Diversity of Xenorhabdus and Photorhabdus spp. and their symbiotic entomopathogenic nematodes from Thailand

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand

Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro.

BACKGROUND: Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays. RESULTS: Morphotype was associated with survival in the presence of H2O2 and antimicrobial peptide LL-37, but not with susceptibility to acid, acidified sodium nitrite, or resistance to lysozyme, lactoferrin, human neutrophil peptide-1 or human beta defensin-2. Incubation under anaerobic conditions was a strong driver for switching of type III to an alternative morphotype. Differences were noted in the survival and replication of the three types following uptake by human macrophages, but marked strain-to strain-variability was observed. Uptake of type III alone was associated with colony morphology switching. CONCLUSIONS: Morphotype is associated with phenotypes that alter the ability of B. pseudomallei to survive in adverse environmental conditions.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

The soil dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis, a serious human infection that occurs in Southeast Asia and northern Australia. The purpose of this study was to evaluate the population genetic structure of B. pseudomallei in the environment. To achieve this, we undertook soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within an area of disused land in northeast Thailand, and undertook detailed genotyping of primary plate colonies isolated from three independent sampling points. Our results demonstrated that multiple B. pseudomallei genotypes were present within a single soil sample, and that different genotypes were present at independent but nearby sampling points. The B. pseudomallei genetic population was unevenly distributed within a given sample, with a predominant genotype co-existing with several genotypes present as a minority population. We discuss the implications of this structuring of genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies

Factors Predicting and Reducing Mortality in Patients with Invasive Staphylococcus aureus Disease in a Developing Country

BACKGROUND: Invasive Staphylococcus aureus infection is increasingly recognised as an important cause of serious sepsis across the developing world, with mortality rates higher than those in the developed world. The factors determining mortality in developing countries have not been identified. METHODS: A prospective, observational study of invasive S. aureus disease was conducted at a provincial hospital in northeast Thailand over a 1-year period. All-cause and S. aureus-attributable mortality rates were determined, and the relationship was assessed between death and patient characteristics, clinical presentations, antibiotic therapy and resistance, drainage of pus and carriage of genes encoding Panton-Valentine Leukocidin (PVL). PRINCIPAL FINDINGS: A total of 270 patients with invasive S. aureus infection were recruited. The range of clinical manifestations was broad and comparable to that described in developed countries. All-cause and S. aureus-attributable mortality rates were 26% and 20%, respectively. Early antibiotic therapy and drainage of pus were associated with a survival advantage (both p<0.001) on univariate analysis. Patients infected by a PVL gene-positive isolate (122/248 tested, 49%) had a strong survival advantage compared with patients infected by a PVL gene-negative isolate (all-cause mortality 11% versus 39% respectively, p<0.001). Multiple logistic regression analysis using all variables significant on univariate analysis revealed that age, underlying cardiac disease and respiratory infection were risk factors for all-cause and S. aureus-attributable mortality, while one or more abscesses as the presenting clinical feature and procedures for infectious source control were associated with survival. CONCLUSIONS: Drainage of pus and timely antibiotic therapy are key to the successful management of S. aureus infection in the developing world. Defining the presence of genes encoding PVL provides no practical bedside information and draws attention away from identifying verified clinical risk factors and those interventions that save lives

Photorhabdus luminescens subsp namnaonensis subsp nov., isolated from Heterorhabditis baujardi nematodes

A lightly yellowish-pigmented, oxidase-negative bacterial strain (PB45.5(T)) isolated from the Nam Nao district of Phetchabun in central Thailand was investigated to determine its taxonomic position. Cells of the isolate showed a rod shaped appearance. The strain stained Gram-negative. Strain PB45.5(T) shared highest 16S rRNA gene sequence similarity with the type strains of Photorhabdus luminescens subsp. akhurstii (99.2 %) and Photorhabdus luminescens subsp. hainanensis (99.1 %) and lower similarities to all other Photorhabdus luminescens subspecies (< 98.0 %). Multilocus sequence analysis (MLSA) based on concatenated partial recA, dnaN, gltX, gyrB and infB gene sequences confirmed the affiliation obtained by 16S rRNA gene sequence analysis but showed a clear distinction of PB45.5(T) from the closest related type strains. Strain PB45.5(T) shared only 96.9% sequence similarity (concatenated nucleotide sequences) with P. luminescens subsp. akhurstii FRG04(T) and 96.8% with P. luminescens subsp. hainanensis C8404(T). The fatty acid profile of the strain consisted of the major fatty acids C-14:0, C-16:0, C-17:0 cyclo, C-16:1 omega 7c and/or iso-C-15 :0 2-OH, and C-18:1 omega 7c. The MLSA results and the differential biochemical and chemotaxonomic properties showed that strain PB45.5(T) represents a novel P. luminescens subspecies, for which the name Photorhabdus luminescens subsp. namnaonensis subsp. nov. (type strain PB45.5(T) = LMG 29915(T) = CCM 8729(T)) is proposed

Identification of entomopathogenic nematodes and symbiotic bacteria from Nam Nao National Park in Thailand and larvicidal activity of symbiotic bacteria against Aedes aegypti and Aedes albopictus.

Entomopathogenic nematodes (EPNs) that are symbiotically associated with Xenorhabdus and Photorhabdus bacteria can kill target insects via direct infection and toxin action. There are limited reports identifying such organisms in the National Park of Thailand. Therefore, the objectives of this study were to identify EPNs and symbiotic bacteria from Nam Nao National Park, Phetchabun Province, Thailand and to evaluate the larvicidal activity of bacteria against Aedes aegypti and Ae. albopictus. A total of 12 EPN isolates belonging to Steinernema and Heterorhabditis were obtained form 940 soil samples between February 2014 and July 2016. EPNs were molecularly identified as S. websteri (10 isolates) and H. baujardi (2 isolates). Symbiotic bacteria were isolated from EPNs and molecularly identified as P. luminescens subsp. akhurstii (13 isolates), X. stockiae (11 isolates), X. vietnamensis (2 isolates) and X. japonica (1 isolate). For the bioassay, bacterial suspensions were evaluated for toxicity against third to early fourth instar larvae of Aedes spp. The larvae of both Aedes species were orally susceptible to symbiotic bacteria. The highest larval mortality of Ae. aegypti was 99% after exposure to X. stockiae (bNN112.3_TH) at 96 h, and the highest mortality of Ae. albopictus was 98% after exposure to P. luminescens subsp. akhurstii (bNN121.4_TH) at 96 h. In contrast to the control groups (Escherichia coli and distilled water), the mortality rate of both mosquito larvae ranged between 0 and 7% at 72 h. Here, we report the first observation of X. vietnamensis in Thailand. Additionally, we report the first observation of P. luminescens subsp. akhurstii associated with H. baujardi in Thailand. X. stockiae has potential to be a biocontrol agent for mosquitoes. This investigation provides a survey of the basic diversity of EPNs and symbiotic bacteria in the National Park of Thailand, and it is a bacterial resource for further studies of bioactive compounds

Insights into mobile genetic elements and the role of conjugative plasmid in transferring aminoglycoside resistance in extensively drug-resistant Acinetobacter baumannii AB329

Acinetobacter baumannii is a major cause of nosocomial infection, and the incidence of extensively drug-resistant A. baumannii (XDRAB) infections has dramatically increased worldwide. In this study, we aimed to explore the complete genome sequence of XDRAB 329, ST1166/98 (Oxford/Pasteur), which is an outbreak clone from a hospital in Thailand. Whole-genome sequencing (WGS) was performed using short-read Illumina and long-read PacBio sequencing, and a conjugation assay of its plasmid was performed. The complete genome sequence of A. baumannii AB329 revealed a circular chromosome 3,948,038 bp in length with 39% GC content. Antibiotic resistance genes (ARGs), including beta-lactam resistance (blaOXA-51, blaADC-25, blaOXA-23, blaTEM-1D), aminoglycoside resistance (aph(3′)-Ia, aph(3″)-Ib, aph(6)-Id, armA), tetracycline resistance (tet(B), tet (R)), macrolide resistance (mph(E), msr(E)), and efflux pumps, were found. Mobile genetic elements (MGEs) analysis of A. baumannii AB329 revealed two plasmids (pAB329a and pAB329b), three prophages, 19 genomic islands (GIs), and 33 insertion sequences (ISs). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. aph(3′)-VIa was detected in pAB329b, and a major facilitator superfamily (MFS) transporter was detected in the prophage. Acinetobacter baumannii resistance island 4 (AbaR4) harboring tetracycline and aminoglycoside resistance was detected in the genome of A. baumannii AB329. pAB329b, which belongs to Rep-type GR6 (plasmid lineage LN_1), is a conjugative plasmid with the ability to transfer an aminoglycoside resistance gene to sodium azide-resistant A. baumannii. This study provides insights into the features of the MGEs of XDRAB, which are the main reservoir and source of dissemination of ARGs

High prevalence of multi-drug resistant Streptococcus pneumoniae among healthy children in Thailand

Summary: Antibiotic resistance in Streptococcus pneumoniae is an emerging health problem worldwide. The incidence of antimicrobial-resistant S. pneumoniae is increasing, and nasal colonization of S. pneumoniae in children increases the risk of pneumococcal infection. In this study, the prevalence of S. pneumoniae nasal colonization was studied in Thai children from three different districts. S. pneumoniae nasal colonization was found in 38 of 237 subjects (16.0%). The carriage rate indicated higher rates in two rural districts (18.2% and 29.8%) than in the urban district (2.8%). The antibiotic susceptibility pattern was determined using the disk diffusion method. Prevalence of multi-drug resistance S. pneumoniae (MDR-SP) was 31.6%. Resistance to commonly prescribed antibiotics was found for ampicillin (5.3%), azithromycin (26.3%), cefepime (2.6%), chloramphenicol (18.4%), clindamycin (18.4%), erythromycin (21.1%), oxacillin (44.7%), trimethoprim/sulfamethoxazole (78.9%) and tetracycline (15.8%). All isolates were sensitive to ceftriaxone. The pulsed-field gel electrophoresis pattern was used to compare genetic diversity of the S. pneumoniae isolates. PFGE demonstrated the variation in genotypes of S. pneumoniae from different areas. High prevalence of multi-drug resistance S. pneumoniae nasal colonization in healthy Thai children was indicated. Effective strategies for appropriate use of antibiotics are therefore needed in the community. Keywords: Antibacterial resistance, Streptococcus pneumoniae, Genotype, Children, Nasal colonizatio