Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis

Streptococcus agalactiae (Group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (Group C Streptococcus, GCS) and Streptococcus uberis are relevant mastitis pathogens, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production. The aims of this study were the evaluation of antimicrobial drug resistance patterns, particularly important for streptococcal mastitis control and the identification of strain molecular features. Antimicrobial resistance was assessed by disk diffusion against amoxicillin–clavulanic acid, cefazolin, cefoperazone, pirlimycin-PRL, rifaximin, streptomycin, chloramphenicol, erythromycin-ERY, gentamicin, tetracycline-TET and vancomycin. Genotypic relationships were identified using pulsed-field gel electrophoresis (PFGE), macrolide and/or tetracycline resistance gene profiling, GBS capsular typing, GBS virulence gene profiling and GBS and S. uberis multi locus sequence typing (MLST).The majority of the isolates were susceptible to all drugs except to aminoglycoside, macrolide, lincosamide and tetracycline. Close to half of the TET resistant isolates have tetO and tetK and almost all ERY–PRL resistant isolates have ermB. A high degree of intra-species polymorphism was found for GCS. The GBS belonged to ST-2, -554, -61, -23 lineages and five new molecular serotypes and human GBS insertion sequences in the cpsE gene were found. Also, GBS of serotype V with scpB and lmb seem to be related with GBS isolates of human origin (same ST-2 and similar PFGE). Overall our results suggested that different therapeutic programs may have been implemented in the different farms and that in most cases clones were herd-specific.     

Multilocus sequence typing of Campylobacter jejuni from broilers

Campylobacter jejuni isolates from a national Swedish Campylobacter monitoring in broilers were characterized by multilocus sequencing typing (MLST) in order to study the genetic diversity of this bacterial population. Isolates were initially characterized by pulsed-field gel electrophoresis (PFGE). One hundred were chosen for MLST genotyping. PFGE identified 69 distinct types compared to 44 different sequence types (STs) identified with MLST. Eighteen STs had not been described previously, while the remaining 26 STs were assigned to previously known clonal complexes. The majority of isolates were of genotypes noted in broilers and in humans in earlier studies. However, three clonal complexes, ST-206 complex, ST-677 complex and ST-1034 complex, previously associated with wild bird and environmental samples, were among the genotypes found. This study shows that most of the Swedish broiler isolates were of genotypes noted as common in broilers. However, it also highlights the potential influence of environmental sources on the broiler C. jejuni genotypes.     

Multilocus sequence typing of Mycoplasma bovis reveals host-specific genotypes in cattle versus bison

Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and the bacterium most frequently isolated from the polymicrobial syndrome known as bovine respiratory disease complex. Recently, M. bovis has emerged as a significant health problem in bison, causing necrotic pharyngitis, pneumonia, dystocia and abortion. Whether isolates from cattle and bison comprise genetically distinct populations is unknown. This study describes the development of a highly discriminatory multilocus sequencing typing (MLST) method for M. bovis and its use to investigate the population structure of the bacterium. Genome sequences from six M. bovis isolates were used for selection of gene targets. Seven of 44 housekeeping genes initially evaluated were selected as targets on the basis of sequence variability and distribution within the genome. For each gene target sequence, four to seven alleles could be distinguished that collectively define 32 sequence types (STs) from a collection of 94 cattle isolates and 42 bison isolates. A phylogeny based on concatenated target gene sequences of each isolate revealed that bison isolates are genetically distinct from strains that infect cattle, suggesting recent disease outbreaks in bison may be due to the emergence of unique genetic variants. No correlation was found between ST and disease presentation or geographic origin. MLST data reported here were used to populate a newly created and publicly available, curated database to which researchers can contribute. The MLST scheme and database provide novel tools for exploring the population structure of M. bovis and tracking the evolution and spread of strains.     

