Salmonellosis - Chula · Salmonellosis outbreak study!Epidemiological investigation!to assess potential risk factors for infection!the department of health conducted a case-control

SalmonellosisSalmonellosis

Salmonella Salmonella sppspp..

!More than 2000 serovar!Gram negative, rod shape,non spore forming bacteria!Facultative anaerobes!Optimum growth at 37 °C and grow well at 43 ° C!Motile (except S. gallinarum and S. pullorum)!Cause Salmonellosis in humans and animals (broad

host ranges)!Worldwide distribution

O antigen

H antigen

SalmonellaSalmonella sppspp

!Widespread occurrence of Salmonellosis in animals,especially in poultry and swine

!Environmental sources of the organism include water,soil, insects, factory surfaces, kitchen surfaces,animal feces, raw meats, raw poultry, and raw sea-foods

!S. typhi and the paratyphoid bacteria are normallycaused septicemia and produce typhoid or typhoid-like fever in humans

!Other forms of Salmonellosis generally producemilder symptoms.


!Salmonella sp. are second only to Campylobacter asa cause of bacterial diarrhea illness in the UnitedStates

! I t is estimated that from 2 to 4 million cases ofSalmonellosis occur in the U.S. annually

!Salmonella enteritidis has ranked first or second infrequency of isolation from humans since 1988 andaccounted for 21% of reported isolates in 1993.

! Each year, an average of 55 outbreaks of SE infections arereported to CDC

!approximately 11% of patients are hospitalized, and 0.3%die

!Salmonella typhimurium and Salmonella dublin


!Unique characteristic

!Mesophiles (Temperature 7 ° C - 45 ° C)

!Catalase positive and oxidase negative

!Infective dose: high infective dose (> 105 CFU/g)

!Lower infective doses for young infants and adult in poor

health

!Bacteria can survive but not grow

!Not grow if water activity less than 0.95

!Not grow if pH less than 4.6

!Heat sensitive: Die if temperature more than 60 ° C


!Foodborne infection

! Incubation period: 6-36 hrs (usually 12-24 hrs)

!Duration of illness: 1-7 days

!Clinical signs! fever

!Acute gastroenteritis: Nausea, Vomit and diarrhea

!Abdominal pain, abdominal cramps

!Dehydration in severe cases (infant or elderly)

!Arthritic symptoms may follow 3-4 weeks after onset ofacute symptoms

!Low fatality rate!S. enteritidis fatality rate 0.3%


!Foodborne infection!Colonization of bacteria in the intestine

!Distal small intestine and colon!Competitive with normal flora (produce organic acid)

!Invasion of the intestinal epithelium!Invade villous tip of the ileum and colon!Penetrate brush borders and enter cell!Stimulate inflammatory response

!Stimulation of fluid secretion into intestinal lumenwith the consequent watery diarrhea!Intestinal mucosa releases prostaglandin which activate

adenylate cyclase!Release vasoactive substances that increase permeability

of mucosal vessel

Salmonella Salmonella sppspp. outbreaks. outbreaks!Most common: raw meats, poultry, eggs,

!Less common: milk and dairy products, fish, shrimp,vegetable

!Yeast, coconut, sauces and salad dressing, cake mixes,cream-filled desserts and toppings, dried gelatin, peanutbutter, cocoa, and chocolate

!High fat content

!Various Salmonella species have long been isolatedfrom the outside of egg shells. The present situationwith S. enteritidis is complicated by the presence ofthe organism inside the egg, in the yolk.

!Vertical transmission, i.e., deposition of the organisms in theyolk by an infected layer hen prior to shell deposition. Foodsother than eggs have also caused outbreaks of S. enteritidisdisease

Salmonella Salmonella sppspp. outbreak. outbreak

!1985 Salmonellosis outbreak

! involving 16,000 confirmed cases in 6 states

! low fat and whole milk from one Chicago dairy.

!Salmonellosis in restaurant

!S. enteritidis was isolated from employees and patrons ofthree restaurants of a chain in Maryland

!At least 71 illnesses resulting in 17 hospitalizations

!Scrambled eggs from a breakfast bar were epidemiologicallyimplicated in this outbreak and in possibly one other of thethree restaurants

!The plasmid profiles of isolates from patients all threerestaurants matched.

