Non-Spore-Forming Non-Spore-Forming Gram-Positive Gram-Positive

BacilliBacilli

Non-Spore-Forming Gram-Positive Non-Spore-Forming Gram-Positive BacilliBacilli

• Corynebacterium C. diphtheriae

DiseaseDiphteria Opportunistic infections by other

Corynebacterium species (diphteroids)

PropertiesProperties• Club-shaped also V- or L-shaped

• Methachromatic granules (Albert staining): Beaded appearance

• Nonmotile, no capsule

• Facultative anaerobic

• Classified in CNMCNM (Corynebacterium, Nocardia, Mycobacterium) group.

BiotypesBiotypes(based on colony shape, biochemical properties and virulence)

GravisMitisIntermediusBelfanti

CultureCulture

• Sheep blood agar: It supports the growth of C.diphtheriae: Granular, gray, irregular edges, alpha hemolysis (sometimes)

• Loeffler serum slope: It is not a selective medium but gives abundant growth and morphology of C.diphtheriae. This medium can also be used as a transport medium.

•

CultureCulture

• Blood tellurite agar: - Tellurite reduction - It enhances the chances of isolation of

C.diphtheriae from clinical specimen in which commensal bacterial flora may also be present

- It differentiate different vars.

Growing colonies of Corynebacterium diphtheria on

Telorit potassium Blood agar

Clinical finding

• Common diphtheria (Nasopharyngitis)Incubation period of 2–5 days.

Fibrinous exudate “pseudomembrane”

- Sore throat - Fever - Enlargement of neck lymph nodes and neck edema - Irregulatory of cardiac rhythm and myocarditis - Difficulties with vision, speech and swallowing

This micrograph reveals an intranuclear This micrograph reveals an intranuclear inclusion body in a heart section from a inclusion body in a heart section from a

patient with diphtheria-related patient with diphtheria-related myocarditis.myocarditis.

Clinical findingsClinical findings

• Corrosion of myelin sheaths in the central and peripheral nervous system leading to degenerating motor control.

Cutanous diphtheria (a secondary infection)

TransmissionTransmission• Humans are the only natural host

• C. diphtheriae reside in the upper respiratory tract

• Transmitted by airborn droplet

• Infection at the site of a pre-existing skin lesion

PathogenesisPathogenesis

• InvasivnessInvasivness- Cord factorA glycolipid inhibits eukaryotic cell oxidation.

- NuraminidaseRemoves N-acetyl nuraminic acid from musine

membranes.

• ExotoxinExotoxin

Exotoxin (Encoded by gen tox from a temperate phage)

Fragment B. Binding of the toxin

Fragment A. Enzymatic activity

A

B

Exotoxin mechanism

Nicotinamide adenine dinucleotide phosphate (NAD)

Exotoxin (A fragment)

Nicotinamide ADPReaction with EF2

ADP-EF2Protein synthesis

inhibition

Testing immunity(Schick’s test)

• Toxin Intradermal injection (0.1 mL):

I. Cause inflammation (4-7 days later): No antitoxin in patient

II. No inflammation: Antitoxin is present (Immune person)

Important points about diagnosis of Important points about diagnosis of diphtheriadiphtheria

• The diagnosis is a laboratory emergency.

• Mild cases resemble pharyngitis and pseudomembrane may be lacking.

• Diphtheria is not diagnosed easily on clinical grounds: Streptococcal pharyngitis & Vincent's pharyngitis

Important points about diagnosis of diphtheria

• Non-toxigenic strains of C.diphtheriae have the potential to cause invasive disease and to undergo lysogenic conversion to toxin producers in vivo leading to disease.

• Specimen should be transported (e.i. by Amies medium)and processed as soon as possible.

Laboratory diagnosis

• Swabs should be taken both from the throat and the nose.

• Preferably two swabs should be collected from the site most affected.

• Microscopic observation (differentiation from streptococcal and Vincent's pharyngitis)

Laboratory diagnosisLaboratory diagnosis

• Isolating the organism

• Demonstrating toxin production- Animal inoculation- Eleck test- ELISA (Enzyme-linked immunosorbent assay)

• PCR to detect tox gene

Elek testElek test

TreatmentTreatment

• Tracheostomy in children (to prevent croup)

• Antitoxin (Intra muscular)

• Penicillin or erythromycin

Prevention• VaccinationDPT vaccine: A combination of diphtheria toxoid,

tetanus toxoid, and killed pertusis organism.

Given at 2, 4 an 6 months of age, with a booster at 1 and 6 years of age and then each 10 years afterward. (DPT or DT)

The toxoid is prepared by treating the exotoxin with 0.3% formaldehyde.

Listeria monocytogenesListeria monocytogenes

• Small rod (Coccobacillus: 1-2 micron) like “chinese character”.

• In old cultures 6-20 micrones. • No capsule, Facultative aerobic.• Tumbling movement in 25 c• Growing in 4c• Resistant to cold, heat, salt, pH extremes, and

bile

In 37 centigrade

In 20 – 25 centigrade

Listeria monocytogenesListeria monocytogenes

• Small, gray and smooth colonies with a narrow zone of beta hemolysis.

• Growth in low O2 and 5-10% CO2

• Fermentater, Catalase +, Oxidase +

TransmissionTransmission

• The organism is distributed worldwide in animals, plants, soil, stream water, sewage, unpasteurized milk, vegetables.

• Transmission to human by contact with animals or their feces.

• Endogenously from gasterointestinal tract.

ListeriosisListeriosis

• Rarely the cause of human disease• Potentially lethal food-borne infectioncase fatality rate: For those with a severe form of infection

is 25%. (Salmonella, in comparison, has a mortality rate estimated at less than 1%)

Virulence FactorsVirulence Factors

• Soluble components:- Hemolysins- Soluble antigens : Lypolytic Ags- Endotoxins (LPS): Cold Aglotinin: Abs react with RBC at low temp

(28-31 c): Lysis of RBC

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EpidemiologyEpidemiology

• Primary reservoir is soil and water; animal intestines. It contaminates foods and grow during refrigeration

• Listeriosis – most cases associated with dairy products, poultry, and meat

• Often mild or subclinical in normal adults. • In immunocompromised, fetuses, and

neonates affects brain and meninges.

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EpidemiologyEpidemiology

• Pregnant women susceptible• Infection passed to fetus prenatally

across placenta• Postnatal neonates acquire

infection in birth canal

DiseaseDisease• Meningitis and sepsis in:1. The fetus or newborn

I. Granulomatis Infantiseptica (during pregnancy)

or

II. Late listeriosis (5 days after giving birth).

2. Immunosuppressed adults (especially renal transplant patients)

3. The infected mother: asymptomatic or influenzalike illness/ Abortion

PathogenesisPathogenesis• Listeria induces macrophage phagocytic uptake:• Internalins & D-galactose (in their teichoic acids

) bound by the macrophage's polysaccharide receptors (E-cadherin)

• Once phagocytosed (as Phagosome), then encapsulated by the host cell's acidic phagolysosome.

• Listeria, escapes the phagolysosome by lysing the vacuole‘s membrane with hemolysin (listeriolysin O).

Pathogenesis

Internalin E-cadherinPhagocytosis

Into macrophages & epithelial cells

PhagocytiosisBy

macrophages and

hepatocytes

Phagolysosome formation

(acidic condition)

Lysteriolysin OsecretionRelease from phagolysosome

Inducing actin polymerization in cytoplasm

Forming filopods

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Figure 19.10 Multiplication cycle of Listeria monocytogenes

Lab. diagnosisLab. diagnosis

• Microscopic observation: Diphtheroids • Isolation by culture: Blood and CSF samples on blood agar• Cold Enrichment• Rapid diagnostic tests using ELISA,

immunofluorescence, and DNA analysis

• Effective sanitation of food contact surfaces: Alcohol & Quaternary ammonium

• 4°C discourages bacterial growth.

