Gene-specific Polymerase Chain Reaction Research Articles

Characterization of Salmonella species from poultry slaughterhouses in South Korea: carry-over transmission of Salmonella Thompson ST292 in slaughtering process.

Salmonella outbreaks linked to poultry meat have been reported continuously worldwide. Therefore, Salmonella contamination of poultry meats in slaughterhouses is one of the critical control points for reducing disease outbreaks in humans. This study examined the carry-over contamination of Salmonella species through the entire slaughtering process in South Korea. From 2018 to 2019, 1,097 samples were collected from the nine slaughterhouses distributed nationwide. One hundred and seventeen isolates of Salmonella species were identified using the invA gene-specific polymerase chain reaction, as described previously. The serotype, phylogeny, and antimicrobial resistance of isolates were examined. Among the 117 isolates, 93 were serotyped into Salmonella Mbandaka (n = 36 isolates, 30.8%), Salmonella Thompson (n = 33, 28.2%), and Salmonella Infantis (n = 24, 20.5%). Interestingly, allelic profiling showed that all S. Mbandaka isolates belonged to the lineage of the sequence type (ST) 413, whereas all S. Thompson isolates were ST292. Moreover, almost all S. Thompson isolates (97.0%, 32/33 isolates) belonging to ST292 were multidrug-resistant and possessed the major virulence genes whose products are required for full virulence. Both serotypes were distributed widely throughout the slaughtering process. Pulsed-field gel electrophoretic analysis demonstrated that seven S. Infantis showed 100% identities in their phylogenetic relatedness, indicating that they were sequentially transmitted along the slaughtering processes. This study provides more evidence of the carry-over transmission of Salmonella species during the slaughtering processes. ST292 S. Thompson is a potential pathogenic clone of Salmonella species possibly associated with foodborne outbreaks in South Korea.

Transposon insertion in Rothia dentocariosa

ObjectivesRothia spp. are emerging as significant bacteria associated with oral health, with Rothia dentocariosa being one of the most prevalent species. However, there is a lack of studies examining these properties at the genetic level. This study aimed to establish a genetic modification platform for R. dentocariosa. MethodsRothia spp. were isolated from saliva samples collected from healthy volunteers. Subsequently, R. dentocariosa strains were identified through colony morphology, species-specific polymerase chain reaction (PCR), and 16S ribosomal RNA gene sequencing. The identified strains were then transformed with plasmid pJRD215, and the most efficient strain was selected. Transposon insertion mutagenesis was performed to investigate the possibility of genetic modifications. ResultsA strain demonstrating high transforming ability, designated as R. dentocariosa LX16, was identified. This strain underwent transposon insertion mutagenesis and was screened for 5-fluoroorotic acid-resistant transposants. The insertion sites were confirmed using arbitrary primed PCR, gene-specific PCR, and Sanger sequencing. ConclusionThis study marks the first successful genetic modification of R. dentocariosa. Investigating R. dentocariosa at the genetic level can provide insights into its role within the oral microbiome.

Bacterial Diversity and Antibiotic Resistance Genes Associated with the Different Farming Systems of Black Tiger Shrimp (Penaeus monodon) in Bangladesh

