Denise - Enterobacterales Flashcards
Write about the enterobacterales species
Gram negative bacilli
Facultatively anaerobic
Ferment glucose
Reduce nitrate to nitirite
Oxidase negative
Weak catalase positive
Most motile
Some capsulated
List the eight enterobacterales
Lactose fermenters:
Escherichia
Citrobacter
Enterobacter
Klebsiella
Non lactose fermenters
Shigella
Proteus
Salmonella
Yersenia
Write about the four factors of enterobacter pathogenesis
Enteropathogens in the GIT
Uropathogens in the UTI
Opportunistice infections
HCAIs
Give some examples of opportunistic infections caused by enterobactet
Central nervous system infection (Escherichia)
Lower respiratory tract infection
Blood stream infection
What enterobacters can cause a lower respiratory tract infection
Klebsiella
Enterobacter
Escherichia
What enterobacters can cause bloodstream infections
Escherichia
Klebsiella
Enterobacter
What enterobacters can infect the GIT
Salmonella
Shigella
Escherichia
Yersinia
What HCAIs are caused by enterobacter
Blood stream infections
Catheter associated UTIs
Ventilator associated pneumonia
Antimicrobial resistance
What infections does Escherichia, Salmonella and Yersinia cause
infection within the GT and outside the GIT
What infections does Shigella cause
Primarily gastrointestinal
What infections does Klebsiella, Enteobacter and Citrobacter cause
Primarily extraintesitinal
what are sources of enterobacterales
(3)
Faeces from humans/animals containing pathogens/toxins
This is then ingested through food or liquid (milk or water) or on your fingers
Ingestion of organism and/or toxins
what happens upon ingestion of organisms and/or toxins
Organisms multiply and toxins produced but infection remains localised -> diarrhoea -> pathogen in faeces
Organisms invade or toxins are absorbed -> dissemination -> symptoms of systemic infection fever
How are faecal specimens processed in the lab
Evaluate specimens macroscopically based on appearance
Direct molecular detection is carried out - if positive then further testing is carried out
Enrichment broths and sub culturing onto selective plates
What investigation must be done for cryptosporidium/giardia faecal specimens
Smear microscopy
What treatment shous clostridium diff/perfringens and B. cereus feaces undergo?
Pretreatment
Alcohol shock
Toxin detection
Explain the conventional micro process of laboratory investigation of faeces
Faeces -> broth for 24 hours -> selective agar for 24 hours -> biochemical ID -> serotyping
Explain the contemporary micro process of laboratory investigation of faeces
Direct detection via Real Time PCR or EntericBio
Same day detection of target gene
Write about E. Coli
Common constituent of mammalian digestive tracts
Predominant facultative anaerobe
Essential for our well being:
- improves digesiton
- produces essential vitamins
- competitive exclusion
Write about the diversity of E. Coli: Seroty[es
Tradition classification made on the basis of presence of three antigens O, K and H antigens
A combination of O and H antigens have been identified and used to serotype E. Coli
Give some examples of E. Coli serotypes
O157:H7
O104:H4
O26
O103
O111
How does E. Coli acquire virulence genes
Horizontal gene transfer
What are the three steps of horizontal gene transfer
Transfer of virulence genes via plasmids
- Conjugation
- Transduction
- Transformation
Or transfer of virulence genes via bacteriophages
Change of vell to virulence
Virulence genes bringing about:
- adherence
- enterotoxins
- invasiveness
- cytotoxicity
Due to intestinal disease
Describe intestinal pathogenic E. Coli, what are the different strains causing intestinal infection
(6)
Enterotocigenic (ETEC)
Enteropathogenic (EPEC)
Enteroaggregative (EAEC)
Enteroinvasive (EIEC)/Shigella
Diffuse adhering (DAEC)
Shiga toxin producin (STEC) and other enterohemorrhagic (EHEC)
What strain of E. Coli causes extraintestinal infection
ExPEC
- uropathogenic
- strains associated with neonatal meningitis
What infections does enterotoxigenic (ETEC) cause
Watery diarrheae
What infections does enteropathogenic (EPEC) cause
Watery diarrhear of lung duration
Mostly in infants, often in developing countries
What infections does Enterohaemorrhagic E. Coli cause
EHEC
