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BACKGROUND: Emerging infectious diseases of zoonotic origin present a critical threat to global population health. As accelerating globalisation makes epidemics and pandemics more difficult to contain, there is a need for effective preventive interventions that reduce the risk of zoonotic spillover events. Public policies can play a key role in preventing spillover events. The aim of this review is to identify and describe evaluations of public policies that target the determinants of zoonotic spillover. Our approach is informed by a One Health perspective, acknowledging the inter-connectedness of human, animal and environmental health. METHODS: In this systematic scoping review, we searched Medline, SCOPUS, Web of Science and Global Health in May 2021 using search terms combining animal health and the animal-human interface, public policy, prevention and zoonoses. We screened titles and abstracts, extracted data and reported our process in line with PRISMA-ScR guidelines. We also searched relevant organisations' websites for evaluations published in the grey literature. All evaluations of public policies aiming to prevent zoonotic spillover events were eligible for inclusion. We summarised key data from each study, mapping policies along the spillover pathway. RESULTS: Our review found 95 publications evaluating 111 policies. We identified 27 unique policy options including habitat protection; trade regulations; border control and quarantine procedures; farm and market biosecurity measures; public information campaigns; and vaccination programmes, as well as multi-component programmes. These were implemented by many sectors, highlighting the cross-sectoral nature of zoonotic spillover prevention. Reports emphasised the importance of surveillance data in both guiding prevention efforts and enabling policy evaluation, as well as the importance of industry and private sector actors in implementing many of these policies. Thoughtful engagement with stakeholders ranging from subsistence hunters and farmers to industrial animal agriculture operations is key for policy success in this area. CONCLUSION: This review outlines the state of the evaluative evidence around policies to prevent zoonotic spillover in order to guide policy decision-making and focus research efforts. Since we found that most of the existing policy evaluations target 'downstream' determinants, additional research could focus on evaluating policies targeting 'upstream' determinants of zoonotic spillover, such as land use change, and policies impacting infection intensity and pathogen shedding in animal populations, such as those targeting animal welfare.
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Doenças Transmissíveis Emergentes , Zoonoses , Animais , Humanos , Zoonoses/prevenção & controle , Zoonoses/epidemiologia , Doenças Transmissíveis Emergentes/prevenção & controle , Saúde Global , Formulação de Políticas , PolíticasRESUMO
Wild mushrooms are a vital source of income and nutrition for many poor communities and of value to recreational foragers. Literature relating to the edibility of mushroom species continues to expand, driven by an increasing demand for wild mushrooms, a wider interest in foraging, and the study of traditional foods. Although numerous case reports have been published on edible mushrooms, doubt and confusion persist regarding which species are safe and suitable to consume. Case reports often differ, and the evidence supporting the stated properties of mushrooms can be incomplete or ambiguous. The need for greater clarity on edible species is further underlined by increases in mushroom-related poisonings. We propose a system for categorizing mushroom species and assigning a final edibility status. Using this system, we reviewed 2,786 mushroom species from 99 countries, accessing 9,783 case reports, from over 1,100 sources. We identified 2,189 edible species, of which 2,006 can be consumed safely, and a further 183 species which required some form of pretreatment prior to safe consumption or were associated with allergic reactions by some. We identified 471 species of uncertain edibility because of missing or incomplete evidence of consumption, and 76 unconfirmed species because of unresolved, differing opinions on edibility and toxicity. This is the most comprehensive list of edible mushrooms available to date, demonstrating the huge number of mushrooms species consumed. Our review highlights the need for further information on uncertain and clash species, and the need to present evidence in a clear, unambiguous, and consistent manner.
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Agaricales , Intoxicação Alimentar por Cogumelos , Alimentos , Humanos , Intoxicação Alimentar por Cogumelos/epidemiologiaRESUMO
OBJECTIVES: To clone and express a neopullulanase gene from Lactobacillus mucosae LM1 in Escherichia coli and characterise the resulting recombinant neopullulanase. RESULTS: An ORF in L. mucosae corresponding to a neopullulanase was cloned and expressed in E. coli. The predicted amino acid sequence of the neopullulanase contained catalytic sites and conserved motifs that are present in members of the neopullulanase subfamily. The resulting recombinant neopullulanase was efficiently purified by Ni-NTA affinity chromatography. The purified enzyme optimally hydrolyses pullulan at 37 °C and pH 6.0, producing panose as the major reaction product. CONCLUSIONS: To the best of our knowledge, this is the first report of the cloning, expression and characterisation of a neopullulanase gene from a lactic acid bacterium.
