Jane Cottin

Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1‐independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/unites/RIF/transcriptdata/.

Impact of Mycobacterium ulcerans biofilm on transmissibility to ecological niches and Buruli ulcer pathogenesis

The role of biofilms in the pathogenesis of mycobacterial diseases remains largely unknown. Mycobacterium ulcerans, the etiological agent of Buruli ulcer, a disfiguring disease in humans, adopts a biofilm-like structure in vitro and in vivo, displaying an abundant extracellular matrix (ECM) that harbors vesicles. The composition and structure of the ECM differs from that of the classical matrix found in other bacterial biofilms. More than 80 proteins are present within this extracellular compartment and appear to be involved in stress responses, respiration, and intermediary metabolism. In addition to a large amount of carbohydrates and lipids, ECM is the reservoir of the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, and purified vesicles extracted from ECM are highly cytotoxic. ECM confers to the mycobacterium increased resistance to antimicrobial agents, and enhances colonization of insect vectors and mammalian hosts. The results of this study support a model whereby biofilm changes confer selective advantages to M. ulcerans in colonizing various ecological niches successfully, with repercussions for Buruli ulcer pathogenesis.

Oral treatment for Mycobacterium ulcerans infection : results from a pilot study in Benin

Mycobacterium ulcerans infection is responsible for severe skin lesions in sub-Saharan Africa. We enrolled 30 Beninese patients with Buruli ulcers in a pilot study to evaluate efficacy of an oral chemotherapy using rifampicin plus clarithromycin during an 8-week period. The treatment was well tolerated, and all patients were healed by 12 months after initiation of therapy without relapse.

Seasonal and regional dynamics of M. ulcerans transmission in environmental context: deciphering the role of water bugs as hosts and vectors.

Background Buruli ulcer, the third mycobacterial disease after tuberculosis and leprosy, is caused by the environmental mycobacterium M. ulcerans. Various modes of transmission have been suspected for this disease, with no general consensus acceptance for any of them up to now. Since laboratory models demonstrated the ability of water bugs to transmit M. ulcerans, a particular attention is focused on the transmission of the bacilli by water bugs as hosts and vectors. However, it is only through detailed knowledge of the biodiversity and ecology of water bugs that the importance of this mode of transmission can be fully assessed. It is the objective of the work here to decipher the role of water bugs in M. ulcerans ecology and transmission, based on large-scale field studies. Methodology/Principal Findings The distribution of M. ulcerans-hosting water bugs was monitored on previously unprecedented time and space scales: a total of 7,407 water bugs, belonging to large number of different families, were collected over one year, in Buruli ulcer endemic and non endemic areas in central Cameroon. This study demonstrated the presence of M. ulcerans in insect saliva. In addition, the field results provided a full picture of the ecology of transmission in terms of biodiversity and detailed specification of seasonal and regional dynamics, with large temporal heterogeneity in the insect tissue colonization rate and detection of M. ulcerans only in water bug tissues collected in Buruli ulcer endemic areas. Conclusion/Significance The large-scale detection of bacilli in saliva of biting water bugs gives enhanced weight to their role in M. ulcerans transmission. On practical grounds, beyond the ecological interest, the results concerning seasonal and regional dynamics can provide an efficient tool in the hands of sanitary authorities to monitor environmental risks associated with Buruli ulcer.

Use of Fine-Needle Aspiration for Diagnosis of Mycobacterium ulcerans Infection

ABSTRACT Noninvasive methods for the bacteriological diagnosis of early-stage Mycobacterium ulcerans infection are not available. It was recently shown that fine-needle aspiration (FNA) could be used for diagnosing M. ulcerans infection in ulcerative lesions. We report that FNA is an appropriate sampling method for diagnosing M. ulcerans infection in nonulcerative lesions.

