First detection and characterization of mcr-1 colistin resistant E. coli from wild rat in Bangladesh.

Colistin resistance is a global concern warning for a one health approach to combat the challenge. Colistin resistant E. coli and their resistance determinants are widely distributed in the environment, and rats could be a potential source of these isolates and resistant determinants to a diverse environmental setting. This study was aimed to determine the presence of colistin resistant E. coli (CREC) in wild rats, their antimicrobial resistance (AMR) phenotypes, and genotypic analysis of mcr-1 CREC through whole genome sequencing (WGS). A total of 39 rats were examined and CREC was isolated from their fecal pellets onto MacConkey agar containing colistin sulfate (1 µg/ mL). AMR of the CREC was determined by disc diffusion and broth microdilution was employed to determine MIC to colistin sulfate. CREC were screened for mcr genes (mcr-1 to mcr-8) and phylogenetic grouping by PCR. Finally, WGS of one mcr-1 CREC was performed to explore its genetic characteristics especially resistomes and virulence determinants. 43.59% of the rats carried CREC with one (2.56%) of them carrying CREC with mcr-1 gene among the mcr genes examined. Examination of seventeen (17) isolates from the CREC positive rats (n = 17) revealed that majority of them belonging to the pathogenic phylogroup D (52.94%) and B2 (11.76%). 58.82% of the CREC were MDR on disc diffusion test. Shockingly, the mcr-1 CREC showed phenotypic resistance to 16 antimicrobials of 8 different classes and carried the ARGs in its genome. The mcr-1 gene was located on a 60 kb IncI2 plasmid. On the other hand, ARGs related to aminoglycosides, phenicols, sulfonamides, tetracyclines and trimethoprims were located on a 288 kb mega-plasmid separately. The mcr-1 CREC carried 58 virulence genes including genes related to adhesion, colonization, biofilm formation, hemolysis and immune-evasion. The isolate belonged to ST224 and closely related to E. coli from different sources including UPEC clinical isolates from human based on cgMLST analysis. The current research indicates that rats might be a possible source of CREC, and the presence of mcr-1 and other ARGs on plasmid increases the risk of ARGs spreading and endangering human health and other environmental components through this infamous pest.

Epidemiology of feline bartonellosis and molecular characteristics of Bartonella henselae in Bangladesh.

Bartonellosis, a neglected vector-borne zoonotic disease transmitted from animals to humans, continues to threaten human and animal health significantly. This study aims to determine the epidemiology of feline bartonellosis and the molecular characteristics of Bartonella spp. in cats. From June 2018 to June 2020, 304 oral swabs were randomly collected from Bangladesh's Dhaka, Mymensingh, and Rajshahi districts. A pre-tested questionnaire was administered to collect data. Oral swabs were subjected to PCR targeting htrA gene to confirm Bartonella spp., which was subsequently validated through sequencing. Risk factors were identified using multivariable logistic regression analysis. The overall prevalence of feline bartonellosis was found to be 15.1 %. The following factors were significantly (p < 0.05) associated with Bartonella infection in risk factor analysis: cats aged ≥ 1 year (OR: 3.23, 95 % CI: 1.38-24.40), local breed cats (OR: 3.37, 95 % CI: 1.05-10.81), cats carrying fleas (OR: 2.33, 95 % CI: 1.93-13.45), antifleacidal drugs inconsistently administered cats (OR: 6.74, 95 % CI: 3.17-14.31), outdoor access cats (OR: 2.54, 95 % CI: 1.16-5.57). Notably, zoonotic B. henselae was confirmed through sequencing, establishing it as the causal agent of cat scratch disease. Phylogenetic analysis showed homology with B. henselae sequences from Brazil, Saint Kitts, and Nevis. We recommend consistent and appropriate flea control measures to curb its spread among Bangladeshi cats. Moreover, limiting outdoor exposure or implementing preventive measures for outdoor cats could reduce the disease burden. The associated human health risk can be decreased by effectively controlling this disease within the cat population.

