Effect of disruption of Staphylococcus aureus PBP4 gene on resistance to beta-lactam antibiotics

Penicillin-binding proteins (PBPs) mediate susceptibility to beta-lactam antibiotics. PBP 4, although not essential for survival, has been associated with low-level resistance to beta-lactam antibiotics. To determine its contribution to survival of Staphylococcus aureus cells exposed to beta-lactams, the PBP 4 gene (pbp4) was disrupted and then complemented in the methicillin-susceptible strain RN4220 and the homogeneous methicillin-resistant strain COL. Depending on the antibiotic tested, the presence or absence of an intact pbp4 has no effect or only a modest effect on growth measured by population analysis. These data indicate that PBP 4 is a relatively unimportant target of beta-lactams not only in methicillin-susceptible but also methicillin-resistant S. aureus.     

Detection of intrinsically resistant (heteroresistant) Staphylococcus aureus with the Sceptor and AutoMicrobic systems.

Modified procedures for the Sceptor Gram-Positive MIC Panel and the Vitek AutoMicrobic System GPS-M Card were evaluated for their ability to detect methicillin-resistant (heteroresistant) Staphylococcus aureus. A total of 398 clinical isolates (including 222 methicillin-resistant S. aureus) obtained from 10 hospitals were tested. Both systems had 2% NaCl in the oxacillin wells. Sceptor MIC panels were inoculated with an organism suspension prepared from an 18- to 24-h blood agar plate and were inoculated for a full 24 h at 35 degrees C before MICs were read. All methicillin-resistant S. aureus isolates were detected as resistant to oxacillin at greater than or equal to 8 micrograms/ml by the Sceptor method and at greater than 2 micrograms/ml by the Vitek method. All 176 oxacillin-susceptible, methicillin-susceptible S. aureus isolates were correctly distinguished from methicillin-resistant S. aureus isolates by Sceptor. However, with the Vitek system 29 methicillin-susceptible S. aureus isolates tested as falsely resistant to oxacillin and four isolates tested as falsely resistant to vancomycin. The modified testing procedure with the Sceptor system can be used reliably for accurate susceptibility testing of methicillin-resistant and methicillin-susceptible S. aureus. The Vitek GPS-M card does not accurately discriminate between methicillin-resistant and methicillin-susceptible S. aureus with an oxacillin breakpoint of greater than 2 micrograms/ml.     

Validation and implementation of the GeneXpert MRSA/SA blood culture assay in a pediatric setting

Blood cultures positive for gram-positive cocci in clusters can pose a dilemma for empiric antimicrobial therapy because they could represent coagulase-negative staphylococcus or Staphylococcus aureus bacteremia. The GeneXpert MRSA/SA BC Assay (Cepheid, Sunnyvale, CA) is a polymerase chain reaction-based method for identifying S aureus and methicillin resistance that has been approved for use in adults, but data on its use in samples from pediatric patients is limited. We validated the Xpert MRSA/SA BC Assay for use with anaerobic and polymicrobial specimens from pediatric patients and implemented it for routine presumptive identification of S aureus in our pediatric hospital. The assay was 100% sensitive and specific for methicillin-resistant S aureus and 100% sensitive and 99.5% specific for methicillin-susceptible S aureus. Time to presumptive identification of S aureus bacteremia and determination of methicillin susceptibility was reduced by more than 24 hours. We found the Xpert MRSA/SA BC Assay to be a rapid, accurate tool for detecting methicillin-resistant and methicillin-susceptible S aureus in positive pediatric blood cultures, including polymicrobial cultures and those recovered in anaerobic blood culture media.     

Usefulness of PCR-restriction fragment length polymorphism typing of the coagulase gene to discriminate arbekacin-resistant methicillin-resistant Staphylococcus aureus strains

We compared the results of two typing methods for 678 strains of methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus. PCR-restriction fragment length polymorphism typing of the coagulase gene was a more reliable method than coagulase serotyping from the viewpoint of arbekacin resistance.     

