Jaap A. Wagenaar

Comparative Genotyping of Campylobacter jejuni by Amplified Fragment Length Polymorphism, Multilocus Sequence Typing, and Short Repeat Sequencing: Strain Diversity, Host Range, and Recombination

ABSTRACT Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.

Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation.

A total of 814 isolates of the foodborne pathogen Campylobacter jejuni were characterized by multilocus sequence typing (MLST) and analysis of the variation of two cell-surface components: the heat-stable (HS) serotyping antigen and the flagella protein FlaA short variable region. We identified 379 combinations of the MLST loci (sequence types) and 215 combinations of the cell-surface components among these isolates, which had been obtained from human disease, animals, food, and the environment. Despite this diversity, 748 (92%) of the isolates belonged to one of 17 clonal complexes, 6 of which contained many (318, 63%) of the human disease isolates. Several clonal complexes exhibited associations with isolation source or particular cell-surface components; however, the latter were poorly predictive of clonal complex. These data demonstrate that the clonal complex, as defined by MLST, is an epidemiologically relevant unit for both long and short-term investigations of C. jejuni epidemiology.

MRSA Transmission between Cows and Humans

We isolated methicillin-resistant Staphylococcus aureus (MRSA) from cows with subclinical mastitis and from a person who worked with these animals. The bovine and human strains were indistinguishable by phenotyping and genotyping methods and were of a low frequency spa type. To our knowledge, this finding indicates the first documented case of direct transmission of MRSA between cows and humans.

Prevalence of Campylobacter jejuni, Campylobacter lari, and Campylobacter coli in different ecological guilds and taxa of migrating birds

ABSTRACT A total of 1,794 migrating birds trapped at a coastal site in southern Sweden were sampled for detection of Campylobacter spp. All isolates phenotypically identified as Campylobacter jejuni and a subset of those identified as non-C. jejuni were identified to the species level by PCR-based techniques. C. jejuni was found in 5.0% of the birds, Campylobacter lari was found in 5.6%, and Campylobacter coli was found in 0.9%. An additional 10.7% of the tested birds were infected with hippurate hydrolysis-negative Campylobacter spp. that were not identified to the species level. The prevalence of Campylobacter spp. differed significantly between ecological guilds of birds. Shoreline-foraging birds feeding on invertebrates and opportunistic feeders were most commonly infected (76.8 and 50.0%, respectively). High prevalence was also shown in other ground-foraging guilds, i.e., ground-foraging invertebrate feeders (11.0%), ground-foraging insectivores (20.3%), and plant-eating species (18.8%). Almost no Campylobacter spp. were found in ground-foraging granivores (2.3%), arboreal insectivores (0.6%), aerial insectivores (0%), or reed- and herbaceous plant-foraging insectivores (3.5%). During the autumn migration, a high proportion of samples from juveniles were positive (7.1% in passerines, 55.0% in shorebirds), indicating transmission on the breeding grounds or during the early part of migration. Prevalence of Campylobacter spp. was associated with increasing body mass among passerine bird species. Furthermore, prevalence was higher in short-distance migrants wintering in Europe than in long-distance migrants wintering in Africa, the Middle East, or Asia. Among ground-foraging birds of the Muscicapidae, those of the subfamily Turdinae (i.e., Turdus spp.) showed a high prevalence of Campylobacter spp., while the organism was not isolated in any member of the subfamily Muscicapinae (i.e., Erithacus and Luscinia). The prevalence of Campylobacter infection in wild birds thus seems to be linked to various ecological and phylogenetic factors, with great variations in carriership between different taxa and guilds.

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

ABSTRACT Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.

Methicillin Resistant Staphylococcus aureus ST398 in Veal Calf Farming : Human MRSA Carriage Related with Animal Antimicrobial Usage and Farm Hygiene

Introduction Recently a specific MRSA sequence type, ST398, emerged in food production animals and farmers. Risk factors for carrying MRSA ST398 in both animals and humans have not been fully evaluated. In this cross-sectional study, we investigated factors associated with MRSA colonization in veal calves and humans working and living on these farms. Methods A sample of 102 veal calf farms were randomly selected and visited from March 2007–February 2008. Participating farmers were asked to fill in a questionnaire (n = 390) to identify potential risk factors. A nasal swab was taken from each participant. Furthermore, nasal swabs were taken from calves (n = 2151). Swabs were analysed for MRSA by selective enrichment and suspected colonies were confirmed as MRSA by using slide coagulase test and PCR for presence of the mecA-gene. Spa types were identified and a random selection of each spa type was tested with ST398 specific PCR. The Sequence Type of non ST398 strains was determined. Data were analyzed using logistic regression analysis. Results Human MRSA carriage was strongly associated with intensity of animal contact and with the number of MRSA positive animals on the farm. Calves were more often carrier when treated with antibiotics, while farm hygiene was associated with a lower prevalence of MRSA. Conclusion This is the first study showing direct associations between animal and human carriage of ST398. The direct associations between animal and human MRSA carriage and the association between MRSA and antimicrobial use in calves implicate prudent use of antibiotics in farm animals.

