Molecular Characterization of Methicillin-Susceptible Staphylococcus aureus Clinical Isolates in the United States, 2004 to 2010

While much is known about the geographic distribution of different clonal types of methicillin-resistant Staphylococcus aureus (MRSA), few studies have assessed the molecular epidemiology of methicillin-susceptible S. aureus (MSSA), despite its continued clinical importance. In each U.S. Census region, reference laboratories collected successive MSSA isolates from patients with invasive or superficial staphylococcal infections for use in the Tigecycline Evaluation and Surveillance Trial. All isolates from the periods of 2004 to 2005 and 2009 to 2010 underwent antimicrobial susceptibility testing and characterization of their staphylococcal protein A (spa) type. Of the 708 isolates analyzed, 274 spa types were identified and divided into 15 genetic clusters. The most common clones were spa t002 (n = 63, 8.9%) and t008 (n = 56, 7.9%). While the distribution of the predominant spa types did not differ by U.S. Census region or time period, spa t008 was nearly twice as common in community skin and soft tissue infections than in nosocomial bloodstream infections (11.1% versus 5.6%, respectively; P = 0.008). Despite such differences, both community and nosocomial settings had diverse staphylococcal clonal types representing all major spa clusters. In contrast to those of MRSA, MSSA infectious isolates show wide genetic diversity without clear geographical or temporal clustering. Notably, the prevalent MSSA strains (spa t002 and spa t008) are analogous to the predominant MRSA clones, further demonstrating the importance of both lineages.     

Catalase-Negative Staphylococcus lugdunensis Strain with a Novel Point Mutation in the Catalase Gene Isolated from a Patient with Chronic Suppurative Otitis Media

This report describes the results of the sequence analysis of a methicillin-susceptible strain of catalase-negative Staphylococcus lugdunensis. Molecular characterization of the deduced sequence revealed a novel point mutation in the catalase gene. To our knowledge, this is the first report of a catalase-negative S. lugdunensis strain, although catalase-negative isolates of Staphylococcus aureus and Staphylococcus epidermidis have been previously reported.     

Molecular characterization of a catalase-negative Staphylococcus aureus subsp. aureus Strain collected from a patient with mitral valve endocarditis and pericarditis revealed a novel nonsense mutation in the katA gene

We report a case of endocarditis and pericarditis caused by catalase-negative Staphylococcus aureus. Molecular characterization revealed a novel nonsense mutation in the katA gene, leading to a loss of 238 amino acids (47% of the wild-type catalase protein), including the heme-binding site, NADPH-binding region, and Tyr-337, essential for catalysis.     

Molecular Characterization of Endocarditis-Associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.     

Twelve aberrant strains of Staphylococcus aureus subsp. aureus from clinical specimens.

A new biovar of Staphylococcus aureus subsp. aureus was isolated from human clinical specimens and described on the basis of studies of 12 isolates that were compared with 11 standard reference strains. Both DNA hybridization experiments and numerical taxonomy analysis demonstrated that these strains were strictly related to S. aureus subsp. aureus; however, they were significantly different from the latter. The atypical strains belonging to the new biovar can be distinguished from typical S. aureus subsp. aureus strains by their alpha-chymotrypsin, alpha-glucosidase, beta-N-acetylglucosaminidase, lipase (C-14), and leucine arylamidase enzymatic activities and novobiocin resistance. Thus, the combination of alpha-glucosidase and beta-N-acetyl-glucosaminidase is more useful for distinguishing these S. aureus strains from the other, typical ones.     

Ward-Specific Rates of Nasal Cocolonization with Methicillin-Susceptible and -Resistant Staphylococcus spp. and Potential Impact on Molecular Methicillin-Resistant Staphylococcus aureus Screening Tests

We report that the rates of nasal cocolonization with methicillin-susceptible Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci can vary widely between patients admitted to different wards within a single hospital. Such cocolonization can greatly influence the performance of molecular methicillin-resistant S. aureus (MRSA) screening tests depending on the methods used and targets selected.     

