Browsing by Author "Ngamwongsatit N."

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Antibiotic resistant Escherichia coli from diarrheic piglets from pig farms in Thailand that harbor colistin-resistant mcr genes
(2022-12-01) Nguyet L.T.Y.; Keeratikunakorn K.; Kaeoket K.; Ngamwongsatit N.; Mahidol University
Antibiotic-resistant Escherichia coli is one of the most serious problems in pig production. This study aimed to determine the antibiotic susceptibility and genotypes profiles of diarrhoeagenic E. coli that causes diarrhea in piglets. Thirty-seven pathogenic E. coli strains were used in this study. These were isolated from rectal swabs of diarrheic piglets from farms in Thailand from 2018 to 2019. Escherichia coli isolates were highly resistant to amoxicillin (100%), followed by oxytetracycline (91.9%), enrofloxacin (89.2%), trimethoprim/sulfamethoxazole (86.5%), amoxicillin: clavulanic acid (81.1%), colistin and gentamicin (75.7%), ceftriaxone and ceftiofur (64.9%), ceftazidime (35.1%) and 97.3% showed multidrug-resistance (MDR). There were 8 (21.6%) mcr-1 carriers, 10 (27.0%) mcr-3 carriers and 10 (27.0%) co-occurrent mcr-1 and mcr-3 isolates. The phenotype-genotype correlation of colistin resistance was statistically significant (performed using Cohen’s kappa coefficient (κ = 0.853; p < 0.001)). In addition, PCR results determined that 28 of 37 (75.7%) isolates carried the int1 gene, and 85.7% int1-positive isolates also carried the mcr gene. Genetic profiling of E. coli isolates performed by ERIC-PCR showed diverse genetics, differentiated into thirteen groups with 65% similarity. Knowledge of the molecular origins of multidrug-resistant E. coli should be helpful for when attempting to utilize antibiotics in the pig industry. In terms of public health awareness, the possibility of transmitting antibiotic-resistant E. coli from diarrheic piglets to other bacteria in pigs and humans should be of concern.
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Antimicrobial activity of cell free supernatants from probiotics inhibits against pathogenic bacteria isolated from fresh boar semen
(2023-12-01) Keeratikunakorn K.; Kaewchomphunuch T.; Kaeoket K.; Ngamwongsatit N.; Mahidol University
The use of antibiotics with semen extender appears to be a practical solution to minimise bacterial growth in fresh boar semen preservation. Unfortunately, the excessive use of antibiotics promotes antimicrobial resistance (AMR). This becomes a worldwide concern due to the antimicrobial resistance genes transmitted to animals, environment, and humans. Probiotics are one of the alternative methods to reduce antibiotic use. They could inhibit pathogenic bacteria by producing antimicrobial substances in cell free supernatants (CFS). Nevertheless, there is no comprehensive study undertaken on inhibitory activity against pathogenic bacteria isolated from boar semen origin. Our study investigated the efficacy of CFS produced from selected probiotics: Bacillus spp., Enterococcus spp., Weissella spp., Lactobacillus spp., and Pediococcus spp. inhibiting pathogenic bacteria isolated from fresh boar semen. Besides, the semen-origin pathogenic bacteria are subjected to identification, antimicrobial resistance genes detection, and antibiotic susceptibility test (AST). Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis are the most common pathogens identified in boar semen with resistance to numerous antibiotics used in pig industry. The CFS with its antimicrobial peptides and/or bacteriocin constituent derived from selected probiotics could inhibit the growth of pathogenic bacteria carrying antimicrobial resistance genes (mcr-3 and int1 genes). The inhibition zones for Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis provided more efficient results in the CFS derived from Lactobacillus spp. and Pediococcus spp. than those of the CFS produced from Enterococcus spp., Weissella spp. and Bacillus spp., respectively. It is worth noted that as the incubation time increased, the antibacterial activity decreased conversely. Our results on CFS with its antimicrobial peptides and/or bacteriocin constituent inhibits semen-origin pathogenic bacteria guide the direction as a promising alternative method used in the semen extender preservation of the pig industry.
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Ca-EDTA restores the activity of ceftazidime-avibactam or aztreonam against carbapenemase-producing Klebsiella pneumoniae infections
(2023-07-21) Wannigama D.L.; Sithu Shein A.M.; Hurst C.; Monk P.N.; Hongsing P.; Phattharapornjaroen P.; Fox Ditcham W.G.; Ounjai P.; Saethang T.; Chantaravisoot N.; Wapeesittipan P.; Luk-in S.; Sae-Joo S.; Nilgate S.; Rirerm U.; Tanasatitchai C.; Kueakulpattana N.; Laowansiri M.; Liao T.; Kupwiwat R.; Rojanathanes R.; Ngamwongsatit N.; Tungsanga S.; Leelahavanichkul A.; Devanga Ragupathi N.K.; Badavath V.N.; Hosseini Rad S.M.A.; Kanjanabuch T.; Hirankarn N.; Storer R.J.; Cui L.; Amarasiri M.; Ishikawa H.; Higgins P.G.; Stick S.M.; Kicic A.; Chatsuwan T.; Abe S.; Mahidol University
Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.
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Canine distemper outbreak and laryngeal paralysis in captive tigers (Panthera tigris)
(2025-01-24) Suwanpakdee S.; Wiratsudakul A.; Chaisilp N.; Prasittichai L.; Skulpong A.; Maneeorn P.; Bhusri B.; Mongkolpan C.; Buddhirongawatr R.; Taowan J.; Wongluechai P.; Arya N.; Suwannaprapha P.; Ngamwongsatit N.; Wiriyarat W.; Sangkachai N.; Suwanpakdee S.; Mahidol University
The canine distemper virus (CDV) could infect various wildlife species worldwide. The viral infection in large felids directly impacts wildlife conservation. This study aimed to understand better the burden of CDV outbreaks in captive tiger populations in Thailand and a novel discovery of their clinical signs with a history of CDV exposure. We followed up on their infection from May 2016 to October 2020 with laboratory testing and veterinary medical records. The cumulative morbidity and mortality rates were relatively high. Moreover, 50% of the tigers survived at 2 years after infection. All suspected and confirmed cases of CDV infections were significantly associated with laryngeal inflammation, which developed into paralysis in almost 50% of cases. Altogether, 50% of all tiger cases with chronic infection developed stridor at 314 days after virus infection [95% CI: 302-320]. Therefore, laryngeal paralysis may result from CDV infection and degeneration, potentially affecting the peripheral and central nervous systems. This condition could pose a life-threatening risk to tigers. The virus could spread quickly by contact with bodily excretion among tigers and fomite contamination once it affects a specific population. Implementation of biosecurity measures and vaccination is essential to mitigate the risk of disease spread and infection rates in tiger populations.
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Cannabidiol Demonstrates Remarkable Efficacy in Treating Multidrug-Resistant Enterococcus Faecalis Infections In Vitro and In Vivo
(2024-09-01) Hongsing P.; Ngamwongsatit N.; Kongart C.; Nuiden N.; Phairoh K.; Wannigama D.L.; Hongsing P.; Mahidol University
The growing prevalence of multidrug-resistant Enterococcus faecalis infection has become a global concern. There is a demand for alternative antibacterial agents, such as herbal alternatives, such as Cannabidiol, that are cost-effective, non-toxic and efficient. This study investigates the Minimum Inhibitory Concentration (MIC) for planktonic cells and the Minimum Biofilm Eradication Concentration (MBEC) for biofilm formation in multidrug-resistance isolates of E. faecalis isolates, particularly focusing cannabidiol activity. E. faecalis isolates with strong biofilm presence, vancomycin, levofloxacin, and daptomycin displayed high MIC and MBEC values. Cannabidiol exhibited a significantly lower MIC (1 μg/mL) for planktonic cells and a low MBEC (2 μg/mL). Moreover, at low concentrations (2 µg/mL), cannabidiol demonstrated notable reductions in biofilm biovolume, bacterial cell viability and colony-forming unit compared to vancomycin, levofloxacin, and daptomycin. The mice treated with cannabidiol (100 mg/kg) exhibited a significant reduction in E. faecalis bacterial load in internal organs and increased the survival. In conclusion, the findings underscore the promising antibiofilm properties of cannabidiol against E. faecalis, indicating its potential as a novel therapeutic agent.
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Cell-free culture supernatants of Lactobacillus spp. and Pediococcus spp. inhibit growth of pathogenic Escherichia coli isolated from pigs in Thailand
(2022-12-01) Kaewchomphunuch T.; Charoenpichitnunt T.; Thongbaiyai V.; Ngamwongsatit N.; Kaeoket K.; Mahidol University
Background: Pathogenic Escherichia coli (E. coli) is an important causative agent for infectious diseases in pigs and causes significant economic loss. The global concern of antimicrobial resistance of bacteria raises awareness of the alternative ways of using antimicrobial peptides (AMPs). The study was aimed to identify and test the efficacy of AMPs from Lactobacillus spp. against the growth of pathogenic E. coli isolated from pigs in Thailand. Briefly, cell-free culture supernatants (CFCS) from 3 strains of lactic acid bacteria (LAB) consisting of Lactobacillus acidophilus (strain KMP), Lactobacillus plantarum (strain KMP), and Pediococcus pentosaceus (strain KMP) were tested against pathogenic E. coli via agar well diffusion assay in quadruplicates. The presence of a zone of inhibition (ZOI) around wells was evaluated at different incubation time. Acid and bile tolerance test was performed for bacterial viability in acid and bile salt conditions. In addition, LAB cross-streaking assay was evaluated for antagonist activity. Results: The study showed that CFCS from L. acidophilus KMP, L. plantarum KMP, and P. pentosaceus KMP could inhibit the growth of pathogenic E. coli isolated from pigs in a time-dependent manner. To exemplify, the ZOI of L. plantarum KMP against E. coli (ETEC) at 8, 10, 12, 14, and 16 h incubation, were 26.6 ± 1.1, 24.9 ± 1.9, 22.5 ± 2.4, 20.3 ± 2.9, and 17.9 ± 3.3 mm, respectively. The ZOI was significantly different between 8, 10, 12, 14 h incubation, and the ZOI of the CFCS from L. plantarum KMP was larger than others (P-value < 0.05). Furthermore, L. acidophilus KMP, L. plantarum KMP, and P. pentosaceus KMP showed viability in pH 3.0, 0.3, and 0.5% (w/v) bile salt concentration. They exhibited no antagonist activity among each other. Conclusions: According to the results, the CFCS from LAB including L. acidophilus KMP, L. plantarum KMP and P. pentosaceus KMP can inhibit the growth of pathogenic E. coli, isolated from pigs in Thailand. The antimicrobial activity observed was incubation time dependent.
