Chapter 19: Gram-Negative Bacteria: E. coli

• Gram-negative bacteria are as capable of opportunistic infections as Gram-positive bacteria, with individual strain traits being crucial for their ability to exploit host defense breaches.
• Gram-negative opportunists can originate from the human microbiota, soil, aquatic environments, or be transmitted by arthropods.

Common Traits of Gram-Negative Opportunists

• These pathogens exploit specific breaches in human defenses, to which they appear to be adapted.
• Many exhibit resistance to multiple antibiotics, making treatment difficult.

E. coli: An Ever-Changing Pathogen

• E. coli is a facultative anaerobe found in the environment and as part of the intestinal microbiota.
• Most E. coli strains are nonpathogenic, but some cause diarrhea, dysentery, bladder/kidney infections, hemolytic uremic syndrome (HUS), meningitis, and septicemia.
• E. coli strains cause a wide range of diseases because they have acquired different sets of virulence factors.
• Serotyping of E. coli is based on O-antigen of lipopolysaccharide (LPS), flagellar H-antigen, and capsular K-antigen and it is not adequate for distinguishing clinical isolates with different virulence properties.
• Virotyping is a classification method based on profiling virulence factors like invasion, adherence, toxin production, and effects on host cells.

Enteroinvasive E. coli (EIEC)

• EIEC strains and Shigella species (Shigella dysenteriae) cause bloody diarrhea.
• Shigella may be classified as a virulent strain of E. coli due to their genetic similarity.
• Virulent strains of Shigella and EIEC carry a large plasmid responsible for their invasive phenotype.
• They spread from cell to cell via actin-based motility.
• Shigella produces Shiga toxin (STx), which damages intestinal cells and can cause HUS.
• EIEC strains do not produce STx.

Enterotoxigenic E. coli (ETEC)

• ETEC causes self-limiting acute infant diarrhea and "traveler's diarrhea."
• ETEC adheres to the small intestinal mucosa via type 1 and type IV pili without being invasive and produces heat-stable toxin (HST) and heat-labile toxin (HLT).
• HST activates guanylate cyclase receptors, increasing cGMP levels and causing diarrhea.
• HLT ADP-ribosylates and activates the α-subunit of the heterotrimeric Gs protein, increasing cAMP levels and causing diarrhea.

Enteropathogenic E. coli (EPEC)

• EPEC causes severe infant and traveler’s diarrhea with pathology different from ETEC or EIEC.
• It does not usually have genes for HST or HLT.
• EPEC causes enterocyte attachment and effacement (EAE), altering the ultrastructure of mucosal epithelial cells.
• EAE involves pedestal formation with actin rearrangement.
• All EPEC genes necessary for the EAE phenotype are found on the locus of enterocyte effacement (LEE) pathogenicity island.
• LEE encodes a type III secretion system (T3SS) and Esp effector proteins.
• EPEC strains produce bundle-forming pili (Bfp) for self-aggregation.

Enterohemorrhagic E. coli (EHEC) / Shiga Toxin-Producing E. coli (STEC)

• EHEC/STEC are food-borne pathogens that cause bloody diarrhea and HUS.
• They adhere to and efface intestinal epithelial cells (EAE phenotype) but do not aggregate like EPEC.
• They produce Shiga-like toxins (SLTs) like Shigella strains.

Uropathogenic E. coli (UPEC)

• UPEC is a common cause of urinary tract infections (UTIs), especially in women.
• They produce hemolysins and colonize the urinary tract instead of the intestinal tract.
• UPEC strains produce type 1 and P pili which are critical for their ability to colonize the urinary tract.
• Type 1 pili's tip protein adhesin (FimH), binds to mannose-containing glycoproteins which mediates invasion
• P pili binds specifically via a tip protein adhesin (PapG) to the disaccharide globobiose
• Attachment and/or invasion can result, causing bladder infection (cystitis), leading to replication inside cellular vacuoles and eventually to lysis and exfoliation (sloughing) of the cells.
• UPEC strains can ascend further up the urinary tract and cause kidney infection (pyelonephritis).

Neonatal Meningitis-causing E. coli (NMEC)

• NMEC causes septicemia and meningitis in infants.
• NMEC is the second most common cause of neonatal meningitis next to group B streptococcal infections,
• E. coli K1 strain infection is most commonly associated with sepsis and meningitis.
• Infants can be exposed to E. coli during birth.
• Progression begins with gastrointestinal colonization, followed by translocation across the intestinal mucosa.
• Delayed treatment can lead to neurological complications.
• LPS is the main virulence factor in adult cases, and the K1 capsule is most important in neonatal meningitis.
• The outer membrane protein A (OmpA) of K1 strains can also interact with an endothelial cell glycoprotein and cause disruption of barrier integrity.
• Colonization by the mother plays an important role in transmission to the neonate.

