A comparison of PCR detection of mecA with oxacillin disk susceptibility testing in different media and sceptor automated system for both Staphylococcus aureus and coagulase-negative staphylococci isolates

PURPOSE: To evaluate three methods for 406 isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS) for the detection of methicillin resistance (MR) using National Committee for Clinical Laboratory Standards (NCCLS) new interpretive criteria. METHODS: We used polymerase chain reaction (PCR) as a gold standard method to evaluate three methods [disk diffusion with Mueller-Hinton agar (MHA) and mannitol salt agar (MSA) and Sceptor system (Becton Dickinson, USA)] for the detection of mecA gene. The isolates that were methicillin-resistant with any of the three tests were evaluated further for MR by E-test. RESULTS: MHA, MSA and Sceptor showed sensitivities of 100, 100 and 99% for S. aureus and 100, 82.6 and 72.1% for CNS, respectively. The specificities of the same methods were found as 100, 90.1 and 99.3% for S. aureus and 79.2, 95.8 and 97.2% for CNS, respectively. E-test showed 100% sensitivity for both S. aureus and CNS. Forty-eight CNS and 16 S. aureus isolates, which presented discrepancies with the three phenotypic methods (MHA disk diffusion method, MSA disk diffusion method and Sceptor), were correctly classified as resistant/susceptible with the E-test when compared with PCR. Only five CNS isolates, which were mecA-negative with PCR were resistant with E-test. Analysis of 248 S. aureus revealed that MHA is superior to other phenotype-based susceptibility testing methods in detecting MR. When we examined the results of 158 CNS, none of the three methods proved efficient in detecting MR. CONCLUSIONS: We conclude that although the accuracy of the MHA disk diffusion test for the detection of MR approaches the accuracy of PCR for S. aureus isolates, the need for easy and reliable methods of detecting MR in CNS still remains.     

Sepsis due to linezolid resistant Staphylococcus cohnii and Staphylococcus kloosii: first reports of linezolid resistance in coagulase negative staphylococci from India

Linezolid, a viable alternative to vancomycin against methicillin resistant staphylococcal isolates, has been in use for a decade around the globe. However, resistance against staphylococci remains extremely rare and unreported from most of the Asian countries. Herein, we report two cases of linezolid resistant, coagulase negative staphylococcal sepsis for the first time from India. The first case was an 18-year-old burn patient, who, after a major graft surgery, landed in sepsis, and linezolid resistant Staphylococcus cohnii with an minimum inhibitory concentration (MIC) of >256 μg/ml by both broth microdilution and Etest, was isolated from multiple blood cultures. The second patient was a 60-year-old male with an intracranial bleed and sepsis, from whose blood cultures, linezolid resistant Staphylococcus kloosii was repeatedly isolated. Linezolid MIC was >32 μg/ml by broth microdilution and >16 μg/ml by Etest.     

Trimethoprim resistance determinants encoding a dihydrofolate reductase in clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci

The molecular and biochemical basis of resistance to high concentrations (MIC greater than or equal to 1000 mg/L) of trimethoprim (Tpr(H] was examined in Australian isolates of Staphylococcus aureus and coagulase-negative staphylococci. The Tpr(H) determinant (dfr A) was located within a 2.75-Kb Bg/II fragment on the S. aureus aminoglycoside-resistance plasmids pSK1 and pSK16 as judged by comparative restriction mapping with two naturally-occurring variants, pSK9 and pSK14, which did not encode trimethoprim resistance. This was confirmed in DNA-DNA hybridisation experiments in which a 0.9-Kb sequence of pSK1 DNA was used as a specific probe for the Tpr(H) gene. Plasmid DNA from three strains of coagulase-negative staphylococci, and the chromosomal material of one other isolate, were found to share homology with the probe DNA. Dihydrofolate reductases produced by these strains were virtually identical to the type S1 enzyme encoded by the S. aureus plasmid pSK1. Interspecies transfer may have been responsible for the conservation of Tpr(H) gene sequences among staphylococci.     

