Open Database Searching Enables the Identification and Comparison of Bacterial Glycoproteomes without Defining Glycan Compositions Prior to Searching.

Mass spectrometry has become an indispensable tool for the characterization of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. Although glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses before database searching. Using this approach, we demonstrate how wide tolerance searching can be used to compare glycan use across bacterial species by examining the glycoproteomes of eight Burkholderia species (B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.

A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides.

In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.

Effect of exposure to stress conditions on propidium monoazide (PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses.

Campylobacter quantification by qPCR is unable to distinguish viable vs. dead cells in contrast to the culture-based ISO 10272-2 reference method. Propidium monoazide (PMA) has been used to overcome this disadvantage. A Campylobacter PMA-qPCR enumeration method was evaluated for its consistency and compared to the culture-based enumeration for both artificially and natural contaminated broiler carcass rinses. The PMA effect was further evaluated on stressed cells. Five conditions, commonly encountered during the slaughter process and storage (acid, heat, cold, oxidation and freezing), were inflicted to the broiler carcass rinses artificially contaminated with Campylobacter jejuni or Campylobacter coli. A better correlation between the reference method and the qPCR enumeration was obtained when PMA was used. The two cultured-based methods used showed a significant CFU reduction for heat, cold and acid stresses although the PMA-qPCR enumeration showed that viable bacteria were underestimated. Freezing showed the highest reduction effect, while the reduction extend was also overestimated by the microbiological enumeration procedure. Exposure to a mild oxidative stress was the only stress condition applied at temperatures permitting adaptation of Campylobacter and did not lead to either reduction in CFU nor in the PMA-qPCR signal.

Influence of measurement and control of microaerobic gaseous atmospheres in methods for Campylobacter growth studies.

Campylobacter is the leading cause of bacterial enteritis in the world. For this reason, this pathogen is widely studied. As a microaerophilic and capnophilic microorganism, this foodborne pathogen requires an atmosphere with reduced oxygen (O2) and elevated carbon dioxide (CO2) concentrations for its optimal growth in vitro. According to the procedure for Campylobacter spp. isolation and cultivation from food products and environmental samples, European and American standards recommend gas proportions of 5% O2 and 10% CO2, complemented with nitrogen (N2). However, in the literature, the reported proportion of O2 for microaerobic growth conditions of Campylobacter spp. can range from 2.5% to 15% and the reason for this variation is usually not explained. The use of different gas generating systems and media to detect and to grow Campylobacter from foodstuff and the lack of information about gas producing systems are the main sources of the loss of consistancy between data. In this review, the relevance, strengths and weaknesses of these methods and their impact on Campylobacter biology are discussed. In conclusion the minimum information concerning microaerobic gaseous atmospheres are suggested in order to better harmonize data obtained from research studies for a better understanding of Campylobacter features.

High-mass matrix-assisted laser desorption ionization-mass spectrometry of integral membrane proteins and their complexes.

Analyzing purified membrane proteins and membrane protein complexes by mass spectrometry has been notoriously challenging and required highly specialized buffer conditions, sample preparation methods, and apparatus. Here we show that a standard matrix-assisted laser desorption/ionization (MALDI) protocol, if used in combination with a high-mass detector, allows straightforward mass spectrometric measurements of integral membrane proteins and their complexes, directly following purification in detergent solution. Molecular weights can be determined precisely (mass error ≤ 0.1%) such that high-mass MALDI-MS was able to identify the site for N-linked glycosylation of the eukaryotic multidrug ABC transporter Cdr1p without special purification steps, which is impossible by any other current approach. After chemical cross-linking with glutaraldehyde in the presence of detergent micelles, the subunit stoichiometries of a series of integral membrane protein complexes, including the homomeric PglK and the heteromeric BtuCD as well as BtuCDF, were unambiguously resolved. This thus adds a valuable tool for biophysical characterization of integral membrane proteins.

Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products.

Using primers and fluorescent probes specific for the most common food-borne Campylobacter species (Campylobacter jejuni and Campylobacter coli), we developed a multiplex, most probable number (MPN) assay using quantitative PCR (qPCR) as the determinant for binomial detection: i.e., number of p positive pathogen growth responses out of n = 6 observations each of 4 mL (V) per dilution. Working with media washes of thrice frozen-thawed chicken pieces which had been spiked with known levels of C. jejuni and C. coli, we found that about 20% of the experiments had a significant amount of error in the form of either greater than 25% MPN calculation error (Δε) and/or a low apparent recovery rate (R less than 1 = MPN observed ÷ CFU spiked). Assuming such errors were exacerbated by an excessively small n, we examined computer-generated MPN enumeration data from the standpoint of stochastic sampling error (Δ) and found that such binomial-based assays behaved identically to Poisson-based methods (e.g., counting data) except that fewer technical replicates (n) appeared to be required for the same number of cells per test volume (µ). This result implies that the qPCR detection-based MPN protocol discussed herein should accurately enumerate a test population with a µ ≥ 1 using n = 6 observations per dilution. For our protocol, this equates to ≥ 8 cells per 400-500 g of sampled product. Based on this analysis, the error rate we saw in spiked experiments (where µ >> 1) implied a non-stochastic source. In other experiments we present evidence that this source was, at least in part, related to the cell concentration step (i.e., centrifugation). We also demonstrate that the error rate lessened (from ~38% to ~13%) at lower Campylobacter levels (µ ≤ 40) as would most likely exist in nature. Using this protocol, we were able to quantify 14 to 1,226 MPN per 450 g of naturally contaminated chicken for skinless pieces and 11 to 244 MPN per 450 g for wings, breasts, legs, and thighs (skin on) whereupon about 50% of the 29 samples tested negative for both species. Four of these chicken wash samples did have substantially lower Campylobacter levels (1 to 6 MPN per 450 g) which might be better enumerated using a larger n. However, we established that the limit of quantification of this protocol diminishes for n > 6 because one is ever more diluting the sample, or lessening V, to achieve the requisite n.

Comprehensive detection and discrimination of Campylobacter species by use of confocal micro-Raman spectroscopy and multilocus sequence typing.

A novel strategy for the rapid detection and identification of traditional and emerging Campylobacter strains based upon Raman spectroscopy (532 nm) is presented here. A total of 200 reference strains and clinical isolates of 11 different Campylobacter species recovered from infected animals and humans from China and North America were used to establish a global Raman spectroscopy-based dendrogram model for Campylobacter identification to the species level and cross validated for its feasibility to predict Campylobacter-associated food-borne outbreaks. Bayesian probability coupled with Monte Carlo estimation was employed to validate the established Raman classification model on the basis of the selected principal components, mainly protein secondary structures, on the Campylobacter cell membrane. This Raman spectroscopy-based typing technique correlates well with multilocus sequence typing and has an average recognition rate of 97.21%. Discriminatory power for the Raman classification model had a Simpson index of diversity of 0.968. Intra- and interlaboratory reproducibility with different instrumentation yielded differentiation index values of 4.79 to 6.03 for wave numbers between 1,800 and 650 cm(-1) and demonstrated the feasibility of using this spectroscopic method at different laboratories. Our Raman spectroscopy-based partial least-squares regression model could precisely discriminate and quantify the actual concentration of a specific Campylobacter strain in a bacterial mixture (regression coefficient, >0.98; residual prediction deviation, >7.88). A standard protocol for sample preparation, spectral collection, model validation, and data analyses was established for the Raman spectroscopic technique. Raman spectroscopy may have advantages over traditional genotyping methods for bacterial epidemiology, such as detection speed and accuracy of identification to the species level.

Highly efficient microscale purification of glycerophospholipids by microfluidic cell lysis and lipid extraction for lipidomics profiling.

This article presents a novel method for small-scale lipidomics of bacterial cells by integrating extraction of glycerophospholipids on a microchip with a nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometer (nanoESI-Q-TOF MS/MS). The standard starting point for typical macroscale lipid analysis is a multiphase liquid-liquid extraction. Working with small populations of cells (1 to about 1000) requires a scaled down process in order to minimize dilution and facilitate the interface with microscale separation methods for sample cleanup and introduction to mass spectrometry. We have developed a microfluidic system that allows for lysis of bacterial cells, capture of lipids, and elution of captured lipids from a solid phase for microscale purification of lipids. The best on-chip extraction efficiency for glycerophospholipids was as high as 83.3% by integrating silica beads as the packing material with methanol as the eluent. A total of 10 successive measurements were evaluated indicating that the microchip packed with fresh silica beads is capable of being reused four times without any loss in the lipid extraction process. The initial screening based on high-resolution tandem mass spectrometry data along with a discovery profiling approach revealed the presence of 173 identified phospholipid species from microfluidic cell extracts. This work demonstrates the potential of incorporating microchip-based lipid extraction into cellular lipidomics research.

