Genomic Landscape Reveals Chromosomally-Mediated Antimicrobial Resistome and Virulome of a High-Risk International Clone II Acinetobacter baumannii AB073 from Thailand.

This study utilized integrative bioinformatics' tools together with phenotypic assays to understand the whole-genome features of a carbapenem-resistant international clone II Acinetobacter baumannii AB073. Overall, we found the isolate to be resistant to seven antibiotic classes, penicillins, ß-lactam/ß-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and folate pathway antagonists. These resistance phenotypes are related to various chromosomal-located antibiotic resistance determinants involved in different mechanisms such as reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efflux. IC2 A. baumannii AB073 could not transfer antibiotic resistance by conjugation experiments. Likewise, mobilome analysis found that AB073 did not carry genetic determinants involving horizontal gene transfer. Moreover, this isolate also carried multiple genes associated with the ability of iron uptake, biofilm formation, immune invasion, virulence regulations, and serum resistance. In addition, the genomic epidemiological study showed that AB073-like strains were successful pathogens widespread in various geographic locations and clinical sources. In conclusion, the comprehensive analysis demonstrated that AB073 contained multiple genomic determinants which were important characteristics to classify this isolate as a successful international clone II obtained from Thailand.

Assessment of the genetic diversity of lymnaeid (Gastropoda: Pulmonata) snails and their infection status with trematode cercariae in different regions of Thailand.

Lymnaeid snails are some of the most widespread snails and are the first intermediate host of trematode parasites that affect human and livestock health. A full understanding of the genetic relationship of hosts and parasites is of paramount importance for effective parasite management. The present study assessed the prevalence of trematode larvae in lymnaeid snails and examined the genetic diversity of these snails collected across Thailand. We collected 672 lymnaeid snails from 39 locations in 22 provinces of six regions in Thailand. Subsequently, cercarial infection in the snails was observed by using the shedding method. Lymnaeid snails released 5 types of trematode cercariae, namely, xiphidiocercariae, echinostome cercariae I, echinostome cercariae II, furcocercous cercariae, and strigea cercariae. The phylogenetic analysis based on ITS2 and 28S rDNA sequences revealed 5 cercaria types assigned to four trematode families, of which two belong to the group of human intestinal flukes. Combination of shell morphology and sequence analysis of the mitochondrial COI and 16S rDNA genes, the lymnaeid snails were classified into two species, Radix rubiginosa and Orientogalba viridis. Moreover, the combined dataset of mtDNA genes (COI + 16S rDNA) from R. rubiginosa and O. viridis revealed 32 and 15 different haplotypes, respectively, of which only a few haplotypes were infected with cercariae. The genetic diversity and genetic structure revealed that R. rubiginosa and O. viridis experienced a bottleneck phenomenon, and showed limited gene flow between populations. Population demographic history analyses revealed that R. rubiginosa and O. viridis experienced population reductions followed by recent population expansion. These findings may improve our understanding of parasite-lymnaeid evolutionary relationships, as well as the underlying molecular genetic basis, which is information that can be used for further effective control of the spread of trematode disease.

Population genetic structure of Indoplanorbis exustus (Gastropoda: Planorbidae) in Thailand and its infection with trematode cercariae.

Indoplanorbis exustus is a freshwater gastropod belonging to the family Planorbidae. This snail is widely distributed across the tropics and plays an important role as the intermediate host for trematodes. However, relatively little is understood regarding the genetic relationship between I. exustus and trematodes. The goals of this study were to investigate the current transmission status of trematode cercariae in I. exustus in Thailand and to examine the genetic diversity, genetic structure, and demographic history of I. exustus. We collected 575 I. exustus from 21 provinces across six regions of Thailand and investigated cercarial infections by using the shedding method. I. exustus from two provinces were infected with cercarial trematodes, and two types of cercarial stages were molecularly identified as furcocercous cercaria and xiphidiocercariae. Phylogenetic tree analysis based on 28S rDNA and ITS2 sequences demonstrated that furcocercous cercaria and xiphidiocercariae were closely clustered with a clade of Euclinostomum sp. and Xiphidiocercariae sp., respectively. Phylogenetic and network analyses of I. exustus haplotypes based on the COI, 16S rDNA, and ITS1 genes demonstrated four main clades. Only snails in clade A were distributed in all regions of Thailand and harbored trematode cercariae. The level of genetic diversity of I. exustus was relatively high, but most populations were not genetically different, thus suggesting the appearance of gene flow within the I. exustus populations. Overall, the haplotype network was star-shaped, thus suggesting the recent demographic expansion of populations. This result was also supported by the unimodal mode of the mismatch distribution graph and the large negative values of the neutrality tests. Therefore, the I. exustus snail was likely another freshwater snail of the invasive species in Thailand. This information will aid in monitoring the spread of the parasitic trematodes carried by I. exustus from different populations.

