Differentiation of Staphylococcus sciuri strains isolated from free-living rodents and insectivores

Twenty-nine Staphylococcus sciuri strains isolated from free-living insectivores and rodents were comparatively analysed for their biochemical capacities and their SmaI macrorestriction patterns. The 29 S. sciuri isolates represented 21 different biotypes and 22 different SmaI macrorestriction types. This observation confirmed that S. sciuri isolates obtained from insectivores and rodents living in natural environments constituted a heterogeneous population. Cluster analysis revealed that the macrorestriction patterns and the biochemical profiles matched in some cases. However, S. sciuri isolates that exhibited the same or closely related biochemical profiles were also found to be associated with different macrorestriction patterns. Analysis of the 29 S. sciuri isolates for their plasmid content and their sensitivity to antimicrobial agents showed that most of the isolates were plasmid-free and susceptible to all antimicrobial agents tested.     

Tetracycline Resistance in Staphylococci from Free‐living Rodents and Insectivores

One hundred and fifty‐eight staphylococcal strains isolated from wild rodents and insectivores were analysed for plasmid‐borne resistance to tetracycline (Tc). Only 10 isolates, six Staphylococcus saprophyticus isolates and single isolates of S. xylosus, S. equorum, S. warneri and S. cohnii subsp. cohnii carried a Tc resistance plasmid of approximately 4.4 kb as confirmed by protoplast transformation. All 10 plasmids harboured a Tc resistance gene of hybridization class K [tet(K)] as confirmed by polymerase chain reaction (PCR). The plasmid was assigned to the pT181 family as it revealed a high degree of restriction map homology to pT181 and other members of this family. Macrorestriction analysis with the enzyme SmaI showed that three of the six isolates identified as S. saprophyticus shared the same pulsed‐field gel electrophoresis (PFGE) pattern.     

Utilization of both phenotypic and molecular analyses to investigate an outbreak of multidrug-resistant Salmonella anatum in horses.

Phenotypic and molecular techniques, including antimicrobial susceptibility testing, plasmid analysis, and pulsed-field gel electrophoresis (PFGE) were used to characterize 15 isolates of multidrug-resistant (MDR) Salmonella anatum cultured during a 16 mo period from horses and a veterinary clinic environment. The isolates were resistant to multiple antimicrobial agents and could be placed into 4 groups based on their antimicrobial resistance patterns. The isolates contained multiple plasmids ranging in size from 2 to > 100 kb that could be grouped into 3 different plasmid profile patterns; these patterns did not correlate with the antimicrobial resistance groupings. Furthermore, antimicrobial resistance was conjugatively transferable. Digestion of genomic DNA from the 15 isolates with 3 different restriction endonucleases, SfiI, SpeI, and XbaI followed by PFGE revealed a highly conserved restriction endonuclease digestion pattern. In contrast, diverse banding patterns were observed with S. anatum obtained from other sources. These observations suggest that the MDR S. anatum isolates represent a common outbreak strain even though they possess different, albeit similar, antibiograms and plasmid profiles. The study showed that PFGE is a useful epidemiological tool for discriminating between unrelated and outbreak-related strains of S. anatum. In conclusion, epidemiological studies of outbreaks caused by MDR isolates of S. anatum should consist of both genotypic and phenotypic methods of analysis.     

Clonal Spread of Salmonella enterica Serovar Infantis in Serbia: Acquisition of Mutations in the Topoisomerase Genes gyrA and parC Leads to Increased Resistance to Fluoroquinolones

