Application of MALDI-TOF mass spectrometry, and DNA sequencing-based SLST and MLST analysis for the identification of Cronobacter spp. isolated from environmental surveillance samples.

Cronobacter spp. are emerging infectious foodborne bacteria that can cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. Although, little is known about its reservoirs or transmission routes, it has been linked to powdered infant formula worldwide. Three Cronobacter spp. (C. sakazakii, C. malonaticus, and C. turicensis) have been described as more virulent, and isolated frequently from infant meningitis cases. The estimated mortality rates are as high as 80% in infants. Thus, surveillance and typing of Cronobacter spp. isolated from food and environmental samples is essential to prevent contamination and spread of this pathogen. In this study, we have characterized 83 Cronobacter isolates recovered from various environmental samples by conventional microbiologic protocols. Species identification was accomplished by VITEK 2 system and real-time PCR analysis. Subsequently, these isolates were analyzed using VITEK MS system. Single locus sequence typing (SLST) was achieved by characterizing the regions of 16S rRNA and rpoB genes. Multilocus sequence typing (MLST) was performed by sequence characterization of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, and pps) using ABI 3500XL Genetic Analyzer. VITEK MS system identified, the majority of isolates as Cronobacter sakazakii with a high confidence value (99.9%). MLST analysis ascertained 12 distinct clonal complexes (CC1, CC4, CC8, CC13, CC17, CC21, CC31, CC40, CC52, CC64, CC73, and CC83) for the recovered C. sakazakii isolates. The results suggest that the MALDI-TOF MS is a reliable diagnostic tool for rapid species identification whereas 7-loci MLST is a powerful technique to discriminate and differentiate Cronobacter spp. isolates.

Infant wheezing and prenatal antibiotic exposure and mode of delivery: a prospective birth cohort study.

Introduction: Assessments on whether prenatal antibiotic exposure and mode of delivery increase the risk of wheezing in infants and toddlers are inconsistent. Our goal is to evaluate the association between prenatal antibiotic use and Cesarean section with three subtypes of wheezing in infancy.Methods: An ongoing prospective three generations cohort study provides data on prenatal antibiotic use and mode of delivery. Respective questionnaire data was used to distinguish three subtypes of wheezing: any wheezing, infectious wheezing, and noninfectious wheezing. Repeated measurements of wheezing at 3, 6, and 12 months were analyzed using generalized estimation equations. Latent transition analysis assessed patterns of infant wheezing development in the first year of life.Results: The prevalence of any wheezing was highest at 12 months (40.1%). The prevalence of infectious wheezing was higher (3 months 23.8%, 6 months 33.5%, 12 months 38.5%) than of noninfectious wheezing (3 months 13.0%, 6 months 14.0%, 12 months 11.1%). About 11-13% of children had both infectious and noninfectious wheezing at 3, 6, and 12 months (3 months 10.7%, 6 months 13.9%, 12 months 13.1%). Children born via Cesarean section have approximately a 70-80% increase in the risk of any wheezing (RR = 1.83, 95% CI 1.29-2.60) and of infectious wheezing (RR = 1.72, 95% CI 1.18-2.50).Conclusions: Analyses of infectious and noninfectious wheezing subtypes suggests that children born by Cesarean sections may be more susceptible to infectious wheezing, warranting investigations into microbial factors of infectious wheezing. No significant associations were found between prenatal antibiotic exposure and wheezing types.

A Comparative Evaluation Study of Growth Conditions for Culturing the Isolates of Campylobacter spp.

