General characteristics:

• Gram-negative, straight or slightly curved rods.
• They do not catabolize glucose, so they are called “non-fermenters”
• Aerobes
  – However, in the lungs of patients with cystic fibrosis, research indicates that formation of biofilms may facilitate anaerobic respiration.
• Ubiquitous in the environment: they are often found in soil and water.
• Flagellar and pili twitching motility
• Oxidase-positive (aids in laboratory identification)
• Fruity, grape-like aroma
• Survive on trace nutrients – for example, they’ve even been found in distilled water.

Pseudomonas aeruginosa
Causes opportunistic infections; as we’ll see, it is especially well-adapted for survival in patients with cystic fibrosis.

• Produces two pigments that produce a distinctive appearance in culture:
  – Pyocyanin, which is a bluish color
  – Pyoverdine, which is a yellowish-green color (occaisionally spelled “pyoverdin”)

Virulence Factors

Some virulence factors are regulated by quorum sensing, and some factors work together in disease pathogenesis.

• Adhesins and pili facilitate adherence to host cells.
• Biofilm production facilitates immune system evasion and, as mentioned earlier, plays an important role in infection of cystic fibrosis patients.
• Endotoxin, which comprises lipopolysaccharide; as in other Gram-negative bacteria, endotoxin produces symptoms of sepsis and shock.
• Polysaccharide capsule with alginate.
  – Capsule is anti-phagocytic and prevents clearance by antibodies, and is upregulated in patients with cystic fibrosis.
  Exotoxins and enzymes are injected into host cells by Type III secretion systems, or into the tissues by Type II secretion systems.
• Exotoxin A inhibits protein synthesis and contributes to tissue necrosis.
• Pyoverdine, which, as described above, lends the bacteria a yellow-green hue, regulates secretion of Exotoxin A.
  – Also acts as a siderophore to “steal” iron from the host.
• Pyocyanin, the blue pigment, increases intracellular levels of cytotoxic superoxide and hydrogen peroxide
  – Also promotes apoptosis of neutrophils, which inhibits the innate immune response.
  – Interferes with respiratory cilia and damages mucosal cells, which are otherwise important mechanisms of microbe clearance.
• Alkaline protease inhibits complement and contributes to tissue destruction.
• Exoenzymes S, T, U, and Y
  – Exoenzymes S and T disrupt host cell actin cytoskeletons and promote cell death.
  – Exoenzyme U is cytotoxic, particularly to alveolar epithelial cells and macrophages.
  – Exoenzyme Y causes edema.
• Elastases* degrade complement and elastin, which is an important component of lung tissue.
  – LasA and LasB.
  – Host produces anti-elastase antibodies that form immune complexes; their deposition in tissues contributes to damage and malfunction.

Opportunistic infections

Infections are associated with hospital settings, especially in wet or moist areas.
Innate immune deficiencies or trauma, especially burn wounds, promote Pseudomonas infections.

Pulmonary infections range from mild tracheobroncitis to severe pneumonia with necrosis.
– Patients with cystic fibrosis are particularly vulnerable.
– Patients who rely on mechanical ventilation are also at a higher risk of infection.

Skin and soft tissue infections, particularly from burn wounds, are common.
– Pseudomonas aeruginosa can also cause folliculitis and nail infections; cases have been linked to contaminated water in hot tubs, spas, and salons.

Urinary tract infections, particularly in patients with in-dwelling catheters.

Osteochondritis can develop after puncture wound infection; This is common in foot wounds caused by stepping on contaminated nails or other sharp objects.
– Be aware that osteochondritis can also occur when infection spreads from other sites.

Otitis externa ranges from mild cases, aka, Swimmer’s ear, to more severe cases, which are associated with diabetics and elderly people.

Corneal infections, which can produce ulcers, can occur after trauma; for example, corneal scratches caused by contact lenses.

Bacteremia with a high mortality rate, often because of multi-drug resistant strains and the fact that infected patients tend to be immunocompromised.
– Ecthyma gangrenosum is characterized by necrotic and hemorrhagic skin lesions.

Antibiotic resistance:

– Intrinsic properties of their cell walls.
– Acquired resistance via horizontal gene transfer.
– Adaptive resistance; for example, environmental triggers in the lungs of cystic fibrosis patients induces upregulation of resistant mechanisms.

Closely related to Pseudomonas

Many of these were once considered members of Pseudomonas until recently.
Burkholderia cepacia Complex comprises multiple species that are associated with Pulmonary infections, urinary tract infections, and bacteremia; they are generally susceptible to treatment with Trimethoprim-sulfamethoxazole (TMP-SMX).

Burkhoderia pseudomallei causes melioidosis, aka, Whitmore’s disease; cutaneous and pulmonary infections can be treated with TMP-SMX.

Stenotrophomonas maltophilia causes pneumonia and bacteremia, and can also be treated with TMP-SMX. (formerly known as Xanthomonas maltophilia)

Acinetobacter species are associated with infections of the respiratory tract, urinary tract, and wounds; unfortunately, these species are resistant to many antibiotics.

Moraxella catahrralis is associated with Bronchial infections, sinusitis, and otitis; it is penicillin-resistant, but susceptible to other types of antibiotics.

HAEMOPHILUS

• Pleiomorphic small rods or coccobacilli
• Facultative anaerobes
• Growth of most species requires addition of X factor and/or V factor on chocolate agar.
• Pili and non-pilus adhesins that attach to host cells are key to pathogenesis.

Haemophilus influenzae

this species derives its name from early suspicions that it was the causative agent of influenza, which we now know is caused by a virus.

• Spread in air droplets and respiratory secretions.
• Endotoxin comprises lipo-oligosaccharide, which impairs ciliary function and damages respiratory epithelium.
  – Allows the bacteria to evade ciliary clearance and to invade pharyngeal tissues to reach the blood.
• IgA 1 protease inhibits humoral immunity, which would otherwise activate complement and destroy the microbes.
• Pili are specifically adapted to adhere to mucus, epithelial cells, and extracellular matrix proteins.

Haeomophilus influenzae Non-Encapsulated

• Aka, Non-typeable
• Common colonizers of the upper respiratory tract
• Opportunistic pathogens that invade epithelial cells and macrophages, which facilitates infection.
• Associated with sinusitis, otitis, bronchitis, and pneumonia.
  – These infections are more likely in individuals with adaptive immune system deficiencies; in healthy individuals, serum is bactericidal and prevents microbial spread.

