The Complete General Secretory Pathway in Gram Negative Bacteria
Gram Negative Bacteria
Transcript of Gram Negative Bacteria
COLLEGE OF MEDICINE, NURSING & HEALTH SCIENCES
MLS 602 : General & Medical Microbiology
LECTURE 5 : Gram Negative Bacteria
Ms. Edwina Razak
Learning outcomes
•Explain why some bacteria stain as Gram Negative based on Gram staining test
•Describe the cell wall structure of Gram negative bacteria.
•Name the different types of Gram Negative bacteria.
•Discuss the biochemical characteristics of common Gram Negative bacteria.
•Describe the purpose and principle of common biochemical tests used to identify gram negative bacteria
•Briefly discuss on the pathogenesis mechanism
Recap: Cell Wall of Bacteria
Gram Negative Bacteria- Cell wall
Gram-negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide (LPS).
• Between the cytoplasmic membrane and the outer membrane is a space called the periplasmic space or periplasm.
• The periplasmic space contains the loose network of peptidoglycan chains referred to as the peptidoglycan layer.
Gram negative bacilli
Family:EnterobacteriaceaeReferred as “Enteric bacilli”
aerobes or and facultative anaerobes
ferment glucose (fermentation of other sugars varies) with the production of acid or acid and gas.
either non-motile or motile with
peritrichous flagella.
oxidase-negative.
Reduce nitrate to nitrites as part of their energy-generating processes.
Intestinal microorganism
All lactose fermenting (LF) enterobacteria, e.g. Escherichia, Klebsiella, Enterobacter and Citrobacter are popularly known as ‘coliform bacilli’ as the most common member of this group is the colon bacillus or Escherichia coli.
The major intestinal pathogens, Salmonella and Shigella are non-Iactose-fermenters (NLF).
Escherichia coliMay have a capsule and most are motile
Habitat:◦ Normal bowel flora of humans and other
animals.
A small proportion of E. coli strains are pathogenic.
The harmless strains produce vitamin K and prevent colonization of the intestine by pathogenic bacteria.
Is a major cause of urinary tract infection
Are Indole and motility and Lysine positive
E.coli pathogenic strains are ◦ Enteropathogenic E.coli (EPEC)◦ Enterotoxigenic E.coli (ETEC)◦ Shiga toxin producing E.coli (STEC)◦ Enteroinvasive E.coli (EIEC)◦ Enteroaggregative E.coli (EAEC)
Gram stained smear E.coli on MacConkey Agar
Klebsiella sppHabitat:
◦ Normal flora of the GIT◦ nonsporing, non-motile bacilli,
produce pink mucoid colonies on MacConkey’s agar.
Ranked among the top ten bacterial pathogen responsible for nosocomial infections
K.pneumoniae is most commonly isolated and can be found in respiratory tract and feces of about 5% of healthy individuals
Transmission:◦ Endogenous, or person to person
especially in hospitalized patients.
are short,plump, gram-negative, non-sporing, capsulated
Lactose fermenters and are urease positive, citrate positive and urease positive
There are 7 species of Klebsiella but we will just look at the 3 main types:
Klebsiella pneumonia
Klebsiella oxytoca
Klebsiella ozaenae
Klebsiella pneumoniae on MCA
Lactose fermenter and mucoid colonies
Positive for string test
Other Lactose Fermenting Entrobacteriacae ENTEROBACTER
Habitat
ofeces, sewage, soil and water
motile, capsulated, non-spore-forming which is indole and citrate positive
Enterobacter aerogenes and E. cloacae have taken on clinical significance as opportunistic bacteria and have emerged as nosocomial pathogens from intensive care patients pathogenic, especially to those who are on mechanical ventilation
SERRATIA
Normal flora of the GIT
S. marcescens is the one most commonly encountered species in clinical specimens.
small, motile, gram-negative bacillus
forms a pink, red or magenta, nondiffusible pigment called prodigiosin.
Ferments Lactose slowly
Non Lactose FermentersProteus
P. mirabilis and P. vulgaris are widely recognized human pathogens
Isolated from urine, wounds, and ear and bacteremic infections
Both produce swarming colonies on non-selective media and have a distinctive “burned chocolate” odor
Both are strongly urease and phenyl alanine positive
Pleomorphism is frequent—short coccobacilli to long filaments. In fresh cultures, many of the bacteria are long, curved and filamentous.
motile with peritrichous flagella
Noncapsulated
Indole is formed by P. vulgaris but is negative in P. mirabilis.
H2S is produced by P. vulgaris and P. mirabilis
Proteus
Non Lactose FermentersSalmonella
Habitat:
◦ S.typhi and paratyphi – only found in humans but not part of the normal flora of the bowel
◦ Other Salmonella spp. – Widely disseminated in nature and associated with various animals
motile with peritrichous flagella, non-capsulated and non spore-forming.
Hydrogen sulfide is produced except by S. paratyphi A
1. Flagellar antigen H
2. Somatic antigen O.
3. Surface antigen Vi, found in some species.
Salmonella typhi on XLDNon lactose fermenter with black centred colonies (due to production of Hydrogen sulphate.
Non Lactose FermentersShigella
Habitat:
◦ Only found in human GIT at times of infections and is not part of the normal bowel flora
nonsporing, noncapsulate,
nonmotile and nonflagellate
S. sonnei, a late fermenter of lactose, become pink when incubation is prolonged beyond 24 hours.
The shigellae are divided into fourgroups: S. dysenteriae,
S. flexneri,
S. boydii
S. sonnei.
Other GNBAeromonas
Curved gram-negative rods and oxidase positive, NLF
strains that are pathogenic to humans are motile
Most significant human pathogens are:
Aeromonas hydrophila
Aeromonas caviae
Aeromonas sobria
Colonies on SBA shows large Beta hemolytic grayish green, foul smelling colonies
◦ gastrointestinal disease - Chronic diarrhea in adults, Self-limited acute severe disease in children.
◦ Wound infections
Vibriosshort, curved, cylindrical rods. The cell is typically comma-shaped. S-shaped or spiral forms may be seen due to two or more cells lying end-to-end.
The motility is of the darting type. They are nonsporing, noncapsulated.
V. cholera causes Cholera and is associated with
oxidase positive
tolerate alkaline media
Pseudomonadsaerobic rod, NLF, Oxidase positive, actively motile usually with a single polar flagellum.
Produce pigments:◦ Pyocynin – blue-green
◦ Pyoverdin – yellow-green, fluorescence (UV)
◦ Pyorubin – red
◦ Pyomeanin – brown
It is nonsporing, noncapsulatedbut many strains have a mucoid slime layer (Psedomonasaeruginosa).
Habitat:◦ The primary habitat of P.
aeruginosa is the environment, chiefly found in soil and water, Also found in 10% people as normal flora of colon.
Transmission:◦ Ingestion of contaminated food and
water, Exposure to contaminated medical devices and solutions
◦ Introduction by penetrating wounds.
Pseudomonas -Oxidase positive MHM- shows green pigmentation Blood Agar with Pseudomonas
(pyocyanin)
Gram negative diplococci
GNDC on MTM agar
Gram negative coccobacilli-pleomorphic
Grams stained smear of Coccobacilli
X & V factors on MHM Hemophilus on CA
Identification of hemin (X factor) and NAD (V factor) as
growth requirements using paper disks. The top strain is
only growing around the disk containing both hemin and
NAD (black arrow), and is presumptively identified as H.
influenzae.
Lab Diagnosis of Hib
Biochemical TestingTRIPLE SUGAR IRON AGAR (TSI)
• It is a composite media used to study different properties of a bacterium –sugar fermentation, gas production and H2S production.
•In addition to peptone, yeast extract & agar, it contains 3 sugars –Glucose, Lactose, Sucrose.
•The Iron salt –Ferric citrate indicates H2S production.
•Phenol red is the indicator.
•It is an orange red medium with a slant and a butt.
•pH of the medium –7.4
TSI ReadingYellow – Acid
Pink -Alkaline
Yellow slant/ Yellow butt (A/A) –Lactose fermenters.
Pink slant/ Yellow butt (K/A) –Non lactose fermenters.
Pink slant / no color change (K/K) –Non fermenters
Black color–H2S production.
Gas bubbles or crack in the medium –gas production.
LF –E.coli, Klebsiella, Citrobacter
NLF –Salmonella, Shigella, Pseudomonas
H2S –Proteus, Salmonella typhi
Lysine Indole Motility Test (LIM)semi-solid medium used in the presumptive identification and differentiation of Enterobacteriaceae.
