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Witawat Tunyong,1 Weewan Arsheewa,2 Sirijan Santajit,3,4 Thida Kong-ngoen,1 Pornpan Pumirat,1 Nitat Sookrung,5,6 Wanpen Chaicumpa,6 Nitaya Indrawattana1 1Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand; 2Department of Microbiology, Phrapokklao Hospital, Chanthaburi, 22000, Thailand; 3School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, 80161, Thailand; 4Research Excellence Center for Innovation and Health Products, Walailak University, Nakhon Si Thammarat, 80161, Thailand; 5Biomedical Research Incubation Unit, Department of Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; 6Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok, 10700, ThailandCorrespondence: Nitaya IndrawattanaDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, ThailandTel +66 2 354 9100 Ext. 1598Fax +66 2 643 5598Email nitaya.ind@mahidol.ac.thBackground: The global spread of carbapenem-resistant Enterobacterales (CRE) inflicts a severe threat to human health. The CRE infections have resulted in an increased mortality rate in hospitals and other health-care settings worldwide. In this study, the antibiotic-resistance pattern and prevalence of carbapenemase-encoding genes among CRE isolated from patients of one hospital in Thailand were investigated.Methods: By using conventional biochemical tests, we identified and isolated all species of Enterobacterales from the clinical samples kept at Prapokklao Hospital, Chanthaburi, Thailand, which were collected during 2016– 2017. Multidrug-resistant (MDR) bacteria were determined by disc diffusion method and minimum inhibitory concentration (MIC) test strips. Carbapenemase genes were detected by PCR and confirmed by Sanger sequencing.Results: Klebsiella pneumoniae complex, Escherichia coli, and Enterobacter spp. were isolated from the specimens. Of 9,564 isolated Enterobacterales, 282 were multidrug-resistance (MDR). The MIC test strips revealed that the MDR CRE were resistant to ertapenem (92.9%) and meropenem (81.3%). All these isolates carried carbapenemase-coding genes, including blaNDM (90%) and blaIMP (71%), the two most commonly found genes among CRE strains. There were 39.2% of the isolates that carried a combination of blaNDM-blaIMP and 22.6% carried combined blaNDM-blaIMP-blaOXA-48-like genes.Conclusion: This study demonstrates a significantly high prevalence of CRE isolates with the MDR phenotypes. A minority of the isolates carried a single carbapenem-resistant gene, while the majority harbored multiple genes in combination. Regular monitoring of MDR CRE and characterization of their drug resistance are important for guiding treatment, intervention and control of the CRE spread and outbreak in a health-care setting.Keywords: carbapenemase, CRE, drug resistant, Enterobacterales, nosocomial infections

Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd), PFGE types, accessory gene regulator (agr) groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1-7 (56 isolates) were methicillin resistant (MRSA) and 8-10 (36 isolates) were methicillin sensitive (MSSA). One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq), and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70%) but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant. [ABSTRACT FROM AUTHOR]

Carbapenemase-producing Enterobacterales (CPE) isolates increasingly carry oxacillinase-48 (OXA-48)-like enzymes encoded by bla OXA-48-like , which can confer high levels of carbapenem resistance. This aims to determine the prevalence of CPE and genetic variation among bla OXA-48-like -carrying isolates recovered from Prapokklao Hospital in Chanthaburi Province, Thailand in 2016-2017. In total, 122 carbapenem-resistant Enterobacterales (CRE) isolates were recovered from clinical samples. CRE were evaluated using standard biochemical tests and MIC test strips. Carbapenemase production was assessed through the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and EDTA-modified carbapenem inactivation method (eCIM). Detection of bla OXA-48-like mutations was conducted via PCR and confirmed by Sanger sequencing. Among these CRE isolates, 72 (59.02%), 44 (36.07%), 3 (2.46%), and 3 (2.46%) were Klebsiella pneumoniae , Escherichia coli , Enterobacter aerogenes , and Enterobacter cloacae , respectively. The MHT identified carbapenemase production in 108 isolates (88.52%). Based on the mCIM, 81 isolates (66.39%) were carbapenemase producers. Seventy-three isolates (59.84%) were eCIM-positive, indicating metallo-β-lactamase production. Three distinct genetic variants of the bla OXA-48-like gene were identified among the isolates, including the wild-type and two point mutation types harboring the mutations E168Q and S171A (mutation type 1) and E168Q, S171A, and R214S (mutation type 2). Multiple-sequence alignment and in silico analysis revealed variation of R214 located in the β5-β6 loop. This study identified bla OXA-48-like point mutation groups and carbapenemase production, predominantly metallo-β-lactamases, among CRE isolates, especially K. pneumoniae and E. coli . These findings highlight the importance of implementing stringent infection control measures and active antimicrobial resistance surveillance to combat the spread of difficult-to-treat, metallo-β-lactamase-producing CRE in healthcare settings., Importance: In this study, we aimed to investigate genetic variation and CPE among bla OXA-48-like carrying isolates recovered from Prapokklao Hospital, Chanthaburi Province, Thailand, during 2016-2017. A total of 122 carbapenem-resistant Enterobacterales (CRE) were recovered from clinical samples in Prapokklao Hospital. All CRE samples were confirmed by standard biochemical tests and minimum inhibitory concentration (MIC) test strips (E-test). The carbapenemase production was determined using the modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM), and EDTA-CIM (eCIM). Three single mutations (E168Q, S171A, and R214S) were characterized in this study. This mutation might reflect the hydrolysis of the modified β-lactam spectrum, especially carbapenem, by OXA-48-like. Our report provides evidence of the bla OXA-48-like point mutation and carbapenemase-producing phenotype of CRE detected in this healthcare setting. Effective control measures and active surveillance of drug resistance in nosocomial pathogens are crucial for controlling diseases associated with difficult-to-treat bacteria.

The quorum sensing (QS) system mediated by the abaI gene in Acinetobacter baumannii is crucial for various physiological and pathogenic processes. In this study, we constructed a stable markerless abaI knockout mutant (ΔabaI) strain using a pEXKm5-based allele replacement method to investigate the impact of abaI on A. baumannii. Proteomic analysis revealed significant alterations in protein expression between the wild type (WT) and ΔabaI mutant strains, particularly in proteins associated with membrane structure, antibiotic resistance, and virulence. Notably, the downregulation of key outer membrane proteins such as SurA, OmpA, OmpW, and BamA suggests potential vulnerabilities in outer membrane integrity, which correlate with structural abnormalities in the ΔabaI mutant strain, including irregular cell shapes and compromised membrane integrity, observed by scanning and transmission electron microscopy. Furthermore, diminished expression of regulatory proteins such as OmpR and GacA-GacS highlights the broader regulatory networks affected by abaI deletion. Functional assays revealed impaired biofilm formation and surface-associated motility in the mutant strain, indicative of altered colonization capabilities. Interestingly, the mutant showed a complex antibiotic susceptibility profile. While it demonstrated increased susceptibility to membrane-targeting antibiotics, its response to beta-lactams was more nuanced. Despite increased expression of metallo-beta-lactamase (MBL) superfamily proteins and DcaP-like protein, the mutant unexpectedly showed lower MICs for carbapenems (imipenem and meropenem) compared to the wild-type strain. This suggests that abaI deletion affects antibiotic susceptibility through multiple, potentially competing mechanisms. Further investigation is needed to fully elucidate the interplay between quorum sensing, antibiotic resistance genes, and overall antibiotic susceptibility in A. baumannii. Our findings underscore the multifaceted role of the abaI gene in modulating various cellular processes and highlight its significance in A. baumannii physiology, pathogenesis, and antibiotic resistance. Targeting the abaI QS system may offer novel therapeutic strategies for this clinically significant pathogen., (© 2024. The Author(s).)