Microbial community inhabiting the intestine of the shrimp (Penaeus monodon) and their surrounding environments (e.g., water and sediment) is considered as a key contributing factor for the sustainable farming of shrimp. Indiscriminate application of antibiotics in aquaculture is a growing concern due to the emergence of antibiotic resistant bacteria (ARB), more specifically the antibiotic resistance genes (ARGs). The present study investigates the microbiome composition and 19 ARGs from four different shrimp farming systems; (i) cluster, (ii) extensive, (iii) semi-intensive, and (iv) improved extensive in the southwest coastal region of Bangladesh. In doing so, the study applied advanced 16S rRNA-based metagenomic sequencing to study the bacterial composition. Moreover, gene specific polymerase chain reaction (PCR) was employed to detect the ARGs in shrimp, water, and sediments of different farming systems. In the current study, bacteria from the phylum Proteobacteria and Firmicutes were predominant among the samples (n = 12) collected from the different farming systems followed by Actinobacteria, Bacteroidetes, and Cyanobacteria. Firmicutes was the predominant phylum in the gut of shrimp cultured in the cluster (relative abundance 53.33%) and semi-intensive (relative abundance 59.2%) culture systems. Results indicated that the bacterial community structure was significantly ( p < 0.05 ) distinct among gut, sediment, and water samples as well as the farming systems. The shared operational taxonomic unit (OTU) in the sediment sample (16,495) was nearly double than the gut (7,931) and water (8,513) bacterial communities. The improved extensive farming system showed 1,289 (11.05%) shared OTUs among gut, sediment, and water followed by semi-intensive (6.87%), cluster (6.27%), and extensive (5.46%) farming system. Among the tested ARGs, sul1, cat, gyrA(C), tetA, tetC, tetX, ere(A), vanR, and dfrA1 were predominant in water and sediment samples. Semi-intensive farming system had the highest prevalence of ARGs (21.05%) while the lowest prevalence was found in extensive (5.26%) farming system. Overall, the study provides a comprehensive scenario of bacterial composition and growing emergence of ARGs in shrimp farming of Bangladesh. Therefore, the production strategy must focus on the alternatives of antibiotic for shaping the shrimp cultivation technique more sustainable.

Prevalence of Methicillin Resistant Staphylococcus aureus Carriage Amongst Healthcare Workers of Critical Care Units in Tertiary Hospitals of Jashore, Bangladesh

Staphylococcus aureus is responsible for numerous mild to severe diseases in humans in both hospital and community settings. This problem is getting even worse for multi-drug resistant S. aureus. Healthcare workers (HCWs) are in a threat of MRSA carriage and can serve as reservoirs. Therefore, this can lead to subsequent infection and cause a nosocomial outbreak of MRSA. This cross-sectional study involved 85 healthcare workers (HCWs) from surgery units and critical care units of five different tertiary hospitals at Jashore, Bangladesh. Nasal swab samples were collected and inoculated on selective Mannitol Salt Agar (MSA) media. S. aureus was confirmed by selective growth on MSA and detection of the presence of the femA gene. Antibiotic susceptibility pattern was determined for all S. aureus isolates. Methicillin resistance was detected using both disc diffusion assay and gene specific polymerase chain reaction. S. aureus specific staphylococcal protein A (Spa) typing was also done by sequence analyses of PCR products. Among the 85 enrolled HCW personnel, 34 (40%) were carrying S. aureus followed by the S. intermedius (N=32). An overall prevalence of MRSA was found to be 3.53% (3/85). However, all the strains were susceptible to vancomycin. Additionally, 100% sensitivity was found against Trimethoprim/sulfamethoxazole and Linezolid. Sequence analysis of Spa PCR product revealed that all S. aureus isolates were of t304 spa type. None of the S. aureus was a biofilm former. This study was only a survey on the prevalence of MRSA among HCWs. Integrated surveillance for MDR MRSA carriage among HCWs is warranted to control bursts of nosocomial infections effectively. It is strongly recommended to maintain good microbiological and personal hygiene practices by HCWs.
 Bangladesh J Microbiol, Volume 39, Number 2, December 2022, pp 60-66

The detection and phylogenetic analysis of Anaplasma phagocytophilum-like 1, A. ovis and A. capra in sheep: A. capra divides into two genogroups

