Using major outer membrane protein typing as an epidemiological tool to investigate outbreaks caused by milk-borne Campylobacter jejuni isolates in California.

We describe using major outer membrane protein (MOMP) typing as a screen to compare the Campylobacter jejuni porA gene sequences of clinical outbreak strains from human stool with the porA sequences of dairy farm strains isolated during two milk-borne campylobacteriosis outbreak investigations in California. The genetic relatedness of clinical and environmental strains with identical or closely related porA sequences was confirmed by multilocus sequence typing and pulsed-field gel electrophoresis analysis. The first outbreak involved 1,644 C. jejuni infections at 11 state correctional facilities and was associated with consumption of pasteurized milk supplied by an on-site dairy (dairy A) at a prison in the central valley. The second outbreak involved eight confirmed and three suspect C. jejuni cases linked to consumption of commercial raw milk and raw chocolate colostrum at another central valley dairy (dairy B). Both dairies bottled fluid milk on the farm and distributed the finished product to off-site locations. Altogether, C. jejuni was isolated from 7 of 15 (46.7%) bovine fecal, 12 of 20 (60%) flush alley water, and 1 of 20 (5%) lagoon samples collected on dairy A. At dairy B, C. jejuni was cultured from 9 of 26 (34.6%) bovine fecal samples. Environmental strains indistinguishable from the clinical outbreak strains were found in five flush alley water samples (dairy A) and four bovine fecal samples (dairy B). The findings demonstrate that MOMP typing is a useful tool to triage environmental isolates prior to conducting more labor-intensive molecular typing methods.

Rapid identification of protein biomarkers of Escherichia coli O157:H7 by matrix-assisted laser desorption ionization-time-of-flight-time-of-flight mass spectrometry and top-down proteomics.

Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALDI, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli O157:H7 and 7 strains of the "near-neighbor" serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, nonpathogenic E. coli by MALDI-TOF-TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z approximately 9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics.

An engineered R-type pyocin is a highly specific and sensitive bactericidal agent for the food-borne pathogen Escherichia coli O157:H7.

Some strains of Pseudomonas aeruginosa produce R-type pyocins, which are high-molecular-weight phage tail-like protein complexes that have bactericidal activity against other Pseudomonas strains. These particles recognize and bind to bacterial surface structures via tail fibers, their primary spectrum determinant. R-type pyocins kill the cell by contracting a sheath-like structure and inserting their hollow core through the cell envelope, resulting in dissipation of the cellular membrane potential. We have retargeted an R-type pyocin to Escherichia coli O157:H7 by fusing a tail spike protein from an O157-specific phage, phiV10, to the pyocin tail fiber. The phiV10 tail spike protein recognizes and degrades the O157 lipopolysaccharide. This engineered pyocin, termed AVR2-V10, is sensitive and specific, killing 100% of diverse E. coli O157:H7 isolates but no other serotypes tested. AVR2-V10 can kill E. coli O157:H7 on beef surfaces, making it a candidate agent for the elimination of this pathogen from food products. All rare AVR2-V10-resistant mutants isolated and examined have lost the ability to produce the O157 antigen and are expected to have compromised virulence. In addition, E. coli O157:H7 exposed to and killed by AVR2-V10 do not release Shiga toxin, as is often the case with many antibiotics, suggesting potential therapeutic applications. The demonstration that a novel R-type pyocin can be created in the laboratory by fusing a catalytic tail spike from the family Podoviridae to a tail fiber of a member of the family Myoviridae is evidence that the plasticity observed among bacteriophage tail genes can, with modern molecular techniques, be exploited to produce nonnatural, targeted antimicrobial agents.

Autoinducer-2 production in Campylobacter jejuni contributes to chicken colonization.

Inactivation of luxS, encoding an AI-2 biosynthesis enzyme, in Campylobacter jejuni strain 81-176 significantly reduced colonization of the chick lower gastrointestinal tract, chemotaxis toward organic acids, and in vitro adherence to LMH chicken hepatoma cells. Thus, AI-2 production in C. jejuni contributes to host colonization and interactions with epithelial cells.

Web-based software for rapid top-down proteomic identification of protein biomarkers, with implications for bacterial identification.

