PFGE diversity within the methicillin-resistant Staphylococcus aureus clonal lineage ST398

<p>Abstract</p> <p>Background</p> <p>Livestock has recently been identified as a new reservoir of methicillin-resistant <it>Staphylococcus aureus </it>(MRSA). Most isolates belong to ST398 and are non-typeable with PFGE using <it>Sma</it>I, making it difficult to study transmission and outbreaks. Therefore, a new PFGE using <it>Cfr</it>9I, a neoschizomer of <it>Sma</it>I was optimized and evaluated to investigate ST398 isolates.</p> <p>Results</p> <p>After optimizing and evaluating the <it>Cfr</it>9I PFGE, clear and reproducible banding patterns were obtained from all previously non-typeable MRSA (NT_{<it>Sma</it>I}-MRSA) isolates. The PFGE patterns of ST398 isolates showed more diversity than with <it>spa</it>-typing and/or MLST. The PFGE results showed diversity within and between the two most prevalent <it>spa</it>-types of NT_{<it>Sma</it>I}-MRSA (t011 and t108). No match was found, when comparing banding patterns of the NT_{<it>Sma</it>I}-MRSA with 700 different PFGE types, obtained with <it>Sma</it>I digestion, in our database of more than 4000 strains. Furthermore, possible transmission among veterinarians and their family members was investigated and an outbreak of ST398 MRSA in a residential care facility was confirmed with the <it>Cfr</it>9I PFGE.</p> <p>Conclusions</p> <p>The adjusted PFGE can be used as a method for selecting important and distinct ST398 isolates for further research. The adjustments in the PFGE protocol using <it>Cfr</it>9I are easy to implement to study the ST398 clonal lineage in laboratories which already have a PFGE facility.</p

Methicillin-Resistant Staphylococcus aureus in a Beauty Salon, the Netherlands

An outbreak of community-associated USA300 methicillin-resistant Staphylococcus aureus occurred in a beautician and 2 of her customers. Eight other persons, who were either infected (n = 5) or colonized (n = 3), were linked to this outbreak, including a family member, a household contact, and partners of customers

Extended-Spectrum β-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands

We determined the prevalence and characteristics of extended-spectrum β-lactamase (ESBL) genes of Enterobacteriaceae in retail chicken meat and humans in the Netherlands. Raw meat samples were obtained, and simultaneous cross-sectional surveys of fecal carriage were performed in 4 hospitals in the same area. Human blood cultures from these hospitals that contained ESBL genes were included. A high prevalence of ESBL genes was found in chicken meat (79.8%). Genetic analysis showed that the predominant ESBL genes in chicken meat and human rectal swab specimens were identical. These genes were also frequently found in human blood culture isolates. Typing results of Escherichia coli strains showed a high degree of similarity with strains from meat and humans. These findings suggest that the abundant presence of ESBL genes in the food chain may have a profound effect on future treatment options for a wide range of infections caused by gram-negative bacteria

Multiple-Locus Variable Number Tandem Repeat Analysis of Staphylococcus Aureus: Comparison with Pulsed-Field Gel Electrophoresis and spa-Typing

(MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology

Rhesus Macaques (Macaca mulatta) Are Natural Hosts of Specific Staphylococcus aureus Lineages

Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa repeat sequencing and multi-locus sequence typing (MLST), and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs). Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention

Quantification of Bacteria Adherent to Gastrointestinal Mucosa by Real-Time PCR

The use of real-time quantitative PCR (5′ nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides vulgatus. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5′ nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to E. coli and B. vulgatus and with eight other gut microflora species. Mucosal samples spiked with known amounts of E. coli or B. vulgatus DNA showed no PCR inhibition. We conclude that the 5′ nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora