Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains.

BACKGROUND: Lumpy skin disease (LSD) is a contagious viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD has recently spread in Asia following outbreaks in the Middle East and Europe. The disease emerged in Bangladesh in July 2019 in the Chattogram district, then rapidly spread throughout the entire country. We investigated six LSD outbreaks in Bangladesh to record the clinical signs and collect samples for diagnostic confirmation. Furthermore, we performed the molecular characterization of Bangladesh isolates, analyzing the full RPO30 and GPCR genes and the partial EEV glycoprotein gene. RESULTS: Clinical observations revealed common LSD clinical signs in the affected cattle. PCR and real-time PCR, showed the presence of the LSDV genome in samples from all six districts. Phylogenetic analysis and detailed inspection of multiple sequence alignments revealed that Bangladesh isolates differ from common LSDV field isolates encountered in Africa, the Middle East, and Europe, as well as newly emerged LSDV variants in Russia and China. Instead, they were closely related to LSDV KSGP-0240, LSDV NI2490, and LSDV Kenya. CONCLUSIONS: These results show the importance of continuous monitoring and characterization of circulating strains and the need to continually refine the strategies for differentiating vaccine strains from field viruses.

The application of bacteriophage to control Cronobacter sakazakii planktonic and biofilm growth in infant formula milk.

The goal was to identify the biofilm-forming ability of Cronobacter sakazakii on surfaces of stainless steel (SS) and silicone rubber (SR) in contact with infant formula milk. Two representative bacteriophages (PBES04 and PBES19) were used to control the growth of C. sakazakii as well as its biofilm forming ability on either SS or SR surfaces. Bacterial growth was confirmed at 20 °C when PBES04 and PBES19 were used, whereas C. sakazakii was not normally detected in infant formula milk treated with both bacteriophages for 6 h. In an additional biofilm reduction experiment, the biofilm on SS or SR surfaces were reduced by 3.07 and 1.92 log CFU cm-2, respectively after PBES04 treatment, and 3.06 and 2.14 log CFU cm-2, respectively, after PBES19 treatment. These results demonstrate that bacteriophages can be effective in inactivating C. sakazakii in biofilms which could potentially increase food safety in commercial facilities.

Molecular characteristics, biofilm-forming abilities, and quorum sensing molecules in Vibrio parahaemolyticus strains isolated from marine and clinical environments in Korea.

Vibrio parahaemolyticus is an inhabitant of marine and estuarine environments and causes seafood-borne gastroenteritis in humans. In this study, an UltraFast LabChip Real-Time PCR assay was evaluated for rapid detection and quantification of pathogenic V. parahaemolyticus isolates. Escherichia coli and Vibrio harveyi were used as negative controls. Twenty-six tdh-positive, biofilm-producing V. parahaemolyticus isolates were analyzed by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). REP-PCR analysis showed that the majority of the V. parahaemolyticus isolates originated from seafood and that clinical specimens formed two major clusters at 92.8% and 32% similarity levels. The presence and quantification of Autoinducer-2 was carried out using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after derivatization of Autoinducer-2 with 2, 3-diaminonaphthalene. The presence of tdh-positive V. parahaemolyticus in marine samples highlights the need for constant environmental monitoring to protect public health.

First identification and genomic features of multidrug-resistant Citrobacter freundii ST669 strain isolated from a domesticated duck in Bangladesh.

