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Foodborne infections and intoxications

Relative risk associated with different aspects of food safety


Aspect Relative risk Agents Ratio between repoted
and estimated
incidences
Microbial 106 Bacteria, Viruses, 1:10 to 1:100
contamination Rickettsia, Protozoa,
Fungi, Algae, Parasites
Nutritional imbalance 106 1:100 to 1:1000
Environmental 100 1:10
pollution
Pesticides 1 1:10 to 1:100
Food additives 1 1:100 or more
Food poisoning Agents
Group of agents Agents
Micro-organisms Bacteria, Viruses, Rickettsia, Protozoa, Fungi,
Algae
Chemicals Intrinsic
Extrinsic-
a. Direct contamination
b. Environmental
Parasites Animal parasites
Fish parasites
Diseases associated with consumption of milk
Illness Causative agent Historical perspective
Brucellosis B. abortus/ B. melitensis Rare after the introduction of
Tuberculosis M. tuberculosis, M. bovis pasterization of milk
Typhoid/ paratyphoid S Typhi and Paratyphi
Dysentery Shigella species

Diphtheria Corynebacterium diphtheriae


Streptococcal infections Streptococcus species
Q fever Coxiella burnetii

Listeriosis Listeria monocytogenes Still going strong


Gastroenteritis
Salmonella serovars
E coli
Yersinia enterocolitica
Camplobacter species
Foodborne diseases due to bacteria
Organism Incubation Symptoms
time
Salmonella 12-16 hrs Dirrhoea, emesis, fever and malaise
Shigella 12-50 hrs Dirrhoea, dysentery, cramping stomach pain and bloody stool
Campylobacter 2-11 days Prodromal fever and malaise, abdominal pain followed by
species profuse diarrhoea
Yersinia 2- 48 hrs Abdominal pain, fever headache, diarrhoea and vomiting
enterocolitica
Vibrio Profuse diarrhoea leading to dehydration, vomiting and fever
parahaemolyticus symptoms for infections vith V. cholerae and V vulnificus
often vary
Areomonas Diarrhoea in normal patients but systemic infection in
immunocompromised
Plesiomonas 48 hrs Dirrhoea, abdominal pain, nausea and vomiting
shigelloides
Staph. aureus 2-6 hrs Vomiting, abdominal pain and diarrhoea leading to
dehydration and death
B. cereus Emesis syndrome or diarrhoeal syndrome
C. botulinum 24-72 hrs Fatigue, lassitude, dizziness, problems of CNS e.g. speech
difficulties and visual disturbances
C. perfringens 8-22 hrs Abdominal pain, nausea, vomiting and occasional diarrhoea
L. monocytogenes Septicemia and meningitis in neonates and abortion and
septicemia in pregnant females

Foodborne diseases due to viruses


Illness Viruses
Gastroenteritis Small round structured viruses e.g. Norwalk, Snow mountain and
Ditchling agents
Astrovirus
Calcivirus
Parvovirus
Rotavirus
Hepatitis Hepatitis A
Hepatitis E
Enterically transmitted non A non B hepatitis
Spongiform encephalopathy Scrapie/ BSE like agents
Other syndromes Echovirus and Coksackievirus

Foodborne diseases due to protozoa


Illness Agents
Gastroenteritis Giardia lamblia
Cryptosporidium parvum
Entamoeba hystolytica
Isospora belli
Balantidium coli
Respiratory symptoms Cryptosporidium parvum
Others Pathogenic free living amoeba

Foodborne acute extraintestinal diseases


Illness Agents
Lungs and respiratory tract infectins Campylobacter jejuni
Salmonella spp.
Cryptosporidium spp.
Enteric viruses
Kidney problems E. coli O 157:h7
Campylobacter spp.
Salmonella spp.
Shigella spp.
Heart and circulatory system problems Yersinia enterocolitica
Salmonella spp.
CNS problems Clostridium botulinum
Salmonella spp.
Listeria monocytogenes
Enteroviruses
Foetal tissues Listeria monocytogenes
Skin and soft tissues Campylobacter spp.
Salmonella spp.
Yersinia enterocolitica
Foodborne chronic extraintestinal diseases
Illness Agents
Joint problems Yersinia enterocolitica
Camplobacter jejuni
Salmonella spp.
Shigella spp.
E. coli
Staphylococcus aureus
Autoimmune thyroid disease Yersinia enterocolitica
Neural and neuromuscular disorders Yersinia enterocolitica
Camplobacter jejuni
E. coli
Heart and vascular diseases Salmonella spp.
Enteric viruses
Foodborne algal diseases
Illness Agents
Ciguatera poisoning Reef fish containing ciuguatoxin accumulatecdue feeding on
Ip: immediately or delayed by Gambierdiscus toxicus, a dinoflagellate algae in tropical and
few hours subtropical regions. The toxin is heat stable.
Symptoms: severe nausea, vomiting, diarrhoea, neurosnsory
disturbances as parasthesia and dysaesthesia
Paralytic shellfish poisoning Saxitoxin containing shellfish (Alaskan butter calm)
IP: instant and death within 24 Saxitoxin in fish when >80mmg/kg (400 mouse units/100gm) is
hours always dangerous to eat.
Toxin is the product of a few dinoflagellates e.g. Gonyaulax
catenella and G. tamarensis.
Symptoms:neurological disorders, tingling, burning and numbness
of lips, finger tips, ataxia, drowsiness deat due to perepheral and
respiratory paralysis.
Neurotoxic shellfish poisoning Brevitoxin B and C produced by dianoflagellates e.g.
IP: as above, few mintes to Ptychodiscus brevis accumulates in red tide seasons in dicated
several hours by massive deaths of fish in the region. At such times shellfish
should not be taken. Symptoms same as above but milder and
death is not so common, Diarrhoea may also be present.
DIARRHOEIC shellfish Symptoms: diarrhoea, nausea, vomiting and abdominal pain. Short
poisoning lived problem amy persist for few days.
IP: 30 min to several hours Toxins such as Dianophysistotoxins, pectenotoxins and ochadaic
acid produced by Dianophysis fortii and D. acuminata, accumulate
in fish organs.
Domoic acid poisoning Poison is produced by a planktonic plant, Nitzschia pungens, it
antagonises glutamate in CNS
Symptoms are burning sensation, memory loss, delerium,
breathing difficulty.
Foodborne fungal diseases (mainly causes cancer and delayed organ damage)
Toxins Moulds Foods at risk- hepatitis, cirrhosis
Aflatoxin Aspergillus flavus Nuts, rice, coffee, cocoa, soya, corn and corn
(Rye syndrome) products and milk and milk products
Citrinin Penicillium citrinum P. Wheat, oats, rye, rice, cheese
viridicatum
Ochartoxin A. ocharceus Nuts, rice, coffee, cocoa, cereal crops, soya,
P. viridicatum cheese
Penicillic acid P. cyclopium Corn, beans, fruits
P. martensii
Sterigmatocystine A. versicolor Grains, coffee and miscellaneous foods
Trichothecenes Fusarium graminaerum Corn and cereal crops Haemorrhagic enteritis,
(Degnala disease) F. tricinctum skin irritation ad desquamation of skin
Zearalenon Fusarium graminaerum Corn and cereal crops
Ergotoxin/ergotamine Claviceps purpurea Millets and rye gangrenous and convulsive
(ergotism) disease
Foodborne diseases caused by parasites of fish and animals
Type of parasites Name of agent
Tapeworms of animal origin Taenia saginata (beef). T. soleum (pork)
Nematodes of animals Trichinella spiralis
Liver flukes of animals Fascilola hepatica
Protozoa of animals Toxoplasma gondii
Sarcocysts Sarccystis bovihominis (beef), S. suihominis
(pork)
Fish nematodes Anisakis simplex and other spp.
Terranota spp. And Phocanema spp.

