Anna H. Bates

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.

ABSTRACT 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.

Norovirus binds to blood group A-like antigens in oyster gastrointestinal cells

Aims:  To determine if histo‐blood group antigens (HBGA) present in oyster gastrointestinal (GI) cells mediate accumulation of human noroviruses (NoV) in oyster GI cells.

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

ABSTRACT 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°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°C. This enteric pathogen also persisted in the rhizosphere of spinach for prolonged periods of time at 16°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°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.

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

ABSTRACT 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, φV10, to the pyocin tail fiber. The φV10 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.

High-Resolution Genotyping of Campylobacter Species by Use of PCR and High-Throughput Mass Spectrometry

ABSTRACT 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 As, Gs, Cs, and Ts). 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.

Web-Based Software for Rapid Top-Down Proteomic Identification of Protein Biomarkers, with Implications for Bacterial Identification

ABSTRACT 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.

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 pathogens genome sequence.

Autoinducer-2 Production in Campylobacter jejuni Contributes to Chicken Colonization

ABSTRACT 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.

Isolation of Campylobacter from feral swine (Sus scrofa) on the ranch associated with the 2006 Escherichia coli O157:H7 spinach outbreak investigation in California.

We report the isolation of Campylobacter species from the same population of feral swine that was investigated in San Benito County, California, during the 2006 spinach‐related Escherichia coli O157:H7 outbreak. This is the first survey of Campylobacter in a free‐ranging feral swine population in the United States. Campylobacter species were cultured from buccal and rectal‐anal swabs, colonic faeces and tonsils using a combination of selective enrichment and antibiotic‐free membrane filtration methods. Matrix‐assisted laser desorption ionization–time of flight mass spectrometry (MALDI‐TOF‐MS, Bruker Daltonics, Inc., Billerica, MA, USA) was used to identify species followed by confirmatory multiplex PCR or 16S rRNA sequencing. Genetic relatedness of Campylobacter jejuni and Campylobacter coli strains was determined by multilocus sequence typing (MLST) and porA allele sequencing. Altogether, 12 (40%) of 30 feral swine gastrointestinal and oral cavity specimens were positive, and six species were isolated: Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalsis, Campylobacter jejuni, Campylobacter lanienae and Campylobacter sputorum. Campylobacter jejuni subtypes were closely related to MLST sequence type 21 (ST‐21) and had identical porA sequences. Campylobacter coli subtypes were unrelated to isolates in the pubMLST/porA database. This feral swine population lived in close association with a ‘grassfed’ beef cattle herd adjacent to spinach and other leafy green row crop fields. The findings underscore the importance of protecting raw vegetable crops from faecal contamination by wild or feral animals. The study also illustrates a potential risk of Campylobacter exposure for hunters during handling and processing of wild swine meat.

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 α-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) ∼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 β-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.