Haemophilus influenzae Encapsulated

• Further classified into serotypes a – f based on the antigens of their polysaccharide capsules
  – These strains are more often associated with invasive disease.
• Prior to the development of an effective vaccine, Haemophilus influenzae type b was a significant cause of pediatric bacterial infections; in regions where immunization programs are not well-established, it remains a major source of childhood illness.
• Meningitis particularly in children 3-18 months of age
• Arthritis
• Cellulitis around the cheeks and periorbital area.
• Epiglottitis is a life-threatening disease that primarily affects children 2-4 years old
  – It is characterized by pharyngitis, fever, and, difficulty breathing that progresses as swelling obstructs the airways.
• Be aware that non-type b encapsulated Haemophlius influenzae infections may be on the rise.

Haemophilus aegyptius occasionally causes acute purulent conjunctivitis.

• Conjunctivitis is more commonly associated with Gram-positive bacteria (specifically, Staphylococcus and Streptococcus).

Be aware that some authors call Haemophilus aegyptius a biogroup of Haemophilus influenzae.

Haemophilus ducreyi

• Causes chancroid, a sexually transmitted infection that is uncommon in the United States.
  Be aware that some authors call Haemophilus aegyptius a biogroup of Haemophilus influenzae.
  – Infection causes genital ulcers that can be treated with erythromycin.

PASTEURELLA

• Pasteurella multocida commonly colonizes the mouths of cats and dogs.
• Pasteurella canis colonizes the mouths of dogs.
  – Pasteurella canis is less commonly associated with human infection.
• Coccobacilli
• Facultative anaerobes
• Polysaccharide capsules that comprise hyaluronic acid.
• Infection usually results from animal bites or scratches.

Pastuerella multocida

• Systemic infection particularly in individuals with immune or liver dysfunctions.
• Exacerbation of underlying chronic respiratory disease.

Pastuerella multocida and Pasteruella canis

• Localized cellulitis and lymphadenitis.

ACTINOBACILLUS

• Colonize the oropharynx of humans and animals; infections are relatively rare.
• Actinobacillus equuli and Actinobacillus ligniereslii can cause bite wound infections, especially bites by farm animals.
• Actinobacillus ureae can cause septicemia, meningitis, and pneumonia.
• Actinobacillus hominis can cause septicemia and pneumonia.

AGGREGATIBACTER

• This genus comprises species that were formerly classified as Actinobacillus or Haemophilus
  – Be aware that some authors continue to refer to these species by their former genus names.
• Aggregatibacter actinomycetemcomitans is often found with Actinomyces in mixed infections, hence its name.
  – Colonies are described as star-shaped or like cigars crossing.
  – It is associated with subacute endocarditis of damaged valves.
  – Some strains cause localized aggressive periodontitis; toxins, including leukotoxins, cause significant damage to the tissues of the oral cavity.
• Aggregatibacter aphrophilus is also associated with endocarditis of damaged valves.
  – This bacteria has also been found in brain abscesses in children with congenital heart disease.
• Endocarditis caused by Aggregatibacter species can be treated with cephalosporins
• Periodontitis treatment includes biofilm debridement and administration of doxycycline or other antibiotics.
• Brain abscesses caused by Aggregatibacter aphrophilusinfection can be treated with meropenem.

RESPIRATORY ILLNESSES CAUSED BY GRAM-NEGATIVE RODS

Haemophilus influenzae

• Transmitted via respiratory droplets.
• Two key types of Haemophilus influenzae: Nonencapsulated and Encapsulated.
• Haemophilus influenzae non-encapsulated, aka, Non-typeable colonizes the upper respiratory tract of many people.
  – The bacteria can migrate to other parts of the respiratory tract and cause:
  Sinusitis and otitis
  Bronchitis and pneumonia; patients can develop bronchial inflammation and/or pneumonia with consolidation. Patients with chronic obstructive pulmonary disease (COPD) are at especially high risk of pneumonia from Haemophilus influenzaemigration.
• Encapsulated Haemophilus influenzae, especially type B, causes serious, life-threatening infections in unvaccinated children.
  Meningitis and septic arthritis
  Epiglottitis is characterized by a swollen epiglottis that can obstruct the airways and esophagus. Patients have a sore throat, and may drool due to the inability to swallow saliva; may be life-threatening.

Bordetella pertussis and Bordetella parapertussis

• Transmitted via respiratory droplets.
• These bacteria cause pertussis, aka, whooping cough.
• Infection is highly contagious, and is most severe in infants, who experience respiratory failure.
• Clinicians are seeing an increasing number of cases in adolescents and adults.
• DTaP vaccine protects children from infection.

Stages of Pertussis

• Incubation period lasts approximately one week.
• Catarrhal stage lasts one to two weeks.
  – This stage is characterized by non-specific symptoms of fever, runny nose, sneezing, and cough.
• Paroxysmal stage can last for several weeks.
  – Characterized by episodes of forceful expirations followed by a loud inspiration – the “whoop!” of “whooping cough”.
  – Because the fits are prolonged, oxygen levels can drop, leading to cyanosis, and post-pertussive vomiting can occur.
• Convalescent stage is characterized by reduced coughing; complications are possible, including pneumonia and encephalopathy.

Legionella pneumophila

• Transmitted via aerosolized water.
  – For example, in the mist from air humidifiers, shower heads, etc.
• Legionnaire’s disease is characterized by severe pneumonia and fever; older and less healthy individuals are more susceptible to disease.
• Pontiac fever is characterized by fever, myalgias, and other flu-like symptoms.

Francisella tularensis

• Transmitted via infected rabbits or bites from ticks and deerflies; thus, tularemia is a zoonotic disease.
• Tularemia can manifest in different forms:
  – Ulceroglandular tularemia produces skin lesions, often with black areas; the lymph nodes also become infected and produce tender, painful bulges in the skin – it is sometimes said to resemble the bubonic plague.
  – Pneumonia*
  – Tularemia can also affect other organs, including the eye, mouth, and throat.

SHARED TRAITS

• Salmonella, Shigella, and Yersinia are members of the family Enterobacteriacea.
  – This family also includes Esherichia coli, Citrobacter, Serratia, Proteus, Klebisiella, and Morganella.
  – To learn more about their general properties and common virulence factors, please see our overview tutorial on E. coli.
• Salmonella, Shigella, and Yersinia gain access to the host by using Peyers’ patch M-cells to cross the intestinal epithelium.

