Construction and validation of a first-generation Bordetella bronchiseptica long-oligonucleotide microarray by transcriptional profiling the Bvg regulon

Abstract Background Bordetella bronchiseptica is a bacterial respiratory pathogen that infects a broad range of mammals, causing chronic and often subclinical infections. Gene expression in Bordetella is regulated by a two-component sensory transduction system, BvgAS, which controls the expression of a spectrum of phenotypic phases transitioning between a virulent (Bvg+) phase and a non-virulent (Bvg-) phase. Results Based on the genomic sequence and using the freely available software ArrayOligoSelector, a long oligonucleotide B. bronchiseptica microarray was designed and assembled. This long-oligonucleotide microarray was subsequently tested and validated by comparing changes in the global expression profiles between B. bronchiseptica RB50 and its Bvg- phase-locked derivative, RB54. Data from this microarray analysis revealed 1,668 Bvg-regulated genes, which greatly expands the BvgAS regulon defined in previous reports. For previously reported Bvg-regulated transcripts, the gene expression data presented here is congruent with prior findings. Additionally, quantitative real-time PCR data provided an independent verification of the microarray expression values. Conclusion The results presented here provide a comprehensive, genome-wide portrait of transcripts encompassing the BvgAS regulon, while also providing data validating the long-oligonucleotide microarray described here for studying gene expression in Bordetella bronchiseptica.</p

Microarray-based genomic surveying of gene polymorphisms in Chlamydia trachomatis

By comparing two fully sequenced genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays, a logarithmic correlation was demonstrated between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes. Variable genes within 14 uncharacterized strains of C. trachomatis were identified by array analysis and verified by DNA sequencing. These genes may be crucial for understanding chlamydial virulence and pathogenesis

Subinhibitory Concentrations of Amoxicillin, Lincomycin, and Oxytetracycline Commonly Used to Treat Swine Increase Streptococcus suis Biofilm Formation

Streptococcus suis is a bacterial swine pathogen with a significant economic burden. It typically colonizes the tonsil and nasal cavity of swine causing a variety of symptoms ranging from asymptomatic carriage to lethal systemic disease. A key barrier toward the development of improved vaccines or interventions for S. suis infections is a gap in our understanding of the mechanisms contributing to persistence in the host, in which colonized pigs continue to shed and transmit S. suis. We hypothesized that exposure to sub-MICs of antibiotics commonly used by the swine industry would increase the biofilm capacity of S. suis strains. Using a 96-well plate MIC protocol, we experimentally determined the MIC for each of 12 antibiotics for a virulent strain of S. suis strain that consistently formed biofilms using a standard crystal violet assay. Using this static biofilm assay, we demonstrated that sub-MICs of bacitracin, carbadox, chlortetracycline, enrofloxacin, gentamicin, neomycin, sulfadimethoxine, tiamulin, and tylosin did not increase S. suis biofilms. In contrast, we demonstrated that sub-MICs of amoxicillin, lincomycin, and oxytetracycline increased overall biofilm formation under both static and flow conditions. The biofilm formation of 11 additional clinical isolates were measured using the relevant concentrations of amoxicillin, lincomycin, and oxytetracycline. Eight of the eleven strains increased the biofilm formation with lincomycin, seven with amoxicillin, and three with oxytetracycline. Collectively, our data demonstrate that exposure to sub-MICs of these commonly used antibiotics contributes to increased biofilm formation of S. suis, thereby potentially increasing survival and persistence within the respiratory tract of swine

Population structure and genetic diversity of Streptococcus suis isolates obtained from the United States

Diseases caused by the zoonotic pathogen Streptococcus suis are an extensive economic problem as well as an animal welfare concern for the global swine industry. Previous studies have evaluated the genomic diversity and population structure of S. suis isolates, however, the majority of these studies utilized isolates obtained from countries other than the U.S. This study applied whole genome sequencing and cgMLST-based typing to evaluate the population structure and genetic relatedness among S. suis isolates obtained within the U.S. The established high-resolution phylogenomic framework revealed extensive genomic variation and diversity among the sampled S. suis isolates, with isolates from the U.S. and from countries outside the U.S. found interspersed in the phylogeny. S. suis isolates obtained within the U.S. did not cluster by state or geographic location, however, isolates with similar serotypes, both obtained from within and outside the U.S., generally clustered together. Average nucleotide identity (ANI) values determined for the S. suis genomes were extensively broad, approaching the recommended species demarcation value, and correlated with the phylogenetic group distribution of the cgMLST-based tree. Numerous antimicrobial resistance (AMR) elements were identified among both U.S. and non-U.S. isolates with ble, tetO, and ermB genes identified as the most prevalent. The epf, mrp, and sly genes, historically used as markers for virulence potential, were also observed in the genomes of isolates that grouped together forming a subclade of clonal complex 1 (CC1) isolates. Collectively, the data in this report provides critical information needed to address potential biosurveillance needs and insights into the genetic diversity and population structure of S. suis isolates obtained within the U.S

Antimicrobial Resistance Distribution Differs Among Methicillin Resistant Staphylococcus aureus Sequence Type (ST) 5 Isolates From Health Care and Agricultural Sources

