Keith A. Lampel

Thermocycling steps and optimization of multiplex PCR

Multiplex PCR (M-PCR), a method that detects more than two target loci in a single reaction, relies on the variables which influence single template specific PCR. We describe here the role of temperature cycles in ensuring the efficiency of detection. We have designed a multi-step protocol, which uses gradients between the temperature steps. This has facilitated the target specific annealing in the developed M-PCR. We have examined various thermocycling steps and optimized the M-PCR protocol using 105 to 101 cells of Escherichia coli, Salmonella typhi, and Vibrio cholera as template in a single reaction. The sensitivity of the detection observed was 102 cells of each pathogen used in the study.

Development of duplex PCR for the detection of Salmonella and Vibrio in drinking water.

We report here a rapid protocol for the detection of Vibrio and Salmonella in drinking water using a duplex PCR reaction. The developed protocol can detect as few as 500 cells in a single reaction, which has been achieved by optimizing the temperature steps and magnesium chloride concentration for the reactions. The described PCR protocol could detect Vibrio and Salmonella spiked in drinking water.

Prevalence of Shiga toxin-producing Shigella species isolated from French travellers returning from the Caribbean: an emerging pathogen with international implications

Shiga toxins (Stxs) are potent cytotoxins that inhibit host cell protein synthesis, leading to cell death. Classically, these toxins are associated with intestinal infections due to Stx-producing Escherichia coli or Shigella dysenteriae serotype 1, and infections with these strains can lead to haemolytic-uraemic syndrome. Over the past decade, there has been increasing recognition that Stx is produced by additional Shigella species. We recently reported the presence and expression of stx genes in Shigella flexneri 2a clinical isolates. The toxin genes were carried by a new stx-encoding bacteriophage, and infection with these strains correlated with recent travel to Haiti or the Dominican Republic. In this study, we further explored the epidemiological link to this region by utilizing the French National Reference Centre for Escherichia coli, Shigella and Salmonella collection to survey the frequency of Stx-producing Shigella species isolated from French travellers returning from the Caribbean. Approximately 21% of the isolates tested were found to encode and produce Stx. These isolates included strains of S. flexneri 2a, S. flexneri Y, and S. dysenteriae 4. All of the travellers who were infected with Stx-producing Shigella had recently travelled to Haiti, the Dominican Republic, or French Guiana. Furthermore, whole genome sequencing showed that the toxin genes were encoded by a prophage that was highly identical to the phage that we identified in our previous study. These findings demonstrate that this new stx-encoding prophage is circulating within that geographical area, has spread to other continents, and is capable of spreading to multiple Shigella serogroups.

Stx-Producing Shigella Species From Patients in Haiti: An Emerging Pathogen With the Potential for Global Spread.

Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Escherichia coli. However, the toxin genes have been detected in additional Shigella species. We recently reported the emergence of Stx-producing Shigella in travelers in the United States and France who had recently visited Hispaniola (Haiti and the Dominican Republic). In this study, we confirm this epidemiological link by identifying Stx-producing Shigella from Haitian patients attending clinics near Port-au-Prince. We also demonstrate that the bacteriophage encoding Stx is capable of dissemination to stx-negative Shigella species found in Haiti, suggesting that Stx-producing Shigella may become more widespread within that region.

Antimicrobial Resistance in Shigella Species

As with many other bacterial pathogens, Shigella species have acquired the genetic determinants to become resistant to many antibiotics used to fight bacterial infections. Antibiotic resistance by Shigella species is now a global issue and the rise in multidrug-resistant strains poses critical concerns for the future treatment of infected individuals. Traditional first-line antibiotic treatment included ampicillin, trimethoprim-sulfamethoxazole (co-trimoxazole), and nalidixic acid but resistance to these antimicrobials by Shigella spp. has become so widespread that they are no longer effective and therefore not recommended. Ciprofloxacin has become the antibiotic of choice for multiple-drug-resistant strains, however, resistance to this antimicrobial has been recorded worldwide. Alternative choices for patient treatment of all ages include other fluoroquinolones, pivmecillinam (amdinocillin pivoxil), and ceftriaxone, whereas azithromycin can be used to treat adults.

Distribution and characterization of Shiga toxin converting temperate phages carried by Shigella flexneri in Hispaniola

Shigella infections account for a considerable burden of acute diarrheal diseases worldwide and remain a major cause of childhood mortality in developing countries. Although, all four species of Shigella (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) cause bacillary dysentery, historically only S. dysenteriae type 1 has been recognized as carrying the genes for Shiga toxin (stx). Recent epidemiological data, however, have suggested that the emergence of stx carrying S. flexneri strains may have originated from bacteriophage-mediated inter-species horizontal gene transfer in one specific geographical area, Hispaniola. To test this hypothesis, we analyzed whole genome sequences of stx-encoding phages carried by S. flexneri strains isolated in Haiti and S. flexneri S. boydii and S. dysenteriae strains isolated from international travelers who likely acquired the infection in Haiti or the Dominican Republic. Phylogenetic analysis showed that phage sequences encoded in the Shigella strains from Hispaniola were bacteriophage φPOC-J13 and they were all closely related to a phage isolated from a USA isolate, E. coli 2009C-3133 serotype O119:H4. In addition, despite the low genetic heterogeneity of phages from different Shigella spp. circulating in the Caribbean island between 2001 and 2014, two distinct clusters emerged in Haiti and the Dominican Republic. Each cluster possibly originated from phages isolated from S. flexneri 2a, and within each cluster several instances of horizontal phage transfer from S. flexneri 2a to other species were detected. The implications of the emergence of stx-producing non-S. dysenteriae type 1 Shigella species, such as S. flexneri, spans not only the basic science behind horizontal phage spread, but also extends to medical treatment of patients infected with this pathogen.

Food industry current status

Abstract To ensure a safe, global food supply, food testing has remained a staple within the food industry. Today’s industry is compelled to adhere to government regulations as well as the demand from the consumer for wholesome, nutritious, and safe foods. Over the past few decades, the emergence of newer techniques to analyze foods (in particular, the polymerase chain reaction) has included the refinement of molecular-based technology, and its transition from the research laboratory to the analytical laboratory. It is important to appreciate that such methods have their strengths and weaknesses. Molecular-based technology via its sensitivity and speed has facilitated the decision-making process, and has found a home in the food analytical laboratory. This chapter describes some of this technology and its application to the food industry, and also some of the future challenges that are on the horizon.

Oligodeoxyribonucleotide Probe Specific For The 230 Kilobase Pair Virulence Plasmid in Enteroinvasive Escherichia Coli and Shigella

Enteroinvasive Escherichia coli and the four Shigella species, S. dysenteriae, S. flexneri, S. sonnei and S. boydii are the causative agents of bacillary dysentery (shigellosis) in higher primates. Shigella-induced infections are due to the ingestion of either contaminated water or, as is now becoming increasingly apparent, foods. The infective dose for humans is 101 to 104 bacterial cells, with an incubation period of 12–50 hours. The illness is characterized by cramps, nausea, diarrhea and bloody stools and usually lasts about 4 days. According to Smith,24 the primary reason for many outbreaks in the developed world is poor personal hygiene of food handlers who contaminate the food that they contact.