Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis). Thin-layer chromatography (TLC) using radiolabeled phosphatidylcholine and sphingomyelin as substrates indicated that cell extracts of M. tuberculosis contain both phospholipase C (PLC) and phospholipase D (PLD) activities. In contrast, only PLD activity was detected in cell extracts of M. smegmatis. Neither activity was detected in cell-free culture supernatants from these organisms. We and others recently identified two open reading frames in M. tuberculosis with the potential to encode proteins which are highly homologous to the nonhemolytic (PlcN) and hemolytic (PlcH) phospholipase C enzymes of Pseudomonas aeruginosa. In contrast to the plc genes in P. aeruginosa, which are considerably distal to each other (min 34 and 64 on the chromosome), the mycobacterial genes, designated mpcA and mpcB, are tandemly arranged in the same relative orientation and separated by only 191 bp. Both the mpcA and the mpcB genes were individually cloned in M. smegmatis, and PLC activity was expressed from each gene in this organism. Hybridization experiments using the mpcA and the mpcB genes as probes under conditions of moderate stringency identified sequences homologous to these genes in M. bovis, M. bovis BCG, and M. marinum but not in several other Mycobacterium species, including M. smegmatis, M. avium, and M. intracellulare. TLC analysis using radiolabeled substrates indicated that M. bovis and M. marinum cell extracts contain PLC and PLD activities, but only PLD activity was detected in M. bovis BCG cell extracts. Sphingomyelinase activity was also detected in whole-cell extracts of M. tuberculosis, M. marinum, M. bovis, and M. bovis BCG, but this activity was not detected in extracts of M. smegmatis. Sphingomyelinase activity was detected in cell extracts from M. smegmatis harboring either recombinant mpcA or mpcB. These data indicate that PLC and sphingomyelinase activities are associated with the most virulent mycobacterial species, while PLD activity was detected in both virulent and saprophytic strains.

Pseudomonas aeruginosa is a persistent pathogen in the airways of patients with cystic fibrosis or bronchiectasis from other causes and appears to have evolved strategies to survive the inflammatory response of the host. We hypothesized that the secreted hemolytic phospholipase C (PLC) of P. aeruginosa (PlcHR) would decrease neutrophil respiratory burst activity. We found that while intact wild-type P. aeruginosa cells stimulated moderate respiratory burst activity from human neutrophils, an isogenic mutant pseudomonas (ΔHR strain) containing a targeted deletion of the plcHR operon induced a much more robust oxidative burst from neutrophils. In contrast, a second pseudomonas mutant (ΔN) containing a disruption in the gene encoding the nonhemolytic PLC (PlcN) was not different from the wild type in stimulating neutrophil O2 ·−production. Readdition of purified PlcHR to the ΔHR strain suppressed neutrophil O2 ·−production to levels stimulated by wild-type bacteria. Interestingly, purified PlcHR decreased phorbol myristate acetate (PMA)- but not formyl methionyl-leucyl-proline (fMLP)-induced respiratory burst activity, suggesting interference by PlcHR with a protein kinase C (PKC)-specific signaling pathway. Accordingly, the PKC inhibitor bisindolylmaleimide inhibited the oxidative burst induced by either PMA or intact pseudomonas, but not by fMLP, whereas the p38 kinase inhibitor SB-203580 fully inhibited the respiratory burst induced by fMLP or the PlcHR-replete wild-type bacteria, but not PMA or the PlcHR-deficient ΔHR bacterial mutant. We conclude that expression of PlcHR by P. aeruginosa suppresses bacterium-induced neutrophil respiratory burst by interfering with a PKC-dependent, non-p38 kinase-dependent pathway.

The aim of this study was to develop a reverse transcription-PCR assay and lateral flow detection protocol for specific identification of Cryptosporidium parvum. The method which we developed is sensitive and specific and has a low limit of detection. In our protocol a solid phase material, the Xtra Bind Capture System, was used for extraction and purification of double-stranded RNA (dsRNA) specific for C. parvum. The Xtra Bind Capture System interfaced with pellets concentrated from water samples collected with previously developed filtration devices. The pellets were resuspended in reagent water (final volume, 0.5 ml), and an equal amount of rupture buffer and the Xtra Bind Capture System was added to the resuspended pellet mixture. The dsRNA target sequences in a 0.5-ml portion were captured by the solid phase material via hybridization. The debris and potential inhibitors were removed by washing the Xtra Bind material several times with buffer. The Xtra Bind material with its bound dsRNA was added directly to an amplification reaction mixture, and the target was amplified without elution from the Xtra Bind material. A PCR was performed in the presence of the Xtra Bind Capture System, which resulted in robust amplification of the target. The detection system which we used was adapted from lateral flow chromatography methods typically used for antigen-antibody reactions. The result was a colored line that was visible if the organism was present. When this method was used, we were able to reproducibly and correctly identify 10 oocysts added to 0.5 ml of reagent water. When the protocol was evaluated with a small set of environmental samples, the level of detection was as low as 1 oocyst/liter. The total time from resuspension of the pellet to detection was about 3 h, which is considerably less than the 5 h required for immunomagnetic separation followed by an indirect immunofluorescence assay and microscopy.In recent studies workers found that Cryptosporidium parvum oocysts were present in 65 to 97% of the surface water tested at locations throughout the United States (29,30,36,37). Outbreaks have occurred in water systems ranging from simple chlorination systems to complete filtration and ozonation systems (5, 7, 9, 14, 15, 17-19, 32, 33, 38). An outbreak in Kitchner-Waterloo, Ontario, Canada, affected 1,000 people, and the water at this location was both filtered and ozonated. There have been more than 30 documented outbreaks of cryptosporidiosis in the United States. An outbreak during 1993 in Milwaukee, Wis., resulted in more than 400,000 cases of illness and 100 deaths (33). Filtration, which physically removes the parasite from contaminated water, is the only effective treatment since oocysts are resistant to chemical disinfectants. However, a filtration system that is not well maintained and operated may not provide absolute protection. Recent surveys in which the researchers examined the occurrence of Cryptosporidium oocysts in fully treated (disinfected and filtered) municipal water demonstrated that s...

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A design-of-experiments approach was used to examine the effect of hexadecylpyridinium chloride (HPC), alone or in combination with the antibiotics vancomycin and natamycin, on the growth of Mycobacterium avium subsp. paratuberculosis (MAP). At concentrations above 74.4 microg/ml, HPC had a highly significant detrimental effect on the growth of MAP, whereas natamycin at 10.8 and 21.6 microg/ml and vancomycin at 5.2 and 10.4 microg/ml did not have such an effect. Titration of the amount of HPC tolerated by MAP indicated that growth can occur in the presence of 24.8 microg/ml or lower. Processing of bovine fecal specimens indicated that reducing the concentration of HPC from 32.22 to 1.07 mg/ml during decontamination may improve detection when cultures are grown on solid medium but not when cultures are grown in liquid medium. Further investigation into optimizing HPC concentration during processing of fecal samples is warranted. Natamycin, in conjunction with vancomycin, may be useful for controlling fungal contamination during isolation of MAP from fecal samples.