Multilocus sequence typing (MLST) of Mycoplasma hyopneumoniae: a diverse pathogen with limited clonality

A multilocus sequence typing (MLST) scheme was established and evaluated for Mycoplasma hyopneumoniae, the etiologic agent of enzootic pneumonia in swine with the aim of defining strains. Putative target genes were selected by genome sequence comparisons. Out of 12 housekeeping genes chosen and experimentally validated, the 7 genes efp, metG, pgiB, recA, adk, rpoB, and tpiA were finally used to establish the MLST scheme. Their usefulness was assessed individually and in combination using a set of well-defined field samples and strains of M. hyopneumoniae. A reduction to the three targets showing highest variation (adk, rpoB, and tpiA) was possible resulting in the same number of sequence types as using the seven targets. The established MLST approach was compared with the recently described typing method using the serine-rich repeat motif-encoding region of the p146 gene. There was coherence between the two methods, but MLST resulted in a slightly higher resolution. Farms recognized to be affected by enzootic pneumonia were always associated with a single M. hyopneumoniae clone, which in most cases differed from farm to farm. However, farms in close geographic or operational contact showed identical clones as defined by MLST typing. Population analysis showed that recombination in M. hyopneumoniae occurs and that strains are very diverse with only limited clonality observed. Elaborate classical MLST schemes using multiple targets for M. hyopneumoniae might therefore be of limited value. In contrast, MLST typing of M. hyopneumoniae using the three genes adk, rpoB, and tpiA seems to be sufficient for epidemiological investigations by direct amplification of target genes from lysate of clinical material without prior cultivation.

Source attribution of human Campylobacter isolates by MLST and fla-typing and association of genotypes with quinolone resistance

Campylobacteriosis is the most frequent zoonosis in developed countries and various domestic animals can function as reservoir for the main pathogens Campylobacter jejuni and Campylobacter coli. In the present study we compared population structures of 730 C. jejuni and C. coli from human cases, 610 chicken, 159 dog, 360 pig and 23 cattle isolates collected between 2001 and 2012 in Switzerland. All isolates had been typed with multi locus sequence typing (MLST) and flaB-typing and their genotypic resistance to quinolones was determined. We used complementary approaches by testing for differences between isolates from different hosts with the proportion similarity as well as the fixation index and by attributing the source of the human isolates with Bayesian assignment using the software STRUCTURE. Analyses were done with MLST and flaB data in parallel and both typing methods were tested for associations of genotypes with quinolone resistance. Results obtained with MLST and flaB data corresponded remarkably well, both indicating chickens as the main source for human infection for both Campylobacter species. Based on MLST, 70.9% of the human cases were attributed to chickens, 19.3% to cattle, 8.6% to dogs and 1.2% to pigs. Furthermore we found a host independent association between sequence type (ST) and quinolone resistance. The most notable were ST-45, all isolates of which were susceptible, while for ST-464 all were resistant.

Low rate of methicillin-resistant coagulase-positive staphylococcal colonization of veterinary personnel in Hong Kong

Elevated rates of methicillin-resistant Staphylococcus aureus (MRSA) carriage have been reported in veterinary personnel, suggesting an occupational colonization risk. Hong Kong veterinary personnel (n = 150) were sampled for coagulase-positive staphylococci (CPS) nasal colonization. Risk factors for colonization were assessed by questionnaire. Isolates were identified and antibiotic susceptibility determined. All CPS isolates were investigated for mecA carriage, SCCmec type and PVL genes. Two subjects were colonized with methicillin-resistant CPS: one with MRSA (spa type t002 (CC5), SCCmec type II) and one with methicillin-resistant Staphylococcus pseudintermedius (MRSP) (MLST type ST71, SCCmec type II-III). MLST type ST71 S. pseudintermedius strain is the predominant MRSP clone circulating in dogs in Europe and in Hong Kong. The low MR-CPS colonization rate may be associated with low levels of large animal exposure or low rates of MRSA colonization of companion animals in Hong Kong. Colonization with non-aureus CPS, which may cause human infection, must also be considered in veterinary personnel.

Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data

OBJECTIVES: We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans. METHODS: Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various beta-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed. RESULTS: Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried bla(OXA-66), bla(ADC-25), bla(TEM-1), aacC2 and IS1133. Strains of ST15 possessed bla(OXA-69), bla(ADC-11), bla(TEM-1) and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of bla(ADC) (one ST10 and one ST12) and/or bla(OXA-66) (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection. CONCLUSIONS: STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumannii.

