- Volume 51, Issue 11, 2002

An outbreak of serious illness and death occurred in injecting drug users during 2000 in Scotland, Ireland and England. National and international collaboration was necessary for the investigation and management of this outbreak. In England and Wales active case-finding was initiated, coupled with standardised data collection and microbiological investigation of cases. Twenty-six definite or probable cases were identified in England between 1 April and 31 Aug. 2000; 17 of these occurred in the North. The overall case fatality was 50% (13/26). The principal apparent risk factor was a history of intramuscular or subcutaneous injection of heroin and the limited duration of the outbreak suggested that the problem might have been related to a particular supply of heroin. Clostridium novyi was isolated from two English cases. Taken in conjunction with contemporaneous microbiological and epidemiological results from Scottish and Irish cases, the probable aetiology for this outbreak was infection with C. novyi associated with both a particular supply of heroin and the method of preparation and injection used. A ‘toolkit’ was distributed in Sept. 2000 to all Consultants for Communicable Disease Control in England and Wales to assist them with the ongoing surveillance, investigation and management of this condition. Lessons learned have been used to produce guidance for the investigation and management of outbreaks of unexplained serious illness of possible infective aetiology.

Pathogenic species of the genus Clostridium may contaminate the materials used in the injection of drugs and under the right conditions may cause serious or life-threatening disease. C. novyi type A was implicated in an outbreak of severe infection with high mortality in injecting drug users who injected heroin extravascularly. The isolation of such highly oxygen-sensitive clostridia from clinical material may require adherence to enhanced methods and, once isolated, commercially available anaerobe identification kits alone may not give an accurate identification. Additional phenotypic tests that are useful in recognising the main pathogenic species are described. Differentiation of C. novyi type A from C. botulinum type C in reference laboratories was based on 16S rDNA sequence data and specific neutralisation of cytopathic effects in tissue culture.

As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C. perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis. Among the 43 C. novyi isolates, 23 different AFLP profiles were detected. The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user. Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988). The 40 C. perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin. Sixteen AFLP types of C. perfringens were distinguished and there was little evidence for commonality among the isolates. The AFLP types of C. perfringens from heroin differed and were unique. Six isolates of B. cereus were from four drug users and two samples of heroin. Four different AFLP patterns were distinguished. Three AFLP types were isolated from four drug users. B. cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user. The AFLP type of the isolate from a second and unrelated heroin sample was unique. The AFLP results showed no or very limited evidence for commonality between the different isolates of B. cereus and C. perfringens. In marked contrast, the C. novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C. novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation.

In 2000, an unusual increase of morbidity and mortality among illegal injecting drug users in the UK and Ireland was reported and Clostridium novyi was identified as the likely source of the serious infection, although infections due to C. botulinum and Bacillus cereus were also reported. Because heroin was a possibile source of infection, this study investigated the microflora of heroin samples seized in England during 2000 and 2002. Two methods were developed for the examination of the microflora of heroin. The first consisted of suspension of the drug in maximum recovery diluent (MRD) which was inoculated directly into Clostridium Botulinum Isolation Cooked Meat Broth (CBI). The second method rendered the heroin soluble in citric acid, concentrated particulate material (and bacterial cells) by filtration and removed heroin residues by washing with citric acid and phosphate-buffered saline before placing the filter in CBI broth. Duplicate CBI broths from both methods were incubated without heating and after heating at 60°C for 30 min. Subcultures were made after incubation for 7 and 14 days on to eight different solid media. The methods were evaluated with heroin samples spiked with either C. botulinum or C. novyi spore suspensions; recovery of 10 spores in the original sample was demonstrated. Fifty-eight heroin samples were tested by citric acid solubilisation and 34 by the MRD suspension technique. Fifteen different gram-positive species of four genera were recognised. No fungi were isolated. Aerobic endospore-forming bacteria (Bacillus spp. and Paenibacillus macerans) were the predominant microflora isolated and at least one species was isolated from each sample. B. cereus was the most common species and was isolated from 95% of all samples, with B. licheniformis isolated from 40%. Between one and five samples yielded cultures of B. coagulans, B. laterosporus, B. pumilus, B. subtilis and P. macerans. Staphylococcus spp. were isolated from 23 (40%) samples; S. warneri and S. epidermidis were the most common and were cultured from 13 (22%) and 6 (10%) samples respectively. One or two samples yielded cultures of S. aureus, S. capitis and S. haemolyticus. The remainder of the flora detected comprised two samples contaminated with C. perfringens and two samples with either C. sordellii or C. tertium. Multiple bacterial species were isolated from 43 (74%) samples, a single species from the remaining 15. In 13 samples B. cereus alone was isolated, in one B. subtilis alone and in one sample B. pumilus alone. C. botulinum and C. novyi were not isolated from any of the heroin samples. Recommendations for the optimal examination of the microflora of heroin are given.