Detection of foodborne pathogens in foods


1. Isolation of micro-organism (resuscitation, enrichment, selective plating)
(Sensitivity and slectivity are two qualities of specific media.
Selectivity: Ability to prevent the growth of bacteria other than those for which medium was
designed.
Sensitivity: Ability of medium to recover the bacteria for which the medium was designed.
Factors affecting the selectivity and sensitivity of medium are
Type of food or sample to be analysed
Contaminating microflora/ inhabitant microbes of the food/sample.)
1a. Magnetic beads coupled with specific antibodies
2. Membrane filtration techniques: used mainly for liquids
Type of membranes:
a. Conventional membrane filters of cellulose acetate/ nitrate, nylone membranes.
b. Hydrophobic grid membranes- being water repellent these limits the spreading of colonies and
size of colony is restricted therefore up to 104 colonies could be counted on one membrane.
3. Microscopic counting techniques: Bread smear used to determine quality of milk was first in this
series. Lateron FAT and DEFT (direct epifluorescent filter techniques) are the newer methds.
4. Electrical methods:
1. Impedence measurement using equipment like Bactometer, Malthus and Rabit. The technique
has been standardized for Salmonella, E. coli, Staph. Aureus and Listeria monocytogenes The
requirement for equipment and media engineering are the prerequisites.
2. Measuring potential difference instead of impedence of current has been envisaged as better
option due to its less sensitivity to changes in temperature and thus need less costly equipment.
Here two electrodes made up of two different metals (aluminium and gold) are put in medium
and voltage is measured.
5. ATP measurement: Like luciferase assay can detect specific microbe (using specific lytic phages
for the bacteria) equivalent to 105 CFU/gm.
6. Serological techniques: Latex coagglutination techniqe, ELISA
7. Genetic methods: PCR, DNA probes, DNA hybridisation, LCR etc.
8. Labelled bacteriophage detection particularly for Salmonella and Listeria. Phages amy be enzyme
labelled, phage attaches to only specific bacteria and then they can be detected either colorimetrically
or fluorogenically. Another technique involve incorporation of lux gene of luminescent bacterium V.
fischeri in to phage DNA and bacteria taking phage then produces luminescence detectible with
photo meter.
Bacterial identification systems available
Bacteria Systems
Salmonella/ Shigella / Yersinia API Z
Staphylococcus API Rapidec Staph
API ATB Staph (based on preformed enzymes)
Non enteric Gram negative bacteria API 20NE
Anaerobes and clostrdia API ATB 32A (Based on preformed enzymes)
API 20A (based on biochemical tests)
Enterobacteriaceae API ATB 32E (based on preformed enzymes)
Biolog GN Micro Plate (based on carbon source utilization)
API 10E, API 20E, Rapid 20E (based on biochemical tests)
Enterotube, Miniteck (based on biochemical tests)
Gram negative oxidase positive Oxi /Ferm (based on biochemical tests)
Streptococcus API 20 Strept (basaed on preformed enzymes)
Bacillus API 50 CHB (based on biochemical tests)

