The CpxRA two-component system represses gene expression of the heat-labile toxin of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strains produce at least one of two types of enterotoxins: the heat-labile (LT) and heat-stable (ST) toxins, which are responsible for the watery secretory diarrhoea that is a hallmark of the human ETEC infection. One regulatory system that controls the transcription of virulence genes in pathogenic bacteria is the CpxRA two-component system (TCS). We reported that the eltAB bicistronic operon, which encodes for the A and B subunits of LT, was repressed for the CpxRA TCS by direct binding of CpxR-P from -12 to +6 bp with respect to the transcription start site of eltAB. Moreover, the Cpx-response activation down-regulated the transcription of eltAB genes, and this negative effect was CpxRA-dependent. Our data show that CpxRA TCS is a negative regulator of the LT, one of the main virulence determinants of ETEC.

The Two-Component System CpxRA Represses Salmonella Pathogenicity Island 2 by Directly Acting on the ssrAB Regulatory Operon.

The acquisition of Salmonella pathogenicity island 2 (SPI-2) conferred on Salmonella the ability to survive and replicate within host cells. The ssrAB bicistronic operon, located in SPI-2, encodes the SsrAB two-component system (TCS), which is the central positive regulator that induces the expression of SPI-2 genes as well as other genes located outside this island. On the other hand, CpxRA is a two-component system that regulates expression of virulence genes in many bacteria in response to different stimuli that perturb the cell envelope. We previously reported that the CpxRA system represses the expression of SPI-1 and SPI-2 genes under SPI-1-inducing conditions by decreasing the stability of the SPI-1 regulator HilD. Here, we show that under SPI-2-inducing conditions, which mimic the intracellular environment, CpxRA represses the expression of SPI-2 genes by the direct action of phosphorylated CpxR (CpxR-P) on the ssrAB regulatory operon. CpxR-P recognized two sites located proximal and distal from the promoter located upstream of ssrA. Consistently, we found that CpxRA reduces the replication of Salmonella enterica serovar Typhimurium inside murine macrophages. Therefore, our results reveal CpxRA as an additional regulator involved in the intracellular lifestyle of Salmonella, which in turn adds a new layer to the intricate regulatory network controlling the expression of Salmonella virulence genes. IMPORTANCE SPI-2 encodes a type III secretion system (T3SS) that is a hallmark for the species Salmonella enterica, which is essential for the survival and replication within macrophages. Expression of SPI-2 genes is positively controlled by the two-component system SsrAB. Here, we determined a regulatory mechanism involved in controlling the overgrowth of Salmonella inside macrophages. In this mechanism, CpxRA, a two-component system that is activated by extracytoplasmic stress, directly represses expression of the ssrAB regulatory operon; as a consequence, expression of SsrAB target genes is decreased. Our findings reveal a novel mechanism involved in the intracellular lifestyle of Salmonella, which is expected to sense perturbations in the bacterial envelope that Salmonella faces inside host cells, as the synthesis of the T3SS-2 itself.

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin.

Klebsiella oxytoca is a resident of the human gut. However, certain K. oxytoca toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the aroX and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of K. oxytoca, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δcrp isogenic mutant. In summary, we found that CRP directly activates the transcription of the aroX and NRPS operons and that the absence of CRP reduced cytotoxicity of K. oxytoca on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.

Bacteriological quality of bottled water obtained from Mexico City small water purification plants: Incidence and identification of potentially pathogenic nontuberculous mycobacteria species.

The aim of this study was to determine the bacteriological quality of bottled water samples obtained from small purification plants located in Mexico City and to identify potentially pathogenic nontuberculous mycobacteria (NTM) species found in these samples. All 111 samples analyzed were positive for aerobic mesophilic bacteria (AMB) and 46 (41.4%) did not comply with Mexico's Official Guidelines. Sixty-nine (62.1%) and 23 (20.7%) water samples were positive for total coliforms (TC) and fecal coliforms (FC), respectively. A total of 81 (72.9%) of the water samples exceeded the maximum allowed limit stipulated in the guideline. Thirty-three (29.7%) of the purified water samples were positive for NTM, being recovered a total of 40 isolates. These NTM isolates were identified using three molecular markers (hsp65, rrs and rpoB genes) which corresponded to the fast-growing mycobacteria M. chelonae (n = 12), M. porcinum (n = 8), M. senegalense (n = 5), M. abscessus (n = 4), M. septicum (n = 4), M. wolinskyi (n = 3), M. mucogenicum (n = 2), M. fortuitum (n = 1) and M. sp. (n = 1). In seven purified water samples, two different NTM species were isolated simultaneously. Overall, these results showed that most of the purified bottled water samples analyzed in this study had unsatisfactory microbiological quality and some harbored NTM associated with illness. Our data could hasten health authorities to intensify efforts in the routine monitoring of activities in the purified bottled water industry in order to supply safe and healthy water to the public.

