- Volume 54, Issue 3, 2005

Cytolethal distending toxin (CDT) is a novel bacterial toxin that is produced by a variety of pathogenic bacteria. The mechanism of cytotoxicity of CDT is unique in that it enters into eukaryotic cells and breaks double-stranded DNA. This initiates the cell's own DNA damage-response mechanisms, resulting in the arrest of the cell cycle at the G2/M boundary. Affected cells enlarge until they finally undergo programmed cell death. This review encompasses recent work on CDT and focuses on the molecular mechanisms used by this toxin to block cell-cycle progression, the benefit to the bacterium of possession of this toxin and the clinical relevance of intoxication.

Human listeriosis is generally caused by consumption of ready-to-eat (RTE) foods that are stored for extended periods of time at refrigeration temperatures and that permit the growth of the causative agent, Listeria monocytogenes. Food-consumption patterns in China are undergoing rapid changes and more regular consumption of refrigerated-storage RTE foods may increase the risk of human listeriosis. In total, 40 L. monocytogenes isolates were obtained from food (n = 32) and sewage (n = 6) samples and from two human listeriosis cases that occurred in China. All isolates were characterized into molecular subtypes by DNA sequencing of the 597 bp 3′-terminal region of the virulence gene actA. Sequence data were used to classify the 40 Chinese L. monocytogenes isolates into sequence types and phylogenetic lineages, and to compare the sequence types of the Chinese isolates with those of isolates from the USA. Phylogenetic analyses showed that the Chinese isolates could be separated into two genetic lineages, with 14 and 26 isolates belonging to lineages I and II, respectively. Lineage II could be subdivided further into two clusters, IIA and IIB. Lineages I and II were identical to the two lineages described previously among US L. monocytogenes isolates. In total, 14 actA sequence types could be differentiated among the 40 Chinese L. monocytogenes isolates; two specific actA sequence types were found among both Chinese and US isolates. Isolates belonging to lineage II showed a significantly lower ability to invade and multiply within human intestinal epithelial Caco-2 cells than lineage I isolates. It was concluded that DNA sequencing of the 3′-terminal region of actA appears to be an effective method for rapid subtype and lineage classification of L. monocytogenes. As strains belonging to lineages I and II have previously been found among isolates from Europe and North America, these results show that L. monocytogenes clonal groups found in China are very similar to those found in the USA. Many L. monocytogenes strains may thus represent globally distributed clonal types. Together with the first description of two human listeriosis cases in China, these data indicate that changes in food-distribution and -consumption patterns in China and other countries will probably lead to the emergence of human listeriosis as a food-safety issue, as virulent strains of this pathogen appear to be present in the Chinese food supply.

Interleukin 8 (IL8) is usually produced in both epithelial and monocytic cells during bacterial infections, causing inflammation. Helicobacter pylori induces production of IL8 from gastric epithelial cells via its cag pathogenicity island (cag PAI) system, LPS and outer-membrane protein. In some bacteria, heat-shock protein 60 (HSP60) also elicits a strong pro-inflammatory response in cells of the innate immune system. Three recombinant H. pylori HSP60 (rHSP60) proteins of different sizes were produced and one of these was used to raise two monoclonal antibodies (2E7 and 7B5). IL8 production was found to be induced in cultured monocytic cells treated with H. pylori cells or rHSP60 proteins, as measured by ELISA, and the amount of IL8 produced was dose-dependent. Pre-incubation of H. pylori cells or rHSP60 preparations with the antibody 2E7 significantly inhibited IL8 production from monocytic cells. These results indicated that HSP60 is closely associated with IL8 production in monocytic cells.