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Clonagem Molecular/métodos , Glucanos/biossíntese , Glucanos/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Lactobacillus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/química , Hidrólise , Lactobacillus/genética , Fases de Leitura Aberta , Especificidade por SubstratoRESUMO
This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs.
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The draft genome sequence of Lactiplantibacillus plantarum BS25, previously isolated from a fermented rice-shrimp mixture (balao-balao) in the Philippines, was analyzed. The genome contains 3,264,139 bp (44.62% GC%), and a total of 3,069 predicted coding sequences, 2 rRNAs, and 52 tRNAs.
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Pediococcus acidilactici is a well-studied fermentative bacterium reported to have potential probiotic properties. Here, we report the draft genome of P. acidilactici 3G3 previously isolated from burong babi, a Philippine fermented pork dish. The 3G3 draft genome has 1,886,498 bp, a GC content of 42.27%, and a completeness of 99.38%.
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Micro-organisms belonging to the Lactobacillus genus complex are often used for oral consumption and are generally considered safe but can exhibit pathogenicity in rare and specific cases. Therefore, screening and understanding genetic factors that may contribute to pathogenicity can yield valuable insights regarding probiotic safety. Limosilactobacillus mucosae LM1, Lactiplantibacillus plantarum SK151, Lactiplantibacillus plantarum BS25, Limosilactobacillus fermentum SK152 and Lactobacillus johnsonii PF01 are current probiotics of interest; however, their safety profiles have not been explored. The genome sequences of LM1, SK151, SK152 and PF01 were downloaded from the NCBI GenBank, while that of L. plantarum BS25 was newly sequenced. These genomes were then annotated using the Rapid Annotation using Subsystem Technology tool kit pipeline. Subsequently, a command line blast was performed against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) to identify potential virulence factors and antibiotic resistance (AR) genes. Furthermore, ResFinder was used to detect acquired AR genes. The query against the VFDB identified genes that have a role in bacterial survivability, platelet aggregation, surface adhesion, biofilm formation and immunoregulation; and no acquired AR genes were detected using CARD and ResFinder. The study shows that the query strains exhibit genes identical to those present in pathogenic bacteria with the genes matched primarily having roles related to survival and surface adherence. Our results contribute to the overall strategies that can be employed in pre-clinical safety assessments of potential probiotics. Gene mining using whole-genome data, coupled with experimental validation, can be implemented in future probiotic safety assessment strategies.
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a novel strain of coronavirus first reported in December 2019 which rapidly spread throughout the world and was subsequently declared a pandemic by the World Health Organization (WHO) in March 2020. Although vaccines, as well as treatments, have been rapidly developed and deployed, these are still spread thin, especially in the developing world. There is also a continuing threat of the emergence of mutated variants which may not be as responsive to available vaccines and drugs. Accessible and affordable sources of antiviral drugs against SARS-CoV-2 offer wider options for the clinical treatment of populations at risk for severe COVID-19. Using in silico methods, this study identified potential inhibitors against the SARS-CoV-2 main protease (Mpro), the protease directly responsible for the activation of the viral replication enzyme, from a consolidated database of 1516 Philippine natural products. Molecular docking experiments, along with in silico ADME predictions, determined top ligands from this database with the highest potential inhibitory effects against Mpro. Molecular dynamic trajectories of the apo and diosmetin-7-O-b-D-glucopyranoside (DG) in complex with the protein predicted potential mechanisms of action for the ligand-by separating the Cys145-His41 catalytic dyad and by influencing the protein network through key intra-signaling residues within the Mpro binding site. These findings show the inhibitory potential of DG against the SARS-CoV-2 Mpro, and further validation is recommended through in vitro or in vivo experimentation.
