- Volume 47, Issue 12, 1998

This review compares the rates of detection of non-O157:H7 enterohaemorrhagic Escherichia coli (EHEC) with EHEC O157:H7 in outbreaks and sporadic cases of human disease by analysing Australian data and the world literature. Numerous outbreaks of disease have been attributed to EHEC O157:H7. in many studies, isolation rates of this organism have been low and attempts to seek other EHEC have not been made. Ease of isolation and identification of the O157:H7 serotype may have given the impression that this serotype was the sole organism responsible for the outbreaks. Careful review and analysis shows that serotypes other than O157:H7 also play an important role in human disease. Evidence is presented from several overseas outbreaks described in the literature, as well as from investigations of the Adelaide O111:H− outbreak, that suggests an association between severity of disease and multiple infecting serotypes. While not diminishing the role of the O157:H7/H− clone, this review indicates that other serotypes can be responsible for outbreaks as well as cases of sporadic human disease. the current focus on O157:H7 has major implications in terms of diagnosis, the food industry and human health.

The susceptibilities of 40 strains of various Mycoplasma species to 10 classes of antimicrobial agents were compared in vitro by a broth microdilution method. the strains tested comprised 20 strains of four AIDS-associated species – M. penetrans (1 strain), M. fermentas (5 strains), M. pirum (6 strains) and M. genitalium (8 strains) – nine strains of the urogenital tract species M. hominis and 11 strains of M. pneumoniae. the results demonstrated wide variation in the susceptibilities of the different Mycoplasma spp. to different classes of antimicrobial agent. All the mycoplasmas were susceptible or highly susceptible to the fluoroquinolones, with sparfloxacin the most active, and to the diterpine antibiotic tiamulin. M. pneumoniae and M. genitalium strains were also highly susceptible to the macrolides, particularly azithromycin and had similar antibiotic susceptibility patterns to most other antimicrobial agents. However, all strains of M. genitalium were resistant to streptomycin (MIC 250–>500 mg/L) whereas all M. pneumoniae isolates, except the MAC strain, were susceptible (MICs 1.25–12.5 mg/L). M. pirum isolates varied considerably in their susceptibility to macrolides (MIC range versus azithromycin 0.0025–>100 mg/L). M. fermentans strains were susceptible to the tetracyclines, lincosamides and mupirocin, but varied in susceptibility to aminoglycosides. Most M. hominis strains were susceptible to the tetracyclines and all were susceptible to clindamycin and mupirocin. M penetrans GTU 54 was susceptible to azithromycin, the tetracyclines and lincosamides as well as to the fluoroquinolones and tiamulin.

To study the aerobic and anaerobic microbiology of liver and spleen abscesses and correlate the results with predisposing factors, potential causes and routes of infection, clinical and laboratory data of 48 patients with liver abscesses and 29 with spleen abscesses treated between 1970 and 1990 were reviewed retrospectively. in liver abscesses, a total of 116 isolates (2.4 isolates/specimen) was obtained; 43 were aerobic and facultative species (0.9 isolates/specimen) and 73 were anaerobic species or micro-aerophilic streptococci (1.5 isolates/specimen). Aerobic bacteria only were isolated from 12 (25%) abscesses, anaerobic bacteria only from eight (17%), and mixed aerobic and anaerobic bacteria from 28 (58%); polymicrobial infection was present in 38 (79%). the predominant aerobic and facultative isolates were Escherichia coli (11 isolates), Streptococcus group D (8), Klebsiella pneumoniae (5) and Staphylococcus aureus (4). the predominant anaerobes were Peptostreptococcus spp. (18 isolates), Bacteroides spp. (13), Fusobacterium spp. (10), Clostridium spp. (10) and Prevotella spp. (4). There were 12 isolates of micro-aerophilic streptococci. S. aureus and β-haemolytic streptococci were associated with trauma; Streptococcus group D, K. pneumoniae and Clostridium spp. with biliary disease; and Bacteroides spp. and Clostridium spp. with colonic disease. in splenic abscesses, a total of 56 isolates (1.9 isolates/specimen) was obtained; 23 were aerobic and facultative species (0.8 isolates/specimen), 31 were anaerobic species or micro-aerophilic streptococci (1.1 isolates/specimen) and two were Candida albicans. Aerobic bacteria only were isolated from nine (31%) abscesses, anaerobic bacteria from eight (28%), mixed aerobic and anaerobic bacteria from 10 (34%) and C. albicans in two (7%); polymicrobial infection was present in 16 (55%). the predominant aerobic and facultative isolates were E. coli (5 isolates), Proteus mirabilis (3), Streptococcus group D (3), K. pneumoniae (3) and S. aureus (4). the predominant anaerobes were Peptostreptococcus spp. (11 isolates), Bacteroides spp. (5), Fusobacterium spp. (3) and Clostridium spp. (3). S. aureus, K. pneumoniae and Streptococcus group D were associated with endocarditis, E. coli with urinary tract and abdominal infection, Bacteroides spp. and Clostridium spp. with abdominal infection and Fusobacterium spp. with respiratory infection.

