Microbiology - Lesson 17 - Gram-negative Bacteria PDF

Lesson 17 Bacteria of dental interest: Gram-negative Microbiology Dra. Sara Mª Martínez Sánchez Dra. Ana Isabel García Guillén Bachelor in Dentistry Neisseriaceae Neisseria N. gonorrhoeae N. meningitidis Moraxella Enterobacteriaceae Eschericheae Escherichia coli Salmonellae Salmonella spp. V...

Lesson 17 Bacteria of dental interest: Gram-negative Microbiology Dra. Sara Mª Martínez Sánchez Dra. Ana Isabel García Guillén Bachelor in Dentistry Neisseriaceae Neisseria N. gonorrhoeae N. meningitidis Moraxella Enterobacteriaceae Eschericheae Escherichia coli Salmonellae Salmonella spp. Veillonella spp. V. parvula Shigellae Parvobacteria Klebsielleae Haemophilus Haemophilus influenzae Bordetella: Bordetella pertussis Pasteurella Aggregatibacter actinomycetemcomitans Eikenella Capnocytophaga HACEK group bacteria Shigella spp. Klebsiella spp. Enterobacter spp. Serratia spp. Pseudomonas Pseudomonas aeruginosa Yersinieae Yersinia pestis Vibrios Vibrio cholerae Vibrio parahaemolyticus Campylobacters Campylobacter spp. Helicobacter pylori Wolinella W. succinogenes As a reminder Gram-positive 3 Gram-negative 4 Neisseriaceae Pathogens Neisseria gonorrhoeae (the gonococcus) N. meningitidis (the meningococcus) Non-pathogens N. sicca Opportunistic Moraxella Kidney-shaped, Gram-negative cocci usually arranged in pairs, and oxidasepositive N. mucosa N. lactamica 5 The genus Neisseria General characteristics • Non-motile, Gram-negative cocci • 0.6 to 1.0 µm • Pairs with concave adjacent sides (bean-shaped) • Tetrads, short chains and clusters occasionally • Nutritionally fastidious, especially on clinical specimens • N. gonorrhoeae is capnophilic • Haemolysed blood and solubilized starch • 36-39oC, non-pathogenic below 24oC The genus Neisseria N. gonorrhoeae • Agent of gonorrhoea • Most frequently diagnosed venereal disease in western Europe and USA • Pelvic inflammatory disease (PID) • Sterility in women • Arthritis and sometimes septicaemia N. gonorrhoeae Habitat and transmission • Human urogenital tract • Oral, nasopharyngeal and rectal carriage is not uncommon • Spread by sexual intercourse or intimate contact N. gonorrhoeae Identification • Non-motile, non-capsulated diplococci • Enriched medium (lysed blood or chocolate agar) in 5-10% CO2 • Small, grey, oxidase-positive colonies • Staining by fluorescent antibody techniques • Acid from glucose but not from maltose or sucrose • Diagnosis by Gram-stained smears urethral exudate from men and cervix in women N. gonorrhoeae Pathogenicity • • • • Pili, adhere and colonize epithelial surfaces IgA proteases (IgA major defence factor on mucosas) β-lactamase (plasmid-mediated) Tracheal cytotoxin (damage ciliated cells of fallopian tube) N. gonorrhoeae Treatment and prevention • Resistant to β-lactams • Treatment: β-lactamase-stable cephalosporins • Prevention requires ’safe sex’, health education and contact tracing The genus Neisseria N. meningitidis • Agent of meningococcal • Meningitis • Highly contagious disease • Associated with a mortality rate approximating 80% when untreated N. meningitidis Habitat and transmission • Nasopharynx • Droplet spread N. meningitidis Characteristics • Resembles the gonococcus • Culture as for N. gonorrhoeae • Gram-negative cocci in pairs in nasopharyngeal discharge, cerebrospinal fluid or blood smears • Organism is found pure in cerebrospinal fluid • Carbohydrate utilization test (acid from glucose and maltose) • Diagnosis by serology N. meningitidis N. gonorrhoeae N. meningitidis Pathogenicity • Septicaemia is accompanied by a rash • Eventual death may be due to meningitis or adrenal haemorrhage (Waterhouse-Friderichsen syndrome) • Antiphagocytic properties of the capsule • Toxic effects due to the meningococcal endotoxin N. meningitidis Diagnosis and treatment • Rash that does not fade under pressure (glass test) • Penicillin or cefotaxime Commensal species Neisseria spp. • Common in the oral cavity, nose and pharynx • Female genital tract • • • • • N. subflava N. mucosa N. sicca Earliest colonizers of a clean tooth surface Consume oxygen facilitating subsequent growth of facultative and obligate anaerobes Opportunistic pathogens Moraxella spp. • Gram-negative cocci • Asaccharolytic • Habitat: human respiratory tract • Opportunistic meningitis, endocarditis, otitis media, maxillary sinusitis, and chronic obstructive pulmonary disease • Produce beta-lactamase, indirectly promoting other pathogens Veillonella