- Volume 56, Issue 7, 2007

Acute hypercytokinaemia represents an imbalance of pro-inflammatory and anti-inflammatory cytokines, and is believed to be responsible for the development of acute respiratory distress syndrome and multiple organ failure in severe cases of avian (H5N1) influenza. Although neuraminidase inhibitors are effective in treating avian influenza, especially if given within 48 h of infection, it is harder to prevent the resultant hypercytokinaemia from developing if the patient does not seek timely medical assistance. Steroids have been used for many decades in a wide variety of inflammatory conditions in which hypercytokinaemia plays a role, such as sepsis and viral infections, including severe acquired respiratory syndromes and avian influenza. However, to date, the results have been mixed. Part of the reason for the discrepancies might be the lack of understanding that low doses are required to prevent mortality in cases of adrenal insufficiency. Adrenal insufficiency, as defined in the sepsis/shock literature, is a plasma cortisol rise of at least 9 μg dl−1 following a 250 μg dose of adrenocorticotropin hormone (ACTH), or reaching a plasma cortisol concentration of >25 μg dl−1 following a 1–2 μg dose of ACTH. In addition, in the case of hypercytokinaemia induced by potent viruses, such as H5N1, systemic inflammation-induced, acquired glucocorticoid resistance is likely to be present. Adrenal insufficiency can be overcome, however, with prolonged (7–10 or more days) supraphysiological steroid treatment at a sufficiently high dose to address the excess activation of NF-κB, but low enough to avoid immune suppression. This is a much lower dose than has been typically used to treat avian influenza patients. Although steroids cannot be used as a monotherapy in the treatment of avian influenza, there might be a potential role for their use as an adjunct treatment to antiviral therapy if appropriate dosages can be determined. In this paper, likely mechanisms of adrenal insufficiency are discussed, drawing from a broad background of literature sources.

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pb18) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pb18a by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein β-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

The Gram-negative intracellular bacteria Rickettsia conorii and Rickettsia typhi are the aetiological agents of Mediterranean spotted fever and endemic typhus, respectively, in humans. Infection of endothelial cells (ECs) lining vessel walls, and the resultant vascular inflammation and haemostatic alterations are salient pathogenetic features of both of these rickettsial diseases. An important consideration, however, is that dramatic differences in the intracellular motility and accumulation patterns for spotted fever versus typhus group rickettsiae have been documented, suggesting the possibility of unique and potentially different interactions with host cells. This study characterized and compared R. conorii- and R. typhi-mediated effects on cultured human ECs. The DNA-binding activity of nuclear transcription factor-κB (NF-κB) and the phosphorylation status of stress-activated p38 kinase were determined as indicators of NF-κB and p38 activation. R. conorii infection resulted in a biphasic activation of NF-κB, with an early increase in DNA-binding activity at 3 h, followed by a later peak at 24 h. The activated NF-κB species were composed mainly of RelA p65–p50 heterodimers and p50 homodimers. R. typhi infection of ECs resulted in only early activation of NF-κB at 3 h, composed primarily of p65–p50 heterodimers. Whilst R. conorii infection induced increased phosphorylation of p38 kinase (threefold mean induction) with the maximal response at 3 h, a considerably less-intense response peaking at about 6 h post-infection was found with R. typhi. Furthermore, mRNA expression of the chemokines interleukin (IL)-8 and monocyte chemoattractant protein-1 in ECs infected with either Rickettsia species was higher than the corresponding controls, but there were distinct differences in the secretion patterns for IL-8, suggesting the possibility of involvement of post-transcriptional control mechanisms or differences in the release from intracellular storage sites. Thus, the intensity and kinetics of host-cell responses triggered by spotted fever and typhus species exhibit distinct variations that could subsequently lead to differences in the extent of endothelial activation and inflammation and serve as important determinants of pathogenesis.

This study evaluated a novel peptide nucleic acid (PNA) probe targeting a region of the 23S rRNA gene of Klebsiella pneumoniae by fluorescence in situ hybridization (FISH). Analytical performance was determined using 39 reference strains and other well-characterized strains of Klebsiella spp. and Enterobacter aerogenes. The probe was found to be specific for the K. pneumoniae complex (K. pneumoniae including Klebsiella ozaenae and Klebsiella variicola). The diagnostic accuracy was evaluated with 264 blood cultures containing Gram-negative rods. Using conventional identification as the reference, performance specifications were as follows: sensitivity 98.8 %, specificity 99.5 %, positive predictive value 98.8 % and negative predictive value 99.5 %. Discrepancies were resolved by PNA FISH retest and phenotypic tests. In conclusion, the K. pneumoniae probe provided an accurate diagnosis within 3 h and may supplement other methods for direct identification of Gram-negative bacteria.