In this study, the presence, prevalence, and genotypes of Anaplasma phagocytophilum, A. ovis, and A. capra in sheep were investigated based on 16S SSU rRNA, groEL, and gtlA gene-specific polymerase chain reaction (PCR), respectively. The sequences of the genes were used for detection of the phylogenetic position of the species. Additionally, a restriction fragment length polymorphism (RFLP) were carried out for discrimination of A. phagocytophilum and related variants (A. phagocytophilum-like 1 and 2). The prevalence of Anaplasma spp. was found as 25.8% (101/391), while it was found that A. ovis, A. phagocytophilum-like 1, and A. capra are circulating in the sheep herds in Kyrgyzstan, according to the PCRs, RFLP and the partial DNA sequencing results. The positivity rates of A. phagocytophilum-like 1, A. ovis, and A. capra genotype-1 were 6.9, 22.5, and 5.3%, respectively. A total of 32 (8.2%) sheep were found to be mix infected. Moreover, phylogenetic analyses and sequence comparison with those available in the GenBank showed that A. capra formed two distinct genetic groups (A. capra genotype-1 and A. capra genotype-2). Considering the zoonotic potential of these species, it may be necessary to make changes in the interpretation of anaplasmosis cases in animals and there is a need for further studies to determine the pathogenicity of the species/genotypes circulating in animals.

Percentage of Asymptomatic Infections among SARS-CoV-2 Omicron Variant-Positive Individuals: A Systematic Review and Meta-Analysis.

Background: Asymptomatic infections are potential sources of transmission for coronavirus disease 2019, especially during the epidemic of the SARS-CoV-2 Omicron variant. We aimed to assess the percentage of asymptomatic infections among SARS-CoV-2 Omicron variant-positive individuals detected by gene sequencing or specific polymerase chain reaction (PCR). Methods: We searched PubMed, EMBASE, and Web of Science from 26 November 2021 to 13 April 2022. This meta-analysis was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and was registered with PROSPERO (CRD42022327894). Three researchers independently extracted data and two researchers assessed quality using pre-specified criteria. The pooled percentage with 95% confidence interval (CI) of asymptomatic infections of SARS-CoV-2 Omicron was estimated using random-effects models. Results: Our meta-analysis included eight eligible studies, covering 7640 Omicron variant-positive individuals with 2190 asymptomatic infections. The pooled percentage of asymptomatic infections was 32.40% (95% CI: 25.30–39.51%) among SARS-CoV-2 Omicron variant-positive individuals, which was higher in the population in developing countries (38.93%; 95% CI: 19.75–58.11%), with vaccine coverage ≥ 80% (35.93%; 95% CI: 25.36–46.51%), with a travel history (40.05%; 95% CI: 7.59–72.51%), community infection (37.97%; 95% CI: 10.07–65.87%), and with a median age < 20 years (43.75%; 95% CI: 38.45–49.05%). Conclusion: In this systematic review and meta-analysis, the pooled percentage of asymptomatic infections was 32.40% among SARS-CoV-2 Omicron variant-positive individuals. The people who were vaccinated, young (median age < 20 years), had a travel history, and were infected outside of a clinical setting (community infection) had higher percentages of asymptomatic infections. Screening is required to prevent clustered epidemics or sustained community transmission caused by asymptomatic infections of Omicron variants, especially for countries and regions that have successfully controlled SARS-CoV-2.

Microbial aetiology of diarrhea in children less than five years of age at tertiary care hospital of Western Uttar Pradesh, India

Introduction and Aim: Diarrhoeal diseases are one of the major causes of childhood morbidity and mortality globally. Multiple aetiologies and associated risk factors are responsible for diarrhoeal diseases in children. The present study aims to determine the microbial aetiology and possible risk factors of diarrhoea in children less than five years of age at Chhatrapati Shivaji Subharti Hospital, Subharti Medical College, Meerut in Western Uttar Pradesh, India. Materials and Methods: A total of 240 children &lt; 5 years of age with history of acute diarrhoea of less than 7 days duration were included in the study. Microbial etiology of diarrhoea in children were looked for. Bacterial etiology was identified by standard bacteriological technique. Diarrheagenic E. coli (DEC) were identified by gene-specific polymerase chain reaction. Rotavirus (RV) antigen was detected by Enzyme Immunoassay (EIA), parasites were identified by wet mount preparation and fungal etiology was identified by microscopy and culture on Sabouraud’s Dextrose Agar. Results: Out of 240 stool samples, 152 were identified as known aetiology of diarrhea. Microbial etiology could be established in 133/240 (55.41%) stool samples. The most common microbial etiology was bacterial 63(47.36%) followed by rotavirus 35 (17.29 %), parasitic etiology in 22 (16.54%) and least common etiology was fungal in 13 (9.77%) cases. Among other etiology, Lactose intolerance was found in 19/240 (7.91%) cases. Mixed aetiology with two or more pathogen causing diarrhoea was seen in 27% cases. Conclusion: Aetiological agents of diarrhoea could be identified in 63.3% cases. Causes of childhood diarrhoea is polymicrobial and preventable. Awareness and education especially to the mothers regarding the disease, safe drinking water, proper food intake and proper sanitation are key to overcome the burden of the preventable infectious diseases among children less than five years of age.

