- Volume 53, Issue 10, 2004

The highly virulent bacterium Francisella tularensis is well adapted to the intracellular habitat but the mechanisms behind its intracellular survival have been elusive. Recently, it was shown that the bacterium is capable of escaping from the phagosome of human and mouse monocytic cells. Here it is shown that this escape is affected by gamma interferon (IFN-γ) treatment of mouse peritoneal exudate cells since in treated cells the proportion that escaped was significantly lower (80 %) than in untreated cells (97 %) as determined by transmission electron microscopy. By contrast, < 1 % of mutant bacteria lacking expression of a 23 kDa protein denoted IglC were able to escape from the phagosome. Infection with the Δ iglC strain complemented with the iglC gene resulted in 60 % of the bacteria escaping from the phagosome. Whereas IFN-γ treatment conferred a static effect on intracellular wild-type bacteria, the treatment had a bactericidal effect on the Δ iglC strain. The results show that the activation status of infected cells affects the escape of F. tularensis from the phagosome. An even more profound effect on this escape is related to expression of IglC by F. tularensis. Its absence rendered the mutant bacteria incapable of escaping from the phagosome and of multiplying intracellularly.

To understand the biological processes within host cells induced by VacA, isogenic strains of Helicobacter pylori (NCTC 11638 or 11638-ΔvacA) were used to stimulate gastric cancer cells SGC7901, and differentially expressed genes in host cells were identified using cDNA microarray technology. More than 300 genes were found to alter their mRNA expression at different time points, among which 68 were related to the cytoskeleton, 87 were associated with cell cycle, cell death and proliferation, IL8 expression was also found to be up-regulated. Cells co-cultured with broth-culture supernatant (BCS) of NCTC 11638 showed more alteration in microtubule cytoskeleton morphology, as observed by laser scanning confocal microscopy, and a lower apoptosis rate, detected by flow cytometry, compared with those co-cultured with BCS of 11638-ΔvacA. The supernatants of cells co-cultured with NCTC 11638 showed significantly higher IL8 expression than those co-cultured with 11638-ΔvacA. It is concluded that VacA disrupts cytoskeletal architecture by influencing the expression of cytoskeleton-associated genes. VacA breaks the balance between cell proliferation and cell death by inducing the maladjustment of genes related to cell cycle. VacA is also able to induce the inflammatory response.

Three specimens from 111 asymptomatic male partners of infertile couples attending the Department of Urology in Amiens, France, were examined by the PCR COBAS AMPLICOR test (Roche Molecular Diagnostics) for the presence of Chlamydia trachomatis. The specimens analysed were: first void urine (FVU), urine obtained after prostatic massage (UPM) and semen specimens. Serum from each patient was also obtained and analysed for the presence of IgG and IgA chlamydial antibodies by in-house microimmunofluorescence (MIF) and pELISA. C. trachomatis was detected by PCR in 5.4 % of FVU samples, 2.7 % of semen specimens and in 0.9 % of UPM samples. Two treatments for processing the samples (storage at −70 °C and heating to 95 °C) were routinely used before initial testing to reduce the effects of inhibitors of PCR. Despite these precautions, the PCR method revealed the presence of inhibitors in 7.3 % of semen specimens and 3.6 % of FVU samples. C. trachomatis was detected by PCR COBAS AMPLICOR in seven of 111 patients (6.3 %) and by serology in five of 111 patients (4.5 %). The detection of C. trachomatis in FVU, UPM and semen specimens can serve as a marker for the presence of this organism in the genital tract, and can be used as a reliable way of detecting asymptomatic carriers of infection.

Streptococcus agalactiae or group B streptococci (GBS) often colonize the gastrointestinal and urogenital tracts of women, who may transmit these organisms to their offspring during the birth process. Using PFGE analysis, the genetic diversity of GBS was studied for strains isolated from pregnant women and their newborn infants in a teaching hospital. A total of 48 different PFGE profiles were obtained from 123 strains, with one profile (S1) appearing to be predominant among both groups studied. There was good overall correlation between the profiles obtained for strains from mother–infant pairs and for strains isolated from different body sites in the same individual. Occasional discrepancies seen in related body sites and among mother–infant pairs suggest concurrent carriage of different strains in the same individual as well as the possibility of an environmental source of organism for the neonate. The overall results demonstrated that many variants of GBS strains occur in Malaysia.

An outbreak of bacteraemia in a haemodialysis unit where 65 episodes of infection involved 35 outpatients is reported. Burkholderia cepacia complex was the agent most frequently recovered from blood. Thirty-three environmental and clinical isolates of B. cepacia complex were characterized by whole-cell protein electrophoresis and recA-RFLP profile. Fourteen isolates were genomovar I and 16 isolates were not classifiable by their recA-RFLP pattern. Ribotyping, random amplification of polymorphic DNA (RAPD) and integron profile were used to explore the clonality of the isolates, and revealed multiple strain genotypes. Four ribotypes and RAPD types and three integron patterns were identified. The water supply was identified as the source of the outbreak, and inappropriate cleaning and a leak in the reverse osmosis tubing connection were the probable causes of contamination. B. cepacia complex was still recovered from blood of patients even after apparently adequate measures were taken and water quality standards were met, suggesting that higher standards for water quality should be adopted in haemodialysis units. The genomovars recovered here were distinct from those commonly reported for cystic fibrosis isolates.

The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20–28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1 : 10 000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1 : 160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood–brain barrier is discussed.

Resistance to antibiotics, especially clarithromycin, is the major cause of the failure to eradicate Helicobacter pylori. There are few studies in children concerning fluoroquinolone activity against H. pylori. Primary resistance to antibiotics including fluoroquinolones was studied in 55 H. pylori strains isolated from Japanese children. DNA sequences of the gyrA gene in fluoroquinolone-resistant strains were determined. Twelve strains (21.8 %) were resistant to clarithromycin and three (5.5 %) were resistant to both levofloxacin and ciprofloxacin. Out of 12 clarithromycin-resistant strains, 11 (91.7 %) were susceptible to levofloxacin and ciprofloxacin. Sequence analysis in three fluoroquinolone-resistant strains showed point mutations of the gyrA gene at G271A, G271T and A272G, indicating mutations of the codon Asp91 in the fluoroquinolone-resistance-determining region of the DNA gyrase. The results suggest that fluoroquinolones should be considered as an option for second- or third-line H. pylori eradication therapy in children.

The data presented show the ability of subinhibitory concentrations of plant essential oils to influence the production of enterotoxins A and B and α-toxin by Staphylococcus aureus. Subinhibitory concentrations of the oils of bay, clove, cinnamon, nutmeg and thyme had no significant effect on the overall quantity of extracellular protein produced. Haemolysis due to α-toxin was significantly reduced after culture with all five plant essential oils. This reduction was greatest with the oils of bay, cinnamon and clove. These three oils also significantly decreased the production of enterotoxin A; the oils of clove and cinnamon also significantly decreased the production of enterotoxin B.

β-Glucuronidase-positive (GUD+) Shiga toxin-producing Escherichia coli (STEC) O157 : H7 was isolated from both an asymptomatic woman and uncooked deer meat in her possession in Hokkaido, Japan. The phenotypic and genotypic characteristics of the two isolates were identical or closely related, indicating probable transmission of the deer isolate to the woman. Moreover, several other GUD+ STEC O157 : H7 strains investigated belonged to the distinct atypical GUD+ STEC O157 : H7 group that has been identified previously. This is the first report that deer can be a reservoir of GUD+ STEC O157 : H7 in Japan.