Influence of cell surface hydrophobicity on attachment of Campylobacter to abiotic surfaces.

This work aimed to investigate the influence of physicochemical properties and prior mode of growth (planktonic or sessile culture) on attachment of 13 Campylobacter jejuni strains and 5 Campylobacter coli strains isolated from chicken samples to three abiotic surfaces: stainless steel, glass and polyurethane. Water contact angle and zeta potential measurements indicated that the strains varied with respect to surface hydrophobicity (17.6 ± 1.5 to 53.0 ± 2.3°) and surface charge (-3.3 ± 0.4 to -15.1 ± 0.5 mV). Individual strains had different attachment abilities to stainless steel and glass (3.79 ± 0.16 to 5.45 ± 0.08 log cell cm(-2)) but did not attach to polyurethane, with one exception. Attachment of Campylobacter to abiotic surfaces significantly correlated with cell surface hydrophobicity (P ≤ 0.007), but not with surface charge (P ≥ 0.507). Cells grown as planktonic and sessile culture generally differed significantly from each other with respect to hydrophobicity and attachment (P < 0.05), but not with respect to surface charge (P > 0.05). Principal component analysis (PCA) clustered strains into three groups (planktonic culture) and two groups (sessile culture) representing those with similar hydrophobicity and attachment. Of the four highly hydrophobic and adherent strains, three were C. coli suggesting that isolates with greater hydrophobicity and adherence may occur more frequently among C. coli than C. jejuni strains although this requires further investigation using a larger number of strains. Assignment of pulsed-field gel electrophoresis profiles to PCA groups using Jackknife analysis revealed no overall relationship between bacterial genotypes and bacterial attachment. No relationship between serotype distribution and bacterial attachment was apparent in this study.

Seasonal influence on the prevalence of thermotolerant Campylobacter in retail broiler meat in Denmark.

In Denmark, the incidence of human campylobacteriosis cases, as well as the Campylobacter prevalence in broiler flocks, is strongly influenced by season with a summer peak in July-August. Therefore, it was considered that the prevalence of Campylobacter in broiler meat sold at retail in Denmark might also be influenced by season. A retrospective survey analysis was performed on 2001-2007 national surveillance data of the prevalence of thermotolerant Campylobacter in all conventional broiler flocks at slaughter, and in randomly sampled broiler meat at retail. There was a significant effect of season on the occurrence of Campylobacter in meat at retail; the largest effect was found for domestic chilled meat. Thus, the Campylobacter prevalence in Danish broiler flocks, which fluctuated with season, was found to be a strong predictor for the occurrence of Campylobacter in fresh, chilled, Danish broiler meat. However, besides flock prevalence, there was also a direct effect of season on the occurrence of Campylobacter in Danish broiler meat at retail.

Differentiation of Campylobacter species by surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry.