We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption ionization (MALDI), mass isolated, and fragmented using a tandem time of flight (TOF-TOF) mass spectrometer. The sequence-specific fragment ions generated were compared to a database of in silico fragment ions derived from bacterial protein sequences whose molecular weights are the same as the nominal molecular weights of the protein biomarkers. A simple peak-matching and scoring algorithm was developed to compare tandem mass spectrometry (MS-MS) fragment ions to in silico fragment ions. In addition, a probability-based significance-testing algorithm (P value), developed previously by other researchers, was incorporated into the software for the purpose of comparison. The speed and accuracy of the software were tested by identification of 10 protein biomarkers from three Campylobacter strains that had been identified previously by bottom-up proteomics techniques. Protein biomarkers were identified using (i) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions with all possible in silico N and C terminus fragment ions (i.e., ions a, b, b-18, y, y-17, and y-18), (ii) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions to residue-specific in silico fragment ions (i.e., in silico fragment ions resulting from polypeptide backbone fragmentation adjacent to specific residues [aspartic acid, glutamic acid, proline, etc.]), and (iii) fragment ion error analysis, which distinguished the systematic fragment ion error of a correct identification (caused by calibration drift of the second TOF mass analyzer) from the random fragment ion error of an incorrect identification.

Engaging people who inject drugs in hepatitis C virus testing and prevention through community-based outreach, in Sydney, Australia.

INTRODUCTION AND AIMS: Increasing treatment uptake among people who inject drugs (PWID) with chronic hepatitis C virus (HCV) infection is integral to eliminating viral hepatitis. This study explored the role of community-based outreach in engaging and retaining Australian PWID in the testing component of the HCV care cascade. DESIGN AND METHODS: Semi-structured interviews were conducted with 28 PWID, including new initiates to injecting and those from culturally and linguistically diverse (CALD) backgrounds, who acquired HCV infection while enrolled in a community-based prospective observational study of hepatitis C vaccine preparedness in Sydney. Participants were interviewed at diagnosis and 12 months later. Transcripts were thematically analysed using constant comparative techniques. RESULTS: Community-based outreach was effective in engaging newly infected participants in HCV monitoring and decision-making about seeking interferon-based treatment. Key factors in the acceptability of outreach were privacy and discretion, and opportunities to build trust with non-judgmental staff. Retaining participants in the HCV cascade of care required more than a one-off session of post-test counselling. Ongoing discussions with staff enabled paced and tailored delivery of information about HCV prevention, testing and treatment. Increased understanding of the role of HCV ribonucleic acid viremia in determining the need for treatment, and access to this testing, was pivotal in making HCV monitoring salient for participants. DISCUSSION AND CONCLUSIONS: Outreach is an effective strategy for engaging new initiates to injecting and CALD PWID in HCV testing and decision-making about treatment. Findings highlight the need to increase availability and access to HCV ribonucleic acid testing for PWID.

High-resolution genotyping of Campylobacter species by use of PCR and high-throughput mass spectrometry.

In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high-performance mass spectrometry, which "weighs" PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of A's, G's, C's, and T's). Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.

Acceptability of hepatitis C virus testing methods among injecting drug users.

INTRODUCTION AND AIMS: The acceptability of testing methods and procedures has implications for uptake of blood-borne virus screening in sentinel samples of injecting drug users (IDUs) likely to participate in surveillance. The aim of the current study was to determine the acceptability of three methods of hepatitis C virus (HCV) testing among injecting drug users (IDUs): oral fluid, capillary blood and venous blood sampling. DESIGN AND METHODS: A cross-sectional survey of IDUs was conducted in inner-city Sydney in 2005 for a laboratory validation study of HCV antibody testing. Participants were tested using the three different specimen collection methods and asked about the acceptability of each method and a particular preference documented. RESULTS: Two-hundred and twenty-nine IDUs participated in the study. Before and after specimen collection, the acceptability of all three collection methods for HCV testing was high (> 85%). Oral fluid remained the preferred method after sample collection, with females (65%) significantly more likely than males (49%) to report a preference (unadjusted odds ratio 2.0; 95% confidence interval 1.1-3.5, p = 0.03) for that method. DISCUSSION AND CONCLUSIONS: Findings suggest that oral fluid testing is an acceptable and preferred alternative for HCV testing among IDUs. However, concerns reported by participants in the study indicate that information and education regarding the nature and diagnostic value of oral fluid testing is necessary prior to its implementation for surveillance purposes among this population.