SALMONELLA

• Be aware that Salmonella nomenclature is somewhat contested and highly confusing. Currently, the full name of an isolate includes italicized genus and species with non-italicized serovar type.
  – So, for example, the full name of the isolate that causes typhoid fever is: Salmonella enterica serovar Typhi; most authors shorten this to Salmonella Typhi.

• Salmonella causes foodborne infections.
• Does not ferment lactose, unlike the other members of Enterobacteriacea;
• Produces hydrogen sulfide.
• Infects both humans and non-human animals
  – Exception: Typhoid-causing strains are strict human pathogens.
• Intracellular pathogens that can invade all tissue types, including the brain.
• Virulence genes are encoded on Pathogenicity islands I and II:
  – Type three secretion systems inject effector proteins into host cells.
  – Salmonella-secreted invasion proteins, which facilitate entry into host cells.
  – Immune evasion proteins.
• Infection is acquired after consumption of contaminated foods, especially eggs, poultry, and dairy.

Salmonellosis

• Gastroenteritis, characterized by inflammation of the intestinal lining, is the most common form of Salmonellosis in the U.S.
  – Symptoms include nausea, vomiting, diarrhea, fever, and cramps.
  – Gastroenteritis is usually self-limiting, and requires only supportive care (i.e., water and ion replenishment).
  – Salmonella Typhimurium, the isolate often associated with gastroenteritis, resides in Salmonella-containing vacuoles (SCVs) within phagocytes; this protective endosome allows the bacteria to survive and replicated safely inside host cells.
• Septicemia causes typical bacteremia symptoms, including fever, chills, and low blood pressure.
  – In a small subset of patients, localized infections can develop, particularly in the joints and cardiovascular endothelium.
  – Salmonella septicemia is most common in young children and older adults, and in individuals with HIV or other immune deficiencies.
  – Be aware that recurrent septicemia is an AIDS-defining condition.
  – Treatment includes Fluoroquinolone or Cephalosporin antibiotics.

• Enteric fevers
• Typhoid fever is associated with Salmonella Typhi
• Paratyphoid fever, a milder form is associated with Salmonellaserotypes Paratyphi A, Paratyphi B, and Paratyphi C
  Be aware that S. Paratyphi B is also referred to as Salmonella Schottmeulleri, and S. Paratyphi C is also referred to as Salmonella Hirshfeldii
• Symptoms & Pathogenesis:
  – Approximately 10 days after ingestion of the bacteria, individuals experience fever, fatigue, and other non-specific symptoms; indicate that some patients develop “rose spots,” which are small red macules on the trunk.
  – Approximately 20 days after ingestion, patients experience gastrointestinal symptoms, which can include constipation or diarrhea, nausea, and vomiting.
  • We show that Typhoid-associated strains, such as SalmonellaTyphi, move through the lining of the intestine and are engulfed by macrophages.
    Macrophages carry the microbes to the bone marrow, liver, and spleen, where bacterial replication occurs.
    This sets the stage for bacteremia and the non-specific symptoms patients experience.
    Infection increases splenic cellularity and leads splenomegaly in some patients; liver enlargement has also been reported.
    Ultimately, the bacteria colonize the gallbladder and re-infect the intestines, producing gastrointestinal symptoms.
• Treatment includes administration of fluoroquinolones or ciprofloxacin.
• Write that asymptomatic carriers maintain typhoid-associated strains within populations; for example, enteric fever is endemic in Southeast Asia and parts of Africa.

SHIGELLA

• So closely related to Escherichia coli, particularly Enteroinvasive E. coli, that some authors call Shigella an E. coli biogroup.
• Shigella is an intercellular pathogen that only infects humans.
• Asymptomatic carriers are a key reservoir.
• Virulence factors are encoded on a plasmid, but regulated by chromosomal genes; this means that the plasmid, alone, is not enough to promote virulence.
  – Type three secretion systems inject effectors
  – Ipa’s A-D, facilitate invasion of epithelial cells and macrophages.
• Within host cells, Shigella lyses phagosomes and replicates within the cytoplasm; notice that this is unlike Salmonella, which replicates within special vacuoles.
• Actin tails facilitate Shigella migration to adjacent cells, which allows the bacteria to spread without exposure to agents of innate immunity.

Shigellosis

• Primarily affects small children; outbreaks are associated with daycares and preschools, and other places where fecal-oral transmission via contaminated hands is likely.
• Shigella has a low infectious dose.
• Upon infection, Shigella enterotoxin initially causes watery diarrhea.
• Then, as bacteria invade the colon mucosa, bloody diarrhea with pus, cramping, and fever occur.
  – The presence of neutrophils, red blood cells, and mucus in the stool is a helpful diagnostic criterion.
• Most patients with Shigellosis recover on their own; supportive care to prevent dehydration is all that is needed.
  – In severe cases, antibiotics can be administered.
• There are multiple pathogenic strains of Shigella:
  – Shigella sonnei is responsible for most cases of Shigellosis in the U.S.
  – Shigella flexneri is responsible for most cases of Shigellosis in developing countries.
  – Shigella dysenteriae causes dysentery, the most severe form of Shigellosis.
  These strains produce Shiga exotoxin, which impairs protein synthesis.
  As a result, damaged intestinal epithelium produces bloody diarrhea.
  Damaged renal endothelial cells can lead to Hemolytic uremia syndrome (HUS); this occurs most often in infected children.

YERSINIA

• Zoonotic infections.
• Short rods, aka, coccobacilli
• Bipolar Gram-staining: their tips stain darker than their middles.
• Type three secretion systems secrete immune-suppressingeffectors into host cells, which allows Yersinia to survive and replicate.