Campylobacter is one of the leading causes of foodborne travelers' diarrhea worldwide. Although a large number cases of campylobacteriosis go undiagnosed or unreported, it is considered as the second most common foodborne illness in the USA affecting over 1.3 million individuals every year. Of various Campylobacter species, C. jejuni, C. coli, and C. lari have been accounted for causing more than 99% of human infections. Thus, there is a need to have efficient isolation method to protect public health on food safety and monitoring the burden of campylobacteriosis. Nevertheless, it is a challenging task as the exposure of environmental stress during isolation process makes Campylobacter species less culturable. Sixteen Campylobacter spp. were used to evaluate the current protocols used in Campylobacter isolation. For optimal recovery, a range of growth media (Bolton, Columbia, Muller Hinton, CVA Campy and mCCDA), incubation temperatures, and additional supplements (including antibiotics) were tested. Blood agars without antibiotics were sufficient for the initial recovery. Afterward, the isolates could grow on agars without any supplements, and in some cases growth was observed in the presence of antibiotics. Incubation at 37 °C was found to be the optimal temperature for the recovery and the growth of most species. Additionally, a food adulteration study was also carried out by artificially contaminating three food matrices that included egg, milk, and infant cereal, with two isolates of C. jejuni and C. coli. Results of this study should provide the insight for culturing and isolation of Campylobacter from food and other sources.

Evaluation of Cronobacter Growth and Phenotypic Variation Under Modified Culture Conditions.

Cronobacter sakazakii is an opportunistic pathogen known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. It has been isolated from a wide range of food and environmental samples, and has been linked to outbreaks associated with powdered infant formula. This study was carried out to assess variations in growth conditions (temperature, pH, and sugar supplement) and to establish how these changes impact phenotypic characteristics for successful recovery and identification of Cronobacter, particularly for routine surveillance purposes. A total of six Cronobacter isolates were tested to evaluate the above growth conditions, including three ATCC Cronobacter reference and three environmental isolates obtained from regulatory sample screening. Although only slight changes in colony-forming units were observed across the pH range and the sugars tested, the morphology was significantly impacted by changes in these growth factors. Incubation between 30 and 50 °C resulted in growth after 24 h, and the growth was slower at ambient temperature and colony formation was most robust at 30 °C. Results of this study suggest that 30 °C may be suitable for recovery of some Cronobacter strains, and minor variations in growth conditions can alter colony morphology and appearance. Expression of unique biological characteristics based on phenotypic observations may be beneficial for differentiating various Cronobacter strains.

Genetic Characterization of Cronobacter sakazakii Recovered from the Environmental Surveillance Samples During a Sporadic Case Investigation of Foodborne Illness.

Cronobacter sakazakii is an opportunistic human-pathogenic bacterium known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. This human-pathogenic microorganism has been isolated from a variety of food and environmental samples, and has been also linked to foodborne outbreaks associated with powdered infant formula (PIF). The U.S. Food and Drug Administration have a policy of zero tolerance of these organisms in PIF. Thus, this agency utilizes the presence of these microorganisms as one of the criteria in implementing regulatory actions and assessing adulteration of food products of public health importance. In this study, we recovered two isolates of Cronobacter from the 91 environmental swab samples during an investigation of sporadic case of foodborne illness following conventional microbiological protocols. The isolated typical colonies were identified using VITEK2 and real-time PCR protocols. The recovered Cronobacter isolates were then characterized for species identification by sequencing the 16S rRNA locus. Further, multilocus sequence typing (MLST) was accomplished characterizing seven known C. sakazakii-specific MLST loci (atpD, fusA, glnS, gltB, gyrB, infB, and pps). Results of this study confirmed all of the recovered Cronobacter isolates from the environmental swab samples to be C. sakazakii. The MLST profile matched with the published profile of the complex 31 of C. sakazakii. Thus, rRNA and 7-loci MLST-based sequencing protocols are robust techniques for rapid detection and differentiation of Cronobacter species, and these molecular diagnostic tools can be used in implementing successful surveillance program and in the control and prevention of foodborne illness.

Indoor Bacterial and Fungal Burden in "Moldy" versus "Non-Moldy" Homes: A Case Study Employing Advanced Sequencing Techniques in a US Metropolitan Area.

