10/20/2017 Learning Objectives Food 1.Use basic biochemical characteristics to identify Salmonella 2.Understand what conditions in foods favor Salmonella growth Chapter14 AN INTRODUCTION 3.Recognize,from symptoms and time of onset,a case of foodborne illness caused by Salmonella Salmonella species 4.Choose appropriate interventions(heat, preservatives,and formulations)to prevent the growth of Salmonella 5.Identify environmental sources of Salmonella 6.Understand the roles of Salmonella toxins and virulence factors in causing foodborne illness Outbreak Outbreak Cheesy chicken and rice frozen entrees Cheesy chicken and rice frozen entrees Spring and summer 2010 Frozen dinners can be cooked in ovens -Unopened packages revealed contamination Most prefer to use the microwave to heat with Salmonella enterica serovar Chester such meals EMLE团 -DNA fingerprint identical to the outbreak Spring and summer 2010 strain -44 cases of salmonellosis Heating meals in microwave ovens due to improperly cooked may not heat foods thoroughly cheesy chicken entre Microwaves vary in strength -40%of patients were hospitalized Microwaves cook foods unevenly No deaths reported Food should reach an internal temperature of 1650F
10/20/2017 1 Chapter 14 Salmonella species Learning Objectives 1. Use basic biochemical characteristics to identify Salmonella 2. Understand what conditions in foods favor Salmonella growth 3. Recognize, from symptoms and time of onset, a case of foodborne illness caused by Salmonella 4. Choose appropriate interventions (heat, preservatives, and formulations) to prevent the growth of Salmonella 5. Identify environmental sources of Salmonella 6. Understand the roles of Salmonella toxins and virulence factors in causing foodborne illness Outbreak Cheesy chicken and rice frozen entrées • Frozen dinners can be cooked in ovens • Most prefer to use the microwave to heat such meals • Spring and summer 2010 – 44 cases of salmonellosis - due to improperly cooked cheesy chicken entré - 40% of patients were hospitalized - No deaths reported Outbreak Cheesy chicken and rice frozen entrées • Spring and summer 2010 - Unopened packages revealed contamination with Salmonella enterica serovar Chester - DNA fingerprint identical to the outbreak strain • Heating meals in microwave ovens may not heat foods thoroughly • Microwaves vary in strength • Microwaves cook foods unevenly • Food should reach an internal temperature of 165OF
10/20/2017 Introduction Cost More Than 19,336 Salmonella species are leading causes of foodborne illness in humans $3.7 Billion Hospitalized Salmonellosis from food consumption is on the rise worldwide in2013 This increase is not a result of increased surveillance programs 1,027,561 378 Major outbreaks of foodborne patients in Died salmonellosis indicate various food types and numerous Salmonella serovars 2013 implicated in illness USDA ERS http/otias Salmonellosis is NOT JUST caused by Poultry Characteristics of the Organism In fact,meat poultry products were the source of only 33%of Salmonella outbreaks from 1998 to 2008 19th century,clinical pathologists in 19% France associated intestinal ulceration Poultry with a contagious infectious agent 67% .20th century,serological detection dairy, advanced for the O-antigen(LPS)and eggs, 14%Beef H-antigen (flagellar) produce, and Pork An antigenic scheme was developed for others the classification Now 2500 serovars of salmonellae
10/20/2017 2 • Salmonella species are leading causes of foodborne illness in humans • Salmonellosis from food consumption is on the rise worldwide • This increase is not a result of increased surveillance programs • Major outbreaks of foodborne salmonellosis indicate various food types and numerous Salmonella serovars implicated in illness Introduction Characteristics of the Organism • 19th century, clinical pathologists in France associated intestinal ulceration with a contagious infectious agent • 20th century, serological detection advanced for the O-antigen (LPS) and H-antigen (flagellar) • An antigenic scheme was developed for the classification • Now > 2500 serovars of salmonellae