A functional human IgM response to HIV-1 Env after immunization with NYVAC HIV C.

Env-specific IgG and IgM were detected in 25 and 60%, respectively, of volunteers immunized with NYVAC expressing clade C gp120. The serum sample with the highest IgM titre but undetectable IgG neutralized the homologous isolate with a reciprocal IC90 titre of 7.8 in the absence of complement, and 24.4 in the presence of complement (P = 0.0003). These results suggest that vaccine-induced, Env-specific IgM may have antiviral activity and should be subjected to further investigation.

Binding of recombinant norovirus like particle to histo-blood group antigen on cells in the lumen of pig duodenum.

Histo-blood group antigens (HBGA) expressed on cells in the human GI tract have been shown to function as receptors for noroviruses. In concordance with earlier reports (Backer et al., 1997; Yamamoto and Yamamoto, 2001), this study found that individual pigs are either HBGA type A positive or type H1 (type O) positive. Recombinant norovirus like particles from a genogroup I (rNVLP) or three genogroup II (rMOH, rVA207, and rVA387) strains bound to plates coated with pig gastro-intestinal washings with similar binding patterns to humans. The binding of human norovirus like particles was inhibited by pre-incubating the wells with MAbs specific for either type A or type H1 HBGA, or by the presence of free HBGAs from human saliva. Co-localization of rNVLP and corresponding HBGA on epithelial cells of pig gastro-intestinal tissue (PGIT) was also observed. These findings suggest that rNVLP binds to HBGAs expressed on PGIT epithelial cells. This is the first report of the specific binding of human rNVLP to HBGAs in epithelial cells of pig gastrointestinal tissue. It highlights the importance of further study of human norovirus incidence and potential infection and residence in non-human animal hosts and suggests the possibility that norovirus may be a zoonotic pathogen.

More than just someone to inject drugs with: Injecting within primary injection partnerships.

BACKGROUND: Studies have shown intimate injection partners engage in higher rates of syringe and injecting equipment sharing. We examined the drug use context and development of injection drug use behaviors within intimate injection partnerships. METHODS: In-depth interviews (n=18) were conducted with both members of nine injecting partnerships in Sydney, Australia. Content analysis identified key domains related to the reasons for injecting with a primary injection partner and development of drug injection patterns. MAIN FINDINGS: Most partnerships (n=5) were also sexual; three were blood-relatives and one a friend dyad. The main drug injected was heroin (66%) with high rates of recent sharing behaviors (88%) reported within dyads. Injecting within a primary injection partnership provided perceived protection against overdose events, helped reduce stress, increased control over when, where, and how drugs were used, and promoted the development of an injecting pattern where responsibilities could be shared. Unique to injecting within primary injection partnerships was the social connection and companionship resulted in a feeling of fulfillment while also blinding one from recognizing risky behavior. CONCLUSIONS: Findings illuminated the tension between protection and risks within primary injection partnerships. Primary injection partnerships provide a potential platform to expand risk reduction strategies.

Condom negotiation across different relationship types by young women engaged in sex work in Phnom Penh, Cambodia.

Cambodia's 100% Condom Use Programme is credited with an increase in consistent condom use in commercial sexual interactions and a decrease in HIV prevalence among female sex workers (FSWs). There has been little improvement in condom use between FSWs and non-commercial partners, prompting calls for more innovative approaches to increasing condom use in these relationships. To understand why condoms are used or not used in sexual interactions involving FSWs, we examined condom negotiation across different types of relationships. We conducted 33 in-depth interviews with young (15 to 29 years) women engaged in sex work in Phnom Penh. There was an important interplay between the meanings of condom use and the meanings of women's relationships. Commercial relationships were characterised as inherently risky and necessitated condom use. Despite a similar lack of sexual fidelity, sweetheart relationships were rarely construed as risky and typically did not involve condom use. Husbands and wives constructed their sexual interactions with each other differently, making agreement on condom use difficult. The lack of improvement in condom use in FSWs' non-commercial sexual relationships needs to be understood in relation to both sex work and the broader Cambodian sexual culture within which these relationships are embedded.

Genome sequence of E. coli O104:H4 leads to rapid development of a targeted antimicrobial agent against this emerging pathogen.