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Produtos Biológicos , Tratamento Farmacológico da COVID-19 , Antivirais/química , Antivirais/farmacologia , Produtos Biológicos/farmacologia , Proteases 3C de Coronavírus , Cisteína Endopeptidases/química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Filipinas , Inibidores de Proteases/química , SARS-CoV-2 , Proteínas não Estruturais ViraisRESUMO
INTRODUCTION: The increasing incidence of pathogen transmission from animals to humans (zoonotic spillover events) has been attributed to behavioural practices and ecological and socioeconomic change. As these events sometimes involve pathogens with epidemic or pandemic potential, they pose a serious threat to population health. Public policies may play a key role in preventing these events. The aim of this review is to identify evaluations of public policies that target the determinants of zoonotic spillover, examining approaches taken to evaluation, choice of outcomes measures and evidence of effectiveness. Our approach to identifying and analysing this literature will be informed by a One Health lens, acknowledging the interconnectedness of human, animal and environmental health. METHODS AND ANALYSIS: A systematic scoping review methodology will be used. To identify articles, we will search Medline, SCOPUS, Web of Science and Global Health in May 2021 using search terms combining animal health and the animal-human interface, public policy, prevention and zoonoses. We will screen titles and abstracts and extract data according to published guidelines for scoping reviews. All evaluations of public policies aiming to prevent zoonotic spillover events will be eligible for inclusion. We will summarise key data from each study, mapping policies along the spillover pathway and outlining the range of policies, approaches to evaluation and outcome measures. Review findings will provide a useful reference for researchers and practitioners, outlining the state of the evaluative evidence around policies to prevent zoonotic spillover. ETHICS AND DISSEMINATION: Formal ethical approval is not required, because the study does not involve primary data collection. The findings of this study will be disseminated through a peer-reviewed publication, presentations and summaries for key stakeholders.
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Projetos de Pesquisa , Zoonoses , Animais , Humanos , Zoonoses/prevenção & controle , Política de Saúde , Avaliação de Resultados em Cuidados de Saúde , Revisões Sistemáticas como AssuntoRESUMO
Probiotic strains from different origins have shown promise in recent decades for their health benefits, for example in promoting and regulating the immune system. The immunomodulatory potential of four Lactobacillus strains from animal and plant origins was evaluated in this paper based on their genomic information. Comparative genomic analysis was performed through genome alignment, average nucleotide identity (ANI) analysis and gene mining for putative immunomodulatory genes. The genomes of the four Lactobacillus strains show relative similarities in multiple regions, as observed in the genome alignment. However, ANI analysis showed that L. mucosae LM1 and L. fermentum SK152 are the most similar when considering their nucleotide sequences alone. Gene mining of putative immunomodulatory genes studied from L. plantarum WCFS1 yielded multiple results in the four potential probiotic strains, with L. plantarum SK151 showing the largest number of genes at around 74 hits, followed by L. johnsonii PF01 at 41 genes when adjusted for matches with at least 30â% identity. Looking at the immunomodulatory genes in each strain, L. plantarum SK151 and L. johnsonii PF01 may have wider activity, covering both immune activation and immune suppression, as compared to L. mucosae LM1 and L. fermentum SK152, which could be more effective in activating immune cells and the pro-inflammatory cascade rather than suppressing it. The similarities and differences between the four Lactobacillus species showed that there is no definitive trend based on the origin of isolation alone. Moreover, higher percentage identities between genomes do not directly correlate with higher similarities in potential activity, such as in immunomodulation. The immunomodulatory function of each of the four Lactobacillus strains should be observed and verified experimentally in the future, since some the activity of some genes may be strain-specific, which would not be identified through comparative genomics alone.
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A novel peptide having free radical scavenging activity was separated, using an on-line high-performance liquid chromatography (HPLC) - ABTS screening method, from bovine skim milk fermented by Lactococcus lactis SL6 (KCTC 11865BP). It was further purified using reverse phase-HPLC (RP-HPLC) and sequenced by RP-HPLC-tandem mass spectrometry. The amino acid sequence of the identified peptide was determined to be Phe-Ser-Asp-Ile-Pro-Asn-Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu-Trp (2,362 Da), which is corresponding to the C-terminal fragment of bovine αs1-casein (f179-199). The hydroxyl radicals scavenging activity (IC50 28.25±0.96 µM) of the peptide chemically synthesized based on the MS/MS data showed a slightly lower than that of the natural antioxidant Trolox (IC50 15.37±0.52 µM). Furthermore, derivatives of the antioxidant peptide were synthesized. The antioxidative activity of the derivatives whose all three proline residues replaced by alanine significantly decreased, whereas replacement of two proline residues in N-terminal region did not affect its antioxidative activity, indicating that 3rd proline in C-terminal region is critical for the antioxidative activity of the peptide identified in this study. In addition, N-terminal region of the antioxidant peptide did not show its activity, whereas C-terminal region maintained antioxidative activity, suggesting that C-terminal region of the peptide is important for antioxidative activity.