Genetic variation of the freshwater snail Indoplanorbis exustus (Gastropoda: Planorbidae) in Thailand, inferred from 18S and 28S rDNA sequences.

Indoplanorbis exustus, a freshwater pulmonate snail, is widely distributed in tropical and subtropical zones and plays a significant role as an intermediate host for trematode parasites. Various genetic markers have been used for species identification and phylogenetic studies of this snail. However, there are limited studies about their molecular genetics based on nuclear ribosomal DNA (rDNA) genes. A genetic analysis of I. exustus in Thailand was conducted based on the nuclear 18S rDNA (339 bp) and 28S rDNA (1036 bp) genes. Indoplanorbis snails were collected from 29 localities in 21 provinces covering six regions of Thailand. Nucleotide sequences from 44 snails together with sequences from the GenBank database were examined for phylogenetic relationships and genetic diversity. All sequences of the selected nucleotide regions exhibited a high level of similarity (99%) to the sequences of I. exustus in the GenBank database. The maximum likelihood tree based on the 18S and 28S rDNA fragment sequences of I. exustus in Thailand revealed only one group with clear separation from another genus in the family Planorbidae. The I. exustus 28S rDNA sequences showed intraspecific genetic divergence ranging from 0 to 0.78% and were classified into 8 different haplotypes. Conversely, the 18S rDNA data showed lower variation than the 28S rDNA data and revealed a single haplotype and intraspecific distances of zero among all sampled individuals. The haplotype network of 28S rDNA sequences of I. exustus in Thailand revealed six unique haplotypes and two haplotypes shared by at least two regions. Overall, both markers were successful in the identification of I. exustus. However, these markers, particularly the 18S rDNA, may not be suitable for genetic analysis within the species, particularly for population genetic studies, due to their limited variation as seen in this study. In summary, this study not only enhances understanding of genetic variation in I. exustus but is also useful for the selection of molecular markers in future genetic research.

Genome mining reveals novel biosynthetic gene clusters in entomopathogenic bacteria.

The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.

Identification and genetic characterization of Angiostrongylus cantonensis isolated from the human eye.

Angiostrongylus cantonensis, or the rat lungworm, is the causative agent of human angiostrongyliasis associated with eosinophilic meningitis or meningoencephalitis. Additionally, this nematode can cause ocular angiostrongyliasis, though this is rare. The worm can cause permanent damage to the affected eye and sometimes even blindness. Genetic characterization of the worm from clinical samples is limited. In the present study, we investigated the genetics of A. cantonensis recovered from a patient's eye in Thailand. We sequenced two mitochondrial genes (cytochrome c oxidase subunit I, or COI, and cytochrome b, or cytb) and nuclear gene regions (66-kDa protein and internal transcribed spacer 2, or ITS2) from a fifth-stage larva of Angiostrongylus sample that was surgically removed from the human eye. All sequences of the selected nucleotide regions were highly similar (98-100%) to the sequences of A. cantonensis in the GenBank database. The maximum likelihood and neighbor-joining trees of the COI gene indicated that A. cantonensis was closely related to the AC4 haplotype, whereas the cytb and 66-kDa protein genes were closely clustered with the AC6 and Ac66-1 haplotypes, respectively. In addition, the phylogeny of the concatenated nucleotide datasets of the COI and cytb revealed that the worm was closely related to the Thai strain and strains from other countries. This study confirms the identification and genetic variation of the fifth-stage larvae of A. cantonensis recovered from a patient's eye in Thailand. Our findings are important for future research on the genetic variation of A. cantonensis that causes human angiostrongyliasis.

Antibacterial activity of vB_AbaM_PhT2 phage hydrophobic amino acid fusion endolysin, combined with colistin against Acinetobacter baumannii.

