Genetic Relatedness Among Streptococcus agalactiae Isolated from Cattle, Fish, and Humans.

Streptococcus agalactiae is well recognized to cause a variety of infections in many animal species and humans. We aimed to investigate genetic relatedness of S. agalactiae strains isolated from humans and animal origins, including cattle and fish, using capsular gene typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing techniques. Our results revealed that S. agalactiae strains with capsular type Ia and ST103 were observed from all bovine isolates (17/17) and one human isolate (1/5). S. agalactiae strains with capsular type III and ST283 were detected among isolates from fish (5/5) and from humans (2/5). Two PFGE clusters containing isolates from mixed origins were demonstrated: one cluster of five fish and one human isolate, and another cluster of one bovine and one human isolate. In conclusion, the close genetic relationship among S. agalactiae strains isolated from humans and animal origins was evident.

Characterization of Erythromycin and Tetracycline Resistance Genes of Streptococcus gallolyticus Subspecies pasteurianus Strains Isolated from Patients with Septicemia and Bacteremia in Thailand.

BACKGROUND: Streptococcus gallolyticus subspecies (subsp.) pasteurianus, previously known as Streptococcus bovis biotype II/2, has been described as a causative agent of endocarditis, neonatal sepsis, meningitis, bacteremia, and colorectal carcinoma in humans. The aim of this study was to characterize the erythromycin and tetracycline resistance genes of S. gallolyticus subsp. pasteurianus strains isolated from patients with septicemia and bacteremia in Thailand. METHODS: The clinical isolates of Streptococcus gallolyticus were identified by using conventional biochemical tests, PCR, and sodA gene sequence analysis. The erythromycin and tetracycline susceptibilities were determined by disk diffusion and agar dilution methods, while the resistance genes were identified by nucleotide sequence analysis. RESULTS: From a total of 108 blood cultures, 36 (33%) were identified as S. gallolyticus subsp. pasteurianus with the nucleotide sequence identities of partial sodA gene with the reference strains ranging from 98.1 to 100%. Of these, 25 (69.4%) contained erythromycin resistance genes and erm(B) was the most predominant gene (30.6%), followed by erm(T) (19.4%) and mef(A) (5.6%). In addition, erm(B) was also detected in combination with lnu(B) (8.3%), erm(T) and mef(A) (2.8%), and mef(A) and lnu(B) (2.8%). It was interesting to note that lnu(B) was detected for the first time in S. gallolyticus subsp. pasteurianus in this study. For tetracycline resistance genes, tet(L) and tet(M) were detected at 13.9% and 11.1%, respectively. However, tet(M) in combination with tet(L) was detected most commonly at 69.4% and with tet(L) and tet(O) at 5.6%. CONCLUSIONS: A number of erythromycin and tetracycline resistance genes were detected in S. gallolyticus subsp. pasteurianus strains circulating in Thailand.

Emergence and Spread of Epidemic Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is one of the most common nosocomial pathogens worldwide. Although the emergence of multidrug-resistant (MDR) P. aeruginosa is a critical problem in medical practice, the key features involved in the emergence and spread of MDR P. aeruginosa remain unknown. This study utilized whole genome sequence (WGS) analyses to define the population structure of 185 P. aeruginosa clinical isolates from several countries. Of these 185 isolates, 136 were categorized into sequence type (ST) 235, one of the most common types worldwide. Phylogenetic analysis showed that these isolates fell within seven subclades. Each subclade harbors characteristic drug resistance genes and a characteristic genetic background confined to a geographic location, suggesting that clonal expansion following antibiotic exposure is the driving force in generating the population structure of MDR P. aeruginosa. WGS analyses also showed that the substitution rate was markedly higher in ST235 MDR P. aeruginosa than in other strains. Notably, almost all ST235 isolates harbor the specific type IV secretion system and very few or none harbor the CRISPR/CAS system. These findings may help explain the mechanism underlying the emergence and spread of ST235 P. aeruginosa as the predominant MDR lineage.

