Molecular Characterization of Staphylococcus aureus Complex Isolated from Free-Ranging Long-Tailed Macaques at Kosumpee Forest Park, Maha Sarakham, Thailand.

The Staphylococcus (S.) aureus complex, including methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA), and S. argenteus are bacterial pathogens that are responsible for both human and animal infection. However, insights into the molecular characteristics of MRSA, MSSA, and S. argenteus carriages in wildlife, especially in long-tailed macaques, rarely have been reported in Thailand. The objective of this study was to assess molecular characterization of MRSA, MSSA, and S. argenteus strains isolated from free-ranging long-tailed macaques (Macaca fascicularis) at Kosumpee Forest Park, Maha Sarakham, Thailand. A total of 21 secondary bacterial isolates (including 14 MRSA, 5 MSSA, and 2 S. argenteus) obtained from the buccal mucosa of 17 macaques were analysed by a Polymerase chain reaction (PCR) to identify several virulence genes, including pvl, tst, hla, hlb clfA, spa (x-region), spa (IgG biding region), and coa. The most prevalent virulence genes were clfA, coa, and the spa IgG biding region which presented in all isolates. These data indicated that MRSA, MSSA, and S. argenteus isolates from the wild macaques at Kosumpee Forest Park possess a unique molecular profile, harbouring high numbers of virulence genes. These findings suggest that wild macaques may potentially serve as carriers for distribution of virulent staphylococcal bacteria in the study area.

Investigation of methicillin-resistant Staphylococcus aureus, methicillin-susceptible Staphylococcus aureus, and Staphylococcus argenteus from wild long-tailed macaques (Macaca fascicularis) at Kosumpee Forest Park, Maha Sarakham, Thailand.

Background and Aim: In the past, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections in both humans and animals has increased across Thailand. Staphylococcus argenteus has been associated with infections among humans, exotic pets, and livestock. Both species have been identified in non-human primate species from geographically diverse locations but not from non-human primates in Thailand. This study aimed to determine the presence of MRSA/methicillin-susceptible S. aureus (MSSA) and S. argenteus isolates collected from buccal swab samples in Macaca fascicularis at Kosumpee Forest Park (KFP), Maha Sarakham, Northeast Thailand. Materials and Methods: Aseptic buccal swab samples were collected from 30 free-ranging macaques in November 2018. All isolates were tested using multiple biochemical tests and S. aureus latex slide agglutination test. Presumptive S. aureus isolates were tested for the presence of the mecA gene using polymerase chain reaction (PCR) assays. The isolates were phenotypically determined to be resistant to a ß-lactam antibiotic using the disk diffusion method with a 30 mg cefoxitin disk. The isolates were analyzed by PCR for the non-ribosomal peptide synthetase (NRPS) gene to distinguish S. argenteus from S. aureus. Results: Fifteen macaques (50%) were colonized with S. aureus and 21 isolates were characterized. Three of the macaques carried both the MRSA and MSSA isolate. One animal carried both MRSA and S. argenteus isolate, and one animal carried only S. argenteus. The NRPS gene analysis confirmed that 2 isolates (9.52%) were S. argenteus and 19 isolates (90.48%) were S. aureus [five MSSA and 14 MRSA]. Conclusion: This study is the first to identify MRSA/MSSA and S. argenteus in wild free-ranging M. fascicularis from Thailand at the KFP in Maha Sarakham. This study is also the first report on the occurrence of S. argenteus carriage in M. fascicularis from Thailand.

Leptospira Seroprevalence in Free-Ranging Long-Tailed Macaques (Macaca fascicularis) at Kosumpee Forest Park, Maha Sarakham, Thailand.

BACKGROUND: Leptospirosis is a zoonotic disease that is ubiquitously distributed and is classified as a re-emerging infectious disease in humans and animals. Many serovars are carried by wildlife; all of them are capable of causing illness in humans. The purpose of this study was to investigate the prevalence of Leptospirosis in wild long-tailed macaques (Macaca fascicularis) at Kosumpee Forest Park, Mahasarakham, Thailand. METHODS: A cross-sectional study was conducted at the park. Blood samples were collected via saphenous vein from 30 free-ranging long-tailed macaques. All samples were tested by the microscopic agglutination test. The LipL32 gene was used to detect pathogenic Leptospira in blood samples by conventional polymerase chain reaction. RESULTS: Screening of the 30 wild macaques showed an overall Leptospira seroreactivity of 13.33%. Three of 30 macaques reacted against Leptospira serovar Shermani and one macaque was infected with Leptospira serovar Sejroe. None of the macaques presented clinical signs of leptospirosis. None of the blood samples showed the detection of the LipL32 gene. CONCLUSIONS: The results indicate that the long-tailed macaques at Kosumpee Forest Park may act as natural reservoirs for Leptospirosis. Further, the results provide evidence-based information indicating that several pathogenic Leptospira serovars are circulating in the wild macaques in the study area.