Empirical therapy for serious Gram-positive infections: making the right choice

It is well established that delaying the administration of effective antimicrobials for the treatment of serious infections has a significant impact on patient outcomes. In this atmosphere of urgency, decision-making regarding therapy is further complicated by the current high rates of drug resistance among important pathogens, such as Staphylococcus aureus . To improve treatment outcomes, decrease the risk of mortality and reduce hospital costs, physicians should always administer the most appropriate antimicrobial for the given scenario. When a staphylococcal infection is suspected but the resistance phenotype is not known, agents that are effective against methicillin-susceptible S. aureus and methicillin-resistant S. aureus provide optimal empirical coverage. However, the number of such empirical monotherapeutic options is limited. Daptomycin has proven clinical efficacy as compared with comparator agents in Gram-positive infections, and could be considered an appropriate therapy for the treatment of infections caused by either methicillin-susceptible S. aureus or methicillin-resistant S. aureus .     

Epidemic methicillin-susceptible Staphylococcus aureus lineages are the main cause of infections at an Iranian university hospital

The majority of Staphylococcus aureus infections from Isfahan, Iran, were caused by epidemic methicillin-susceptible S. aureus (MSSA) lineages, sequence type 8 (ST8), ST22, ST30, and ST6. The predominant methicillin-resistant S. aureus strain was ST239. We observed a high prevalence of Panton-Valentine leukocidin-positive MSSA strains (19.7%), which is a matter of considerable concern, since these strains have the ability to cause severe infections.     

Incidence of constitutive and inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci in a community and a tertiary care hospital

The incidences of inducible clindamycin resistance at two hospitals (an inner-city hospital and a suburban community hospital) were 7 and 12% for methicillin-resistant Staphylococcus aureus, 20 and 19% for methicillin-susceptible S. aureus, and 14 and 35% for coagulase-negative staphylococci, respectively. Given the variability of inducible resistance to clindamycin found in our two hospitals, we conclude that susceptibility testing of staphylococci should include the disk diffusion induction test (D-test).     

Reliability of two novel methods, Alamar and E test, for detection of methicillin-resistant Staphylococcus aureus.

E test strips (AB Biodisk, Culver City, Calif.) tested on 2% NaCl-supplemented Mueller-Hinton agar and Alamar panels (Alamar, Sacramento, Calif.) correctly characterized 127 of 127 methicillin-resistant Staphylococcus aureus isolates and 100 of 100 methicillin-susceptible S. aureus isolates. These overnight antimicrobial susceptibility test systems reliably detect this clinically important resistance.     

Incidence of inducible clindamycin resistance in staphylococci: first results from Turkey

In total, 408 staphylococcal isolates were tested for inducible clindamycin resistance (ICR) by the disk-diffusion induction test (D-test). ICR was detected in 5.7% of 105 methicillin-resistant Staphylococcus aureus (MRSA) isolates, 3.6% of 111 methicillin-susceptible S. aureus isolates, 30.8% of 94 methicillin-resistant coagulase-negative staphylococcal (CoNS) isolates, and 11.2% of 98 methicillin-sensitive CoNS isolates. All MRSA isolates that were erythromycin-resistant and clindamycin-susceptible were positive by the D-test. The same results were obtained with an azithromycin instead of an erythromycin disk. All isolates were susceptible to quinupristin-dalfopristin. The cost-benefit of the d-test should be evaluated locally after determining the incidence of the different resistance phenotypes.     

A high frequency of macrolide-lincosamide-streptogramin resistance determinants in Staphylococcus aureus isolated in South Korea

Genes conferring resistance to one of the macrolide-lincosamide-streptogramin (MLS) antibiotics may confer cross-resistance to others, because they have similar effects on bacterial protein synthesis. In Korea, over 70% of Staphylococcus aureus isolates are methicillin-resistant and erythromycin-resistant methicillin-resistant S. aureus (MRSA) is also prevalent. We investigated the frequency of MLS resistance in erythromycin-resistant S. aureus isolates. A total of 682 isolates of S. aureus were collected in a nationwide antibiotic resistance survey. Susceptibility to erythromycin, clindamycin, and quinupristin/dalfopristin was tested by disk diffusion. In all, 37% of the methicillin-susceptible S. aureus (MSSA) and 97% of the MRSA isolates were resistant to at least one of the MLS antibiotics, whereas all were susceptible to quinupristin/dalfopristin. Out of 518 strains that were resistant to erythromycin, 60 clindamycin-susceptible (30 MSSA, 30 MRSA) and 44 clindamycin-resistant isolates (14 MSSA, 30 MRSA) were selected at random from these strains. Thirteen genes related to MLS resistance were detected in these isolates by PCR. Of the 104 MSSA and MRSA strains tested, 98 harbored one or more erm gene. The most common was erm(A), with erm(C) next. But, msr(A), lnu(A), and mef(A) were rare and no resistance to streptogramin A was encountered.     