Serotype- and virulence-associated gene profile of Streptococcus suis isolates from pig carcasses in Chiang Mai Province, Northern Thailand.

In this present study, the serotype of 40 Streptococcus suis isolates from submaxillary glands of pig carcasses sold in wet markets in Chiang Mai Province, northern Thailand, was investigated. Eleven serotypes, including types 2, 3, 4, 5, 7, 8, 9, 17, 21, 22 and 31, were found in the isolates by a Multiplex PCR combined with serum agglutination. Of the eleven serotypes present, type 3 was the most prevalent, while types 2, 4, 5 and 21 were of primary interest due to their human isolate serotype. The mrp+/epf - /sly - genotype was found to be the most prevalent genotype. This study indicates the importance of effective control of human S. suis infection due to raw pork or pig carcass handling in northern Thailand.

Occurrence and characterization of livestock-associated methicillin-resistant Staphylococcus aureus in pig industries of northern Thailand.

This study was conducted to determine the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs, farm workers, and the environment in northern Thailand, and to assess LA-MRSA isolate phenotypic characteristics. One hundred and four pig farms were randomly selected from the 21,152 in Chiang Mai and Lamphun provinces in 2012. Nasal and skin swab samples were collected from pigs and farm workers. Environmental swabs (pig stable floor, faucet, and feeder) were also collected. MRSA was identified by conventional bacterial culture technique, with results confirmed by multiplex PCR and multi locus sequence typing (MLST). Herd prevalence of MRSA was 9.61% (10 of 104 farms). Among pigs, workers, and farm environments, prevalence was 0.68% (two of 292 samples), 2.53% (seven of 276 samples), and 1.28% (four of 312 samples), respectively. Thirteen MRSA isolates (seven from workers, four from environmental samples, and two from pigs) were identified as Staphylococcal chromosomal cassette mec IV sequences type 9. Antimicrobial sensitivity tests found 100% of the MRSA isolates resistant to clindamycin, oxytetracycline, and tetracycline, while 100% were susceptible to cloxacillin and vancomycin. All possessed a multidrug-resistant phenotype. This is the first evidence of an LA-MRSA interrelationship among pigs, workers, and the farm environment in Thailand.

Latest developments on Streptococcus suis: an emerging zoonotic pathogen: part 2.

First International Workshop on Streptococcus suis, Beijing, China, 12-13 August 2013. This second and final chapter of the report on the First International Workshop on Streptococcus suis follows on from Part 1, published in the April 2014, volume 9, issue 4 of Future Microbiology. S. suis is a swine pathogen and a zoonotic agent afflicting people in close contact with infected pigs or pork meat. Although sporadic cases of human infections had been reported worldwide, deadly S. suis outbreaks emerged in Asia. The severity of the disease underscores the lack of knowledge on the virulence and zoonotic evolution of this human-infecting agent. The pathogenesis of the infection, interactions with host cells and new avenues for treatments were among the topics discussed during the First International Workshop on S. suis (China 2013).

Latest developments on Streptococcus suis: an emerging zoonotic pathogen: part 1.

First International Workshop on Streptococcus suis, Beijing, China, 12-13 August 2013 The first international workshop on Streptococcus suis, which is an important swine pathogen and emerging zoonotic agent, took place in Beijing, jointly organized by the Faculty of Veterinary Medicine, University of Montreal, Canada and the National Institute for Communicable Disease Control and Prevention, China CDC. The aim of the meeting was to gather together, for the first time, more than 80 researchers working on S. suis, from countries including China, Canada, Japan, The Netherlands, Germany, Thailand, the UK and Vietnam. This article, the first of a two-part report on this First International Workshop, reviews current aspects of the epidemiology and population genomics of S. suis, covers public health concerns and discusses questions about S. suis serotyping and molecular diagnostics.

Correlation between PFGE Groups and mrp/epf/sly Genotypes of Human Streptococcus suis Serotype 2 in Northern Thailand.

