Low prevalence of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in Belgian hospitals

Staphylococcus aureus strains with decreased susceptibility to glycopeptides (GISA) have been associated with increased risk of glycopeptide treatment failure. To assess the prevalence of these strains in hospitalised patients in Belgium, 455 methicillin-resistant S. aureus (MRSA) isolates collected in 2001 were screened by two assays: (i) growth on vancomycin agar screen (VAS; brain heart infusion agar (BHI) + vancomycin 6 mg/L); and (ii) a synergy/antagonism test with aztreonam/cefazolin on Mu3 agar (BHI + vancomycin 3 mg/mL). Isolates growing on VAS or Mu3 agar were characterised further by analysis of population susceptibility profiles. MICs of glycopeptides were determined by agar dilution, broth microdilution and Etest (low and high inocula) methods. The isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and determination of staphylococcal cassette chromosome mec (SCCmec) type. No GISA isolates were found. Three (0.7%) hetero-vancomycin intermediate S. aureus (hVISA) and ten (2.2%) hetero-teicoplanin intermediate S. aureus (hTISA) isolates were identified by population analysis. All but one hetero-GISA isolate belonged to either epidemic PFGE group A/SCCmec type I (69%) or PFGE group D/SCCmec type I (23%), both of which were resistant to gentamicin. The sensitivity and specificity for the detection of hetero-GISA by the two assays were 15.4% and 99.8%, respectively, for VAS, and 84.6% and 95.9%, respectively, for Mu3. The data indicated that hetero-GISA strains were uncommon among Belgian MRSA isolates from hospitalised patients. Use of Mu3 agar was more sensitive, but less specific, than VAS as a screening method.     

Development of glycopeptide-intermediate resistance by Staphylococcus aureus leads to attenuated infectivity in a rat model of endocarditis

Glycopeptide-intermediate resistant Staphylococcus aureus (GISA) are characterized by multiple changes in the cell wall and an altered expression of global virulence regulators. We investigated whether GISA are affected in their infectivity in a rat model of experimental endocarditis. The glycopeptide-susceptible, methicillin-resistant S. aureus M1V2 and its laboratory-derived GISA M1V16 were examined for their ability to (i) adhere to fibrinogen and fibronectin in vitro, (ii) persist in the bloodstream after intravenous inoculation, (iii) colonize aortic vegetations in rats, and (iv) compete for valve colonization by co-inoculation. Both GISA M1V16 and M1V2 adhered similarly to fibrinogen and fibronectin in vitro. In rats, GISA M1V16 was cleared faster from the blood (P < 0.05) and required 100-times more bacteria than parent M1V2 (10⁶ versus 10⁴ CFU) to infect 90% of vegetations. GISA M1V16 also had 100 to1000-times lower bacterial densities in vegetations. Moreover, after co-inoculation with GISA M1V16 and M1V2Rif, a rifampin-resistant variant of M1V2 to discriminate them in organ cultures, GISA M1V16 was outcompeted by the glycopeptide-susceptible counterpart. Thus, in rats with experimental endocarditis, GISA showed an attenuated virulence, likely due to a faster clearance from the blood and a reduced fitness in cardiac vegetations. The GISA phenotype appeared globally detrimental to infectivity.     

Methods for the detection of resistance to oxacillin in strains of Staphylococcus aureus

Methods suitable for detection of resistance of staphylococci to oxacillin were tested in a group of 77 strains of Staphylococcus aureus (39 strains sensitive and 38 strains resistant to oxacillin). The influence of the composition of the medium, the growth phase of the inoculum and time of incubation on detection of resistant strains was investigated. By none of the methods resistance to oxacillin was proved in the control group of sensitive strains. For evidence of oxacillin resistance after 24 hours' incubation the standard micro-method is suitable and the diffuse method with 1 microgram disc of oxacillin which both detected 100% of resistant strains, and the screening method with a yield of 97% strains. The growth phase of the inoculum and the incubation period do not influence the results of these methods. The micromethod in MH broth, the diffuse method with a 10 micrograms oxacillin disc and the dilution plate method were influenced to a considerable extent by the composition of the medium, the growth phase of the inoculum, the incubation period and they revealed a small number of resistant strains.     