Typing of foodborne pathogens


Bacteria Methods
Salmonella Serotyping (based on somatic/ flagellar/ capsular antigens)
Phagetyping, Bacteriocin typing, Biotyping, toxinotyping,
haemolysin typing, plasmid profiling, RAPD, RFLP, AFLP,
ribotyping, PFGE, BRENDA (Bacterial restriction
endonuclease analysis), antibiograms
Staphylococcus Phagetyping, plasmid profiling, RAPD, RFLP, AFLP,
ribotyping, PFGE, BRENDA (Bacterial restriction
endonuclease analysis)
E. coli Serotyping (based on somatic/ flagellar/ capsular antigens)
Bacteriocin typing, Biotyping, plasmid profiling, RAPD,
RFLP, AFLP, ribotyping, PFGE, BRENDA (Bacterial
restriction endonuclease analysis), toxinotyping
Shigella Serotyping (based on somatic/ flagellar/ capsular antigens),
phage typing, colicin typing, Bacteriocin typing, Biotyping,
plasmid profiling, RAPD, RFLP, AFLP, ribotyping, PFGE,
BRENDA (Bacterial restriction endonuclease analysis)
Proteus Serotyping (based on somatic/ flagellar), Dienes typing,
Phagetyping, Bacteriocin typing, Biotyping, plasmid profiling,
RAPD, RFLP, AFLP, ribotyping, PFGE, BRENDA (Bacterial
restriction endonuclease analysis)
Toxins produced by different foodborne pathogens
Type of toxin Microbes producing toxins
Enterotoxins similar to cholera toxin V. cholerae, E. coli, Campylobacter spp., Salmonlla,
Aeromonas, Giardia lamblia, Klebsiella sp., Edwardsiella sp.
Shiga and Shiga like toxins Shigella spp., E. coli, Salmonella, Klebsiella, Serratia,
Edwardsiella
ST toxins like of E. coli E. coli, Citrobacter, Klebsiella
Tetradotoxin Vibro spp. And Aeromonas spp.
Haemolysins Streptococcus spp., Stahp spp., Salmonella, E.coli,
Klebsiella, Clostridia , Listeria spp., Bacillus spp.,
Lecithinase and phospholipases Salmonella, Bacillus, Clostridia, Listeria
Emetic toxin and diarrhoeic toxins Bacillus cereus
Botulinal toxins Clostridium botulinum, C. butyricum, C. barati and other
clostridia
Heat stable Enterotoxins Staph aureus and other Staph. Spp.
Toxins and their receptors
Bacteria Toxin Receptor Mechnism
E.coli LT GM1 ganglioside, cAMP pathway
galactoproteins
Sta & ST Nonganglioside cGMP pathway
receptors
STb
VT/SLT
CNF/dermonecrotoxin
Shigella Shiga toxin (entero, Gb3 Ricin like activity,
neuro &cyto toxic), inhibit protein
secreted as zymogen synthesis
HeLa cell cytotoxin
CHO elongation factor
LT
Microbes causing foodborne diseases in different countires
Country Agents/ disease Share % Reference
Poland Salmonella 87 Gerigk, K (1992)
Staph aureus 1.4
Clos. Botulinum 0.7
Trichinella 0.22
Others 11
United Salmonella 77 Sharp et. al. 1992
Kingdom/Scotland
Campylobacter 9.4
Rotavirus 5
Clo. perfringens 3
Bacillus spp. 1
Staph aureus 1
Others 4.6
Finland Staph aureus 21.02 Hirn et.al. 1992
Clos perfringens 13.35
Salmonella 17.61
Bacillus cereus 8.24
Yersinia enetrocolitica 0.57
E. coli and others 33.24
France Salmonella 45
Clos. Perfringens 13
Staph. Aureus 10
Others 32
Hungary Salmonella 50 Biro, G 1992 (figure are for year
1990)
Staph aureus 6.6
C. botulinum 1.4
Others 45
Spain Salmonella 50 Hernandaez et. al. 1992
Staph aureus 5
Others (Brucella and C. 45
perfringens)
Why Yersinia differs from other members of Enterobacteriaceae
Characteristics Yersinia Other Enterobacteriaceae
Size of colony Small Medium to large
Morphology of colony Not affected by Affected by supplement
supplement
Cell size and shape Small coccoid Medium to large
cocobacillary
Preferred grwth temerature <30oC 37oC
Dependent of major characteristics on temperature High Low
of incubation
Loss of plasmid At 37oC At .42oC
Dissociation of colonies on 48 hrs of incubation in Common on all Rare
to small (0.5 mm) and large type (2 mm), can be medium
used as presumptive test

Differential Media used for isolation of Enterobacteriaceae


SN Media Differential agents Bacteria and their colonies
2 Brilliant green agar and Brilliant green, Salmonella-pink white/ red surrounded by red
its modified formula lactose and medium
sucrose Proteus-Red colonies with swarming
Pseudomamns- crenated red colonies
E. coli and other lactose fermenters-yellow/
green
3 Bismith sulphite agar Bismith sulphie & Salmonella Typhi_Rabbit eye colony in 48 hrs
brilliant green Other Salmonella- black with greenish grey
periphery & metallic sheen, E. coli & others-
brown/ green or black
4 Brilliant green bile agar Brilliant green and Salmonella- colourless to light pink
bile E. coli and other lactose fermenters- pink/ deep
red with precipitated bile under the colony
5 CLED agar (cystine cystine lactose & E. coli- Bright pink colonies surrounded by
lactose electrolyte electrolyte pink medium, Proteus mirabilis- blue green
deficient agar) transluscent, Proteus vulgaris- not swarms,
blue/ blue green, Staphylococcus- pail pink,
Klebsiella, grey green mucoid,yellow, Strept-
small opaque grey
6 DCLS agar Desoxycholate, E. coli- poor growth- Red colonies
(Desoxycholate citrate lacvtose, sucrose Salmonella- Colourless to pink
lactose sucrose agar) and citrate Shigella- Colourless to pink transluscent
7 Lysine decarboxylase Decaroxylation of Salmonella, E. coli, Klebsiella, Edwardsiella-
medium lysine Purple colour + ve
Proteus/ Shigella/- yellow ve. Also differ-
entiate Vibrio, Aeromanas and Plesiomonas
8 Desoxycholate agar Lactose, E. coli- pink umblicated 1-2 mm colonies with
(Sodium deoxycholate deoxycholate, white precititate, Klebsiella- domed and
agar) Deoxycholate citrate, ferric mucoid, S. Paratyphi B- domed centrally
lactose agar & citrate black2-4 mm transparent, S. Typhi- up tp 1 mm
Deoxycholate citrate pale pink, flat, conical colourless slightly
agar(DCA) opaque, other Salmonella, Psedomonas and
Shigella- colourless 1-2 mm as od S. Paratyphi
B, Proteus- large central black, fishy odour
9 EC medium Lactose and bile E. coli and other coliform- gas with acid
10 EC medium with MUG Lactose & MUG E. coli- Fluorescent growth with gas
11 Endo agar Lactose E. coli and coliforms- deep or pink red with
golden metallic sheen with red surrounded
medium, Salmonella and non lactose
fermenters- colourless to light pink translucent
with pink or colourless background
12 EE broth Dextrose & Enterobacteriaceae members yellow colour
brilliant green
13 EMB agar Lactose/ sucrose Enterococcus and Salmonella- colourless to
and eosine amber transluscent, Candida albicans- spidery
methyline blue or feathery, other Candida form smooth yeast
like, E. coliblue black with dark centre and
green metallic sheen, Klebsiella and
Enterobacter-4-6 mm raised mucoid confluent
grey brown centred without metallic sheen
14 mFC agar W or W/O Lactose, rosolic E. coli and other coliforms- blue (with white
MUG acid, aniline blue fluorescent if medium is with MUG)
14 Hektoen enteric agar Lactose, sucrosae, E. coli- salmon orange with bile precipitate,
salicin, Shigella & Providencia- greenish blue moist
thiosulphate, ferric raised, Salmonella- blue green with or without
ammonium citrate black centres, Pseudomonas- green or brownish
flat irregular, Commensals- Salmon coloured
16 MacConkey agar Lactose E. coli & coliforms- pink raised, Proteus-
colourless, no swarming, Salmonella-
translucent colourless, Klebsiella &
Enterobacter- red mucoid, Enterococcus- small
round opaque
17 Rappaport Vassiliadis Malachite green Citrobacter and Pseudomonas are inhibited
Semi-solid medium and motility Salmonella grow and produce halo of
decolouration of blue malachite green
17 SS agar Lactose, Ferric Gram positives and coliforms are inhibited due
citrate and to bile salt, lactose fermenters produce pink to
thiosulphate, red colonies, non lactose fermenters produce
Briiliant green colourless colonies. Salmonella- colourless
with black centres
18 Tergitiol-7 agar Lactoswe, E. coli and other lactose fermenters-yellow
tergitiol-7, colonies with yellow background
bromothymol blue Non lactose fermenters, Salmonella-
Transparent colonies with blue background
19 VRBA ( can be added Lactose, crystal E. coli, Klebsiella and Enterobacter- dark red
with MUG to see violet & neutral with red precipitate around colonies, Non
fluorescence) red lactose fermenters- colourless small colonies
20 VRB dextrose agar Lactose, glucose Only Enterobacteriaceae members grow, those
crystal violet & can grow only aerobically are unable to grow,
neutral red further differentiation can be obtained by
oxidase test, oxidase negative and every colony
with a purple halo on a purple background is of
Enterobacteriaceae
21 XLD medium Xylose, lysine, Salmonella, Edwardsiella- Red colonies with
ferric citrate, black centres, Shigella, Providencia & H2S
thiosulphate negative Salmonella, Pseudomonas and a few
proteus- Red colonies
E. coli-Enterobacter, Klebsiella, Citrobacter,
Proteus, Serratia- yellow opaque colonies
22 XLT4 agar Xylose, lactose, Proteus, E. coli and other lactose and sucrose
sucrose, lysine, fermenters- yellow colonies
ferric citrate, Salmonella yellow to red colonies with black
thiosulph, tergitol4 centres
23 Yersinia selective agar Crystal violet, Yersinia- colourless colonies with dark pink
(CIN agar) irgasan, mannitol centres, may be with bile precipitate