Antimicrobial resistance patterns of Staphylococcus aureus isolated from apparently healthy pet cats of Bangladesh.

Objective: This study sought to determine the occurrence, molecular identification, antimicrobial-resistant trends, and gene distribution of Staphylococcus aureus in pet cats and their owners' hand swabs. Materials and Methods: From different places and clinics in Mymensingh and Dhaka, 168 pet cat samples and 42 hand swab samples from cat owners were obtained. The organisms were scrutinized by assessing the outcomes using conventional and molecular techniques. The disc diffusion technique was applied to find the resistance pattern against 12 antibiotics, and genes were discovered by targeting specific genes using PCR. Results: The occurrence of pathogenic S. aureus in pet cats was 7.74%, while it was 9.50% in pet owners' hand swabs, and 25.0% of the pet owner's hand swabs contained these genes. Staphylococcus aureus was utterly resistant to amoxicillin, ampicillin, cefixime, erythromycin, and imipenem in both pet cat and hand swabs of pet owner samples. All S. aureus isolates had a multidrug-resistant phenotype, and 1 from pet cats (O19) and 1 from pet owner hand swabs (H9) were resistant to all 12 antibiotics in the 7 antimicrobial classes. Several antibiotic-resistance genes were detected by PCR. Conclusion: The study confirmed multidrug-resistant pathogenic S. aureus in pet cats and their owners in Bangladesh, indicating a major health risk to both people and cats. Thus, a holistic and integrated one-health approach between veterinary and medical specialists is needed to mitigate the global distribution of these zoonotic antibiotic-resistant S. aureus strains.

Epidemiology and molecular characterization of Feline panleukopenia virus from suspected domestic cats in selected Bangladesh regions.

Feline panleukopenia (FPL) is a highly contagious cat disease and is endemic in Bangladesh. The study aims to describe the epidemiology and molecular characterization of the Feline panleukopenia virus from the suspected domestic cats in selected Bangladesh regions. Randomly, 161 rectal swabs were collected from the pet hospitals between July 2021 and December 2022. A structured questionnaire was administered through face-to-face interviews with cat owners in order to collect data on potential risk factors for FPL, such as age, sex, sharing litter boxes and every day utensils in multicat households, vaccination history, hospital visits for other diseases, and season. The rectal swabs were tested by PCR targeting the VP2 capsid protein gene, and six PCR-positive samples were further sequenced for molecular characterizations. The risk factors for FPLV were identified using multivariable logistic regression analysis. The overall prevalence of FPL among suspects was 22.9%. The mortality and case fatality were 10.6%, and 45.9%, respectively. However, mortality in kittens was significantly higher (16.4%) than younger cats. The odds of FPL were 8.83 times (95% CI: 3.14-24.85) higher among unvaccinated cats than vaccinated cats. The winter season had almost six times (95% CI: 1.38-24.40) higher odds of FPL than rainy season. In a multicat house, the odds of FPL was about five times (95% CI: 1.93-13.45) higher for cats that shared a litter box and food utensils compared to those that did not engage in such sharing. Visiting hospitals for other reasons nearly triples the odds of FPL (OR: 2.80, 95% CI: 1.04-7.54) compared to cats that do not visit hospitals. Analysis of partial sequence of the VP2 gene revealed genetic variations among the isolates from different regions. Among these isolates, four were identical to FPLV isolates from South Korea and China, while one showed complete homology with FPLV isolates from Thailand. In contrast, the remaining one was 100% identical to Carnivore protoparvovirus-1 isolated from a feline sample in Italy. Our isolates were classified into three distinct clades alongside Feline panleukopenia virus and Carnivore protoparvovirus-1. One in every three suspected cats was infected with Feline panleukopenia. Regular vaccination of the cats, especially those that share common litter box and food utensils and visit hospitals for other purposes, will help reduce the prevalence of FPL in Bangladesh. Besides, it is worth emphasizing the existence of genetic diversity among the circulating Feline panleukopenia viruses in Bangladesh.

Domestic cats are potential reservoirs of multidrug-resistant human enteropathogenic E. coli strains in Bangladesh.

