Dynamics and determinants of Staphylococcus aureus carriage in livestock veterinarians: a prospective cohort study.

BACKGROUND: Since 2003, a new clade of methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex (CC) 398 and associated with animal husbandry has emerged in the Netherlands. The purpose of this study was to determine the dynamics of carriage in persons with direct contact to livestock. METHODS: A 2-year prospective cohort study was performed in which the anterior nares and oropharynx of 137 livestock veterinarians were sampled for the presence of S. aureus every 4 months during the first year and again 1 year later. All S. aureus isolates were genotyped by staphylococcal protein A (spa) typing and with multilocus variable-number tandem repeat analysis (MLVA). RESULTS: The mean prevalence of MRSA CC398 carriage was 44% (range, 42%-46%), and for S. aureus the prevalence was 72% (range, 69%-75%). Thirty-two veterinarians (23%) were always carrying MRSA CC398 and 18 of those (56%, 13% of all veterinarians) had identical MLVA types at all sampling moments. CONCLUSIONS: A high proportion of veterinarians had persistent MRSA CC398 carriage during the 2-year study period, indicating that this variant may colonize humans for prolonged periods. Furthermore, prevalence of S. aureus carriage was extremely high, indicating that MRSA CC398 is not replacing the susceptible strains, but comes on top of it.

Optimization of human immunodeficiency virus type 1 envelope glycoproteins with V1/V2 deleted, using virus evolution.

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens.

Livestock-associated MRSA in household members of pig farmers: transmission and dynamics of carriage, a prospective cohort study.

This prospective cohort study describes carriage of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in household members from 49 farrowing pig farms in the Netherlands (2010-2011). Of 171 household members, 4% were persistent MRSA nasal carriers, and the MRSA prevalence on any given sampling moment was 10% (range 7-11%). Working in the stables (of which 98% was MRSA-positive, prevalence ratio (PR) = 2.11 per 10 hours), working with sows (PR=1.97), and living with an MRSA-positive pig farmer (PR=4.63) were significant determinants for MRSA carriage. Significant protective factors were carriage of methicillin-susceptible Staphylococcus aureus (MSSA) (PR=0.50), and wearing a facemask when working in the stables (37% decreased prevalence). All MRSA strains during the study period were known livestock-associated types. The bacteriophage φ3 was not found in household members. Transmission from pigs and the environment appeared to be important determinants; human-to-human transmission could not sufficiently be differentiated. Wearing a facemask when working in the stables and carriage of MSSA are potential interventional targets.

Livestock-associated Staphylococcus aureus CC398: animal reservoirs and human infections.

Over the past 15 years the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has changed significantly. Being initially a nosocomial pathogen, other clones have been detected in the community, leading to infections in relatively young and healthy individuals lacking contact with healthcare. More recently, a specific clone of MRSA CC398 emerged, which has spread extensively in livestock animals and is also found in retail meat. People in contact with food production animals are at high risk of colonization. The ways in which MRSA CC398 can be transmitted to humans are direct contact with animals, environmental contamination, and eating or handling contaminated meat. The role of MRSA CC398 as a food pathogen needs further research. Recently, whole genome sequencing and other genetic analyses have shown that livestock-associated strains are distinct from human-derived strains. However, there is also an exchange of strains between the reservoirs. Livestock-associated and human-associated strains of CC398 share some virulence factors, but there are also distinct virulence factors that appear to be important in host adaptation. Exchange of genes encoding these virulence factors between strains may expand the host range and thereby threaten public health. Since the emergence of MRSA CC398 in humans, approximately 10 years ago, this clone has shown a remarkable evolution, which is described in this review.

Transmission of methicillin-resistant Staphylococcus aureus CC398 from livestock veterinarians to their household members.

