The role of extracellular matrix in mouse and human corneal neovascularization.

Corneal neo-vascularization (CNV) is a highly prevalent medical condition which impairs visual acuity. The role of specific proteins in modulating CNV has been extensively reported, although no studies have described the entire human proteome in CNV corneas. In this paper, we performed a proteomic analysis of vascularized vs healthy corneal stroma, in a CNV mouse model and in CNV-affected patients, with a specific focus on extracellular matrix (ECM) proteins. We identified and quantified 2315 murine proteins, 691 human proteins and validated 5 proteins which are differentially expressed in vascularized samples and conserved in mice and humans: tenascin-C and fibronectin-1 were upregulated, while decorin, lumican and collagen-VI were downregulated in CNV samples. Interestingly, among CNV patients, those affected with Acanthamoeba keratitis showed the highest levels of fibronectin-1 and tenascin-C, suggesting a specific role of these two proteins in Acanthamoeba driven corneal CNV. On a broader picture, our findings support the hypothesis that the corneal stroma in CNV samples is disorganized and less compact. We are confident that the dissection of the human corneal proteome may shed new light on the complex pathophysiology of human CNV, and finally lead to improved treatments.

Identification of a new variable number tandem repeat locus in Mycobacterium ulcerans for potential strain discrimination among African isolates.

Intra-species discrimination in the highly clonal pathogen Mycobacterium ulcerans was studied in a diverse collection of isolates by PCR amplification of a short sequence repeat locus containing heterogeneous arrays of tri-nucleotide repeats with an ACC consensus pattern. Post-amplification analysis indicated excellent typeability and identified five M. ulcerans alleles, including a unique Angolan type differentiated for the first time among a genetically conserved cluster of African isolates. These results are consistent with previously investigated independent markers, and provide an additional locus for variable number tandem repeat-based typing of M. ulcerans.

Characterization of cold-active pectate lyases from psychrophilic Mrakia frigida.

AIMS: This study was conducted to determine optimal conditions for pectate lyase (PL) production by two psychrophilic yeast strains and to compare the properties of the cold-active enzymes using mesophilic PL as reference enzyme. METHODS AND RESULTS: Two psychrophilic yeasts isolated from remote geographical locations (European Alps, north Siberia) produced extracellular cold-active PL. Both strains were identified as Mrakia frigida by analysis of ITS and large subunit (LSU) rRNA sequences. Maximum enzyme production occurred at a cultivation temperature of 1 or 5 degrees C. The apparent optimum for enzyme activity was observed at 30 degrees C and pH 8.5-9. The enzymes were thermolabile, but were resistant to repeated freezing and thawing. CONCLUSION: We describe for the first time alkaline PL-producing representatives of the yeast species M. frigida. The two strains produce cold-active PL with similar properties, but have a different enzyme production pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzymes described in this study could be useful for a wide range of applications, such as low-temperature pretreatment of wastewater containing pectic substances.

A one-year survey of candidemia in Belgium in 2002.

A total of 211 episodes of bloodstream yeast infections in 207 patients, hospitalized in 28 Belgian hospitals participating in a National Surveillance Program, were evaluated. A total of 81% of the patients were more than 50 years of age. Candida albicans was the cause of infections in 55% of patients, 22% were due to C. glabrata and 13% to C. parapsilosis. The most common predisposing factors were antibacterial therapy (42%), residence in an intensive care unit (32.9%) and presence of an intravascular catheter (29.7%). Most patients had more than one predisposing factor. Fluconazole alone or in association with another antifungal agent was the treatment of choice for 89.7% of the cases. In vitro susceptibility testing of the isolates revealed that 99% were susceptible to amphotericin B, 95% to 5-fluorocytosine, 82% to fluconazole and 69% to itraconazole. Resistance to azoles was more common among C. glabrata isolates in the elderly. We conclude that the frequency of C. albicans infection is decreasing in Belgium and this is associated with the emergence of other species, most notably, C. glabrata.