Streptococcus suis infection is a severe zoonotic disease commonly found in Northern Thailand where people often consume raw pork and/or pig's blood. The most frequent clinical presentations are meningitis, sepsis, and endocarditis with higher rate of mortality and hearing loss sequelae. To clarify the correlation between pulsed-field gel electrophoresis (PFGE) groups and mrp/epf/sly genotypes of S. suis serotype 2, 62 patient and 4 healthy pig isolates from Northern Thailand were studied. By PFGE analysis, at 66% homology, most human isolates (69.4%) and 1 pig isolate were in group A, whereas 14.5% of human isolates and 3 out of 4 pig isolates were in group D. According to mrp/epf/sly genotypes, 80.6% of human isolates were identified in mrp (+) epf (-) sly (-) and only 12.9% were in mrp (-) epf (-) sly (+) genotypes; in contrast, 1 and 3 pig isolates were detected in these two genotypes, respectively. Interestingly, all isolates of S. suis serotype 2 classified in PFGE groups A, B, and E were set in mrp (+) epf (-) sly (-) genotypes. These data show a close correlation between PFGE groups and mrp/epf/sly genotypes of human S. suis serotype 2.

Detection of environmental sources of Histoplasma capsulatum in Chiang Mai, Thailand, by nested PCR.

Histoplasmosis is a systemic mycosis caused by inhaling spores of Histoplasma capsulatum, a dimorphic fungus. This fungus grows in soil contaminated with bat and avian excreta. Each year, patients with disseminated histoplasmosis have been diagnosed in Chiang Mai, northern Thailand. No published information is currently available on the environmental sources of this fungus in Chiang Mai or anywhere else in Thailand. The aim of this study was to detect H. capsulatum in soil samples contaminated with bat guano and avian droppings by nested PCR. Two hundred and sixty-five samples were collected from the following three sources: soil contaminated with bat guano, 88 samples; soil contaminated with bird droppings, 86 samples; and soil contaminated with chicken droppings, 91 samples. Genomic DNA was directly extracted from each sample, and H. capsulatum was detected by nested PCR using a primer set specific to a gene encoding 100-kDa-like protein (HcI, HcII and HcIII, HcIV). Histoplasma capsulatum was detected in seven of 88 soil samples contaminated with bat guano, one of 21 soil samples contaminated with pigeon droppings and 10 of 91 soil samples contaminated with chicken droppings. The results indicate the possibility of the association of bat guano and chicken droppings with H. capsulatum in this area of Thailand.

Postmortem diagnosis of sudden unexpected death from Streptococcus suis type 2 infection: a case report.

Streptococcus suis causes meningitis, septicemia, arthritis, endocarditis and death in both pigs and humans. Sudden death is rarely documented in the forensic field and almost all cases were diagnosed pre-mortem. Here we report a 49-year-old man who died from S. suis type 2 infection. S. suis was identified as the causative pathogen using bacterial culture, standard biochemical and coagglutination tests, specific DNA amplification by polymerase chain reaction, and histopathologic examination. We discuss the postmortem investigation of a suspected S. suis infection.

Acinetobacter baumannii nosocomial pneumonia in tertiary care hospitals in Thailand.

