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Disk with high oxacillin content discriminates between methicillin-resistant and borderline methicillin-susceptible Staphylococcus aureus strains in disk diffusion assays using a low salt concentration.

A separation between mecA+ strains of Staphylococcus aureus and strains lacking mecA was achieved by the disk diffusion assay and the agar dilution method, utilizing disks containing 5 microg of oxacillin and inocula of approximately 5 x 10(5) CFU/spot, respectively, provided that agar with 0 to 0.5% NaCl and incubation at 30 degrees C were employed. The 5-microg oxacillin disks clearly discriminated between borderline methicillin-susceptible and mecA+ strains. The oxacillin MICs were more affected by the inoculum density and salt concentration than were the methicillin MICs, and oxacillin MICs of 4 to 16 microg/ml were obtained for strains lacking mecA. Significantly higher levels of beta-lactamase production and reduced oxacillin susceptibilities were recorded for strains lacking mecA, in particular strains of phage group V, when agar with >/=2% NaCl was used than when agar with 0 to 0.5% NaCl was employed. The results indicate that the borderline methicillin-susceptible phenotype is a salt-dependent in vitro phenomenon of questionable clinical relevance.

Culture Media↗

Interactions between methicillin and vancomycin in methicillin-resistant Staphylococcus aureus strains displaying different phenotypes of vancomycin susceptibility.

Vancomycin-sensitive, -intermediate, and -heterointermediate methicillin-resistant Staphylococcus aureus isolates were tested by using E-tests to explore the interaction of methicillin and vancomycin. For the vancomycin-intermediate and -heterointermediate strains both drugs showed antagonism at concentrations below their MICs but synergy at methicillin concentrations near the MIC. This property could be used to screen for heterointermediate S. aureus strains.

Anti-Bacterial Agents↗

Effect of beta-lactamase on minimum inhibitory concentrations of methicillin in methicillin-resistant Staphylococcus aureus.

A study was undertaken to determine whether the production of penicillin-binding protein-2' (PBP-2') and the production of beta-lactamase were related to the minimum inhibitory concentrations of methicillin (DMPPC) in various strains of methicillin-resistant Staphylococcus aureus. The amount of PBP-2' produced by the low-level resistant strain L20A was small and the strain became resistant to DMPPC as a result of inactivation of DMPPC by beta-lactamase. The largest amount of PBP-2' produced was in a moderately resistant strain L21A but the MIC was not high since the strain was not capable of producing beta-lactamase and the population of cells was heterogeneous. The amount of PBP-2' produced in the high-level resistant strain L457A was lower than that in strain L21A but the MIC was high as a result of the production of beta-lactamase. Production of PBP-2' was essential for resistance to methicillin but production of beta-lactamase had a major effect on the MIC.

Bacterial Proteins↗

Use of commercial particle agglutination systems for the rapid identification of methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

The performance of a recently introduced Staphylococcus aureus identification system (Slidex Staph-Kit) was compared with that of currently available systems (Immuno Scan Staphlatex, Staphyloslide, Staphaurex and SeroSTAT II) for the identification of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus. The new system, which detects a capsular antigen common in MRSA, performed with equal or greater sensitivity than the other systems. None of the commercial systems was adversely affected by the methicillin susceptibility of the staphylococci when isolates were recovered from non-selective media. The greatest advantage of the anti-capsular monoclonal reagent was its improved performance on isolates recovered from selective media.

Humans↗

Methicillin-resistant Staphylococcus aureus: interstate spread of nosocomial infections with emergence of gentamicin-methicillin resistant strains.

A methicillin-resistant strain of Staphylococcus aureus (MRSA, phage type 84/85) was introduced into City of Memphis Hospital by a burn patient who had recently been treated for MRSA bacteremia in another institution 500 miles distant. Despite prompt recognition of the problem and institution of isolation procedures, six other patients developed secondary colonization during the ensuing six months, and five of these experienced clinically significant infections with MRSA. Three of the patients originally infected with MRSA, as well as two additional patients, subsequently developed colonization with staphylococcal strains of phage type 84/85 that were resistant to both methicillin and gentamicin (MRGRSA). Spread of the staphylococcal strains was most likely accomplished primarily via passive transfer from person to person. The hydrotherapy unit, which became contaminated with both MRSA and MRGRSA, may have played a secondary role. As illustrated by this outbreak, patients carrying potentially dangerous bacterial strains should be identified and informed of the problems posed by such carriage. It may be imprudent to admit such patients to hospitals that are free of the potential pathogen. The outbreak described here exemplifies a number of potential problems associated with control of nosocomial staphylococcal infections: (a) interhospital spread of methicillin-resistant strains; (b) secondary patient-to-patient intrahospital spread; and (c) emergence of even more resistant strains, possibly associated with selective pressures exerted by widespread use of broad-spectrum antimicrobial agents.