An appropriate animal model is essential to study Helicobacter pylori infection. the aim of this study was to investigate if H. pylori can colonise the guinea-pig stomach and whether the infection causes gastritis and a serological response similar to that observed in man. Guinea-pigs were infected either with fresh H. pylori isolates from human gastric biopsies or with a guinea-pig passaged strain. When the animals were killed, 3 and 7 weeks after inoculation, samples were taken for culture, histopathology and serology. H. pylori was cultured from 22 of 29 challenged animals. All culture-positive animals exhibited a specific immune response against H. pylori antigens in Western blotting and gastritis in histopathological examination. Antibody titres in enzyme immunoassay were elevated among animals challenged with H. pylori. the inflammatory response was graded as severe in most animals and consisted of both polymorphonuclear leucocytes and lymphocytes. Erosion of the gastric epithelium was found in infected animals. These results suggest that the guinea-pig is suitable for studying H. pylori-associated diseases. Moreover, guinea-pigs are probably more similar to man than any other small laboratory animal as regards gastric anatomy and physiology.

Fifteen randomly selected nasopharyngeal (NP) swab specimens (culture-negative for influenza A virus) were spiked with influenza A virus and the nucleic acids were extracted and subjected to PCR amplification with Thermus aquaticus (Taq) and T. thermophilus (Tth) DNA polymerases. Products of the expected size, and giving equivalent band intensities, were obtained from four specimens with both polymerases. Fox six specimens, less products were obtained with Taq DNA polymerase than with Tth DNA polymerase. Products were detected from five NPs only by PCR with Tth DNA polymerase. the transport medium and the calcium alginate swab fibre of the specimens were shown not to be the source of the inhibitors. the incorporation of 32P-dCTP into cDNA, and the yield of PCR products of cDNA made from control RNA template (purified from H2O spiked virus suspension) were decreased in the presence of inhibitory extracts, showing that both the reverse transcription (RT) and PCR steps in amplification with Taq DNA polymerase were sensitive to the inhibitors. in contrast, Tth DNA polymerase was more resistant to the inhibitors and viral nucleic acid from all the specimens examined could be amplified and detected in a single step by RT-PCR with Tth DNA polymerase.

The sequence of the non-typable Haemophilus influenzae (NTHi) P5 outer-membrane protein from a range of clinical isolates is presented and represents the first analysis of the heterogeneity in P5 from NTHi isolates from diverse anatomical sites. the basis of the previously observed inter-strain variation in the electrophoretic mobility is attributed to heterogeneity in three hypervariable regions. Alignment of the P5 sequences identified regions which are highly conserved and align with the transmembrane region predicted for the homologous Escherichia coli protein, OmpA. Variable regions correspond to surface-exposed loops, of which the first loop falls into subclasses. However, these subclasses fail to correlate with anatomical predisposition. Although P5 has been proposed as a fimbrial protein composed of coiled coils, both structural analysis by circular dichroism of purified P5 and computer analysis of the multiply aligned sequences predict a high proportion of β strand with no evidence of coiled coil structure. A detailed model of P5 is presented.