spp. Generalities • Obligate anaerobic Gram-negative bacteria • Cocci shape • Frequent in oral samples • V. parvula (the type species) • V. dispar • V. atypica Veillonella spp. V. parvula • Small anaerobic cocci • Mostly in dental plaque • ‘Benevolent organisms’ • Metabolize the lactic acid into weaker acids (acetic and propionic) • Unknown pathogenic potential J b n Parvobacteria Generalities • Small size • Gram-negative bacilli • Heterogeneous group: • Haemophilus* • Bordetella* • Pasteurella* (includes Aggregatibacter spp.) • Eikenella* • Brucella • Francisella • Gardnerella Haemophilus spp. Generalities • Tiny, non-motile, aerobic, Gram-negative cocco-bacilli • Some capsulated • Two growth factors: • X factor - haematin present in blood • V factor - nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) H. influenza H. parainfluenza Haemophilus Habitat and transmission • Upper respiratory tract commensal • Major aetiological agent of upper respiratory tract infections and acute exacerbations of chronic bronchitis • Common secondary colonizer after influenza • Gram-negative, non-sporing, non-motile rods • Virulent strains are capsulated H. influenzae Characteristics • Requirements of two growth factors: • X factor: haematin present in blood • V factor: nicotinamide adenine dinucleotide (NAD or NADP) • Large colonies around colonies of other organism (satellitism) Blood agar plate streaked with S. aureus (V factor) H. influenzae Pathogenicity • Meningitis, acute epiglottitis, osteomyelitis, arthritis Polysaccharide capsule, IgA protease • Factor causes slowing and incoordination of ciliary beating • Outer membrane and endotoxin H. influenzae Treatment and prevention • Ampicillin (beta-lactamase-negative strains) • Vaccination against invasive type b infection Bordetella pertussis Characteristics • Respiratory tract in diseased individuals • Spread is by the airborne route. • Healthy carriers are not known • Gram-negative rods, encapsulated • Fastidious growth requirements • Confirmed serologically Bordetella pertussis Pathogenicity • Whooping cough, preschool children • Paroxysmal coughs followed by the ‘whoop’ of rapid inhalation after coughing • Tracheal cytotoxin, fimbrial antigen and endotoxin • Treatment: • Erythromycin • Immunization diphtheria–tetanus–pertussis (DTP) Aggregatibacter actinomycetemcomitans Characteristics • Primary habitat is likely to be subgingival • Infection is endogenous • Small, short, straight or curved rods • Bleb-like structures on the cell surface • Fresh isolates possess fimbriae Aggregatibacter actinomycetemcomitans Identification • White, translucent, smooth, non-haemolytic colonies in blood agar • Best aerobically with 5–10% carbon dioxide • White, translucent colonies with a star-shaped or crossed cigar-shaped (selective media) Aggregatibacter actinomycetemcomitans Pathogenicity • A number of virulence factors, including lipopolysaccharide • (endotoxin), a leukotoxin, collagenase, cytolethal distending toxin (cdt), epitheliotoxin-bone resorption inducing factor and a protease-cleaving IgG • Together with other coagents, A. actinomycetemcomitans is involved in localized aggressive periodontitis and destructive periodontal disease in adults. • Also isolated from cases of infective endocarditis, and from brain and subcutaneous abscesses. • Treatment with Tetracycline Eikenella Generalities • Resemble Haemophilus spp. • Commensals of oral cavity and intestine • Capnophilic, Gram-negative, short coccobacilli • Corrode agar surface • Predisposition factors: trauma mucosal surface • Brain and abdominal abscesses, peritonitis, endocarditis, osteomyelitis and meningitis • Human bites or fist-fight injuries E. corrodens Capnocytophaga Characteristics • Subgingival area • Long, thin fusiform • Gliding motility • Facultative anaerobes • Opportunistic pathogens (gingivitis immunocompromised patients) HACEK group bacteria HACEK acronym: Haemophilus spp. Aggregatibacter actinomycetemcomitans Cardiobacterium hominis Eikenella corrodens Kingella kingae Fastidious slow growing bacteria Resident in mouth Can cause severe infections, particularly endocarditis Capnophilic, take 7-30 days to become positive Antibiotic sensitivity tests (common drug resistance) Some questions to fix concepts Which of the following statements on Neisseriaceaceae are true? Note: they can be more than one A they possess a capsule B they are commensals of the oral cavity C they demonstrate motility D Neisseria gonorrhoeae causes syphilis