Susceptibility of methicillin-resistant Staphylococcus aureus to lysostaphin.

One hundred and eleven isolates of methicillin-resistant Staphylococcus aureus recovered from patients at the Olin E. Teague Veterans Center from March 1983 to April 1987 were as susceptible to lysis by lysostaphin as methicillin-susceptible S. aureus controls were.     

Epidemiologic typing of Staphylococcus aureus by DNA restriction fragment length polymorphisms of rRNA genes: elucidation of the clonal nature of a group of bacteriophage-nontypeable, ciprofloxacin-resistant, methicillin-susceptible S. aureus isolates.

Analysis of DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) was employed to assist in the epidemiologic investigation of the emergence and spread of ciprofloxacin-resistant Staphylococcus aureus at the Atlanta VA Medical Center because many isolates of interest were nontypeable by phages and harbored few plasmids useful as strain markers. Chromosomal DNAs of selected S. aureus isolates were digested initially with 20 different restriction enzymes. EcoRI appeared to give the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Overall, 15 different ribotypes were seen among the 50 S. aureus isolates studied (7 ribotypes among 13 methicillin-susceptible S. aureus [MSSA] isolates and 9 ribotypes among 37 methicillin-resistant S. aureus [MRSA] isolates). Seven of eight ciprofloxacin-resistant MSSA (CR-MSSA) patient isolates had identical antibiograms, were nontypeable by phages, and had a single 22-MDa plasmid. Six of these seven CR-MSSA isolates had an identical ribotype pattern. Ribotyping distinguished this CR-MSSA strain or clone from MRSA and other MSSA isolates, including nontypeable isolates that contained a 22-MDa plasmid. Five ciprofloxacin-susceptible MSSA isolates studied had five ribotypes; one pattern was identical to the CR-MSSA clone. Twenty-three CR-MRSA isolates recovered from the Atlanta VA Medical Center had four different ribotypes. Ribotyping proved to be a useful molecular epidemiologic tool in the study of S. aureus because it differentiated isolates which were indistinguishable by more traditional methods. In addition, this technique demonstrated that at our institution, ciprofloxacin resistance emerged in multiple strains of MRSA, as opposed to primarily a single strain or clone of MSSA.     

Validation of virulence and epidemiology DNA microarray for identification and characterization of Staphylococcus aureus isolates

The human pathogen Staphylococcus aureus is isolated and characterized using traditional culture and sensitivity methodologies that are slow and offer limited information on the organism. In contrast, DNA microarray technology can provide detailed, clinically relevant information on the isolate by detecting the presence or absence of a large number of virulence-associated genes simultaneously in a single assay. We have developed and validated a novel, cost-effective multiwell microarray for the identification and characterization of Staphylococcus aureus. The array comprises 84 gene targets, including species-specific, antibiotic resistance, toxin, and other virulence-associated genes, and is capable of examining 13 different isolates simultaneously, together with a reference control strain. Analysis of S. aureus isolates whose complete genome sequences have been determined (Mu50, N315, MW2, MRSA252, MSSA476) demonstrated that the array can reliably detect the combination of genes known to be present in these isolates. Characterization of a further 43 S. aureus isolates by the microarray and pulsed-field gel electrophoresis has demonstrated the ability of the array to differentiate between isolates representative of a spectrum of S. aureus types, including methicillin-susceptible, methicillin-resistant, community-acquired, and vancomycin-resistant S. aureus, and to simultaneously detect clinically relevant virulence determinants.     

High prevalence of Panton-Valentine leukocidin among methicillin-sensitive Staphylococcus aureus colonization isolates in rural Iowa

Recent studies have shown that livestock can carry Staphylococcus aureus and transmit it to human caretakers. We conducted a pilot study to determine the prevalence and molecular epidemiology of S. aureus among rural Iowans, including individuals with livestock contact. Nasal and throat swabs were collected and plated onto selective media to isolate methicillin-susceptible and methicillin-resistant S. aureus (MRSA), followed by antibiotic resistance testing and molecular analysis of the isolates. While no MRSA was detected, overall, 23.7% (31/131) of participants were found to harbor S. aureus in their nose, throat, or both. Fifteen isolates displayed resistance to one or more tested antibiotics, and the Panton-Valentine leukocidin (PVL) genes were present at a high level (29% [9/31] of S. aureus-positive participants). Younger age and tobacco use were associated with increased risk of S. aureus carriage. Our results suggest that carriage of PVL-positive S. aureus is common among rural Iowans, even in the absence of detectable MRSA colonization.     