Diminished in vitro antibacterial activity of oxacillin against clinical isolates of borderline oxacillin-resistant Staphylococcus aureus

Since it is unknown whether β-lactam antimicrobial agents can be used effectively against borderline oxacillin-resistant Staphylococcus aureus (BORSA) with oxacillin MICs ≥4 mg/L, the in vitro bactericidal activity and pharmacodynamic effect of oxacillin against clinical BORSA isolates was evaluated. Time-kill experiments with oxacillin were performed and the results compared with those obtained with vancomycin, daptomycin and linezolid against BORSA with oxacillin MICs ≥4 mg/L and BORSA with oxacillin MICs ≤2 mg/L. Furthermore, the effect of β-lactamase production and plasmid profile analysis were taken into account to clarify responses to oxacillin. Oxacillin killing activity was attenuated against BORSA compared with ATCC 29213 since the pharmacodynamic parameters revealed that the potency of oxacillin was markedly reduced (c. ten-fold) against BORSA with oxacillin MICs ≥4 mg/L. pBORa53-like plasmid-containing BORSA with oxacillin MICs ≤2 mg/L showed markedly more regrowth. In conclusion, oxacillin was non-effective in the eradication of either (i) BORSA with oxacillin MICs ≥4 mg/L or (ii) β-lactamase-hyperproducing BORSA (MICs ≤2 mg/L). Further investigation into β-lactam dosing strategies against different BORSA strains is warranted in order to avoid possible therapy failure.     

Synergistic effect of linezolid with fosfomycin against Staphylococcus aureus in vitro and in an experimental Galleria mellonella model

Background/purposesTreatment of Staphylococcus aureus infections is challenging owing to widespread multidrug resistance. There is now considerable interest in the potential of combination therapies. Although linezolid/fosfomycin combination appears to be a promising treatment option based on in vitro data, further preclinical work is needed. In this study, the Galleria mellonella system was employed to study the in vivo efficacy of this combination in order to determine whether it should be explored further for the treatment of S. aureus infections.MethodsThe antimicrobial activity of linezolid and fosfomycin alone and in combination was assessed versus four S. aureus. Synergy studies were performed using the microtitre plate chequerboard assay and time-kill methodology. The in vivo activity of linezolid/fosfomycin combination was assessed using a G. mellonella larvae model.ResultsThe combination of linezolid and fosfomycin was synergistic and bacteriostatic against four tested strains. Treatment of G. mellonella larvae infected with lethal doses of S. aureus resulted in significantly enhanced survival rates when low-dose of combination has no significant differences with high-dose combination (P > 0.05), G. mellonella hemolymph burden of S. aureus suggest that combination therapy with rapid and sustained bacteriostatic activity compared monotherapy.ConclusionThis work indicated that linezolid combination with fosfomycin has synergistic effect against S. aureus in vitro and in an experimental G. mellonella model, and it suggests that high-dose of linezolid and fosfomycin may not necessary.     

Impact of psm-mec in the mobile genetic element on the clinical characteristics and outcome of SCCmec-II methicillin-resistant Staphylococcus aureus bacteraemia in Japan

