Gram Negative Bacteria PDF

Medically important Gram negative Bacteria General Introduction Shafik Habal, MD Associate Professor, Medical Microbiology and Immunology Philadelphia College of Osteopathic Medicine Georg...

Medically important Gram negative Bacteria General Introduction Shafik Habal, MD Associate Professor, Medical Microbiology and Immunology Philadelphia College of Osteopathic Medicine Georgia Campus  Learning Objectives Describe general characteristics of Neisseria spp and Moraxella spp. including morphology, distinguishing features, common diseases and major virulence factors List general characteristics of members of the Enterobacteriaceae family – oxidase test, lactose fermentation, opportunistic vs primary pathogens – Describe the basis of the serological classification (O, H, K antigens) of the Enterobacteriaceae Describe the morphology, distinguishing features, common diseases and major virulence factors associated with the following Gram negative organisms: – Pseudomonas aeruginosa, and related bacteria (Burkholderia cepacia, Acinetobacter baumannii) – Vibrio spp, Campylobacter spp, and Helicobacter pylori – Haemophilus spp. and Bordetella pertussis Murray’s Medical Microbiology. 9th edition Chapters: 23, 24, 25, 26, 27, 28, 29  Gram (+ve) and Gram (-ve) Bacteria Gram Positive Bacteria Gram Negative Bacteria  Gram (+ve) and Gram (-ve) Bacteria Gram Negative Rods Gram Positive Rods  Gram negative Major Gram Negative Bacteria Obligate anaerobe bacteria Bacteroides Prevotella Pasturella Cocci Coccobacilli Rods Brucella N. meningitidis H. influenzae N. gonorrhoeae Bordetella Moraxella Yersinia Francesella Lactose Fermenters Lactose Non fermenters Fast Slow Oxidase + Oxidase – E. coli Citrobacter Pseudomonas aeruginosa Shigella sp. Enterobacter Serratia Burkholderia cepacia Salmonella sp. Klebsiella Others Proteus Providencia  GRAM NEGATIVE CLASSIFICATION Pseudomonas Bacteroides Haemophilus GRAM NEGATIVE OTHER Bordetella Brucella SPIROCHETES COCCI RODS CURVED CAMPLOBACTERIACEAE NEISSERIACEAE ENTEROBACTERIACEAE VIBRIONACEAE HELYCOBACTERIACEAE Campylobacter jujuni Neisseria meningitidis Vibrio parahaemolyticus Yersinia Klebsiella Campylobacter fetus Neisseria gonnorheae Vibrio vulnificus Serratia Enterobacter Helycobacter pylori Moraxella catarrhalis Vibrio cholerae Citrobacter Proteus Salmonella Shigella Escherihia GRAM NEGATIVE COCCI  Neisseria species Gram negative diplococci – bean shaped Non spore forming intracellular pathogen Aerobic Capnophilic (3 – 7% CO2 for optimal growth) Most are oxidase positive Catalase positive All metabolize glucose, only N. meningitidis metabolizes maltose Grow on modified “Thayer Martin Agar” Neisseria meningitidis General properties and identification: 2nd most common cause of community acquired meningitis Transmitted through respiratory, pharyngeal droplets Grows on “Thayer Martin Agar” Encapsulated Capnophillic – 5% CO2 Metabolizes (oxidation) Glucose and Maltose 5-10% of Humans are carriers (nasopharynx) Neisseria meningitidis Virulence and pathogenesis: Polysaccharide capsule 🡪 Antiphagocytic – 5 Serotypes (A, C, W-135, Y, and B) – B is the most common type and least immunogenic Pili 🡪 Attachment to respiratory epithelium Respiratory transmission IgA protease 🡪 Colonize mucosal membrane RTI Lipooligosaccharide (LOS) endotoxin 🡪 Fever, shock C6 – C9 complement deficiency 🡪 Neisserial infections Bacteremia Meningitis  Neisseria meningitidis Clinical signs and manifestations: Meningitis: – Fever – Headache + Vomiting – Nuchal rigidity Complications: – Septicemia – Waterhouse – Fridrichesn Syndrome Meningiococcemia Adrenal insufficiency Intravascular coagulation Diagnosis: Gram stain and culture of CSF and rapid PCR  Neisseria gonorrhoeae General properties: Gram negative diplococci Intracellular pathogen Doesn’t metabolize Maltose Fastidious Symptomatic in 90% of males, 50% of females More prevalent in southern states Infections are underreported by a factor of 2  Neisseria gonorrhoeae Virulence and pathogenesis: Pili 🡪 Attachment, antigenic variation Outer membrane proteins 🡪 Antigenic variation Urethral exudate showing PMN’s LipoOligosaccharide (LOS) modified endotoxin IgA protease 🡪 Hydrolyze secretory IgA – Invades mucosal surfaces  Neisseria gonorrheae Clinical signs and manifestations: Males 🡪 Urethritis (Dysuria, purulent discharge) Females 🡪 Endocervicities (vaginal discharge), salpengitis Homosexual males 🡪 Rectalitis and proctitis Adult conjunctivitis and pharyngitis Newborn 🡪 Ophthalmia neonatorum (conjunctivitis) – It leads to blindness – Prophylactic Erythromycin or silver nitrate ointment Complications: Gonococcemia 🡪 Arthritis and PID Diagnosis: Culture and NAAT (nucleic acid amplification test) Treatment: Ceftriaxone (IM) and Azithromycin (oral) GRAM NEGATIVE BACILLI  GRAM NEGATIVE RODS Non Enteric Gram Negative Rods Enteric Gram Negative Rods Bacteroides – Aerobes Campylobacter Helicobacter Psuedomonas aeruginosa Vibrionaceae Burkholderia cepacia Enterobacteriacae Bordetella pertussis – Facultative Anaerobes Haemophilus influenzae Yersinia pestis  Pseudomonas aeruginosa – Overview Small Gram negative rod with flagellum Ubiquitous, frequently found in soil and water. Often isolated from water sources in hospitals Strict aerobe, opportunistic organism Oxidase positive Non lactose fermenter Resistant to some common disinfectants. Antibiotic resistance is common including multidrug resistance. Risk factor for pneumonia in cystic fibrosis (CF) and ventilated patients  Pseudomonas aeruginosa – Virulence Factors Exotoxin A: Acts on EF2 🡪 Inhibits protein synthesis, tissue damage Phospholipase C: Hemolysin 🡪 break lecthin 🡪 tissue damage Alginate: Mucoid exopolysaccharide forming a biofilm Pyocyanin (blue pigment): Inhibits ciliary movement Pyoveridin: Iron binding green pigment. Ciliastatic Elastase: Destroys elastin and collagen  Pseudomonas aeruginosa Clinical signs and manifestations: 3rd most common cause of UTIs, specially indwelling catheters Pneumonia in hospitalized and cystic fibrosis patients Burn victims infections 🡪 soft tissue Hot tub and swimming pool infections 🡪 Folliculitis like Otitis externa “swimmer’s ear” Ocular infections associated with contaminated contact lenses Endocarditis  Acinetobacter baumannii Gram-negative, aerobic rods Commonly found in the soil, water, and skin (flora) Commonly isolated from hospitalized patients Rarely known to cause disease Clinical manifestations include: Who is at risk? – Respiratory tract infections People most at risk include patients in hospitals, especially – Urinary tract infections those who: – Wound infections – Septicemia are on breathing machines (ventilators) have devices such as catheters have open wounds from surgery Hospital strains are highly drug resistant are in intensive care units have prolonged hospital stays  Burkholderia cepacia [burk-hōld–er–ee-uh si–pay–shee–uh] (also called B. cepacia) Gram negative rod which does not ferment lactose Catalase positive Can be found in soil and water (in hospital irrigation solutions, IV fluids) Serious respiratory infections, especially in patients with cystic fibrosis Opportunistic Can also be spread to susceptible persons by: – Person-to-person contact – Contact with contaminated surfaces – Exposure to B. cepacia in the environment B. cepacia can be resistant to many common antibiotics  Bordetella pertussis General properties and identification: Human is the only natural host Disease is highly communicable (person – to – person) Non motile Encapsulated Gram-negative coccobacillus Fastidious 🡪 Require Nicotinamide Grows on “Bordet – Gengou” or “Regan- Lowe” agars