Plague

• Yersinia Pestis causes plague.
• Infected fleas and lice transmit the bacteria to humans.
• Virulence factors and their genes:
  – Antiphagocytic protein capsule (Factor 1 gene).
  – Outer membrane proteins degrade complement proteins C3b and C5a; this prevents bacterial opsonization and phagocyte migration (Plasminogen activator gene).
  – **Plasminogen activator proteases **also degrade fibrin clots, which may facilitate bacterial spread in the host (Plasminogen activator gene).
• Bubonic plague is characterized by fever, headache, chills, and weakness; replication in the lymph nodes produces swelling and tenderness – “buboes” refers to the visible lymph node swellings that typically appear in the axilla or groin.
• Septicemic plague occurs when infection spreads throughout the body via the bloodstream or lymph; it produces fever, chills, and extreme weakness, as well as abdominal pain, and, possibly, shock or bleeding into the skin or other organs.
• Pneumonic plague can quickly lead to respiratory failure and death; and, because is spread in respiratory droplets, transmission is human–to-human.
• Treatment includes streptomycin or gentamycin; because disease progression is rapid, and infection can spread to the lungs, early administration of these antibiotics is crucial.

Yersiniosis

• Yersinia enterocolitica and Yersinia pseudotuberculosiscause enterocolitis, especially in children.
• They are associated with *undercooked pork.
• Young children tend to experience bloody diarrhea, cramps, and fever.
• Older children tend to experience fever, and a pain in the right side of the abdomen that may be confused with appendicitis.
• In most cases, supportive care is sufficient.

ENTEROPATHIC E. COLI (EPEC)

• Watery diarrhea and vomiting.
• Occurs most often in toddlers and infants in developing countries.

EPEC gastroenteritis

• Adhesion and effacing lesions formed on the epithelial cells of the small intestine.
  – The lesions destroy the microvillus brush border, which is where the final stages of carbohydrate and protein digestion and absorption take place.
• Virulence factors and gene locations:
  – Bundle-forming pili (BFP)* mediate initial attachment to host and bacterial cells and form microcolonies.
  BFP genes are found on the EPEC adherence factor plasmid in typical strains.
  – Interactions between intimin and the translocated intimin receptor (Tir) mediate more intimate attachment; their genes are found on the Locus of Enterocyte Effacement (LEE) pathogenicity island.
  – The Type 3 Secretion System (T3SS) translocates Tir and various cytotoxic effectors into the host cell cytoplasm; EPEC cytotoxic effectors are encoded by pathogenicity islands and phages.

ENTEROTOXIGENIC E. COLI (ETEC)

• Watery diarrhea with vomiting, fever, and abdominal cramps.
• Travelers and infants in developing countries.

ETEC gastrotenteritis

• Attaches to epithelial cells of the small intestine via colonization factor antigens I-III; the genes for CFAs are carried on plasmids.
• Flagellar adhesions and outer membrane proteins Tia and TibA also promote adherence; these traits are carried on pathogenicity islands.
• Heat-stable and heat-labile enterotoxins cause water and ion loss by altering activity in the cells of the small intestine; as a result, water and ions are flushed out of the body in diarrhea. The genes for both enterotoxins are on plasmids.
  – Heat-stable enterotoxin (STa) increases cGMP in host cells; As a result, sodium absorption decreases and water secretion increases.
  – Heat-labile enterotoxin increases cAMP; as a result, chloride secretion increases and sodium and chloride absorption decreases.

ENTEROAGGREGATIVE E. COLI (EAEC)

• Watery diarrhea with inflammatory cell infiltration, vomiting, and dehydration.
• Causes gastroenteritis in travelers and infants; in some children, it can lead to chronic diarrhea and growth retardation.

EAEC gastroenteritis

• Characterized by aggregative adherence to each other and the epithelial cells of the small and large intestines; this results in a “stacked brick” aggregation pattern
  – Attachment is mediated via aggregative adherence fimbriae; the genes for AAF are carried on a plasmid.
• AAF also trigger mononuclear cell infiltration and inflammation.
• Cytotoxins damage the intestinal mucosa.
  – The specific cytotoxins vary by strain.
  – Plasma-encoded toxin (Pet), which is coded for by plasmid genes, and induces exfoliation.
  – Be aware that some isolates produce Shigella enterotoxin 1 and EAST1.

SHIGA TOXIN-PRODUCING E. COLI (STEC)

• Watery diarrhea that can progress to hemorrhagic colitis with severe abdominal cramping; some patients develop hemolytic uremic syndrome.
• Children younger than 5 years old are most commonly infected by STEC.
• Enterohemorragic E. coli (EHEC) is generally considered to be a subset of STEC, and there is phenotypic variation even within EHEC isolates.
• Animals, particularly cattle, are key reservoirs for STEC.
• Humans are infected upon ingestion of foods, especially undercooked meat and raw fruits and vegetables; water is also a source of infection.
• Undercooked hamburger was once the main cause of STEC infection in the U.S.; increased awareness of proper handling has reduced the rate of hamburger-associated infections.
• Because STEC has a low infectious dose, person-to-person transmission is also possible.
• Antibiotics increase the risk of HUS complications, and should NOT be given to patients with STEC infections; it is thought that antibiotics cause phage lysis and increase toxin release.

STEC gastroenteritis

• Some strains produce adhesion and effacing lesions that damage microvilli brush border; as in EPEC strains, this is mediated by intimin and Tir;
• Hemorrhagic coli pilus is a type IV pilus.
• Cytotoxins cause necrosis and cell death.
  – More specifically Shiga toxins damage the microvasculature; the genes for these toxins are on phages.
• In some cases of severe infection, Shiga toxins destroy red blood cells resulting in hemolytic uremia syndrome, aka, HUS.
  – HUS is characterized by microvascular thrombi, thrombocytopenia, hemolytic anemia and renal damage that leads to renal failure.

ENTEROINVASIVE E. COLI (EIEC)

• Watery, then bloody, diarrhea and abdominal cramps.
• Fever helps to distinguish EIEC from STEC.
• EIEC infection is rare, and the bacteria are closely related to Shigella. In fact, many authors consider them to be members of the same species, and much of what we know about EIEC comes from research on Shigella.
• Unlike most other strains of E. coli, EIEC are non-motile, and do not ferment lactose.

EIEC gastroenteritis

• EIEC bacteria are intracellular pathogens that invade cells of the colon.
• Genes for the following invasion virulence factors are on the Invasion plasmid (InvP):
  – The type 3 secretion system and IpaC effector protein promote bacterial entry into cells
  – IpgD, IpaA, and VirA effectors facilitate bacterial invasion of phagosomes
  – IpaB, IpaC, and IpaD effectors facilitate phagosome escape
  – OpsB, OpsF, and OpsG effectors inhibit host immune responses
• Intra- and intercellular movement is facilitated by VirG, which promotes actin polymerization; these actin tails push the bacteria within and between host cells.
• Severe infection can lead to Shigellosis, aka, bacillary dysentery, which is characterized by colon epithelium destruction and inflammation. However, the EIEC-associated form is rare.