Other Hybrid E. coli Virotypes

• By changing their complement of virulence factors, E. coli strains can dramatically change their virulence and pathological profiles.
• Enteroaggregative E. coli (EAEC) have virulence plasmids that encode aggregative adherence fimbriae (AAF) for biofilm formation.
• Adherent-invasive E. coli (AIEC) can attach and invade through a macropinocytosis-like uptake process involving actin microfilaments and microtubules and replicate inside large vacuoles triggering strong pro-inflammatory responses.
• The potential for new virotypes to emerge is high.

Klebsiella pneumoniae Nosocomial Infections

• Klebsiella are nonmotile, encapsulated, rod-shaped bacteria found in nature.
• They reside as part of the commensal microbiota of the nasal, oral, and gastrointestinal tracts of animals and humans.
• Major virulence factors of K. pneumoniae are production of the iron-chelating siderophore aerobactin and the presence of a highly mucoid phenotype due to production of a capsule.
• In immunocompromised individuals, certain strains can lead to nosocomial infections, including pneumonia, neonatal infection, meningitis, diarrhea, UTIs, and septicemia.
• The inability to treat the infections with antibiotics due to extensive drug resistance.

Hypervirulence

• Over the past couple of decades, a new, highly virulent, invasive strain of K. pneumoniae has emerged, predominantly in the form of pyogenic liver abscesses that are not limited to immunocompromised individuals.
• These community-acquired hypervirulent K. pneumoniae isolates have pronounced hypermucoviscous phenotype.
• Most hypervirulent clinical isolates belong to a single, recently evolved clonal lineage, called sequence type 23 (ST23) or clonal complex 23 (CC23).
• ST23 is distinct from nonhypermucoviscous, nonhypervirulent strains, such as ST258, due to acquisition of a single virulence plasmid and a chromosomal genomic island.

Multidrug Resistance

• Multidrug resistant strains of K. pneumoniae have emerged as some of the most-feared pathogens in the hospital setting, the so-called ESKAPE pathogens.
• Given the relatively high frequency of horizontal gene transfer (HGT) involving antibiotic resistance-conferring plasmids among K. pneumoniae, there is mounting fear that MDR isolates of hypervirulent strains will soon appear on the health scene.
• An exciting strategy that is gaining momentum is the use of K. pneumoniae-specific lytic bacteriophage.

Bacteroides fragilis: The Bad Sheep of the Family

• Bacteroides is a major group of bacteria in the colonic microbiota.
• Bacteroides are Gram-negative, obligately anaerobic, rod-shaped bacteria.

Enterotoxigenic B. fragilis

• A subset of B. fragilis strains (enterotoxigenic B. fragilis) causes diarrheal disease and colitis in animals and humans.
• These strains produce one of three subtypes of a 45-kDa proenzyme enterotoxin (BFT or fragilysin), BFT1 through BFT3.
• Long-term colonization with enterotoxigenic B. fragilis is associated with increased risk of colorectal cancer

Nonenterotoxigenic B. fragilis

• Non-BFT-producing strains of B. fragilis (NTBF) are responsible for causing the main types of infection associated with Bacteroides, namely intra-abdominal abscesses and bacteremia.
• The cause of B. fragilis infections is trauma to the abdominal area, primarily from wounds or during surgery, that leads to breaches in the colon wall that allow the bacteria to enter tissue and blood.
• Regions of dead tissue become highly anoxic due to loss of their blood supply and NTBF is able to colonize such dead tissue and form abscesses that can leak bacteria into the bloodstream.
• The major virulence factor of NTBF is its unusual, complex polysaccharide capsules.
• A clinically important feature of B. fragilis and other Bacteroides species that can cause opportunistic human infections is their increasing resistance to a variety of antibiotics.

Porphyromonas gingivalis: A Keystone Pathogen

• P. gingivalis is related to Bacteroides species, but is located in the mouth.
• The black-pigmented P. gingivalis is an obligate anaerobe located in the periodontal pocket.
• It has multiple ways of evading host immune responses.

Periodontal Disease

• P. gingivalis is involved in the inflammatory gum disease gingivitis.
• P. gingivalis can have a disproportionate impact on the microbial community, eventually leading to a shift in the microbial composition and exploits the resulting dysbiotic conditions to grow which causes inflammation.
• The two major virulence factors are: fimbriae and proteases.
• Rgp-mediated cleavage of the C5 protein into C5a and C5b releases C5a, a chemoattractant for neutrophils and The result is inflammation, which causes a deepening of the periodontal pocket, bleeding gums, resorption of bone, and even progression to tooth loss because of the altered microbiota.
• Most cases of periodontal disease are still treated by surgery and new laser-based surgery techniques are starting to gain popularity.

Oral Bacteria and Coronary Artery Disease

• P. gingivalis can occasionally cause internal abscesses, but the most serious internal condition associated with invasive P. gingivalis is cardiovascular disease
• Once P. gingivalis binds via its adhesins, such as fimbriae FimA, to cellular receptors it can invade human coronary artery endothelial cells through receptor-mediated endocytosis.

Fusobacterium nucleatum: A Cause of Preterm Birth?

• Oral anaerobes, such as Fusobacterium nucleatum, may be involved in a wide range of diseases, including periodontitis, oral cancer, and preterm birth.
• Environmental Inhabitants Weigh in as Opportunists

Pseudomonas aeruginosa: A Versatile Opportunist of the Highest Order

• P. aeruginosa is a soil microorganism that can utilize a variety of carbon and energy sources.
• P. aeruginosa can cause a variety of infections which can range from bloodstream infections in immunocompromised patients to UTIs.
• P. aeruginosa is best known for its ability to infect damaged, exposed tissue in wound and burn victims and to cause lung infections in people with cystic fibrosis (CF) and in hospital patients with ventilator-associated pneumonia.

Wound and Burn Infections

• The predilection of P. aeruginosa for damaged wound or burned tissue is easy to understand since it is likely to be on the scene due to its ubiquitous presence in the environment.
• It is essential to treat P. aeruginosa infections as early as possible because it is well known for its intrinsic and HGT-acquired resistance to a variety of antibiotics.
• P. aeruginosa infections are the most likely cause of death of patients who have survived the initial trauma of severe burns.

Lung Infections in Cystic Fibrosis Patients

• P. aeruginosa causes particularly problematic infections in the lungs of CF patients.
• CF is an inherited chronic lung disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR).
• The mutation causes misfolding of the protein that leads to unregulated Cl– (and consequently Na+) ion efflux and the The resulting water adsorption causes, among other things, dehydration of the airway surface and a thickening of the mucus layer in the lungs and airways.
• An important contributor to early tissue damage in the CF lung appears to be release of neutrophil extracellular traps (NETs).
• Widespread efforts over the past couple of decades to eradicate initial acquisition of P. aeruginosa in childhood have greatly reduced the prevalence of P. aeruginosa in CF patients.

Virulence Factors

• There are a number of secreted tissue damaging toxins and enzymes, such as proteases, hemolysins, exotoxin (ExoA), toxic T3SS effector proteins (ExoS, ExoT, ExoU, and ExoY), and the nonprotein redox-active toxic pigment, pyocyanin (see chapter 12).
• A different type of cytotoxin is the pigment pyocyanin which gives colonies of P. aeruginosa their characteristic blue-green color on agar medium.
• Large amounts of pyocyanin are produced in the lungs of CF patients.
• P. aeruginosa employs two-partner cooperation between the type IV pili, which attach to the host cells, and a two-component toxin system, comprised of exolysin A (ExlA) and exolysin B (ExlB).

Resistance to Antibiotics

• P. aeruginosa is one of the notorious ESKAPE pathogens.
• P. aeruginosa often employs a variety of different resistance mechanisms that act in concert
• Because the minimum inhibitory concentration (MIC) needed to kill the bacteria within the biofilm matrix is about 1,000-fold greater than that for planktonic bacteria, current efforts for treating infections are directed toward finding ways to disperse biofilms so that antibiotics can work more effectively.
• A new and exciting approach toward solving the CF problem that is under development is the application of targeted gene therapy for delivery of gene-editing technologies to lungs for correction of the CFTR mutation.
• BCC is a large group of related Burkholderia species, including B. cepacia, B. cenocepacia, and about 20 other species, typically found in aquatic or moist soil environments.

Burkholderia cepacia pneumonia

• B. cenocepacia has been and continues to be a serious infectious disease problem in CF patients and in patients with nosocomial respiratory infections.
• As in the case of P. aeruginosa, the resistance of BCC to a variety of antibiotics is a serious and growing problem, which appears to be due in large part to efflux pumps that eject many types of antibiotics from Burkholderia cells.

Acinetobacter baumannii: A Deadly Threat Emerges from the Iraq War

• A. baumannii has decidedly earned its mark as an ESKAPE pathogen.
• A. baumannii rapidly became one of the most feared nosocomial disease-causing opportunists, spreading first to other military hospitals and then to civilian hospitals around the country.

Antibiotic Resistance

• A. baumannii strains so dangerous is the fact that they are also remarkably difficult to treat due to extensive inherent and acquired antibiotic resistance.
• There are still a few first-line and some second-line antibiotics that work, but their number is small and dwindling.

Ehrlichia spp.

• Even though most of these pathogens are not usually considered opportunists because they cause infections in otherwise healthy people, some of them could be considered opportunists in the sense that they only cause serious disease in immunocompromised individuals.
• An example of such an arthropod-borne opportunistic pathogen is E. chaffeensis, which is transmitted by ticks, principally the lone star tick (Amblyomma americanum).
• The reason for calling E. chaffeensis an opportunist is that HME is seen almost exclusively in older people and in people with compromised immune systems.
• In humans, E. chaffeensis invades monocytes or macrophages, somehow prevents phagosome-lysosome fusion, and multiplies within the phagosome.