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Combination of curcumin or chitosan with photodynamic therapy as an effective alternative therapy for overcoming wound infection associated with multidrug-resistant Acinetobacter baumannii
(2025-06-01) Yonpiam M.; Wannigama D.L.; Shein A.M.S.; Liao T.; Hurst C.; Monk P.N.; Amarasiri M.; Phattharapornjaroen P.; Ounjai P.; Saethang T.; Badavath V.N.; Luk-in S.; Nilgate S.; Rirerm U.; Srisakul S.; Ngamwongsatit N.; Leelahavanichkul A.; Ragupathi N.K.D.; Kanjanabuch T.; Narkban P.; Miyanaga K.; Cui L.; Boontaveeyuwat E.; Higgins P.G.; Kicic A.; Hamamoto H.; Abe S.; Hongsing P.; Chatsuwan T.; Yonpiam M.; Mahidol University
The increasing prevalence of multidrug-resistant Acinetobacter baumannii as an opportunistic pathogen in wound infections raises significant concerns due to its antibiotic resistance and biofilm-mediated antibiotic tolerance. This underscores the urgent need to explore an alternative approach to effectively managing wound infections caused by MDR A. baumannii. In this study, combining subinhibitory concentrations of curcumin (25 µg/ml) or chitosan (156 µg/ml) with photodynamic therapy-PDT (10 J/cm2) resulted in a significant reduction of planktonic viabilities and biofilm biovolume of MDR A. baumannii clinical isolates in vitro. In murine A. baumannii-associated wound infection, a combination of curcumin (25 µg/ml) or chitosan (156 µg/ml) with PDT (10 J/cm2) resulted in complete bacterial eradication with wound healing at Day 15 of post-treatments with these combination therapies. Our study demonstrated that combining curcumin or chitosan with PDT at their subinhibitory concentrations represents a promising alternative strategy as localized therapy for effectively managing A. baumannii-associated wound infection.
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Community-based mpox and sexually transmitted disease surveillance using discarded condoms in the global south
(2024-01-01) Wannigama D.L.; Amarasiri M.; Phattharapornjaroen P.; Hurst C.; Modchang C.; Besa J.J.V.; Miyanaga K.; Cui L.; Fernandez S.; Huang A.T.; Ounjai P.; Werawatte W.K.C.P.; Ali A.H.; Vatanaprasan P.; Jay D.J.; Saethang T.; Luk-in S.; Storer R.J.; Kanthawee P.; Al-Dois M.; Bani R.; Thuptiang W.; Khan S.; Tacharoenmuang R.; Cynthia B.; Vitharana S.P.H.S.; Ngamwongsatit N.; Ishikawa H.; Singer A.C.; Ragupathi N.K.D.; Furukawa T.; Sei K.; Nanbo A.; Leelahavanichkul A.; Kanjanabuch T.; Chatsuwan T.; Higgins P.G.; Sano D.; Kicic A.; Rockstroh J.K.; Siow R.; Trowsdale S.; Hongsing P.; Khatib A.; Shibuya K.; Abe S.; Wannigama D.L.; Mahidol University
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Development and Validation of an Efficient Multiplex PCR Assay for Simultaneous Detection of Six Common Foodborne Pathogens and Hygiene Indicators
(2023-06-01) Ngamwongsatit N.; Chaturongakul S.; Aunpad R.; Mahidol University
Microbial contamination in foods could lead to illnesses and substantial losses in both food industry and public health sectors. Rapid detection of microbial hazards (i.e., pathogens, hygiene indicator microorganisms) can accelerate surveillance and diagnostic processes reducing transmission and minimizing undesirable consequences. This study developed a multiplex PCR (m-PCR) for the detection of six common foodborne pathogens and hygiene indicators using specific primers for uidA of Escherichia coli, stx2 of Escherichia coli O157:H7, invA of Salmonella spp., int of Shigella spp., ntrA of Klebsiella pneumoniae, and ail of Yersinia enterocolitica and Yersinia pseudotuberculosis. Sensitivity of the m-PCR was 100 fg or ∼20 bacterial cells. Each primer set amplified only the targeted strain, and specificity was demonstrated by lack of nonspecific bands with DNA from 12 other bacterial strains. Following ISO 16140-2:2016, the relative limit of detection of the m-PCR was comparable to that of the gold-standard method; however, the processing time was five times faster. The m-PCR was applied to detect the six pathogens in 100 natural samples (50 pork meat and 50 local fermented food samples) and compared to results of the gold-standard method. Positive cultures for Klebsiella, Salmonella, and E. coli were 66%, 82%, and 88%, respectively, of meat samples and 78%, 26%, and 56%, respectively, of fermented food samples. Escherichia coli O157:H7, Shigella, and Yersinia were not detected in any of the samples by both standard and m-PCR methods. The developed m-PCR assay showed comparable results with the traditional culture technique proving its rapid and reliable detection of six foodborne pathogens and hygiene indicators in food.