10/20/2017 Biochemical Identification Biochemical Identification Facultative anaerobic gram-negative rod- ·Oxidase negative shaped bacteria ·Catalase negative Enterobacteriaceae family Produce hydrogen sulfide Most are motile but non-motile strains exist Decarboxylate lysine and ornthine Utilizes a wide range of carbon substrates Does not hydrolyze urea Grow optimally at 370C Grows on citrate as sole carbon source Catabolizes glucose and other carbohydrates with acid and gas production Biochemical Identification Most of these traits are the basis for presumptive identification of Salmonella ·Typically produce acid and gas from glucose but do not Mixed culture of Salmonella enterica on Hektoen enteri ferment lactose or sucrose in TSI,Hektoen Salmonella on XLD agar ogar.Note the black center of the roge agar,XLD agar ting hyof carbohydrate fermentation. Produce an alkaline reaction from decarboxylation of lysine to cadaverine in LIA slants Produce hydrogen sulfide gas in TSI and LIA
10/20/2017 3 Biochemical Identification • Facultative anaerobic gram-negative rodshaped bacteria • Enterobacteriaceae family • Most are motile but non-motile strains exist • Utilizes a wide range of carbon substrates • Grow optimally at 37OC • Catabolizes glucose and other carbohydrates with acid and gas production Biochemical Identification • Oxidase negative • Catalase negative • Produce hydrogen sulfide • Decarboxylate lysine and ornthine • Does not hydrolyze urea • Grows on citrate as sole carbon source Biochemical Identification • Most of these traits are the basis for presumptive identification of Salmonella • Typically - produce acid and gas from glucose but do not ferment lactose or sucrose in TSI, Hektoen agar, XLD agar - Produce an alkaline reaction from decarboxylation of lysine to cadaverine in LIA slants - Produce hydrogen sulfide gas in TSI and LIA Mixed culture of E coli and Salmonella on XLD agar Salmonella enterica on Hektoen enteric agar. Note the black center of the transparent colonies, indicating hydrogen sulfide production in the absence of carbohydrate fermentation
10/20/2017 Biochemical Identification ·eit6amaorofccelstasngeoC5c great concern in the clinic escapes detection in standard assays used in hospitals and industry lron Agar (M37 sucrose and lactose positive strains acquired gar Iron Agar (M021) richia coll ATCC 25922 genes for this phenotype via plasmids ol ATCC 25922 teu almonella Typhimurium ATCC 14028 Bismuth sulfite agar remains a medium of choice ris ATC A due to... high selectivity phiaeteysineandofomhydrogen -responds effectively to low levels of hydrogen sulfide Biochemical Identification Taxonomy and Nomenclature WHO Collaborating Centre for Reference and Research Variability in biochemical traits has lead to the conclude that Salmonella includes 2 species with replacement of traditional testing with multiple serovars as below molecular testing protocols Salmonella species and subspecies No.of Targets of molecular tests are stable genes or serovars gene products that are unique to Salmonella S.enterica subspecies enterica(I) 1,454 S.enterica subspecies salamae (II) 489 S.enterica subspecies arizonae(IIIa) 94 S.enterica subspecies diarizonae(IIIb) 324 S.enterica subspecies houtenae (IV) 70 S.enterica subspecies indica(VI) 2 S.bongori(V) 20 Total 2.463
10/20/2017 4 Differentiates enteric organisms based on their ability to decarboxylate or deaminate lysine and to form hydrogen sulphide Biochemical Identification • Genetic variability of clinical strains may occur as a result of mutations or plasmid exchange and is of great concern in the clinic - escapes detection in standard assays used in hospitals and industry - sucrose and lactose positive strains acquired genes for this phenotype via plasmids • Bismuth sulfite agar remains a medium of choice due to … - high selectivity - responds effectively to low levels of hydrogen sulfide Biochemical Identification • Variability in biochemical traits has lead to the replacement of traditional testing with molecular testing protocols • Targets of molecular tests are stable genes or gene products that are unique to Salmonella Taxonomy and Nomenclature WHO Collaborating Centre for Reference and Research conclude that Salmonella includes 2 species with multiple serovars as below Salmonella species and subspecies No. of serovars S. enterica subspecies enterica (I) 1,454 S. enterica subspecies salamae (II) 489 S. enterica subspecies arizonae (IIIa) 94 S. enterica subspecies diarizonae (IIIb) 324 S. enterica subspecies houtenae (IV) 70 S. enterica subspecies indica (VI) 12 S. bongori (V) 20 Total 2,463