E most gonococci are resistant to penicillin Some questions to fix concepts Which of the following statements on Neisseriaceaceae are true? A they possess a capsule TRUE B they are commensals of the oral cavity TRUE C they demonstrate motility FALSE D Neisseria gonorrhoeae causes syphilis FALSE E most gonococci are resistant to penicillin TRUE Virulence factors of Haemophilus influenzae include: Note: they can be more than one A the polysaccharide capsule B immunoglobulin A (IgA) protease C an exotoxin D an endotoxin E a pyrogenic factor causing influenza Some questions to fix concepts Virulence factors of Haemophilus influenzae include: A the polysaccharide capsule B immunoglobulin A (IgA) protease C an exotoxin D an endotoxin E a pyrogenic factor causing influenza Some questions to fix concepts Which of the following statements on Aggregatibacte actinomycetemcomitans are true? Note: they can be more than one A it is a key pathogen in localized aggressive periodontitis B it possesses an IgG protease C it can cause deep-seated abscesses D it can be presumptively identified by star-shaped colonies in selective media E it is susceptible to tetracycline Some questions to fix concepts Which of the following statements on Aggregatibacter actinomycetemcomitans are true? A it is a key pathogen in localized aggressive periodontitis B it possesses an IgG protease C it can cause deep-seated abscesses D it can be presumptively identified by star-shaped colonies in selective media E it is susceptible to tetracycline Neisseriaceae Neisseria N. gonorrhoeae N. meningitidis Moraxella Enterobacteriaceae Eschericheae Escherichia coli Salmonellae Salmonella spp. Veillonella spp. V. parvula Shigellae Parvobacteria Klebsielleae Haemophilus Haemophilus influenzae Bordetella: Bordetella pertussis Pasteurella Aggregatibacter actinomycetemcomitans Eikenella Capnocytophaga HACEK group bacteria Shigella spp. Klebsiella spp. Enterobacter spp. Serratia spp. Pseudomonas Pseudomonas aeruginosa Yersinieae Yersinia pestis Vibrios Vibrio cholerae Vibrio parahaemolyticus Campylobacters Campylobacter spp. Helicobacter pylori Wolinella W. succinogenes Enterobacteria (Enterobacteriaceae) Habitat • Human gut • Density of approximately 109 cells per gram of faeces • Bacteroides (15%) Predominant species • Mostly transient commensals • The oral carriage may increase in old age and xerostomia Enterobacteria Characteristics • Commensal Gram-negative rods • Facultative anaerobes that ferment glucose • Many species are motile and possess a capsule • Endotoxigenic because of lipopolysaccharide outer cell wall • Pili (adhesion) and flagella (locomotion) E. coli Enterobacteria Culture and identification • • • • • • Ordinary media Circular, convex and glistening/mucoid colonies Some motile species form swarming patterns on agar cultures Ferment a large number of carbohydrates Lactose fermenters and non-fermenters API 10E and API 20E • All species have the somatic (O) antigen • Most have the flagellar (H) antigen • Some species have capsular (K) antigen Enterobacteria Culture and identification Circular, convex and glistening/mucoid colonies Swarming of Proteus on an agar plate Enterobacteria Pathogenicity • Potentially pathogenic • Immunosuppressed, most susceptible to infection • Endotoxin shock releases when they are destroyed • Toxic lipopolysaccharide comprises lipid A + Core polysaccharide and the O antigen • Lipid A responsible for symptoms such: fever, hypotension, intravascular coagulation, effects on the immune system • Large doses may cause death Enterobacteria Treatment • Readily acquire resistance-coding plasmids • Ampicillin/amoxicillin, cephalosporins, aminoglycosides, trimethoprim, chloramphenicol and ciprofloxacin Escherichia coli Characteristics • • • • • Gram-negative rods Motile Facultative anaerobe Sometimes capsulate Bile-tolerant E. coli Escherichia coli Pathogenicity Major agent of sepsis Urinary tract infection Young women and elderly adults Simple urethritis to serious pyelonephritis Escherichia coli Pathogenicity Diarrhoeal diseases Enterotoxins (coded by plasmids) Heat-labile (LT) similar to cholera toxin Heat-stable (ST) Enteroinvasiveness Invade intestinal epithelial cells and cause inflammation Adhesive factors (plasmid-coded pili) ’Colonization factor antigens’ Vero cytotoxicity Cytopathic effects on Vero cells (grown in tissue culture) Diarrhoea with haemorrhagic symptoms Escherichia coli Pathogenicity Neonatal meningitis Septicaemia Wound