The ultrastructural features of cells of Calymmatobacterium granulomatis from monocyte co-cultures and tissue biopsy specimens were compared. in cultures the bacteria were mainly extracellular, i.e., not within membrane-bound vacuoles. the bacterial body was surrounded by a uniformly extensive homogeneous layer with a relatively high electron density. This layer varied considerably in tissue biopsy specimens, having either homogeneously electron-dense or delicate web-like structures with varying density and thickness. in tissue specimens the bacteria were located predominantly within vacuoles of varying sizes in the cytoplasm of the macrophages and, occasionally, extracellularly within the intercellular spaces of the stroma. the bacterial cytoplasm contained ribosomes scattered throughout with electron-dense granules located peripherally. the trilaminar cell-wall structure was typical of a gram-negative organism, comprising an outer membrane, a middle electron-opaque layer and an inner plasma membrane. Surface structures such as fimbriae, flagella and bacteriophages were not identified in specimens from either source.

The ability of the Lactobacillus acidophilus RC14 biosurfactant ‘surlactin’ to inhibit the initial adhesion of various uropathogenic bacteria and two yeast strains to silicone rubber was investigated in a parallel-plate flow chamber in filter-sterilised pooled human urine. A parallel-plate flow chamber with a silicone rubber bottom plate was filled with a 1.0 mg/ml biosurfactant solution for adsorption overnight (18 h). Subsequently, the adhesion of the bacterial or yeast cells from a urine suspension under low flow (shear rate 15 s−1) was followed in situ by automated image analysis. Control tests were with untreated silicone rubber. Initial deposition rates and numbers of adhering cells after 4 h of flow were determined. Surlactin layers caused a marked inhibition of the initial deposition rates and adhesion numbers after 4 h for the majority of the bacteria (11 of 15 strains tested) and this inhibition was particularly effective against Enterococcus faecalis, Escherichia coli and Staphylococcus epidermidis. Although the initial deposition rates of the two Candida albicans strains were reduced by c. 50% in comparison with the controls, the numbers of yeast cells adhering after 4 h were similar.

Capnocytophaga, one of the genera of oral bacteria, has been implicated in the pathogenesis of several diseases, including endocarditis, septicaemia and disorders of the oral cavity such as abscesses and periodontal disease. This study examined sonic extracts (SE) of Capnocytophaga strains for their ability to alter lymphocyte function. the SE of tested Capnocytophaga caused dose-dependent suppression of spleen cells in response to mitogen. This suppressive effect was heat-labile and sensitive to the proteolytic enzyme pronase E. the suppressive factor (SF) was purified from SE of C. ochrasea by a combination of ultrogel-AcA34, high-pressure liquid DEAE ion-exchange chromatography and hydroxyapatite columns, which revealed a single band of 14 kDa by SDS-PAGE. Rabbit anti-serum against the purified SF inhibited the immunosuppression induced by SE of C. ochracea with the recovery of lymphocyte proliferation.

Recent revision of the O serotyping scheme for Serratia marcescens has allowed the definitive serological identification of a collection of 511 epidemiologically distinct strains in terms of both lipopolysaccharide (O) antigens and capsular (K) antigens. High levels of typability were achieved, 88% and 91% respectively, with only 2% failing to type with either method. in most cases, non-typability was due to a lack of antigen, i.e., the strains produced only rough LPS or were acapsular, suggesting that typability would be little improved by the discovery of additional serotypes. the distribution of the 58 O:K serotypes was very uneven, with O14:K14 accounting for 30% of the 423 clinical strains in the collection, but only 5% of the 88 non-clinical, environmental strains. Thus, the prevalence of O14:K14 strains in hospitals is not reflected in the environment. Similar conclusions were valid for O27:K14, O21:K3 and O21:K14 strains, as well as those with rough lipopolysaccharide. Conversely, the proportions of O6:K3, O6:K14, O8:K14 or O28:K28 strains were significantly lower among the clinical collection than among their environmental counterparts (12% in total rather than 65%). This suggests that O14:K14 may have a selective advantage in colonising or infecting hospitalised patients and, therefore, that the O14 and K14 polysaccharides themselves may contribute towards the apparent pathogenicity of these serotypes.

