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Bacteriology Review Part I Alyson Yee 5/4/16 Hints • Lectures (slides, notes, recordings) are high yield • Work on understanding the basic biology of each bacterium and how that contributes to its pathogenesis • Practice questions on Chalk in with lectures • Think about each answer choice • Even if you get a question right, write out an explanation of WHY and/or try to alter the stem to make the statement true Things to know • About each bug: • Classification (G+/-, shape) • Clinical picture • What makes this bug unique? Think protein virulence factors or intracellular lifestyle, etc • What about the biology helps explain the associated disease? • About treatment/prevention: • Know if there are vaccines • Know major Abx and resistance mechanisms Basic Principles Prokaryotes vs. Eukaryotes • Bacteria: • Lack membrane bound organelles (including ER, nucleus) • Have cell walls (exception : MTb) • Have motility structures like flagella • Can undergo horizontal gene transfer • Can carry plasmids • Have smaller ribosomes Pathogens: Koch’s postulates • Organism is regularly found in the lesions of the disease (diseased, not healthy) • The organism can be isolated in pure culture on artificial media • Inoculation of this culture produces a similar disease in experimental animals • The organism can be recovered from the lesions in these reinfected animals When might Koch’s postulates not apply? • No animal model (ex. Syphilis) • Can’t be grown in culture • Opportunistic pathogens (found in healthy individuals but not disease-causing) Peptidoglycan • Backbone composed of GlcNAc and MurNAc repeats • Tetra or pentapeptide substituent on each repeat • Peptide bridges linking the carboxylic acid of D-alanine to an amino group of a neighboring chain. • Gram negatives use diaminopimelic acid for their NH2 group • Gram positives use either lysine or diaminopimelic acid Peptidoglycan biosynthesis 1. Water soluble nucleotide linked precursor in cytoplasm 2. Transfer from the nucleotide to a membrane lipid (C55 AKA bactoprenol AKA undecaprenol AKA lipid I) 3. Addition of this building block to the linear glycan chain (transglycosylation) 4. Cross-linking to an adjacent chain (transpeptidation) Staining and Cell Shapes Gram Stain • Crystal violet is washed out of thin peptidoglycan layer, cells only stain with safranin -> pink cells = gram negative • Crystal violet embeds deep into thick peptidoglycan, not fully washed out -> purple cells = gram positive • Some bacteria have a thick, waxy cell wall which is resistant to Gram staining • • Mycobacteria, Nocardia, Cryptosporidium Ziehl-Neelson stain: use heat and acid to drive lipid-soluble stain into cell wall Shapes •Spherical: coccus, cocci • Staphylococcus, streptococcus •Rod-shaped: bacillus, bacilli • Bacillus and Clostridium spp. •Comma shaped and spiral • Vibrios, spirilla and spirochetes Gram positive envelope Envelope structure of Gram-positive and Gram-negative bacteria Unique to Gram Positives Wall teichoic acid = attached to cell wall, give structure and resistance to antimicrobials Lipoteichoic acid = attached to lipid membrane, give division plane and anchor wall LPXTG motif proteins are anchored to the wall by sortase **Remember: Gram positive bacteria have a THICK cell wall Unique to Gram Negatives Lipopolysaccharide = LPS activates immune response Gram negative sepsis 3 components: 1. O Antigen = variable among different species, seen by immune system 2. Core= sugar, common to all Gram negatives 3. Lipid A= outer half of outer membrane bilayer, common to all Gram negatives Periplasm= Space b/w outer and inner membranes in Gram negatives. Contains many hydrolytic enzymes (βlactamases Braun’s lipoprotein: links the peptidoglycan (via diaminopimelic acid) to the outer membrane. Allows the firm attachment of the OM to the cell envelope **Remember: Gram negative bacteria have: 1) two membranes 2) a THIN cell wall 1) Which of the following statements concerning bacterial envelope structure is not correct? (A) Many Gram-positive bacteria elaborate both wall teichoic acid and lipoteichoic acid (B) Peptidoglycan is a constituent of the envelope of Gram-positive and Gram-negative bacteria (C) The outer membrane, an asymmetrical lipid bilayer containing lipopolysaccharide, is found in Gram-negative bacteria but not in Gram-positive bacteria (D) Lipoproteins, containing thioether linked diacylglyceride, are found in Gram-negative bacteria but not in Gram-positive bacteria (E) Flagella, an envelope embedded rotary filament conferring motility, are found in Gram-positive and in Gram-negative bacteria 1) Which of the following statements concerning bacterial envelope structure is not correct? (A) Many Gram-positive bacteria elaborate both wall teichoic acid and lipoteichoic acid (B) Peptidoglycan is a constituent of the envelope of Gram-positive and Gram-negative bacteria (C) The outer membrane, an asymmetrical lipid bilayer containing lipopolysaccharide, is found in Gram-negative bacteria but not in Gram-positive bacteria (D) Lipoproteins, containing thioether linked diacylglyceride, are found in Gram-negative bacteria but not in Gram-positive bacteria (E) Flagella, an envelope embedded rotary filament conferring motility, are found in Gram-positive and in Gram-negative bacteria 3) Which of the following statements concerning bacterial envelope assembly is not correct? A) Lipopolysaccharide synthesis begins in the bacterial cytoplasm/cytoplasmic membrane and generates the lipid X intermediate. B) Peptidoglycan synthesis requires undecaprenol (bactoprenol) as a lipid carrier C) Surface proteins of Gram-negative bacteria are inserted into the outer membrane, typically using a beta-barrel structure D) Sortases insert surface proteins into the lipid bilayer by forming a linkage with glycolipids. E) D-alanylation of wall teichoic acids proceeds via a lipoteichoic acid intermediate. 