Green Synthesized Chitosan Nanoparticles for Controlling Multidrug-Resistant mecA- and blaZ-Positive Staphylococcus aureus and aadA1-Positive Escherichia coli.

Antimicrobial resistance has recently been considered an emerging catastrophe globally. The public health and environmental threats were aggravated by the injudicious use of antibiotics in animal farming, aquaculture, and croup fields, etc. Consequently, failure of antibiotic therapies is common because of the emergence of multidrug-resistant (MDR) bacteria in the environment. Thus, the reduction in antibiotic spillage in the environment could be an important step for overcoming this situation. Bear in mind, this research was focused on the green synthesis of chitosan nanoparticles (ChiNPs) using Citrus lemon (Assam lemon) extract as a cross-linker and application in controlling MDR bacteria to reduce the antibiotic spillage in that sector. For evaluating antibacterial activity, Staphylococcus aureus and Escherichia coli were isolated from environmental specimens, and their multidrug-resistant pattern were identified both phenotypically by disk diffusion and genotypically by detecting methicillin- (mecA), penicillin- (blaZ), and streptomycin (aadA1)-resistance encoding genes. The inhibitory zone's diameter was employed as a parameter for determining the antibacterial effect against MDR bacteria revealing 30 ± 0.4 mm, 34 ± 0.2 mm, and 36 ± 0.8 mm zones of inhibition against methicillin- (mecA) and penicillin (blaZ)-resistant S. aureus, and streptomycin (aadA1)-resistant E. coli, respectively. The minimum inhibitory concentration at 0.31 mg/mL and minimum bactericidal concentration at 0.62 mg/mL of yielded ChiNPs were used as the broad-spectrum application against MDR bacteria. Finally, the biocompatibility of ChiNPs was confirmed by showing a negligible decrease in BHK-21 cell viability at doses less than 2 MIC, suggesting their potential for future application in antibiotic-free farming practices.

Characterization, histopathology and immunogenicity of the lumpy skin disease virus isolated during 2019-20 in Bangladesh.

Introduction: Lumpy skin disease (LSD) is a highly contagious vector-borne viral disease of cattle. LSD has emerged in Bangladesh in 2019, causing significant economic losses due to its high morbidity and mortality. This research was designed to isolate, identify, and assess the immunogenicity of LSD virus (LSDV) using nodular tissue samples obtained from affected cattle during the 2019-20 outbreak across nine districts of Bangladesh. Methods: To determine the presence of LSDV in nodular tissues, we initially used iiPCR and PCR, followed by histopathological examination. 151 were positive via iiPCR and PCR among the 180 collected samples. The PCR positive 151 samples were then inoculated into 10-day-old embryonated chicken eggs via the CAM route to isolate LSDV, confirmed through PCR. Subsequently, partial sequencing and phylogenetic analysis of the P32 gene were performed to determine the origin of the circulating LSDV strain. The immunogenicity of selected LSDV strains was assessed through an ELISA test. Results: The PCR results revealed a distinct positive band at 192 bp in both the nodular tissue samples and the LSDV isolated from chicken embryo inoculations. Microscopic analysis of the nodular lesions revealed thickening of the epidermis, ballooning degeneration of keratinocytes, and proliferation of follicular epithelia. Additionally, mononuclear infiltration was observed at the demarcation line between infected and healthy tissue, with necrosis of muscular tissues beneath the epidermis. The LSDV isolate from Bangladesh exhibited a close genetic relationship with LSDV strains isolated from neighboring and other regional countries including India, Myanmar, and Mongolia. This observation strongly suggests the possibility of a transboundary spread of the LSD outbreak in Bangladesh during 2019-2020. The results of the immunogenicity test showed that the serum antibody titer remained at a protective level for up to 18 months following secondary immunization with inactivated LSDV antigen. This finding suggests that the inactivated LSDV antigen could be a potential vaccine candidate to protect cattle in Bangladesh against LSDV. Conclusion: In conclusion, our research successfully isolated, identified, and characterized LSDV in cattle nodular tissues from the 2019-20 outbreak in Bangladesh. Furthermore, it provided insights into the probable origin of the circulating strain and investigated a potential vaccine candidate to protect cattle in the region from LSDV.

