WQ-3810, a fluoroquinolone with difluoropyridine derivative as the R1 group exerts high potency against quinolone-resistant Campylobacter jejuni.

Quinolone-resistant Campylobacter jejuni have been increasing worldwide. Quinolones exert their antibacterial activity by inhibiting DNA gyrase, but most of the isolates acquire quinolone resistance via an amino acid substitution in the A subunit of DNA gyrase. WQ-3810 is a quinolone antibiotic that has been reported to have high potency even to DNA gyrase with amino acid substitutions in several bacterial species; however, there was no information on C. jejuni. Hence, this study aimed to evaluate the activity of WQ-3810 to inhibit wild-type/mutant DNA gyrases of C. jejuni and the bacterial growth for accessing the potency for the treatment of quinolone-resistant C. jejuni infection. The inhibitory activity of WQ-3810 was assessed and compared with ciprofloxacin and nalidixic acid by calculating the half maximal inhibitory concentration (IC50) against wild-type/mutant DNA gyrases. Next, the minimum inhibitory concentration (MIC) of WQ-3810 and five other quinolones was determined for C. jejuni including quinolone-resistant strains with amino acid substitutions in GyrA. Furthermore, the interaction between WQ-3810 and wild-type/mutant DNA gyrase was speculated using docking simulations. The IC50 of WQ-3810 against wild-type DNA gyrase was 1.03 µg/mL and not different from that of ciprofloxacin. However, those of WQ-3810 against mutant DNA gyrases were much lower than ciprofloxacin. The MICs of WQ-3810 ranged <0.016-0.031 µg/mL and were the lowest against both quinolone-susceptible and quinolone-resistant strains among the examined quinolones. The results obtained by the docking simulation agreed well with this observation. WQ-3810 seems to be a promising antimicrobial agent for the infections caused by quinolone-resistant C. jejuni. IMPORTANCE: WQ-3810, a relatively new quinolone antibiotic, demonstrates exceptional antibacterial properties against certain pathogens in previous studies. However, its efficacy against quinolone-resistant Campylobacter jejuni was not previously reported. The prevalence of quinolone-resistant C. jejuni as a cause of foodborne illnesses is increasing, prompting this investigation into the effectiveness of WQ-3810 as a countermeasure. This study revealed high inhibitory activity of WQ-3810 against both wild-type and mutant DNA gyrases of C. jejuni. WQ-3810 was equally efficacious as ciprofloxacin against wild-type DNA gyrases but showed superior effectiveness against mutant DNA gyrases. WQ-3810 also demonstrated the lowest minimum inhibitory concentrations, highlighting its enhanced potency against both susceptible and resistant strains of C. jejuni. This observation was well supported by the results of the in silico analysis. Consequently, WQ-3810 exhibits a higher level of bactericidal activity compared to existing quinolones in combating both susceptible and resistant C. jejuni isolates.

Chlamydia trachomatis L2 434/Bu readily activates glycolysis under hypoxia for efficient metabolism.

To understand why Chlamydia trachomatis (Ct) (L2/434/Bu) favors hypoxia, we examined the dynamics of infected cells using a glycolysis-related PCR array and metabolomic analysis, along with the perturbation of nucleotide synthesis. Our findings revealed that, compared to normoxia, hypoxia with infection significantly and selectively upregulates the expression of genes related to glycolysis, glycogen degradation, and the pentose phosphate pathway. Furthermore, hypoxia induced a significant decrease in metabolite levels, particularly methionine-related metabolites, independent of infection, indicating efficient metabolism under hypoxia. Additionally, the perturbation of nucleotide synthesis with adenosine derivatives impaired Ct growth. Collectively, our results suggest that Ct favors a hypoxic environment with efficient metabolism, in which Ct readily activates glycolysis responsible for stable nucleotide synthesis as well as ATP supply.

Epidemiology and molecular characterization of Mycobacterium tuberculosis including a drug-resistant strain associated with mortality of Asian elephants in Nepal 2019-2022.

