Identification of source and sink populations for the emergence and global spread of the East-Asia clone of community-associated MRSA.

BACKGROUND: Our understanding of the factors influencing the emergence, dissemination and global distribution of epidemic clones of bacteria is limited. ST59 is a major epidemic clone of community-associated MRSA in East Asia, responsible for extensive morbidity and mortality, but has a much lower prevalence in other parts of the world. The geographic origin of ST59 and its international routes of dissemination are unclear and disputed in the literature. RESULTS: To investigate the origin and spread of the ST59 clone, we obtained whole genome sequences of isolates from four continents, sampled over more than a decade, and carried out a time-scaled phylogeographic analysis. We discover that two distinct ST59 clades emerged concurrently, in East Asia and the USA, but underwent clonal expansion at different times. The East Asia clade was strongly enriched for gene determinants associated with antibiotic resistance, consistent with regional differences in antibiotic usage. Both clones spread independently to Australia and Europe, and we found evidence of the persistence of multi-drug resistance following export from East Asia. Direct transfer of strains between Taiwan and the USA was not observed in either direction, consistent with geographic niche exclusion. CONCLUSIONS: Our results resolve a longstanding controversy regarding the origin of the ST59 clone, revealing the major global source and sink populations and routes for the spread of multi-drug resistant clones. Additionally, our findings indicate that diversification of the accessory genome of epidemic clones partly reflects region-specific patterns of antibiotic usage, which may influence bacterial fitness after transmission to different geographic locations.

Resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus isolates from broiler chickens at slaughter and abattoir workers.

OBJECTIVES: To comparatively investigate the resistance phenotypes and genotypes of various methicillin-resistant Staphylococcus aureus (MRSA) isolates from broilers at slaughter and workers at the respective poultry slaughterhouses. METHODS: Forty-six MRSA isolates (28 from broilers and 18 from humans) obtained at four different slaughterhouses were included. In addition to previously determined sequence types (STs) and spa types, the isolates were characterized by dru typing, SCCmec typing and PFGE. Resistance phenotypes were determined by broth microdilution. Resistance genes and clonal complexes (CCs) were detected by DNA microarray or specific PCR assays. RESULTS: MRSA of CC398, spa type t011 and varying dru types represented 23/28 broiler isolates and 12/18 human isolates. Three ST9/t1430/dt10a isolates were each seen among the isolates from the abattoir workers and the broilers. In addition, two human CC398/ST1453/t4652/dt3c isolates, a single human CC398/t034/dt6j isolate and two chicken CC398/t108/dt11a isolates were detected. All CC398 isolates (including ST1453) and some of the ST9 isolates from chickens and humans showed resistance to four to nine classes of antimicrobial agents and carried a wide range of resistance genes. While the resistance phenotypes and genotypes of the chicken isolates of the same flock were closely related, they usually differed from the resistance phenotypes and genotypes of the isolates from the workers at the respective slaughterhouse. CONCLUSIONS: The apparent homogeneity of MRSA isolates from the same flock suggests exchange of isolates between the respective animals. The apparent heterogeneity of MRSA isolates from abattoir workers might reflect their occupational contact with animals from numerous chicken flocks.

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.

Extended-spectrum ß-lactamase genes of Escherichia coli in chicken meat and humans, The Netherlands.

We determined the prevalence and characteristics of extended-spectrum ß-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.

Prevalence of livestock-associated MRSA in communities with high pig-densities in The Netherlands.

BACKGROUND: Recently, livestock-associated methicillin-resistant Staphylococcus aureus CC398 has been discovered in animals, livestock farmers and retail meat. This cross-sectional study aimed to determine the spread to persons not in direct contact with livestock in areas with a high density of pig farms. METHODOLOGY/PRINCIPAL FINDINGS: With a random mailing in 3 selected municipalities in The Netherlands, adult persons were asked to fill in a questionnaire and to take a nose swab. In total, complete information was obtained on 583 persons. Of the 534 persons without livestock-contact, one was positive for MRSA (0.2%; 95% confidence interval, <0.01-1.2). Of the 49 persons who did indicate to be working at or living on a livestock farm, 13 were positive for MRSA (26.5%; 95% confidence interval, 16.1-40.4). All spa-types belonged to CC398. CONCLUSIONS/SIGNIFICANCE: Livestock-associated MRSA has a high prevalence in people with direct contact with animals. At this moment it has not spread from the farms into the community.

