Molecular characterization of community‐associated methicillin‐resistant Staphylococcus aureus from pet dogs

Community‐associated methicillin‐resistant Staphylococcus aureus (MRSA) is a serious public health concern and in Australia, one that disproportionately affects Aboriginal people. Paralleling MRSA in human medicine, methicillin‐resistant S. pseudintermedius (MRSP) is an increasingly prevalent pathogen in veterinary medicine. We aimed to characterize the carriage of MRSA and MRSP in dogs and cats from predominantly Aboriginal communities in a very remote region of New South Wales (NSW), Australia. Pets (303 dogs and 80 cats) were recruited from six communities in western NSW. Three swabs were collected from each animal (anterior nares, oropharynx and perineum) and from skin lesions or wounds (if present) and cultured on selective media for methicillin‐resistant staphylococci. Human host‐adapted community‐associated MRSA representing four multilocus sequence types (ST1‐IV, ST5‐IV, ST72‐IV, ST93‐IV) were isolated from eight dogs (prevalence 2.6%, 95% confidence interval 1.3%–5.1%). Two ST5‐IV isolates from a single dog were phenotypically trimethoprim‐resistant, harbouring trimethoprim‐resistant gene dfrG within the SCCmec type IVo mobile genetic element. MRSA was not isolated from any cats and MRSP was not isolated from any dogs or cats. This study estimated a high prevalence of human host‐adapted community‐associated MRSA carriage in dogs despite an absence of MRSP. This suggests MRSA carried by dogs in remote NSW originate from human hosts. The cycle of transmission between people, dogs and common environmental sources warrants further investigation. To our knowledge, this is the first report of trimethoprim‐resistant ST5‐IV in eastern Australia and the first report of trimethoprim‐resistant ST5‐IV from a dog.     

Detection of methicillin‐resistant Staphylococcus aureus (MRSA) in the microbial flora from the conjunctiva of healthy donkeys from Sicily (Italy)

Objective To describe the bacterial flora present in the normal conjunctiva of donkeys from Sicily (Italy). Animals studied A total of 46 healthy donkeys housed in 3 locations within the territory of Palermo (Sicily, Italy) were studied. Donkeys ranged from 2 to 13 years of age, with a median age of 6 years. Procedures Forty‐six conjunctival swabs were obtained from both eyes of each animal, and specimens were cultured for aerobic bacteria. Furthermore, the antimicrobial activity of methicillin (1 μg) and oxacillin (5 μg) on Staphylococcus spp. isolates was evaluated, and a specific PCR assay, which allows the detection of mecA gene specific for methicillin‐resistant Staphylococcus aureus (MRSA) strains, was performed. Results Forty of 46 (86.9%) donkeys were positive for bacteria. Eighty bacterial isolates, representing 9 bacteria genera, were successfully cultured. The most frequently recovered bacterial genus was Staphylococcus (52/80 isolates; 65%). Several strains (20/80 isolates; 25%) belonging to the Enterobacteriaceae family were also isolated, among which the most frequently isolated genus was Enterobacter (eight isolates). Of the 52 Staphylococcus spp. isolates, 14 (26.9%) strains were oxacillin/methicillin resistant. The mecA gene was detected in 6/52 (11.5%) strains. Conclusions This study contributes to the knowledge about normal ocular flora and MRSA occurrence in donkey farms in Sicily.     

A bovine myeloid antimicrobial peptide (BMAP‐28) kills methicillin‐resistant Staphylococcus aureus but promotes adherence of the bacteria

The cathelicidin family is one of the several families of antimicrobial peptides (AMPs). A bovine myeloid antimicrobial peptide (BMAP‐28) belongs to this family. Recently, the emergence of drug‐resistant bacteria such as methicillin‐resistant Staphylococcus aureus (MRSA) has become a big problem. AMPs are expected to be leading compounds of new antibiotics against drug‐resistant bacteria. In this study, we focused on the activity of BMAP‐28 against bacterial cell surfaces. First, we observed morphological change of MRSA caused by BMAP‐28 using a scanning probe microscope. We also studied activities of BMAP‐28 against adherence of S. aureus to fibronectin, collagen type I, collagen type IV. We confirmed whether BMAP‐28 can bind to lipoteichoic acid (LTA) of S. aureus. BMAP‐28 was indicated as damaging the cell surface of MRSA. In a particular range of concentrations, BMAP‐28 promoted adherence of S. aureus against fibronectin and collagens. It was revealed that BMAP‐28 and LTA of S. aureus bound with each other. Our study showed the potential of BMAP‐28 which can damage MRSA and interact with LTA of S. aureus but promote its adherence in some concentrations. This study provides new points of which to take notice when we use AMPs as medicines.     

Prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma

Currently, antimicrobial-resistant staphylococci, particularly methicillin-resistant Staphylococcus pseudintermedius (MRSP), are frequently isolated from canine superficial pyoderma in Japan. However, little is known regarding the nasal prevalence of MRSP in pet dogs. Here, we determined the prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma. Of the 125 nares and 108 affected sites of pet dogs with superficial pyoderma, 107 (13 species) and 110 (eight species) staphylococci strains, respectively, were isolated. The isolation rate of S. pseudintermedius from pyoderma sites (82/110 strains, 74.5%) was significantly higher than that from nares (57/107 strains, 53.3%) (P<0.01). Notably, the prevalence of MRSP (18/57 strains, 31.6%) in nares was equivalent to that in pyoderma sites (28/82 strains, 34.1%). Furthermore, the phenotypes and genotypes of antimicrobial resistance in MRSP strains from nares were similar to those from pyoderma sites. Our findings revealed that the prevalence of antimicrobial-resistant staphylococci in the nares of pet dogs with superficial pyoderma is the same level as that in affected sites. Therefore, considerable attention should be paid to the antimicrobial resistance of commensal staphylococci in companion animals.     

Carriage of methicillin-resistant Staphylococcus aureus by veterinarians in Australia

Objective To estimate the prevalence of carriage of methicillin‐resistant Staphylococcus aureus (MRSA) among Australian veterinarians. Methods Individuals attending veterinary conferences in Australia in 2009 were recruited to provide nasal swabs and complete a questionnaire about their professional activities. Swabs were processed by standard methods for detecting MRSA and questionnaire responses were used to group veterinarians according to their areas of major work emphasis (species and practice type). Prevalence was estimated for each of these grouping and contingency tables and regression tree analysis used to explain the variation in MRSA carriage. Results Among the 771 respondents ‘industry and government veterinarians’ (controls) had the lowest prevalence of MRSA carriage at 0.9%. Veterinarians with horses as a major area of work emphasis had a prevalence of 11.8% (13‐fold that of controls) and those whose only major emphasis was horses had a prevalence of 21.4% (23‐fold that of controls). Veterinarians with dogs and cats as a major activity had a 4.9% prevalence (5‐fold that of controls). Prevalence rates for other major activities (pigs, dairy and beef cattle, avian and wildlife) were also increased, but were estimated from smaller numbers of respondents. Regression tree analysis clearly isolated equine veterinarians and dog and cat practitioners as groups at increased risk of carriage of MRSA. Conclusion Carriage of MRSA is a notable occupational health issue for veterinarians in clinical practice in Australia, particularly those who work with horses.     

Methicillin-resistant Staphylococcus aureus in a population of horses in Australia

Objective To evaluate if methicillin‐resistant Staphylococcus aureus (MRSA) is present in the horse population in Australia. Design A two‐part retrospective study of laboratory submissions of microbial culture results from horses. Methods Part A: medical records of 216 horses that had MRSA screening performed on nasal swabs collected over a 30‐day period at admission to the Scone Equine Hospital Clovelly Intensive Care Unit were retrieved. Part B: laboratory records from 2004 to 2009 of culture submissions to the Scone Veterinary Laboratory were reviewed and cultures that grew MRSA were identified. The MRSA isolates from Parts A and B were genotyped over an 18‐month period. Results MRSA screening of 216 horses identified eight (3.7%) positive samples. MRSA was isolated from cultures of 80 (0.002%) clinical bacteriology samples over a 6‐year period. Genotypic analysis was performed on 36 isolates. All MRSA characterised had the same pulse field gel electrophoresis pattern (type 1), with eight closely related subtypes identified (subtypes A–F and H) and 66% of isolates classified as subtype D, which multilocus sequence and staphylococcal cassette chromosome mec typing analysis identified as ST612‐MRSA‐IVa, a clonal complex (CC) 8 S. aureus strain. Antimicrobial resistance to more than two classes of antimicrobials was common. Conclusions MRSA was present in a population of horses in Australia. Genotypic analysis of the isolates identified the MRSA strain as CC8 S. aureus. Further research needs to be undertaken to evaluate MRSA infection and colonisation of horses and personnel in Australia.     