Genetic diversity and methicillin resistance of Staphylococcus aureus originating from buffaloes with mastitis in Iran

The aims of the present study were to investigate the genetic diversity and methicillin resistance in S. aureus isolates recovered from mastitis-affected buffaloes. Five hundred seventy-eight milk samples were obtained from buffaloes with mastitis in three provinces, Iran. Ninety-one of the 578 tested samples contained S. aureus (15.74%), in two cases were methicillin resistant S. aureus (MRSA). Isolates were typed by spa typing, followed by MLST on some representative isolates and SCCmec typing for MRSA strains. The presence of genes encoding Panton–Valentine leukocidin (PVL) was also tested by PCR. Eight spa types were identified, with t3576 (n = 18), t7311 (n = 18) and t937 (n = 17) were the most common, followed by t304 (n = 11), t7308 (n = 9), t521 (n = 7), t267 (n = 6), and t527 (n = 5). MLST revealed four different sequence types (STs) including ST97 (related to t521 and t527 spa types), ST352 (related to t267), ST291 (related to t304 and t937) and ST522 (related to t7338, t7311 and t3576). Two MRSA were identified as t304-ST291-SCCmecIV and t7311-ST522-SCCmecIV. No PVL-positive S. aureus were found. A significant difference in geographical distribution of genotypes was observed, with some types being prevalent in all studied provinces (P < 0.001). The results demonstrated genetic diversity among the S. aureus strains involved in mastitis in buffaloes. This study also provides evidence of the presence of MRSA belonging to genotypes which have been earlier reported in human infections, emphasizing the need for their epidemiological monitoring.     

Population structure and virulence content of avian pathogenic Escherichia coli isolated from outbreaks in Sri Lanka

Avian pathogenic Escherichia coli (APEC) causes economically significant infections in poultry. The genetic diversity of APEC and phylogenetic relationships within and between APEC and other pathogenic E. coli are not yet well understood. We used multilocus sequence typing (MLST), PCR-based phylogrouping and virulence genotyping to analyse 75 avian E. coli strains, including 55 isolated from outbreaks of colisepticaemia and 20 from healthy chickens. Isolates were collected from 42 commercial layer and broiler chicken farms in Sri Lanka. MLST identified 61 sequence types (ST) with 44 being novel. The most frequent ST, ST48, was represented by only six isolates followed by ST117 with four isolates. Phylogenetic clusters based on MLST sequences were mostly comparable to phylogrouping by PCR and MLST further differentiated phylogroups B1 and D into two subgroups. Genotyping of 16 APEC associated virulence genes found that 27 of the clinical isolates and one isolate from a healthy chicken belonged to highly virulent genotype according to previously established classification schemes. We found that a combination of four genes, ompT, hlyF, iroN and papC, gave a comparable prediction to that of using five and nine genes by other studies. Four STs (ST10, ST48, ST117 and ST2016) contained APEC isolates from this study and human UPEC isolates reported by others, suggesting that these STs are potentially zoonotic. Our results enhanced the understanding of APEC population structure and virulence association.     

Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing,China

Clostridium perfringens is an important zoonotic pathogen. This study was designed to explore the prevalence and toxin types of C. perfringens in retail beef collected from Beijing, China. Among 221 beef samples collected, 53 samples were positive for C. perfringens, resulting in the average prevalence as 23.98%. By toxin gene-based typing, the most C. perfringens strains belong to type A (96.23%, 51/53), only 2 strains were identified as type D. By a multi-locus sequence typing (MLST)-based analysis, a total of 36 sequence types (STs) were detected, and the most STs (n=30) represented just a single strain. These finding suggested that the prevalence of C. perfringens in retail beef in Beijing was considerably high and these bacteria displayed extreme diversity in genetics.     

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A–D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.     

Genetic diversity among Campylobacter jejuni isolates from healthy livestock and their links to human isolates in Spain

This study was aimed at determining the genetic diversity of Campylobacter jejuni from healthy ruminants and poultry, and study by multilocus sequence typing (MLST) their links to human isolates in Spain. MLST analysis of 160 animal isolates generated 45 sequence types (STs, nine of them new to this study), that clustered into 18 clonal complexes (CC) and nine singletons. The 71 isolates from humans generated 28 STs (13 CC plus four singletons). Only 11 STs and nine CCs were shared by humans and animals (particularly from dairy cattle and sheep), mainly corresponding to sporadic cases rather than outbreaks, probably as an adaptation of the general human population to the types commonly circulating in livestock. PCR analysis of the distribution of four virulence-associated genes detected the cdtABC gene cluster in all 160 isolates but with a 700-bp deletion in four of them, and amplified the virB11, cgtB and wlaN genes in 4.7%, 21.3% and 21.9% respectively. A subset of 87 C. jejuni animal isolates analysed using flaA PCR-RFLP, MLST and pulsed-field gel electrophoresis generated 31, 38 and 55 types respectively. The combined typing approach used provided reliable inter-strain relationships, confirming the co-existence of several strains in some farms, but also identifying identical genotypes sampled over a wide temporal span in different environments and hosts. Typing results confirmed a high genetic diversity of C. jejuni in our region and suggested that ruminants are also important sources for human infection. MLST data provided will help to obtain a more comprehensive image of the population structure of C. jejuni and establish reliable source attribution schemes.     

Antimicrobial Resistance and Multilocus Sequence Types of Finnish Campylobacter jejuni Isolates from Multiple Sources

Antimicrobial susceptibility was determined for 805 domestic Campylobacter jejuni isolates obtained from broilers (n = 459), bovines (n = 120), human patients (n = 95), natural waters (n = 80), wild birds (n = 35) and zoo animals/enclosures (n = 16) with known multilocus sequence types (MLST) for 450 isolates. The minimum inhibitory concentration (MIC) values for erythromycin, tetracycline, streptomycin, gentamicin and the quinolones ciprofloxacin and nalidixic acid were determined with the VetMIC method. MICs were compared with MLST types to find possible associations between sequence type and resistance. The proportions of resistant isolates were 5% (broilers), 6.3% (natural waters), 11.4% (wild birds), 11.6% (human patients), 16.7% (bovines) and 31.3% (zoo). The most common resistance among the human and bovine isolates was quinolone resistance alone while resistance to streptomycin alone was most often detected among the broiler isolates and tetracycline resistance was most commonly observed in the wild bird, water and zoo isolates. No or negligible resistance to erythromycin or gentamicin was detected. In all data, 12/26 of the tetracycline‐resistant isolates were also resistant to streptomycin (P < 0.001) and the clonal complex (CC) ST‐1034 CC showed a high proportion of 75% (9/12) of tetracycline‐resistant isolates, most originating from the zoo and broilers with closely associated MLST types from these sources. No association between quinolone resistance and MLST type was seen. The low percentage of resistant isolates among the domestic Campylobacter infections is most probably due to the long‐term controlled use of antimicrobials. However, the higher percentage of tetracycline resistance observed among the zoo isolates could present a risk for zoo visitors of acquisition of resistant C. jejuni. The resistance pattern of tetracycline and streptomycin most often found in ST‐1034 CC could indicate a common resistance acquisition mechanism commonly present in this CC. Overall, MLST typing was found to be a useful method in recognition of potential genetic lineages associated with resistance.     

Multilocus sequence typing as a tool for studying the molecular epidemiology and population structure of Brachyspira hyodysenteriae

The purpose of this study was to develop and apply a multilocus sequence typing (MLST) scheme to study the molecular epidemiology of Brachyspira hyodysenteriae, the aetiological agent of swine dysentery. Sequences of seven conserved genomic loci were examined in 111 B. hyodysenteriae strains. Fifty-eight of these previously had been analysed by multilocus enzyme electrophoresis (MLEE), and for some the results of pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis (REA) and/or serotyping also were available. The discriminatory power of these methods was compared. The strains were divided into 67 sequence types (STs) and 46 amino acid types (AATs) by MLST. The Index of Association value was significantly different from zero, indication that the population was clonal. Eleven clonal complexes (Cc) comprising between 2 and 10 STs were recognised. A population snapshot based on AATs placed 77.5% of the isolates from 30 of the AATs into one major cluster. The founder type AAT9 included 13 strains from nine STs that were isolated in Australia, Sweden, Germany and Belgium, including one from a mallard. The MLST results were generally comparable to those produced by MLEE. The MLST system had a similar discriminatory power to PFGE, but was more discriminatory than REA, MLEE or serotyping. MLST data provided evidence for likely transmission of strains between farms, but also for the occurrence of temporal “micro-evolution” of strains on individual farms. Overall, the MLST system proved to be a useful new tool for investigating the molecular epidemiology and diversity of B. hyodysenteriae.     