Tuberculosis (TB) is an emerging threat to the survival of elephants in Nepal. We investigated the lung tissue samples from nine elephants that died from 2019 to 2022 in Nepal using culture, conventional PCR, and loop-mediated isothermal amplification (LAMP) and then performed genotyping of five PCR-positive isolates to understand the possible transmission dynamics of Mycobacterium tuberculosis (Mtb). Results showed that two-thirds (6/9) of elephants were confirmed to be infected from Mtb by LAMP, 5/9 by PCR, and 4/9 by culture. Genotyping of Mtb isolates showed that elephants were infected with the Indo-Oceanic and Beijing lineages including an isoniazid-resistant Beijing lineage. MIRU-VNTR-based phylogeny, gyrA, and katG sequencing showed the possibility of ongoing transmission of Indo-Oceanic lineages and likely transmission of the drug-resistant Beijing lineage from human to elephant. Implementation of comprehensive surveillance and preventive measures are urgently needed to address this zoonotic disease and protect elephants from TB in Nepal.

Wide distribution of plasmid mediated quinolone resistance gene, qnrS, among Salmonella spp. isolated from canal water in Thailand.

AIMS: This research focused on assessing the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and antimicrobial susceptibility in Salmonella strains isolated from Thai canal water. METHODS AND RESULTS: From 2016 to 2020, 333 water samples were collected from six canals across Bangkok, Thailand. Salmonella spp. was isolated, PMQR genes were detected through polymerase chain reactions, and the antimicrobial susceptibility was examined using the disk diffusion method. The results indicated a 92.2% prevalence of Salmonella spp. in canal water, being serogroups B and C the most frequently detected. Overall, 35.3% of isolates harbored PMQR genes, being qnrS the most prevalent gene (97.2%, n = 137/141). Other PMQR genes, including qnrB, qnrD, oqxAB, and aac(6')-Ib-cr, were detected. Notably, six isolates harbored multiple PMQR genes. Furthermore, 9.3% and 3.8% of the overall isolates were resistant to nalidixic acid (NAL) and ciprofloxacin (CIP), respectively. PMQR-positive isolates showed higher rates of non-susceptibility to both NAL (48.2%, n = 68/141) and CIP (92.2%, n = 130/141) compared to PMQR-negative isolates (NAL: 8.9%, n = 23/258; CIP: 11.2%, n = 30/258). CONCLUSIONS: The high prevalence of Salmonella spp., significant PMQR-positive, and reduced susceptibility isolates in canal water is of public health concern in Bangkok.

Interplay between Amino Acid Substitution in GyrA and QnrB19: Elevating Fluoroquinolone Resistance in Salmonella Typhimurium.

Globally, there have been increasing reports of antimicrobial resistance in nontyphoidal Salmonella (NTS), which can develop into severe and potentially life-threatening diarrhea. This study focuses on the synergistic effects of DNA gyrase mutations and plasmid-mediated quinolone resistance (PMQR) genes, specifically qnrB19, on fluoroquinolone (FQ) resistance in Salmonella Typhimurium. By utilizing recombinant mutants, GyrAS83F and GyrAD87N, and QnrB19's, we discovered a significant increase in fluoroquinolones resistance when QnrB19 is present. Specifically, ciprofloxacin and moxifloxacin's inhibitory concentrations rose 10- and 8-fold, respectively. QnrB19 was found to enhance the resistance capacity of mutant DNA gyrases, leading to high-level FQ resistance. Additionally, we observed that the ratio of QnrB19 to DNA gyrase played a critical role in determining whether QnrB19 could protect DNA gyrase against FQ inhibition. Our findings underscore the critical need to understand these resistance mechanisms, as their coexistence enables bacteria to withstand therapeutic FQ levels, posing a significant challenge to treatment efficacy.

Genomic analysis of Salmonella isolated from canal water in Bangkok, Thailand.

