Pseudomonas aeruginosa biofilm formation

Pseudomonas aeruginosa expolysacharides promote biofilms and protect against phagocytic clearance

Pseudomonas aeruginosa pneumonia pathogenesis requires its type III secretion system PcrV (T3S needle cap), ExoT & ExoS (Rho GAP, ADP-ribosyltransferase), ExoY (adenylyl cyclase) and ExoU (phospholipase)

Pseudomonas aeruginosa type III effectors and pneumonia pathogenesis

Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis patients (>5 yoa)

Meningitis & Veneral Diseases

Causes of bacterial meningitis in the United States (1998-2007) Microbe Age group Preventive vaccine US cases per year Case fatality S. pneumoniae Adolescents, young adults yes 58% 13% S. agalactiae (GBS) Newborns no 18% 15% N. meningitidis 14% H. influenzae Infants 7% 6% L. monocytogenes Newborns, immune comp. 3% 21% E. coli <1% NA C. fetus Immune- compromised All causes 4,100 500 (15%)

Analysis of cerebrospinal fluid aids in the differential diagnosis of meningitis Cells/ml Cell Type Protein (g/L) Glucose (mmol/L) Bacterial 200-20,000 neutrophils 1-10 <2.5 Aseptic 100-1,000 lymphocytes 0.5-1.0 3.3 M.tb 100-500 1-2 <2.8 Fungal 10-200 0.5-2 <2.2

Neisseria meningitidis (Meningococcus) Gram-negative diplococcus, capsulating with serotypes: A, B, C, W135, Y and X (very rare) Colonizes the nasopharynx of approximately 10% of the human population (carrier state) Spreads by respiratory or throat secretions (spit, kissing), and spread is promoted by changes in housing and close quarters (soldiers, college students) 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%)

Capsular serotypes of Neisseria meningitidis

Stages in the pathogenesis of N. meningitidis The capsule and lacto-N-neotetraose (LNT) of lipopolysaccharide (LPS) are spontaneously phase variable in Neisseria meningitidis at high frequencies (10-3–10-4 per generation). During colonization, a non-encapsulated, non-sialylated phenotype is frequently present. The loss of capsulation and of sialic acid on LPS may help to establish long-term nasopharyngeal carriage by becoming intracellular, thereby avoiding host complement and phagocytic defenses. Dissemination from the site of colonization capsulation, as non-encapsulated bacteria cannot survive in the blood.

Stages in the pathogenesis of N. meningitidis Neisseria meningitidis may be acquired through the inhalation of respiratory droplets. The organism establishes intimate contact with non-ciliated mucosal epithelial cells of the upper respiratory tract, where it may enter the cells briefly before migrating back to the apical surfaces of the cells for transmission to a new host. Asymptomatic carriage is common in healthy adults in which bacteria that enter the body by crossing the epithelial barrier are eliminated. Besides transcytosis, N. meningitidis can cross the epithelium either directly following damage to the monolayer integrity or through phagocytes in a 'Trojan horse' manner. In susceptible individuals, once inside the blood, N. meningitidis may survive, multiply rapidly and disseminate throughout the body and the brain. Meningococcal passage across the brain vascular endothelium (or the epithelium of the choroid plexus) may then occur, resulting in infection of the meninges and the cerebrospinal fluid.

Envelope components of N. meningitidis that interact with host cells a | Pili traverse the capsule and are the most prominent adhesins of encapsulated Neisseria meningitidis. In addition, the integral outer membrane (OM) adhesins, Opa and Opc, are known to mediate interactions with specific host-cell receptors in appropriate phenotypes. Lipopolysaccharide may interfere with the adhesion functions of OM proteins, but can also contribute to cellular interactions by interacting with various cellular receptors. The OM of N. gonorrhoeae differs in two important aspects: gonococci are non-encapsulated and Opc expression has not been shown at the protein level. b | A cross-section of a pilus fibre showing that variable domains (V) and glycans (G) as well as other substitutions (not shown) are located externally, whereas the constant domains are buried within the fibre, protected from the host environment.

Invasion mechanisms of N. meningitidis Opc protein a | Colony opacities of Opc phase variants of Neisseria meningitidis. Colonies of N. meningitidis expressing Opc (or Opa) generally appear opaque when viewed using oblique substage lighting, whereas Opc-deficient bacteria give rise to transparent colonies. b | Opc interacts with serum factors to target integrins that recognize an Arg–Gly–Asp motif, and this results in efficient invasion of host cells, as can be seen in the scanning electron microscopy (EM) image of a fractured endothelial cell with a large number of intracellular N. meningitidis (indicated by the arrow). c | This also leads to transcytosis of N. meningitidis, as shown by the transmission EM image, in which a cross-section of a human endothelial cell with invading N. meningitidis (indicated by arrows) emerging from its basolateral surface is depicted.

CEACAM1 as a receptor of Neisseria meningitidis Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is the main receptor targeted by Neisseria meningitidis Opa proteins. It belongs to the immunoglobulin (Ig) superfamily and contains an amino-terminal Ig variable (IgV)-like domain that is targeted by N. meningitidis Opa, Haemophilus influenzae P5 and Moraxella catarrhalis UspA1 adhesins. rD-7 binding to human epithelial cells that express high levels of CEACAM1 in vitro prevents host-cell invasion of N. meningitidisexpressing Opa and pili that is mediated by the Opa–CEACAM1 pathway (b), but does not abrogate the binding of N. meningitidis through pili as depicted in c.

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, spontaneus bleeding, necrosis of extremities Rapid progression toward lethal outcome CSF: neutrophils with Gram-negative diplococci

Subcutaneous purpura (ecchymosis) Coma & necrosis of limbs Ocular petechiae Subcutaneous purpura (ecchymosis) Loss of limb Coma & necrosis of limbs

Neisseria meningitidis Diagnosis and treatment CSF sediment microscopy: Gram-negative diplococci (cocci bacilli=H. influenzae, Gram-positive diplococci=S. pneumoniae), protein, glucose and immune cell counts for differential diagnosis CSF and blood culture to isolate causative agent, antimicrobial sensitivity testing and MLST typing CSF & blood sample PCR tests – distinguish N. meningitidis, S. pneumoniae, H. influenzae and identify their serotypes Waterhouse-Friderichsen Syndrome: necrosis of adrenal glands, adrenal insufficiency, shock, DIC, multiorgan failure – symptomatic therapy Immediately intramuscular ceftriaxone or cefotaxime, then iv administration; rifampin (or ciprofloxacin or ceftriaxone) for close contacts

Meningococcal meningitis Prevention through vaccination Quadrivalent capsule vaccine (A, C, W135, Y) MCV4 (Menactra® and Menveo® conjugate-vaccine: 11-18 & >55yoa (prime booster) +>9 month yoa (high risk individuals) MPSV4 (Menomune® polysaccharide vaccine): >55 yoa Group B meningococcal vaccine (10-25 yoa; factor H binding protein (fHbp) as key ingredient Pfizer’s Trumenba® (three injections 0, 2 and 6 months with two fHbp (A &B); coverage may not include all group B isolates. Novartis’ (GSK) Bexsero ® (two injections 0 & 2 months with outer membrane vesicles + fHbp, NhbA, NadA); used at Princeton University and UC St. Barbara outbreaks Emil Gotschlich

Factor H binding to fHBP on the surface of Neisseria meningitidis Prevents complement-mediated killing of meningococci; likely target of vaccine escape variants fH binding on fHBP. Shown is the cocrystal structure of human fH67 bound to B24 fHBP. fH67 is colored in orange, while fHBP is colored by domain: green, N-terminal domain; blue, C-terminal domain; pink, linker between the β-structures of two domains. (B) Neighbor-joining tree of 569 fHBP variants from Neisseria isolates. The tree was generated in ClustalW with 500 bootstraps and drawn using MEGA 5.05. Subfamily designations are defined by Murphy et al. and Masignani et al. (20, 29). Subfamily A (variants 2 and 3) can be further subdivided into 4 major groupings based on the N- and C-terminal domains of fHBP (N1C1, N2C2, N1C2, and N2C1), and subfamily B can be divided into 3 groupings (N4, N5, and N6). Pajon et al. have made similar groupings based on five variable segments, dividing all fHBP variants into 9 modular groups (31). The six major modular groups are indicated by the roman numerals (the minor groups VIII and IX, each represented by a single variant, are not labeled on the tree). Not shown are several hybrid sequences that are composed of both subfamily A and subfamily B regions. The bar indicates genetic distance.

Population genetics and epidemiology of Neisseria meningitidis

Haemophilus influenzae Gram-negative coccobacillus, facultative anaerobe colonizing the human nasopharynx (75% of the human population) Auxotroph for heme and NAD, grows on supplemented chocolate agar with CO2; catalase and oxidase positive Encapsulated (a, b, c, d, e, f serotypes) and non-typable (non-encapsulated) H. influenzae 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 tetanus-toxoid= Hib vaccine (3 injections first six months, 1 year booster)

Immune response to capsular polysaccharide with and without conjugation to protein a | Polysaccharides from the encapsulated bacteria that cause disease in early childhood stimulate B cells by cross-linking the B-cell receptor (BCR) and drive the production of immunoglobulins. This process results in a lack of production of new memory B cells and a depletion of the memory B-cell pool, such that subsequent immune responses are decreased. b | The carrier protein from protein–polysaccharide conjugate vaccines is processed by the polysaccharide-specific B cell, and peptides are presented to carrier-peptide-specific T cells, resulting in T-cell help for the production of both plasma cells and memory B cells. CD40L, CD40 ligand; TCR, T-cell receptor.

Decline of childhood H. influenza type b disease in the United States following vaccination

H. influenza epiglottitis

Listeriosis caused by Listeria monocytogenes Gram-positive, motile rod colonizing the gastrointestinal tract of 1% of the human population Foodborne pathogen crossing the intestinal mucosa, the blood brain barrier and the placental barrier Causes meningitis, meningoencephalitis, septicemia, abortions and gastroenteritis In adults (approximately 550 cases per year), infection occurs predominantly in immune compromised individuals During pregnancy and in neonates (75 cases per year), early onset (granulomatosis infantiseptica) and late onset 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 Listeria monocytogenes, a facultative intracellular pathogen, enters host cells, lysis the vacuole, replicates and uses actin-based motility to spread from cell to cell. In red are the Listeria factors responsible for specific steps in its life-cycle.

Haemophilus ducreyi – chancroid STD that is difficult to diagnose In men, ulcerative lesion (painful!) on the penis; in 2010, 24 cases in the US Sexually transmitted diseases; only 10% of infected women are symptomatic Incubation period 5-7 days Tender papular lesion with erythema that disintegrates into a non-indurated ulceration (DD 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 Gram-negative diplococcus that infects humans to cause gonorrhea; in 2012: 334,826 cases (107.5/100,000) in the US Rates highest among young women (15-19y, 519; 20-24y, 578.5) and men (20-24y, 462.8) 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 In the US, N. gonorrheae developed broad antibiotic resistance including fluoroquinolon and cefixime (cephalosporin). Dual therapy with ceftriaxone and either azithromycin or doxycycline is now the only CDC recommended treatment

Gonorrhea in the United States

Stages in the pathogenesis of N. gonorrhoeae Neisseria gonorrhoeae is acquired through sexual contact and establishes infection in the urogenital tracts by interacting with non-ciliated epithelial cells; this results in cellular invasion. Although different molecular mechanisms are involved during the establishment of gonococci on the mucosal surfaces of males and females, infection often leads to inflammation and polymorphonuclear leukocyte (PMN) influx. However, infection of the lower female genital tract is typically asymptomatic. N. gonorrhoeae engulfed by PMN are secreted in PMN-rich exudate. Both tumour necrosis factor (TNF) from phagocytes and gonococcal products, such as peptidoglycan and lipopolysaccharide (LPS), also cause toxic damage to ciliated epithelial cells of mucosal surfaces. ECM, extracellular matrix.

Gonococcal urethritis in male patients and, less frequently, epididymitis

Recombinant conversion of the pilE locus as a mechanism of antigenic variation

Chromosomal organization of pilin genes in Neisseria gonorrhoea Chromosomal organization of pilin genes in Neisseria gonorrhoeae. A representative map of pilE and pilS loci arranged according to chromosomal position. Colored shapes indicate pilE and pilS gene copies, with the pointed end showing direction of translation. The coding sequence of pilS copies ranges from 300 to 400 bp in length, and the pilE coding region is 500 bp in length. The small black arrow in front of pilE indicates a functional promoter. The black oval represents the conserved Sma/Cla repeat. ori is the predicted origin of replication, experimentally determined for FA1090 (185). Numbers indicate chromosomal position in kilobase pairs. (a) Position and orientation of pilin loci with respect to the replication origin in strain MS11. (b) Position of pilin loci in strain FA1090. The presence and orientation are similar between the strains, except FA1090 does not have pilS5 but does have a pilS3 locus, more silent copies within pilS2, and only one upstream silent copy (uss). (c) Graphic representation of silent copies within each locus and the relative distances between them for strain MS11. 

Gonorrhea in women Asymptomatic with cervicitis, urethritis Cervicitis with abnormal vaginal discharge, inter-menstrual bleeding, dysuria, lower abdominal pain or dyspareunia (painful intercourse) Accessory gland infections (Bartholin’s or Skene’s gland) Pelvic Inflammatory Disease (PID): asymptomatic, lower abdominal pain, discharge, dyspareunia, irregular menstrual bleeding and fever Fitz-Hugh-Curtis Syndrome (perihepatitis) Infertility Tubular pregnancy Fitz-Hugh-Curtis Syndrome

Neisseria gonorrhoeae, cervicitis and inflammation of the bartholini’s gland

Neisseria gonorrhoeae, cutaneous lesions following urethritis and conjuctivits

Neisseria gonorrhoeae, ophthalmitis in a newborn

Extragenital gonorrhea in men and women Anorectal infection acquired by anal intercourse Usually asymptomatic Symptoms: anal irritation, painful defecation, constipation, scant rectal bleeding, painless mucopurulent discharge, tenesmus, and anal pruritus Signs: mucosa may appear normal, or purulent discharge, erythema, or easily induced bleeding may be observed with anoscopic exam Pharyngeal infection May be sole site of infection if oral-genital contact is the only exposure Most often asymptomatic, but symptoms, if present, may include pharyngitis, tonsillitis, fever, and cervical adenitis

Extragenital gonorrhea in men and women (continued) Conjunctivitis Usually a result of autoinoculation in adults Symptoms/signs: eye irritation with purulent conjunctival exudate Disseminated gonococcal infection (DGI) Systemic gonococcal infection Occurs infrequently. More common in women than in men Associated with a gonococcal strain that produces bacteremia without associated urogenital symptoms Clinical manifestations: skin lesions, arthralgias, tenosynovitis, arthritis, hepatitis, myocarditis, endocarditis,and meningitis

Invasion and transcytosis of gonococci in urinary or endometrial epithelia

Phase-variable genes with polymeric sequences in the genome of Neisseria Types of phase variation. The blue arrow represents the coding region of the gene in the direction of translation. The small black arrow is the promoter. Orange rectangles represent a single nucleotide repeat, and yellow rectangles represent a more complex repeat structure. Wavy blue lines represent mRNA transcript. (a) Homopolymeric repeats. The addition or deletion of a single nucleotide changes the reading frame of the gene. In this example, a normally translated gene is shifted out of frame and truncates early. (b) Short tandem repeats. The addition or deletion of a short DNA tract changes the reading frame of the gene if the number of nucleotides is not a multiple of three. In this example, the gene is out of frame. Deletion of one pentanucleotide repeat shifts the reading frame but not to the correct one, whereas addition of the repeat restores the proper frame. (c) Homopolymeric repeats in the promoter region alter the distance between the −10 and −35 polymerase recognition sites, affecting the degree of binding. In this example, changes to the promoter length alter transcription levels. Addition of one nucleotide causes more efficient transcription, whereas removal of one nucleotide leads to loss of transcription, denoted by X. Abbreviation: CDS, coding sequence.

Gonorrhea and chlamydial urethritis

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 Earlier tests (DFA, EIA, hybridization, serology) are no longer recommended. Treatment: ceftriaxone & azithromycin or ceftriaxone & doxycycline