Over-expression of alpha-phenol-soluble modulins (PSMs) results in high virulence of community-associated methicillin-resistant Staphylococcus aureus (MRSA). The psm-mec gene, located in the mobile genetic element SCCmec-II, suppresses PSMαs production. Fifty-two patients with MRSA bacteraemia were enrolled. MRSA isolates were evaluated with regard to the psm-mec gene sequence, bacterial virulence, and the minimum inhibitory concentration (MIC) of vancomycin and teicoplanin. Fifty-one MRSA isolates were classified as SCCmec-II, and 10 had one point mutation in the psm-mec promoter. We compared clinical characteristics and outcomes between mutant MRSA and wild-type MRSA. Production of PSMα3 in mutant MRSA was significantly increased, but biofilm formation was suppressed. Wild-type MRSA caused more catheter-related bloodstream infections (30/41 vs. 3/10, p 0.0028), whereas mutant MRSA formed more deep abscesses (4/10 vs. 3/41, p 0.035). Bacteraemia caused by mutant MRSA was associated with reduced 30-day mortality (1/10 vs. 13/41, p 0.25), although this difference was not significant. The MIC₉₀ of teicoplanin was higher for wild-type MRSA (1.5 mg/L vs. 1 mg/L), but the MIC of vancomycin was not different between the two groups. The 30-day mortality of MRSA with a high MIC of teicoplanin (≥1.5 mg/L) was higher than that of strains with a lower MIC (≤0.75 mg/L) (6/10 vs. 6/33, p 0.017). Mutation of the psm-mec promoter contributes to virulence of SCCmec-II MRSA, and the product of psm-mec may determine the clinical characteristics of bacteraemia caused by SCCmec-II MRSA, but it does not affect mortality.     

In vitro activity of quinupristin/dalfopristin in comparison with five antibiotics against worldwide clinical isolates of staphylococci

Objective   To evaluate the in vitro activity of quinupristin/dalfopristin (Q/D), a streptogramin combination, in comparison with five antibiotics against worldwide clinical isolates of staphylococci.
Methods   A multicenter in vitro study was performed using the E test during a period of 3 months (April to June) in 1997 on fresh, clinically significant, non repetitive strains of staphylococci from patients hospitalized in 23 different hospitals in 18 countries tested.
Results   A total of 2132 staphylococcal isolates including methicillin resistant (MR), methicillin susceptible (MS) S. aureus (1003 MS, 462 MR), S. epidermidis (169 MS, 251 MR), S. haemolyticus (28 MS, 46 MR), S. hominis (28 MS, 16 MR), and coagulase negative staphylococci (86 MS, 43 MR) were analyzed.
Q/D was highly active against all species tested. MIC₉₀ (mg/L) ranged from 0.5 to 2 depending on the species. Strains had MIC≤1 mg/L in 97.6%. For S. aureus , S. epidermidis , S. hominis and other coagulase-negative staphylococci no differences in MIC₉₀ were observed for MS or MR. One dilution difference was observed for S. haemolyticus , which overall was the less susceptible species. Erythromycin resistance was observed among 57– 87% of MR-strains and was lower among MS-strains (18–56%). Erythromycin resistance had no or little influence on MIC of Q/D. In comparison to vancomycin, Q/D was two to four times more active.
Conclusions   The streptogramin combination Q/D showed an excellent in vitro activity against all staphylococcal species tested regardless of the resistance pattern to other drug classes, particularly resistance to methicillin. Q/D was two to four times more active than vancomycin and MIC values varied from 0.5–2 according to the species. The synergy of Q/D was well conserved in macrolide-resistant strains.     

Comparison of PCR detection of mecA with agar dilution and Etest for oxacillin susceptibility testing in clinical isolates of coagulase-negative staphylococci

Oxacillin-resistant staphylococci are heterogeneous in their expression of resistance to beta-lactam antibiotics. Different recommendations regarding screening methods for routine use have been published. In this study, the susceptibility to oxacillin of 232 coagulase-negative staphylococci (CoNS) was determined by agar dilution, Etest and presence of the mecA gene. When an oxacillin resistance breakpoint of > or = 0.5 mg/L was used, the sensitivity and specificity for agar dilution were 97.6% and 100%, and those for Etest were 100% and 95.4%. The current National Committee for Clinical Laboratory Standards oxacillin breakpoint recommendation will categorise accurately the CoNS species encountered commonly.     