BACKGROUND: Nosocomial pneumonia (NP) is an important cause of morbidity and mortality in hospitalized patients. Acinetobacter baumannii is one of the common causative pathogens in NP. The prevalence of multi-drug resistance in A. baumannii has been increasing. The information on clinical features and clinical courses of A. baumannii NP in Thai patients are limited. OBJECTIVE: To determine the clinical features, risk factors and clinical courses of A. baumannii NP in Thai patients hospitalized in tertiary care hospitals in Thailand. MATERIAL AND METHOD: This was a prospective, hospital-based, active surveillance study on hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) in adults hospitalized in 12 tertiary care hospitals in Thailand between 2008 and 2009. RESULTS: There were 651 NP patients. A. baumannii was the most common cause of NP in 198 patients (30.4%). Most of NP patients were males with median age of 71 years. About 80% had late onset NP with the median duration of 10 days after admission in both A. baumannii and non-A. baumannii NP. Most of NP occurred in patients hospitalized in general medical wards. Most of the features of NP in A. baumannii NP and non-A. baumannii NP were not significantly different. The initial antibiotics prescribed were concordant in about 50% of the patients in both groups. Colistin was usually prescribed to the patients who received antibiotic modifications. The initial clinical responses in A. baumannii NP were less favorable than those in non-A. baumannii NP. The mortality rate in A. baumannii NP seemed to be more than that in non-A. baumannii NP. There was a trend of more persistence of pathogen in A. baumannii NP. Most isolates of A. baumannii were resistant to antibiotics including carbapenems. The patients with extensive drug resistant A. baumannii NP had less favorable responses than NP due to other bacteria, including non-extensive drug resistant A. baumannii. VAP, NP developed in medical ICU and NP with bilateral lung involvements on chest X-ray were associated with A. baumannii as the isolated pathogen. CONCLUSION: A. baumannii is the most common causative pathogen for NP in tertiary care hospitals in Thailand and most of A. baumannii isolates were resistant to many antibiotics including carbapenems. The hospitalized patient in tertiary care hospitals with VAP, or NP that was developed in medical ICU, or NP with bilateral lung involvements on chest x-ray was likely to be due to A. baumannii. Many NP patients received inappropriate initial antibiotic regimens leading to a high mortality.

Phenotypic and PCR-based identification of bacterial strains isolated from patients with suspected streptococcus suis infection in northern Thailand.

Twenty bacterial strains isolated from the blood of patients with suspected Streptococcus suis infection based on clinical symptoms in northern Thailand between 2009 and 2010 were subjected to phenotypic and genotypic identification. Commercial identification kits and a PCR-based assay targeting the S. suis-specific 16S rDNA sequence correctly identified S. suis isolated from patients in northern Thailand; however, there was a risk of misidentifying S. gallolyticus as S. suis using a PCR assay targeting the S. suis-specific house keeping gene encoding glutamate dehydrogenase. This is the first paper to report S. gallolyticus infection in humans in Thailand.

Methicillin-resistant Staphylococcus aureus ST9 in pigs in Thailand.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial and community-associated pathogen. Recently, livestock-associated MRSA (LA-MRSA) has emerged and disseminated in Europe and North America and now constitutes a considerable zoonotic burden in humans with risk factors of pig exposure, whereas the extent of the livestock reservoir is relatively unknown on other continents. METHODOLOGY/PRINCIPAL FINDINGS: From March through April 2011, MRSA was identified in pigs from 3 out of 30 production holdings in Chang Mai Province, Thailand. Representative isolates were subjected to molecular characterization and antimicrobial susceptibility testing; all isolates had genotypic and phenotypic characteristics of LA-MRSA previously characterized in the region: they belonged to ST9, lacked the lukF-lukS genes encoding Panton-Valentine leukocidin, and were resistant to multiple non-ß-lactam antimicrobials. However, unlike other Asian LA-MRSA-ST9 variants, they were spa type t337 and harbored a different staphylococcal cassette chromosome mec IX. CONCLUSIONS/SIGNIFICANCE: A novel MRSA-ST9 lineage has been established in the pig population of Thailand, which differs substantially from LA-MRSA lineages found in other areas of the continent. The emergence of novel LA-MRSA lineages in the animal agriculture setting is worrisome and poses a serious threat to global public health.

Lineage and virulence of Streptococcus suis serotype 2 isolates from North America.

We performed multilocus sequence typing of 64 North American Streptococcus suis serotype 2 porcine isolates. Strains were sequence type (ST) 28 (51%), ST25 (44%), and ST1 (5%). We identified nonrandom associations between STs and expression of the virulence markers suilysin (SLY), muramidase-relased protein (MRP), and extracellular factor (EF). Expression of pili encoded by the srtF and srtG pilus clusters was also nonrandomly associated with STs. ST1 strains were SLY+ EF+ MRP+ srtF pilus+ srtG pilus-. ST25 strains were SLY- EF- MRP- srtF pilus- srtG pilus+, and most ST28 strains were SLY- MRP+ EF- srtF pilus+ srtG pilus+. ST28 isolates proved essentially nonvirulent in a mouse infection model; ST25 strains showed moderate virulence and ST1 isolates were highly virulent. ST1 is responsible for a high proportion of S. suis disease in humans worldwide. Its presence in North America indicates that potential zoonotic S. suis outbreaks in this continent cannot be disregarded.

Clinical study concerning the relationship between community-acquired pneumonia and viral infection in northern Thailand.

OBJECTIVE: The etiological agents associated with community-acquired pneumonia (CAP) in Thailand have been studied extensively in bacterial pathogens, but not in viral pathogens. To clarify the association of viral pathogens with CAP, we conducted a comprehensive study of viral and bacterial pathogens in patients with CAP. METHODS: We enrolled 119 hospitalized patients with CAP in Nakornping Hospital, Chiang Mai, Thailand between 2006 and 2008. The severity of pneumonia was classified and the risk factors for death were estimated. Bacterial and fungal pathogens were determined from specimens taken from blood and sputum, and viral pathogens were identified from nasopharyngeal specimens by RT-PCR using primers specific for 7 respiratory viruses. RESULTS: Overall, 29 patients were HIV-infected and 90 patients were non-HIV-infected. The microbial pathogens most commonly isolated among HIV-infected patients were: 4 Klebsiella pneumoniae, 4 Mycobacterium tuberculosis and 3 Haemophilus influenzae. Among non-HIV infected patients, predominant microbial pathogens were: 6 Pseudomonas aeruginosa, 5 Haemophilus influenzae and 4 Klebsiella pneumoniae. As for viral pathogens for CAP, influenza virus was identified from 2 HIV-infected patients and 5 non-HIV infected patients. In addition, human rhinovirus (HRV) and respiratory syncytial virus (RSV) were identified from 2 patients each among non-HIV-infected patients. CONCLUSION: Our study demonstrates that the most common viral agent was influenza virus (5%), followed by HRV (2%) and RSV (2%) among CAP patients in northern Thailand. The underlying chronic obstructive pulmonary disease (COPD) seems to be correlated with the severity of illness.

Application of a non-species dependent ELISA for the detection of antibodies in sera of Burkholderia pseudomallei-immunized goats.

A modified, non-species dependent ELISA was performed to detect antibodies in sera of Burkholderia pseudomallei-immunized goats using protein G- or protein A-peroxidase conjugates. The rise of antibody titers during the immunization period exhibited corresponding results by modified ELISA comparison to conventional ELISA and the IHA. Regarding the increase of antibody levels from the pre-immunized baseline to the post-immunized status, the antibody titer detected by modified ELISA was higher than IHA but lower than conventional ELISA. Further efforts are needed to improve the modified ELISA for the diagnosis of melioidosis in animals.

Incidence and presence of virulence factors of Streptococcus suis infection in slaughtered pigs from Chiang Mai, Thailand.

This study was designed to determine the incidence of Streptococcus suis infection in slaughtered pigs raised in industrial facility and backyard system in Chiang Mai City, Thailand. A total of 90 tonsils and submaxillary salivary gland/lymph node samples from slaughtered pigs raised in industrial facility and 122 samples from slaughtered pigs raised in backyard system were collected. Isolation and identification of S. suis were conducted using standard bacteriological methods. Farm management and risk factor data were collected by a questionnaire. Serotyping and presence of virulence factor genes, epf, mrp and sly, were determined by multiplex PCR assay. The overall incidence of S. suis in this study was 9% (n = 212) and the incidence is significantly higher in districts located at a greater distance south of Chiang Mai City. S. suis serotype 2 was present more in healthy pigs (43%) than ill pigs (10%). Every S. suis isolate carried mrp and sly and ill pigs carried epf (80%) more than healthy pigs (57%). However, the probability of S. suis serotype 2 with epf+ (0.245) detected in healthy pigs was higher than in ill pigs (0.08) indicating people may have a higher risk of being infected with S. suis from healthy than ill pigs.