Phage lytic enzymes are promising antimicrobial agents. In this study, an endolysin derived from vB_AbaM_PhT2 (vPhT2), was identified. This endolysin represented the conserved lysozyme domain. Recombinant endolysin (lysAB- vT2) and hydrophobic fusion endolysin (lysAB-vT2-fusion) were expressed and purified. Both endolysins showed lytic activity against bacterial crude cell wall of Gram-negative bacteria. The MIC of lysAB-vT2-fusion was 2 mg/ml corresponding to 100 µM, while the MIC of lysAB-vT2 was more than 10 mg/ml (400 µM). Combination of lysAB-vT2-fusion with colistin, polymyxin B or copper was synergistic against A. baumannii (FICI value as 0.25). Antibacterial activity of lysAB-vT2-fusion plus colistin at the fractional inhibitory concentrations (FICs) revealed that it can inhibit Escherichia coli, Klebsiella pneumoniae and various strains of extremely drug-resistant A. baumannii (XDRAB) and phage resistant A. baumannii. The lysAB- vT2-fusion still retained its antibacterial activity after incubating the enzyme at 4, 20, 40 and 60 °C for 30 min. The lysAB-vT2-fusion could inhibit the mature biofilm, and incubation of lysAB-vT2-fusion with T24 human cells infected with A. baumannii led to a partial reduction of LDH release from T24 cells. In summary, our study highlights the antimicrobial ability of engineered lysAB-vT2-fusion endolysin, which can be applied for the control of A. baumannii infection.

Entomopathogenic nematodes isolated from agricultural areas of Thailand and their activity against the larvae of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae).

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have been considered to be effective biological control agents for several insects. In this study, we isolated and identified EPNs from soil samples in agricultural areas of northern Thailand and evaluated their efficacy for controlling larvae of three mosquito vector species, Aedes aegypti, Ae. albopictus and Culex quinquefasciatus. A total of 51 of 1,000 soil samples were positive (5.1% prevalence) for EPNs, which were identified through sequencing of the rDNA and ITS to 37 Steinernema isolates (3.7%) and 14 Heterorhabditis isolates (1.4%). For the bioassay, the larvae of mosquitoes were exposed to Steinernema surkhetense (eALN6.3_TH), Steinernema lamjungense (eALN11.5_TH), Heterorhabditis indica (eACM14.2_TH) and Heterorhabditis bacteriophora (eALN18.2_TH). Heterorhabditis bacteriophora showed the highest efficacy against Ae. aegypti and Cx. quinquefasciatus. At 96 h after exposure, the mortality rates were 60.0 and 91.7%, respectively. The EPNs were observed in the dead mosquito larvae, which were mostly found in the thorax followed by the head and abdomen. Some EPNs were dead with melanization, and some were able to survive in the cavity of mosquito larvae. Our results show the low prevalence of EPN in agricultural areas of Thailand. Moreover, H. bacteriophora may be considered an alternative biocontrol agent for managing and controlling these vector mosquitoes.

Genetic analysis of a 66-kDa protein-encoding gene of Angiostrongylus cantonensis and Angiostrongylus malaysiensis.

The rat lungworm Angiostrongylus cantonensis is globally known to be the cause of oeosinophilic meningitis in humans. Another congener, Angiostrongylus malaysiensis, is closely related to A. cantonensis and has been described as a potential human pathogenic parasite. These 2 worms are similar in terms of life cycle, host range and morphological and genetic information. However, there are limited studies about their genetic diversity based on the 66-kDa protein-encoding gene. The objective of this study was to explore the 66-kDa protein sequence variation of A. cantonensis and A. malaysiensis collected from Thailand. Two adult and 53 third-stage larval specimens of Angiostrongylus from 4 geographic locations in Thailand were molecularly identified using the 66-kDa protein gene. The phylogenetic trees (Bayesian inference tree and maximum-likelihood tree) showed that Angiostrongylus formed a monophyletic clade with a clear separation between A. cantonensis and A. malaysiensis. The genetic distance between A. cantonensis and A. malaysiensis varies from 0.82 to 2.86%, with a total of 16 variable sites. The analysis of genetic diversity revealed 1 and 5 new haplotypes of A. cantonensis and A. malaysiensis, respectively, and showed genetic differences between the populations of A. cantonensis and A. malaysiensis. The haplotype networks of A. cantonensis and A. malaysiensis populations in Thailand are similar to those of populations in some countries, indicating the range expansion of genomic origin between populations in different areas. In conclusion, the 66-kDa protein gene was a good genetic marker for studying genetic diversity and discriminating between A. cantonensis and A. malaysiensis.

Entomopathogenic Nematodes and Their Symbiotic Bacteria from the National Parks of Thailand and Larvicidal Property of Symbiotic Bacteria against Aedes aegypti and Culex quinquefasciatus.