Antimicrobial resistance poses a global health threat, with Staphylococcus aureus emerging as a notorious pathogen capable of forming stubborn biofilms and regulating virulence through quorum sensing (QS). In the quest for novel therapeutic strategies, this groundbreaking study unveils the therapeutic potential of Paederia foetida Linn., an Asian medicinal plant containing various bioactive compounds, contributing to its antimicrobial activities, in the battle against S. aureus . Through a comprehensive approach, we investigated the effect of ethanolic P. foetida leaf extract on S. aureus biofilms, QS, and antimicrobial activity. The extract exhibited promising inhibitory effects against S. aureus including the biofilm-forming strain and MRSA. Real-time PCR analysis revealed significant downregulation of key virulence and biofilm genes, suggesting interference with QS. Biofilm assays quantified the extract's ability to disrupt and prevent biofilm formation. LC-MS/MS analysis identified quercetin and kaempferol glycosides as potential bioactive constituents, while molecular docking studies explored their binding to the QS transcriptional regulator SarA. Computational ADMET predictions highlighted favorable intestinal absorption but potential P-glycoprotein interactions limiting oral bioavailability. While promising anti-virulence effects were demonstrated, the high molecular weights and excessive hydrogen bond donors/acceptors of the flavonoid glycosides raise concerns regarding drug-likeness and permeability. This integrated study offers valuable insights for developing novel anti-virulence strategies to combat antimicrobial resistance.

Background and Aim: Staphylococci, which inhabit skin and mucous membranes in humans and animals, are opportunistic pathogens. Coagulase-positive and coagulase-negative staphylococci (CoNS) are the two main groups. Clinical abscesses in rabbits often harbor Staphylococcus aureus and CoNS. This study estimated S. aureus and CoNS prevalence, resistance profiles, antimicrobial-resistant genes, and the accessory gene regulator ( agr ) group in rabbit clinical abscesses., Materials and Methods: Sixty-seven abscesses were gathered from 67 rabbits who visited Prasu-Arthorn Animal Hospital in Nakornpathom, Thailand, from January 2014 to October 2015. Thirty-four subcutaneous, 29 dental, 2 ocular, 1 mammary gland, and 1 uterine abscess were present. Conventional methods, including Gram staining, mannitol fermentation, hemolysis on blood agar, catalase testing, and coagulase production, identified and isolated S. aureus and CoNS from all abscesses. All S. aureus and CoNS isolates underwent antimicrobial susceptibility testing using the disk diffusion method. Polymerase chain reaction was used to detect the presence of bla Z, aac A- aph D, msr A, tet K, gyr A, grl A, dfr G, and cfr antimicrobial-resistant genes. Methicillin resistance was identified through the detection of a cefoxitin-resistant phenotype and the presence of mec A gene. Further investigation was conducted on the agr group of S. aureus isolates., Results: In 67 abscesses, we found 19 S. aureus isolates in 9 abscesses (13.43%) and 37 CoNS isolates in 18 abscesses (26.87%), both majorly located at subcutaneous sites. About 59.46% of CoNS isolates were methicillin-resistant compared to 5.26% of S. aureus isolates. Methicillin-resistant S. aureus (MRSA) and methicillin-resistant CoNS (MRCoNS) both displayed multidrug resistance (MDR). Both MRSA and MRCoNS expressed multiple antimicrobial resistance genes, including bla Z, aac A- aph D, gyr A, grl A, msr A, tet K, and dfr G. Approximately 73.68% of the agr groups were agr I, 15.79% were agr III, and 10.53% were agr II., Conclusion: This study found a high prevalence of MRCoNS with antimicrobial resistance and multiple antimicrobial-resistant genes in rabbits with clinical abscesses. The effectiveness of antibiotics against infections caused by such strains is a matter of concern. Owners should be educated about the importance of good hygiene practices and judicious antibiotic use to prevent widespread antimicrobial resistance., Competing Interests: The authors declare that they do not have any competing interests., (Copyright: © Suriyakhun, et al.)

The Gulf of Thailand provides many services to the Thai population, and human activities may influence the diversity of microorganisms in the seawater. Information of the microorganisms' profile which inhabit the coastline can be used to monitor the water quality. This study aimed to investigate the bacterial community in the waters along the coastline provinces, including Rayong, Chonburi, Prachuap Kiri Khan, and Nakhon Sri Thammarat. Seawater samples were collected at each site, and the conductivity, pH, salinity, temperature, and turbidity were measured. The samples were subjected to whole DNA extraction, 16S rRNA amplification, next-generation sequencing, and statistical analysis to identify the bacterial diversity and analyse the effects of water parameters on the bacterial community. The dominant bacterial phyla found were Proteobacteria, Bacteroidota, and Cyanobacteria. Spearman rank correlation analysis revealed a high correlation of Pseudoalteromonas , the NS5 marine group, Lachnospiraceae , Marinobacterium , Mariviven , and Vibrio with the seawater parameters. The predatory bacteria Peredibacter and Halobacteriovorax were reported among these bacterial communities for the first time in the Gulf of Thailand. Interestingly, Akkermansia , a novel candidate for next-generation probiotics to improve human health, was also found in the sample from Nakhon Sri Thammarat Province. Although the rich-ness and diversity of the bacterial communities differed among sampling sites, it is a possible source of many valuable bacteria for future use., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) emphasises the urgent need for novel antimicrobial agents as alternatives to antibiotics. Bacteriophage therapy is one of the most promising antimicrobial strategies. Here, we isolated and comprehensively characterized a novel Staphylococcus phage, vB_SauM_VL10 (VL10), from urban sewage. The VL10 genome displays 141,746 bp of linear double-stranded DNA, containing 193 open reading frames and lacking tRNA, virulence, or antibiotic resistance genes. Phylogenetic analysis categorizes VL10 as a novel species within the Silviavirus genus, Twortvirinae subfamily. VL10 exhibits lytic behaviour characterized by efficient adsorption, a short latent period, and substantial burst size, with environmental stability. It demonstrates lytic activity against 79.06% of tested S. aureus strains, highlighting its species specificity. Additionally, VL10 effectively targets MRSA biofilms, reducing biomass and viable cells. In MRSA-infected G. mellonella larvae, VL10 enhances survival rates, supporting its potential for phage therapy applications. Moreover, the emergence of VL10-resistant S. aureus strains associated with fitness trade-offs, including reduced growth, biofilm formation, and virulence. Altogether, these findings emphasize VL10 as a promising candidate for developing therapeutic agents against MRSA infections, providing insights into phage biology and resistance dynamics., (© 2024. The Author(s).)