• pasteurization and well cooking

• Cell-mediated immunity is active (but no immunization)

Prevention

Treatment• PenicillinResistant are rare• Ampicillin and trimethoprim/

sulfamethoxazole

LactobacilliLactobacilli

LactobacillusLactobacillus

• Methachromatic granules (Albert staining)

• Nonmotile • no capsule

• Facultative anaerobic, microaerophilic or aerotolerant anaerobic

• L. casei L. acidophilus, L. salivarius, L. reuteri Are the representatives of distinct subclades.

Lactobacillus are a major part of lactic acid bacteria group: mostly

convert lactose and other sugars to lactic acid.

They are common and usually benign.

In humans they are present in vagina and gastrointestinal tract

inhibiting the growth of some harmful bacteria.

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ErysipelothrixErysipelothrix rhusiopathiaerhusiopathiae(causing (causing Erysipeloid)

• Gram-positive rod (sometimes looks like GNB)

• Single, streptobacilli, sometimes filamentous.

• Catalase -, Oxidase -, Indol –• SH2 in TSI

• Microaerophilic needed 5% co2

• Small shiny or white colonies• Alpha hemolytic on BA

• Differentiated from Listeria by Alpha hemolysis and SH2 & catalase.

TransmissionTransmission• in environment and normal flora in sheep,

chickens, and fish, duck (indigenous zoonotic Infectious disease)

• Transmission from animals or their products to human

• Primary reservoir – tonsils of healthy sheep & pigs.

• On the surface of fresh fish

Erysipeloid on hand

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• Enters through skin abrasion, multiplies to produce erysipeloid, dark red lesions without systemic symptoms.

• Latent period: 2-7 days• Sever pain, inflamation with purple color

lesions on skin.• No pus (differentiation from Staphyloccocal

and Streptococcal infections)• Endocardit

Pathogenesis & Clinical findingsPathogenesis & Clinical findings

Control and treatmentControl and treatment

• Penicillin or erythromycin• Treated after 3-4 weeks• Always resistant to vancomycin• Vaccine for sheep and pigs

Lab diagnosisLab diagnosis

• Biopsy or aspirated liquids from the edge of lesions.

• Blood sample in case of endocardit.

Chronic swimming pool granuloma

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• In severe cases, they may penetrate the surrounding bone and muscle to the skin, where they break open and leak large amounts of pus. The purulent leakage via the sinus cavities contains "sulfur granules," not actually sulfur-containing but resembling such particles. These granules contain progeny bacteria.

Diagnosis

• The disease is suggested by a supportive lesion with Gram-positive filaments in the exudate.

• Sulfur granules may be present.• Bacterial isolation and identification

Control

• Good oral hygiene• Antibiotic trapy: Penicillin• Drainage of lesion

Actinobacteria

• They include some of the most common soil life, freshwater life, and marine life, playing an important role in decomposition of organic materials, such as cellulose and chitin, and thereby playing a vital part in organic matter turnover and carbon cycle.

• Actinobacteria are well known as secondary metabolite producers and hence of high pharmacological and commercial interest. In 1940 Selman Waksman discovered that the soil bacteria he was studying made actinomycin, a discovery for which he received a Nobel Prize.

• Since then, hundreds of naturally occurring antibiotics have been discovered in these terrestrial microorganisms, especially from the genus Streptomyces.

• Some Actinobacteria form branching filaments, which somewhat resemble the mycelia of the unrelated fungi, among which they were originally classified under the older name Actinomycetes. Most members are aerobic,

• a few, such as Actinomyces israelii, can grow under anaerobic conditions. Unlike the Firmicutes, the other main group of Gram-positive bacteria, they have DNA with a high GC-content, and some Actinomycetes species produce external spores.

• Some types of Actinobacteria are responsible for the peculiar odor emanating from the soil after rain, mainly in warmer climates.[7