The presence of fungi in the indoor environment is associated with allergies and other respiratory symptoms. The aim of this study was to use sequencing and molecular methods, including next-generation sequencing (NGS) approaches, to explore the bacterial and fungal communities and their abundance in the indoor environment of houses (n = 20) with visible "moldy" (HVM) and nonvisible "non-moldy" (HNM) in Memphis, TN, USA. Dust samples were collected from air vents and ground surfaces, and the total DNA was analyzed for bacteria and fungi by amplifying 16S rRNA and ITS genes on the Illumina Miseq. Results indicated that Leptosphaerulina was the most abundant fungal genus present in the air vent and ground samples from HNM and HVM. At the same time, the most abundant bacterial genera in the air vent and ground samples were Propionibacterium and Streptococcus. The fungi community diversity was significantly different in the air vent samples. The abundance of fungal species known to be associated with respiratory diseases in indoor dust samples was similar, regardless of the visibility of fungi in the houses. The existence of fungi associated with respiratory symptoms was compared with several parameters like dust particulate matter (PM), CO2 level, temperature, and humidity. Most of these parameters are either positively or negatively correlated with the existence of fungi associated with respiratory diseases; however, none of these correlations were significant at p = 0.05. Our results indicate that implementing molecular methods for detecting indoor fungi may strengthen common exposure and risk assessment practices.

A Single-Laboratory Performance Evaluation of MALDI-TOF MS in Rapid Identification of Staphylococcus aureus, Cronobacter sakazakii, Vibrio parahaemolyticus, and Some Closely Related Bacterial Species of Public Health Importance.

BACKGROUND: Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains has caused a major health care burden worldwide. Cronobacter is a group of Gram-negative bacteria that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp., and Vibrio spp. is crucial for the source tracking of contaminated food, as well as to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. OBJECTIVE: This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like isolates of public health importance. METHOD: A total of 226 isolates recovered from various food, environmental surveillance samples, and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates, whole genome sequencing (WGS) and DNA sequencing of 16S rRNA partial region were also performed for species identification. RESULTS: The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system were comparable to data obtained by the VITEK MS system. CONCLUSIONS: The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates and bacterial isolates from additional sources, in most cases. HIGHLIGHTS: The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

MALDI-TOF Mass Spectrometry and 16S rRNA Gene Sequence Analysis for the Identification of Foodborne Clostridium Spp.

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) have been 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 matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and rRNA sequencing methods. METHOD: Thirty-three Clostridium-like isolates were recovered from various baby food and surveillance samples. Species identification of these isolates was accomplished using the 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 identification was observed for the remaining five isolates. Nucleotide sequencing of the 16S rRNA region identified all 33 Clostridium-like isolates. Furthermore, while characterizing the 16S rRNA gene, 11 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 isolates was identified as 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 using the VITEK MS for the Clostridium-like isolates was comparable to that from the 16S rRNA sequencing-based data. CONCLUSIONS: The VITEK MS and 16S rRNA sequence analysis can be implemented in the species identification of Clostridium spp. isolates of public health importance. HIGHLIGHTS: MALDI-TOF MS and 16S rRNA sequencing can be used in the species identification of Clostridium species.

Application of Ribosomal Internal Transcribed Spacer 1, Internal Transcribed Spacer 2, and Large-Subunit D1-D2 Regions as the Genetic Markers to Identify Fungi Isolated from Different Environmental Samples: A Molecular Surveillance Study of Public Health Importance.

BACKGROUND: In September 2012, a multistate fungal meningitis outbreak started across 20 states in the United States. It affected 753 individuals and caused 64 deaths who received contaminated spinal injections. In a previous study, we analyzed 26 environmental samples collected from the manufacturing premises of a compounding company to determine the possible cause of an outbreak and identified 14 distinct fungal species. OBJECTIVES: In this follow-up study, we have analyzed 198 environmental samples collected from three additional compounding company premises located in the United States for the presence of pathogenic fungi. METHODS: Environmental swab samples were initially examined by standard microbiological methods. Subsequently, DNA sequencing was performed on all of the 25 recovered fungal isolates at the D1-D2 domain of the large subunit (LSU) ribosomal RNA (rRNA) and the internal transcribed spacer (ITS) regions. RESULTS: Sequence analysis of the ITS1, ITS2, and LSU rRNA regions confirmed the presence of the following fungal species in the environmental samples analyzed: (i) Pestalotiopsis cocculi from the region Ia; (ii) Epicoccum nigrum and Trichaptum biforme from the region Ib; (iii) Nigrospora sphaerica and Fusarium sp. from the region II; and (iv) Curvularia sp., Fusarium sp., Penicillium sp., and Preussia sp. from the region III. Species identification of 25 recovered fungal isolates matched, in most cases, at 3 sequenced loci (ITS1, ITS2, and LSU). HIGHLIGHTS: DNA sequencing of ITS1, ITS2, and LSU D1-D2 regions can be used to perform fungal typing and in implementing effective environmental monitoring programs of public health importance.