Antimicrobial resistance (AMR) poses an escalating global public health threat. Canals are essential in Thailand, including the capital city, Bangkok, as agricultural and daily water sources. However, the characteristic and antimicrobial-resistance properties of the bacteria in the urban canals have never been elucidated. This study employed whole genome sequencing to characterize 30 genomes of a causal pathogenic bacteria, Salmonella enterica, isolated from Bangkok canal water between 2016 and 2020. The dominant serotype was Salmonella Agona. In total, 35 AMR genes and 30 chromosomal-mediated gene mutations were identified, in which 21 strains carried both acquired genes and mutations associated with fluoroquinolone resistance. Virulence factors associated with invasion, adhesion, and survival during infection were detected in all study strains. 75.9% of the study stains were multidrug-resistant and all the strains harbored the necessary virulence factors associated with salmonellosis. One strain carried 20 resistance genes, including mcr-3.1, mutations in GyrA, ParC, and ParE, and typhoid toxin-associated genes. Fifteen plasmid replicon types were detected, with Col(pHAD28) being the most common type. Comparative analysis of nine S. Agona from Bangkok and 167 from public databases revealed that specific clonal lineages of S. Agona might have been circulating between canal water and food sources in Thailand and globally. These findings provide insight into potential pathogens in the aquatic ecosystem and support the inclusion of environmental samples into comprehensive AMR surveillance initiatives as part of a One Health approach. This approach aids in comprehending the rise and dissemination of AMR and devising sustainable intervention strategies.IMPORTANCEBangkok is the capital city of Thailand and home to a large canal network that serves the city in various ways. The presence of pathogenic and antimicrobial-resistant Salmonella is alarming and poses a significant public health risk. The present study is the first characterization of the genomic of Salmonella strains from Bangkok canal water. Twenty-two of 29 strains (75.9%) were multidrug-resistant Salmonella and all the strains carried essential virulence factors for pathogenesis. Various plasmid types were identified in these strains, potentially facilitating the horizontal transfer of AMR genes. Additional investigations indicated a potential circulation of S. Agona between canal water and food sources in Thailand. The current study underscores the role of environmental water in an urban city as a reservoir of pathogens and these data obtained can serve as a basis for public health risk assessment and help shape intervention strategies to combat AMR challenges in Thailand.

Effect of WQ-3334 on Campylobacter jejuni carrying a DNA gyrase with dominant amino acid substitutions conferring quinolone resistance.

INTRODUCTION: Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of quinolones-resistant Campylobacter jejuni poses a significant challenge. The predominant mechanism of quinolones-resistance in this bacterium involves point mutations in the gyrA, resulting in amino acid substitution from threonine to isoleucine at 86th position, representing more than 90% of mutant DNA gyrase, and aspartic acid to asparagine at 90th position. WQ-3334, a novel quinolone, has demonstrated strong inhibitory activity against various bacteria. This study aims to investigate the effectiveness of WQ-3334, and its analogues, WQ-4064 and WQ-4065, with a unique modification in R1 against quinolones-resistant C. jejuni. METHODS: The structure-activity relationship of the examined drugs was investigated by measuring IC50 and their antimicrobial activities were accessed by MIC against C. jejuni strains. Additionally, in silico docking simulations were carried out using the crystal structure of the Escherichia coli DNA gyrase. RESULT: WQ-3334 exhibited the lowest IC50 against WT (0.188 ± 0.039 mg/L), T86I (11.0 ± 0.7 mg/L) and D90 N (1.60 ± 0.28 mg/L). Notably, DNA gyrases with T86I substitutions displayed the highest IC50 values among the examined WQ compounds. Moreover, WQ-3334 demonstrated the lowest MICs against wild-type and mutant strains. The docking simulations further confirmed the interactions between WQ-3334 and DNA gyrases. CONCLUSION: WQ-3334 with 6-amino-3,5-difluoropyridine-2-yl at R1 severed as a remarkable candidate for the treatment of foodborne diseases caused by quinolones-resistant C. jejuni as shown by the high inhibitory activity against both wild-type and the predominant quinolones-resistant strains.

