Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.

Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.

Re-evaluation, optimization, and multilaboratory validation of the PulseNet-standardized pulsed-field gel electrophoresis protocol for Listeria monocytogenes.

The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms.

Evaluation and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping Vibrio parahaemolyticus: an international multicenter collaborative study.

The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.

Laboratory-based surveillance of paratyphoid fever in the United States: travel and antimicrobial resistance.

BACKGROUND: The incidence of paratyphoid fever, including paratyphoid fever caused by antimicrobial-resistant strains, is increasing globally. However, the epidemiologic and laboratory characteristics of paratyphoid fever in the United States have never been studied. METHODS: We attempted to interview all patients who had been infected with laboratory-confirmed Salmonella serotypes Paratyphi A, Paratyphi B, or Paratyphi C in the United States with specimens collected from 1 April 2005 through 31 March 2006. At the Centers for Disease Control and Prevention (CDC), isolates underwent serotype confirmation, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis typing. RESULTS: Of 149 patients infected with Salmonella Paratyphi A, we obtained epidemiologic information for 89 (60%); 55 (62%) of 86 were hospitalized. Eighty-five patients (96%) reported having travel internationally, and 80 (90%) had traveled to South Asia. Of the 146 isolates received at the CDC, 127 (87%) were nalidixic acid resistant; nalidixic acid resistance was associated with travel to South Asia (odds ratio, 17.0; 95% confidence interval, 3.8-75.9). All nalidixic acid-resistant isolates showed decreased susceptibility to ciprofloxacin (minimum inhibitory concentration, > or = 0.12 microg/mL). Of 49 patients infected with Salmonella Paratyphi B, only 12 (24%) were confirmed to have Paratyphi B when tested at the CDC. Four (67%) of 6 patients were hospitalized, and 5 (83%) reported travel (4 to the Andean region of South America). One case of Salmonella Paratyphi C infection was reported in a traveler to West Africa with a urinary tract infection. CONCLUSIONS: Physicians should be aware of the increasing incidence of infection due to Salmonella Paratyphi A and treatment options given its widespread antimicrobial resistance. A paratyphoid fever vaccine is urgently needed. Continued surveillance for paratyphoid fever will help guide future prevention and treatment recommendations.

Human Salmonella and concurrent decreased susceptibility to quinolones and extended-spectrum cephalosporins.

The National Antimicrobial Resistance Monitoring System monitors susceptibility among Enterobacteriaceae in humans in the United States. We studied isolates exhibiting decreased susceptibility to quinolones (nalidixic acid MIC >32 microg/mL or ciprofloxacin MIC > or =0.12 microg/mL) and extended-spectrum cephalosporins (ceftiofur or ceftriaxone MIC > or =2 microg/mL) during 1996-2004. Of non-Typhi Salmonella, 0.19% (27/14,043) met these criteria: 11 Senftenberg; 6 Typhimurium; 3 Newport; 2 Enteridis; and 1 each Agona, Haifa, Mbandaka, Saintpaul, and Uganda. Twenty-six isolates had gyrA mutations (11 at codon 83 only, 3 at codon 87 only, 12 at both). All Senftenberg isolates had parC mutations (S801 and T57S); 6 others had the T57S mutation. The Mbandaka isolate contained qnrB2. Eight isolates contained bla(CMY-2); 1 Senftenberg contained bla(CMY-23). One Senftenberg and 1 Typhimurium isolate contained bla(SHV-12); the Mbandaka isolate contained bla(SHV-30). Nine Senftenberg isolates contained bla(OXA-1) contained bla(OXA-9). Further studies should address patient outcomes, risk factors, and resistance dissemination prevention strategies.

Development of a universal probe for electronic microarray and its application in characterization of the Staphylococcus aureus polC gene.

Electronic microarray technology is an exceptionally accurate and effective technique for detecting and defining single nucleotide polymorphisms (SNPs) in DNA sequences. Target oligonucleotides are electronically addressed to a gel matrix containing streptavidin to which biotinylated polymerase chain reaction (PCR) amplicons are bound. Typically, a fluorescent-labeled reporter oligonucleotide specific for each locus of interest is hybridized and reported. We detail the development of a universal reporter system to replace the standard method that is used to detect many different sequences accurately. The universal reporter eliminates the need to synthesize specific labeled reporters for each SNP sequence thereby dramatically reducing the cost and time required for assay development. The feasibility of this approach was demonstrated by successfully analyzing eight SNPs distributed within a highly variable 1-kb region of the polC gene from six isolates of Staphylococcus aureus.