Familial Furunculosis Associated with Community-Acquired Leukocidin-Positive Methicillin-Susceptible Staphylococcus aureus ST152

Panton-Valentine leukocidin (PVL) is associated with staphylococcal skin and pulmonary infections. Community dissemination of PVL-producing Staphylococcus aureus strains constitutes a public health concern. Family transmission and spread of community-acquired leukocidin-positive methicillin-susceptible S. aureus ST152 isolates associated with severe clinical symptoms are herein described. Remarkably, ST152 isolates usually are methicillin resistant.     

Molecular Surveillance and Population Structure Analysis of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in High-Risk Wards

In this study we report the results of analysis of 253 isolates of Staphylococcus aureus (132 methicillin [meticillin]-resistant S. aureus [MRSA] isolates and 121 methicillin-susceptible S. aureus [MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureus infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureus species, maximizing benefits and reducing the cost of infection control strategies.Surveillance of hospital-acquired infections (HAIs), as a critical part of any infection control program, is an indispensable instrument for identification of the dimensions of the problem, for early recognition of changes in infection patterns, and for monitoring of infection trends and rates. Furthermore, surveillance programs allow one to evaluate the effectiveness of interventions, reinforcing good practices and influencing key hospital staff and decision makers (3, 16).Molecular typing techniques greatly improve the quality of epidemiological information obtained by surveillance programs, allowing more-accurate differentiation of strains. Molecular typing techniques are very useful for recognizing sporadic, unrelated strains and endemic, persistent strains (1, 30) and for determining if a single strain or different unrelated strains are the cause of observed increases in the frequency of HAIs by a microbial species.Staphylococcus aureus is one of the main etiologic agents of HAIs, particularly in high-risk wards such as intensive care units (ICUs), and methicillin (meticillin)-resistant S. aureus (MRSA) strains are more frequently involved than methicillin-susceptible S. aureus (MSSA) strains (12, 35). This situation turns out to be particularly serious due to the diffusion of highly pathogenic and multidrug-resistant strains (6).The low degree of genetic variability reported for MRSA populations (30) is a major limitation to strain identification, especially when a short time period and a limited area, such as a single hospital, are monitored. Different molecular typing techniques have been used to point out minor but epidemiologically significant genetic differences between MRSA strains (7, 23, 32, 33, 34). No single technique is clearly superior to others in the resolution of MRSA populations, and a combination of two or more methods has been suggested to be the most efficacious approach (23, 34).Unlike MRSA strains, which have been the subject of several studies of virulence, pathogenesis, development of new antibiotic resistances, strain diffusion worldwide and in hospital settings, and genome analysis (5, 11, 14, 21, 28), MSSA strains, because of their susceptibility to first-line antibiotics, have only occasionally been the subject of molecular epidemiological studies in hospital settings (7, 38). Recent studies performed by multilocus sequence typing have shown a strong genetic relationship between MRSA and MSSA strains, suggesting that MRSA clones arise on multiple occasions from successful hospital MSSA clones by horizontal acquisition of the methicillin resistance (mec) gene (8).In this work, we report the results obtained from an extended molecular surveillance program for S. aureus carried out for 18 high-risk wards of six hospitals in Florence, Italy, over an 8-month period. Our aim was to study the population structure and the diffusion of the MRSA and MSSA strains that colonize and infect patients admitted to the wards under observation. With this aim, amplified fragment length polymorphism (AFLP) analysis was utilized to type MRSA and MSSA isolates, whereas multiplex PCR was used to subtype MRSA isolates falling into the same AFLP group. The Simpson index was employed to evaluate the discriminatory powers of the two molecular techniques and to analyze the structures of both the MRSA and the MSSA populations.     

Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Strains Isolated from Patients with Hospital Readmissions

It is unclear whether patients colonized with methicillin-resistant Staphylococcus aureus (MRSA) continue to harbor the same genotype during hospital readmissions. We characterized 140 MRSA strains isolated from 33 persistent MRSA carriers with hospital readmissions. Nearly half of the patients continued to harbor the same genotype, and the rest acquired different genotypes. Among 25 patients who received eradication therapy, 16 (64%) were colonized with MRSA strains exhibiting different genotypes from the preexisting one.     