Evaluation of PCR-restriction profile analysis and IS2404 restriction fragment length polymorphism and amplified fragment length polymorphism fingerprinting for identification and typing of Mycobacterium ulcerans and M. marinum.

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marinum (n = 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3' end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.

The use of IS2404 restriction fragment length polymorphisms suggests the diversity of Mycobacterium ulcerans from different geographical areas.

Buruli ulcer, caused by Mycobacterium ulcerans, has been reported in five continents: Africa, Asia, Australia, and North and South America. In the present study, restriction fragment length polymorphism with the recently described M. ulcerans specific insertion sequence IS2404 as a probe, was applied to Mycobacterium shinshuense, Mycobacterium marinum, and 14 clinical M. ulcerans isolates originating from six geographic areas: Africa (n = 6), Australia (n = 2), Mexico (n = 1), south Asia (n = 2), Asia (n = 1), and South America (n = 2). Using this probe, six subtypes of M. ulcerans, related to the six geographic origins of the isolates were distinguished, confirming that M. ulcerans can be divided into subgroups corresponding to different geographic variants of the same species.

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity.

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

First reported case of Mycobacterium ulcerans infection in a patient from China.

Buruli ulcers have not been previously described in China, and only once at higher latitudes on the northern hemisphere. A patient who travelled in the Shan Dong Province in the People's Republic of China developed an ulcer which was proven to be a Buruli ulcer. The clinical picture and histopathological findings from biopsy specimens are characteristic for a Buruli ulcer, and also the growth in culture (Coletsos medium) at a restricted temperature of 30 degrees C. A multiplex polymerase chain reaction (PCR) based on the amplification of the gene encoding for 16S ribosomal RNA and a nested PCR based on the Mycobacterium ulcerans specific repeated sequence 2404 were performed. These PCR investigations identified the bacteria as M. ulcerans, subspecies shinshuense. The patient was initially treated with clarithromycin and rifampicin, which was changed to ciprofloxacin and rifabutin when rifampicin resistance of the first isolate was established. There were no signs of reactivation of the disease 6 months after the end of treatment. M. ulcerans infection occurs above 30 degrees latitude on the northern hemisphere in China and is caused by M. ulcerans, subspecies shinshuense. This case appears to be cured by chemotherapy alone, in contrast to the general experience that surgical treatment is indicated. The granulomatous reaction with only fragments of acid-fast bacteria in the biopsy at the end of treatment many indicate the development of an adequate cell-mediated immune response leading to resistance to the infection.

Biochemical and genetic evidence for phospholipase C activity in Mycobacterium ulcerans.

This study reports the existence of phospholipase C and D enzymatic activities in Mycobacterium ulcerans cultures as determined by use of thin-layer chromatography to detect diglycerides in hydrolysates of radiolabeled phosphatidylcholine. M. ulcerans DNA sequences homologous to the genes encoding phospholipase C in Mycobacterium tuberculosis and Pseudomonas aeruginosa were identified by sequence analysis and DNA-DNA hybridization. Whether or not the phospholipase C and D enzymes of M. ulcerans plays a role in the pathogenesis of the disease needs further investigation.

Correlation of pncA sequence with pyrazinamide resistance level in BACTEC for 21 mycobacterium tuberculosis clinical isolates.

Mutations in the pncA gene, encoding pyrazinamidase, are considered the major mechanism of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis, but resistant strains containing the wild-type gene have been described. The correlation of pncA sequence with PZA resistance level was examined for 21 M. tuberculosis clinical isolates. Susceptibility patterns were determined for 100, 300, and 900 microg/ml concentrations of the drug in BACTEC. Insertions and deletions and a substitution in the putative promoter region led to high-level resistance, whereas substitutions within the open reading frame seemed to confer variable levels of resistance. Variable resistance levels and PZase activities were also observed among isolates lacking pncA mutations. The high-level resistance (900 microg/ml) in pncA wild-type isolates highlights the clinical significance of these isolates. These data also suggest that there may still be more than one alternative mechanism leading to PZA resistance in M. tuberculosis isolates.

The production of nitric oxide and tumor necrosis factor by murine macrophages infected with mycobacterial strains differing by hemolytic activity.