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Early treatment with fluvoxamine, bromhexine, cyproheptadine, and niclosamide to prevent clinical deterioration in patients with symptomatic COVID-19: a randomized clinical trial
(2024-04-01) Wannigama D.L.; Hurst C.; Phattharapornjaroen P.; Hongsing P.; Sirichumroonwit N.; Chanpiwat K.; Ali A.H.; Storer R.J.; Ounjai P.; Kanthawee P.; Ngamwongsatit N.; Kupwiwat R.; Kupwiwat C.; Brimson J.M.; Devanga Ragupathi N.K.; Charuluxananan S.; Leelahavanichkul A.; Kanjanabuch T.; Higgins P.G.; Badavath V.N.; Amarasiri M.; Verhasselt V.; Kicic A.; Chatsuwan T.; Pirzada K.; Jalali F.; Reiersen A.M.; Abe S.; Ishikawa H.; Tanasatitchai C.; Amphol S.; Nantawong L.; Sangchan P.; Sinkajarern V.; Phoonakh T.; Utenpattanun P.; Sithu Shein A.M.; Vitoonpong T.; Chongthavonsatit N.; Mankong Y.; Chaichana P.; Yaithet J.; Pongprajak D.; Traimuangpak S.; Saksirisampant G.; Lamloeskittinon P.; Hamdy A.A.; Kosasih S.S.; Luk-in S.S.; Wannigama D.L.; Mahidol University
Background: Repurposed drugs with host-directed antiviral and immunomodulatory properties have shown promise in the treatment of COVID-19, but few trials have studied combinations of these agents. The aim of this trial was to assess the effectiveness of affordable, widely available, repurposed drugs used in combination for treatment of COVID-19, which may be particularly relevant to low-resource countries. Methods: We conducted an open-label, randomized, outpatient, controlled trial in Thailand from October 1, 2021, to June 21, 2022, to assess whether early treatment within 48-h of symptoms onset with combinations of fluvoxamine, bromhexine, cyproheptadine, and niclosamide, given to adults with confirmed mild SARS-CoV-2 infection, can prevent 28-day clinical deterioration compared to standard care. Participants were randomly assigned to receive treatment with fluvoxamine alone, fluvoxamine + bromhexine, fluvoxamine + cyproheptadine, niclosamide + bromhexine, or standard care. The primary outcome measured was clinical deterioration within 9, 14, or 28 days using a 6-point ordinal scale. This trial is registered with ClinicalTrials.gov (NCT05087381). Findings: Among 1900 recruited, a total of 995 participants completed the trial. No participants had clinical deterioration by day 9, 14, or 28 days among those treated with fluvoxamine plus bromhexine (0%), fluvoxamine plus cyproheptadine (0%), or niclosamide plus bromhexine (0%). Nine participants (5.6%) in the fluvoxamine arm had clinical deterioration by day 28, requiring low-flow oxygen. In contrast, most standard care arm participants had clinical deterioration by 9, 14, and 28 days. By day 9, 32.7% (110) of patients in the standard care arm had been hospitalized without requiring supplemental oxygen but needing ongoing medical care. By day 28, this percentage increased to 37.5% (21). Additionally, 20.8% (70) of patients in the standard care arm required low-flow oxygen by day 9, and 12.5% (16) needed non-invasive or mechanical ventilation by day 28. All treated groups significantly differed from the standard care group by days 9, 14, and 28 (p < 0.0001). Also, by day 28, the three 2-drug treatments were significantly better than the fluvoxamine arm (p < 0.0001). No deaths occurred in any study group. Compared to standard care, participants treated with the combination agents had significantly decreased viral loads as early as day 3 of treatment (p < 0.0001), decreased levels of serum cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) as early as day 5 of treatment, and interleukin-8 (IL-8) by day 7 of treatment (p < 0.0001) and lower incidence of post-acute sequelae of COVID-19 (PASC) symptoms (p < 0.0001). 23 serious adverse events occurred in the standard care arm, while only 1 serious adverse event was reported in the fluvoxamine arm, and zero serious adverse events occurred in the other arms. Interpretation: Early treatment with these combinations among outpatients diagnosed with COVID-19 was associated with lower likelihood of clinical deterioration, and with significant and rapid reduction in the viral load and serum cytokines, and with lower burden of PASC symptoms. When started very soon after symptom onset, these repurposed drugs have high potential to prevent clinical deterioration and death in vaccinated and unvaccinated COVID-19 patients. Funding: Ped Thai Su Phai (Thai Ducks Fighting Danger) social giver group.
