Pt Care 2: Module 2.1 Interactions of Bacteria with the Immune System + Different Species of Bacteria

Interactions of Bacteria with the Immune System

Interactions of Bacteria with the Immune System

Defense against microbes

• Innate immunity

• Adaptive immunity

• Vaccines

• Antibody therapy

• Antibiotics

Innate Immunity against Bacteria

A. To prevent bacteria from entering the body

• Physical barrier: intact skin

• Mechanical Removal: urination, mucous membrane

• Chemical microbicide: stomach acid

• Biological microbicide: lysozyme in body fluids,

• Commensal / mutual existence: microbiota in GI track

B. To destroy bacteria inside the body

• Macrophages and neutrophils: Use pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) of microbes to phagocytose and kill bacteria.

• Complement system: recognizes and forms membrane pores to lyse bacteria.

PRRs recognize _______.

PAMPs (Pathogen associated molecular patterns)

Adaptive Immunity against Bacteria

• T cells mediated adaptive immune response plays a major role in defense

against intracellular bacteria.

- CD8+ T cells

- CD4+ T cells

A: Antibody-mediated responses:

• Antigen presenting cells, CD4+ helper T cells and B cells work together to produce antibodies.

• Antibodies will

- Inactivate virulence factors

- Inactivate toxins

- Increase phagocytosis and removal

- Provide antigen-specific IgA for mucosal defense

B: Cell-mediated responses: Cytotoxic T cell-mediated lysis

Role of Inflammatory Responses

• Acute (local) inflammation

- Local vasculature response to limit injury/infection

- Neutrophil infiltration to kill bacteria

• Systemic Response:

- Acute phase protein production to tag or kill the invading bacteria (e.g. C reactive protein-CRP)

- Release of cytokines to further activate immune cells

- Stimulation of WBC production

- Fever generation to reduce bacterial activity.

- Increased serum transferrin concentration to bind iron

- Increased tissue iron storage

Pathogenic Extracellular Bacteria

(See Image)

Why is clostridium tetani considered G-???

Extracellular Bacteria and Host Immunity

• Extracellular Bacteria

- Rapid growth, if uncontrolled

- Toxins can cause local tissue damage, inflammation, septic shock in a very short time span.

• Host Immune Responses

- Innate immunity plays a major role

- At the body’s surface, various means to prevent and control bacterial growth

- Once inside the body, bacteria are quickly recognized and destroyed via multiple mechanisms.

Is extracellular bacteria associated with innate or adaptive immunity?

Innate

Intake Skin's Defense

Keratinized skin (body surface)

- A relatively impermeable

physical barrier

- Has glands secreting

lysozymes (breaks bacterial cell wall), toxic lipids (sebum), and hydrogen irons (H+)

Mucosal Defense - The Environment

• The mucosal epithelium

- Including GI, nasopharynx, upper airway,

urinary and reproductive track

- Not keratinized

- Moist and rich in nutrients

Mucosal Defense - Mechanical

• Local bacterial proliferation is controlled by mechanical cleaning, soluble bactericidal factors and lack of available free iron.

- GI track:

Peristaltic mobility, mucus secretion, stomach acid, bile as a detergent

- Lower respiratory track

A sterile environment

Mucus movement of cilia lining to remove aspirated bacteria

- Urinary track

A sterile environment

Urination serves to flush out bacteria

- Cigarette smoking, viral or bacterial infections and reduced mucociliary clearance in the elderly comprise immune defense and promote opportunistic infections

Mucosal Defense - Bactericidal

• Bactericidal Agents in Mucosal Fluids:

(Saliva, tears, nasal, gastric, cervical and

bronchial mucus)

- Lactoferrin: binds free iron

- Defensin: forms pores in bacterial membrane to allow efflux of irons and nutrients

- Lysozyme: breaks bacterial peptidoglycan

- IgA: interferes with bacterial adhesion,

inactivates toxins, promotes bacterial aggregation for removal by mucus

Lactoferrin

Binds free iron, inhibits bacterial growth

Defensin

Forms pores in bacterial membrane

to allow efflux of irons and nutrients

Why is important to limit the amount of free iron in the blood?

