Natural and Artificial Strategies To Control the Conjugative Transmission of Plasmids.

Conjugative plasmids are the main carriers of transmissible antibiotic resistance (AbR) genes. For that reason, strategies to control plasmid transmission have been proposed as potential solutions to prevent AbR dissemination. Natural mechanisms that bacteria employ as defense barriers against invading genomes, such as restriction-modification or CRISPR-Cas systems, could be exploited to control conjugation. Besides, conjugative plasmids themselves display mechanisms to minimize their associated burden or to compete with related or unrelated plasmids. Thus, FinOP systems, composed of FinO repressor protein and FinP antisense RNA, aid plasmids to regulate their own transfer; exclusion systems avoid conjugative transfer of related plasmids to the same recipient bacteria; and fertility inhibition systems block transmission of unrelated plasmids from the same donor cell. Artificial strategies have also been designed to control bacterial conjugation. For instance, intrabodies against R388 relaxase expressed in recipient cells inhibit plasmid R388 conjugative transfer; pIII protein of bacteriophage M13 inhibits plasmid F transmission by obstructing conjugative pili; and unsaturated fatty acids prevent transfer of clinically relevant plasmids in different hosts, promoting plasmid extinction in bacterial populations. Overall, a number of exogenous and endogenous factors have an effect on the sophisticated process of bacterial conjugation. This review puts them together in an effort to offer a wide picture and inform research to control plasmid transmission, focusing on Gram-negative bacteria.

Altered antigenic profiling and infectivity of Porphyromonas gingivalis in smokers and non-smokers with periodontitis.

BACKGROUND: Cigarette smokers are more susceptible to periodontal diseases and are more likely to be infected with Porphyromonas gingivalis than non-smokers. Furthermore, smoking is known to alter the expression of P. gingivalis surface components and compromise immunoglobulin (Ig)G generation. The aim of this study is to evaluate whether the overall IgG response to P. gingivalis is suppressed in smokers in vivo and whether previously established in vitro tobacco-induced phenotypic P. gingivalis changes would be reflected in vivo. METHODS: The authors examined the humoral response to several P. gingivalis strains as well as specific tobacco-regulated outer membrane proteins (FimA and RagB) by enzyme-linked immunosorbent assay in biochemically validated (salivary cotinine) smokers and non-smokers with chronic periodontitis (CP: n = 13) or aggressive periodontitis (AgP: n = 20). The local and systemic presence of P. gingivalis DNA was also monitored by polymerase chain reaction. RESULTS: Smoking was associated with decreased total IgG responses against clinical (10512, 5607, and 10208C; all P <0.05) but not laboratory (ATCC 33277, W83) P. gingivalis strains. Smoking did not influence IgG produced against specific cell-surface proteins, although a non-significant pattern toward increased total FimA-specific IgG in patients with CP, but not AgP, was observed. Seropositive smokers were more likely to be infected orally and systemically with P. gingivalis (P <0.001), as determined by 16S RNA analysis. CONCLUSION: Smoking alters the humoral response against P. gingivalis and may increase P. gingivalis infectivity, strengthening the evidence that mechanisms of periodontal disease progression in smokers may differ from those of non-smokers with the same disease classification.

Genetic and antigenic analyses of Porphyromonas gingivalis FimA fimbriae.

The periodontal pathogen Porphyromonas gingivalis generally expresses two distinct fimbriae, FimA and Mfa1, which play a role in biofilm formation. The fimA gene that encodes FimA fimbrilin is polymorphic, and polymerase chain reaction analysis has identified six genotypes called types I-V and Ib. We found recently that fimbriae exhibit antigenic heterogeneity among the genotypes. In the present study, we analysed the fimA DNA sequences of 84 strains of P. gingivalis and characterized the antigenicity of FimA fimbriae. Strains analysed here comprised 10, 16, 29, 13, 10 and 6 strains of types I, Ib, II, III, IV and V, respectively. DNA sequencing revealed that type Ib does not represent a single cluster and that type II sequences are remarkably diverse. In contrast, the fimA sequences of the other types were relatively homogeneous. Antigenicity was investigated using antisera elicited by pure FimA fimbriae of types I-V. Antigenicity correlated generally with the respective genotype. Type Ib strains were recognized by type I antisera. However, some strains showed cross-reactivity, especially, many type II strains reacted with type III antisera. The levels of fimbrial expression were highly variable, and expression was positively correlated with ability of biofilm formation on a saliva-coated plate. Further, two strains without FimA and Mfa1 fimbriae expressed fimbrial structures, suggesting that the strains produce other types of fimbriae.