One Health Approach of Melioidosis and Gastrointestinal Parasitic Infections from Macaca fascicularis to Human at Kosumpee Forest Park, Maha Sarakham, Thailand.

BACKGROUND AND OBJECTIVES: Gastrointestinal parasitic and melioidosis infections are major causes of morbidity and mortality from infectious disease in rural areas, especially in northeastern Thailand. Both diseases are zoonotic giving rise to health problems in both long-tailed macaques and in humans. In Thailand, macaques have adapted to live and share space with humans and can spread some zoonoses to humans. Therefore, this research aimed to measure the prevalence of gastrointestinal parasitic infections and melioidosis in long-tailed macaques at Kosumpee Forest Park and measure associated risk factors of their diseases among people in this area. METHODS: This study was conducted at Kosumpee Forest Park, Maha Sarakham, Thailand. Twenty-eight blood samples and 135 fecal samples were collected from free-ranging long-tailed macaques. Blood samples were tested by indirect hemagglutination test and fecal samples were analyzed by formalin-ethyl acetate concentration technique. A cross-sectional study was conducted among 350 respondents who were involved with the Forest Park using a multi-stage stratified random sampling method and performed to measure knowledge, attitude, and practice toward the zoonoses among the respondents. RESULTS: It was found that seroprevalence of melioidosis was 57.1% from macaque samples. The prevalence of gastrointestinal parasites infection was 35.11% from fecces samples, including Strongyloides spp. (15.27%), Trichuris spp. (22.9%), hookworm (4.58%) and Ascarid spp. (1.53%). KAP study indicated that the level of knowledge related to melioidosis and gastrointestinal parasites of people in the area was very low and moderate, respectively. The attitude of respondents who were aware of the diseases was at a moderate level for melioidosis and a high level for parasitic infection. CONCLUSION: The study therefore emphasizes the importance of one health approach for diagnosis, surveillance and management of zoonotic diseases to promote the development of hygiene measures and to educate people in the community around Kosumpee Forest Park.

Staphylococcus spp. associated with subclinical bovine mastitis in central and northeast provinces of Thailand.

BACKGROUND: Staphylococcus spp. are major cause of bovine mastitis (BM) worldwide leading to economic damage to dairy farms and public health threat. Recently, a newly emerged Staphylococcus argenteus has been found as a human and animal pathogen. Molecular characteristics, virulence and antibiotic resistant phenotypes of bacteria causing BM in Thailand are rare. This study aimed to investigated Staphylococcus spp. associated with subclinical bovine mastitis (SCM) in Thailand. METHODS: Milk samples were collected from 224 cows of 52 dairy herds in four central and northeast provinces. Total somatic cell counts (SCC) and California mastitis test (CMT) were used to identify SCM cows. Milk samples were cultured for Staphylococcus spp. Coagulase-positive isolates were subjected to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Organisms suspected as S. argenteus were verified by detecting nonribosomal peptide synthetase gene. All isolates were checked for antibiograms and the presence of various virulence genes. RESULTS: From the 224 milk samples of 224 cows, 132 (59%) were positive for SCM by SCC and CMT and 229 staphylococcal isolates were recovered. They were 32 coagulase-positive (24 S. aureus and eight S. argenteus) and 197 coagulase-negative. PFGE of the S. aureus and S. argenteus revealed 11 clusters and a non-typeable pattern. MLST of representatives of the 11 PFGE clusters, three PFGE non-typeable S. aureus isolates from different locations and S. argenteus showed 12 sequence types. The eight S. argenteus isolates belonged to ST1223 (three isolates), ST2250 (two isolates), and ST2793 (two isolates). The antimicrobial tests identified 11 (46%) methicillin-resistant S. aureus and 25 (13%) methicillin-resistant coagulase-negative isolates, while seven S. argenteus were methicillin-susceptible and one isolate was methicillin-resistant. All of the 229 isolates were multiply resistant to other antibiotics. The most prevalent virulence genes of the 24 S. aureus isolates were clfA, coa and spa (X and IgG-binding region) (100%), hla (96%), pvl (96%) and sec (79%). Six S. argenteus isolates carried one enterotoxin gene each and other virulence genes including coa, clfA, hla/hlb, spa, tsst and pvl, indicating their pathogenic potential. CONCLUSION AND PERSPECTIVE: This is the first report on the S. argenteus from cow milk samples with SCM. Data on the molecular characteristics, virulence genes and antibiograms of the Staphylococcus spp. obtained from the present study showed a wide spread and increasing trend of methicillin-resistance and multiple resistance to other antibiotics. This suggests that the "One Health" practice should be nurtured, not only at the dairy farm level, but also at the national or even the international levels through cooperation of different sectors (dairy farmers, veterinarians, medical and public health personnel and scientists) in order to effectively combat and control the spread of these pathogens.