Microbiological Quality and Occurrence of Nontuberculous Mycobacteria in Fresh-Squeezed Orange Juice Samples Purchased from Street Vendors in Mexico City.

Nontuberculous mycobacteria (NTM) are potentially pathogenic agents commonly found in a natural ecosystem. For this reason, food is considered another source of NTM transmission for humans. The aims of this study were to evaluate the microbiological quality and the occurrence of NTM in fresh-squeezed orange juice samples purchased from street vendors. All 102 samples analyzed were positive for aerobic mesophilic bacteria (AMB), with limits ranging from 1.8 to 6.2 log CFU/ml. A total of 55 (54%), 25 (25%), and 13 (13%) orange juice samples were positive for total coliforms (TC), fecal coliforms (FC), and Escherichia coli , respectively. TC, FC, and E. coli were present with limits ranging from <3 to >1,100 most probable number (MPN)/ml, <3 to 460 MPN/ml, and <3 to 11 MPN/ml, respectively. Six orange juice samples harbored NTM. These NTM were identified by using three molecular markers (hsp65, rrs, and rpoB genes) and corresponded to the fast-growing mycobacteria: Mycobacterium fortuitum (n = 3), Mycobacterium rhodesiae (n = 1), Mycobacterium obuense (n = 1), and a mixture of M. fortuitum and Mycobacterium mucogenicum in an additional sample (n = 1). No correlation was found between the presence NTM in orange juice samples with the presence and concentration of the indicator microorganisms (aerobic mesophilic bacteria, TC, and FC). Overall, these results suggest that fresh-squeezed orange juice might represent a vehicle for NTM transmission in humans. Therefore, prevention of contamination by humans (proper handling and washing of oranges) during juice preparation should be recommended.

Microbiological Quality of Ready-to-Eat Vegetables Collected in Mexico City: Occurrence of Aerobic-Mesophilic Bacteria, Fecal Coliforms, and Potentially Pathogenic Nontuberculous Mycobacteria.

The aims of this study were to evaluate the microbiological quality and the occurrence of nontuberculous mycobacteria (NTM) in a variety of salads and sprouts from supermarkets and street vendors in Mexico City. Aerobic-mesophilic bacteria (AMB) were present in 100% of RTE-salads samples; 59% of samples were outside guidelines range (>5.17 log10 CFU per g). Although fecal coliforms (FC) were present in 32% of samples, only 8% of them exceeded the permissible limit (100 MPN/g). Regarding the 100 RTE-sprouts, all samples were also positive for AMB and total coliforms (TC) and 69% for FC. Seven NTM species were recovered from 7 salad samples; they included three M. fortuitum, two M. chelonae, one M. mucogenicum, and one M. sp. Twelve RTE-sprouts samples harbored NTM, which were identified as M. porcinum (five), M. abscessus (two), M. gordonae (two), M. mucogenicum (two), and M. avium complex (one). Most RTE-salads and RTE-sprouts had unsatisfactory microbiological quality and some harbored NTM associated with illness. No correlation between the presence of coliforms and NTM was found. Overall, these results suggest that RTE-salads and RTE-sprouts might function as vehicles for NTM transmission in humans; hence, proper handling and treatment before consumption of such products might be recommendable.

Primary thrombophilia in Mexico IX: the glycoprotein IIIa PLA1/A2 polymorphism is not associated with the sticky platelet syndrome phenotype.

INTRODUCTION: The sticky platelet syndrome (SPS) seems to be a common cause of thrombosis, although no molecular substrate to explain platelet hyperaggregability has been found. OBJECTIVE: To analyze an association between the SPS phenotype and the platelet glycoprotein (GP) IIIa PL(A1/A2) (human platelet antigen [HPA]-1a/b) gene polymorphism. METHODS: Along an 18-month period, Mexican mestizo thrombophilic patients were prospectively accrued. The SPS phenotype was assessed by aggregometry, whereas a tetra-primer amplification refractory mutation system (ARMS) polymerase chain reaction analysis was used to detect the PLA1 and PLA2 alleles. RESULTS: A total of 95 individuals with SPS and 127 healthy donors were studied; in 11 of the donors and 16 of the patients with SPS the A2 allele of the GP IIb/IIIA was found, yielding a weak and nonsignificant association (odds ratio 2.14, 95% CI 0.94-4.85). CONCLUSION: In Mexican mestizo patients, the platelet GP IIIa PL(A1/A2) gene polymorphism does not lead to the SPS phenotype.