A B Outbreak

Cut with restriction enzymes

plasmid

Plasmid Plasmid profilesprofilesBacterial plasmid from

food sample

A B Outbreak

plasmid

Salmonella Salmonella sppspp. outbreak. outbreak!1984 Salmonellosis outbreak

!186 cases of Salmonellosis (S. enteritidis) were reported on29 flights to the United States on a single internationalairline

!An estimated 2,747 passengers were affected overall

!No specific food item was implicated, but food ordered fromthe first class menu was strongly associated with disease

!1994 S. typhimurium outbreak,!Wisconsin: 17 patients with acute gastrointestinal illness

characterized by diarrhea and abdominal cramps

!At least 14 patients reported having eaten raw ground beefthat was either plain or seasoned with onions and an herbmix during the 72 hours before illness onset

! Stool samples for culture were obtained from 11 patients;Salmonella serotype Typhimurium was isolated from sevenspecimens.

Salmonella Salmonella sppspp. outbreak. outbreak

!The CDC estimates that 75% of those outbreaks areassociated with the consumption of raw orinadequately cooked Grade A whole shell eggs

!The U.S. Department of Agriculture publishedRegulations on February 16, 1990, in the FederalRegister establishing a mandatory testing programfor egg-producing breeder flocks and commercialflocks implicated in causing human illnesses

!This testing should lead to a reduction in cases ofgastroenteritis caused by the consumption of Grade Awhole shell eggs.

Salmonellosis outbreak studySalmonellosis outbreak study

Outbreak of Salmonella enteritidis Associated withNationally Distributed Ice Cream Products

! From September 19 through October 10, 1994,

! a total of 80 confirmed cases of Salmonella enteritidis (SE) infection

were reported

! Cases were characterized by diarrhea, abdominal cramps, and fever

! Recent increases in SE cases also were reported from South Dakota

(14 cases during September 6-October 7, compared with 20 casesduring all of 1993) and Wisconsin (48 cases during September 6-

October 7, compared with 187 during all of 1993).

! This report summarizes preliminary findings from the outbreakinvestigation.


!Epidemiological investigation

! to assess potential risk factors for infection

! the department of health conducted a case-control study of 15

cases and 15 age- and neighborhood-matched controls.

!A case was defined as culture-confirmed SE in a person with

onset of illness during September. Eleven case-patients (73%)

and two controls (13%) reported consumption of Schwan's

ice cream within 5 days of illness onset for case-patients and

a similar period for controls

!Odds ratio= 10.0; 95% confidence interval= 1.4-434.0


!Epidemiological investigation

! Press releases informing the public of this problem

! Advised persons who had been ill since September 1 and who had

consumed Schwan's ice cream to contact the health department

! During October 8-11, a total of 2014 persons who had consumed

suspected products and had been ill with diarrhea contacted the DH

by telephone

! Samples of ice cream from households of ill persons grew SE

! These products had production dates in August and September

! The implicated products are distributed nationwide, primarily by directdelivery to homes, and are sold only under the Schwan's label

! Investigations to examine the extent and causes of the outbreak areunder way.


!S. enteritidis contamination in ice-Cream

!Plasmid profile

!copy number

!cut with restriction enzyme

!etc

!Compare isolates from patients, ice-cream, raw-

eggsPatients I ce-cream Egg

Cut with restriction enzymesPlasmid


!Recall product

!On October 7, the company voluntarily stopped distribution andproduction at the Marshall plant pending further findings fromthese investigations

!Product recall

!Find source of contamination

!Source of contamination?

!Check HACCP (critical control point)

!Pasteurization? Milk/ Ingredient

!Transportation? Milk/ Ice-cream

!Milk was pasteurized at one place and transported toice-cream plant by truck


!Source of contamination?

!Milk was pasteurized at one place and transported toice-cream plant by truck

!Truck tank clean?

!Sanitation before and after transportation

!What is the source of S. enteritidis ?

!Some trucks also transport liquid egg

!Cracking of tank seal

!SE survived from sanitation

!Contamination of pasteurized milk

!Contamination of S. enteritidis in ice-cream

!Source of S. enteritidis is liquid egg

Role of VeterinarianRole of Veterinarian

!Salmonella free farms

!Prevent horizontal and vertical transmission

!Slaughter house hygiene

!Meat hygiene and inspection

!Meat quality&Trade barrier

!Outbreak investigation

!Department of health

!Diagnosis and researches

!Education


!Control of Salmonella spp. infection

!Education: Good hygiene practices!Consumer!Food handler

!Reduce incidence of microorganism at farmlevel and raw food

!Reduce incidence of microorganism atprocessing plant

!Proper storage!Temperature < 5 °C : not grow

!Proper food handling!Heat treatment: Cooking temperature

Isolation ofIsolation of Salmonella from foodSalmonella from food

!At least 4 days to isolate Salmonella from food

!5 steps to isolate Salmonella

!Nonselective pre-enrichment

!Selective enrichment

!Selective plating

!Biochemical confirmation

!Serology confirmation


!Nonselective pre-enrichment

!25 g of sample + 225 ml of Lactose broth

! Incubate 24 hrs at 35 °C

!Purpose: to facilitate the isolation

!Lactose broth, nutrient broth and tryptic soy broth (TSB)