Performance of different methods of oxacillin resistance detection in atypic strains of Staphylococcus aureus

Seventy-three of aminoglycoside-susceptible methicillin-resistant Staphylococcus aureus (AS-MRSA) and 12 kanamycin-tobramycin-resistant methicillin-susceptible S. aureus (KTR-MSSA) isolates were phenotypically and genotypically examined for methicillin susceptibility. The AS-MRSA profile represents 8.3% of MRSA strains and the KTR-MSSA profile represents 1.38% of MSSA strains. The diffusion method using the 5 microg oxacillin and 30 microg cefoxitin discs on Mueller-Hinton Agar (MHA) with and without NaCl, the incubation at 35 degrees C or 30 degrees C for 24 or 48 hours respectively, and the determining oxacillin MICs by E-test (AES, Combourg, France) were performed and used as phenotypic methods. We also used the mecA gene PCR which was considered as the "gold standard" for methicillin resistance detection, and the Slidex MRSA Detection (bioMérieux) that detect the presence of mecA gene product (PBP 2a). To increase the level of PBP 2a expression, the 30 microg cefoxitin disc was used as an inducer. All the AS-MRSA strains (100%) were detected by the cefoxitin disc in all conditions and by the oxacillin disc on MHA with 2% of NaCl at 35 degrees C. Without NaCl, the sensitivity fell to 97,2% by oxacillin disc. The oxacillin MICs for these isolates ranged from 2 to 128 mg/l. The mecA gene determinant and its product PBP 2a were detected in all AS-MRSA strains. All KTR-MSSA strains were phenotypically methicillin-susceptible and oxacillin MICs were below or borderline of breakpoint (< or =2 mg/l). The mecA gene determinant and its product were detected in one strain which was considered to be the most heterogeneous of those tested.     

Emergence of Staphylococcus aureus strains resistant to pristinamycin in Sfax (Tunisia)

AimWe report the emergence of Staphylococcus aureus resistant to pristinamycin in Tunisia, and the characterization of the mechanisms of resistance to macrolides and streptogramins.Methods and resultsFive strains of S. aureus resistant to pristinamycin were recovered from the department of dermatology in a Tunisian university hospital from skin samples after oral use of pristinamycin between 2004 and 2007. Susceptibility testing showed that all isolates were resistant to quinupristin–dalfopristin (MIC = 4–32 mg/L), lincomycin, gentamicin, kanamycin, tobramycin, tetracycline and rifampin. One isolate was susceptible to erythromycin. All five strains were closely related after analysis by pulsed-field gel electrophoresis. erm(C) was amplified from three strains and erm(A) from one strain. vga and vat genes were amplified from all strains. None of the isolates carried the vgb gene. The vga and vat genes were typed as vga(B) and vat(B) by restriction profiles analysis after electrophoresis.ConclusionThis is the first report of clonal emergence of S. aureus resistant to pristinamycin carrying vga and vat genes in Tunisia. The role of selective pressure of pristinamycin use is certainly the main explanation of this emergence. So we must reduce the utilisation of this antibiotic for the treatment of cutaneous and bone infectious disease caused by multidrug resistant bacteria.     

Staphylococcus aureus: new detection of intrinsic resistance using the diffusion method

Because of an heterogeneously-expressed resistance among methicillin-resistant Staphylococcus aureus strains the conditions for antibiogram determination had rapidly to be modified so as to improve their detection. The newly recommended conditions (incubation at +30 degrees C or on hypersalted agar medium) remain widely used at the moment, although they appear to be more and more often badly adapted, particularly because of the recently-observed renewed outbreak of wild strains with a weak in vitro phenotypic expression. It is the reason why we searched for a new and more reliable phenotypic method although still accessible for any laboratory. Sixty-five strains of Staphylococcus aureus entered the study. The absence or presence of mecA gene was previously investigated by gene amplification. These strains were of various origins and had often caused difficulties for the detection of intrinsic resistance to methicillin on the antibiogram. Our results confirm the failures of the classical methods (false negative results at +30 degrees C, false negative or positive results on hypersalted agar medium incubated at +37 degrees C). They also allow to propose a new method which relies on the determination of the susceptibility to cefoxitin using the usual conditions for antibiogram determination. In our series of strains, this new method proved to widely improve both the sensitivity and the susceptibility for the detection of methicillin-resistance by diffusion on the antibiogram.     

Multiple antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis: plasmids in strains associated with nosocomial infection

The plasmid DNA profiles were compared to phenotypically-similar, antibiotic-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis associated with nosocomial infections in a Melbourne hospital. Whereas resistance to gentamicin, tobramycin and kanamycin was encoded by one of 3 plasmids [pSK1, 18 megadalton (Md); pSK4, 22 Md; pSK9, 17 Md] in S. aureus, no similar plasmids were detected in S. epidermidis. Mediated exclusively by the chromosome in S. aureus, tetracycline resistance was encoded either by the chromosome or by a 2.8 Md plasmid in strains of S. epidermidis. The inability to detect common resistance plasmids in strains of S. aureus and S. epidermidis recovered from this outbreak is in contrast to recent observations with staphylococci from other geographic areas; nevertheless, on the basis of restriction endonuclease analyses of 3 Md chloramphenicol resistance plasmids, it is suggested that a common gene pool does exist within isolates of S. aureus and S. epidermidis from Melbourne hospitals.     