Bacteriocins produced by mutans streptococci are known as mutacins. In this study 16 broadly active mutacin-producing Streptococcus mutans strains from New Zealand, North America and Europe were classified into four groups (A–D) on the basis of differences in their activity in deferred antagonism tests against either the homologous producer strain (to test for presence of self-immunity) or indicator strains Staphylococcus aureus 46 and Enterococcus faecium TE1. Two of the strains included in the study (UA140 and UA96) were representatives of the group I and II mutacin producer strains previously described by Caufield and co-workers. One of the New Zealand isolates of group A (S. mutans strain N) appeared to produce inhibitory activity similar to that of the group I prototype strain UA140. Four other New Zealand isolates of group B (S. mutans strains M19, M34, B34 and D14) had mutacin II-like activity. The group B mutacin producers differed from the group A mutacin producers in their additional activity against Staph. aureus 46. Seven S. mutans strains (M46, B46, B57, M12, M28, B28 and 13M) were distinguished from the group A and group B mutacin producers in that they inhibited E. faecium TE1. These were called group C mutacin producers. Strains H7 and H23 resembled the group C strains in their action on both indicator strains TE1 and 46. However, these two strains failed to exhibit immunity to their own inhibitory products in the deferred antagonism test and were separately classified as group D mutacin producers. Phylogenetic analysis of the strains by several genotypic and phenotypic characteristics revealed that the mutacin groups were associated with distinct evolutionary lineages of S. mutans.

Peptostreptococci are gram-positive, strictly anaerobic bacteria which, although regarded as members of the commensal human microflora, are also frequently isolated from sites of clinical infection. The study of this diverse group of opportunist pathogens has been hindered by an inadequate taxonomy and the lack of a valid identification scheme. Recent re-classification of the Peptostreptococcus family into five distinct genus groups has helped to clarify the situation. However, this has been on the basis of 16S rRNA sequence determinations, which are both time-consuming and expensive. The aim of the present study was to evaluate the use of PCR-amplified ribosomal DNA spacer polymorphisms for the rapid differentiation of the currently recognised taxa within the group of anaerobic gram-positive cocci. A collection comprising 19 reference strains with representatives of each of the 15 species, two close relatives and two of the well-characterised groups, together with 38 test strains was studied. All strains were identified to species group level by phenotypic means. Amplification of the 16S–23S intergenic spacer region (ISR) with universal primers produced distinct banding patterns for all the 19 reference strains and the patterns could be differentiated easily visually. However, of the 38 test strains, less than half could be speciated from ISR analysis alone. Only five groups produced correlating banding patterns for all members tested (Peptoniphilus lacrimalis, P. ivorii, Anaerococcus octavius, Peptostreptococcus anaerobius and Micromonas micros). For other species, either the type strain differed significantly from other species members (e.g., A. hydrogenalis) or there appeared to be considerable intra-species variation (e.g., A. vaginalis). Partial 16S rRNA gene sequences for the ‘trisimilis’ and ‘βGAL’ groups showed that both are most closely related to the Anaerococcus group. This work highlights the heterogeneous nature of a number of Peptostreptococcus species and hence the need for still further revision of the taxonomy of this important group of pathogens.

The hypothesis that Candida albicans isolate (CR1) from an HIV-infected individual induced apoptosis of macrophages was examined by optical microscopy, binding of annexin V-FITC and analyses of DNA degradation (TUNEL tests and agarose gel electrophoresis). Resident murine peritoneal macrophages co-incubated for 5–15 min with C. albicans CR1 bound annexin V, whereas macrophages incubated with either heat-inactivated strain CR1, C. albicans 577 (isolated from a patient with mucocutaneous candidiasis) or C. albicans FCF14 (a mutant that did not produce proteases and phospholipases) did not bind annexin for up to 2 h of observation. However, macrophages exposed to C. albicans CR1 did not present the pattern of DNA degradation typical of apoptosis. Macrophages became increasingly permeable to propidium iodide from 30 min to 2 h after their exposure to C. albicans CR1. Most of the phagocytosed C. albicans CR1 yeast cells switched to germ-tubes inside the macrophages after incubation for 1–2 h. These results show that macrophages exposed to C. albicans CR1 presented early signs of apoptosis but progressed to necrosis, and suggest that Candida strains that readily switch to germ-tubes inside those apoptotic cells might have a competitive advantage in vivo because released germ-tubes resist further attack by macrophages.