Companion animals serve as our best friends, confidants, and family members. Thus, disease and antibiotic resistance gene transmission in pets and humans must be sought out. The study aimed to identify the common pathogenic Escherichia coli (E.coli) in pet cats and the antibiotic resistance patterns and resistant gene distribution. Samples (n = 210) were collected from different veterinary clinics in Bangladesh's cities of Mymensingh and Dhaka. Pathogenic E. coli was identified using conventional and molecular approaches. The disc diffusion method assessed the resistance profile against 12 antibiotics, and PCR was used to identify the beta-lactam resistance genes. The prevalence of the stx-1 gene was found to be 2.86%, whereas the rfbO157 prevalence was found to be 1.90% in cats. The stx-1 gene (n = 6) was 100% resistant to erythromycin and imipenem, whereas 100% sensitive to chloramphenicol. In turn, the rfbO157 gene (n = 4) exhibited 100% resistance to erythromycin, imipenem, cefixime, and azithromycin. In addtion, we identified genes that exhibit resistance to beta-lactam antibiotics (100% blaTEM, 40% blaCTX-M, 40% blaSHV2). This study found shiga-toxin producing and extended-spectrum beta-lactamase (ESBL) producing E. coli for the first time in pet cats of Bangladesh. Furthermore, the antimicrobial resistance (AMR) profile of the isolated strains refers to the occurrence of multidrug, which concerns cats and their owners. The existence of these genes in non-diarrheic pet animal isolates indicates that domestic pets may serve as a reservoir for human infection. Thus, one health strategy comprising animal and human health sectors, governments, together with stakeholders is needed to confront multidrug-resistant E. coli infections in Bangladesh.

Diversity of Streptococcus spp. and genomic characteristics of Streptococcus uberis isolated from clinical mastitis of cattle in Bangladesh.

Introduction: Streptococci are the major etiology in mastitis in dairy cattle, a cause of huge economic losses in the dairy industries. This study was aimed to determine the diversity of Streptococcus spp. isolated from clinical mastitis of cattle reared in Bangladesh. Methods: A total of 843 lactating cattle reared in four prominent dairy farms and one dairy community were purposively included in this study where 80 cattle were positive to clinical mastitis (CM) based on gross changes in the udder (redness, swelling, and sensitive udder) and/or milk (flakes and/or clots). Milk samples were collected from all the eighty cattle with clinical mastitis (CCM) and twenty five apparently healthy cattle (AHC). Samples were enriched in Luria Bertani broth (LB) and one hundred microliter of the enrichment culture was spread onto selective media for the isolation of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli and Corynebacterium spp., the major pathogen associated with mastitis. Isolates recovered from culture were further confirmed by species specific PCR. Results and Discussion: Out of 105 samples examined 56.2% (59/105), 17.14% (18/105), 9.52% (10/105) and 22.9% (24/105) samples were positive for Staphylococcus, Streptococcus, Enterococcus faecalis and E. coli, respectively. This study was then directed to the determination of diversity of Streptococcus spp. through the sequencing of 16S rRNA. A total of eighteen of the samples from CCM (22.5%) but none from the AHC were positive for Streptococcus spp. by cultural and molecular examination. Sequencing and phylogenetic analysis of 16S rRNA identified 55.6, 33.3, 5.6 and 5.6% of the Streptococcus isolates as Streptococcus uberis, Streptococcus agalactiae, Streptococcus hyovaginalis and Streptococcus urinalis, respectively. Considering the high prevalence and worldwide increasing trend of S. uberis in mastitis, in-depth molecular characterization of S. uberis was performed through whole genome sequencing. Five of the S. uberis strain isolated in this study were subjected to WGS and on analysis two novel ST types of S. uberis were identified, indicating the presence of at least two different genotypes of S. uberis in the study areas. On virulence profiling, all the isolates harbored at least 35 virulence and putative virulence genes probably associated with intramammary infection (IMI) indicating all the S. uberis isolated in this study are potential mastitis pathogen. Overall findings suggest that Streptococcus encountered in bovine mastitis is diverse and S. uberis might be predominantly associated with CM in the study areas. The S. uberis genome carries an array of putative virulence factors that need to be investigated genotypically and phenotypically to identify a specific trait governing the virulence and fitness of this bacterium. Moreover, the genomic information could be used for the development of new genomic tools for virulence gene profiling of S. uberis.