This study investigated the prevalence and antimicrobial resistance (AMR) of Escherichia coli (E. coli) in Nile tilapia from fresh markets and supermarkets. A total of samples (n = 828) were collected from Nile tilapia including fish flesh (n = 276), liver and kidney (n = 276), and intestine (n = 276). Overall prevalence of fecal coliforms (61.6%) and E. coli (53.0%) were observed. High prevalence of E. coli was found in the intestine (71.4%), followed by the liver and kidney (45.7%). The highest prevalence of resistance was commonly found against tetracycline (78.5%), ampicillin (72.8%), and sulfamethoxazole (45.6%) with resistance to only tetracycline (15.2%) as the most common antibiogram. The prevalence of multidrug resistance (MDR) (54.4%) and Extended-spectrum beta-lactamases (ESBLs) (5.7%) were examined. The predominant virulence genes (n = 158) were st (14.6%), followed by eaeA (0.6%). The blaTEM (73.4%), tetA (65.2%), and qnrS (57.6%). There is statistical significance between Nile tilapia from fresh markets and supermarkets. Based on logistic regression analysis, ampicillin-resistant E. coli was statistically associated with the phenotypic resistance to tetracycline and trimethoprim, and the presence of blaTEM and tetA (p < 0.05). Further investigation of AMR transference and their mechanisms is needed for AMR control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Central nervous system (CNS) melioidosis caused by Burkholderia pseudomallei is being increasingly reported. Because of the high mortality associated with CNS melioidosis, understanding the underlying mechanism of B. pseudomallei pathogenesis in the CNS needs to be intensively investigated to develop better therapeutic strategies against this deadly disease. The type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells to benefit the infection process. In this study, the role of the T6SS accessory protein TagAB-5 in B. pseudomallei pathogenicity was examined using the human microglial cell line HCM3, a unique resident immune cell of the CNS acting as a primary mediator of inflammation. We constructed B. pseudomallei tagAB-5 mutant and complementary strains by the markerless allele replacement method. The effects of tagAB-5 deletion on the pathogenicity of B. pseudomallei were studied by bacterial infection assays of HCM3 cells. Compared with the wild type, the tagAB-5 mutant exhibited defective pathogenic abilities in intracellular replication, multinucleated giant cell formation, and induction of cell damage. Additionally, infection by the tagAB-5 mutant elicited a decreased production of interleukin 8 (IL-8) in HCM3, suggesting that efficient pathogenicity of B. pseudomallei is required for IL-8 production in microglia. However, no significant differences in virulence in the Galleria mellonella model were observed between the tagAB-5 mutant and the wild type. Taken together, this study indicated that microglia might be an important intracellular niche for B. pseudomallei, particularly in CNS infection, and TagAB-5 confers B. pseudomallei pathogenicity in these cells.

The bacterial pathogen Burkholderia pseudomallei causes human melioidosis, which can infect the brain, leading to encephalitis and brain abscesses. Infection of the nervous system is a rare condition but is associated with an increased risk of mortality. Burkholderia intracellular motility A (BimA) was reported to play an important role in the invasion and infection of the central nervous system in a mouse model. Thus, to gain insight of the cellular mechanisms underlying the pathogenesis of neurological melioidosis, we explored the human neuronal proteomics to identify the host factors that are up- and downregulated during Burkholderia infection. When infected the SH-SY5Y cells with B. pseudomallei K96243 wild-type (WT), 194 host proteins showed a fold change of >2 compared with uninfected cells. Moreover, 123 proteins showed a fold change of >2 when infected with a knockout bimA mutant (Δ bimA ) mutant compared with WT. The differentially expressed proteins were mainly associated with metabolic pathways and pathways linked to human diseases. Importantly, we observed the downregulation of proteins in the apoptosis and cytotoxicity pathway, and in vitro investigation with the Δ bimA mutant revealed the association of BimA with the induction of these pathways. Additionally, we disclosed that BimA was not required for invasion into the neuron cell line but was necessary for effective intracellular replication and multinucleated giant cell (MNGC) formation. These findings show the extraordinary capacity of B. pseudomallei in subverting and interfering with host cellular systems to establish infection and extend our understanding of B. pseudomallei BimA involvement in the pathogenesis of neurological melioidosis. IMPORTANCE Neurological melioidosis, caused by Burkholderia pseudomallei, can result in severe neurological damage and enhance the mortality rate of melioidosis patients. We investigate the involvement of the virulent factor BimA, which mediates actin-based motility, in the intracellular infection of neuroblastoma SH-SY5Y cells. Using proteomics-based analysis, we provide a list of host factors exploited by B. pseudomallei. The expression level of selected downregulated proteins in neuron cells infected with the Δ bimA mutant was determined by quantitative reverse transcription-PCR and was consistent with our proteomic data. The role of BimA in the apoptosis and cytotoxicity of SH-SY5Y cells infected by B. pseudomallei was uncovered in this study. Additionally, our research demonstrates that BimA is required for successful intracellular survival and cell fusion upon infection of neuron cells. Our findings have significant implications for understanding the pathogenesis of B. pseudomallei infections and developing novel therapeutic strategies to combat this deadly disease., Competing Interests: The authors declare no conflict of interest.

(1) Background: Acinetobacter baumannii is well known as a causative agent of severe hospital-acquired infections, especially in intensive care units. The present study characterised the genetic traits of biofilm-forming carbapenem-resistant A. baumannii (CRAB) clinical isolates. Additionally, this study determined the prevalence of biofilm-producing A. baumannii isolates from a tertiary care hospital and investigated the association of biofilms with the distribution of biofilm-related and antibiotic resistance-associated genotypes. (2) Methods: The 995 non-duplicate A. baumannii isolates were identified, and their susceptibilities to different antibiotics were determined using the disk diffusion method. Using the modified microtiter plate assay, the CRAB isolates were investigated for their biofilm formation ability. Hemolysin and protease activities were determined. CRABs were subjected to polymerase chain reaction (PCR) assays targeting bla VIM , bla NDM , bla IMP , bla OXA-23-like , bla OXA-24-like , bla OXA-51-like , csu E and pga B genes. Individual CRAB isolates were identified for their DNA fingerprint by repetitive element sequence-based (REP)-PCR. (3) Results: Among all A. baumannii isolates, 172 CRABs were identified. The major antibiotic resistance gene among the CRAB isolates was bla OXA-51-like (100%). Ninety-nine isolates (57.56%) were biofilm producers. The most prevalent biofilm gene was pga B (79.65%), followed by csu E (76.74%). Evidence of virulence phenotypes revealed that all CRAB exhibited proteolytic activity; however, only four isolates (2.33%) were positive for the hemolytic-producing phenotype. REP-PCR showed that 172 CRAB isolates can be divided into 36-DNA fingerprint patterns. (4) Conclusions: The predominance of biofilm-producing CRAB isolates identified in this study is concerning. The characterisation of risk factors could aid in controlling the continual selection and spreading of the A. baumannii phenotype in hospitals, thereby improving patient care quality.