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Effect of Antimicrobial Peptide BiF2_5K7K on Contaminated Bacteria Isolated from Boar Semen and Semen Qualities during Preservation and Subsequent Fertility Test on Pig Farm
(2024-07-01) Keeratikunakorn K.; Chanapiwat P.; Aunpad R.; Ngamwongsatit N.; Kaeoket K.; Keeratikunakorn K.; Mahidol University
The purpose of this study was to determine the impact of an antimicrobial peptide, BiF2_5K7K, on semen quality and bacterial contamination in boar semen doses used for artificial insemination. A key factor affecting semen quality and farm production is bacterial contamination in semen doses. Using antibiotics in a semen extender seems to be the best solution for minimizing bacterial growth during semen preservation. However, concern regarding antibiotic-resistant microorganisms has grown globally. As a result, antimicrobial peptides have emerged as interesting alternative antimicrobial agents to replace the current antibiotics used in semen extenders. BiF2_5K7K is an antimicrobial peptide that can inhibit Gram-negative and Gram-positive bacteria isolated from boar semen and sow vaginal discharge. In this study, ten fresh boar semen samples were collected and diluted with one of two types of semen extender: with (positive control) or without (negative control) an antibiotic (i.e., gentamicin). The semen extender without an antibiotic contained antimicrobial peptide BiF2_5K7K at different concentrations (15.625, 31.25, 62.5, and 125 µg/mL). The samples were stored at 18 °C until use. Semen quality parameters were assessed on days 0, 1, 3, and 5, and the total bacterial count was also evaluated at 0, 24, 36, 48, and 72 h after storage. A fertility test on a pig farm was also performed via sow insemination with a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. No significant difference was found in terms of semen quality on days 0 or 1. On days 3 and 5, the total motility, progressive motility, and viability remained normal in the 15.625 and 31.25 µg/mL groups. However, the sperm parameters decreased starting on day 3 for the 125 µg/mL group and on day 5 for the 62.5 µg/mL group. For total bacterial count at 0, 24, 36, 48, and 72 h, the lowest bacterial count was found in the positive control group, and the highest bacterial count was found in the negative control group compared with the other groups. Comparing antimicrobial peptide groups from 0 to 48 h, the lowest bacterial count was found in the 125 µg/mL group, and the highest bacterial count was found in the 15.625 µg/mL group. For the fertility test, artificial insemination was conducted by using a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. The results showed a superior pregnancy rate, farrowing rate, and total number of piglets born compared with artificial insemination conducted using a commercial extender plus antibiotic. In conclusion, BiF2_5K7K can inhibit bacterial growth in extended boar semen for 24 h, and thereafter, the bacterial count slightly increases. However, the increase in the number of bacterial counts from days 0 to 3 had no negative effect on sperm quality in the positive control, 15.625, or 31.25 µg/mL groups. This indicates that BiF2_5K7K might be an antimicrobial peptide candidate with potential for use as an alternative antimicrobial agent to replace the conventional antibiotic used in boar semen extenders.
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Enhancement by pyrazolones of colistin efficacy against mcr-1-expressing E. coli: an in silico and in vitro investigation
(2023-01-01) Hanpaibool C.; Ounjai P.; Yotphan S.; Mulholland A.J.; Spencer J.; Ngamwongsatit N.; Rungrotmongkol T.; Mahidol University
Owing to the emergence of antibiotic resistance, the polymyxin colistin has been recently revived to treat acute, multidrug-resistant Gram-negative bacterial infections. Positively charged colistin binds to negatively charged lipids and damages the outer membrane of Gram-negative bacteria. However, the MCR-1 protein, encoded by the mobile colistin resistance (mcr) gene, is involved in bacterial colistin resistance by catalysing phosphoethanolamine (PEA) transfer onto lipid A, neutralising its negative charge, and thereby reducing its interaction with colistin. Our preliminary results showed that treatment with a reference pyrazolone compound significantly reduced colistin minimal inhibitory concentrations in Escherichia coli expressing mcr-1 mediated colistin resistance (Hanpaibool et al. in ACS Omega, 2023). A docking-MD combination was used in an ensemble-based docking approach to identify further pyrazolone compounds as candidate MCR-1 inhibitors. Docking simulations revealed that 13/28 of the pyrazolone compounds tested are predicted to have lower binding free energies than the reference compound. Four of these were chosen for in vitro testing, with the results demonstrating that all the compounds tested could lower colistin MICs in an E. coli strain carrying the mcr-1 gene. Docking of pyrazolones into the MCR-1 active site reveals residues that are implicated in ligand–protein interactions, particularly E246, T285, H395, H466, and H478, which are located in the MCR-1 active site and which participate in interactions with MCR-1 in ≥ 8/10 of the lowest energy complexes. This study establishes pyrazolone-induced colistin susceptibility in E. coli carrying the mcr-1 gene, providing a method for the development of novel treatments against colistin-resistant bacteria.