First detection of African swine fever (ASF) virus genotype X and serogroup 7 in symptomatic pigs in the Democratic Republic of Congo

BackgroundAfrican swine fever (ASF) is a highly contagious and severe hemorrhagic viral disease of domestic pigs. The analysis of variable regions of African swine fever virus (ASFV) genome led to more genotypic and serotypic information about circulating strains. The present study aimed at investigating the genetic diversity of ASFV strains in symptomatic pigs in South Kivu province of the Democratic Republic of Congo (DRC).Materials and methodsBlood samples collected from 391 ASF symptomatic domestic pigs in 6 of 8 districts in South Kivu were screened for the presence of ASFV, using a VP73 gene-specific polymerase chain reaction (PCR) with the universal primer set PPA1-PPA2. To genotype the strains, we sequenced and compared the nucleotide sequences of PPA-positive samples at three loci: the C-terminus of B646L gene encoding the p72 protein, the E183L gene encoding the p54 protein, and the central hypervariable region (CVR) of the B602L gene encoding the J9L protein. In addition, to serotype and discriminate between closely related strains, the EP402L (CD2v) gene and the intergenic region between the I73R and I329L genes were analyzed.ResultsASFV was confirmed in 26 of 391 pigs tested. However, only 19 and 15 PPA-positive samples, respectively, were successfully sequenced and phylogenetically analyzed for p72 (B646L) and p54 (E183L). All the ASFV studied were of genotype X. The CVR tetrameric repeat clustered the ASFV strains in two subgroups: the Uvira subgroup (10 TRS repeats, AAAABNAABA) and another subgroup from all other strains (8 TRS repeats, AABNAABA). The phylogenetic analysis of the EP402L gene clustered all the strains into CD2v serogroup 7. Analyzing the intergenic region between I73R and I329L genes revealed that the strains were identical but contained a deletion of a 33-nucleotide internal repeat sequence compared to ASFV strain Kenya 1950.ConclusionASFV genotype X and serogroup 7 was identified in the ASF disease outbreaks in South Kivu province of DRC in 2018–2019. This represents the first report of ASFV genotype X in DRC. CVR tetrameric repeat sequences clustered the ASFV strains studied in two subgroups. Our finding emphasizes the need for improved coordination of the control of ASF.

Altered Fecal Microbiome Years after Traumatic Brain Injury.

