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

Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis

The Role of the County Professional Council in Advanced Training and Professional Development of Class Teachers

Stručno usvršavanje je obvezan dio učiteljskog posla koji se provodi na četiri osnovne razine: individualnoj, školskoj, županijskoj i državnoj. Brojne su prednosti i nedostaci svakog oblika usavršavanja, a svi su oni na putu profesionalnog razvoja učitelja jednako važni i korisni. U radu je prikazan model stručnog usvršavanja učitelja na županijskoj razini (Županijsko stručno vijeće učitelja razredne nastave – Grad Sisak) pri čemu veliku ulogu imaju upravo županijski voditelji koji su poveznica između školske i državne razine stručnog usavršavanja učitelja.Advanced training is a mandatory part of teacher’s job and it is being carried out at four basic levels: individual, school, county and state level. There are numerous advantages and disadvantages of any form of training, and they are all equally important and useful in the course of professional development of teachers. This study presents the model of advanced training of class teachers at county level (County professional council of class teachers – the Town of Sisak), where the very county leaders, who are the link between the school and state level of advanced training of teachers, have a great role

Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis

Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections

Molecular Characteristics of Methicillin-Resistant Staphylococci Clinical Isolates from a Tertiary Hospital in Northern Thailand

Methicillin-resistant staphylococci are now recognized as a major cause of infectious diseases, particularly in hospitals. Molecular epidemiology is important for prevention and control of infection, but little information is available regarding staphylococcal infections in Northern Thailand. In the present study, we examined antimicrobial susceptibility patterns, detection of antimicrobial resistance genes, and SCCmec types of methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from patients in a hospital in Northern Thailand. The species of MRSA and MR-CoNS were identified using combination methods, including PCR, MALDI-TOF-MS, and tuf gene sequencing. The susceptibility pattern of all isolates was determined by the disk diffusion method. Antimicrobial resistance genes, SCCmec types, and ST239 were characterized using single and multiplex PCR. ST239 was predominant in MRSA isolates (10/23). All MR-CoNS (N=31) were identified as S. haemolyticus (N=18), S. epidermidis (N=3), S. cohnii (N=3), S. capitis (N=6), and S. hominis (N=1). More than 70% of MRSA and MR-CoNS were resistant to cefoxitin, penicillin, oxacillin, erythromycin, clindamycin, gentamicin, and ciprofloxacin. In MRSA isolates, the prevalence of ermA (78.3%) and ermB (73.9%) genes was high compared to that of the ermC gene (4.3%). In contrast, ermC (87.1%) and qacA/B genes (70.9%) were predominant in MR-CoNS isolates. SCCmec type III was the dominant type of MRSA (13/23), whereas SCCmec type II was more present in S. haemolyticus (10/18). Ten MRSA isolates with SCCmec type III were ST239, which is the common type of MRSA in Asia. This finding provides useful information for a preventive health strategy directed against methicillin-resistant staphylococcal infections

Prevalence and genetic diversity of Burkholderia pseudomallei isolates in the environment near a patient's residence in Northeast Thailand.

BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease in tropical regions. It is necessary to understand the risk of acquiring this infection from the environment.Methodology /principal findingsThe prevalence, concentration and genetic diversity of B. pseudomallei isolates collected from two sites in Buriram, Northeast Thailand were investigated. Forty-four environmental samples (18 from soil, 14 from rice rhizosphere, and 12 from water) were collected; of those 44 samples, 19 were collected from near a patient's residence and 25 from suspected exposure sites and compared with 10 clinical isolates of the patient. Quantitative culture was performed, and B. pseudomallei was identified using the latex agglutination test and matrix-laser absorption ionisation mass spectrometry. Genotyping was performed in 162 colonies from clinical (N = 10) and environmental samples (N = 152) using pulse-field gel electrophoresis (PFGE) followed by multi-locus sequence typing (MLST) of the clinical strain. B. pseudomallei was detected in 11 of the 44 environmental samples (1 from soil, 4 from rice rhizosphere, and 6 from water). The bacterial count in the positive soil sample was 115 CFU/g. The mean concentrations ± SDs of B. pseudomallei in the positive water and rhizosphere samples were 5.1 ± 5.5 CFU/ml and 80 ± 49 CFU/g, respectively. Six water samples with positive results were collected from a pond and water sources for drinking and daily use. All colonies isolated from the patient shared the same PFGE type (PT) indicating monoclonal infection of ST99. Although the 152 colonies from environmental isolates exhibited 25 PTs, none were identical to the patient's isolates. PT5 and PT7 were most common genotype among the environmental samples.Conclusions/significanceDiverse genotypes of B. pseudomallei were prevalent in the environment. However, the patient may have been infected with a low-density genotype. Intervention strategies for preventing B. pseudomallei infection are required

Survival of Burkholderia pseudomallei in distilled water for 16 years

Burkholderia pseudomallei was examined after being maintained in distilled water at 25 °C for 16 years. The Gram stain was atypical (pale pink cocci or coccobacilli). The estimated number of live and dead B. pseudomallei was 3.8 × 107 cells/ml and 1.4 × 105 cells/ml, respectively. A colony count on agar of 1.0 × 106 cfu/ml suggested that a proportion of cells were in a viable but non-culturable state. Colony morphology was different from the parental isolate for 84% of colonies. Pulsed-field gel electrophoresis analysis of AvrII DNA restriction fragments revealed six different but related banding patterns, which may represent genomic rearrangement