Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Hepatitis C Virus (HCV) Sequence Variation Induces an HCV-Specific T-Cell Phenotype Analogous to Spontaneous Resolution

Hepatitis C virus (HCV)-specific CD8⁺ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127⁺ T cells is driven by viral variation.Hepatitis C virus (HCV) persists in the majority of acutely infected individuals, potentially leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The cellular immune response has been shown to play a significant role in viral control and protection from liver disease. Phenotypic and functional studies of virus-specific T cells have attempted to define the determinants of a successful versus an unsuccessful T-cell response in viral infections (10). So far these studies have failed to identify consistent distinguishing features between a T-cell response that results in self-limiting versus chronic HCV infection; similarly, the impact of viral persistence on HCV-specific memory T-cell formation is poorly understood.Interleukin-7 (IL-7) receptor alpha chain (CD127) is a key molecule associated with the maintenance of memory T-cell populations. Expression of CD127 on CD8 T cells is typically only observed when the respective antigen is controlled and in the presence of significant CD4⁺ T-cell help (9). Accordingly, cells specific for persistent viruses (e.g., HIV, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) have been shown to express low levels of CD127 (6, 12, 14) and to be dependent on antigen restimulation for their maintenance. In contrast, T cells specific for acute resolving virus infections, such as influenza virus, respiratory syncytial virus (RSV), hepatitis B virus (HBV), and vaccinia virus typically acquire expression of CD127 rapidly with the control of viremia (5, 12, 14). Results for HCV have been inconclusive. The expected increase in CD127 levels in acute resolving but not acute persisting infection has been found, while a substantial proportion of cells with high CD127 expression have been observed in long-established chronic infection (2). We tried to reconcile these observations by studying both subjects with acute and chronic HCV infection and identified the presence of antigen as the determinant of CD127 expression.Using HLA-peptide multimers we analyzed CD8⁺ HCV-specific T-cell responses and CD127 expression levels in acute and chronic HCV infection. We assessed a cohort of 18 chronically infected subjects as well as 9 individuals with previously resolved infection. In addition, we longitudinally studied 9 acutely infected subjects (5 individuals who resolved infection spontaneously and 4 individuals who remain chronically infected) (Tables ​(Tables11 and ​and2).2). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval from the local institutional review boards. HLA-multimeric complexes were obtained commercially from Proimmune (Oxford, United Kingdom) and Beckman Coulter (CA). The staining and analysis procedure was as described previously (10). Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies: CD3 from Caltag; CD8, CD27, CCR7, CD127, and CD38 from BD Pharmingen; and PD-1 (kindly provided by Gordon Freeman). Primer sets were designed for different genotypes based on alignments of all available sequences from the public HCV database (http://hcvpub.ibcp.fr). Sequence analysis was performed as previously described (8).

TABLE 1.

Patient information and autologous sequence analysis for patients with chronic and resolved HCV infection

Single Nucleotide Polymorphism-Based Diagnostic System for Crop-Associated Sclerotinia Species

A molecular diagnostic system using single nucleotide polymorphisms (SNPs) was developed to identify four Sclerotinia species: S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. DNAs of samples are hybridized with each of five 15-bp oligonucleotide probes containing an SNP site midsequence unique to each species. For additional verification, hybridizations were performed using diagnostic single nucleotide substitutions at a 17-bp sequence of the calmodulin locus. The accuracy of these procedures was compared to that of a restriction fragment length polymorphism (RFLP) method based on Southern hybridizations of EcoRI-digested genomic DNA probed with the ribosomal DNA-containing plasmid probe pMF2, previously shown to differentiate S. sclerotiorum, S. minor, and S. trifoliorum. The efficiency of the SNP-based assay as a diagnostic test was evaluated in a blind screening of 48 Sclerotinia isolates from agricultural and wild hosts. One isolate of Botrytis cinerea was used as a negative control. The SNP-based assay accurately identified 96% of Sclerotinia isolates and could be performed faster than RFLP profiling using pMF2. This method shows promise for accurate, high-throughput species identification.Sclerotinia is distinguished morphologically from other genera in the Sclerotiniaceae (Ascomycota, Pezizomycotina, Leotiomycetes) by the production of tuberoid sclerotia that do not incorporate host tissue, by the production of microconidia that function as spermatia but not as a disseminative asexual state, and by the development of a layer of textura globulosa composing the outer tissue of apothecia (8). Two hundred forty-six species of Sclerotinia have been reported, most distinguished morphotaxonomically (Index Fungorum [www.indexfungorum.org]). These include the four species of agricultural importance now recognized plus many that are imperfectly known, seldom collected, or apparently endemic to relatively small geographic areas (2, 5, 6, 7, 8, 9, 17).The main species of phythopathological interest in the genus Sclerotinia are S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. Sclerotinia species 1 is an important cause of disease in vegetables in Alaska (16) and has been found in association with wild Taraxacum sp., Caltha palustris, and Aconitum septentrionalis in Norway (7). It is morphologically indistinguishable from S. sclerotiorum, but it was shown to be a distinct species based on distinctive polymorphisms in sequences from internal transcribed spacer 2 (ITS2) of the nuclear ribosomal repeat (7). The other three species have been delimited using morphological, cytological, biochemical, and molecular characters (3, 8, 9, 10, 12, 15). Interestingly, given that the ITS is sufficiently polymorphic in many fungal genera to resolve species, in Sclerotinia, only species 1 and S. trifoliorum are distinguished by characteristic ITS sequence polymorphisms; S. sclerotiorum and S. minor cannot be distinguished based on ITS sequence (2, 7).Sclerotinia sclerotiorum is a necrotrophic pathogen with a broad host range (1). S. minor has a more restricted host range but causes disease in a variety of important crops such as lettuce, peanut, and sunflower crops (11). S. trifoliorum has a much narrower host range, limited to the Fabaceae (3, 8, 9). Sclerotial and ascospore characteristics also serve to differentiate among the three species. Sclerotinia minor has small sclerotia that develop throughout the colony in vitro and aggregate to form crusts on the host, while the sclerotia of S. sclerotiorum and S. trifoliorum are large and form at the colony periphery in vitro, remaining separate on the host (8, 9). The failure of an isolate to produce sclerotia or apothecia in vitro is not unusual, especially after serial cultivation (8). The presence of dimorphic, tetranucleate ascospores characterizes S. trifoliorum, while S. sclerotiorum and S. minor both have uniformly sized ascospores that are binucleate and tetranucleate, respectively (9, 14).With the apparent exception of Sclerotinia species 1, morphological characteristics are sufficient to delimit Sclerotinia species given that workers have all manifestations of the life cycle in hand. In cultures freshly isolated from infected plants, investigators usually have mycelia and sclerotia but not apothecia. Restriction fragment length polymorphisms (RFLPs) in ribosomal DNA (rDNA) are diagnostic for Sclerotinia species (3, 10), but the assay requires cloned probes (usually accessed from other laboratories) hybridized to Southern blots from vertical gels, an impractical procedure for large samples. We have analyzed sequence data from previous phylogenetic studies (2) and have identified diagnostic variation for the rapid identification of the four Sclerotinia species. The single nucleotide polymorphism (SNP) assay that we report here is amenable to a high throughput of samples and requires only PCR amplification with a standard set of primers and oligonucleotide hybridizations to Southern blots in a dot format.The SNP assay was performed using two independent sets of species-specific oligonucleotide probes, all with SNP sites shown to differentiate the four Sclerotinia species (Fig. ​(Fig.1).1). A panel of 49 anonymously coded isolates (Table ​(Table1)1) was screened using these species-specific SNP probes, as outlined in Fig. ​Fig.1.1. The assay was validated by comparison to Southern hybridizations of EcoRI-digested genomic DNA hybridized with pMF2, a plasmid probe containing the portion of the rDNA repeat with the 18S, 5.8S, and 26S rRNA cistrons of Neurospora crassa (4, 10).Open in a separate windowFIG. 1.Protocol for the SNP-based identification of Sclerotinia species, with diagnostic SNP sites underlined and in boldface type for each hybridization probe.

TABLE 1.

Isolates and hybridization results for all SNP-based oligonucleotide probes^f

Genetic Detection of Extended-Spectrum β-Lactamase-Containing Escherichia coli Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal

Extended-spectrum β-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).The high and sometimes excessive use of antibiotics is directly related to the great spread and development of bacterial antibiotic resistance, a problem in public health nowadays. Thus, many studies have been done in order to understand the mechanisms of bacterial drug resistance, as many multidrug-resistant bacterial strains have been detected in domestic animals and humans (12). Recent studies from our research group have focused on wild animals, trying to understand how they acquire resistance to antibiotics without direct contact (11, 17, 20). One of the most clinically relevant antimicrobial resistance mechanisms is constituted by extended-spectrum β-lactamases (ESBLs) (3, 16) harbored by Enterobacteriaceae, such as Escherichia coli, commensals of the gastrointestinal tract of animals and humans, serving as a reservoir for resistances, namely, to β-lactam antibiotics (1, 15). Most ESBLs are derivatives of TEM or SHV enzymes, but a new family of plasmid-mediated ESBLs, CTX-M, has also been reported worldwide in E. coli and other Enterobacteriaceae. Different variants of the β-lactamase TEM, as in the case of TEM-1, are often reported in cases of plasmid-mediated β-lactamase resistance (2, 5, 8, 14, 21, 22). In this study, fecal samples from wild birds of prey at the Serra da Estrela Natural Reserve were analyzed in order to detect ESBL-containing E. coli isolates and characterize the type of ESBL-encoding genes and other associated resistance genes and the corresponding phylogenetic groups.One hundred nineteen fecal samples from birds of prey were recovered from April to July 2008 and studied for the presence of ESBL-containing E. coli strains (Table ​(Table1).1). All the fecal samples were collected individually from each animal and obtained in collaboration with CERVAS (Center of Ecology, Collecting, Welcome and Handling of Wild Animals). This Center receives injured animals found in its natural environments, is located at the Serra da Estrela Natural Reserve, and belongs to the Portuguese Institute of the Nature Management. None of the animals had been previously fed by humans or had received antibiotics. Each bird that arrives at CERVAS is placed in an individual cage to be treated and released back into the environment. Sampling was performed by recovering the fecal material in the cloaca by using a sterile swab. They were immediately transferred to peptone water and manipulated during the first 24 h of arrival to the laboratory. Samples were seeded in Levine agar supplemented with cefotaxime (2 mg/liter), and colonies with typical E. coli morphology were selected, identified by classical biochemical methods and by the API 20E system (bioMérieux, La Balme Les Grottes, France), and further studied. Susceptibility to 16 antibiotics (ampicillin, amoxicillin plus clavulanic acid [AMC], cefoxitin, cefotaxime [CTX], ceftazidime [CAZ], aztreonam [ATM], imipenem, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulfamethoxazole-trimethoprim, tetracycline, and chloramphenicol) was tested by the disc diffusion method (7) for all recovered E. coli isolates. E. coli ATCC 25922 was used as a quality control strain. The double-disc diffusion test (CTX, CAZ, and ATM in the presence or absence of AMC) was performed to detect ESBL production (7).

TABLE 1.

Animal species in which ESBL-positive E. coli isolates were recovered

Searching for Mesophilic Thermotogales Bacteria: “Mesotogas” in the Wild

All cultivated Thermotogales are thermophiles or hyperthermophiles. However, optimized 16S rRNA primers successfully amplified Thermotogales sequences from temperate hydrocarbon-impacted sites, mesothermic oil reservoirs, and enrichment cultures incubated at <46°C. We conclude that distinct Thermotogales lineages commonly inhabit low-temperature environments but may be underreported, likely due to “universal” 16S rRNA gene primer bias.Thermotogales, a bacterial group in which all cultivated members are anaerobic thermophiles or hyperthermophiles (5), are rarely detected in anoxic mesothermic environments, yet their presence in corresponding enrichment cultures, bioreactors, and fermentors has been observed using metagenomic methods and 16S rRNA gene amplification (6) (see Table S1 in the supplemental material). The most commonly detected lineage is informally designated here “mesotoga M1” (see Table S1 in the supplemental material). PCR experiments indicated that mesotoga M1 sequences amplified inconsistently using “universal” 16S rRNA gene primers, perhaps explaining their poor detection in DNA isolated from environmental samples (see text and Table S2 in the supplemental material). We therefore designed three 16S rRNA PCR primer sets (Table ​(Table1)1) targeting mesotoga M1 bacteria and their closest cultivated relative, Kosmotoga olearia. Primer set A was the most successful set, detecting a wider diversity of Thermotogales sequences than set B and being more Thermotogales-specific than primer set C (Table ​(Table22).

TABLE 1.

Primers targeting mesotoga M1 bacteria constructed and used in this study

Detection and Identification of tdh- and trh-Positive Vibrio parahaemolyticus Strains from Four Species of Cultured Bivalve Molluscs on the Spanish Mediterranean Coast

Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.Gastroenteritis caused by Vibrio parahaemolyticus has been reported worldwide, though only sporadic cases have been reported in Europe (7, 14). The bacterium can be naturally present in seafood, but pathogenic isolates capable of inducing gastroenteritis in humans are rare in environmental samples (2 to 3%) (15) and are often not detected (10, 19, 20).The virulence of V. parahaemolyticus is based on the presence of a thermostable direct hemolysin (tdh) and/or the thermostable direct hemolysin-related gene (trh) (1, 5). Both are associated with gastrointestinal illnesses (2, 9).Spain is not only the second-largest producer in the world of live bivalve molluscs but also one of the largest consumers of bivalve molluscs, and Catalonia is the second-most important bivalve producer of the Spanish Autonomous Regions. Currently, the cultivation of bivalves in this area is concentrated in the delta region of the Ebro River. The risk of potentially pathogenic Vibrio spp. in products placed on the market is not assessed by existing legislative indices of food safety in the European Union, which emphasizes the need for a better knowledge of the prevalence of diarrheal vibrios in seafood products. The aim of this study was to investigate the distribution and pathogenic potential of V. parahaemolyticus in bivalve species exploited in the bays of the Ebro delta.Thirty animals of each species of Mytilus galloprovincialis, Crassostrea gigas, Ruditapes decussatus, and Ruditapes philippinarum were collected. They were sampled from six sites of the culture area, three in each bay of the Ebro River delta, at the beginning (40°37′112"N, 0°37′092"E [Alfacs]; 40°46′723"N, 0°43′943"E [Fangar]), middle (40°37′125"N, 0°38′570"E [Alfacs]; 40°46′666"N, 0°45′855"E [Fangar]), and end (40°37′309"N, 0°39′934"E [Alfacs]; 40°46′338"N, 0°44′941"E [Fangar]) of the culture polygon. Clams were sampled from only one site per bay as follows: in the Alfacs Bay from a natural bed of R. decussatus (40°37′44"N, 0°38′0"E) and in the Fangar Bay from an aquaculture bed of R. philippinarum (40°47′3"N, 0°43′8"E). In total, 367 samples were analyzed in 2006 (180 oysters, 127 mussels, 30 carpet shell clams, and 30 Manila clams) and 417 samples were analyzed in 2008 (178 oysters, 179 mussels, 30 carpet shell clams, and 30 Manila clams).All animals were individually processed and homogenized, and 1 ml of the homogenate was inoculated into 9 ml of alkaline peptone water (Scharlau, Spain). Following a 6-h incubation at 37°C, one loopful of the contents of each tube of alkaline peptone water was streaked onto CHROMagar vibrio plates (CHROMagar, France) and incubated for 18 h at 37°C. Mauve-purple colonies were purified, and each purified isolate was cryopreserved at −80°C (135 isolates in 2006 and 96 in 2008). From the initial homogenate portion, 100 μl was inoculated onto marine agar (Scharlau, Spain) and onto thiosulfate citrate-bile salts-sucrose agar (Scharlau, Spain) for total heterotrophic marine bacteria counts and total vibrio counts, respectively (Table ​(Table11).

TABLE 1.

Vibrio parahaemolyticus isolates, serotypes, and origins and total number of vibrios/heterotrophic bacteria contained in the bivalve^a

One- and Two-Locus Population Models With Differential Viability Between Sexes: Parallels Between Haploid Parental Selection and Genomic Imprinting

A model of genomic imprinting with complete inactivation of the imprinted allele is shown to be formally equivalent to the haploid model of parental selection. When single-locus dynamics are considered, an internal equilibrium is possible only if selection acts in the opposite directions in males and females. I study a two-locus version of the latter model, in which maternal and paternal effects are attributed to the single alleles at two different loci. A necessary condition for the allele frequency equilibria to remain on the linkage equilibrium surface is the multiplicative interaction between maternal and paternal fitness parameters. In this case the equilibrium dynamics are independent at both loci and results from the single-locus model apply. When fitness parameters are additive, analytic treatment was not possible but numerical simulations revealed that stable polymorphism characterized by association between loci is possible only in several special cases in which maternal and paternal fitness contributions are precisely balanced. As in the single-locus case, antagonistic selection in males and females is a necessary condition for the maintenance of polymorphism. I also show that the above two-locus results of the parental selection model are very sensitive to the inclusion of weak directional selection on the individual''s own genotypes.PARENTAL genetic effects refer to the influence of the mother''s and father''s genotypes on the phenotypes of their offspring, not attributable just to the transfer of genes. Examples have been documented across a wide range of areas of organism biology; see, for example, Wade (1998) and ​and22 in Rasanen and Kruuk (2007). Parental selection is a more formal concept used in theoretical modeling and concerns situations where the fitness of the offspring depends, besides other factors, on the genotypes of its parent(s) (generalizing from Kirkpatrick and Lande 1989).