Iron is a vital nutrient for bacterial growth and is often the factor that limits growth in vivo. Iron is essential for many physiological processes in bacteria and fungi, including DNA replication, transcription, metabolism, and energy generation. Iron is also a co-factor for various proteins and can influence other cell components. For example, in Escherichia coli, iron is needed for the functioning of ribo-nucleotide diphosphate reductase subunit B2, which is responsible for synthesizing deoxy-ribotides for DNA synthesis.

Lysozyme

Breaks bacterial peptidoglycan (so probably contributes to why gram-negative bacteria are more difficult to deal with)

IgA

Interferes with bacterial adhesion, inactivates toxins, promotes bacterial aggregation for removal by mucus

How extracellular bacteria work around skin and mucosal defense mechanisms?

- Physical damage to the skin allows bacterial entry into tissue

- Mucosal bacteria can also actively invade deeper tissues through multiple mechanism including producing enzymes to destroy host tissues.

- H. pylori produces urease to generate ammonia to neutralize stomach acid.

- Inflammation caused by initial bacterial infection could lead to tissue damage, allowing additional bacterial entry.

How does H. pylori survive in the stomach?

Produces ammonia from urea to neutralize the acid + they bind specifically to gastric-type epithelium to prevent being shed

Once entering the body, bacteria

encounter:

• Innate Immunity: Rapid Response for Removal

- Blood Stream: Complement System

- Blood Stream: Neutrophils

- Tissue

▪ phagocytosis of bacteria by macrophages

▪ subsequent activation of both cell types release

inflammatory factors to induce neutrophil infiltration

▪ Neutrophils phagocytose bacteria

• Adaptive Immunity: Long-term Control

- Tissues: Dendritic Cells, Macrophages

- B cells

- End Result: antibody production

How extracellular bacteria work around immune defense mechanisms?

• Increase chance of colonization and growth

- Use surface molecules (e.g., adhesins) as ligands to host’s receptors to establish strong adhesion to the surfaces of host

epithelial cells

- Compete with host cells for iron in mucosa and blood as a vitalnutrient:

- N. meningitidis use a complex surface transport system to steal

iron from host’s transferrin

- E. coli secrete high affinity iron chelators (siderophores) to steal

soluble iron in the local environment before taking it up and releasing it into their cytoplasm.

Adhesions

Protein complexes that bind to protein receptors on the host cell surface

Bacterial capsule: Reduce immune cell recognition

- Mimicry: make bacterial capsule components more like eukaryotic

cell surface (polysaccharides)

- Mask: cover up surface antigenic epitope

- Inaccessibility: Antibodies and complements bound to antigenic structures deep beneath surface are less effective for phagocytosis

- Phase Variation: Dynamically express capsular polysaccharides (CPS) in response in stages of invasion:

* high (on) when needed for evading host immune response in initial invasion,

* low or no (off) when needing to adhere to or enter host cell,

* high (on) again after gaining access in deeper tissue with high abundance of complement and phagocytes.

**Means to reduce tagging and phagocytosis:**

• Proteins in the outer layer that inhibit complement deposition

- PspA/C (pneumococcal surface protein A/C)

- C3 binding protein (S. pneumoniae)

• Proteins in the outer layer that prevent antibody binding:

- protein A of Staphylococcus aureus binds to Fc region (the constant bottom region) of Ig and Fc prevent opsonization and phagocytosis. Then the bacteria is able to release itself.

• Bacterial Ig A proteases (e.g., Neisseria G/M) removes Fc fragment

of bound antibody (stops neutrophil phagocytosis & prevent immune detection by covering up its antigen---very clever!!!)

Means to reduce chance of being killed:

• Use pneumolysin (S. pneumoniae) to deplete complement

proteins

• Use C5a peptidase (S. pyogenes, group B streptococci) to

inactivate C5a (chemoattractant) and reduce neutrophil

infiltration

• Produces antioxidants (S. aureus) to inactivate neutrophil free radicals.