Cross Infection↗

Enterotoxin and toxic shock syndrome toxin-1 production of methicillin resistant and methicillin sensitive Staphylococcus aureus strains.

In this study the production of enterotoxin A-D and toxic shock syndrome toxin-1 (TSST-1) of 181 methicillin resistant (MRSA) and 100 methicillin sensitive (MSSA) Staphylococcus aureus first isolates from different patients was investigated. All the MRSA- and MSSA isolates in the study were collected in a period between 1993 and 1995 from specimens sent from 11 different acute care hospitals in the greater Düsseldorf area. As far as possible the isolates were matched according to ward and hospital. The isolates were collected in the same time period and matched for specimen from which isolated. Furthermore, only first isolates were analysed in both groups. No significant difference in the production of toxin of any type between MRSA and MSSA could be detected (51 and 40% respectively). When the individual toxins were analysed, again no significant difference between MRSA and MSSA was demonstrable (enterotoxin production by MRSA 40% and MSSA 36%, and TSST-1 16% and 8% respectively). Despite this, a slight tendency for MRSA to produce enterotoxin A and B and for MSSA to produce enterotoxin C was observed. In addition, generation of TSST-1 by both groups was independent of enterotoxin A-D production. Interestingly, no increase in the proportion of TSST-1- or enterotoxin-producing MRSA and MSSA isolates was observed in strains isolated from blood cultures from patients with a clinical diagnosis of sepsis. Genotypical pulsed-field-gel-electrophoresis (PFGE) and phenotypical (bacteriophage typing, lysotyping) characterization of the 181 MRSA isolates resulted in 28 different PFGE patterns (of which 19 were toxin producers) and 22 lysotyping groups (18 of which produced toxin). In summary, the investigated clinical S. aureus isolates showed no difference in their ability to produce toxin and this was independent of their sensitivity to methicillin.

Bacterial Toxins↗

Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis.

A meta-analysis was performed to summarize the impact of methicillin-resistance on mortality in Staphylococcus aureus bacteremia. A search of the MEDLINE database for studies published during the period of 1 January 1980 through 31 December 2000 and a bibliographic review identified English-language studies of S. aureus bacteremia. Studies were included if they contained the numbers of and mortality rates for patients with methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) bacteremia. Data were extracted on demographic characteristics of the patients, adjustment for severity and comorbid illness, source of bacteremia, and crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for in-hospital mortality. When the results were pooled with a random-effects model, a significant increase in mortality associated with MRSA bacteremia was evident (OR, 1.93; 95% CI, 1.54-2.42; P<.001); significant heterogeneity was present. We explored the reasons for heterogeneity by means of subgroup analyses. MRSA bacteremia is associated with significantly higher mortality rate than is MSSA bacteremia.

Bacteremia↗

Is methicillin-resistant Staphylococcus aureus more contagious than methicillin-susceptible S. aureus in a surgical intensive care unit?

BACKGROUND AND OBJECTIVE: In the Netherlands, the prevalence of methicillin resistance among Staphylococcus aureus isolates has been kept to less than 1% by using active screening programs and isolation. At the University Medical Center Utrecht (UMCU), an active screening program for methicillin-resistant S. aureus (MRSA) in the surgical intensive care unit (ICU) was implemented in 1986. Between 1992 and 2001, only 6 patients with MRSA were admitted to the surgical ICU. However, 4 of these 6 strains were able to spread to 23 other patients and 15 healthcare workers (HCWs). We were surprised by the epidemic behavior of these strains and wondered whether this was exceptional for S. aureus or whether methicillin-susceptible S. aureus (MSSA) was also spreading in the ICU. DESIGN: A 2-month, prospective, observational study to investigate the incidence and spread of MSSA in the surgical ICU of UMCU and historical data collected during a 10-year period regarding MRSA. SETTING: A 10-bed surgical ICU in a 1,042-bed teaching hospital. RESULTS: Weekly swabs revealed the presence of MSSA in 11 (24%) of 45 patients and 16 (22%) of 72 HCWs. Of all 4,105 patient-HCW contacts, there were only 21 episodes in which both the patient and the HCW were found to carry MSSA. With the use of pulsed-field gel electrophoresis, no identical strains could be identified. CONCLUSION: In our surgical ICU, MRSA seems to spread more easily than MSSA, probably because of selection under antibiotic pressure or a still unknown intrinsic factor within MRSA.