GENERAL PRINCIPLES

• Gram-negative Rods
• Colon microbiome
  – E. coli are common members of the colon microbiome.
  – Opportunistic pathogens: upon transfer to new anatomical niches, these strains can cause endogenous infections.
• Found in soil and water
  – As a result of animal or human fecal contamination.
  – Upon ingestion, pathogenic strains cause exogenous infections in humans.
• Because they reside in the intestines of animals and humans, E. coli are “enteric” bacteria.
• E. coli common pilus
  – Aids in adhesion to host surface and biofilm formation.
• Flagellar motility facilitates movement.
• Rapid growth
  – Simple nutritional requirements
• Facultative anaerobe
• Catalase-positive
• Oxidase-negative
• Reduce nitrate
• Ferments glucose
• Ferments lactose
  – Coliforms: Gram-negative enteric lactose fermenters.
  – Also includes strains from Klebisella, Serratia, Klebsiella, and Citrobacter.
  – Eosin methylene blue agar and MacConkey’s agar are two types of selective media used to identify coliforms; both inhibit Gram-positive bacterial growth and signify acid production from lactose fermentation.
  EMB agar: lactose fermentation and strong acid production yield a characteristic greenish hue.
  MacConkey’s agar: E. coli lactose fermentation produces a pinkish-purple color.

COMMON VIRULENCE FACTORS

Many virulence factors are acquired via horizontal gene transfer of plasmids, bacteriophages, and pathogenicity islands.

• E. coli endotoxin comprises heat-stable lipopolysaccharide:
  – Its A-antigen has classic endotoxin activity: it triggers the host inflammatory responses, disseminated intravascular coagulation, and hypovolemic shock.
  – The core polysaccharide is common to all Enterobacteriaceae.
  – The O-antigen is used to classify E. coli serotypes.
• Exotoxins promote cell death or fluid loss.
• Adhesins are present on pili, fimbriae, or strain-specific surface antigens.
• Type III secretion systems inject virulence effector proteins into host cells.
  – Virulence effectors promote attachment, invasion, and cell destruction.
• Phase variation alters the expression of O, K, and flagellar H antigens and pili.
  – Alteration of surface antigens allows E. coli to avoid host immune system and, possibly, to adjust energy expenditure according to environmental factors.
• Antibiotic resistance
  – Resistant phenotypes are rapidly spread via HGT, which makes antibiotic treatment of E. coli infections particularly difficult.
• Siderophores, special receptors, and transporters
  – Acquire iron from the host; recall that iron is necessary for bacterial growth.
• Polysaccharide capsule lends repels phagocytes and avoids serum killing.

E. COLI INFECTIONS

Extraintestinal Infections

• Causative strains are called Extraintestinal pathogenic E. coli (ExPEC).
• ExPEC are often commensals that act as opportunistic pathogens when exposed to new anatomical sites.

• Urinary Tract Infections
  – Uropathic E. coli (UPEC) causes most urinary tract infections.
  – Adhesins: Type 1 and P pili and Dr fimbriae attach to urothelium.
  – Hemolysin A causes cell damage and facilitates bacterial movement through superficial cell layers.
  – Strains lacking these virulence factors are easily removed from the body in the urine.

• Neonatal Meningitis
  – E. coli is the second most common cause of bacterial neonatal meningitis.
  – Neonatal Meningitis-causing E. coli (NMEC) have special virulence factors that enable translocation across the blood-brain barrier:
  Type I pili and Outer Membrane Protein A (OmpA) attach to brain microvascular endothelial cells.
  Ibe (invasion of brain endothelial cells) proteins, CNF1 (cytotoxic necrotizing factor 1) and other mediators facilitate invasion of the blood brain barrier.
  K1 capsular antigens are thought to prevent lysosome fusion to allow successful movement of live bacteria across the barrier.

• Septicemia
  – Septicemia can occur when urinary or gastrointestinal tract infections spread to the blood; this can happen, for example, upon trauma to the abdomen.

Gastroenteritis

• E. coli pathotypes responsible for gastroenteritis are collectively referred to as “Intestinal Pathogenic E. coli” (IPEC).
  Infection typically follows ingestion of contaminated food or water; human or animal feces is often the source of the bacteria.
• IPEC can affect the small and/or large intestine:
  – Small intestine: Enteropathogenic E. coli and Enterotoxigenic E. coli
  – Small and Large intestines: Enteroaggregative E. coli
  – Large intestine: Enteroinvasive E. coli and Shiga toxin-producing E. coli
  Virulence factors specific to each pathotype determine the pathogenesis of infection.

PATHOGENIC ENTEROBACTERIACEAE

Be aware that we will not include all pathogenic members, and that details can be found elsewhere.

Common features of enterobacteriaceae:

• They are ubiquitous in the environment, and some are commensals.
• They are a significant cause of disease, particularly urinary tract and gastrointestinal infections.
  The following characteristics are helpful in identification:
• The bacteria in this family share a common surface antigen.
• They are facultative anaerobes.
• The ferment glucose and reduce nitrate
• They are catalase positive and oxidase negative
• They have common virulence factors, including endotoxin, antibiotic resistance, and others.

Ferments lactose: Escherichia coli

• When grown in MacConkey’s agar, lactose-fermenters, including most strains of E. coli, produce bright pinkish-purple colonies; in Eosin methylene blue agar, lactose-fermenters produce colonies with a bright green sheen.
  Escherichia coli produce extra-intestinal and gastrointestinal infections, depending on the bacterial strain.

• Extra-intestinal infections are typically caused by opportunistic pathogens, such as commensals that migrate to new anatomical niches.
  – Primary cause of urinary tract infections.
  – Significant cause of neonatal meningitis.

