2024 Gram Negative ESKAPE Pathogens MIIM30011 PDF

Professor Christopher McDevitt Email: http://mcdevittlab.org Department of Microbiology & Immunology Peter Doherty Institute for Infection and Immunity  To develop an understanding of Acinetobacter baumannii and Pseudomonas aeruginosa To l...

Professor Christopher McDevitt Email: http://mcdevittlab.org Department of Microbiology & Immunology Peter Doherty Institute for Infection and Immunity  To develop an understanding of Acinetobacter baumannii and Pseudomonas aeruginosa To learn how these Gram-negative bacterial pathogens impact human Learning health Objectives To learn about bacterial drug efflux pumps and apply knowledge to discuss how they contribute to protection against antibiotic therapy  Gram-negative, short rod-shaped bacterium Motile – lacks flagella, but may use Type IV pili or secreted exopolysaccharide Strict aerobe Classified in the Gamma-proteobacteria Was a low-virulence commensal bacterium Evolved to be successful opportunistic pathogen and is a major cause of nosocomial infections Environmental reservoir? Some studies suggest that it is a ubiquitous environmental bacterium, while other argue that it is solely present hospital environments  Relatively large bacterial genome Average genome is ~3.5 Mbp Clinical isolates range from 3.0 – 4.0 Mbp Genomes encodes between 2,500 and 4,000 open reading frames (strain dependent) Core genome is only 1,344 genes Accessory genome and unique genes make up about 60% of the genomic content Pan-genome contains large amounts of non-conserved (xenogeneic) material component, i.e. isolate-specific, genes indicating horizontal gene transfer  Clinically significant opportunistic, nosocomial pathogen Pneumonia, UTIs, wound infections, meningitis and bacteremia ICUs and aged-care facilities Post-trauma infections (Iraqibacter/natural disasters/Bali bombings) Adverse lifestyles Treatment options Imipenem or meropenem was used, but carbapenem-resistance is increasingly occurring Polymyxins, such as colistin, but have adverse side effects Phage-therapy has had some success  First isolated by Carle Gessard in 1882 from wound bandages that had discolouration Gram-negative, encapsulated, rod-shaped bacterium Unipolar motility (flagella and Type IV pili) Classified in the Gamma-proteobacteria Secretes pigments including: pyocyanin ( ), pyoverdine ( ), pyorubin ( ), and pyomelanin ( ) Ubiquitous environmental bacterium present in soil, water, skin flora, and most man-made environments  Large bacterial genome PAO1 is ~6.2 Mbp Other clinical isolates range from 5.5–7.5 Mbp Genomes encodes between 5,500 and 6,500 open reading frames (strain dependent) 50% of encoded proteins have no known function Core genome is only 665 genes Represents only 10% of the genome Close to the size of a minimal genome Horizontal gene transfer accounts for much of the pan-genome (complete and fragmented plasmids)  Large genome confers broad metabolic capacity and can proliferate in conditions of partial or total oxygen depletion Facultative anaerobe Able to grow under microaerophilic or anaerobic conditions Anaerobic growth with nitrate or nitrite as a terminal electron acceptor. Able to ferment arginine and pyruvate by substrate-level phosphorylation Growth in a diversity of environments Able to grow on a range of different carbon and nitrogen sources Able to grow in diesel and jet fuels causing microbial corrosion Able to grow in cleaning agents and disinfectants  Clinically significant opportunistic, nosocomial pathogen of immunocompromised individuals Infects the airway, urinary tract, burns, and wounds, and also causes other blood infections ~10% of all hospital-acquired infections are P. aeruginosa Frequent colonizer of aseptic medical devices, e.g. catheters and ventilators Most common infections of burn injuries Case mortality rates associated with P. aeruginosa can reach: Up to 60% for pneumonia Up to 50% for bacteraemia Site Disease Respiratory tract Pneumonia Blood Bacteraemia Heart Endocarditis Central nervous system Meningitis and brain abscess Ear Chronic otitis media Eye Bacterial keratitis, scleral abscess, endophthalmitis and ophthalmia neonatorum Bones and joints Vertebral osteomyelitis , pyoarthrosis Gastrointestinal tract Pseudomonal diarrhea Urinary tract infections Pseudomonal UTIs Skin Green nail syndrome, secondary wound infections - burn wound sepsis  Ubiquitous – i.e. everywhere No flowers in burns wards  Clinically significant opportunistic, nosocomial pathogen of immunocompromised individuals Infects the airway, urinary tract, burns, and wounds, and also causes other blood infections ~10% of all hospital-acquired infections are P. aeruginosa Frequent colonizer of aseptic medical devices, e.g. catheters and ventilators Most common infections of burn injuries Case mortality rates associated with P. aeruginosa can reach: Up to 60% for pneumonia Up to 50% for bacteraemia Immunocompromising conditions exacerbate likelihood of infection, including: Diabetes - Malignant otitis externa Drug addiction - Endocarditis, osteomyelitis Leukaemia – Sepsis Cancer - Pneumonia, sepsis Burn wound - Cellulitis, sepsis Cystic fibrosis - Pneumonia Surgery involving CNS - Meningitis Tracheostomy - Pneumonia Neonatal period - Diarrhoea Corneal ulcer - Panophthalmitis Vascular catheterization - Bacteraemia, suppurative thrombophlebitis Urinary catheterization - UTI  Autosomal recessive genetic disease Occurrence of ~1 in 2,500 Recessive mutation in a transmembrane chloride channel (CFTR) Heterozygote advantage? Diagnosis Infants born with lung inflammation Blood sample - genetic screening Identifies ‘high-risk’ of CF. Not a definitive test and doesn’t identify carrier status Sweat test to measure chloride in the sweat Two to five times the normal amount of chloride in sweat ~3,700 Australians were registered with the Australian CF Data Registry  Genetic disorder affects multiple organs Primarily affects the lungs, but also the gut, pancreas, liver, kidneys, and intestines Lung impact manifests as decreased mucociliary clearance and mucus accumulation P. aeruginosa is the leading cause of morbidity and mortality associated with cystic fibrosis  Treatments No cure Gene therapy Organ transplantation – resolves primary lung impact, but not a cure Modulators – chemical chaperones/reversers Managing the symptoms >90% of CF patients received antibiotic treatment (IV, inhaled, or oral) >24% of adults were on continuous oral treatment regimes Antibiotics show poor penetration and/or efficacy within biofilms In 1950s, median life expectancy was 6 months In 2022, median life expectancy was 44.2 years of age  Management is the primary approach and includes: Antibiotics to control infection(s) Intensive daily airway clearance Exercise to maintain lung function Enzyme replacement tablets each day to aid digestion High energy diets with added vitamins and salt CFTR modulator combinations effectively eliminate these requirements in the majority of individuals ~80% of patients use at least one CFTR modulator  Cystic fibrosis transmembrane conductance regulator (CFTR) gene, encodes a chloride (Cl−) channel in secretory epithelia CF is associated with a loss of CFTR function Mutation ΔF508 (87.4% of cases) causes misfolding and degradation of CFTR  CFTR secretes Cl- ions and regulates ENac expression (Na+ importer) Airway epithelia with defective CFTR cannot secrete Cl- ions. Dysregulation of ion flow to and from airway surface liquid (ASL) Loss of CFTR function results in ENac overexpression which leads to Na+ and H2O hyper-reabsorption, leading to accumulation of thick mucus Cilia are matted down by the thick mucus Mucus is an ideal medium for opportunistic pathogens  P. aeruginosa lung infections occur early in life P. aeruginosa rapidly adapts to the mucus Chronic infection in CF is never eradicated 56% of patients have a P. aeruginosa infection By adulthood up to 80% of CF patients had lung infections with ~61% having moderate to severely compromised lung function  Lung mucus becomes an ideal medium for opportunistic pathogens Strains isolated from CF lungs have dramatic genetic, morphological and physiological changes Characteristics that are rarely seen in individuals that do not have CF CF clinical P. aeruginosa isolate phenotypes: Mucoidy phenotype grow as biofilms Fail to express virulence factor genes Show hyper-mutability Resistance to multiple classes of antibiotics  A. baumannii and P. aeruginosa have low antibiotic susceptibility due to: Multidrug efflux pumps Antibiotic-degrading and antibiotic-inactivating enzymes Low permeability of the cellular membranes Rapid acquisition of new resistance determinants Biofilm barriers and altered metabolism Resistance to common first-line antibiotics: β-lactams Carbapenems Polymyxins Aminoglycosides Limits treatment options in many cases  Decreasing pipeline of new antibiotics  Resistance is multifactorial Four main mechanisms 1. Drug inactivation or modification 2. Alteration of drug target site 3. Alteration of cellular metabolic pathway 4. Active extrusion  Resistance-nodulation-division family Ubiquitous in bacteria, archaea and eukaryotes Efflux pumps that are highly prevalent in many Gram-negative bacterial species Located in the cytoplasmic membrane Function as proton/substrate antiporters 7-8 phylogenetic families: o Hydrophobe/amphiphile efflux (HAE) – drug resistance o Heavy metal efflux (HME) – Zn and Cu resistance P. aeruginosa contain 13 RND transport systems (one HME-RND and the remaining HAE-RNDs) A. baumannii contains 3-4 RND transport systems (HAE-RNDs)  RND systems involved in drug efflux are: AdeABC, AdeFGH and AdeIJK in A. baumannii MexAB-OprM and MexXY-OprM in P. aeruginosa Homologs of the archetypal E. coli RND efflux pump AcrABZ-TolC  AcrB/MexB/AdeB subunit confers drug specificity Multi-drug specificity is provided by different residues (combinations of aromatic residues) in AcrB/MexB/AdeB AcrB/MexB/AdeB is an asymmetric homotrimer  Key Learning Concepts A. baumannii and P. aeruginosa are a clinically important opportunistic ESKAPE pathogens particularly in the immunocompromised P. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis Antibiotic resistance is multifactorial Polyspecific drug efflux pumps, such as RND transporters, contribute to the drug resistance of the Gram-negative ESKAPE pathogens