Detection of methicillin resistance in coagulase-negative staphylococci by cefoxitin disc diffusion and oxacillin Etest. A study of consecutive bacteraemia isolates

Coagulase-negative staphylococci (CoNS) are a significant cause of nosocomial bacteraemia and their susceptibility to beta-lactamase-stabile penicillins is unpredictable. To ensure appropriate antibiotic therapy reliable methods for detection of methicillin resistance (MR) are needed. The objectives of this study were to determine the frequency of MR in a set of CoNS from cases of monomicrobial bacteraemia and to evaluate two phenotypic assays for detection of MR, the 10 microg cefoxitin disk test on Iso-Sensitest agar using a semiconfluent inoculum and the oxacillin Etest. MR was determined by a commercial genomic mecA assay. Of 110 CoNS, 75 were mecA positive and 35 mecA negative. Using interpretive zone diameters R < 22 mm and S > or = 27 mm, the cefoxitin disk test had a sensitivity and specificity of 100%. A correct prediction was obtained for 86 isolates, while 23 were indeterminate (> or = 22 mm; <27 mm). Using CLSI's guidelines, sensitivity and specificity of the oxacillin Etest were 100% and 80%, respectively. A correct prediction was obtained for 102 isolates, while 7 mecA negative isolates were classified as resistant. Thus, the cefoxitin disk test and the oxacillin Etest performed with high accuracy and both seem to be suitable for routine use.     

Identification of clinical coagulase-negative staphylococci,isolated in microbiology laboratories,by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and two automated systems

A study was performed to compare matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), linked to a recently engineered microbial identification database, and two rapid identification (ID) automated systems, BD Phoenix (Becton Dickinson Diagnostic Systems, France) and VITEK-2 (bioMérieux, Marcy L'Etoile, France), for the ID of coagulase-negative staphylococci (CoNS). Two hundred and thirty-four clinical isolates of CoNS representing 20 species were analyzed. All CoNS isolates were characterized by sodA gene sequencing, allowing interpretation of the ID results obtained using the respective database of each apparatus. Overall correct ID results were obtained in 93.2%, 75.6% and 75.2% of the cases with the MALDI-TOF-MS, Phoenix and VITEK-2 systems, respectively. Mis-ID and absence of results occurred in 1.7% and 5.1% of the cases with MALDI-TOF-MS, in 23.1% and 1.3% with the Phoenix, and in 13.7% and 0.9% with the VITEK-2 systems, respectively. In addition, with the latter automate, 10.3% of the IDs were proposed with remote possibility. When excluding the CoNS species not included in the databases of at least one of the three systems, the final percentage of correct results, Mis-ID and absence of ID were 97.4%, 1.3% and 1.3% with MALDI-TOF-MS, 79%, 21% and 0% with the Phoenix, and 78.6%, 10.3% and 0.9% with the VITEK-2 system, respectively. The present study demonstrates the robustness and high sensitivity of our microbial identification database used with MALDI-TOF-MS technology. This approach represents a powerful tool for the fast ID of clinical CoNS isolates.     

Molecular characterization and susceptibility of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from hospitals and the community in Vladivostok,Russia

A prospective study was conducted during an 8-month period, from August 2006 to April 2007, to describe the epidemiology of Staphylococcus aureus-associated infections. In addition, the molecular characteristics, antimicrobial susceptibilities and antibiotic resistance determinants were identified in S. aureus isolates from hospitals and the community in Vladivostok, Russia. Among the 63 S. aureus isolates eligible for this study, methicillin resistance was observed in 48% (n = 30). Hospital-acquired strains accounted for 93% (28/30) of all methicillin-resistant S. aureus (MRSA) isolates. The major MRSA clone (sequence type (ST) 239, staphylococcal cassette chromosome mec (SCCmec) type III, Panton--Valentine leukocidin (PVL)-negative, with two related staphylococcal protein A gene (spa) types (types 3 and 351)) represented 90% of all of the MRSA isolates. This clone was multidrug-resistant, and 41% of isolates showed resistance to rifampicin. Community-acquired MRSA isolates (n = 2) were categorized as ST30, SCCmecIV, spa type 19, and PVL--positive, and as ST8, SCCmecIV, of a novel spa type 826, and PVL-negative. Eight different STs were detected among methicillin-susceptible S. aureus (MSSA) isolates, of which 55% were PVL--positive. One MSSA clone, which was categorized as ST121, spa type 273, and PVL--positive, caused fatal community-acquired pneumonia infections. The strains predominantly isolated in hospitals in Russia belonged to the multidrug-resistant Brazilian/Hungarian ST239 MRSA clone; however, this clone has new antibiotic susceptibilities. Additionally, the emergence of PVL--positive MSSA strains with enhanced virulence was observed, warranting continued surveillance.     