Characterization of ß-lactamase and virulence genes in Pseudomonas aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh.

The emergence and spread of multidrug-resistant pathogens like Pseudomonas aeruginosa are major concerns for public health worldwide. This study aimed to assess the prevalence of P. aeruginosa in clinical, environmental, and poultry sources in Bangladesh, along with their antibiotic susceptibility and the profiling of ß-lactamase and virulence genes using standard molecular and microbiology techniques. We collected 110 samples from five different locations, viz., BAU residential area (BAURA; n = 15), BAU Healthcare Center (BAUHCC; n = 20), BAU Veterinary Teaching Hospital (BAUVTH; n = 22), Poultry Market (PM; n = 30) and Mymensingh Medical College Hospital (MCCH; n = 23). After overnight enrichment in nutrient broth, 89 probable Pseudomonas isolates (80.90%) were screened through selective culture, gram-staining and biochemical tests. Using genus- and species-specific PCR, we confirmed 22 isolates (20.0%) as P. aeruginosa from these samples. Antibiogram profiling revealed that 100.0% P. aeruginosa isolates (n = 22) were multidrug-resistant isolates, showing resistance against Doripenem, Penicillin, Ceftazidime, Cefepime, and Imipenem. Furthermore, resistance to aztreonam was observed in 95.45% isolates. However, P. aeruginosa isolates showed a varying degree of sensitivity against Amikacin, Gentamicin, and Ciprofloxacin. The blaTEM gene was detected in 86.0% isolates, while blaCMY, blaSHV and blaOXA, were detected in 27.0%, 18.0% and 5.0% of the P. aeruginosa isolates, respectively. The algD gene was detected in 32.0% isolates, whereas lasB and exoA genes were identified in 9.0% and 5.0% P. aeruginosa isolates. However, none of the P. aeruginosa isolates harbored exoS gene. Hence, this study provides valuable and novel insights on the resistance and virulence of circulating P. aeruginosa within the clinical, environmental, and poultry environments of Bangladesh. These findings are crucial for understanding the emergence of ß-lactamase resistance in P. aeruginosa, highlighting its usefulness in the treatment and control of P. aeruginosa infections in both human and animal populations.

Isolation, adaptation, and characterization of lumpy skin disease virus from cattle in Bangladesh.

Objective: The research aimed to isolate, adapt to cell culture, and characterize the lumpy skin disease virus (LSDV) from clinically infected cattle in Bangladesh. Materials and Methods: From September 2019 to June 2020, 37 skin nodules and skin swabs were aseptically collected from afflicted cattle in the outbreak regions of Jhenaidah and Kishoreganj in Bangladesh. The LSDV was isolated from embryonated specific pathogen-free (SPF) chicken eggs along the chorioallantoic membrane (CAM) route and the Vero cell line after several blind passages. The viral attachment protein was targeted for molecular detection using polymerase chain reactions (PCR). For phylogenetic analysis, PCR-positive products were partially sequenced. Results: The virus was evident in the cell line, showed cytopathic effects after the 13 blind passage, and on the CAM of SPF chicken eggs, exhibited thickening of the CAM with pock-like lesions. A total of 12 samples (32.43%) tested positive for LSDV by PCR. Phylogenetic analysis of the present isolates (accession numbers MN792649 and MN792650) revealed 100% similarity with strains from India (MN295064), Kenya (AF325528, MN072619, KX683219), Greece (KY829023), Serbia (KY702007), and Kazakhstan (MN642592); moreover, 99.43% to 100% similarity to the sheep pox virus. Conclusion: Partially sequenced LSDV was developed as a vaccine seed and was first isolated in Bangladesh and characterized at the molecular level.