Zinc Oxide Therapy Increases Prevalence and Persistence of Methicillin‐Resistant Staphylococcus aureus in Pigs: A Randomized Controlled Trial

There is concern that therapeutic use of zinc oxide (ZnO) in swine production may select for methicillin‐resistant Staphylococcus aureus (MRSA) due to co‐location of the zinc resistance gene (czrC) and methicillin resistance gene (mecA) within the staphylococcal cassette chromosome mec (SCCmec). The objective of this investigation was to determine whether MRSA carriage in pigs is influenced by exposure to therapeutic doses of in‐feed ZnO (3000 mg/kg) when compared to the recommended dietary levels (100 mg/kg). A randomized controlled trial was completed using 110 pigs that were naturally colonized with czrC‐positive MRSA. The pigs were followed from birth to weaning (21 d), at which point they were randomized into 8 pens and exposed to either a control feed (100 mg ZnO/kg feed; n = 49 pigs) or a treatment feed (3000 mg ZnO/kg feed; n = 50 pigs); neither feed contained additional antimicrobials. MRSA carriage was monitored weekly in each group for 4 weeks post‐weaning. The prevalence of MRSA was significantly higher in the treatment group at 1‐week (OR = 18.1; P < 0.01) and 2 weeks (OR = 3.01; P = 0.01) post‐weaning when compared to the control group, but there was no difference later in the nursery phase. Persistent MRSA carriage (testing positive ≥2 times post‐weaning) was observed in 2% (1/49) of control pigs and 22% (11/50) of treated pigs (P < 0.01). All MRSA isolates (spa types t034 and t3075) carried czrC and showed uniform resistance to zinc. These findings demonstrate that the prevalence and persistence of MRSA in nursery pigs can be affected by high levels of in‐feed ZnO in the absence of antibiotics.     

Methicillin‐resistant Staphylococcus aureus in urban Norway rat (Rattus norvegicus) populations: Epidemiology and the impacts of kill‐trapping

Urban Norway rat (Rattus norvegicus) populations can carry the bacteria methicillin‐resistant Staphylococcus aureus (MRSA). There are numerous knowledge gaps in the epidemiology of MRSA in these populations that limit understanding of its ecology in urban environments. For example, fecal shedding of MRSA, which may increase environmental contamination, has been reported in other species; however, it is unknown whether Norway rats carry the bacteria rectally. Furthermore, while intermittent MRSA shedding has been shown in other species and may dictate when the risk of transmission is highest, duration of carriage has not been examined for Norway rats. Previous work has shown that lethal animal‐control methods may increase the level of pathogens within reservoir populations, possibly by disrupting ecological patterns. However, the impact of rodent‐control on potentially environmentally acquired pathogens like MRSA has not been tested. Using capture‐mark‐recapture methods in an inner‐city neighborhood in Vancouver, Canada, we show that rats intermittently carry MRSA both in the rectum and oropharynx. By assessing the prevalence of MRSA before and after enacting a pest‐control intervention, we report that kill‐trapping had no impact on the prevalence of carriage of this environmentally‐acquired agent.     

Methicillin‐resistant Staphylococcus aureus in Resident Animals of a Long‐term Care Facility

Animals provide benefits to elderly and chronically ill people by decreasing loneliness, increasing social interactions, and improving mental health. As a result, many hospitals and long‐term care facilities allow family pets to visit ill or convalescing patients or support animal‐assisted therapy programs. These include programs that have resident animals in long‐term care facilities. Despite the benefits, there are concerns about disease transmission between pets and patients. Antibiotic‐resistant bacteria, such as methicillin‐resistant Staphylococcus aureus (MRSA), are a recognized problem in healthcare settings leading to refractory infections and potentially life‐threatening illnesses. MRSA has been isolated from numerous animal species, yet few studies are available on the carriage of this pathogen in animals residing in long‐term care facilities. Our objective was to characterize MRSA carriage among resident animals in a long‐term care facility. Methods: To document MRSA colonization, nasal swabs from 12 resident animals (one dogs and 11 cats) of a long‐term care facility were collected weekly for 8 weeks. Staphylococcus isolates were characterized by antimicrobial susceptibility and MRSA isolates were further characterized by pulsed‐field gel electrophoresis (PFGE). PFGE isolate patterns were compared with an existing database of MRSA isolate patterns at the Minnesota Department of Health. Results: Two of 11 cats were colonized with MRSA. MRSA was recovered from five of eight weekly samples in one cat and two of eight weekly samples in the other cat. All isolates were classified as USA100 (healthcare‐associated strains). Discussion: Long‐term care resident animals may acquire MRSA. Clonally related strains were identified over the 8‐week sampling period. It is unclear if pets serve as an on‐going source of infection to their human companions in long‐term care facilities.     

