- Volume 53, Issue 6, 2004

Increased recognition of parvovirus B19 (B19), an erythrovirus, as a significant human pathogen that causes fetal loss and severe disease in immunocompromised patients has resulted in intensive efforts to understand the pathogenesis of B19-related disease, to improve diagnostic strategy that is deployed to detect B19 infection and blood-product contamination and, finally, to elucidate the nature of the cellular immune response that is elicited by the virus in diverse patient cohorts. It is becoming clear that at least three related erythrovirus strains (B19, A6/K71 and V9) are circulating in the general population and that viral entry into target cells is mediated by an expanding range of cellular receptors, including P antigen and β-integrins. Persistent infection by B19 is emerging as a contributory factor in autoimmune disease, a hypothesis that is constrained by the detection of B19 in the skin of apparently healthy individuals. B19 infection during pregnancy may account for thousands of incidences of fetal loss per annum in Europe, North America and beyond, yet there is currently only minimal screening of pregnant women to assess serological status, and thereby risk of infection, upon becoming pregnant. Whilst major advances in diagnosis of B19 infection have taken place, including standardization of serological and DNA-based detection methodologies, blood donations that are targeted at high-risk groups are only beginning to be screened for B19 IgG and DNA as a means of minimizing exposure of at-risk patients to the virus. It is now firmly established that a Th1-mediated cellular immune response is mounted in immunocompetent individuals, a finding that should contribute to the development of an effective vaccine to prevent B19 infection in selected high-risk groups, including sickle-cell anaemics.

Aeromonas veronii is an opportunistic human pathogen that causes diarrhoea and extraintestinal infections, such as wound infection and septicaemia. An A. veronii protease (AVP) from a biovar sobria strain, AeG1, was partially purified and characterized. Mature AVP hydrolysed casein but not elastin, and protease activity was inhibited by metalloprotease inhibitors. Nucleotide sequence analysis showed that AVP belongs to the thermolysin family of proteases. An AVP-deficient mutant was constructed to investigate the role of AVP in aerolysin activation. Western blot analysis using anti-aerolysin antisera revealed that proaerolysin (52 kDa) in the AVP-deficient mutant was not completely activated to mature aerolysin (47 kDa) as seen in the wild-type strain. The AVP-deficient mutant showed lower cytotoxic and haemolytic activities than wild type. AVP and proaerolysin had no haemolytic activity; however, activity appeared after incubating both proteins. Taken together, these results suggested that AVP plays an indirect role in virulence through activating aerolysin, which is an essential step for cytotoxic activity.

Therapeutic activity against oral candidiasis of orally administered bovine lactoferrin (LF), a multifunctional milk protein, was shown in a previous report using an immunosuppressed murine model. In the present study, the influence of orally administered LF on immune responses relevant to this therapeutic effect was examined. Because mice were immunosuppressed with prednisolone 1 day before and 3 days after the infection with Candida, the numbers of peripheral blood leukocytes (PBL) and cervical lymph node (CLN) cells were reduced. LF feeding prevented the reduction in the numbers of PBL on day 1 and CLN cells on days 1, 5 and 6 in the Candida-infected mice. The number of CLN cells of individual mice on days 5 and 6 was inversely correlated with the Candida c.f.u. in the oral cavity. Increased production of IFN-γ and TNF-α by CLN cells stimulated with heat-killed Candida albicans on day 6 was observed in LF-treated mice compared with non-treated mice. Concanavalin A (ConA)-stimulated CLN cells from LF-treated mice also showed a significant increase in the production of IFN-γ and IL12 on day 5 and a tendency for increased production of IFN-γ and TNF-α on day 6. The levels of cytokine production by ConA-stimulated CLN cells on day 6 were inversely correlated with the Candida c.f.u. in the oral cavity. In conclusion, the alleviation of oral candidiasis by LF feeding in this model may correlate with the enhancement of the number of leukocytes and their cytokine responses in regional lymph nodes against Candida infection.