Complete genome sequence analysis of the multidrug resistant Aeromonas veronii isolated for the first time from stinging catfish (Shing fish) in Bangladesh.

Objective: Whole genome sequencing (WGS) of Aeromonas veronii Alim_AV_1000 isolated from ulcerative lesions of Shing fish (stringing catfish; Heteropneustes fossilis) was performed during the outbreak year 2021. Materials and Methods: Using next-generation sequencing (Illumina) technology, WGS was accomplished, resulting in the sequencing, assembly, and analysis of the entire genome of the A. veronii strain. Moreover, the genomic features, virulence factors, antimicrobial resistome, and phylogenetic analysis for the molecular evolution of this strain were also examined. Results: The genome size of the A. veronii Alim_AV_1000 strain was 4,494,515 bp, with an average G+C content of 58.87%. Annotation revealed the known transporters and genes linked to virulence, drug targets, and antimicrobial resistance. Conclusion: The findings of the phylogenetic analysis revealed that the strain of the present study has a close relationship with the China strain TH0426 and strain B56. This study provides novel information on A. veronii isolated from Shing fish in Bangladesh.

Antimicrobial Resistance, Biofilm Formation, and Virulence Determinants in Enterococcus faecalis Isolated from Cultured and Wild Fish.

Fish has always been an integral part of Bengali cuisine and economy. Fish could also be a potential reservoir of pathogens. This study aimed to inquisite the distribution of virulence, biofilm formation, and antimicrobial resistance of Enterococcus faecalis isolated from wild and cultivated fish in Bangladesh. A total of 132 koi fish (Anabas scandens) and catfish (Heteropneustes fossilis) were collected from different markets in the Mymensingh district and analyzed to detect E. faecalis. E. faecalis was detected by conventional culture and polymerase chain reaction (PCR), followed by the detection of virulence genes by PCR. Antibiotic susceptibility was determined using the disk diffusion method, and biofilm-forming ability was investigated by crystal violet microtiter plate (CVMP) methods. A total of 47 wild and 40 cultured fish samples were confirmed positive for E. faecalis by PCR. The CVMP method revealed four per cent of isolates from cultured fish as strong biofilm formers, but no strong producers were found from the wild fish. In the PCR test, 45% of the isolates from the wild and cultivated fish samples were found to be positive for at least one biofilm-producing virulence gene, where agg, ace, gelE, pil, and fsrC genes were detected in 80, 95, 100, 93, and 100% of the isolates, respectively. Many of the isolates from both types of samples were multidrug resistant (MDR) (73% in local fish and 100% in cultured fish), with 100% resistance to erythromycin, linezolid, penicillin, and rifampicin in E. faecalis from cultured fish and 73.08, 69.23, 69.23, and 76.92%, respectively, in E. faecalis from wild fish. This study shows that E. faecalis from wild fish have a higher frequency of virulence genes and biofilm-forming genes than cultivated fish. However, compared to wild fish, cultured fish were found to carry E. faecalis that was more highly multidrug resistant. Present findings suggest that both wild and cultured fish could be potential sources for MDR E. faecalis, having potential public health implications.

Antimicrobial Resistance Profiles, Virulence Determinants, and Biofilm Formation in Enterococci Isolated from Rhesus Macaques (Macaca mulatta): A Potential Threat for Wildlife in Bangladesh?

