Proteome and immunogenicity differences in BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c during planktonic growth in 7H9 and Proskauer Beck media.

Bacillus Calmette-Guèrin (BCG) remains as the only vaccine employed to prevent tuberculosis (TB) during childhood. Among factors likely contributing to the variable efficacy of BCG is the modification in its antigenic repertoire that may arise from in vitro growth conditions. Our vaccine candidate, BCGΔBCG1419c, improved protection against TB in mice and guinea pigs with bacteria grown in either 7H9 OADC Tween 80 or in Proskauer Beck Tween 80 media in independent studies. Here, we compared the proteomes of planktonic cultures of BCG and BCGΔBCG1419c, grown in both media. Further to this, we compared systemic immunogenicity ex vivo elicited by both types of BCG strains and cultures when used to vaccinate BALB/c mice. Both the parental strain BCG Pasteur ATCC 35734, and its isogenic mutant BCGΔBCG1419c, had several medium-dependent changes. Moreover, ex vivo immune responses to a multiantigenic (PPD) or a single antigenic (Ag85A) stimulus were also medium-dependent. Then, not only the presence or absence of the BCG1419c gene in our strains under study affected the proteome produced in vitro but also that this was affected by culture medium, potentially leading to changes in the capacity to induce ex vivo immune responses.

BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice.

The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c, encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c, encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain.

hilD Is Required for the Active Internalization of Salmonella Newport into Cherry Tomatoes.

Salmonella enterica is a foodborne pathogen that can be internalized into fresh produce. Most of the Salmonella virulence genes are clustered in regions denominated Salmonella Pathogenicity Islands (SPI). SPI-1 encodes a Type Three Secretion System (T3SS-1) and effector proteins that allow the internalization of Salmonella into animal cells. HilD is a transcriptional regulator that induces the expression of SPI-1 genes and other related virulence genes located outside of this island. Here, we assessed the role of hilD in the internalization of Salmonella Newport and Typhimurium into cherry tomatoes, by evaluating either an isolate from an avocado orchard, S. Newport-45 or the laboratory strain S. Typhimurium SL1344 and their isogenic mutants in hilD. The internalization of these bacteria was carried out by using a temperature gradient of 12°C. The transcription of hilD and invA was tested by qRT-PCR experiments. Our results show that S. Newport-45 hilD mutant viable cells obtained from the interior of the fruit were decreased (2.7-fold), compared with those observed for S. Typhimurium SL1344. Interestingly, at 3 days postinoculation, the cells recovered from S. Newport-45 hilD mutant were similar to those recovered from all the strains evaluated, suggesting that hilD is required only for the initial internalization of S. Newport.

Pathogenesis of Salmonella Newport to BALB/c mice when internalized into cherry tomatoes.

Salmonella Newport is a serovar frequently associated with outbreaks caused by consumption of raw tomatoes. This study tested the internalization of S. Newport-45 into cherry tomatoes and its resulting pathogenicity in vivo. Pathogenicity of S. Newport-45 was tested in BALB/c mice inoculated orally with either LB grown or cherry tomatoes homogenates internally contaminated with S. Newport-45. CFU of S. Newport-45 was recovered from the gastrointestinal tract, liver and spleen of the inoculated animals. Similar loads (p > 0.05) were recovered from the GI tract of BALB/c mice inoculated with S. Newport-45 grown in LB or with cherry tomato homogenates internally contaminated. Spread of S. Newport-45 to the liver of mice increased (p < 0.05) when they were inoculated with homogenates of cherry tomatoes internally contaminated with S. Newport-45 stored for 3 days compared with bacteria grown in LB. Salmonella Newport-45 hilA and rpoS genes were transcribed when the bacteria were inside the cherry tomato. The results obtained in this study show S. Newport-45 pathogenicity when it is internalized in a raw consumption fruit such as cherry tomato.

Genetic diversity and antimicrobial resistance of Salmonella serotypes recovered throughout the beef production chain and from patients with salmonellosis.