Assessment of the usefulness of performing bacterial identification and antimicrobial susceptibility testing 24 h a day in a clinical microbiology laboratory

The impact of inoculating agar media with positive blood cultures and of performing bacterial identification and antimicrobial susceptibility testing (AST) for positive urine cultures, blood cultures and certain fluid cultures after day hours (night service (NS)) was evaluated in a clinical microbiology laboratory. The impact of the NS was assessed in terms of decreases in the delays from the time of sampling to the time at which results became available and of the consequences for patient management and antimicrobial treatment. Two major benefits were obtained: initiation of earlier appropriate treatment, and change to a reduced-spectrum but still efficient regimen. The hours of laboratory testing and the availability and transmission of results to the clinical staff were recorded. Concurrently, these hours were estimated as though laboratory tests had been performed in the absence of NS. Reductions in delay were defined as the differences between the hours actually spent and the estimated hours. Economic concerns were also considered. Overall, 430 samples for which an identification and/or AST were performed during the NS were included in the study. The NS led to the implementation of earlier appropriate therapy in 97 cases (22.6%), and to the change to reduced-spectrum but still efficient regimens in 23 additional cases (5.3%). In conclusion, there appeared to be benefits from a system providing bacterial identification and AST overnight, but a study of the cost-effectiveness of the NS would be useful to back up this observation.

Buruli Ulcer in Long-Term Traveler to Senegal

To the Editor: Buruli ulcer (BU) is caused by infection of subcutaneous fat with the environmental pathogen Mycobacterium ulcerans. BU has been reported or suspected in more than 30 countries. It has never been reported in Senegal and Guinea-Bissau (1). We report a case of travel-associated BU in a French traveler to Senegal. The patient was a 24-year-old Caucasian man who came to the University Hospital of Bordeaux, France, with a nonhealing lesion on the anterior left leg that had been present for ≈12 weeks. The patient had traveled in Senegal to the border of Guinea-Bissau from September 2006 through August 2007. His trip had begun in Dakar and proceeded south to the districts of Kaolack, Toubacouta, and Casamance. The patient stayed in Casamance during the rainy season from June 2007 through August 2007. He had been working on construction of wood dugouts, had been bare-legged regularly, and had been in contact with stagnant water. He first noticed a lesion during June 2007, which had gradually increased to a small, centrally crusted ulcer. By the end of August 2007 (week 8 of the lesion), skin examination showed a 3 × 6-cm necrotizing ulcer with central crusting and an erythematous border (Figure). The lesion was not warm or tender but generated a seropurulent discharge. Concurrently, palpable left inguinal lymph nodes were observed. Figure Ulcer (3 × 6 cm) on anterior side of the left leg of the patient, showing an erythematous border. Bacteriologic swabs identified Staphylococcus aureus and group A Streptoccocus pyogenes. Two punch-biopsy specimens were taken from the border of the lesion. Histologic analysis showed nonspecific acute and chronic dermal inflammation with necrotizing granulomas that extended into the subcutaneous tissues, suggestive of infection with atypical Mycobacterium spp. Bacteriologic examination did not identify acid-fast bacilli (negative direct smear result after Ziehl-Neelsen staining) or other specific microorganisms (negative direct smear results after periodic acid–Schiff, Giemsa, and Gram staining). Tissue specimens were placed into BACTEC 12B broth (Becton Dickinson, Franklin Lakes, NJ, USA) (incubated at 35°C) and onto Lowenstein-Jensen slants (incubated at 30°C). No growth was detected after 42 days. On the basis of clinical findings, we suspected a diagnosis of BU. Taq-Man real-time quantitative PCR that used primers for 2 M. ulcerans–specific genes (insertion sequence 2404 and ketoreductase B gene) (2,3) and negative controls showed positive results for DNA from both biopsy specimens. A normalized standard curve was constructed, which indicated a bacterial load of ≈6 × 103 organisms/g of tissue. Laboratory investigations indicated a total leukocyte count of 16,400 cells/μL (reference range 3,600–10,000 cells/μL) and a C-reactive protein level of 0.59 mg/mL (reference value <0.01 mg/mL). Results of radiologic investigations were normal. The patient was treated with rifampin (600 mg/day) and moxifloxacin (400 mg/day) for 12 weeks. Additional surgical excision was planned 4 weeks after treatment was begun. Unfortunately, 15 days later, the patient was lost to follow-up. BU has been reported in many West African countries, with Guinea being the northern limit of reported cases. Detection of this case of BU suggests that the region in West Africa endemic for this disease has been underestimated or is expanding. Infection in the traveler may have occurred in Casamance, if one assumes an incubation period of 6 weeks to 3 months. Further cases should be actively sought in this region and adjoining districts visited to evaluate the geographic extent of the disease. The environmental reservoir and mode of transmission of BU in our patient are unknown. Exposure of unprotected skin with stagnant or slow-flowing water is linked with BU. Our patient reported prolonged contact with water during his occupation. Recent studies implicating aquatic predator insects (4,5) and mosquitoes (6) in transmission of BU suggest that use of insect repellents and protective clothing may help prevent infection. The diagnosis of BU in this patient relied on the PCR detection of 2 M. ulcerans–specific genes; this procedure is considered adequate (7,8). The relatively low number of organisms detected may explain the negative acid-fast bacilli smear and culture results (9). Our report of M. ulcerans infection from Senegal is not surprising because southern Senegal shares similar ecologic features with neighboring affected countries, especially during the heavy rainy season. Although BU is a disease that affects mainly persons in recognized disease-endemic areas, this case emphasizes that tropical skin ulcers should be considered in differential diagnosis of BU in travelers returning from disease-endemic countries (1,10). Diagnostic delays can be avoided by use of M. ulcerans–specific PCR, a test available from World Health Organization collaborating laboratories, which enables rapid confirmation of diagnosis of BU.