Prophage and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus strains in Iran

A total of 114 isolates of methicillin-resistant Staphylococcus aureus (MRSA) were collected from hospitals in Tehran, Iran. A multiplex PCR was designed to examine the presence of six different prophage classes. The results showed high diversity of bacteriophages, with four different prophage types and eight prophage patterns. An important S. aureus phage coding for several virulence factors, Φ-77-like phage, was detected in 97?% of the isolates. We found a high rate of resistance of MRSA isolates to penicillin, ciprofloxacin, tobramycin and kanamycin. This is the first study showing high prevalence and diverse bacteriophage populations in MRSA strains in Iranian hospitals.     

Genetic background and antibiotic resistance of Staphylococcus aureus strains isolated in the Republic of Georgia

The genetic composition and antibiotic sensitivities of 50 clinical isolates of Staphylococcus aureus obtained from various clinics in the Republic of Georgia were characterized. S. aureus strains ATCC 700699 and ATCC 29737 were included as reference standards in all analyses. All 52 strains had identical 16S rRNA profiles. In contrast, pulsed-field gel electrophoresis (PFGE) identified 20 distinct PFGE types among the 52 strains examined, which indicates that PFGE is more discriminating than is 16S rRNA sequence analysis for differentiating S. aureus strains. The results of our PFGE typing also suggest that multiple genetic subpopulations (related at the ca. 85% similarity level, based on their SmaI PFGE patterns) exist among the Georgian S. aureus strains. Twenty-two of the 50 Georgian strains were methicillin resistant and PCR positive for mecA, and 5 strains were methicillin sensitive even though they possessed mecA. None of the strains were vancomycin resistant or contained vanA. The nucleotide sequences of mecA fragments obtained from all mecA-containing strains were identical. Our data indicate that the population of S. aureus strains in Georgia is fairly homogeneous and that the prevalence of methicillin-resistant, mecA-positive strains is relatively high in that country.     

Induction of resistance with heat-killed compact-type strains of Staphylococcus aureus against challenge with the diffuse variant of the Smith strain of Staphylococcus aureus.

Active immunization of mice with high doses of heat-killed and autoclaved vaccine of unencapsulated strains of Staphylococcus aureus, which was grown in brain heart infusion media, protected against challenge with the Smith diffuse strain of Staphylococcus aureus. These organisms were capable of absorbing the protective antibody in rabbit hyperimmune sera prepared with the Smith diffuse strain. Also, mice treated with rabbit hyperimmune sera prepared with these strains (four out of six strains) protected against challenge with the Smith diffuse strain. Protective activities of these rabbit hyperimmune sera were assumed to be essentially identical to the protective antibody induced by the Smith diffuse strain.     

Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus

The probe-based Velogene Rapid MRSA Identification Assay (ID Biomedical Corp., Vancouver, British Columbia, Canada) and the latex agglutination MRSA-Screen (Denka Seiken Co., Tokyo, Japan) were evaluated for their ability to identify methicillin-resistant Staphylococcus aureus (MRSA) and to distinguish strains of MRSA from borderline oxacillin-resistant S. aureus (BORSA; mecA-negative, oxacillin MICs of 2 to 8 microgram/ml). The Velogene is a 90-min assay using a chimeric probe to detect the mecA gene. MRSA-Screen is a 15-min latex agglutination test with penicillin-binding protein 2a antibody-sensitized latex particles. We compared these assays with the BBL Crystal MRSA ID System (Becton Dickinson, Cockeysville, Md.) and with PCR for mecA gene detection. A total of 397 clinical isolates of S. aureus were tested, consisting of 164 methicillin-susceptible strains, 197 MRSA strains, and 37 BORSA strains. All assays performed well for the identification of MRSA with sensitivities and specificities for Velogene, MRSA-Screen, and BBL Crystal MRSA ID of 98.5 and 100%, 98.5 and 100%, and 98.5 and 98%, respectively. Three MRSA strains were not correctly identified by each of the Velogene and MRSA-Screen assays, but repeat testing with a larger inoculum resolved the discrepancies. The BBL Crystal MRSA ID test misclassified four BORSA strains as MRSA. Both the Velogene and the MRSA-Screen assays are easy to perform, can accurately differentiate BORSA isolates from MRSA isolates, and provide a rapid alternative for the detection of methicillin resistance in S. aureus in clinical laboratories, especially when mecA PCR gene detection is unavailable.     