Objectives: Citrobacter freundii is a prevalent source of nosocomial infections and a well-known cause of diarrheal diseases. In recent years, it has also become increasingly resistant to various antimicrobials. In this study, we screened and characterized a multidrug-resistant (MDR) C. freundii isolate obtained from a domesticated diseased duck to better understand the genetic features, molecular epidemiology, and underlying factors linked to the antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs) of the isolate. Methods: The C. freundii BAU_TM8 strain was isolated using culturing, staining, biochemical, polymerase chain reaction, and Matrix-assisted laser desorption/ionization-time of flight methods. The MDR properties of the strain were determined by a disk diffusion test. The genomic sequence of C. freundii BAU_TM8 was performed using the Illumina NextSeq2000 platform. The ARGs, VFGs, and genomic functional characteristics of the C. freundii BAU_TM8 strain were identified using several open-source databases. Results: The sequence type of this strain was ST669, and the pathogenicity index of the strain was 0.919. Moreover, the strain had an estimated genome length of 5,797,806 bp, harboring 62 contigs, a G + C content of 54.32 %, and five contig L50s with an N50 value of 443,947 bp. Using phylogenetic analysis, this strain was closely related to two strains isolated from human and environmental samples in the USA and China despite huge geographical distances. The C. freundii BAU_TM8 strain consisted of 40 AGRs encoding resistance to 19 antimicrobial categories, e.g., fluoroquinolones, macrolides, folate pathway antagonists, aminoglycosides, tetracyclines, cephalosporins, and others. According to the phenotypic assay and genome sequence, the sensitivity and specificity of resistance profiles of the strain were 100 % and 20 %, respectively. Moreover, the virulence factor database detected 66 VFGs in this strain. This strain contained 1581 subsystems, having 33 % subsystem coverage and 2275 genes encoding amino acid derivatives, carbohydrate metabolism, protein metabolism, cofactors, vitamins, prosthetic groups, pigments, respiration, motility and chemotaxis, stress response, DNA metabolism, nucleosides and nucleotides, and others. Conclusions: To the best of our knowledge, this is the first WGS report of C. freundii from a domesticated duck in Bangladesh. The ubiquitous occurrence of ARGs and VFGs in the C. freundii BAU_TM8 strain detected in this study highlights the growing concern about antimicrobial resistance in humans, animals, and environments.

The Prevalence, Risk Factors, and Antimicrobial Resistance Determinants of Helicobacter pylori Detected in Dyspeptic Patients in North-Central Bangladesh.

Chronic infection of Helicobacter pylori represents a key factor in the etiology of gastrointestinal diseases, with high endemicity in South Asia. The present study aimed to determine the prevalence of H. pylori among dyspeptic patients in north-central Bangladesh (Mymensingh) and analyze risk factors of infection and antimicrobial resistance (AMR) determinants in the pathogen. Endoscopic gastrointestinal biopsy samples were collected from dyspeptic patients for a one-year period from March 2022 and were checked for the presence of H. pylori via the rapid urease test and PCR and further analyzed for the status of virulence factors vacA/cagA and genetic determinants related to AMR via PCR with direct sequencing or RFLP. Among a total of 221 samples collected, 80 (36%) were positive for H. pylori, with the vacA+/cagA+ genotype being detected in almost half of them. H. pylori was most prevalent in the age group of 41-50-year-olds, with it being more common in males and rural residents with a lower economic status and using nonfiltered water, though the rates of these factors were not significantly different from those of the H. pylori-negative group. Relatively higher frequency was noted for the A2147G mutation in 23S rRNA, related to clarithromycin resistance (18%, 7/39). Amino acid substitutions in PBP-1A (T556S) and GyrA (N87K and D91N) and a 200 bp deletion in rdxA were detected in samples from some patients with recurrence after treatment with amoxicillin, levofloxacin, and metronidazole, respectively. The present study describes the epidemiological features of H. pylori infection in the area outside the capital in Bangladesh, revealing the spread of AMR-associated mutations.

Genomic characterisation of Escherichia coli isolated from poultry at retail through Sink Surveillance in Dhaka, Bangladesh reveals high levels of multi-drug resistance.