Evolution of multidrug-resistant Staphylococcus aureus infections in horses and colonized personnel in an equine clinic between 2005 and 2010

A total of 70 Staphylococcus aureus isolates from postoperative infections in hospitalized horses were isolated between January 2005 and January 2011. Among them, 12 isolates were methicillin-susceptible S. aureus (MSSA), 18 were borderline-oxacillin-resistant S. aureus (BORSA), and 40 were methicillin-resistant S. aureus (MRSA). During the same period, the equine clinic personnel were screened for nasal carriage of BORSA and MRSA. Genotyping revealed that BORSA ST1(MLST)-t2863(spa) isolates were responsible for most equine infections and were the main isolates found in colonized members of the personnel between 2005 and 2007, and that in 2007, MRSA ST398-t011-IVa(SCCmec) emerged in infection sites and personnel, replacing BORSA. Besides decreased susceptibility to oxacillin, all MRSA and BORSA of these two major clonal lineages displayed resistance to gentamicin and kanamycin conferred by the aac(6')-Ie-aph(2')-Ia gene and to trimethoprim conferred by dfr(K) in MRSA and dfr(A) in BORSA. All MRSA had additional resistance to tetracycline conferred by tet(M), whereas BORSA generally also display resistance to streptomycin conferred by str. The number of hospital-acquired MRSA infections in horses could be limited after the introduction of basic hygiene measures and personnel decolonization. Two MRSA carriers could not be decolonized using mupirocin, and a year after decolonization, additional members were recolonized with MRSA. Hygiene measures should, therefore, be maintained to limit the transmission of S. aureus between personnel and horses.

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

AIMS: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. METHODS AND RESULTS: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which - in combination with MLST - increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37.5, 33.1 and 8.1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone-sensitive human isolates showed overlapping MLST-flaB types with those of chicken origin, resistant strains showed only 39% of matching types. CONCLUSION: Mainly quinolone-sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.

Genotyping of Mycoplasma hyopneumoniae in wild boar lung samples

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia (EP), an important cause of disease-associated losses in swine production and a role of wild boar in recurrent infections can be supposed. Genotypes of M. hyopneumoniae from wild boar are unknown but could indicate its role as a potential reservoir. Therefore, 34 lung samples being PCR-positive for M. hyopneumoniae from wild boar from the Geneva region in Switzerland were assayed by genotyping using the p146 and multi-locus sequence typing (MLST) approaches and compared to data from outbreak cases from domestic swine in Switzerland. Successful genotyping was dependent on a sufficiently high concentration of M. hyopneumoniae DNA in the samples as assessed by different real-time PCR assays. The p146 genotyping was more successful with 24 samples (70.5%) being typeable whereas only 6 samples (17.6%) could be genotyped using the MLST approach. Variability of genotypes was high but identical types were found in geographically related animals. Genotypes from wild boar showed phylogenetic relatedness to those from domestic pigs but no matching types could be identified. Results show that direct genotyping from wild boar lung samples is possible and provides a promising approach to investigate future EP outbreak related samples from wild boar. (C) 2011 Elsevier B.V. All rights reserved.

The increase of methicillin-resistant Staphylococcus aureus (MRSA) and the presence of an unusual sequence type ST49 in slaughter pigs in Switzerland

Background: In years past, methicillin-resistant S. aureus (MRSA) has been frequently detected in pigs in Europe, North America and Asia. Recent, yet sporadic studies have revealed a low occurrence of MRSA in Switzerland. In 2009, a monitoring survey of the prevalence and genetic diversity of methicillin-resistant S. aureus (MRSA) in slaughter pigs in Switzerland was conducted using methods recommended by the EU guidelines, and using a sampling strategy evenly distributed throughout the year and representative of the Swiss slaughter pig population. Monitoring should determine if the overall prevalence of MRSA in the entire country is increasing over the years and if specific multi-resistant MRSA clones are spreading over the country.;Results: In 2009, the nasal cavities of eight out of 405 randomly selected pigs were positive for MRSA, representing a prevalence of 2.0% (95% CI 0.9-3.9). The following year, 23 out of 392 pigs were positive for MRSA [5.9% prevalence (95% CI 3.8-8.7)]. Three multilocus sequence types (ST), four spa types and two types of staphylococcal cassette chromosome mec (SCCmec) elements were detected. The most frequent genotypes were ST398 (MLST)-(spa)t034-V (SCCmec) (n = 18) and ST49-t208-V (n = 7), followed by ST398-t011-V (n = 4), ST398-t1451-V (n = 1), and ST1-t2279-IVc (n = 1). The isolates displayed resistance to beta-lactams [mecA, (31/31); blaZ, (19/31)]; tetracycline [tet(M), (31/31); tet(K), (30/31)] (n = 31); macrolides and lincosamides [erm(C) (4/31) or erm(A) (18/31)] (n = 22); tiamulin [vga(A)v (9/31) or unknown mechanism (18/31)] (n = 27); trimethoprim [dfr(G) (18/31); spectinomycin [ant(9)-Ia (19/31) or unknown mechanism (3/31)] (n = 22); streptomycin [str(19/31)]; sulphamethoxazole (7/31) and ciprofloxacin (n = 1) (mechanisms not determined).;Conclusions: This study is the first to describe the presence of MRSA ST49 in slaughter pigs, and to demonstrate a significant and nearly three-fold increase of MRSA prevalence in pigs within two years. The presence of a specific clonal lineage of MRSA from Switzerland suggests that it has been selected in Swiss pig husbandry. Effective hygiene measures should be enhanced within the entire pig production chain to suppress the spread of these pathogens into the community.

Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

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.

Multiplex strategy for multilocus sequence typing, fla typing, and genetic determination of antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates collected in Switzerland

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.