Tracing genomic variations in two highly virulent Yersinia enterocolitica strains with unequal ability to compete for host colonization.

BACKGROUND: Yersinia enterocolitica is a gastrointestinal foodborne pathogen found worldwide and which especially affects infants and young children. While different bioserotypes have been associated with varying pathogenicity, research on Y. enterocolitica is mainly conducted on the highly virulent mouse-lethal strains of biotype 1B and serotype O:8. We demonstrate here that two Y. enterocolitica bioserotype 1B/O:8 strains, 8081 and WA-314, display different virulence and fitness properties in a mouse model. In vivo co-infection experiments revealed that strain WA-314 overcomes strain 8081 in the colonization of spleen and liver. To trace the reasons of this incongruity, we present here the first high-quality sequence of the whole genome of strain WA-314 and compare it to the published genome of strain 8081. RESULTS: Regions previously accepted as unique to strain 8081, like the YAPI and YGI-3 genomic islands, are absent from strain WA-314, confirming their strain-specificity. On the other hand, some fitness- and bacterial competition-associated features, such as a putative colicin cluster and a xenobiotic-acyltransferase-encoding gene, are unique to strain WA-314. Additional acquisitions of strain WA-314 are seven prophage-like regions. One of these prophages, the 28-kb P4-like prophage YWA-4, encodes a PilV-like protein that may be used for adhesion to and invasion of the intestinal cells. Furthermore, a putative autotransporter and two type 1 fimbrial proteins of strain WA-314 show a sequence similarity <50% with the orthologous proteins in strain 8081. The dissimilar sequences of these proteins indicate possible different functions or interaction modes, reflecting the specific adhesion properties of Y. enterocolitica strains 8081 and WA-314 and thus the different efficiency of host colonization. Further important differences were found in two pYV plasmid-encoded virulence factors, YopM and YscP. The impact of these differences on virulence is discussed. CONCLUSIONS: Our study emphasizes that the virulence of pathogens can be increased, by acquiring new genes and/or improving the function of essential virulence proteins, resulting in permanently hyper-virulent strains. This work also highlights the importance of addressing genetic and phenotypic variations among closely related bacterial strains, even those belonging to the same bioserotype.

The pathogenic potential of Yersinia enterocolitica 1A.

Yersinia enterocolitica 1A strains are generally considered apathogenic. However, besides environmental sources, foods and animals, they are repeatedly isolated from patients with gastrointestinal symptoms typical to those evoked by Yersinia of the virulent 1B and 2-4 biotypes. Also, at least 2 gastrointestinal outbreaks associated with 1A strains have been reported. There is a general controversy concerning the pathogenic potential of 1A isolates of clinical and non-clinical origin. To address the 1A puzzle, we have determined the genome sequences of 2 1A strains, a nosocomial O:5 and environmental O:36 isolates, and compared them to each other and to O:8/1B and O:3/4 representatives of the virulent serobiotypes. 1A isolates have mosaic genomes and share genes both with serobiotypes O:8/1B and O:3/4 that implies their common descent. Besides the pYV virulence plasmid, 1A strains lack the classical virulence markers, like the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. However, they still possess genes encoding such known and suspect virulence-associated determinants like the YstB enterotoxin, the InvA invasin, the mucoid Yersinia factor MyfA, and the enterochelin utilisation fepBDGC/fepA/fes gene cluster. In contrast to previous studies, we have found that the strains of the 1A group possess the MyfA antigen although with limited similarity to the highly conserved MyfA in the virulent serobiotypes. In turn, the MyfB chaperone coevolved with the MyfA fibrillae, while the MyfC usher retains 90% identity to its MyfC counterparts in O:3/O:8 group. The only notable difference between clinical and non-clinical 1A strains was the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital O:5 isolate. Taken together, Y. enterocolitica BT 1A group represents opportunistic pathogens whose opportunity to establish infection seems to rely mainly on the state of the host defence system. However, presence of known and putative virulence-associated features shared with the pathogenic serobiotypes compels to reconsider properly the pathogenic potential of this group of emerging pathogens.

Complement C1q and C3 are critical for the innate immune response to Streptococcus pneumoniae in the central nervous system.