Patients with chronic traumatic brain injury (TBI) requiring long-term, permanent care suffer a myriad of clinical symptoms (i.e., impaired cognition, fatigue, and other conditions) that persist for years beyond the acute brain injury. In addition to these comorbid clinical symptoms, chronic TBI patients exhibit altered amino acid and hormonal profiles with distinct cytokine patterns suggesting chronic inflammation. This metabolic link suggests a role of the gut-brain axis in chronic TBI. Thus, we utilized a two-site trial to investigate the role of the gut-brain axis in comorbidities of chronic TBI. The fecal microbiome profile of 22 moderate/severe TBI patients residing in permanent care facilities in Texas and California was compared to 18 healthy age-matched control subjects working within the participating facilities. Each fecal microbiome was characterized by 16S(V4) ribosomal RNA (rRNA) gene sequencing and metagenomic genome sequencing approaches followed by confirmatory full 16S rRNA gene sequencing or focused tuf gene speciation and specific quantitative polymerase chain reaction evaluation of selected genera or species. The average chronic TBI patient fecal microbiome structure was significantly different compared to the control cohort, and these differences persisted after group stratification analysis to identify any unexpected confounders. Notably, the fecal microbiome of the chronic TBI cohort had absent or reduced Prevotella spp. and Bacteroidies spp. Conversely, bacteria in the Ruminococcaceae family were higher in abundance in TBI compared to control profiles. Previously reported hypoaminoacidemia, including significantly reduced levels of l-tryptophan, l-sarcosine, ß-alanine, and alanine, positively correlated with the reduced levels of Prevotella spp. in the TBI cohort samples compared to controls. Although the sequelae of gut-brain axis disruption after TBI is not fully understood, characterizing TBI-related alterations in the fecal microbiome may provide biomarkers and therapeutic targets to address patient morbidity.

Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India.

Background & objectives:Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube (MGIT) for the determination of OFX resistance as an alternative and cost-effective method.Methods:A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively.Results:Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P<0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene.Interpretation & conclusions:These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance.

Molecular characterization and phylogenetic analysis of fowl adenovirus serotype-4 from Guangdong Province, China.

Aim:Our aim in this study was to isolate potentially novel strains of fowl adenovirus serotype-4 (FAdV-4) that is currently circulating in broiler chicken flocks in Guangdong Province, China, and to compare nucleotide and amino acid (AA) sequences of their respective hexon genes.Materials and Methods:The experiment was carried out on poultry farms experiencing outbreaks of FAdV-4-associated hydropericardium syndrome (HPS). Tissue samples from the hearts and livers of deceased chickens were screened for FAdV-4 infection using hexon gene-specific polymerase chain reaction (PCR).Results:New virus isolates were used to infect 7-day-old chicks, which went onto reproduce typical HPS signs. The hypervariable region of the FAdV-4 hexon gene was PCR-amplified and sequenced. The hexon nucleotide and deduced AA sequence identities were 99.8-99.9% and 99.5-99.8%, respectively, among the four novel isolates. In addition, the new isolates were 97-100% and 96.4-99.9% identical to the nucleotide and deduced AA sequences, respectively, of FAdV-4 hexon genes available in the National Center for Biotechnology Information GenBank database. Phylogenetic analyses, based on the hexon gene sequence, revealed that the new isolates, clustered with FAdV-C; the FAdV-A, FAdV-B, FAdV-D, and FAdV-E viruses, were more distantly related.Conclusion:New FAdV-4 isolates from Guangdong Province are similar to those identified in other regions of the world. This information provides critical insight into HPS epidemiology and provides a perspective for monitoring outbreaks and developing strategies for disease prevention.

Emergence of colistin-resistant Escherichia coli in poultry, house flies, and pond water in Mymensingh, Bangladesh.

Objective:Emergence of colistin-resistant Escherichia coli (CREC) has generated a sense of public alarm. The objective of this study was to detect the CREC and identification of the gene responsible for such resistance.Materials and Methods:A total of 150 samples comprising poultry cloacal swab, house flies (Musca domestica), and pond water were collected randomly from Mymensingh, Bangladesh and analyzed. Isolation and identification of E. coli were done based on culture and E. coli 16S rRNA gene-specific polymerase chain reaction (PCR). Phenotypic detection of CREC was done by disk diffusion method. Finally, colistin resistance genes were detected by PCR by using colistin resistant gene mcr3 specific primers.Results:Among the 150 samples, phenotypically 18.00% (n = 27/150) isolates were found as colistin resistant. By PCR, 8.00% of the E. coli isolates were found positive for the presence of mcr3 gene.Conclusions:Colistin resistant E. coli carrying mcr3 are detected in poultry, house flies and water that are of great public health concern.