Buruli Ulcer, Central African Republic

To the Editor: Buruli ulcer, the third most common mycobacterial disease of humans after tuberculosis and leprosy, is an important disfiguring and disabling cutaneous infection disease caused by Mycobacterium ulcerans. Buruli ulcer was declared an emerging skin disease of public health concern by the World Health Organization (WHO) in 1998. Although the disease is known to be associated with swampy areas and environmental changes, the mode of transmission is not yet clearly understood. A possible role for water bugs in the transmission has been postulated in the last 10 years. In this direction, several researchers have proposed that biting water bugs could be vectors for M. ulcerans (1). M. ulcerans produces a potent toxin known as mycolactone (2), which lyses dermal cells, leading to the development of continuously expanding ulcers with undermined edges. Surgery is the only treatment for late lesions, which involves excision of necrotic tissues, followed by skin grafting. After such treatment, patients suffer from functional limitations, social stigmatization, and the loss of livelihood (3). Antimicrobial drug treatment is available (a combination of rifampin and streptomycin), but it is effective only for early lesions (4). The disease is endemic in rural wetlands of tropical countries of Africa, the Americas, and Asia. Over the past decade, the prevalence of Buruli ulcer was highest in western Africa (3,5), with an alarming increase in detected cases. In central Africa, foci of Buruli ulcer have been reported in Gabon, Equatorial Guinea, Cameroon, Congo, the Democratic Republic of Congo, and Sudan (6), which are all neighboring countries of the Central African Republic (CAR). Surprisingly, in CAR, no cases of Buruli ulcer have been reported so far, even though its presence in this country was suspected in 2006, although not confirmed. This situation motivated us to begin a passive survey in the hospitals of Bangui, the capital of CAR. We report here 2 confirmed cases of Buruli ulcer that were found through this survey. The 2 patients were admitted in April 2007 to Hopital de l’Amitie, Bangui, CAR, with extensive skin ulcers, which might correspond to Buruli ulcer according to WHO guidelines (7). Both patients were farmers from the Ombella M’poko region. They lived on the border of the M’poko River and carried out daily activities in an aquatic environment. The first patient was a 62-year-old man who had a large ulceration of the right limb (Figure, panel A). Differential diagnosis eliminated other ulcerative diseases such as drepanocytosis, and the patient was HIV negative. For bacteriologic diagnosis, 4 samples were taken with sterile cotton swabs from beneath the undermined edges of the ulcer. Proteus mirabilis was isolated from the lesion, and a few acid-fast bacilli were shown by Ziehl-Neelsen (ZN) staining. Unfortunately, 1 week later, the patient died of an unknown cause. Figure Patient 1: extensive ulcer of the right limb (A). Patient 2: ulcer of the left ankle before treatment (B) and 8 weeks after specific antimicrobial drug therapy (C). Scale bars = 12 cm (A), 5 cm (B), and 2 cm (C). The second patient was a man of the same age who had an ulceration 6.5 cm in diameter on the left ankle (Figure, panel B). His condition had been treated with various antimicrobial agents without any result. Blood testing showed minor anemia (hemoglobin 12.4 g/dL) and that the patient was HIV negative. Bacteriologic analysis found no gram-positive and gram-negative bacteria, and ZN staining showed the presence of acid-fast bacilli. He received the specific recommended treatment for M. ulcerans