Enterococci are commensal bacteria that inhabit the digestive tracts of animals and humans. The transmission of antibiotic-resistant genes through human-animal contact poses a potential public health risk worldwide, as zoonoses from wildlife reservoirs can occur on every continent. The purpose of this study was to detect Enterococcus spp. in rhesus macaques (Macaca mulatta) and to investigate their resistance patterns, virulence profiles, and biofilm-forming ability. Conventional screening of rectal swabs (n = 67) from macaques was followed by polymerase chain reaction (PCR). The biofilm-forming enterococci were determined using the Congo red agar plate assay. Using the disk diffusion test (DDT), antibiogram profiles were determined, followed by resistance and virulence genes identification by PCR. PCR for bacterial species confirmation revealed that 65.7% (44/67) and 22.4% (15/67) of the samples tested positive for E. faecalis and E. faecium, respectively. All the isolated enterococci were biofilm formers. In the DDT, enterococcal isolates exhibited high to moderate resistance to penicillin, rifampin, ampicillin, erythromycin, vancomycin, and linezolid. In the PCR assays, the resistance gene blaTEM was detected in 61.4% (27/44) of E. faecalis and 60% (9/15) of E. faecium isolates. Interestingly, 88.63 % (39/44) of E. faecalis and 100% (15/15) of E. faecium isolates were phenotypically multidrug-resistant. Virulence genes (agg, fsrA, fsrB, fsrC, gelE, sprE, pil, and ace) were more frequent in E. faecalis compared to E. faecium; however, isolates of both Enterococcus spp. were found negative for the cyl gene. As far as we know, the present study has detected, for the first time in Bangladesh, the presence of virulence genes in MDR biofilm-forming enterococci isolated from rhesus macaques. The findings of this study suggest employing epidemiological surveillance along with the one-health approach to monitor these pathogens in wild animals in Bangladesh, which will aid in preventing their potential transmission to humans.

Modulation of growth performance, gut morphometry, and cecal microbiota in broilers by clove (Syzygium aromaticum) and tulsi (Ocimum sanctum) supplementation.

In an epoch of the growing risk of antibiotic resistance, there is a dire need to establish an effective novel feeding practice for broiler nutrition as an alternative to antibiotics. Hence, the aim of the current study was to evaluate the impact of clove powder and tulsi extract on the growth performance, gut morphologic and morphometric indices, and cecal microbial status of broiler, as an alternative to antibiotic growth promoters (AGPs). Sixty day-old chicks of Cobb-500 strain were randomly divided into 4 groups, each having 15 birds. Chicks of the control group (T0) were fed commercial broiler feed with no additional supplementation. The treatment groups were offered commercial broiler feed and received clove powder and tulsi extract with drinking water at the rate of 0.5% + 2% (T1), 1% + 3% (T2), and 1.5% + 4% (T3), respectively. Results showed a nonlinear relationship with the dosage of clove and tulsi. All the growth parameters substantially (P < 0.05) improved in T2 while T1 and T3 showed no significant improvement compared to T0. The final body weight was significantly (P < 0.05) higher in T2. Giblet and offal weights showed no noticeable differences except in the intestine and heart where intestine weight markedly (P < 0.05) decreased in T3 and heart weight significantly (P < 0.05) increased in T1 and T2. Clove and tulsi supplementation substantially improved the villus height and villus surface area of the small intestine in T2 while the large intestine remained mostly unaffected by the treatment. Cecal microbial status significantly improved in all the treatment groups having increased (P < 0.05) Lactobacillus spp. count and decreased (P < 0.05) E. coli count compared to T0. Based on the aforementioned findings, it can be concluded that the combination of clove and tulsi can improve the growth performance and gut health of broilers which is largely dose-dependent and might be supplied as a potential alternative to AGPs.

Identification of peste des petits ruminants virus along with co-infecting diseases of goats in Bangladesh.

