Chitrita DebRoy

DIFFERENTIATION OF MICROORGANISMS BY FTIR-ATR AND NIR SPECTROSCOPY

Identification and differentiation of different species of microorganisms were explored through spectroscopy. The nmid-infrared (MIR) and near-infrared (NIR) spectral profiles of five different microorganisms could be classified into separate ngroups when spectral data were compressed using principal component analysis (PCA) and processed by canonical variate nanalysis (CVA). Fourier transform mid-infrared (FT-MIR) spectroscopy was found to be a rapid method for identification nand differentiation of not only closely related Escherichia coli strains but also pathogenic and non-pathogenic E. coli strains. nResults clearly demonstrate the potential of FT-MIR spectroscopy as a tool for microbial strain identification and nclassification. The method is rapid, inexpensive, reproducible, and requires minimum sample preparation.

Incidence of Shiga toxin–producing Escherichia coli strains in beef, pork, chicken, deer, boar, bison, and rabbit retail meat:

The objective of the current study was to determine the incidence of contamination by the top 7 Shiga toxin–producing Escherichia coli (STEC) O-groups, responsible for the majority of E. coli infections in human beings, in retail meat from different animal species. Samples from ground beef (n = 51), ground pork (n = 16), ground chicken (n = 16), and game meat (deer, wild boar, bison, and rabbit; n = 55) were collected from retail vendors for the detection of 7 STEC O-groups (O26, O45, O103, O111, O121, O145, and O157). Meat samples were tested by using a multiplex polymerase chain reaction assay targeting the wzx gene of O antigen gene clusters of the 7 STEC O-groups. The positive samples were further tested for Shiga toxin genes (stx1 and stx2). Out of a total of 83 ground beef, pork, and chicken samples, 17 (20%) carried O121, 9 (10%) carried O45, 8 (9%) carried O157, 3 (3%) carried O103, and 1 (1%) carried O145. None of the samples were positive for O26, O111, or the stx gene. All 3 white-tailed deer samples (100%) were positive for O45, O103, or both, 2 (10%) out of 20 red deer samples exhibited the presence of O103, and all 3 bison samples were contaminated with either O121, O145, or O157. One sample from ground deer, contaminated with E. coli O45, carried the stx1 gene. This preliminary investigation illustrates the importance of microbiological testing of pathogens in meat products, as well as the recognized need for increased surveillance and research on foodborne pathogens.

Comparative Analysis of Super-Shedder Strains of Escherichia coli O157:H7 Reveals Distinctive Genomic Features and a Strongly Aggregative Adherent Phenotype on Bovine Rectoanal Junction Squamous Epithelial Cells

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as “super-shedders” (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

DIFFERENTIATION OF FOOD PATHOGENS USING FTIR AND ARTIFICIAL NEURAL NETWORKS

FTIR absorbance spectra in conjunction with artificial neural networks (ANNs) were used to differentiate selected nmicroorganisms at the generic and serogroup levels. The ANN consisted of three layers with 595 input nodes, 50 nodes at the nhidden layer, and 5 output nodes (one for each microorganism or strain). Ten replications of each experiment were conducted, nand 70% of the data was used for training and 30% for validation of the network. Results indicated that differentiation could nbe achieved at an accuracy of 80% to 100% at the generic level and 90% to 100% at the serogroup level at 103 CFU/mL nconcentration.