Previous studies suggest that the complement system can contribute to limiting pneumococcal outgrowth within the CNS. In this study, we evaluated the role of the complement system in the activation of the innate immune response and the development of the prognosis-relevant intracranial complications in a murine model of pneumococcal meningitis. Thereby, we used mice deficient in C1q, lacking only the classical pathway, and C3, lacking all three complement activation pathways. At 24 h after intracisternal infection, bacterial titers in the CNS were almost 12- and 20-fold higher in C1q- and C3-deficient-mice, respectively, than in wild-type mice. Mean CSF leukocyte counts were reduced by 47 and 73% in C1q- and C3-deficient-mice, respectively. Intrathecal reconstitution with wild-type serum in C3-deficient mice restored both the ability of mice to combat pneumococcal infection of the CSF and the ability of leukocytes to egress into the CSF. The altered recruitment of leukocytes into the CSF of C3-deficient mice was paralleled by a strong reduction of the brain expression of cytokines and chemokines. The dampened immune response in C3-deficient mice was accompanied by a reduction of meningitis-induced intracranial complications, but, surprisingly, also with a worsening of short-term outcome. The latter seems to be due to more severe bacteremia (12- and 120-fold higher in C1q- and C3-deficient-mice, respectively) and, consecutively, more severe systemic complications. Thus, our study demonstrated for the first time that the complement system plays an integral role in mounting the intense host immune response to Streptococcus pneumoniae infection of the CNS.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response.

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Chronic perivascular inoculation with Chlamydophila pneumoniae results in plaque formation in vivo.

Hypercholesterolemic and normocholesterolemic rabbit models of chronic arterial Chlamydophila (Chlamydia) pneumoniae (CPN) inoculation were established and the role of both viable and inactivated bacteria was investigated in atherogenesis. A total of 29 rabbits were randomized to four groups. Groups A and B were fed a cholesterol-enriched diet, and groups C and D were fed a normal diet. Arterial segments of group A and C animals were inoculated in vivo using viable CPN chronically using repeated perivascular applications. Contralateral arteries were treated using heat-inactivated CPN. Group B and D animals were treated with repeated perivascular injections of bacterial lipopolysaccharide (LPS) and saline (control). Additional hypercholesterolemic rabbits were treated by repeated injections using viable and inactivated CPN, each controlled by saline injections. To compare the effects of this chronic inoculation model, additional animals received single injections of either viable CPN, inactivated CPN, LPS, or saline. Vascular tissues (n=162 treated arteries of 29 rabbits) were analyzed using morphometry at histology. CPN was detected by fluorescence-immunohistochemistry and nested polymerase chain reaction. Only in hypercholesterolemic, but not in normocholesterolemic rabbits, chronic perivascular infection of all bacterial components, viable and heat-inactivated CPN, as well as LPS resulted in a significant increase in atheromatous lesion formation (lesion area index: 0.23+/-0.08, 0.25+/-0.09, and 0.15+/-0.05) when compared to controls (lesion area index 0.01+/-0.01, P=0.002). CPN persisted in atheromatous lesions and vascular tissues. Single perivascular infection using CPN or inactivated CPN was not able to induce lesion formation (lesion area index: 0.03+/-0.03, 0.03+/-0.02 vs 0.03+/-0.02 after single saline inoculation, P=0.965). In conclusion, chronic vascular infection with CPN or CPN components acts as a cofactor requiring other major atherogenic stimuli, rather than as a causative agent.

MyD88 is required for mounting a robust host immune response to Streptococcus pneumoniae in the CNS.

Myeloid differentiation factor 88 (MyD88) is an essential intracellular signal transducer in Toll-like receptor (TLR) and interleukin (IL)-1 receptor family member-mediated cell activation. In order to characterize the role of MyD88 in pneumococcal meningitis we used gene-targeted mice lacking functional MyD88 expression. At 24 h after intracisternal infection, MyD88- deficient mice displayed a markedly diminished inflammatory host response in the CNS, as evidenced by reduced CSF pleocytosis and expression of cytokines, chemokines and complement factors. The reduced CNS inflammation was paralleled by a marked reduction in the prognostic relevant CNS complications, such as brain oedema formation. Nevertheless, MyD88 deficiency was associated with a worsening of disease which seemed to be attributable to severe bacteraemia. This notion was supported by the unexpected observation that infected MyD88-deficient mice displayed enhanced mRNA expression of inflammatory mediators [such as the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) and the CXC chemokine macrophage inflammatory protein (MIP-2)] in the lung and consequently increased cell influx in the bronchoalveolar lavage fluid, compared with infected wild-type mice. Thus, the present study demonstrated for the first time an important role of MyD88 in immune activation to bacterial pathogens within the CNS. The role played by MyD88 in mounting an immune response to Streptococcus pneumoniae, however, seems to be dependent on the anatomical compartment involved.