A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

Cronobacter spp. are emerging infectious bacteria that can cause acute meningitis and necrotizing enterocolitis in neonatal and immunocompromised individuals. Although this opportunistic human-pathogenic microorganism has been isolated from a wide variety of food and environmental samples, it has been primarily linked to foodborne outbreaks associated with powdered infant formula. The U.S. Food and Drug Administration use the presence of these microbes as one of the criteria to assess food adulteration and to implement regulatory actions. In this study, we have examined 195 aliquots of enrichments from the nine major categories of foods (including baby and medical food, dairy products, dried food, frozen food, pet food, produce, ready-to-eat snacks, seafood, and spices) from 44 countries using conventional microbiological and molecular techniques. The typical colonies of Cronobacter were then identified by VITEK2 and real-time PCR. Subsequently, sequence typing was performed on the 51 recovered Cronobacter isolates at the 16S rRNA, rpoB and seven O-antigen loci for species identification in order to accomplish an effective surveillance program for the control and prevention of foodborne illnesses.

Background: Lysinibacillus fusiformis is a Gram-positive, rod-shaped bacterium that can cause tropical ulcers, severe sepsis, and respiratory illnesses in humans. Objective: In this study, we analyzed cosmetic samples for the presence of human pathogenic microorganisms. Methods: Five unopened jars of exfoliating cream were examined initially by microbiological methods. Afterward, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS and 16S ribosomal RNA (rRNA) sequencing techniques were applied to characterize the recovered isolates. Results: Of the eight recovered Gram-positive bacterial subs, the VITEK® MS could provide genus-level identification to five subs and species-level identification to two subs (L. fusiformis with a 99.9% confidence value); one sub was unidentified. Subsequently, the deoxyriboneucleic acid sequencing of the 16S rRNA gene was done on an ABI 3500XL Genetic Analyzer for the confirmation of species identification. An analysis of sequencing data revealed a complete absence of genetic variation among the eight subs sequenced at this locus and confirmed the eight bacterial subs to be L. fusiformis, as their respective 16S rRNA sequences were identical to the available sequence in public domain (GenBank accession No. KU179364). Conclusions: Our results suggest that the VITEK MS and the 16S rRNA sequencing can be used for the identification of human pathogenic bacteria of public health importance. Highlights: We characterized eight isolates of Lysinibacillus spp. from cosmetics by MALDI-TOF MS and 16S rRNA sequence analyses.

Background Clostridium is a genus of Gram-positive, spore-forming, anaerobic bacteria comprising approximately 100 species. Some Clostridium spp. (C. botulinum, C. perfringens, C. tetani and C. difficile) were recognized to cause acute food poisoning, botulism, tetanus, and diarrheal illness in humans. Thus, rapid identification of Clostridium spp. is critical for source tracking of contaminated food and to understand the transmission dynamics of these foodborne pathogens. Objective This study was carried out to rapidly identify Clostridium-like isolates by MALDI-TOF MS and rRNA sequencing methods. Methods Thirty-three Clostridium-like isolates were recovered from various baby food and surveillance samples. Species identification of these isolates was accomplished using VITEK MS system. Sequence characterization of the 16S rRNA region was done on an ABI 3500XL Genetic Analyzer. Results The VITEK MS system identified 28 of the 33 Clostridium-like isolates with a high confidence value (99.9%); no ID was observed for the rest of the five isolates. Nucleotide sequencing of 16S rRNA region identified all 33 Clostridium-like isolates. Furthermore, while characterizing the 16S rRNA gene, eleven distinct Clostridium spp. (Clostridium aciditolerans, Clostridium aerotolerans, Clostridium argentinense, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum, Clostridium cochlearium, Clostridium difficile, Clostridium perfringens, Clostridium sporogenes, and Clostridium subterminale) were recognized among the 33 Clostridium-like isolates. One of the Clostridium-like isolate was identified as the Citrobacter amalonaticus by both diagnostic methods. The generated 16S rRNA sequences matched completely (100%) with sequences available in GenBank for Clostridium and Citrobacter species. Species identification attained by the VITEK MS for the Clostridium-like isolates was comparable to the 16S rRNA sequencing based data. Highlights MALDI-TOF mass spectrometry and 16S rRNA sequencing can be used in the species identification of Clostridium species.