Adriana E. Rosato

Unusual form of oxacillin resistance in methicillin-resistant Staphylococcus aureus clinical strains

The mechanisms by which there is differential expression of resistance to oxacillin within the populations of a single strain remains to be fully understood. The purpose of this study was to evaluate and characterize 25 GOA48 methicillin-resistant Staphylococcus aureus (MRSA) oxacillin-susceptible mecA-positive strains, which were obtained by screening consecutively 832 S. aureus isolates. These 25 isolates (3% of the total strains investigated) were uniformly detected by extending the 24-h oxacillin agar screen plate to 48 h (namely, GOA48-MRSA). Twenty-two isolates tested positive for penicillin-binding protein 2a, whereas the remaining 3 isolates were inconsistently mecA positive. Inconsistent detection of mecA by polymerase chain reaction (PCR) in the mentioned 3 isolates was investigated by colony hybridization using a mecA probe (> or = 80% of colonies hybridized poorly to the probe). A PCR product that amplified the empty SCCmec insertion site (attB), present only if the element was excised, resulted positive in all 3 isolates before oxacillin exposure, whereas integrated elements were positive only for oxacillin-grown isolates. The remaining 22 strains did not reveal excision demonstrating stable mecA. We concluded that resistance to beta-lactams in MRSA-positive mecA strains susceptible to oxacillin is associated to an extreme heterogeneous expression of resistance combined in some cases to oxacillin SCCmec excision.

Identification and Phenotypic Characterization of a β-Lactam-Dependent, Methicillin-Resistant Staphylococcus aureus Strain

ABSTRACT Methicillin resistance in Staphylococcus aureus is primarily mediated by the acquired penicillin-binding protein PBP 2a, which is encoded by mecA. PBP 2a acts together with native PBP 2 to mediate oxacillin resistance by contributing complementary transpeptidase and transglycosylase activities, respectively. In this study, we have investigated a phenotype of β-lactam dependence in a clinical methicillin-resistant S. aureus strain (strain 2884D) obtained by in vitro selection with ceftobiprole. 28884D, which grew very poorly in blood agar, required the presence of the β-lactam antibiotics to grow. On the basis of this observation, we hypothesized that a gene or genes essential for growth were dependent on oxacillin induction. Identification and analysis of genes regulated by oxacillin were performed by both real-time reverse transcription-PCR and spotted microarray analysis. We found that mecA was constitutively expressed in strain 2884D and that the constitutive expression resulted from perturbations in the two systems involved in its regulation, i.e., MecI/MecR1 (staphylococcal chromosome cassette mec type I) and BlaI/BlaR1 (nonfunctional penicillinase operon). PBP 2 appeared to be poorly induced by oxacillin in 2884D. Further analysis of the PBP 2 two-component VraSR regulatory system showed that it was nonfunctional, accounting for the lack of response to oxacillin. Together, these results support the notion that limited PBP 2 availability may have led 2884D to become dependent on oxacillin-mediated mecA induction as a required survival mechanism.

Community-acquired methicillin resistant Staphylococcus aureus in a women's collegiate basketball team.

Community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) infections are becoming increasingly frequent, and cutaneous disease with this organism is often seen in otherwise healthy organized sports participants. A case of CA-MRSA skin and soft tissue infection in a female collegiate basketball player is presented, and screening and management of her team is discussed. Interestingly, multiple MRSA strains were discovered on testing of the team, raising concern that the prevalence of colonization in this population may be high.

Exposure of Clinical MRSA Heterogeneous Strains to β-Lactams Redirects Metabolism to Optimize Energy Production through the TCA Cycle

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a β-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to β-lactam (HeR) in which only a small portion (≤0.1%) of the population expresses resistance to oxacillin (OXA) ≥10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of β-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with β-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished β-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.

Nasal carriage of inducible dormant and community-associated methicillin-resistant Staphylococcus aureus in an ambulatory population of predominantly university students

BACKGROUND We studied risk factors for nasal colonization with inducible dormant methicillin-resistant Staphylococcus aureus (ID-MRSA) and community-associated MRSA (CA-MRSA) in a cohort of predominantly university students. METHODS Nasal surveillance cultures were performed in student health and ambulatory clinics. Molecular features were identified and risk factors for CA-MRSA and ID-MRSA colonization were determined by logistic regression. RESULTS Of the 1000 participants, 89% (n = 890) were university students. Sixty-four percent were female, 59% Caucasian. The mean age was 23.5 years; 1.6% (n = 16) were CA-MRSA and 1.4% (n = 14) were ID-MRSA colonized. Fifteen (94%) of the CA-MRSA strains were PFGE type IV. pvl (Panton-Valentine leukocidin gene) positivity was 75% in CA-MRSA and 57% in ID-MRSA. ID-MRSA isolates were pulsed-field gel electrophoresis (PFGE) type I, 7%; type II, 14%; type V, 7%; and type IV, 71%. CA-MRSA SCCmec classification was 94% type IV and 6% type V. Risk factors for carriage of CA-MRSA were older age (OR 1.046, p=0.040) and dog ownership (OR 1.450, p=0.019). Single family home (OR 0.040, p=0.007) was a protective factor. There were no significant variables of association found for ID-MRSA colonization. CONCLUSIONS ID-MRSA/CA-MRSA colonization was low. Most isolates were PFGE types IV and II, pvl-positive and susceptible to several antibiotics. Older age and dog ownership were risk factors for CA-MRSA. Future studies are needed to assess the impact of ID-MRSA carriage.

Combination antibiotic exposure selectively alters the development of vancomycin intermediate resistance in staphylococcus aureus

ABSTRACT Invasive methicillin-resistant Staphylococcus aureus (MRSA) treated with vancomycin (VAN) is associated with reduced VAN susceptibility and treatment failure. VAN combination therapy is one strategy to improve response, but comprehensive assessments of combinations to prevent resistance are limited. This study identifies optimal combinations to prevent the emergence of VAN-intermediate Staphylococcus aureus (VISA). Two standard MRSA and two heterogeneous VISA (hVISA) strains were exposed for 28 days in vitro to VAN alone, VAN with cefazolin (CFZ), fosfomycin, gentamicin, meropenem, rifampin, piperacillin-tazobactam (TZP), or trimethoprim-sulfamethoxazole. In addition to VAN susceptibility testing, cell wall thickness (CWT), carotenoid content, and membrane fluidity were determined for Mu3. VAN plus any β-lactam limited the VAN MIC increase to 1 to 4 mg/liter throughout the 28-day exposure, with CFZ and TZP being the most effective agents (VAN MIC = 1 to 2 mg/liter). Similar MIC trends occurred with the lipo-/glycopeptide agents daptomycin and telavancin, where β-lactam combinations with VAN prevented MIC increases to these agents as well. Combinations with non-β-lactams were ineffective in preventing VAN MIC increases with VAN MICs of 4 to 16 mg/liter emerging during weeks 2 to 4 of treatment. VAN plus β-lactam decreased CWT significantly, whereas VAN plus other antibiotics significantly increased the CWT. No correlation was observed between carotenoid content or membrane fluidity and antibiotic exposure. Only the combination exposures of VAN plus β-lactam suppress the development of VISA. Rational selection of VAN plus β-lactam should be further explored as a long-term combination treatment of MRSA infections due to their ability to suppress VAN resistance.