The surveillance of antimicrobial resistance (AMR) in commensal Escherichia coli from livestock at slaughter is widely employed to assess the potential for risk to humans. There is currently a limited understanding of AMR in Bangladesh poultry at retail in live bird markets, with studies focussing solely on phenotypic characterisation of resistance. To address this evidence gap we performed antimicrobial susceptibility testing and whole genome sequencing on E. coli obtained from chickens from live bird markets in Dhaka in 2018 (n = 38) and 2020 (n = 45). E. coli were isolated from caeca samples following ISO guidelines and sequenced using short and long read methods. Multidrug resistance was extremely common (n = 77) and there was excellent concordance between AMR phenotype and the presence of corresponding AMR genes or mutations. There was considerable genomic diversity, with 43 different sequence types detected. Public health considerations included the high occurrence of resistance to ciprofloxacin (n = 75) associated with plasmid-residing qnrS or mutations in the gyrA and parC chromosomal genes; and the detection of a tigecycline resistant isolate harbouring tet(X4) on an IncHI1A/B-IncFIA mosaic plasmid. Thirty-nine isolates were resistant to azithromycin and harboured mphA, with a significant increase in the incidence of resistance between 2018 and 2020. Although azithromycin is banned for veterinary use in Bangladesh it remains an important treatment option for humans. Interestingly, mphA confers high-level resistance to azithromycin and erythromycin, and the latter is commonly used on poultry farms in Bangladesh. Seven isolates were colistin resistant and carried mcr1. For two isolates hybrid assemblies revealed that mcr1 resided on a highly conserved IncHI2 plasmid that had 93% nucleotide identity to a plasmid from the published genome of an E. coli isolate of Bangladeshi human origin. Six isolates had resistance to third generation cephalosporins, associated with plasmid-residing bla CTX-M-55, bla CTX-M-65, or bla DHA-1. By employing phenotypic and genomic approaches for AMR surveillance we have provided new insights into the potential for One Health AMR linkages in Bangladesh. Employing similar approaches in human and environmental sectors will help inform the One Health approach to addressing AMR, and generate evidence to support mitigation measures such as improved antimicrobial stewardship.

Characterization of the dominant strain (G-VII) of Newcastle disease viruses isolated from commercial chickens in Bangladesh during recent outbreaks.

Objective: Newcastle disease virus genotype VII (NDV-GVII), an extremely infectious pathogen, has been causing severe economic consequences for the chicken industry. The current study aimed to isolate and characterize NDV-GVII from commercial chickens in Bangladesh during a recent outbreak. Materials and Methods: From clinically suspected chickens from 70 commercial poultry farms, a total of 420 samples (trachea, lungs, and brain tissue) were collected. The samples were cultivated in 9-10 day-old seronegative embryonated chicken eggs (ECEs) after evaluating them using the rapid Newcastle disease virus (NDV) antigen detection kit. The hemagglutination (HA) inhibition test, agar gel immune diffusion (AGID) test, molecular detection by reverse transcription-polymerase chain reaction (RT-PCR), and phylogenetic studies using gene sequences of fusion (F) protein. The HA pattern of isolated NDV was determined using different avian and mammalian red blood cells (RBCs). The pathogenicity of the isolated virus was evaluated using mean death time (MDT), intravenous pathogenicity index (IVPI), and intracerebral pathogenicity index (ICPI). Results: The study found 87 NDV samples positive using the rapid NDV Ag detection kit and then 60 positives for virus isolation in ECEs. All 60 isolates were positive for NDV by HI, AGID, and RT-PCR. Phylogenetic tree analysis indicated that recent NDV isolates belong to genotype VII and exhibit a similarity of 99.7%-98.5% with isolates from Bangladesh, Iran, and India. The new isolates, identified as velogenic strains of NDV, possess an F protein cleavage site with 112-R-T-K-R-F-117 amino acid motifs. The isolated NDV showed diversified HA activity while using RBCs from birds and mammals. The results of ICPI, IVPI, and MDT indicated that the recent NDV isolates were very virulent. Conclusion: This study concluded that NDV-GVII is prevalent in commercial poultry farms in Bangladesh.

Characterization of infectious laryngotracheitis virus isolated from commercial layer chickens in Bangladesh during the year 2021-2022.

