BackgroundStreptococcus suis is a major swine pathogen worldwide that causes meningitis, septicemia, arthritis, and endocarditis. Using animal models, a surface-associated subtilisin-like protease (SspA) has recently been shown to be an important virulence factor for S. suis. In this study, we hypothesized that the S. suis SspA subtilisin-like protease may modulate cytokine secretion by macrophages thus contributing to the pathogenic process of meningitis.ResultsPhorbol 12-myristate 13-acetate-differentiated U937 macrophages were stimulated with recombinant SspA prior to monitor cytokine secretion by ELISA. Our results indicated that the recombinant SspA was able to dose-dependently induce IL-1β, IL-6, TNF-α, CXCL8 and CCL5 secretion in macrophages. The heat-inactivated protease was still able to induce cytokine secretion suggesting a non-proteolytic mechanism of macrophage activation. Using specific kinase inhibitors, evidence were bought that cytokine secretion by macrophages stimulated with the recombinant SspA involves the mitogen-activated protein kinase signal transduction pathway. While stimulation of macrophages with low concentrations of recombinant SspA was associated to secretion of high amounts of CCL5, the use of recombinant SspA at a high concentration resulted in low amounts of CCL5 detected in the conditioned medium. This was found to be associated with a proteolytic degradation of CCL5 by SspA. The ability of SspA to induce cytokine secretion in macrophages was confirmed using a mutant of S. suis deficient in SspA expression.ConclusionIn conclusion, this study identified a new mechanism by which the S. suis SspA may promote central nervous system inflammation associated with meningitis.

BackgroundSalmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids.ResultsThe blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations.ConclusionsThe ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the insertion and deletion of DNA stretches have shaped their evolutionary histories.

BackgroundRapid identification (ID) and antibiotic susceptibility testing (AST) of the causative micro-organism of bloodstream infections result in earlier targeting of antibiotic therapy.In order to obtain results of ID and AST up to 24 hours earlier, we evaluated the accuracy of direct inoculation of the Phoenix system from positive blood cultures (BACTEC) by using Serum Separator Tubes to harvest bacteria from positive blood cultures. Results were compared to those of standard Phoenix procedure. Discrepancies between the two methods were resolved by using the API system, E-test or microbroth dilution.ResultsID with the direct method was correct for 95.2% of all tested Enterobacteriaceae (n = 42) and 71.4% of Pseudomonas aeruginosa strains (n = 7).AST with the direct method showed a categorical agreement for Gram-negative rods (GNR) of 99.0%, with 0.7% minor errors, 0.3% very major errors and no major errors. All antibiotics showed an agreement of >95%.The direct method for AST of Staphylococcus (n = 81) and Enterococcus (n = 3) species showed a categorical agreement of 95.4%, with a minor error rate of 1.1%, a major error rate of 3.1% and a very major error rate of 0.4%. All antibiotics showed an agreement of >90%, except for trimethoprim-sulfamethoxazole and erythromycin.ConclusionsInoculation of Phoenix panels directly from positive blood cultures can be used to report reliable results of AST of GNR a day earlier, as well as ID-results of Enterobacteriaceae. For Staphylococcus and Enterococcus species, results of AST can also be reported a day earlier for all antibiotics, except for erythromycin and trimethoprim-sulfamethoxazole.

BackgroundSince 1991 several outbreaks of acute coccidioidomycosis (CM) were diagnosed in the semi-arid Northeast of Brazil, mainly related to disturbance of armadillo burrows caused by hunters while digging them for the capture of these animals. This activity causes dust contaminated with arthroconidia of Coccidioides posadasii, which, once inhaled, cause the mycosis. We report on the identification of C. posadasii in soil samples related to outbreaks of CM.ResultsTwenty four soil samples had their DNA extracted and subsequently submitted to a semi-nested PCR technique using specific primers. While only 6 (25%) soil samples were positive for C. posadasii by mice inoculation, all (100%) were positive by the molecular tool.ConclusionThis methodology represents a simple, sensitive and specific molecular technique to determine the environmental distribution of Coccidioides spp. in endemic areas, but cannot distinguish the species. Moreover, it may be useful to identify culture isolates. Key-words: 1. Coccidioidomycosis. 2. Coccidioides spp. 3. C. posadasii. 4. Semi-arid. 5. Semi-nested PCR

BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models.ResultsB. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD) values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis.ConclusionsWe have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.

BackgroundSimian Immunodeficiency Viruses (SIVs) are the precursors of Human Immunodeficiency Viruses (HIVs) which have lead to the worldwide HIV/AIDS pandemic. By studying SIVs in wild primates we can better understand the circulation of these viruses in their natural hosts and habitat, and perhaps identify factors that influence susceptibility and transmission within and between various host species. We investigated the SIV status of wild West African chimpanzees (Pan troglodytes verus) which frequently hunt and consume the western red colobus monkey (Piliocolobus badius badius), a species known to be infected to a high percentage with its specific SIV strain (SIVwrc).ResultsBlood and plasma samples from 32 wild chimpanzees were tested with INNO-LIA HIV I/II Score kit to detect cross-reactive antibodies to HIV antigens. Twenty-three of the samples were also tested for antibodies to 43 specific SIV and HIV lineages, including SIVwrc. Tissue samples from all but two chimpanzees were tested for SIV by PCRs using generic SIV primers that detect all known primate lentiviruses as well as primers designed to specifically detect SIVwrc. Seventeen of the chimpanzees showed varying degrees of cross-reactivity to the HIV specific antigens in the INNO-LIA test; however no sample had antibodies to SIV or HIV strain - and lineage specific antigens in the Luminex test. No SIV DNA was found in any of the samples.ConclusionsWe could not detect any conclusive trace of SIV infection from the red colobus monkeys in the chimpanzees, despite high exposure to this virus through frequent hunting. The results of our study raise interesting questions regarding the host-parasite relationship of SIVwrc and wild chimpanzees in their natural habitat.