Genome-wide identification, annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila

Published ArticleCytochrome P450 monooxygenases (P450s) are ubiquitous heme-thiolate proteins that have potential biotechnological application. Thermostable-P450s that can withstand hostile industrial conditions, such as high temperatures, extremes of pH and organic solvents, are needed for biotechnological usage. Here, for the first time, we report a large number of thermostable-P450s from two thermophilic biomass-degrading fungi, Myceliophthora thermophila and Thielavia terrestris. Genome-wide P450 analysis revealed the presence of 79 and 70 P450s (P450ome) in T. terrestris and M. thermophila. Authentic P450s containing both the P450 signature domains (EXXRand CXG) were classified as follows: T. terrestris (50 families and 56 subfamilies) and M. thermophila (49 families and 53 subfamilies). Bioinformatics analysis of P450omes suggested the presence of a large number of thermostable-P450s. Based on aliphatic index cut-off ([90), 14 and 11 P450s were determined to be thermostable in T. terrestris and M. thermophila. Among the thermostable P450s, six P450s from T. terrestris and three from M. thermophila had a melting temperature (Tm) of [65 C, suggesting their hyperthermal tolerance. Analysis of the instability index of two ascomycete P450omes revealed the presence of 12 and 19 in vitro stable P450s in T. terrestris and M. thermophila. Overall, six P450s from T. terrestris and four from M. thermophila showed both thermal tolerance and in vitro stability. Thermophilic ascomycetes P450s are of potential interest from a structural, mechanistic and biotechnological point of view, as five P450s showed higher thermal tolerance and five showed higher in vitro stability compared to the wellcharacterized thermostable-P450s CYP175A1 (bacteria) and CYP119 (archaea)

Coronavirus disease 2019 and future pandemics: Impacts on livestock health and production and possible mitigation measures

The World Health Organization declared coronavirus disease 2019 (COVID-19) a pandemic on March 11, 2020. COVID-19, the current global health emergency, is wreaking havoc on human health systems and, to a lesser degree, on animals globally. The outbreak has continued since the first report of COVID-19 in China in December 2019, and the second and third waves of the outbreak have already begun in several countries. COVID-19 is expected to have adverse effects on crop production, food security, integrated pest control, tourism, the car industry, and other sectors of the global economy. COVID-19 induces a range of effects in livestock that is reflected economically since human health and livelihood are intertwined with animal health. We summarize the potentially harmful effects of COVID-19 on livestock and possible mitigation steps in response to this global outbreak. Mitigation of the negative effects of COVID-19 and future pandemics on livestock requires the implementation of current guidelines

Prevalence and molecular detection of fluoroquinolone-resistant genes (qnrA and qnrS) in Escherichia coli isolated from healthy broiler chickens

Aim: The present study was carried out to determine the prevalence and molecular detection of fluoroquinolone-resistant Escherichia coli carrying qnrA and qnrS genes in healthy broiler chickens in Mymensingh, Bangladesh, and also to identify the genes responsible for such resistance. Materials and Methods: A total of 65 cloacal swabs were collected from apparently healthy chickens of 0-14 days (n=23) and 15-35 days (n=42) old. The samples were cultured onto Eosin Methylene Blue Agar, and the isolation and identification of the E. coli were performed based on morphology, cultural, staining, and biochemical properties followed by polymerase chain reaction (PCR) targeting E. coli 16S rRNA genes. The isolates were subjected to antimicrobial susceptibility test against five commonly used antibiotics under fluoroquinolone (quinolone) group, namely gatifloxacin, levofloxacin, moxifloxacin, ofloxacin, and pefloxacin by disk diffusion method. Detection of qnrA and qnrS genes was performed by PCR. Results: Among the 65 cloacal samples, 54 (83.08%) were found to be positive for E. coli. Antibiotic sensitivity test revealed that, of these 54 isolates, 18 (33.33%) were found to be resistant to at least one fluoroquinolone antibiotic. The highest resistance was observed against pefloxacin (61.11%). By PCR, of 18 E. coli resistant to fluoroquinolone, 13 (72.22%) were found to be positive for the presence of qnrS. None of the isolates were found positive for qnrA. Conclusion: Fluoroquinolone-resistant E. coli harboring qnrS genes is highly prevalent in apparently healthy broiler chickens and possesses a potential threat to human health

PreS1 Mutations Alter the Large HBsAg Antigenicity of a Hepatitis B Virus Strain Isolated in Bangladesh

