Molecular and epidemiological characterization of recurrent Mycobacterium ulcerans infections in Benin

BackgroundBuruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans, an environmental mycobacterium. Although transmission of M. ulcerans remains poorly understood, the main identified risk factor for acquiring Buruli ulcer is living in proximity of potentially contaminated water sources. Knowledge about the clinical features of Buruli ulcer and its physiopathology is increasing, but little is known about recurrence due to reinfection.Methodology/Principal findingsWe describe two patients with Buruli ulcer recurrence due to reinfection with M. ulcerans, as demonstrated by comparisons of DNA from the strains isolated at the time of the first diagnosis and at recurrence. Based on the spatial distribution of M. ulcerans genotypes in this region and a detailed study of the behavior of these two patients with respect to sources of water as well as water bodies and streams, we formulated hypotheses concerning the sites at which they may have been contaminated.Conclusions/SignificanceSecond episodes of Buruli ulcer may occur through reinfection, relapse or a paradoxical reaction. We formally demonstrated that the recurrence in these two patients was due to reinfection. Based on the sites at which the patients reported engaging in activities relating to water, we were able to identify possible sites of contamination. Our findings indicate that the non-random distribution of M. ulcerans genotypes in this region may provide useful information about activities at risk.     

Biochemical characterization of resistance determinants cloned from antibiotic-producing streptomycetes.

Determinants of antibiotic resistance have been cloned from four antibiotic-producing streptomycetes into Streptomyces lividans. Biochemical analyses of resistant clones revealed the presence of enzymes that had previously been characterized as likely resistance determinants in the producing organisms. These included: 23S rRNA methylases from S. azureus and S. erythreus, which confer resistance to thiostrepton and erythromycin, respectively; viomycin phosphotransferase from S. vinaceus; and aminoglycoside phosphotransferase and acetyltransferase from the neomycin producer S. fradiae. In general, the levels of antibiotic resistance of the clones were similar to those of the producing organisms. Although the two aminoglycoside-modifying enzymes from S. fradiae could independently confer only low-level resistance to neomycin, the presence of both enzymes in the same strain resulted in a level of resistance comparable with that of the producing organism.     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.     

A taxonomic and ecological study of candidosis

A total of 450 yeast isolates were obtained from up to 34 sites on 59 human subjects. The yeasts were characterized using morphological features and assimilation tests. Ten species were identified but, of these,Candida albicans andCandida parapsilosis were the most common and accounted for 84% of the isolates. An examination of the biotypes of the various species indicated a much greater diversity in the yeast microflora than that detected by species identifications alone. Fifty-five biotypes were differentiated and it is suggested that these could be regarded as distinct taxonomic or ecological entities.     

Structure analysis of bacterial community and their heavy-metal resistance determinants in the heavy-metal-contaminated soil sample

In this study we performed the phylogenetic analysis of non-cultivable bacteria from anthropogenically disturbed soil using partial sequences of the 16S rRNA (16S rDNA) and the heavy-metal resistance genes. This soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and also a trace amount of cadmium (<0.25 mg/kg). The 16S rDNA sequences from a total of 74 bacterial clones were distributed into four broad taxonomic groups, Acidobacteria, Actinobacteria, Bacteroidetes and Gemmatimonadetes, and some of them were unidentified. Comparing our clone sequences with those from the GenBank database, only 9 clones displayed high similarity to known bacteria belongig to actinomycetes; others were identified as uncultured ones. Among clones evidently Actinobacteria predominated. Sixteen clones from soil sample carried only the nccA-like heavy-metal-resistance genes and all sequences showed too low similarity to known proteins encoded by these genes. However, our results suggested that the heavy-metal-contaminated soil is able to present very important reservoir of the new and until now unknown partly bacteria, partly heavy-metal-resistance determinants and their products. Bacteria and nccA-like genes identified in this study could represent the objects of interest as bioremediation agents because they can be potentially used in different transformation and immobilization processes.     

Oral treatment with ketoconazole in systemic candidosis of guinea-pigs: microbiology, hematology and histopathology

Non-pretreated Albino guinea-pigs were infected intravenously with Candida albicans and treated orally either with placebo or ketoconazole. The 17-day follow-up of the fungal dissemination was based upon hematology, on gross and microscopic lesions and on the demonstration of the fungus by culture techniques. The efficacy of ketoconazole, both with regard to the quantity and the morphology of fungi in various organs and the tissue-healing process are discussed. The treatment of human systemic candidosis will also be considered. No side-effects due to the therapy were observed in these experiments.     

Pheochromocytoma presenting as recurrent urinary tract infections : a case report

Introduction

Idiopathic purpura fulminans is a cutaneous thrombotic disorder usually caused by autoimmune-mediated protein C or S deficiency. This disorder typically presents with purpura and petechiae that eventually slowly or rapidly coalesce into extensive, necrotic eschars on the extremities. We present the first known case of idiopathic purpura fulminans consistent with prior clinical presentations in the setting of a prothrombotic genetic mutation, but without hallmark biochemical evidence of protein C or protein S deficiency. Another novel feature of our patient's presentation is that discontinuation of anti-coagulation has invariably led to recurrence and formation of new lesions, which is unexpected in idiopathic purpura fulminans because clearance of autoimmune factors should be followed by restoration of anti-coagulant function. Although this disease is rare, infants with suspected idiopathic purpura fulminans should be rapidly diagnosed and immediately anti-coagulated to prevent adverse catastrophic outcomes such as amputation and significant developmental delay.