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

BACKGROUND: Livestock has recently been identified as a new reservoir of methicillin-resistant Staphylococcus aureus (MRSA). Most isolates belong to ST398 and are non-typeable with PFGE using SmaI, making it difficult to study transmission and outbreaks. Therefore, a new PFGE using Cfr9I, a neoschizomer of SmaI was optimized and evaluated to investigate ST398 isolates. RESULTS: After optimizing and evaluating the Cfr9I PFGE, clear and reproducible banding patterns were obtained from all previously non-typeable MRSA (NT(SmaI) -MRSA) isolates. The PFGE patterns of ST398 isolates showed more diversity than with spa-typing and/or MLST. The PFGE results showed diversity within and between the two most prevalent spa-types of NT(SmaI) -MRSA (t011 and t108). No match was found, when comparing banding patterns of the NT(SmaI) -MRSA with 700 different PFGE types, obtained with SmaI 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 Cfr9I PFGE. CONCLUSIONS: 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 Cfr9I are easy to implement to study the ST398 clonal lineage in laboratories which already have a PFGE facility.

Unexpected sequence types in livestock associated methicillin-resistant Staphylococcus aureus (MRSA): MRSA ST9 and a single locus variant of ST9 in pig farming in China.

In October 2008 nine farrow-to-finish pig farms were visited in Shuangliu County in Sichuan Province, China. One farm was empty for one month but not cleaned after depopulation. Dust samples were collected at each farm and analysed for the presence of methicillin-resistant Staphylococcus aureus (MRSA). Dust samples from four farms were also analysed for the presence of methicillin-susceptible S. aureus (MSSA). On 5/9 farms MRSA was isolated and on 2/4 farms MSSA was isolated. On two farms, including the empty farm, no MRSA or MSSA could be detected. All MRSA isolates (n=43) belonged to spa type t899. MSSA isolates belonged to spa type t899 (n=12) and spa type t034 (n=2). From 4/9 farms the MRSA isolates of spa type t899 were assigned to multilocus sequence type (MLST) ST9 whereas on one farm the MRSA spa type t899 isolates belonged to a single locus variant of MLST ST9 (ST1376). MSSA isolates with spa type t899 belonged to MLST ST9 and the MSSA with spa type t034 belonged to MLST ST398. This is the first report on MRSA in pig farms in China and the first time that MRSA ST9 and a single locus variant of ST9 are detected in pig farms. This study shows that livestock associated MRSA is not restricted to clonal lineage ST398 as found in Europe and Northern America in commercial pigs but that other MRSA lineages are able to spread in livestock as well. The study confirms that livestock may act as a reservoir for MRSA.

Multiple-locus variable number tandem repeat analysis of Staphylococcus aureus: comparison with pulsed-field gel electrophoresis and spa-typing.

BACKGROUND: Molecular typing of methicillin-resistant Staphylococcus aureus (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. METHODOLOGY/PRINCIPAL FINDINGS: A new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-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. CONCLUSIONS: The MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus 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.

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.

Methicillin-resistant and -susceptible Staphylococcus aureus sequence type 398 in pigs and humans.

Methicillin-resistant Staphylococcus aureus sequence type 398 (ST398 MRSA) was identified in Dutch pigs and pig farmers. ST398 methicillin-susceptible S. aureus circulates among humans at low frequency (0.2%) but was isolated in 3 human cases of bacteremia (2.1%; p = 0.026). Although its natural host is probably porcine, ST398 MRSA likely causes infections in humans.

Emergence of methicillin-resistant Staphylococcus aureus of animal origin in humans.