• Use catalase (E. Coli) to convert neutrophil H2O2 to water.

Intracellular Bacteria: Overview

- Invade host cells

- Survive and replicate inside host cells

- Spread to other cells/tissues within the host

- Cause systemic infection

A limited number of “strategically positioned” immune and non-immune cells can be used a target host cells.

- “First encounter” barrier cells: epithelial cells

(mucosa, GI and respiratory)

- “First encounter” immune cells: macrophages

(monocytes), neutrophils

- “Barrier/lining” cells: endothelial cells

- “Others”: hepatocytes, erythrocytes, Schwann cells

(PNS glia)

Immune Cells as Target Host Cells

- Immune cells (macrophages, neutrophils) are designed to recognize extracellular bacterial

PAMPs via PRR as well as Ig or complement- tagged bacteria via Fc Receptors or Complement Receptors.

- Recognition by immune cells leads to phagocytosis and degradation of the ingested bacteria.

- Intracellular bacteria that are able to remain and

replicate inside a host immune cells usually stay

in vacuoles, avoiding being killed.

Endocytic (Exogenous) Pathway

The usual Process:

• Extracellular microbe

phagocytized by an

immune cell results in the

breakdown of the microbe

and its presentation by

MHC-II.

• The end result is antibody

production against the

microbial epitopes.

MHC

• Major Histocompatability complex, a set of proteins found on the plasma membranes of cells that help display antigen to T cells.

• MHC I is found on all cells and displays bits of proteins from within the cell; this allows T cells to monitor cell contents and if abnormal peptides are displayed on the surface, the cell is destroyed by killer T cells.

• MHC II is found only on macrophages and B cells. This class of MHC allows these cells (known as antigen presenting cells) to display bitts of "eaten" (phagocytosed or internalized) proteins on their surface, allowing the activation of helper Ts --> thus further activating immune response.

Evasion of the Endocytic Pathway

• Prevention of phagolysosome formation

- Mycobacterium tuberculosis can impair

lysosomal acidification, a process required for

proper fusion of endosome with lysosome.

• Prevention of free radical mediated killing in

neutrophils

- Anaplasma phagocytophylum can prevent the production and proper assembly of NADPH

oxidase cytosolic and membrane subunits so

free radicals can not be generated to enable

the killing of the ingested bacteria.

• Escape from phagosome and enter cytosol

- Listeria monocytogenes secretes, listeriolysin O (LLO), a protein that ruptures phagosome.

Advantage:

a) Able to rapidly replicate due to available ingredients in cytosol

b) Facilitates repeated infection of neighboring host cells

Disadvantage:

Bacterial proteins in the cytosol of the host cells can be processed and presented by MHC-I resulting in the recognition

and killing of the host cell by a CD8+ cytotoxic T cell.

Non-immune Cells as Target Host Cells

• Non immune cells can not actively recognize and phagocytose bacteria.

• Bacteria, on the other hand, enter non-immune cells by inducing their own

phagocytosis.

• Non-immune cells are also poorly equipped to kill intracellular bacteria.

• Hence, invasion of non-immune cells enables bacteria to proliferate and invade more cells.

**Example: Orally introduced Salmonella enterica “crossing” GI epithelial cell membrane to eventually “enter” the epithelial cell.**

- A bacterium encounters GI epithelial cell surface

- It assembles a multiprotein needle complex (type 3 secretion system, T3SS), crossing bacterial inner and outer membrane and epithelial plasma membrane, to form a channel between bacterial cytosol and the epithelial cytosol

- It then injects “effector proteins” into the epithelial cells to induce cytoskeleton rearrangement that results in the engulfment of the bacterium.

- It can remain in the original vacuole formed by host cell membrane to replicate and infect neighboring cells.

- Similar mechanisms are believed to be used by other bacteriato invade non-immune cells.