! to give organisms that may have been injured in food

processing steps a chance to repair any injuries before being

exposed to selective agent


!Selective enrichment

!portion of nonselective broth is transferred to selective

enrichment (1 ml of Lactose broth to SC and TT)

! two selective enrichment broth

!Selenite Cystine (SC)

!Tetrathionate (TT)

! Incubate 35 °C 24 hrs


!Purpose: to facilitate the isolation

!Selenite Cystine (SC)

!Selenite: Reduces the growth of fecal coliforms and enterococci

!Tetrathionate (TT)

!Tetrathionate: Inhibits or kills coliforms and enhances the

growths of S. typhi and S. paratyphi

!Iodine and Brilliant green: Inhibits non-Salmonellae


!Selective plating

!Streak for isolation from the SC and TT broth onto 3 agar

media

!XLD agar

!HE agar

!BS agar

! Incubate 35 °C 24 hrs

!Purpose: to obtain isolated colonies of Salmonella

and to differentiate Salmonella from other organisms

!Salmonella not ferment lactose

!Salmonella produce hydrogen sulfide


!XLD agar (Xylose lysine desoxycholate)!Desoxycholate: selective for enteric organism

!Xylose: fermented rapidly to acid by most enteric organisms

!Lysine: selective for salmonella

!Salmonella red colonies with or without blackcenters (Coliforms develop as yellow colonies)

!HE agar (Hektoen enteric agar)!Bromthymal blue and fuschin: Indicators of lactose

fermentation

!Thiosulfate and ferric ammonium citrate: Hydrogen sulfideorganisms

!Salmonella blue-green colony (Coliforms develop pinkto orange colony


!BS agar (Bismuth sulfite agar)!Highly selective

!Use to isolate S. typhi from feces and sewage

!Bismuth sulfite and brilliant green: Inhibits gram-positiveand coliform organisms

!Salmonella brown to black color with metallic sheen(non-Salmonella develop brown green colonies)

!Other selective plating media!BGA (Brilliant green agar)

!SS (Salmonella Shigella agar)


!Biochemical confirmation

!Select one typical colony from XLD, HE, BS

! Inoculate (Streak and stab technique)

!Triple sugar iron (TSI)

!Lysine iron agar (LIA)

! Incubate at 35 °C 24 hrs


!Purpose: non selective differential media!TSI (Triple sugar iron agar): Fermentation of dextrose,

lactose and sucrose is detected by conversion of phenol redindicator

!Salmonella acid butt (yellow) and alkaline surface(red)

!Purpose: non selective differential media

!LIA (Lysine iron agar): Dextrose is rapidly fermented by

enteric organisms and Bromcresol purple as indicator

!Salmonella decarboxylate lysine change pH

purple color both butt and surface

!LIA: Hydrogen sulfide detection


TSIOrganisms Slant Surface Gas H2SE. coli A A + -S. enteritidis K A + +LAIOrganisms Slant Surface Gas H2SE. coli K K - -S. enteritidis K K + +


!Serological confirmation! Inoculate growth from slant into BHI broth

! Incubate at 35 °C 6 hrs

!0.5 ml of sample + 0.5 ml of Salmonella polyvalentantiserum (One loopful for slide agglutination)

! Incubate at 48-50 °C

!Observe every 15 mins

!Results: Positive Agglutination

!Purpose: Use to confirm that Salmonella has beenisolated or to determine what species of Salmonella ispresent

Lactose broth 225 ml

Sample 25 ml

Non selective pre-enrichmentNon selective pre-enrichment Selective enrichmentSelective enrichment

SC TT

37 C 24 hrs

Selective plating Selective plating

37 C 24 hrs

XLD HE BS

Biochemical confirmationBiochemical confirmation

TSI LI A37 C 24 hrs

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Salmonellosis - Chula · Salmonellosis outbreak study!Epidemiological investigation!to assess potential risk factors for infection!the department of health conducted a case-control