The virulence-associated V Ag (LcrV) of pathogenic Yersinia species is part of the translocation apparatus, required to deliver antihost effector proteins (Yersinia outer proteins) into host cells. An orthologous protein (denoted as PcrV) has also been identified in the ExoS regulon of Pseudomonas aeruginosa. Additionally, it is known that LcrV is released by yersiniae into the environment and that LcrV causes an immunosuppressive effect when injected into mice. In this study, we demonstrate for the first time that rLcrV, but not PcrV, is capable of suppressing TNF-α production in zymosan A-stimulated mouse macrophages and the human monocytic Mono-Mac-6 cell line. The underlying mechanism of TNF-α suppression could be assigned to LcrV-mediated IL (IL)-10 production, because 1) LcrV induces IL-10 release in macrophages, 2) anti-IL-10 Ab treatment completely abrogated TNF-α suppression, and 3) TNF-α suppression was absent in LcrV-treated macrophages of IL-10-deficient (IL-10−/−) mice. The relevance of LcrV-mediated immunosuppression for the pathogenicity of yersiniae became evident by experimental infection of mice; in contrast to wild-type mice, IL-10−/− mice were highly resistant against Yersinia infection, as shown by lower bacterial load in spleen and liver, absent abscess formation in these organs, and survival.

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia,Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 × 105 CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, andP. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.

The V antigen is a 37-kDa secreted polypeptide encoded on the 70-kb virulence plasmid of pathogenic Yersinia spp. Besides having regulatory functions, it is known to be a virulence factor and a protective antigen. DNA sequencing of the most common serotypes of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis revealed that two evolutionary distinct types of V antigen exist in Yersinia spp. One type is represented by Y. enterocolitica serotype O8 strains WA, WA-314, and NCTC 10938 (designated LcrV-YenO8); the other type comprises Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serotypes O3, O9, and O5,27 (LcrV-Yps). A hypervariable region between amino acids 225 and 232 represents the main difference between the two types. By raising monospecific antisera against both types of V antigen (anti-rVO8 and anti-rVO3), we were able to demonstrate that, in general, passive immunization of mice against a challenge with yersiniae was possible with both anti-Y. enterocolitica V antigen sera. However, anti-V antigen serum was protective only if the immunizing V antigen was the same type as the V antigen produced by the infective strain. The failure of the American V antigen type represented by Y. enterocolitica serotype O8 to protect against Yersinia spp. carrying the other V antigen type (LcrV-Yps) could be an explanation for the presence of plague foci in American countries.

No abstract

A total of 137 sputa from 95 consecutive cystic fibrosis (CF) patients undergoing routine bacteriological surveillance were analyzed for Pneumocystis carinii colonization using nested PCR. Seven of 95 patients (7.4%) were PCR positive, suggesting that P. carinii carriage may exist among CF patients due to their underlying pulmonary disease.Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disorder in Caucasians; it is caused by different mutations in the CF transmembrane conductance regulator gene, which is on chromosome 7 (4). Since the CF transmembrane conductance regulator gene encodes a protein functioning as a cyclic AMP-regulated chloride channel in the apical membrane of epithelial cells, several systems, including the sinopulmonary system, the gastrointestinal tract, and the male urogenital tract, can be affected in CF. The chronic bronchopulmonary manifestation of the disease poses the most serious clinical problem and causes most morbidity and mortality in CF patients. One major factor contributing to bronchopulmonary disease in CF patients is the persistent colonization and infection with typical CF bacterial pathogens, like mucoid and nonmucoid Pseudomonas aeruginosa, Staphylococcus aureus, nontypeable Haemophilus influenzae, and Burkholderia cepacia.Pneumocystis carinii is an opportunistic pathogen causing serious and even life-threatening pneumonia (P. carinii pneumonia [PCR]) in immunosuppressed patients. PCP is speculated to result either from a de novo infection or from reactivation of latent childhood infection. Seroconversion usually happens during early childhood, leading to high seroprevalence rates (3). Autopsy studies using microscopy or immunofluorescence, however, revealed no or only a very low prevalence of P. carinii, less than 1%, in adults without predisposing diseases (1, 2, 10). More sensitive methods like PCR may be able to detect even low numbers of P. carinii organisms in clinically asymptomatic but colonized persons. In a recent study it was shown that a considerable percentage (19%) of immunocompetent adult patients with primary pulmonary disease but without overt PCP were colonized by P. carinii, as established by primary and nested PCR on bronchoalveolar lavage (BAL) specimens (6). This suggests that P. carinii carriage may exist in immunocompetent patients with underlying pulmonary disease. Since it is conceivable that preexisting lung tissue damage may favor colonization by P. carinii, we hypothesized that P. carinii DNA might be detected in respiratory samples from CF patients. In a previous study on P. carinii carriage among CF patients, however, Varela et al. were not able to detect P. carinii using four different staining methods on sputa from 45 consecutive CF patients (8).To evaluate the prevalence of P. carinii colonization in CF patients, 137 sputa from 95 consecutive CF patients (43 females and 52 males; median age, 23.2 years) who were not receiving trimethoprim-sulfamethoxazole for treatment of underlying bacterial infections were exami...