Reduction of Escherichia coli O157:H7 on produce by use of electrolyzed water under simulated food service operation conditions.

Treatment of fresh fruits and vegetables with electrolyzed water (EW) has been shown to kill or reduce foodborne pathogens. We evaluated the efficacy of EW in killing Escherichia coli O157:H7 on iceberg lettuce, cabbage, lemons, and tomatoes by using washing and/or chilling treatments simulating those followed in some food service kitchens. Greatest reduction levels on lettuce were achieved by sequentially washing with 14-A (amperage) acidic EW (AcEW) for 15 or 30 s followed by chilling in 16-A AcEW for 15 min. This procedure reduced the pathogen by 2.8 and 3.0 log CFU per leaf, respectively, whereas washing and chilling with tap water reduced the pathogen by 1.9 and 2.4 log CFU per leaf. Washing cabbage leaves for 15 or 30 s with tap water or 14-A AcEW reduced the pathogen by 2.0 and 3.0 log CFU per leaf and 2.5 to 3.0 log CFU per leaf, respectively. The pathogen was reduced by 4.7 log CFU per lemon by washing with 14-A AcEW and 4.1 and 4.5 log CFU per lemon by washing with tap water for 15 or 30 s. A reduction of 5.3 log CFU per lemon was achieved by washing with 14-A alkaline EW for 15 s prior to washing with 14-A AcEW for 15 s. Washing tomatoes with tap water or 14-A AcEW for 15 s reduced the pathogen by 6.4 and 7.9 log CFU per tomato, respectively. Application of AcEW using procedures mimicking food service operations should help minimize cross-contamination and reduce the risk of E. coli O157:H7 being present on produce at the time of consumption.

Buruli ulcer and schistosomiasis: no association found.

Helminth infections elicit an immune response potentially enhancing susceptibility to mycobacterial diseases. Schistosomiasis and infection with Mycobacterium ulcerans show a remarkable similarity in epidemiologic characteristics in Ghana. In 2000, a case-control study was conducted in three districts in Ghana endemic for M. ulcerans. One hundred six patients with confirmed M. ulcerans disease and 106 matched community controls were included. Schistosome infection of these patients and controls was measured by an enzyme-linked immunosorbent assay that detected circulating anodic antigen in serum. Fifty percent of the participants tested positive for schistosomiasis. There was no difference in detection rates among patients and matched controls. Similarly, there were no differences in worm burden between patients and controls. These results do not support the hypothesis that susceptibility to M. ulcerans disease is driven by a co-infection with schistosomes.

Demonstration of spread by Mycobacterium tuberculosis bacilli in A549 epithelial cell monolayers.

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.

Cloning, expression, and functional characterization of the Mycobacterium tuberculosis secA gene.

To better understand the protein secretion mechanisms involved in the growth and pathogenesis of Mycobacterium tuberculosis, we examined the secA gene from M. tuberculosis (tbsecA; cosmid sequence accession No. z95121.gb_ba). We generated plasmids containing the full-length tbsecA gene or a fusion containing the 5' sequence from the M. tuberculosis secA gene and the remainder from the Escherichia coli secA gene and evaluated the ability of each construct to complement the defective SecA protein in E. coli MM52ts when grown at the non-permissive temperature. The full-length tbsecA gene was unable to compensate for the temperature-sensitive defect, whereas E. coli MM52ts that has been transformed with plasmid pMF8TB226 containing a chimeric secA gene was able to grow at 42 degrees C. This work confirms that the topography of SecA and its ATP binding sites are highly conserved, whereas its membrane insertion domains are species specific.

Analysis of an IS2404-based nested PCR for diagnosis of Buruli ulcer disease in regions of Ghana where the disease is endemic.

Mycobacterium ulcerans causes Buruli ulcer disease (BUD), an ulcerative skin disease emerging mainly in West Africa. Laboratory confirmation of BUD is complicated as no "gold standard" for diagnosis exists. A nested primer PCR based on IS2404 has shown promise as a diagnostic assay. We evaluated the IS2404-based PCR to detect M. ulcerans DNA in tissue specimens from 143 BUD patients diagnosed according to the World Health Organization BUD clinical case definition in Ghana. Comparisons were made with culture and histopathology results. Variables influencing detection rate tested in this PCR protocol included the amount of tissue used and the stage of disease. The nested PCR was repeated on DNA extracted from a different part of the same biopsy specimen of 21 culture-positive samples. Of all 143 specimens, 107 (74.8%; 95% confidence interval, 68 to 82%) showed the presence of M. ulcerans DNA by PCR. Of the 78 histology-confirmed BUD patient samples, 64 (83%) were PCR positive. Detection rates were influenced neither by the amount of tissue processed for PCR nor by the stage of disease (preulcerative or ulcerative). Taken together, the two nested PCR tests on the subset of 21 culture-positive samples were able to detect M. ulcerans DNA in all 21 culture-confirmed patients. For future studies, small tissue samples, e.g., punch biopsy samples, might be sufficient for case confirmation.

Immunoglobulin M antibody responses to Mycobacterium ulcerans allow discrimination between cases of active Buruli ulcer disease and matched family controls in areas where the disease is endemic.

Buruli ulcer disease (BUD) is an emerging disease caused by Mycobacterium ulcerans. In the present study we have characterized the serological reactivities of sera from volunteer case patients with laboratory-confirmed BUD and controls living in three different regions of Ghana where the disease is endemic to determine if serology may be useful for disease confirmation. Our results showed highly reactive immunoglobulin G (IgG) responses among patients with laboratory-confirmed disease, healthy control family members of the case patients, and sera from patients with tuberculosis from areas where BUD is not endemic. These responses were represented by reactivities to multiple protein bands found in the M. ulcerans culture filtrate (CF). In contrast, patient IgM antibody responses to the M. ulcerans CF (MUCF) proteins were more distinct than those of healthy family members living in the same village. A total of 84.8% (56 of 66) of the BUD patients exhibited strong IgM antibody responses against MUCF proteins (30, 43 and 70 to 80 kDa), whereas only 4.5% (3 of 66) of the family controls exhibited such responses. The sensitivity of the total IgM response for the patients was 84.8% (95% confidence interval [CI], 74.3 to 91.6%), and the specificity determined with sera from family controls was 95.5% (95% CI, 87.5 to 98.4%). These studies suggest that the IgM responses of patients with BUD will be helpful in the identification and production of the M. ulcerans recombinant antigens required for the development of a sensitive and specific serological assay for the confirmation of active BUD.

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.

Histopathologic features of Mycobacterium ulcerans infection.

Because of the emergence of Buruli ulcer disease, the World Health Organization launched a Global Buruli Ulcer Initiative in 1998. This indolent skin infection is caused by Mycobacterium ulcerans. During a study of risk factors for the disease in Ghana, adequate excisional skin-biopsy specimens were obtained from 124 clinically suspicious lesions. Buruli ulcer disease was diagnosed in 78 lesions since acid-fast bacilli (AFB) were found by histopathologic examination. Lesions with other diagnoses included filariasis (3 cases), zygomycosis (2 cases), ulcerative squamous cell carcinomas (2 cases), keratin cyst (1 case), and lymph node (1 case). Thirty-seven specimens that did not show AFB were considered suspected Buruli ulcer disease cases. Necrosis of subcutaneous tissues and dermal collagen were found more frequently in AFB-positive specimens compared with specimens from suspected case-patients (p<0.001). Defining histologic criteria for a diagnosis of Buruli ulcer disease is of clinical and public health importance since it would allow earlier treatment, leading to less deforming sequelae.