Objective: Peste des petits ruminants (PPR) virus is the main infectious cause of goat mortality in Bangladesh, and co-infection may make diseases more severe. This study aimed to detect PPR and co-infecting diseases in goats. Materials and Methods: One hundred goats suspected to be infected with the PPR virus were collected from various areas of Mymensingh district, Bangladesh. A systemic post-mortem examination was carried out on PPR-suspected goats. Lungs, spleen, and lymph nodes (pre-scapular) were used for ribonucleic acid extraction, whereas lungs and mesenteric lymph nodes were used for deoxyribonucleic acid extraction. Seven-pair primer sets were used for molecular detection of pathogens specific for PPR, goat pox, contagious ecthyma (Orf), foot and mouth disease (FMD) virus, Klebsiella sp., and Mycobacterium sp. Reverse transcriptase-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) were used to find the exact cause. Results: Out of 100 PPR-suspected goats examined, 55 goats were confirmed as PPR-detected by RT-PCR. Among the 55 PPR-positive goats, 2 were co-infected with goat pox, 2 with tuberculosis, 10 with Klebsiella sp. infection, and 6 with FMD as detected by PCR and RT-PCR. Moreover, 12 goats were co-infected with PPRV and fascioliasis. Conclusion: About 58% of PPR virus-infected goats were co-infected with other organisms. There is a need to design technology to detect the state of co-infectivity at its early onset and future preventive and therapeutic strategies for co-infecting diseases. This is the first study in Bangladesh to describe co-infecting diseases of goats along with PPR.

Phenotypic and Genotypic Detection of Biofilm-Forming Staphylococcus aureus from Different Food Sources in Bangladesh.

Staphylococcus aureus is a major foodborne pathogen. The ability of S. aureus to produce biofilm is a significant virulence factor, triggering its persistence in hostile environments. In this study, we screened a total of 420 different food samples and human hand swabs to detect S. aureus and to determine their biofilm formation ability. Samples analyzed were meat, milk, eggs, fish, fast foods, and hand swabs. S. aureus were detected by culturing, staining, biochemical, and PCR. Biofilm formation ability was determined by Congo Red Agar (CRA) plate and Crystal Violet Microtiter Plate (CVMP) tests. The icaA, icaB, icaC, icaD, and bap genes involved in the synthesis of biofilm-forming intracellular adhesion compounds were detected by PCR. About 23.81% (100/420; 95% CI: 14.17−29.98%) of the samples harbored S. aureus, as revealed by detection of the nuc gene. The CRA plate test revealed 20% of S. aureus isolates as strong biofilm producers and 69% and 11% as intermediate and non-biofilm producers, respectively. By the CVMP staining method, 20%, 77%, and 3% of the isolates were found to be strong, intermediate, and non-biofilm producers. Furthermore, 21% of S. aureus isolates carried at least one biofilm-forming gene, where icaA, icaB, icaC, icaD, and bap genes were detected in 15%, 20%, 7%, 20%, and 10% of the S. aureus isolates, respectively. Bivariate analysis showed highly significant correlations (p < 0.001) between any of the two adhesion genes of S. aureus isolates. To the best of our knowledge, this is the first study in Bangladesh describing the detection of biofilm-forming S. aureus from foods and hand swabs using molecular-based evidence. Our findings suggest that food samples should be deemed a potential reservoir of biofilm-forming S. aureus, which indicates a potential public health significance.

Efficacy of bi-valent whole cell inactivated bacterial vaccine against Motile Aeromonas Septicemia (MAS) in cultured catfishes (Heteropneustes fossilis, Clarias batrachus and pangasius pangasius) in Bangladesh.

