Novel class of mutations of pilS mutants, encoding plasmid R64 type IV prepilin: interface of PilS-PilV interactions

The type IV pili of plasmid R64 belonging to the type IVB group are required only for liquid mating. They consist of the major and minor components PilS pilin and PilV adhesin, respectively. PilS pilin is first synthesized as a 22-kDa prepilin from the pilS gene and is then processed to a 19-kDa mature pilin by PilU prepilin peptidase. In a previous genetic analysis, we identified four classes of the pilS mutants (T. Horiuchi and T. Komano, J. Bacteriol. 180:4613-4620, 1998). The products of the class I pilS mutants were not processed by prepilin peptidase; the products of the class II mutants were not secreted; in the class III mutants type IV pili with reduced activities in liquid mating were produced; and in the class IV mutants type IV pili with normal activities were produced. Here, we describe a novel class, class V, of pilS mutants. Mutations in the pilS gene at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesin, which were inactive in liquid mating. Residues 56 and 57 of PilS pilin are suggested to function as an interface of PilS-PilV interactions.     

Structural basis of typhoid: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N‐terminal 25 amino acid deleted S. typhi native PilS protein (ΔPilS), which makes the pilus, was determined at 1.9 Å resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of ΔPilS and a target CFTR peptide, determined at 1.8 Å, confirms that residues 113–117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

The burden and characteristics of enteric fever at a healthcare facility in a densely populated area of Kathmandu

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.     

Type IV‐pili dependent motility is co‐regulated by PilSR and PilS2R2 two‐component systems via distinct pathways in Myxococcus xanthus

Myxococcus xanthus is an environmental bacterium with two forms of motility. One type, known as social motility, is dependent on extension and retraction of Type‐IV pili (T4P) and production of extracellular polysaccharides (EPS). Several signaling systems have been linked to regulation of T4P‐dependent motility. In particular, expression of the pilin subunit pilA requires the PilSR two‐component signaling system (TCS). A second TCS, PilS2R2, encoded within the same locus that encodes PilSR, has also been linked to M. xanthus T4P‐dependent motility. We demonstrate that PilSR and PilS2R2 regulate M. xanthus T4P‐dependent motility through distinct pathways. Consistent with known roles of PilSR, our results indicate that the primary function of PilSR is to regulate expression of pilA. In contrast, PilS2 and PilR2 have little to no affect on PilA protein levels. However, deletion of pilR2 resulted in a reduction of assembled pili, significant decreases in EPS production and loss of T4P‐dependent motility. Furthermore, the pilR2 mutation led to increased production of outer membrane vesicles (OMV). Collectively, we propose that PilS2R2 is required for proper assembly of T4P and regulation of OMV production, and hypothesize that production of these vesicles is related to M. xanthus motility.     

Differential Epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu,Nepal: A Matched Case Control Investigation in a Highly Endemic Enteric Fever Setting

Background

Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.

Methodology and principal findings

A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.

Conclusions and significance

Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.     

Bloodstream Infection among Children Presenting to a General Hospital Outpatient Clinic in Urban Nepal

Background

There are limited data on the etiology and characteristics of bloodstream infections in children presenting in hospital outpatient settings in South Asia. Previous studies in Nepal have highlighted the importance of murine typhus as a cause of febrile illness in adults and enteric fever as a leading bacterial cause of fever among children admitted to hospital.

Methods

We prospectively studied a total of 1084 febrile children aged between 2 months and 14 years presenting to a general hospital outpatient department in Kathmandu Valley, Nepal, over two study periods (summer and winter). Blood from all patients was tested by conventional culture and by real-time PCR for Rickettsia typhi.

Results

Putative etiological agents for fever were identified in 164 (15%) patients. Salmonella enterica serovar Typhi (S. Typhi) was identified in 107 (10%), S. enterica serovar Paratyphi A (S. Paratyphi) in 30 (3%), Streptococcus pneumoniae in 6 (0.6%), S. enterica serovar Typhimurium in 2 (0.2%), Haemophilus influenzae type b in 1 (0.1%), and Escherichia coli in 1 (0.1%) patient. S. Typhi was the most common organism isolated from blood during both summer and winter. Twenty-two (2%) patients were PCR positive for R. typhi. No significant demographic, clinical and laboratory features distinguished culture positive enteric fever and murine typhus.

Conclusions

Salmonella infections are the leading cause of bloodstream infection among pediatric outpatients with fever in Kathmandu Valley. Extension of immunization programs against invasive bacterial disease to include the agents of enteric fever and pneumococcus could improve the health of children in Nepal.     

Estimating the Burden of Paratyphoid A in Asia and Africa

Despite the increasing availability of typhoid vaccine in many regions, global estimates of mortality attributable to enteric fever appear stable. While both Salmonella enterica serovar Typhi (S. Typhi) and serovar Paratyphi (S. Paratyphi) cause enteric fever, limited data exist estimating the burden of S. Paratyphi, particularly in Asia and Africa.We performed a systematic review of both English and Chinese-language databases to estimate the regional burden of paratyphoid within Africa and Asia. Distinct from previous reviews of the topic, we have presented two separate measures of burden; both incidence and proportion of enteric fever attributable to paratyphoid. Included articles reported laboratory-confirmed Salmonella serovar classification, provided clear methods on sampling strategy, defined the age range of participants, and specified the time period of the study.A total of 64 full-text articles satisfied inclusion criteria and were included in the qualitative synthesis. Paratyphoid A was commonly identified as a cause of enteric fever throughout Asia. The highest incidence estimates in Asia came from China; four studies estimated incidence rates of over 150 cases/100,000 person-years. Paratyphoid A burden estimates from Africa were extremely limited and with the exception of Nigeria, few population or hospital-based studies from Africa reported significant Paratyphoid A burden.While significant gaps exist in the existing population-level estimates of paratyphoid burden in Asia and Africa, available data suggest that paratyphoid A is a significant cause of enteric fever in Asia. The high variability in documented incidence and proportion estimates of paratyphoid suggest considerable geospatial variability in the burden of paratyphoid fever. Additional efforts to monitor enteric fever at the population level will be necessary in order to accurately quantify the public health threat posed by S. Paratyphi A, and to improve the prevention and treatment of enteric fever.     

Molecular modelling, docking and interaction studies of human-plasmogen and salmonella enolase with enolase inhibitors

Salmonella enteric serovar Typhi Ty2 is a human specific pathogen and an etiological agent for typhoid fever. Most of Salmonella serotypes produce glycogen which has a comparatively minor role in virulence and colonization, but has a more significant role in survival. Enzymes present in glycolytic pathway of bacteria help bacteria to survive by activating other factors inside host. Numerous pathogenic bacteria species intervene with the plasminogen system, and this plasminogen-enolase association may play a critical role in the virulence of S. Typhi by causing direct damage to the host cell extracellular matrix, possibly by enzymic degradation of extracellular matrix proteins or other protein constituents. In this study, molecular modelling of enolase of Salmonella has been accomplished in silico by comparative modelling; we have then analyzed Human alpha enolase which is a homodimer and serves on epithelial cells with our model. Both Structures were docked by D-tartronate semialdehyde phosphate (TSP) and 3-aminoenolpyruvate phosphate (AEP) enolase inhibitors. Our study shows that salmonella enolase and human enolase have different active sites in their structure. This will help in development of new ligands, more suitable for inhibiting bacterial survival inside host as vaccines for typhoid fever are not fully protective. The study also confirmed that enolase Salmonella and Human Plasminogen suggested direct physical interaction between both of them as the activation loop of plasminogen residues showed conformational changes similar to the tissue type plasminogen activator. Various computational biology tools were used for our present study such as Modeller, Molegro Virtual Docker, Grommacs.     

ChIP‐seq and transcriptome analysis of the OmpR regulon of Salmonella enterica serovars Typhi and Typhimurium reveals accessory genes implicated in host colonization

OmpR is a multifunctional DNA binding regulator with orthologues in many enteric bacteria that exhibits classical regulator activity as well as nucleoid‐associated protein‐like characteristics. In the enteric pathogen Salmonella enterica, using chromatin immunoprecipitation of OmpR:FLAG and nucleotide sequencing, 43 putative OmpR binding sites were identified in S. enterica serovar Typhi, 22 of which were associated with OmpR‐regulated genes. Mutation of a sequence motif (TGTWACAW) that was associated with the putative OmpR binding sites abrogated binding of OmpR:6×His to the tviA upstream region. A core set of 31 orthologous genes were found to exhibit OmpR‐dependent expression in both S. Typhi and S. Typhimurium. S. Typhimurium‐encoded orthologues of two divergently transcribed OmpR‐regulated operons (SL1068–71 and SL1066–67) had a putative OmpR binding site in the inter‐operon region in S. Typhi, and were characterized using in vitro and in vivo assays. These operons are widely distributed within S. enterica but absent from the closely related Escherichia coli. SL1066 and SL1067 were required for growth on N‐acetylmuramic acid as a sole carbon source. SL1068–71 exhibited sequence similarity to sialic acid uptake systems and contributed to colonization of the ileum and caecum in the streptomycin‐pretreated mouse model of colitis.     

Evaluation of an Electricity-free,Culture-based Approach for Detecting Typhoidal Salmonella Bacteremia during Enteric Fever in a High Burden,Resource-limited Setting

Background

In many rural areas at risk for enteric fever, there are few data on Salmonella enterica serotypes Typhi (S. Typhi) and Paratyphi (S. Paratyphi) incidence, due to limited laboratory capacity for microbiologic culture. Here, we describe an approach that permits recovery of the causative agents of enteric fever in such settings. This approach involves the use of an electricity-free incubator based upon use of phase-change materials. We compared this against conventional blood culture for detection of typhoidal Salmonella.

Methodology/Principal Findings

Three hundred and four patients with undifferentiated fever attending the outpatient and emergency departments of a public hospital in the Kathmandu Valley of Nepal were recruited. Conventional blood culture was compared against an electricity-free culture approach. Blood from 66 (21.7%) patients tested positive for a Gram-negative bacterium by at least one of the two methods. Sixty-five (21.4%) patients tested blood culture positive for S. Typhi (30; 9.9%) or S. Paratyphi A (35; 11.5%). From the 65 individuals with culture-confirmed enteric fever, 55 (84.6%) were identified by the conventional blood culture and 60 (92.3%) were identified by the experimental method. Median time-to-positivity was 2 days for both procedures. The experimental approach was falsely positive due to probable skin contaminants in 2 of 239 individuals (0.8%). The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% (95% CI: 80.0%–97.0%) and 96.0% (92.7%–98.1%), respectively. After initial incubation, Salmonella isolates could be readily recovered from blood culture bottles maintained at room temperature for six months.

Conclusions/Significance

A simple culture approach based upon a phase-change incubator can be used to isolate agents of enteric fever. This approach could be used as a surveillance tool to assess incidence and drug resistance of the etiologic agents of enteric fever in settings without reliable local access to electricity or local diagnostic microbiology laboratories.     

Linkage of avian and reproductive tract tropism with sequence divergence adjacent to the 5S ribosomal subunit rrfH of Salmonella enterica

The 183 bp between the end of the 23S rrlH rRNA gene and the start of the 5S rrfH rRNA gene (ISR-1) and the 197 bp between the end of the rrfH rRNA gene and the start of the transfer RNA aspU (ISR-2) of Salmonella enterica ssp. enterica serotypes Enteritidis, Typhimurium, Pullorum, Heidelberg, Gallinarum, Typhi and Choleraesuis were compared. ISR-1s of D1 serotypes (Pullorum, Gallinarum and Enteritidis), B serotypes (Typhimurium and Heidelberg) and the C2 serotype Newport and the enteric fever pathogens serotype A Paratyphi and serotype D1 Typhi formed three clades, respectively. ISR-2 further differentiated the avian-adapted serotype Gallinarum from avian-adapted Pullorum and Salmonella bongori from S. enterica. The results suggest that serotypes Heidelberg and Choleraesuis share some evolutionary trends with egg-contaminating serotypes. In addition, ISR-1 and ISR-2 sequences that confirm serotype appear to be linked to clinically relevant host associations of the Salmonellae.     

Increased persistence of Salmonella enterica serovar Typhi in the presence of Acanthamoeba castellanii

Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of the systemic disease typhoid fever. Transmission occurs via ingestion of contaminated food or water. S. Typhi is specific to humans, and no animal or environmental reservoirs are known. As the free-living amoeba Acanthamoeba castellanii is an environmental host for many pathogenic bacteria, this study investigates interactions between S. Typhi and A. castellanii by using cocultures. Growth of both organisms was estimated by cell count, viable count, flow cytometry, and fluorescence microscopy. Results indicate that S. Typhi can survive at least 3 weeks when grown with A. castellanii, as opposed to less than 10 days when grown as singly cultured bacteria under the same conditions. Interestingly, growth rates of amoebae after 14 days were similar in cocultures or when amoebae were singly cultured, suggesting that S. Typhi is not cytotoxic to A. castellanii. Bacteria surviving in coculture were not intracellular and did not require a physical contact with amoebae for their survival. These results suggest that S. Typhi may have a selective advantage when it is associated with A. castellanii and that amoebae may contribute to S. Typhi persistence in the environment.     

The plasmid R64 thin pilus identified as a type IV pilus.

The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.     

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Summary Phages with high affinity to the S protein obtained from rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of biopanning. 9 different peptides from the enriched library were selected by ELISA. Circular dichroism (CD) spectra of the GST-S fusion protein with binding phages and non-binding phages showed that structure of the S protein was changed after it bound to each of these 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of S protein. Thus, those peptides might be used to develop plant resistance and disrupt virus transmission. 3 of the 12-mer peptide genes were fused with the GST gene in pGEX 3X. The fusion proteins were also obtained usingE. coli expression system and purified.     

The three-dimensional structure of CFA/I adhesion pili: traveler's diarrhea bacteria hang on by a spring

To survive the harsh environment of a churning intestinal tract, bacteria attach to the host epithelium via thin fibers called pili (or fimbriae). Enterotoxigenic Escherichia coli bacteria expressing colonization factor antigen I (CFA/I) pili and related pili are the most common known bacterial cause of diarrheal disease, including traveler's diarrhea. CFA/I pili, assembled via the alternate chaperone pathway, are essential for binding and colonization of the small bowel by these pathogenic bacteria. Herein, we elucidate unique structural features of CFA/I pili that appear to optimize their function as bacterial tethers in the intestinal tract. Using transmission electron microscopy of negatively stained samples in combination with iterative three-dimensional helical reconstruction methods for image processing, we determined the structure of the CFA/I pilus filament. Our results indicate that strong end-to-end protein interactions and weak interactions between the coils of a sturdy spring-like helix provide the combination of strength, stability, and flexibility required to sustain bacterial adhesion and incite intestinal disease. We propose that CFA/I pili behave like a spring to maintain attachment to the gut lining during vortex mixing and downward flow of the intestinal contents, thereby persisting long enough for these bacteria to colonize the host epithelium and cause enteric disease.     

Identification of novel vaccine candidate against Salmonella enterica serovar Typhi by reverse vaccinology method and evaluation of its immunization

Salmonella enterica serovar Typhi (S. Typhi) is an essential enteric fever causing bacterium worldwide. Due to the emergence of multidrug-resistant strains, urgently attention is needed to prevent the global spread of them. Vaccination is an alternative approach to control these kinds of infections. Currently available commercial vaccines have significant limitations such as non-recommendation for children below six years of age and poor long-term efficacy. Thus, the development of a new vaccine overcoming these limitations is immediately required. Reverse Vaccinology (RV) is one of the most robust approaches for direct screening of genome sequence assemblies to identify new protein-based vaccines. The present study aimed to identify potential vaccine candidates against S. Typhi by using the RV approach. Immunogenicity of the best candidate against S. Typhi was further investigated. The proteome of S. Typhi strain Ty2 was analyzed to identify the most immunogenic, conserved, and protective surface proteins. Among the predicted vaccine candidates, steD (fimbrial subunit) was the best for qualifying all the applied criteria. The synthetic steD gene was expressed in E.coli, and the mice were immunized with purified recombinant steD protein and then challenged with a lethal dose of S. Typhi. Immunized animals generated high protein-specific antibody titers and demonstrated 70% survival following lethal dose challenge with S. Typhi. Pretreatment of the S. Typhi cells with immunized mice antisera significantly decreased their adhesion to Caco-2 cells. Altogether, steD as a protective antigen could induce a robust and long term and protective immunity in immunized mice against S. Typhi challenge.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding.

Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.     

Pili and the interaction of Aeromonas species with humanperipheral blood polymorphonuclear cells

Abstract The interaction of differentiallu piliated Aeromonas strains expressing pili of two broadly different morphologic types (short, rigid (S/R) and/or long, wavy (L/W)) with human peripheral blood mononuclear leukocytes (PMN) was investigated to determine whether host defense cells might exert a selective pressure on pili expression in vivo accounting for the different pili phenotypes of clinical and environmental strains. A majority of Aeromonas veronii biotype sobria strains from water (6/6) and faeces (8/11) readily associated with PMN (>60% PMN with adherent and/or internalised bacteria), irrespective of their degree, or predominant type, of piliation. Rigid pili of Aeromonas species did not promote interaction with PMN. However, the majority (55%) of strains which interacted well with PMN were adherent to HEp-2 cells. Interactio with PMN is unlikely to be the reason few S/R pili are seen on faecal strains, but it may be a selective pressure on L/W adhesive pili, or other OMP adhesins, resulting in the shedding of strains which have lost critical adhesins.