Quinolone‐resistant Salmonella Infantis (n = 64) isolated from human stool samples, food and poultry during the years 2006–2011 were analysed for their resistance phenotypes, macrorestriction patterns and molecular mechanisms of decreased susceptibility to fluoroquinolones. Minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were determined by the agar dilution procedure, and the susceptibility to additional antimicrobial agents was determined by the disc diffusion method. To assess the influence of enhanced efflux activity, MICs were determined in the presence and absence of the inhibitor PAβN. The results of pulsed‐field gel electrophoresis (PFGE) typing revealed that quinolone‐resistant S. Infantis in Serbia had similar or indistinguishable PFGE profiles, suggesting a clonal spread. All S. Infantis showed combined resistance to NAL and tetracycline, whereas multiple drug resistance to three or more antibiotic classes was rare (2 isolates of human origin). The MICs ranged between 512 and 1024 μg/mL for NAL and 0.125–2 μg/mL for CIP. A single‐point mutation in the gene gyrA leading to a Ser83→Tyr exchange was detected in all isolates, and a second exchange (Ser80→Arg) in the gene parC was only present in eight S. Infantis isolates exhibiting slightly higher MICs of CIP (2 μg/mL). The inhibitor PAβN decreased the MIC values of CIP by two dilution steps and of NAL by at minimum 3–6 dilution steps, indicating that enhanced efflux plays an important role in quinolone resistance in these isolates. The plasmid‐mediated genes qnr, aac(6′)‐lb‐cr and qepA were not detected by PCR assays.     

Antimicrobial susceptibilities and population structure of Staphylococcus epidermidis associated with ovine mastitis

Intramammary infections are a serious problem for dairy sheep farms, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 131 S. epidermidis isolates, collected from 2201 dairy Sarda sheep belonging to 14 flocks with high somatic cell count scores, were studied. The flocks were located in diverse geographical areas of Sardinia, Italy. The aim of study was to assess the susceptibility of isolates to 13 antimicrobial agents, many of which are frequently used in mastitis therapy. Oxacillin was used for detecting methicillin-resistant S. epidermidis (MRSE) by disk diffusion test. Thirty-eight percent of the isolates (n = 50) were resistant to penicillin, 7.6% (n = 10) were resistant to tetracycline, and 2.3% (n = 3) were resistant to both penicillin and tetracycline (PTRSE). Two isolates were resistant to five antimicrobials including methicillin. Analysis of staphylococcal cassette chromosome mec (SCCmec) elements showed that both MRSE isolates harbored SCCmec type IVa. Based on pulsed-field gel electrophoresis (PFGE) typing by SmaI macrorestriction, S. epidermidis isolates were grouped into four clusters at 75% similarity level. The two multi-drug resistant MRSE isolates displayed distinct PFGE patters. This study indicates that S. epidermidis isolates from sheep milk samples may accumulate resistance markers for different antimicrobial agents. Furthermore, the occurrence of PTRSE and MRSE suggests to adopt adequate hygienic measures when handling animals with intramammary infections, in order to prevent spreading PTRSE and MRSE strains to humans through direct contact and/or consumption of contaminated food.     

First Report of Molecular Characterization of Argentine Isolates of Streptococcus equi subsp. equi by Pulsed-Field Gel Electrophoresis

Strangles is one of the most frequently diagnosed equine respiratory infectious diseases in the world. It is caused by Streptococcus equi subsp. equi (S. equi), and it is an acute infection characterized by pyrexia, nasal discharge, pharyngitis, and abscessation of lymph nodes. Frequently, healthy horses might continue to harbor S. equi after clinical recovery. Although the genetic distance between S. equi isolates is short, strains can be differentiated by pulsed-field gel electrophoresis (PFGE) and single locus sequence typing for epidemiological studies. The aim of this study was to characterize by PFGE Argentine isolates of S. equi obtained from horses with acute strangles and those that had recovered. Bacterial isolation and identification of 80 S. equi isolates by phenotypic and genotypic tests were performed using samples from 29 horses with acute strangles and 95 from healthy animals. Also, the isolates were characterized by PFGE using Bsp120I and SmaI. Visual comparison of macrorestriction patterns generated with both enzymes revealed three different DNA fragment profiles with variations of one or two bands. Interestingly, an identical profile was found in isolates from the same horse and from horses that were infected at the same time, and the horses recovered from strangles continue to carry the same strain. Some vaccinated horses have been mild infected for a different strain from that of carriers suggesting other source of infection. This is the first molecular characterization of Argentine isolates of S. equi, which shows the presence of three strains between 2010 and 2013 in Buenos Aires.     