Associated with Lymphocytosis (toxin increases IL4, IgE) Pertussis is nationally notifiable and clinicians should notify the appropriate health department of all patients with suspected pertussis. Similarly, diagnostic laboratories should notify health departments of all positive pertussis laboratory results National Notifiable Diseases Surveillance System (NNDSS) Bordetella pertussis Virulence and Pathogenesis: Polysaccharide capsule Adenylate cyclase/hemolysin: Inhibits phagocytosis Tracheal cytotoxin destroys ciliated epithelial cell – Induces host cells to produce IL-1 – IL – 1 🡪 NO 🡪 Death of respiratory cells 🡪 ciliastatic – Vasoconstriction, inflammation, dermonecrotic lesions Filamentous Hemagglutnins (FHA): RBC agglutination – Bacterial attachment to epithelial cells Pertussis (AB) Toxin: 5 (S1-S5) subunits (4 B involved in binding)  Bordetella pertussis S4 Pertussis Exotoxin: S2 S5 S4 calmodulin S3 Inhibition of chemotaxis S1 Increased IgE synthesis 🡪 Increased histamine Inhibition of phagocytosis G Gi Inhibition of superoxide generation - - Induction of apoptosis in macrophages Host Host Adenylate Adenylate Cylase Cylase Mechanism: ADP ribosylation of G🡪 Adenylate cyclase 🡪 cAMP ADP cAMP ADP cAMP Increased cAMP 🡪 inhibits negative feedback 🡪 Increased cAMP Whooping cough 4.9%  Bordetella pertussis Management: Two types of vaccines protect against pertussis and other disease: 1. Diphtheria, tetanus, and pertussis (DTaP) vaccines (7 years) DTaP: 2, 4, 6, 12 – 15 months, and booster 4 – 6 years. DTaP: Acellular FHA and pertussis toxoid In the past DTP (whole killed – longer immunity) Tdap has a reduced dose of the diphtheria and pertussis vaccines  Haemophilus influenzae General properties and identification: Gram negative, non motile pleomorphic coccobacilli Strict human pathogen (nasopharynx) Majority Facultative anaerobe of serious infections Polysaccharide capsule 🡪 6 serotypes (a, b, c, d, e, f) Diagnosis: (polysaccharide antigens) – Grows on heated blood agar, require factors V and X – Latex agglutination test – Counterimmunoelectrophoresis – PCR  Haemophilus influenzae Clinical signs and manifestations: Person – to – person transmission People with compromised CMI are at risk (Asplenics, HIV, infants and the elderly) There are two types: – Typable (Hib): Infantile meningitis, epiglottitis, cellulites, pneumonina. + Quellung – Non typable: Otitis media, sinusitis , bronchitis, conjunctivitis (pink eye). (Commonly found in URT of people) Vaccine: Hib conjugate vaccine (2, 4, 6, and 12 – 15 months)  Seagull wing Campylobacter jejuni appearance General properties and Identification: MCC of infectious diarrhea (> Shigella & Salmonella combined) Gram negative curved “comma or S shaped” rod Motile, polar flagellum at one or both ends Microaerophilic (5%O2), Capnophilic (10%CO2) Campylobacters are thermophilic (42°C) except C. fetus (25°C) Oxidase positive and Catalase positive Grows on CAMPY agar  Campylobacter jejuni Virulence factors and pathogenesis: Fecal oral transmission Associated with ingestion of undercooked poultry Low ID50 Adhesions mediate mucosal attachment Inflammatory diarrhea (pus, blood in stool)  Campylobacter jejuni Clinical sings and manifestations: Gastroenteritis – Fever, abdominal pain and diarrhea (often bloody) – Management: Replacement of fluids and electrolytes is the mainstay of therapy Guillain Barre Syndrome (o:19 serotype) – Possibly due to O-antigens cross reacting with components of Schwann cells or myeline resulting in demyelination – An example of molecular mimicry – Management: Supportive Helicobacter pylori Epidemiology: A common cause of gastritis and duodenal ulcers (up to 90%) Maybe associated with gastric adenocarcinoma Infections are common w/ worldwide distribution Transmission thought to be person-to-person b/c