Topic+31+32+-+Microbiology+-+Gram+Negative+Bacteria+1+AND+Gram+Negative+Bacteria+II.pdf

Gram Negative Bacterial Pathogens Vineet K. Singh, Ph.D. Department of Microbiology/Immunology ATSU/Missouri School of Dentistry and Oral Health  Bacteria G...

Gram Negative Bacterial Pathogens Vineet K. Singh, Ph.D. Department of Microbiology/Immunology ATSU/Missouri School of Dentistry and Oral Health  Bacteria Gram (+)ve Gram (-)ve Cocci Rods Rods Cocci Aerobic Anaerobic Cells in pairs Clusters Pairs/chains Catalase (+)ve Catalase (-)ve Neisseria, Moraxella Bacillus (spores), Clostridium (spores) Corynebacterium Actinomyces Staphylococcus Streptococcus Listeria (branching) Aerobic Anaerobic Coagulase (+)ve S. aureus Bacteroides Fusobacterium (-)ve S. epidermidis S. saprophyticus Simple growth Fastidious requirement growth S. pyogenes (A-S) b S. agalactiae (A-R) Hemolysis Haemophilus (X & V factors) S. pneumoniae (O-S) Legionella Bordetella a Campylobacter (Seagull Viridans’ streps (O-R) shape, 43°C) Enterococci Non- Lactose fermenters Lactose non-fermenters Others Bile resistant hemolytic Escherichia Salmonella, Shigella Pseudomonas [Oxidase (+)ve] Klebsiella (Mucoid) Proteus [Urease (+)ve] Vibrio (comma shape) Mycobacterium (acid fast) Organisms that Treponema, Leptospira, Borrelia stain poorly or not Mycoplasma (no cell wall) at all with Gram’s Rickettsia, Coxiella, Chlamydia stain (intracellular bacteria)  Bacteria Gram (-)ve Rods Cocci Cells in pairs Neisseria, Moraxella Aerobic Anaerobic Bacteroides Fusobacterium Simple growth Fastidious requirement growth Haemophilus (X & V factors) Legionella Bordetella Campylobacter (Seagull shape, 43°C) Lactose fermenters Lactose non-fermenters Others Escherichia Salmonella, Shigella Pseudomonas [Oxidase (+)ve] Klebsiella (Mucoid) Proteus [Urease (+)ve] Vibrio (comma shape) Organisms that Treponema, Leptospira, Borrelia stain poorly or not Mycoplasma (no cell wall) at all with Gram’s Rickettsia, Coxiella, Chlamydia stain (intracellular bacteria) Gram Negative Cocci Neisseria Neisseria meningitidis Neisseria gonorrhoeae Neisseria meningitidis (meningococcus)  Gram (-)ve, capsulated, fastidious, oxidase (+)ve.  Humans the only known host, colonizes nasopharynx.  Transmitted by respiratory droplets.  Prevalence rate 1-40%; highest in school aged children.  Disease highest in 80% UTIs.  Also enteric fever, meningitis and food poisoning.  Common Enterobacteriaceae  Escherichia coli  Shigella  Proteus  Salmonella  Yersinia  Klebsiella  All are oxidase negative and all ferment glucose.  Some ferment lactose (important in differentiation).  Enterobacteriaceae  Facultative anaerobic rods and some ferment lactose.  Lactose - fermenters: Escherichia, Klebsiella  Non-fermenters: Proteus, Salmonella, Shigella, Yersinia.  All have LPS, some are motile and some are not (SKY). Virulence of Enterobacteriaceae Capsule (K-antigen):Antiphagocytic Flagella (H-antigen): Role in motility and adherence Pili (F-antigen): Help in adherence & genetic transfer Antigenic variation: K&H antigens, evasion of host defense  Enterobacteriaceae species Growth on MacConkey Agar:  Bile salts & crystal violet inhibit growth of Gram positive bacteria.  Lactose is a fermentable sugar for differentiation.  Neutral dye is red at pH 6.8. Lactose-fermenters Lactose-non-fermenters E. coli; K. pneumoniae Salmonella; Shigella; Proteus Escherichia coli  Escherichia coli  Gram (-)ve, many serotypes based on O, H, F and K antigens.  Can result in 50-100K serotypes in various combinations.  Normal intestinal flora, may also be in lower urethra & vagina.  Pili (CFAI, CFAII, CFAIII) associated with GI infections.  P fimbriae (pili) - associated with urinary tract infections.  E. coli - Diseases #1 cause of urinary tract Infection (UTI):  Dysuria, frequency, suprapubic pain.  Fever & back pain progression to kidneys (pyelonephritis). Neonatal meningitis (K1 capsule associated):  2nd leading cause in neonates.  E. coli Gastroenteritis Caused by 5 groups of strains: 1. Enterotoxigenic (ETEC) 2. Enteropathogenic (EPEC) affects small intestine 3. Enteroaggregative (EAEC) 4. Shiga toxin producing (STEC) affects large intestine 5. Enteroinvasive (EIEC) E. coli  E. coli Gastroenteritis 1. Enterotoxigenic E. coli (ETEC)  ~220 M cases (~75 million cases in children 18K in children below 5)  ~80K in US travelers (Traveler’s diarrhea).  From fecally contaminated food/water, produce cholera-like toxins.  In 1-2 d - watery diarrhea, cramps, vomiting. Persists for 3-4 days.  E. coli Gastroenteritis 2. Shiga Toxin producing E. coli (STEC)  Enterohemorrhagic E. coli (EHEC) is a subset of STEC.  From undercooked meat, vegetables, unpasteurized milk, fruits.  Most common in developed countries.  265K illnesses, >3.6K hospitalizations, 30 deaths each year in USA.  >80 serotypes identified (O157:H7 most common in the US).  Strains produce shiga toxin that disrupts protein synthesis.  E. coli Gastroenteritis 2. Shiga Toxin producing E. coli (STEC)  ~100 bacteria can produce disease (person to person).  Destruction of intestinal villus (A/E lesions) causes fluid secretion.  Mild to bloody diarrhea, severe abdominal pain.  In ~8% - may lead to HUS and kidney failure (3-5% with HUS die). Identification of E. coli MacConkey agar Oxidase negative and lactose fermenting motile rod  Salmonella  2 species [S. enterica (clinically more significant) & S. bongori].  >2,500 serovars of S. enterica.  Serotypes - incorrectly named as species (S. typhimurium, S. typhi).  Salmonella enterica serovar Typhimurium or Salmonella Typhimurium.  Salmonella diseases 1. Salmonellosis (gastroenteritis) – most salmonella 2. Enteric fever (typhoid fever) – Salmonella Typhi Gastroenteritis (Salmonellosis)  ~1.35 m salmonellosis, 26,500 hospitalizations, 420 deaths/y in USA.  More common in children (60yr).  Acquired from poultry & dairy products and undercooked meat.  6-48 h incubation, resolves in 2d-1wk.  Symptoms: fever, vomiting, watery (non-bloody) diarrhea, cramps.  Usually self limiting, antibiotic treatment is not recommended.  Typhoidal Salmonella – Enteric Fever  Mainly cause by Salmonella Typhi - a strict human pathogen.  9m. global enteric fever cases annually (110K deaths), ~350 in US.  Low inoculum - Initially a bacteremia (fever) then intestinal ulceration.  Consider in febrile patients with recent travel to endemic areas.  Typhoidal Salmonella – Enteric Fever  S. Typhi initially invades intestinal epithelial cells.  Disseminates throughout the body in Mf via the lymphatics.  Colonize the reticuloendothelial tissues (liver, spleen, bone marrow).  Re-infect Peyer's patches (PP) via gall bladder.  Re-exposure of GI tract leads to intestinal ulcers & necrosis.  Other symptoms: slow heart rate, rose spots, enlarged liver & spleens. Salmonella Typhi - Enteric Fever Rose spots  Identification of Salmonella Culture on SS Agar:  Contain lactose, bile salt (selects enteric rod), ferric citrate, neutral red.  Brilliant green, ox bile, high thiosulfate and citrate inhibit the normal flora.  H2S production detected by using thiosulfate & iron - colonies turn black.  Shigella  A Gram negative rod, resistant to gastric acid.  4 species (dysenteriae; boydii; flexneri; sonnei).  Actually four E. coli biotypes - still called Shigella.  Shigella  >450k shigellosis/y in US; (150m worldwide).  Primarily pediatric disease (~70% in children 90% of Proteus infections.  Colonize human genitourinary tract.  Significant cause of urinary tract infections.  Shows swarming motility in culture, a lactose non-fermenter.  Infect kidneys more commonly than E. coli from urinary tract.  Gram Negative Anaerobic Rods  Bacteroides, Fusobacterium,Tannerella, Aggregatibacter, Porphyromonas & Prevotella These genera are obligate anaerobic, short Gram-negative rods or coccobacilli.  Bacteroides fragilis Characteristics: Small pleomorphic anaerobic Gram negative rod. Epidemiology: predominant in the intestine (1011 cells/g of feces). Virulence: LPS & proteases. Clinical infections if it escapes from GIT. Diseases: Commonly intra-abdominal abscess type infections.  Fusobacterium nucleatum Characteristics: Small pleomorphic anaerobic Gram negative rod. Epidemiology: Indigenous to the human oral cavity. Virulence: Not well known. Spreads from oral cavities. Diseases: Periodontal plaques & frequent in polymicrobial infections.  Tannerella forsythia Nonmotile, pleomorphic, spindle-shaped Gram (-)ve anaerobic rods.  in supragingival & subgingival sites (more common in the latter). Degree of isolation directly correlated to increasing pocket depth. T. forsythia, Treponema denticola and Porphyromonas gingivalis 3 red complex bacteria - always associated with periodontal disease. Aggregatibacter actinomycetemcomitans Often found in association with localized aggressive periodontitis. Also suspected to be involved in chronic periodontitis.  Prevotella Predominant site for these species is the human oral cavity. Prevotella intermedia is associated more with periodontal disease. These organisms are classified as belonging to the orange complex. Associated with the developmental stages of periodontal disease. Precede the arrival of the red complex group of bacteria.  A 24-yo, sexually-active woman experienced dysuria & urgency. Urine culture on MacConkey produced fermenting growth. Agent? 1. Enterococcus faecalis 2. Escherichia coli 3. Proteus mirabilis 4. Salomonella Typhi 5. Staphylococcus saprophyticus A 24-year-old, sexually-active woman experienced dysuria. Urine culture yielded Gram positive and catalase positive coccus. Agent? 1. Enterococcus faecalis 2. Escherichia coli 3. Klebsiella pneumoniae 4. Proteus mirabilis 5. Staphylococcus saprophyticus Bacterium that causes bloody stool? 1. Enterococcus faecalis 2. Enterotoxigenic Escherichia coli 3. Shigella dysenteriae 4. Salmonella Typhimuriam 5. Proteus vulgaris The patient presents with fever, GI symptoms and rose spots on the abdomen. Agent? 1. Enterococcus faecalis 2. Escherichia coli 3. Shigella dysenteriae 4. Salmonella Typhi 5. Yersinia enterocolitis Likely to cause periodontitis? 1. Eschericihia coli 2. Fusobacterium nucleatum 3. Proteus vulgaris 4. Pseudomonas aeruginosa 5. Salmonella Typhimuriam Likely to cause periodontitis? 1. Aggregatibacter actinomycetemcomitans 2. Eschericihia coli 3. Pseudomonas aeruginosa 4. Salmonella Typhimuriam 5. Proteus vulgaris Likely to cause periodontitis? 1. Eschericihia coli 2. Pseudomonas aeruginosa 3. Salmonella Typhimuriam 4. Proteus vulgaris 5. Tannerella forsythia Likely to cause periodontitis? 1. Eschericihia coli 2. Prevotella intermedia 3. Pseudomonas aeruginosa 4. Salmonella Typhimuriam 5. Proteus vulgaris Curved Gram Negative Rods ⚫ Pseudomonas ⚫ Vibrio ⚫ Helicobacter ⚫ Campylobacter  Pseudomonas aeruginosa  Small, aerobic, motile, pigmented Gram negative rod.  Can grow at 4-42˚C, non-fastidious (even in tap water).  Oxidase positive (distinct from Enterobacteriaceae).  Some are mucoid because of a polysaccharide capsule.  #1 colonizers of cystic fibrosis patients.  Pseudomonas aeruginosa  Ubiquitous (soil, water, vegetation), moist hospital locations.  But uncommon to be the part of the normal flora.  Transient URT & GIT colonization of hospitalized patients.  Patient to patient spread in nosocomial situations.  ~51K healthcare-associated infections each year in US hospitals.  >6,000 due to MDR strains with about 440 deaths per year.  P. aeruginosa - Virulence Virulence Factors Biological Roles STRUCTURAL COMPONENTS Capsule Adhesin, anti-phagocytic, inhibits antibiotic killing. Pili Adhesin LPS Endotoxin activity Pyocyanin Impairs ciliary fn, stimulates inflammation & production of ROI TOXINS & ENZYMES Exotoxin A Inhibits protein synthesis, tissue damage, immunosuppressive. Exotoxin S/T Inhibits protein synthesis, immunosuppressive. Cytotoxin Cytotoxic for host membrane (disrupts leukocyte function). Elastase (LasA & LasB) Degrades tissue (blood cells, lung, skin), collagen, IgGs, complement. Alkaline protease Destruction of tissues, inactivation of INF and TNF-a. Phospholipase C Heat labile hemolysin, mediates tissue damage and inflammation. Rhamnolipid Heat-stable Hly, disrupts lecithin in tissues, inhibits ciliary activity. Antibiotic resistance Complicates therapy. No other GNB produces these many toxins & other virulence factors.  P. aeruginosa pneumonia  Common cause of ventilation associated pneumonia.  Tracheobronchitis to necrotizing bronchopneumonia.  High fever, chills and purulent sputum, high mortality. P. aeruginosa - Diseases Bacteremia:  >50% mortality, more common in ICU patients.  High mortality – most are immuno-compromised.  May cause ecthyma gangrenosum (pathognomonic). Small or large painful maculopapular lesions. Initially pink purple black and necrotic. P. aeruginosa - Ecthyma gangrenosum Early lesions Late lesions P. aeruginosa - Eye infections  Infection after trauma to cornea (e.g., abrasion from contact lens).  Acute irritation, rapid corneal perforation, may cause vision loss.  P. aeruginosa - Ear infections Otitis externa (swimmer's ear) ⚫ #1 cause of outer ear infections. ⚫ Mild irritation of external ear to destruction of cranial bones. ⚫ Itching, pain (may be severe), mucopurulent exudate.  Other P. aeruginosa infections ⚫ Hot tub folliculitis and other skin lesions. ⚫ Acute right sided infective endocarditis among drug users. ⚫ Osteomyelitis after puncture wound through gym shoes. ⚫ UTI in hospitalized patients with urinary catheters.  P. aeruginosa - Identification Positive oxidase helps discriminate from Enterobacteriaceae. 1 2 3 1. Lactose non-fermenter on MacConkey agar. 2. Sweet grape like odor & a green color on nutrient agar. 3. Gram negative slightly curved aerobic rod; polar flagella.  Vibrio cholerae  A highly motile, curved, oxidase positive, Gram(-)ve rod.  Acquired from water contaminated with human feces.  Acid sensitive, high ID50, no person-to-person transmission.  >200 LPS serotypes, O1 & O139 make cholera toxin.  Cholera cases and deaths vary from year to year. Vibrio cholerae - Virulence  Cholera toxin (an AB5 toxin).  Toxin binds GM1, endocytosed & travels to ER.  Through ER degradasome, A1 reaches the cytosol.  A1 ADP-ribosylates Gs, stimulates adenylate cyclase (AC).  AC raises cAMP leading to activation of PKA.  PKA phosphorylates the major chloride channel, CFTR. Cholera toxin mode of action  Cholera Rice-water stool  In 1-3d - severe diarrhea (10-20 l/d), no fever.  Metabolic acidosis, hypokalemia, hypovolemic shock.  Muscle cramp due to electrolyte disturbances.  Rice water stool because of flecks of mucus.  If fluid & electrolyte not replaced quickly – shock & death.  Campylobacter  Microaerophilic and motile, grows better at 42˚C.  From contaminated poultry/other meat, water, milk.  Person-to-person – a possibility, but mostly from dairy & meat.  1.5 million illnesses each year in the United States  #1 bacterial gastroenteritis in U.S (>Salmonella + Shigella).  C. jejuni - Gastroenteritis  Subclinical in developing nations, immunity from prior infection.  Incubation period 2-4d, impacts jejunum, ileum and colon.  Prodrome of fever, headache, myalgia and malaise in 12-48h.  Followed by foul smelling diarrhea, abdominal pain and fever.  Symptoms from several loose stools to grossly bloody diarrhea.  Usually self limited, however symptoms may last for a weak.  Helicobacter pylori  Spiral Gram negative rod with 4-6 polar flagella.  Stomach colonization by the ability to adhere to the mucosa.  Produces urease, increases the juxta-mucosal pH and survive.  Colonizes interface of gastric epithelium & overlying mucous layer.  Helicobacter pylori  A human pathogen, clustering in families suggest human-to-human.  Spread by close personal contact (fecal-oral & oral-oral).  ~90% colonized in developing nations by age 10.  About 2/3rd in the world infected (more in developing countries).  Low in the US in healthy but increases with age (>50% over 60 yr).  Once infection is established in the host’s stomach – lasts for life.  H. pylori - Virulence urea H. pylori penetrates mucus layer, adheres to gastric mucosa. Urease neutralizes the gastric acid - H. pylori proliferates.  H. pylori – Diseases  Mostly a chronic gastritis - no clinically significant disease.  ~10-20% may present with gastric and duodenal ulcers.  2-6-fold higher risk of gastric cancer and MALT lymphoma. H. pylori – Diseases Symptoms:  Burning epigastric pain  Discomfort in the upper abdomen  Bloating  Feeling full after eating a small meal  Lack of appetite  Nausea or vomiting  Dark or tar-colored stools  H. pylori - Identification Urea breath test:  Patient drinks a solution of 13C/14C-urea.  Air breathed out from patients shows digestion of urea. A curved bacillus was isolated from a patient with rice-water like stool. Pathogen? 1. Campylobacter jejuni 2. Escherichia coli 3. Helicobacter pylori 4. Pseudomonas aeruginosa 5. Vibrio cholerae #1 cause of gastroenteritis in the US? 1. Campylobacter jejuni 2. Helicobacter pylori 3. Salmonella Typhimurium 4. Shigella flexineri 5. Vibrio vulnificus Pathogen associated with gastric ulcer and a positive urea breath test? 1. Campylobacter jejuni 2. Helicobacter pylori 3. Pseudomonas aeruginosa 4. Salmonella Typhimurium 5. Vibrio cholerae Cystic fibrosis patients are frequently colonized with? 1. Campylobacter jejuni 2. Helicobacter pylori 3. Pseudomonas aeruginosa 4. Vibrio cholerae 5. Staphylococcus epidermidis Fastidious Gram Negative Rods Haemophilus HACEK group Bordetella Legionella Bacteria with complex or particular nutritional requirements and they grow only when specific nutrients are provided.  Haemophilus influenzae Gram negative, fastidious coccobacilli (pleomorphic). Human are the only hosts for H. influenzae. Colonizers of nasopharynx; airborne transmission.  Haemophilus influenzae Mostly non-capsulated (non-typeable strains). ~5% are encapsulated; 6 capsular serotypes (a-f); Serotype b is responsible for invasive diseases. Non-typeable strains colonize URT of ~70% healthy adults.  H. influenzae – invasive diseases A preventable disease caused by H. influenzae serotype ‘b’. Rare in the US, Hib vaccination reduces morbidity/mortality. H. influenzae – Invasive diseases Meningitis: In unimmunized children – spread from nasopharynx. Initially a mild URT disease - then fever & systemic signs. High level bacteremia – direct gram stain of blood or CSF. Mortality is currently 63K reported cases in 2022 (true number ~300,000).  White footed mouse are the primary reservoirs.  Transmitted to humans by the nymph of the Ixodes tick.  The tick must attach 36-48 h to transmit the spirochete. B. burgdorferi - Lyme Disease  Spirochete present in low numbers in the skin lesion.  Present at the border regions of erythema migrans.  Rarely seen in clinical material from late stage disease.  Early and late stages in Lyme disease.  B. burgdorferi - Early Disease  Progressively enlarging circular rash.  3-30d after tick bite, clear in the center (like bull's eye).  Rash (erythema migrans) may be >12 inches in diameter.  Erythema migrans (not itchy) is hallmark of Lyme disease.  Flu-like - fever, chills, fatigue, malaise when rash present. B. burgdorferi - Early Disease Erythema chronicum migrans  B. burgdorferi - Late Disease  Appears in untreated patients (weeks to months later).  Erythema migrans in other areas of the body.  Bouts of severe migratory joint pain/swelling (mostly knee).  Mo to yrs later, meningitis, Bell's palsy, limb weakness, etc.  Leptospira  Obligate aerobe and stains poorly.  Pathogenic strains in past were referred to as L. interrogans.  Spiral with hooked end like a question mark (gives the name).  Many species – species names are confusing.  Leptospira  Usual hosts: rodents, bats, cattle, sheep, goats.  Develop chronic kidney infection in these animals.  Infected animals excrete leptospira in urine.  Remain viable in alkaline water or wet soil for months.  Leptospira - Pathogenesis  From ingestion or exposure to contaminated food/water.  At risk – farmers, sewage workers, water sports enthusiasts.  ~ 1 million global infection every year (~200 in the US/year).  Mainly infect the kidneys (but any organs can be affected).  Leptospirosis  Incubation period 1-4 week but may be asymptomatic.  Symptom from mild anicteric disease to a fatal outcome.  >90% anicteric (a mild febrile, influenza like illness).  In