A Rapid Immunochromatography Test Based on Hcp1 Is a Potential Point-of-Care Test for Serological Diagnosis of Melioidosis.

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.

Quantification of Fel d 1 in house dust samples of cat allergic patients by using monoclonal antibody specific to a novel IgE-binding epitope.

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.

Proteome, Allergenome, and Novel Allergens of House Dust Mite, Dermatophagoides farinae.

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.

A novel IgE-binding epitope of cat major allergen, Fel d 1.

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.

Allergenicity of native and recombinant major allergen groups 1 and 2 of Dermatophagoides mites in mite sensitive Thai patients.

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.

Proteome and allergenome of Asian wasp, Vespa affinis, venom and IgE reactivity of the venom components.

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.

Antimicrobial resistance, virulence profile, and genetic analysis of ESBL-producing Escherichia coli isolated from Nile tilapia in fresh markets and supermarkets in Thailand.

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.

Investigation of the marine bacterial community along the coastline of the Gulf of Thailand.

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.

Isolation and characterisation of a novel Silviavirus bacteriophage promising antimicrobial agent against methicillin-resistant Staphylococcus aureus infections.

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.

Intranasal, liposome-adjuvanted cockroach allergy vaccines made of refined major allergen and whole-body extract of Periplaneta americana.

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.

In Vitro Roles of Burkholderia Intracellular Motility A (BimA) in Infection of Human Neuroblastoma Cell Line.

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 (ΔbimA) 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 ΔbimA 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 ΔbimA 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.

Phenotypic and Genotypic Investigation of Carbapenem-Resistant Acinetobacter baumannii in Maharaj Nakhon Si Thammarat Hospital, Thailand.

(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 blaVIM, blaNDM, blaIMP, blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, csuE and pgaB 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 blaOXA-51-like (100%). Ninety-nine isolates (57.56%) were biofilm producers. The most prevalent biofilm gene was pgaB (79.65%), followed by csuE (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.

Type VI Secretion System Accessory Protein TagAB-5 Promotes Burkholderia pseudomallei Pathogenicity in Human Microglia.

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.

Comparison of allergenic components and biopotency in whole body extracts of wild and laboratory reared American cockroaches, Periplaneta americana.

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.

Convenient, rapid and economic detection and semi-quantification of American cockroach allergen in the environment.

BACKGROUND: Measuring allergen levels in the environment provides useful information to guide the management of allergic patients. A laboratory-based test kit sandwich ELISA for quantification of Per a 9, the major allergen of Periplaneta americana was recently developed. However, it is not suitable for screening. OBJECTIVE: To develop a simple, rapid, and economic format for semi-quantification of Per a 9 assay using dot-blot ELISA technique. METHODS: The efficacy of direct dot-blot ELISA and sandwich dot-blot ELISA was evaluated. Direct dot-blot ELISA was selected for further modification into 6 protocols. The selected protocol of direct dot-blot was further compared with the laboratory-based test kit, sandwich ELISA. RESULTS: The lowest detection limits in protocols no. 1-6 were 3.9, 15.6, 15.6, 62.5, 125 and 62.5 microg/ml of native Per a 9 whereas time required for each protocol was 145, 45, 30, 26, 18 and 26 minutes, respectively. The sensitivity of direct dot-ELISA was 3.9 microg/ml of Per a 9. Protocol no. 3 was the most suitable assay because its detection limits were as low as 15.6 microg/ml of CR allergen and the total process took only 30 minutes. In comparison with the 2 days required for laboratory sandwich ELISA, the selected protocol provided a similar yield of allergen detection but it offers significant savings of time. Additionally, this method could be easily interpreted by various groups of people. CONCLUSION: This modified direct dot-blot ELISA is the first membrane ELISA which is a semiquantitative test appropriate for screening American cockroach allergen owing to its simplicity, speed and good yield.

Quorum Sensing in ESKAPE Bugs: A Target for Combating Antimicrobial Resistance and Bacterial Virulence.

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.

Antimicrobial Resistance and Virulence of Non-Typhoidal Salmonella from Retail Foods Marketed in Bangkok, Thailand.

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 ß-Lactamase. Polymerase chain reaction indicated that 32.76, 81.03, 39.66, and 5.17% of the isolates carried blaTEM-1, tetA, sul2, and dfrA7, 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.

Occurrence, antimicrobial resistance, virulence, and biofilm formation capacity of Vibrio spp. and Aeromonas spp. isolated from raw seafood marketed in Bangkok, Thailand.

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 hlyA 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 hlyA 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.

Occurrence of antimicrobial resistance and antimicrobial resistance genes in methicillin-resistant Staphylococcus aureus isolated from healthy rabbits.

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 (blaZ, mecA, aacA-aphD, msrA, tetK, gyrA, grlA, and dfrG) 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, blaZ (47.83%), followed by gyrA (36.17%) and tetK (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.

GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus.

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.