there has been no isolation from food or water Untreated infections: can become chronic/life-long  Helicobacter pylori General properties and identification: Gram negative, microaerophilic curved rod Motile (4-6 polar flagella) 🡪 Corkscrew motility Oxidase, Urease and Catalase positive Urea breath test NH2CONH2 + H2O → 2NH3 + CO2 Radiolabeled Mesophile 🡪 Grows well at (37°C) not 42°C Grows on Campy agar Helicobacter pylori  H. pylori pathogenesis and the inflammatory response Clinical Science (2006) 110, 305-314 - www.clinsci.org  Helicobacter pylori Clinical signs and manifestations: Gastritis and peptic ulcer disease GERD Not associated with bacteremia Treatment: First line Quadruple Therapy (if no history of antibiotic use) – Proton pump inhibitor (omeprazole) – Antibiotic (Clarithromycin, Amoxicillin) – Bismuth compound – If resistant 🡪 Tetracycline + Metronidazole Recurrence if treatment is incomplete  VIBRIONACEAE General properties and identification: Gram negative curved rods Facultative anaerobes Single polar flagellum Oxidase positive All Vibrio species can grow in water with high salinity Grows on Trisulphate Citrate Bile Sucrose TCBS agar Alkaline growth media  VIBRIONACAE Vibrio vulnificus Vibrio parahaemolyticus Ingestion or injury by shellfish (shucking oysters) Ingestion of undercooked seafood, Underlying liver disease (EtOH) especially shellfish, oysters Lactose fermenting Hemolytic and Halophilic (3%) Halophile Gastroenteritis, self-limiting watery INVASIVE 🡪 Cellulitis, Bacteremia diarrhea lasting for 3 days. Treatment: Tetracycline  Vibrio cholerae General properties: Human reservoir (colon) Grows small yellow colonies on TCBS Outbreaks associated with contaminated water 6 serotypes, only serotype O1 and O139 is associated with epidemics Serotype O1 is divide to Classical and El tor (both cause disease)  Vibrio cholerae Cholera toxin and pathogenesis: Heat labile enterotoxin composed of 6 subunits ADP ribosylation of Gαs 🡪 adenylate cyclase 🡪 cAMP 🡪 Loss of water, electrolytes Similar to Pertussis toxin (Gα unable to inactivate adenylate cyclase 🡪 cAMP)  Vibrio cholerae Clinical signs and manifestations: CHOLERA Ingestion of contaminated food and water Perfuse watery non bloody diarrhea (Rice water stool) – Up to 20L of fluids/day 🡪 Shock – Colorless, odorless, no blood, some mucus Loss of K 🡪 Hypokalemia 🡪 Cardiac arrhythmias Loss of H2CO3- 🡪 Metabolic acidosis Management: Fluid replacement, antibiotics, sewage treatment 60% fatal if not treated  ENTEROBACTERIACEAE (OX-) (OX+) General properties and characteristics: Gram negative rods (short, 1–5 μm in length) Human pathogens There are always exceptions They don’t form spores Citrobacter All are facultative anaerobes 🡪 Ferment glucose, reduce Nitrate Enterobacter Eschericia Shigella All are Oxidase negative (OX-) Klebsiella Proteus All are motile except; Shigella, Yersinia and Klebsiella Salmonella Yersinia NO3- 🡪 NO2- ENTEROBACTERIACEAE In Salmonella enterica, the capsular antigen is called Vi antigen O antigen  ENTEROBACTERIACEAE The Enteropluri ® Tube: Manufactured by Becton – Dickinson to identify Gram negative rods associated with enteric infections. Used for rapid identification of Enterobacteriaceae. Composed of 15 tests. Reagents are added as needed glucose, gas production, lysine decarboxylase, ornithine decarboxylase, H2S, indole, adonitol, lactose, arabinose, sorbitol, Voges-Proskauer, dulcitol, phenylalanine deaminase, urea and citrate.  