The Motile Aeromonas Septicemia (MAS) is an important disease of cultured catfishes (Heteropneustes fossilis, Clarias batrachus and Pangasius pangasius), caused by different species of Aeromonas bacteria which have been documented to be higher death rates (≤70%) in Bangladesh since 2016. Present study was conducted to develop bi-valent vaccine using A. hydrophila and A. veronii, and to validate their efficacy via intra-muscular (IM) and oral-routes of immunization in selected species of fishes. Brood fishes of the three species were immunized with three doses of inactivated vaccine (107 CFU /2.3 mg/ml). Hematological parameters of brood fishes and antibody levels (IgM) of broods, their larvae and eggs were determined by ELISA. Additionally, Relative Percent Survivability (RPS) and the IgM levels of the larvae after challenge with virulent A. hydrophila and A. veronii were also evaluated. Findings of this study showed that the lymphocytes, monocytes, granulocytes counts and antibody (IgM) titre of brood fishes, larvae and eggs from the vaccinated fishes were found significantly higher (p< 0.05) compared to the un-vaccinated control groups. Alternatively, antibody levels (IgM) in the larvae of vaccinated group of brood fishes fed with antigen coated feed was exhibited to be remarkably higher (p< 0.05) than the antigen non-fed group. The RPS of larvae of Shing (91.24 ± 2.00%), Magur (88.09 ± 2.88%) and Pangas (93.17 ± 1.52%) was found to be higher in the larvae at 20-day age of vaccinated group compared to non-vaccinated brood fishes group. Findings of this study indicated that the active immunization of brood fishes followed by oral immunization of their larvae feeding with antigen coated feed showed synergistic effect in protecting cultured Shing, Magur and Pangas fishes from frequent attack with Aeromonas spp at any age of their lifetime.

Detection of blaTEM, blaCTX-M, blaCMY, and blaSHV Genes Among Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolated from Migratory Birds Travelling to Bangladesh.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli cause severe health hazards. Migratory birds are reservoirs and transmitters of many pathogens including ESBL-producing E. coli. To examine migratory birds as potential carriers of ESBL-producing E. coli and E. coli-carrying antibiotic resistance genes, 55 PCR-positive E. coli isolates were screened using the disk diffusion method, double-disk synergy test, and further polymerase chain reaction (PCR) tests. Genes encoding resistance to tetracycline [tetA, 100% (35/35); tetB, 31.43% (11/35)], fluoroquinolone [qnrA, 35.71% (10/28); qnrB, 25% (7/28)], and streptomycin [aadA1, 90.24% (37/41)] were detected in the isolated E. coli. Of the 55 E. coli isolates, 21 (38.18%) were ESBL producers, and all of them were multidrug resistant. All the ESBL-producing E. coli isolates harbored at least two or more beta-lactamase genes, of which blaTEM, blaCMY, blaCTX-M, and blaSHV were detected in 95.24%, 90.48%, 85.71%, and 42.86% of isolates, respectively. All the beta-lactamase genes were present in four of the ESBL-producing E. coli isolates. Furthermore, 95.24% of ESBL-producing E. coli isolates were positive for one or more antibiotic resistance genes. To the best of our knowledge, this is the first study to detect E. coli-carrying antibiotic resistance genes including beta-lactamase blaCMY and blaSHV originating from migratory birds in Bangladesh. These results suggest that migratory birds are potential carriers of ESBL-producing E. coli along with other clinically important antibiotic resistance genes which may have detrimental impacts on human health.

groEL gene-based molecular detection and antibiogram profile of Riemerella anatipestifer from duck in Bangladesh.

Objectives: This study was designed to detect Riemerella anatipestifer through polymerase chain reaction (PCR) from duck farming areas of the Mymensingh and Sylhet divisions and to determine the antibiogram profile of the PCR-positive isolates using the disc diffusion method. Materials and Methods: Fifty two samples were collected, comprising clinically sick (32 ducks) and dead ducks (20). PCR confirmation was accomplished, and consistent findings were observed, employing R. anatipestifer groEL (271-bp) gene as appropriate molecular markers. For further clarification, see R. anatipestifer specific PCR assay (546-bp) and gyrB-based PCR (162-bp) were also done. The disc diffusion method was followed for the antibiotic susceptibility test of the isolates against commonly used antibiotics. Results: A total of 21 samples, 8 from clinically sick birds and 13 from dead birds, showed positive results in both conventional and molecular assays out of 52 samples. High occurrences were found in oropharyngeal swabs from sick ducks and the liver and heart from dead ducks. Antibiotic susceptibility testing revealed that the isolates were 100% resistant to penicillin G, cefradine, streptomycin, neomycin, gentamycin, meropenem, and erythromycin, but 100% sensitive to -cotrimoxazole, florfenicol, and levofloxacin. Conclusion: For diverse duck-populated areas in Bangladesh, this study shows the severity of R. anatipestifer infection among ducks.