In 2003 in the Netherlands, a new methicillin-resistant Staphylococcus aureus (MRSA) strain emerged that could not be typed with Sma1 pulsed-field gel electrophoresis (NT-MRSA). The association of NT-MRSA in humans with a reservoir in animals was investigated. The frequency of NT-MRSA increased from 0% in 2002 to >21% after intensified surveillance was implemented in July 2006. Geographically, NT-MRSA clustered with pig farming. A case-control study showed that carriers of NT-MRSA were more often pig or cattle farmers (pig farmers odds ratio [OR] 12.2, 95% confidence interval [CI] 3.1-48.6; cattle farmers OR 19.7, 95% CI 2.3-169.5). Molecular typing showed that the NT-MRSA strains belonged to a new clonal complex, ST 398. This study shows that MRSA from an animal reservoir has recently entered the human population and is now responsible for >20% of all MRSA in the Netherlands.

Methicillin-resistant Staphylococcus aureus in Dutch soccer team.

An outbreak of community-acquired methicillin-resistant Staphylococcus aureus occurred among members and close contacts of a soccer team. Typing of the isolates showed the outbreak was caused by the well-known European ST80-IV strain. To our knowledge, this is the first report of an outbreak of this strain among members of a sports team.

Community-acquired MRSA and pig-farming.

BACKGROUND: Sporadic cases of CA-MRSA in persons without risk-factors for MRSA carriage are increasing. CASE PRESENTATION: We report a MRSA cluster among family members of a pig-farmer, his co-workers and his pigs. Initially a young mother was seen with mastitis due to MRSA. Six months later her baby daughter was admitted to the hospital with pneumococcal otitis. After staying five days in hospital, the baby was found to be MRSA positive. At that point it was decided to look for a possible source, such as other family members and house-hold animals, including pigs on the farm, since those were reported as a possible source of MRSA earlier. Swabs were taken from the throat and nares of family members and co-workers. A veterinarian obtained swabs from the nares, throat and perineum of 10 pigs. Swabs were cultured following a national protocol to detect MRSA that included the use of an enrichment broth. Animal and human strains were characterized by PFGE, spa-typing, MLST analysis, SSCmec, AGR typing, and the detection for PVL, LukM, and TSST toxin genes. Three family members, three co-workers, and 8 of the 10 pigs were MRSA positive. With the exception of the initial case (the mother) all persons were solely colonized, with no signs of clinical infections. After digestion with SmaI, none of the strains showed any bands using PFGE. All isolates belonged to spa type t108 and ST398. CONCLUSION: 1. This report clearly shows clonal spread and transmission between humans and pigs in the Netherlands. 2. MLST sequence type 398 might be of international importance as pig-MRSA, since this type was shown earlier to be present in epidemiologically unrelated French pigs and pig-farmers. 3. Research is needed to evaluate whether this is a local problem or a new source of MRSA, that puts the until now successful Search and Destroy policy of the Netherlands at risk.

Detection of Helicobacter species DNA by quantitative PCR in the gastrointestinal tract of healthy individuals and of patients with inflammatory bowel disease.

In many animal species different intestinal Helicobacter species have been described and a few species are associated with intestinal infection. In humans, the only member of the Helicobacter family which is well described in literature is Helicobacter pylori. No other Helicobacter-associated diseases have definitely been shown in humans. We developed a sensitive quantitative PCR to investigate whether Helicobacter species DNA can be detected in the human gastrointestinal tract. We tested gastric biopsies (including biopsies from H. pylori positive persons), intestinal mucosal biopsies and fecal samples from healthy persons, and intestinal mucosal biopsies from patients with inflammatory bowel disease (IBD) for the presence of Helicobacter species. All gastric biopsies, positive for H. pylori by culture, were also positive in our newly developed PCR. No Helicobacter species were found in the mucosal biopsies from patients with IBD (n = 50) nor from healthy controls (n = 25). All fecal samples were negative. Our study suggests that Helicobacter species, other than H. pylori, are not present in the normal human gastrointestinal flora and our results do not support a role of Helicobacter species in IBD.

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.