Genotypes and antibiotic resistance of canine Campylobacter jejuni isolates

Campylobacter jejuni is the most important cause of bacterial gastroenteritis in humans. It is a commensal in many wild and domestic animals, including dogs. Whereas genotypes of human and chicken C. jejuni isolates have been described in some detail, only little information on canine C. jejuni genotypes is available. To gain more information on genotypes of canine C. jejuni and their zoonotic potential, isolates from routine diagnostics of diarrheic dogs as well as isolates of a prevalence study in non-diarrheic dogs were analyzed. Prevalence of thermophilic Campylobacter among non-diarrheic dogs was 6.3% for C. jejuni, 5.9% for Campylobacter upsaliensis and 0.7% for Campylobacter coli. The C. jejuni isolates were genotyped by multi locus sequence typing (MLST) and flaB typing. Resistance to macrolides and quinolones was genetically determined in parallel. Within the 134 genotyped C. jejuni isolates 57 different sequence types (ST) were found. Five STs were previously unrecognized. The most common STs were ST-48 (11.2%), ST-45 (10.5%) and ST-21 (6.0%). Whereas no macrolide resistance was found, 28 isolates (20.9%) were resistant to quinolones. ST-45 was significantly more prevalent in diarrheic than in non-diarrheic dogs. Within the common time frame of isolation 94% of the canine isolates had a ST that was also found in human clinical isolates. In conclusion, prevalence of C. jejuni in Swiss dogs is low but there is a large genetic overlap between dog and human isolates. Given the close contact between human and dogs, the latter should not be ignored as a potential source of human campylobacteriosis.     

Identification and characterization of methicillin-resistant Staphylococcus aureus (MRSA) from Austrian companion animals and horses

The aim of this study was to investigate the antimicrobial resistance, resistance gene patterns and genetic relatedness of a collection of Austrian methicillin-resistant Staphylococcus aureus (MRSA) isolates from companion animals and horses.A total of 89 non-repetitive MRSA isolates collected during routine veterinary microbiological examinations from April 2004 to the end of 2012, and one isolate from 2013 were used for this study. The presence of mecA and other resistance genes was confirmed by PCR. Isolates were genotyped by spa typing, two multiple-locus variable-number tandem repeat analyses (MLVA) analyses, SCCmec typing and multilocus sequence typing (MLST). PCR targeting Panton-Valentine leukocidin (PVL) and detection of staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST) was performed using PCR assays. Antimicrobial susceptibility testing was performed.Five sequence types (STs—ST398, ST254, ST22, ST5 and ST1), SCCmec types II, IVa, V, and non-type-abele, 8 spa-types (t003, t011, t036, t127, t386, t1348, and t4450), and two isolates could not be assigned, 21 MLVA-14Orsay types Multiplex-PCR MLVA (mMLVA) displayed 17 different MLVA types.The present study is the most comprehensive dealing with MRSA from Austrian companion animals and horses. The results confirm that MRSA ST398 is present in a wide range of animal species and is predominant especially in horses. In other companion animals it is unclear whether the infections with the different MRSA isolates investigated in the present study truly represents a rare phenomenon or may be an emerging problem in companion animals.     

Characterization of 26 isolates of Staphylococcus aureus, predominantly from dairy sheep, using four different techniques of molecular epidemiology.