• Gastrointestinal infections are caused by pathogenic strains in the GI tract; they are usually introduced via contaminated food or water.
• Pathogenesis and symptoms vary by pathotype
• Infants and children are most likely to suffer long-term consequences of dehydration due to severe diarrhea and/or vomiting.
• Pathotypes that tend to produce their effects in the small intestine and cause watery diarrhea and vomiting (illness is often referred to as “traveler’s diarrhea):
  – Enteropathogenic, Enterotoxigenic, and Enteroaggregative; Enteroaggregative E. coli also affects the large intestine.
• Pathotypes that primarily affect the large intestine and produce watery, then bloody diarrhea:
  – Shiga toxin-producing Escherichia coli, as its name suggests, produces Shiga-like toxin.
  Enterohemorrhagic Escherichia coli (EHEC) is usually considered a subset of Shiga toxin-producing Escherichia coli.
  In severe infection, a condition called hemolytic uremic syndrome (HUS) can develop when red blood cells and platelets are destroyed; the damaged cells can obstruct the renal filtration system and cause renal failure.
  Antibiotics increase the risk of HUS in patients with Shiga toxin-producing Escherichia coli infection.
  – Enteroinvasive Escherichia coli is similar to Shigella, which produces inflammatory lesions and ulcers in the colon.

Enterobacteriaceae that do not ferment lactose (or do so very slowly)

• They produce colorless colonies on MacConkey’s agar, as opposed to the bright pink colonies of E. coli.

Salmonella

• Transmitted via the fecal-oral route, usually via contaminated foods.
• Causes typhoid and paratyphoid fevers, which are characterized by flu-like, nonspecific and gastrointestinal symptoms.
  – More specific signs of typhoid and paratyphoid fever include *gallbladder infection due to bacterial replication, liver and spleen enlargement, and the development of so-called rose spots over the skin of the torso.
• Also causes gastroenteritis without typhoid fever; this is the most common form of infection in the United States.
• Can cause extra-intestinal problems, such as osteomyelitis and septicemia; recurrent septicemia is an AIDS-defining condition.

Shigella

• Transmitted via the fecal-oral route, and show that it is an intracellular pathogen.
• Shigellosis is most common in children, and is easily transmitted due to its low infectious dose.
• Associated with watery, then bloody diarrhea.
• Shigella strains that produce the Shiga toxin can cause Hemolytic uremia syndrome (HUS), often in children.

Yersinia

• Zoonotic infections.
• Fleas transmit the strain of Yersinia that causes the plague, which is characterized by fever, chills, and weakness.
  Three key forms of the plague: bubonic, system, and pneumonic.
• Bubonic plague is characterized by infection of the lymphatic system, and that bacterial replication in the lymph nodesproduces buboes.
  – Localized lymph node destruction produces these inflamed, raised areas that are tender or even painful.
• Septicemic plague occurs when the bacteria enter the bloodstream. Patients experience extreme weakness, as well as pain in the abdominal area.
  – Disseminated intravascular coagulation occurs, producing necrosis and even organ failure without treatment.
• Pneumonic plague can occur if bubonic plague spreads to the lungs, or, in other cases, as the result of human-human transmission via respiratory droplets.
  – Inflammation and necrosis in the lungs can rapidly lead to respiratory failure.

• Entercolitis is caused by other strains of Yersinia
• Transmitted via consumption of infected animals, especially pork products.
  – Whereas younger children are likely to have bloody diarrhea, older children tend to experience pain the right abdomen that may be mistaken for appendicitis.

ACTINOMYCES ISRAELII

• A. israelii is anerobic, slow-growing, and has low virulence.
• Opportunistic pathogen:
  – It is a common colonizer of the oral cavity and upper respiratory tract, and is sometimes present in the gastrointestinal and urogenital tracts.
• Branching, fungus-like morphology.
• Colonies, aka, sulfur granules
  – Granules comprise bacteria and calcium phosphate, and have a dimpled, molar-like appearance.
  – They do not contain sulfur.
• Most A. israelii infections occur when the bacteria invade deeper tissues of the oral cavity after trauma or surgery. From there, infections can spread.

Actinomyces:

• Chronic, slow forming granulomatous lesions that become abscesses that drain pus with sulfur granules.
• Infection most commonly occurs in the cervicofacial region following dental trauma.
  – Produces localized swelling, often in the mandibular region: “lumpy jaw.”
  – The abscess may form sinus tracts that erupt on the face.
• When infection occurs in other sites, more serious complications can arise.
  – Central nervous system infection can manifest as a single abscess with headache and focal neurological signs. In the image of an abscess removed from the brain, we see examples of “dust bunny” formation; this reflects aggregation of the filamentous bacteria.
  – Thoracic infection typically produces nonspecific symptoms such as fever and non-productive cough, and lung abscesses may form.
  – Abdominal cavity infection can affect any organ, and can produce fever and fatigue. Be aware that digestion problems and inflammation may be mistaken for signs of Crohn’s diseases, and masses have been mistaken for tuberculosis and cancerous tumors.
  Abdominal cavity infection can affect any organ, and can produce fever and fatigue.
  – Pelvic actinomycosis has been associated with long-term use of intra-uterine devices; masses can easily be mistaken for tumors.

• Prevention of actinomyces includes good oral hygiene, and, in the case of dental procedures, prophylactic antibiotics.
• Treatment includes drainage or surgical debridement when necessary and administration of penicillin.

Be aware that other species of Actinomcyes are also associated with actinomcyosis.

NOCARDIA

Includes several species that can infect and cause nocardiosis.

Be aware that Nocardia nomenclature and classification has changed dramatically over the years. As a result, some isolates that were once commonly associated with nocardiosis no longer are. For example, several isolates formerly identified as Nocaria asteroides have been reclassified.

• Nocardia are weakly acid-fast, with a delicate “beaded”appearance.
• Aerobic and catalase-positive, with slow growth.
• Unique aerial hyphae, with filaments that grow upward from the colony.
• Nocardia are not considered part of the normal human microflora.
• Found in soil.
• Gain entry to human hosts via inhalation and aspiration.
• Individuals with defective cellular immunity are particularly susceptible to infection.
• Pathogenic Nocarida have multiple ways to avoid phagocytic destruction, which, as you may recall, is key to innate immunity.
• Enzymes catalase and superoxide dismustase protect them from the harmful effects of phagocytic reactive oxygen species.
• Cord factor: When phagocytosed by macrophages, Nocarida cord factor (aka, trehalose dimycolate) prevents phagosome-lysosome fusion, which means the bacteria avoid bactericidal molecules.
  – Nocardia can survive and replicate within macrophages, which travel throughout the body.

Nocardiosis

Overall, Nocardiosis is rare, and manifests as non-specific symptoms.
However, it should be ruled out early to avoid delayed diagnosis and treatment. Fortunately, the bacteria’s weakly acid-fast nature and aerial hyphae make it easy to identify.