Mycobacterium leprae cannot be cultured, so ascertaining viability of the organism remains a major obstacle, impeding many avenues of investigation. This study tested a two-colour, Syto9 and propidium iodide, fluorescence assay, which scores for membrane damage in individual bacilli, to determine if a rapid direct-count viability-staining technique can be reliably applied to M. leprae. A variety of experimental conditions were employed to validate this technique. This technique was also used to correlate the viability of M. leprae with the course of athymic mouse foot pad infection to optimize the provision of viable M. leprae as a research reagent. The data show that in untreated suspensions of M. leprae there is a good correlation between the metabolic activity of leprosy bacilli and their membrane damage. Fixation of M. leprae with ethanol, paraformaldehyde and gluteraldehyde completely suppressed their metabolic activity but showed little effect on their membrane integrity. The present study also showed that the metabolic activity of M. leprae declines more than the extent of membrane damage at 37 °C within 72 h, but that they are not significantly affected at 33 °C. Irradiation at 104 Gy showed high numbers of dead bacilli by the staining method. The results show that the reliability of metabolic-activity data as well as viability-staining data is dependent on the method by which M. leprae is killed. This staining method helped us predict reliably that the smaller M. leprae-infected athymic mouse foot pad seen early in infection, between 4 and 5 months, yields markedly better quality leprosy bacilli than older, larger foot pad infections, as defined by their metabolic activity and membrane integrity.

Multi-drug-resistant tuberculosis (MDR-TB) is a major public-health problem, because treatment is complicated and patients remain infectious for months or years, despite receiving the best available therapy. To gain better understanding of MDR-TB, a retrospective study was initiated to determine the level of drug resistance among patients in a chest-disease institute in India. Two hundred and sixty-three isolates from treatment-failure pulmonary tuberculosis patients (20–70 years) were studied. Drug-sensitivity testing was performed by the modified-proportion method. First- and second-line drugs, along with two quinolone drugs (ofloxacin and ciprofloxacin), were tested. Patients included in this study did not improve with therapy; however, 151 isolates (57.5 %) were susceptible to all four first-line antituberculosis drugs. This study reports low resistance to fluoroquinolones among the strains present in these patients.

It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-α) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-α levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-α antibody. However, exogenous TNF-α could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-α antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-α may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans-induced apoptosis of human immune cells may be important in terms of initiation and progression of periodontal diseases.

Dynamics of anti-M antibody response following intranasal infection with group A Streptococcus (GAS) M-18 were investigated in a Swiss albino mouse model. Mice arranged in three groups were inoculated intranasally with 2.0 × 107 c.f.u. ml−1 of GAS M-18 on 1, 2 alternate and 3 alternate days. Plasma collected from the retro-orbital plexus was tested for antibodies by an in-house indirect ELISA. The antibody titres of the plasma samples varied from 1 : 8 to 1 : 1024 in the 1 day dose, from 1 : 4 to 1 : 256 in the 2 day dose and from 1 : 4 to 1 : 128 in the 3 day dose. Peak titres were seen on day 42 or 56 and in all cases the titres had declined by day 84. Swiss albino mouse can thus serve as a useful animal model to study different aspects of type-specific anti-M immune responses against GAS disease when designing candidate streptococcal vaccines.

Eradication treatment for Helicobacter pylori is known to cause mild but relatively frequent adverse effects. Some adverse effects such as diarrhoea and soft stools are related to disruption of the composition of the intestinal microflora. This study investigated the microfloral changes resulting from administration of an eradication regimen using proton pump inhibitor (PPI), amoxycillin and clarithromycin. Twenty-eight laboratory-bred Japanese macaques either were administered eradication treatment by this regimen for 7 days or received no medication. Faecal samples were collected for analysis on days 0, 8 and 15, and both aerobic and anaerobic cultures were performed. Among aerobic bacteria, Streptococcus had significantly decreased by day 8, while Enterococcus and Enterobacteriaceae had significantly increased. However, the total number of aerobic bacteria was not significantly decreased from pretreatment levels 1 day after completion of treatment. The number of anaerobic bacteria did not change significantly by day 8. However, the number of Lactobacillus and the detection rate of Bifidobacterium, Peptostreptococcus and Veillonella significantly decreased by day 8, although the number of Bifidobacterium, Peptostreptococcus and Veillonella had almost recovered up to the pretreatment levels 1 week after completion of treatment (day 15). These results suggest that the alterations in the composition of the intestinal microflora caused by the antimicrobial regimen that excludes metronidazole are different from those caused by the regimen including this drug. However, the alterations in bacterial microflora had almost reversed 7 days after completion of treatment in these macaques, which supports clinical findings that diarrhoea or soft stools in humans resolve relatively quickly after a similar treatment.