Academic Medical Centers↗

In-vitro effects of vancomycin and rifampicin, alone and in combination, against methicillin-sensitive and methicillin-resistant staphylococci.

The in-vitro inhibitory and bactericidal activities of vancomycin and rifampicin were tested against several Staphylococcus strains recently isolated from clinical material. All strains were first tested for their susceptibility to methicillin, and typed by lyogroup. With vancomycin, minimum inhibitory concentrations (MICs) fell within a narrow range (0.62-2.5 mg/l), and significant differences were not observed both among different lyogroups and between methicillin-sensitive (M-S) and methicillin-resistant (M-R) strains. Minimum bactericidal concentrations (MBCs) of vancomycin either equalled or exceeded by twofold the MICs. With rifampicin, MICs were found to be distributed over a wide range (0.002 to greater than 5 mg/l), and susceptibility appeared to be less in M-R than in M-S isolates. Although MICs greater than or equal to mg/l were only observed in three of the six lyogroups, data appeared insufficient to substantiate definite interspecies differences in staphylococcal susceptibility to rifampicin. MBCs of rifampicin either equalled or exceeded by two- to eightfold the MICs. In-vitro interactions between vancomycin and rifampicin, were investigated both by checkerboard and time-kill tests. By both methods, indifference was observed with the great majority of isolates. Synergism was a rare event, and antagonism was never observed.

Drug Combinations↗

Development of new antibiotic resistance in methicillin-resistant but not methicillin-susceptible Staphylococcus aureus.

The frequency of infections caused by multidrug-resistant Staphylococcus aureus continues to increase while the numbers of alternative therapeutic agents remain limited. To investigate the changing patterns of in-vitro susceptibility of S. aureus to 16 antibiotics, 190 clinical isolates from two different years were studied. The MICs of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) strains isolated in 1987 were compared with those of similar numbers of strains isolated in 1989. For MRSA > or = 90% of isolates from both years were resistant to clindamycin, gentamicin and erythromycin. These strains remained highly susceptible to vancomycin (100%), minocycline (90%) and rifampicin (100%). The greatest increase in resistance was observed for ofloxacin (2% in 1987 vs 62% in 1989); cross-resistance to all of the quinolones tested was demonstrated. MSSA strains remained susceptible to vancomycin (100%), minocycline (98%), rifampicin (100%), clindamycin (90%), gentamicin (90%) and ciprofloxacin (98%). It is concluded that methicillin susceptibility is a useful marker for selecting potential agents for the treatment of infections caused by S. aureus. A combination of minocycline and rifampicin may be a useful alternative to vancomycin for treating MRSA infections.

Drug Resistance, Microbial↗

Clindamycin treatment of invasive infections caused by community-acquired, methicillin-resistant and methicillin-susceptible Staphylococcus aureus in children.

BACKGROUND: Community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) is an established pathogen in several areas of the United States, but experience with clindamycin for the treatment of invasive MRSA infections is limited. We compared the outcome of therapy for MRSA with that of methicillin-susceptible (MSSA) invasive infections in children treated with clindamycin, vancomycin or beta-lactam antibiotics. METHODS: The demographics, hospital course and outcome of children at Texas Children's Hospital between February and November 2000 and between August 2001 and August 2002 with invasive S. aureus infections were reviewed from medical records in this retrospective study. RESULTS: CA-MRSA and community-acquired methicillin-susceptible S. aureus (MSSA) caused invasive infections in 46 and 53 children, respectively. The median ages (range) of the children were: MRSA, 3.5 years (2 months to 18.6 years); MSSA, 4.8 years (3 months to 19.8 years). The sites of infection for MRSA vs. MSSA isolates, respectively, were: bacteremia, 3 vs. 6; osteomyelitis, 14 vs. 14; septic arthritis, 5 vs. 7; pneumonia, 11 vs. 3; lymphadenitis, 7 vs. 14; other, 5 vs. 8. Among MRSA patients 39 (20 received clindamycin only, 18 had vancomycin initially and 8 were treated with a beta-lactam initially) received clindamycin and 6 received vancomycin as primary therapy. Among MSSA patients, clindamycin, nafcillin or other beta-lactam antibiotics were used in 24, 18 and 9, respectively. The median number of febrile days was 3 (0 to 14) and 2 (0 to 6) for MRSA and MSSA patients, respectively (P = 0.07). The median number of days with positive blood cultures was 2 for the MRSA (n = 16) and 1 for the MSSA (n = 18) patients (P = 0.04). CONCLUSION: Clindamycin was effective in treating children with invasive infections caused by susceptible CA-MRSA isolates.