Immune Response

against Intracellular Bacteria

• Immune responses to intracellular

bacteria are not as effective as that to extracellular bacteria.

• Primarily mediated by cellular immunity.

Innate Immunity: Neutrophils and

Macrophage

• Effective in infections that cause the death of host

cells.

Examples:

- Infection of epithelial cells by L. monocytogenes

causes the apoptosis of the infected epithelial cells

and the release of chemoattractant to recruit

neutrophil infiltration and the phagocytosis of the

released bacteria.

- Released bacteria will also cause macrophage

activation and phagocytosis and the release of

cytotoxic factors. If the phagocytosed bacteria can

not be killed, granuloma is formed to contain the

bacteria.

CD8+ T Cells and Intracellular Bacteria

• When intracellular bacteria replicate inside the host cell, some of its components will be processed as antigens and presented by MHC-I host P / G cells which will be killed by CD8+ T cells.

• Are also known a cytotoxic T cells

CD4+ T Cells and Granuloma

• Formation of granuloma serves to contain intracellular bacteria if

they can not be completely eliminated.

- Example: L. monocytogenes, M. tuberculosis

- Structure: a core of pathogen-containing macrophages and

scattered CD4+ T cells, surrounded by a layer of CD8+ T cells; in

most cases, cells in the core eventually die of apoptosis and the outer layer T cells become calcified and fibrotic.

Process:

• Macrophages fail to kill phagocytosed bacteria instead release IL-12.

• IL-12 activates T-helper cells to release IFN-γ that hyperactivate microphages to release more ROS and RNS to enhance killing

• Hyperactivated macrophages fuse together to form the core

• Remanent CD4+ and CD8+ T cells form the shell.

**Granuloma Formation Process**

• Macrophages fail to kill phagocytosed bacteria instead release IL-12.

• IL-12 activates T-helper cells to release IFN-γ that hyperactivate microphages to release more ROS and RNS to enhance killing

• Hyperactivated macrophages fuse together to form the core

• Remanent CD4+ and CD8+ T cells form the shell.

Antibody and Intracellular Bacteria

Limited participation

• Blocking bacteria and host receptor for

entry into host cells

• Opsonization of extracellular bacteria to

prevent entry into host cells

• Antibodies taken into the phagosome, together with the bacteria can act in situ

Example: Anti-LLO (listeriolysin O) antibody

protects against L. monocytogenes infection

Do gram negative bacteria have an outer membrane?

Yes, it has both an inner and outer membrane. It also has LPS on that outer membrane.

LPS-induced inflammatory response

• Lysed LPS binds to TLR4 leading to a cascade of events that lead to a. cytokine storm + cytotoxic peroxynitrite + TNF-alpha + interleukins + interferons, the cycle will continue as more immune cells are recruited, leading to sepsis

TLR4-related signaling pathway

• LPS: lipopolysaccharide

• CD14:

• MD-2: Myeloid Differentiation

factor 2

• Adaptor protein: MyD88

• Kinases: MAPK (mitogen-

activated protein kinases).

• AP-1, NF-kB: gene transcription

factors

Between innate and adaptive immunity, which plays a bigger role in defense

against extracellular bacteria?

Innate immunity

Between innate and adaptive immunity, which plays a bigger role in defense

against intracellular bacteria? Which cell type-mediated immunity is most

important?

Adaptive immunity, innate immunity is effective for infections that cause the death of host cells.

T-cell mediated adaptive immune response is most important.

Can you cite examples of host and bacterial proteins used to keep or steal free iron?

Too Keep by Host

1. Transferrin binds to iron

2. Increase iron storage

3. Lactoferrin binds to free iron

Too Steal by Bacteria

1. N. meningitidis use a complex surface transport system to steal iron from host’s transferrin

2. E.coli secrete high affinity iron chelators (siderophores) to steal

soluble iron

Can you cite examples of host and bacterial proteins used to facilitate

phagocytosis of bacteria or disruption of it?

