.

Vancomycin resistance of Staphylococcus aureus NY-VRSA and VRSA-5 is due to acquisition of a vanA operon located in a Tn1546-like element. The vanA gene cluster of NY-VRSA contained one copy of insertion sequences IS1251 and IS1216V relative to that of VRSA-5. As evidenced by the nature of the late peptidoglycan precursors and by quantification of d,d-peptidase activities, the vancomycin resistance genes were efficiently expressed in both strains. Study of the stability and inducibility of glycopeptide resistance suggested that low-level glycopeptide resistance of NY-VRSA was most probably due to plasmid instability combined with a long delay for resistance induction. The activity of combinations of vancomycin or teicoplanin with oxacillin against the four VanA-type S. aureus strains already reported was tested by single and double disk diffusion, E-test on agar alone or supplemented with antibiotics, the checkerboard technique, and by determining time-kill curves. A strong synergism against the four clinical isolates, with fractional inhibitory concentration indexes from 0.008 to 0.024, was reproducibly observed between the two antibiotics by all methods. These observations indicate that cell wall inhibitors of the beta-lactam and glycopeptide classes exert strong and mutual antagonistic effects on resistance to each other against VanA-type methicillin-resistant S. aureus.

Heterologous expression of the enterococcal vanA operon in methicillin-resistant Staphylococcus aureus.

Two methicillin- and vancomycin-resistant Staphylococcus aureus strains, MI-VRSA and PA-VRSA, and Enterococcus faecalis DMC83006B, considered to be the potential donor of glycopeptide resistance to MI-VRSA, were studied. MI-VRSA is highly resistant to both glycopeptides, whereas PA-VRSA displays low-level resistance to vancomycin and reduced susceptibility to teicoplanin. We have analyzed the expression of the vanA operon in the three clinical isolates. Determination of the relative amounts of late peptidoglycan precursors and quantification of the d,d-peptidase activities, in the absence or after induction by glycopeptides, revealed that the resistance genes were expressed at similarly high levels in the three strains. Glycopeptide resistance stability in the three strains was studied by replica plating. Resistance was lost at high frequency, ca. 50%, after overnight growth of PA-VRSA in the absence of antibiotics, whereas it was fully stable in MI-VRSA and E. faecalis DMC83006B. Induction of resistance by vancomycin was significantly delayed in PA-VRSA relative to MI-VRSA. Low-level glycopeptide resistance of S. aureus PA-VRSA is thus likely due to instability of the genetic element, plasmid or transposon, carrying the vanA operon associated with a longer lag phase before growth resumes after induction by vancomycin.

Le point sur les staphylocoques dorés de moindre sensibilité aux glycopeptides en réanimation

The emergence of Staphylococcus aureus exhibiting intermediate levels of resistance to glycopeptides (GISA) has been expected for a long time. After 45 years of vancomycin use, low-level glycopeptide resistance emerged in S. aureus after emergence in Enterococcci and in coagulase-negative staphylococci. If the transfer of vancomycin resistance genes from Enterococci to S. aureus is limited to day, in 1997 a strain of methicillin resistant S. aureus (MRSA) with intermediate resistance to vancomycin (VISA) that was associated with therapeutic failure was reported in Japan. This description was followed by reports in Europe, USA and Asia. Difficulties to detect this resistance mechanism are in relation with a heterogeneous phenotype and can be a potential source of treatment failure. The true significance of these isolates is not clear. Some studies indicate outbreaks in hospital units and patients with a poor outcome. Their detection may be of clinical relevance and the finding of GISA cannot be ignored.

Cloning of the Staphylococcus aureus ddh gene encoding NAD+-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate.

The mechanism of low-level glycopeptide resistance among staphylococci is not known. A cytoplasmic protein, provisionally called Ddh (W. M. Milewski, S. Boyle-Vavra, B. Moreira, C. C. Ebert, and R. S. Daum, Antimicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript that contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the susceptible parent isolate, 523. Sequence analysis had previously revealed that Ddh is related to NAD+-dependent D-lactate dehydrogenase (D-nLDH) and VanH. This latter protein is essential for high-level glycopeptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-terminating peptidoglycan precursors with low affinity for vancomycin. We now provide the direct evidence that the ddh gene product is Staphylococcus aureus D-nLDH and hereafter refer to the protein as D-nLDH. However, overproduction of this protein in isolate 523k did not result in production of D-lactate-containing peptidoglycan precursors, and susceptibility testing of ddh mutants of 523k demonstrated that S. aureus D-nLDH is not necessary for glycopeptide resistance in this isolate. We conclude that the mechanism of glycopeptide resistance in this isolate is distinct from that in enterococci.