Phenotypic and genotypic mupirocin resistance among Staphylococci causing prosthetic joint infection

Mupirocin MICs and mupA presence were determined in 108 staphylococci causing prosthetic joint infection. Zero of 35 isolates (0%) of methicillin-susceptible Staphylococcus aureus, 4/15 (27%) methicillin-resistant S. aureus isolates, 3/16 (19%) methicillin-susceptible coagulase-negative staphylococci, and 11/42 (26%) methicillin-resistant coagulase-negative staphylococci were mupirocin resistant. mupA was detected in all five high-level mupirocin-resistant staphylococci and one mupirocin-susceptible staphylococcus.     

Staphylococcus aureus sepsis and the Waterhouse-Friderichsen syndrome in children

Staphylococcus aureus has increasingly been recognized as a cause of severe invasive illness. We describe three children who died at our institution after rapidly progressive clinical deterioration from this infection, with necrotizing pneumonia and multiple-organ-system involvement. The identification of bilateral adrenal hemorrhage at autopsy was characteristic of the Waterhouse-Friderichsen syndrome, a constellation of findings usually associated with fulminant meningococcemia. The close genetic relationship among the three responsible isolates of S. aureus, one susceptible to methicillin and two resistant to methicillin, underscores the close relationship between virulent methicillin-susceptible S. aureus and methicillin-resistant S. aureus isolates now circulating in the community.     

Identification of factors contributing to T-cell toxicity of Staphylococcus aureus clinical isolates

We examined the ability of 206 clinical isolates of Staphylococcus aureus to lyse T cells and found differences between Agr groups. We found that the beta and delta hemolysins are involved and that methicillin-resistant S. aureus strains are less toxic than methicillin-susceptible S. aureus strains.     

Rapid Detection of Methicillin Resistance in Staphylococcus aureus Isolates by the MRSA-Screen Latex Agglutination Test

The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecA PCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n = 106), as well as for Micrococcus spp. (n = 10), members of the family Enterobacteriaceae (n = 10), Streptococcus pneumoniae (n = 10), and Enterococcus spp. (n = 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecA PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory.     

Screening method for rapid detection of methicillin-resistant (heteroresistant) Staphylococcus aureus.

A broth screening method was developed to detect methicillin-resistant (heteroresistant) strains of Staphylococcus aureus from primary plates. Lyophilized vials containing cation-supplemented Mueller-Hinton broth, 2% NaCl, and 10 micrograms of methicillin and 6 micrograms of oxacillin per ml were hydrated and tested with 129 methicillin-resistant and 35 methicillin-susceptible strains of S. aureus. With the addition of a tetrazolium indicator after 5 h of incubation, 96.9% of resistant strains were detected. No false-positive results occurred.     

Rapid Slide Latex Agglutination Test for Detection of Methicillin Resistance in Staphylococcus aureus

The MRSA screen test (Denka Seiken Co., Ltd.), a commercially available, rapid (20-min) slide latex agglutination test for the determination of methicillin resistance by detection of PBP 2a in Staphylococcus aureus, was compared with the oxacillin agar screen test and PCR detection of the mecA gene. A total of 563 S. aureus isolates were tested. Two hundred ninety-six of the isolates were methicillin-susceptible isolates from cultures of blood from consecutive patients. Also, 267 methicillin-resistant isolates that comprised 248 different phage types were tested. Methicillin resistance was defined as the presence of the mecA gene. Of the 267 mecA gene-positive isolates, 263 were positive by the MRSA screen test (sensitivity, 98.5%), and all the mecA-gene negative strains were negative by the MRSA screen test (specificity, 100%). The oxacillin agar screen test detected methicillin resistance in 250 of the mecA gene-positive isolates (sensitivity, 93.6%). The sensitivity of the MRSA screen test was statistically significantly higher than the sensitivity of the oxacillin agar screen test (P < 0. 05). The MRSA screen test is a highly sensitive and specific test for the detection of methicillin resistance. Also, it offers results within half an hour and is easy to perform, which makes this test a valuable tool in the ongoing battle against methicillin-resistant S. aureus.