Standard Analytical Protocol for Non-Typhoidal in Drinking and Surface Water - USEPA

Jeremy Olstadt WI State Laboratory of Hygiene, Madison, WI Background

• Prolonged survival in water • 1.2 million illnesses in the US per year (CDC)- most are foodborne • Non-typhoidal Salmonella genus is comprised of Salmonella bongori and • 2500 know serotypes – all of which are potential human pathogens • Only a few serotypes cause the majority of illness

• Haley (2009) Background

• Salmonellosis

• Symptoms: diarrhea, vomiting, abdominal cramps • Develops 12-72 hours after infection • Lasts 4-7 days Standard Analytical Procedure - Salmonella • BSL-2 Laboratory

• USEPA Method 1682: Salmonella in Sewage Sludge (Biosolids)

• Support of EPA Homeland Security Efforts Summary of Method

• Identification of Salmonella by: Non-Selective and Selective Media

Morphological characteristics

Biochemical characteristics

Serological characteristics Non-Selective Media – XLD Plate, Triple Sugar Iron and Salmonella O antiserum agglutination

XLD TSI Antibody Test Selective Media – MSRV Plate-Modified Semisolid Rappaport- Vassiliadis Agar Quantitative Sample Analysis

• 15 Tube MPN (most probable number)Method  3 Rows of 5 tubes  10mL of 3X (TSB), 5ml of 3X(TSB) and 10mL of 1X(TSB) Inoculate 10mL 3X TSB tubes with 20 mL of sample Inoculate 5mL 3X TSB with 10mL of sample Inoculate 10mL of 1X TSB with 1 mL of sample Incubate at 36.0oC for 24+ 2 hours Arrangement of TSB Tubes for initial enrichment of Salmonella on MSRV Plates

• Select TSB tubes exhibiting turbidity • Inoculate MSRV plate with six 30uL drops • Each tube uses a separate MSRV plate • Space drops evenly across plate to prevent overlap of spots • Allow plates to absorb to media for 1 hour • Incubate at 42oC + 0.5oC for 16-18 hours Selective Media – MSRV Plate – Modified Semisolid Rappaport Vassiliadis Agar Isolation on XLD Plates

• Examine MSRV plates for motility (whitish halo) • Using an inoculating loop, stab into halo and streak for isolation onto XLD plates • Incubate at 36.0oC + 1.5oC for 18-24 hours • Following incubation look for black and/or pink to red colonies with black center on XLD plates Pure and Mixed Cultures on XLD

Pure culture Mixed culture Biochemical Confirmation of Salmonella • Triple Sugar Iron Slant (TSI) • Inoculate a TSI slant with an inoculating needle containing a portion a colony from the XLD plate • Stab into butt of slant and streak the slant • Incubate “loose-capped” at 36.0oC+ 1.5oC for 24+ 2 hours • Positive for Salmonella will have acid(yellow) butt and alkaline (red) slant and possible H2S production XLD Plate, Triple Sugar Iron and Salmonella O antiserum agglutination Testing

XLD TSI Antibody Test Biochemical Confirmation of Salmonella • (LIA) • Similar to TSI inoculate an LIA slant • Positive Salmonella have alkaline (purple) butt

and alkaline (purple) slant and possible H2S production Biochemical Confirmation of Salmonella • Urea Broth • Use a sterile loop to inoculate the broth with a portion of a potential positive colony • Salmonella are -negative • If Salmonella, the result will be no color change in urea broth Urea broth

Control Salmonella Serological Analysis - Confirmation

• Inoculate a portion of bacteria from the TSI tube into sterile saline • Place two drops onto a slide • Add one drop of antisera to one and one drop of saline to the other • View under magnification for agglutination reaction • Perform a + and – ctrl along side at the same time for comparison XLD Plate, Triple Sugar Iron and Salmonella O antiserum agglutination Testing

XLD TSI Antibody Test MPN Methodology

• Count the number of positive or confirmed Salmonella positive TSB tubes from the initial step • Apply the number of positive tubes to the MPN table • The volumes analyzed will determine which MPN table you will use • Ex. 20mL,10mL or 1mL vs. 10mL, 1mL or 0.1mL Example MPN Calculation

• Three rows of TSB tubes used for 20mL, 10ml and 1mL volumes • Result was turbidity/growth in: – 5 tubes positive containing 20mL of sample – 3 tubes positive containing 10mL of sample – 0 tubes positive containing 1 mL of sample – Designation on chart would be 5-3-0 – 5-3-0 MPN= 0.1151/mL or 11.51/100mL of sample Most Probable Number (MPN) Chart Qualitative Sample Analysis

• Samples diluted 1:1 in 2X TSB (Tryptic Soy Broth) • Sample volume of 100mL would use 100mL of 2X TSB • Incubated at 36oC + 1.5oC for 24 + 2 Hours • Followed by isolation using MSRV, XLD, TSI, LIA, Urea Broth and Salmonella O antiserum for confirmation Strengths and Weaknesses of the Method • Strengths – Detects live (potentially infective) cells – Quantitative abilities – Multiple layers of specificity • Selectivity of MSRV • Selectivity of XLD – Distinctive positive reactions

• Weakness – TSB permissive growth conditions, need for increased selectivity in initial step – Interference by ubiquitous bacteria and toxic substances – Numerous tubes and plates needed for one quantification – Time to completed test – five days Thanks for your attention

• Jeremy Olstadt • Wisconsin State Laboratory of Hygiene • [email protected] • 608 224-6262