The genus Campylobacter contains several, widespread pathogens causing food-borne diseases of zoonotic nature in humans. In case of outbreaks, the differentiation of closely related Campylobacter is essential for epidemiological studies, which investigate the routes of geographical spread and ways of transmission. Recent advances in mass spectrometry (MS) have shown that matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS is a valuable tool for speciation of bacteria such as Campylobacter. Surface-enhanced laser desorption/ionization (SELDI)-TOF-MS is a specific MALDI-TOF application that combines a chip-based chromatographic enrichment of proteins with TOF-MS. This pilot study aims at investigating for the first time whether SELDI-TOF-MS can be applied for discrimination of Campylobacter at the level of species and even strains. Campylobacter type-strains and isolates from different regions were cultured and subsequently subjected to physicochemical lysis. Protein lysates were then applied on CM10 and IMAC30 ProteinChip Array surfaces and analyzed using a PCS 4000 SELDI Protein Chip System (Bio-Rad Laboratories). By comparison of the spectra from Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis, and Campylobacter lari, 166 and 160 different protein peaks were observed (p<0.05) using CM10 and IMAC30 chips, respectively. Development of classification trees, comprising 2-4 of these peaks, allows for discrimination of different Campylobacter species and even strains. Moreover, species and strains can be sufficiently separated from each other by hierarchical cluster analysis. Thus, SELDI-TOF-MS is a promising tool to differentiate Campylobacter species and even strains. Species/strain-specific ions observed in addition to well-established markers identified by MALDI-TOF might be of value for future Campylobacter-identifying algorithms. To further clarify the potential advantages of this method, our results have to be validated against several independent test datasets of, preferably, a multitude of prospectively collected different isolates and compared with other typing techniques.

Campylobacter phages use hypermutable polyG tracts to create phenotypic diversity and evade bacterial resistance.

Phase variation is a common mechanism for creating phenotypic heterogeneity of surface structures in bacteria important for niche adaptation. In Campylobacter, phase variation occurs by random variation in hypermutable homonucleotide 7-11 G (polyG) tracts. To elucidate how phages adapt to phase-variable hosts, we study Fletchervirus phages infecting Campylobacter dependent on a phase-variable receptor. Our data demonstrate that Fletcherviruses mimic their host and encode hypermutable polyG tracts, leading to phase-variable expression of two of four receptor-binding proteins. This creates phenotypically diverse phage populations, including a sub-population that infects the bacterial host when the phase-variable receptor is not expressed. Such population dynamics of both phage and host promote co-existence in a shared niche. Strikingly, we identify polyG tracts in more than 100 phage genera, infecting more than 70 bacterial species. Future experimental work may confirm phase variation as a widespread strategy for creating phenotypically diverse phage populations.

Plastic-adherent DNA aptamer-magnetic bead and quantum dot sandwich assay for Campylobacter detection.

DNA aptamers were developed against MgCl(2)-extracted surface proteins from Campylobacter jejuni. The two highest affinity aptamers were selected for use in a magnetic bead (MB) and red quantum dot (QD)-based sandwich assay scheme. The assay was evaluated using both heat-killed and live C. jejuni and exhibits detection limits as low as an average of 2.5 colony forming unit (cfu) equivalents in buffer and 10-250 cfu in various food matrices. The assay exhibits low cross-reactivity with bacterial species outside the Campylobacter genus, but exhibits substantial cross-reactivity with C. coli and C. lari. The assay was evaluated with a spectrofluorometer and a commercially available handheld fluorometer, which yielded comparable detection limits and ranges. Remarkably, the sandwich assay components adhere to the inside face of polystyrene cuvettes even in food matrices near neutral pH, thereby enabling a rapid homogeneous assay, because fluorescence is concentrated to a small, thin planar area and background fluorescence from the bulk solution is minimized. The plastic cuvette-adherent technology coupled to a sensitive handheld fluorometer may enable rapid (15-20 min), portable detection of foodborne pathogens from "farm-to-fork" by obviating the slow enrichment culture phase used by other food safety tests.

Enhanced enrichment and detection of thermotolerant Campylobacter species from water using the Portable Microbe Enrichment Unit and real-time PCR.

An enhanced enrichment using the Portable Microbe Enrichment Unit (PMEU) with the microaerobic bubbling of broths was applied for the detection of thermotolerant Campylobacter species from water. This PMEU enrichment was compared with the conventional static enrichment of the international standard ISO 17995:2005. In addition, Campylobacter detection after enrichment using a real-time PCR detection was compared with colony counts. The tests with stressed Campylobacter jejuni cells in drinking water indicated that the PMEU enrichment yielded a significantly higher number of Campylobacter cells in the Bolton broth compared with the conventional static incubation. Application of the real-time PCR technique shortened the Campylobacter detection time. This combination of method modifications can be used for Campylobacter detection from water and adds methodological repertoire for the rapid survey and management of waterborne outbreaks.

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.

Occurrence of Campylobacter species in poultry forms in Lagos area of Nigeria.