TABLE 1

Frequencies of genotypes and fitness parameterizations in model 1

Impact of Viral Dose and Major Histocompatibility Complex Class IB Haplotype on Viral Outcome in Mauritian Cynomolgus Monkeys Vaccinated with Tat upon Challenge with Simian/Human Immunodeficiency Virus SHIV89.6P

The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6P_cy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID₅₀]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID₅₀ (P < 0.0001), and contained acute CD4 loss at 15 MID₅₀ (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.Advances in typing of the major histocompatibility complex (MHC) of Mauritian cynomolgus macaques (14, 20, 26) have provided the opportunity to address the influence of host factors on vaccine studies (13). Retrospective analysis of 22 macaques vaccinated with Tat or a Tat-expressing adenoviral vector revealed that monkeys with the H6 or H3 MHC class IB haplotype were overrepresented among aviremic or controller animals, whereas macaques with the H2 or H5 haplotype clustered in the noncontrollers (12). More recently, the H6 haplotype was reported to correlate with control of chronic infection with simian immunodeficiency virus (SIV) mac251, regardless of vaccination (18).Here, we performed a retrospective analysis of 112 Mauritian cynomolgus macaques, which included the 22 animals studied previously (12), to evaluate the impact of the challenge dose and class IB haplotype on the acquisition and severity of simian/human immunodeficiency virus (SHIV) 89.6P_cy243 infection in 45 control monkeys and 67 monkeys vaccinated with Tat from different protocols (Table ​(Table11).

TABLE 1.

Summary of treatment, challenge dose, and outcome of infection in cynomolgus monkeys

Association of Campylobacter jejuni Cj0859c Gene (fspA) Variants with Different C. jejuni Multilocus Sequence Types

Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and responsible for more than 90% of Campylobacter infections (7). Case-control studies have identified consumption or handling of raw and undercooked poultry meat, drinking unpasteurized milk, and swimming in natural water sources as risk factors for acquiring domestic campylobacteriosis in Finland (7, 9). Multilocus sequence typing (MLST) has been employed to study the molecular epidemiology of Campylobacter (4) and can contribute to virulotyping when combined with known virulence factors (5). FspA proteins are small, acidic, flagellum-secreted nonflagellar proteins of C. jejuni that are encoded by Cj0859c, which is expressed by a σ²⁸ promoter (8). Both FspA1 and FspA2 were shown to be immunogenic in mice and protected against disease after challenge with a homologous strain (1). However, FspA1 also protected against illness after challenge with a heterologous strain, whereas FspA2 failed to do the same at a significant level. Neither FspA1 nor FspA2 protected against colonization (1). On the other hand, FspA2 has been shown to induce apoptosis in INT407 cells, a feature not exhibited by FspA1 (8). Therefore, our aim was to study the distributions of fspA1 and fspA2 among MLST types of Finnish human, chicken, and bovine strains.In total, 367 human isolates, 183 chicken isolates, and 119 bovine isolates (n = 669) were included in the analyses (3). PCR primers for Cj0859c were used as described previously (8). Primer pgo6.13 (5′-TTGTTGCAGTTCCAGCATCGGT-3′) was designed to sequence fspA1. Fisher''s exact test or a chi-square test was used to assess the associations between sequence types (STs) and Cj0859c. The SignalP 3.0 server was used for prediction of signal peptides (2).The fspA1 and fspA2 variants were found in 62.6% and 37.4% of the strains, respectively. In 0.3% of the strains, neither isoform was found. Among the human and chicken strains, fspA1 was significantly more common, whereas fspA2 was significantly more frequent among the bovine isolates (Table ​(Table1).1). Among the MLST clonal complexes (CCs), fspA1 was associated with the ST-22, ST-45, ST-283, and ST-677 CCs and fspA2 was associated with the ST-21, ST-52, ST-61, ST-206, ST-692, and ST-1332 CCs and ST-58, ST-475, and ST-4001. Although strong CC associations of fspA1 and fspA2 were found, the ST-48 complex showed a heterogeneous distribution of fspA1 and fspA2. Most isolates carried fspA2, and ST-475 was associated with fspA2. On the contrary, ST-48 commonly carried fspA1 (Table ​(Table1).1). In our previous studies, ST-48 was found in human isolates only (6), while ST-475 was found in both human and bovine isolates (3, 6). The strict host associations and striking difference between fspA variants in human ST-48 isolates and human/bovine ST-475 isolates suggest that fspA could be important in host adaptation.

TABLE 1.

Percent distributions of fspA1 and fspA2 variants among 669 human, poultry, and bovine Campylobacter jejuni strains and their associations with hosts, STs, and CCs