Irshad M. Sulaiman

Genetic characterization of human-pathogenic Cyclospora cayetanensis parasites from three endemic regions at the 18S ribosomal RNA locus

Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Understanding the biology and epidemiology of C. cayetanensis is difficult because little is known about its origin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we developed a 70kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. cayetanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetanensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR amplified products of previously characterized C. cayetanensis isolates from three endemic regions at HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus characterized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically distinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at the18S rRNA loci.

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.

Characterization of methicillin-resistant Staphylococcus aureus isolates from fitness centers in the Memphis metropolitan area, Tennessee.

Indoor skin-contact surfaces of public fitness centers may serve as reservoirs of potential human transmission of methicillin-resistant Staphylococcus aureus (MRSA). We found a high prevalence of multidrug-resistant (MDR) MRSA of clonal complex 59 lineage harboring a variety of extracellular toxin genes from surface swab samples collected from inanimate surfaces of fitness centers in the Memphis metropolitan area, Tennessee. Our findings underscore the role of inanimate surfaces as potential sources of transmission of MDR MRSA strains with considerable genetic diversity.

Campylobacteriosis: An Emerging Infectious Foodborne Disease

Abstract Campylobacter is considered one of the leading causes of diarrheal illness in the USA and in international travelers. Even though a large number of cases of campylobacteriosis go undiagnosed or unreported, the Foodborne Diseases Active Surveillance Network (FoodNet) estimates that it is the second most common foodborne illness in the USA, affecting over 1.3 million individuals every year. Furthermore, Campylobacter makes up 8.4% of all diarrheal diseases and ranks fourth among identified pathogen that cause diarrhea worldwide. The genus Campylobacter comprises of microaerophilic, oxidase-positive, nonfermentative, Gram-negative bacteria with corkscrew motility. To date, almost a dozen species of Campylobacter have been identified as human pathogenic; Campylobacter jejuni is recognized for causing over 90% of human infection. Epidemiologic surveys have indicated that Campylobacter is frequently found in raw chicken meat in retail stores in the USA. Recovering these emerging infectious bacteria from food is still a difficult task. Molecular typing has been effective in characterizing Campylobacter isolated from food, outbreak, sporadic cases, surveillance, and environmental samples. Currently, multilocus sequence typing (MLST) and whole genome sequencing (WGS) is increasingly applied in epidemiologic investigations and transmission dynamics of bacteria causing foodborne diseases.

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.

Foodborne Pathogens in Milk and Dairy Products: Genetic Characterization and Rapid Diagnostic Approach for Food Safety of Public Health Importance

Abstract A food produced from the milk of mammals, predominantly cows, water buffalos, goats, sheep, horses, camels, and yaks, is considered as a dairy product. It contains several essential nutrients (including calcium, vitamins, and proteins) that are recognized to boost human health and reduce the risk factor of some diseases. Nevertheless, in a recent comprehensive report published by the Centers for Disease Control, the consumption of raw milk products was linked as a cause for foodborne illnesses. Dairy foods were also found as the second leading source of human-pathogenic microorganisms causing 14% of foodborne illness and 10% of deaths with 128,000 hospitalizations of Americans during the period of 1998–2008. Thus, there is a need to have effective surveillance program and robust diagnostic methods to prevent and control dairy foodborne illnesses and outbreaks. Sequence characterization of partial or full-length regions of conserved and regulatory genes can be utilized in understanding inter- and intra-species genetic variations and transmission dynamics of pathogenic microorganisms causing foodborne illness of public health importance. To date, the whole genome sequencing is considered and used by the Food and Drug Administration and others as the gold standard to ascertain pathogens isolated from food samples during foodborne outbreaks and sporadic cases of illness worldwide.

Novel Microbial Diagnostic Methods for Clinical, Environmental, and Food Samples

1Division of Epidemiology, Biostatistics, and Environmental Health Science, School of Public Health, University of Memphis, Memphis, TN, USA 2Southeast Regional Laboratory, U.S. Food and Drug Administration, Atlanta, GA, USA 3Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary 4Royal Hobart Hospital, University of Tasmania School of Medicine, Hobart, TAS, Australia 5Nestlé Research Center, Lausanne, Switzerland