Background and Aim: Domestic and wild animals are important reservoirs for antibiotic-resistant bacteria. This study aimed to isolate Escherichia coli from feces of domestic and wild animals at an agricultural land interface area of Salaphra Wildlife Sanctuary, Thailand, and study the phylogenic characteristics and antibiotic resistance in these isolates., Materials and Methods: In this cross-sectional, descriptive study, we randomly collected ground feces from free-ranging wild animals (deer and elephants) and domestic animals (cattle and goats). All fecal samples were inoculated onto MacConkey agar plates, and lactose-fermenting colonies were identified as E. coli . Antibiotic susceptibility of the E. coli isolates was determined using the disc diffusion method. Polymerase chain reaction assays were used to detect antibiotic resistance and virulence genes., Results: We obtained 362 E. coli isolates from the collected fecal samples. The E. coli isolates were categorized into four phylogenetic groups according to the virulence genes ( chuA , vjaA , and TspE4C2 ). Phylogenetic Group D was predominant in the deer (41.67%) and elephants (63.29%), whereas phylogenetic Group B1 was predominant in the cattle (62.31%), and phylogenetic Groups A (36.36%) and B2 (33.33%) were predominant in the goats. Antibiotic susceptibility testing revealed that most antibiotic-resistant E. coli were isolated from domestic goats (96.96%). Among the 362 E. coli isolates, 38 (10.5%) were resistant to at least one antibiotic, 21 (5.8%) were resistant to two antibiotics, and 6 (1.66%) were resistant to three or more antibiotics. Ampicillin (AMP) was the most common antibiotic (48.48%) to which the E. coli were resistant, followed by tetracycline (TET) (45.45%) and trimethoprim-sulfamethoxazole (3.03%). One isolate from an elephant was resistant to five antibiotics: AMP, amoxicillin, sulfisoxazole, TET, and ciprofloxacin. Determination of antibiotic resistance genes confirmed that E. coli isolates carried antibiotic resistance genes associated with phenotypic resistance to antibiotics. Most antibiotic-resistant E. coli belonged to phylogenic Groups A and B1, and most non-resistant E. coli belonged to phylogenic Groups B2 and D., Conclusion: Monitoring E. coli isolates from wild and domestic animals showed that all four phylogenic groups of E. coli have developed antibiotic resistance and are potential sources of multidrug resistance. High levels of antibiotic resistance have been linked to domestic animals. Our results support strengthening surveillance to monitor the emergence and effects of antibiotic-resistant microorganisms in animals., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Duangurai, et al.)

Background and Aim: Methicillin-resistant globally, Staphylococcus aureus (MRSA) is a major cause of disease in both humans and animals. Several studies have documented the presence of MRSA in healthy and infected animals. However, there is less information on MRSA occurrence in exotic pets, especially healthy rabbits. This study aimed to look into the antimicrobial resistance profile, hidden antimicrobial-resistant genes in isolated bacteria, and to estimate prevalence of MRSA in healthy rabbits., Materials and Methods: Two-hundreds and eighteen samples, including 42 eyes, 44 ears, 44 oral, 44 ventral thoracic, and 44 perineal swabs, were taken from 44 healthy rabbits that visited the Prasu-Arthorn Animal Hospital, in Nakornpathom, Thailand, from January 2015 to March 2016. The traditional methods of Gram stain, mannitol fermentation, hemolysis on blood agar, catalase test, and coagulase production were used to confirm the presence of Staphylococcus aureus in all specimens. All bacterial isolates were determined by antimicrobial susceptibility test by the disk diffusion method. The polymerase chain reaction was used to identify the antimicrobial-resistant genes ( bla Z, mec A, aac A -aph D, msr A, tet K, gyr A, grl A, and dfr G) in isolates of MRSA with a cefoxitin-resistant phenotype., Results: From 218 specimens, 185 S. aureus were isolated, with the majority of these being found in the oral cavity (29.73%) and ventral thoracic area (22.7%), respectively. Forty-seven (25.41%) MRSAs were found in S. aureus isolates, with the majority of these being found in the perineum (16, 34.04%) and ventral thoracic area (13, 27.66%) specimens. Among MRSAs, 29 (61.7%) isolates were multidrug-resistant (MDR) strains. Most of MRSA isolates were resistant to penicillin (100%), followed by ceftriaxone (44.68%) and azithromycin (44.68%). In addition, these bacteria contained the most drug-resistance genes, bla Z (47.83%), followed by gyr A (36.17%) and tet K (23.4%)., Conclusion: This study revealed that MRSA could be found even in healthy rabbits. Some MRSAs strains were MDR-MRSA, which means that when an infection occurs, the available antibiotics were not effective in treating it. To prevent the spread of MDR-MRSA from pets to owners, it may be helpful to educate owners about effective prevention and hygiene measures., Competing Interests: The study was funded by HUVEPHARMA; however, no person from HUVEPHARMA were involved at any stage of the study., (Copyright: © Jangsangthong, et al.)

A clique of Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter spp. (ESKAPE) bugs is the utmost causative agent responsible for multidrug resistance in hospital settings. These microorganisms employ a type of cell-cell communication termed 'quorum sensing (QS) system' to mediate population density and synchronously control the genes that modulate drug resistance and pathogenic behaviors. In this article, we focused on the present understanding of the prevailing QS system in ESKAPE pathogens. Basically, the QS component consisted of an autoinducer synthase, a ligand (e.g., acyl homoserine lactones/peptide hormones), and a transcriptional regulator. QS mediated expression of the bacterial capsule, iron acquisition, adherence factors, synthesis of lipopolysaccharide, poly- N -acetylglucosamine (PNAG) biosynthesis, motility, as well as biofilm development allow bacteria to promote an antimicrobial-resistant population that can escape the action of traditional drugs and endorse a divergent virulence production. The increasing prevalence of these harmful threats to infection control, as well as the urgent need for effective antimicrobial strategies to combat them, serve to highlight the important anti-QS strategies developed to address the difficulty of treating microorganisms.

Burkholderia pseudomallei is a pathogenic bacterium that causes human melioidosis, which is associated with a high mortality rate. However, the underlying mechanisms of B. pseudomallei pathogenesis are largely unknown. In this study, we examined the infection of human neuronal SH-Sy5y cells by several clinically relevant B. pseudomallei strains. We found that all tested B. pseudomallei strains can invade SH-Sy5y cells, undergo intracellular replication, cause actin-tail formation, and form multinucleated giant cells. Additionally, a deletion mutant of B. pseudomallei cycle-inhibiting factor (cif) was constructed that exhibited reduced invasion in SH-Sy5y cells. Complementation of cif restored invasion of the B. pseudomallei cif-deleted mutant. Our findings enhance understanding of B. pseudomallei pathogenicity in terms of the virulence factor Cif and demonstrate the function of Cif in neurological melioidosis. This may eventually lead to the discovery of novel targets for treatment and a strategy to control the disease.

Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 10 2 TCID 50 /mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

Background and Aim: Bacteria of the genera Vibrio and Aeromonas cause seafood-borne zoonoses, which may have a significant impact on food safety, economy, and public health worldwide. The presence of drug-resistant and biofilm-forming phenotypes in the food chain increases the risk for consumers. This study aimed to investigate the characteristics, virulence, biofilm production, and dissemination of antimicrobial-resistant pathogens isolated from seafood markets in Bangkok, Thailand., Materials and Methods: A total of 120 retail seafood samples were collected from 10 local markets in Bangkok and peripheral areas. All samples were cultured and the Vibrio and Aeromonas genera were isolated using selective agar and biochemical tests based on standard protocols (ISO 21872-1: 2017). The antibiotic susceptibility test was conducted using the disk diffusion method. The presence of hemolysis and protease production was also investigated. Polymerase chain reaction (PCR) was used to determine the presence of the hly A gene. Furthermore, biofilm formation was characterized by microtiter plate assay and scanning electron microscopy., Results: The bacterial identification test revealed that 35/57 (61.4%) belonged to the Vibrio genus and 22/57 (38.6%) to the Aeromonas genus. The Kirby-Bauer test demonstrated that 61.4% of the isolates were resistant to at least one antibiotic and 45.61% had a high multiple antibiotic resistance index (≥0.2). PCR analysis indicated that 75.44% of the bacteria harbored the hly A gene. Among them, 63.16% exhibited the hemolysis phenotype and 8.77% showed protease activity. The biofilm formation assay demonstrated that approximately 56.14% of all the isolates had the potential to produce biofilms. The moderate biofilm production was the predominant phenotype., Conclusion: The results of this study provide evidence of the multiple drug resistance phenotype and biofilm formation capacity of Vibrio and Aeromonas species contaminating raw seafood. Effective control measures and active surveillance of foodborne zoonoses are crucial for food safety and to decrease the occurrence of diseases associated with seafood consumption., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Santajit, et al.)

Nontyphoidal- Salmonella bacteria cause foodborne gastroenteritis that may lead to fatal bacteremia, osteomyelitis, and meningitis if not treated properly. The emergence of multidrug-resistant Salmonella strains is a global public health threat. Regular monitoring of genotypes and phenotypes of Salmonella isolated from humans, animals, foods, and environments is mandatory for effective reduction and control of this food-borne pathogen. In this study, antimicrobial-resistant and virulent genotypes and phenotypes of Salmonella isolated from retail food samples in Bangkok, Thailand, were investigated. From 252 raw food samples, 58 Salmonella strains that belonged only to serotype Enteritidis were isolated. Disc diffusion method showed that all isolates were still sensitive to amikacin and carbapenems. More than 30% of the isolates were resistant to ampicillin, tetracycline, and ciprofloxacin. Twenty isolates resist at least three antibiotic classes. Minimum inhibitory concentration tests showed that 12.07% of the isolates produced extended-spectrum β-Lactamase. Polymerase chain reaction indicated that 32.76, 81.03, 39.66, and 5.17% of the isolates carried bla TEM-1 , tet A, sul 2, and dfr A7, respectively. All isolates were positive for invasion-associated genes. Effective prevention and control of Salmonella (as well as other food-borne pathogens) is possible by increasing public awareness and applying food hygienic practices. Active and well harmonised "One Health" co-operation is required to effectively control food-borne zoonosis.

A point-of-care diagnostic for early and rapid diagnosis of scrub typhus caused by Orientia tsutsugamushi is required for prompt and proper treatment of patients presenting with undifferentiated febrile illnesses. In this study, an immunochromatographic antigen detection test kit (ICT AgTK) that targets the highly conserved O. tsutsugamushi 60 kDa GroEL chaperonin (heat shock protein 60) was developed. E. coli -derived recombinant GroEL expressed from DNA coding for the consensus sequence of 32 GroEL gene sequences extracted from the GenBank database was used to immunize rabbits and mice. Rabbit polyclonal antibodies (pAb) were used for preparing a gold-pAb conjugate, and the rGroEL-specific mouse monoclonal antibody was used as the antigen detection reagent at the ICT test line. In-house validation revealed that the ICT AgTK gave 85, 100 and 95% diagnostic sensitivity, specificity and accuracy, respectively, compared to the combined clinical features and standard IFA when tested on 40 frozen serum samples. The test kits correctly identified 10 scrub typhus samples out of 15 fresh plasma/buffy coat samples of patients with febrile illnesses. For independent laboratory validation, the ICT AgTK was sent to one provincial hospital. The ICT AgTK utilized by the hospital medical technologist correctly identified six scrub typhus samples out of 20 serum samples of patients with fever, as confirmed by specific IgM/IgG detection by IFA. The ICT AgTK is easy to perform with rapid turn-around time. It has the potential to be used as an important tool for on-site and early scrub typhus diagnosis by allowing testing of freshly collected samples (serum, plasma or buffy coat), especially in resource-limited healthcare settings.

LasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting Pseudomonas aeruginosa . LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immune modulation and evasion. LasB digests a variety of the host tissue proteins, extracellular matrices, as well as components of both innate and adaptive immune systems, including immunoglobulins, complement proteins, and cytokines. Thus, this enzyme is an attractive target for disarming the P. aeruginosa. This study generated human single-chain antibodies (HuscFvs) that can neutralize the elastolytic activity of native LasB by using phage display technology. Gene sequences coding HuscFvs ( huscfvs ) isolated from HuscFv-displaying phage clones that bound to enzymatically active LasB were sub-cloned to expression plasmids for large scale production of the recombinant HuscFvs by the huscfv -plasmid transformed Escherichia coli . HuscFvs of two transformed E. coli clones, i.e., HuscFv-N42 and HuscFv-N45, neutralized the LasB elastolytic activities in vitro . Computer simulation by homology modeling and molecular docking demonstrated that antibodies presumptively formed contact interfaces with the LasB residues critical for the catalytic activity. Although the LasB neutralizing mechanisms await elucidation by laboratory experiments, the HuscFvs should be tested further towards the clinical application as a novel adjunctive therapeutics to mitigate severity of the diseases caused by P. aeruginosa .

The quorum sensing (QS) signaling molecule, N -(3-oxododecanoyl)- L -homoserine lactone (3O-C12-HSL), contributes to the pathogenesis of Pseudomonas aeruginosa by regulating expression of the bacterial virulence factors that cause intense inflammation and toxicity in the infected host. As such, the QS molecule is an attractive therapeutic target for direct-acting inhibitors. Several substances, both synthetic and naturally derived products, have shown effectiveness against detrimental 3O-C12-HSL activity. Unfortunately, these compounds are relatively toxic to mammalian cells, which limits their clinical application. In this study, fully human single-chain variable fragments (HuscFvs) that bind to P. aeruginosa haptenic 3O-C12-HSL were generated based on the principle of antibody polyspecificity and molecular mimicry of antigenic molecules. The HuscFvs neutralized 3O-C12-HSL activity and prevented mammalian cells from the HSL-mediated apoptosis, as observed by Annexin V/PI staining assay, sub-G1 arrest population investigation, transmission electron microscopy for ultrastructural morphology of mitochondria, and confocal microscopy for nuclear condensation and DNA fragmentation. Computerized homology modeling and intermolecular docking predicted that the effective HuscFvs interacted with several regions of the bacterially derived ligand that possibly conferred neutralizing activity. The effective HuscFvs should be tested further in vitro on P. aeruginosa phenotypes as well as in vivo as a sole or adjunctive therapeutic agent against P. aeruginosa infections, especially in antibiotic-resistant cases., (Copyright © 2020 Santajit, Seesuay, Mahasongkram, Sookrung, Pumirat, Ampawong, Reamtong, Chongsa-Nguan, Chaicumpa and Indrawattana.)