MacConkey Agar Lactose fermenting Lactose non- fermenting Escherichia Shigella Klebsiella Salmonella Enterobacter Proteus Citrobacter Yersinia EMB Agar  Escherichia coli General properties and identification: Most common cause of UTI Present in GI normal flora Usually motile Doesn’t produce H2S Tests: Urease -, Citrate -, Indole +, lactose +  E. coli (Pathotypes) Dysentery (inflammatory) 🡪 pus and blood (rare in the US – strains O124) – closely related to Shigella Enteroinvasive (EIEC) NON-MOTILE Leading cause of diarrhea in children and traveler’s to 3rd world countries – Self limiting “Traveler’s diarrhea” Enterotoxogenic (ETEC) Heat labile toxin (LT) 🡪 ADP ribosylation of Gs 🡪 ⇧ cAMP (similar to Cholera toxin) diarrheagenic E. coli Heat stable toxin (ST) 🡪 Increase guanylate cyclase Infantile watery diarrhea (less than 6 months) – rare in US Enteropathogenic (EPEC) Has locus of enterocyte effacement (LEE) pathogenicity island Although all EHEC are STEC, not all STEC are EHEC Enterohemorrhagic (EHEC) Associated with ingestion of undercooked meat, unpasteurized milk Shiga toxin producing (STEC) Strain: O157:H7 (common in the US – unlike other strains) Verotoxin producing (VTEC) Verotoxin (shiga-like toxin) Interfere with 60s subunit 🡪 ⇩ Protein synthesis Associated with hemorrhagic (bloody) colitis and HUS EnteroAggregative Pediatric diarrhea in developing countries 🡪 Persistent watery diarrhea Diffusely –adherent (DAEC) Less likely to be associated with diarrhea and may be linked to urinary tract infections Extra intestinal E. coli Uropathogenic (UPEC) Associated with UTI, pyelonephritis More common in females. A common cause of meningitis in newborns Meningitis (NMEC) Sepsis associated strain Sepsis associated (SEPEC)  Escherichia coli Clinical signs and manifestations: UTI 🡪 Acute cystitis and pyelonephritis Gastroenteritis Hemorrhagic colitis (bloody) Major cause of neonatal meningitis Hemolytic uremic syndrome associated with O157:H7  Klebsiella pneumoniae - Overview Gram-negative encapsulated, non-motile rods Lactose fermenter Common pathogen of the GIT Frequent cause of nosocomial infections Intubated patients and alcoholics are at risk of pneumonia Antibiotic resistance: – Many strains produce “extended-spectrum beta lactamase” – Many strains are resistant to fluoroquinolones and aminoglycosides – Carbapenem resistant strains  Klebsiella pneumoniae Clinical presentation: Community acquired pneumonia – (Lobar) – Thick blood tinged sputum – currant jelly – Malnourished and hospitalized – VA Pneumonia – Liver disease and EtOH – aspiration pneumonia UTI’s and septicemia  Serratia marcescens General properties and Identification: Opportunistic Gram negative, motile rods Lactose fermenter Produce red pigments (Prodigiocin) The only Serratia species with clinical relevance Associated with nosocomial UTI’s Associated with catheter septicemia and burn infections Drug resistance increasing  Proteus spp. Culture and Identification: Gram negative rods with several flagella 🡪 “Swarming motility” Urease +, lactose non fermenters Produces H2S (Hydrogen sulfide) Normal microbiota of human GI tract. Two species of Proteus differentiated based on Tryptophan metabolism – P. vulgaris: Can metabolizing tryptophan 🡪 Indole positive (often hospital associated) – P. mirabilis: Can’t metabolize tryptophan 🡪 Indole negative (most common strain)  Proteus spp. Virulence and pathogenesis: Associated with nosocomial infections The majority of the infections are P. miribilis Clinically: UTI’s – Urease production 🡪 precipitation of organic and inorganic compounds – Obstruction and Renal stones – Associated with pyelonephritis, cystitis  am o n ex Urease producing species and Struvite Stones t No About 75% of all staghorn stones are struvite stones Struvite stones composed of magnesium, ammonium, and phosphate crystals Staghorn stones are either all calcium or have calcium added to struvite crystals. Urease producing bacteria 🡪 struvite stones. UREA Urease Ammonia 2NH3 + CO2 Urease Urease producing bacteria Proteus NH4+ + OH- Ammonium Pseudomonas Providencia Klebsilla Staphylococcus Alkaline urine Crystals formation Mycoplasma  Salmonella