- Volume 43, Issue 2, 1995

Amplification of the region separating the genes coding for the two rRNA species 16S and 23S was performed with 56 strains of several mycobacterial species, including 21 clinical isolates of Mycobacterium kansasii and the M. kansasii type strain ATCC 12478. On the basis of PCR product profiles, the previously suggested heterogeneity of M. kansasii species was confirmed. Three subgroups were identified; members of the first subgroup showed the same PCR profile as the reference strain. Different profiles were obtained for the two other subgroups. Amplification of the 16S–23S spacer is rapid and simple and, consequently, may be a helpful tool for identification and characterisation of M. kansasii isolates in epidemiological analysis.

Since the mid-1980s, the rate of decline in reported cases of tuberculosis (TB) has reached a plateau or reversed because of a combination of poverty and increased homelessness, immigration and displacement, poorly managed and supplied TB control programmes and, particularly in the developing world, the emergence of human immunodeficiency virus (HIV) infection. TB in HIV-positive patients may present atypically, both clinically and radiologically, with a lower probability of sputum positivity, greater difficulty in diagnosis, and a more rapid clinical deterioration than TB in HIV-seronegative patients. The emergence of multiple-drug-resistant strains of Mycobacterium tuberculosis, particularly in patients infected by HIV, carries a high mortality and has been associated with outbreaks in Europe and the USA. Microscopy and culture form the basis of diagnosis, but there is a need for more rapid diagnostic techniques and novel methods of drug susceptibility testing. Prolonged supervised treatment programmes and the development of new chemotherapeutic agents and regimens are essential prerequisites for successful TB therapy in AIDS patients. This review examines the clinical, microbiological and epidemiological issues associated with TB in HIV-infected individuals.

Experimental mastitis induced with a single intramammary injection of Listeria monocytogenes was compared with two naturally occurring cases. Four strains of L. monocytogenes, two of serotype 1/2a and two of serotype 4b were used for the experimental infections and two diametrically opposed quarters of four cows were inoculated with 300 cfu. Bacteriological examination and somatic cell counts of quarter foremilk samples were performed weekly for at least 6 months after challenge. All the inoculated quarters developed chronic subclinical mastitis with occasional clinical episodes. The results were similar to those observed in natural listeria mastitis. Four experimentally infected quarters were treated during lactation (gentamicin and cloxacillin) or at “drying-off” (cloxacillin), or at both times. Only one of four quarters was cured after treatment only at “drying-off”. All experimental and naturally infected animals were slaughtered and bacteriological examination was performed on liver, spleen and supramammary, iliac and mesenteric lymph nodes. L. monocytogenes strains were isolated from the supramammary lymph nodes of two experimentally and two naturally infected cows and from an iliac lymph node from one of the naturally infected cows. The epidemiological data were supported by serotyping, lysotyping and DNA macro-restriction analysis. The experimental model of listeria mastitis mimics spontaneous cases and should be useful in further studies of listeria mastitis.

Adhesion of Staphylococcus epidermidis NCTC 11047 to the external surface of polyurethane catheters was quantified by a radiolabelling assay. Maximum adhesion was achieved with an initial cell concentration of 3 × 108/ml after incubation for 120 min. The assay was tested for reproducibility by analysis of variance. Adhesion of clinical strains of S. epidermidis and S. aureus to uncoated polyurethane and hydrogel (Hydromer®)-coated polyurethane catheters was compared. Hydrogel coating significantly reduced adhesion for both S. epidermidis and S. aureus (mean percentage reduction 71% for S. epidermidis, 69% for S. aureus). Clinical isolates were also tested for adhesion to polystyrene by a modified microtitration well adhesion assay; there was no correlation between staphylococcal adhesion to polyurethane catheters and adhesion to polystyrene. Cell surface hydrophobicity values varied widely for both species. Positive correlations were found between cell surface hydrophobicity and adhesion to polystyrene and uncoated polyurethane catheters for S. epidermidis but not for S. aureus.

Cyclophosphamide (CY) is used in many animal studies, including models of bacteraemia, to deplete peripheral neutrophils and induce a compromised state. Although CY also influences lymphocyte function, the protective role of lymphocytes in bacteraemia is unclear. Therefore, CY (200 mg/kg) was administered to ddY mice and its influence on the number, cellular composition, and function of lymphocytes in the spleen and Peyer’s patches was examined. A single dose of CY reduced the number of lymphocytes in a time-dependent fashion. Flow cytometry showed that B cells carrying B220 antigen decreased significantly. The production of IgA in Peyer’s patches, as measured by enzyme-linked immunosorbent assay, was also suppressed in a time-dependent fashion. Blastogenic responses of splenic lymphocytes to Concanavalin-A, lipopolysaccharide and heat-killed Pseudomonas aeruginosa were suppressed 48 h after CY administration. The results suggest that CY suppresses the number and function of lymphocytes, especially B cells. This may lead to bacterial overgrowth in the gut and result in bacteraemia. Intravenous transfusion of normal lymphocytes or oral inoculation of IgA to mice with P. aeruginosa D4 endogenous bacteraemia significantly increased survival rates, indicating that lymphocytes and their products have a protective role in bacteraemia in mice.