Mutations in the hepatitis B virus (HBV) genome can potentially lead to vaccination failure, diagnostic escape, and disease progression. However, there are no reports on viral gene expression and large hepatitis B surface antigen (HBsAg) antigenicity alterations due to mutations in HBV isolated from a Bangladeshi population. Here, we sequenced the full genome of the HBV isolated from a clinically infected patient in Bangladesh. The open reading frames (ORFs) (P, S, C, and X) of the isolated HBV strain were successfully amplified and cloned into a mammalian expression vector. The HBV isolate was identified as genotype C (sub-genotype C2), serotype adr, and evolutionarily related to strains isolated in Indonesia, Malaysia, and China. Clinically significant mutations, such as preS1 C2964A, reverse transcriptase domain I91L, and small HBsAg N3S, were identified. The viral P, S, C, and X genes were expressed in HEK-293T and HepG2 cells by transient transfection with a native subcellular distribution pattern analyzed by immunofluorescence assay. Western blotting of large HBsAg using preS1 antibody showed no staining, and preS1 ELISA showed a significant reduction in reactivity due to amino acid mutations. This mutated preS1 sequence has been identified in several Asian countries. To our knowledge, this is the first report investigating changes in large HBsAg antigenicity due to preS1 mutations

Isolation and molecular detection of Avipoxvirus from field outbreaks in Mymensingh, Bangladesh

Objective: The present study was performed for isolation, identification, and molecular detection of Avipoxvirus [Turkeypox virus (TPV), Fowlpox virus (FPV), and Pigeonpox virus (PPV)] from field outbreaks in some selected areas of Mymensingh division, Bangladesh. Materials and Methods: A total of 60 suspected cutaneous nodular samples (10 TPV, 20 PPV, and 30 FPV) were collected. The samples were then subjected to isolation and identification by chicken embryo propagation followed by confirmation using polymerase chain reaction (PCR). Results: The TPV, FPV, and PPV were successfully isolated and identified from the nodular samples using embryo propagation and PCR technique targeting pox virus p4b gene. Out of 10 Turkeypox suspected field samples, five (50%) were positive for TPV. Similarly, among 30 Fowl pox suspected field samples, 12 (40%), and out of 20 Pigeonpox suspected field samples, eight (40%) were found to be positive for FPV and PPV, respectively. The overall prevalence of avipox (TPV, FPV, and PPV) virus infections in Mymensingh division was 41.67% (n = 25/60). Conclusion: This study has shown that TPV, FPV, and PPV are circulating in Mymensingh division. The isolated TPV, FPV, and PPV field isolates can be used as vaccine candidates to develop an effective vaccine for effective controlling of the avipox in Mymensingh division and surrounding areas. [J Adv Vet Anim Res 2019; 6(1.000): 54-59

Prevalence and characterization of multi-drug resistant Salmonella Enterica serovar Gallinarum biovar Pullorum and Gallinarum from chicken

Aim: Salmonella is an important zoonotic pathogen responsible for animal and human diseases. The aim of the present study was to determine the prevalence and stereotyping of Salmonella isolates isolated from apparently healthy poultry. Furthermore, the clonal relatedness among the isolated Salmonella serovars was assessed. Materials and Methods: A total of 150 cloacal swab samples from apparently healthy chickens were collected, and were subjected for the isolation and identification of associated Salmonella organisms. The isolated colonies were identified and characterized on the basis of morphology, cultural characters, biochemical tests, slide agglutination test, polymerase chain reaction, and pulsed-field gel electrophoresis (PFGE). Antibiotic sensitivity patterns were also investigated using commonly used antibiotics. Results: Of the 150 samples, 11 (7.33%) produced characteristics pink colony with black center on XLD agar medium, and all were culturally and biochemically confirmed to be Salmonella. All possessed serovar-specific gene SpeF and reacted uniformly with group D antisera, suggesting that all of the isolates were Salmonella Enterica serovar Gallinarum, biovar Pullorum and/or Gallinarum. Antimicrobial susceptibility testing revealed that 54.54% of the isolated Salmonella Enterica serovars were highly sensitive to ciprofloxacin, whereas the 81.81% isolates were resistant to amoxycillin, doxycycline, kanamycin, gentamycin, and tetracycline. Pulsed-field gel electrophoresis of the XbaI-digested genomic DNA exhibited identical banding patterns, suggesting that the multidrug resistant Salmonella Enterica serovars occurring in commercial layers are highly clonal in Bangladesh. Conclusion: The present study was conducted to find out the prevalence of poultry Salmonella in layer chicken and to find out the clonal relationship among them. The data in this study suggest the prevalence of Salmonella Enterica, which is multidrug resistant and highly clonal for commercial layers of Bangladesh

Antibiotic resistance profile of bacteria isolated from raw milk samples of cattle and buffaloes