Staphylococcus argenteus: An emerging subclinical bovine mastitis pathogen in Thailand.

BACKGROUND AND AIM: Staphylococcus argenteus is an emerging species of the Staphylococcus aureus complex. It has usually been misidentified as S. aureus by conventional methods and its characteristics. S. argenteus is potentially emerging in both humans and animals with an increasing global distribution. This study aimed to differentiate and identify S. argenteus from S. aureus collected and isolated from milk samples of subclinical bovine mastitis cases in Maha Sarakham Province, Northeastern Thailand. MATERIALS AND METHODS: Forty-two isolates of S. aureus were studied from 132 individual milk samples collected from subclinical bovine mastitis cases of 15 dairy farms in three districts of Maha Sarakham, Thailand. The identification was confirmed by conventional and immune-agglutination methods. Fifteen representative isolates which were suspected as being S. argenteus were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The result from MALDI-TOF MS confirmed that seven from 15 isolates were S. argenteus and eight isolates were S. aureus. CONCLUSION: This study indicated that MALDI-TOF MS used as an identification and classification method could accurately differentiate the novel species, S. argenteus, from the S. aureus complex which is usually misdiagnosed. In addition, the identification of S. argenteus seems to be very limited in technical difficulty despite the fact that it may be the important causative pathogen in bovine mastitis as well as a pathogenic bacterium in food and milk. Therefore, it is essential for both bovine medicine and veterinary public health to emphasize and recognize this bacterial pathogen as an emerging disease of staphylococcal bacteria that there is a need for further study of S. argenteus infections.

Staphylococcus argenteus from rabbits in Thailand.

Staphylococcus argenteus, a novel species of the genus Staphylococcus or a member of the S. aureus complex, is closely related to S. aureus and is usually misidentified. In this study, the presence of S. argenteus in isolated S. aureus was investigated in 67 rabbits with abscess lesions during 2014-2016. Among 19 S. aureus complex isolates, three were confirmed to be S. argenteus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, nonribosomal peptide synthetase gene amplification, and multilocus sequence type. All S. aureus complex isolates, including the S. aureus isolates, were examined for their antimicrobial resistance phenotype by disk diffusion and for their resistance genotype by PCR assays. Among the S. argenteus isolates, one was susceptible to all antimicrobial drugs and the other two were resistant to penicillin and doxycycline. In contrast, most S. aureus isolates were resistant to penicillin (37.5%), and gentamicin (12.5%). Moreover, S. aureus isolates harbored the blaZ, mecA, aacA-aphD, and mrs(A) as well as mutations of gyrA and grlA, but S. argenteus isolates carried solely the blaZ. S. argenteus isolates were investigated for enterotoxin (sea-sed) and virulence genes by PCR. One isolate carried sea, sec, and sed, whereas the other two isolates carried only sea or sed. No isolate carried seb and see. All three S. argenteus isolates carried hla, hlb, and clfA, followed by pvl, whereas coa, spa (IgG-binding region), and spa (x region) were not detected in the three isolates. This paper presents the first identification of S. argenteus from rabbits in Thailand. S. argenteus might be pathogenic because the isolates carried virulence genes. Moreover, antimicrobial resistance was observed. Investigations of this new bacterial species should be conducted in other animal species as well as in humans.