What infections does Enterohaemorrhagic E. Coli (EHEC) cause
Bloody diarrhea
Haemorrhagic colitis and haemolytic uremic syndrome (HUS)
What does infection enteronvasive (EIEC) cause
Blood diarrhea
What infection does enteroadherant E. Coli (EAEC) cause
Persistent watery diarrhea in children and patients infected with HIV
Write about enteropathogenic E. Coli
Induces the formation of pedestals on the host cell surface
Causes infantile diarrhoea mostly in the tropics
Caused by poor hygiene
Explain the pathogenesis of Enteropathogenic E. Coli
Induces the formation of pedestals on the host cell surface
Causes infantile diarrhoea mostly in the tropics
Caused by poor hygiene
Non invasive
Does not produce toxins
Adherence to intestinal mucosa
Small intestine effacement
Plasma encoded protein
What does enteropathogenic E. Coli secrete
Intimin and TIR
What does secretion of intimin and TIR by EPEC do
Destroys microvilli
Loss of absorbtive surface
Leads to diarrhoeae
Write about EnteroInvasive E. Coli (EIEC)
Inflammatory dysentry
Bloody diarrhoea
Large intestine
Intracellular pathogen
fever
Explain the pathogenesis of EIECs
Invades the basolateral surface
- invasion plasmid antigen
- Ipa gene
Lateral spread
Food, water and person-to- person transmission
Write about enterotoxigenic E. Coli
Non inflammatory
Diarrhoae in children in developing countries
Travellers Diarrhoea
Write about the pathogenicity of ETEC
Colonisation factor antigen
LT heat-labile toxin activates adenylate cyclase-increases production of cAMP
ST heat-stable toxin activates guanylate cyclase- increased production of cGMP
Increased fluid secretion = diarrhoae
Write about EHEC
E. Coli 0157
Shiga toxin production
Hemolysin
LEE
Mobile genetic elemets:
- phages
- genomic islands
- plasmid
What infections does E. Coli 0157:H7 cause
Intestinal
- asymptomatic
- watery diarrhoea
- haemorrhagic colitis
Systemic
- haemlytic uremic syndrom HUS
Causes:
- Thrombotic thrombocytopenic purpura (TTP)
- kidney failure
- haemolytic anaemia
What does infection of 0157:H7 cause
- Thrombotic thrombocytopenic purpura (TTP)
- kidney failure
- haemolytic anaemia
What is E. Coli 0157 called
EHEC
STEC
VTEC
Write about the epedemiology of STEC/VTEC infections
Ireland had the highest annual incidence of VTEC between 2009 and 2018 with the exception of 2011
Write about the day 1 investigation of E. Coli
Clinical sample = faeces
Direct detection of shiga toxin genes by entericBio real time PCR
Selective culture on day 1 is positive
What selective media should E. Coli be put up on
MacConkey for all E. Coli
STEC
- STEC chromagar
- CTSMAC (Cefixime Tellurite sorbitol MacConkey)
Explain the use of Enteric Bio real time PCR for detection of VTEC/EHEC
Real-time detection of pathogens directly from faeces
Without pre-enrichment DNA
DNA extraction amplification and detection
Rapid result 3 hours Day 1
Write about Enteric Bio Panels for Bacteria, what can we test for
Shiga toxin (1)
Shiga toxin 2
Salmonella/Shigella/Campylobacter
Yersinia
Vibrio
Clostridium difficile
What parasites can we detect with enteric bio
Cryptosporidium/Giardia
Entamoeba
What viruses can be detected with Enteric Bio
Norovirus, Rotavirus, Adenovirus
Where is the reference lab for VTEC/EHEC
Cherry Orchard
Write about VTEC isolation on CT SMAC agar
Sorbitol fermenters = pink e.g. non 0157H7
Non sorbitol fermenter = colourless e.g. O157H7
Write about STEC Chromagar
Detects most common Shiga-toxin E. Coli serotypes -> mauve colonies
Other enterobacterales are coulerless, blue or inhibited
Write about the day 2 investigation of E. Coli
GNBs
Oxidase negative
Weak catalase positive
Biochemical screen: IMVic Test
What is meant by the IMVic test
I: indole
M: methyl red
V: Voges-Proskauer
iC: citrate
What are the biochemical IMVic results for E. Coli
I: produces indole
M: methyl red positive
V: negative
iC: negative
Write about the indole test
decompose the amino acid tryptophan to indole
accumulates in the medium
Inoculate a peptone water
Incubate 24 hrs @ 37
When indole is combined with Kovac’s Reagent
yellow to cherry red
an oily layer at the top of the broth.
Write about the methyl red test
Methyl Red Test
detection of end products from the
metabolism of glucose.
Inoculate a buffered glucose
the fermentation of glucose.