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Exploring indoor and outdoor dust as a potential tool for detection and monitoring of COVID-19 transmission
(2024-03-15) Anupong S.; Chadsuthi S.; Hongsing P.; Hurst C.; Phattharapornjaroen P.; Ali A.H.; Fernandez S.; Huang A.T.; Vatanaprasan P.; Saethang T.; Luk-in S.; Storer R.J.; Ounjai P.; Devanga Ragupathi N.K.; Kanthawee P.; Ngamwongsatit N.; Badavath V.N.; Thuptimdang W.; Leelahavanichkul A.; Kanjanabuch T.; Miyanaga K.; Cui L.; Nanbo A.; Shibuya K.; Kupwiwat R.; Sano D.; Furukawa T.; Sei K.; Higgins P.G.; Kicic A.; Singer A.C.; Chatsuwan T.; Trowsdale S.; Abe S.; Ishikawa H.; Amarasiri M.; Modchang C.; Wannigama D.L.; Anupong S.; Mahidol University
This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.
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Feasibility of crude F4 fimbriae extract as a vaccine candidate for preventing Escherichia coli-induced diarrhea in piglets
(2023-10-01) Nguyet L.T.Y.; Ounjai P.; Kaeoket K.; Ngamwongsatit N.; Nguyet L.T.Y.; Mahidol University
Background and Aim: Enterotoxigenic Escherichia coli (ETEC) poses a substantial risk of neonatal diarrhea and postweaning diarrhea among piglets, with F4+ ETEC strains emerging as a particularly challenging issue within the pig farming industry. This study aimed to introduce a straightforward approach for generating a crude extract of F4 fimbriae that shows promise as an antigenic determinant for potential vaccination strategies. Materials and Methods: A crude F4 fimbriae extract was obtained from F4+ ETEC using a combination of heat shock and homogenization techniques. Subsequently, three 4-week-old piglets were immunized with a primary dose of 150 µg and a booster dose 2 weeks later. Blood samples were collected to evaluate the level of serum F4-specific antibodies using an enzyme-linked immunosorbent assay. Results: Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography tandem-mass spectrometry techniques unveiled crucial insights into the composition of the crude F4 fimbriae extract. Notably, a distinct prominent band (~24 kDa) was identified, corresponding to the size of FaeG, the major subunit of F4 fimbriae. Regarding antibody response, there was a remarkable disparity between the levels of serum immunoglobulin (Ig)G and IgA antibodies targeting F4 compared with other E. coli strains (F18+ ETEC, F41+ ETEC, and F4−F18−F41− EC), as well as with the unvaccinated control group (p < 0.01). Specifically, the levels of IgG antibodies against other E. coli strains were also significantly higher than those observed in the unvaccinated control group (p < 0.05). Conclusion: Our findings suggest that the crude F4 fimbriae extracts obtained using our simple extraction method induce specific immune responses against F4+ E. coli and stimulate cross-immunity against other E. coli strains. Therefore, our method shows potential for use in future vaccine development against diarrhea in pigs caused by E. coli.
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First detection of multidrug-resistant and toxigenic Pasteurella aerogenes in sow vaginal discharge: a novel threat to swine health in Thailand
(2024-10-26) Keeratikunakorn K.; Kaeoket K.; Ounjai P.; Wannigama D.L.; Chatsuwan T.; Ngamwongsatit N.; Keeratikunakorn K.; Mahidol University
Pasteurella aerogenes has been implicated in reproductive disorders in sows, yet its prevalence and characteristics in vaginal discharge are not well understood. This study aimed to detect P. aerogenes in sow vaginal discharge samples and investigate its antibiotic resistance profile, toxin genes, and toxicity. P. aerogenes was isolated from 40% (8/20) of samples. Antimicrobial susceptibility testing revealed universal resistance to amoxicillin-clavulanate (4:1), with 87.5% of isolates also resistant to oxytetracycline, amoxicillin, ceftriaxone, and enrofloxacin. The colistin resistance gene mcr-2 was detected in 75% of isolates, while class 1 integron (int1) was found in 12.5%. The pax toxin gene cluster was present in 75% of isolates. Toxicity assays using Panagrellus redivivus demonstrated dose-dependent effects of P. aerogenes supernatant containing pax toxins. This study represents the first report of P. aerogenes isolation from sow vaginal discharge in Thailand. The high prevalence of antibiotic resistance, presence of the mcr-2 gene, and toxicity of pax toxin-positive isolates suggest that P. aerogenes may be an underestimated factor in swine reproductive health. These findings highlight the need for further investigation into the role of P. aerogenes in sow reproductive disorders and its potential impact on swine production.