Cryptosporidiosis: A zoonotic disease concern.

Cryptosporidiosis is considered to be a crucial zoonotic disease caused by worldwide distributing parasitic protozoa called Cryptosporidium spp. Cryptosporidiosis becomes a major public health and veterinary concern by affecting in human and various host range species of animals. Essentially, its importance of infection is increasing because of the high incidence in young children, immunocompromised persons, or immunodeficiency syndrome patients, especially in HIV/AIDS, and it is also one of the most causes of mortality in those patients who infected with Cryptosporidium spp. as well as young animals. All domestic animal, livestock, wildlife, and human can be potential reservoirs that contribute Cryptosporidium spp. to food and surface waters and transmitted to other hosts through fecal-oral route. The oocyst stage of Cryptosporidium spp. can remain infective and resistant to various environmental exposure and also resistant to many general disinfecting agents including chlorination which normally used in water treatment. Therefore, the understanding of these zoonotic pathogens is very essential in both animal and human health. This review focuses on the biology, life cycle, transmission, diagnosis, treatment, prevention, and control of this protozoan infection to emphasize and remind as the significant One Health problem.

Screening method for Staphylococcus aureus identification in subclinical bovine mastitis from dairy farms.

BACKGROUND: Staphylococcus aureus is one of the most important contagious bacteria causing subclinical bovine mastitis. This bacterial infection is commonly identified by determine the pathogen in bovine milk samples through conventional technique including coagulase test. However, this test has several disadvantages as low sensitivity, risk of biohazard, cost expensive, and limited preparation especially in local area. AIM: Aim of this study was to compare and assess the screening method, Mannitol fermentation test (Mannitol salt agar [MSA]), and deoxyribonuclease (DNase) test, for S. aureus identification in milk samples. MATERIALS AND METHODS: A total of 224 subclinical bovine mastitis milk samples were collected from four provinces of Thailand and determined S. aureus using conventional method and also subjected to the screening test, MSA and DNase test. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) among both tests were analyzed and compared to the tube coagulase test (TCT), as reference method. Immunological test by latex agglutination and molecular assay by determined spa gene were also used to identify and differentiate S. aureus. RESULTS: A total of 130 staphylococci were isolated by selective media, Gram-stain, and catalase test. The number of S. aureus which identified using TCT, MSA and DNase test were 32, 102, and 74 isolates, respectively. All TCT results were correlated to results of latex agglutination and spa gene which were 32 S. aureus. MSA showed 100% sensitivity, 28.57% specificity, 31.37% PPV, and 100% NPV, whereas DNase showed 53.13% sensitivity, 41.84% specificity, 22.97% PPV, and 73.21% NPV. DNase test showed higher specificity value than MSA but the test presented 26.79% false negative results whereas no false-negative result from MSA when comparing to TCT. CONCLUSION: MSA had a tendency to be a good preference for screening S. aureus because of its high sensitivity and NPV. The result from this study will improve a choice to use a screening test to diagnose S. aureus of veterinary field for prompt disease controlling and effective treatment.

Detection and drug resistance profile of Escherichia coli from subclinical mastitis cows and water supply in dairy farms in Saraburi Province, Thailand.

Subclinical mastitis is a persistent problem in dairy farms worldwide. Environmental Escherichia coli is the bacterium predominantly responsible for this condition. In Thailand, subclinical mastitis in dairy cows is usually treated with various antibiotics, which could lead to antibiotic resistance in bacteria. E. coli is also a reservoir of many antibiotic resistance genes, which can be conveyed to other bacteria. In this study, the presence of E. coli in milk and water samples was reported, among which enteropathogenic E. coli was predominant, followed by enteroaggregative E. coli and enterohemorrhagic E. coli, which was found only in milk samples. Twenty-one patterns of antibiotic resistance were identified in this study. Ampicillin- and carbenicillin-resistant E. coli was the most common among the bacterial isolates from water samples. Meanwhile, resistance to ampicillin, carbenicillin, and sulfamethoxazole-trimethoprim was the pattern found most commonly in the E. coli from milk samples. Notably, only the E. coli from water samples possessed ESBL phenotype and carried antibiotic resistance genes, blaTEM and blaCMY-2. This indicates that pathogenic E. coli in dairy farms is also exposed to antibiotics and could potentially transfer these genes to other pathogenic bacteria under certain conditions.