- Volume 53, Issue 3, 2004

Melanins, or melanin-like compounds, may play a role in the pathogenesis of a number of human fungal infections. This study investigated the production of melanin by the important opportunistic pathogen Aspergillus fumigatus. Conidia from A. fumigatus were harvested and treated with proteolytic enzymes, denaturant and hot, concentrated acid; this yielded dark particles which were similar in size and shape to the original propagules. Electron spin resonance spectroscopy revealed that the conidial-derived particles were stable free radicals consistent with an identification as melanin. Melanin particles were used to immunize BALB/c mice in order to produce a total of five anti-melanin monoclonal antibodies (mAbs). The latter mAbs were strongly reactive both with intact conidia and with extracted melanin particles by ELISA and immunofluorescence reactivity. Immunofluorescence labelling with the novel mAbs was used to examine the temporal expression of melanin during in vitro culture of A. fumigatus –melanization was confined to conidial structures and was absent from hyphae. SDS-PAGE l-3,4-dihydroxyphenylalanine (l-DOPA) substrate analysis confirmed the presence of a laccase-type activity in conidial extracts, but not in hyphae. Melanin-binding mAbs were used to detect the presence of melanized conidia in three patients with nasal aspergilloma, indicating that in vivo melanization may occur during infection.

Laboratory results of 67 cases of legionnaires’ disease caused by Legionella pneumophila serogroup (Sg) 1 spanning a 6-year period were analysed by both phenotypic and genotypic methods. The methods compared were urinary antigen enzyme immunoassay (EIA), an immunofluorescent antibody (IFA) test, direct fluorescent antibody (DFA), culture and a 5S rRNA PCR with Southern blotting confirmation. Urine was available in 53 cases, of which 35 (66 %) were positive, with an antigen peak observed at 5–10 days after onset of disease symptoms. The IFA test was positive in 62 (92.5 %) cases, with 56 (90.3 %) cases producing a greater than fourfold rise in titre and 6 (9.7 %) giving presumptive high titres of ⩾1 : 128. There were two antibody peaks, one at 10–15 days and another at >25 days after onset. In 23 cases where samples were available, DFA and culture were respectively positive in 5 (22 %) and 10 (48 %) cases. There was a peak in culture-positives 5–10 days after onset of disease. A Legionella-specific 5S rRNA PCR on patient serum was positive in 54 (80.5 %) cases, with a peak in PCR positivity at 6–10 days after disease onset. In 22 of the 67 cases, the full panel of diagnostic methods was available for comparison. The relative sensitivity and specificity of the urinary antigen EIA and the serum PCR was 100 %. The IFA gave relative sensitivity and specificity values of 93.8 and 95 %. DFA and culture, although 100 % specific, produced only low sensitivities, of 19 and 42.8 %, respectively. This study has shown that urinary antigen and serum PCR are valuable tests in the acute phase of disease, with excellent sensitivity and specificity values. At present, the Legionella species causing infection requires to be verified by IFA serology and/or culture, but this could become unnecessary as new antigen and L. pneumophila Sg 1-specific PCR tests become available.

This study examines the role of the penicillin-binding proteins (PBPs) of Bacteroides fragilis in the mechanism of resistance to different β-lactam antibiotics. Six of the eight strains used were β-lactamase-positive by the nitrocefin assay. These strains displayed reduced susceptibility to imipenem (MIC, 2–16 mg l−1) and some of them were resistant to the actions of ampicillin, cefuroxime, cephalexin, cefoxitin and piperacillin. When studying specific enzymic activity, the capacity to degrade cefuroxime was only detected in strains AK-4, R212 and 0423 and the capacity to degrade cephalexin was only detected in strains R212 and 2013E; no specific activity was detected on imipenem. Metallo-β-lactamase activity was only detected in strains AK-2 and 119, despite the fact that the cfiA gene was identified in four strains (AK-2, 2013E, 119 and 7160). The cepA gene was detected in six of the eight strains studied. Three high-molecular-mass PBPs were detected in all strains; however, in some cases, PBP2Bfr and/or PBP3Bfr appeared as a faint band. PBP4Bfr and PBP5Bfr were detected in six strains. PBP6Bfr only was detected in B. fragilis strains AK-2, 0423, 119 and 7160. By analysis of the sequence of B. fragilis chromosomal DNA and comparison with genes that are known to encode PBPs in Escherichia coli, six genes that encode PBP-like proteins were detected in the former organism. The gene that encodes the PBP2 orthologue of E. coli (pbpABfr, PBP3Bfr) was sequenced in six of the eight strains and its implications for resistance were examined. Differences in the PBP3Bfr amino acid sequences of strains AK-2 and 119 and their production of β-lactamases indicate that these differences are not involved in the mechanism of resistance to imipenem and/or cephalexin.