Genetic organization and preferential distribution of putative pilus gene clusters in Streptococcus suis.

Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis (srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A-L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection.

Clinical and microbiological characteristics of community-acquired pneumonia among human immunodeficiency virus-infected patients in northern Thailand.

Human immunodeficiency virus (HIV) infections are prevalent in Thailand. However, the clinical and microbiological characteristics of community-acquired pneumonia (CAP) in such patients are not completely clear at present. In the present study, we analyzed the characteristics of CAP in 191 HIV-infected patients (192 episodes, 130 males and 61 females, mean age 32.9 years, range: 20-62) who had been admitted to Nakornping Hospital in northern Thailand between December 1996 and January 2002. The mean peripheral blood CD4 lymphocyte count was 68.5/mm3 (range: 0-791). The most common organisms detected in the blood of the subjects were as follows: Penicillium marneffei, 13, Salmonella spp., 5, Cryptococcus neoformans, 4, Staphylococcus aureus, 3, and Rhodococcus equi, 3, and the most common organisms detected in sputum included Haemophilus influenzae, 38, P. marneffei, 10, Streptococcus pneumoniae, 10, R. equi, 9, and S. aureus, 9. Life-threatening meningitis in 5 (cryptococcal in 3 and tuberculous in 2), pneumothorax in 2, and tuberculous lymphadenitis in 1 were also noted, resulting in 21 fatalities (10.9%). The mean peripheral blood CD4 lymphocyte count for cases in which the subject died was 74.8/mm3 (range: 0-340). Logistic regression analysis demonstrated that high age (odds ratio of over 40 years: 15.62) and R. equi infection (odds ratio: 8.14) are related to death of HIV-infected patients with CAP. The above findings indicate that various types of organisms, including mixed organisms, cause CAP in HIV-infected patients in northern Thailand, and high age and R. equi infection seem to be risk factors for death.

Allelic variation and prevalence of serum opacity factor among the Streptococcus suis population.

Serum opacity factor of Streptococcus suis (OFS) has recently been identified as a virulence determinant of an S. suis strain. In this study, we investigated the prevalence and variations of the ofs gene among 108 S. suis isolates from diseased and healthy pigs, and human patients. PCR screening and sequencing analysis showed that besides the ofs gene reported already (designated type 1), there were three allelic variants of ofs (designated types 2 to 4). Type-1 and type-2 ofs genes were expected to encode functional OFS, and SDS extracts of the isolates with type-1 ofs and type-2 ofs opacified horse serum. Culture supernatants of the isolates with type-2 ofs also showed strong serum opacification activity. In contrast, type-3 ofs was interrupted by a point mutation and type-4 ofs was disrupted by either insertion of an IS element or genetic rearrangement, and therefore the SDS extracts and culture supernatants of the isolates with type-3 ofs and type-4 ofs did not show serum opacification activity. Regardless of their origins, approximately 30 % of the isolates possessed functional OFSs, although type-2 ofs was found only in three isolates from healthy pigs. Multilocus sequence typing analysis showed that most of the isolates with type-1 ofs belonged to the sequence type (ST)1 complex, and most of the isolates with type-3 ofs and type-4 ofs belonged to the ST27 complex. The isolates with type-2 ofs were not assigned to a major ST complex. These results suggest that type-1 OFS contributes to the virulence of a limited number of S. suis isolates, i.e. those of the ST1 complex type, whereas other S. suis may not possess this category of virulence factor; the importance of type-2 OFS is obscure.