• Lung infection is most common.
  – Illness onset is associated with nonspecific symptoms.
  – Lung abscesses and necrosis can develop, and dissemination to other organs can occur.
• CNS infection is the most serious form of nocardiosis, and results in abscesses with non-specific symptoms (such as fever and headache); meningitis is possible but infrequent.
• Cutaneous infections manifest as granulomas, ulcers, or cellulitis, and may involve nearby lymphatics. Infection can be primary or secondary.

• Treatment
• Nocardia response to antibiotics varies, so testing for antimicrobial susceptibility is crucial.
• In general, pulmonary infections are treated with trimethoprim-sulfamethoxazole (TMP-SMX) and amikacin;
• CNS infections are treated with trimethoprim-sulfamethoxazole and imipenem or cephalosporin.
• Prolonged antibiotic treatment is recommended to avoid relapse.

CORYNEBACTERIUM DIPHTHERIAE

Causative agent of diphtheria.

• Club-shaped, and often arranged in “L” or “V”- shaped formations.
• Pathogenic C. diphtheriae contain granules with volutin, aka, polyphosphate, which provide intracellular energy storage and stain metachromatically.
• Non-motile
• Airborne transmission, human-human is typical.
• Bacteria colonize the oropharynx and skin of asymptomatic carriers, which maintains their presence within populations.
• Four subtypes of C. diphtheriae; the mitis biotype is most often responsible for human disease.

Diptheria Toxin

• Lysogenic bacteriophages introduce the Diphtheria toxin gene into the bacteria.
• The A subunit acts via ADP-ribosylation of elongation-factor 2to inactivate host cell protein synthesis.
• The B subunit has two regions:
  – Binding region binds heparin-binding EGF-like growth factor, which is anchored to the membranes of many host cells, particularly heart and nerve cells.
• Translocation region facilitates movement of diphtheria toxin into the host cell.

Respiratory diphtheria

• Sudden onset of fever, sore throat, and adenopathy.
• Pharyngeal exudate forms a pseudomembrane that can extend to the larynx.
  – Comprises immune cells, bacteria, and fibrin, and, unique to diphtheria.
  – It is firmly adhered to the underlying tissue.
• Complications:
  – Obstructed airways.
  – Neurotoxicity, which tends to manifest as cranial nerve weakness beginning in the pharynx.
  – Myocarditis is common in diphtheria, and tends to appear a week or two after illness onset.
  – Edema with inflammation in the myocardium.
  – Arrhythmia, heart failure, and death.

Cutaneous diphtheria

• Occurs after skin contact with an infected person.
• Characterized by chronic ulcers, which may be covered by gray membranes.

Prevention with DPT Vaccination:

• Typically, in the U.S, children are given a series of injections with a combined preparation of diphtheria, pertussis, and tetanus antigens.
• Booster immunizations should be given every 10 years after the last childhood injection.

Treatment:

• Antitoxin should be given immediately because the toxins bind irreversibly and cause cell death. – Test for hypersensitivity because serum sickness can occur.
• Antibiotics such as Penicillin G or erythromycin should be prescribed.
• Vaccinate after recovery.
  – Many people do not develop protective antibodies in response to natural diphtheria infections.

LISTERIA MONOCYTOGENES

Causes febrile gastroenteritis and meningitis.

• Animals, plants, and soil.
• Also grows readily in cold temperatures.
• In the U.S., infectious outbreaks are associated with contaminated unpasteurized dairy products and deli meats.
• Short rods; some authors categorize them as coccobacilli.
• Gray, weakly beta-hemolytic colonies on blood agar plates.
• Unique tumbling motility, which makes it appear as if it’s doing summersaults in broth media.
• Facultative intracellular anaerobes.

Virulence factors

Adhesion and invasion, vacuole escape, and movement are regulated by positive regulatory factor (PrfA), a transcription factor that is activated upon host cell infection.
These virulence factors allow L. monocytogenes to move from the lumen of the gut to infect the meninges and other body tissues.

• Internalins A & B facilitate attachment and entry into host cells:
  – Internalin A recognizes receptors on host enterocytes.
  – Internalin B interacts with a wider range of cells, including endothelial cells, fibroblasts, and enterocytes.
• Listeriolysin O and Phospholipase C
  – Upon entry into the host cell, bacteria release listeriolysin O and phospholipase C to escape from vacuoles.
  – This promotes microbial evasion of phagolysosome destruction and access to the cytosol for reproduction.
• Actin assembly-inducing protein (ActA) facilitates intra- and intercellular movement via “comet tails”.
  – ActA is also associated with aggregation and biofilm formation, and avoidance of autophagy.

L. monocytogenes meningitis pathogenesis:

1. Ingestion of L. monocytogenes-contaminated foods.
2. Interalins facilitate entry to enterocytes from the lumen of the GI tract.
3. Actin tails push the bacteria out of enterocytes and to macrophages, where replication occurs.
4. The parasitized macrophages disseminate the bacteria throughout the body.
5. Ultimately, L. monocytogenes can cross the blood-brain barrier and cause meningitis.

Listeriosis

Infection with L. monocytogenes can induce a range of dysfunction: some individuals are asymptomatic carriers, while others, particularly those with defective cellular immunity, experience disease.

• Healthy adults: flu-like symptoms and gastroenteritis with watery diarrhea, fever, aches, and abdominal cramps.
• Immunocompromised adults, including pregnant women, the elderly, and transplant recipients: bacteremia and meningitiscan develop.
  – L. monocytogenes meningitis is associated with high mortality.
• Neonate: Early and late onset diseases:
  – Early onset infection occurs when the bacteria cross the placenta; this can result in spontaneous abortion, pre-term birth, or granulomatosis infantiseptica, which is characterized by rash, abscesses and granulomas in the liver, lungs, spleen, and other organs.
  – Late-onset infection is acquired during or soon after birth, and can result in meningitis that appears up to a month after birth.

Prevention

• Because L. monocytogenes is ubiquitous in the environment, and there is no vaccine, prevention is difficult.
• At-risk populations, including pregnant women, are advised to avoid unpasteurized dairy foods and cold deli foods.

Treatment

• Invasive infections can be treated with ampicillin and gentamicin.

