Benchmarking the empirical accuracy of short-read sequencing across the M. tuberculosis genome.

MOTIVATION: Short-read whole-genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences and sequencing bias reduces the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias and GC content. RESULTS: Reference-based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results, we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems and more generally for WGS applications in other organisms. AVAILABILITY AND IMPLEMENTATION: All relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation.

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches.

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis ( Mtb ) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants.

BACKGROUND: Pre-genomic and post-genomic studies demonstrate that chlamydiae actively recombine in vitro and in vivo, although the molecular and cellular biology of this process is not well understood. In this study, we determined the genome sequence of twelve Chlamydia trachomatis recombinants that were generated in vitro under antibiotic selection. These strains were used to explore the process of recombination in Chlamydia spp., including analysis of candidate recombination hotspots, and to correlate known C. trachomatis in vitro phenotypes with parental phenotypes and genotypes. RESULTS: Each of the 190 examined recombination events was the product of homologous recombination, and no candidate targeting motifs were identified at recombination sites. There was a single deletion event in one recombinant progeny that resulted in the removal of 17.1 kilobases between two rRNA operons. There was no evidence for preference for any specific region of the chromosome for recombination, and analyses of a total of over 200 individual recombination events do not provide any support for recombination hotspots in vitro. Two measurable phenotypes were analyzed in these studies. First, the efficiency of attachment to host cells in the absence of centrifugation was examined, and this property segregated to regions of the chromosome that carry the polymorphic membrane protein (Pmp) genes. Second, the formation of secondary inclusions within cells varied among recombinant progeny, but this did not cleanly segregate to specific regions of the chromosome. CONCLUSIONS: These experiments examined the process of recombination in C. trachomatis and identified tools that can be used to associate phenotype with genotype in recombinant progeny. There were no data supporting the hypothesis that particular nucleotide sequences are preferentially used for recombination in vitro. Selected phenotypes can be segregated by analysis of recombination, and this technology may be useful in preliminary analysis of the relationship of genetic variation to phenotypic variation in the chlamydiae.

Lineage classification and antitubercular drug resistance surveillance of Mycobacterium tuberculosis by whole-genome sequencing in Southern India.

Whole-genome sequencing has created a revolution in tuberculosis management by providing a comprehensive picture of the various genetic polymorphisms with unprecedented accuracy. Studies mapping genomic heterogeneity in clinical isolates of Mycobacterium tuberculosis using a whole-genome sequencing approach from high tuberculosis burden countries are underrepresented. We report whole-genome sequencing results of 242 clinical isolates of culture-confirmed M. tuberculosis isolates from tuberculosis patients referred to a tertiary care hospital in Southern India. Phylogenetic analysis revealed that the isolates in our study belonged to five different lineages, with Indo-Oceanic (lineage 1, n = 122) and East-African Indian (lineage 3, n = 80) being the most prevalent. We report several mutations in genes conferring resistance to first and second line antitubercular drugs including the genes rpoB, katG, ahpC, inhA, fabG1, embB, pncA, rpsL, rrs, and gyrA. The majority of these mutations were identified in relatively high proportions in lineage 1. Our study highlights the utility of whole-genome sequencing as a potential supplemental tool to the existing genotypic and phenotypic methods, in providing expedited comprehensive surveillance of mutations that may be associated with antitubercular drug resistance as well as lineage characterization of M. tuberculosis isolates. Further larger-scale whole-genome datasets with linked minimum inhibition concentration testing are imperative for resolving the discrepancies between whole-genome sequencing and phenotypic drug sensitivity testing results and quantifying the level of the resistance associated with the mutations for optimization of antitubercular drug and precise dose selection in clinics. IMPORTANCE Studies mapping genetic heterogeneity of clinical isolates of M. tuberculosis for determining their strain lineage and drug resistance by whole-genome sequencing are limited in high tuberculosis burden settings. We carried out whole-genome sequencing of 242 M. tuberculosis isolates from drug-sensitive and drug-resistant tuberculosis patients, identified and collected as part of the TB Portals Program, to have a comprehensive insight into the genetic diversity of M. tuberculosis in Southern India. We report several genetic variations in M. tuberculosis that may confer resistance to antitubercular drugs. Further wide-scale efforts are required to fully characterize M. tuberculosis genetic diversity at a population level in high tuberculosis burden settings for providing precise tuberculosis treatment.