Entomopathogenic nematodes (EPNs) are insect parasitic nematodes of the genera Het-erorhabditis and Steinernema. These nematodes are symbiotically associated with the bacteria, Photorhabdus and Xenorhabdus, respectively. National parks in Thailand are a potentially rich resource for recovering native EPNs and their symbiotic bacteria. The objectives of this study are to isolate and identify EPNs and their bacterial flora from soil samples in four national parks in Thailand and to evaluate their efficacy for controlling mosquito larvae. Using a baiting method with a Galleria mellonella moth larvae and a White trap technique, 80 out of 840 soil samples (9.5%) from 168 field sites were positive for EPNs. Sequencing of an internal transcribed spacer resulted in the molecular identification of Heterorhabditis nematode isolates as H. indica, H. baujardi and Heterorhabditis SGmg3, while using 28S rDNA sequencing, Steinernema nematode species were identified as S. guang-dongense, S. surkhetense, S. minutum, S. longicaudum and one closely related to S. yirgalemense. For the symbiotic bacterial isolates, based on recA sequencing, the Photorhabdus spp. were identified as P. luminescens subsp. akhurstii, P. luminescens subsp. hainanensis and P. luminescens subsp. australis. Xenorhabdus isolates were identified as X. stockiae, X. indica, X. griffiniae, X. japonica and X. hominickii. Results of bioassays demonstrate that Photorhabdus isolates were effective on both Aedes aegypti and Culex quinquefasciatus. Therefore, we conclude that soil from Thailand's national parks contain a high diversity of entomopathogenic nematodes and their symbiotic bacteria. Photorhabdus bacteria are larvicidal against culicine mosquitoes and may serve as effective biocontrol agents.

Genome analysis of secondary metabolite­biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Larvicidal activity of Photorhabdus and Xenorhabdus bacteria isolated from insect parasitic nematodes against Aedes aegypti and Aedes albopictus.

Aedes aegypti and Aedes albopictus are important vectors for several arboviruses such as the dengue virus. The chemical control of Aedes spp., which is usually implemented, affects both humans and the environment. The biological control of Aedes spp. with entomopathogenic bacteria such as Photorhabdus and Xenorhabdus may be an alternative method that can overcome such issues. This study aimed to isolate and identify Photorhabdus and Xenorhabdus bacteria from entomopathogenic nematodes (EPNs) collected in Thailand and evaluate their larvicidal properties in controlling A. aegypti and A. albopictus. Colony morphology and recA sequencing of the 118 symbiotic isolated bacteria indicated that most were P. luminescens subsp. akhurstii and X. stockiae with minor prevalence of P. luminescens subsp. hainanensis, P. asymbiotica subsp. australis, X. indica, X. griffiniae, X. japonica, X. thuongxuanensis, and X. eapokensis. The larvicidal bioassay with the third- and fourth-instar mosquito larvae suggested that a whole-cell suspension of X. griffiniae (bMSN3.3_TH) had the highest efficiency in eradicating A. aegypti and A. albopictus, with 90 ± 3.71% and 81 ± 2.13% mortality, respectively, after 96 h exposure. In contrast, 1% of ethyl acetate extracted from X. indica (bSNK8.5_TH) showed reduced mortality for A. aegypti of only 50 ± 3.66% after 96 h exposure. The results indicate that both X. griffiniae (bMSN3.3_TH) and X. indica (bSNK8.5_TH) could be used as biocontrol agents against Aedes larvae.

Prevalence and genetic analysis of Enterobius vermicularis in schoolchildren in lower northern Thailand.

Enterobius vermicularis, a nematode parasite with a global distribution causes enterobiasis in schoolchildren and is considered a neglected parasite. An understanding of the prevalence and genetic diversity of enterobiasis is crucial for appropriate control measures. Therefore, the objectives of this research were to study the prevalence and genetic diversity of E. vermicularis in schoolchildren from lower northern Thailand, based on cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) sequences. Using the scotch tape technique, 7.4% (188/2544) of schoolchildren from 21 primary schools were found positive for E. vermicularis eggs, which is a relatively low infection rate. Phylogenetic trees of partial COI sequences (397 bp) revealed similar topologies using maximum likelihood (ML) and neighbor-joining (NJ) methods and identified E. vermicularis type A (105 sequences) and B (1 sequence). Haplotype network analysis of the COI sequences demonstrated a high haplotype diversity (Hd = 0.9028). In contrast, phylogenetic analysts of a 343 bp region of the ITS2 locus (52 sequences) revealed a monophyletic group. More sequence analyses of E. vermicularis from humans and other hosts in Thailand are necessary to better understand the genetic diversity of this parasite.