10/20/2017 Serological Identification Gram-negative cell wall O-polysaccharide Aim:to determine the complete antigenic formulas for individual Salmonella isolates 3 main antigenic determinants 1.O-antigen associated with lipopolysaccharides heat stabile polysaccharide 与A ran Serological Identification 2.H-antigen associated with peritrichous flagella heat-labile protein antigens 3.Vi(virulence)-antigen associated with capsular antigen Weak or no only in serovars Readily agglutinates Typhi,Paratyphi C, with antibodies agglutination with and Dublin antibodies
10/20/2017 5 Serological Identification Aim: to determine the complete antigenic formulas for individual Salmonella isolates 3 main antigenic determinants 1. O-antigen associated with lipopolysaccharides - heat stabile polysaccharide Gram-negative cell wall Readily agglutinates with antibodies Weak or no agglutination with antibodies Serological Identification 2. H-antigen associated with peritrichous flagella - heat- labile protein - may produce 1 or 2 sets of flagellar antigens 3. Vi (virulence)- antigen associated with capsular antigen - only in serovars Typhi, Paratyphi C, and Dublin
10/20/2017 Physiology Physiology Temperature Strain can adapt to extreme environmental Temperature conditions ·Heat resistance -20C to 540c growth range increases in stationary phase cells pre-conditioning strains to cold or hot -increases when grown with high femperatures increases survival nutrients serious problems for the food industry increases as water activity of the Survives extended periods in food at food matrix decreases freezing and room temps and several factors will influence this Physiology Physiology Temperature pH Example:complex interaction of food, Grows at pH values ranging from 4.5-9.5 temperature,and organism ·Optimum pH6.5-7.5 -Salmonella survive on dry-roasted cocoa beans Pre-conditioned strains grown on pH gradient leads to contamination of in-line and finished plates can grow at a lower pH than the starting products cultures thermal inactivation of Salmonella in molten Raises concerns for fermented foods such as chocolate is difficult cured sausages and raw fermented milk products time needed would result in an unacceptable Starter culture used to acidify the foods product provide an environment that enhances Salmonella Salmonella survives for years in the finished survival in the read-to-eat product product Also promotes survival of Salmonella in the host
10/20/2017 6 Physiology Temperature • Strain can adapt to extreme environmental conditions - 2OC to 54OC growth range - pre-conditioning strains to cold or hot temperatures increases survival - serious problems for the food industry • Survives extended periods in food at freezing and room temps and several factors will influence this Physiology Temperature • Heat resistance - increases in stationary phase cells - increases when grown with high nutrients - increases as water activity of the food matrix decreases Physiology Temperature • Example: complex interaction of food, temperature, and organism - Salmonella survive on dry-roasted cocoa beans - leads to contamination of in-line and finished products - thermal inactivation of Salmonella in molten chocolate is difficult - time needed would result in an unacceptable product - Salmonella survives for years in the finished product Physiology pH • Grows at pH values ranging from 4.5-9.5 • Optimum pH 6.5-7.5 • Pre-conditioned strains grown on pH gradient plates can grow at a lower pH than the starting cultures • Raises concerns for fermented foods such as cured sausages and raw fermented milk products - Starter culture used to acidify the foods provide an environment that enhances Salmonella survival in the read-to-eat product - Also promotes survival of Salmonella in the host