Little information is available regarding the molecular epidemiology of Staphylococcus aureus-induced mastitis in dairy sheep. In this study, 4 different typing techniques were compared in typing 26 S. aureus isolates, predominantly from cases of subclinical mastitis in dairy ewes. The 4 techniques were pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) on 2 genes (coagulase and clumping factor B), randomly amplified polymorphic DNA-polymerase chain reaction (PCR) (RAPD-PCR), and multilocus sequence typing (MLST). On the basis of discriminatory power as the key parameter of typing systems, MLST and PFGE were found to be the most powerful techniques. The MLST and PFGE could contribute to epidemiological surveillance and evaluation of mastitis control programs, by documenting prevalence and dissemination of endemic clones in infected populations. The results of this study show that a single clone of S. aureus is widely distributed in infected ewe mammary glands.     

Genetic diversity and associated pathology of Pasteurella multocida isolated from porcine pneumonia

Pasteurella multocida is a widespread respiratory pathogen in pigs associated with atrophic rhinitis and contributing to aggravation of the pulmonary lesions. The aims of the present study were to characterize isolates of P. multocida from porcine bronchopneumonia by pulsed-field gel electrophoresis (PFGE), PCR based capsular typing and multilocus sequence typing (MLST) and to compare clonal complexes outlined with the type of histological lung lesions to investigate if a correlation between clonal lineages and lesions might exist. Isolates of P. multocida were obtained from cases of cranioventrally located porcine bronchopneumonia. All lung lesions were described and classified according to histological lesions. A total of 139 isolates, from lung (n=111), pericardial sac (n=21) and kidney (n=7) of 111 pigs were described using PFGE with ApaI as the restriction enzyme. Furthermore, 20 and 29 isolates were characterized by capsular serotyping and multilocus sequence typing, respectively. PFGE demonstrated 15 different clusters showing 50% or more similarity. All selected isolates were of capsular serotype A and only three main sequence types (ST) were detected among the isolates. Associations were not found between histopathology and clonal complexes of P. multocida. In conclusion, PFGE demonstrated a high diversity of genotypes of P. multocida associated with porcine bronchopneumonia. However, isolates obtained mainly belonged to few STs, indicating that isolates of P. multocida associated with porcine bronchopneumonia originates from a limited number of clonal lineages and therefore might have adapted to porcine hosts. No correlation was demonstrated between genotypes and types of lesions, and extra-pulmonary spreading was only rarely demonstrated.     

Sequence types and pleuromutilin susceptibility of Brachyspira hyodysenteriae isolates from Italian pigs with swine dysentery: 2003–2012

Swine dysentery is a mucohaemorrhagic colitis of pigs caused by infection with Brachyspira hyodysenteriae. The disease can be controlled by treatment with antimicrobial agents, with the pleuromutilins tiamulin and valnemulin being widely used. In recent years, the occurrence of B. hyodysenteriae with reduced susceptibility to these drugs has been increasing. The aim of this study was to determine temporal changes in genetic groups and pleuromutilin susceptibility amongst B. hyodysenteriae isolates from Italy. Multilocus sequence typing (MLST) was performed on 108 isolates recovered from 87 farms in different regions of Italy from 2003 to 2012, and their minimum inhibitory concentrations (MICs) for tiamulin and valnemulin were determined. Logistic regression was performed to assess associations between susceptibility to the two antimicrobial agents and genetic group, year and region of isolation. The isolates were allocated to 23 sequence types (STs), with five clonal clusters (Ccs) and seven singletons. More than 50% of isolates were resistant to both pleuromutilins (MIC >2.0 µg/mL for tiamulin and >1.0 µg/mL for valnemulin). All 10 isolates in ST 83 were resistant; these were first isolated in 2011 and came from nine farms, suggesting recent widespread dissemination of a resistant strain. Significant associations were found between the proportion of pleuromutilin susceptible isolates and the genetic group and year of isolation. Although resistant isolates were found in all Ccs, isolates in Ccs 2 and 7 were over five times more likely to be susceptible than those in the other Ccs. A significant trend in the reduction of susceptibility over time also was observed.     

