Isolation and Identification of the Causative Agent for a Salmonellosis Outbreak from White Pepper

Abstract

In March 2009, epidemiologist in several western states (California, Nevada, Oregon and Washington) noted a spike in the number of cases of an extremely rare Salmonella serotype – Salmonella Rissen (mean for U.S. < 10 cases/year). A coordinated multi-state investigation ensued, but despite intensive structured interviews, no common source became apparent. Spices were considered as a vehicle from the outset because a matching isolate had been recovered from a sample of imported black pepper tested by the FDA in 2006. The purpose of this study was to isolate and quantify Salmonella in epidemiologically outbreak-associated food products.

Spice and other food samples collected from restaurants visited by cases were tested using a multiplex polymerase chain reaction assay and confirmed following the FDA-BAM method (Chapter 5). Salmonella loads in positive samples with sufficient mass were determined by FDA-BAM. Salmonella isolates were analyzed by pulsed-field gel electrophoresis (PFGE) on restriction DNA digest using Xbal and BlnI enzymes.

Introduction

The number of salmonellosis cases reported to the Centers for Disease Control (CDC) has risen to about 40,000 annually. The most common serotypes of Salmonella spp. reported each year are S. Typhimurium, S. Enteriditis, and S. Newport, comprising nearly 50 percent of the reported cases. The remainder are distributed among the approximately 2500 other serotypes. Salmonella Rissen has been reported in the US only 85 times between 1996 and 2006, an average of just under 8 per year.

In March of 2009, Health Departments in Oregon and California noted a significant increase in reported cases of S. Rissen. Extensive structured epidemiological interviews were unable to definitively identify a common source, but spices were suspected as a possible vehicle based on the interview results and the fact that the Food and Drug Administration (FDA) had isolated a strain of S. Rissen from black pepper in 2006.

Methods

Samples were collected by OR Health Department at the restaurants associated with the index cases and sent by overnight courier to IEH Laboratories in Washington State. Samples included: Mungbean, basil, Jalapeno peppers, white ground peppercorns, black pepper (two brands), Asian spice mix 1 (Pho seasoning), dried green onions, cilantro, Asian spice mix 2 ( seasoning herbs), and black pepper powder.

Subsequent sampling from restaurants in different cities throughout the Portland Metropolitan area included: white and black ground peppers (different brands), garlic powder, almonds in white pepper container, black pepper in white pepper container, curry powder, and garlic.

Samples were analyzed by screening with a Salmonella multiplex assay (IEH E. coli O157, Stx-producing E. coli (STEC) with Intimin and Salmonella Test Sytem [AOAC Certificate 100701]) and by the conventional FDA-BAM method for Salmonella.

Samples showing multiplex-PCR positive bands for Salmonella were directly and immediately streaked onto selective agar media for isolation of suspect Salmonella colonies. Suspect colonies were picked and evaluated as cell suspensions by multiplex-PCR and presumptive Salmonella identified were tested fro purity and analyzed by PFGE.

Samples were weighed into 25g portions and 225ml Trypticase Soy Broth (TSB) added and homogenized. The pre-enrichment broth was allowed to stand at room temperature for 60 +/-5 minutes, before mixing again and incubating at 35 +/-2C for 24 +/-2hr (mungbeans and cilantro were inoculated to Lactose Broth (LB) as the pre enrichment medium without a 60 minute holding period). At the end of the pre-enrichment period, 0.1ml and 1.0 ml of pre-enrichment was transferred to Rappaport Vassiliadis (RV) and Tetrathionate (TT) broths respectively. RV broth was incubated at 42+/-0.2C and TT was incubated at 35+/-2.0C for 24 +/-2hr.

The pre-enrichments and selective enrichments were tested by the IEH Multiplex kit and the selective enrichments were inoculated onto freshly prepared Xylose Lysine Deoxycholate, Hektoen and Bismuth Sulfite agars, incubated at 35 +/-0.5C. (Bismuth Sulfite agar was evaluated at 24 hours and allowed to incubate an additional 48 hours).

Typical and atypical colonies were picked for biochemical screening in Lysine Iron Agar (LIA) and Triple Sugar Iron (TSI) Agar slants, and Urea Agar. Those organisms which could not be excluded according to the FDA-BAM screening reactions were tested further by Vitek (GNI) (AOAC Method 991.13) for biochemical identification. Organisms were classified as non-Salmonella and discarded only if they were properly identified as another genus.

Isolates classified as Salmonella spp. were analyzed by pulsed-field gel electrophoresis (PFGE) on restriction DNA digests using Xba1 and Bln1 enzymes.

Samples testing culturally positive for Salmonella were retested quantitatively using the MPN approach (FDA-BAM Appenix 2) applied to the FDA-BAM Salmonella method as described above.

Sample Types and Results of Multiplex-PCR and Conventional FDA-BAM Culture Methods for Isolation of Salmonella spp.

table1

 Results of Muliplex-PCR for Salmonella spp. from white pepper sample 65726-04

figure1

 

Pulse Field Gel Electrophoresis Analysis of Salmonella Isolates Recovered from White Pepper

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Results

Thirty-eight samples were received for analysis: 13 of ground black pepper, 11 ground white pepper, and 14 other spices and condiments (mungbean sprouts, basil, Jalapeno peppers; and powdered curry and garlic). Seven of the white pepper samples (all brand X) tested positive for Salmonella. By PFGE, all the white pepper isolates were indistinguishable from clinical isolates of S. Rissen from an eventual total of 87 outbreak-associated human cases. MPN enumeration of Salmonellain five samples yielded counts from 2.8 to 1,490,000 MPN/100 g.

Multiplex-PCR analysis facilitated more rapid isolate recovery and PFGE analysis linking contaminated food source with outbreak strain.