Pheno- and genotyping of Rhodococcus equi isolated from faeces of healthy horses and cattle

The present study was designed to comparatively investigate 21 Rhodococcus equi isolates from the faeces of clinically healthy horses and cattle. The isolates were identified by cultural and biochemical properties and by PCR analysis. The latter, targeted to the gene coding for the 16S ribosomal RNA, revealed a species specific PCR product.The isolates were further characterised by serotyping with two typing systems, by haemagglutination tests and by plasmid and virulence protein profiling. Among the 21 cultures, four cultures contained plasmids, two of the four cultures expressed 15–17 kDa virulence proteins, no cultures contained 20 kDA virulence proteins. The 21 cultures were further analysed by DNA-finger-printing. This was performed by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). The DNA-restriction patterns were different for most of the isolates indicating a clone heterogeneity among isolates from single farms.Serotyping, determination of virulence marker and PFGE analysis of R equi appeared to be useful for further characterisation of this species, possibly of importance for virulence estimation of single R equi isolates and for epidemiological studies.     

Antimicrobial resistance of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius, Staphylococcus intermedius and Staphylococcus delphini together comprise the S. intermedius group (SIG). Within the SIG, S. pseudintermedius represents the major pathogenic species and is involved in a wide variety of infections, mainly in dogs, but to a lesser degree also in other animal species and humans. Antimicrobial agents are commonly applied to control S. pseudintermedius infections; however, during recent years S. pseudintermedius isolates have been identified that are meticillin‐resistant and have also proved to be resistant to most of the antimicrobial agents approved for veterinary applications. This review deals with the genetic basis of antimicrobial resistance properties in S. pseudintermedius and other SIG members. A summary of the known resistance genes and their association with mobile genetic elements is given, as well as an update of the known resistance‐mediating mutations. These data show that, in contrast to other staphylococcal species, S. pseudintermedius seems to prefer transposon‐borne resistance genes, which are then incorporated into the chromosomal DNA, over plasmid‐located resistance genes.     

Molecular Characterization of Phenotypically CAMP‐Negative Streptococcus agalactiae Isolated from Bovine Mastitis

In the present study three phenotypically CAMP‐negative Streptococcus agalactiae, isolated from three cows with mastitis, were characterized by molecular analysis. An identification of the S. agalactiae was performed by conventional methods and by PCR amplification of species specific parts of the 16S rRNA gene and the 16S‐23S rDNA intergenic spacer region. In addition all three phenotypically CAMP‐negative isolates harboured a normal sized CAMP‐factor encoding cfb gene indicating a reduced expression of CAMP‐factor or a gene defect elsewhere along the pathway of expression. The clonal identity of the three isolates could be demonstrated by macrorestriction analysis of their chromosomal DNA.     

Treating Animal Bites: Susceptibility of Staphylococci from Oral Mucosa of Cats

Infected wounds determined by cats’ bites represent high costs to public health, and their adequate treatment relies on the knowledge of the antimicrobial susceptibility of bacterial agents found in the oral microbiota. Members of the genus Staphylococcus sp. belong to the microbiota of the oral mucosa of cats and are frequently involved in secondary infections of these wounds. This study aimed to evaluate the antimicrobial susceptibility of Staphylococcus species isolated from oral mucosa of cats. Samples were collected from 200 clinically healthy cats and processed by standard bacteriological methods and tested for susceptibility to a panel of 16 antimicrobials. A total of 212 staphylococci isolates were obtained from 141 of the 200 cats (70.5%), and more than one colony was recognized in 53 cases. Coagulase‐negative species were most frequently found (89.6%) distributed among Staphylococcus xylosus (50.9%), Staphylococcus felis (27.4%), Staphylococcus simulans (6.1%) and Staphylococcus sciuri (5.2%). Coagulase‐positive species (10.4%) were distributed among Staphylococcus aureus (4.7%) and Staphylococcus intermedius group (SIG) (5.7%). Regarding to antimicrobial resistance, 178 isolates (83.9%) were resistant to at least one antimicrobial, and rifampicin showed the best results with 100% of sensitive strains. Conversely, high rates of resistance were observed for penicillin and tetracycline (56.1%). The 212 staphylococci isolates and 30 (14.1%) strains were resistant to methicillin (on the disc susceptibility test) and may be preliminarily considered as methicilin‐resistant staphylococci. In conclusion, this study reports important rates of antimicrobial resistance among the species of Staphylococcus isolated from clinical specimens of cats, which must be considered for the treating of cats’ bites in humans.     