Objective: Infectious laryngotracheitis virus (ILTV) is responsible for causing infectious laryngotracheitis (ILT), which is a rapidly spreading and extremely transmissible disease in chickens. The current research aims to isolate and characterize ILTV from layer chickens in Bangladesh. Materials and Methods: A total of 345 samples (trachea, larynx, and lungs) were collected from ILT-suspected dead and sick layer chickens of 32 ILT-suspected farms in three different outbreak districts (Gazipur, Tangail, and Mymensingh) of Bangladesh during the outbreak year 2021-2022. Rapid detection kits examined the samples for avian influenza virus (AIV) and Newcastle disease virus (NDV). ILTV-specific primers were used to screen 72 NDV- and AIV-negative samples by polymerase chain reaction (PCR). Using chorioallantoic membrane (CAM), the study isolated the ILT virus from 9 to 10-day-old seronegative embryonated chicken eggs (ECEs) using selected PCR-positive samples. The virus was confirmed using nucleotide sequencing, agar gel immunodiffusion test (AGIDT), viral neutralization test (VNT), and pathogenicity evaluations using mortality index for chicken embryos (MICEs) and intra-tracheal pathogenicity index (ITPI). Results: The results indicated that among the PCR-positive 10 samples, only two (Alim_ILT_1001 and Alim_ILT_1,000) were found positive using ECEs. There were two field isolates of ILTVs, as shown by the amplicon size of the ICP4 gene-based PCR. A phylogenetic study of the ICP4 gene revealed that the recent isolates have a close similarity with the ILTV isolates of Turkey, Bangladesh, and Australia. AGIDT revealed strong precipitation lines due to ILTV-specific antibodies reacting with field viruses, while VNT neutralized both isolates with conventional ILTV antibodies. The pathogenicity testing indicated that Alim_ILT_1001 had MICE and ITPI values of 0.77 and 0.63, whereas Alim_ILT_1,000 had 0.71 and 0.57. Conclusion: Both the ILTV isolates have similarities with the isolates of Turkey, Bangladesh, and Australia, and they are highly virulent for chickens.

Characterization of predominant genotype (QX) of avian infectious bronchitis virus isolated from layer chickens in Bangladesh.

Avian infectious bronchitis (AIB) is a highly transmissible infection that affects the poultry industry globally. This study aims to isolate and characterize emerging strains of infectious bronchitis virus (IBV) from field samples of layer chickens in Bangladesh. A total of 108 samples (trachea, lung, and kidney) were taken from dead and sick layer chickens from 18 farms in 4 areas detecting outbreaks in Bangladesh. The samples were processed and inoculated in embryonated chicken eggs (ECEs) and finally screened by the trypsin-induced hemagglutination (THA) test. Using various techniques such as hemagglutination inhibition (HI), agar gel immuno-diffusion (AGID), virus neutralization test (VNT), reverse transcription-polymerase chain reaction (RT-PCR), and nucleotide sequencing, we were able to identify and confirm the isolated IBV viruses. The study also determined the hemagglutination (HA) pattern of isolated virus using avian and mammalian red blood cells. The pathogenicity of the isolated IBV was determined using embryonated chicken eggs and day-old chicks. The study found that 8 samples were positive for IBV using ECEs, and 4 were positive by the THA test. These isolates were confirmed using HI, AGID, and VN tests. S1 gene-based RT-PCR confirmed all four isolates as IBV, with the recent isolates belonging to the genotype-QX and being similar to IBV isolates from Thailand, Saudi Arabia, and India. The HA pattern of the recent isolates showed that the isolated IBV was virulent. The pathogenicity test also revealed that the four isolates were highly pathogenic. The study indicated that the prevalent genotype (QX) of the IBV strain is present in the layer chicken population of Bangladesh.

Draft Genome Sequence of the Multidrug-Resistant Citrobacter freundii 132-2 Strain Isolated from a Domestic Duck in Bangladesh.

We sequenced a multidrug-resistant strain of Citrobacter freundii, 132-2, isolated from a cloacal swab sample of a domestic duck. The whole genome of the C. freundii 132-2 strain had a length of 5,097,592 bp, 62 contigs, two plasmids, and an average G+C content of 51.85%, with a 105.0× genome coverage.