Differential identification media for enterobacteriaceae


S Media Differential Bacteria and their colonies
N agents
1 Acetate Aceatate E. coli- Blue butt and slant +ve test
differenti uisability Shigella- Green butt and slant ve test
al agar Salmonella Paratyphi B ve others are +ve
Proteus & Providencia ve; Klebsiella, Enterobacter, Citrobacter &
Serratia +ve
2. Kligler Lactose, Bacteria Slant/butt Gas H2S
iron agar glucose, Citobacter A/A + +
thiosulphate, E. coli A/A + -
ferric Proteus Alk/A - +
ammonium Salmonella Alk/A + +
citrate Klebsiella enterobacter Alk/A + -
Shigella -/A - -
3 Kohn Mannitol, Bact Medium-1 Medium-2
two tube glucose, urea, Glu Man Ureas Sucr/ Motil H2S Indol
medium sucrose, sicin, e Sali ity e
motility, H2S, Salmonella A/A A - - + + -
indole G
Shigella A A - - - - V
Proteus V V + AG/- + V V
4 Koser Citate E. coli & Shigella- -ve grren, Klebsiella & Citrobacter- +ve blue,
citrate Salmonella- V
medium
5 Lysine Glucose, L- Bacteria Butt (lysine Slant (lysine H2S
iron agar lysine, Ferric decarboxylation) deamination)
Arizona +, purple - purple + Black
citrate
Salmonella +, purple - purple + Black
Proteus -, yellow + Red + black
Providencia -, yellow + RED -
Citrobacter -, yellow - purple + black
E. coli +, purple - purple -
Shigella -, yellow - purple -
Klebsiella +, purple - purple -
6 MIL Dextrose, L- Bacteria Lysine Motility Lysine Indole
medium lysine, Ferric decarboxy Deaminase
citrate & E. coli + + - +
bromocresol Providencia - + + -
purple Salmonella + + - -
Shigella - - - -
7 MOI Dextrose, L- Bacteria Ornthine decarboxy Motility Indole
ornithine & Enterobacter + + -
bromocresol E. coli + + +
purple Klebsiella - - -
Proteus + + -
8 Malonat Sodium Enterobacter, Salmonella arizona- blue +,
e broth amlonate, E. coli & Salmonella- green,
bromothymol Salmonella paratyphi B- Blue
blue
9 Motility TTC E.coli-motile, reduce TTC
medium- reduction, Klebsiella- nonmotile, reduce TTC
S motility Shigella flexneri- nonmotile, not reduce TTC

10 MRVP Dextrose For doing methyl red test and VP test as E. coli is MR +ve, acidic
medium end product pH <4.4, acetic, lactic and formic acids & Klebsiella is
VP +ve neutral end product pH> 6.0, acetyl methyl carbinol, most
enterobacteria gives either MR or VP test positive, but some as
Hafnia alvei and Proteus mirabilis can give both tests +ve
11 Nitrate Pot. Nitrate Enterobacteriaceae, vibrionaceae- positive, cherry red
broth Acinetobacter- negative
12 OF Glucose Bacteria Open Closed
medium Oxidation Enterobacteriaceae Acid gas or Acid Acid gas or Acid
fermentation Pseudomonadaceae Acid Alkaline
Acinetobacter Acid Alkaline
Vibrionaceae Acid gas or Acid Acid gas or Acid
13 Peptone Ferric citrate, H2S producer- Black coloration of medium in tube
iron agar Thiosulphate Non H2S producer- medium remains yellow

