COLLEGE OF MEDICINE, NURSING & HEALTH SCIENCES

MLS 602 : General & Medical Microbiology

LECTURE 5 : Gram Negative Bacteria

Ms. Edwina Razak

Edwina.Razak@fnu.ac.fj

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