Microbiomes Associated With Foods From Plant and Animal Sources.

Food microbiome composition impacts food safety and quality. The resident microbiota of many food products is influenced throughout the farm to fork continuum by farming practices, environmental factors, and food manufacturing and processing procedures. Currently, most food microbiology studies rely on culture-dependent methods to identify bacteria. However, advances in high-throughput DNA sequencing technologies have enabled the use of targeted 16S rRNA gene sequencing to profile complex microbial communities including non-culturable members. In this study we used 16S rRNA gene sequencing to assess the microbiome profiles of plant and animal derived foods collected at two points in the manufacturing process; post-harvest/pre-retail (cilantro) and retail (cilantro, masala spice mixes, cucumbers, mung bean sprouts, and smoked salmon). Our findings revealed microbiome profiles, unique to each food, that were influenced by the moisture content (dry spices, fresh produce), packaging methods, such as modified atmospheric packaging (mung bean sprouts and smoked salmon), and manufacturing stage (cilantro prior to retail and at retail). The masala spice mixes and cucumbers were comprised mainly of Proteobacteria, Firmicutes, and Actinobacteria. Cilantro microbiome profiles consisted mainly of Proteobacteria, followed by Bacteroidetes, and low levels of Firmicutes and Actinobacteria. The two brands of mung bean sprouts and the three smoked salmon samples differed from one another in their microbiome composition, each predominated by either by Firmicutes or Proteobacteria. These data demonstrate diverse and highly variable resident microbial communities across food products, which is informative in the context of food safety, and spoilage where indigenous bacteria could hamper pathogen detection, and limit shelf life.

Salmonella Serotyping Using Whole Genome Sequencing.

Until recently, traditional serology and the Kauffmann White Scheme (KWS) have been the gold standard for Salmonella serotyping. Whole Genome Sequencing (WGS) has now emerged as an alternative in this field. Serotype information remains a cornerstone in food safety and public health activities to reduce the burden of salmonellosis. At the same time, recent advances in WGS have improved the ability to perform advanced pathogen characterization while improving trace back investigations to determine the source of foodborne illness during outbreaks. Serovar prediction based on WGS can be performed using in silico data analysis tools. Three such tools have been developed: (a). Salmonella in silico Typing Resource (SISTR), (b). SeqSero, and (c). in silico 7-gene MLST ST (Multilocus Sequence Typing Sub-Typing) which was generated using the SISTR platform. Public health officials around the world are diligently working to validate these tools for replacing traditional surveillance methods to provide a more powerful approach for molecular epidemiology in support of public health investigations. In this study, we report a retrospective analysis of our laboratory inventory of 1,041 Salmonella isolates collected between 1999 and 2017. These isolates are of public health significance since they all came from either food, feed or environmental swabs. They were all serotyped by both traditional serology and WGS using an in silico SeqSero tool for serovar prediction. Both predicted identical Salmonella serotypes in 899 isolates (86.4% of the 1,041 Salmonella isolates). SeqSero assignments differed from traditional serological testing in 80 isolates (7.7%) and no serotype prediction was ascertained from 62 isolates (5.9%). This retrospective study is an excellent example of using WGS and SeqSero as a data analysis tool to predict Salmonella serotypes that can provide numerous advantages including molecular and genetic details regarding the characteristics of the Salmonella isolates compared to traditional KWS serotyping. In conclusion, it is evident that using WGS and in silico tools for Salmonella serotyping might someday replace traditional serotyping.

Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis.

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.

Fibular fixation as an adjuvant to tibial intramedullary nailing in the treatment of combined distal third tibia and fibula fractures: a biomechanical investigation.

OBJECTIVE: Distal third tibia fractures have classically been treated with standard plating, but intramedullary (IM) nailing has gained popularity. Owing to the lack of interference fit of the nail in the metaphyseal bone of the distal tibia, it may be beneficial to add rigid plating of the fibula to augment the overall stability of fracture fixation in this area. This study sought to assess the biomechanical effect of adding a fibular plate to standard IM nailing in the treatment of distal third tibia and fibula fractures. METHODS: Eight cadaveric tibia specimens were used. Tibial fixation consisted of a solid titanium nail locked with 3 screws distally and 2 proximally, and fibular fixation consisted of a 3.5 mm low-contact dynamic compression plate. A section of tibia and fibula was removed. Testing was accomplished with an MTS machine. Each leg was tested 3 times; with and without a fibular plate and with a repetition of the initial test condition. Vertical displacements were tested with an axial load up to 500 N, and angular rotation was tested with torques up to 5 N*m. RESULTS: The difference in axial rotation was the only statistically significant finding (p = 0.003), with fibular fixation resulting in 1.1 degrees less rotation through the osteotomy site (17.96 degrees v. 19.10 degrees ). Over 35% of this rotational displacement occurred at the nail-locking bolt interface with the application of small torsional forces. CONCLUSION: Fibular plating in addition to tibial IM fixation of distal third tibia and fibula fractures leads to slightly increased resistance to torsional forces. This small improvement may not be clinically relevant.

The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue.

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.

Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV.

An immunization strategy using attenuated bacteria to deliver DNA vaccine plasmids to mucosal sites may induce protective T cell responses against sexual HIV transmission. In a murine intranasal (i.n.) immunization model, we demonstrate that transiently persistent Deltaasd Shigella flexneri strain 15D harboring DNA vaccines induces HIV- and SIV-specific gamma interferon (IFN-gamma) producing CD8+ T cells among splenocytes more efficiently than either a longer persisting DeltaaroD Salmonella typhimurium strain SL7207 or transiently persistent S. typhi strain Ty21a harboring DNA vaccines. Also, the frequency of antigen-specific gamma interferon (IFN-gamma) producing cells induced by Shigella 15D harboring a DNA vaccine were comparable to that induced by intramuscular (i.m.) immunization with purified DNA vaccine. Moreover, the magnitude of mucosal and systemic antigen-specific IgA and IgG responses after immunization were dependent upon the route (i.m. vs. i.n.) of inoculation, with i.n. Shigella 15D DNA vaccines generating higher levels of HIV-specific IgA in vaginal washings than i.m. purified DNA vaccine. Deltaasd S. flexneri is a promising vector for mucosal DNA vaccine immunization against HIV.