Blinded comparison of repetitive-sequence PCR and multilocus sequence typing for genotyping methicillin-resistant Staphylococcus aureus isolates from a children's hospital in St. Louis, Missouri

We performed a blinded study to compare repetitive-sequence PCR and multilocus sequence typing for genotyping hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA). The MRSA strains that were sequence type 8 (ST8), staphylococcal cassette chromosome mec (SCCmec) type IV, and Panton-Valentine leukocidin-positive clustered separately from those that were ST5 and SCCmec type II.     

Characterisation of isolates of Staphylococcus aureus from acute, chronic and subclinical mastitis in cows in Norway

Eighty-six Staphylococcus aureus isolates from cases of bovine mastitis were characterised biochemically and with respect to serotype, multilocus enzyme electrophoresis genotypes, antibiotic sensitivity, and production of enterotoxins A through D (SEA-D) and toxic shock syndrome toxin-1 (TSST-1). The samples were obtained from 81 different cows from 79 Norwegian dairy herds in 10 different counties in southern Norway. There was an equal representation of isolates from cases of acute, chronic and subclinical mastitis. Multilocus enzyme electrophoresis using 13 genetic loci showed that 69 of 86 isolates had the same electrophoretic type. This common electrophoretic type comprised isolates that differed in the expression of other phenotypical characteristics studied. Fifty-eight percent of the isolates produced one or more enterotoxins, predominantly a combination of SEC and TSST-1. Capsular serotyping revealed that 95% of the isolates belonged to serotype 8. No correlation was found between the factors studied and the clinical classification of mastitis. It appears that the majority of S. aureus isolates recovered from cases of bovine mastitis in Norway are genetically closely related and express common phenotypical characteristics.     

Comparative bactericidal activities of daptomycin, glycopeptides, linezolid and tigecycline against blood isolates of Gram-positive bacteria in Taiwan.

In-vitro MICs and minimum bactericidal concentrations (MBCs) of daptomycin, linezolid, tigecycline, vancomycin and teicoplanin against Gram-positive bacteria were determined using the broth microdilution method for ten blood isolates each of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), including two vancomycin-intermediate S. aureus (VISA), vancomycin-resistant Enterococcus faecium and Enterococcus faecalis. One strain of VISA was tested in a time-kill synergism assay of daptomycin combined with oxacillin, imipenem, rifampicin and isepamicin. Daptomycin showed excellent in-vitro bactericidal activity against all the isolates tested, with no tolerance or synergism effects when combined with other agents, except with rifampicin against VISA. Vancomycin had better bactericidal activity against MRSA and MSSA than did teicoplanin. Linezolid had the poorest bactericidal activity against the isolates tested, with 100% tolerance by the MSSA and VRE isolates, and 80% tolerance by the MRSA isolates. Tolerance towards tigecycline was exhibited by 40% of the MRSA isolates, 100% of the MSSA and vancomycin-resistant E. faecalis isolates, and 90% of the vancomycin-resistant E. faecium isolates.     