Preliminary Survey of Larval Trematodes in Freshwater Snails of Phitsanulok Province in Lower Northern Thailand.

Background: Freshwater snails serve as intermediate hosts for a variety of trematodes that cause illness in the human and animal populations. Several species of freshwater snails in Thailand have been found to have larval trematode infections. We aimed to investigate a freshwater snail in Phitsanulok Province and report on its current status of larval trematode infection. Methods: Freshwater snails were collected from six localities (rice field and irrigation canal) by handpicking and using a count per unit of time sampling approach. The snails were identified by their external shell morphology. The shedding method was applied to observe the cercariae, which were photographed under a light microscope to determine their morphological types. Results: A total of 211 snails were classified into seven genera. The most abundant snail species was Lymnaea sp., representing 31.3% of the sample, followed by Physella sp., Bithynia sp., Pomacea canaliculata, Filopaludina martensi, Indoplanorbis exustus, and Melanoides tuberculata, in that order. From the sample, 21 snails (9.95%), including Bithynia sp., Lymnaea sp., I. exustus, and M. tuberculata, were infected with cercarial trematodes, which could be categorized into four types, namely amphistome, parapleurolophocercous, echinostome, and xiphidiocercaria. Amphistome emerged from Bithynia sp., and I. exustus was the most common cercaria to be recovered, representing 80.9% of all infected snails. Conclusion: This study presents the current prevalence of cercariae in infected snails within the studied area. It is important to manage intermediate host snails in order to restrict trematode life cycle completion.

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 (bla OXA-51, bla ADC-25, bla OXA-23, bla TEM-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.

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria.

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes generate natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the XP biosynthetic gene clusters (BGCs) has limited the understanding of how natural products affect interactions between the organisms. Here we combine pangenome and sequence similarity networks to analyse BGCs from 45 XP strains that cover all sequenced strains in our collection and represent almost all XP taxonomy. The identified 1,000 BGCs belong to 176 families. The most conserved families are denoted by 11 BGC classes. We homologously (over)express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, virulence factors against insects, metallophores and insect immunosuppressants. These findings explain the functional basis of bacterial natural products in this tripartite relationship.

Comparative genome analysis of Escherichia coli bacteriophages isolated from sewage and chicken meat.

Escherichia coli, a bacterium that causes severe foodborne diseases, is transmitted to humans primarily through the consumption of contaminated foods. These foodborne pathogens are causing a public health problem that requires alternative control approaches, such as bacteriophage (phage) biocontrol. In this study, we characterized vB_EcoM_Tw01 (vTw01) isolated from sewage and vB_EcoM_Tcm05 (vTcm05) isolated from chicken meat. Both vTw01 and vTcm05 were assigned to the family Myoviridae based on their morphology, with the former exhibiting a narrow host range with low minimum inhibitory multiplicity of infection (miMOI), and the latter exhibiting a broad host range with high miMOI. The latent periods of these phages were 20 and 30 min for vTw01 and vTcm05, while the burst sizes were ∼140 and ∼300 PFU/cell, respectively. They were relatively stable over a wide range of pH values and temperatures. The bioinformatics analysis of the genomic sequence suggests that vTw01 and vTcm05 have double-stranded DNA with genome sizes of 170,107 bp and 149,059 bp, respectively. Bacteriophage encoded enzymes, such as tail-lysozyme, spanin Rz, holin, cell wall hydrolase (CWH), and endolysin, were identified in the genome of both phages. In conclusion, this study investigated the morphological, physiological, and genomic features of two E. coli phages isolated from different sources. It was confirmed that these phages and their enzymes can serve as potential candidates for phage biocontrol.

Molecular identification and phylogeny of Steinernema and Heterorhabditis nematodes and their efficacy in controlling the larvae of Aedes aegypti, a major vector of the dengue virus.