The first insight into Mycobacterium tuberculosis complex isolates in the lower northern region in Thailand.

BACKGROUND: Tuberculosis (TB) remains an important infectious disease and different genotypes have been reported. This study aimed to investigate the genetic diversity and molecular epidemiology of TB in the lower northern region of Thailand, where genotyping data are limited. METHODS: A total of 159 Mycobacterium tuberculosis complex (MTBC) isolates from this region were genotyped by spoligotyping and the major spoligotypes were further subdivided by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method. RESULTS: Spoligotyping identified 34 types and classified them into 14 clusters. East African-Indian (EAI) groups were the most frequent (44.7%), followed by Beijing (36.5%), with a higher prevalence of drug resistance. By 15-loci MIRU-VNTR typing, the major groups of the Beijing and EAI2_NTB were further differentiated into 44 and 21 subtypes forming 9 and 5 subclusters with cluster rates of 0.26 and 0.44, respectively. The Hunter-Gaston Discriminatory Index among the Beijing and EAI2_NTB groups were 0.987 and 0.931, respectively, indicating high diversity. CONCLUSIONS: This is the first look at the MTBC genotypes in the lower northern region of Thailand, which could aid in understanding the distribution and potential spread of MTBC and Mycobacterium bovis in the target region to support TB control in Thailand.

Whole-Genome Investigation of Zoonotic Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolated from Pigs and Humans in Thailand.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been widespread globally in pigs and humans for decades. Nasal colonization of LA-MRSA is regarded as an occupational hazard to people who are regularly involved in livestock production. Our previous study suggested pig-to-human transmission caused by LA-MRSA clonal complex (CC) 398, using traditional molecular typing methods. Instead, this study aimed to investigate the zoonotic transmission of LA-MRSA CC398 using whole genome sequencing (WGS) technologies. A total of 63 LA-MRSA isolates were identified and characterized in Thailand. Further, the 16 representatives of LA-MRSA CC9 and CC398, including porcine and worker isolates, were subjected to WGS on the Illumina Miseq platform. Core-genome single nucleotide polymorphism (SNP)-based analyses verify the zoonotic transmission caused by LA-MRSA CC398 in two farms. WGS-based characterization suggests the emergence of a novel staphylococcal cassette chromosome (SCC) mec type, consisting of multiple cassette chromosome recombinase (ccr) gene complexes via genetic recombination. Additionally, the WGS analyses revealed putative multi-resistant plasmids and several cross-resistance genes, conferring resistance against drugs of last resort used in humans such as quinupristin/dalfopristin and linezolid. Significantly, LA-MRSA isolates, in this study, harbored multiple virulence genes that may become a serious threat to an immunosuppressive population, particularly for persons who are in close contact with LA-MRSA carriers.

Exploration of the novel fluoroquinolones with high inhibitory effect against quinolone-resistant DNA gyrase of Salmonella Typhimurium.

IMPORTANCE: Quinolone-resistant nontyphoidal Salmonella is a pressing public health concern, demanding the exploration of novel treatments. In this study, we focused on two innovative synthetic fluoroquinolones, WQ-3034 and WQ-3154. Our findings revealed that these new compounds demonstrate potent inhibitory effects, even against mutant strains that cause resistance to existing quinolones. Hence, WQ-3034 and WQ-3154 could potentially be effective therapeutic agents against quinolone-resistant Salmonella Typhimurium. Furthermore, the data obtained in this study will be baseline information for antimicrobial drug development.

Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

Molecular Characterisation of Mycobacterium bovis Isolates from Cattle Slaughtered in Adamawa and Gombe States, North-Eastern Nigeria.

