Biphasic Medium Using Nicotinamide for Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis.

Reliable drug susceptibility testing of pyrazinamide (PZA) is technically difficult, since PZA activity is pH sensitive. The aim of this study was to evaluate a biphasic medium assay (BMA) for the reliable detection of PZA resistance in Mycobacterium tuberculosis (MTB) using nicotinamide (NIC) as a surrogate for PZA and identifying the appropriate cut-off value for the assay. The PZA susceptibility of 122 multidrug-resistant tuberculosis (MDR-TB) isolates and 39 drug-susceptible tuberculosis (DS-TB) isolates was examined using the BMA with NIC at four different concentrations (250, 500, 1000, and 2000 mg/L) and comparing the results with results from the BACTEC MGIT 960 reference method. Out of 122 MDR-TB isolates, 40 were identified as resistant by the BACTEC MGIT 960 system, of which 92.5% contained mutations within their pncA gene plus promoter region. A minimum inhibitory concentration of NIC ≥ 1000 mg/L was used as the cut-off concentration to define resistance in correlation with the MGIT 960 outcomes. NIC-BMA had a sensitivity of 90.91%, a specificity of 100%, and an accuracy of 97.52% compared with the MGIT 960 method. NIC-BMA is a promising assay to screen PZA resistance in microbiological laboratories without automation or advanced molecular instruments.

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.

Application of Genomic Epidemiology of Pathogens to Farmed Yellowtail Fish Mycobacteriosis in Kyushu, Japan.

To investigate mycobacterial cases of farmed yellowtail fish in coastal areas of western Japan (Kagoshima, Kyushu), where aquaculture fisheries are active, Mycobacterium pseudoshottsii, the causative agent, was isolated from six neighboring fishing ports in 2012 and 2013. A phylogenetic ana-lysis revealed that the strains isolated from one fishing port were closely related to those isolated from other regions of Japan, suggesting the nationwide spread of a single strain. However, strains from Japan were phylogenetically distinct from those from the Mediterranean and the United States; therefore, worldwide transmission was not observed based on the limited data obtained on the strains exami-ned in this study. The present results demonstrate that a bacterial genomic ana-lysis of infected cases, a mole-cular epidemiology strategy for public health, provides useful data for estimating the prevalence and transmission pathways of M. pseudoshottsii in farmed fish. A bacterial genome ana-lysis of strains, such as that performed herein, may play an important role in monitoring the prevalence of this pathogen in fish farms and possible epidemics in the future as a result of international traffic, logistics, and trade in fisheries.

Association of toxin-producing Clostridioides difficile with piglet diarrhea and potential transmission to humans.

The pathogenicity of Clostridioides difficile in piglets remains controversial. It is unknown whether C. difficile control helps protect piglet health. To clarify the association between C. difficile presence and piglet diarrhea, isolates were obtained from piglets with and without diarrhea. In addition, to determine the genetic relationship of C. difficile from pigs and humans, we performed whole-genome sequencing (WGS) of C. difficile isolates. Diarrheal and non-diarrheal stool samples were collected from neonatal piglets from five farms in Japan in 2021. To clarify the relationship between C. difficile derived from pigs and those from human clinical cases, WGS of C. difficile isolates was performed. Toxin-positive C. difficile were significantly more prevalent in piglets with diarrhea, although the overall frequency of C. difficile did not differ between piglets with and without diarrhea. This observation indicates an association between toxin-positive C. difficile and diarrhea in piglets. However, further studies are needed to establish a direct causal relationship and to explore other contributing factors to diarrhea in piglets. WGS results showed that C. difficile sequence type (ST) 11 including the hypervirulent PCR ribotype 078 isolates derived from Japanese pigs were closely related to ST11 of overseas strains (human clinical and animal-derived) and a Japanese human clinical strain. Toxin-positive C. difficile may cause diarrhea in piglets and hypervirulent C. difficile are spreading among pigs and human populations worldwide.

Antimicrobial Use Survey and Detection of ESBL-Escherichia coli in Commercial and Medium-/Small-Scale Poultry Farms in Selected Districts of Zambia.

Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers' usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum ß-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88-20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00-0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response.

Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.

Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.

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.

Carbapenem and colistin-resistant hypervirulent Klebsiella pneumoniae: An emerging threat transcending the egyptian food chain.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a great public health problem and is associated with many disease outbreaks and high mortality rates. Alarmingly, K. pneumoniae has been isolated from food in several recent studies. This study aimed to investigate the prevalence and characteristics of CRKP in food samples from Egypt. METHODS: A total of 311 food samples (including 116 minced meat, 92 chicken meat, 75 diced meat, and 28 mutton) were collected from local markets in Egypt and were screened for CRKP with the determination of their antimicrobial resistance profiles. The whole genome sequence was done for 23 CRKP isolates to clarify the relationship between CRKP from food and human cases in Egypt using the SNP core genome. The conjugation probability of the blaNDM-5 harboring plasmid was identified using oriTfinder RESULTS: CRKP was isolated from 11% (35/311) of the samples, with 45.71% (16/35) of them showing resistance to colistin, one of the last-resort options for treating CRKP-mediated infections. In addition to the carbapenem and colistin resistance, the CRKP isolates frequently exhibited resistance to multiple antimicrobials including ß-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and chloramphenicol. In addition, most of the CRKP were potentially hypervirulent K. pneumoniae (HvKP) identified as phylogroup Kp1 and of high-risk groups as detected in STs reported in many human outbreaks globally, such as ST383 and ST147. The core-genome phylogeny showed similarities between the isolates from this study and those previously isolated from clinical human samples in Egypt. In addition, analysis of the plasmid on which blaNDM is encoded revealed that several antimicrobial resistance genes such as blaOXA-9, blaCTX-M-15, aac(6')-Ib, qnrS1, and several virulence genes are encoded on the same plasmid. CONCLUSIONS: This study is significant for food safety and public health and is important to further identify the change in the epidemiology of CRKP infections, especially the consumption of contaminated food products.

Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae from Diseased Broiler Chickens in Lusaka District, Zambia.

Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.

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.

Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaques.

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection.

Transferable linezolid resistance genes (optrA and poxtA) in enterococci derived from livestock compost at Japanese farms.

OBJECTIVES: Linezolid is a last-resort antimicrobial in human clinical settings to treat multidrug-resistant Gram-positive bacterial infections. Mobile linezolid resistance genes (optrA, poxtA, and cfr) have been detected in various sources worldwide. However, the presence of linezolid-not-susceptible bacteria and mobile linezolid resistance genes in Japan remains uncertain. Therefore, we clarified the existence of linezolid-not-susceptible bacteria and mobile linezolid resistance genes in farm environments in Japan. METHODS: Enterococci isolates from faeces compost collected from 10 pig and 11 cattle farms in Japan in 2021 were tested for antimicrobial susceptibility and possession of mobile linezolid resistance genes. Whole-genome sequencing of optrA and/or poxtA genes positive-enterococci was performed. RESULTS: Of 103 enterococci isolates, 12 from pig farm compost were not-susceptible (2 resistant and 10 intermediate) to linezolid. These 12 isolates carried mobile linezolid resistance genes on plasmids or chromosomes (5 optrA-positive Enterococcus faecalis, 6 poxtA-positive E. hirae or E. thailandicus, and 1 optrA- and poxtA-positive E. faecium). The genetic structures of optrA- and poxA-carrying plasmids were almost identical to those reported in other countries. These plasmids were capable of transferring among E. faecium and E. faecalis strains. The optrA- and poxtA-positive E. faecium belonged to ST324 (clade A2), a high-risk multidrug-resistant clone. The E. faecalis carrying optrA gene on its chromosome was identified as ST593. CONCLUSIONS: Although linezolid is not used in livestock, linezolid-not-susceptible enterococci could be indirectly selected by frequently used antimicrobials, such as phenicols. Moreover, various enterococci species derived from livestock compost may serve as reservoirs of linezolid resistance genes carried on globally disseminated plasmids and multidrug-resistant high-risk clones.

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.

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.

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.

Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours.

Immune checkpoint inhibitors (ICIs) have been developed for canine tumour treatment, and pilot clinical studies have demonstrated their antitumour efficacy in dogs with oral malignant melanoma (OMM). Although ICIs have been approved for various human malignancies, their clinical benefits in other tumour types remain to be elucidated in dogs. Here, we conducted a clinical study of c4G12, a canine chimeric anti-PD-L1 antibody, to assess its safety and efficacy in dogs with various advanced malignant tumours (n = 12) at the Veterinary Teaching Hospital of Hokkaido University from 2018 to 2023. Dogs with digit or foot pad malignant melanoma (n = 4), osteosarcoma (n = 2), hemangiosarcoma (n = 1), transitional cell carcinoma (n = 1), nasal adenocarcinoma (n = 1), B-cell lymphoma (n = 1), or undifferentiated sarcoma (n = 2) were treated with 2 or 5 mg/kg c4G12 every 2 weeks. Treatment-related adverse events of any grade were observed in eight dogs (66.7%), including elevated aspartate aminotransferase (grade 3) in one dog (8.3%) and thrombocytopenia (grade 4) in another dog (8.3%). Among dogs with target disease at baseline (n = 8), as defined by the response evaluation criteria for solid tumours in dogs (cRECIST), one dog with nasal adenocarcinoma and another with osteosarcoma experienced a partial response (PR), with an objective response rate of 25.0% (2 PR out of 8 dogs; 95% confidence interval: 3.2-65.1%). These results suggest that c4G12 is safe and tolerable and shows antitumor effects in dogs with malignant tumours other than OMM. Further clinical studies are warranted to identify the tumour types that are most likely to benefit from c4G12 treatment.

Retrospective identification of pathogenic mycobacterial species in fish: Mycobacterium pseudoshottsii YM-3, isolated from a yellowtail fish in 1986 in Kochi, Japan.

The complete genome sequence of mycobacterial strain YM-3, isolated from cultured yellowtail in 1986, was determined. The strain was Mycobacterium pseudoshottsii, a closely related subspecies of Mycobacterium marinum, so the strain was isolated earlier than the first report of the subspecies in 2005.