Species Identification of Campylobacter jejuni and Campylobacter coli Isolates from Raw Poultry Products by MALDI-TOF MS and rRNA Sequence Analysis.

BACKGROUND: Campylobacter is a genus of Gram-negative bacteria. It is considered one of the leading cause of diarrheal illness worldwide. Incidence of human campylobacteriosis has increased significantly during recent decades; the increase has been directly linked with the advancement made in Campylobacter detection methods. C. jejuni and C. coli are the two major human-pathogenic species that can colonize poultry and cause foodborne illness and outbreak. OBJECTIVE: This study was carried out to rapidly identify Campylobacter-like isolates recovered from raw poultry products by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS and DNA sequencing of 16S and 23S ribosomal RNA (rRNA) regions. METHODS: Twenty-seven isolates of Campylobacter-like organisms were isolated from raw poultry products and cultured on blood agars for MALDI-TOF MS analysis and DNA isolation. For each isolate, one to two colonies were directly spotted on the VITEK MS analysis. Genomic DNA was extracted from overnight bacterial culture. Afterward, two-directional Sanger sequencing of rRNA gene was performed to confirm species identification on an ABI 3500xL Genetic Analyzer. RESULTS: The VITEK MS could provide species-level identification for all 27 Campylobacter-like bacterial isolates (including 13 isolates as C. jejuni and 14 isolates as C. coli). Species identification attained by the VITEK MS matched completely with the rRNA sequence characterization data. CONCLUSIONS: Based on the restricted number of target strains and agar plates, MALDI-TOF MS and rRNA sequence analysis can be used for rapid identification of C. jejuni and C. coli isolates. HIGHLIGHTS: MALDI-TOF MS and rRNA sequencing can provide species identification of C. jejuni and C. coli isolates.

Association of Virulence and Antibiotic Resistance in Salmonella-Statistical and Computational Insights into a Selected Set of Clinical Isolates.

The acquisition of antibiotic resistance (AR) by foodborne pathogens, such as Salmonella enterica, has emerged as a serious public health concern. The relationship between the two key survival mechanisms (i.e., antibiotic resistance and virulence) of bacterial pathogens is complex. However, it is unclear if the presence of certain virulence determinants (i.e., virulence genes) and AR have any association in Salmonella. In this study, we report the prevalence of selected virulence genes and their association with AR in a set of phenotypically tested antibiotic-resistant (n = 117) and antibiotic-susceptible (n = 94) clinical isolates of Salmonella collected from Tennessee, USA. Profiling of virulence genes (i.e., virulotyping) in Salmonella isolates (n = 211) was conducted by targeting 13 known virulence genes and a gene for class 1 integron. The association of the presence/absence of virulence genes in an isolate with their AR phenotypes was determined by the machine learning algorithm Random Forest. The analysis revealed that Salmonella virulotypes with gene clusters consisting of avrA, gipA, sodC1, and sopE1 were strongly associated with any resistant phenotypes. To conclude, the results of this exploratory study shed light on the association of specific virulence genes with drug-resistant phenotypes of Salmonella. The presence of certain virulence genes clusters in resistant isolates may become useful for the risk assessment and management of salmonellosis caused by drug-resistant Salmonella in humans.

Genetic Characterization of Fungi Isolated from Environmental Samples: A Molecular Surveillance Study of Public Health Importance.