Draft genome sequence of multidrug-resistant Citrobacter portucalensis BAU_133-2 strain isolated from a domestic duck (Anas platyrhynchos domesticus) in Bangladesh.

We announce the genome sequence of the Citrobacter portucalensis BAU_133-2 strain isolated from a domestic duck. Our assembled genome contained a length of 4.8 Mb, 110.0× genome coverage, 51.91% of an average GC content, 1 plasmid, 1 CRISPR array, 8 prophages, 27 antibiotic resistance genes, and 75 virulence factor genes.

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.

Application of bacteriophages for the inactivation of Salmonella spp. in biofilms.

Microbial biofilms pose a serious threat to food industry, as they are difficult to inactivate or remove owing to their inherent resistance to traditional physical and antimicrobial treatments. Bacteriophages have been suggested as promising biocontrol agents for eliminating biofilms within the food industry. The efficacy of phages (BP 1369 and BP 1370) was evaluated against Salmonella spp. in biofilms. Biofilms were grown on food (lettuce), food contact surfaces (stainless steel and rubber), and MBEC biofilm devices. The efficacy of these phages in reducing biofilms was examined following phage (108 PFU/mL) treatment for 2 h. Bacteriophage treatment reduced biofilm cells by 3.0, 2.0, and 3.0 log CFU/cm2 on stainless steel, rubber, and an MBEC device, respectively. The adhered viable cells on lettuce were reduced by more than 1.0 log CFU/cm2 with phage treatment.

Probiotics as potential alternative biocontrol agents in the agriculture and food industries: A review.

Pathogenic microorganisms are a potential threat to the agriculture and food industries. Food contamination can be happened in the production levels at any point in the chain by pathogenic microorganisms. Conventional methods, such as those involving antibiotics, disinfectants, and physical methods, are commonly used as microbial control strategies. Owing to the limitations of these methods, such as emergence of resistance, low effectiveness, high cost, and detrimental effects on food, health, and the environment, many countries have adopted laws and regulations restricting their use. To overcome these problems, an environmentally friendly, cost-effective alternative approach is urgently needed. Probiotics are live microorganisms that offer health benefits to the host, when consumed in adequate amounts, by providing pathogen protective action and nutritional benefits. From a food microbiological point of view, to use probiotics in animals, there is a reduction of zoonotic pathogens in the gastro-intestinal tract (GIT) among animals which prevent the transmission of these pathogens through food. Therefore, probiotics have been proposed as an alternative antimicrobial means to protect against pathogenic microorganisms for better healthcare and food safety. In this review, we discuss probiotics, their selection criteria, mechanisms of action, and their prospects as alternative biocontrol agents, with special emphasis on the agriculture (livestock and aquaculture sectors), and food industries.

Reduction of Escherichia coli O157:H7 in Biofilms Using Bacteriophage BPECO 19.

Biofilm formation is a growing concern in the food industry. Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persists in food and food-related environments and subsequently produce biofilms. The efficacy of bacteriophage BPECO 19 was evaluated against three E. coli O157:H7 strains in biofilms. Biofilms of the three E. coli O157:H7 strains were grown on abiotic (stainless steel, rubber, and minimum biofilm eradication concentration [MBECTM ] device) and biotic (lettuce) surfaces at different temperatures. The effectiveness of bacteriophage BPECO 19 in reducing preformed biofilms on these surfaces was further evaluated by treating the surfaces with a phage suspension (108 PFU/mL) for 2 h. The results indicated that the phage treatment significantly reduced (P  < 0.05) the number of adhered cells in all the surfaces. Following phage treatment, the viability of adhered cells was reduced by ≥3 log CFU/cm2 , 2.4 log CFU/cm2 , and 3.1 log CFU/peg in biofilms grown on stainless steel, rubber, and the MBECTM device, respectively. Likewise, the phage treatment reduced cell viability by ≥2 log CFU/cm2 in biofilms grown on lettuce. Overall, these results suggested that bacteriophages such as BPECO 19 could be effective in reducing the viability of biofilm-adhered cells.