[Outbreak of Campylobacter fetus infection after consumption of unpasteurized sheep's milk cheeses: how to trace the source?] / Een uitbraak met Campylobacter fetus na het eten van rauwmelkse schapenkaas.

Campylobacter fetus is a species of gram-negative bacteria whose primary reservoir is the gastrointestinal tracts of cattle and sheep. Human infections are rare, though often invasive and sometimes fatal. In this paper, we studied an outbreak of six patients with a C. fetus infection and outlined their disease histories. In each case we were able to identify factors that led to a reduced resistance, including pre-existing illnesses and old age. Because of the unusually high number of patients that presented in a time period of only five months, the Community Health Services were commissioned to identify the source of infection. Using whole genome sequencing, we showed that 5 out of 6 patients belonged to the same cluster. This One Health approach resulted in the conclusion that the infection originated from unpasteurized sheep's milk processed into unripened cheese. Finally, various measures were put into place to prevent any further outbreaks.

Dynamics of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus carriage in pig farmers: a prospective cohort study.

Our purpose was to determine the dynamics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) carriage and its determinants in persons working at pig farms, in order to identify targets for interventions. This prospective cohort study surveyed 49 pig farms in the Netherlands on six sampling dates in 1 year (2010-11). Nasal and oropharyngeal swabs were collected, as well as environmental surface samples from stables and house. Of 110 pig farmers, 38% were persistent MRSA nasal carriers. The average cross-sectional MRSA prevalence was 63%. Methicillin-susceptible S. aureus (MSSA) nasal carriage was associated with fewer MRSA acquisitions (prevalence rate (PR) = 0.47, p 0.02). In multivariate analysis, an age of 40-49 years (PR = 2.13, p 0.01), a working week of ≥40 h (PR=1.89, p 0.01), giving birth assistance to sows (PR=2.26, p 0.03), removing manure of finisher pigs (PR=0.48, p 0.02), and wearing a facemask (PR = 0.13, p 0.02) were significantly related with persistent MRSA nasal carriage. A higher MRSA exposure in stables was associated with MRSA in pig farmers (p <0.0001). This study describes a very high prevalence of LA-MRSA carriage in pig farmers, reflecting extensive exposure during work. We identified the possible protective effects of MSSA carriage and of continuously wearing a facemask during work.

Real-time PCR to distinguish livestock-associated (ST398) from non-livestock-associated (methicillin-resistant) Staphylococcus aureus.

BACKGROUND: The Netherlands is one of the most densely populated countries in the world, with extensive livestock of pigs. In 2005, the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) was a fact, with a relatively high MRSA colonisation among pig farmers. These MRSA isolates mostly belonged to sequence type 398 (ST398). Compared to hospital-associated MRSA (HA-MRSA), severe infections due to LA-MRSA and transmission between individuals are still relatively rare. Therefore, LA-MRSA may warrant less stringent containment measures than HA-MRSA in hospital settings. RESULTS: The aim of this study was to develop a rapid diagnostic tool to distinguish LA-MRSA from non-LA-MRSA in aid of infection control. Here, we show that ST398 strains can be readily detected with real-time polymerase chain reaction (PCR). Analysis of a large panel of related and unrelated microorganisms confirmed that the real-time ST398 PCR (ST398-qPCR) assay does not cross-react with other microorganisms or with non-LA-S. aureus strains. ST398-qPCR analysis of MRSA isolates collected in 2010, 2011 and 2012 at the Jeroen Bosch Hospital (n = 275) showed that an average of 78 % of MRSA belonged to sequence type ST398. CONCLUSION: We conclude that the ST398 real-time PCR is a reliable assay to detect LA-S. aureus and anticipate that the use of this assay can prevent the unnecessary closing of hospital wards, which may lead to substantial savings for the health care system.

Multilocus sequence typing for characterization of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.

Transmission of methicillin-resistant Staphylococcus pseudintermedius between infected dogs and cats and contact pets, humans and the environment in households and veterinary clinics.

The objective of this study was to investigate the prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in people, pets and the environment in households with a pet with a clinical MRSP-infection within the past year. Personnel and the environment at veterinary clinics were also screened. Nasal swabs (humans), nasal and perineal swabs (pets) and environmental wipes were examined using selective culturing. Twenty households were enrolled; 10/20 index cases still had clinical signs of infection at the start of the study and all were MRSP-positive. Of the remaining 10 index cases five were MRSP-positive in nasal and/or perineal samples. Five of 14 (36%) contact dogs and four of 13 (31%) contact cats were found MRSP-positive. In the households with an index case with clinical signs of infection 6/7 (86%) contact animals were MRSP-positive. MRSP was cultured from 2/45 (4%) human nasal samples. Domestic contamination was widespread as positive samples were found in 70% of the households and 44% of all environmental samples were MRSP-positive. In all but one of these MRSP-positive households the index case was still MRSP positive. Among the personnel in veterinary clinics 4/141 (3%) were MRSP-positive. MRSP was cultured from 31/200 environmental samples in 7/13 clinics at the first sampling and in 3/6 clinics the environment remained MRSP-positive after cleaning and disinfection indicating that current cleaning procedures often were unable to eliminate MRSP. These results show that transmission of MRSP between infected or colonized dogs and cats and healthy people does occur but is relatively uncommon, while transmission to contact pets occurs frequently, especially when the index case still has clinical signs of MRSP-infection.

Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis.