Antibiotic resistance of Staphylococcus aureus at north Lebanon: place of the methicillin resistance and comparison of detection methods

The aim of this study was to evaluate the susceptibility of 100 Staphylococcus aureus strains isolated from the laboratory of Microbiology of the Islami Hospital of Tripoli (Lebanon) to 19 antibiotics, and to determine the prevalence of methicillin resistant strains. 30% of strains studied were methicillin resistant, 96% were resistant to the penicillin G. Clavulanic acid restaurated the amoxicillin activity to 29%. The resistance level was 34% for amikacin, 3% for gentamycin and tobramycin, 10% for chloramphenicol, 44.33% for tetracyclin, 7% for erythromycin, 4.04% for clindamycin, 20% for trimethoprim-sulfametoxasol and 0% for vancomycin and teicoplanin. The methicillin-resistant Staphylococcus aureus possess more important resistant level in comparison with the methicillin sensitive strains. We compared the ability of latex agglutination test (Slidex(R) SARM, bioMérieux, France) to detect the production of penicillin-binding protein 2' (PBP 2') in 100 clinical isolates of S. aureus with two reference methods: the oxacillin disk diffusion test and the MIC determination by the E-test (AB BIODISK, Sweden). The two reference methods give the same results for the detection of methicillin resistant S. aureus. The Slidex test was positive for all 30 isolates determined to be methicillin resistant by the reference methods (sensitivity 100%). The latex test was negative for 42 of 70 isolates determined to be methicillin susceptible by the reference methods, and the latex test was positive for 28 isolates determined to be susceptible (specificity 60%).     

Lack of evidence for mutation to erythromycin resistance in clinical strains of Staphylococcus aureus.

The properties of 100 erythromycin resistant strains of Staphylococcus aureus obtained from clinical material have been compared with the properties of mutants selected in vitro for resistance to erythromycin. The properties, including inducibility of the resistance and cross-resistance to spiramycin and lincomycin, of the two groups of isolates were always different. The risk that staphylococci will mutate to erythromycin resistance during therapy with this antibiotic is remote.     

Incidence of Staphylococcus aureus with reduced susceptibility to glycopeptides in two French hospitals

Objectives   To determine the incidence of Staphylococcus aureus isolates with reduced susceptibility to glycopeptides among all clinical isolates collected consecutively in two French hospitals between November 1998 and April 1999.
Methods   Methicillin-resistant and -susceptible S. aureus isolates were screened on vancomycin- or teicoplanin-supplemented agar plates. Glycopeptide MICs were determined by the E test procedure with a high inoculum and by an agar dilution technique. Glycopeptide-intermediate S. aureus isolates were identified as homogeneously or heterogeneously resistant to vancomycin by performing population analysis.
Results   Of the 640 isolates recovered from 518 patients, three from the same patient and two from two different patients showed homogeneous or heterogeneous intermediate resistance to vancomycin.
Conclusion   The incidence of glycopeptide-intermediate S. aureus (homogeneously or heterogeneously resistant) in a non-selected patient population, i.e. regardless of predisposing factors and glycopeptide therapeutics, remains low in the two French hospitals involved in the study, representing 0.6% of isolates.     

Surveillance of antimicrobial resistance and antimicrobial use in a university-affiliated hospital: implementation of a computerized system

After half a century of antibiotic use, the increasing problem of the emergence and spread of antimicrobial-resistant pathogens has created a problem of public health. The causes of this problem are multifactorial, but the excessive and inappropriate use of antimicrobials is the principal cause. The current guidelines for the control of antimicrobial resistance in hospitals recommend the implementation of a surveillance system of antimicrobial use and antimicrobial resistance data. AIM OF THE STUDY: The objective of our project was to develop a computerised tool to survey the antibiotic consumption data and the antimicrobial resistance. MATERIALS ET METHODS: We have collected antimicrobial resistance data from the software of the bacteriology laboratory, antibiotic use data from the pharmacy and demographical data from the hospital's admission department. These data were integrated in a database server and available with a web application. Antimicrobial resistance data of 15 major microorganisms were extracted and expressed as a frequency with elimination of repeats by using time criteria (7, 14 or 28 days). Antibiotic use data were converted into defined daily doses (DDD) and expressed per 1000 patient-days. RESULTS: Data are available for consultation in the form of tables or graphs per unit, type of units (medicine, surgery, pediatrics, intensive care units) or in the whole hospital. The system allows the confrontation on the same graph of antimicrobial resistance and antibiotic use data. CONCLUSION: Our surveillance system constitutes a needed prerequisite to the implementation of a global strategy of antibiotic use improvement in our hospital.