The intercellular adhesin locus ica is present in clinical isolates of Staphylococcus aureus from bacteremic patients with infected and uninfected prosthetic joints

Although polysaccharide intercellular adhesin (PIA) is thought to be crucial in the pathogenesis of prosthetic device infections caused by Staphylococcus epidermidis, its role in prosthetic device infections caused by Staphylococcus aureus is unknown. To assess the clinical impact of PIA production, isolates from 15 prospectively identified cases of S. aureus bacteremia in patients with prosthetic joints (8 infected, 7 uninfected) were characterized for biofilm production, hemagglutination, and the presence of a 419-bp amplification product within icaA. Although icaA was present in all 15 isolates, none of the isolates produced hemagglutination and only one isolate (from a patient with an uninfected prosthetic device) weakly produced biofilm in vitro. These results support the observation that the ica locus is conserved between S. epidermidis and S. aureus and that PIA may be expressed only under in vivo conditions. Future investigations should include animal models to approximate the complex milieu surrounding implanted prosthetic medical devices. Received: 19 June 2000     

Chlorhexidine and octenidine use,carriage of qac genes,and reduced antiseptic susceptibility in methicillin-resistant Staphylococcus aureus isolates from a healthcare network

ObjectivesWith the widespread use of antiseptics in healthcare facilities for the prevention of methicillin-resistant Staphylococcus aureus (MRSA) transmission, there are concerns for antiseptic tolerance and resistance. We sought to understand the use of chlorhexidine and octenidine, carriage of qac genes, and reduced antiseptic susceptibilities.MethodsA serial cross-sectional study was conducted in an acute care hospital and three extended-care facilities of a healthcare network in June–July, 2014–2016. Two of the extended-care facilities were exposed to intranasal octenidine and universal daily chlorhexidine/octenidine bathing. The minimum inhibitory concentration (MIC) levels and qac genes were determined by broth microdilution tests and whole genome sequencing respectively. Multivariable logistic regression was used to assess for the independent associations between antiseptic exposures, qac genes, and reduced antiseptic susceptibilities.ResultsA total of 878 MRSA isolates were obtained. There were associations between qacA/B carriage and chlorhexidine (adjusted odds ratio (aOR) 7.80; 95% confidence interval (CI) 3.25–18.71) and octenidine (aOR 11.79; 95% CI 5.14–27.04) exposures. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility (MIC ≥4 mg/L) (aOR 3.15; 95% CI 1.14–8.74). Carriage of qacA/B (aOR 10.65; 95% CI 4.14–27.40) or qacC (aOR 2.55; 95% CI 1.22–5.32) had an association with reduced chlorhexidine susceptibility; while MRSA sequence type modified the association. However, we found no direct association between (i) antiseptics use and qacC carriage, (ii) octenidine exposure and reduced susceptibility, and (iii) reduced octenidine susceptibility and qacA/B or qacC carriage.ConclusionsAntiseptic exposures were associated with carriage of qac genes. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility, requiring continued surveillance for the emergence of resistance.     

Encapsulation of Staphylococcus aureus isolates from mastitic milk: relationship between capsular polysaccharide types 5 and 8 and colony morphology in serum-soft agar, clumping factor, teichoic acid, and protein A.

A total of 193 Staphylococcus aureus isolates from bovine, caprine, and ovine mastitis producing type 5 or 8 capsular polysaccharides were investigated for colony morphology in serum-soft agar and agglutinability by an anti-teichoic acid serum, after cultivation in modified staphylococcus medium no. 110. Also, 40 of these strains were cultivated in brain heart infusion and submitted to clumping factor and protein A detection tests. Considering capsular serotyping as a reference method, diffuse growth in serum-soft agar and inagglutinability by anti-teichoic acid serum identified, respectively, 57.5 and 45% of encapsulated strains cultivated in brain heart infusion and 85.5 and 77.2% of those cultivated in modified staphylococcus medium 110. Consequently, these indirect techniques underestimated encapsulation and were greatly influenced by culture conditions. Whatever the medium used, diffuse colony morphology in serum-soft agar was generally characterized by a masking of teichoic acid and protein A. By contrast, these surface antigens were detected in association with compact morphology; the presence of a thin or discontinuous capsular material could explain this result. Moreover, the masking of teichoic acid and the removal of capsular polysaccharide by washing in saline suggest that type 8 capsular polysaccharide is more abundant and labile than type 5.