Genotypes,Antibiotic Resistance Profiles and Microarray‐Based Characterization of Methicillin‐Resistant Staphylococcus aureus Strains Isolated from Livestock and Veterinarians in Switzerland

Using different typing methods (MLST, spa‐, SCCmec‐ and agr‐typing), PFGE and DNA microarray‐based chip analysis, we characterized 20 MRSA strains isolated from livestock and veterinarians. PFGE analysis after macrorestriction with EagI provided seven different band patterns, which could be grouped into four clusters. One cluster consisted of all MRSA ST398 strains isolated from pigs, calves, mastitis milk and two veterinarians. One strain of ST398 from a veterinarian and the two strains of ST1 and ST8 formed the three other clusters. Antimicrobial susceptibility testing showed that 15 of 20 strains were resistant to ampicillin, cefoxitin, clindamycin, erythromycin, oxacillin, penicillin and tetracycline. All strains were susceptible to rifampin and vancomycin, 19 were susceptible to ciprofloxacin and 18 were susceptible to sulphamethoxazole/trimethoprim. Genes encoding different enterotoxins, leukotoxins and haemolysins were found in certain strains.     

A combinational approach of multilocus sequence typing and other molecular typing methods in unravelling the epidemiology of Erysipelothrix rhusiopathiae strains from poultry and mammals

Erysipelothrix rhusiopathiae infections re-emerged as a matter of great concern particularly in the poultry industry. In contrast to porcine isolates, molecular epidemiological traits of avian E. rhusiopathiae isolates are less well known. Thus, we aimed to (i) develop a multilocus sequence typing (MLST) scheme for E. rhusiopathiae, (ii) study the congruence of strain grouping based on pulsed-field gel electrophoresis (PFGE) and MLST, (iii) determine the diversity of the dominant immunogenic protein SpaA, and (iv) examine the distribution of genes putatively linked with virulence among field isolates from poultry (120), swine (24) and other hosts (21), including humans (3). Using seven housekeeping genes for MLST analysis we determined 72 sequence types (STs) among 165 isolates. This indicated an overall high diversity, though 34.5% of all isolates belonged to a single predominant ST-complex, STC9, which grouped strains from birds and mammals, including humans, together. PFGE revealed 58 different clusters and congruence with the sequence-based MLST-method was not common. Based on polymorphisms in the N-terminal hyper-variable region of SpaA the isolates were classified into five groups, which followed the phylogenetic background of the strains. More than 90% of the isolates harboured all 16 putative virulence genes tested and only intI, encoding an internalin-like protein, showed infrequent distribution. MLST data determined E. rhusiopathiae as weakly clonal species with limited host specificity. A common evolutionary origin of isolates as well as shared SpaA variants and virulence genotypes obtained from avian and mammalian hosts indicates common reservoirs, pathogenic pathways and immunogenic properties of the pathogen.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0216-x) contains supplementary material, which is available to authorized users.     

Difference in virulence between Staphylococcus aureus isolates causing gangrenous mastitis versus subclinical mastitis in a dairy sheep flock

Staphylococcus aureus mastitis in dairy sheep ranges from subclinical mastitis to lethal gangrenous mastitis. Neither the S. aureus virulence factors nor the host-factors or the epidemiological events contributing to the different outcomes are known. In a field study in a dairy sheep farm over 21 months, 16 natural isolates of S. aureus were collected from six subclinical mastitis cases, one lethal gangrenous mastitis case, nasal carriage from eight ewes and one isolate from ambient air in the milking room. A genomic comparison of two strains, one responsible for subclinical mastitis and one for lethal gangrenous mastitis, was performed using multi-strain DNA microarrays. Multiple typing techniques (pulsed-field-gel-electrophoresis, multiple-locus variable-number, single-nucleotide polymorphisms, randomly amplified polymorphic DNA, spa typing and sas typing) were used to characterise the remaining isolates and to follow the persistence of the gangrenous isolate in ewes’ nares. Our results showed that the two strains were genetically closely related and they shared 3 615 identical predicted open reading frames. However, the gangrenous mastitis isolate carried variant versions of several genes (sdrD, clfA-B, sasA, sasB, sasD, sasI and splE) and was missing fibrinogen binding protein B (fnbB) and a prophage. The typing results showed that this gangrenous strain emerged after the initial subclinical mastitis screening, but then persisted in the flock in the nares of four ewes. Although we cannot dismiss the role of host susceptibility in the clinical events in this flock, our data support the hypothesis that S. aureus populations had evolved in the sheep flock and that S. aureus genetic variations could have contributed to enhanced virulence.     