DETECTION OF BACTERIA WITH LOW-RESOLUTION RAMAN SPECTROSCOPY

Bacteria detection methods that are presently used in laboratories and quality control inspections, such as nserological testing, biological enrichment, culturing, and gas chromatograph mass spectroscopy (GCMS), are expensive, nlabor intensive, and time consuming. Therefore, in order to ensure that consumers receive a safe and high-quality product, nrapid and reliable methods need to be developed for detection of pathogens. Raman spectroscopy, an optical technique based non light scattering, was investigated as a means of rapid on-site produce safety assessment. In this study, a dispersive system nspectrophotometer, with a 785 nm diode laser, was employed. Chemometric methods such as partial least squares (PLS) nregression and classification analysis were used to evaluate low-concentration suspensions of Erwinia carotovora pv. ncarotovora (ECC) and Clavibacter michiganense (CBM). The pathogens chosen represent Gram-positive and Gram-negative nbacteria. A clear distinction between samples containing bacteria and clean samples was obtained by this method. The system nwas able to determine bacterial concentrations within 2% of the level in the basic bacteria suspension, based on PLS nregression models. Classification analysis enables researchers to detect the presence of each of the tested bacteria in nmixed-bacteria suspensions that contain between 10 and 100 cells/mL of ECC and CBM.

A high-throughput antibody-based microarray typing platform.

Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the “Big Six” non-O157 Shiga toxin-producing E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers), this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.

Spectroscopic Quantification of Bacteria Using Artificial Neural Networks

Fourier transform-infrared spectroscopy, in conjunction with artificial neural networks, has been used for identification and classification of selected foodborne pathogens. Five bacterial species (Enterococcus faecium, Salmonella Enteritidis, Bacillus cereus, Yersinia enterocolitica, Shigella boydii) and five Escherichia coli strains (O103, O55, O121, O30, O26) suspended in phosphate-buffered saline were enumerated to provide seven different concentrations ranging from 10(9) to 10(3) CFU/ ml. The trained artificial neural networks were then validated with an independent subset of samples and compared with the traditional plate count method. It was found that the concentration-based classification of the species was 100% correct and the strain-based classification was 90 to 100% accurate.

Necrotizing pneumonia and pleuritis associated with extraintestinal pathogenic Escherichia coli in a tiger (panthera tigris) cub

Extraintestinal pathogenic Escherichia coli (ExPEC) cause diseases in humans and animals, affecting organs outside the alimentary canal. In recent years, ExPEC have been reported as a cause of fatal pneumonia in dogs, cats, and in a horse. In the current report, a fatal case of pneumonia and pleuritis is described in a 4-week-old tiger (Panthera tigris) cub associated with ExPEC. The cub was presented with a sudden-onset respiratory illness and died after a few hours. Postmortem examination of the cub revealed an acute necrotizing pneumonia. The alveolar spaces were filled with large numbers of inflammatory cells (predominantly macrophages), edema, fibrin strands, and short bacillary bacteria. Escherichia coli O6:H31 was isolated in pure culture from the affected lung. It carried virulence genes cnf-1, sfa, fim, hlyD, and papG allele III, which are known to be associated with ExPEC strains. No evidence of infection by any other agent was detected. This is the first report, to the authors knowledge, in which ExPEC has been associated with pneumonia in tigers.

Escherichia coli O-Antigen Gene Clusters of Serogroups O62, O68, O131, O140, O142, and O163: DNA Sequences and Similarity between O62 and O68, and PCR-Based Serogrouping.

The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources.

Hemolytic-uremic syndrome in a postpartum mare concurrent with encephalopathy in the neonatal foal

A postpartum mare and foal were presented for evaluation of fever and lethargy in the mare. The mare was diagnosed with endometritis and initially responded well to treatment. On the second day of hospitalization, the mare developed renal insufficiency characterized by oliguria, azotemia, hemolysis, and thrombocytopenia. Concurrently, the foal developed rapidly progressive central nervous system signs culminating in refractory seizures. Both animals failed to respond to treatment and were euthanized. Thrombotic microangiopathy involving glomeruli was evident on microscopic examination of the mares kidneys. Microscopic evidence of brain edema was the principal postmortem finding in the foal. No specific etiology was confirmed in either case. Notably, Escherichia coli 0103:H2 was isolated from the mares uterus and the gastrointestinal tracts of both animals. To the authors knowledge, this is the first report in which an organism implicated as a cause of hemolytic-uremic syndrome was isolated from an animal with clinical signs and postmortem findings consistent with the disease.