A recent widespread outbreak of Escherichia coli O104:H4 in Germany demonstrates the dynamic nature of emerging and re-emerging food-borne pathogens, particularly STECs and related pathogenic E. coli. Rapid genome sequencing and public availability of these data from the German outbreak strain allowed us to identify an O-antigen-specific bacteriophage tail spike protein encoded in the genome. We synthesized this gene and fused it to the tail fiber gene of an R-type pyocin, a phage tail-like bacteriocin, and expressed the novel bacteriocin such that the tail fiber fusion was incorporated into the bacteriocin structure. The resulting particles have bactericidal activity specifically against E. coli strains that produce the O104 lipopolysaccharide antigen, including the outbreak strain. This O-antigen tailspike-R-type pyocin strategy provides a platform to respond rapidly to emerging pathogens upon the availability of the pathogen's genome sequence.

Motivators and barriers influencing willingness to participate in candidate HCV vaccine trials: perspectives of people who inject drugs.

BACKGROUND: A safe and efficacious vaccine may be the most efficient and cost-effective strategy for controlling the hepatitis C virus (HCV) epidemic among people who inject drugs (PWID) and several candidates are in development. However, little is known about the factors that influence willingness to participate (WTP) in candidate HCV vaccine trials among this group. METHODS: HCV seronegative PWID recruited between 2008 and 2010 as part of a prospective observational cohort study in Sydney, Australia were asked whether they would be willing to participate in a future candidate hepatitis C vaccine trial and to provide reasons to explain their decision. RESULTS: Of 113 participants, 74% indicated WTP, 15% were unwilling to participate and 11% reported WTP that was contingent on vaccine characteristics and trial design issues. The most commonly reported motivator for hypothetical trial participation was altruism, followed by potential health benefits, financial remuneration, and knowledge gain. Barriers to hypothetical participation included fears about possible harms to health, such as concerns about vaccine safety, side effects, and acquiring HCV from the vaccine; other barriers included mistrust of biomedical research and time constraints. CONCLUSIONS: These results may be useful in designing strategies to enhance HCV vaccine trial recruitment and retention and have ethical implications for developing informed consent processes and standards of care.

Using ethnographic fieldwork to inform hepatitis C vaccine preparedness studies with people who inject drugs.

BACKGROUND: Feasibility studies are an important component of preparations for field trials of biomedical prevention interventions, including vaccines. METHODS: We conducted ethnographic fieldwork to assess feasibility, including eligibility and willingness to participate, prior to recruitment of a prospective observational study of hepatitis C negative people who inject drugs (PWID) in Sydney, Australia. Five staff conducted ethnographic fieldwork in 16 locations during 2008. Observations and interactions with PWID were recorded as field notes and data were used iteratively to guide targeting of locations and the follow-up of networks and individuals. RESULTS: Findings informed the development of the study protocol, resulting in changes in the amount and type of participant reimbursement and the quantity of blood collected at screening, as well as highlighting the need for increased emphasis on communicating eligibility and exclusion criteria and study remuneration procedures. CONCLUSION: Results illustrate the value of ethnographic research in facilitating consultation and discussion with potential participants in natural settings, identifying motivations and concerns prior to study commencement and providing affected community input into the development of research protocols.

Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry.

We report covalent attachment via a thiol ester linkage of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) to cysteine-containing protein biomarkers from bacterial cell lysates of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry when using SA as the matrix. Evidence to support this conclusion is the appearance of additional peaks in the MS spectra when using SA, which are absent when using alpha-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) approximately 208 greater to the m/z of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight (TOF-TOF) mass spectrometer and top-down proteomics. Three protein biomarkers, HdeA, HdeB, and homeobox or YbgS (each containing two cysteine residues) were identified as having reactivity to SA. Non-cysteine-containing protein biomarkers showed no evidence of reactivity to SA. MS ions and MS/MS fragment ions were consistent with covalent attachment of SA via a thiol ester linkage to the side-chain of cysteine residues. MS/MS of a protein biomarker ion with a covalently attached SA revealed fragment ion peaks suggesting dissociative loss SA. We propose dissociative loss of SA is facilitated by a pentacyclic transition-state followed by proton abstraction of the beta-hydrogen of the bound SA by a sulfur lone pair followed by dissociative loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal. The apparent reactivity of SA to cysteine/disulfide-containing proteins may complicate identification of such proteins, however the apparent differential reactivity of SA and HCCA toward cysteine/disulfide-containing proteins may be exploited for identification of unknown cysteine-containing proteins.