OBJECTIVES: Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. METHODS: Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). RESULTS: The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. CONCLUSIONS: This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

Chlamydophila psittaci infections in The Netherlands.

Psittacosis, caused by Chlamydophila psittaci, is a well described but sporadically occurring clinical entity, which mainly presents as community-acquired pneumonia. Diagnosis used to be relatively difficult. However, new molecular techniques, such as real-time polymerase chain reaction, increased detection of cases. Furthermore, genotyping of the ompA gene can be used as a tool to trace the possible source of an outbreak or to link a specific bird to a particular patient.

Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates by Luminex xMAP technology.

A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.

Widely distributed and predominant CTX-M extended-spectrum beta-lactamases in Amsterdam, The Netherlands.

Three hundred sixty Enterobacteriaceae and nonfermenting gram-negative bacilli, isolated during one week in May 2004 at five hospitals in Amsterdam, The Netherlands, were evaluated for the presence of extended-spectrum beta-lactamases (ESBLs). A prevalence of 7.8% was found, in contrast to the 1% observed in 1997. CTX-M ESBLs dominated, and four types were identified in 18 isolates.

Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system.

A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6,250 copies/mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.

Multicenter validation of the cppB gene as a PCR target for detection of Neisseria gonorrhoeae.

The cppB gene is often used as a target for detection of Neisseria gonorrhoeae by PCR. Using a coded panel of 500 DNA samples, we determined that the cppB gene is missing in 5.8% of N. gonorrhoeae strains, and therefore we consider the cppB gene to be an unsuitable target.

Simultaneous presence of multiple Campylobacter species in dogs.

The prevalence of coinfection of Campylobacter species in dogs was determined using four isolation methods. In 26% of the positive-testing stools, multiple Campylobacter species were identified. The use of multiple isolation methods as well as the time lapse between sampling and processing are important for detection of coinfection.

Comparative study using amplified fragment length polymorphism fingerprinting, PCR genotyping, and phenotyping to differentiate Campylobacter fetus strains isolated from animals.

A collection of Campylobacter fetus strains, including both C. fetus subsp. fetus and C. fetus subsp. venerealis, were phenotypically identified to the subspecies level and genotypically typed by PCR and amplified fragment length polymorphism (AFLP) analysis. Phenotypic subspecies determination methods were unreliable. Genotyping of the strains by PCR and AFLP showed a clear discrimination between the two subspecies.

Genomic relatedness within five common Finnish Campylobacter jejuni pulsed-field gel electrophoresis genotypes studied by amplified fragment length polymorphism analysis, ribotyping, and serotyping.

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.

Neonatal sepsis by Campylobacter jejuni: genetically proven transmission from a household puppy.

We report a case of neonatal Campylobacter jejuni sepsis in a 3-week-old infant who acquired the infection through transmission from a recently acquired household puppy. Genotyping of Campylobacter strains obtained from puppy and child resulted in highly homogeneous findings. This represents the first genetically proven C. jejuni dog-human transmission.

Evidence for a genetically stable strain of Campylobacter jejuni.

The genetic stability of selected epidemiologically linked strains of Campylobacter jejuni during outbreak situations was investigated by using subtyping techniques. Strains isolated from geographically related chicken flock outbreaks in 1998 and from a human outbreak in 1981 were investigated. There was little similarity in the strains obtained from the different chicken flock outbreaks; however, the strains from each of three chicken outbreaks, including strains isolated from various environments, were identical as determined by fla typing, amplified fragment length polymorphism (AFLP) analysis, and pulsed-field gel electrophoresis, which confirmed the genetic stability of these strains during the short time courses of chicken flock outbreaks. The human outbreak samples were compared with strain 81116, which originated from the same outbreak but has since undergone innumerable laboratory passages. Two main AFLP profiles were recognized from this outbreak, which confirmed the serotyping results obtained at the time of the outbreak. The major type isolated from this outbreak (serotype P6:L6) was exemplified by strain 81116. Despite the long existence of strain 81116 as a laboratory strain, the AFLP profile of this strain was identical to the profiles of all the other historical P6:L6 strains from the outbreak, indicating that the genotype has remained stable for almost 20 years. Interestingly, the AFLP profiles of the P6:L6 group of strains from the human outbreak and the strains from one of the recent chicken outbreaks were also identical. This similarity suggests that some clones of C. jejuni remain genetically stable in completely different environments over long periods of time and considerable geographical distances.

Amplified fragment length polymorphism analysis of Campylobacter jejuni strains isolated from chickens and from patients with gastroenteritis or Guillain-Barré or Miller Fisher syndrome.

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni strains infecting chickens (n = 54) and those causing gastroenteritis in humans (n = 53). In addition, C. jejuni strains associated with the development of Guillain-Barré syndrome (GBS) (n = 14) and Miller Fisher syndrome (MFS) (n = 4), two related acute paralytic syndromes in human, were included. Strains were isolated between 1989 and 1998 in The Netherlands. The AFLP banding patterns were analyzed with correlation-based and band-based similarity coefficients and UPGMA (unweighted pair group method using average linkages) cluster analysis. All C. jejuni strains showed highly heterogeneous fingerprints, and no fingerprints exclusive for chicken strains or for human strains were obtained. All strains were separated in two distinct genetic groups. In group A the percentage of human strains was significantly higher and may be an indication that genotypes of this group are more frequently associated with human diseases. We conclude that C. jejuni from chickens cannot be distinguished from human strains and that GBS or MFS related strains do not belong to a distinct genetic group.