10/20/2017 Acid survival responses in enteric microorganisms Acid stress enhances resistance to other adverse Physiology environmental conditions Osmolarity Lysine Foods with water activity <0.93 do not support growth of salmonellae Growth is inhibited in 3-4%NaCl +Inducible decarboxylases Salt tolerance though increases with increasing temperature 'a→RpoS→ASPs Salt in acidified foods can reduce the PhoP一AsPs tect/Repa antibacterial action of organic acids →ASPs Anaerobic conditions associated with vacuum packed foods may enhance salt tolerance in ASPs Salmonella FE5 Lett19971471-80 Reservoirs Reservoirs Poultry meat and eggs are predominant Salmonella will continue to be a significant reservoirs of Salmonella spp.in many countries human pathogen in the global market for USDA-FSIS requires meat and poultry several reasons industries to implement Hazard Analysis and 1.Presence in the environment Critical Control Point (HACCP)plans in all 2.Intensive farming practices in the meat. facilities fish,and shellfish industries FSIS conducts testing to verify that the 3.Recycling of slaughterhouse by-products HACCP plan is helping to control Salmonella on finished products into animal feeds Rate of Salmonella in meat has been reduce due to HACCP plans
10/20/2017 7 Acid survival responses in enteric microorganisms Acid stress enhances resistance to other adverse environmental conditions FEMS Microbiol Lett 1997;147:173-180 Physiology Osmolarity • Foods with water activity < 0.93 do not support growth of salmonellae • Growth is inhibited in 3-4% NaCl • Salt tolerance though increases with increasing temperature • Salt in acidified foods can reduce the antibacterial action of organic acids • Anaerobic conditions associated with vacuum packed foods may enhance salt tolerance in Salmonella Reservoirs Salmonella will continue to be a significant human pathogen in the global market for several reasons 1. Presence in the environment 2. Intensive farming practices in the meat, fish, and shellfish industries 3. Recycling of slaughterhouse by-products into animal feeds Reservoirs • Poultry meat and eggs are predominant reservoirs of Salmonella spp. in many countries • USDA – FSIS requires meat and poultry industries to implement Hazard Analysis and Critical Control Point (HACCP) plans in all facilities • FSIS conducts testing to verify that the HACCP plan is helping to control Salmonella on finished products • Rate of Salmonella in meat has been reduce due to HACCP plans
10/20/2017 Reservoirs Human illnesses caused by Typhimurium and Pandemic of human strain Salmonella serovar Heidelberg since 1986 Enteritidis associated with eating of raw or HACCP begins lightly cooked eggs or egg-containing products Typhimurium Epidemiology of SE tells the story of a pathogen that has found a biological niche in table eggs Heidelberg Viability of SE is unaffected by egg surface sanitation 每日量n的社起创可金的的社的的的的四■的 Transovarian transmission of SE via the egg Year Pathogenesis of egg contamination by Salmonella Reservoirs Aquaculture industry in developing countries feeds raw meat scraps that may harbor Salmonella to reared fish species Aquaculture relies heavily on antibiotics to control bacteria diseases This creates a serious public health concern
10/20/2017 8 Reservoirs • Pandemic of human strain Salmonella serovar Enteritidis associated with eating of raw or lightly cooked eggs or egg-containing products • Epidemiology of SE tells the story of a pathogen that has found a biological niche in table eggs • Viability of SE is unaffected by egg surface sanitation • Transovarian transmission of SE via the egg Pathogenesis of egg contamination by Salmonella Inne Gantois et al. FEMS Microbiol Rev 2009;33:718-738 Reservoirs • Aquaculture industry in developing countries feeds raw meat scraps that may harbor Salmonella to reared fish species • Aquaculture relies heavily on antibiotics to control bacteria diseases • This creates a serious public health concern
10/20/2017 Environmental Impacts of Open-Ocean Aquaculture Reservoirs Fruits and vegetables in recent years Increased due to global export of fresh and dehydrated fruits and vegetables from tropica countries ·Hygienic conditions during production, harvesting,and distribution do not always meet standards Education and improvec hygienic handling would improve food safety Characteristics of the Disease Characteristics of the Disease Symptoms and treatment Salmonella infections and clinical conditions Symptoms and treatment Enteric fever Salmonella infections and clinical conditions strains:typhoid and paratyphoid strains Non-typhoid Salmonella infections incubation:7-28 days Incubation:8-72 hours after ingestion Symptoms:diarrhea,prolonged,spiking fever, Symptoms:non-bloody diarrheal stools, abdominal pain,headache,and extreme abdominal pain within 5 days weakness Treatment:supportive therapy only fluid Diagnosis:isolate bacteria from blood or stools and electrolyte replacement Asymptomatic carrier state follows acute phase Antibiotics prolong carrier state Treatment:supportive therapy and antibiotics Asymptomatic carrier state is likely due to although antibiotic resistance limits the use of antibacterial drugs to treat enteric fever antibiotic repression of normal flora