Samples of poultry droppings were collected from fifty poultry farms in Lagos area of Nigeria. Campylobacter species were isolated from serially diluted samples using Bolton selective enrichment medium under microaerophilic condition at 42 degrees C. Samples of water, both treated and untreated used in the farms were also analysed in the same manner. Fifteen Campylobacter strains were isolated from the droppings. All the strains were identified using biochemical characteristics differentiating Campylobacter species. They were identified as hippurate positive (C. jejuni) and negative species (C. coli). They were biotype' using the extended scheme described by Lior (1984). Campylobacter coli biotype 'I was the most prevalent (66.66%) followed by C. jejuni biotype 1 (20.00%). The biotype ii of the two species accounted for 6.67% each. The C. jejuni biotype III and IV were not isolated during the study. The preponderance of C. coli indicates that poultry can be a major reservoir for the spread of human campylobacteriosis. Poultry farmers, therefore, must ensure prompt and adequate means of disposal of waste materials as well as maintaining good sanitary conditions.

Cloning, refolding, purification and preliminary crystallographic analysis of the sensory domain of the Campylobacter chemoreceptor for multiple ligands (CcmL).

A periplasmic sensory domain of the Campylobacter jejuni chemoreceptor for multiple ligands (CcmL) has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 as a precipitating agent. A complete data set was collected to 1.3 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belonged to space group P21, with unit-cell parameters a = 42.6, b = 138.0, c = 49.0 Å, ß = 94.3°.

Direct detection of thermotolerant campylobacters in chicken products by PCR and in situ hybridization.

We have evaluated the use of PCR and fluorescent in situ hybridization (FISH) techniques for the detection of thermotolerant campylobacters in naturally contaminated chicken products. 16S rRNA sequence data was used to design two specific primers and an oligonucleotide probe for PCR and FISH analyses, respectively. The PCR protocol amplified a 439-bp fragment corresponding to a portion of specific 16S RNA gene from thermotolerant campylobacters. The detection range of the PCR assay varied between 10 cells (after enrichment) to 10(2) cells per mL (without enrichment). FISH probes were able to identify thermotolerant Campylobacter species in 'spiked' and 'unspiked' naturally contaminated samples. PCR and FISH were performed on naturally contaminated samples and compared with the isolation of cells on selective media. The in situ hybridization technique was less sensitive than PCR, although its sensitivity of detection was increased considerably after 22 h of enrichment. These results confirm the usefulness of 16S rRNA-based techniques for the direct detection of campylobacters in food samples.

Outer membrane components of Campylobacter hyointestinalis.

Outer membranes were isolated, by sodium lauryl sulphate extraction, from the American type strain, five Australian, and four English isolates of Campylobacter hyointestinalis. On SDS-PAGE examination, the protein profiles of seven strains (including the type strain) were similar, and were dominated by two major proteins of 47 and 50 kDa. Three other isolates had unique major protein profiles. The largest of these proteins was heat-modifiable in these isolates, and in the type strain. The flagellin of three isolates screened was of similar M(r) to that of Campylobacter jejuni/Campylobacter coli. The lipopolysaccharides of C. hyointestinalis isolates were heterogeneous in structure; 5/10 isolates synthesised material of M(r) value greater than that of the low M(r) C. jejuni/C. coli lipopolysaccharide. By gel excision and re-electrophoresis, it was shown that the higher M(r) materials of one isolate were not artifactual aggregates of lower M(r) species.

Comparative study of lipopolysaccharides from Wolinella recta, W. curva, W. succinogenes and Campylobacter sputorum ssp. sputorum.

Lipopolysaccharides (LPS) were extracted from cells of Wolinella recta ATCC 33238, W. curva ATCC 33224, W. succinogenes ATCC 29543 and Campylobacter sputorum ssp. sputorum A 3563 by a hot phenol-water method and purified by nuclease treatment and by repeated ultracentrifugation. Chemical compositions of the purified LPS including fatty acid and sugar composition were examined and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. All LPS preparations contained a monosaccharide identified as L-glycero-D-mannoheptose, and another heptose isomer identified as D-glycero-D-mannoheptose was a typical constituent of the LPS from all three Wolinella species.