A targeted fimA DNA vaccine prevents alveolar bone loss in mice after intra-nasal administration.

AIM: To construct a dendritic cell (DC)-targeted DNA vaccine against FimA of Porphyromonas gingivalis and evaluate the immunogenicity and protection in mice. MATERIALS AND METHODS: A targeted DNA plasmid pCTLA4-FimA, which encodes the signal peptide and extracellular regions of mouse cytotoxic T lymphocyte-associated antigen 4 (CTLA4), the hinge and Fc regions of human Igγ1 and FimA of P. gingivalis, was constructed. Mice were immunized with pCTLA4-FimA, the non-targeted DNA plasmid pFimA, which contains only fimA gene, or pCI vector intra-nasally. Serum and saliva antibody responses were detected by enzyme-linked immunosorbent assay. The protection against P. gingivalis-induced periodontitis was evaluated by measuring alveolar bone loss in mice. RESULTS: Mice immunized with pCTLA4-FimA showed elevated levels of specific serum IgG and salivary IgA antibody responses compared with mice immunized with pFimA (p<0.01). Both pFimA and pCTLA4-FimA immunized groups showed significantly lower alveolar bone loss, with the magnitude protection greater in the latter (p<0.01), compared with the pCI immunized group. CONCLUSIONS: The DC-targeted DNA construct pCTLA4-FimA enhanced both systemic and mucosal immunity following intra-nasal immunization. A DNA-based immunization strategy may be an effective way to attenuate periodontitis induced by P. gingivalis.

Porphyromonas gingivalis fimbriae-dependent interleukin-6 autocrine regulation by increase of gp130 in endothelial cells.

BACKGROUND AND OBJECTIVE: Local persistent infection by Porphyromonas gingivalis leads to inflammatory systemic diseases, such as atherosclerosis. We have reported previously that avirulent P. gingivalis fimbriae-dependent invasion into endothelial cells might be involved in progression of atherosclerosis. Although interleukin-6 (IL-6) regulates progression of atherosclerosis, little is known about the relationship of P. gingivalis fimbriae-dependent invasion to IL-6 regulation in endothelial cells. MATERIAL AND METHODS: We examined the secretion of IL-6 and the expression of the IL-6 signal transducer gp130 in human umbilical vein endothelial cells (HUVEC) infected with the wild-type FDC381 strain of P. gingivalisand a fimbriae-deficient mutant (fimA) by enzyme-linked immunosorbent assay, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry (fluorescence-activated cell sorting, FACS) analysis. RESULTS: Coculture of HUVEC with P. gingivalis resulted in increase of IL-6 secretion at 24 h postinfection. Interestingly, the increase was inhibited significantly in HUVEC infected with the P. gingivalis fimA mutant. In addition, the increase of IL-6 secretion induced by P. gingivalis infection was significantly impaired by the meiosis specific kinase 1 inhibitor, PD98059, or the nuclear factor kappaB inhibitor, Bay11-7082. Furthermore, we demonstrated that gp130 expression increased with P. gingivalis infection. Importantly, gp130 expression was significantly impaired by P gingivalis fimA mutant infection compared with wild-type P. gingivalis infection, as assessed by both quantitative RT-PCR and FACS analysis. CONCLUSION: Our findings indicate that P. gingivalis fimbriae are important factors in the autocrine regulation of IL-6, by increasing gp130 in endothelial cells.

Identification of the Pseudomonas aeruginosa 1244 pilin glycosylation site.

Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.

Role for fimbriae and lysine-specific cysteine proteinase gingipain K in expression of interleukin-8 and monocyte chemoattractant protein in Porphyromonas gingivalis-infected endothelial cells.