Burkholderia pseudomallei is a Gram-negative bacillus that causes melioidosis and is recognized as an important public health problem in southeast Asia and northeast Australia. The treatment of B. pseudomallei infection is hampered by resistance to a wide range of antimicrobial agents and no vaccine is currently available. At present, the underlying mechanisms of B. pseudomallei pathogenesis are poorly understood. In our previous study, we reported that a B. pseudomallei short-chain dehydrogenase (SDO; BPSS2242) mutant constructed by deletion mutagenesis showed reduced B. pseudomallei invasion and initial intracellular survival. This indicated that SDO is associated with the pathogenesis of melioidosis. In the present study, the role of B. pseudomallei SDO was further investigated using the SDO deletion mutant by a proteomic approach. The protein profiles of the SDO mutant and wild-type K96243 were investigated through gel-based proteomic analysis. Quantitative intensity analysis of three individual cultures of the B. pseudomallei SDO mutant revealed significant down-regulation of five protein spots compared with the wild-type. Q-TOF MS/MS identified the protein spots as a glutamate/aspartate ABC transporter, prolyl-tRNA synthetase, Hsp70 family protein, quinone oxidoreductase and a putative carboxypeptidase. Functional assays were performed to investigate the role of these differentially expressed proteins in adhesion to host cells, biofilm induction and survival under heat stress conditions. The SDO deletion mutant showed a decreased ability to adhere to host cells. Moreover, biofilm formation and the survival rate of bacteria under heat stress conditions were also reduced in the mutant strain. Our findings provide insight into the role of SDO in the survival and pathogenesis of B. pseudomallei at the molecular level, which may be applied to the prevention and control of B. pseudomallei infection., Competing Interests: The authors declare there are no competing interests., (©2020 Reamtong et al.)

Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.

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., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that they have no competing interests.

Allergen-specific immunotherapy (AIT) facilitates long-term resolution of allergic morbidity resulting in reduced drug use and increased refractoriness to new sensitization. AIT effectiveness has been demonstrated in seasonal and perennial allergies, and insect stings. However, data and studies in AIT relative to cockroach (CR) allergy are relatively scarce. In this study, mice allergic to American CR (Periplaneta americana) were treated with a liposome (L)-entrapped vaccine made of mouse Tregitope289-Per a 9 of the CR, Tregitope167-Per a 9, or Per a 9 alone - or placebo. Allergic mice that received an individual vaccine intranasally had reduced Th2 response, reduced lung inflammation, and reduced respiratory tissue remodeling. However, only L-Tregitope289-Per a 9 and L-Tregitope167-Per a 9 induced expression of immunosuppressive cytokine genes (IL-10, TGF-β, and IL-35 for L-Tregitope289-Per a 9, and IL-10 and TGF-β for L-Tregitope167-Per a 9) and increment of idoleamine-2,3-dioxygenase 1 (IDO1), indicating that these vaccines caused allergic disease suppression and reversal of respiratory tissue remodeling via generation of regulatory lymphocytes. Liposome entrapped-recombinant Per a 9 (L-Per a 9) did not cause upregulation of immunosuppressive cytokine genes and IDO1 increment; rather, L-Per a 9 induced high expression of IFN-γ in lungs of treated mice, which resulted in mitigation of allergic manifestations. This study provides compelling evidence that both liposome-entrapped vaccines made of single refined major allergen alone and single refined major allergen linked with Tregitopes are effective for reducing allergen-mediated respiratory tissue inflammation and remodeling, but through different mechanisms.

Strong evidence of high potency of rhodomyrtone as a promising antibacterial agent against pathogenic gram-positive bacteria has been clearly demonstrated in our previous work. The aim of this study was to provide insight into early action of rhodomyrtone, an acylphloroglucinol, on membrane damage in multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA). Early effects of rhodomyrtone on the bacterial membrane integrity were detected in a time-course study. Flow cytometry revealed a reduction in green fluorescent emission and increase in uptake of propidium iodide in rhodomyrtone-treated bacterial cells in a concentration- and time-dependent manner. Disruption of cytoplasmic membrane was further monitored by measuring cellular adenosine triphosphate (ATP) and potassium ion (K + ). Leakage of both ATP and K + and significant decrease in intracellular ATP in MRSA were observed following treatment. Pronounced changes in the bacterial ultrastructure and morphology were confirmed by transmission electron microscopy and scanning electron microscopy. Bacterial cell disruption, holes in cell surface, and bulge formations were noted in rhodomyrtone-treated cells. In this study, we provided relevant data to clarify that rhodomyrtone is a bacterial cell membrane-damaging agent. A possible early effect of this novel compound involves bacterial membrane disruption.

Melioidosis is a fatal infectious disease caused by the environmental bacterium Burkholderia pseudomallei It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations, and definitive diagnosis requires bacterial culture, which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by enzyme-linked immunosorbent assay (ELISA) that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects, including the following: (i) 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; (ii) 202 healthy donors from northeast Thailand; (iii) 90 U.S. healthy donors; and (iv) 207 patients infected with other organisms. Compared to culture results as a gold standard, the sensitivity of ICT for all hospitals was 88.3%. The specificities for Thai donors and U.S. donors were 86.1% and 100%, respectively, and the specificity for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA results with a kappa value of 0.829, indicating that the method is much improved compared with the current serological method, the indirect hemagglutination assay (IHA) (69.5% sensitivity and 67.6% specificity for Thais). The Hcp1-ICT represents a potential point-of-care (POC) test and may be used to replace the IHA for screening of melioidosis in hospitals as well as in resource-limited areas., (Copyright © 2018 Phokrai et al.)

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, which can be fatal in humans. Melioidosis is prevalent in the tropical regions of Southeast Asia and Northern Australia. Ecological data have shown that this bacterium can survive as a free-living organism in environmental niches, such as soil and water, as well as a parasite living in host organisms, such as ameba, plants, fungi, and animals. This review provides an overview of the survival and adaptation of B. pseudomallei to stressful conditions induced by hostile environmental factors, such as salinity, oxidation, and iron levels. The adaptation of B. pseudomallei in host cells is also reviewed. The adaptive survival mechanisms of this pathogen mainly involve modulation of gene and protein expression, which could cause alterations in the bacteria's cell membrane, metabolism, and virulence. Understanding the adaptations of this organism to environmental factors provides important insights into the survival and pathogenesis of B. pseudomallei , which may lead to the development of novel strategies for the control, prevention, and treatment of melioidosis in the future.

Background: Avoidance of allergen exposure is an effective measure for preventing naÏve and allergic individuals from sensitization (primary intervention) and disease aggravation (secondary intervention), respectively. Regular monitoring of the allergens in the environment is required for the effective intervention. Thus, there is a need for cost-effective test kits for environmental allergen quantifications., Objective: To invent a test kit for quantification of cat major allergen, Fel d 1., Methods: A mouse monoclonal antibody (MAb) specific to the newly identified IgE-binding conformational epitope of the cat major allergen (Fel d 1) and rabbit polyclonal IgG to recombinant Fel d 1 were used as allergen capture and detection reagents, respectively. Native Fel d 1 was used in constructing a standard curve., Results and Conclusion: Sixteen of 36 dust samples collected from houses of cat allergic subjects in Bangkok contained Fel d 1 above 0.29 μg/gram of dust which is considered as a novel threshold level for causing cat allergy sensitization or symptoms. Among them, 7 samples contained the allergen exceeding 2.35 μg/gram of dust which is the level that would aggravate asthma. Results of the allergen quantification using the locally made test kit showed strong correlation (r = 0.923) with the allergen quantification using commercialized reagents. The assay using MAb to Fel d 1 IgE-binding epitope of this study has potential application as an economic and practical tool for cat allergy intervention measure especially in localities where health resources are relatively limited.