Serratia marcescens serotypes O6:K14, O8:K14 and O28:K28 are common in the natural environment, but rare in hospitals. Serotypes O14:K14 and O27:K14 predominate among clinical strains, but not in the environment, suggesting that the latter serotypes may be more suited for survival in the clinical setting. Consequently, 469 epidemiologically distinct strains of S. marcescens were tested for various putative virulence factors and analysed for associations with serotype. the factors positively associated with serotype O14:K14 were agglutination of five different species of red blood cells and expression of type 1 fimbriae. These were found in 63% and 53% of O14:K14 strains, respectively, compared with 7% and 12% of the three ‘environmental serotypes’. Almost a quarter of the collection expressed the mannose-resistant haemagglutinin indicative of type 3 fimbriae, but this was not associated with any serotype. the production of DNAase, haemolysin, lipase, lecithinase, proteases and siderophores was almost universal and showed no serotype correlations. Almost half of the strains (46%) were resistant to serum and serotypes O27:K14 and O6:K14 were strongly associated with this characteristic. Serotype O27:K14 was also associated with higher proportions of antibiotic-resistant strains than other serotypes, but the same was not true of serotype O14:K14. All three ‘environmental serotypes’ were associated with low frequencies of antibiotic resistance; <12% were resistant to gentamicin, carbenicillin or piperacillin, or any combination of these three, compared with 20–25% of O14:K14 strains and > 42–51% of O27:K14 strains. Pigment production was strongly associated with serotype. None of the O14:K14 or O27:K14 strains produced prodigiosin, but frequencies for the three ‘environmental serotypes’ ranged from 31% of O28:K28 strains to 85% of O6:K14 strains. the results of this study suggest that the adherence capability of S. marcescens strains may play a role in the colonisation of hospital patients, while the production of prodigiosin is a marker of environmental origin.

The relative pathogenicity of Candida krusei and C. albicans was investigated by assessing their colonisation and infectivity of the Sprague-Dawley rat oral mucosa. During an initial 21-week period with intermittent oral inoculation, both Candida spp. demonstrated variable surface colonisation of the oral mucosa. After 3 days of oral inoculation, both yeast species were recovered from all animals. During the 21-week period the mean oral load of C. albicans in the control group of rats varied between (26–274) × 101 cfu/ml whereas the two test groups of rats carrying C. krusei CK9 and CK13 had a mean load of (2–10) × 101 cfu/ml. Although oral colonisation by C. albicans was greater than that of C. krusei, neither species induced candidal infection during this period. Subsequent immunosuppression of the rats by intramuscular cyclophosphamide (40 mg/kg body weight) initiated C. albicans infection of the dorsal tongue (around the conical papillae area) after 4 weeks, in all of three animals while similar lesions due to C. krusei were seen – albeit after 5–7 weeks – in three of eight animals. Characteristic histological changes of mucosal candidosis were discernible on the lingual mucosa of rats infected with both Candida spp. including parakeratosis, absence of a stratum granulosum, thickened rete ridges, micro-abscess formation and polymorph infiltration of the lingual epithelium. Although both species produced fungal hyphae that penetrated the epithelium up to the prickle cell layer, C. albicans hyphae tended to be relatively more profuse. Taken together these results substantiate, for the first time in an animal model, the clinical evidence that C. krusei, once considered an innocuous commensal, is capable of transforming into an invasive pathogen under conditions of immunosuppression.