Bovine tuberculosis is endemic in Nigeria with control measures as provided by the laws of the country being minimally enforced mostly at the abattoirs only. This study focused on bovine tuberculosis in Adamawa and Gombe States. Tuberculosis lesions were observed in 183 of 13,688 slaughtered cattle in the regions between June and December 2020. Analysis of tissue samples resulted in 17 Mycobacterium bovis isolates, predominantly from Gombe State. Spoligotyping identified four spoligotypes, including SB0944, SB1025, SB1104, and one novel pattern. MIRU-VNTR analysis further differentiated these spoligotypes into eight profiles. All isolates belonged to the Af1 clonal complex. The study emphasises the need for broader coverage and more isolates to comprehensively understand the molecular epidemiology of bovine tuberculosis in Nigeria. To enhance research and surveillance, a cost-effective approach is proposed, utilising a discriminatory VNTR panel comprising five or nine loci. The five-locus panel consists of ETR-C, QUB26, QUB11b, MIRU04, and QUB323. Alternatively, the nine-locus panel includes ETR-A, ETR-B, QUB11a, and MIRU26. Implementing this approach would provide valuable insights into the genetic diversity of M. bovis strains in Nigeria. These findings are crucial for developing effective control measures and minimising the impact of bovine tuberculosis on both animal and human health.

Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia.

The emergence of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) is a threat to TB control programs in developing countries such as Zambia. Studies in Zambia have applied molecular techniques to understand drug-resistance-associated mutations, circulating lineages and transmission patterns of multi-drug-resistant (MDR) Mycobacterium tuberculosis. However, none has reported genotypes and mutations associated with pre-XDR TB. This study characterized 63 drug-resistant M. tuberculosis strains from the University Teaching Hospital between 2018 and 2019 using targeted gene sequencing and conveniently selected 50 strains for whole genome sequencing. Sixty strains had resistance mutations associated to MDR, one polyresistant, and two rifampicin resistant. Among MDR strains, seven percent (4/60) had mutations associated with pre-XDR-TB. While four, one and nine strains had mutations associated with ethionamide, para-amino-salicylic acid and streptomycin resistances, respectively. All 50 strains belonged to lineage 4 with the predominant sub-lineage 4.3.4.2.1 (38%). Three of four pre-XDR strains belonged to sub-lineage 4.3.4.2.1. Sub-lineage 4.3.4.2.1 strains were less clustered when compared to sub-lineages L4.9.1 and L4.3.4.1 based on single nucleotide polymorphism differences. The finding that resistances to second-line drugs have emerged among MDR-TB is a threat to TB control. Hence, the study recommends a strengthened routine drug susceptibility testing for second-line TB drugs to stop the progression of pre-XDR to XDR-TB and improve patient treatment outcomes.

Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.

The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Prevalence and risk factors of bovine tuberculosis in slaughtered cattle, Malawi.

Bovine tuberculosis (bTB) is an infectious disease with significant socioeconomic, animal, and public health impacts. However, the prevalence of bTB remains largely unclear in Malawi due to a paucity of information. Additionally, the existence of multiple risk factors is postulated to enhance bTB transmission in animals. A cross-sectional survey to estimate the prevalence of bTB, animal characteristics and identify associated risk factors was conducted from slaughtered cattle at three major regional abattoirs (southern, central and northern regions) in Malawi. Out of a total of 1547 cattle examined, 154 (9.95%) had bTB-like lesions in various visceral organs and lymph nodes; one sample per animal was collected, processed, and cultured in the in the BACTEC Mycobacterial growth indicator tube (MGIT) 960 system. From the 154 cattle that showed tuberculous like lesions, only 112 were positive on MGIT and 87 were confirmed to have M. bovis based on multiplex PCR. Cattle from the southern region (odds ratio (OR) = 1.96, 95% CI: 1.03-3.85) and central region (OR = 2.00, 95% CI: 1.16-3.56) were more likely presented with bTB-like lesions at slaughter than from the northern region. The risk of having bTB-like lesions was higher in females (OR = 1.51, CI: 1.00-2.29), older cattle (OR = 2.17, CI: 1.34-3.37), and crossbreeds (OR = 1.67, 95% CI: 1.12-2.47) than in males, younger animals, and Malawi Zebu breed, respectively. The high prevalence of bTB is of critical concern and necessitates active surveillance and strengthening of the current control strategies under a One Health (OH) approach at the animal-human interface.

Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole.

Although IFN-γ depletes tryptophan (Trp) as a defense against intracellular Chlamydia trachomatis (Ct) infected to hypoxic vagina, the presence of indole, a precursor of Trp, enables Ct to infect IFN-γ-exposed culture cells. Meanwhile, Trp-derived indole derivatives interact the aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor involved in the cellular homeostasis with tubulin dynamics. Here, the amounts of IFN-γ and indole in cervical swabs with known Ct infection status were measured, and Ct growth in the presence of indole was determined from the perspective of the AhR axis under hypoxia. A positive correlation between the amounts of IFN-γ and indole was found, and both of these amounts were lower in Ct-positive swabs than in Ct-negative ones. Indole as well as other AhR ligands inhibited Ct growth, especially under normoxia. Ct prompted the expression of detyrosinated tubulin (dTTub), but indole inhibited it. Indole did not stimulate the translocation of AhR to nucleus, and it blocked AhR activation in AhR-reporter cells. Ct growth was reduced more effectively under normoxia in AhR-knockdown cells, an effect that was enhanced by indole, which in turn diminished dTTub. Thus, Ct growth relies on the scavenger role of cytosolic AhR responsible for promoting dTTub expression.

Detection of Mutations in pncA in Mycobacterium tuberculosis Clinical Isolates from Nepal in Association with Pyrazinamide Resistance.

Without the proper information on pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis (MTB), PZA is inappropriately recommended for the treatment of both susceptible and multidrug-resistant tuberculosis (MDR-TB) in Nepal. This study aimed to collect information regarding PZA susceptibility in MTB isolates from Nepal by analyzing pncA and its upstream regulatory region (URR). A total of 211 MTB isolates were included in this study. Sequence analysis of pncA and its URR was performed to assess PZA resistance. First-line drug susceptibility testing, spoligotyping, and sequence analysis of rpoB, katG, the inhA regulatory region, gyrA, gyrB, and rrs were performed to assess their association with pncA mutation. Sequencing results reveal that 125 (59.2%) isolates harbored alterations in pncA and its URR. A total of 57 different mutation types (46 reported and 11 novel) were scattered throughout the whole length of the pncA gene. Eighty-seven isolates (41.2%) harbored mutations in pncA, causing PZA resistance in MTB. There was a more significant association of pncA alterations in MDR/pre-extensively drug-resistant (Pre-XDR) TB than in mono-resistant/pan-susceptible TB (p < 0.005). This first report on the increasing level of PZA resistance in DR-TB in Nepal highlights the importance of PZA susceptibility testing before DR-TB treatment.

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines.

The role of lymphocytes as a cornerstone of the inflammatory response in the invasive pathogenesis of Chlamydia trachomatis (Ct) LGV (L1-3) infection is unclear. Therefore, we assessed whether the adaptation of CtL2 to immortal lymphoid Jurkat cells under hypoxic conditions occurred through proinflammatory cytokine profile modification. The quantities of inclusion-forming units with chlamydial 16S rDNA confirmed that CtL2 grew well under hypoxic rather than normoxic conditions in the cells. Confocal microscopic imaging and transmission electron microscopy revealed the presence of bacterial progeny in the inclusions and showed that the inclusions were larger under hypoxic rather than normoxic conditions; this was supported by the results of 3D image construction. Furthermore, PCR-based analysis of proinflammatory cytokines revealed that the gene expression levels under hypoxic conditions were significantly higher than those under normoxic conditions. In particular, the expression of two genes (CXCL8 and CXCR3) was significantly diminished under normoxic conditions. Taken together, the results indicated that hypoxia promoted CtL2 growth in Jurkat cells while maintaining the levels of proinflammatory cytokines. Thus, Ct LGV infection in lymphocytes under hypoxic conditions might be crucial to a complete understanding of the invasive pathogenesis.