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Four New Sequence Types and Molecular Characteristics of Multidrug-Resistant Escherichia coli Strains from Foods in Thailand
(2024-10-01) Thadtapong N.; Chaturongakul S.; Tangphatsornruang S.; Sonthirod C.; Ngamwongsatit N.; Aunpad R.; Thadtapong N.; Mahidol University
The presence of antibiotic-resistant Escherichia coli in food is a serious and persistent problem worldwide. In this study, 68 E. coli strains isolated from Thai food samples were characterized. Based on antibiotic susceptibility assays, 31 of these isolates (45.59%) showed multiple antibiotic resistance (MAR) index values > 0.2, indicating high exposure to antibiotics. Among these, strain CM24E showed the highest resistance (it was resistant to ten antibiotics, including colistin and imipenem). Based on genome sequencing, we identified four isolates (namely, CF25E, EF37E, NM10E1, and SF50E) with novel Achtman-scheme multi-locus sequence types (STs) (ST14859, ST14866, ST14753, and ST14869, respectively). Clermont phylogrouping was used to subtype the 68 researched isolates into five Clermont types, mainly A (51.47%) and B1 (41.18%). The blaEC gene was found only in Clermont type A, while the blaEC-13 gene was predominant in Clermont type B1. A correlation between genotypes and phenotypes was found only in Clermont type B1, which showed a strong positive correlation between the presence of an afa operon and yersiniabactin-producing gene clusters with the colistin resistance phenotype. Strain SM47E1, of Clermont type B2, carried the highest number of predicted virulence genes. In summary, this study demonstrates the pressing problems posed by the prevalence and potential transmission of antimicrobial resistance and virulence genes in the food matrix.
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High prevalence of mgrB-mediated colistin resistance among carbapenem-resistant Klebsiella pneumoniae is associated with biofilm formation, and can be overcome by colistin-EDTA combination therapy
(2022-12-01) Shein A.M.S.; Wannigama D.L.; Higgins P.G.; Hurst C.; Abe S.; Hongsing P.; Chantaravisoot N.; Saethang T.; Luk-in S.; Liao T.; Nilgate S.; Rirerm U.; Kueakulpattana N.; Srisakul S.; Aryukarn A.; Laowansiri M.; Hao L.Y.; Yonpiam M.; Ragupathi N.K.D.; Techawiwattanaboon T.; Ngamwongsatit N.; Amarasiri M.; Ounjai P.; Kupwiwat R.; Phattharapornjaroen P.; Badavath V.N.; Leelahavanichkul A.; Kicic A.; Chatsuwan T.; Mahidol University
The global prevalence of colistin-resistant Klebsiella pneumoniae (ColRkp) facilitated by chromosomal and plasmid-mediated Ara4N or PEtN-remodeled LPS alterations has steadily increased with increased colistin usage for treating carbapenem-resistant K. pneumoniae (CRkp). Our study demonstrated the rising trend of ColRkp showing extensively and pandrug-resistant characteristics among CRkp, with a prevalence of 28.5%, which was mediated by chromosomal mgrB, pmrB, or phoQ mutations (91.5%), and plasmid-mediated mcr-1.1, mcr-8.1, mcr-8.2 alone or in conjunction with R256G PmrB (8.5%). Several genetic alterations in mgrB (85.1%) with increased expressions of Ara4N-related phoPQ and pmrK were critical for establishing colistin resistance in our isolates. In this study, we discovered the significant associations between extensively drug-resistant bacteria (XDR) and pandrug-resistant bacteria (PDR) ColRkp in terms of moderate, weak or no biofilm-producing abilities, and altered expressions of virulence factors. These ColRkp would therefore be very challenging to treat, emphasizing for innovative therapy to combat these infections. Regardless of the underlying colistin-resistant mechanisms, colistin-EDTA combination therapy in this study produced potent synergistic effects in both in vitro and in vivo murine bacteremia, with no ColRkp regrowth and improved animal survival, implying the significance of colistin-EDTA combination therapy as systemic therapy for unlocking colistin resistance in ColRkp-associated bacteremia.
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High prevalence of multidrug-resistant Salmonella enterica in Thailand food markets: Insights from complete genome and phenotypic characterization of ESBL-producing strains
(2025-12-02) Phaophu P.; Thadtapong N.; Wirth S.E.; Gray A.S.; Dangsuk S.; Ngamwongsatit N.; Aunpad R.; Chaturongakul S.; Phaophu P.; Mahidol University
Salmonella contamination in the food chain is a common root of foodborne outbreaks. The transmission of antimicrobial resistance among Salmonella and food bacteria raises global public health concern. As a major food exporter and travel hub in Southeast Asia, Thailand is particularly susceptible. To better understand the circulating Salmonella, we characterized 74 non-typhoidal Salmonella isolates previously obtained from Thailand food markets. Whole genome sequencing, drug susceptibility testing, and host invasion efficiency assays were performed. The most prevalent serotype was Salmonella serotype Rissen (32.43%) followed by Derby (12.16%). Fifty-two isolates were multidrug-resistant (MDR), with their primary resistance pattern being ampicillin, streptomycin, and tetracycline resistance. The most resistant strain was S. Rissen exhibiting resistance to 13 tested drugs, including third-generation cephalosporin. Six cephalosporin-resistant isolates were identified, four of which were extended-spectrum β-lactamase (ESBL)-producing strains harboring the blaCTX-M-14 or blaCTX-M-55 ESBL gene. Complete genomes showed that three ESBL-producing isolates contained plasmid-mediated ESBL, and the other was chromosomal-mediated. In vitro invasion efficiencies of ESBL-producing strains into mucous-producing human colorectal adenocarcinoma HT29-MTX-E12 cells were compared to the reference strain Salmonella Typhimurium LT2. Statistically significant higher invasion efficiency in the chromosomal-mediated ESBL strain was found. Interestingly, the ESBL plasmid detected during this study was highly similar to a plasmid previously isolated from a Thai patient. This study highlights that the majority of Salmonella isolated from Thai foods are MDR and potentially ESBL-producing strains, highlighting the need for continued surveillance and intervention strategies. IMPORTANCE In Thailand, fermented foods are typically consumed raw, and pork is considered a delicacy of Thai cuisine. The presence of multidrug resistant (MDR) foodborne pathogens in these food types raises concern and presents a risk to public health. Here, we report that Salmonella Rissen was the most prevalent serotype isolated from these food samples in Thailand. All isolates carried virulence gene clusters crucial for pathogenesis, and more than 70% of isolates were MDR strains. Four of the MDR strains were ESBL-producing. Whole genome sequence analysis and phenotypic characterizations revealed that chromosome-mediated ESBL strains possessed higher in vitro invasion efficiency than plasmid-mediated ESBL strains. This study highlights two key public health threats: the risk of acquiring difficult-to-treat MDR Salmonella infections from undercooked food and the circulation of AMR plasmids in fresh markets in Thailand.