Recent cross-sectional and prospective epidemiological studies have demonstrated an association between periodontal disease and atherosclerosis and human coronary heart disease. Previously, we have established that the periodontal pathogen Porphyromonas gingivalis is capable of invading aortic, heart, and human umbilical vein endothelial cells (HUVEC). Since atherosclerosis is a chronic inflammatory response initiated at the vascular wall, interactions of P. gingivalis with endothelial cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis. In this study we examined the consequences of P. gingivalis infection of HUVEC on the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1). HUVEC were found to constitutively produce low levels of IL-8 and MCP-1. The addition of P. gingivalis fimbrillin-specific peptides, lipopolysaccharides (LPS), or heat-killed whole cell preparations to HUVEC stimulated modest IL-8 and MCP-1 responses. In contrast, coculture of HUVEC with live P. gingivalis strain A7436, 33277, or 381 abolished the IL-8 and MCP-1 responses. Inhibition of IL-8 and MCP-1 production was not dependent on bacterial adherence since similar results were obtained with the nonadherent P. gingivalis fimA mutant DPG3 or when P. gingivalis was preincubated with fimbrillin peptide antisera prior to the addition to HUVEC. Furthermore, treatment of P. gingivalis-infected HUVEC with cytochalsin D, which prevented P. gingivalis invasion, also abolished the constitutive IL-8 and MCP-1 responses. Treatment of HUVEC with E. coli LPS stimulated robust IL-8 and MCP-1 responses that were abolished when stimulated cells were cocultured with live P. gingivalis. Analysis of P. gingivalis-infected HUVEC cultures by an RNase protection assay revealed an increase in the IL-8 transcript relative to uninfected HUVEC. Pretreatment of P. gingivalis with protease inhibitors prior to the addition to HUVEC prevented the inhibition of IL-8 and MCP-1 production in P. gingivalis-infected HUVEC, indicating that the inhibition was proteolytically mediated. Coculture of HUVEC with a P. gingivalis mutant deficient in lysine-specific cysteine proteinase (gingipain K [Kgp]) resulted in an increase in both IL-8 transcription and protein expression relative to that observed in HUVEC cocultured with the P. gingivalis wild-type strain. These results indicate that P. gingivalis can temporally modulate the chemokine response in endothelial cells through both fimbriae and gingipain-mediated mechanisms.

A rapid strip immunoblot assay for the specific detection of Salmonella enteritidis infection in chickens.

A rapid strip immunoblot assay (RSIA) was developed using recombinant SEF14 antigen. The rSEF14-RSIA was very specific in detecting antibodies to Salmonella enteritidis in chickens. When serum samples obtained from groups of chickens (N = 5) inoculated with six different Salmonella serovars were tested in rSEF14-RSIA, only serum samples obtained from S. enteritidis inoculated birds reacted with rSEF14 antigen except for a group of chickens that had been inoculated with S. dublin. To assess the sensitivity of the rSEF14-RSIA, groups of chickens were inoculated with either 10(4) cfu or 10(10) cfu of S. enteritidis and the serum and egg yolk were analyzed for SEF14 antibodies. By 1 week after infection 66-78% of chickens were found positive for SEF14 antibodies in the serum and the number of positive birds increased subsequently to 89-100%. The S. enteritidis specific antibodies appeared as early as 6 days after infection in the egg yolk of infected chickens. The antibodies to SEF14 in both the serum and egg yolk persisted for at least 7 weeks after infection in a significant proportion of chickens. Our results suggest that rSEF14-RSIA can be a practical and efficient screening test for identifying S. enteritidis infected chickens.

The phosphorylcholine epitope undergoes phase variation on a 43-kilodalton protein in Pseudomonas aeruginosa and on pili of Neisseria meningitidis and Neisseria gonorrhoeae.

Phosphorylcholine (ChoP) is a component of the teichoic acids of Streptococcus pneumoniae and has been recently identified on the lipopolysaccharide of Haemophilus influenzae, also a major pathogen of the human respiratory tract. Other gram-negative pathogens that frequently infect the human respiratory tract were surveyed for the presence of the ChoP epitope as indicated by binding to monoclonal antibodies (MAbs) recognizing this structure. The ChoP epitope was found on a 43-kDa protein on all clinical isolates of Pseudomonas aeruginosa examined and on several class I and II pili of Neisseria meningitidis. The specificity of the anti-ChoP MAb was demonstrated by the inhibition of binding in the presence of ChoP but not structural analogs. As in the case of H. influenzae, the expression of this epitope was phase variable on these species. In P. aeruginosa, this epitope was expressed at detectable levels only at lower growth temperatures. Expression of the ChoP epitope on piliated neisseriae displayed phase variation, both linked to pilus expression and independently of fully piliated bacteria.

Selective phage infection mediated by epitope expression on F pilus.

Proteins and peptides can be displayed on bacterial and bacteriophage surfaces as fusions to bacterial integral membrane proteins or phage coat proteins. We now report on the expression of peptide antigens on the surface of F pili, elaborated by F+ strains of Escherichia coli. The peptides were expressed as fusions to F pilin, the building block of the F pilus that is encoded by the traA gene on the F plasmid. Filamentous bacteriophage infection of E. coli is normally mediated by phage binding to pilin at the F pili tip. Expression of 13 to 15 amino acid long peptides on the F pilus completely blocked infection by derivatives of wild-type infectious M13 phage. However, when a phage displaying a specific recombinant antibody fragment was allowed to interact with F pili displaying an antigenic peptide a bacterial infection could be demonstrated. This infection, mediated by the antibody-antigen interaction, resulted in bacterial cells containing plasmids encoding both the protein and the ligand. In a model library, where a scFv antibody against the human cytomegalovirus AD-2 epitope was selected we achieved an enrichment of 2500 of phage carrying the specific antibody, indicating an efficient selective infection.

Enteropathogens associated with diarrhea among military personnel during Operation Bright Star 96, in Alexandria, Egypt.

This study investigated the microbial causes of diarrheal disease among U.S. troops deployed near Alexandria, Egypt, during October 1995. Bacterial causes associated with 19 cases of diarrhea included: enterotoxigenic Escherichia coli (ETEC), 42% (21% heat-stable, 11% heat-labile, and 11% heat-stable/ heat-labile producers); enteropathogenic E. coli (5.3%); and enteroadherent E. coli (42%). Four cases of diarrhea were associated with enteroaggregative E. coli based on probe analysis for enteroaggregative heat-stable enterotoxin 1. Protozoan causes included; Entamoeba histolytica (11%), E. hartmanni (5%), E. nana (5%), Blastocystis hominis (5%), Chilomastix mesnili (11%), Dientamoeba fragilis (5%), Entamoeba coli (5%), and Cryptosporidium (5%). Shigella, Aeromonas, Plesiomonas, Vibrio, Campylobacter, and Salmonella were not detected. Of the eight ETEC cases, one was colonization factor antigen (CFA)/I only, one was both CFA/I and CFA/III, three were CFA/II, two were CFA/IV, and two were CFA-negative. Antibiograms of the ETEC and enteroadherent E. coli strains showed that all isolates were susceptible to norfloxacin, ciprofloxacin, and nalidixic acid but resistant to ampicillin, tetracycline, chloramphenicol, and sulfamethoxazole.

Phenotypic variants of meningococci and their potential in phagocytic interactions: the influence of opacity proteins, pili, PilC and surface sialic acids.

In previous studies we have examined the roles of meningococcal surface structures (capsule, lipopolysaccharides, pili and opacity proteins: Opa and Opc) in bacterial interactions with human epithelial, endothelial and mononuclear phagocytic cells. In the current investigations, using defined derivatives of a serogroup A strain C751 and a serogroup B strain MC58, we studied the roles of these structures with human polymorphonuclear phagocytes (PMN). In addition, we examined the potential influence of the pilus-associated protein, PilC, previously known to affect epithelial cell interactions. The data show, that, as with monocytes, opacity proteins affect bacterial interactions with PMN and require surface sialic acids (on capsule and LPS) to be down-modulated in order to function. Also, in contrast to their role in human epithelial and endothelial adherence, neither pili nor PilC expression had any effect on phagocytic cell interactions with respect to induction of chemiluminescence as well as phagocytic killing. Examination of the relative influence of Opa and Opc indicated that Opa proteins are more effective than Opc in PMN interactions whereas the reverse was the case with monocytes. These results suggest that Opa and Opc mediate interactions with phagocytic cells via distinct mechanisms. Observations presented here and reported previously collectively show that the structural requirements of meningococci for interacting with phagocytes, in the absence of opsonins, are present in the phenotype which is often isolated from the nasopharynx (asialylated, piliated, Opa/Opc+) whereas the phenotype prevalent in the blood (sialyted, piliated, Opa/Opc+) retains the ability to adhere to endothelial cells (via pili) but appears to be refractory to interactions with phagocytic cells.

Antagonistic effect of synthetic peptides corresponding to the binding regions within fimbrial subunit protein from Porphyromonas gingivalis to human gingival fibroblasts.

