- Volume 60, Issue 11, 2011

The pathogenesis of the primary stage of lymphogranuloma venereum (LGV) is poorly understood. There is no skin cell model and LGV pathogenesis studies are therefore carried out on cells of different origin. Moreover, such studies usually use reference strains, which may have evolved over the years in culture. In this study, a model was developed in which Chlamydia trachomatis enters and grows in human keratinocytes at 37 and 33 °C. Keratinocytes were infected with fresh clinical isolates and the three LGV reference strains L1, L2 and L3. Growth was monitored for 5 days post-infection using fluorescence microscopy and image analysis software. Chlamydial replication was quicker at 37 than at 33 °C, despite 33 °C being the temperature of human skin. The serovar L2 reference strain grew significantly faster than the other strains, although the fresh clinical isolates were also serovar L2. When grown in keratinocytes at 33 °C, the L2 and L3 reference strains produced much larger inclusions than the other strains tested. This model, which utilizes keratinocytes, better simulates the conditions present at the initial site of infection in LGV than previously published literature, making it a useful tool for future LGV pathogenesis studies. In addition, the results indicate that fresh clinical isolates should be included in LGV pathogenesis studies.

Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide, or 3-morpholinosydnonimine hydrochloride, which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50–100-fold. F. tularensis-infected J774 cells were incapable of secreting TNF-α in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-α secretion of J774 cells. Strong staining of nitrotyrosine was observed in SNAP-treated bacteria, and MS identified nitration of two ribosomal 50S proteins, a CBS domain pair protein and bacterioferritin. The results demonstrated that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in modification by nitration of several F. tularensis proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used in clinical microbiological laboratories to identify bacteria and fungi at a species level and to subtype them. The cfiA gene encoding the unique carbapenemases found in Bacteroides is restricted to division II Bacteroides fragilis strains. The aim of this study was to evaluate whether MALDI-TOF MS is suitable for differentiating B. fragilis strains which harbour the cfiA gene from those that do not. A well-defined collection of 40 B. fragilis isolates with known imipenem MICs (0.062–>32 mg l−1) were selected for this study. Twelve B. fragilis strains with known cfiA status, including NCTC 9343 (division I) and TAL3636 (division II), were measured by means of microflex LT MALDI-TOF MS and well-defined differences in mass spectra between the cfiA-positive and cfiA-negative strains were found in the interval 4000–5500 Da. A further 28 strains were selected for the blind measurements: 9 cfiA-positive clinical isolates with different imipenem MICs ranging between 0.06 and >32 mg l−1 (different expressions of the metallo-β-lactamase gene) were clearly separated from the 19 cfiA-negative isolates. The presence or absence of the selected peaks in all tested strains clearly differentiated the strains belonging to B. fragilis division I (cfiA-negative) or division II (cfiA-positive). These results suggest a realistic method for differentiating division II B. fragilis strains (harbouring the cfiA gene) and to determine them at a species level at the same time. Although not all cfiA-positive B. fragilis strains are resistant to carbapenems, they all have the possibility of becoming resistant to this group of antibiotics by acquisition of an appropriate IS element for full expression of the cfiA gene, leading to possible treatment failure.

In contrast to most modern pharmaceuticals, probiotics are used in many parts of the world with little or no research data on the complex system of interactions that each strain may elicit in the human body. Research on probiotics has recently become more significant, as probiotics have begun to be prescribed by clinicians as an alternative for some gut infections, especially when antibiotics are contraindicated. This study attempted to elucidate the inhibitory interaction between the Japanese probiotic strain Clostridium butyricum MIYAIRI 588 (CBM588) and the hospital pathogen Clostridium difficile, which is responsible for a large proportion of antibiotic-associated diarrhoea and colitis. CBM588 has previously shown effectiveness against C. difficile in vivo, and here it was found that the toxicity of C. difficile in in vitro co-culture with CBM588 was greatly decreased or absent. This was dependent on the inoculation ratio and was not accounted for by the small degree of growth and mRNA inhibition observed. CBM588 and its cell-free supernatant also had no effect on toxin already secreted into the culture medium, and culture of the two strains separated by a semi-permeable membrane resulted in loss of the inhibition. Therefore, it was concluded that the detoxification probably occurred by the inhibition of toxin protein production and that this required close proximity or contact between the two species. The low-pH conditions caused by organic acid secretion were also observed to have inhibitory effects on C. difficile growth, metabolism and toxicity.