Antimicrobial resistance (AMR) is an expanding public health concern and methicillin resistant Staphylococcus aureus (MRSA) is a notable example. Since the discovery of livestock associated MRSA (LA-MRSA), public health concerns have arisen surrounding the potential of LA-MRSA isolates to serve as a reservoir for AMR determinants. In this study, we compare swine associated LA-MRSA ST5 and human clinical MRSA ST5 isolates for phenotypic antimicrobial susceptibilities determined via broth microdilution and genotypic determinants of AMR using whole genome sequencing and comparative genomic analysis to identify AMR elements. Swine associated LA-MRSA ST5 isolates exhibited phenotypic resistance to fewer antibiotics than clinical MRSA ST5 isolates from humans with no swine contact. Distinct genomic AMR elements were harbored by each subgroup, with little overlap in shared AMR genes between swine associated LA-MRSA ST5 and clinical MRSA ST5 isolates. Our results demonstrate that phenotypic antimicrobial susceptibilities and genotypic determinants of AMR among swine associated LA-MRSA ST5 and clinical MRSA ST5 isolates are separate and distinct

The contribution of BvgR, RisA, and RisS to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation in Bordetella bronchiseptica

Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation

The Presence of Alpha Interferon at the Time of Infection Alters the Innate and Adaptive Immune Responses to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating and costly diseases to the swine industry worldwide. Overall, the adaptive immune response to PRRS virus (PRRSV) is weak, which results in delayed elimination of virus from the host and inferior vaccine protection. PRRSV has been shown to induce a meager alpha interferon (IFN-α) response, and we hypothesized that elevated IFN-α levels early in infection would shorten the induction time and increase elements of the adaptive immune response. To test this, we measured both antibody and cell-mediated immunity in pigs after the administration of a nonreplicating human adenovirus type 5 vector expressing porcine IFN-α (Ad5–pIFN-α) at the time of PRRSV infection and compared the results to those for pigs infected with PRRSV alone. Viremia was delayed, and there was a decrease in viral load in the sera of pigs administered the Ad5–pIFN-α. Although seroconversion was slightly delayed in pigs receiving Ad5–pIFN-α, probably due to the early reduction in viral replication, little difference in the overall or neutralizing antibody response was seen. However, there was an increase in the number of virus-specific IFN-γ-secreting cells detected in the pigs receiving Ad5–pIFN-α, as well as an altered cytokine profile in the lung at 14 days postinfection, indicating that the presence of IFN-α at the time of infection can alter innate and adaptive immune responses to PRRSV

Blood or Serum Exposure Induce Global Transcriptional Changes, Altered Antigenic Profile, and Increased Cytotoxicity by Classical Bordetellae

The classical bordetellae sense and respond to a variety of environments outside and within their mammalian hosts. By causing inflammation and tissue damage, we reasoned that bordetellae are likely to encounter components of blood and/or serum during the course of a respiratory infection, and that detecting and responding to these would be advantageous. Therefore, we hypothesized that classical bordetellae have the ability to sense and respond to blood or serum. Blood or serum exposure resulted in substantial transcriptional changes in Bordetella bronchiseptica, including enhanced expression of many virulence-associated genes. Exposure to blood or serum additionally elicited production of multiple antigens not otherwise detectable, and led to increased bacterial cytotoxicity against macrophages. Transcriptional responses to blood/serum were observed in a Bvg− phase-locked mutant, indicating that the response is not solely dependent on a functional BvgAS system. Similar transcriptional responses to blood/serum were observed for the other classical bordetellae, Bordetella pertussis and Bordetella parapertussis. These data suggest the classical bordetellae respond to signals present in blood and serum by changing their behavior in ways that likely contribute to their remarkable success, via effects on pathogenesis, persistence and/or transmission between hosts

The SPRY domain–containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation

Macrophages lacking SPSB2 have increased NO production and enhanced pathogen-killing capabilities due to decreased ubiquitin-mediated destruction of iNOS

Single Nucleotide Polymorphism Analysis Indicates Genetic Distinction and Reduced Diversity of Swine-Associated Methicillin Resistant Staphylococcus aureus (MRSA) ST5 Isolates Compared to Clinical MRSA ST5 Isolates

Livestock associated methicillin resistant S. aureus (LA-MRSA) are lineages adapted to livestock species. LA-MRSA can be transmitted to humans and public health concerns exist because livestock may be the largest MRSA reservoir outside of hospital settings. Although the predominant European (ST398) and Asian (ST9) lineages of LA-MRSA are considered livestock adapted, North American swine also harbor ST5, a globally disseminated and highly pathogenic lineage. This study applied whole genome sequencing and single nucleotide polymorphism (SNP) typing to compare the population structure and genetic relatedness between swine associated and human clinical MRSA ST5 isolates. The established high-resolution phylogenomic framework revealed that LA-MRSA and human clinical MRSA ST5 are genetically distinct. LA-MRSA isolates were found to be clonal within farms, while greater genome diversity was observed among sampled clinical MRSA ST5. Analysis of the accessory genome demonstrated that LA-MRSA ST5 isolates and clinical MRSA ST5 isolates harbor different AMR genes and virulence factors, consistent with the SNP analysis. Collectively, our data indicate LA-MRSA and clinical MRSA ST5 isolates are distinct and the swine reservoir is likely of minimal significance as a source of clinical MRSA ST5 infections