Comparison of agar-based media for primary isolation of glycopeptide-resistant enterococci

OBJECTIVE: To compare four vancomycin-containing agar media for the isolation of glycopeptide-resistant enterococci (GRE) from clinical fecal specimens: kanamycin---aesculin---azide (KAA) agar; bile---aesculin---polymixin (BAP) agar; aztreonam---amphotericin blood (CBAA) agar; and neomycin blood (CBN) agar. METHODS: Fecal specimens from 125 patients were inoculated onto each medium. Media were examined for enterococci after incubation for up to 48 h. Enterococci were identified to species level, and glycopeptide phenotypes were determined by measuring minimum inhibitory concentrations of vancomycin and teicoplanin. RESULTS: GRE were isolated from 44/125 samples. Enterococcus faecalis and Enterococcus faecium isolates, expressing glycopeptide resistance of the VanA or VanB phenotypes, were recovered from 27/33 (82%) specimens on BAP medium, 26/33 (79%) on KAA medium, and 21/33 (64%) on CBN and CBAA media. Enterococcus gallinarum and Enterococcus casseliflavus isolates expressing low-level glycopeptide resistance (VanC phenotype) were recovered from 14/15 (93%) specimens on CBAA medium, 7/15 (47%) on KAA and CBN media, and 6/15 (40%) on BAP medium. CONCLUSIONS: The media tested in this study, with the exception of CBN medium, detected at least 75% of patients colonized by GRE. Further development of BAP, CBAA and KAA media is warranted to improve growth and selectivity.

Current perspectives on glycopeptide resistance.

In the last 5 years, clinical isolates of gram-positive bacteria with intrinsic or acquired resistance to glycopeptide antibiotics have been encountered increasingly. In many of these isolates, resistance arises from an alteration of the antibiotic target site, with the terminal D-alanyl-D-alanine moiety of peptidoglycan precursors being replaced by groups that do not bind glycopeptides. Although the criteria for defining resistance have been revised frequently, the reliable detection of low-level glycopeptide resistance remains problematic and is influenced by the method chosen. Glycopeptide-resistant enterococci have emerged as a particular problem in hospitals, where in addition to sporadic cases, clusters of infections with evidence of interpatient spread have occurred. Studies using molecular typing methods have implicated colonization of patients, staff carriage, and environmental contamination in the dissemination of these bacteria. Choice of antimicrobial therapy for infections caused by glycopeptide-resistant bacteria may be complicated by resistance to other antibiotics. Severe therapeutic difficulties are being encountered among patients infected with enterococci, with some infections being untreatable with currently available antibiotics.

Current perspectives on glycopeptide resistance.

In the last 5 years, clinical isolates of gram-positive bacteria with intrinsic or acquired resistance to glycopeptide antibiotics have been encountered increasingly. In many of these isolates, resistance arises from an alteration of the antibiotic target site, with the terminal D-alanyl-D-alanine moiety of peptidoglycan precursors being replaced by groups that do not bind glycopeptides. Although the criteria for defining resistance have been revised frequently, the reliable detection of low-level glycopeptide resistance remains problematic and is influenced by the method chosen. Glycopeptide-resistant enterococci have emerged as a particular problem in hospitals, where in addition to sporadic cases, clusters of infections with evidence of interpatient spread have occurred. Studies using molecular typing methods have implicated colonization of patients, staff carriage, and environmental contamination in the dissemination of these bacteria. Choice of antimicrobial therapy for infections caused by glycopeptide-resistant bacteria may be complicated by resistance to other antibiotics. Severe therapeutic difficulties are being encountered among patients infected with enterococci, with some infections being untreatable with currently available antibiotics.

The emergence of vancomycin resistance in renal dialysis

Intraperitoneal vancomycin is used in the treatment of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). We describe the emergence of low-level glycopeptide-resistance in five Gram-positive species over a one-year period. Isolation of these organisms was associated with vancomycin treatment failure in four patients who had had numerous episodes of peritonitis. Clinicians and microbiologists should be aware that repeated administration of glycopeptides to such patients might lead to the emergence of organisms resistant to these antibiotics.