Aedes aegypti is the mosquito vector of several arboviruses, especially the dengue virus. Aedes aegypti strain resistant to chemical insecticides have been reported worldwide. To tackle this, an entomopathogenic nematode (EPN) may be an alternative bio-control agent. To this end, this study aims to isolate, identify, and analyze the phylogeny of EPNs in Thailand and evaluate their efficacy for controlling the Ae. aegypti larvae. From 12 provinces in Thailand, soil samples were randomly collected, with 118 out of 1,100 them being positive for EPNs (10.73% prevalence) in genera Steinernema (4.46%) and Heterorhabditis (6.27%). Then, molecular discrimination of these two genus was performed based on the sequencing and phylogenetic analysis of the 28S rDNA and internal transcribed spacer regions. The most abundant species of EPN were Heterorhabditis indica, with minor species of Heterorhabditis sp. SGmg3, H. baujardi, S. surkhetense, S. kushidai, S. siamkayai, Steinernema sp. YNd80, Steinernema sp. YNc215, S. guangdongense, and S. huense. The larvicidal activity of five selected EPN isolates were tested against Ae. aegypti. Ten larvae of Ae. aegypti were incubated with different concentration (80, 160, 320, and 640 IJs/larva) of the infective juveniles of EPN in a 24-well and 6-well plates for 4 days. The mortality rates of the larvae were observed daily. Steinernema surkhetense (ePYO8.5_TH) showed the potential to kill mosquito larvae, with the highest mortality rate of 92 ± 9.37% and 89 ± 9.91% after it was treated with 640 IJs/larva in a 24-well plate and 1600 IJs/larva in a 6-well plate, respectively. There is an abundant distribution of EPNs across the country, and S. surkhetense ePYO8.5_TH may be used as a biocontrol agent against Ae. aegypti larvae.

Population Genetics Analysis of a Pomacea Snail (Gastropoda: Ampullariidae) in Thailand and its Low Infection by Angiostrongylus cantonensis.

Pomacea is a freshwater snail in family Ampullariidae that is native to South and Central America. This snail is among the more important intermediate hosts for Angiostrongylus cantonensis and agricultural pests. Herein, we investigated the prevalence of A. cantonensis larvae and the genetic diversity of Pomacea samples collected across Thailand based on mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. The larval-infection rate was 1.7% in Pomacea canaliculata specimens collected from the Uttaradit Province of northern Thailand. We randomly selected specimens of P. canaliculata and P. maculata for genetic analysis. We analyzed 244 COI sequences, including 49 sequences from samples collected from Thailand and a publicly accessible database of snails in their native and non-native ranges. A maximum-likelihood tree of P. canaliculata and P. maculata revealed two main clades. The genetic diversity analysis identified seven P. canaliculata haplotypes and six P. maculata haplotypes, and showed genetic differences between the populations of P. canaliculata and P. maculata. The haplotype networks of P. canaliculata and P. maculata populations in Thailand are similar to those of populations in multiple countries, indicating that this species spread widely to many parts of the world.

A study on Xenorhabdus and Photorhabdus isolates from Northeastern Thailand: Identification, antibacterial activity, and association with entomopathogenic nematode hosts.

Xenorhabdus and Photorhabdus are gram negative bacteria that can produce several secondary metabolites, including antimicrobial compounds. They have a symbiotic association with entomopathogenic nematodes (EPNs). The aim of this study was to isolate and identify Xenorhabdus and Photorhabdus species and their associated nematode symbionts from Northeastern region of Thailand. We also evaluated the antibacterial activity of these symbiotic bacteria. The recovery rate of EPNs was 7.82% (113/1445). A total of 62 Xenorhabdus and 51 Photorhabdus strains were isolated from the EPNs. Based on recA sequencing and phylogeny, Xenorhabdus isolates were identified as X. stockiae (n = 60), X. indica (n = 1) and X. eapokensis (n = 1). Photorhabdus isolates were identified as P. luminescens subsp. akhurstii (n = 29), P. luminescens subsp. hainanensis (n = 18), P. luminescens subsp. laumondii (n = 2), and P. asymbiotica subsp. australis (n = 2). The EPNs based on 28S rDNA and internal transcribed spacer (ITS) analysis were identified as Steinernema surkhetense (n = 35), S. sangi (n = 1), unidentified Steinernema (n = 1), Heterorhabditis indica (n = 39), H. baujardi (n = 1), and Heterorhabditis sp. SGmg3 (n = 3). Antibacterial activity showed that X. stockiae (bMSK7.5_TH) extract inhibited several antibiotic-resistant bacterial strains. To the best of our knowledge, this is the first report on mutualistic association between P. luminescens subsp. laumondii and Heterorhabditis sp. SGmg3. This study could act as a platform for future studies focusing on the discovery of novel antimicrobial compounds from these bacterial isolates.