No abstract

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.

The mouse and rabbit intradermal injection models have been used to define factors that may be important in Haemophilus ducreyi pathogenesis. We used H. ducreyi strains with diverse geographic origins and phenotypic characteristics to evaluate the experimental models. Injection of live and heat-killed bacteria caused skin abscesses in both models. Semiquantitative cultures of skin injected with live bacteria showed that H. ducreyi failed to replicate in animal tissue. These data suggested that the experimental lesions were caused by a heat-stable substance such as lipooligosaccharide (LOS). In mice, injection of H. ducreyi and Haemophilus influenzae LOS and Escherichia coli lipopolysaccharide caused mild to moderate inflammation. In rabbits, injection of H. ducreyi LOS caused intradermal abscesses that were histologically similar to those caused by live and heat-killed bacteria. H. ducreyi and Neisseria gonorrhoeae LOS caused significantly larger lesions than equivalent amounts of H. influenzae LOS and E. coli lipopolysaccharide in the rabbit model. We conclude that the intradermal injection models are not valid models to study the growth of H. ducreyi in vivo. However, these data indicate that H. ducreyi LOS may play an important role in the pathogenesis of chancroid and that the rabbit model should be useful in studying H. ducreyi LOS toxicity at the cellular level.

The P2 porin protein is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae (NTHI). Analysis of sequences of P2 from different strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. The present study was undertaken to test the hypothesis that antibodies to a conserved surface-exposed loop are bactericidal for multiple strains of NTHI and could thus form the basis of vaccines to prevent infection due to NTHI. Polyclonal antiserum to a peptide corresponding to loop 6 was raised and was immunopurified over a loop 6 peptide column. Analysis of the antibodies to whole organisms and peptides corresponding to each of the eight loops of P2 by immunoassays revealed that the antibodies were highly specific for loop 6 of P2. The immunopurified antibodies bound to P2 of 14 of 15 strains in immunoblot assays. These antibodies to loop 6 demonstrated complement-mediated bactericidal killing of 8 of 15 strains. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.Nontypeable Haemophilus influenzae (NTHI) is a small, gram-negative bacillus which causes otitis media in children and lower respiratory infections in adults with chronic obstructive pulmonary disease (COPD). In both otitis media and COPD, patients routinely suffer recurrent episodes of disease (15,21). Factors such as health care costs, pain and suffering, and lost work time underscore the need for a vaccine against NTHI (10,14,22).The ability of NTHI to cause recurrent infections is in part attributable to antigenic variability in several surface-exposed loops of major outer membrane protein P2 (2, 5, 26). The P2 protein is a homotrimeric porin which constitutes approximately one-half of the total outer membrane protein of the organism. The loop 5 region is highly heterogeneous among strains and contains almost all of the epitopes to which an antibody response is mounted when animals are immunized with the whole organism (30). Adults with COPD make new antibodies to strain-specific epitopes on P2 following infection by NTHI (31). Thus, immunity against NTHI is most often strain specific, leaving the patient vulnerable to reinfection by other strains.One approach to vaccine development for NTHI has been to study antigenically conserved outer membrane proteins as potential vaccine antigens. In view of the abundant expression of P2 on the bacterial surface, identification of a conserved region on the P2 molecule to which immune responses could be directed would be a significant step towards developing a vaccine against NTHI.In this study, antibodies to a conserved loop of the P2 molecule of NTHI (loop 6) were raised and studied for their ability to recognize the P2 molecules of heterologous strains. Since bactericidal antibody is associated with protection from otitis media due to NTHI (8, 25), antibodies to loop 6 were also assessed for their ability to direct killing of heterologous strains. MATERIALS AND METHODSBacterial s...