First report of Aliarcobacter cryaerophilus in ready-to-cook chicken meat samples from super shops in Bangladesh.

Objective: This study aimed to isolate Aliarcobacter cryaerophilus in ready-to-cook poultry meat in Bangladesh. Materials and Methods: Thirty drumstick samples were collected from super shops in Dhaka city (n = 10), Mymensingh city (n = 10), and Patuakhali town (n = 10). After sample processing, they were cultured in Blood agar media with Campylobacter base using a microfilter (0.42 nm). Suspected colonies were subjected to DNA extraction and PCR assay targeting 16SrRNA genes. Then, sequencing was performed for confirmation. Results: Of 30 samples, 3 (10%) were positive for A. cryaerophilus. Phylogenetic analysis shows that our isolate has strong similarities with one of the isolates from China. Conclusion: The presence of this organism in ready-to-cook poultry meat is a significant concern for consumers as it bears zoonotic importance.

Molecular detection of Babesia and Theileria from crossbred cattle in Sirajganj and Rangpur districts of Bangladesh.

BACKGROUND: Babesia and Theileria are potential threats to the livestock industry, causing considerable economic losses. These tick-borne blood parasites are more prevalent in crossbred cattle than local cattle in Bangladesh. OBJECTIVES: To confirm the species of Babesia and Theileria in crossbred cattle from the northern part of Bangladesh using conventional and molecular tools. METHODS: A total of 385 crossbred cattle blood samples were subjected to DNA extraction and PCR. For molecular detection, B. bigemina rhoptry-associated protein 1a, B. bovis spherical body protein-4, and Theileria spp. 18S rRNA were used as the marker genes. RESULTS: Using PCR, only 72 (18.7%) samples were found piroplasm positive, of which 12.2% Theileria, 4.7% Babesia, and 1.8% mixed infections. Both Babesia (7.3%), Theileria (7.7%) and mixed (2.8%) infections were detected in Sirajganj, and only Theileria (20.4%) was detected in Rangpur district. By PCR and nPCR we detected B. bigemina and T. annulata in Sirajganj district, and Theileria sp. in Rangpur district. The target gene sequences of isolated pathogens confirmed B. bigemina and T. annulata, and Theileria sp from these samples. Blood smears of all samples were also examined microscopically for Babesia and/or Theileria spp. and 14.3% of samples were found positive, of which 5.9% Babesia and 8.3% Theileria. Generally, the pathogens detected in Sirajgang and Rangpur were genetically related to South Asia, particularly South East Asian isolates. CONCLUSIONS: These findings provide information for a better understanding of the epidemiology of Babesia and Theileria as well as to improve the approaches for diagnosis and control of tick-borne diseases in Bangladesh.

Molecular detection of vancomycin and methicillin resistance in Staphylococcus aureus isolated from food processing environments.