Resistance to a novel antichlamydial compound is mediated through mutations in Chlamydia trachomatis secY.

A novel and quantitative high-throughput screening approach was explored as a tool for the identification of novel compounds that inhibit chlamydial growth in mammalian cells. The assay is based on accumulation of a fluorescent marker by intracellular chlamydiae. Its utility was demonstrated by screening 42,000 chemically defined compounds against Chlamydia caviae GPIC. This analysis led to the identification of 40 primary-hit compounds. Five of these compounds were nontoxic to host cells and had similar activities against both C. caviae GPIC and Chlamydia trachomatis. The inhibitory activity of one of the compounds, (3-methoxyphenyl)-(4,4,7-trimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-C]quinolin-1-ylidene)amine (MDQA), was chlamydia specific and was selected for further study. Selection for resistance to MDQA led to the generation of three independent resistant clones of C. trachomatis. Amino acid changes in SecY, a protein involved in Sec-dependent secretion in Gram-negative bacteria, were associated with the resistance phenotype. The amino acids changed in each of the resistant mutants are located in the predicted central channel of a SecY crystal structure, based on the known structure of Thermus thermophilus SecY. These experiments model a process that can be used for the discovery of antichlamydial, anti-intracellular, or antibacterial compounds and has led to the identification of compounds that may have utility in both antibiotic discovery and furthering our understanding of chlamydial biology.

Prokaryotic Genome Annotation.

In the last decade, the high-throughput and relatively low cost of short-read sequencing technologies have revolutionized prokaryotic genomics. This has led to an exponential increase in the number of bacterial and archaeal genome sequences available, as well as corresponding increase of genome assembly and annotation tools developed. Together, these hardware and software technologies have given scientists unprecedented options to study their chosen microbial systems without the need for large teams of bioinformaticists or supercomputing facilities. While these analysis tools largely fall into only a few categories, each may have different requirements, caveats and file formats, and some may be rarely updated or even abandoned. And so, despite the apparent ease in sequencing and analyzing a prokaryotic genome, it is no wonder that the budding genomicist may quickly find oneself overwhelmed. Here, we aim to provide the reader with an overview of genome annotation and its most important considerations, as well as an easy-to-follow protocol to get started with annotating a prokaryotic genome.

Patterns of genomic interrelatedness of publicly available samples in the TB portals database.

The TB Portals program is an international collaboration for the collection and dissemination of tuberculosis data from patient cases focused on drug resistance. The central database is a patient-oriented resource containing both patient and pathogen clinical and genomic information. Herein we provide a summary of the pathogen genomic data available through the TB Portals and show one potential application by examining patterns of genomic pairwise distances. Distributions of pairwise distances highlight overall patterns of genome variability within and between Mycobacterium tuberculosis phylogenomic lineages. Closely related isolates (based on whole-genome pairwise distances and time between sample collection dates) from different countries were identified as potential evidence of international transmission of drug-resistant tuberculosis. These high-level views of genomic relatedness provide information that can stimulate hypotheses for further and more detailed research.

Self-Hybridization in Leishmania major.

Genetic exchange between different Leishmania strains in the sand fly vector has been experimentally demonstrated and is supported by population genetic studies. In nature, opportunities for Leishmania interstrain mating are restricted to flies biting multiply infected hosts or through multiple bites of different hosts. In contrast, self-mating could occur in any infected sand fly. By crossing two recombinant lines derived from the same Leishmania major strain, each expressing a different drug-resistance marker, self-hybridization in L. major was confirmed in a natural sand fly vector, Phlebotomus duboscqi, and in frequencies comparable to interstrain crosses. We provide the first high resolution, whole-genome sequencing analysis of large numbers of selfing progeny, their parents, and parental subclones. Genetic exchange consistent with classical meiosis is supported by the biallelic inheritance of the rare homozygous single nucleotide polymorphisms (SNPs) that arose by mutation during the generation of the parental clones. In contrast, heterozygous SNPs largely failed to be transmitted in Mendelian ratios for reasons not understood. SNPs that were heterozygous in both parents, however, recombined to produce homozygous alleles in some hybrids. For trisomic chromosomes present in both parents, transmittal to the progeny was only altered by self-hybridization, involving a gain or loss of somy in frequencies predicted by a meiotic process. Whole-genome polyploidization was also observed in the selfing progeny. Thus, self-hybridization in Leishmania, with its potential to occur in any infected sand fly, may be an important source of karyotype variation, loss of heterozygosity, and functional diversity. IMPORTANCE Leishmania are parasitic protozoa that cause a wide spectrum of diseases collectively known as the leishmaniases. Sexual reproduction in Leishmania has been proposed as an important source of genetic diversity and has been formally demonstrated to occur inside the sand fly vector midgut. Nevertheless, in the wild, opportunities for genetic exchange between different Leishmania species or strains are restricted by the capacity of different Leishmania strains to colonize the same sand fly. In this work, we report the first high resolution, whole-genome sequence analysis of intraclonal genetic exchange as a type of self-mating in Leishmania. Our data reveal that self-hybridization can occur with comparable frequency as interstrain mating under experimental lab conditions, leading to important genomic alterations that can potentially take place within every naturally infected sand fly.