infection (after surgery of the lower intestinal tract) Salmonella spp. Characteristics Poultry products (e.g., flesh and eggs) and pet turtles Occupational salmonellosis (veterinary and slaughterhouse workers) By ingestion of contaminated food or person-to-person (faecal- oral) Carrier state Gram-negative, motile, rods, facultative anaerobes All except S. typhi are non-capsulate Salmonella spp. Pathogenicity Enteric fever (typhoid fever) S. typhi or S. paratyphi Gastroenteritis S. enteritidis serotypes Septicaemia S. dublin or S. choleraesuis Treatment and prevention Proper cooking of foods derived from animal sources. Typhoid vaccine, a killed suspension of S. typhi, is available for those travelling to or living in areas where typhoid fever is endemic Salmonella spp. Typhoid Mary Mary Mallon Irish-born cook Typhoid fever asymptomatic carrier It is not known how many people she infected over the course of her career as a cook. She was quarantined twice by public health authorities, and died after nearly three decades in total quarantine. Shigella spp. Characteristics • Reservoir is the human intestine • Faecal-oral route (minute dose can cause disease) • Gram-negative, non-motile rods, non-capsulated c Shigella spp. Pathogenicity and treatment Pathogenicity Locally invade the intestinal epithelium Not systemically like Salmonella Ileum and colon Inflammatory response Bloody, mucopurulent diarrhoea (dysentery) Neurotoxic exotoxin Treatment Fluid and electrolyte replacement. Antibiotics should be avoided due to resistance. Improving sanitation and personal hygiene to interrupt faecal–oral transmission c Klebsielleae • • • • • • Number of species belonging to this tribe Klebsiella Enterobacter Serratia Indigenous to the human intestinal and respiratory tracts Occasionally isolated from the oral cavity • Transient oral commensals • Serious disease in immunocompromised patients (nosocomial infection) Klebsiella spp. Klebsiella pneumoniae • Severe destructive pneumonia • Nosocomial urinary tract infection • Antiphagocytic capsule • Isolated from the oropharynx or gastrointestinal tract • 5% of healthy people • higher in hospitalized Enterobacter spp. • Indigenous to the intestinal tract • Also on plants and as free-living saprophytes • Nosocomial urinary tract infection • Transient of oral cavity: • E. cloacae and E. aerogenes Serratia spp. S. marcescens Occasionally fatal disease in neonates Immunosuppressed and debilitated individuals Pseudomonas • • • • • • Species are not enterobacteria Gram-negative rods with somewhat similar properties Large number of species, but only a few are human pathogens Widely distributed in the environment and may cause disease Nosocomial infections Pseudomonas aeruginosa special problem in burns patients Pseudomonas spp. P. aeruginosa • Human intestine in few healthy individuals • Large proportion of hospitalized patients • Environmental surfaces, especially under moist conditions • Dental unit water lines, as harmless saprophytes • Gram-negative rods, motile by means of polar flagella Some questions Enterobacteria: (mark if they are true or false) A are frequently implicated in periodontal infections B are Gram-variable C are an important cause of hospital-acquired infections D are found in the oral cavity of up to 25% of the population E are associated with ventilator-associated pneumonia Some questions to fix concepts Enterobacteria: A are frequently implicated in periodontal infections B are Gram-variable C are an important cause of hospital-acquired infections D are found in the oral cavity of up to 25% of the population E are associated with ventilator-associated pneumonia Some questions to fix concepts Which of the following organisms has a polysaccharide capsule? A Shigella sonnei B Klebsiella pneumoniae C Escherichia coli D Salmonella paratyphi E Bacillus anthracis Some questions to fix concepts Which of the following organisms has a polysaccharide capsule? A Shigella sonnei B Klebsiella pneumoniae C Escherichia coli D Salmonella paratyphi E Bacillus anthracis- Polyglutamic acid capsule Some questions to fix concepts Pseudomonas aeruginosa: A is an important agent of nosocomial infections B is resistant to most antimicrobial agents C in dental unit water lines cause significant morbidity D produces an extracellular slime that resists phagocytosis Some questions to fix concepts Pseudomonas aeruginosa: A is an important agent of nosocomial infections B is resistant to most antimicrobial agents