infection (antimicrobial drug regimen: rifampin, 10 mg/kg, and streptomycin, 15 mg/kg), and the lesions had receded 2 months later (Figure, panel C). The identification of M. ulcerans was confirmed by PCR on the basis of the IS2404 repeated insertion sequences of M. ulcerans as described by Stinear et al. (8). The positive results were confirmed by quantitative real-time PCR, in the Laboratory of Bacteriology at Central Hospitalier Universistaire, Angers, France, on 2 specific sequences: IS2404 sequence and ketoreductase B domain of the mycolactone polyketide synthase gene from the plasmid pMUM001 (9). According to WHO criteria, 2 confirmative test results should be obtained of 4 laboratory tests (ZN staining, positive culture of M.ulcerans, specific gene amplification, pathognomonic histopathologic features) to establish a definitive diagnosis (7). Concerning the 2 patients in this study, results of ZN staining and PCR were positive, thus confirming the diagnosis of Buruli ulcer. Samples were inoculated on Lowenstein-Jensen (LJ) media and incubated at 30°C for 2 months, but the culture did not grow the organism. This result could be accounted for by the paucity of bacilli in the samples. In conclusion, our study confirms that, although infrequently diagnosed, Buruli ulcer is an endemic disease in CAR. Identification and control of Buruli ulcer remain difficult in CAR, where this disease is often not considered. Even with evocative clinical signs, confirmation of diagnosis by biological analysis is still not easy. It is therefore of high importance that the public health authorities are fully informed and properly trained to identify this neglected disease in the early stages so patients can be cured before the onset of functional impairment and the appearance of extensive lesions. Further investigation to isolate strains present in CAR is also essential.

Évaluation des services rendus par le système de fonctionnement en continu d’un laboratoire de bactériologie

UNLABELLED Since 2005, the clinical microbiology laboratory of the Angers teaching hospital has implemented after hours service requiring the overnight presence of a technician specialized in bacteriology. During that time, bacterial identifications and antibiotic susceptibility testing to antimicrobial agents can be performed for critical samples. OBJECTIVES The authors wanted to evaluate the impact of the after-hours service on the decrease of delay from sampling to results, and from sampling to the implementation of an appropriate antimicrobial therapy. A therapy could be initiated, changed for more efficient agents, or changed to narrower-spectrum agents (major benefits). METHODS A 4-month prospective study was made. All samples for which identification and/or susceptibility testing were performed during after-hours service (continued analyses) were included in the study. Delays observed were compared with theoretical delays estimated in the absence of the after-hours service. RESULTS A minimum 24 hour-decrease of the delay for results was observed for 97 % of the 430 samples included. Overall, a major benefit was obtained for more than 25 % of the analyses, representing a cumulated 111-day benefit in days of efficient treatments and a cumulated 27-day benefit in days of prescription of narrower-spectrum agents. DISCUSSION This organization, unique in French hospitals, is directly related to the improvement of antimicrobial treatments, like antibiotic practice guidelines or infection disease specialists. It was evaluated as a relevant strategy, potentially cost saving, with a significant impact on both the efficiency of treatments and microbial ecology.