Insect glutathione S-transferases (GSTs) play important roles in insecticide/drug resistance and stress response. Medically, GSTs of house dust mites (Dermatophagoides pteronyssinus and Blomia tropicalis) and German cockroach (Blattella germanica) are human allergens. In this study, classes, isoforms and B-cell and allergenic epitopes of GST of American cockroach, Periplaneta americana, the predominant species in the tropics and subtropics were investigated for the first time. Enzymatically active native and recombinant P. americana-GSTs bound to IgE in sera of all P. americana allergic patients that were tested. By gel-based proteomics and multiple sequence alignments, the native GST comprises three isoforms of delta and sigma classes. All isoforms interacted with serum IgE of the cockroach allergic subjects. Molecularly, the protein contains six B-cell epitopes; two epitopes located at β1-α1 and β4-α3 regions bound to patients' serum IgE, indicating that they are allergenic. P. americana are ubiquitous and their GST can sensitize humans to allergic diseases; thus, the protein should be included in the allergen array for component resolved diagnosis (CRD) of allergic patients, either by skin prick test or specific IgE determination. The GST is suitable also as a target of environmental allergen detection and quantification for intervention of cockroach sensitization and allergic morbidity.

Allergen specific immunotherapy (AIT) can modulate the allergic response causing a long-term symptom subsidence/abolishment which leads to reduced drug use and prevention of new sensitization. AIT of house dust mite allergy (HDM) using the mite crude extract (CE) as the therapeutic agent is not only less effective than the AIT for many other allergens, but also frequently causes adverse effects during the treatment course. In this study, mouse model of Dermatophagoides pteronyssinus (Dp) allergy was invented for testing therapeutic efficacies of intranasally administered liposome (L) encapsulated vaccines made of single Dp major allergens (L-Der p 1, L-Der p 2), combined allergens (L-Der p 1 and Der p 2), and crude Dp extract (L-CE). The allergen sparing intranasal route was chosen as it is known that the effective cells induced at the nasal-associated lymphoid tissue can exert their activities at the lower respiratory tissue due to the common mucosal traffic. Liposome was chosen as the vaccine delivery vehicle and adjuvant as the micelles could reduce toxicity of the entrapped cargo. The Dp-CE allergic mice received eight doses of individual vaccines/placebo on alternate days. All vaccine formulations caused reduction of the Th2 response of the Dp allergic mice. However, only the vaccines made of single refined allergens induced expressions of immunosuppressive cytokines (TGF-β, IL-35 and/or IL-10) which are the imperative signatures of successful AIT. The data emphasize the superior therapeutic efficacy of single refined major allergen vaccines than the crude allergenic extract vaccine.

Burkholderia pseudomallei is an environmental saprophyte and the causative agent of melioidosis, a severe infectious disease prevalent in tropical areas, including southeast Asia and northern Australia. In Thailand, the highest incidence of melioidosis is in the northeast region, where saline soil and water are abundant. We hypothesized that B. pseudomallei develops an ability to thrive in saline conditions and gains a selective ecological advantage over other soil-dwelling microorganisms. However, little is known about how an elevated NaCl concentration affects survival and adaptive changes in this pathogen. In this study, we examined the adaptive changes in six isolates of B. pseudomallei after growth in Luria-Bertani medium containing different concentrations of NaCl at 37°C for 6 hr. The bacteria were then investigated for resistance to heat at 50°C and killing by hydrogen peroxide (H 2 O 2 ). In addition, flagellar production, biofilm formation, and the plaque formation efficiency of B. pseudomallei after culture in saline conditions were observed. In response to exposure to 150 and 300 mmol L -1 NaCl, all B. pseudomallei isolates showed significantly increased thermal tolerance, oxidative resistance, and plaque-forming efficiency. However, NaCl exposure notably decreased the number of B. pseudomallei flagella. Taken together, these results provide insight into the adaptations of B. pseudomallei that might be crucial for survival and persistence in the host and/or endemic environments with high salinity., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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.

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, bla TEM and bla CMY-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., Competing Interests: The authors declare there are no competing interests.

Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vβ region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application.

Background and Objective: Natural allergenic extracts using for diagnosis and immunotherapy may have batch-to-batch variations and contaminations with unrefined allergens or non-allergenic components. Thus, recombinant allergen is believed to overcome these shortcomings. In this study, native and recombinant allergens of group 1 and 2 of Dermatophagoides mites were produced and their allergenicities were compared., Methods: Native allergens were prepared by MAb affinity chromatography. All recombinant allergens were produced in E. coli expression system. IgE reactivities of these allergens were determined by IgE-ELISA., Results: The native and recombinant Der p 1, Der p 2, Der f 1, Der f 2 had molecular weights of approximately 25, 15, 25 and 15 kDa, respectively. IgE reactivities of nDer p 1, nDer f 1, rDer p 1 and rDer f 1 were 96.67%, 90%, 43.33% and 46.67%, respectively. Allergenicities of nDer p 2, nDer f 2, rDer p 2 and rDer f 2 were 86.67%, 96.43%, 76.67% and 89.29%, respectively. The findings indicated that recombinant group-1 products were minor allergens which revealed no correlation with their native forms. In contrast, recombinant group-2 allergens were major allergens and showed a significant correlation to their native allergens., Conclusion: We successfully produced native and recombinant group-1 and group-2 allergens. According to their allergenicities, recombinant Der p 2 and rDer f 2 have potential to replace native allergen in diagnostic and therapeutic extracts. Moreover, they can employ as a standard reagent to measure the amount of group 2 allergen in the environment by sandwich-ELISA and utilise this as an immunogen for MAb production.

Information on the antigenic repertoire, especially the IgE-binding epitopes of an allergen is important for understanding the allergen induced immune response and cross-reactivity, as well as for generating the hypoallergenic variants for specific component resolved immunotherapy/diagnosis (CRIT and CRD). Data on the IgE-binding epitopes of cat allergens are scarce. In this study, a novel IgE-binding epitope of the cat major allergen, Fel d 1, was identified. Mouse monoclonal antibody (MAb) specific to the Fel d 1 was produced. Computerized intermolecular docking was used for determining the residues of the Fel d 1 bound by the specific MAb. The presumptive surface exposed residues of the Fel d 1 intrigued by the MAb are located on the chain 1. They are: L34 and T37 (helix 1); T39 (between helices 1 and 2); P40, E42 and E45 (helix 2); R61, K64, N65 and D68 (helix 3); and E73 and K76 (helix 4). The MAb competed efficiently with the cat allergic patients' serum IgE for Fel d 1 binding in the competitive IgE binding assay, indicating allergenicity of the MAb epitope. The newly identified allergenic epitope of the Fel d 1 is useful in a design of the CRIT and CRD for cat allergy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dermatophagoides farinae mite is a predominant source of indoor allergens causing high incidence of allergy worldwide. People with different genetic background respond differently to the mite components, and thus the component-resolved diagnosis (CRD) is preferred to the conventional allergy test based on crude mite extract. In this study, proteome and culprit components in the D. farinae whole body extract that sensitized the allergic patients were studied by using SDS-PAGE (1DE) and 2DE-IgE immunoblotting followed by LC-MS/MS and database search for protein identification. From the 1DE, the mite extract revealed 105 proteins that could be classified into seven functionally different groups: allergens, structural components, enzymes, enzyme inhibitor, receptor proteins, transporters, and binding/regulatory/cell signaling proteins. From the 2DE, the mite extract produced 94 spots; 63 were bound by IgE in sera of 20 D. farinae allergic patients. One more protein that was not revealed by the 2DE and protein staining reacted with IgE in 2 allergic patients. Proteins in 40 spots could be identified as 35 different types. Three of them reacted to IgE of >50% of the allergic patients, and hence they are major allergens: tropomyosin or Der f 10 (75%), aconitate hydratase (70%), and one uncharacterized protein (55%). Aconitate hydratase is a novel D. farinae major allergen unraveled in this study. Several mite minor allergens that have never been previously reported are also identified. The data have clinical applications in the component-resolved diagnosis for tailor-designed allergen-specific immunotherapy.