Molecular detection of virulent Mannheimia haemolytica and Pasteurella multocida in lung tissues of pneumonic sheep from semiarid tropics, Rajasthan, India

The present study was planned to detect the genetic elements of Mannheimia haemolytica and Pasteurella multocida in pneumonic sheep lungs. Pneumonia was diagnosed on the basis of gross pathological lesions. Lung tissues were collected at necropsy of sheep (n = 96) and subjected to isolation of total DNA. The M. haemolytica -specific PHSSA and Rpt2 genes, and the P. multocidaspecific KMT1 and the Omp87 genes were amplified using polymerase chain reaction (PCR). A housekeeping gene targeting the sheep cellular mitochondrial 12S ribosomal DNA was used as an internal control. PCR reactions were optimized using the positive and negative controls. Gene-specific PCR products were subjected to nucleotide sequencing for confirmation. The pneumonic lungs showed congestion and hemorrhagic changes with consolidation, which was most evident in the whole of the apical lobes and parts of the diaphragmatic lobes. PCR amplification showed detection of PHSSA (327 bp) and Rpt2 (~1022 bp) genes specific to M. haemolytica in 52 (54.1%), and the KMT1 (457 bp) and Omp87 (2627 bp) genes specific to P. multocida in 16 (16.6%) lung samples. Sequence analysis confirmed the PCR products for specific genes. This study highlighted the culture-independent, rapid, and confirmatory diagnosis of ovine pneumonic pasteurellosis caused by M. haemolytica and/or P. multocida .

Target-Enriched Endosymbiont Sequencing (TEEseq): A New High-Throughput Sequencing Approach Applied to the Comprehensive Characterization of Endosymbionts.

Intracellular bacteria are ubiquitous in the insect world, with perhaps the best-studied example being the alphaproteobacterium, Wolbachia. Like most endosymbionts, Wolbachia cannot be cultivated outside of its host cells, hindering traditional microbial characterization techniques. Furthermore, multiple Wolbachia strains can be present within a single host, and certain strains can be present in densities below the detection limit of current methods. To date, Wolbachia has most commonly been studied using polymerase chain reaction (PCR) amplification and Sanger DNA sequencing by targeting specific genes in the bacterium's genome. PCR amplification and Sanger sequencing of multiple Wolbachia strains requires analysis of individually cloned sequences, which is resource and labor intensive. To help mitigate these difficulties, we present a modified double digest restriction site associated DNA sequencing (ddRADseq) approach to target and sequence in parallel multiple genes by adding restriction enzyme recognition sites to gene-specific PCR primers. Adopting this strategy allows us to uniquely tag and sequence amplicons from multiple hosts simultaneously on an Illumina MiSeq platform. Our approach represents an efficient and cost-effective method to characterize multiple target genes in population surveys.

Shifts on Gut Microbiota Associated to Mediterranean Diet Adherence and Specific Dietary Intakes on General Adult Population.

There is increasing evidence for the interaction between gut microbiome, diet, and health. It is known that dysbiosis is related to disease and that most of the times this imbalances in gut microbial populations can be promoted through diet. Western dietary habits, which are characterized by high intakes of calories, animal proteins, saturated fats, and simple sugars have been linked with higher risk of obesity, diabetes, cancer, and cardiovascular disease. However, little is known about the impact of dietary patterns, dietary components, and nutrients on gut microbiota in healthy people. The aim of our study is to determine the effect of nutrient compounds as well as adherence to a dietary pattern, as the Mediterranean diet (MD) on the gut microbiome of healthy adults. Consequently, gut microbiota composition in healthy individuals, may be used as a potential biomarker to identify nutritional habits as well as risk of disease related to these habits. Dietary information from healthy volunteers (n = 27) was recorded using the Food Frequency Questionnaire. Adherence to the MD was measured using the PREDIMED test. Microbiota composition and diversity were obtained by 16S rRNA gene sequencing and specific quantitative polymerase chain reaction. Microbial metabolic activity was determined by quantification of short chain fatty acids (SCFA) on high performance liquid chromatography (HPLC). The results indicated that a higher ratio of Firmicutes–Bacteroidetes was related to lower adherence to the MD, and greater presence of Bacteroidetes was associated with lower animal protein intake. High consumption of animal protein, saturated fats, and sugars affected gut microbiota diversity. A significant higher presence of Christensenellaceae was found in normal-weight individuals compared to those who were overweight. This was also the case in volunteers with greater adherence to the MD compared to those with lower adherence. Butyricimonas, Desulfovibrio, and Oscillospira genera were associated with a BMI <25 and the genus Catenibacterium with a higher PREDIMED score. Higher bifidobacterial counts, and higher total SCFA were related to greater consumption of plant-based nutrients, such as vegetable proteins and polysaccharides. Better adherence to the MD was associated with significantly higher levels of total SCFA. Consequently, diet and specific dietary components could affect microbiota composition, diversity, and activity, which may have an effect on host metabolism by increasing the risk of Western diseases.