There are indications that livestock-associated MRSA CC398 has a reduced human-to-human transmissibility, limiting its impact on public health and justifying modified control measures. This study determined the transmissibility of MRSA CC398 from livestock veterinarians to their household members in the community as compared to MRSA non-CC398 strains. A one-year prospective cohort study was performed to determine the presence of MRSA CC398 in four-monthly nasal and oropharyngeal samples of livestock veterinarians (n  =  137) and their household members (n  =  389). In addition, a cross-sectional survey was performed to detect the presence of MRSA non-CC398 in hospital derived control patients (n  =  20) and their household members (n  =  41). Staphylococcus aureus isolates were genotyped by staphylococcal protein A (spa) typing and multiple-locus variable-number tandem repeat analysis (MLVA). Mean MRSA CC398 prevalence over the study period was 44% (range 41.6-46.0%) in veterinarians and 4.0% (range 2.8-4.7%) in their household members. The MRSA CC398 prevalence in household members of veterinarians was significantly lower than the MRSA non-CC398 prevalence in household members of control patients (PRR 6.0; 95% CI 2.4-15.5), indicating the reduced transmissibility of MRSA CC398. The impact of MRSA CC398 appears to be low at the moment. However, careful monitoring of the human-to-human transmissibility of MRSA CC398 remains important.

Livestock-associated MRSA carriage in patients without direct contact with livestock.

BACKGROUND: Livestock-associated MRSA (MC398) has emerged and is related to an extensive reservoir in pigs and veal calves. Individuals with direct contact with these animals and their family members are known to have high MC398 carriage rates. Until now it was assumed that MC398 does not spread to individuals in the community without pig or veal calf exposure. To test this, we identified the proportion of MC398 in MRSA positive individuals without contact with pigs/veal calves or other known risk factors (MRSA of unknown origin; MUO). METHODS: In 17 participating hospitals, we determined during two years the occurrence of MC398 in individuals without direct contact with livestock and no other known risk factor (n = 271) and tested in a post analysis the hypothesis whether hospitals in pig-dense areas have higher proportions of MC398 of all MUO. RESULTS: Fifty-six individuals (20.7%) without animal contact carried MC398. In hospitals with high pig-densities in the adherence area, the proportion of MC398 of all MUO was higher than this proportion in hospitals without pigs in the surroundings. CONCLUSIONS: One fifth of the individuals carrying MUO carried MC398. So, MC398 is found in individuals without contact to pigs or veal calves. The way of transmission from the animal reservoir to these individuals is unclear, probably by human-to-human transmission or by exposure to the surroundings of the stables. Further research is needed to investigate the way of transmission.

High Resolution Typing by Whole Genome Mapping Enables Discrimination of LA-MRSA (CC398) Strains and Identification of Transmission Events.

After its emergence in 2003, a livestock-associated (LA-)MRSA clade (CC398) has caused an impressive increase in the number of isolates submitted for the Dutch national MRSA surveillance and now comprises 40% of all isolates. The currently used molecular typing techniques have limited discriminatory power for this MRSA clade, which hampers studies on the origin and transmission routes. Recently, a new molecular analysis technique named whole genome mapping was introduced. This method creates high-resolution, ordered whole genome restriction maps that may have potential for strain typing. In this study, we assessed and validated the capability of whole genome mapping to differentiate LA-MRSA isolates. Multiple validation experiments showed that whole genome mapping produced highly reproducible results. Assessment of the technique on two well-documented MRSA outbreaks showed that whole genome mapping was able to confirm one outbreak, but revealed major differences between the maps of a second, indicating that not all isolates belonged to this outbreak. Whole genome mapping of LA-MRSA isolates that were epidemiologically unlinked provided a much higher discriminatory power than spa-typing or MLVA. In contrast, maps created from LA-MRSA isolates obtained during a proven LA-MRSA outbreak were nearly indistinguishable showing that transmission of LA-MRSA can be detected by whole genome mapping. Finally, whole genome maps of LA-MRSA isolates originating from two unrelated veterinarians and their household members showed that veterinarians may carry and transmit different LA-MRSA strains at the same time. No such conclusions could be drawn based spa-typing and MLVA. Although PFGE seems to be suitable for molecular typing of LA-MRSA, WGM provides a much higher discriminatory power. Furthermore, whole genome mapping can provide a comparison with other maps within 2 days after the bacterial culture is received, making it suitable to investigate transmission events and outbreaks caused by LA-MRSA.

Lifestyle-Associated Risk Factors for Community-Acquired Methicillin-Resistant Staphylococcus aureus Carriage in the Netherlands: An Exploratory Hospital-Based Case-Control Study.

