Genotypes and antibiotic resistance of Campylobacter coli in fattening pigs

Campylobacter coli is a food-borne zoonotic pathogen causing human gastroenteritis worldwide. The organism is a commensal in the intestine of many food production animals including fattening pigs. The role of the pig as a potential reservoir for C. coli affecting human either directly or via poultry has hardly been investigated and genetic characterization of porcine strains is needed to address this question. For this aim multilocus sequence typing (MLST) and flaB typing was applied to 256 C. coli isolates from faeces of fattening pig collected during 2009 at different slaughterhouses in Switzerland. In addition genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. Of the 67 sequence types (STs) obtained by MLST, 37 were found for the first time. flaB typing revealed 46 different types with 14 of them being novel and was useful to further differentiate strains with an identical ST. Quinolone resistance was detected in 33.6% and macrolide resistance was found in 10.6% of isolates. Comparison with 99 C. coli pig isolates from 2001 revealed a significant decrease in antibiotic resistance towards both groups of antibiotics and there was high overlap between genotypes of 2001 and 2009. Little overlap of porcine genotypes was found with 97 C. coli isolates from poultry collected 2008, however, macrolide resistance was significantly higher in pig isolates. In conclusion, C. coli from Swiss pig are heterogeneous containing many novel STs, findings that could reflect the partitioned Swiss pig production with almost no international breed exchange. The antibiotic resistance echoes the use of corresponding drugs in the Swiss livestock production and indicates the efficacy of restrictive application of antibiotics in order to reduce resistances.

Comparison of genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli on chicken retail meat and at slaughter

Multilocus sequence typing (MLST) and antibiotic resistance patterns of Campylobacter jejuni and Campylobacter coli from retail chicken meat showed high overlap with isolates collected at slaughterhouses, indicating little selection along the production chain. They also showed significant common sequence types with human clinical isolates, revealing chicken meat as a likely source for human infection.

Genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli isolates from domestic and travel-associated human cases

Multilocus sequence typing (MLST) extended with flaB typing of 425 Campylobacter jejuni isolates and 42 Campylobacter coli isolates revealed quite a low overlap between human isolates from travel-associated and domestic cases in Switzerland. Men were more frequently affected by Campylobacter than women, but strains from women and, overall, from travel-associated cases showed mutations conferring quinolone resistance more frequently than strains from men and domestic cases, respectively.

Molecular epidemiology of Flavobacterium psychrophilum from Swiss fish farms

Bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) caused by Flavobacterium psychrophilum are 2 of the major diseases causing high fish mortality in salmonid fish farms. The molecular epidemiology of F. psychrophilum is still largely unknown. Multilocus sequence typing (MLST) has been previously used for this pathogen and underscored a correlation between clonal complexes and host fish species. Here we used MLST to study the relationships among 112 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta fario and S. t. lacustris in Swiss fish farms between 1993 and 2012. The isolates belonged to 27 different sequence types (STs). Most of the Swiss outbreaks were associated with strains belonging to clonal complexes CC-ST2 and CC-ST90, found in both rainbow trout and brown trout and represented by several STs. Eight ST singletons could not be connected to any known clonal complex. Already reported from other parts of Europe and North America, CC-ST2 was the most frequent clonal complex observed, and it caused the majority of outbreaks in Switzerland, with CC-ST90 being the second most important type. In the tightly interconnected Swiss fish farms, no association between clonal complex and host fish was detected, but a temporal evolution of the frequency of some STs was observed. The occurrence of sporadic STs suggests high F. psychrophilum diversity and may reflect the presence of different sequence types in the environment.

The origin of the 'Mycoplasma mycoides cluster' coincides with domestication of ruminants.

The 'Mycoplasma mycoides cluster' comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the 'M. mycoides cluster'. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the 'M. mycoides cluster' dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.

Molecular epidemiology of Bacillus anthracis: determining the correct origin

We analyzed and compared strains of Bacillus anthracis isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human B. anthracis strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurred between 1978 and 1981 in a factory processing cashmere wool from the Indian subcontinent. We interpret the worldwide occurrence of B. anthracis strains of cluster A4 to be due to the extensive global trade of untreated cashmere wool during the last century.

Molecular epidemiology of Mycoplasma hyopneumoniae from outbreaks of enzootic pneumonia in domestic pig and the role of wild boar.

