- Volume 51, Issue 7, 2002

The word ‘complex’ has several meanings and synonyms such as composite, obsession, heterogeneous, mixed and network, can all be used in its place. Our obsession with bacteria from the Burkholderia cepacia complex started in the early 1990s. In less than 10 years, we have seen the status of this bacterium move from: (i) a lesser known pseudomonad opportunist pathogen, (ii) to devastating infections transmitted between patients with cystic fibrosis (CF), (iii) through divisions into several new species, and (iv) now on towards one of the largest gram-negative genome sequencing projects. For microbiologists, hospital infection control officers, caregivers, and most of all the CF community, the changes in our understanding of the taxonomy, epidemiology and pathogenesis of the bacterium ‘B. cepacia’ are complex.

The aim of this study was to determine the role of macrophages in the Actinobacillus actinomycetemcomitans-induced murine immune response. BALB/c mice were given carrageenan solution by intraperitoneal injection before immunisation with heat-killed A. actinomycetemcomitans. Mice immunised with antigens and phosphate-buffered saline served as positive and negative controls, respectively. One week after the last immunisation, the delayed-type hypersensitivity (DTH) response was assessed by measurement of footpad swelling. Serum IgG and IgM anti-A. actinomycetemcomitans antibody levels and culture supernate levels of interferon (IFN)-γ were determined by ELISA. The diameter of abscess formation was determined every 5 days. Sham-immunised spleen cells were transferred to carrageenan-untreated recipients (groups A and B) and to carrageenan-treated recipients (group D). Antigen-immunised spleen cells were transferred to carrageenan-untreated (group C) and carrageenan-treated (group E) recipients. The carrageenan-treated recipients in groups F and G received macrophages from antigen- and sham-immunised mice respectively. All mice except those in group A were immunised with antigen 24 h after cell transfer. After 1 week, a partial suppression of DTH response, reduced levels of IFN-γ, serum IgG and IgM anti-A. actinomycetemcomitans antibodies and delayed healing were seen in carrageenan-treated mice when compared with the positive control. The immune response to A. actinomycetemcomitans in groups A, B and D was lower than that in groups C and E. Healing of the lesion in the former groups was also delayed when compared with the latter groups. The immune response and the healing of the lesion could be partially restored in carrageenan-treated mice that received antigen-pulsed macrophages (group F) but not in those that received naïve macrophages (group G). These results suggest that macrophages play a partial role in the induction of the murine immune response to A. actinomycetemcomitans.

Human papillomaviruses (HPVs) are major pathogens associated with the development of cancer of the uterine cervix, the most common malignant tumour of women world-wide. Reliable diagnosis of HPV infection, particularly the ‘high-risk’ types (16/18), may facilitate early identification of ‘high-risk’ populations for developing cervical cancer and may augment the sensitivity and specificity of primary cervical cancer screening programmes by complementing the conventional Pap test. A simple paper smear method has been developed for dry collection, transport and storage of cervical smears/scrapes at room temperature for subsequent detection of HPV DNA by PCR assay. Imprint biopsies, blood and fine-needle aspirates were also collected by this method. The cervical scrapes or other body fluids were smeared (within 0.5–1 cm diameter) and dried on to sterile small slides made of Whatman 3MM filter paper, and stored individually at room temperature or at 4°C. A small piece (2–3 mm) of the paper smear was punched or cut out with a sterile surgical blade, boiled in an eppendorf tube containing 50 μl of distilled water for 5 min and used directly for PCR amplification. The quality and quantity of DNA derived from paper smears and the results of PCR amplifications for HPV type 16, BRCA1 and p53 genes were identical to those obtained from the same samples following collection in PBS, storage (−70°C) and phenol-chloroform-based DNA extraction. DNA was stable in the paper smears for up to a year, whether stored at room temperature or at 4°C. This method is simple, rapid and cost-effective, and can be effectively employed for large-scale population screening, especially for regions where the specimens are to be transported from distant places to the laboratory.

Mechanisms of tetracycline resistance were investigated in two recent Listeria monocytogenes isolates from food, with L. innocua 52P tetr as a control. Tetracycline resistance was transferred conjugatively from all three strains to L. ivanovii and from one isolate and the control to Enterococcus faecalis. Molecular analysis demonstrated a chromosomal location for the tet determinant, which was identified as tetM in all cases. These studies are the first to show that L. monocytogenes from food could be a source of tetracycline resistance genes able to spread to other micro-organisms.

Serotyping and genotyping are important tools in epidemiological studies of group B streptococcal (GBS) infections, which are important diseases in man, particularly in newborns. In the present study, 241 GBS isolates from Zimbabwe, comprising 124 carrier isolates from pregnant women and 117 isolates from patients hospitalised for various diseases, were serotyped. Antibodies specific for the capsular polysaccharide antigens (CPAs) Ia, Ib and II–V and antibodies specific for the surface-localised proteins, cα, cβ, R1, R3 and R4 were used for serotyping. Strains of the CPA types Ia (17%), III (47.7%) and V (23.2%) predominated. Of the various protein antigens, cα and R4 were expressed with highest frequency, cα by 100% of the CPA type Ia strains and R4 by 92% of the CPA type III strains. The R3 protein occurred frequently (24%), especially in type V strains (84%). A total of 25 serovariants was detected in the strain collection with the variants Ia/cα (16%), III/R4 (43.5%) and V/cα, R3 (14.1%) occurring with the highest frequency. Serotype and subtype distribution of the carrier isolates were essentially similar to those of the disease-associated isolates. Genomic heterogeneity was demonstrated by pulsed-field gel electrophoresis of type III/R4 and type V/cα, R3 isolates, but to a much lesser extent than recorded with Norwegian strains. These results demonstrate that many variants of GBS occur in the Zimbabwean population. The data obtained may assist in the formulation of a possible future GBS vaccine for Zimbabwe and perhaps for other African countries.