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are the leading cause of nosocomial infections throughout the world. Most of them are multidrug resistant isolates, which is one of the greatest challenges in clinical practice. Multidrug resistance is amongst the top three threats to global public health and is usually caused by excessive drug usage or prescription, inappropriate use of antimicrobials, and substandard pharmaceuticals. Understanding the resistance mechanisms of these bacteria is crucial for the development of novel antimicrobial agents or other alternative tools to combat these public health challenges. Greater mechanistic understanding would also aid in the prediction of underlying or even unknown mechanisms of resistance, which could be applied to other emerging multidrug resistant pathogens. In this review, we summarize the known antimicrobial resistance mechanisms of ESKAPE pathogens.

The influence of temperature on bacterial virulence has been studied worldwide from the viewpoint of climate change and global warming. The bacterium enteroaggregative Escherichia coli (EAEC) is the causative agent of watery diarrhea and shows an increasing incidence worldwide. Its pathogenicity is associated with the virulence factors aggregative adherence fimbria type I and II (AAFI and AAFII), encoded by aggA and aafA in EAEC strains 17-2 and 042, respectively. This study focused on the effect of temperature increases from 29 °C to 40 °C on fimbrial gene expression using real-time PCR, and on its virulence using an aggregative adherence assay and biofilm formation assay. Incubation at 32 °C caused an up-regulation in both EAEC strains 17-2 and strain 042 virulence gene expression. EAEC strain 042 cultured at temperature above 32 °C showed down-regulation of aafA expression except at 38 °C. Interestingly, EAEC cultured at a high temperature showed a reduced adherence to cells and an uneven biofilm formation. These results provide evidence that increases in temperature potentially affect the virulence of pathogenic EAEC, although the response varies in each strain.

Purpose: Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105., Methods: Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining., Results: The rPer a 1.0105 (~13 kDa) had 100%, 98% and ≥90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues(99) QDLLLQLRDKGV(110) contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 ℃g per gram of feces., Conclusions: The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.

Vespa affinis (Asian wasp, Thai banded tiger wasp, or local name: Tor Hua Seua) causes the most frequent incidence of medically important Hymenoptera sting in South and Southeast Asia. However, data on the venom components attributable to the sting derived-clinical manifestations (local reactions, IgE mediated-anaphylaxis, or systemic envenomation) are lacking. This study provides the first set information on V. affinis venom proteome, allergenome, and IgE reactivity of individual venom components. From 2DE-gel based-proteomics, the venom revealed 93 protein spots, of which proteins in 51 spots could be identified and classified into three groups: typical venom components and structural and housekeeping proteins. Venom proteins in 32 spots reacted with serum IgE of wasp allergic patients. Major allergenic proteins that reacted to IgE of >50% of the wasp allergic patients included PLA1 (100%), arginine kinase (73%), heat shock 70 kDa protein (73.3%), venom allergen-5 (66.7%), enolase (66.7%), PLA1 magnifin (60%), glyceraldehyde-3-phosphate dehydrogenase (60%), hyaluronidase (53.3%), and fructose-bisphosphate aldolase (53.3%). The venom minor allergens were GB17876 transcript (40%), GB17291 transcript (20%), malic enzyme (13.3%), aconitate hydratase (6.7%), and phosphoglucomutase (6.7%). The information has diagnostic and clinical implications for future improvement of case diagnostic sensitivity and specificity, component-resolve diagnosis, and design of specific Hymenoptera venom immunotherapy.

Background: Cockroach (CR) allergens frequently cause severe asthma in CR-sensitized subjects. Allergen-specific immunotherapy causes a shift of allergic Th2 responses towards Th1 and/or regulatory T cell (Treg) responses which reduce airway inflammation and prevent disease progression. Data are relatively limited on immunotherapy via CR allergy vaccine., Methods: The therapeutic efficacy of an intranasal liposome-adjuvant vaccine made of a refined Periplaneta americana arginine kinase (AK) was compared to the liposome-entrapped P. americana crude extract (CRE) vaccine. Adult BALB/c mice were rendered allergic to CRE. Three allergic mouse groups were immunized intranasally on alternate days with 8 doses of liposome-entrapped CRE (L-CRE), liposome-entrapped AK and placebo, respectively. One week later, all mice received a nebulized CRE provocation. Evaluation of vaccine efficacy was performed 1 day after provocation., Results: Liposome-entrapped native AK attenuated airway inflammation after the CRE provocation and caused a shift of allergic Th2 to Th1 and Treg responses. The L-CRE also induced a shift from the Th2 to the Th1 response but did not induce a Treg response and could not attenuate the airway inflammation upon allergen reexposure., Conclusions: Intranasal liposome-adjuvant CR allergy vaccine containing native AK (Per a 9) is better than L-CRE in attenuating allergic airway inflammation. The findings of this study not only document a more comprehensive and beneficial immune response induced by the refined allergen vaccine but also raise the point that the shift from the Th2 to the Th1 response alone might not correlate with improved airway histopathology, clinical outcome and quality of life., (Copyright © 2013 S. Karger AG, Basel.)

Background and Objective: Most allergen extracts/vaccines used today in clinical practice are derived from natural allergen sources. Therefore, their allergic components may vary as these are prone to natural variation. This study aims to compare the allergenic components and biological potency of crude extracts from wild and laboratory reared American cockroaches., Methods: Crude extracts of male and female of wild and laboratory reared American CR, were prepared by the same method. Their allergenic components were evaluated by in vitro assays such as protein contents, protein profiles, quantification of major allergens (Per a 1 and Per a 9) and IgE inhibition ELISA assay., Results and Discussion: There was no statistically significant difference between the protein contents and the concentrations of Per a 1 in the crude extracts from both groups. However, the Per a 9 levels in extracts of wild CR were significantly higher than those from the extracts of laboratory reared CR. The protein patterns of the extracts of laboratory reared CR exhibited more consistency in the number of bands with higher intensity than those of wild CR. Pooled extracts of laboratory reared CR could inhibit IgE binding to that of wild CR up to 78%. The endotoxin content of extracts of laboratory reared CR were ten times less than those of the the wild CR. We have successfully determined the allergenic potency of the extracts of laboratory reared CR versus those of the wild CRs by in vitro assays. Further studies should be performed to determine the biological potency of CR extracts by in vivo assays for clinical application., Conclusion: Our finding indicates that the laboratory reared CR would be the better source of material in vaccine production than the wild CR.