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Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen-probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.
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Aderência Bacteriana , Carboidratos/química , Jejuno/microbiologia , Lactobacillus/fisiologia , Mucinas/metabolismo , Probióticos/análise , Animais , Linhagem Celular , Mucosa Intestinal/microbiologia , Jejuno/metabolismo , Mucinas/química , Ligação Proteica , SuínosRESUMO
In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.
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Linhagem Celular , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Jejuno/citologia , Proteoma/análise , Suínos , Actinas/isolamento & purificação , Animais , Anexina A2/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Genoma , Proteínas de Choque Térmico/isolamento & purificação , Homeostase , Mucosa Intestinal/imunologia , Jejuno/imunologia , Jejuno/metabolismo , Espectrometria de Massas , Redes e Vias Metabólicas , Proteoma/isolamento & purificação , Transdução de SinaisRESUMO
We isolated a Bacillus strain, RX7, with inhibitory activity against Listeria monocytogenes from soil and identified it as Bacillus amyloliquefaciens based on 16S rRNA gene sequencing. The inhibitory activity was stable over a wide range of pH and was fully retained after 30 min at 80°C, after which it decreased gradually at higher temperatures. The activity was sensitive to the proteolytic action of α-chymotrypsin, proteinase-K, and trypsin, indicating its proteinaceous nature. This bacteriocin was active against a broad spectrum of bacteria and the fungus Candida albicans. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of approximately 5 kDa. Ammonium sulfate precipitation and anion-exchange and gel permeation chromatography integrated with reverse phase-high-performance liquid chromatography were used for bacteriocin purification. Automated N-terminal Edman degradation of the purified RX7 bacteriocin recognized the first 15 amino acids as NH2-X-Ala-Trp-Tyr-Asp-Ile-Arg-Lys-Leu-Gly-Asn-Lys-Gly-Ala, where the letter X in the sequence indicates an unknown or nonstandard amino acid. Based on BLAST similarity search and multiple alignment analysis, the obtained partial sequence showed high homology with the two-peptide lantibiotic haloduracin (HalA1) from Bacillus halodurans, although at least two amino acids differed between the sequences. A time-kill study demonstrated a bactericidal mode of action of RX7 bacteriocin.
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Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Bacillus amyloliquefaciens/química , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Bacillus amyloliquefaciens/genética , Candida albicans/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Listeria monocytogenes/efeitos dos fármacos , Peso Molecular , Proteólise/efeitos dos fármacos , RNA Ribossômico 16S/genética , Microbiologia do Solo , TemperaturaRESUMO
The high level of genetic diversity in the microflora of the gastrointestinal tract has the potential to provide numerous beneficial functions to the host. Thus it is now acknowledged that the complexity in animal functioning is linked to the interacting microbiome in the gut. Despite the importance of gut microbiome, there is a lack of information concerning the microbial communities in the pig gut during the weaning transition. This study describes the fecal microbial shifts of healthy piglets during the weaning transition using barcoded pyrosequencing of the prokaryotic 16S rRNA gene. Fecal samples were obtained from 15 piglets during the pre-weaning period (fourth week after birth) and post-weaning (sixth week after birth) and were subjected to community genomic DNA extraction for pyrosequencing analysis. As the piglets underwent the weaning transition a trend toward increased bacterial diversity was observed, based on species abundance as measured by the Shannon-Weaver index. Firmicutes (54.0%) and Bacteroidetes (59.6%) were the most dominant phyla during pre-weaning and post-weaning, respectively. During the weaning transition a distinct shift from Bacteroides to Prevotella as the most abundant genus was observed. Additionally, we detected a number of abundant gut bacterial species that have not been reported previously. Clostridium rectum, C. clostridioforme, C. lactatifermentans and Butyricimonas virosa were uniquely detected prior to weaning while Roseburia cecicola and Blautia wexlerae were detected during the post-weaning period only.