Livestock-associated methicillin-resistant Staphylococcus aureus in animals and humans

Since 2004 MRSA emerged in animals, particularly in pigs and veal calves. This new MRSA variant was since its first appearance referred to as Livestock Associated-MRSA (LA-MRSA). In Europe and Northern America, LA-MRSA belongs predominantly to clonal complex (CC) 398 whereas in Asia ST9 seems to be dominant in pigs. Persons in direct contact with LA-MRSA-positive animals have an increased risk of becoming MRSA positive. The risk of carriage is mainly related with the intensity of animal contact and with MRSA prevalence among animals on the farm. In contrast with its success in animals, it seemed that MRSA CC398 is a poor persistent colonizer in humans. MRSA ST398 can, however, cause serious (invasive) infections and outbreaks, although, only incidentally reported so far. Farm hygiene and antimicrobial use contributed to MRSA occurrence in animals. Therefore these two determinants should in principle be incorporated into MRSA-control programmes in animal production. Like any other microorganism, LA-MRSA is expected to be able to adapt to new hosts and may change over time in the potential to colonize and to produce toxins. Also, the current circulating clone CC398 may be replaced by another clone in Western countries or emerge in countries where this clone is currently low-prevalent. Ongoing MRSA surveillance in humans and animals is needed to detect changes in epidemiology and to implement effective control measures.

Host-Pathogen Interactions in Campylobacter Infections: the Host Perspective

SUMMARY Campylobacter is a major cause of acute bacterial diarrhea in humans worldwide. This study was aimed at summarizing the current understanding of host mechanisms involved in the defense against Campylobacter by evaluating data available from three sources: (i) epidemiological observations, (ii) observations of patients, and (iii) experimental observations including observations of animal models and human volunteer studies. Analysis of available data clearly indicates that an effective immune system is crucial for the host defense against Campylobacter infection. Innate, cell-mediated, and humoral immune responses are induced during Campylobacter infection, but the relative importance of these mechanisms in conferring protective immunity against reinfection is unclear. Frequent exposure to Campylobacter does lead to the induction of short-term protection against disease but most probably not against colonization. Recent progress in the development of more suitable animal models for studying Campylobacter infection has opened up possibilities to study the importance of innate and adaptive immunity during infection and in protection against reinfection. In addition, advances in genomics and proteomics technologies will enable more detailed molecular studies. Such studies combined with better integration of host and pathogen research driven by epidemiological findings may truly advance our understanding of Campylobacter infection in humans.

Generation of Campylobacter jejuni genetic diversity in vivo

Molecular epidemiology studies suggest that horizontal genetic exchange is a major cause of pathogen biodiversity. We tested this concept for the bacterial enteropathogen Campylobacter jejuni by seeking direct in vivo evidence for the exchange of genetic material among Campylobacter strains. For this purpose, two antibiotic resistance markers were inserted into the hipO or htrA gene of genetically distinct and naturally transformable C. jejuni strains. Genetic exchange of the resistance markers was analysed after co‐cultivation of homologous and heterologous strains in vitro and in vivo during experimental infection of chickens. Double‐resistant recombinants were obtained both in vitro and from the chicken intestine for all combinations of strains tested. Bidirectional genetic exchange of DNA between homologous and heterologous strains was confirmed by Southern blotting in combination with flaA polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP), amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE). Extensive PFGE analyses of isolated recombinants indicated the frequent occurrence of genetic rearrangements during the experimental infection, in addition to the homologous recombination of the antibiotic resistance genes. Together, the data indicate unequivocally that interstrain genetic exchange as well as intragenomic alterations do occur in vivo during C. jejuni infection. These events probably explain the genome plasticity observed for this pathogen.

Persistence of Livestock Associated MRSA CC398 in Humans Is Dependent on Intensity of Animal Contact

Introduction The presence of Livestock Associated MRSA (LA-MRSA) in humans is associated with intensity of animal contact. It is unknown whether the presence of LA-MRSA is a result of carriage or retention of MRSA-contaminated dust. We conducted a longitudinal study among 155 veal farmers in which repeated nasal and throat swabs were taken for MRSA detection. Periods with and without animal exposure were covered. Methods Randomly, 51 veal calf farms were visited from June - December 2008. Participants were asked to fill in questionnaires (n = 155) to identify potential risk factors for MRSA colonisation. Nasal and throat swabs were repeatedly taken from each participant for approximately 2 months. Swabs were analysed for MRSA and MSSA by selective bacteriological culturing. Spa-types of the isolates were identified and a ST398 specific PCR was performed. Data were analyzed using generalized estimation equations (GEE) to allow for correlated observations within individuals. Results Mean MRSA prevalence was 38% in farmers and 16% in family members. Presence of MRSA in farmers was strongly related to duration of animal contact and was strongly reduced in periods with absence of animal contact (−58%). Family members, especially children, were more often carriers when the farmer was a carrier (OR = 2, P<0.05). Only 7% (n = 11) of the participants appeared to be persistent carriers. A large heterogeneity in spa-types was detected, however 92.7% belonged to LA-MRSA CC398. A surprisingly high fraction of the spa-types (7.3%) did not belong to CC398. Conclusion The presence of LA-MRSA in farmers is strongly animal-exposure related. The rapidly decreasing MRSA prevalence during absence of animal contact suggests that LA-MRSA is a poor persistent colonizer in most humans. These results are of relevance for MRSA control strategies.