PREVALENCE AND CHARACTERIZATION OF SHIGATOXIGENIC ESCHERICHIA COLI IN BROILER BIRDS IN MYMENSINGH

Shigatoxigenic Escherichia coli (STEC) are major food-borne pathogens. They transmit to human through contaminated meat and meat products of animals and poultry, and frequently associated with various types of human illness including haemolytic uremic syndrome. This preliminary study showed the prevalence of STEC in 60 cloacal swab samples of live healthy broiler chickens collected randomly when sold at a wholesale market in Mymensingh district of Bangladesh. Isolation and identification of E. coli was carried out using Eosin Methylene Blue (EMB) agar media and 16S rRNA gene specific polymerase chain reaction (PCR). Among the 60 samples, 49 (81.67%) were found positive to E. coli. These E. coli isolates were screened for the detection of STEC by PCR using stx1 and stx2 gene specific primers. Among the 49 positive samples, 5 (10.20%) were found positive for stx1 gene, and 26 (53.06%) were positive for stx2 gene. In addition, 6 (12.24%) isolates were found positive to both stx1 and stx2 genes, and the remaining 12 (22.46%) were negative. The high prevalence ofSTEC in the broiler chicken alarms the public health impact as the people are always in close contact with these live broiler chickens in the open market as well as processing of meat at home before cooking. However, further studies are required to uncover the major source(s) for the transmission of STEC to human in ruralBangladesh.

A Study of Molecular Characterization of the Trimethoprim Resistant Salmonella typhi Strains Prevalent in Himachal Pradesh

Typhoid is a major public health problem in tropical and subtropical countries including India. Salmonella typhi, the causative agent of typhoid fever, is a gram-negative, motile, rod shaped, facultative anaerobe. It is solely a human pathogen and there is no animal reservoir. Antibiotic therapy is the mainstay for the treatment of typhoid fever and the complications associated with it. Nowadays, emerging multidrug resistance among Salmonella typhi strains has become a major public health problem. Present research work was carried out for the identification and molecular characterization of Trimethoprim resistant Salmonella enterica serovar typhi strains from individuals suffering with typhoid fever by means of various techniques i.e.; biochemicals, phenotypical and drug resistant gene specific polymerase chain reaction (PCR). A total of 14 blood specimen of infected patients were collected from Solan district of Himachal Pradesh with varying age groups and were processed via broth enrichment methods for primary isolation and identification of typhoid bacilli. Microbiological and biochemical investigations revealed the presence of S. typhi in all 14 specimens. The antibiotic susceptibility assay was carried out for 11 antimicrobial to study the MDR pattern of the identified bacilli. It was observed that 14/14 S. typhi strains were 100% resistance to Trimethoprim, Co-trimoxazole, Sulfanilamide, Penicillin, Ampicillin, Oxacillin, Tetracycline and Erythromycin, and 70-100% susceptible to Levofloxacin, Amikacin and Amoxicillin. The PCR analysis of these MDR strains showed the presence of dhfr a7 (365 bp) gene in only 10/14 isolates. This study confirmed that Trimethoprim resistance in these strains were due to the presence of dhfr a7 gene and also that PCR based diagnosis could be very useful for the rapid detection of drug resistant S. typhi strains. Present study emphasize that Trimethoprim drug is no longer useful for the treatment of typhoid fever as its MIC optimization was very high (750μg/ml). This study may further help the researchers in selecting the appropriate therapeutic approaches targeting Trimethoprim resistant Salmonella typhi strains.