Specific binding region within fimbrial subunit protein (fimbrilin) from Porphyromonas gingivalis strain 381 was studied in cultured human gingival fibroblasts. Fluorescent micrographs visualised FITC-labelled fimbriae of P. gingivalis specifically bound to normal human fibroblast cell line (Gin-1) along the cell surface. Flow cytometric analysis also revealed the binding of FITC-labelled fimbriae to Gin-1 cells. Synthetic peptides composed of residues 1-20 (AFGVGDDESKVAKLTVMVYN) of the fimbrilin from P. gingivalis, FP381 (1-20), FP381 (69-80; ALTTELTAENQE) and FP381 (171-181; DA-NYLTGSLTT) definitely inhibited P. gingivalis fimbria-binding to Gin-1 cells by enzyme-linked immunosorbent assay (ELISA). Furthermore, based on the Scatchard plot analysis of the binding of 125I-labelled P. gingivalis fimbriae to Gin-1 cells, the apparent dissociation constant (Kd) was calculated as 15.9 pM, and the number of binding sites (Rt) was estimated as 150 sites/cell. Binding studies of 125I-labelled FP381(171-181) also revealed the presence of a non-interacting, single class of affinity binding sites: the apparent Kd and Rt were 29.2 nM and 18440 sites/cell on Gin-1 cells, respectively. These results demonstrate that specific binding regions on P. gingivalis fimbriae to human gingival fibroblasts are present, and certain corresponding peptides clearly inhibited the binding of P. gingivalis fimbriae to human gingival fibroblasts.

[Continuous and common epitopes present in fimbriae of enterotoxigenic Escherichia coli (ETEC)]. / Epítopos continuos y comunes presentes en las fimbrias de Escherichia coli enterotoxigénica (ETEC).

Colonization factor antigens (CFAs and PCFs) are important virulence factors of Escherichia coli (ETEC) diarrhea. Antibodies to CFAs produced after ETEC infection are protective; however, the CFA epitopes which induce protective antibodies have not yet been characterized. This study is the characterization of the immune response to CFAs at molecular level and identification of the epitopes associated with inhibition of cell-adherence and protection that will lead to the development of methods to prevent ETEC infection and disease. The aim of this study was the characterization of the linear epitopes of CFA/I that react with sera from acute and convalescent phase of ETEC-in-fected children, with adult sera from endemic and non-endemic areas, with monoclonal antibodies (Mabs) and with hyperimmune antiserum to CFAs and PCFs different from CFA/I. Three linear and common epitopes were recognized among the CFA/I in child sera and adult sera from endemic areas and with hyperimmune sera against other known CFAs and putative ETEC colonization factors.

Questions about gonococcal pilus phase- and antigenic variation.

Pathogenic organisms inhabit one of several defined locations within a host where temperature, pH, and nutrients are relatively constant. While the microorganism must adapt to different environments within the host, the host immune system is the most formidable predator that can limit the growth of a pathogen. Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of gonorrhoea, and has evolved several systems for varying the antigenicity of different surface antigens, presumably to help evade the effects of the human immune system. The On/Off/On phase variation of surface structure expression also alters the antigenic characteristics of the bacterial cell surface. Antigenic variation of the major subunit of the pilus, pilin, occurs by unidirectional, homologous recombination between a silent locus and the expression locus. The silent loci lie from 1 to 900 kb from the expression locus in the chromosome yet all can donate their sequences to the expression locus. The genetic composition of the pilin loci of two Gc strains has been elucidated, and the types of changes that lead to altered forms of the pilus have been extensively characterized. However, little is known about the precise molecular mechanisms used to allow high-frequency, non-reciprocal, chromosomal recombination between pilin loci or about what regulates the process of maintaining chromosome fidelity.

Effect of onion extract on immune response in rabbits.

A total of 40 NZW rabbits were selected for this study to see the effect of onion extract on immune response following antigenic challenge. These animals were randomly divided into four groups, each composed of ten rabbits. Group I and II were challenged with typhoid H (TH) antigen and groups III and IV with sheep red blood cells (SRBC). Groups I and III were considered as control and II and IV as treated groups. The latter two groups were treated with onion extract orally. The immunosuppressive effect of onion extract was evaluated by estimating antibody levels by Widal test and hemolysin titer. It was found that mean antibody titers were significantly lower in the treated groups than in controls. The weights of thymus and lymph nodes were higher and of adrenal glands were lower in the control groups than in the treated groups. It appeared from the current study that onion extract has an inhibitory effect on immune response.