10/20/2017 9 Reservoirs • Fruits and vegetables in recent years • Increased due to global export of fresh and dehydrated fruits and vegetables from tropical countries • Hygienic conditions during production, harvesting, and distribution do not always meet standards • Education and improved hygienic handling would improve food safety Characteristics of the Disease Symptoms and treatment • Salmonella infections and clinical conditions • Enteric fever – strains: typhoid and paratyphoid strains - incubation: 7-28 days - Symptoms: diarrhea, prolonged, spiking fever, abdominal pain, headache, and extreme weakness - Diagnosis: isolate bacteria from blood or stools - Asymptomatic carrier state follows acute phase - Treatment: supportive therapy and antibiotics although antibiotic resistance limits the use of antibacterial drugs to treat enteric fever Characteristics of the Disease Symptoms and treatment • Salmonella infections and clinical conditions • Non-typhoid Salmonella infections – Incubation: 8-72 hours after ingestion - Symptoms: non-bloody diarrheal stools, abdominal pain within 5 days - Treatment: supportive therapy only fluid and electrolyte replacement - Antibiotics prolong carrier state - Asymptomatic carrier state is likely due to antibiotic repression of normal flora
10/20/2017 Characteristics of the Disease Characteristics of the Disease Preventive measures Symptoms and treatment Killed-bacteria vaccines Salmonella can induce chronic conditions phenol-and heat-killed vaccines widely used today aseptic reactive arthritis against typhoid and paratyphoid salmonellae -Reiter's syndrome Cause adverse reactions:fever,headache,pain, Ankylosing spondylitis and swelling at site of injection Prerequisites for chronic diseases ·Pandemic poultry and egg-borne serovar >ability of the bacteria to infect mucosal Enteritidis infections surfaces underscore benefits of >presence of outer membrane LPS vaccines >ability to invade host cells Live attenuated vaccines continue to generate interest Salmonella infection Characteristics of the Disease Antibiotic resistance Global increase in the numbers of antibiotic- How salmonella abdominal cramps resistant Salmonella is alarming progesses Bacetieele 4-7 daysnsang Major public health concern is that Salmonella will become resistant to antibiotics used in human medicine Liberal use in human medicine and in animal feed contributes to the resistance Virulence genes and resistance genes can Treatment occur on the same plasmid,which increases the disease potential 10
10/20/2017 10 Characteristics of the Disease Symptoms and treatment • Salmonella can induce chronic conditions – aseptic reactive arthritis - Reiter’s syndrome - Ankylosing spondylitis - Prerequisites for chronic diseases ability of the bacteria to infect mucosal surfaces presence of outer membrane LPS ability to invade host cells Characteristics of the Disease Preventive measures • Killed-bacteria vaccines – phenol- and heat-killed vaccines widely used today against typhoid and paratyphoid salmonellae - Cause adverse reactions: fever, headache, pain, and swelling at site of injection • Pandemic poultry and egg-borne serovar Enteritidis infections underscore benefits of vaccines • Live attenuated vaccines continue to generate interest Characteristics of the Disease Antibiotic resistance • Global increase in the numbers of antibioticresistant Salmonella is alarming • Major public health concern is that Salmonella will become resistant to antibiotics used in human medicine • Liberal use in human medicine and in animal feed contributes to the resistance • Virulence genes and resistance genes can occur on the same plasmid, which increases the disease potential