Objectives: The objective of this study was to isolate and identify Staphylococcus aureus and Escherichia coli from raw milk samples of cattle and buffalo, and to evaluate the antibiotic sensitivity pattern. Materials and methods: A total of 34 milk samples were collected twice from 17 different healthy cattle (n=14) and buffaloes (n=3) at one-month interval, and analyzed in laboratory by staining, cultural and biochemical characteristics followed by polymerase chain reaction targeting nuc gene of S. aureus and 16 S rRNA of E. coli. Antibiotic sensitivity pattern of the isolated bacteria was assessed using the disc diffusion method. Results: Confirmation of the isolates as S. aureus and E. coli were carried out by PCR using nuc gene, 16S rRNA gene specific primers specific for S. aureus and E. coli respectively. A total of 12 samples (35.29%; 11 from cattle, 1 from buffalo) were found to be positive for S. aureus; 5 and 7 during first and second month, respectively. The E. coli were found in three samples (2 from cattle, 1 from buffaloe); one in first month and two in the second month. The antibiotic sensitivity test using 4 commonly used antibiotics indicated that the most of the isolates were resistant to Gatifloxacin and one isolate showed intermediate resistance to Ofloxacin while sensitive to Ciprofloxacin and Levofloxacin. Conclusion: Two different species of bacteria i.e., S. aureus and E. coli are contaminating with milk samples. The pathogenic bacteria can be controlled effectively by using Ciprofloxacin and Levofloxacin in the case of mastitis in cattle and buffaloes in Bangladesh. [J Adv Vet Anim Res 2016; 3(1.000): 62-67

Molecular Detection of Multidrug Resistant Staphylococcus aureus Isolated from Bovine Mastitis Milk in Bangladesh

The current study was conducted to isolate and identify multidrug-resistant Staphylococcus aureus (MDR-SA) from mastitis milk samples and to determine their antimicrobial susceptibility pattern. A total of 48 bovine mastitis (BM) milk samples were collected from different parts of the Rangpur division, Bangladesh. After the collection of milk samples, mastitis was confirmed using the California mastitis test. Isolation and identification of Staphylococcus aureus were performed using conventional cultural and biochemical tests as well as using molecular methods of PCR. Nucleotide sequence analysis of the 23S rRNA gene of Staphylococcus aureus was determined. The antibiogram of the isolated bacteria was conducted using the disc diffusion method. Phylogenetic analysis of 23S rRNA was done using MEGA 7, ClustalW multiple sequence alignment, and NCBI-BLAST tools, where the sequence of the isolate showed 98% to 99% identity. Antibiogram test using 15 antimicrobial agents showed that all of the Staphylococcus aureus isolates were classified as multidrug-resistant (MDR). It was found that the isolates were resistant to tetracycline, novobiocin, methicillin, vancomycin, and cephradine, and the isolates were sensitive to ciprofloxacin, azithromycin, norfloxacin, levofloxacin, gentamicin, and amoxicillin. The detection of MDR-SA in mastitis milk is alarming and represents a great public health concern. The findings of the present study help identify Staphylococcus aureus at the molecular level using 23S rRNA gene sequencing and will help select the appropriate and effective antimicrobial agent to control BM in the northern part of Bangladesh

Molecular detection of Salmonella spp. isolated from apparently healthy pigeon in Mymensingh, Bangladesh and their antibiotic resistance pattern

Objectives: Here we determined the prevalence of Salmonella in cloacal swabs and pharyngeal swabs of apparently healthy pigeons sold in the live bird markets and villages in and around Bangladesh Agricultural University Campus, Mymensingh, Bangladesh. Materials and methods: A total of 50 samples, comprised of cloacal swabs (n=24) and pharyngeal swabs (n=26) were collected. The samples were processed, and Salmonella was isolated through a series of conventional bacteriological techniques and biochemical tests followed by polymerase chain reaction (PCR). Results: The prevalence rate of Salmonella was found to be 37.5% (n=9/24) in cloacal swabs and 30.77% (n=8/26) in pharyngeal swabs with an overall prevalence rate of 34% (n=17/50). The prevalence rate of Salmonella pigeon varied slightly among locations; 34.62% (n=9/26) in live bird markets, and 33.33% (n=8/24) in villages. Molecular detection of 17 Salmonella isolates obtained from biochemical test was performed by genus specific PCR, where all of them amplified a region of 496-bp segment of the histidine transport operon gene. Antibiogram study revealed multi-drug resistant traits in most of the isolates tested. The highest resistance was found against Ampicillin (88.23%) followed by Cephalexin (82.35%). The rate of sensitivity of the isolates to Ciprofloxacin was 100% followed by Azithromycin (82.35%), Gentamicin (76.47%) and Nalidixic acid (76.47%). Conclusion: Our findings suggest that pigeons carry multi-drug resistant Salmonella that may transfer to the humans and animals. [J Adv Vet Anim Res 2016; 3(1.000): 51-55

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 blaTEM, 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