CLOSTRIDIUM OVERVIEW

• Rapid-growing
• Thrive in anaerobic conditions, such as the intestinal tract, sewage, water, and soil.
• Pathogenic strains produce histolytic toxins, enterotoxins, and neurotoxins.
• Four strains that cause infection in humans:
  – Clostridium difficile causes diarrhea and colitis.
  – Clostridium perfringens causes gas gangrene and food poisoning.
  – Clostridium tetani causes tetanus.
  – Clostridium botulinum causes botulism.

CLOSTRIDIUM DETAILS

Clostridium difficile

• Transmitted via the fecal-oral route
• Common colonizer of the human colon.
• Historically associated with hospitalized patients, we’ve seen a rise in community-acquired cases.
• A major cause of C. difficile infection is antibiotics that suppress the non-pathogenic colonic flora, which allows the opportunistic C. difficile to flourish.

Virulence factors include potent toxins:

• Toxin A is an enterotoxin that attracts neutrophils, which release cytokines.
  – Toxin A also increases intestinal wall permeability by attacking colonic epithelial tight junctions.
• Toxin B is a cytotoxin that acts on enterocyte actin to destroy cytoskeletal integrity.
• Binary toxin: Some strains of C. difficile produce Binary toxin, aka, C. difficile transferase (CDT).
  – The role of Binary toxin in C. difficile infection is uncertain: Some studies suggest that Binary toxin increases bacterial adherence to host cells and promotes cell death; others report that the toxin suppresses eosinophil activity. It is possible that the toxin has multiple virulence effects.

Infection:

• C. difficile infection acts on the colon, where it induces a range of gastrointestinal issues, from mild diarrhea to severe colitis.
  – Pseudomembranous colitis is the most serious form of C. difficile infection; characterized by yellowish-white exudate on the mucosal surface of the colon. The pseudomembrane comprises fibrin and inflammatory cells in mucus.

Treatment & Prevention:

• Withdrawal of the associated antibiotic often suffices to treat diarrheal symptoms.
• Metronidazole or vancomycin can be administered in more serious cases.
• Colitis relapse is common. Patients with multiple relapses may benefit from fecal microbiota transfer from a healthy donor, which replenishes the healthy bacteria in the colon.

Clostridium perfringens

• Spores are rarely seen clinically, and, that colonies are flat and irregular; it is beta-hemolytic.
• C. perfringens can be divided into subtypes based on toxin production.

Virulence factors:

• Alpha toxins cause hemolysis, vascular leakage, liver toxicity, and cardiac dysfunction.
  – Present in all C. perfringens subtypes.
• Several other toxins form pores and/or induce necrosis.
• Food poisoning enterotoxins alter the intestinal membrane permeability, leading to fluid and ion loss; like some other bacterial toxins, this enterotoxin is a superantigen.

Infection:

• Range of soft tissue infections, including cellulitis, fasciitis, myositis, and myonecrosis.
  – Myonecrosis, aka, gas gangrene, which is a life-threatening disease that destroys muscle tissues. Bacterial metabolic activity produces characteristic gas bubbles, which appear as purlplish-black bulges under the skin. Tissue necrosis and other complications can lead to death.
• C. perfringens food poisoning typically results from consumption of contaminated meat products; the enterotoxin acts on the small intestine to produce abdominal cramps and watery diarrhea without fever or vomiting.

Treatment & Prevention:

• Soft tissue infections:
  – Treatment for C. perfringens soft-tissue infections includes antibiotics and surgical debridement of necrotic tissues.
  – Proper wound care is essential for prevention of infection.

• Food poisoning:
  – Treatment for food poisoning includes rehydration; antibiotics are not recommended because this type of food poisoning is self-limiting.
  – Prevention is achieved by refrigerating and thorough reheating of foods to at least 74 degrees Celsius.

Clostridium tetani

• Distinctive tennis-racket shape during spore formation.
• It is extremely oxygen sensitive, and spores can survive for extended periods of time in the soil.
• Infection typically occurs when wounds encounter contaminated soil.
• Wounds provide ideal necrotic and anaerobic environments for C. tetani growth.

Virulence factors:

• Tetanospasmin, is a heat-labile neurotoxin that blocks the release of inhibitory neurotransmitters, such as GABA and glycine
  – The neurotoxin is endocytosed, then transported along axons to reach neuronal somas in the spinal cord.
  – Because it blocks inhibitory neurotransmitter release, neuronal excitatory activity is unregulated.
• Tetanolysin is another toxin produced by C. tetani.
  – It is an oxygen-labile hemolysin thought to promote tissue necrosis.

Infection:

• Tetanus is characterized by spastic paralysis:
  – This can manifest systemically or locally as lockjaw, grimace (aka, risus sardonicus), and opisthotonos, in which spasms of the extensors of the head, neck, and back produce extreme back extension. Tetanus can also cause fever and sweating.
  – Cephalic tetanus involves the cranial nerves.
  – Maternal tetanus is associated with pregnancy, specifically, contamination during medical or spontaneous abortion and delivery.
  – Neonatal tetanus occurs when infection spreads from the umbilical stump.

Treatment & Prevention:

• Vaccination effectively prevents tetanus.
• Treatment for infection involves wound debridement and administration of metronidazole and antitoxins are necessary to prevent death.

Clostridium botulinum

• Foodborne disease: the spores tend to contaminate vegetables and meat.
• Toxins are protected from degradation within the gastrointestinal tract.

Virulence factors:

• C. botulinum produces exotoxins A-G.
• Types A, B, and E are responsible for most human infections.
• Toxins are pre-formed, particularly in canned goods, which provide ideal alkaline and anaerobic environments.
• From the gut, the neurotoxins are absorbed and delivered to motor neurons; upon endocytosis, neurotoxins block acetylcholine release at peripheral motor junctions.

Infection:

• Botulism
  – Descending flaccid paralysis, blurred vision with dilated pupils, dry mouth, and constipation.
  – Respiratory failure leads to death.
  – Wound infection produces similar symptoms; in the US, this form is associated with injection drug use.

Treatment & Prevention:

• Treatment includes metronidazole or penicillin and antitoxins.
  – Respiratory support may be necessary, and gastric irrigation can aid in toxin removal.
• Prevention of botulism involves boiling of home-canned goods and discarding damaged cans
• Honey can contain C. botulinum organisms, so it should not be given to infants because they have not yet acquired the competitive microflora that prevents C. botulinum survival in adults.