MR Positive: When the culture medium turns red
after addition of methyl red
pH at or below 4.4
Write about Voges-Proskauer test
Voges-Proskauer Test
detection of end products from
the metabolism of glucose.
produce acetoin as the chief end
product of glucose metabolism
Heavy inoculum
Add 40% KoH and alpha Napthol
Shake vigorously
Colour development within 15 mins
Voges-Proskauer negative
Write about Simmons Citrate
Simmons Citrate Test
use citrate as sole carbon source
Growth usually results in the bromothymol blue
indicator, turning from green to blue.
The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6
Write about all of the biochemical test results for E. Coli
produces indole
methyl red positive
Voges-Proskauer negative
cannot use citrate as sole carbon source
Lactose Fermenter
produces lysine decarboxylase
ferments sorbitol except E. coli O157 H7
produce β-glucuronidase except E. coli O157 H7
What two methods of automated identification can be used for E. Coli
Vitek
MaldiTOF
Write about use of Vitek for E. Coli
ID and sensitivity
ID 64 individual substrate tests
Colourmetric
Write about use of MaldiTOF for E. COli
Cannot differentiate between E. Coli and Shigella
93% of their genes are common
What are the four species of shigella studied
Shigella dysenteriae - serogroup A
Shigella flexneri - serogroup B
Shigella boydii - serogroup C
Shigella sonnei - serogroup D
Which is the most serious shigella species
Shigella flexneri
Shigella boydii
Shigella sonnei is the most severe
Write about shigella species
Reservoir is humans
Food = chicken, tuna and potato salad
Spread via faecal-oral route through food, faeces, fingers, flies and fomites
Fragile in human faeces
Incubation time of 24-50 hours
What are the symptoms of shigella
Diarrhoeae
Abdominal pain
Fever
Might become invasive
Write about shigella infection
Toxin mediated infection
Shiga toxin (AB toxin)
Bloody diarrhoea
Infective does < 200 cells
Write about shigella epidemiology
8 shigellosis outbreaks in 2018
Large outbreak of shigellosis in 2017 in HSE
Write about the day 1 laboratory investigation of shigella
Faeces sample
Direct Detection
Selective culture
How is direct detection of shigella carried out
Real Time PCR
EntericBio
BD Max
Syndromic Testing
BioFire GI panel
Shigella Ipa gene DNA detected on Day 1
What selective culture is used for shigella
XLD
DCA
What is the day 2,3 investigation of shigella
Day 2 = reading XLD and DCA agar
Day 3 = biochemical ID, serotyping
Write about deoxycholate citrate agar
Selective factors include sodium deoxycholate and sodium citrate
Differential factors are lactose and sodium ferric chloride
Lactose fermenters = commensals = pink
Shigella = NLF = pale
Salmonella = NLF = pale with H2S positive black centre
Write about XLD media
Selective factor is sodium deoxycholate
Differential factors are Xylose, lysine, H2S
Lactose fermenters = commensals = yellow
Shigella = xylose lysine = red
Salmonella = red with black centre
Write about shigella on chrom GTI
Teal coloured colonies
Non-H2S producing salmonellas will look the same
What are the basic characteristics of Shigella
GNBs
Oxidase negative
Weak catalase positive
Non motile
Late lactose fermenter
Write about biochemical results for shigella
Indole negative
Methyl red positive
Vogues negative
Citrate negative
urease negative
Lysine decarboxylase negative
Where is the shigella reference lab
University Hospital Galway
Salmonella and Shigella Listeria Reference Lab
What are the main reservoirs for salmonella
Foodborne - eggs
Zoonotic - lizards
Human - typhoid mary
What are the main species of salmonella studies
S. Typhimurium
S. Enteritidis
S. Typhi
S. Paratyphi
S. cholerae suis
What infection does S. cholerae suis cause
Septicaemia
What infection does S. enteriditis and S. typhimurium cause
Gastroenteritis
What infection does S, paratyphi and S, typhi cause
Parathyphoid fever or thyphoid fever
Leads to enteric fever
What are the virulence factors of Salmonella
Peptidoglycan
Fimbrae/Pilli
Salmonella Pathogenicity islands
Type 3 secretion system
Flagellin
Vi antigen of S, typhi
What is special about S. typhi
Vi antigen
How does S. enteritidis and S, typhimurium cause gastroenteritis
Invasion of M cell of intestine
Passes into tissue
Uptake by Neutrophils, dendritic cells and macrophages
Limited dissemination i.e. they can’t spread out of intestine
How does S. typhi and S. paratyphi cause typhoid fever
Invasion through M cells
Macrophage uptake
Intramacrophage replication
System dissemination i.e. spread to other organs such as the gallbladder
How does S. typhi cause enteric fever
Uptake through small intestine
Spread to other organs such as gallbladder and lymph nodes
Gallbladder infection leads to cholecystitis and a carrier states
Infection of small intestine caused inflammation and ulceration of intestinal which causes diarrheae and hemorrhage or performation
Explain what happens when S. typhi infects the lymphatic system
(5)
S, typhi passes from small intestine into lymph
Travels to lymph nodes
Enters circulation via thoracic duct (bacteremia) + fever + malasie
Infection of liver, spleen ,kidney, bone marrow etc (secondary bacteremia)
Causes fever, slow pulse, enlarged liver and spleen ,rose spots and low WBC count
Write about Salmonella epidemiology
11 cases of S, Typhi many associated with travel
5 cases of S, paratyphi associated with India
Write about day 1 investigation of salmonella
Direct detection of Salmonella Inv gene
Selective enrichment on selenite broth
How is direct detection of salmonella carried out
Real Time PCR
EntericBio
Syndromic testing
BioFire GI panel
Inv gene DNA detected
What selective agar is used for Salmonella
Selenite broth
What is the day 3 investigation for salmonella
XLS
DCA
Chromagar
What is the day 4 investigation of salmonella
Biochemical ID
Serotyping
Write about samples for Salmonella
Cat 3 facilities required
Enteric fever:
- blood culture
- faeces
- urine
- bone marrow aspirate
Gastroenteritis
- faeces
Write about selenite broth for Salmonella
Selenite broth
Enhances culture yield by reducing growth of faecal coliforms and faecal streptococci.