In this study, we compared the secretion of nitric oxide (NO) and tumor necrosis factor (TNF-alpha) by murine macrophages infected in vitro with hemolytic or unhemolytic mycobacteria isolates. We observed that unhemolytic mycobacteria induced more intensive NO production by macrophages and were more susceptible to bactericidal effect of mononuclear phagocytes than hemolytic mycobacterial strains. In contrast, the high-virulence hemolytic isolates induced significantly stronger TNF-alpha production by infected macrophages than the low-virulence unhemolytic bacilli.

Relationship between pyrazinamide resistance, loss of pyrazinamidase activity, and mutations in the pncA locus in multidrug-resistant clinical isolates of Mycobacterium tuberculosis.

Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing of pncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-type pncA might possess alternative resistance mechanisms.

Comparison of two PCRs for detection of Mycobacterium ulcerans.

Two nested PCRs for the detection of Mycobacterium ulcerans were compared by using a collection of 65 clinical specimens. The first method amplifies the gene coding for 16S rRNA, and the second method amplifies a repetitive DNA sequence. The sensitivities of bacterioscopy, culture, 16S rRNA gene PCR, and repetitive-sequence PCR were 29, 34, 80, and 85%, respectively. Compared to the 16S rRNA gene PCR, the repetitive-sequence PCR was faster, easier to perform, and less expensive.

Detection of fastidious mycobacteria in human intestines by the polymerase chain reaction.

The aim of this study was to determine whether difficult-to-grow mycobacteria are present in human intestines. Intestinal tissue samples were subjected to both mycobacterial culture and a polymerase chain reaction (PCR) assay. After detection by PCR, species identity was determined by hybridizing the amplified 16S rRNA gene fragments with species-specific oligonucleotides. Intestinal biopsies from 63 patients with noninflammatory bowel diseases (n = 22), Crohn's disease (n = 31), or ulcerative colitis (n = 10) were analyzed. Culture and PCR revealed mycobacteria in four (6%) and 25 (40%) samples, respectively. Samples positive by PCR were negative with all probes specific to nine common cultivable species but were positive with Mycobacterium genavense-specific probe in 68% of cases. Mycobacterial isolates were identified as Mycobacterium gordonae and Mycobacterium chelonae. Findings were similar in Crohn's disease samples compared to non-Chron's disease samples. This study shows that difficult-to-grow mycobacteria can be detected by PCR in large and similar proportions of inflamed intestinal tissue from patients with inflammatory bowel disease and intestinal tissue that appears normal from patients with noninflammatory bowel disease.

Direct detection and identification of Mycobacterium ulcerans in clinical specimens by PCR and oligonucleotide-specific capture plate hybridization.

We compared various diagnostic tests for their abilities to detect Mycobacterium ulcerans infection in specimens from patients with clinically active disease. Specimens from 10 patients from the area of Zangnanado (Department of Zou, Benin) with advanced, ulcerated active M. ulcerans infections were studied by direct smear, histopathology, culture, PCR, and oligonucleotide-specific capture plate hybridization (OSCPH). A total of 27 specimens, including 12 swabs of exudate collected before debridement and 15 fragments of tissue obtained during debridement, were submitted to bacteriologic and histopathologic analysis. The histopathologic evaluation of tissues from all six patients so tested revealed changes typical of those caused by M. ulcerans infection. Five specimens were contaminated, and M. ulcerans was cultivated on Löwenstein-Jensen medium from 12 of the remaining 22 (54.5%) specimens. Detection of mycobacteria was performed by PCR, and M. ulcerans was detected by OSCPH with a new probe (5'-CACGGGATTCATGTCCTGT-3') reacting with M. ulcerans and Mycobacterium marinum. In 10 of 22 (45.5%) specimens, M. ulcerans was identified by PCR-OSCPH. There was no statistically significant difference between the detection of M. ulcerans by culture and by PCR-OSCPH (P > 0.05). This is the first demonstration of an amplification system (PCR-OSCPH) with a sensitivity similar to that of culture for the direct and rapid recognition of M. ulcerans in clinical specimens. This system is capable of identifying M. ulcerans, even in paucibacillary lesions. Our findings suggest that PCR-OSCPH should be used in the quest for the elusive environmental reservoir(s) of M. ulcerans.