Facilitate Phagocytosis of Bacteria by Host

1. Opsonization of foreign bodies by antibodies or complement proteins or Ig

2. Recognization of PAMPS by PRRs macrophages and neutrophils

3.

Disruption by Bacteria

1. Mycobacterium tuberculosis can impair

lysosomal acidification

2. Prevent recognition through mimicry, mask or inaccessibility or phase variation

3. Proteins in the outer layer that inhibit complement deposition

- PspA/C (pneumococcal surface protein A/C)

- C3 binding protein (S. pneumoniae)

4. Protein A of S. Aureus binds to Fc to prevent opsonization and phagocytosis

5. Ig A proteases (Neisseria G/M) removes Fc fragment of bound antibody

6. Anaplasma phagocytophylum can prevent

the production and proper assembly of NADPH

oxidase cytosolic

7. Listeria monocytogenes secretes, listeriolysin O (LLO), a

protein that ruptures phagosome.

Intracellular Bacteria Pathway

1. Invade host cell

2. Survive and replicate inside host cells

3. Adapt a way to transfer to other cells

4. Infect new cell

5. Repeat process to systemic infection

Bacterial Evasion: Taking charge ofhost proteins e.g. L. Monocytogenes

1. Invade Macrophages via vacuoles

2. Break out of vacuoles into the cytoplasm

3. ActA protein recruits host Actin filaments to move bacteria to cell membrane and push a protrusion into neighboring cells

4. Neighboring cells take up protrusions as vacuoles

5. Repeat process to systemic infection

Bacterial Evasion: Taking over ormimicking cell organelles e.g. example Legionella pneumophila

1. Invade host cell via Vacuoles

2. L. pneumophila dodge the cell’s immune defenses by interacting with Golgi bodies and ER. Taking some of their proteins and changing their normal vesicular traffic.

3. Newly formed membranes become studded with ribosomes

- To potentially make certain host proteins or could be a result of the membrane’s ER-like identity

4. L. pneumophila replicate inside compartment before bursting out of the cell

5. Repeat process to systemic infection

Bacterial Evasion: Change host cell gene expression e.g. M. Leprae

• M. leprae reprogram cell gene expression reverting them to stem-cell-like state.

• The new stem cell phenotype can differentiate into other cell types like muscle cells. Leading to infection of neighboring tissues.

• Reprogrammed cells also attract macrophages which phagocytose the stem cell-like cell and take up the infection and spread it.

What is LPS? How does LPS induce the production of various proinflammatory

and cytotoxic factors in an immune cell?

LPS is a lipopolysaccharide endotoxin found on the outer membrane of gram negative cells. It is a PAMP recognize by immune cells.

Where is all the toxicity contained in LPS?

Lipid A; O antigen is immunogenic and highly variable

Enterobacterales Overview

Enterobacterales Overview

Enterobacterales

• Family

– Domain: Bacteria

– Phylum: Proteobacteria

– Class: Gammaproteobacteria

– Order: Enterobacterales

– Family: Enterobacteriaceae

• 63 genera with 25+ species

• Natural habitat (normal microbiota)

– Lower gastrointestinal tract (human and animals)

– Ubiquitous (water and soil)

• Gram-negative facultative anaerobe, bacilli, lactase +, oxidase -

• Has a flagella

• Mucoid

– Found on the blood agar plate

– Mix with KOH solution à viscous form

• Voges-Proskauer

– Biochemical test

– Glucose fermentation producing products – acetoin/butylene glycol

• Hydrogen sulfide

– Biochemical test: to detect colorless gas H2S

Lactose Test

• Checks to see if a bacteria can ferment lactose

• Ferment sugar (glucose or lactose) à create acidic byproducts +/– gas à

pH indicator

• Can use the MacConkey agar

Oxidase Test

Identifies bacteria that have cytochrome oxidase

– Possess cytochrome oxidase or

indophenol oxidase

– Catalyze the transport of electrons from donors to electron acceptors

– Reagent: artificial electron acceptor

Surface Antigenic Feature: Cell Envelope

• Inner Membrane (cytoplasmic membrane)