Staphylococcus aureus is a well-known foodborne pathogen. The aim of this study was to investigate the presence of S. aureus isolated from serving utensils in food processing environments in Mymensingh city, Bangladesh and to determine their antibiogram and resistance determinants. A total of 120 environmental samples were collected from different food settings. Isolation and identification were conducted using conventional biochemical tests. Molecular identification of isolates and detection of methicillin and vancomycin resistance were done using primer-specific polymerase chain reaction (PCR) targeting Tuf, nuc, mecA, and mecC genes. Antibiotic sensitivity tests were performed, and resistance genes were also detected by amplifying bla TEM , vanA, vanB, and vanC genes. Among the 120 samples, 81 (67.5%) were positive for Staphylococcus spp. and 41 (50.62%) were positive for the nuc-gene. Among the 41 isolates, 5 (12.20%) were positive for mecA, but none were positive for the mecC gene. A total of 12.2% of the isolates were vanC-positive, of which 4 isolates (9.76%) were also positive for the mecA gene. Antibiotic sensitivity testing revealed that all S. aureus isolates (100%) from hotel samples were sensitive to ciprofloxacin and chloramphenicol, 90.32% were sensitive to doxycycline, and 80.65% were sensitive to streptomycin. Conversely, all isolates (100%) were resistant to ampicillin, and 29.03% were resistant to vancomycin. All S. aureus isolates obtained from non-hotel samples were susceptible to chloramphenicol, ceftriaxone, ciprofloxacin, doxycycline, meropenem, and vancomycin; however, 40% of isolates were resistant to novobiocin. Among the hotel isolates, 29 (93.55%) of the ampicillin-resistant isolates harbored the blaTEM gene while 5 (55.55%) of the vancomycin-resistant isolates harbored the vanC gene. Four of the five vanC positive isolates were also positive for the mecA gene. The presence of methicillin-resistant S. aureus (MRSA) which is also vancomycin-resistant in food processing environments is a threat to public health. This is the first report on the molecular detection of methicillin and vancomycin-resistant S. aureus isolated from food processing environments in Bangladesh.

Isolation and Identification of Natural Colorant Producing Soil-Borne Aspergillus niger from Bangladesh and Extraction of the Pigment.

Natural colorants have been used in several ways throughout human history, such as in food, dyes, pharmaceuticals, cosmetics, and many other products. The study aimed to isolate the natural colorant-producing filamentous fungi Aspergillus niger from soil and extract pigments for its potential use specially for food production. Fourteen soil samples were collected from Madhupur National Park at Madhupur Upazila in the Mymensingh district, Bangladesh. The Aspergillus niger was isolated and identified from the soil samples by following conventional mycological methods (cultural and morphological characteristics), followed by confirmatory identification by a polymerase chain reaction (PCR) of conserved sequences of ITS1 ribosomal DNA using specific oligonucleotide primers. This was followed by genus- and species-specific primers targeting Aspergillus niger with an amplicon size of 521 and 310 bp, respectively. For pigment production, a mass culture of Aspergillus niger was conducted in Sabouraud dextrose broth in shaking conditions for seven days. The biomass was subjected to extraction of the pigments following an ethanol-based extraction method and concentrated using a rotary evaporator. Aspergillus niger could be isolated from three samples. The yield of extracted brown pigment from Aspergillus niger was 0.75% (w/v). Spectroscopic analysis of the pigments was carried out using a UV-VIS spectrophotometer. An in vivo experiment was conducted with mice to assess the toxicity of the pigments. From the colorimetric and sensory evaluations, pigment-supplemented products (cookies and lemon juice) were found to be more acceptable than the control products. This could be the first attempt to use Aspergillus niger extracted pigment from soil samples in food products in Bangladesh, but for successful food production, the food colorants must be approved by a responsible authority, e.g., the FDA or the BSTI. Moreover, fungal pigments could be used in the emerging fields of the food and textile industries in Bangladesh.

Whole genome analysis of Black Bengal goat from Savar Goat Farm, Bangladesh.

OBJECTIVES: Single nucleotide polymorphisms (SNPs) play critical roles in genetic diversity and disease. Many traits and diseases are linked with exonic SNPs that are significant for gene function, regulation or translation. This study focuses on SNPs that potentially act as the genetic basis for desirable traits in the Black Bengal Goat. This variety of goat is native to South Asia, and is identified as one of the most commercially important meat producing animals in the world. The aim of this study was to sequence the genome of Black Bengal Goats and identify SNPs that might play a significant role in determining meat quality in the organism. The study focuses on exonic SNPs for their greater likelihood of affecting the final translated protein product. RESULTS: Approximately 76,000 exonic variants were identified in the study. After filtration using a Wilcoxon test based score, the number came down to 49, 965 which were found to be distributed in 11,568 genes. The functional pathways affected by these variations included fatty acid metabolism and degradation, which are important processes that influence meat quality.