Sub-speciating Campylobacter jejuni by proteomic analysis of its protein biomarkers and their post-translational modifications.

We have identified several protein biomarkers of three Campylobacter jejuni strains (RM1221, RM1859, and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectra (TOF MS) of bacterial cell lysate supernatants ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were: DNA-binding protein HU, translation initiation factor IF-1, cytochrome c553, a transthyretin-like periplasmic protein, chaperonin GroES, thioredoxin Trx, and ribosomal proteins: L7/L12 (50S), L24 (50S), S16 (30S), L29 (50S), and S15 (30S), and conserved proteins similar to strain NCTC 11168 proteins Cj1164 and Cj1225. The protein biomarkers identified appear to represent high copy, intact proteins. The significant findings are as follows: (1) Biomarker mass shifts between these strains were due to amino acid substitutions of the primary polypeptide sequence and not due to changes in post-translational modifications (PTMs). (2) If present, a PTM of a protein biomarker appeared consistently for all three strains, which supported that the biomarker mass shifts observed between strains were not due to PTM variability. (3) The PTMs observed included N-terminal methionine (N-Met) cleavage as well as a number of other PTMs. (4) It was discovered that protein biomarkers of C. jejuni (as well as other thermophilic Campylobacters) appear to violate the N-Met cleavage rule of bacterial proteins, which predicts N-Met cleavage if the penultimate residue is threonine. Two protein biomarkers (HU and 30S ribosomal protein S16) that have a penultimate threonine residue do not show N-Met cleavage. In all other cases, the rule correctly predicted N-Met cleavage among the biomarkers analyzed. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms for protein/pathogen identification. (5) There were fewer biomarker mass shifts between strains RM1221 and RM1859 compared to strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (and thus underlying genetic variations), this suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782 (in addition, a protein biomarker prominent in the spectra of RM1221 and RM1859 was absent from the RM3782 spectrum due to a nonsense mutation in the gene of the biomarker). These observations were confirmed by a nitrate reduction test, which showed that RM1221 and RM1859 were C. jejuni subsp. jejuni whereas RM3782 was C. jejuni subsp. doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms may easily subspeciate bacterial microorganisms. (6) Finally, the number and variation of PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more possible PTMs than suggested previously in the literature.

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within +/-5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.

Genomic and proteomic identification of a DNA-binding protein used in the "fingerprinting" of campylobacter species and strains by MALDI-TOF-MS protein biomarker analysis.

We have identified a prominent approximately 10-kDa protein biomarker observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobacter: jejuni, coli, lari, upsaliensis, and helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari(2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MALDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a approximately 10.5-kDa protein biomarker of a C. concisus strain as an HU protein.

Comparison of survival of Campylobacter jejuni in the phyllosphere with that in the rhizosphere of spinach and radish plants.

Campylobacter jejuni has been isolated previously from market produce and has caused gastroenteritis outbreaks linked to produce. We have tested the ability of this human pathogen to utilize organic compounds that are present in leaf and root exudates and to survive in the plant environment under various conditions. Carbon utilization profiles revealed that C. jejuni can utilize many organic acids and amino acids available on leaves and roots. Despite the presence of suitable substrates in the phyllosphere and the rhizosphere, C. jejuni was unable to grow on lettuce and spinach leaves and on spinach and radish roots of plants incubated at 33 degrees C, a temperature that is conducive to its growth in vitro. However, C. jejuni was cultured from radish roots and from the spinach rhizosphere for at least 23 and 28 days, respectively, at 10 degrees C. This enteric pathogen also persisted in the rhizosphere of spinach for prolonged periods of time at 16 degrees C, a temperature at which many cool-season crops are grown. The decline rate constants of C. jejuni populations in the spinach and radish rhizosphere were 10- and 6-fold lower, respectively, than on healthy spinach leaves at 10 degrees C. The enhanced survival of C. jejuni in soil and in the rhizosphere may be a significant factor in its contamination cycle in the environment and may be associated with the sporadic C. jejuni incidence and campylobacteriosis outbreaks linked to produce.