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Intranasal phage therapy overcomes antibody neutralization challenges in pulmonary Pseudomonas aeruginosa infections
(2025-12-01) Wannigama D.L.; Shao J.; Sun H.; Wang Y.; Hurst C.; Monk P.N.; Amarasiri M.; Phattharapornjaroen P.; Ditcham W.G.F.; Htun T.S.; Luk-in S.; Shimotai Y.; Ngamwongsatit N.; Ishikawa H.; Ragupathi N.K.D.; Pletzer D.; Kanjanabuch T.; Khatib A.; Miyanaga K.; Cui L.; Shibuya K.; Higgins P.G.; Kicic A.; Hongsing P.; Zhao J.; Abe S.; Hamamoto H.; Wannigama D.L.; Mahidol University
Phage therapy is a promising approach against multidrug-resistant infections, yet systemic administration can lead to incomplete cures. We investigated the distribution, immune responses, and efficacy of the therapeutic phage KPP10 delivered via intranasal or intraperitoneal (IP) routes in murine Pseudomonas aeruginosa lung infection models. Intranasal pre-treatment achieved markedly higher localization of KPP10 in the lungs and bronchoalveolar compartment compared to IP delivery. Intranasal administration elicited minimal systemic antibody responses, whereas IP injection triggered significant IgG, IgM, and IgA production. Antibody responses did not differ significantly between doses. In acute and chronic infection models, intranasal KPP10 significantly improved survival (p < 0.01) and reduced lung bacterial loads relative to IP injection. Importantly, IP treatment was associated with bacterial rebound after day 14 in chronic infection, whereas intranasal dosing sustained bacterial clearance. These findings demonstrate that intranasal delivery enhances pulmonary localization, minimizes antibody-mediated neutralization, and provides superior therapeutic efficacy, highlighting its potential as a more effective route for phage therapy against P. aeruginosa lung infections.
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Lactiplantibacillus plantarum dfa1 outperforms enterococcus faecium dfa1 on anti-obesity in high fat-induced obesity mice possibly through the differences in gut dysbiosis attenuation, despite the similar anti-inflammatory properties
(2022-01-01) Ondee T.; Pongpirul K.; Janchot K.; Kanacharoen S.; Lertmongkolaksorn T.; Wongsaroj L.; Somboonna N.; Ngamwongsatit N.; Leelahavanichkul A.; Mahidol University
Fat reduction and anti-inflammation are commonly claimed properties of probiotics. Lactiplan-tibacillus plantarum and Enterococcus faecium were tested in high fat-induced obesity mice and in vitro experiments. After 16 weeks of probiotics, L. plantarum dfa1 outperforms E. faecium dfa1 on the anti-obesity property as indicated by body weight, regional fat accumulation, serum cholesterol, inflammatory cytokines (in blood and colon tissue), and gut barrier defect (FITC-dextran assay). With fecal microbiome analysis, L. plantarum dfa1 but not E. faecium dfa1 reduced fecal abundance of pathogenic Proteobacteria without an alteration in total Gram-negative bacteria when compared with non-probiotics obese mice. With palmitic acid induction, the condition media from both probiotics similarly attenuated supernatant IL-8, improved enterocyte integrity and down-regulated cholesterol absorption-associated genes in Caco-2 cell (an enterocyte cell line) and reduced supernatant cytokines (TNF-α and IL-6) with normalization of cell energy status (extracellular flux analysis) in bone-marrow-derived macrophages. Due to the anti-inflammatory effect of the condition media of both probiotics on palmitic acid-activated enterocytes was neutralized by amylase, the active anti-inflammatory molecules might, partly, be exopolysaccharides. As L. plantarum dfa1 out-performed E. faecium dfa1 in anti-obesity property, possibly through the reduced fecal Proteobacteria, with a similar anti-inflammatory exopolysaccharide; L. plantarum is a potentially better option for anti-obesity than E. faecium.

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