Background: A multistate fungal meningitis outbreak began in September 2012 that affected 751 individuals who received contaminated spinal injections across 20 states in the United States, which led to 64 deaths. In our previous study, we examined 26 environmental swab samples collected from various locations of the manufacturing premises of the compounding company to determine the possible cause of this outbreak and identified 14 novel species of fungi. Objective: In this follow-up study, a total of 198 environmental surveillance samples were collected and analyzed to detect pathogenic fungal species from other compounding company premises located in three regions of the United States. Methods: DNA sequencing was performed at the large subunit ribosomal RNA (LSU rRNA) and internal transcribed spacer (ITS) regions on the 25 positive fungal isolates. Results: Sequence analysis of the ITS1, the ITS2, and the LSU rRNA regions confirmed the presence of the following fungal species in the samples analyzed: (1) Pestalotiopsis cocculi from the region I; (2) Epicoccum nigrum and Trichaptum biforme from the region I; (3) Nigrospora sphaerica and Fusarium sp. from the region II; and (4) Curvularia sp., Fusarium sp., Penicillium sp., and Preussia sp. from the region III. Conclusions: Our results suggest that the LSU and ITS regions are good genetic markers to perform fungal typing. Highlights: DNA sequencing technology can be used in the implementation of effective environmental monitoring programs of public health importance.

Smallpox virus resequencing GeneChips can also rapidly ascertain species status for some zoonotic non-variola orthopoxviruses.

We recently developed a set of seven resequencing GeneChips for the rapid sequencing of Variola virus strains in the WHO Repository of the Centers for Disease Control and Prevention. In this study, we attempted to hybridize these GeneChips with some known non-Variola orthopoxvirus isolates, including monkeypox, cowpox, and vaccinia viruses, for rapid detection.

Rapid Detection of Staphylococcus aureus and Related Species Isolated from Food, Environment, Cosmetics, a Medical Device, and Clinical Samples Using the VITEK MS Microbial Identification System.

Staphylococcus spp. is considered as one of the most common human-pathogenic bacteria, causing illnesses ranging from nonthreatening skin infections to lethal diseases, including sepsis, pneumonia, bloodstream infections, and food poisoning. The emergence of methicillin-resistant Staphylococcus aureus strains has increased morbidity and mortality and resulted in a major healthcare burden worldwide. Single and multilocus sequence typing have been extensively used in the identification of Staphylococcus species. Nevertheless, these assays are relatively time-consuming and require high-quality DNA. Matrix-assisted laser desorption ionization-time-of-flight has been used recently for the rapid identification of several bacterial species. In this study, we have examined 47 Staphylococcus isolates recovered from food, environment, clinical samples, cosmetic products, and a medical device and 3 American Type Culture Collection Staphylococcus reference isolates using bioMérieux VITEK MS and VITEK 2 systems to determine isolate identity. Sequencing of the 16S ribosomal RNA gene was performed to confirm and compare the species identification data generated by VITEK 2 and VITEK MS systems. Although the VITEK 2 system could not identify one of the isolates, VITEK MS identified all 50 Staphylococcus spp. isolates tested. Results of this study clearly suggest that VITEK MS can be used in the rapid identification of Staphylococcus isolates of public health importance.

Application of high-throughput pyrosequencing in the analysis of microbiota of food commodities procured from small and large retail outlets in a U.S. metropolitan area - A pilot study.

With the advent of high-throughput sequencing technologies, it is possible to comprehensively analyze the microbial community of foods without culturing them in the laboratory. The estimation of all microbes inhabiting a food commodity (food microbiota) therefore may shed light on the microbial quality and safety of foods. In this study, we utilized high-throughput pyrosequencing of 16S rRNA genes as well as traditional microbiological methods to evaluate the bacterial diversity and the predicted metabolic pathways associated with the bacterial communities of selected foods (romaine lettuce, cabbage, deli meat, and chicken legs, total 200 samples) procured from small and large retail outlets located in Memphis-Shelby County, Tennessee, USA. For high-throughput sequencing, microbial genomic DNA was directly extracted from the food products and subjected to genetic sequencing. Aerobic plate count of all food samples was also performed. Foods from small stores (such as corner stores) were found to contain higher bacterial counts as compared to large stores (such as supermarkets). High-throughput pyrosequencing in tandem with bioinformatics analyses revealed a comprehensive picture of the bacterial ecology of foods at different taxonomic levels. Firmicutes and Proteobacteria were the most abundant phyla across all products. At the genus level, Enterobacter and Pantoea in vegetables, and Bacillus and Aeromonas in animal products were found to be the most abundant. The bacterial predicted metabolic pathways such as inosine-5'-phosphate biosynthesis I, methylglyoxal (MG) degradation pathways, urea cycle, dTDP-l-rhamnose biosynthesis I, and mevalonate pathway I differed in foods procured from small stores as compared to large groceries or supermarkets. The results from this study revealed that the bacterial ecology (both in terms of numbers and types of bacteria) of food commodities might differ based on the vending outlet type (large vs. small) of retail stores. The overall estimation bacterial communities in foods by high-throughput sequencing method may be useful to identify potential taxa responsible for food spoilage. Moreover, the data from pyrosequencing of 16S rRNA genes can also be applied to infer major metabolic pathways in bacteria inhabiting different foods. This may reflect the role of these pathways in food-bacteria interaction and adaptation.