14 Phenol Phenol red, Sugar fermenters- pink/red colouration


red agar carbohydrate No sugar fermentation- medium remains yellow
15 Phenyl Phenyl- Proteus & providencia- positive, gives blue-green colour on pouring
alanine alanine ferric chloride (10 % in 0.1 N HCl, no colour change means
agar negative test
10 Simmon Citrate in E. coli, S. Typhi, S. Paratyphi, Shigella, Morganella morganii-
s citrate defined negative, i.e. no growth and media remains light green
medium Klebsiella, Enterobacter, Citrobacter, Salmonella, Proteus,
Providencia and Serratia produces characteristic blue colour
11 SIM Ferrous Differentiation is based on H2S and indole production and motility
medium ammonium of the bacterium
sulphate and Bacteria Motility H2S Indole
thisulphate Salmonella + + -
Shigella - - V
Proteus + V V
12 TSI agar Glu, lactose, Bacteria Butt Slant H2S
sucrose, ferric Klebsiella AG A -
citrate, E. coli, Enterobacter AG A -
thiosulphate Proteus spp. AG A +
Salmonella spp. AG Alaline +
Shigella spp. A Alkaline +
S. Paratyphi & Chleraresuis AG Alkaline -
S. Typhi A Alkaline +
13 Urea Urea, Phenol All Salmonella, Shigella and E. coli do not produce +ve, pink color
agar/ red Urease produces as Klebsiella & Proteus group produces pink
broth colour within 5-6 hours
14

Media used for enterobacteriaceae


S.N. Bacteria Enrichment Plating
1 E. coli and Brilliant green bile broth, Lactose Endo Adar, MacConkey agar, Eosine
coliforms ricinoleate broth, MacConkeys methylene blue agar, Levine EMB agar,
broth, GN broth, Lactose broth, Violet red bile agar, Hektoen enteric agar,
lactose peptone broth, laural trptose Modified EC medium with novobiocin,
broth, modified EC medium, A-1 Nutrient agar with MUG, SOB medium,
broth medium mineral modified glutamate agar, mFC agar
medium
2 Salmonella Mannitol selenite broth, selenite Rambach agar, Brilliant green agar, Bismith
broth, selenite cystin broth, sulphite agar, DCLS agar, DCA, HEA,
tetrathionate broth, Rappaport MSRV medium, Tergitiol 7 agar,
Vassiliadis medium, RV R10 broth, MacConkey agar, XLD, XLT4 agar
GN broth, Lactose broth, M broth,
TT broth
3 Yersinia GN broth CIN agar, Yersinia selective agar
4 Shigella GN broth DCA, EMB agar, HEA, MacConkey agar,
XLD agar

Media used for MPN count


S.N. Bacteria MPN
1 E. coli and Lactose broth, Laural tryptose broth, MacConkey broth (purple),
coliforms Minerals modified glutamate broth, Presence absence broth( purple
medium turns yellow), Purple broth medium, mTEC agar, Tryptone
water, Brilliant green bile broth
2 E. coli O157:H7 Modified EC medium

Media use for TPC (total palte count) of Enterobacteriaceae


1. Plate count agar
2. Nutrient agar
Media used for Oxiadation-Fermentation (OF) test
1. Decarcoxylase medium
2. OF basal medium
3. Phenol red broth base
4. Purple agar or broth base
Sterility testing media
1. Fluid thioglycollate medium withor without beef extract
2. Aseptic commissioning medium
3. Agar medium No. F
4. NIH thioglycollate medium
5. Tryptic soy broth
Media used for antibiotic testing of Enterobacteriaceae
1. Muller Hinton agar
2. Antibiotic sensitivity assay medium
3. Antibiotic medium
Media used for transportation of Samples/ swabs of Enterobacteriaceae
1. Amies medium without charcoal
2. Cary & Blair medium
3. Stuarts medium
Media for testing purity of cultures of Enterobacteriaceae
1. TSA
2. BHIA
3. Blood agar
4. Veal infusion agar
Media used for resuscitation (recovery of injured bacteria) of Enterobacteriaceae
1. Buffered peptone water
2. m T7 agar
3. Universal pre-enrichment broth
4. mTEC agar
5. R2A agar
Media used for Recombinant strains of Enterobacteriaceae
1. LB medium
2. NZM broth
3. NZYM broth
4. NZCYM broth
5. Terrific broth

Common microbes in foods


S.N. Food Microbes
1 Fresh meats Staphylococcus spp., Streptococcus spp., Enterococcus spp.,
Clostridia spp. and Enterobacteriaceae members
2 Soy extended ground Escherichia coli and Staphylococcus aureus in addition to all above
meats at No. 1
3 De-boned meat Clostridium perfringens, Staph aureus and all above in no. 1
4 Hot boned meat Staphylococcus spp. and clostridium spp.
5 Vacuum packaged meat Pseudomonas spp., Cl. Perfringens, Bacillus thermophillus
6 Organ and variety meat Morexella spp., Acenetobacter spp., Pseudomonas spp., Cl.
Perfringens
7 Sausages and Bacon Cl. botulinum, Satphyloccus spp. and Streptococcus spp.