Antimicrobial susceptibility of Staphylococcus intermedius and Staphylococcus schleiferi isolated from dogs

The susceptibility to 23 antimicrobial agents was determined in 114 isolates of Staphylococcus intermedius and eight isolates of Staphylococcus schleiferi of canine origin. Overall, 73% of S. intermedius isolates and 37.5% of S. schleiferi isolates were susceptible to all the 23 antimicrobials tested. The large majority of S. intermedius strains retained susceptibility to antimicrobials currently employed in treatment of pyoderma (cephalosporins, cotrimoxazole and association amoxicillin–clavulanic acid) as well as to those effective against staphylococci (fusidic acid, rifampicin and fluoroquinolones). Resistance in S. intermedius was observed mainly against macrolides, chloramphenicol and lincosamides, while S. schleiferi isolates retained susceptibility to all antimicrobials except three of six fluoroquinolones. Although, our results confirm susceptibility to antimicrobials currently employed in pyoderma treatment, the several different resistance patterns observed for S. intermedius emphasize the importance of antimicrobial susceptibility testing of canine staphylococci to choose the most appropriate treatment of infections and to allow the prudent use of antimicrobial drugs in companion animals.     

Molecular Typing and Anti‐microbial Susceptibility of Clinical Isolates of Streptococcus equi ssp. zooepidemicus from Equine Bacterial Endometritis

The anti‐microbial susceptibility and genetic diversity of 65 strains of Streptococcus equi ssp. zooepidemicus (Sez) isolated from mares presenting clinical signs of endometritis was determined by disk agar diffusion and pulsed field gel electrophoresis (PFGE) methods, respectively. Overall, Sez isolates were susceptible to β‐lactams, enrofloxacin, trimethoprim‐sulfamethoxazole and gentamicin. These anti‐microbials could be recommended as empiric anti‐microbial therapy in cases of endometritis caused by Sez. Pulsed field gel electrophoresis typing revealed a great genetic diversity (56 different PFGE macrorestriction profiles) and a low level of genetic relatedness amongst the isolates.     

Distribution and Genotypic Characterization of Campylobacter jejuni Isolated from Poultry in Split and Dalmatia County,Croatia

Consumption of poultry contaminated with Campylobacter jejuni has been recognized worldwide as the leading cause of campylobacteriosis. Therefore, the aim of our study was to investigate the prevalence and genotype diversity of Campylobacter jejuni in poultry meat intended for consumption in Split and Dalmatia County, which is the second biggest County in Croatia. Furthermore, we also wanted to discover possibly stable clones of C. jejuni appearing in different samples and periods of time, which would indicate their ability to persist in or adapt to poultry. In the period from March 2008 until June 2010, 834 samples of poultry from various sources were examined using a surface swab technique. Isolation of C. jejuni was performed by Preston broth and Karmali agar. Identification of the isolates was carried out using biochemical tests. C. jejuni was found in 84 of 574 chicken samples (14.6%) and in nine of 260 samples of turkey (3.5%). Pulse‐field gel electrophoresis (PFGE) was used to analyse 61 obtained isolates using SmaI and KpnI. Of 22 different macrorestriction profiles (MRP) that were found, five were detected in poultry from both different locations and periods of time. Samples from 11 locations were found to be contaminated with more than two different genotypes of C. jejuni. Interestingly, the same MRP were found both in poultry declared to be of domestic origin and in the poultry imported from abroad. The prevalence of C. jejuni in poultry samples was in accordance with previously reported results. Genotypic analysis indicated that the population of C. jejuni in Split and Dalmatia County was diverse and that multiple strains of C. jejuni could be found in the same poultry samples. Furthermore, the same genotypes were identified from the samples obtained from different locations and periods of time, which could support the theory of a global existence of certain MRP that are able to persist in or adapt to poultry.     