Characterization of Mycobacterium avium complex related mycobacteria isolated from an African environment and patients with AIDS.

Thirteen isolates from African AIDS patients and from the environment in Zaire were identified as members of the Mycobacterium avium complex by phenotypic tests. RFLP analysis showed that the isolates belong to a genetically homogeneous cluster. The 16S rRNA sequence analysis suggests a close relationship with the P-49 strain (ATCC 35847), a reference strain for the serotype 7 of M. avium complex. This work shows the close relationship between certain M. avium complex strains responsible for disseminated infection in AIDS patients and M. avium complex strains isolated from the environment in Zaire. Further, our findings confirm that atypical mycobacteria may disseminate in AIDS patients in Africa and suggest that infection in these patients probably originates in their environment.

No Mycobacterium paratuberculosis found in Crohn's disease using polymerase chain reaction.

M. paratuberculosis has been considered as a putative causative factor of Crohn's disease. However, its detection in diseased tissue samples using the polymerase chain reaction yielded conflicting results. We validated this technique for the detection of mycobacteria (any species) and M. paratuberculosis before applying it to 72 intestinal biopsies from patients with Crohn's disease (N = 36), ulcerative colitis (N = 13), and control subjects (N = 23). Possible polymerase chain reaction inhibitors were detected by spiking template DNA with the equivalent of two M. paratuberculosis genomes. Mycobacteria were found in 17/36 (47%), 6/13 (46%), and 13/23 (57%) tissue samples of Crohn's disease, ulcerative colitis, and controls, respectively. No M. paratuberculosis were detected in any sample. It is concluded that mycobacteria are present with a similar frequency in the intestinal tissues or luminal inclusions of patients with inflammatory bowel disease and of those unaffected by the disease. Our data do not support a role for M. paratuberculosis in Crohn's disease.

Cervical lymphadenitis caused by a fastidious mycobacterium closely related to Mycobacterium genavense in an apparently immunocompetent woman: diagnosis by culture-free microbiological methods.

Fastidious mycobacteria usually infect immunocompromised hosts (human immunodeficiency virus-infected or otherwise immunosuppressed patients). We here describe severe lymphadenitis, caused by a fastidious mycobacterium closely related to Mycobacterium genavense, in an apparently immunocompetent woman, whose brother had died from an unidentified mycobacterial infection in 1969. A variety of techniques, including inoculation of nude mice, histopathology, electron microscopy, lipid analysis, ATP measurements, and molecular biology, were used to characterize this mycobacterium. All attempts to culture the etiological agent on many different media failed. The organism multiplied only in congenitally athymic nude mice. Although phenotypically similar to M. genavense, the mycobacterium differs from M. genavense by three nucleotides of the 16S rRNA gene sequence. Various antimycobacterial drugs were administered, including gamma interferon, but multiple relapses occurred. Finally, therapy with a combined regimen of clarithromycin, clofazimine, rifabutin, and ethambutol was curative. To our knowledge, this is the first report of lymphadenitis in an apparently immunocompetent patient, caused by a noncultivable Mycobacterium sp. closely related to M. genavense. This study emphasizes the importance of employing a variety of diagnostic approaches such as the inoculation of laboratory animals, histopathology, electron microscopy, lipid analysis, ATP measurements, and molecular biology to characterize novel microorganisms that cannot be cultured in vitro.

Species-specific Mycobacterium genavense DNA in intestinal tissues of individuals not infected with human immunodeficiency virus.

Mycobacterium genavense species-specific DNA was detected in intestinal tissues from two of nine individuals not infected with human immunodeficiency virus. This newly described microorganism is well documented as a causative agent of disseminated infections in AIDS patients. Our results suggest that it may colonize the guts of individuals not infected with human immunodeficiency virus.