growing black SALMONELLA W nom hat a encl colonies showing classical H2S production a mes ture General properties and Identification: s Gram negative motile rods Lactose non fermenter Hektoen agar H2S producing Classification of Salmonella species varies (2500 serotypes) Genus Species Serovar XLD agar Salmonella enterica Salmonella enterica Enteritidis Non-typhoidal Salmonella Salmonella enterica Typhimurium Salmonella bongori Salmonella enterica Typhi  SALMONELLA Enteritidis Typhi and Paratyphi Gastroenteritis Enteric Fever “Typhoid” Eggs, undercooked poultry Fecal oral, person to person (human is the only Incubation depends on the dose (requires a large known host) infective does) Low infective dose Incubation 10 – 14 days Symptoms begin within 6 – 48 hrs Myalgia, headache, sustained fever, abdominal pain N&V, diarrhea, abdominal pain Rose spots (macular rash) on abdomen Intestinal hemorrhage, cholecystitis Mild to watery, mucopurulent and bloody diarrhea GI symptoms may not be seen in all patients TAB vaccine and Antibiotics Duration: 2 – 7 days Self-limiting Osteomyelitis in patients with sickle cells or underlying disease  SHIGELLA General properties and identification: Gram negative non motile rods Non lactose fermenters Doesn’t produce H2S Small infective dose ID50 Humans are the only reservoir (fecal oral) Fecal-oral, person-to-person transmission Mainly a pediatric infection (school aged children)  Shieglla species Four species (genetic sequencing) – Shigella sonnei (most common in industrial world and the US) – Shigella flexneri (most common in developing countries) – Shigella boydii (uncommon) – Shigella dysenteriae (most pathogenic, mostly outside the US ) Exotoxin 🡪 Shiga toxin (produced by shigella dysenteriae, similar to EHEC) – AB Toxin (B5 binds specific glycosides on cell surface) – A subunit causes irreversible inactivation of the 60S ribosomal subunit causing: Inhibition of protein synthesis Cell death Hemorrhage Unlike Salmonella, Shigella doesn’t multiply inside phagcytic vacuole Doesn’t invade epithelial cells beyond lamina propria 🡪no septicemia  Yersinia enterocolitica Epidemiology: Found in colder cultures – Northern US Spread via contaminated animal products (Milk, meat) Blood transfusions (Human – human) Culture and Identification: Negative urea test Enriched broth (cold temperature 28°C) Gram negative, facultative rods Motile at 25°C and non motile at 37°C Positive urea test Urease + Doesn’t ferment lactose (NH2)2CO + H2O → CO2 + 2NH3 Clinically: Enterocolitis and postinfective arthritis in adults  Yersinia pestis Long before PPE Very small Gram negative coccobacilli Non motile Facultative intracellular Grows on MacConkey agar. Lab must be notified Areas affected include Southwester US Reservoir: Rodents (primarily Prairie dogs in the US) Vector: Fleas  Yersinia pestis Virulence factors and pathogenesis: Capsular antigen “F1” Yersinia outer membrane protein “YOP” Anti phagocytic V and W antigens: Unknown mechanism, intracellular growth All virulence factors are activated at 37°C Coagulase is secreted by the flea at 25°C Sylvatic Cycle: Transmission by fleas from rodents, chipmonks (wild) Urban Cycle: Transmission by fleas from domestic/semidomestic animals  Yersinia pestis Clinical signs and manifestations: Bubonic Plague – Pain and swelling of lymph nodes (bite side) 🡪 Bubo – High fever and pain (extremities) Pneumonic Plague – Inhalation 🡪 person to person – Fever, edema and cyanosis – Fatal within 24 hours if not treated Septecemic Plague Treatment: Tetracycline + Streptomycin Yersinia pestis  Enterobacteriaceae Identification Property Organisms Lactose Fermenters E. coli, Klebsiella, Enterobacter, possibly Citrobacter H 2S + Proteus, Salmonella, Citrobacter Nonmotile Klebsiella, Shigella Voges- Proskauer + Klebsiella, Enterobacter, Serratia