Patrick Chong

Evaluation of MALDI-TOF mass spectroscopy methods for determination of Escherichia coli pathotypes

It is rapidly becoming apparent that many E. coli pathotypes cause a considerable burden of human disease. Surveillance of these organisms is difficult because there are few or no simple, rapid methods for detecting and differentiating the different pathotypes. MALDI-TOF mass spectroscopy has recently been rapidly and enthusiastically adopted by many clinical laboratories as a diagnostic method because of its high throughput, relatively low cost, and adaptability to the laboratory workflow. To determine whether the method could be adapted for E. coli pathotype differentiation the Bruker Biotyper methodology and a second methodology adapted from the scientific literature were tested on isolates representing eight distinct pathotypes and two other groups of E. coli. A total of 136 isolates was used for this study. Results confirmed that the Bruker Biotyper methodology that included extraction of proteins from bacterial cells was capable of identifying E. coli isolates from all pathotypes to the species level and, furthermore, that the Bruker extraction and MALDI-TOF MS with the evaluation criteria developed in this work was effective for differentiating most pathotypes.

Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate.

Background Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15–35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. Methodology/Principal Findings Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. Conclusions/Significance This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.

Proteomic Analysis of a NAP1 Clostridium difficile Clinical Isolate Resistant to Metronidazole

Background Clostridium difficile is an anaerobic, Gram-positive bacterium that has been implicated as the leading cause of antibiotic-associated diarrhea. Metronidazole is currently the first-line treatment for mild to moderate C. difficile infections. Our laboratory isolated a strain of C. difficile with a stable resistance phenotype to metronidazole. A shotgun proteomics approach was used to compare differences in the proteomes of metronidazole-resistant and -susceptible isolates. Methodology/Principal Findings NAP1 C. difficile strains CD26A54_R (Met-resistant), CD26A54_S (reduced- susceptibility), and VLOO13 (Met-susceptible) were grown to mid-log phase, and spiked with metronidazole at concentrations 2 doubling dilutions below the MIC. Peptides from each sample were labeled with iTRAQ and subjected to 2D-LC-MS/MS analysis. In the absence of metronidazole, higher expression was observed of some proteins in C. difficile strains CD26A54_S and CD26A54_R that may be involved with reduced susceptibility or resistance to metronidazole, including DNA repair proteins, putative nitroreductases, and the ferric uptake regulator (Fur). After treatment with metronidazole, moderate increases were seen in the expression of stress-related proteins in all strains. A moderate increase was also observed in the expression of the DNA repair protein RecA in CD26A54_R. Conclusions/Significance This study provided an in-depth proteomic analysis of a stable, metronidazole-resistant C. difficile isolate. The results suggested that a multi-factorial response may be associated with high level metronidazole-resistance in C. difficile, including the possible roles of altered iron metabolism and/or DNA repair.

MicroRNA abundance is altered in synaptoneurosomes during prion disease

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimers disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).

MALDI-TOF MS detection of carbapenemase activity in clinical isolates of Enterobacteriaceae spp., Pseudomonas aeruginosa, and Acinetobacter baumannii compared against the Carba-NP assay

MALDI-TOF MS detection of carbapenemase-activity in Gram-negative bacteria was compared against the Carba-NP assay. MALDI-TOF MS detected activity from 99% of the strains, from all types of carbapenemase (200/202), while Carba-NP assays detected activity from 85% (45/53) of the tested isolates and could not consistently identify OXA- or GES carbapenemase activity.