Case presentation

A six-month-old Caucasian boy was brought to our pediatric hospital service with a low-grade fever and subacute, symmetric, serpiginous, stellate, necrotic eschars on his forearms, legs and feet that eventually spread non-contiguously to his toes, thighs and buttocks. In contrast to his impressive clinical presentation, his serologic evaluation was normal, and he was not responsive to corticosteroids and antibiotics. Full-thickness skin biopsies revealed dermal vessel thrombosis, leading to a diagnosis of idiopathic purpura fulminans and successful treatment with low-molecular-weight heparin, which was transitioned to warfarin. Long-term management has included chronic anti-coagulation because of recurrence of lesions with discontinuation of treatment.

Conclusion

In infants with necrotic eschars, it is important to first consider infectious, inflammatory and hematologic etiologies. In the absence of etiology for protracted idiopathic purpura fulminans, management should include tissue biopsy, in which thrombotic findings warrant a trial of empiric anti-coagulation. Some infants, including our patient, may need long-term anti-coagulation, especially when the underlying etiology of coagulation remains unidentified and symptoms recur when treatment is halted. Given that our patient still requires anti-coagulation, he may have a yet to be identified autoimmune-mediated mechanism for his truly idiopathic case of protracted purpura fulminans.     

Biochemical characterization of two cloned resistance determinants encoding a paromomycin acetyltransferase and a paromomycin phosphotransferase from Streptomyces rimosus forma paromomycinus.

The mechanism conferring resistance to paromomycin in Streptomyces rimosus forma paromomycinus, the producing organism, was studied at the level of both protein synthesis and drug-inactivating enzymes. Ribosomes prepared from this organism grown in either production or nonproduction medium were fully sensitive to paromomycin. A paromomycin acetyltransferase and a paromomycin phosphotransferase, both characteristic of the producer, were highly purified from extracts prepared from two Streptomyces lividans transformants harboring the relevant genes inserted in pIJ702-derived plasmids. In vitro, paromomycin was inactivated by either activity. In vivo, however, S. lividans clones containing the gene for either enzyme inserted in the low-copy-number plasmid pIJ41 were resistant to only low levels of paromomycin. In contrast, an S. lividans transformant containing both genes inserted in the same pIJ41-derived plasmid displayed high levels of resistance to paromomycin. These results indicate that both genes are required to determine the high levels of resistance to this drug in the producing organism. Paromomycin is doubly modified by the enzymes. However, whereas acetylparomomycin was a poorer substrate than paromomycin for the phosphotransferase, phosphorylparomomycin was modified more actively than was the intact drug by the acetyltransferase. These findings are discussed in terms of both a permeability barrier to paromomycin and the possible role(s) of the two enzymes in the biosynthetic pathway of this antibiotic.     

Heterogeneity of tetracycline resistance determinants

We have found that tetracycline resistance on different naturally occurring bacterial plasmids is encoded by more than one genetic determinant. Using restriction enzyme analyses and DNA-DNA hybridization to specific ³²P-labeled genetic probes, we can define at least four genetically distinct tetracycline resistance determiants: Class A (the determinant on prototype plasmid RP1), Class B (that on R222), and Class C (that on plasmid pSC101). At least one other determinant, encoded by plasmid RA1, belongs to none of these three groups and has been designated Class D. These genetic classes confirm phenotypic differences in expression of resistance to tetracycline and tetracycline analogs encoded by the different plasmids.     

The pattern of physical comorbidity and the psychosocial determinants of depression: a prospective cohort study on a representative sample of family practice attendees in Slovenia

Objectives This study aims to present the patterns of physical comorbidity in depressed patients and factors strongly associated with depression in a representative sample of Slovenian family practice attendees.Methods Medical data was obtained for 911 general practice attendees. Of them, 221 (24.3%) were diagnosed as depressed. The depressive states of the subjects were evaluated using the Composite International Diagnostic Interview (CIDI). Physical comorbidity was assessed with a questionnaire covering the most common health problems in the Slovenian adult population. Several psycho-social factors were also analysed.Results Those variables significantly related to ICD depression were included in multivariate binary logistic regression analysis, adjusted by age, gender and education. The calculation included the chi-square, odds ratio (OR) with confidence interval (95% CI) and P-value. A P-value < 0.05 was marked as statistically significant.Conclusions There was no significant difference in the number of concurrent chronic diseases in depressed and non-depressed subjects. The risk of depression was increased by the presence of several concomitant factors. The burden of somatic co-morbidity was shown to be smaller than the impact of psychosocial determinants, which also acted as protective factors: the feeling of safety at home and the absence of problems in intimate relationships. The abuse of alcohol and drugs by a family member and current poor financial situation were strongly associated with depression. The impact of concurrent incontinence and chronic bowel disease was also important, though somewhat weaker.     

Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections

Small colony variants constitute a slow-growing subpopulation of bacteria with distinctive phenotypic and pathogenic traits. Phenotypically, small colony variants have a slow growth rate, atypical colony morphology and unusual biochemical characteristics, making them a challenge for clinical microbiologists to identify. Clinically, small colony variants are better able to persist in mammalian cells and are less susceptible to antibiotics than their wild-type counterparts, and can cause latent or recurrent infections on emergence from the protective environment of the host cell. This Review covers the phenotypic, genetic and clinical picture associated with small colony variants, with an emphasis on staphylococci, for which the greatest amount of information is available.     

pUB2380: characterization of a ColD-like resistance plasmid

A detailed analysis of the mobilizable, ColE1-like resistance plasmid, pUB2380, is reported. The 8.5-kb genome encodes six (possibly seven) major functions: (1) a ColD-like origin of replication, oriV, with associated replication functions, RNAI and RNAII; (2) a set of active mobilization functions highly homologous to that of ColE1, including the origin of transfer, oriT; (3) a ColE1-like multimer resolution site (cer); (4) a kanamycin-resistance determinant, aph, encoding an aminoglycoside-3'-phosphotransferase type 1; (5) an insertion sequence, IS1294; and (6) two genes, probably cotranscribed, of unknown function(s). The GC content of the various parts of the genome indicates that the plasmid is a hybrid structure assembled from DNA from at least three different sources, of which the replication region, the mobilization functions, and the resistance gene are likely to have originated in the enterobacteriaceae.     

Staphylococcus aureus in chronic and recurrent infections

Ninety-fourStaphylococcus aureus strains isolated from chronic and recurrent skin and respiratory tract infections were investigated for several virulence factor expressions. Production of protein A was noticed in all of the tested strains in amounts from less than 0.1 to more than 2.5 ng per 10⁶ bacterial cells. The percentage of the extracellularly produced protein A was found to lie between 4.5 and 27.8%. Two strains (both from the respiratory tract) produced more than 50 % of protein A in the extracellular form and one strain did not produce any detectable amount of the extracellular protein A; 99 % of the tested strains produced the clumping factor, 96% staphylocoagulase, 79 % staphylokinase and 90 % gelatinolytic activity; 79 % produced α-toxin exclusively or in combination with δ- or β-toxin; 8 % of strains produced β-toxin. There were differences in β-toxin production between strains from the respiratory tract (5 %) and skin infections (25 %). δ-Toxin was produced by 53 % of the strains. In each of the tested strains a complex of virulence factors was detected. The importance of inactivated extracellular products (especially α- and δ-toxin and in the case of skin infections also β-toxin) as components of staphylococcal whole-cell vaccine was suggested. Dedicated to Professor C. John on the occasion of his 75th birthday     

Can a concentrated cranberry extract prevent recurrent urinary tract infections in women? A pilot study

BACKGROUND: Urinary tract infections (UTIs) are extremely prevalent and despite treatment with antibiotics, reoccurrences are common causing frustration in the patient and the potential for developing antibiotic resistance. The use of cranberry products to prevent UTIs has recently become popular and more clinical studies are needed to explore this use. OBJECTIVE: This open label pilot study examined the ability of a concentrated cranberry preparation to prevent UTIs in women with a history of recurrent infections. SUBJECTS: Women between the ages of 25 and 70 years old were included with a history of a minimum of 6 UTIs in the proceeding year. INTERVENTION: The women took one capsule twice daily for 12 weeks containing 200 mg of a concentrated cranberry extract standardized to 30% phenolics. DESIGN: A questionnaire was used initially to determine the patient's medical history and they were asked at monthly intervals if any of the information had changed. All of the women in the study had urinalysis within 24h before starting on the study preparation and once a month after that for 4 months. Subjects were followed-up approximately 2 years later. RESULTS: All 12 subjects participated in the 12-week study and were available for follow up 2 years later. During the study none of the women had a UTI. No adverse events were reported. Two years later, eight of the women who continue to take cranberry, continue to be free from UTIs. CONCLUSION: A cranberry preparation with a high phenolic content may completely prevent UTIs in women who are subject to recurrent infections.     

Oral infections as predictors of mortality

Objective: Oral infections may increase the levels of cytokines in the blood which, in turn, are associated with early mortality in the elderly. We investigated the possible association between oral infections and mortality. Design: Prospective cohort study over a 5‐year follow‐up. Setting: Research laboratory. Participants: A cohort born in 1910 (n = 94) was examined in the year 1995. Five years later mortality data were obtained from the population register for 49 deceased subjects. Main outcome measures: Urgent need of dental treatment, lifetime, erythrocyte sedimentation rate (ESR). Results: The multivariate analysis adjusted for general health and lifestyle factors showed that the risk for death of subjects in urgent need of dental treatments was 3.9 times higher than that of the other subjects. Among men ESR correlated significantly with urgent need of dental treatment. Conclusions: Oral infections among frail elderly people may be a sign of early mortality.     

Microarray analysis of erythromycin resistance determinants

AIMS: To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. METHODS AND RESULTS: We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). CONCLUSIONS: Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.