Clinical Outcomes with Rapid Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Routine Blood Cultures

Staphylococcus aureus is a common cause of bacteremia, with a substantial impact on morbidity and mortality. Because of increasing rates of methicillin-resistant Staphylococcus aureus, vancomycin has become the standard empirical therapy. However, beta-lactam antibiotics remain the best treatment choice for methicillin-susceptible strains. Placing patients quickly on the optimal therapy is one goal of antimicrobial stewardship. This retrospective, observational, single-center study compared 33 control patients utilizing only traditional full-susceptibility methodology to 22 case patients utilizing rapid methodology with CHROMagar medium to detect and differentiate methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains hours before full susceptibilities were reported. The time to targeted therapy was statistically significantly different between control patients (mean, 56.5 ± 13.6 h) and case patients (44.3 ± 17.9 h) (P = 0.006). Intensive care unit status, time of day results emerged, and patient age did not make a difference in time to targeted therapy, either singly or in combination. Neither length of stay (P = 0.61) nor survival (P = 1.0) was statistically significantly different. Rapid testing yielded a significant result, with a difference of 12.2 h to targeted therapy. However, there is still room for improvement, as the difference in time to susceptibility test result between the full traditional methodology and CHROMagar was even larger (26.5 h). This study supports the hypothesis that rapid testing plays a role in antimicrobial stewardship by getting patients on targeted therapy faster.     

Molecular Characterization of Methicillin-Sensitive Staphylococcus aureus Isolates from Bacteremic Patients in a Norwegian University Hospital

Staphylococcus aureus bacteremia is common in both nosocomial and community settings, and the pathogenicity of the microbe depends upon a large repertoire of virulence factors. S. aureus bacteremia isolates (n = 126) were characterized using DNA microarrays. Clonal complexes 5, 8, 15, 30, and 45 accounted for 74.6% of the isolates. We identified geographical differences in dominating clones and toxin gene profiles. One isolate was methicillin resistant. Potential associations between age and genotype were detected.     

Molecular population genetic analysis of Staphylococcus aureus recovered from cows.

Staphylococcus aureus is one of the most common causes of bovine mastitis. To estimate genetic relationships among S. aureus strains recovered from cows, 357 isolates from milk samples from worldwide localities were examined for electrophoretic variation at 13 metabolic-enzyme loci. Thirty-nine electrophoretic types which represented distinctive multilocus enzyme genotypes were identified, and nearly 90% of all isolates were assigned to one of eight clones. Genetic heterogeneity was found among organisms recovered from dairy herds from which multiple isolates were obtained, indicating that the S. aureus population in a single herd can be multiclonal. Although humans and cows shared 7 of the 39 S. aureus clones, each clone was predominantly associated with one of these host species. These results are consistent with the concept of host specialization among S. aureus clones and imply that successful transfer of bacteria between humans and cows is limited.     

Molecular epidemiologic analysis of Staphylococcus aureus isolated from clinical specimens.

Nosocomial infections caused by Staphylococcus aureus are clinically serious and control of such infections requires strain typing to identify the source of contamination. Recently, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) assay have been introduced and have provided a high level of strain discrimination of S. aureus isolated from clinical specimens. This study was performed to classify 82 strains of S. aureus isolated from 4 hospitals in the Kwangju-Chonnam area by PFGE and RAPD assay. Methicillin-resistant S. aureus (MRSA) was identified by disk diffusion method using the oxacillin disk and polymerase chain reaction of mecA gene was done in 69 strains. Eight-three strains including S. aureus ATCC 25923 were classified into 10 groups by RAPD assay, and into 8 groups by PFGE. Classified groups were not related to area or hospital. Classification was not characteristic between MRSA and methicillin-susceptible strains. Nosocomial infections due to outbreak were suggested because some strains disclosed identical band patterns by PFGE. These results indicate that medical personnels and instruments are routes of nosocomial infections caused by MRSA. PFGE and RAPD assay are powerful tools for the epidemiological study of S. aureus, but PFGE is more effective than RAPD assay. RAPD assay needs optimal combination of primers.     

