pathogens exam 4

biofilms are inherently tolerant to antibiotic treatment (various mechanisms)

-antibiotic tolerance: growth of bacteria stops in the presence of antibiotic, but cells are not killed 1. failure to penetrate the biofilm; impairment of antibiotic in areas of waste accumulation or altered environment (pH, pCO2, pO2) 2. trapped/destroyed by matrix enzymes 3. altered growth rate 4. biofilm-specific resistance genes 5. stress response to hostile environmental conditions --> expression of enzymes

side effects

-antimicrobial agent specificity for bacterial target without affecting mammalian cells (differential toxicity) -risk vs. reward

Lux system - V. fischeri

-at low cell density, vibrio species have low expression of LuxI (enzyme responsible for making AI-1, which is hydrophobic and can freely move outside of the cell); also low level of expression of LuxR (the target for AI-1 binding; a transcription factor that regulates downstream genes) -at high cell density, LuxI moves back into the cell and binds LuxR, upregulating expression of itself (LuxR expression) and divergent LuxI; also downstream genes required for luciferase formation and light production

Bacteriostatic vs. bacteriocidal

-bacteriostatic: antimicrobial compounds that inhibit bacterial growth -bacteria can frequently recover once treatment is removed; best used in patients with intact immune systems -bacteriocidal: antimicrobial compounds that kill bacteria - best in patients with compromised immune systems **bacteriocidal vs. bacteriostatic can be blurred by properties of bacteria (i.e. fast-growing vs. slow growing, biofilm growth)** --> bacteriostatic antibiotics work best on fast-growing bacteria; may act more like a bacteriostatic agent in slow-growing or biofilm-forming bacteria -minimum inhibitory concentration (MIC) - lowest antibiotic concentration that will prevent bacterial growth -dilution susceptibility assay -minimum bacteriocidal concentration (MBC) - lowest antibiotic concentration that will kill bacteria -tube dilution method for MIC determination: -a series of increasing concentrations of antibiotic is prepared in the culture medium. Each tube is inoculated, and incubation is allowed to proceed -growth (turbidity) occurs in those tubes with antibiotic concentrations below the MIC -first tube where no turbidity (looks clear) = MIC

cell wall synthesis inhibitors

-beta-lactams - bactericidal - inhibit penicillin-binding proteins; allows autolysins to become hyperactive and break down their own cell wall -vancomycin

tetracyclines

-bind 16S rRNA in 30S subunit of bacterial ribosome; disrupt bacterial membrane -inactivation of antibiotic (?); ribosome protection; efflux system -broadly bacteriostatic; some protozoa -tetracycline, doxycycline

streptogramins

-bind 23S rRNA in 50S subunit of bacterial ribosome -inactivation of antibiotic by removing grups -bacteriostatic individually; bactericidal in combination; used for multi-drug resistant enterococcal infections -synercid

macrolides/lincosamides

-bind 23S rRNA in 50S subunit of bacterial ribosome -methylation of target; efflux -bacteriostatic for most; bactericidal for some gram-positive bacteria -erythromycin (macrolide); lincomycin, clindamycin (lincosamides)

fluoroquinolones

-bind DNA gyrase -efflux (?); reduced uptake (?); mutation in DNA gyrase -broadly bactericidal; can enter phagocytes, kill intracellular bacteria -ciprofloxacin, norfloxacin

rifampin

-binds beta subunit of bacterial RNA polymerase -mutation in RNA polymerase -broadly antibacterial; effective against mycobacteria -rifampin, rifadin

Biofilm formation by S. pneumoniae

-biofilm formation associated with nasopharynx colonization, otitis media, fomite transmission -in vitro biofilm formation most enhanced in mutants lacking capsular polysaccharide (CPS) -CPS needed for virulence (prevention of opsonophagocytosis) -mice infected with planktonic bacteria progressed to bacteremia; most challenged with biofilm bacteria did not -biofilm inoculum also induced weaker immune response

Biosecurity

-bioterrorism: the intentional release of viruses, bacteria, or other germs that can sicken or kill people, livestock, or crops -dual-use agents: those biological agents (bacteria, virus, toxin) that are used in non-malicious ways (therapeutic development, educational purposes) but could also be used as bioweapons (i.e. botulinum toxin, staphylococcal enterotoxins, B. anthracis, many others)

EIEC: enteroinvasive E. coli (bloody diarrhea/dysentery) **binds and invades M cells, lateral spread through enterocytes (similar to Shigella)

-invasion --> phagosomal rupture --> intracellular movement --> lateral spread to adjacent cell -adherence via type I pili -large intestine, colon

Beta-lactams (penicillins, cephalosporins, carbapenems, monobactams)

-mech: inhibit transpeptidation step in peptidoglycan synthesis; bind penicillin-binding proteins; stimulate autolysis -resistance mech: gram-negative outer membrane; porin mutations; beta-lactamase; modify target (alteration of penicillin-binding protein) -spectrum: gram-positive and/or gram-negative bacteria (depends on agent) -penicillin, ampicillin, Cefobid, Augmentin

M. tuberculosis

-multi-drug resistant strains a BIG problem -a 4 drug regiment (incl. rifampin) is recommended for persons in US to moderate risk for infection with multi-drug TB -examples of genetic resistance: -rifampin-resistance: mutations in beta subunit of RNA polymerase -streptomycin-resistance: mutations in genes encoding S12 protein and rRNA -fluoroquinolone-resistance: mutations in DNA gyrase gene -other factors that makes TB difficult to treat and/or promotes drug resistance: -slow growth of organism -intracellular -patient compliance (treatment time can be from 2-9 months, depending on resistance profile)

S. pneumoniae: other interesting "Factoids"

-naturally competent --> genetic diversity -genomes of different serotype strains differ by ~10%, these differing regions often contain pathogenic islands with genes that mediate invasive diseases -was one of the first model bacterial systems used to study fundamental molecular genetic processes -Avery, MacLeod and McCarty used the natural transformation of S. pneumoniae to demonstrate that DNA is the genetic material -more than 1/2 of deaths in the great 1918 influenza epidemic died of invasive pneumococcal disease -most people are usually colonized by only one serotype at a time, due to intraspecies competition (partly mediated by bacteriocin/pneumocin production --> regulated by QS)

regulation of erm expression (translational attenuation)

-no erythromycin: erm gene - rbs, AUG inaccessible -erythromycin: erm gene - rbs, AUG exposed --> alternative RNA stem-loop -low level formation of alternative stem-loop in the absence of antibiotic allows basal expression of methylase (keeps some ribosomes functional when initially encountering Erm) -erythromycin - prevents translocation of ribosomes, peptide not translated

regulation of tetB (efflux pump) expression

-no tetracycline: TetR binding to promoter represses transcription; tetR expression is autoregulated -tetracycline: Mg2+ + tetracyline --> TetR binding --> falls off promoter, allows tetB transcription

Gram positive opportunistic pathogens: general characteristics

-normal commensal inhabitants of the human body (either majority of the population (i.e. S. epidermidis) or a portion of the adult population at any one time (i.e. S. aureus, S. pneumoniae, Clostridium difficile) -like the gram negative opportunistic pathogens, multidrug resistance can be a problem (i.e. MRSA) -the "line" between opportunistic pathogen and other categories of disease can be blurred (i.e. community-acquired MRSA, C. difficile, S. mutans) -are all strains created equal in terms of their pathogenic potential? -each bacterial species is actually composed of many subgroups that contain profound differences in their genomes and virulence properties -pangenome or "total composite genome": core set of genes shared by all (~80%) + 20% extrachromosomal elements (transposons, pathogenicity islands, prophage, plasmids) that tend to carry specific virulence genes, toxin genes, antibiotic resistance genes, etc. -these 20% "accessory" genes usually dictates the pathogenic potential of a particular subgroup of a given species (commensal --> opportunistic pathogen --> disease severity)

Regulation of natural competence in S. pneumoniae

-not all members are competent -ComD = sensor kinase, comE = regulator (two-component system) -ComE upregulates comABCDE and comX1, comX2, and various autolysins which degrade the cell wall of bacteria -allolysis = fratricide - killing of neighboring cells -pneumolysin - pore-forming toxin that targets eukaryotic cells -sublytic concentrations: induction of host cell apoptosis, activation of host complement, induction of proinflammatory reaction in immune cell -higher lytic concentration: potential for widespread direct cellular and tissue damage via pore forming properties -increased bacterial fitness: genome plasticity, DNA repair?

Porphyromonas gingivalis

-obligate anaerobic bacterium located in oral cavity -periodontal pocket (region between teeth and gums) -involved in gingivitis (inflammation of gums) increase anaerobes (including P. gingivalis) in periodontal pocket --> inflammation --> deepening periodontal pockets --> bleeding gums --> tooth loss -can also occasionally cause internal abscesses -association between P. gingivalis and coronary heart disease, as well as various autoimmune diseases (diabetes, arthritis)

natural biological threat sources

-persistent, ever-present infectious diseases -high-impact infectious diseases (major health, ecological, and economic consequences) -new and reemerging infectious diseases -multidrug resistant pathogens -human-to-human transmission -vector-borne, epizoonotic transmission -foreign animal, zoonotic transmission -food-borne transmission -water-borne transmission -increasing populations of people with weakened immune systems due to infectious diseases, chronic diseases, and aging -invasive alien species: plants, animals, insects

Extended spectrum beta-lactamase producing enterobacteriaceae

-19% of healthcare associated enterobacteriaceae infections are caused by ESBL-producing enterobacteriaceae -patients with bloodstream infections caused by ESBL-producing enterobacteriaceae are about 57% more likely to die than those with bloodstream infections caused by a non ESBL-producing strain

Previous instances of intentional anthrax

-1979 epidemic in Sverdlovsk (former Soviet Union): 68 confirmed deaths, largest documented outbreak of human inhalational anthrax, later investigation revealed outbreak was likely due to widespread inhalation of anthrax spores accidentally released from a military facility -2001 outbreak in USA -spore-containing envelopes mailed to news and government offices; general consensus is that this was "non-weaponized" (i.e. not mixed with silica/dispersal agents) -11 inhalational and 11 cutaneous cases reported. All 5 patients who developed shock with inhalational disease died -prophylactic antibiotic treatment for many potentially exposed individuals likely mitigated a much higher fatality rate -specific source of the contaminated envelopes is still debated -two suspects investigated, no one ever formally charged/prosecuted

vancomycin-resistant enterococci (VRE)

-20,000 drug resistant enterococcus infections -1300 deaths from drug-resistant enterococcus infections -66,000 enterococcus infections per year -some enterococcus strains are resistant to vancomycin leaving few or no treatment options -represents about 30% of all hospital-acquired Enterococcus infections -enterococcus sp. intrinsically resistant to various beta-lactams, fluoroquinolones, aminoglycosides -VRE infections usually treated with newer antibiotics such as daptomycin -horizontal gene transfer a concern: VRE --> MRSA = VRSA

Translation inhibitors

-50S subunit of bacterial ribosome: chloramphenicol, macrolides, lincosamides, streptogramins, everninomycins, oxazolidinones, lincosamides -30S subunit: aminoglycosides, tetracyclines

cutaneous anthrax

-6 days post-exposure - extensive edema and hemorrhagic vesicles typical of cutaneous anthrax; appear prior to the formation of the ulcer with black center

Multi-drug resistant acinetobacter

-7300 multidrug resistant infections -500 deaths -12000 infections/year -at least 3 different classes of antibiotics no longer cure resistant acinetobacter infections -threat level: serious -some acinetobacter strains are resistant to nearly all or all antibiotics including carbapenems, often considered antibiotics of last resort -about 63% of acinetobacter is considered multidrug-resistant, meaning at least three different classes of antibiotics no longer cure Acinetobacter infections -approx. 2% of healthcare-associated infections reported to CDC's national healthcare safety network are caused by Acinetobacter, but the proportion is higher among critically ill patients on mechanical ventilators (about 7%)

Bacterial QS systems

-AI-1 (autoinducer 1) - within the same species -primarily gram negative, intraspecies communication -AHLs are small, hydrophobic, membrane-diffusible (no transporter) -binding of AHL to receptor regulator protein within cell --> protein dimerization --> regulation of target genes

regulation of blaZ (beta-lactamase) expression

-BlaR1 serine residue covalently binds to beta-lactam -BlaR2 = protease that cleaves BlaI -intact BlaI represses blaZ transcription

DNA replication and transcription inhibitors

-DNA gyrase inhibitors: replication inhibitors = ciprofloxacin, novobiocin -RNA pol inhibitor: transcription inhibitors = rifampin

examples of breaches in human defenses that are used by opportunists to cause infections

-E. coli K1 - infant meningitis - infants exposed to colonized vaginal tract during birth -P. gingivalis - periodontal disease, internal infections - a shift in the microbiota of the gums, giving rise to inflammation -P. aeruginosa - lung infections - CF causing aberrant mucus in the lungs -A. baumannii - wound infections - traumatic war wounds in Iraq veterans

V. cholerae QS

-El Tor Pandemic biotype: -responsible for 7th pandemic (1960s-early 1990s) -milder disease compared to classical strain -AI-1 system requires two-component system -when quorum sensing molecules bind at high densities, inhibit phosphorylation and incites phosphatase activity -at low concentrations - LuxU phosphorylates LuxO - retains phosphorylase activity; produces small RNAs which stabilize production of HapR (when you don't produce enough HapR, allow high levels of cyclic di-GMP to be produced in the cell) -at high concentrations, quorum sensing factors bind and dephosphorylation occurs; turn off small RNA production; not enough HapR - high levels of cyclic di-GMP -LuxS/PQ system = AI2 -CqsA/S system = AHL/AI1 (CAI-1) -classical biotype -responsible for 6 pandemics (1817-1923) -VieS/AB system -bypasses HapR, more directly regulates cyclic di-GMP production -high levels ci-di-DMP = increased biofilm, increased CT; decreased virulence genes involved in exit of host (transmission)

virulence factors of P. aeruginosa

-ExoA - AB toxin that ADP-ribosylates EF-2; inhibits host cell protein synthesis; causes cell death -ExoS, ExoT, ExoU, ExoY - T3SS effector proteins - cytotoxicity, inflammation -MDR efflux system (MexEF, OprMN, and MexT) - antibiotic efflux pump (MexEF-OprMN) and its regulator (MexT); confers antibiotic resistance -quorum sensing - LasR (3-oxo-C12-HSL), RhIR (C4-HSL) - key role in controlling virulence factor production, biofilm formation, swarming motility, and expression of antibiotic efflux pumps

Extended spectrum beta-lactamase (ESBL) producing enterobacteriaceae

-Klebsiella pneumoniae -healthcare-associated infections, including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis -part of normal GI microbiota - opportunistic pathogen -E. coli -variety of pathogenic strains and diseases (diarrhea, dysentery), UTIs, meningitis, respiratory tract, others) -fecal-oral, contaminated food, birth canal -in addition to penicillin, ESBLs can hydrolyze the beta-lactam ring of various cephalosporins -usually associated with active site mutations of plasmid-encoded beta-lactamase genes -these plasmids often encode other antibiotic-resistance genes (i.e. aminoglycosides), making treatment choices limited -carbapenems usually used to treat these infections

Beta-lactam resistance

-PBPs in bacteria facilitate transpeptidation reactions and cell wall synthesis -beta-lactamase = secreted enzyme that cleaves beta-lactam ring; allows attach by H2O molecules -"serine beta-lactamase": covalent bond formation between active-site serine and ring (analogous to covalent bond formed between serine residues in PBPs) -clavulanic acid - prevents beta-lactamase from inactivating antibiotic -high level beta-lactamase expression (i.e. gene duplication) can overcome clavulanic acid supplementation (binding/sequestering) -zinc beta-lactamases use a catalytic mechanism that does not involve active-site serine residues to cleave beta-lactam ring; resistant to clavulanic acid -clavulanic acid only inhibits serine beta-lactamases -beta-lactam = kills bacteria -betalactam+beta-lactamase = beta-lactam inactivated --> no effect on bacteria -beta-lactam + clavulanic acid + beta-lactamase = inactivated beta-lactamase --> beta-lactam unaffected --> kills bacteria

Fig 18-10

-PCV-7 (pneumococcal conjugated seven valent vaccine) has been highly successful in the US and other developed countries -invasive pneumococcal diseases have dropped about 70-80% for infants under 2 years of age, and most infants in the US now routinely receive this vaccine

EHEC: enterohemorrhagic E. coli (bloody diarrhea, hemolytic uremic syndrome (HUS); "hamburger disease") **produces SLT (Shiga-like toxin)

-ST: kills cells by removing adenine base ("depurination") from ribose backbone of host 28S rRNA --> blocks protein synthesis -initial adherence type I pili -delivery of shiga toxin (encoded by lysogenic phage) -intimate attachment, pedestal formation, microvillous effacement (EAE) -O157:H7 -delivery of shiga toxin to HUS (irreversible kidney failure in children, elderly) -O antigen from LPS, H antigen from flagella -large intestine, colon

other S. pneumoniae virulence factors

-SpxB --> hydrogen peroxide - stimulates cytokine production by surrounding host cells; Niche competition; ATP production (energy) -surface adhesins - adherence to epithelial cells -pneumolysin - assembles in the cholesterol-containing membranes of the host cells; large transmembrane pores. Increased concentration --> inhibition of ciliary movement, chemotaxis of immune cells -LTA -PAF receptor binding, inflammation - modified phosphocholine (ChoP) groups **phosphocholine groups help better binding to PAF receptor which also has these groups (molecular mimicry)

E. coli

-UPEC: uropathogenic E. coli: causes UTIs, especially in women; hemolysins; tropism for UT/bladder due to Type I pili containing FimH and P pili containing PapG; adherence --> invasion/intracellular replication --> host cell lysis/exfoliation -NNMEC: neonatal-meningitis-causing E. coli: -adults: abdominal trauma --> bloodstream --> inflammation/septic shock --> organ damage -modified LPS main virulence factor (endotoxin): poorly recognized by TLRs/innate immune cells -infants: abdominal trauma or translocation across GI mucosa --> bloodstream --> cerebral spinal fluid --> neurological complications (cerebral palsy, learning disability, hearing loss) -K1 polysialic acid capsule (mimics host sialic acid), confers serum resistance, helps bacteria cross the blood-brain barrier

biological threats originated by people

-accidental release of infectious agents -unintentional release through mishap or malfunction -anthropogenic impact on environment or ecology that shifts natural habitats or exposes new sources -nonmalicious but intentional release or introduction that results in an unintended outcome -sloppiness or negligent hygiene during food preparation of processing -deliberate (the "terror effect") -state-sponsored bioweapons: WMD, or "weapons of mass destruction" -bioterrorism acts by groups or individuals: WMD, or "weapons of mass disruption"

ETEC: enterotoxigenic E. coli (traveler's diarrhea) **labile and/or stable toxin-mediated

-adherence via type I pili, type IV (TCP-like) pili -delivery of enterotoxins HST (heat stable toxin) and HLT (heat labile toxin) -stimulates chloride ion channels --> cell leakage -small intestine

Common disinfectants and their mode of killing

-alcohols (ethanols, isopropanol) = denature proteins -alkylating agents (formaldehyde, ethylene oxide) = form epoxide bridges that inactivate proteins -halides (I-, Cl-, NaClO) = oxidizing agents -heavy metals (Hg2+, Ag+) = bind -SH groups, thus denaturing proteins -phenols - denature proteins; disrupt cell membranes by intercalating in them -QACs = disrupt cell membranes by intercalating in them -UV radiation = blocks DNA replication and transcription by damaging DNA

treatment and prevention

-all forms of disease treatable with antibiotics' the sooner the better (especially for inhalational or GI anthrax) -a vaccine is also available, but only recommended for certain at-risk adult groups (i.e. military, researchers) -anti-toxin strategies still under investigation

P. aeruginosa

-all wild-type P. aeruginosa strains are capable of synthesizing alginate -mucoid phenotype is not observed outside the human host -conversion to an overtly mucoid phenotype appears to depend on host environmental pressures

antibiotic resistance in bacteria

-although a trend for decades, public first began to really take notice of antibiotic-resistant bacteria in 1990s (hospital-acquired MRSA**, MDR TB infections) -read box 16-1 -prohibitive treatment costs (insurance companies, HMOs) -agricultural use of antibiotics in feed -discovery of new antibiotics nearly ceased; not a big $$ maker for industry -1980s research effort (mechanisms of resistance, regulation of resistance genes) can be viewed as "old-fashioned" --> de-emphasis of work and funding -nowadays, multi-drug resistant (MDR) bacteria is a huge concern, as treatment options dwindle and research/drug development catches up

anthrax as a bioweapon

-anthrax spores are easily found in nature, can be produced in a lab, and can last a long time in the environment -could be released quietly and without anyone knowing. Microscopic spores could be put into powders, sprays, food, and water. Invisible, odorless, tasteless -anthrax has been used as a "weapon" before...

B. anthracis virulence factors

-capsule: poly-y-D-glutamic acid polypeptide; protects against phagocytosis -exotoxins: -edema factor: adenylate cyclase --> high levels of intracellular cAMP --> disturbance of H2O homeostasis, disruption of intracellular signaling pathways -lethal factor: zinc metalloprotease --> selectively inactivates mitogen-activated protein kinase kinases (MAPKKs) --> deregulation of numerous cell processes --> abnormal ion transport, loss of electrolytes and water, blockage of cellular signal transduction pathways, improper functioning of immune response -net result: shock and lethality (precise mechanisms still under investigation) -highly-purified toxins administered to animals cause symptoms typical for anthrax -ability to achieve high microbial concentrations in infected hosts

Pyocyanin production

-characteristic blue-green pigment of PA on agar plate

biofilm infections

-chronic sinusitis, burn-related infection, intravascular catheter infection, prosthetic valve endocarditis, pacemaker infection, dialysis catheter infection, pulmonary infection in CF patient

Factors that contribute to Acinetobacter baumannii environmental persistence and host infection and colonization

-colonization of respiratory tract, skin -colonization/infection of wounds -UTI -invasion/infection of bloodstream -pneumonia -colonization: adherence to host cells; resistance to inhibitory agents and conditions of skin and mucosal surfaces; biofilm formation; quorum sensing -environmental survival: resistance to desiccation, disinfectants and antibiotics; use of various substrates for growth; biofilm formation on surfaces, equipment and devices; quorum sensing for regulation of (ex: biofilm formation) -invection: elicitation of inflammatory responses, cytotoxicity, iron acquisition, resistance to serum and complement activation

gram negative opportunists

-come from either normal microbiota, soil, insects (controversial) -usually requires a specific type of "breach" to initiate infection (i.e. they respond to a more limited range of opportunities relative to gram positive opportunists) -many are multi-drug resistant --> difficult to treat

P. aeruginosa pathogenesis in CF

-disease of abnormal electrolyte transport and mucous secretion from exocrine glands and secretory epithelia -inherited as an autosomal recessive trait at a rate of 1 in 2000 live births; 5% of population are estimated to be heterozygous carriers -"CF gene" located on chromosome 7; mutation results in production of a defective transmembrane conductance regulator protein -CF airways characterized by inadequate iron transport and defective mucociliary clearance --> predisposition to persistent microbial colonization

Disinfectants/antiseptics

-disinfectants: applied to non-living objects or surfaces; bleach, alcohols, phenolics -primarily bacteriocidal; too toxic for external/internal treatment of patients -antiseptics: applied to living skin or tissue; often contain diluted disinfectants -both of these categories: -often also active against viruses, fungi, protozoa -often several targets in bacterial cell -most effective against actively growing bacteria -in general, resistance mechanisms poorly understood relative to antibiotics -membrane-active agents less effective against gram negative bacteria -membrane pumps (Quaternary Ammonium Cation resistance in staphylococci)

C. difficile infection

-disruption of normal enteric flora; acquisition of toxigenic Clostridium difficile -protective factors: high serum antibody response to toxin A, mild underlying disease --> Asymptomatic C. difficile colonization (carrier state) --> low likelihood of C. difficile diarrhea (may act as reservoir for nosocomial spread of C. difficile) -risk factors: low serum antibody response to toxin A, severe underlying disease --> C. difficile diarrhea --> additional protective factors: age <65 years, no exposure to additional antibiotics **see image**

scary facts (2013 CDC report)

-each year in the US, at least 2 million people become infected with drug-resistant microbes -at least 23,000 people die each year as a direct result of these infections

Virulence factors of P. aeruginosa

-flagellum (polar) - motility, dissemination, initiation of the innate immune response -type IV pili (polar) - adherence, twitching motility -siderophores - iron acquisition -phospholipases - hydrolysis of phospholipids in host membranes, tissue damage, phosphate acquisition -elastase, alkaline protease, and other proteases - proteolytic degradation of elastin, collagen, immunoglobulins, complement proteins, immune evasion, and nutrient acquisition; tissue damage -LPS - endotoxic shock, sepsis, serum resistance -alginate - adherence, protection from dehydration, antiphagocytic, protection of biofilm from host immune system -hemolysins - pore-forming toxins that damage host cell membranes -pyocyanin - small-molecule toxic blue-green pigment; redox-active compound that generates reactive oxygen species; disrupts action of cilia and phagocytic function

antibiotics "agents against life"

-general characteristics: -low-medium MW compounds that kill or inhibit bacterial growth -can be ingested or injected into humans or animals with minimal side effects -generally interfere with a specific cellular process or enzyme -characteristics of a "good" antibiotic -few or no side effects to the host "differential toxicity" -broad spectrum of activity (but can have drawbacks...i.e. C. difficile) -appropriate bioavailability (rate of amount of drug that reaches the infection site) and pharmokinetics to get to site(s) of infection -pill/ingested form -cost

development of antibiotic resistance in bacteria

-genomic plasticity of bacteria (core genome mutation acquisition/accumulation, intra-/inter-species genetic exchange, mobile genetic elements) -50% of antibiotic use in U.S. estimated to be inappropriate -antibiotics prescribed for viral or inappropriate bacteria -use of antibiotics to enhance growth of livestock -crowding, homelessness, poor nutrition/sanitation -international travel

Gingipains

-gingipains aid in the destruction of host antibodies and complement, and contribute to localized tissue destruction -i.e. reductions in cytokine production interferes with neutrophil recruitment to the periodontal pocket. Modification of local host responses to the biofilm flora results in the skewed immune response that drives periodontitis progression -gingipains = proteases

EPEC: enteropathogenic E. coli (diarrhea) **alters epithelial cell function by binding via bundle-forming pili (BFP)

-initial adherence via type I pili -colony formation via BFP pili (plasmid-encoded) -pedestal - actin -intimate attachment, pedestal formation, microvillous effacement (EAE) -altered ultrastructure - bacterial factors encoded by a 35-kb pathogenicity island -sometimes - delivery of CLDT, CNF (toxins that cause cell enlargement and cytoskeletal rearrangements)

A. baumannii

-gram negative rod, aerobic, non-motile -high incidence among immunocompromised individuals, especially those who have experienced a prolonged (over 90 day) hospital stay -ubiquitous in soil and aquatic environments; can also colonize skin -became well-known during Iraq war -both colonized and lethal wound infections (soldiers) --> Iraq strains brought to US via colonized wounded soldiers --> Walter Reed --> other US hospitals -wound infections (primarily), also bloodstream and pneumonia -has been categorized as a "superbug" (along with TB and MRSA) because they are extensively drug resistant -also inherently resistant to disinfectants and various environmental conditions --> hampers decontamination efforts -aggressive sterilization and patient isolation required (1 patient = 1 doctor/1 nurse rule)

Escherichia coli

-gram negative rod, motile -pink colonies on MacConkey agar (E. coli ferments lactose) -many strains!! classification via serotyping (O antigen - LPS, H antigen - flagella, K capsule type) -serotyping often not sensitive enough to detect differences between clinical strains (i.e. same serotype, different virulence properties), therefore virotyping can be used (profiling based on virulence factor production, effects on host cells, clinical symptoms)

vibrio cholerae

-gram negative, motile, halophilic; exists in the ocean and different bodies of water -causative agent of cholera - voluminous watery diarrhea; life-threatening (dehydration) -fecal-oral transmission; aquatic environments natural reservoir (algae, copepods, crustacean shells) -penetration of intestinal epithelium facilitated by flagellar motility, mucinase -pili-mediated attachment to GI epithelium, co-regulated with cholera toxin production (ADP ribosylase --> Gs protein (host cell adenylate cyclase protein; increase cyclic AMP levels in eukaryotic epithelial cells --> loss of ion flow control --> extreme water loss --> diarrhea, dehydration) -"rice water stool" - mucus, epithelial cells, high concentration of V. cholerae -mortality rate without treatment 25-50% (keep patients hydrated! also use of antibiotics) -stool-sample testing - gram staining -disulfate sucrose agar plate - yellow colonies -elisa or eukaryotic cell culture based test looking for toxin

Regulation of natural competence in S. pneumoniae

-gram positive - uses peptide-based quorum sensing --> peptide is CSP (ComC) -ComC encodes the peptide -comAB encodes a transporter that processes and transports comC from inside to outside the cell -as culture grows, peptide accumulates, binds to comD (sensor kinase), which then phosphorylates comE and turns on the whole system through positive feedback -cibAB - encodes bacteriocin -lytA - hydrolase - allolysis

Bacterial QS systems - overview

-gram positive and gram negative interspecies communication -AI1 or AI1-dependent system -AI2: furanosyl borate diester; byproduct of SAM degradation -common to all bacteria; clear indicator of presence of other bacteria - can help bacteria detect colonization of areas by competing species and produce a response to clear them out (i.e. toxins/bacteriocins, etc.) -small, hydrophobic; diffusible across cell membrane -binding of AI2 to membrane receptor protein (HK - histidine kinase protein) --> phosphorelay to RR --> regulation of target genes

Streptococcus pneumoniae

-gram positive diplococci, inhabits upper respiratory tract of up to 40% humans -pneumonia, bacterial meningitis, otitis, various upper respiratory infections -~60% of all bacterial pneumonia -over 83 types of polysaccharide capsule; major contributor to invasive potential (bloodstream, cerebral spinal fluid) -at risk populations have less natural antibodies (6mo-2yrs, elderly) or immunocompromised, recovering from viral infection -aerosol spread (community-pneumonia) -PPSV-23 vaccine (23 capsule types) recommended for at-risk adults, elderly -other vaccines available for <2yr olds -primarily antibiotic treatment (~15% of strains resistant to beta-lactam antibiotics) -alpha-hemolytic

Streptococcus pneumoniae

-gram positive diplococci, normal inhabitant of upper respiratory tract of up to 40% of humans -pneumonia; bacterial meningitis, otitis, various upper respiratory infections -~60% of all bacterial pneumonia - over 83 types of polysaccharide capsule (inhibition of phagocytic uptake/complement); major contributor to invasive potential (bloodstream, cerebrospinal fluid) -at risk populations have less natural antibodies (6mo-2yrs, elderly) or immunocompromised, recovering from viral infection -aerosol spread (community) -PPSV-23 vaccine (23 capsule types) recommended for at risk adults, elderly -other vaccines available for <2-years old -primarily antibiotic treatment (~15% of strains resistant to beta-lactam antibiotics)

Enterococcus faecalis

-gram positive facultative anaerobe -causative agent of UTIs, abdominal/pelvic wound infections, bacteremia -environment, GI tract -opportunistic pathogen --> usually infections are hospital-acquired -bile esculin agar: esculin hydrolysis + ferric citrate = iron salts (black)

Clostridium difficile

-gram positive rod; spore former -endogenous or environmental (spores) -bloody diarrhea; pseudomembranous colitis -overgrowth in the colon a result of normal flora disruption (antibiotics) -recurrence common; little protective immunity -deposition of fibrin, neutrophil debris, mucin (pseudomembranous plaques)

Bacillus anthracis

-gram positive, non-motile, aerobic, facultative anaerobic, large rod-shaped bacteria capable of forming dormant spore -found in soil --> herbivore mammals <-- humans -several forms of disease: -cutaneous (most common but least fatal; ~95% of reported cases) -inhalation -gastrointestinal -injectional (drug use) (2009-2010 outbreaks in UK and Germany - 54 cases, 18 deaths)

lessons learned from the 2001 anthrax attacks

-important for there to be an official organized network of scientific experts to communicate with media and the public -minimize hype and propagation of misinformation (i.e. needless destruction of anthrax strains at ISU) -appreciation that a bioweapon attack does not have to be large scale exposure with catastrophic outcomes (pre-2001 era scenario) -increased scrutiny, reporting requirements for obtaining and working with select agents -better medical infrastructure and crisis management plans now in place: -in 2002, decision made to acquire enough smallpox vaccine for the entire U.S. population -currently more than 300 million doses of stockpiled FDA-approved vaccine, which was made using modern cell culture methods

glycopeptides

-inhibit transglycosylation and transpeptidation steps in peptidoglycan synthesis by binding D-ala-D-ala -gram-negative outer membrane; modify target (substitute D-Ala-D-lactate for D-Ala-D-Ala) -most effective against gram-positive bacteria -vancomycin, teichoplanin, daptomycin

Quorum "group" sensing

-population density based method of intercellular bacterial communication -small, diffusible signaling molecules "autoinducers" -acyl-homoserine lactones (AHLs; primarily gram negative) -autoinducing peptides (AIPs; primarily gram positive) - linear or cyclic peptides -acyl-homoserine lactone autoinducers (AI-1): V. fischeri/LuxI, P. aeruginosa/LasI, V. harveyi/LuxLM -oligopeptide autoinducers (AIP): B. subtilis/ComX, B. subtilis/CSF, S. aureus/subgroup 1 -usually produced under positive regulatory feedback control -can be used to sense population densities of "self" (within same species) as well as non-self (other species)

Bacterial QS systems - general overview

-primarily gram positive, intraspecies communication -AIPs are small, hydrophilic post-translationally modified peptides --> transported across cell membrane - bind to sensor molecule embedded in membrane and sticks out into the external environment -retained in extracellular environment -binding of AIP to membrane receptor protein (HK) --> phosphorelay to RR --> regulation of target genes -Two-component system (TCS) -sensor kinase: membrane-embedded protein with binding pocket for AIP (binds at critical concentration) - phosphorylates response regulator which targets genes to upregulate or downregulate target gene expression

Anthrax toxins EF and LF

-protective antigen required for binding host cell receptors - docks the toxin to the cell; also important for delivery of toxins (edema factor and lethal factor) into the host cell -cascade of proteolytic cleavage events - ends up making heptameric complex which binds up to 3 EF/LF subunits at one time; in this heptameric form it induces receptor-mediated endocytosis into the cell -initially localized in the endosome that forms -acidification of endosome results in transfer of LF/EF across membrane -lethal factor - Zn2+-dependent protease --> cleaves MAPKKs, lethality ensues -edema factor - adenylate cyclase - causes high level accumulation of cyclic AMP in the cell - leads to edema

CPS is a primary virulence factor

-resists opsonophagocytosis -low-level capsule production, increased net negative charge of certain serotypes, helps evade entrapment by mucus (mucopolysaccharides highly negatively charged due to sialic acid and other anionic residues) by electrostatic repulsion -invasive strains of S. pneumoniae are encapsulated; unencapsulated strains are infrequent, usually only associated with topical eye infection -**CPS production also required for efficient in vivo colonization; suggests that S. pneumoniae must strike a balance between CPS hindrance of biofilm formation and resistance to host defense -infection prevention by vaccination: -adults: vaccine consists of the 23 most common antigenic capsular types. Very safe, routinely given to the elderly (at most risk of contracting pneumonia), efficacy ~60%. This vaccine is useless in infants (lack T-cell independent responses to generate antibodies to longchain polysaccharides) -infants: a protein conjugated-capsule vaccine (7 most prevalent serotypes "PCV-7") was introduced in 2000. Covalently linking carbohydrates to protein segments can induce a major histocompatibility complex type II-mediated T-cell-dependent response to the carbohydrates, even in infants

P. aeruginosa pathogenesis in burn/chronic wounds

-seepage of plasma = rich nutrient source -PA is the leading cause of infection of burn wounds -bacteria leak into bloodstream --> systemic infection -extremely difficult to treat due to PA antibiotic resistance (biofilm growth, drug efflux pumps) -biofilms impair wound healing by stimulating chronic inflammation, leading to elevated levels of proteases and ROS that degrade proteins that are essential for healing -important virulence factors similar to those required for CF lung infection (biofilm formation, toxins, efflux pumps, etc.) -pyocyanin production thought to contribute to tissue necrosis ; blue pus fluoresces under long-wave UV light

developing resistance

-some antibiotic resistance trends arose quicker than others

mechanisms of tetracycline resistance

-tetracycline efflux pump (cytoplasmic membrane): intracellular tetracyline concentration remains too low; prevents binding to 16S rRNA -ribosome modification/protection: TetM GTPase activity perturbs helix in 16S region bound by tetracycline; prevents binding to 16S rRNA

multi-drug resistant pseudomonas aruginosa

-threat level: serious -6700 multidrug resistant pseudomonas infections -440 deaths -51000 pseudomonas infections per year -some strains of pseudomonas aeruginosa have been found to be resistant to nearly all or all antibiotics including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems -approx. 8% of all healthcare-associated infections reported to CDC's national healthcare safety network are caused by Pseudomonas aeruginosa -about 13% of severe healthcare-associated infections caused by Pseudomonas aeruginosa are multidrug resistant, meaning several classes of antibiotics no longer cure these infections

Pharmacokinetics (distribution of antimicrobial compound in host body)

-toxicity dictates external vs. internal use -absorption, dosage, distribution/dissemination, route of administration, stability

persister cell hypothesis (subpopulation of dormant/non-growing cells that survive antibiotic treatment)

-toxin-antitoxin modules, ppGpp induced in subpopulation --> inhibit macromolecular synthesis processes --> dormancy -an initial treatment with antibiotic kills planktonic cells and the majority of biofilm cells. The immune system kills planktonic persisters, but the biofilm persister cells are protected from host defenses by the exopolysaccharide matrix. After the antibiotic concentration drops, persisters resurrect the biofilm and the infection relapses

Mycobacterium tuberculosis

-tuberculosis (TB) -"acid-fast" bacillus (Ziehl-Neelsen stain of sputum) -identified from sputum culture -chest x-ray -TB skin test (indicates exposure; does not necessarily mean infection) - positive result indicated by red welt greater than 20 mm in diameter -aerosols from infected individuals (**efficient!) --> alveoli --> internalized by macrophage --> inhibition of lysosomal fusion --> SLOW replication -potential outcomes dictated by health status of infected individual -infecting microbes are killed/cleared -infecting microbes remain viable but controlled in granuloma for many years -growth, lung damage, dissemination and destruction of other organs

threats

-urgent: Clostridium difficile -serious: multidrug-resistant Acinetobacter, extended spectrum beta-lactamase producing enterobacteriaceae (ESBLs), vancomycin-resistant enterococcus (VRE), methicillin-resistant staphylococcus aureus (MRSA), drug-resistant tuberculosis -concerning: vancomycin-resistant staphylococcus aureus (VRSA)

Mechanism of vancomycin resistance

-vancomycin binds to the C-terminal acyl-D-Ala-D-Ala of the undecaprenol-diphosphate MurNAc-pentapeptide intermediate and inhibits transglycosylation and transpeptidation reactions in cell wall peptidoglycan polymerization and cross-linking -there have been 11 VRSA (vancomycin-resistant S. aureus) strains isolated in the USA; HGT of VanA/B, VanX from VRE via a transposon -gram negative bacteria naturally-resistant to vancomycin (bulky --> cannot diffuse through outer membrane porins) Resistant bacteria: 1. one enzyme (VanH) catalyzes conversion of pyruvate to D-lactate 2. second enzyme (VanA or VanB) leads to formation of D-Ala-D-lactate; vancomycin cannot bind D-Ala-D-Lactate 3. third enzyme (VanX) cleaves any D-Ala-D-Ala formed by usual pathway back to D-Ala, thus preventing incorporation of D-Ala-D-Ala into cell wall peptides

PA in CF

-virulence factors of note: -pyocyanin - blue-green pigment that generates ROS --> interferes with cilia action and phagocyte function -alginate capsule - adherence, resistance against phagocytosis, protection of biofilm, anti-desiccant -biofilm growth - variety of factors -quorum sensing -multi-drug efflux pump -variety of toxins (cytotoxicity, inflammation, antiphagocytic)

B. anthracis strains

-virulent strain (has capsule plasmid pX02 and toxin plasmid pX01 which encodes for PA, LF, EF) - cannot form protective vaccine (lethal) -avirulent strain: only has capsule plasmid pX02 - can't form protective vaccine (strains lacking either pX01 or pX02 display greatly reduced virulence) -avirulent strain: only has toxin plasmid pX01 - can form protective vaccine - attenuated live vaccine strain developed by Sterne in 1937 is still the basis of most anthrax vaccines for livestock; lacks pX02 and is therefore Cap-Tox+. protection related primarily to antibodies specific for the protective antigen component of the toxin -avirulent strain (Cap-/Tox-) = no virulence factors, does not form protective vaccine

"Top Four" Bioterror Agents

-yersinia pestis - "fear factor" (black death); requires flea as a vehicle for transmission; bacterium sensitive to antibiotics; was used as a bioweapon during WW2 -botulinum neurotoxin - introduction into water or food supplies; difficulty in initial diagnosis; relative ease in acquisition -smallpox - naive population; extremely contagious and lethal; has been used as a bioweapon in the past; ability to vaccinate exposed persons key in control of disease spread (disease prevented if vaccination occurs within 3-4 days post-exposure) -B. anthracis - spores are extremely stable outside the host; can be produced in large quantities; spores require refinement

symptoms, prognosis of B. anthracis

1. cutaneous (working with infected animals or products) - small blisters or bumps (may itch) --> painless skin sore (ulcer) with a black center (often on face, neck, arms, or hands) --> swelling --> dissemination and death (20%) if left untreated 2. inhalation (working with infected animals or products) - anthrax spores less than 5 micrometers in size are inhaled and reach the lower respiratory tract. Fever and chills, chest discomfort, shortness of breath, nausea, vomiting, or stomach pains, sweats (often drenching), extreme tiredness --> shock --> can be highly fatal 3. GI (consuming raw/undercooked meat from infected animals) - fever and chills, swelling of neck or neck glands, sore throat, painful swallowing, hoarseness, nausea and bloody vomiting, flushing (red face) and red eyes, stomach pain, swelling of abdomen (stomach) 4. injectional (heroin/drug use) - similar to cutaneous, but much more rapid disease progression

how antibiotic resistance happens

1. lots of germs. a few are drug resistant. 2. antibiotics kill bacteria causing the illness, as well as good bacteria protecting the body from infection. 3. the drug-resistant bacteria are now allowed to grow and take over. 4. some bacteira give their drug-resistance to other bacteria, causing more problems -antibiotic-resistant infections can happen anywhere -data show that most happen in the general community -however, most deaths related to antibiotic resistance happen in healthcare settings, such as hospitals and nursing homes

Bacillus anthracis

1. the bacteria produce spores that are dormant (not active) and can live in the environment, like soil, for a long time even decades --> High O2, low temp = sporulation 2. when spores get into the body of an animal or person (a place rich with water, sugars, and other nutrients), they can be "activated" and turn into active growing cells --> high CO2, high temp = germination 3. when they become active, the bacteria can multiply, spread out in the body, produce toxins (poisons) and cause severe illness and death --> high CO2, high temp = germination

P. aeruginosa (PA)

diseases caused: lung (CF patients), skin (burn wounds, chronic wounds), eye, bone, heart, UTI, meningitis -ubiquitous in soil and water, plants -some humans colonized -diagnosed by culturing on King's medium (blue and/or yellow pigment production) -motile, gram negative rod, capable of growth at 42 degrees C -a frequent cause of nosocomial infections! (environmental reservoirs in hospitals - potable water, taps, showers, disinfectants, ice makers, flower vases, medication, bath toys, etc.)

Opportunistic Pathogens 1: Pseudomonas aeruginosa

opportunistic pathogens -classical definition: microorganism that rarely causes infection in healthy people -requires impairment of host defense(s) to initiate infection -physical breach (burns, cuts, surgery) -immunocompromised (cancer, immunosuppressive drugs/Co-infection w/ AIDs, CF, etc.) -definitions can be blurry, there are exceptions... -community-acquired infections (MRSA, C. difficile) -strains with increased virulence properties (S. pyogenes "flesh-eating disease") -even in the case of "true" pathogens, there exists wide variation in host susceptibility (i.e. TB - asymptomatic infection) -our view of "pathogen" is evolving -hard and fast distinctions --> continuum with wide range of outcomes

antibiotic resistance part II: -specific examples of resistance mechanisms and their regulation, antibiotic tolerance vs. genetic resistance

overview of resistance mechanisms: -limiting access of the antibiotic (to the target in the cell) -active efflux of antibiotic (antiporters, energy-dependent ABC transporters) -enzymatic inactivation of the antibiotic -beta-lactamase (penicillin resistance), (chloramphenical/streptogramin acetyltransferases) -modification of protection of target -PBPs (S. aureus methicillin resistance), glycopeptide resistance (VRE), tetracycline resistance (widespread), macrolide resistance (widespread)