3) Which of the following statements concerning bacterial envelope assembly is not correct? A) Lipopolysaccharide synthesis begins in the bacterial cytoplasm/cytoplasmic membrane and generates the lipid X intermediate. B) Peptidoglycan synthesis requires undecaprenol (bactoprenol) as a lipid carrier C) Surface proteins of Gram-negative bacteria are inserted into the outer membrane, typically using a beta-barrel structure D) Sortases insert surface proteins into the lipid bilayer by forming a linkage with glycolipids. E) D-alanylation of wall teichoic acids proceeds via a lipoteichoic acid intermediate. Pattern recognition receptors (PRRs) sense pathogen associated microbial patterns (PAMPs) PRRs Location PAMP Adaptors Effectors Toll like receptors (TLRs) TLR1 Surface Triacylated lipopeptides MyD88 IL-6, TNF-α IL-6, TNF-α, IL-8, MCP-1, RANTES TLR2 Surface Di/triacylated lipopeptides MyD88, TIRAP TLR4 Surface LPS MyD88, TRIF, TIRAP, TRAM IL-6, TNF-α, IFNβ, IP-10 TLR5 Surface Flagellin MyD88 TNF-α TLR6 Surface Diacylated lipopeptides MyD88, TIRAP TNF-α, IL-6, IL-8, MCP-1, RANTES TLR11 Endosomes Profilin, flagellin MyD88 IL-12, TNF-α TLR12 Endosomes Profilin MyD88 IL-12p40, IFN-α TLR13 Endosomes 23s rRNA MyD88 IL-6, IL-12p40 IPS1 IFN-β Cytosolic DNA sensors cGAS Cytoplasm AT rich dsDNA STING Cytoplasm dsDNA, c-di-GMP, c-di-AMP STING IFN-β, IP-10, IL-6, IL-1β Cytosolic Nod factors and inflammasome Nod2 Cytoplasm muropeptides ASC IL-1β, IL-18 NAIP5/6 Cytoplasm flagellin ASC IL-1β, IL-18 NAIP2 Cytoplasm T3/4S substrates ASC IL-1β, IL-18 2) Which of the following statements concerning recognition of bacterial invaders by the mammalian immune system is correct? A) Nod signaling activates the inflammasome in mammalians cells and can respond to the lipopolysaccharide of Gram-negative bacteria. B) Toll like receptors are positioned in the cytoplasm of mammalian cells where they perceive lipoprotein and peptidoglycan signals from bacterial invaders C) cGAS is an enzyme that generates 2’-3’ cGAMP from DNA to activate STING-mediated immune signaling of microbial invaders D) Flagellin, a component of flagella, can only be recognized by the NAIP2/NLRC4 inflammasome of mammalian cells, which activates caspase 1 and IL-1beta production. E) TLR4 recognizes the 23S rRNA of bacterial invaders 2) Which of the following statements concerning recognition of bacterial invaders by the mammalian immune system is correct? A) Nod signaling activates the inflammasome in mammalians cells and can respond to the lipopolysaccharide of Gram-negative bacteria. B) Toll like receptors are positioned in the cytoplasm of mammalian cells where they perceive lipoprotein and peptidoglycan signals from bacterial invaders C) cGAS is an enzyme that generates 2’-3’ cGAMP from DNA to activate STING-mediated immune signaling of microbial invaders D) Flagellin, a component of flagella, can only be recognized by the NAIP2/NLRC4 inflammasome of mammalian cells, which activates caspase 1 and IL-1beta production. E) TLR4 recognizes the 23S rRNA of bacterial invaders Gram positive Cocci: Staphylococcus spp. Streptococcus spp. Pathogenic staphylococci Trait S. aureus S. epidermidis S. saprophyticus Many Infections / Intoxications Positive Opportunistic Infections Negative Urinary Tract Infections Negative Habitat Skin & Nares Skin Colonies Yellow (aureus) White (albus) Skin, Urinary Tract Often white Catalase Plus Plus Plus Protein A / Clumping F. Mannitol Plus Negative Negative Plus Negative Plus Novobiocin Negative Negative Plus Disease Coagulase Staphylococcus aureus • CLASSIFICATION: G+ cocci in clusters • CLINICAL: infections (skin, trauma-related, hematogenous, nosocomial) and toxinoses (food poisoning, exfoliative skin disease, Toxic shock) • IMPORTANT PROTEINS: exoproteins: hemolysins, hydrolases, exotoxins, surface protein A • OTHER KEY CONCEPTS: Forms abscesses (sequestered from immune system), Quorum sensing via agr locus • DIAGNOSIS: culture, coagulase test • TREATMENT: surgical drainage, Abx (vancomycin) • RESISTANCE: B-lactamases, PBP2a, transposon-based vancomycin resistance Staphylococcal abscesses enable persistence in host tissues S. aureus diseases • SSTIs: • 30%/30%/30% stably/intermittently/not colonized • >1 million per year, high recurrence • Carbuncle, furunculosis, impetigo • Invasive diseases: • Following trauma or other disease • Fasciitis, cellulitis, osteomyelitis • Hematogenous: • Leading cause of sepsis, lethal infectious disease • Endocarditis, abscesses, septic arthritis S. aureus toxinoses • Food poisoning • T cell superantigens • SSSS • Exfoliatin A and B • Toxic shock • tsst-1 or -2 S. aureus virulence factors (There are a TON but these are the most famous) Protein A Ag-binding Fc Coagulase Blocks Fc region from binding complement or receptors for phagocytosis, tricks B cells (super Ag) Prothrombin +++ Thrombin Fibrinogen Alpha-hemolysin Lysis Other surface proteins: IsdB: Fe acquisition FnBP: binding to mucosa Fibrin Protein A blocks opsonization and phagocytosis as well as the development of adaptive immunity S. aureus treatment Treating S. aureus is difficult because it’s so good at acquiring resistance. Vancomycin is used a lot because of MRSA, but VRSA appearing now too Penicillin resistance Methicillin resistance 1. Betalactam is sensed 2. Causes production of altered PBP (PBP2a) 1. Beta-lactam is sensed 2. Causes production of beta-lactamase 3. Beta-lactamase hydrolyzes beta-lactam 3. Methicillin can’t block PBP2a from building cell wall S. aureus and MRSA diagnosis and therapy • Surgical debridement/drainage of SSTI or deep seated abscess; removal of infected foreign material • Culture tissue or blood sample for S. aureus/MRSA isolation and antibiotic resistance analysis • Antibiotic treatment for S. aureus/MRSA begins without culture diagnosis of the bacterial infection • clindamycin (macrolide, inhibits protein synthesis) • vancomycin (glycopeptide, inhibits cell wall synthesis) • linezolid (oxazolidinones, inhibits protein synthesis) • daptomycin (lipopeptide, unknown mechanism) • trimethoprim-sulfomethazole (folic acid synthesis inhibition) • S. aureus persisters, small colony variants that escape antibiotic killing, occur frequently and cause recurrent disease while acquiring drugresistance. 4) Which of the following statements concerning immunity to S. aureus infection is not correct? A) Recurrent disease following antibiotic and surgical therapy of skin and soft tissue infections (SSTI) occurs frequently (≤9%) B) If left untreated, S. aureus infection culminates in an immunological crisis and in the development of immune responses that protect against subsequent infection. C) As clinical trials for candidate vaccines and antibody therapeutics have failed, an FDA licensed vaccine is currently not available. D) Individuals who are colonized with S. aureus are more likely to acquire S. aureus infections as compared to non-colonized individuals. E) Risk factors for S. aureus infection includes indwelling catheters, endotracheal intubation, medical implantation of foreign bodies, trauma, surgical procedures, hemodialysis, immunosuppressive therapy, diabetes, increased age and low birth weight. 4) Which of the following statements concerning immunity to S. aureus infection is not correct? A) Recurrent disease following antibiotic and surgical therapy of skin and soft tissue infections (SSTI) occurs frequently (≤9%) B) If left untreated, S. aureus infection culminates in an immunological crisis and in the development of immune responses that protect against subsequent infection. C) As clinical trials for candidate vaccines and antibody therapeutics have failed, an FDA licensed vaccine is currently not available. D) Individuals who are colonized with S. aureus are more likely to acquire S. aureus infections as compared to non-colonized individuals. E) Risk factors for S. aureus infection includes indwelling catheters, endotracheal intubation, medical implantation of foreign bodies, trauma, surgical procedures, hemodialysis, immunosuppressive therapy, diabetes, increased age and low birth weight. 