– Impermeable to polar molecules

• Periplasmic Space

– Between the inner and outer membrane

– Peptidoglycan

• Outer Membrane

– Phospholipids inner leaflet

– Lipopolysaccharide in the outer leaflet which serve as antigens (PAMP)

Surface Antigenic Feature: Lipopolysaccharides (LPS)

• Lipid A (endotoxin)

- Recognized by host pattern

recognition receptor

• Core phosphyorylated

oligosaccharide

• Repeating oligosaccharide

side chains (O antigen)

• Extremely potent virulence factor

Immunoglobulins

Antibodies such as IgA, IgE, IgG, IgM, and IgD; secreted by plasma cells (mature B cells) in response to the presence of an antigen.

Surface Antigenic Feature: Capsule

• Envelope of surface polysaccharide

• Repeating trisaccharide

– Bonded to LPS and/or phosphoglyceride

• Ability to mask the O antigen from antibodies

Virulence/Virulence Factors

• Lipopolysaccharide and capsule

• Adhesins

• Secretion systems and Toxins

• Iron acquisition

• Plasmids

Adhesins

• Bind selectively to receptors on host

– Overcome electrostatic repulsion

• Fimbriae

– Rigid rod (pili) of FimA protein

– Binds to mannose residues on glycoprotein/glycolipids on host

• Outer membrane proteins: serve as adhesin

• Surface carbohydrates – adhesive properties

Toxins

• Released into the environment or

directed to the host cells

• SPATE: serine protease autotransporters

• Capable of inducing lysis of host cells

– Type I secretion system: hemolysins

– Type II secretion system: variety of enzymes e.g. chitinase

– Type III secretion system: exports and injects into cell

Plasmid

• Extrachromosomal autonomously replicating DNA

• Genes may play major roles in pathogenesis

– Type III secretion system: invade cells

– Pilus

– Type II: protease/hemolysins

• Transfer between different genera (conjugation)

• Resistance genes: antibiotic resistance

Antibiotic Resistance

• Produce beta-lactamase

– Intrinsic resistance

• Penicillinase – enzyme that degrades

– Break down penicillin

– Break down ampicillin

• AmpC producers – stronger enzyme

– Break down most cephalosporins (e.g. cefazolin, cefuroxime, and ceftriaxone)

– Extrinsic resistance (plasmid-mediated)

• Extended-spectrum beta-lactamase (ESBL)

• Mutational alterations of targeted sites

– Fluoroquinolones (ciprofloxacin)

– Aminoglycosides (gentamicin)

Escherichia

Escherichia

Discovery of Escherichia

• 1885 – Theodore Escherich

– Fecal microbiota in neonates

• One of most common genera

– Most commonly encountered pathogen

• This very distinguishable

– Motile (flagella)

– Produce indole from tryptophan (indole +)

• Poorly adapted to cause disease in healthy

- Opportunistic

• Includes E. Coli

Hemolysis

Destruction of red blood cells

E. Coli

• Gram stain: negative rod

• Motile

• Facultative anaerobes

• Lactose fermenter: positive

• Oxidase test: negative

• Hemolysis on sheep’s blood agar: positive

• Voges-Proskauer reaction test: negative

• Commonly found – Normal Microbiota (Normal Flora)

– Gastrointestinal

Pathogenesis: E. coli Diarrhea

• Very common worldwide

• Virulence Classification

- Enteropathogenic E. coli

- Enterotoxigenic E. coli

- Shiga toxin-producing E. coli

- Enteroinvasive E. coli

- Enteroaggregative E. coli

Enteropathogenic E. coli (EPEC)

• Developing countries (infants)

• Watery diarrhea with vomiting and fever

• Pilus: adherence factor (EAF)

– Adherence to

mucosal cells

of small intestine

• Inject effector

proteins

• Form actin

pedestal to bind/anchor to mucosal cells

Enterotoxigenic E. coli (ETEC)