Methicillin‐Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types

The aim of this study was to investigate the phenotypic and genotypic diversity and anti‐microbial resistance among staphylococci of dairy herds that originated from Paraiba State, north‐eastern Brazil, a region where such studies are rare. Milk samples (n = 552) were collected from 15 dairy farms. Isolates were evaluated for anti‐microbial susceptibility by Kirby–Bauer disc diffusion method. Confirmation of methicillin‐resistant Staphylococcus aureus (MRSA) was performed using multiplex PCR targeting mecA and nuc genes in addition to phenotypic assay based on PBP‐2a latex agglutination. Clonal relatedness of isolates was determined by pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) genotyping. Staphylococci were detected in 269 (49%) of the samples. Among these, 65 (24%) were S. aureus. The remaining 204 isolates were either coagulase‐negative staphylococci (n = 188; 70%) or coagulase positive other than S. aureus (n = 16; 6%). Staphylococci were cultured in seven (35%) of the 20 hand swab samples, from which five isolates were S. aureus. The isolates were most commonly resistant against penicillin (43%), ampicillin (38%) and oxacillin (27%). The gene mecA was detected in 21 S. aureus from milk and in one isolate from a milker's hand. None of the isolates were resistant to vancomycin. PFGE findings showed high clonal diversity among the isolates. Based on MLST, we identified a total of 11 different sequence types (STs 1, 5, 6, 83, 97, 126, 1583, 1622, 1623, 1624 and 1625) with four novel STs (ST1622‐ST1625). The findings show that MRSA is prevalent in milk from semi‐extensive dairy cows in north‐eastern Brazil, and further investigation on its extent in various types of milk production systems and the farm‐to‐table continuum is warranted.     

Identical genotypes of community‐associated MRSA (ST59) and livestock‐associated MRSA (ST9) in humans and pigs in rural China

This study investigated the prevalence of MRSA in samples taken in households, with and without backyard pigs in villages in a rural area of Shandong Province, China. Community‐associated MRSA and livestock‐associated MRSA, belonging to ST59 and ST9, respectively, were identified in both humans and pigs. The genotypic and phenotypic comparison of isolates indicates that bidirectional transmission of MRSA has occurred between humans and pigs in the villages.     

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP‐28) against methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus

A bovine myeloid antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP‐28 has bactericidal activity against methicillin‐resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin‐susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5–20 µg/mL). It was also revealed that MSSA (MIC range; 1.25–20 µg/mL) had two levels of susceptibility to BMAP‐28. We also examined the effect of BMAP‐28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP‐28 is a promising candidate for medicine against drug‐resistant bacteria.     

Susceptibility difference between methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus to a bovine myeloid antimicrobial peptide (BMAP‐28)

A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin‐resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP‐28. This study aims to clarify how a few amino acid substitutions of BMAP‐28 are related to its antimicrobial activity using four analog peptides of BMAP‐28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP‐28 do not change antimicrobial activity. It was also revealed that the percentage of cis‐11‐eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP‐28.     

Genome analysis of methicillin‐resistant Staphylococcus aureus isolated from pigs: Detection of the clonal lineage ST398 in Cameroon and South Africa