Sources of microbes in foods


S. N Source Organisms
1 Soil and water Bacteria: Acenetobacter, Alkaligens group ( Klebsiella and Enterobacter),
Bacillus, Citrobacter, Clostridia, Corynebacterium, Enterococcus,
Micrococcus, Pseudomonas, Serratia, Streptomyces
Yeast and moulds: Aspergillus, rhizopus, Penicillium, Trichthecium,
Botrytis and Fusarium
2 Plants and plant Bacteria: Acetobacter, Erwinia, Flavobacter, Kurthia, Lactobacillus,
products Leuconostoc, Listeria, Pediococcus, and Streptococcus
Yeast and Moulds: Saccharomyces, Rhodotorula,, Torula
3 Utensils All as above from plants and water
4 Gastrointestinal Bacteria: Bacteroides, Escherichia, Lactobacillus, Proteus, Salmonella,
tract of man and Shigella, Staphylococcus, Streptococcus, Klebsiella, Enterobacter,
animals Clostridium, Citrobacter, Pseudomonas
5 Food handlers Micrococcus, Staphylococcus, Salmonella, Shigella and all others those
can come in faeces and urine.
6 Animal feeds Salmonella, E. coli, Listeria
7 Animal hides Bacillus anthracis
8 Air and dust Staphylococcus, Salmonella, Bacillus, Micrococcus, Torula and
Rhodotorula

Special Risk groups in spreading foodborne diseases


S. No. Group
1 Food handlers handling wrapped foods to be consumed without further cooking
2 Health care, nursery and other staff having direct or indirect contact through food with
highly susceptible people as aged, infants, patients with AIDS/TB and other wasting diseases
3 Children < 5 years of age attending nurseries and play groups
4 Older children and adults with poor standards of personal hygiene e.g. mentally ill,
handicapped, infirm, aged, in refugee camps, children in infant schools

Criteria for clearance of food handlers after infection diseases


Causative agent Criteria
Salmonella Typhi and Paratyphi 12 consecutive negative stool samples
Other Salmonella None after complete recovery if personal hygiene is of high
standard or 3 consecutive negative stool samples
Shigella, Vibrio cholerae and E. coli 3 consecutive negative stool samples
(outbreaks only)
Staphylococcus aureus- Vomiting or None after recovery
Diarrhoea or both
Staphylococcus aureus- skin lesions Until treated and healed
Foodborne viral diseases (Small strured 2 days after recovery
viruses)
Hepatitis A 7 days after appearance of jaundice
Rotavirus 7 days after recovery
Entamoeba histolytica 3 consecutive negative stool samples
Thread worms and pork tape worms Until treated
If the food handler also take care of young children, infants and infirm dependents then the
diseases in dependents also warrants the similar rules.

Media used for different bacteria


S.N. Bacteria Enrichment Plating
1 Vibrio cholerae Sodium gelatine phosphate broth, alkaline peptone TCBSA
water (APW),Trpticase soy broth with salt (TSBS)
all at 37oC for 6-8 hours
2 V. Glucose salt teepol broth (GSTB), Salt colistin TCBSA, Colistin
parahaemolyticus broth (STB) or Salt polymyxin B broth (SPB), polymyxinB cellobiose
APW with atleast 3-7% salt all at 37oC for 6-8 hr agar (CPCA)
3 V. vulnificus Starch gelatinase polymyxin B broth (SGPB) and TCBSA
APW all at 37oC for 6-8 hours
4 V. hollisae APW at 37oC for 6-8 hours Sucrose tellurite teepol
agar (STTA)

Vibrios as foodborne pathogen


Vibrio are said to be comma shaped but there are not comma shaped always.
There flagella are sheathed in a sheath which is merely an extension of cell wall.
They can go under sleep in unfavourable conditions, these sleeping vibrios are uncultivable in
laboratory but are viable and this phase appears to be the normal phase of their life.
Types:
1. V. cholerae O:1 ElTor and classical types (Aseatic cholera or King cholera or cholera gravis)
2. V. cholerae non O:1
3. V. cholerae O:139
4. V. parahaemolyticus (40-70% cases of total food poisoning cases in fish eating communities)
5. V. vulnificus (New terror of deep)-one of the most invasive bacteria

Differentiation of Vibrios

Test Vibrio Aeromonas Plesiomonas


Na+ requirement + - -
Effect of O/129 (150g) + + -
Sensitivity to 1% methyline blue + - -
Gas from glucose - (except V. Furnissii) v -
Ornithine decarboxylase V - (except A. veronii) +
Acid from inositol - (except V. metschnikovii) - +
Acid from mannitol V V -

Differentiation of Classical and ElTor varities of V. cholerae


S. No. Test name Classic ElTor
1 Haemolysis - V
2 VP test - +
3 Haemagglutination - +
4 Sensitivity to 1. Polymyxine B (50IU) + -
2. Classic phage IV + -
3. ElTor phage V - +