• Traveler's Diarrhea and children < 5 yrs

• Adherence to

epithelial cells of

small intestine

• Enterotoxins

- Heat labile (LT)

• Mostly found

- Heat stable (STa)

• Some

• Plasmid mediated

Shiga toxin-producing E. coli (STEC)

Most often associated w/ foodborne outbreaks, HUS-related

• Diarrhea → Death

– Mild (non-bloody) to hemorrhagic colitis

• Virulence: cytotoxin (Shiga-like toxin-1 and toxin-2)

– Block protein synthesis and induce host cell apoptosis

– Released in stressful conditions e.g. exposure to antibiotics

• O157:H7 most common (serotype of E. coli)

– Responsible for large outbreaks

• Normal microbiota: ruminant (cattle)

– Under cooked meat and unpasteurized food

How do you treat STEC?

Supportive care

O157:H7 Outbreak in 1992

• Infected 732 people in the US

- 6 western states

- Strain was detected in stool 38 days after illness onset

- 23% were hospitalized → 4 children died

• Contaminated beef patties

- 11 lots of patties and 5 slaughter plants in the US and 1 in Canada

• Undercooked burgers and cooked burgers

- Since toxins are heat stable

Penicillinase

Enzyme produced by bacteria that deactivates penicillin

Antibiotic Resistance of E. Coli

• Intrinsic resistance: penicillinase

– Resistant to penicillin

– Resistant to ampicillin

• Some may have more resistance

– Depends on acquisition of plasmids

Infectious Diseases Attributable to E. coli

• Diarrhea

– Traveler’s diarrhea

– Food poisoning to hemorrhagic colitis

• Urinary tract infection

– Cystitis

– Pyelonephritis

– Bloodstream infection

• Intra-abdominal infections

Treatment options for E. Coli

• Ampicillin/sulbactam* - Beta - lactamase

• Cefazolin* - 2nd gen ceph

• Cefuroxime - 3rd gen ceph

• Ceftriaxone

• Ciprofloxacin*

Klebsiella

Klebsiella

Discovery of Klebsiella

• 1882: Carl Friedländers

– Lobar pneumonia caused by Klebsiella

– Sweats, fever, pleuritic pain with bloody sputum

– Bulging interlobar fissures on x-ray

• 1885: V. Trevisan: honored Theodor Albrecht Edwin Klebs

• Common in the community and in the hospital setting for infections

Voges-Proskauer Test

Designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butanediol; adding VP reagents to the medium oxidizes the acetoin to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color

Klebsiella: pneumoniae, oxytoca, aerogenes

• Gram stain: negative

• Facultative anaerobes

• Lactose fermenter: positive

• Oxidase test: negative

• Hemolysis on sheep’s blood agar: negative

• Voges-Proskauer reaction test: positive

• Exhibit mucoid growth and non-motile (except aerogenes: motile)

• Commonly found – Normal Microbiota (Normal Flora)

– Gastrointestinal

Klebsiella: Virulence Factor

• Polysaccharide capsule

- 70 antigenic varieties

- Responsible for the mucoid phenotype

- Inhibit phagocytosis

• Fimbrial types - pili

- Adherence to host cells

• LPS

• Plasmids

Klebsiella: Antibiotic Resistance

• Chromosomal gene encoding penicillinase

– Resistant to ampicillin and amoxicillin

• Nosocomial (originating in hospital) isolates may be resistant to other antibiotics

– Plasmid mediated

• Extended spectrum beta-lactamase production (ESBL)

– Resistant to all cephalosporin

• Klebsiella producing carbapenemase (KPC)

– Resistant to cephalosporins and carbapenems

• Resistant to fluoroquinolones (ciprofloxacin)

• Resistant to aminoglycosides (gentamicin)

Infectious Diseases Attributable to Klebsiella sp.