Identification of Lysinibacillus fusiformis Isolated from Cosmetic Samples Using MALDI-TOF MS and 16S rRNA Sequencing Methods.

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.

Application of MALDI-TOF MS Systems in the Rapid Identification of Campylobacter spp. of Public Health Importance.

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.

Multilocus Genetic Characterization of Lactobacillus fermentum Isolated from Ready-to-Eat Canned Food.

The primary mission of the U.S. Food and Drug Administration is to enforce the Food, Drug, and Cosmetic Act and regulate food, drug, and cosmetic products. Thus, this agency monitors the presence of pathogenic microorganisms in these products, including canned foods, as one of the regulatory action criteria and also ensures that these products are safe for human consumption. This study was carried out to investigate the effectiveness of pathogen control and integrity of ready-to-eat canned food containing Black Bean Corn Poblano Salsa. A total of nine unopened and recalled canned glass jars from the same lot were examined initially by conventional microbiologic protocols that involved a two-step enrichment, followed by streaking on selective agar plates, for the presence of gram-positive and gram-negative bacteria. Of the eight subsamples examined for each sample, all subsamples of one of the containers were found positive for the presence of slow-growing rod-shaped, gram-positive, facultative anaerobic bacteria. The recovered isolates were subsequently sequenced at rRNA and gyrB loci. Afterward, multilocus sequence typing (MLST) was performed characterizing 11 additional known MLST loci (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). Analyses of the nucleotide sequences of rRNA, gyrB, and 11 MLST loci confirmed these gram-positive bacteria recovered from canned food to be Lactobacillus fermentum . Thus, the DNA sequencing of housekeeping MLST genes can provide species identification of L. fermentum and can be used in the canned food monitoring program of public health importance.

Molecular Surveillance of Cronobacter spp. Isolated from a Wide Variety of Foods from 44 Different Countries by Sequence Typing of 16S rRNA, rpoB and O-Antigen Genes.

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.

Evaluation of Two Standard and Two Chromogenic Selective Media for Optimal Growth and Enumeration of Isolates of 16 Unique Bacillus Species.

The genus Bacillus is a group of gram-positive endospore-forming bacteria that can cause food poisoning and diarrheal illness in humans. A wide range of food products have been linked to foodborne outbreaks associated with these opportunistic pathogens. The U.S. Food and Drug Administration recommends (in their Bacteriological Analytical Manual) the use of Bacara or mannitol egg yolk polymyxin (MYP) agar plates and the most-probable-number (MPN) method for enumeration and confirmation of Bacillus cereus and related species isolated from foods, sporadic cases, outbreaks, and routine environmental surveillance samples. We performed a comparative analysis of two chromogenic media (Bacara and Brilliance) and two traditional media (MYP and polymyxin egg yolk mannitol bromothymol blue agar [PEMBA]) for the isolation and enumeration of 16 Bacillus species under modified growth conditions that included pH, temperature, and dilution factor. A total of 50 environmental, food, and American Type Culture Collection reference isolates from 16 distinct Bacillus species were evaluated. A food adulteration experiment also was carried out by artificially adulterating two baby food matrices with two isolates each of B. cereus and Bacillus thuringiensis . Our results clearly indicated that chromogenic plating media (Bacara and Brilliance) are better than conventional standard media (MYP and PEMBA) for the detection and enumeration of B. cereus in foods and other official regulatory samples. The comparison of the two chromogenic media also indicated that Brilliance medium to be more efficient and selective for the isolation of Bacillus.