Development and application of enzyme-linked immunosorbent assay for specific detection of Salmonella enteritidis infections in chickens based on antibodies to SEF14 fimbrial antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to the SEF14 fimbrial antigen (SEF14-DAS ELISA) and was evaluated for its use in the specific detection of chicken flocks infected with Salmonella enteritidis. The SEF14-DAS ELISA successfully discriminated between chickens experimentally infected with S. enteritidis and those infected with S. panama or S. typhimurium, although the SEF14 responses in adult birds infected with S. enteritidis were detectable but low. In contrast, ELISAs used to detect antibodies to lipopolysaccharide (LPS) and flagella were unable to discriminate between the infected groups of chicks and adult birds infected with different Salmonella serotypes. LPS and flagellar responses were low and variable in chicks, whereas in adult hens they were found to be consistently strong. When flocks naturally infected with S. enteritidis were tested by the SEF14-DAS ELISA and ELISAs to detect LPS and flagellar antibodies, it was found that they could all identify the infected flocks, although there was little correlation between individual serum samples. The study shows that the SEF14-DAS ELISA may offer advantages over existing assays with comparable sensitivities coupled with higher specificities for the serological detection of S. enteritidis-infected chicken flocks.

Development of an anti-adhesive vaccine for Pseudomonas aeruginosa targeting the C-terminal region of the pilin structural protein.

This study describes the development of passive and active vaccines directed at the Pseudomonas aeruginosa pilus adhesin. Passive immunization studies were carried out with P. aeruginosa strain K pilus-specific (PK3B, PK99H) and cross-reactive (PAK-13) monoclonal antibodies (MAbs). When A.BY/SnJ mice were passively immunized with a pilus-specific MAb (PK99H), which inhibited pilus-mediated adherence to respiratory epithelial cells, mice challenged with 5 x LD 50 of P. aeruginosa were completely protected while mice were not protected when animals were passively immunized with a pilus specific MAb (PK3B), which did not inhibit pilus adherence to epithilial cells. MAb PAK-13 was found to cross-react with the C-terminal portion of pili of different strains of P. aeruginosa. When mice were passively immunized with MAb PAK-13, subsequent challenge with KB7 (3 x LD50), PAO (8 x LD50) and PAK (3 x LD50) strains of P. aeruginosa resulted in a 70%, 60% and 90% protection of the mice, respectively. MAb PK99H has been previously shown to recognize a linear antigenic epitope consisting of the sequence DEQFIPK. This epitopic peptide was conjugated to protein carriers using different coupling strategies. Use of an appropriate adjuvant and the correct conjugation strategy were critical for raising high affinity antipeptide antisera. In a comparison of Freund's, alum, and Adjuvax, as adjuvants for a peptide-tetanus toxoid conjugate vaccine, highest titers for the synthetic peptide component of the conjugate were obtained with Adjuvax, while highest titers for the carrier protein components were obtained with Freund's. Of the four peptide-conjugates used in this study, only the C-terminal conjugated peptide failed to produce antibodies that bind to native antigen and did not protect mice in active immunization experiments (no survivors at 80 h in the mouse infection model). Conformationally restricted peptide conjugates in which the peptide was conjugated to the carrier at both ends provided better protection in mice challenged with lethal doses of P. aeruginosa than either N- or C-terminal linked peptide-conjugates. The pilus adhesin plays a critical role in P. aeruginosa pathogenesis and this is an excellent vaccine target for either active or passive immunization strategies.

Antigenic phenotypes of Escherichia coli in urine from patients with urinary tract infections.

Various antigenic phenotypes of Escherichia coli in urine were analysed using monoclonal antibody against pyelonephritis-associated P-pili (PAP-pili), and polyvalent O- and K1-antisera, and the results were compared with the clinical diagnosis. PAP-pili, O1- and K1-positive E. coli were isolated more frequently in urine from patients with acute pyelonephritis. E. coli found in urine from patients with recurrent pyelonephritis were frequently PAP positive. Based on the antigenic phenotypes of strains in urine, it is suggested that pyelonephritopathogenic strains may originate from a small number of clones.