Take sub-cultures of broth from the upper third of the broth column should be at least 5 cm in depth.
Better for enrichment of Salmonella Typhi
37 not exceeding 18 hours
Write about XLD agar for Salmonella
Lactose Fermenters Commensals yellow
Shigella xylose Lysiene red
Salmonella red with black centres
Write about DCA agar for Salmonella
Lactose Fermenters Commensals pink
Non-lactose fermenters are colourless pale
Shigella NLF pale
Salmonella NLF pale
Many Salmonella spp H2 S pos
Write about Bismith Sulphite agar for Samonella
S Typhi and Paratyphi
BS and Brilliant Green
Selective
Write about Salmonella on Chromagar
Teal coloured colonies with black centres
What are the basic characterisations for Salmonella
Motile
Non Lactose Fermenter
Oxidase Negative
Weak Catalase Positive
GNB
Write about the biochemical results for Salmonella
I : negative
M: positive
V: negative
iC: positive
urease negative
lysine decarboxylase positive
What are the components of the lysine decarboxylase test
Glucose
Lysine
Bromocresol Purple
Write about the Kauffman-White Classification of Salmonella
Differentiates isolates based on surface antigens
First “O” antigen type is determined- associated with LPS of outer membrane
Then the “H” antigen is determined- associated with flagella
Salmonella exist in two phases; a motile (specific) phase and a non-motile (nonspecific phase
Different “H” antigens are produced depending on the phase in which the salmonella is found
What are the three main Salmonella antigens
O-somatic cell wall antigens
H-flagellar antigens
Vi-antigens (enteric fever strains)
What do we do with a confirmed salmonella case
Whole genome sequencing
Antimicrobial resistance
Write about whole genome sequencing for salmonella
Done for:
- serology
- antimicrobial susceptibility
- epidemiological typing
- outbreak investigation
Write about the antimicrobial resistance in Salmonella
21% MDR
3 or more antimicrobials
1/3 profile of resistance
to ampicillin, sulphonamide and tetracycline (ASuT) were mainly monophasic S. Typhimurium
1 ESBL
S.Brandenburg
What are the three most important yersinia species
Yersinia pestis
Yersinia enterocolitica (foodborne)
Yersinia pseudotuberculosis
Write about yersinia pestis infection
Plague - caused by yersinia pestis
Bubonic plague
Write about bubonic plague
Swollen and painfil axillary (arm pit) and inguinal (groin) lymph nodes (buboes)
Transmitted from mammalian reservoirs by flea bites or contact with contaminated animal tissues
If infection spread to lung from lymph it caused pneumonic plague
Person-to person spread
Write about yersinia enterocolitica infection
Enterocolitis
Causes enterocolitis with diarrhoeae fever abdominal pain
Invasion of the ileum
Necrosis of the Peyers Patches
Inflammation of the mesenteric lymph nodes
1/4 of children develop blood diarrhoea
Appendicitis type syndrome in children
BSI in elderly
6 cases in 2018
Write about yersinia spread
Reservoir: animals, rats, pets, soil, lakes
Food: milk, PORK, poultyr, shellfish, vegetables
Psychotrophic growth at 4 degrees
Cold chain food products hazards
Enteric zoonosis
Incubation time 24-36 hours
What are symptoms of Enteric zoonosis
Diarrhoea
Vomiting
Fever
Headache
Pseudoappendicitis
Write about day 1 yersinia investigation
Direct detection via:
- rela time PCR
- BD max
- Syndromic testing
- BioFire GI panel
Yersenia gene DNA detected
Selective culture on CIN agar
What are the basic characteristics of yersinia
GNBs
Oxidase negative
Weak catalase positive
Motile at 25 not 37
Non lactose fermenter
What is CIN agar
Selective for yersinia
Cefsulodin, irgasan, novobiocin agar
What are the biochemical results for Yersinia
Indole positive
Methyl red negative
Vogues positive