Epstein-Barr virus in oral shedding of children with multiple sclerosis

Objective: To investigate Epstein-Barr virus (EBV) oral shedding frequency and EBV genetic diversity in pediatric patients with multiple sclerosis (MS). Methods: This was a prospective case-control study. We used PCR-based assays to detect viral DNA in the monthly mouth swabs of 22 pediatric patients with MS and 77 age- and sex-matched healthy controls. EBV-positive samples were further analyzed for sequence variation in the EBV BCRF1 (ebvIL-10) gene using direct DNA sequencing methods, and in the EBV LMP1 gene by mass spectrometry. Results: Nineteen of the 22 (86.4%) children with MS were seropositive for remote EBV infection compared to 35 out of 77 (45.5%) healthy controls (p = 0.008). Baseline analysis of mouth swabs revealed a higher proportion of EBV-positive samples from EBV-seropositive patients with MS compared to EBV-seropositive healthy controls (52.6% vs 20%, p = 0.007). Longitudinal analysis of monthly swabs revealed average EBV detection rates of 50.6% in patients with MS and 20.4% in controls (p = 0.01). The oral shedding frequencies of Herpesviruses herpes simplex virus–1, cytomegalovirus, human herpesvirus (HHV)-6, and HHV-7 did not differ between groups. Changes in the predominant EBV genetic variants were detected more frequently in patients with MS; however, no specific EBV genetic variant was preferentially associated with MS. Conclusion: Children with MS demonstrate abnormally increased rates of EBV viral reactivation and a broader range of genetic variants, suggesting a selective impairment in their immunologic control of EBV.

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight-Based Peptide Mass Fingerprinting

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

A simple shotgun proteomics method for rapid bacterial identification

Bacterial pathogens were rapidly identified by shotgun proteomics using a novel, easy to implement database search strategy. Peptide sequence data from nano-LC-MS/MS was searched against a database represented by concatenated proteomes of completed genome sequences. Select bacterial species, including BSL-3 select agents, were used to demonstrate this method.

Highly Pathogenic H5N1 and Novel H7N9 Influenza A Viruses Induce More Profound Proteomic Host Responses than Seasonal and Pandemic H1N1 Strains

Influenza A viruses (IAV) are important human and animal pathogens with potential for causing pandemics. IAVs exhibit a wide spectrum of clinical illness in humans, from relatively mild infections by seasonal strains to acute respiratory distress syndrome during infections with some highly pathogenic avian influenza (HPAI) viruses. In the present study, we infected A549 human cells with seasonal H1N1 (sH1N1), 2009 pandemic H1N1 (pdmH1N1), or novel H7N9 and HPAI H5N1 strains. We used multiplexed isobaric tags for relative and absolute quantification to measure proteomic host responses to these different strains at 1, 3, and 6 h post-infection. Our analyses revealed that both H7N9 and H5N1 strains induced more profound changes to the A549 global proteome compared to those with low-pathogenicity H1N1 virus infection, which correlates with the higher pathogenicity these strains exhibit at the organismal level. Bioinformatics analysis revealed important modulation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response in infection. Cellular fractionation and Western blotting suggested that the phosphorylated form of NRF2 is not imported to the nucleus in H5N1 and H7N9 virus infections. Fibronectin was also strongly inhibited in infection with H5N1 and H7N9 strains. This is the first known comparative proteomic study of the host response to H7N9, H5N1, and H1N1 viruses and the first time NRF2 is shown to be implicated in infection with highly pathogenic strains of influenza.

The CJIE1 prophage of Campylobacter jejuni affects protein expression in growth media with and without bile salts

BackgroundThe presence of Campylobacter jejuni temperate bacteriophages has increasingly been associated with specific biological effects. It has recently been demonstrated that the presence of the prophage CJIE1 is associated with increased adherence and invasion of C. jejuni isolates in cell culture assays.ResultsQuantitative comparative proteomics experiments were undertaken using three closely related isolates with CJIE1 and one isolate without CJIE1 to determine whether there was a corresponding difference in protein expression levels. Initial experiments indicated that about 2% of the total proteins characterized were expressed at different levels in isolates with or without the prophage. Some of these proteins regulated by the presence of CJIE1 were associated with virulence or regulatory functions. Additional experiments were conducted using C. jejuni isolates with and without CJIE1 grown on four different media: Mueller Hinton (MH) media containing blood; MH media containing 0.1% sodium deoxycholate, which is thought to result in increased expression of virulence proteins; MH media containing 2.5% Oxgall; and MHwithout additives. These experiments provided further evidence that CJIE1 affected protein expression, including virulence-associated proteins. They also demonstrated a general bile response involving a majority of the proteome and clearly showed the induction of almost all proteins known to be involved with iron acquisition. The data have been deposited to the ProteomeXchange with identifiers PXD000798, PXD000799, PXD000800, and PXD000801.ConclusionThe presence of the CJIE1 prophage was associated with differences in protein expression levels under different conditions. Further work is required to determine what genes are involved in causing this phenomenon.