C in dental unit water lines cause significant morbidity D produces an extracellular slime that resists phagocytosis Neisseriaceae Neisseria N. gonorrhoeae N. meningitidis Moraxella Enterobacteriaceae Eschericheae Escherichia coli Salmonellae Salmonella spp. Veillonella spp. V. parvula Shigellae Parvobacteria Klebsielleae Haemophilus Haemophilus influenzae Bordetella: Bordetella pertussis Pasteurella Aggregatibacter actinomycetemcomitans Eikenella Capnocytophaga HACEK group bacteria Shigella spp. Klebsiella spp. Enterobacter spp. Serratia spp. Pseudomonas Pseudomonas aeruginosa Yersinieae Yersinia pestis Vibrios Vibrio cholerae Vibrio parahaemolyticus Campylobacters Campylobacter spp. Helicobacter pylori Wolinella W. succinogenes Yersinieae Y. pestis Alexandre Yersin (1894) Y. pestis (the causative agent of plague) bubonic, pneumonic and septicemic plague (’black death’) Y. enterocolitica (enteric and systemic disease) Vibrio, Campylobacter and Wolinella • Morphologically similar, being Gram-negative curved bacilli • Enteric pathogens of humans or part of the normal microbiota • Unusual growth requirements Vibrio spp. V. cholerae • Water contaminated with faeces of patients or carriers • Life-threatening, watery diarrhoea (rice-water stools) • Gram-negative slender bacilli, comma-shaped with pointed ends • Highly motile by means of a single polar flagellum • Diagnosis: Directly by dark-field microscopy V. cholerae Pathogenicity • Colonize intestinal tract in very high numbers 108 cells/mL faeces • Attach but do not invade the intestinal mucosa • Secretion of an enterotoxin • Lag period of 15-45 min V. cholerae Pathogenicity V. cholerae Treatment and prevention • Intravenous administration of fluids and electrolytes • Oral rehydration therapy • Tetracycline Vibrio spp. V. parahaemolyticus • Requires a relatively high salt concentration • Distributed worldwide in marine environments (South-East Asia) • Acute enteritis associated with the consumption of improperly cooked seafood • Half of all cases of food poisoning in Japan • No specific treatment for diarrhoea Campylobacter Campylobacter spp. • Natural reservoir in animals • Acquired from contaminated food and milk • Curved, seagull-shaped, Gram-negative Rods • Mobile with a single polar flagellum Campylobacter spp. Pathogenicity • Gastroenteritis, especially in children • Usually self-limiting, but may last for several days • Heat-labile enterotoxin of C. fetus • C. jejuni agent of Guillain-Barre syndrome • C. rectus isolated from periodontal disease, cytotoxin Helicobacter pylori • Previously classified as Campylobacter • Significant proportion of gastritis and duodenal ulcers • May play a role in gastric cancer • Antimicrobial therapy eradicates the bacteria from the stomach • Small numbers in human supragingival plaque Helicobacter pylori Curiosities: Breath test for diagnostic: H. pylori produces an enzyme called urease, which breaks urea down into ammonia and carbon dioxide. During the test, a tablet containing urea is swallowed and the amount of exhaled carbon dioxide is measured. This indicates the presence of H. pylori in the stomach. Treatment: omeprazole+ clarithromycin + amoxicillin Wolinella spp. W. succinogenes • Curved or helical Gram-negative rods • Motility due to a polar flagellum • High correlation between presence and periodontal disease activity • Pathogenic role is not clear • Can induce alveolar bone loss in gnotobiotic rats Some questions to fix concepts Some questions Vibrio cholerae: A are Gram-negative, highly motile slightly curved rods B grow well in alkaline media C pathogenicity is by means of invasion of the intestinal mucosa D cause dysentery E whole-cell vaccine is effective in preventing the disease Some questions to fix concepts Vibrio cholerae: A are Gram-negative, highly motile slightly curved rods B grow well in alkaline media C pathogenicity is by means of invasion of the intestinal mucosa D cause dysentery E whole-cell vaccine is effective in preventing the disease Some questions to fix concepts Campylobacter spp.: A are implicated in food poisoning B are isolated from active sites of periodontal infection C are not motile D are Gram-negative curved bacilli Some questions t fix concepts Campylobacter spp.: A are implicated in food poisoning B are isolated from active sites of periodontal infection C are not motile D are Gram-negative curved bacilli Ana Isabel García Guillén [email protected] UCAM Universidad Católica de Murcia © UCAM © UCAM