Adolescent↗

Rapid discrimination between methicillin-sensitive and methicillin-resistant Staphylococcus aureus by intact cell mass spectrometry.

Rapid, accurate discrimination between methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving intact cell mass spectrometry (ICMS) is presented that shows promise for identification, discrimination of MSSA from MRSA and typing. In ICMS, cells from a bacterial colony are emulsified in a chemical matrix, added to a sample slide, dried and analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). This technique examines the chemistry of the intact bacterial cell surface, yielding spectra consisting of a series of peaks from 500 to 10000, which represent the mass:charge (m:z) ratios. Each peak corresponds to a molecular fragment released from the cell surface during laser desorption. Specimens can be prepared in a few seconds from plate cultures and a spectrum can be obtained within 2 min. ICMS spectra for 20 staphylococcal isolates showed characteristic peaks, some of which were conserved at species level, some at strain level and some were characteristic of the methicillin susceptibility status of the strain. ICMS may have potential for MRSA identification and typing, and may improve infection control measures.

Cluster Analysis↗

Tn554 inserts in methicillin-resistant Staphylococcus aureus from Australia and England: comparison with an American methicillin-resistant group.

We have compared methicillin-resistant (Mcr) Staphylococcus aureus isolates from Australia, the UK and the USA with regard to chromosomal inserts of the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon Tn554. The American isolates were known to have a distinctive Tn554 insert, designated insert 6, which was closely associated epidemiologically with the methicillin-resistance phenotype. Southern blots of DNA from Australian and London, UK Mcr isolates were hybridized with a range of probes related to Tn554. The isolates had similar or identical Tn554 inserts, and we consider them to be a single group, designated 'Australondon'. Australondon isolates were compared in detail with a deletion mutant, ANS62, that had lost the methicillin-resistance determinant mec, plus other resistance determinants resident in the mec region of the chromosome, and with an American Mcr isolate containing Tn554 insert 6. The Australondon isolates had three Tn554 inserts. Sequence analysis with the polymerase chain reaction showed that all of these inserts differed from classical Tn554 in that the 3'-terminal residues of the transposons were reverse complements of the usual GATGTA. One of the Australondon inserts, designated 6B, closely resembled Tn554 insert 6 in the sequence of its left flanking chromosomal DNA. This insert was found to abut the deletion from the mec region which results in strain ANS62. We infer that Tn554 insert 6B is part of the mec region of the chromosome in Australondon isolates, supporting the idea that insert 6 of the American isolates is also part of this chromosomal region.

Australia↗

Subinhibitory concentrations of imipenem induce increased resistance to methicillin and imipenem in vitro in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant (MR) Staphylococcus aureus that was susceptible to less than 0.75 micrograms of imipenem per ml demonstrated inducible resistance. MR S. aureus preincubated with 0.05 microgram of imipenem per ml grew in medium with an imipenem concentration of 32 micrograms/ml, and methicillin MICs increased 20-fold. Non-MR S. aureus exhibited no induction. Preincubation with methicillin produced no effect. Induction appeared to be a unique interaction of imipenem with MR S. aureus.

Enzyme Induction↗

Comparative activity of CGP 31608, nafcillin, cefamandole, imipenem, and vancomycin against methicillin-susceptible and methicillin-resistant staphylococci.

