Albert J. de Neeling

Emergence of Methicillin-Resistant Staphylococcus aureus of Animal Origin in Humans

MRSA from an animal reservoir has recently entered the human population and is now responsible for >20% of all MRSA in the Netherlands.

Community-acquired MRSA and pig-farming.

BackgroundSporadic cases of CA-MRSA in persons without risk-factors for MRSA carriage are increasing.Case presentationWe 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 Sma I, none of the strains showed any bands using PFGE. All isolates belonged to spa type t108 and ST398.Conclusion1. 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.

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.

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.

Epidemic and Nonepidemic Multidrug-Resistant Enterococcus faecium

The epidemiology of vancomycin-resistant Enterococcus faecium (VREF) in Europe is characterized by a large community reservoir. In contrast, nosocomial outbreaks and infections (without a community reservoir) characterize VREF in the United States. Previous studies demonstrated host-specific genogroups and a distinct genetic lineage of VREF associated with hospital outbreaks, characterized by the variant esp-gene and a specific allele-type of the purK housekeeping gene (purK1). We investigated the genetic relatedness of vanA VREF (n=108) and vancomycin-susceptible E. faecium (VSEF) (n=92) from different epidemiologic sources by genotyping, susceptibility testing for ampicillin, sequencing of purK1, and testing for presence of esp. Clusters of VSEF fit well into previously described VREF genogroups, and strong associations were found between VSEF and VREF isolates with resistance to ampicillin, presence of esp, and purK1. Genotypes characterized by presence of esp, purK1, and ampicillin resistance were most frequent among outbreak-associated isolates and almost absent among community surveillance isolates. Vancomycin-resistance was not specifically linked to genogroups. VREF and VSEF from different epidemiologic sources are genetically related; evidence exists for nosocomial selection of a subtype of E. faecium, which has acquired vancomycin-resistance through horizontal transfer.

The Carbapenem Inactivation Method (CIM), a Simple and Low-Cost Alternative for the Carba NP Test to Assess Phenotypic Carbapenemase Activity in Gram-Negative Rods

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

Penicillin-Resistant Streptococcus pneumoniae in the Netherlands: Results of a 1-Year Molecular Epidemiologic Survey

The molecular epidemiologic characteristics of penicillin-resistant pneumococci in the Netherlands were investigated in 1995. Dutch electronic surveillance data showed that 0.7% of all pneumococci were intermediately resistant and 0.4% were highly resistant to penicillin. From March 1995 to March 1996, 89 penicillin-resistant isolates were collected by 39 medical microbiology laboratories. Thirty different genotypes were observed by restriction fragment end labeling. Twenty-one DNA types were unique, whereas 9 distinct genotypes were shared by > or = 2 isolates. Different serogroups were found within 6 of the 9 genetically identical clusters of penicillin-resistant isolates, suggesting that horizontal transfer of capsular genes is common. Finally, nosocomial transmission of penicillin-resistant pneumococci was observed among 21 elderly adults with chronic obstructive pulmonary disease. This study demonstrates that multiple clones of penicillin-resistant pneumococci have been introduced in the Netherlands, a country with a low prevalence of pneumococcal infection. Some clones spread among the population in and outside hospitals.

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

BackgroundLivestock 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 Sma I, making it difficult to study transmission and outbreaks. Therefore, a new PFGE using Cfr 9I, a neoschizomer of Sma I was optimized and evaluated to investigate ST398 isolates.ResultsAfter optimizing and evaluating the Cfr 9I PFGE, clear and reproducible banding patterns were obtained from all previously non-typeable MRSA (NTSma I-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 NTSma I-MRSA (t011 and t108). No match was found, when comparing banding patterns of the NTSma I-MRSA with 700 different PFGE types, obtained with Sma 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 Cfr 9I PFGE.ConclusionsThe 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 Cfr 9I are easy to implement to study the ST398 clonal lineage in laboratories which already have a PFGE facility.

Validation of syndromic surveillance for respiratory pathogen activity.

The studied respiratory syndromes are suitable for syndromic surveillance because they reflect respiratory pathogen activity patterns

Low prevalence of methicillin-resistant Staphylococcus aureus in Dutch nursing homes.

ACKNOWLEDGMENTS Conflict of Interest: The editor in chief has reviewed the conflict of interest checklist provided by the authors and has determined that the authors have no financial or any other kind of personal conflicts with this paper. Dr. Mellman has recently been engaged as a consultant to Eisai pharmaceuticals. The activity has no relationship to the study report. Dr. Mellman’s activity as a mentor to novice investigators was supported in part by National Institutes of Health Frant K24 MH001917, a midcareer award in patient-oriented research. Author Contributions: Everyone who contributed significantly to the work has been included in the authorship, and all authors contributed to the conception and design, acquisition of data, analysis and interpretation of data; drafting the article or revising it critically for important intellectual content; and final approval of the submitted manuscript. Sponsor’s Role: There was no external sponsorship of the study.