BACKGROUND: Community-acquired MRSA (CA-MRSA) is rapidly increasing. Currently, it is unknown which reservoirs are involved. An exploratory hospital-based case-control study was performed in sixteen Dutch hospitals to identify risk factors for CA-MRSA carriage in patients not belonging to established risk groups. METHODS: Cases were in- or outpatients from sixteen Dutch hospitals, colonised or infected with MRSA without healthcare- or livestock-associated risk factors for MRSA carriage. Control subjects were patients not carrying MRSA, and hospitalised on the same ward or visited the same outpatients' clinic as the case. The presence of potential risk factors for CA-MRSA carriage was determined using a standardised questionnaire. RESULTS: Regular consumption of poultry (OR 2⋅40; 95% CI 1⋅08-5⋅33), cattle density per municipality (OR 1⋅30; 95% CI 1⋅00-1⋅70), and sharing of scuba diving equipment (OR 2⋅93 95% CI 1⋅19-7⋅21) were found to be independently associated with CA-MRSA carriage. CA-MRSA carriage was not related to being of foreign origin. CONCLUSIONS: The observed association between the consumption of poultry and CA-MRSA carriage suggests that MRSA in the food chain may be a source for MRSA carriage in humans. Although sharing of scuba diving equipment was found to be associated with CA-MRSA carriage, the role played by skin abrasions in divers, the lack of decontamination of diving materials, or the favourable high salt content of sea water is currently unclear. The risk for MRSA MC398 carriage in areas with a high cattle density may be due to environmental contamination with MRSA MC398 or human-to-human transmission. Further studies are warranted to confirm our findings and to determine the absolute risks of MRSA acquisition associated with the factors identified.

Outbreak of methicillin-resistant Staphylococcus aureus ST398 in a Dutch nursing home.

We describe an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in a nursing home in the Netherlands. Seven residents and 4 healthcare workers were identified with MRSA ST398, but 2 of the healthcare workers carried other strains. This study demonstrates that MRSA ST398 can spread in nursing homes.

Recent emergence of Staphylococcus aureus clonal complex 398 in human blood cultures.

BACKGROUND: Recently, a clone of MRSA with clonal complex 398 (CC398) has emerged that is related to an extensive reservoir in animals, especially pigs and veal calves. It has been reported previously that methicillin-susceptible variants of CC398 circulate among humans at low frequency, and these have been isolated in a few cases of bloodstream infections (BSI). The purpose of this study was to determine the prevalence of S. aureus CC398 in blood cultures taken from patients in a geographic area with a high density of pigs. METHODOLOGY/PRINCIPAL FINDINGS: In total, 612 consecutive episodes of S. aureus BSI diagnosed before and during the emergence of CC398 were included. Three strains (2 MSSA and 1 MRSA) that were isolated from bacteremic patients between 2010-2011 were positive in a CC398 specific PCR. There was a marked increase in prevalence of S. aureus CC398 BSI isolated between 2010-2011 compared to the combined collections that were isolated between 1996-1998 and 2002-2005 (3/157, 1.9% vs. 0/455, 0.0%; p = 0.017). CONCLUSIONS/SIGNIFICANCE: In conclusion, in an area with a relative high density of pigs, S. aureus CC398 was found as a cause of BSI in humans only recently. This indicates that S. aureus CC398 is able to cause invasive infections in humans and that the prevalence is rising. Careful monitoring of the evolution and epidemiology of S. aureus CC398 in animals and humans is therefore important.

Clinical evaluation of Oxoid Brilliance MRSA Agar in comparison with bioMerieux MRSA ID medium for detection of livestock-associated meticillin-resistant Staphylococcus aureus.

Oxoid Brilliance MRSA Agar and bioMérieux MRSA ID medium were evaluated for their ability to identify meticillin-resistant Staphylococcus aureus (MRSA) in clinical samples. Nasal and throat samples (n = 629) were taken from veterinarians and their household members. The sensitivities of Brilliance MRSA Agar and MRSA ID medium after 20 h of incubation were 63.6 and 64.5 %, and the specificities were 94.1 and 99.4 %, respectively. After an enrichment step, the sensitivities increased to 96.3 and 97.2 %, but the specificities decreased to 88.7 and 98.5 %, respectively. Brilliance MRSA Agar and MRSA ID medium are both sensitive methods for the screening of MRSA in combination with broth enrichment, but positive results require confirmation.

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.