Molecular characteristics of new clonal complexes of Staphylococcus pseudintermedius from clinically normal dogs

Background: High prevalence of methicillin resistance among clinical isolates of Staphylococcus pseudintermedius obtained from dogs was reported in Seoul metropolitan area, South Korea. However, no information on genetic lineage and clonal spread is currently available.

Objective: The aim is to identify the genetic diversity of methicillin-resistant or -susceptible S. pseudintermedius (MRSP and MSSP, respectively) from healthy dogs.

Animals and methods: From 119 healthy dogs, 29 isolates consisting of 20 MRSP and 9 MSSP were collected from June 2013 to February 2014. Phenotypic features, antibiogram, multilocus sequence type (MLST), Staphylococcal cassette chromosome mec (SCCmec) type and spa gene type were analyzed.

Results: MLST showed 24 sequence types (STs), including 20 new STs that were genetically distinct from the previous STs in other geographic areas. SCCmec typing revealed that all isolates had SCCmec type V, a predominant type in North America. spa gene typing was successful in only 13 isolates (10 MRSP and 3 MSSP) and revealed two known types (t02 and t06), as well as one novel type (t73).

Conclusion: Our cumulative data indicate the presence of various populations of S. pseudintermedius in clinically normal dogs in Seoul metropolitan area.     

Molecular Epidemiology and Antimicrobial Resistance Mechanisms of Methicillin‐Resistant Staphylococcus aureus Isolated from Bovine Milk

The aim of this study was to identify methicillin‐resistant Staphylococcus aureus (MRSA) strains gathered from 2002 to 2006 from milk samples in Aydin region in Turkey. Among 93 S. aureus strains isolated from bovine milk with mastitis, 16 were resistant to methicillin. Methicillin‐resistant S. aureus strains were studied further for their staphylococcal cassette chromosome mec (SCCmec) types, pulsotypes, spa and MLST types, antimicrobial susceptibilities, mechanisms of resistance and presence of Panton–Valentine leucocidin (PVL) toxin gene. The MRSA strains were multi‐drug resistant. The susceptibility rates to antimicrobials tested were 0%, 0%, 0%, 0%, 6.25%, 16.25% and 56.25% for erythromycin, clindamycin, chloramphenicol, gentamicin, tetracyclin, ciprofloxacin and vancomycin, respectively. All tetracycline and gentamicin resistant strains carried tet(M) and aac(6)‐aph(2) gene, respectively. Among macrolide‐resistant isolates, nine had erm(A), and seven had both erm(A) and erm(B) genes. The molecular characterization by pulsed‐field gel electrophoresis showed presence of three pulsotypes with their variants. The pulsotype B strains were type IV with SCCmec typing, and representative of pulsotype B was t190 by spa typing and ST8 by MLST typing. The strains with pulsotype A and C were SCCmec III, and representative of these pulsotypes was t030 by spa typing. The MLST type of pulsotype A was ST239 and pulsotype C was one allele variant of ST239. None of the isolates harboured the PVL gene. Presence of hospital‐related MRSA strains may indicate transmission of these strains between human and animals. In case of clonal spread beside the infected animals’ treatment of MRSA carrier, farm workers should also be considered. Hygienic measures and rational antibiotic use may avoid resistance selection, clonal dissemination of resistant strains and decrease losses because of mastitis in dairy herds.