5) Which of the following is not a suitable therapeutic for MRSA infections? A) oxacillin B) clindamycin C) daptomycin D) linezolid E) trimethoprim-sulfomethoxazole 5) Which of the following is not a suitable therapeutic for MRSA infections? A) oxacillin B) clindamycin C) daptomycin D) linezolid E) trimethoprim-sulfomethoxazole • 6) Which of the following statements concerning S. aureus virulence factors is correct? • A) Coagulase is a protease that cleaves fibrinogen to form a fibrin deposit. • B) α-hemolysin (Hla) cleaves phospholipids to destabilize host cell membranes including the membranes of red blood cells. • C) von Willebrand Factor binding protein associates with von Willebrand Factor to block the formation of a fibrin meshwork during S. aureus bloodstream infections. • D) Staphylococcal enterotoxins bind to the GSα subunit of intestinal epithelial cells to synthesize the second messenger cAMP. • E) Protein A crosslinks B cell receptors (VH3 clan IgM) and triggers proliferation of B lymphocytes. • 6) Which of the following statements concerning S. aureus virulence factors is correct? • A) Coagulase is a protease that cleaves fibrinogen to form a fibrin deposit. • B) α-hemolysin (Hla) cleaves phospholipids to destabilize host cell membranes including the membranes of red blood cells. • C) von Willebrand Factor binding protein associates with von Willebrand Factor to block the formation of a fibrin meshwork during S. aureus bloodstream infections. • D) Staphylococcal enterotoxins bind to the GSα subunit of intestinal epithelial cells to synthesize the second messenger cAMP. • E) Protein A crosslinks B cell receptors (VH3 clan IgM) and triggers proliferation of B lymphocytes. Other Staph (coagulase -) • Epidermidis • • • • • Opportunistic pathogen (nosocomial) Common cause of sepsis, NEC in neonates Highly resistant to antibiotics Colonize human skin Treatment: Vancomycin, remove catheter/valve/IV • Saprophyticus • UTI • Second (only to E. coli) leading cause of UTI in sexually active, young women • Treatment: Penicillin, TMP-SMX (TrimethoprimSulfomethoxazole) Streptococcus classification: Two ways Hemolysis: zone of clearance on blood agar • Alpha = green (reduced hemoglobin) : S. pneumoniae, Viridans group • Beta = clear : S. pyogenes, S. agalactiae • Gamma = no hemolysis: Viridans group • Enterococci can be alpha, beta or gamma Lancefield: cell wall Ccarbohydrate • Group A: S. pyogenes • Group B: S. agalactiae (Notice that these are both beta-hemolytic) **All are Gram positive cocci in chains, catalase negative** Streptococci and their human diseases GAS β-hemolytic S. pyogenes GBS GDS β-hemolytic β-hemolytic S. agalactiae E. faecalis Viridans S. α- or γ-hemol. S. pneumoniae α-hemolytic Pharyngitis Neonatal meningitis Endocarditis (subacute) Endocarditis (Subacute) Pneumonia Scarlet fever Puerperal fever UTI Otitis media Erysipelas, Pyoderma Nosocomial infections Sinusitis Pneumonia Peritonitis Bacteremia Sinusitis, Otitis Endocarditis (subacute) Toxinoses Meningitis Acute rheumatic fever* Glomerulonephritis* GAS = Streptococcus pyogenes • Virulence factors • M protein: critical determinant. Responsible for resisting phagocytosis. Immunity to infection is M type specific. • Hyaluronic acid capsule resembles hyaluronic acid in host (synovial fluid, joints) so hidden from immune system • Streptolysin O: pokes hole in mammalian cells (ASO is a lab to indicate strep infection) S. pyogenes diseases • Infections • Pharyngitis (strep throat) • *the differential for pharyngitis is enormous. Understand findings for and against strep. • Diagnosis: culture (gold standard), rapid antigen-detection test (>95% sensitivity) • Skin infections (impetigo, cellulitis) • Can progress to otitis media, abscesses, meningitis, pneumonia • Toxigenic • Scarlet fever: erythrogenic toxin (SpeA), centrifugal rash (trunk to extremities), spares palms and soles, strawberry tongue Erythrogenic toxin (SpeA) • Structural gene is located on bacteriophage (T12 or other) that lysogenizes GAS • Secreted SpeA binds to the T cell receptor and crosslinks with MHC class II receptor of antigen presenting cells • Triggers T cell proliferation and cytokine storm • Increases disease severity of GAS pharyngitis • Formerly used to assess susceptibility by intradermal injections with and without neutralizing antibody (Dick test) Post-infectious complications of S. pyogenes • Acute rheumatic fever (assoc w/ strep throat): anti-strep Ab attack heart proteins. High probability of RF if evidence of GAS infection and two major manifestations or one major and two minor. • Jones Criteria supporting evidence of antecedent GAS infection (positive throat culture or rapid streptococcal antigen test), elevated or rising streptococcal antibody titer: • Major Criteria • • • • • Carditis Polyarthritis Chorea Erythema Marginatum Subcutaneous nodules • Minor criteria: • Clinical findings: • • • Arthralgia Fever Elongated PR interval • Laboratory findings • Elevated acute phase reactants (ESR, CRP) • Acute glomerulonephritis (associated with skin infections): immune complexes deposit in glomerulus. Causes hematuria, hypertension Group B Strep: S. agalactiae • Causative agent of neonatal meningitis & sepsis • Give Penicillin/Amp to pregnant GBS + women • Diagnosis: latex agglutination with antibodies recognizing GBS group carbohydrate antigen or CAMP test • Classified by T pili serotypes CAMP+ GBS S. aureus (hlb+) Group D Strep: Enterococcus faecalis • Commensal of the human and animal gastrointestinal tract • Diseases: subacute bacterial endocarditis (SBE), UTIs, biliary tract infections, nosocomial infections in surgical, immunocompromised, cancer and transplant patients • Diagnosis: culture, antibody against group specific carbohydrate, PCR, antibiotic susceptibility testing • Most hospital E. faecalis isolates are HIGHLY resistant to antibiotics even ampicillin and vancomycin (aka: vancomycin-resistant enterococci (VRE)). • Treatment: ampicillin, vancomycin + aminoglycosides, linezolid, daptomycin Viridans Group Strep • This is a group. Not a species. • Found on teeth, cause caries • Prophylactic antibiotics (penicillin) for dental patients with hx of heart valve disease • Alpha hemolytic, optochin resistant Strep pneumoniae = Pneumococcus • No Lancefield group • -hemolysis due to pneumolysin, optochin sensitive • Pneumonia • Otitis media • Sinusitis • Bacteremia—found in 80% of meningitis (adults/children), can cause subacute endocarditis Symptoms & signs of pneumococcal pneumonia • Sudden onsets of chills, fever 40°C and malaise • Cough, tachypnea, dyspnea and chest pain • Bloody sputum • Auscultation & percussion – loss of vesicular sound (normal sound heard over the lung field), crackling, loss of resonance, egophony (increased resonance) • Day 5-10 of illness, crisis, sudden drop in fever and improvement or fatal infection (development of opsonizing antibodies) • Pathology: phases of red and white (or grey) hepatization. Polysaccharide Capsule in Strep pneumo • The single most important virulence factor • antiphagocytic properties • prevents complement deposition • more than 100 serotypes • Capsular “Opsonizing antibodies” "lysis" after "crisis" of pneumococcal pneumonia. • 25 μg of capsular polysaccharides = vaccine against S. pneumoniae Pneumovax/Prevnar • Vaccine has led to serotype replacement • 7) Which of the following statements concerning the Lancefield classification of streptococcal species is not correct? • A) Group A streptococci (GAS), also designated Streptococcus pyogenes, cause pharyngitis in humans and can be detected in throat swab samples with a quick test specific for the group A carbohydrate • B) Group B streptococci (GBS), also designated Streptococcus agalactiae, cause neonatal meningitis and can be detected in vaginal swab samples of pregnant women 35-37 weeks of gestation. • C) Group D streptococci, also designated Enterococcus faecalis, colonize the intestinal tract and are able to cause nosocomial infections in cancer patients. • D) Streptococcus pneumoniae is not subject to the Lancefield classification and its C-polysaccharide is detected with the C-reactive protein in human plasma. • E) Group (wall) carbohydrates are subject to antigenic variation, which is why new Lancefield groups of streptococcal species are uncovered frequently. • 7) Which of the following statements concerning the Lancefield classification of streptococcal species is not correct? • A) Group A streptococci (GAS), also designated Streptococcus pyogenes, cause pharyngitis in humans and can be detected in throat swab samples with a quick test specific for the group A carbohydrate • B) Group B streptococci (GBS), also designated Streptococcus agalactiae, cause neonatal meningitis and can be detected in vaginal swab samples of pregnant women 35-37 weeks of gestation. • C) Group D streptococci, also designated Enterococcus faecalis, colonize the intestinal tract and are able to cause nosocomial infections in cancer patients. • D) Streptococcus pneumoniae is not subject to the Lancefield classification and its C-polysaccharide is detected with the C-reactive protein in human plasma. • E) Group (wall) carbohydrates are subject to antigenic variation, which is why new Lancefield groups of streptococcal species are uncovered frequently. • 8) Which of the following statements concerning the acute rheumatic fever (ARF) is not correct? • A) Subcutaneous nodules and polyarthritis can be manifestations of ARF. • B) Fewer than 10,000 cases of ARF occur world-wide each year, which is why the development of a protective vaccine would not be economical. • C) Myocarditis with Aschoff body lesions and heart murmur can be a manifestation of ARF. • D) ARF is associated with GAS (S. pyogenes) pharyngitis. • E) Chorea Sydenham and Erythema marginatum can be manifestations of ARF • 8) Which of the following statements concerning the acute rheumatic fever (ARF) is not correct? • A) Subcutaneous nodules and polyarthritis can be manifestations of ARF. • B) Fewer than 10,000 cases of ARF occur world-wide each year, which is why the development of a protective vaccine would not be economical. • C) Myocarditis with Aschoff body lesions and heart murmur can be a manifestation of ARF. • D) ARF is associated with GAS (S. pyogenes) pharyngitis. • E) Chorea Sydenham and Erythema marginatum can be manifestations of ARF • 9) Which of the following statements concerning the prevention of streptococcal diseases is not correct? • A) Neonatal meningitis caused by GBS is prevented by vaccination of pregnant women with capsular conjugate vaccine. • B) Otitis media caused by Streptococcus pneumoniae is prevented by immunization with capsular conjugate vaccine. • C) The further development of acute rheumatic fever is prevented with penicillin prophylaxis. • D) Pneumococcal pneumonia is prevented with a 23-valent capsular polysaccharide vaccine without polysaccharide conjugation to protein. • E) Nosocomial infection with GDS (Enterococcus faecalis) cannot be prevented with vancomycin because of drug resistance genes. • 9) Which of the following statements concerning the prevention of streptococcal diseases is not correct? • A) Neonatal meningitis caused by GBS is prevented by vaccination of pregnant women with capsular conjugate vaccine. • B) Otitis media caused by Streptococcus pneumoniae is prevented by immunization with capsular conjugate vaccine. • C) The further development of acute rheumatic fever is prevented with penicillin prophylaxis. • D) Pneumococcal pneumonia is prevented with a 23-valent capsular polysaccharide vaccine without polysaccharide conjugation to protein. • E) Nosocomial infection with GDS (Enterococcus faecalis) cannot be prevented with vancomycin because of drug resistance genes. • 10) Which of the following statements concerning the pneumococcal conjugate vaccine (PCV13) is not correct? • A) PCV13 is comprised of 13 capsular polysaccharide types conjugated to protein adjuvant. • B) PCV13 immunization is recommended for all children (<5 yoa) and for adults (>65 yoa). • C) PCV13 immunization is recommended for high risk individuals (sickle cell anemia, CSF leak, cochlear implant, asplenia, malignancy). • D) PCV13 immunization in the United States has diminished the colonization and carrier state with S. pneumoniae. • E) PCV13 immunization in the United States has diminished pneumococcal disease caused by PCV13-serotype S. pneumoniae strains. • 10) Which of the following statements concerning the pneumococcal conjugate vaccine (PCV13) is not correct? • A) PCV13 is comprised of 13 capsular polysaccharide types conjugated to protein adjuvant. • B) PCV13 immunization is recommended for all children (<5 yoa) and for adults (>65 yoa). • C) PCV13 immunization is recommended for high risk individuals (sickle cell anemia, CSF leak, cochlear implant, asplenia, malignancy). • D) PCV13 immunization in the United States has diminished the colonization and carrier state with S. pneumoniae. • E) PCV13 immunization in the United States has diminished pneumococcal disease caused by PCV13-serotype S. pneumoniae strains. Rules of thumb for bacterial pathogens • If you want to invade a host - stick to it • Adherins: ex Vibrio cholerae • If you don’t want to get killed - bring your weapons and fight • Proteins that mess with the immune system, ex S. aureus superantigen Protein A • If you want to invade another host - get out if you can • Think about modes of transmission (contact, sexual, IV, insects, etc) • If you want to get all of it done - bring your proteins out • Secretion systems Genetic structure in bacteria • Most bacterial chromosomes are circular and doublestranded • Plasmids are also DNA that is circular and double-stranded but they are smaller than the chromosome. They often carry genes for resistance and virulence • Bacteria have little junk DNA – tight packed genes • Genes can be clumped together into a polycistronic operon, which means that they are all regulated and transcribed together. The proteins these genes encode usually work together Transformation • DNA is the “transforming principle” • Transformation = taking up DNA from the environment • Free DNA can be taken up homologous recombination with a section of chromosomal DNA • Plasmid DNA can be taken up and exist separately in the cell • Competent bacteria take up DNA naturally Transposons • These are jumping pieces of DNA. Sometimes when they jump, they take a bit of the surrounding chromosomal DNA, which is one mechanism of creating bacterial genetic diversity • They often carry antibiotic resistance genes and provide an easy way for lots of bacteria to pick up resistance Conjugation 1. One bacterium has F plasmid (F+). The other doesn’t (F-). F is for fertility 2. F+ cell extends sex pilus (encoded on F plasmid) to bring F- cell closer 3. F plasmid in F+ gets cut, and as it starts to replicate to fix the error, the extending strand projects into F-. 4. Complementary strands are synthesized and both are now F+ Hfr: the F plasmid can integrate into the chromosome. Then, the F plasmid starts conjugation as above. There is not enough time for all of the F plasmid and then the rest of the chromosome to be transferred before they get interrupted, so only the F plasmid contents and then whatever bits of chromosome follow are transferred. Phage • Virus that infects bacteria: made up of genetic material and protein coat • Often carry virulence or abx resistance genes (ex. cholera toxin, diptheria toxin) • The phage DNA incorporates into the bacterial chromosome • The phage monitors nutritional status of the bacterium • Bounty of food--> phage in lysogenic phase (doesn’t hurt bacterium, lets bacterium replicate phage DNA as the bacterium grows and divides) • Starvation--> lytic cycle- the virus assembles its protein coat, lyses the bacterial cell and goes out to find a new home Transduction • Transfer of bacterial DNA from one bacterium to another by way of a phage • Two forms (not specified in lecture) • “Generalized transduction” = when bacterium is infected with phage, some of its DNA gets chopped up and accidentally packaged as the phage capsid is forming • “Specialized transduction”: as the phage DNA that is incorporated in the chromosome comes out for the lytic cycle, some downstream genes from the chromosome get carried along too (analogous to Hfr conjugation) Adhesins • Pili/fimbriae • Membrane bound proteins These enable the bacteria to bind to an epithelium so that they can invade/not get washed away/etc. For your interest: Pili identified in B. cereus by Jon Budzik, a former MSTP in Olaf’s lab (Budzik et al 2007) – Now at UCSF for ID Toxins • Endotoxin: lipid A is toxic part of LPS. Innate immune system recognizes TNF and IL-1 are produced shock (fever, hypotension, systemic clotting) • Exotoxins: secreted molecules with specific effect on the host. These are the various toxins we’ve learned (ex. Clostridium toxin, shiga toxin, etc.) • Superantigens: an exotoxin that binds to the T cell receptor and MHC but NOT through the antigen-binding domain so that any T cell can be stimulated, regardless of its specificity. As a result, you have an over-activated immune system, which is eventually depleted Secretion systems (overview) • Secretion in Gram positives: simple—protein that needs to be exported has a signal that directs it to the membrane, it is transported across plasma membrane, and then either gets covalently linked to the cell wall or floats away • Secretion in Gram negatives: More complicated… Secretion Systems: Sec Pathway A peptide sequence at the front of the protein tells the protein to go to the Sec proteins in membrane The Sec proteins transport the protein through the membrane (How much of a barrier cell wall poses to these proteins seems to be a point of contention among microbiologists, but beyond the scope of this class…) Gram negative bacteria can use homologous systems to get proteins through the inner membrane. Secretion Systems: Autotransporters 4. The protein cleaves itself, separating the soluble part from the beta-barrel 3. The cross-hatched part folds into a beta barrel and inserts in membrane 2. Now protein precursor is in the periplasm and starts to fold. 1. Protein has signal peptide that sends it to Sec system like on last slide Secretion Systems: Type I secretion (ABC Transporter) Three proteins spanning the inner and outer membrane form a channel for secretion of target protein. One of those proteins (ATP-binding cassette) creates the energy to allow the transport in one step. Membrane-spanning channel Protein to be exported ATP-binding cassette Secretion Systems: Type II Secretion (aka “General Secretory pathway”) 2. Proteins in periplasm fold and assemble 1. Protein has signal peptide that sends it to Sec system 3. Secreted through outer membrane transport protein in a second step Secretion Systems: Type III Secretion (T3SS) Gram negative bacteria assemble a complex structure composed of many proteins that extend a needle into a target eukaryotic cell. “Effector” proteins of the bacteria then travel through the needle into the eukaryotic cell where they have pathogenic effects Ex. Chlamydia, Salmonella, Yersinia Secretion Systems: Type IV Secretion Modification of bacterial conjugation system. Ex. Legionella 2. Multimers assembled in periplasm 1. Protein has signal peptide that sends it to Sec system • 11) Which of the following statements concerning the secretion systems of Gram-negative bacteria is correct? • A) Signal-peptide bearing precursors are transported via the Sec pathway into the extracellular medium. • B) The type I secretion pathway transports proteins across the bacterial inner and outer membrane into the extracellular medium. • C) The type II secretion pathway transports proteins across the plasma membrane of host cells. • D) The type III secretion pathway secretes activated toxins, after proteolysis and sulfhydryl oxidation, into the extracellular medium. • E) The type IV secretion pathway occurs only in bacteria that are pathogenic to plants. • 11) Which of the following statements concerning the secretion systems of Gram-negative bacteria is correct? • A) Signal-peptide bearing precursors are transported via the Sec pathway into the extracellular medium. • B) The type I secretion pathway transports proteins across the bacterial inner and outer membrane into the extracellular medium. • C) The type II secretion pathway transports proteins across the plasma membrane of host cells. • D) The type III secretion pathway secretes activated toxins, after proteolysis and sulfhydryl oxidation, into the extracellular medium. • E) The type IV secretion pathway occurs only in bacteria that are pathogenic to plants. Iron Acquisition • Bacteria need iron. We hide most of our Fe inside hemoglobin (Hb) inside RBCs • Bacteria have various methods to get Fe: • Siderophores: Fe chelators (not a protein) that bind free Fe3+ and then get transported back into bacteria • Hemolysins: toxins lyse RBCs to release the Hb • Hemophores: proteins that bind Hb, bind bacterial receptor, and get taken up into the bacterium, thereby acquiring Fe • Host fights back with siderocalins (bind siderophore and prevents Fe acquisition) • 12) Which of the following statements concerning iron restriction/nutritional immunity is not correct? • A) Under physiological conditions, iron is sequestered in human tissues and is not available to bacterial invaders. • B) Lysis of red blood cells triggers haptoglobin- and hemopexin-mediated sequestration of hemoglobin and heme, respectively. • C) Lipocalins are host proteins that sequester bacterial siderophore:iron complexes. • D) Hemochromatosis, whether of adult or juvenile disease onset, perturbs the nutritional immunity of human tissues. • E) Intracellular or endosomal iron is freely available to bacterial pathogens as long as they can enter the host cell. • 12) Which of the following statements concerning iron restriction/nutritional immunity is not correct? • A) Under physiological conditions, iron is sequestered in human tissues and is not available to bacterial invaders. • B) Lysis of red blood cells triggers haptoglobin- and hemopexin-mediated sequestration of hemoglobin and heme, respectively. • C) Lipocalins are host proteins that sequester bacterial siderophore:iron complexes. • D) Hemochromatosis, whether of adult or juvenile disease onset, perturbs the nutritional immunity of human tissues. • E) Intracellular or endosomal iron is freely available to bacterial pathogens as long as they can enter the host cell. Other pathogenic elements • Flagella: complex protein structures for motility • Capsule: generally a polysaccharide coating that protects bacterium from phagocytosis • Exception: Bacillus anthracis (D-glutamic acid coat) The human microbiome Hypotheses on how to perturb the interactions between microbiota, host and pathogen Respiratory Infections Gram + • Corynebacterium diphtheriae Gram • Pseudomonas aeruginosa • Klebsiella • Legionella • Bordetella pertussis • Haemophilus Gram + Non-spore forming rod Corynebacterium diphtheriae • This would still cause disease if we didn’t vaccinate everyone--it hasn’t gone away! • Diphtheria toxin: AB toxin that ADP-ribosylates EF-2 so proteins aren’t made, encoded on a phage • Causes croup: pharyngitis & laryngitis with greyishwhite pseudomembrane, swollen neck, respiratory stridor, asphyxia, mildly elevated temperature 100.4°F, cardiomyopathy • Toxin also affects cardiac conduction, nerves • Culture: Loeffler’s medium, potassium tellurite • Treatment: diphtheria antitoxin (DAT), abx, Vaccine: diphtheria toxoid (inactivated toxin) Diphtheria (tetanus and pertussis) vaccine Emil von Behring Balto & Gunnar Kaasen of the Iditarod race • Behring used heat inactivated diphtheria toxin to immunize guinea pigs & protect against C. diphtheriae; also immunized humans against diphtheria and used diphtheria-immune serum for therapy (1901 Nobel Prize) • DT=diphtheria & tetanus toxoid, no pertussis vaccine • Td= tetanus & diphtheria vaccine for adults (booster immunization following exposure) • DTaP= DT & acellular pertussis vaccine • Children should get five doses of DTaP: 2, 4, 6, 12-15 months and 4-6 years • Tdap= Td & acellular pertussis vaccine • Tdap for 7-10 yo and for women should receive Tdap during each pregnancy • 13) Which of the following statements concerning the pathogenesis of diphtheria is not correct? • A) Diphtheria is typically an upper respiratory tract infection caused by toxigenic C. diphtheriae. • B) Diphtheria is rare and in the past three years not a single case has been reported in the United States. • C) Because of low disease incidence, CDC no longer recommends diphtheria immunization for Americans. • D) Individuals who suffered from diphtheria acquire life-long immunity against this disease. • E) Without therapy, the mortality of diphtheria is high (>15%) and caused by suffocation (asphyxia) and/or cardiomyopathy. • 13) Which of the following statements concerning the pathogenesis of diphtheria is not correct? • A) Diphtheria is typically an upper respiratory tract infection caused by toxigenic C. diphtheriae. • B) Diphtheria is rare and in the past three years not a single case has been reported in the United States. • C) Because of low disease incidence, CDC no longer recommends diphtheria immunization for Americans. • D) Individuals who suffered from diphtheria acquire life-long immunity against this disease. • E) Without therapy, the mortality of diphtheria is high (>15%) and caused by suffocation (asphyxia) and/or cardiomyopathy. Bordetella pertussis http://www.buddycom.com/bacteria/gnr/acinetc1259.jpg Lab: Aerobic, Bordet-Gengou (potato) agar, requires nicotinamide, slow growing *Most bacteria* Complications: 2° infection, damage from intrathoracic pressure Whole cell vaccine: better immunity, worse reactogenicity (only used in adults under special circumstances for control of hospital outbreaks. ) Acelluar vaccine: several components, including PT. Immunity lasts ~12 years --> we’re susceptible! Rx: erythromycin or other macrolides Pertussis cases must be reported through the National Notifiable Diseases Surveillance System B. Pertussis pathogenesis Highly contagious, airborne, infects respiratory tract Survivors acquire immunity Whooping cough can be caused by B. pertussis or B. parapertussis (lacks PT) Toxins: Pertussis Toxin: ADP-ribosylating AB5 toxin modifies α-subunit of heterotrimeric Gi proteins, increases cAMP synthesis and suppressing pro-inflammatory cytokine production ACT: increases cAMP synthesis and suppresses all kinds of immune response FHA/Fim/Prn: Filamentous haemagglutinin (FHA) and Fim proteins (fimbriae) are involved in adhesion, required for persistence. Prn (pertactin) may help resist neutrophil clearance Pertussis treatment and prevention • Erythromycin or trimethoprim-sulfamethoxazole for pertussis cases and as a post-exposure prophylaxis (new CDC recommendation) • Antibiotic therapy has little effect at the time of diagnosis (clinical pertussis disease): the patient is already intoxicated! It prevents dissemination. • Supportive therapy and pertussis immune globin (PT specific antibodies) • Surveillance for apnea and vomiting • whole cell vaccine safety was controversial and no longer recommended by CDC • acellular pertussis (subunit) vaccine recommended by CDC • 14) Which of the following has a proven therapeutic effect for infants with manifest pertussis disease (paroxysmal phase)? • A) intravenous pertussis immune globin • B) trimethoprim-sulfamethoxazole • C) erythromycin • D) acellular subunit vaccine • E) whole cell pertussis vaccine • 14) Which of the following has a proven therapeutic effect for infants with manifest pertussis disease (paroxysmal phase)? • A) intravenous pertussis immune globin • B) trimethoprim-sulfamethoxazole • C) erythromycin • D) acellular subunit vaccine • E) whole cell pertussis vaccine Legionella • This is the air conditioning bug • Difficult to Gram stain, grow on BCYE buffered charcoal yeast extract (rapid detection methods available for Dx) • Pathogenesis: • Adherence (Properly working cilia in host respiratory tree are key to prevention) • Prevent phagolysosomal fusion • Multiplies inside monocytes and then causes rupture • Diseases: • Pontiac fever: flu-like, no pneumonia, self-limited • Legionnaire’s Disease: Pneumonia • Rx: antibiotics that get intracellular (not beta-lactams): think azithromycin • No human-to-human transmission Ventilator associated pneumonia (VAP) • Critically ill patients in the ICUs of hospitals that are unable to sustain breathing by themselves and receive mechanical ventilation • Rate of infection increases with the duration of mechanical ventilation (>5 days) • VAP: new chest infiltrate on chest X-ray, body temperature >100.5°F, blood leukocytosis (>109 ml-1) , purulent sputum, hypoxemia, positive culture [endotracheal aspirate or BAL, 2 blood cultures (25% sensitivity], CPIS=clinical pulmonary infection score • ~300,000 cases in the US per year; 5-10 per 1,000 hospital admissions • Case fatality 15-50% Ventilator associated pneumonia (VAP) • Pseudomonas aeruoginosa (20-40%), S. aureus/ MRSA (15-30%), S. pneumoniae, K. pneumoniae, S. marcescens, Enterobacter, Citrobacter, A. baumannii, S. maltophila • Treatment is initiated before culture diagnosis is established as mortality for VAP is as high as 50% • Pick antibiotics from three lists (A-C) to cover (A) drug-resistant P. aeruginosa, (B) drug-resistant enterobacteria, and (C) MRSA • List A: cephalosporin (Cefepime, Ceftazidime), carbapenem (Imipenem, Meropenem), β-lactam/ β-lactamase inhibitor (Piperacillin-tazobactam) • List B: fluoroquinolone (Ciprofloxacin), aminoglycoside (Amikacin, Gentamycin, Tobramycin) • List C: Vancomycin or Linezolid Pseudomonas aeruginosa • Most common cause of VAP • Forms biofilms: expolysacharides promote biofilms and protect against phagocytic clearance • Type III secretion system • Causes chronic lung infections in cystic fibrosis patients • 15) Which of the following statements concerning the diagnosis and therapy of ventilator associated pneumonia (VAP) is correct? • A) High incidence of VAP infection with drug-resistant bacteria mandates that antibiotic therapy commence only after culture diagnosis (and antibiotic-resistance spectrum) has been established. • B) Streptococcus pneumoniae is a frequent cause of VAP. • C) Risk for VAP is correlated with the duration of assisted ventilation, with a sharp increase in the incidence of VAP after 5 days. • D) A single broad spectrum antibiotic can be used as an early therapeutic against the most frequent causes of VAP. • E) Mortality associated with VAP is less than that caused by Legionella pneumophila, the agent of Legionnaires disease. • 15) Which of the following statements concerning the diagnosis and therapy of ventilator associated pneumonia (VAP) is correct? • A) High incidence of VAP infection with drug-resistant bacteria mandates that antibiotic therapy commence only after culture diagnosis (and antibiotic-resistance spectrum) has been established. • B) Streptococcus pneumoniae is a frequent cause of VAP. • C) Risk for VAP is correlated with the duration of assisted ventilation, with a sharp increase in the incidence of VAP after 5 days. • D) A single broad spectrum antibiotic can be used as an early therapeutic against the most frequent causes of VAP. • E) Mortality associated with VAP is less than that caused by Legionella pneumophila, the agent of Legionnaires disease. Causes of bacterial meningitis in the United States (1998-2007) Microbe Age group Preventive US cases Case vaccine per year fatality S. pneumoniae Adolescents, young adults yes 58% 13% S. agalactiae (GBS) Newborns no 18% 15% N. meningitidis Adolescents, young adults yes 14% 15% H. influenzae Infants yes 7% 6% L. monocytogenes Newborns, immune comp. no 3% 21% E. coli Newborns no <1% NA C. fetus Immunecompromised no <1% NA All causes NA NA 4,100 500 (15%) • N. gonorrhea (no capsule, local) • N. meningitidis (capsule, disseminates) • LOS (short LPS) looks like self sphingolipid • Antigenic variation Neisseria Gram negative cocci • Pili are turned on and off by recombination • Opas are on/off by polymerase slippage Species Phenotype Sequence N. gonorrhoeae opa (CTCTT)n N. meningitis capsule (siaD) (C )n N. meningitis Capsule (siaA) IS1301 N. gonorrhoeae pili conserved repeats Neisseria meningitidis • G- diplococcus • Produces polysaccharide capsule (serotypes A, B, C, W, Y) • Spread thru respiratory droplets/close quarters (soldiers, college students) • Enters epithelium from damage or in phagocytes (unencapsulated) • Disseminates thru blood (encapsulated) Brain and crosses vascular epithelium into meninges and CSF • Colonizes the nasopharynx of approximately 10% of the human population (carrier state) • Can invade healthy individuals to establish bacteremia and meningitis; cases are typically sporadic (95%) • Outbreaks (5%) occur in close communities over a short period of time and are caused by the same isolate. • Colonization involves pili and Opc/Opa proteins, invasion requires capsule and factor H binding protein (fHBP), molecules that are antigenically variable • Meningitis is a severe disease with high case fatality (up to 15%) and long term disabilities (retardation, loss of limb) in survivors (15-30%) Symptoms and signs of meningococcal disease • Headache, fever, nuchal rigidity, obtunded sensorium • Rash, petechia and/or ecchymosis • Shock • Neurological signs, vanishing reflexes, coma • Disseminated intravascular coagulation, spontaneous bleeding, necrosis of extremities • Rapid progression toward lethal outcome CSF: neutrophils with Gram-negative diplococci Meningitis • Nuchal rigidity • Make pt try to put their chin on their chest • Fever, headache, confusion • Lumbar puncture • If it’s infected it’s cloudy • If it’s not infected it’s clear • Gram stain/Giemsa stain the CSF • PCR tests to distinguish bacteria • Meningococcus often also produces skin petechiae, may progress to DIC, destruction of adrenals (Waterhouse-Friderichsen) • Therapy = IV or IM penicillin • Vaccine = polysaccharide conjugated to diphtheria toxin, doesn’t hit type B (33% of cases) Meningococcal vaccines Quadrivalent capsule vaccine (A, C, W135, Y) • MCV4 (Menactra® and Menveo® conjugate-vaccine: Emil Gotschlich 11-18 & >55yoa (prime booster) +>9 month yoa (high risk individuals) Group B meningococcal vaccine (10-25 yoa; factor H binding protein (fHbp) as key ingredient • The group B capsule is NOT immunogenic Haemophilus influenzae • G- coccobacillus, colonizes most people • Auxotroph for heme and NAD, grows on supplemented chocolate agar with CO2; catalase and oxidase positive • Causative agent of pediatric diseases: otitis media, sinusitis, pneumonia, meningitis, epiglottitis • Therapy: for severe cases i.v. cefotaxime or ceftriaxone; oral ampicillin & sulbactam (β-lactamase inhibitor) or ciprofloxacine • Prevention: poly-ribosyl-ribitol-conjugated to tetanustoxoid= Hib vaccine (3 injections first six months, 1 year booster) Listeriosis caused by Listeria monocytogenes • G+ rod • Foodborne pathogen that can cross the intestinal mucosa, the blood brain barrier and the placental barrier • Causes meningitis, meningoencephalitis, septicemia, abortions and gastroenteritis • Usually not a problem in adults • Can be a problem in pregnancy and neonatal meningitis • Prevention: Food safety is the key prevention of L. monocytogenes infections; in pregnant women, monitoring of fetal growth; no vaccine available • Therapy: ampicillin; alternatively trimethoprim-sulfamethoxazole or erythromycin Listeria monocytogenes pathogenesis • 16) Which of the following statements concerning the causative agents of bacterial meningitis is not correct? • A) Listeria monocytogenesis, a food-born pathogen, can infect the placenta of pregnant women and cause fetal meningitis with high mortality. • B) Hib immunization has reduced the incidence of infant meningitis caused by Haemophilus influenza. • C) PCV13 immunization has reduced the incidence of pneumococcal meningitis caused by PCV13 vaccine serotypes. • D) Antibiotic prophylaxis has reduced the incidence of early onset bacterial meningitis caused by Group B streptococci (Streptococcus agalactiae). • E) E. coli is a frequent cause of neonatal meningitis. • 16) Which of the following statements concerning the causative agents of bacterial meningitis is not correct? • A) Listeria monocytogenesis, a food-born pathogen, can infect the placenta of pregnant women and cause fetal meningitis with high mortality. • B) Hib immunization has reduced the incidence of infant meningitis caused by Haemophilus influenza. • C) PCV13 immunization has reduced the incidence of pneumococcal meningitis caused by PCV13 vaccine serotypes. • D) Antibiotic prophylaxis has reduced the incidence of early onset bacterial meningitis caused by Group B streptococci (Streptococcus agalactiae). • E) E. coli is a frequent cause of neonatal meningitis. • 17) Which of the following statements concerning the prevention of meningococcal meningitis is not correct? • A) MCV4 (Menactra® or Menveo®), quadrivalent capsule conjugate vaccines (serotypes A, C, W135, Y), are recommended for 11-18 yoa and >55yoa Americans as prime-booster vaccines. • B) MCV4 vaccines cannot protect against meningitis caused by N. meningitidis serotype B. • C) N. meningitidis serotype B capsular polysaccharide [(α2-8) Nacetylneuraminic acid] is not immunogenic. • D) Protective immunity against N. meningitidis requires antibodies that promote phagocytic uptake, not complement mediated killing of bacteria. • E) Group B meningococcal vaccines (Trumenba® and Bexsero®) include factor H binding protein (fHbp) as a key ingredient. • 17) Which of the following statements concerning the prevention of meningococcal meningitis is not correct? • A) MCV4 (Menactra® or Menveo®), quadrivalent capsule conjugate vaccines (serotypes A, C, W135, Y), are recommended for 11-18 yoa and >55yoa Americans as prime-booster vaccines. • B) MCV4 vaccines cannot protect against meningitis caused by N. meningitidis serotype B. • C) N. meningitidis serotype B capsular polysaccharide [(α2-8) Nacetylneuraminic acid] is not immunogenic. • D) Protective immunity against N. meningitidis requires antibodies that promote phagocytic uptake, not complement mediated killing of bacteria. • E) Group B meningococcal vaccines (Trumenba® and Bexsero®) include factor H binding protein (fHbp) as a key ingredient. STDs • Chlamydia trachomatis (intracellular pathogen) • N. gonorrhoaea • Treponema pallidum (spirochete) • Haemophilus ducreyi Haemophilus ducreyi – chancroid STD that is difficult to diagnose • • • • In men, chancroid on the penis Usually asymptomatic in women Incubation period 5-7 days Tender papular lesion with erythema that disintegrates into a non-indurated ulceration (differential diagnosis: syphilis) with lymphadenopathy • Swab culture to establish culture (difficult) or PCR diagnosis • Treatment: oral azithromycin or erthyromycin Neisseria gonorrheae (gonorrhea) frequent, notifiable STD complicated by antibiotic resistance • G- diplocci • Transmitted by sexual contact or perinatally; primarily affects the mucous membranes of the lower genital tract and less frequently those of the rectum, oropharynx, and conjunctivae • Due to Abx resistance, dual therapy (ceftriaxone and either azithromycin or doxycycline) • In women: cervicitis, urethritis, accessory gland infection, PID, perihepatitis, infertility, tubular pregnancy • Extragenital: • • • • Anorectal infection Pharyngeal infection Conjunctivitis Disseminated infection Diagnosis and therapy of gonorrhea • Culture diagnosis through isolation of N. gonorrheae on laboratory media (chocolate agar with hemin and antibiotics incubated with CO2 for 2 days); oxidase + and biochemical tests; allows antibiotic susceptibility testing • Methylene blue smear of secretions for microscopic detection of diplococci and neutrophils (picture) • NAATs (nucleic acid amplification tests): PCR detection of N. gonorrheae; great for first urine and vaginal swabs, for rectal, oropharyngeal and conjunctival testing; useful for detection of Chlamydia trachomatis and DD of mixed infections • Treatment: ceftriaxone & azithromycin or ceftriaxone & doxycycline • 18) Which of the following statements concerning the diagnosis and treatment of gonorrhea is not correct? • A) Cefixime is recommended by CDC as a monotherapy for gonorrhea. • B) Culture isolation of Neisseria gonorrhea enables antibiotic sensitivity testing. • C) Gonorrhea is a diagnosis that must be reported to CDC. • D) NAATs (nucleic acid amplification tests) allow detection of N. gonorrheae in urine and vaginal, rectal, oropharyngeal as well as conjunctival swabs. • E) NAATs are useful for the detection of mixed infections with N. gonorrheae and Chlamydia trachomatis. • 18) Which of the following statements concerning the diagnosis and treatment of gonorrhea is not correct? • A) Cefixime is recommended by CDC as a monotherapy for gonorrhea. • B) Culture isolation of Neisseria gonorrhea enables antibiotic sensitivity testing. • C) Gonorrhea is a diagnosis that must be reported to CDC. • D) NAATs (nucleic acid amplification tests) allow detection of N. gonorrheae in urine and vaginal, rectal, oropharyngeal as well as conjunctival swabs. • E) NAATs are useful for the detection of mixed infections with N. gonorrheae and Chlamydia trachomatis.