Syndrome and symptoms of foodborne diseases


Disease and Symptoms and syndromes Infective Persons at Specific
causative dose IP special risk foods
organism and involved
Course
Salmonellosis- 1.Enteric fever-anorexia, headache, non 103or Very young, Raw foo
S.Typhi, S. productive cough, remitted fever, rose less elderly, persons of anim
Paratyphi, S. spots, ddiarrhoea or constipation, myelgia, some with chronic origin
Schottmullerii stiff neck and rarely photophobia. times illness and those milk, be
and S . Sendii more with turkey,
also, malignancies icecream
depends MDR strains pork,
2. Gastroenteritis (food poisoning on affect more to chicken,
syndrome- strains. those under some dressings
8 hours antibiotic containin
Thousands of to few treatment raw eg
other Salmonella days for Persons handling unpasteri
FIRN strains 3. Extraintestinal infections- septicemia, typhoid, meat, poultry, d cream
(Fimbriae, HUS, Erythema nodosum, Meningitis, no high Vets, sewer
inositol, osteomyelitis, septic arthritis, pneumonia, body workers, tourists,
rhamnose cholecystitis, peritonitis, pylonephritis, temp. persons in
negative strains) cystitis, endocarditis, pericarditis, vasculitis 10-15 asylum, prisons,
Typhi, days for refugee camps,
Choleraesuis, paratyph ICUs,
Dublin, Virchow, oid, high hospitalised,
Panama & body sanitoria, and
London temp. child care units
For food
poisonin
g, 8
hours to
a few
days,
course
of
disease
is short
For ETI,
often
very
long and
chronic
course
Shigellosis 1.Classical-frequent liquid stools with ID-10 to Elderly, with Salds
Shigella blood and mucus, fever, abdominal 10000 underlying grown
dysenteriae cramps 12- diseases contamin
(common), 2. Diarrhoea , travellers diarrhea 50hours Neonates are ed so
flexneri & boydii In neonates only septicemia of comparatively washed
In children diarrhoea, emesis & dehydration infection resistant contamin
All above (rare), In older childrens and adults dysentery (3-4 Male ed wat
S. sonnei 2. HUS days) homosexuals, handled
(common) 3. Extraintestinal- Rheumatoid diseases course:1 Travellers in carriers,
(Reiters syndrome) and reactive 0-14 countries with drinking
arthritis), retarted height days poor personal water a
hygiene icecream
In army barracks, Milk a
camps, religious milk
communities products
(largest outbreak Potato,
in a Jews, 1986- tuna, e
87, >13000 salads,
affected, urban lettuce,
poor shrimps
Escherichia coli 1. Infantile diarrhoea-watery stool or rice 12-16 Neonates ands Any fo
1. EPEC- water stool, emesis, low grade fever, hrs infants <18 contamin
enteropathogenic dehydration, acidosis and shock Chronic months of age ed w
class I and II alsoType I causes life threatening persistent faeces
called as AEEC diarrhoea. animal
(attaching 2. Severe cholera like disease, watery 12-36 Travellers, all human,
effacing E. coli) diarrhoea & dehydration hrs age groups, sewage,
2. ETEC- ST induces emesis besides above Course elderly salads
enterotoxigenic 3. Dysentery like-bloody, mucoid stool with 1-2 days grown
neutrophils, tenesmus & fever & colitis. to a Aged, children contamin
3. EIEC-shigella 4. enterohaemorrhagic colitis (erosion, edema, week <5 years ed soil.
like haemorrhages of colonic mucosa) sudden For ST Milk
enteroinvasive severe pain followed by diarrhoea strains Patients having milk
5. HUS-May start as watery, haemorrhagic IP-4hrs gastrectomy, products
4. EHEC- diarrhoea followed by acute kidney failure, IP-2-3 antibiotic Raw milk
enterohaemorrhag micro angiopathic haemolytic anaemia, days therapy, H2 Meat
ic throbocytopenia, haemorrhagic cystitis, blocking drugs washed
5. VTEC- balanitis, convulsion, sepsis, iatrogenic with
verotoxigenic- haemorrhages IP 3-4 Children <16 contamin
colohaemorrhagic days years of age and ed awter.
6. EA-AggEC- some elderly, those Beef m
entero-adherent- times 1- under antibiotic have
aggregative 14 days therapy intrinsic
O157
Yersiniosis Not a foodborne diasesa Underco
Yersinia pestis Bubonic plague transmitted by flea ed me
Black death- by many means (pork),
Foodborne in Japan and elsewhere also unpasteu
Y. Typhoid like fatal septicemiaabdiminal pain, Contact with zed mi
pseudotuberculo nausea, fever, emesis absence of diarrhoea animals unchlorin
sis often confuse with appendicitis. IP 2-3d ed water
Terminal ileitis is confused with Crohns And oth
disease raw food
HUS, Erythema nodosum, arthritis, renal
deficiency cuasing confusion with Kawasaki
syndrome (mucocutaneous lymph node Elderly, children
syndrome in Japanese children. up to 14 yrs of
Foodborme disease all over globe, age &
gastroenteritis, abdominal pain, diarrhoea, immunocompro
enterocolitis with ulcerations, fever, nausea & mised
emesis Infants <1year of
Acute terminal ileitis leads to appendicitis age
some times pain in right fossa iliaca i.e. lower Those consume
right quadrant of trunk, due to mesenteric more Bantu beer
lymphedinitis may confuse with appendicitis. having more
Y. enterocolitica In children up to 18 years. amount of
With Painful arthritis with heart, pereicardial and ionisable iron.
autoimmune eye lesions. Overdosing with
diseases O:3 Erythema nodosum with painful swelling in iron tablets (in
serovar is the women of aged 40 to 70 years pregnancy)
main cause. Sjogrens syndrome- in middle aged women- Contact with
O:8 with soft inflammation of cornea, conjuctiva, pharynx, dogs and cat
tissue infection, glands and polyarthritis Nosocomial
conjunctivitis & Graves disease (exopthalmic goitre due to infections are
ostomyelitis excessive secretion of thyroid harmone also common
Foodborne pathogens
Pathogen Mini-Optim- Mini- Moisture Chemical Heat inactivation
Maxi pH Optim- requirement toxicity
Maxi temp as aw Radiation
and Eh & toxicity
aw
(--)
Salmonella 4 - 6.6 to 5.3 -35- >0.94 at >9% salt More with low aw and
8.2- 9 42- 47 neutral pH, >0.5 Mrad, high fat content
Acetic acid is (Eh 30 mV high at acidic more with 54.4oC with D value
more effective & aw 0.93) pH low aw, of 2.5 min, D60 0.2-
than citric or 3Kgy for 6.5 min. D70 in milk
HCl, Succinic chicken on chocolate 6-8 h for
acid is most ice Senftenberg and 12-
effective as 1% 18 h for
hot or cold dip Typhimurium
to meats
E. coli 4.4 -6.6-7.4- 4 -30-37- >0.95 >6% salt D55, 5 min D60, 0.1
9 46oC 200ppm min
nitrite at 10-
15oC and
400ppm at
20oC
3Kgy for
chicken on
ice
E.coli O157 4 to 45oC 3Kgy for D60oC 45 second in
chicken on ground beef
ice 9 months at 20oC
Shigella 5.5 6 to 7- 7---37---46 3Kgy for D63 is 5 min
7.5 S. sonnei chicken on
inactivated in 10-37-44 ice
30 min at pH S. flexneri nitrite
3.5 and 4 hours >700ppm S.
at pH 4 sonnei,
>450 ppm
S. flexneri
Y. 4.0 to 9.0 0.5 -28- >5% salt D62.8oC 0.7 to 17
enterocolitica sensitive to 29- >200ppm seconds, 0.96 min
tartaric acid 42oC nitrite
0.7 to 1.2
Kgy in TSB,
double dose
in beef at
25oC
>0.1 to 0.2
% sorbate at
pH 5.5