The activity of CGP 31608 for 53 strains of Staphylococcus aureus and 48 strains of S. epidermidis, both methicillin susceptible and resistant, was compared with that of vancomycin, nafcillin, cefamandole, and imipenem. Microdilution MICs were determined in Mueller-Hinton broth with or without 2.5% NaCl at an inoculum of 3 x 10(5) CFU/ml with a 20-h, 37 degrees C incubation. The MICs of imipenem and CGP 31608 for methicillin-resistant strains were lower than the MICs of nafcillin or cefamandole for these strains; these differences diminished in the presence of 2.5% NaCl. Subpopulations were detected in strains of methicillin-resistant S. aureus and S. epidermidis that were resistant to all the beta-lactam antibiotics tested at 5 micrograms/ml. This resistant subpopulation produced progeny that were uniformly resistant to high concentrations of each of the beta-lactams.

Anti-Bacterial Agents↗

Treatment of experimental endocarditis due to methicillin-susceptible or methicillin-resistant Staphylococcus aureus with trimethoprim-sulfamethoxazole and antibiotics that inhibit cell wall synthesis.

Using two strains of Staphylococcus aureus, one susceptible and one heterogeneously resistant to methicillin, for which MICs and MBCs of trimethoprim-sulfamethoxazole (TMP-SMX) were 0.06 and 0.06 micrograms/ml and 0.06 and 0.25 microgram/ml, respectively (concentrations are those of TMP), we studied the efficacies of TMP-SMX and cloxacillin, teicoplanin, and vancomycin for treatment of experimental staphylococcal endocarditis. Rabbits were treated with dosages of TMP-SMX selected to achieve concentrations in serum equivalent to that obtained in humans treated for Pneumocystis carinii pneumonia. The overall mortality rate of rabbits treated with TMP-SMX was 84% at day 3, not different from that of the control groups (P > 0.1). No sterile vegetations were observed to be present in control groups or in animals treated with TMP-SMX. However, 26, 60, and 75% of rabbits treated with teicoplanin, cloxacillin, and vancomycin, respectively, showed sterile vegetations. For methicillin-susceptible S. aureus (MSSA), the mean vegetation counts were not significantly different between the control group and the group treated with TMP-SMX (P > 0.1). For methicillin-resistant S. aureus (MRSA), treatment with TMP-SMX was more effective than no therapy, decreasing the number of organisms in vegetations (P < 0.01). For both strains, therapy with cloxacillin and therapy with teicoplanin or vancomycin were significantly more effective than therapy with TMP-SMX. Despite high concentrations of teicoplanin in serum which exceeded MBCs for staphylococci more than 50 times at the peak and 10 times at the trough, therapy with cloxacillin or vancomycin was superior to therapy with teicoplanin against both MSSA and MRSA. These data do not support the use of TMP-SMX in treatment of endocarditis and other severe staphylococcal infections with high bacterial counts.

Animals↗

DNA polymorphisms in methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus.

Restriction fragment length polymorphisms in methicillin-susceptible and methicillin-resistant (MRSA) strains of Staphylococcus aureus isolated in the same hospital over a 4-month period were studied by using SmaI and ApaI digestion of genomic DNA and pulsed-field gel electrophoresis. Each of the 20 methicillin-susceptible strains had a unique SmaI pattern, but the 27 MRSA strains showed only seven SmaI patterns. More than half of the SmaI fragments in all of these seven patterns were identical, as were those in the patterns from two unrelated MRSA strains. Digestion with ApaI, which cuts staphylococcus DNA into at least twice as many fragments, confirmed the results obtained with SmaI. Lastly, the plasmid contents of MRSA strains showing identical SmaI and ApaI electrophoretic patterns were not identical. These results are interpreted as supporting the hypothesis that all MRSA strains arose from a single clone and emphasize the need to use several methods in epidemiological investigations of MRSA outbreaks.

Bacterial Typing Techniques↗

Real-time PCR can rapidly detect methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles.

We developed, validated, and implemented real-time polymerase chain reaction (PCR) detection of the femA gene for Staphylococcus aureus and the mecA gene for methicillin resistance directly from BACTEC (Becton Dickinson, Sparks, MD) blood culture bottles showing gram-positive cocci in clusters. For the 332 positive blood cultures tested, the assay had 100% sensitivity and specificity for identifying methicillin-susceptible (n=28) and methicillin-resistant (n=28) S aureus, and overall was 98% sensitive and 94% specific, with 3 uninterpretable test results when identification of coagulase-negative staphylococci was included. PCR detection yields rapid (2-3 hours) results and accurate identification of S aureus directly from signal-positive blood culture bottle samples.

Bacteremia↗