Eighty-one bronchoalveolar lavage (BAL) specimens obtained from 26 HIV-infected, 45 non-HIV immunosuppressed and 10 immunocompetent patients with primary pulmonary diseases were analysed for the presence of Pneumocystis carinii by staining and by P. carinii 5S rDNA determined by PCR. P. carinii was observed by staining of BAL specimens from HIV-infected patients significantly more frequently than those from immunocompromised hosts without HIV infection (57.7% versus 20.0%, respectively). P. carinii 5S rDNA was detected by PCR assay in seven (26.9%) HIV-infected individuals, which was significantly more frequent than for four (8.9%) immunosuppressed patients without HIV infection, for whom staining was negative. None of these patients developed P. carinii pneumonia (PCP) within the follow-up period. BAL specimens from 10 immunocompetent patients with pulmonary disorders were negative for PCP by both staining and PCR assay.

Pyogenic sacro-iliitis is an uncommon osteo-articular infection that occurs usually in immunocompromised patients and is associated with gram-positive cocci. It is very rarely linked with a gram-negative aetiology. The first case of Pseudomonas aeruginosa sacro-iliitis is described, which occurred in a previously healthy young man, without history of prior traumatic events, hospitalisation or chronic underlying disease.

The present study characterised 73 Hafnia alvei isolates and five Escherichia isolates (originally identified as H. alvei) isolated from cases of diarrhoeal disease by the International Centre for Diarrhoeal Disease Research Branch (ICDDRB) in Bangladesh. Based upon the hydrolysis of arbutin and aesculin and the fermentation of salicin and d-arabinose, four distinct biotypes could be recognised among the 73 H. alvei isolates tested; biotype 1 (d-(-)-arabinose-positive only) accounted for 75% of all isolates analysed. Hydrolysis of aglycone compounds such as arbutin, salicin and aesculin appeared to be associated with expression of β-glucosidase activity. ICDDRB isolates, when compared with type or reference strains of H. alvei, were shown not to belong to the genus Hafnia based upon resistance to Hafnia-specific bacteriophage 1672, possession of the phoE gene, expression of glutamate decarboxylase activity and significant 16S rDNA sequence divergence (∼8%) from the type strain, ATCC 13337T. True H. alvei strains, implicated in outbreaks of diarrhoeal disease in Canada, lacked the eaeA gene in contrast to ICDDRB isolates. Twenty-two H. alvei isolates were selected for further study. Based upon partial 16S rDNA sequencing, these 22 isolates fell into two genomic groups (genomospecies), identical to DNA groups previously established by DNA hybridisation studies. Markers such as motility, biotype, or enzymic or carbohydrate fermentation patterns did not correlate totally with DNA grouping, although malonate utilisation appeared to be the single best discriminatory phenotype. The results indicate that the genus Hafnia is heterogeneous and there do not appear to be any laboratory data available specifically linking these organisms to gastro-enteritis.

A Burkholderia pseudomallei mutant which was attenuated in a mouse model of melioidosis was identified by a signature tagged mutagenesis approach. The transposon was shown to be inserted into a gene within the capsular biosynthetic operon. Compared with the wild-type bacteria this mutant demonstrated a 105-fold increase in the median lethal dose in a mouse model and it did not react with a monoclonal antibody against high mol. wt polysaccharide of B. pseudomallei. To determine the kinetics of infection, mice were dosed intraperitoneally (i.p.) and intravenously (i.v.) with mutant and wild-type bacteria. After i.p challenge, the number of mutant bacteria in the peritoneal cavity declined, whereas wild-type bacteria proliferated. When administered by the i.v. route, the mutant was able to cause disease but the time to death was increased compared with the wild type. Mice were dosed with the mutant and subsequently challenged with wild-type B. pseudomallei, but the mutant failed to induce a protective immune response.

A prospective study was conducted in 1999 at the National Cancer Institute, Cairo University, to estimate the incidence, morbidity and mortality of fungal infections along with the evaluation of risk factors influencing outcome of infections among paediatric cancer patients. Of 1917 infectious episodes, the fungal infection rate as documented both clinically and microbiologically was 3.7% (70 cases). Fungal pathogens isolated were yeasts in 55 patients (78.6%) and moulds in 15 patients (21.11%). Among yeasts, Candida parapsilosis was the commonest, followed by C. tropicalis. Pneumonia was the most common fungal infection (n=25, 35.7%), followed by fungaemia (n=18, 25.7%). The overall mortality rate was 40% (n=28), with an infection-related mortality of 28.5% (n=20). Risk factors that accompanied mortality were relapsing or recurrent disease, profound neutropenia, ADE (Ara-C, daunorubocin and etoposide) protocol of chemotherapy, C. tropicalis isolated and fungaemia as a site of infection. Early use of empirical antifungal therapy (day 4) was not associated with a better outcome. In the light of the poor outcome of patients with fungaemia and fungal pneumonia, every effort should be made to prevent these infections in paediatric cancer patients.