Salmonella is one of the major foodborne pathogens worldwide. The antimicrobial resistance (AMR) of this foodborne pathogen has raised a great concern in recent years. Studies on the frequency and characterization of Salmonella serotypes can help to improve our knowledge on the epidemiology of this pathogen. The purpose of this study was to compare the serotypes, AMR and genetic profiles of Salmonella isolates recovered from raw beef throughout the beef production chain and from human feces associated with clinical cases of salmonellosis. The serotype, AMR and pulsed-field gel electrophoresis profile of 243 Salmonella enterica isolates recovered from beef carcasses (n = 78), ground beef (n = 135), and human feces from clinical cases of salmonellosis (n = 30) were compared. Forty-three different Salmonella serotypes were identified and regardless of the source, the top five corresponded to Typhimurium, Give, Group B (partially serotyped), Infantis and Anatum. Twelve serotypes from beef carcasses were also found in ground beef, showing their presence throughout the beef production chain. Salmonella Typhimurium, Infantis, Anatum and Montevideo were the only serotypes identified in all sample types. Resistance to tetracyclines was the most frequent (41.2%) followed by resistance to aminoglycosides (37%), folate pathway inhibitors (21%), quinolones (20.2%), phenicols (17.1%), penicillins (15.6%) and cephems (7%). Multidrug resistance was observed in 28.8% of the isolates, and those from human feces showed resistance to a larger number of antimicrobials. Great concern arises from the resistance and reduced susceptibility observed to quinolones and cephalosporins because these drugs are the first line of treatment for invasive Salmonella infections. Twenty-seven distinct pulse-types were detected among 238 isolates. Clustering analysis for the most frequent serotypes identified groups of isolates with similar AMR profiles. Multidrug resistance spreading throughout the food production chain should be continually monitored and its importance emphasized.

Determination of extended spectrum ß-lactamases/AmpC ß-lactamases and plasmid-mediated quinolone resistance in Escherichia coli isolates obtained from bovine carcasses in Mexico.

Food-borne bacterial infections have worldwide importance, and a great variety of antibiotic resistance mechanisms, mainly of the chromosome type, have rapidly developed. Antimicrobial resistance was determined in this study in terms of the presence of extended-spectrum ß-lactamases (ESBLs), plasmid AmpC ß-lactamases (pAmpC), and plasmid-mediated quinolone resistance (PMQR) from 155 Escherichia coli isolates obtained from bovine carcasses from two states in Mexico (states of Mexico and Jalisco). Isolates were challenged with ß-lactam antimicrobials (ampicillin, ceftazidime, and cefotaxime) and quinolones (nalidixic acid and ciprofloxacin). The presence of the bla TEM, bla SHV, bla CTX-M, bla OXA , bla CMY, bla MOX, bla LAT, bla BIL, qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA genes was examined by PCR. Clonal relationship was determined using pulsed-field gel electrophoresis (PFGE). The highest resistance was found to be to nalidixic acid (64 %), followed by ampicillin (32 %), ciprofloxacin (10 %), and ceftazidime and cefotaxime (both 1.3 %). bla CMY (n = 1), bla TEM (n = 24), qnrB (n = 9), and qnrS (n = 7) genes were detected. PFGE analysis showed that the majority of isolates had a different genotypic profile. To our knowledge, this is the first report of the presence of the qnrB, qnrS, and bla CMY genes in E. coli isolated from bovine meat in Mexico.

Genetic characterization of Escherichia coli isolated from cattle carcasses and feces in Mexico state.

Meat of bovine origin is one of the major vehicles in the transmission of verotoxigenic Escherichia coli (VTEC) to human consumers. This pathogen can produce serious human illness, including bloody diarrhea and hemolytic uremic syndrome. The aim of the current study was to characterize E. coli isolates (mainly VTEC strains) belonging to several serotypes in samples from cattle carcasses and feces of three municipal slaughter plants from Mexico State. The genetic diversity and molecular relatedness among the isolates was evaluated with multiple-locus variable-number tandem repeat analysis (MLVA). To our knowledge, and with the exception of E. coli O157:H7, this is the first time that serotypes analyzed here have been subtyped by MLVA in Mexico. MLVA typing grouped the 37 strains from this study into 30 distinct genotypes, 26 of which were unique. These findings indicate that cattle carcasses and feces from slaughter plants in Mexico are a source of VTEC that are genetically diverse in terms of serotypes and virulence profiles. The presence of these pathogens in carcasses indicates the high probability of the spread of VTEC strains during slaughter and processing.