Variation in Growth, Colonization of Maize, and Metabolic Parameters of GFP- and DsRed-Labeled Fusarium verticillioides Strains.

Autofluorescent proteins are frequently applied as visual markers in the labeling of filamentous fungi. Genes gfp and DsRed were transformed into the genome of Fusarium verticillioides via the Agrobacterium tumefaciens-mediated transformation method. The selected transformants displayed a bright green or red fluorescence in all the organelles of the growing fungal mycelia and spores (except for the vacuoles) both in cultures and in the maize (Zea mays) roots they colonized. The results of gene-specific polymerase chain reaction (PCR) analysis and the thermal asymmetrical interlaced (TAIL)-PCR analysis demonstrated that gfp and DsRed were integrated on different chromosomes of the fungus. Reductions in the colony growth on the plates at pH 4.0 and 5.5 was observed for the green fluorescent protein (GFP)-transformant G3 and the DsRed-transformant R4, but transformants G4 and R1 grew as well as the wild-type strain at pH 4.0. The speed of growth of all the transformants was similar to the wild-type strain at pH ≥ 7. The insertion of gfp and DsRed did not alter the production of extracellular enzymes and fumonisin B by F. verticillioides. The transformants expressing GFP and DsRed proteins were able to colonize maize roots. However, the four transformants examined produced fewer CFU in the root samples than the wild-type strain during a sampling period of 7 to 28 days after inoculation.

Mutation detection of CYP21A2 gene in nonclassical congenital adrenal hyperplasia patients with premature pubarche.

Background:Congenital adrenal hyperplasia (CAH) due to mutations in the gene encoding 21-hydroxilase is one of common disease with an autosomal recessive form. In this study, our aim is to detect the prevalence of eight common mutations in nonclassical congenital adrenal hyperplasia (NCAH).Materials and Methods:A total of 30 patients with clinical and laboratory evidence of NCAH was selected. Gene-specific polymerase chain reaction (PCR) without contamination of pseudogene was carried out, and PCR product of this step was used to amplification-refractory mutation system PCR on eight common mutations in CYP21A2 gene.Results:Two heterozygote patients for I2G mutation and six heterozygote patients for Q318X mutation is reported in our study. These mutations associated with the classic form of CAH, and heterozygotes presented with NC symptom, including premature pubarche and hirsutism.Conclusion:There are some data about the association of the mutation with the clinical form of CAH including classic (salt-wasting and simple virilizing) and NC form. I2G and Q318X mutations were reported in classic form in homozygote state, but the heterozygote form associated with NC form. CAH diagnosis with NC symptom and with measurement of 17-hydroxyprogestrone as NCAH is not a trusted assessment and require to molecular analysis for accurate diagnosis.

Demonstration of virulent genes within Listeria and Klebsiella isolates contaminating the export quality frozen shrimps

In the previously studied export quality shrimp samples, presence of a range of pathogenic bacteria including Listeria spp. and Klebsiella spp. were detected. The presence of eae gene in Escherichia coli, aero specific gene in Aeromonas spp., and sodB gene in Vibrio spp. were observed which might confer the associated virulence. Present study further attempted to detect the existence of virulent genes in Listeria spp. and Klebsiella isolates from the same shrimp samples through the gene specific polymerase chain reaction (PCR). Visualization of bands of iap (457 bp) and gyrB2 (711 bp) genes after PCR amplification referred the presence of virulence strains of Listeria spp. and Klebsiella spp., respectively in the export quality shrimp samples. Together with the previous findings of virulence genes of several pathogenic bacteria, the outcomes of current study further conferred the possible detrimental impact of the export quality frozen shrimps as and when consumed.