In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells.

T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.

Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC.

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.

Complete genome sequence and annotation of the Treponema pallidum subsp. pallidum Chicago strain.

In syphilis research, the Nichols strain of Treponema pallidum, isolated in 1912, has been the most widely studied. Recently, important differences among T. pallidum strains emerged; therefore, we sequenced and annotated the Chicago strain genome to facilitate and encourage the use of this strain in studying the pathogenesis of syphilis.

Genome sequencing of recent clinical Chlamydia trachomatis strains identifies loci associated with tissue tropism and regions of apparent recombination.

The human pathogen Chlamydia trachomatis exists as multiple serovariants that have distinct organotropisms for different tissue sites. Culture and epidemiologic data have demonstrated that serovar G is more prevalent, while serovar E is less prevalent, for rectal isolates from men having sex with men (MSM). The relative prevalence of these serovars is the opposite for isolates from female cervical infections. In contrast, the prevalence of serovar J isolates is approximately the same at the different tissue sites, and these isolates are the only C-class strains that are routinely cultured from MSM populations. These correlations led us to hypothesize that polymorphisms in open reading frame (ORF) sequences correlate with the different tissue tropisms of these serovars. To explore this possibility, we sequenced and compared the genomes of clinical anorectal and cervical isolates belonging to serovars E, G, and J and compared these genomes with each other, as well as with a set of previously sequenced genomes. We then used PCR- and restriction digestion-based genotyping assays performed with a large collection of recent clinical isolates to show that polymorphisms in ORFs CT144, CT154, and CT326 were highly associated with rectal tropism in serovar G isolates and that polymorphisms in CT869 and CT870 were associated with tissue tropism across all serovars tested. The genome sequences collected were also used to identify regions of likely recombination in recent clinical strains. This work demonstrated that whole-genome sequencing along with comparative genomics is an effective approach for discovering variable loci in Chlamydia spp. that are associated with clinical presentation.

Exposure of Mycobacterium avium subsp. homonissuis to Metal Concentrations of the Phagosome Environment Enhances the Selection of Persistent Subpopulation to Antibiotic Treatment.

Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic intracellular pathogen causing infections in individuals with chronic lung conditions and patients with immune-deficient disorders. The treatment of MAH infections is prolonged and outcomes many times are suboptimal. The reason for the extended treatment is complex and reflects the inability of current antimicrobials to clear diverse phenotypes of MAH quickly, particularly, the subpopulation of susceptible but drug-tolerant bacilli where the persistent fitness to anti-MAH drugs is stimulated and enhanced by the host environmental stresses. In order to enhance the pathogen killing, we need to understand the fundamentals of persistence mechanism and conditions that can initiate the drug-tolerance phenotype in mycobacteria. MAH can influence the intracellular environment through manipulation of the metal concentrations in the phagosome of infected macrophages. While metals play important role and are crucial for many cellular functions, little is known how vacuole elements influence persistence state of MAH during intracellular growth. In this study, we utilized the in vitro model mimicking the metal concentrations and pH of MAH phagosome at 1 h and 24 h post-infection to distinguish if metals encountered in phagosome could act as a trigger factor for persistence phenotype. Antibiotic treatment of metal mix exposed MAH demonstrates that metals of the phagosome environment can enhance the persistence state, and greater number of tolerant bacteria is recovered from the 24 h metal mix when compared to the viable pathogen number in the 1 h metal mix and 7H9 growth control. In addition, bacterial phenotype induced by the 24 h metal mix increases MAH tolerance to macrophage killing in TNF-α and IFN-γ activated cells, confirming presence of persistent MAH in the 24 h metal mix condition. This work shows that the phagosome environment can promote persistence population in MAH, and that the population differs dependent on a concentration of metals.

Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations.

Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

Draft Genome Sequences of Three Coxiella burnetii Strains Isolated from Q Fever Patients.

In the current study, we determined the draft genome sequences of three Coxiella burnetii human disease isolates. The Coxiella burnetii Turkey (RSA315) and Dyer (RSA345) strains were isolated from acute Q fever patients, while the Ko (Q229) strain was isolated from a Q fever endocarditis patient.

Draft Genome Sequences of the Avirulent Coxiella burnetii Dugway 7D77-80 and Dugway 7E65-68 Strains Isolated from Rodents in Dugway, Utah.

Here, we present the draft genome sequences of the Coxiella burnetii Dugway 7D77-80 and Dugway 7E65-68 strains, which were isolated from rodents in Dugway, UT, in the 1950s. The strains reside in a distinct genomic group of C. burnetii and are considered avirulent despite having the largest genomes of the Coxiella genus.

Comparative analysis of the genomes of clinical isolates of Mycobacterium avium subsp. hominissuis regarding virulence-related genes.

PURPOSE: Mycobacterium avium subsp. hominissuis is a member of the M. avium complex, a heterogeneous group of bacteria that cause lung infection in immunocompetent patients or disseminated infection in patients with immunosuppression. The bacteria belonging to this complex have variable virulence, depending on the strain considered, and therefore a representative of the most common clinical phenotype was analysed. METHODOLOGY: The genomic sequences of four M. avium subsp. hominissuis isolates obtained from clinical specimens were completed. Mav101, Mav100 and MavA5 were isolated from the blood of patients with AIDS. MavA5 was disseminated from the lung, while Mav3388 was isolated from the lungs of a patient with chronic lung disease. The sequences were annotated using the published Mav104 genome as a blueprint. Functional and virulence analyses of the sequences were carried out. Mice studies comparing the virulence of the strains were performed. RESULTS: Findings showed that while Mav101 was very similar to Mav104, there were numerous differences between Mav104 and the remaining strains at nucleotide and predicted protein levels. The presence of genes associated with biofilm formation and several known virulence-related genes were sometimes differentially present among the isolates, suggesting overlapping functions by different genetic determinants. CONCLUSIONS: The sequences provided important information about M. avium heterogenicity and evolution as a pathogen. The limitation is the lack of understanding on possible overlapping functions of genes/proteins.

Draft Genome Sequences of Historical Strains of Coxiella burnetii Isolated from Cow's Milk and a Goat Placenta.

Here, we report draft genome sequences of historical strains of Coxiella burnetii derived from cow's milk and the placenta of a goat that had aborted. The California and Ohio milk strains display a different sequence type than do contemporary milk strains.

Identification of Prophages within the Mycobacterium avium 104 Genome and the Link of Their Function Regarding to Environment Survival.

Mycobacterium avium is an opportunistic bacterium associated with pathogenic behavior in both humans and animals. M. avium has evolved as a pathogen by having an environmental component in its life style. Prophages are the integrated viral forms in bacterium genome. They constitute about 10% - 20% of genome of many bacteria and they contribute to pathogenicity of microbes. We investigated whether the M. avium 104 genome contained prophages and evaluated the genes/proteins for putative functions. Three prophage genes were identified in the M. avium 104 database, and sequences were analyzed for specific motifs. The prophage sequences were then cloned in Mycobacterium smegmatis and the bacterial phenotype was evaluated in gain of function assays for environmental stresses, such as tolerance to extreme temperatures, UV light, biofilm formation and resistance to acid as well as macrophage survival. The results indicate that two of the prophage genes, MAV_0696 and MAV_2265, confer M. smegmatis with enhanced ability to produce biofilm. Using a Real-Time PCR, it was determined that MAV_0696 and MAV_2265 transcripts were upregulated upon biofilm formation by M. avium. The expression of MAV_2265 gene was significantly higher at all selected time points. In addition, the expression of MAV_2265 in M. smegmatis also led to significantly greater survival rate at pH 5.0 compared to the wild-type control. None of the other physical abilities were altered by overexpressing the prophage genes in M. smegmatis. In summary, we identified three prophage sequences in M. avium 104, from which two of them were found to be associated with biofilm formation and one with resistance to the acidic environment. Future studies will identify the mechanisms involved in the prophages function.