Clostridium difficile-associated diarrhoea (CDAD) is the most common hospital-acquired diarrhoea, and is a major type of gastroenteritis infection in nursing homes and facilities for the elderly. In this study the antimicrobial activity of the two-component lantibiotic, lacticin 3147, against a range of genetically distinct C. difficile isolates was studied. The bacteriocin exhibited an MIC50 of 3.6 μg ml−1 for 10 genetically distinct C. difficile strains isolated from healthy subjects, inflammatory bowel disease patients and culture collection strains. In time-kill studies, 106 c.f.u. ml−1 C. difficile ATCC 42593 and CDAD isolate DPC 6220 were killed within 120 or 20 min incubation, respectively, at a concentration of 6 μg lacticin ml−1. Interestingly, addition of lacticin 3147 to exponentially growing cells of C. difficile ATCC 43593 caused rapid lysis of the cells after an initial lag phase, as measured by the concomitant release of the intracellular enzyme, acetate kinase. The addition of a food-grade, milk-based lacticin containing powder to faecal fermentation demonstrated that lacticin is effective in completely eliminating 106 c.f.u. C. difficile ml−1 from a model faecal environment within 30 min when present at concentrations as low as 18 μg ml−1. While other culturable microflora such as total anaerobes, bacteroides, total non-spore-forming anaerobes and total Gram-negative anaerobes were unaffected, populations of lactobacilli and bifidobacteria were reduced by 3 log cycles at bacteriocin levels sufficient to eliminate over 106 C. difficile. In light of these findings, the potential of lacticin 3147 for treatment of CDAD is discussed.

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 18S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n=33 sets) and the results were 100 % concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from ∼72 to <3 h, and consequently allows optimization of the antifungal regimen at an earlier stage.

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis. The complexity of mechanisms involved in dermatophytic infections makes relevant in vivo studies particularly difficult to perform. The aim of this study was to develop a new in vitro model of M. canis dermatophytosis using feline fetal keratinocytes in reconstructed interfollicular epidermis, and to investigate its relevance in studying the host–pathogen relationship. Histological analysis of reconstructed interfollicular feline epidermis (RFE) revealed a fully differentiated epidermis. A proliferation assay showed replicating cells only in the basal layer, indicating that RFE is a well-stratified living tissue, leading to the formation of a horny layer. Histopathological analysis of RFE infected by M. canis arthroconidia revealed that the fungus invades the stratum corneum and produces SUB3, a keratinase implicated in the infectious process. In view of these results, an M. canis dermatophytosis model on RFE seems to be a useful tool to investigate mechanisms involved in natural M. canis feline infections.

Oral epithelia are constantly exposed to non-pathogenic (commensal) bacteria, but generally remain healthy and uninflamed. Fusobacterium nucleatum, an oral commensal bacterium, strongly induces human β-defensin-2 (hBD2), an antimicrobial and immunomodulatory peptide, in gingival epithelial cells (GECs). hBD2 is also expressed in normal oral tissue leading to the hypothesis that oral epithelia are in an activated state with respect to innate immune responses under normal in vivo conditions. In order to test this hypothesis, global gene expression was evaluated in GECs in response to stimulation by an F. nucleatum cell wall (FnCW) preparation and to hBD2 peptide. FnCW treatment altered 829 genes, while hBD2 altered 209 genes (P<0.005, ANOVA). Many induced genes were associated with the gene ontology categories of immune responses and defence responses. Consistent with the hypothesis, similar responses were activated by commensal bacteria and hBD2. These responses included up-regulation of common antimicrobial effectors and chemokines, and down-regulation of proliferation markers. In addition, FnCW up-regulated multiple protease inhibitors, and suppressed NF-κB function and the ubiquitin/proteasome system. These global changes may protect the tissue from inflammatory damage. Both FnCW and hBD2 also up-regulated genes that may enhance the epithelial barrier. The findings suggest that both commensal bacteria and hBD2 activate protective responses of GECs and play an important role in immune modulation in the oral cavity.

Streptococcus mutans, considered to be a pathogen for dental caries, is known to cause bacteraemia and infective endocarditis. Herein, an unusual case of repeated bacteraemia caused by S. mutans identified in a 71-year-old male is described. The patient visited Itami City Hospital with the major complaint of a fever, and a subsequent clinical examination led to a diagnosis of possible infective endocarditis without specific vegetation formation around the heart valve. A bacteriological examination of blood taken at the first visit showed the presence of S. mutans. Antimicrobial treatment was provided, which successfully eliminated the pathogenic bacteria from the blood. However, the patient returned and was hospitalized twice more with a recurrent fever, and S. mutans was again detected. Analyses of the biological properties of the S. mutans isolates showed that they possessed cariogenic properties and had a low susceptibility to phagocytosis by human polymorphonuclear leukocytes. Since the patient had Sjögren's syndrome, in which a reduction of saliva secretion is a characteristic feature, a great number of dental caries lesions were identified. The findings indicated that S. mutans present in those dental caries lesions caused repeated bacteraemia in this case.