Isolation of an Acidic Surface Antigen from a Conventional Strain of Staphylococcus aureus

Staphylococcus aureus strain 7007, a prototype isolated from a hospital burn unit, was shown to exhibit a significant degree of resistance to ingestion by mouse polymorphonuclear leukocytes. An acidic surface antigen was isolated from strain 7007 by a combination of 10% trichloroacetic acid extraction, ion-exchange chromatography, and gel filtration. Chemical analysis indicated that the surface antigen consists of an unknown aminouronic acid and an amino sugar. Immunochemical analysis suggested that the 7007 antigen is a common feature of all the strains collected from the burn unit. No cross-reactivity was observed between the carbohydrate preparations of various hospital staphylococcal strains and the 7007 heteropolymer. These results suggested the possibility that the surface antigen of strain 7007 represents a strain- or type-specific antigen.     

Molecular Characterization of Staphylococcus aureus and Influenza Virus Coinfections in Patients with Fatal Pneumonia

Molecular techniques were used to characterize genetic features of Staphylococcus aureus in 66 fatal cases of pneumonia caused by S. aureus and influenza A or B viruses. Nucleic acids were extracted from formalin-fixed, paraffin-embedded tissues. The majority of cases revealed genetic markers for Panton-Valentine leukocidin, mecA, and spa type t008.     

Development of Oxacillin Resistance in a Patient with Recurrent Staphylococcus aureus Bacteremia

Whole-genome sequencing was used to compare longitudinal isolates of Staphylococcus aureus that developed resistance to oxacillin (MIC up to 16 μg/ml). The mecA gene was absent. A novel 5-bp TATCC frameshift insertion in a gene encoding an ABC transporter similar to that of the teichoic acid translocation ATP-binding protein TagH and a 3-bp GCT nonframeshift insertion in the pdhA pyruvate dehydrogenase gene were detected in the oxacillin-resistant isolates.     

Strain relatedness of meticillin-resistant Staphylococcus aureus isolates recovered from patients with repeated bacteraemia

Information on the relatedness of isolates causing repeated meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia is limited. An observational study of 177 patients with MRSA bacteraemia, admitted to the emergency department of National Taiwan University Hospital, was conducted from January 2001 to June 2006. Among these patients, 28 had a previous episode of MRSA bacteraemia and 59 died during the index episode of bacteraemia. Until December 2007, among the 118 patients who survived the index episode (101 without previous bacteraemia and 17 with previous bacteraemia), 24 (20.3%) had repeated MRSA bacteraemia. The duration from discontinuation of antimicrobial therapy to repeat episodes was in the range 35–854 days (median 86 days). Eight patients (33.3%) died as a result of the second bacteraemic episode. Clinical characteristics associated with repeated bacteraemia included the diagnosis of infective endocarditis and active malignancy. Pulsed-field gel electrophoresis and multilocus sequence typing analysis were performed for 32 pairs of available isolates recovered from patients with repeated bacteraemia and revealed that 29 of them (90.6%) were genetically closely-related strains. The majority of patients with repeated MRSA bacteraemia had recurrent infections and a high mortality rate.     

Characterization of clinical strains of Staphylococcus aureus associated with pneumonia.

A total of 5 Staphylococcus aureus strains from patients with postinfluenzal staphylococcal pneumonia, 7 from burn patients with staphylococcal pneumonia, and 21 from the nasopharynx of carriers were phenotypically characterized. All or most strains produced coagulase, clumping factor, DNase, thermostable DNase, protease, gelatinase, lipase, and pigment; the strains were low to moderate producers of extracellular protein A, fibrinolysin, and alpha-hemolysin. All strains were sensitive to mercury, half were sensitive to arsenate and cadmium, and 67 to 92% were resistant to penicillin. Differences between strains were not statistically significant. Cell surface hydrophobicity was determined by measuring percent adsorption to hexadecane. Hydrophobicity of postinfluenzal staphylococcal pneumonia strains was significantly lower than that of pneumonia strains from burn patients and carriers (P less than 0.005). Immunoblot experiments with sera immune to one clinical test strain allowed the separation of all strains into three groups based on probe-positive reactions with primarily four staphylococcal polypeptides (154,200, 130,000, 77,100, and 64,400 molecular weight). The difference in distribution of clinical and carrier strains was highly significant (P = 0.007).