Citrate negative
Urease positive
Lysine negative
Ornithine decarboxylase positive
What three things caus urinary tract infections
UroPathogens
Escherichia
Proteus
Give an example of a urpathogen
E. Coli UPEC
Write about EPEC
80% of UTIs
Endogenous infection
How can EPEC cause an infection
Contamination of urethral with EPEC from gut
Colonisation of urethra with migration towards bladder
Colonisation and invasion of bladder mediated by pili and adhesion
Inflammatory response in bladder and fibrinogen accumulation in the catheter
Neutrophil infiltration
Bacterial multiplication and immune system subverison
Biofilm formation
Epithelial damage by bacterial toxins and proteases
Ascension to the kidneys
Colonisation of the kidneys
Host tissue damage by bacterial toxins
Bacteraemia
Write about Chromagar
E. coli - dark pink to red
Enterococcus - turquoise blue
Klebsiella, Enterobacter, Citrobacter - metallic blue
Proteus - brown hali
Pseudomonas - cream/translucent
S. aureus -> golden/opaque
S. saprophyticus -> pink small
Write about UPEC biochemical tests
Indole positive
Methyl red positive
Vogees negative
Citrate negative
urease negative
Lysine dacarb positive
Dulcitol positive
Lactose positive
Write about proteus
Second most common cause of UTI
Proteus mirabilis
Sample = mid stream urine
Write about isolation of proteus
Not fastidious
Highly motile-form ‘swarm’
No swarm on macConkey (NLF)
Chromogenic UTI medium brown
Write about the biochemical results for proteus mirabilis
Indole negative
MR positive
VP negative
Citrate positive
Urease positive
Lysine decarboxylase -
Dulcitol -
Lactose -
Phenylalanine postiive
Write about extra intestinal E. Coli
Neonatal meningitis
- 30% caused by E. Coli not HCAI
Bloodsteam infection
- progression from UTI
What are the two main enterobacters involved in HCAI
Klebsiella pneumonia
Enterobacter species
How do you remember the HCAIs
ESKAPE
Enterococcus faecium
S. aureus
K. pneumoniae
Acinetobacter baumannii
P. aeruginosa
Enterobacter species
What are the four main HCAIs
Central line associated blood stream infections
Catheter associated UTI
Ventilator associated pneumonia
Antimicrobial resistance
Write about Klebsiella species
Commensal but frequently an opportunistic pathogen causing BSI, HCAI UTI, VAP pneumoniae
Specimens: blood, urine, sputum
Screen for carriage -> rectal swabs and faeces
Write about Klebsiella antimicrobial resistance
Carbapenemase producing enterobacterales CPE
What are the basic characteristics of Klebsiella
Not fastidious
Mucoid colonies
Basic characterisation (GNBs Ox negative weak cat positive)
Write about the Klebsiella Biochemical tests
Indole negative
MR negative
VP positive
Citrate postiive
Urease V positive (K. pneumo)
Lysine decarboxylase +
Dulcitol +
Write about enterobacter species
Opportunistic Infections
Health Care Acquired Infection
Antimicrobial Treatment
Catheters
Invasive Procedures
Burns/ wounds
Pneumonia
UT
Write about Enterobacter ID
LF on MacConkey
Non fastidious
GNB
Oxidase negative
Weak catalase postiive
API or Vitek can be used
What are the biochemical results for enterobacter
Indole negative
MR negative
VP positive
Citrate postiive
Urease negative
Lysine decarboxylase negative
Dulcitol negative
Write about citrobacter species
Inhabitant of soil and water and found in GIT
Citrobacter freundii- main pathogen
Healthcare-associated infection, UTI, bacteraemia
What are the biochemical results for citrobacter species
I : produces indole -
M: methyl red positive +
V: Voges-Proskauer negative -
iC: cannot use citrate as sole carbon source +
Urease -
Lysine Decarboxylase -
Dulcitol –
Lactose + LF
Look at slide 100 to learn biochemical tests
Look at slide 100 to learn biochemical tests