Antimicrobial resistance patterns and plasmid profiles of Streptococcus suis isolates.

Streptococcus suis isolates recovered from diseased animals in Quebec and western Canada and from human cases in Europe were tested for their susceptibility to different antimicrobial agents and screened for their plasmid content. Most isolates from Quebec were clindamycin, erythromycin, and tetracycline resistant; animal isolates from western Canada were notably less resistant to clindamycin and erythromycin, whereas human isolates were considerably more susceptible to most antimicrobials tested. More than 60% of isolates had plasmids that ranged from 1.5 to 35 kilobases (kb). Of the 7 plasmid profiles found, 2 were particularly frequent in isolates from Quebec and western Canada, suggesting the presence of epidemic strains in the swine population. A particular plasmid band of about 5 kb was present in most Canadian isolates. When this band was used as a probe in colony and Southern blot hybridization, most isolates harboring the 5-kb plasmid hybridized, even though their plasmid profiles were different. Human isolates from Europe differed in their plasmid content from Canadian isolates of animal origin. Although a high degree of antimicrobial resistance was associated with the presence of plasmids in most isolates, it was not possible to establish a causative relationship.     

Prevalence and Antimicrobial Resistance of Salmonella spp. in Non‐diarrhoeic Dogs in Trinidad

The estimated prevalence and antimicrobial resistances of Salmonella spp. in non‐diarrhoeic dogs across Trinidad was determined. The serotypes of Salmonella spp. isolated were also identified. Of a total of 1391 dogs sampled, 50 (3.6%) were positive for Salmonella spp. with 28 different serotypes, the predominant serotypes were Javiana (12), Newport (6), Arechavaleta (5) and Heidelberg (5). Fifty‐seven (85.1%) of 67 isolates exhibited resistance to one or more antimicrobial agents. Of eight antimicrobial agents tested, resistance was exhibited to streptomycin (80.6%), cephalothin (37.3%), neomycin (38.8%) and gentamicin (9.0%). All isolates were sensitive to ampicillin, norfloxacin, choramphenicol and sulphamethoxazole/trimethoprim. It was concluded that the isolation of the Salmonella spp. from non‐diarrhoeic dogs could pose health hazard to their owners as most serotypes are known to be virulent. Furthermore, the prevalence of resistance to antimicrobial agents amongst the Salmonella isolates from these animals indicates susceptibility testing may influence chemotherapeutic choices when treating these isolates.     

High‐Level Ciprofloxacin‐Resistant Campylobacter jejuni Isolates Circulating in Humans and Animals in Incheon,Republic of Korea

Campylobacter jejuni is one of the major causative pathogens of outbreaks or sporadic cases of diarrhoeal diseases worldwide. In this study, we compared the phenotypic and genetic characteristics of C. jejuni isolates of human and food‐producing animal origins in Korea and examined the genetic relatedness between these two groups of isolates. Regardless of isolation source, all C. jejuni isolates harboured four virulence genes, cadF, cdtB, ciaB and racR, whereas the wlaN and virB11 genes were more frequently observed in human isolates. Antimicrobial susceptibility testing showed that the majority of C. jejuni isolates displayed high‐level resistance to fluoroquinolone (95.2%) or tetracycline (76.2%) antibiotics, and 12.4% of isolates exhibited multidrug resistance (more than three classes of antibiotics tested). Pulsed‐field gel electrophoresis (PFGE) of all Campylobacter isolates revealed 51 different SmaI‐PFGE patterns and six major clusters containing both human and animal isolates. These results indicate that genetically diverse strains of C. jejuni with antimicrobial drug‐resistance and virulence properties have prevailed in Incheon. Nevertheless, some particular populations continue to circulate within the community, providing the evidence for an epidemiological link of C. jejuni infections between humans and food‐producing animals. Therefore, the continued monitoring and surveillance of C. jejuni isolates of human and food‐producing animal origins are required for public health and food safety.     

Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates

Recently, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has gained some importance in the epidemiology of salmonellosis in Poland. Since biotyping, resistance typing, and plasmid profiling were insufficient for strain differentiation, genome macrorestriction by means of pulse-field gel electrophoresis (PFGE) was applied and proved to be the method of choice in S. Mbandaka epidemiological studies. XbaI and BcuI macrorestriction produced 15 and 14 pulse-field profiles (PFP), respectively, but in the case of each enzyme one profile was prevalent. When macrorestriction profiles were combined, a total 24 patterns were found. Based on the similarity of the profiles, four clonal lineages were identified. One clonal lineage contained the majority of poultry, feed and human isolates. Poultry was concluded to be an important source of S. Mbandaka for humans in Poland. Complementary use of various typing techniques improved efficacy of epidemiological studies giving possibility to subdivide S. Mbandaka into 35 types and the index of discrimination reached 0.947.     

Phenotypic and genotypic characteristics of antimicrobial resistant Escherichia coli isolated from symbovine flies, cattle and sympatric insectivorous house martins from a farm in the Czech Republic (2006-2007)

The prevalence of antimicrobial resistant Escherichia coli was tested in symbovine flies and sympatric house martins (Delichon urbica) at a dairy farm. Antimicrobial resistant E. coli was detected in 89% (n = 147) of isolates from flies within a calf barn. Isolates with the same antimicrobial resistance phenotypes, genes, and pulsotypes were found between both fly and calf E. coli isolates, suggesting that the calves were the initial source of the antimicrobial resistant strains in fly isolates. Symbovine flies were considered as important reservoirs of antimicrobial resistant E. coli strains at a dairy farm, due to their intensive contact with cattle feces and manure. House martin fecal samples from the same farm contained 4.5% (n = 393) of antimicrobial resistant E. coli. House martin isolates displayed different macrorestriction profiles than fly isolates and the significance of house martins as a reservoir and vector of antimicrobial resistant E. coli appears low.     

Species distribution and antimicrobial susceptibility of staphylococci isolated from canine otitis externa

The diversity of species of the genus Staphylococcus sp. and the antimicrobial resistance of isolates from 151 unmedicated dogs of both sexes with a clinical diagnosis of otitis were recorded. Ninety‐one isolates of Staphylococcus spp. were identified by biochemical reactions and tested for susceptibility to 15 antimicrobials. Coagulase‐positive species were most common; S. pseudintermedius (38.4%), S. schleiferi schleiferi (15.4%), S. aureus (14.3%), S. epidermidis (11%), S. simulans (11%), S. schleiferi coagulans (8.8%) and S. saprophyticus (1.1%). All the isolates showed resistance to at least one drug and 89% were multiresistant. Amoxicillin combined with clavulanic acid and oxacillin were the most effective, while resistance was widely observed for neomycin and erythromycin. The results highlight the recognition and the potential need for bacterial culture with species identification and antimicrobial susceptibility tests for appropriate antimicrobial therapy.     

Molecular epidemiology of Salmonella Heidelberg in an equine hospital

From 1992 to 1997, multi-drug resistant (MDR) Salmonella Heidelberg isolates were cultured from a number of horses hospitalised in a veterinary hospital in Victoria, Australia. To examine the relationships between the cases, 28 isolates from the hospital were compared by pulsed field gel electrophoresis (PFGE), IS200 element profiles, antimicrobial resistance patterns, plasmid profiles and phage typing. The PFGE patterns following digestion with XbaI and BlnI restriction endonucleases showed that the isolates from the veterinary hospital originated from a common source. These isolates also had indistinguishable IS200 profiles. However, PFGE was more discriminatory than IS200 profiles. All the veterinary hospital isolates and one independent isolate had the same antimicrobial resistance pattern and had at least one plasmid in common. Localisation of antimicrobial resistance genes indicated that the veterinary hospital isolates had more than one plasmid carrying resistance genes and that the genes encoding sulphathiazole and trimethoprim resistance were not on these plasmids. Phage typing was ineffective as 22 of the 28 isolates were untypeable. In conclusion, the combination of different methods used for epidemiological studies suggested that a single strain of MDR S. Heidelberg was isolated from horses admitted to the hospital for 6 years and caused salmonellosis in susceptible horses within that period with no apparent correlation between the antimicrobials used and retention of its MDR phenotype.