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Fezes/microbiologia , Trato Gastrointestinal/microbiologia , Consórcios Microbianos/genética , Microbiota/genética , Suínos/microbiologia , Animais , Animais Lactentes/microbiologia , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Sequência de Bases , Biodiversidade , Clostridium/genética , Clostridium/isolamento & purificação , DNA Bacteriano/genética , Variação Genética , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Prevotella/genética , Prevotella/isolamento & purificação , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , DesmameRESUMO
This study examined the fecal bacterial diversity of 15-week-old pigs from three purebred lines: Duroc, Landrace, and Yorkshire. Taxon-dependent and -independent analyses were performed to evaluate differences in the fecal bacterial communities and to identify bacterial genera that can be used to discriminate breeds, following high-throughput pyrosequencing of 16S rRNA genes. Among the breeds evaluated, Landrace had the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detected in all samples regardless of breed. On the other hand, Catenibacterium, Blautia, Dialister, and Sphaerochaeta were differentially detected among breeds, as demonstrated by the canonical loading plot. The discriminant analysis of principal components plot also showed clear separation of the three purebred pig lines, with a certain degree of similarity between Landrace and Yorkshire pigs and a distinct separation between Duroc pigs and the other two breeds. Other factors not related to breed, such as season or time of sampling and pen effects, may contribute to shaping the gut microbiota of pigs.
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Fezes/microbiologia , Microbiota , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Sus scrofa/microbiologia , Animais , Cruzamento , Genes de RNAr , Variação Genética , Análise de Componente PrincipalRESUMO
Background. Emergence of multidrug-resistant tuberculosis (MDR-TB) poses a major challenge to prevailing disease management. MDR-TB arises from mutations in several genes comprising the resistance determining regions, including rpoB, katG and gyrA. Objective. To detect and characterize mutations in rpoB, katG and gyrA. Methods. Thirty selected Mycobacterium tuberculosis isolates from the IDS-PGH were subjected to PCR amplification and sequencing. Sequences were compared to the wild type strain H37Rv. Results. Mutations were detected in codons 512, 513, 516, 522, 526, 531 and 533 of rpoB, codons 280, 281, 315 and 333 of katG, and codons 90 and 94 of gyrA sequences. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94, respectively. A clustering analysis of the sequences showed occurrence of seven, four and three clusters for the genes rpoB, katG and gyrA, respectively. The eight clusters obtained from the concatenated sequences of the three genes represent the eight potential genotypes of local strains. One cluster represents the wild type strain genotype, another cluster represents the XDR strain genotype, and six clusters represent the MDR strain genotypes. Conclusion. These findings indicate the utility of multiple RDR sequence analysis in both identifying specific drug resistance mutation and genotyping of various M. tuberculosis isolates.
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Tuberculose , Terapêutica , TerapêuticaRESUMO
BACKGROUND: Emergence of multidrug-resistant tuberculosis (MDR-TB) poses a major challenge to prevailing disease management. MDR-TB arises from mutations in several genes comprising the resistance determining regions, including rpoB, katG and gyrA. OBJECTIVE: To detect and characterize mutations in rpoB, katG and gyrA. METHODS: Thirty selected Mycobacterium tuberculosis isolates from the IDS-PGH were subjected to PCR amplification and sequencing. Sequences were compared to the wild type strain H37Rv. RESULTS: Mutations were detected in codons 512, 513, 516, 522, 526, 531 and 533 of rpoB, codons 280, 281, 315 and 333 of katG, and codons 90 and 94 of gyrA sequences. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94, respectively. A clustering analysis of the sequences showed occurrence of seven, four and three clusters for the genes rpoB, katG and gyrA, respectively. The eight clusters obtained from the concatenated sequences of the three genes represent the eight potential genotypes of local strains. One cluster represents the wild type strain genotype, another cluster represents the XDR strain genotype, and six clusters represent the MDR strain genotypes. CONCLUSION: These findings indicate the utility of multiple RDR sequence analysis in both identifying specific drug resistance mutation and genotyping of various M. tuberculosis isolates.