Chronic Prosthetic Hip Infection Caused by a Small-Colony Variant of Escherichia coli

From two different specimens of a chronic prosthetic hip infection taken at an interval of 2 months a slow-growing gram-negative bacterium was isolated in pure culture. The strain grew with the typical features of a small-colony variant (SCV). 16S rRNA sequencing identified the bacterium as Escherichia coli. Biochemical characterization demonstrated multiple phenotypic alterations of a mutant carrying a defect in the heme biosynthetic pathway (Hem⁻): (i) catalase and nitrate reductase reactions were both negative, (ii) a negative benzidine reaction demonstrated the lack of heme-containing cytochromes, and (iii) growth stimulation under anaerobic conditions as well as gentamicin resistance indicated defective aerobic respiration. PCR and Southern hybridization demonstrated that the mutation of the SCV of E. coli was localized in the hemB gene and was most likely due to a deletion of the hemB gene. On blood agar plates revertants were recognized growing as normal-sized colonies between the dominant small colonies of the strain. Feeding experiments indicated that the revertants but not the small colonies were permeable for hemin. A strong antibody response against the infecting SCV of E. coli was found. To our knowledge, this is the first report of a Hem⁻ E. coli strain as the etiological agent of a chronic bacterial infection.     

The gene slyA of Salmonella typhimurium is required for destruction of M cells and intracellular survival but not for invasion or colonization of the murine small intestine.

Recent studies have shown that Salmonella typhimurium invades the M cells of Peyer's patches (PP) of the murine ileum. The slyA gene of S. typhimurium has also recently been reported to affect virulence of this pathogen in mice and survival in macrophages. We therefore compared the effect on PP tissue of four strains of S. typhimurium: a wild-type strain, two slyA insertion mutants, and a recombinant S. typhimurium derivative carrying multiple copies of slyA. Invasion assays performed 2 and 7 days after orogastric infection revealed significantly lower numbers of bacteria of the slyA mutants and of the SlyA-overproducing strain in PP than of the wild type. However, similar numbers of bacteria of all strains were still present in the lumen of the small intestine after these times. Invasion assays of PP tissue after 90-min ileal loop infection yielded comparable numbers of bacteria of all strains in PP. Transmission and scanning electron microscopy of PP tissue after ileal loop infection demonstrated that the two slyA mutants and the SlyA-overproducing strain were able to attach to, induce membrane ruffling of, and invade M cells in a way morphologically and quantitatively similar to that of the wild type. In contrast to the wild type, both slyA mutants and, to a lesser extent, the SlyA-overproducing strain were significantly impaired in their ability to destroy M cells and adjacent enterocytes. Taken together, these data suggest that slyA is involved in intracellular survival and M-cell cytotoxicity but not in the invasion process and that the amount of SlyA needs to be precisely balanced for virulence.     

Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures

Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients.     

Ambiguous Role of Interleukin-12 in Yersinia enterocolitica Infection in Susceptible and Resistant Mouse Strains

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-γ) production in NK and CD4⁺ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-γ-receptor-deficient (IFN-γR^−/−) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55^−/−) mice. IFN-γR^−/− mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-γR^+/+ mice. Administration of IL-12 was protective in IFN-γR^+/+ mice but not in IFN-γR^−/− mice, suggesting that IFN-γR-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor β (TGF-β). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-β. While administration of IL-12 alone increased TNF-α levels, administration of TGF-β or TGF-β plus IL-12 decreased both TNF-α and IFN-γ levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55^−/− mice, suggesting that TNF-α accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.     

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF- or anti-IFN- antibodies (serotherapy) prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF- treatment. The later treatment with anti-TNF- or anti-IFN- antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF- and IFN- is required for the restriction of a primary Y. enterocolitica infection in mice.     

Angiogenesis is an early event in the development of chemically induced skin tumors

In this study we have analyzed the vascular response induced in the two- stage carcinogenesis model in SENCAR mice. The role of angiogenesis has not been explored in this model, which is the paradigm of multistage carcinogenesis and a model for neoplastic lesions derived from exophytic premalignant lesions (e.g. colon carcinoma, bladder papilloma). We investigated if angiogenesis is involved in the formation of papillomas and in the progression from papilloma to carcinoma. To this end we analyzed the vasculature of normal and hyperplastic skin, focal epidermal hyperplasias that are precursors of papillomas, papillomas at different stages and squamous cell carcinomas. We also analyzed the vascularization of papillomas induced in two strains of mice that differ in their susceptibility to malignant progression. We show here that angiogenesis is turned on in the earliest stages of papilloma formation. In late stages, regardless of state of progression, the predominant response is an increase in the size of blood vessels. Thus, in the SENCAR mouse model, representative of exophytic tumors, the angiogenesis switch is a very early event, probably mechanistically related to the development of the primarily exophytic lesions. Therefore, the density of blood vessels cannot be used as a predictor of malignant progression in this model.      

Evaluation of the ICT Malaria Pf test for rapid post-mortem diagnosis of Plasmodium falciparum malaria in corpses examined for forensic reasons

To test the diagnostic value of a rapid and simple immunochromatographic test (ICT) based on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) for post-mortem examination, blood samples from 30 consecutive corpses were analysed by ICT and Giemsa-stained blood films. Compared to microscopy, ICT had 100% sensitivity and 100% specificity even after a considerable time had passed between the presumed time of death and testing or after prolonged storage of whole blood samples. The ICT yielded positive results for four travellers who had returned from Kenya and died from Pl. falciparum malaria. The ICT might therefore serve as an additional tool for rapid malaria diagnosis, especially in non-endemic countries where experience with microscopic malaria detection is limited. Received: 5 August 1999 / Received in revised form: 7 October 1999     

Prognostic impact of PD-1 and its ligands in renal cell carcinoma

Programmed death-1 receptor (PD-1) and programmed death-1 receptor-ligand (PD-L1) have been suggested to play a role as prognostic markers in clear cell renal cell carcinoma (ccRCC). The association between PD-L1 and prognosis seems to be more robust than for PD-1. Further, preliminary analyses suggest that neither PD-1 nor its ligands play a role as prognostic markers in non-clear cell RCC, while the prognostic role of PD-L2 in ccRCC as well as in non-clear cell RCC remains unclear.