Mycoplasma hyopneumoniae is the major cause of enzootic pneumonia (EP) in domestic pigs, a disease with low mortality but high morbidity, having a great economic impact for producers. In Switzerland EP has been successfully eradicated, however, sporadic outbreaks are observed with no obvious source. Besides the possibility of recurrent outbreaks due to persisting M. hyopneumoniae strains within the pig population, there is suspicion that wild boars might introduce M. hyopneumoniae into swine herds. To elucidate possible links between domestic pig and wild boar, epidemiological investigations of recent EP outbreaks were initiated and lung samples of pig and wild boar were tested for the presence of specific genotypes by multilocus sequence typing (MLST). Despite generally different genotypes in wild boar, outbreak strains could be found in geographically linked wild boar lungs after, but so far not before the outbreak. Recurrent outbreaks in a farm were due to the same strain, indicating unsuccessful sanitation rather than reintroduction by wild boar. In another case outbreaks in six different farms were caused by the same strain never found in wild boar, confirming spread between farms due to hypothesized animal transport. Results indicate the presence of identical lineages of wild boar and domestic pig strains, and possible transmission of M. hyopneumoniae between wild boar and pig. However, the role of wild boar might be rather one as a recipient than a transmitter. More important than contact to wild boar for sporadic outbreaks in Switzerland is apparently persistence of M. hyopneumoniae within a farm as well as transmission between farms.

Outbreak of Morel's disease in a Swiss goat flock

Staphylococcus aureus subsp. anaerobius is the causative agent of Morel’s disease in goats and sheep. We report the first outbreak of Morel’s disease in a Swiss goat flock. Multilocus sequence typing revealed that the Swiss isolates belong to sequence type (ST)1464, which is the ST responsible for outbreaks worldwide.

Evolution of methicillin-resistant Staphylococcus aureus (MRSA)

In 2009, a monitoring survey on the prevalence and genetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) in slaughter pigs in Switzerland was started and revealed the presence of an unusual Multi Locus Sequence Type (ST49), which was detected for the first time in slaughter pigs worldwide. In addition to ST49, pigs also harboured ST398 and ST1. The prevalence of MRSA in slaughter pigs in Switzerland increased from 2.0% (95%CI 0.9-3.9) in 2009 to 5.9% (95% CI 3.8-8.7) in 2010, and remained stable in 2011 with 5,6% (95% CI 3.6-8.4). The increase was mainly due to the spread of ST398-t034, whereas ST49 was less frequently detected in 2010 and 2011. The presence of a specific sequence type (ST49) in MRSA from Switzerland suggests that it has been selected in local pig husbandry. However, MRSA of this lineage seems to have a lower potential for dissemination than ST398, which is increasingly detected in Swissslaughter pigs.

Genotypes and antibiotic resistance of bovine Campylobacter and their contribution to human campylobacteriosis

Campylobacter jejuni and Campylobacter coli are the most important bacterial causes of human gastroenteritis. Chicken has been recognized as a major source for human infection, whereas cattle might also contribute to a lesser extent. However, there is a paucity of information available regarding Campylobacter in Swiss cattle and their role for human campylobacteriosis. To gain more information on genotypes and antibiotic resistance of bovine C. jejuni and C. coli and on their contribution to human disease, 97 cattle isolates were analysed. Multilocus sequence typing (MLST) and flaB typing were applied and the gyrA and 23S rRNA genes were screened for point mutations responsible for quinolone and macrolide resistance, respectively. A total of 37 sequence types (STs) and 44 flaB types were identified, including two sequence types and five flaB types not previously described. Most common sequence types were ST21 (21%), ST61 (12%) and ST48 (11%). Only one isolate was macrolide resistant while 31% (n = 30) were quinolone resistant. Source attribution indicated chicken as the main source of human infection with cattle being second. In conclusion, cattle should not be underestimated as a potential source of human campylobacteriosis.

Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies using ribosomal spacer (RS)-PCR, however, demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infections are genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. Furthermore, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. In addition to RS-PCR, other methods for subtyping Staph. aureus are known, including spa typing and multilocus sequence typing (MLST). They are based on sequencing the spa and various housekeeping genes, respectively. The aim of the present study was to compare the 3 analytic methods using 456 strains of Staph. aureus isolated from milk of bovine intramammary infections and bulk tanks obtained from 12 European countries. Furthermore, the phylogeny of animal Staph. aureus was inferred and the zoonotic transfer of Staph. aureus between cattle and humans was studied. The analyzed strains could be grouped into 6 genotypic clusters, with CLB, CLC, and CLR being the most prominent ones. Comparing the 3 subtyping methods, RS-PCR showed the highest resolution, followed by spa typing and MLST. We found associations among the methods but in many cases they were unsatisfactory except for CLB and CLC. Cluster CLB was positive for clonal complex (CC)8 in 99% of the cases and typically positive for t2953; it is the cattle-adapted form of CC8. Cluster CLC was always positive for t529 and typically positive for CC705. For CLR and the remaining subtypes, links among the 3 methods were generally poor. Bovine Staph. aureus is highly clonal and a few clones predominate. Animal Staph. aureus always evolve from human strains, such that every human strain may be the ancestor of a novel animal-adapted strain. The zoonotic transfer of IMI- and milk-associated strains of Staph. aureus between cattle and humans seems to be very limited and different hosts are not considered as a source for mutual, spontaneous infections. Spillover events, however, may happen.

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

Sequence capture and next-generation resequencing of multiple tagged nucleic acid samples for mutation screening of urea cycle disorders

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.

The mutation causing the black-and-tan pigmentation phenotype of Mangalitza pigs maps to the porcine ASIP locus but does not affect its coding sequence

The gene for agouti signaling protein (ASIP) is centrally involved in the expression of coat color traits in animals. The Mangalitza pig breed is characterized by a black-and-tan phenotype with black dorsal pigmentation and yellow or white ventral pigmentation. We investigated a Mangalitza x Piétrain cross and observed a coat color segregation pattern in the F2 generation that can be explained by virtue of two alleles at the MC1R locus and two alleles at the ASIP locus. Complete linkage of the black-and-tan phenotype to microsatellite alleles at the ASIP locus on SSC 17q21 was observed. Corroborated by the knowledge of similar mouse coat color mutants, it seems therefore conceivable that the black-and-tan pigmentation of Mangalitza pigs is caused by an ASIP allele a(t), which is recessive to the wild-type allele A. Toward positional cloning of the a(t) mutation, a 200-kb genomic BAC/PAC contig of this chromosomal region has been constructed and subsequently sequenced. Full-length ASIP cDNAs obtained by RACE differed in their 5' untranslated regions, whereas they shared a common open reading frame. Comparative sequencing of all ASIP exons and ASIP cDNAs between Mangalitza and Piétrain pigs did not reveal any differences associated with the coat color phenotype. Relative qRT-PCR analyses showed different dorsoventral skin expression intensities of the five ASIP transcripts in black-and-tan Mangalitza. The a(t) mutation is therefore probably a regulatory ASIP mutation that alters its dorsoventral expression pattern.

Allelic Heterogeneity at the Equine KIT Locus in Dominant White (W) Horses

White coat color has been a highly valued trait in horses for at least 2,000 years. Dominant white (W) is one of several known depigmentation phenotypes in horses. It shows considerable phenotypic variation, ranging from approximately 50% depigmented areas up to a completely white coat. In the horse, the four depigmentation phenotypes roan, sabino, tobiano, and dominant white were independently mapped to a chromosomal region on ECA 3 harboring the KIT gene. KIT plays an important role in melanoblast survival during embryonic development. We determined the sequence and genomic organization of the approximately 82 kb equine KIT gene. A mutation analysis of all 21 KIT exons in white Franches-Montagnes Horses revealed a nonsense mutation in exon 15 (c.2151C>G, p.Y717X). We analyzed the KIT exons in horses characterized as dominant white from other populations and found three additional candidate causative mutations. Three almost completely white Arabians carried a different nonsense mutation in exon 4 (c.706A>T, p.K236X). Six Camarillo White Horses had a missense mutation in exon 12 (c.1805C>T, p.A602V), and five white Thoroughbreds had yet another missense mutation in exon 13 (c.1960G>A, p.G654R). Our results indicate that the dominant white color in Franches-Montagnes Horses is caused by a nonsense mutation in the KIT gene and that multiple independent mutations within this gene appear to be responsible for dominant white in several other modern horse populations.