Isolation of Various foodborne pathogens


Bacteria MPN Media for Resuscitati Selective Selective plating Differential Specific
Count Impediomet on enrichment plating different
ry media
Salmonel Morgan- Selenite- BPW/ RV, MSRV, BGA, SSA, BSA, HEA, MIL,
la Jones cystine- lactose TTB, Salmonella DCLS, Lysine
Lysine- trimethylam broth, Selenite selective agar, MacConke agar, U
Iron- ine oxide BPW with Rambach agar ys agar test,
Cystine (SCTO) 0.01% phenylal
neutral dulcitol; malachite agar, O
red broth SCTO- green, test
(LICNR) mannitol; 0.002%
, with Lysine BG or
novobioc decarboxyla 0.004%
in se broth; CV
Alkaline Lactalbumin
reaction hydrolysate-
with glucose-
black lysine-
precipitat selenite
e
E. coli TSB (4-6 EE broth, VRBA, EMBA, HEA, TSI,
hrs) laural MacComkeys DCA, MIL
tryptose agar XLDA
broth, With and without
Lactose MUG (4-methyl-
bile salt umbelliferyl-B-
broth, GN D-glucuronide),
(gram Tryptose bile salt
negative) agar
broth at
41-45oC,
TSB at
44oC
E.coli EC broth MacConkey
O157 containing sorbitol agar at
novobiocin 42oC overnight,
reduced some strains do
bile salt at not grow at 42oC
37 oC, 6 hrs and lower temp is
shaking or needed
24 hrs at
35oC static
Shigella BPW, Gram SS agar, DCLS Congo
Trptic negative added with dye bin
phytone broth (pH sodium pyruvate agar
glucose 6-7),
agar Selenite
Cystine
broth for
18-24 hrs
at 35oC
Yersinia Alkali Cold CIN (Cefsulodin- Tempera
treatment: enrichment irgasan- depende
0.5 ml as PBS novobiocin) agar characte
BOS with with 2% (not suitable for Yersinia
4.5 ml of peptone at Y. pestis and ONPG
0.5% pH 8 to 8.3 pseudotuberculosi ornithine
KOH, at 4oC for s), MLA decarbox
0.5% NaCl 21 to 28 e, indole
for 15 days or acid
seconds 9oC for 7 cellobio
and plate days, raffinose
immediatel modified sorbitol
y RV with
Or irgasan &
0.5 ml ticarcillin
sample and Pot/.
homogenat Chlorate or
e with 0.5 bile-
ml dilute oxalate-
aqueous sorbate
alkaline (BOS)
solution broth at 22
(0.72% - 25oC for
KOH: 5 days.
0.5% Alkali
NaCl) for treatment
30 seconds of sample
and plate before
on to CIN processing
or MLA also
reduces
contamina
nts

Differentiation of Shigella from related bacteria


Tests Shigella (subgroup) Escherichia Hafnia Providen
S. S. flexneri(B) S. S.
dysenteriae(A) boydii sonnei
(C) (D)
B- Type-1 strains - V + + (except + -
galactosidase always + inactive
strains)
Gas from - - Only type 6 + Only - + (except + -
glucose type 13 inactive
& 14 + strains)
Ornthine - - - (Type + V + -
decarboxylase 13 +)
Lysine - - - - V + -
decarboxylase
Indole + (type 1-ve) + (type 6 ve) V - + - +
Phenyalanone - - - - - - +
Urease - - - - - - V
Acid from - -(type2a variable) - (type 9 + V - -
Lactose variable)
Xylose - - V - + + -
Rhamnose - - - + V + V
Motility - - - - V + +
Serotypes 1-10 1a,1b,2a,2b,3a,3b,3c, 1-15 Phase
4, 5, 6 1 and
2

Differentiation of Escherichiafrom related bacteria


Tests Escherichia spp Salmonella Klebsiella Enterobacter
Coli Adecarboxylata Hermani Vulneris
B- + + + + -(arizonae V +
galactosidase +)
Lysine V - - V + V V
decaroxylase
Ornithine V - + - + V +
decarboxylase
Indole + + + - - V -
Simmons - - - - + V +
citrate
VP - - - - - V +
Aesculin - + - - - V +
hydrolysis
Acid from + + + + -(aerizonae V +
lactose +)
Raffinose - - - - - + +
Salicin V V V V - + +
Sucrose V V V V - V V
Adonitol - + - - + V V
Amygdalin - + + V
Cellobiose - + + +
Sorbitol - - - -
(except
of
O157)
Differentiation of Yersinia and related genera

Character Yersinia Es
Pseudotu- Entero- Fredrikseni Intermedia Kristenseni Mollaretti Bercovieri
berculosis colitica
Colony size <1 mm <1 mm <1 mm <1 mm <1 mm <1 mm <1 mm >1
Lysine - - - - - - - V
decraboxylase
Ornithine - - + + + - - V
decarboxylase
Simmons citrate - except - V + - - - -
For Yersinia at serogroup
25oC IV
Urease + + + + + + + -
Indole - V + + V - - +
Phenyalanine - - - - - - - -
Gas from - V V V V V V +
glucose
Motility at 25oC + + + + + + + +
o
At 37 C - - - - - - - +
O
VP test at 25 C - + + + - - - -
Acid from - + + + + + +
cellobiose
Methyl-d- - - - + - - -
glucoside
Mucate - - V V - + +
Raffinose V - - + - - -
Rhamnose + - + + - - -
Sorbitol - + + + + + +
Sorbose - + + + + + -
Sucrose - + + + - + +
Pyrazinamidase* - V + + + + +
Pyrazinamidase test: A slant is made with TSA containing pyrezinecarboxamide and inoculated tubes
are incubated at 28oC for 48hr and then flooded with ferrous ammonium sulphate, which reacts with
pyrazinoic acid to give pink colour.
Wauters serotyping scheme is for Y. enterocolitica-18 serovars with 20 O antigens.
Biovars of Y. enterocolitica
Test 1e (environmental) 1a (american) 2 3
Hydrolysis of tween + + - -
Aesculin V + - -
Indole productiuon + + + -
Acid from xylose + + + +
Ornthine decarboxylase, nitrate + + + +
reduction and acid from trehalose
Pyrazinmidase + - - -
DNAse - - - -
Reservoir/ animal host Human and animals Humans, rats Human/pig/cat Chin
& fleas tle and sheep
Pathogenicity None (environmental) Human Human Chin
O Serovars associated with disease 4; 8; 13a; 13b; 9; 5,27 1,2,3
18; 20;21