Multilocus sequence typing (MLST) of 48 methicillin-sensitive Staphylococcus aureus isolates from intravenous drug user abscesses/soft-tissue infections revealed 12 sequence types (STs) belonging to eight genetically distinct lineages. Only two novel STs were recovered (one isolate of each), indicating that isolates in this study were similar to those from previous studies of disease and carriage. However, ST59, the most common genotype recovered (from six individuals), may be adept at causing subcutaneous lesions in this patient population, as it is rare in carriage and disease. PCR detection of 22 toxin genes revealed a high prevalence of the gene for staphylococcal enterotoxin B compared with previous studies, indicating that this toxin may promote infections in this patient group.

Escherichia coli isolates (n = 2629) were collected between 1996 and 2000 from 2100 Thai children less than 12 years of age with acute diarrhoea. Enterotoxigenic (ETEC), enteroinvasive (EIEC), Shiga-toxin-producing (STEC), enteropathogenic (EPEC) and enteroaggregative (EAEC) E. coli were identified by their virulence marker profiles, as determined by multiplex PCR, and HeLa cell-adherence patterns. Serogroups of isolates were determined using 43 monovalent O antisera. Of 2629 isolates, 16.9 % were identified as diarrhoeagenic E. coli, and the mean isolation rates per year were 10.2 % for EAEC (range 8–12.5 %), 3.2 % for EPEC (0–8 %), 3.0 % for ETEC (2–5.4 %), 0.5 % for EIEC (0–1 %) and 0.04 % for STEC (0–0.1 %). The isolation rates of pathotypes from four different age groups (0–5 months, 6–11 months, 1–2 years and 2–12 years) in 905 children whose ages were recorded were respectively 19.3, 18.2, 9.1 and 8.1 % for EAEC, 3.1, 4.3, 1.7 and 2.2 % for EPEC and 2.6, 2.3, 1.3 and 5 % for ETEC. About 38 % of diarrhoeagenic E. coli, including 55.1, 66.7, 100, 45.9 and 29 %, respectively, of ETEC, EIEC, STEC, EPEC and EAEC, and 24 % of non-diarrhoeagenic E. coli were O-antigen typable. Only four serogroups (9.3 %) were restricted to single pathotypes, whereas 27 serogroups (62.8 %) were not restricted to any pathotype. This study shows that EAEC are the most prevalent diarrhoea-associated pathotype in Thai children.

Oxalobacter formigenes, an anaerobic bacterium that inhabits the mammalian gastrointestinal tract, has an important symbiotic relationship with its vertebrate hosts by regulating oxalic acid homeostasis. Epidemiological studies of O. formigenes colonization in man have shown that colonization occurs in young children, that every child can become colonized naturally, that >20 % lose colonization during adolescence or as adults and that stable colonization can be disrupted by antibiotic use or changes in diet, greatly affecting subsequent health. As O. formigenes is a fastidious anaerobe that seldom re-colonizes adults, the question arises as to how initial colonization occurs. To investigate this question, non-colonized female laboratory rats were placed on diets high in oxalate and were colonized by oesophageal gavage with O. formigenes either before or after being impregnated. Faecal specimens from their offspring were tested for the presence of O. formigenes. Although the bacterium was first detected in a few neonates as early as 7 days post-partum, colonization of all the offspring did not occur until after weaning. In each case, the offspring were colonized with the bacterial strain carried by their mothers. To determine whether O. formigenes colonization occurs vertically or horizontally, newborn rats were placed with foster mothers that were either non-colonized or colonized with an O. formigenes strain different from that of their natural mothers. Colonization occurred temporally in a manner similar to natural colonization but all offspring became colonized only with the O. formigenes strain of the foster mothers. These data indicate that intestinal colonization occurs horizontally, but does not answer the question of how O. formigenes survives the aerobic environment in order to be transmitted.

Cyclospora cayetanensis, the parasitic agent responsible for human cyclosporiasis, is an emerging worldwide cause of diarrhoea in immunocompetent people as well as in immunocompromised patients, such as those with AIDS. Reported here is the case of a 30-year-old Turkish woman, a lawyer, who was admitted to hospital in July 2002 with complaints of watery diarrhoea, anorexia, nausea, vomiting, abdominal pain and weight loss over a period of 1 week. Cyclospora sp. oocysts were determined by using modified Kinyoun's acid-fast stain. The patient was treated with trimethoprim/sulfamethoxazole (160/800 mg) b.i.d. for 7 days. This report is the first example of autochthonous cyclosporiasis in an immunocompetent patient in Turkey.