Analysis of growth characteristics, multilocus enzyme electrophoresis, restriction digest pattern (RDP) typing and multilocus sequence typing have identified clonotypes of serotype III group B Streptococcus agalactiae (GBS) associated with invasive infection in neonates. This study sought to unify phenotypic and genotypic classifications of type III GBS strains associated with increased virulence in newborns. High-virulence clonotype (HVC) strains possessed the translation initiation factor 2 (infB) C allele, found in RDP type III-3 strains, and hybridized with the RDP type III-3-specific probe AA3.6, whereas non-HVC strains shared the infB A allele and genomic DNA from these strains did not hybridize with the AA3.6 probe. The characteristic growth lag of HVC GBS at 40 °C has been attributed to the presence of a heat-labile fructose-1,6-bisphosphate aldolase (Fba) enzyme in these strains. The deduced amino acid sequence of fba genes of both HVC and non-HVC strains, however, were identical. HVC and RDP type III-3 represent the same genetically related group of bacteria. The characteristic growth differences of virulent strains of type III GBS, however, are not directly attributable to differences in fba.

A comprehensive study was performed to examine incidence, species distribution, drugs sensitivity, virulence genes and molecular fingerprints of Aeromonas species isolated from patients with acute diarrhoea over a period of 2 years in Kolkata, India. Following the Aerokey II scheme, more than 95 % of strains were identified to species level. Seven different species were encountered in this study, with Aeromonas caviae being dominant, followed by Aeromonas hydrophila and Aeromonas veronii biovar sobria. Thirty different serotypes were encountered, with O16, O83 and O85 being dominant, but no serotype was associated specifically with a single species. The majority of Aeromonas strains exhibited multidrug resistance. The alt and act genes, which encode heat-labile cytotonic and cytotoxic enterotoxins, were respectively found in 71.9 and 20.1 % of strains examined. Only 2.4 % of strains carried the heat-stable cytotonic enterotoxin (ast) gene. The hlyA gene was found in 28 % of Aeromonas strains. With few exceptions, genomic diversity of Aeromonas strains belonging to the same serotype was observed by random amplification of polymorphic DNA PCR and ribotyping. Different species of Aeromonas and different clones of Aeromonas species seem to be associated with hospitalized cases of diarrhoea in Kolkata, India.

Staphylococcus epidermidis has become one of the most important causes of nosocomial infections in recent years. The staphylococcal accessory gene regulator (agr) is the most important locus responsible for the regulation of virulence factors, and it has been shown to be polymorphic. The aim of this study was to investigate the agr locus and its genetic polymorphisms in different Chinese S. epidermidis isolates and the relationship between genetic polymorphisms and pathogenicity. Specific PCR was used to amplify the different agr groups. Results were confirmed by restriction enzyme digestion and sequence analysis. agr mutations were detected and three agr groups of S. epidermidis were determined. Of the isolates, 12 % were pathogenic S. epidermidis and 17 % had naturally occurring agr mutations (P > 0.05). Pathogenic S. epidermidis isolates comprised 68.2 % agr group I, 19.3 % group II and 12.5 % group III, while isolates from healthy controls comprised 39 % agr group I, 51 % group II and 10 % group III (P < 0.01). The percentages of agr locus mutants and the three agr groups in different hospitals showed no significant differences (P > 0.05). The percentage of agr group I S. epidermidis isolated from catheters and blood was higher than that isolated from the other clinical specimens. This is the first study to investigate the genetic polymorphism of agr in S. epidermidis in China. The mean percentage of agr locus mutants was 14.9 % (12 % in clinical specimens; 17.7 % in controls). Genetic polymorphism of agr in S. epidermidis was linked to its pathogenicity; group I was associated with pathogenicity, while most isolates from healthy subjects were group II. The mechanism is to be investigated.