This study examined the pattern of colonization of the neonatal gut by aerobic Gram-negative bacilli (GNB) and evaluated the association between gut colonization and sepsis in the developing world. This deserves attention because of the high incidence of sepsis and the differences in hygienic environments in developing countries compared with the developed world. The study was carried out on neonates in a tertiary-care government hospital. Serial gut samples were analysed (gastric aspirates and stool samples) for GNB. Blood samples of cases showing clinical signs of sepsis were also analysed for septic screening and culture positivity. Antibiograms, serotyping and PFGE were carried out to evaluate the relatedness of the gut and blood isolates. A diverse array of GNB was isolated from the gut of the neonates, Klebsiella pneumoniae being most common, followed by Escherichia coli. The rate of isolation of GNB was consistently higher in stool samples compared with gastric aspirate samples. Colonization was influenced by a stay in the neonatal intensive care unit and by the prolonged use of a feeding tube. GNB were the cause of sepsis in the majority of cases, with K. pneumoniae being the most frequently isolated GNB from the blood. Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae and Burkholderia cepacia were the other GNB recovered from the blood of the neonates. Neonates with GNB in the gut had a higher incidence of clinical sepsis than those without. In 50 % of cases, the genotypes of the organisms found in the blood were indistinguishable from their gut counterpart. These results show that the neonatal gut is colonized with a diverse array of GNB, and an association between gut colonization and neonatal sepsis was observed.

Coagulase-negative staphylococci (CoNS) have become increasingly recognized as important agents of nosocomial infection. One of the characteristics of CoNS is their resistance to multiple antimicrobial agents commonly used for the treatment of staphylococcal infections. CoNS strains (n = 745) isolated from a university teaching hospital in China between 2004 and 2009 were tested for antibiotic resistance. The antibiotics were placed into three categories based on resistance levels of the CoNS strains to these antibiotics: high resistance (resistance rate >70 %), including penicillin G, oxacillin and erythromycin; medium resistance (resistance rate between 30 and 70 %), including tetracycline, clindamycin, ciprofloxacin, trimethoprim/sulfamethoxazole and chloramphenicol; and low resistance (resistance rate <30 %), including rifampicin, ceftizoxime and gentamicin. We also found that the prevalence of strains non-susceptible to teicoplanin increased from 4.5 to 6.7 % between 2008 and 2009. A one-step vancomycin agar selection experiment and subsequent population analysis revealed potentially vancomycin-resistant subpopulations that have been selected from the teicoplanin-non-susceptible strains. Vigilant surveillance of nosocomial isolates of CoNS is needed to determine their resistance to glycopeptides.

Streptococcus suis, particularly serotype 2, is a pathogen of both pigs and humans associated with a wide range of diseases, including meningitis, septicaemia and endocarditis. Among the genes in the capsular polysaccharide biosynthesis (cps) locus, cps2J exists only in the serotype 2 and 1/2 strains; therefore, cps2J-positive strains are suspected to have capsules of serotype 2 or 1/2. Coagglutination using antiserotype 1 and antiserotype 2 sera and/or transmission electron microscopy analysis of 288 cps2J-positive isolates from pigs showed that 32 (100 %) isolates from meningitis were encapsulated, whereas 86 (34 %) of 256 isolates from endocarditis were unencapsulated, indicating that capsule loss often occurred in the isolates from endocarditis. To investigate the genetic backgrounds, we randomly selected 43 unencapsulated isolates and analysed their cps loci by PCR scanning. Among them, 8 and 10 isolates apparently had deletions and insertions, respectively, in cps loci. In addition, a representative unencapsulated isolate and an unencapsulated strain showed adherence to porcine and human platelets, a major virulence determinant for infective endocarditis, to a significantly greater extent than the encapsulated strains. Although the capsule is considered to be an important virulence factor in S. suis, these results suggest that loss of capsular production is beneficial to S. suis in the course of infective endocarditis.

Streptococcus mutans is one of the oral pathogens associated with infective endocarditis (IE). With respect to bacterial binding ability to the extracellular matrix, the Cnm protein, a cell surface collagen-binding adhesin of S. mutans, is known as one of the possible virulence factors with regard to IE. In this study, we aimed to determine the distribution of the cnm gene, which encodes Cnm, in a large number of clinical isolates of S. mutans from Thai subjects. Then, the cnm-positive strains were classified using a multilocus sequence typing (MLST) scheme, which we constructed previously. In addition, the data were analysed together with our previous MLST data of cnm-positive strains from Japan and Finland in order to evaluate the clonal relationship among S. mutans strains harbouring the cnm gene. The cnm gene was detected in 12.4 % of all 750 Thai isolates, and serotype f showed the highest rate of detection (54.5 %). According to the MLST data, two clonal complex groups were revealed as the important clones related to cnm-positive S. mutans from various origins of isolation. Moreover, the collagen-binding properties of S. mutans strains with the cnm gene were significantly greater than those of strains without the gene, although four cnm-negative strains classified into two sequence types (STs), ST110 and ST136, showed extremely high collagen-binding rates suggesting the presence of additional genes involved with collagen binding in these STs. Taken together, these results provided information on both epidemiological as well as evolutional aspects of S. mutans possessing the cnm gene.

We report a case of concurrent diphtheria and infectious mononucleosis in an 11-year-old Brazilian child. Two days after specific treatment for diphtheria was started the patient was discharged following clinical recovery. This case highlights the difficulties in the clinical diagnosis of diphtheria in partially immunized individuals, and for the management and control of diphtheria in developing countries.