The addition of the amino acid, lysine, along with the pH indicator, bromcresol purple, allows for the detection of the bacterial enzymes lysine decarboxylase and lysine deaminase.
Lysine positive is purple in color
Lysine negative is no color change
Motility
Positive (+): Diffuse growth radiating outside of the stab line/cloudy/turbidity
Negative (–): Growth only along the stab line/clear
INDOLE TEST They produce indole from tryptophan present in peptone water.
Some bacteria are capable converting tryptophan (an amino acid) to indole and pyruvic acid by using the enzyme tryptophanase.
After overnight incubation, a few drops of indole reagent (Kovac’s reagent) is added.
Positive test is indicated by a pink ring.
Positive indole test –pink ring
Negative indole test –yellow ring
Indole positive –E.coli
Indole negative –Klebsiella, Salmonella.
Used to detect indole production by the organism.
CITRATE UTILIZATION
Done in Simmon’sCitrate medium.
To detect the ability of certain bacteria to utilize citrate as the sole source of carbon.
Contains Sodium citrate and bromothymol blue as the indicator.
If citrate is utilized, alkali is produced which turns the medium to blue.
Citrate positive –blue color
Citrate negative –green colour
Positive –Klebsiella
Negative –E.coli
Phenylalanine Test To determine the presence of phenylalanine deaminase.
This enzyme removes the amine group fromthe amino acid phenylalanine and producesphenylpyruvic acid (PPA) and ammonia
After incubation, 10% ferric chloride is addedto the media; if phenylpyruvic acid wasproduced, it will react with the ferric chlorideand turn dark green.
This is a positive result. This is a negative result.
UREASE TEST
This test is used to detect organisms that produce urease.
Urease produced by the organisms split urea into ammonia and CO2.
Urease positive –pink color
Urease negative –yellow color
Positive –Proteus, Klebsiella
Negative –E.coli, Salmonella
Oxidase TestDetects the presence of an enzyme “oxidase” produced by certain bacteria which will reduce the dye – tetramethyl-pphenylenediamine dihydrochloride. Positive test is indicated by the development of a purple colour. Oxidase positive – Pseudomonas, Vibrio, Neisseriae Oxidase negative – Salmonella, Shigella
Nitrate Reductase Test
Used to detect the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or some other nitrogenous compound, such as molecular nitrogen (N3) using the enzyme nitrate reductase
THE UNDERLYING MECHANISMSOF BACTERIAL PATHOGENICITYTwo broad qualities of pathogenic bacteria underlie the means by which they cause disease:
Invasiveness is the ability to invade tissues. It encompasses mechanisms for colonization (adherence and initial multiplication), production of extracellular substances which facilitate invasion (invasins) and ability to bypass or overcome host defense mechanisms.
Toxigenesis is the ability to produce toxins. Bacteria may produce two types of toxins called exotoxins and endotoxins
the initiation of the Infectious process and the mechanisms leading to the development of signs and symptoms of bacterial disease
VIRULENCE FACTORS1. ADHERENCE
Interaction between organism and tissue cell surface is complex
For example: once pili allows attachment on the host cells, other outer membrane proteins specifically attach to receptors of host cells, tightening the adherence preventing any fluid such as tears, mucus or urine from sweep the organism away
2. TOXINSExotoxins are released from bacterial cells and may act at tissue sites removed from the site of bacterial growth.
Endotoxins are cell-associated substance. (In a classic sense, the term endotoxin refers to the lipopolysaccharide component of the outer membrane of Gram-negative bacteria).
However, endotoxins may be released from growing bacterial cells and cells that are lysed as a result of effective host defense (e.g. lysozyme) or the activities of certain antibiotics (e.g. penicillin and cephalosporin)
Hence, bacterial toxins, both soluble and cell-associated, may be transported by blood and lymph and cause cytotoxic effects at tissue sites remote from the original point of invasion or growth.
Toxins produced by some bacteria can cause food poisoning, paralysis, uncontrolled muscle contraction, diarrhea and even toxic shock syndrome.
3. ENZYMESEnzymes causing cells lysis usually act on the cell membrane by insertion into the membrane (forming a pore that results in cell lysis), or by enzymatic attack on phospholipids, which destabilizes the membrane.
THE END