• Urinary tract infections

• Respiratory tract infections

• Intra-abdominal infections

– Liver abscess

– Biliary tract infections

– Peritonitis

• Wound infections

ESBL

Resistant to all cephalosporins : O

Klebsiella: Treatment

• Cefazolin*

• Cefuroxime*

• Ceftriaxone*

• Cefepime*

• Piperacillin/tazobactam*

• Meropenem

- These cephalosporins will not be effective if the bacteria is ESBL producing, so do not use if there is a risk that they might be ESBL

Enterobacter

Citrobacter

Serratia

Enterobacter

Citrobacter

Serratia

Discovery of Enterobacter

• Widely identified as a pathogen in 1970

– Nationwide outbreak of septicemia

– 378 patients @ 25 hospitals

– Contaminated IV solutions from Abbott Laboratories

• Hospital-acquired infection

– Received antibiotics previously

– Admitted in the intensive care units

Enterobacter species: cloacae

• Gram stain: negative

• Facultative anaerobes

• Lactose fermenter: positive

• Oxidase test: negative

• Hemolysis on sheep’s blood agar: negative

• Voges-Proskauer reaction test: positive

• Motile and exhibit mucoid growth

• Commonly found – Normal Microbiota (Normal Flora)

– Gastrointestinal

• All the same characteristics of Klebsiella except it is motile (although aerogenes is motile)

Hydrogen Sulfide Test

Tests bacteria with abilty to turn H2S from amino acids or other sulfur-containing compounds. Gives off a black color

Discovery of Citrobacter

• 1932: Werkman and Gillen

– Urinary pathogen in a hospitalized patient

• Named after their ability to use citrate as their sole carbon source

• Produce H2S (positive test)

– Not many Enterobacterales produce this

Citrobacter species: Koseri and fruendii

• Gram stain: negative

• Facultative anaerobes'

• Lactose fermenter: positive

• Oxidase test: negative

• Voges-Proskauer reaction test: negative

• Motile

• Sulfide Test: positive

• Commonly found - Normal Microbiota (Normal Flora)

- Soil and water

- Gastrointestinal tract in animals and humans

• Very similar to E. Coli, except no mention of hemolysis and it has a positive sulfide test

Discovery of Serratia

• Wide spread in the environment

– Less common in the human fecal flora

– Saprophytic bacteria

• Produce exotoxin: Dnase

• Produce a red pigment- prodigosin

– Polenta cultivated with red discoloration

• Named after Serafino Serrati

– Actually invented the steamboat engine

– Species marcescens Latin term: to decay

Serretia Species: marcesens

• Gram stain: negative

• Facultative anaerobes

• Lactose fermenter: positive

• Oxidase test: negative

• Hemolysis on sheep’s blood agar: negative

• Voges-Proskauer reaction test: positive

• Motile

• Commonly found – Normal Microbiota (Normal Flora)

– Environment

• All the same characteristics of Enterbactor cloacae OR of Klebsiella except it is motile (although aerogenes is motile)

Virulence Factors: Serratia sp

• Chromosomal inducible ampC genes encoding beta-lactamases to degrade certain antibiotics

– Resistance to

• Ampicillin

• Cefazolin

• Cefuroxime

• Ceftriaxone

• Carry plasmids encoding resistance to multiple antibiotics

• Fimbriae

– Allows adherence to uroepithelial cells

– Cytotoxic to tissue cells

• Able to survive in harsh conditions

– Thrive with disinfectants

– Spread through hospital personnel hands

• ampC gene produce higher levels of beta-lactamases during

therapy

ampC

Gene codes for beta lactamase

-regulated by AmpR

– Resistance to

• Ampicillin

• Cefazolin

• Cefuroxime

• Ceftriaxone

**Infectious Diseases Attributable to Enterobacter sp.**

• Respiratory tract infection

– Pneumonia

• Urinary tract infections

– Indwelling catheter

• Intra-abdominal infections

• Wound or burn related skin infections

Infectious Diseases Attributable to Citrobacter sp.

• Urinary tract infections

– Indwelling catheter

• Respiratory tract infection

– Pneumonia vs. colonization

• Intra-abdominal infections

• Wound infection

• Osteomyelitis