Food animals are considered reservoirs of methicillin‐resistant Staphylococcus aureus (MRSA) and are implicated in their zoonotic transmission in the farm‐to‐plate continuum. LA‐MRSA has been reported as a zoonotic agent that has the potential to spread to humans and may cause infections in at‐risk groups. In this study, whole genome sequencing was used to describe the genetic environment (resistance mechanisms, virulence factors and mobile genetic elements) and investigate the genetic lineages of MRSA isolates from pigs in Cameroonian and South African abattoirs. During March–October 2016, 288 nasal and rectal pooled samples from 432 pigs as well as nasal and hand swabs from 82 humans were collected. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were de novo‐assembled using the Qiagen CLC Genomics Workbench and SPAdes. The assembled contigs were annotated, and antibiotic resistance genes, virulence factors, plasmids, SCCmec and phage elements were identified with ResFinder, Virulence Finder, PlasmidFinder, SCCmec Finder and PHAST, respectively. Core genome single nucleotide analysis was undertaken to assess clonal relatedness among isolates. A lower MRSA prevalence was observed in pigs in Cameroon (n = 1/13; 0.07%) compared with South Africa (n = 4/22; 18.18%), and none of the workers were colonized by MRSA. Genome analysis identified various antibiotic resistance genes along with six virulence factors in all isolates. All MRSA isolates belonged to the clonal lineage ST398 (spa‐type t011) and harboured the type Vc SCCmec and several plasmids. Our study shows that the livestock‐associated MRSA clonal lineage ST398 is already present in both Cameroon and South Africa and is probably underestimated in the absence of molecular epidemiological studies. It reveals the serious food safety and public health threat associated with this animal strain and underscores the need for interventions to contain this resistant clone.     

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.     

Seasonal prevalence of fungi in the conjunctival fornix of healthy cows during a 2‐year study

Objective The aims of the present paper were to: (i) identify and quantify conjunctival fungi isolated from healthy cows; (ii) verify the influence of different methods of farm management on the prevalence (percentage of positive cultures for each fungal species per farm) of conjunctival fungi. Material and methods Forty Friesian and twenty Limousin female cows aged 1–10 years stabled in three farms with different managements (farm 1: cows housed strictly indoors; farm 2: cows housed outside during the day and inside the stall during the night; farm 3: cows housed strictly outdoors) were investigated for conjunctival fungal flora. Air and food were also tested. Specimens were collected every season during a 2‐year study. Identification of colonies of filamentous fungi was achieved to the genus level on the basis of macro‐ and microscopic features. Results The total number of eyes positive for fungi ranged from 85 to 100% at farm 1, from 65 to 95% at farm 2, and from 55 to 95% at farm 3. Fungi most frequently isolated from conjunctival fornix were Cladosporium spp. and Penicillium spp. Statistical analysis did not show any differences in fungal prevalences among the three farms during the same season. Some fungal species were consistently isolated while others were intermittently isolated. Conclusions Fungi found in the conjunctival fornix of cows might represent transient seeding from the environment, as suspected in other species. The prevalence of conjunctival fungal organisms is not different in cattle housed indoors vs. outdoors.     

Characterization of Methicillin‐Resistant Staphylococcus aureus Isolates from Pig Carcasses in Hong Kong

This study describes the isolation and characterization of methicillin‐resistant Staphylococcus aureus (MRSA) from slaughtered pigs sampled from local markets in Hong Kong. The nares of 400 slaughtered pigs were cultured and MRSA isolates characterized for the presence of antibiotic‐resistance determinants, toxins and SCCmec and spa types using PCR. Clonality was investigated using PFGE and MLST. The prevalence of MRSA colonization of slaughter pigs was 39.3%, the majority (92%) harbouring SCCmec type IVb. Of the 157 samples yielding MRSA, 13 had two distinct MRSA strains present. Spa type t899 was predominant, with only 5/170 isolates displaying closely related types (t4474, t1939, t2922 and t5390). PFGE with sma1 and MLST confirmed the strains as ST9. Most isolates were multidrug resistant. Tetracycline resistance (97%) was mainly attributable to tet(K) with only 3% of isolates additionally harbouring tet(M). Resistance to erythromycin (89%) and chloramphenicol (71%) was associated with the presence of erm(C), and fex( A), respectively. No strains carried cfr and there was no resistance to linezolid, although minimum inhibitory concentration (MICs) were close to the resistance break point. Resistance to clindamycin (99%), ciprofloxacin(78%), quinopristin–dalfopristin (44%) and cotrimoxazole (32%) was common, but remained low for fusidic acid (4%) and rifampicin (2%). All strains were negative for PVL, exfoliative, and enterotoxins. This survey confirmed the uniformity of MRSA isolates in pigs from several regions of China, in contrast to more diversified characteristics reported in European studies. Colonization rates were higher than previously reported. Isolates were resistant to a wide range of antibiotics, but resistance was not detected to linezolid, nitrofurantoin, vancomycin or tigecycline. Although the clinical importance of ST9 in humans is uncertain, continued surveillance, in particular of those occupationally‐exposed, is recommended.