Dietary supplementation of Bacillus-based probiotics on the growth performance, gut morphology, intestinal microbiota and immune response in low biosecurity broiler chickens.

A feeding trial was conducted to evaluate the effects of Bacillus-based probiotics on growth performance, intestinal histo-morphology, gut microbial population and immune response in broilers. A total of 2000 Hubbard Classic day-old chicks were randomly enrolled in four experimental groups and 4 replicates of 500 birds in each group, and reared for 35 days under a low- level of biosecurity measures. The trial groups were assigned treatment-1 (T1): basal diet(control), treatment-2 (T2): basal diet plus Bacillus licheniformis (DSM17236), treatment-3 (T3): basal diet plus Bacillus subtilis (PB6), and treatment-4 (T4) basal diet plus 4% Flavomycin. All four groups were fed with maize-soybean based prepared feeds (starter, grower and finisher). Dietary inclusion of B. licheniformis significantly improved body weight gain and lessened FCR in T2 compared to other groups (p < 0.05). Probiotics increased the population of Bacillus spp. and decreased the population of Clostrium perfringens, Salmonella spp. and Escherichia coli in the jejunum and ileum in broiler birds on day 21 and 35 (p < 0.05). The highest antibody production was observed in B. licheniformis treated group (T2) compared to other probiotic treated group (T1). Taken together, the study findings suggest that B. licheniformis probiotics could be used as a feasible alternative to antimicrobials in the broiler production considering beneficial impacts at low biosecurity broiler farms.

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.

Isolation and molecular detection of Streptococcus agalactiae from popped eye disease of cultured Tilapia and Vietnamese koi fishes in Bangladesh.

OBJECTIVE: Present research aims to isolate, identify, and determine the virulence of the Streptococcus agalactiae (group B Streptococcus; GBS), isolated from popped eye disease affected Tilapia and Vietnamese Koi (V. Koi) fishes. MATERIALS AND METHODS: A total of 330 fish samples were collected, of which Tilapia (n = 180) and V. Koi (n = 150), were collected from 35 affected ponds of four selected districts of Bangladesh. Isolation of the bacterium was done using different culture media (Nutrient broth, Plate count agar, Tryptic Soy Agar, and Blood agar), and identification by using various biochemical tests (conventional and using API 20 Strep kit) and polymerase chain reaction (PCR) using primers against 16S rRNA gene of S. agalactiae. Antibiotic susceptibility of the bacteria was performed using seven different antibiotics disc (Tetracycline, Oxytetracycline, Chlortetracycline, Streptomycin, Ciprofloxacin, Gentamicin, and Neomycin). Virulence of the isolated S. agalactiae was determined by infecting healthy Tilapia and V. Koi fishes through experimental infection. RESULTS: Isolated bacteria were found Gram-positive paired and chained cocci, ß-hemolytic and non-motile. Findings of biochemical and serological tests indicate that the isolated bacterium belongs to Group B Streptococcus of Lancefield classification. PCR result also confirmed that the bacteria were S. agalactiae. The bacterial isolates possessed resistance property against all the seven antibiotics used in this study. The isolated GBS was found highly virulent and showed 80%-90% mortality for Tilapia and V. Koi fishes in experimental infection within 1-6 days of post-infection. CONCLUSION: From the findings of this study, it may be concluded that isolated GBS from the Tilapia and V. Koi fishes were highly virulent and possessed multidrug-resistance properties.

Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes.

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65 °C for 30 min, 63 °C for 30 min, and 63 °C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics.