Gram Negative Aerobes Pt 1 PDF

Gram Negative Aerobes Kayla Antosz, PharmD Lead Antimicrobial Stewardship Pharmacist, Antimicrobial Stewardship Collaborative of South Carolina (ASC-SC) Clinical Instructor, University of South Carolina College of Pharmacy 11/1...

Gram Negative Aerobes Kayla Antosz, PharmD Lead Antimicrobial Stewardship Pharmacist, Antimicrobial Stewardship Collaborative of South Carolina (ASC-SC) Clinical Instructor, University of South Carolina College of Pharmacy 11/14/24 Slides modified from: Julie Justo, PharmD Objectives Compare key features of gram-negative aerobic bacteria including morphology, growth characteristics, and classification Interpret biochemical tests used to identify gram-negative aerobic bacteria List clinically relevant species of gram-negative aerobic bacteria Describe the pathogenesis and clinical syndromes associated with each of these pathogens Recognize resistance mechanisms gram-negatives express against common antibiotic classes Enterobacterales Order of gram-negative bacteria Share an enterobacterial common antigen Previously classified under the family Enterobacteriaceae Moderate sized, short bacilli (rod-shaped) Facultative anaerobes Other characteristics: Ferment glucose (some ferment lactose) Reduce nitrate to nitrite Catalase (+) Oxidase (-) Some resistant to bile salts Gram Stain Review  Gram-positive vs. Gram-negative Gram-Positive Bacteria Gram-Negative Bacteria Thick peptidoglycan layer in cell Thin peptidoglycan layer in cell wall wall No lipopolysaccharide membrane Lipopolysaccharide membrane Produces exotoxins Produces endotoxins Stained purple by gram-staining Stained red/pink by gram staining https://biologydictionary.net/gram-positive-vs-gram-negative/ Pathogenesis and Immunity Common Virulence Factors Associated with Enterobacterales Endotoxin Capsule Antigenic phase variation Type III secretion systems Sequestration of growth factors Resistance to serum killing Antimicrobial resistance Pathogenesis and Immunity Common Virulence Factors Associated with Enterobacterales Endotoxin Capsule The lipopolysaccharides in the outer Antigenic phase variation membrane of gram-negative bacteria Capable of causing lethal shock to cell Type III secretion systems Activation of complement system, release of cytokines, leukocytosis, Sequestration of growth factors thrombocytopenia, fever, decreased Resistance to serum killing peripheral circulation, shock, death Antimicrobial resistance Pathogenesis and Immunity Common Virulence Factors Associated with Enterobacterales Endotoxin Capsule Encapsulated Enterobacterales are Antigenic phase variation protected by phagocytosis by hydrophilic capsular antigens Type III secretion systems Antigens interfere with the binding of antibodies to the bacteria Sequestration of growth factors Protective role of capsule is diminished if patient develops specific anticapsular Resistance to serum killing antibodies Antimicrobial resistance Pathogenesis and Immunity Common Virulence Factors Associated with Enterobacterales Endotoxin Capsule Antigenic phase variation Extended-spectrum beta-lactamases Type III secretion systems (ESBLs) DNA gyrase and topoisomerase mutations Sequestration of growth factors Resistance to serum killing Antimicrobial resistance Common Enterobacterales Modified from Murray et al. 2016. Chapter 25: Enterobacteriaceae. Box 25-1 & Table 25-1.  https://emerypharma.com/blog/infection-by-site/ Sites of Infection How Can We Identify Organisms? Gram stain Growth media Sterile specimens: non-selective media Blood agar Non-sterile specimens: selective media MacConkey agar Biochemical tests Automated systems (Vitek 2, Microscan, Phoenix) Benchtop tests (spot indole, oxidase) Rapid Diagnostic Tests Multiplex PCR (FilmArray, Verigene) MALDI-TOF MS MacConkey Agar Selective and differential agar Selective: only grows gram negative bacteria Ingredients: bile salts and crystal violet dye Limit growth of gram-positive bacteria Differential: can further differentiate organisms based on lactose metabolism Ingredients: lactose Lactose fermentation decreases pH → vibrant pink / red color MacConkey Agar Lactose-fermenters Escherichia coli Klebsiella spp. Enterobacter spp. Citrobacter spp. (except C. koseri) Nonlactose-fermenters Salmonella spp. Shigella spp. Serratia spp. Proteus spp.  Spot Indole Test Detects ability of bacteria to metabolize tryptophan to produce indole Indole (-) Indole (+) 1. Drop indole reagent onto filter paper 2. Using inoculating loop, pick up fresh colonies of bacteria and rub colonies over reagent on paper 3. Observe color change (within 20 seconds) https://microbiologynote.com/spot-indole-test/  Spot Indole Test Indole (-) Enterobacter cloacae Klebsiella aerogenes Klebsiella pneumoniae Indole (-) Indole (+) Citrobacter freundii Indole (+) Escherichia coli Klebisella oxytoca https://microbiologynote.com/spot-indole-test/ Oxidase Test Used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain 1. Drop reagent (tetramethyl-p- phenylenediamine) onto filter paper 2. Using inoculating loop, pick up fresh colonies of bacteria and rub colonies over reagent on paper 3. Observe color change (within 10 seconds) Oxidase Test Used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain Oxidase (+): Pseudomonas spp. Vibrio spp. Aeromonas spp. Moraxella spp. Oxidase (-): Salmonella spp. Acinetobacter spp. Shigella spp. Proteus spp. Providencia spp. Serratia spp. Morganella spp. Escherichia coli https://www.istockphoto.com/photos/e-coli-bacteria Escherichia coli Most common and clinically significant Enterobacterales Part of the normal flora of GI tract, may colonize lower end of urethra/vagina Associated with Urinary tract infections (UTI) - #1 leading organism Gastroenteritis Meningitis (leading cause in neonatal meningitis) Sepsis Different serotypes with varying virulence Also member of normal intestinal microbiota Morphology Circular, smooth, flat, nonviscous colonies with distinct edges Hemolysis on blood agar Metallic sheen on different agars Lactose fermenter Indole positive  Clinical Syndromes- UTI UPEC= uropathogenic E.coli Flores-Mireles, et al. Nat Rev Microbiol. 2015. Clinical Syndromes- UTI Treatment Antimicrobials that only achieve concentrations in the urine are recommended Fosfomycin, nitrofurantoin Trimethoprim/sulfamethoxazole, fluoroquinolones for more complicated and/or resistant infections Clinical Syndromes- Escherichia coli Diarrhea Very common worldwide Strains of E. coli that cause gastroenteritis are divided into a number of groups Enterotoxigenic (ETEC) Enteropathogenic (EPAC) Small intestine- secretory diarrhea Enteroaggregative (EAEC) Shiga toxin-producing (STEC) Large intestine Enteroinvasive (EIEC) Enterotoxigenic Escherichia coli (ETEC) Traveler’s diarrhea (30% of cases) Infant diarrhea in developing countries Transmission: consumption of fecally contaminated food or water Symptoms: watery nonbloody diarrhea, abdominal cramps, nausea/vomiting Pathogenesis: plasmid-mediated enterotoxins (heat-stable and heat- liable) that stimulate hypersecretion of fluids and electrolytes Enteropathogenic Escherichia coli (EPEC) Infant diarrhea in developing countries (rare in the US) Transmission: fecal-oral exposure to contaminated food/surfaces Symptoms: watery diarrhea, vomiting Pathogenesis: plasmid-mediated attachment & effacement (disruption of normal microvillus structure resulting in malabsorption and diarrhea), invade epithelial cells lining the colon w/ destruction of normal mucosal epithelial cells Enteroaggregative Escherichia coli (EAEC) Infant diarrhea in developing and some developed countries Can be chronic → growth retardation Traveler’s diarrhea Transmission: fecal – oral Symptoms: persistent watery diarrhea, vomiting, dehydration, low- grade fever Pathogenesis: autoagglutination; adherence of rods (“stacked bricks”) over epithelium of small intestine → cytokine release, shortening of microvilli, hemorrhage, decreased fluid absorption Shiga Toxin-Producing Escherichia coli (STEC) Consumption of undercooked ground beef/meat products, water, unpasteurized milk/fruit juices 90% of these species 5-30% risk of inducing resistance while on antibiotics Biggest risk with Enterobacter spp. exposed to third-generation cephalosporins Include E. cloacae complex and K. aerogenes For Citrobacter: mainly species C. freundii or C. youngae Rare acquisition: Plasmid-mediated AmpC gene Always resistant even in absence of antibiotics Aka “stably derepressed AmpC” Proteus spp., Morganella spp., Providencia spp. Motile, slow/non-lactose fermenters Proteus can ________ on media due to its motility Proteus & Morganella: Urease (+) Generates CO2 and ammonia (distinctive ammonia smell) Increases urine pH → Mg2+ + Ca2+ precipitate → kidney stones Providencia: Urease (-) Associated with UTIs, pneumonia, bacteremia Salmonella & Shigella Salmonella spp. Motile Ferment glucose and mannose Resistant to bile salts Non-lactose/non-sucrose fermenter Produces H2S Not generally part of the human microbiome Transmission: fecal to oral spread, ingestion of contaminated foods Person to person w/ low inoculum possible for Salmonella Typhi Associated with gastroenteritis, bacteremia, enteric fever, asymptomatic colonization Hektoen Enteric Agar Represents both selective and differential media Divides Salmonella, Shigella, and “everything else” Selective ingredients: bile salts, lactose, sucrose, salicin, peptone Bile salts: limit growth of gram positive bacteria Lactose, sucrose, salicin: preferred by other gram-negative bacteria, not used by Salmonella and Shigella Peptone: for Salmonella and Shigella Differential ingredients: Lactose, sucrose, and salicin fermentation decreases pH → vibrant yellow- orange color Thiosulfate or ferric ammonium citrate → black precipitate with H2S production Hektoen Enteric Agar Salmonella Classification Salmonella: Gastroenteritis Most common form of salmonellosis AKA “food poisoning” Transmission: contaminated food/water Highest incidence in summer/fall (outdoor gatherings) Common sources: poultry, eggs, dairy, contaminated cutting boards Symptoms: N/V, nonbloody diarrhea, fever, cramps, myalgias, headaches Onset: 6-48 hours Duration: 2-7 days Salmonella: Bacteremia Most common: Salmonella Typhi, Salmonella Paratyphi, and Salmonella Choleraesuis Higher risk in pediatric and immunocompromised patients Clinical presentation similar to that of other gram-negative infections, however localized suppurative infections (e.g. osteomyelitis, endocarditis, arthritis) can occur in ~10% of patients Transmission: contaminated food or water Salmonella: Enteric Fever (Typhoid Fever) Severe, systemic illness caused by Salmonella Typhi or Salmonella Paratyphi Less frequently seen in developed countries (~300 cases/yr in US, mostly associated with foreign travel) Reservoir: exclusively humans Transmission: contaminated food or water Symptoms: fever, headache, myalgias, malaise, anorexia → GI symptoms → bacteremic phase → colonization of gall bladder → re- infection of intestines Salmonella: Asymptomatic Colonization Chronic colonization: Salmonella detected in the bodily specimens (e.g. stool, urine) >12 months after acute infection Generally occurs with Typhoid and Paratyphoid Occurs in 1-5% of patients Gallblader is typical reservoir Recurrent infection unlikely Can shed high amounts of bacteria and infect others Salmonella Typhoid vaccines (2 types) Inactivated typhoid vaccine, injection: ViCPS Live typhoid vaccine, oral: Ty21a Treatment: Rehydration Antibiotics (in some cases) → check susceptibilities Recommended for: Travelers to parts of the world where typhoid is common People in close contact with typhoid carrier Laboratory workers who work with Salmonella typhi bacteria Salmonella: Prevention Hand washing!! “Boil it, cook it, peel it, or forget it” Tips from CDC, especially when traveling to developing countries Skip ice water Peel fresh fruit and veggies after washing hands Avoid street vendors Shigella spp. Slender, non-motile bacilli Convex, circular, transparent colonies Resistant to bile salts Non-lactose fermenter Does not produce H2S Most clinically important: S. dysenteriae (most serious) S. sonnei (most common in US) S. flexneri (most common in underdeveloped countries) Hektoen Enteric Agar Shigella spp. S. dysenteriae: Produces Shiga toxin (exotoxin) → disrupts protein synthesis Primary manifestation of toxin activity is damage to the intestinal epithelium Can also damage glomerular endothelial cells, resulting in renal failure (HUS) Causes dysentery (bloody diarrhea) Most severe form, mortality rate 5-15% Shigella spp. Transmission: fecal-oral Low inoculum can cause disease (100-200 bacteria) Daycares, children, close quarters Pathogenesis: Invade M cells in Peyer’s patches of intestine, engulfed by cell, replicate within cell, undergo cell-to-cell passage, eventually leads to cell apoptosis Symptoms: watery diarrhea → mucuous/bloody diarrhea, fever, abdominal cramps, tenesmus (cramping rectal pain, feeling of incomplete defecation) Treatment: usually self limiting, rehydration, antibiotics if severe Yersinia Yersinia spp. Zoonotic infection Usually associated with contact with rats /rodents Most clinically important: Y. pestis: highly virulent pathogen, causes disease known as the plague Y. entercolitica, Y. pseudotuberculosis: primarily enteric pathogens, relatively uncommon Yersinia pestis: plague Bubonic plague Transmission: bite from infected flea Incubation period: 7 days Affects the lymphatic system, inflammation in lymph nodes, high fever → bacteremia If not treated, 75% of patients die Pneumonic plague Transmission: respiratory droplets, does not have to involve fleas/animals Incubation period: 2-3 days More virulent and rare than bubonic plague (1 bacilli can cause disease) Pulmonary symptoms, fever, severe hemorrhaging (death in hours) If not treated, ~95% of patients die Black Death- Bubonic Plague Bubonic plague pandemic occurring in Wester Eurasia and North Africa from 1331 – 1770 (peaking in 1347-1351) Most fatal pandemic in human history During the 14th century, killed more than 1/3 of Europe 200M Black Death (Bubonic Plague) Yersinia entercolitica Enterocolitis Transmission: ingestion of contaminated food/water Incubation period: 1-10 days, average 4-6 days Symptoms: diarrhea, fever, abdominal pain, “pseudo-appendicitis” Chronic form of the disease can develop and persist for months  Gram Negative Aerobes (Part 1) Kayla Antosz, PharmD Lead Antimicrobial Stewardship Pharmacist, Antimicrobial Stewardship Collaborative of South Carolina (ASC-SC) Clinical Instructor, University of South Carolina College of Pharmacy 11/14/24 Slides modified from: Julie Justo, PharmD