Terminal RFLP (T-RFLP) analysis was used to investigate changes in the oral microbiota in saliva and subgingival plaque samples from one patient with aggressive periodontitis (subject A) and two patients with chronic periodontitis (subjects B and C) before and 3 months after periodontal treatment. Substantial changes in the T-RFLP patterns of subgingival plaque samples of subjects B and C were noted after 3 months of improved oral hygiene and full-mouth supra- and subgingival scaling and root planing. However, there was little change in the subgingival microbiota of subject A. Although the proportions of terminal restriction fragments (T-RFs) larger than 1000 bp were notable in the T-RFLP patterns generated after digestion with HhaI of the samples from two subjects before treatment (subject B, 35.5 %; subject C, 29.6 %), the proportions of these T-RFs were significantly reduced or not detected after treatment (subject B, none; subject C, 4.1 %). Real-time PCR showed a significant change in the proportions of target bacteria in subgingival plaque samples of subject B. After 3 months, the Porphyromonas gingivalis population was markedly reduced (3.1 × 10−3 %), whereas the proportion of Porphyromonas gingivalis before treatment was 7.6 %. The proportions of Tannerella forsythensis, Treponema denticola and Treponema socranskii were also markedly diminished after treatment. Similarly, the proportion of the T-RF presumed to represent Porphyromonas gingivalis was 5.9 % and became undetectable after 3 months. Analysis of 16S rRNA gene clone libraries from subgingival plaque samples of subject B before and after treatment showed a notable change in the subgingival microbiota. These results were in agreement with the T-RFLP analysis data and showed that the T-RFs larger than 1000 bp represent Peptostreptococcus species. Our results indicate that T-RFLP analysis is useful for evaluation of the effects of medical treatment of periodontitis.

Ruminants are a major reservoir of enterohaemorrhagic Escherichia coli (EHEC), which cause acute gastroenteritis in humans with potentially life-threatening sequelae. The mechanisms underlying EHEC persistence in ruminant hosts are poorly understood. EHEC produce several cytotoxins that inhibit the proliferation of bovine lymphocytes in vitro and influence EHEC persistence in calves, suggesting that bacterial suppression of mucosal inflammation may be important in vivo. In order to address this hypothesis, intraepithelial lymphocytes (IEL) obtained from ligated intestinal loops of five 9–14 day old calves were characterized 12 h after inoculation with E. coli strains. Loops were inoculated with an EHEC O103 : H2 strain, an isogenic Δstx1 mutant incapable of producing Shiga toxin 1 (Stx1) and a porcine non-pathogenic E. coli strain. The IEL mainly comprised activated CD2+ CD3+ CD6+ CD8α+ T cells and resembled IEL obtained from the intestinal mucosa of orally challenged calves. Forty per cent of all IEL were potentially sensitive to Stx1 in that they expressed the receptor for Stx1. Nevertheless, analysis of IEL from inoculated loops failed to detect a significant effect of the different E. coli strains on proliferative capacity, natural killer cell activity or the cytokine mRNA profile. However, the EHEC wild-type strain reduced the percentage of CD8α+ T cells in the ileal mucosa compared with loops inoculated with the Δstx1 mutant. This shift in IEL composition was not associated with inhibition of IEL proliferation in situ, since the majority of the IEL from all loops were in the G0/G1 phase of the cell cycle. These studies indicate that the ligated ileal loop model will be a useful tool to dissect the mechanisms underlying suppression of mucosal inflammation by EHEC in the reservoir host.

Clostridial myonecrosis or gas gangrene occurs most frequently in contaminated wounds following trauma or surgery. It is caused by a wide variety of Clostridium species, the most common being Clostridium perfringens. Spontaneous, non-traumatic clostridial myonecrosis is uncommon and is usually associated with gastrointestinal and haematological malignancy, diabetes mellitus and peripheral vascular disease. The case of a previously healthy 16-year-old boy with acute onset of gastrointestinal symptoms, who died of bacterial sepsis without apparent preceding trauma, is presented here. Clostridium fallax was identified as the most probable causative agent. As far as is known, this is the first report of fatal sepsis in humans due to C. fallax, which has been described only rarely as a cause of gas oedema in animals.