Clinical (101) and collection (26) strains of gram-positive anaerobic cocci were examined in conventional tests and pyrolysis mass spectrometry (PMS). Numerical classifications based upon conventional test reaction patterns (CTRPs) and PMS showed 27 and 22 clusters, respectively. Cross-tabulation of cluster membership in the two classifications showed excellent correlation, with the combined classifications showing clear groups corresponding to the currently recognised species Peptostreptococcus anaerobius, P. helio-trinreducens, P. hydrogenalis, P. indolicus, P. lactolyticus, P. magnus, P. micros and Peptococcus niger. Strains of P. prevotii and P. tetradius clustered together in a heterogeneous group of saccharolytic organisms. However, strains previously identified as P. asaccharolyticus were divided into three distinct groups in PMS, two of which differed only in indole-associated pyrolysis products. A further four groups and several single-member clusters were distinct from these species. PMS data supported the validity of identification by pre-formed enzyme profiles and confirmed that Hare group III is synonymous with P. hydrogenalis, Hare group IV with P. magnus, and the “ADH group” with P. vaginalis. There is clearly a need for a taxonomic revision of the genus Peptostreptococcus, which probably encompasses several generic groups.

From 1974 to 1990, 336 Bacteroides isolates were obtained from 312 specimens from 274 patients. They comprised 180 (54%) B. fragilis isolates, 55 (16%) B. thetaiotaomicron, 36 (11%) B. vulgatus, 34 (10%) B. distasonis, 21 (6%) B. ovatus and 10 (3%) B. uniformis. Infections in 253 (92%) patients were polymicrobial, but in 21 (8%) children, a Bacteroides sp. was isolated in pure culture. Most Bacteroides isolates were from peritoneal fluid (114), abscesses (110), wound infections (20), blood cultures (10) and from patients with pneumonia (14) or chronic otitis media (8). Predisposing conditions were present in 145 (53%) children; these were previous surgery (46), trauma (28), malignancy (21), prematurity (19), immunodeficiency (18), steroid therapy (12) foreign body (10), diabetes (9) and sickle cell disease (7). The micro-organisms isolated most commonly mixed with Bacteroides spp. were anaerobic cocci (221), Escherichia coli (122), Fusobacterium spp. (38) and Clostridium spp. (30). All patients received antimicrobial therapy in conjunction with surgical drainage or correction of pathology in 197 (72%) cases. All but 12 (5%) patients recovered. These data illustrate the importance of Bacteroides spp. in infections in children.

A monospecific polyclonal antiserum, prepared against Bacteroides fragilis common polysaccharide antigen purified by polyacrylamide gel immunoblot detected B. fragilis, B. thetaiotaomicron, B. ovatus and Prevotella melaninogenica in pus samples from various anatomical sites by immunofluorescence microscopy of the pus. With standard clinical laboratory culture methods, 36% of 147 samples were positive for one or more of the above bacteria. Of these, B. fragilis accounted for 33%. By immunofluorescent labelling of pus with the common antigen antiserum the detection of these bacteria in the samples increased to 50%. All nine of the blood cultures in which B. fragilis was detected by culture contained bacteria positive for the common antigen. Immunofluorescent labelling of pus samples with a selection of monoclonal antibodies specific for surface polysaccharides which are known to be antigenically variable in culture in vitro and in an animal model of infection showed that these polysaccharides are also variable in natural infection. The results indicate that the common polysaccharide antigen, in contrast to the variable surface polysaccharides, is a suitable target for the immunodetection of B. fragilis in clinical samples from a range of anatomical sites.

Early diagnosis of leptospirosis is important because severe leptospiral infection can run a fulminant course. The polymerase chain reaction (PCR) was evaluated for the detection of leptospires in clinical samples from patients with acute leptospiral infection. Blood and urine samples from 71 patients with leptospirosis were examined by PCR, culture or serology. Samples from 44 (62%) patients with the diagnosis of leptospirosis were positive by PCR as compared to 34 (48%) by culture. The presence of leptospires was demonstrated by PCR in 13 patients before the development of antibodies, as well as in two patients who were seronegative during their illness and at autopsy. Samples from 16 patients without leptospirosis were seronegative and culture negative, and also negative by PCR. We conclude that PCR is a rapid, sensitive and specific means of diagnosing leptospiral infection, especially during the first few days of the disease.

The involvement of the central nervous system (CNS) in lepromatous leprosy (LL) patients was investigated; 33 patients were examined clinically in detail and upper motor neuron involvement was observed in eight and lower motor neuron in three of these patients. Anti-Mycobacterium leprae antibodies could be detected in the CSF by PGL-1 enzyme-linked immunosorbent assay (ELISA) and monoclonal antibody (MAb) based competitive assays against defined epitopes on the 35-kDa protein and 30–40-kDa polysaccharide (lipo-arabinomannan) antigens with MAbs ML04 and ML34, respectively. Antibodies against PGL-1 and 35-kDa protein were observed in more subjects than antibodies against the 30-40-kDa antigen. Some correlation was observed between the upper motor neuron signs and antibody positivity for 35-kDa and PGL-1 antigens in the CSF of these patients.

Molecular characterisation is an important pre-requisite for post-vaccine studies of Haemophilus influenzae type b (Hib). Three capsular genotyping patterns, b(S), b(G) and b(V), have been described in the major phylogenetic lineage of Hib. However, in a recent series of prospective studies, three new hybridisation patterns were observed among 425 strains of Hib. Four pairs of polymerase chain reaction (PCR) primers were used to identify the capsular gene (cap) structure of these Hib strains. This showed that the strains possessed simple DNA re-arrangements. In two instances a change in restriction enzyme recognition site was the most likely cause of the new hybridisation pattern. The third strain possessed a cap b locus consisting of intact tandem repeats of cap b in a b(S) background. It was reasoned that a similar cap b locus would not be readily recognised by hybridisation in a b(G) background, and b(G) strains were therefore characterised by the PCR method. This showed one of 35 b(G) strains to possess a cap locus with intact tandem repeat copies of cap b. The novel capsular genotypes described here are rare, but can be detected rapidly and accurately by a combination of PCR and capsular genotyping hybridisation patterns.