The normal gastrointestinal flora. Gram-negative facultative anaerobic rods. Miklos Fuzi

Transcription

1 The normal gastrointestinal flora Gram-negative facultative anaerobic rods Miklos Fuzi

2 The normal human gastrointestinal flora Comprises of microbes/gram feces Not just commensal but symbiotic relationship with host - fermentation of unused energy substrates - training of the immune system - preventing the growth of harmful, pathogenic microbes - producing vitamins - trophic effects - preventing allergy Can become harmful - cause infections (if immune system is compromised) - increase cancer risk

3 The normal human gastrointestinal flora Acquisition and composition of the normal flora: - following birth infants quickly acquire Escherichia coli and streptococci which generate an anaerobic environment - in breast-fed infants a bifidobacterium flora establishes itself - in formula fed infants and in older children on solid diet other anaerobes: bacteroides (30%!), clostridia, fusobactria, peptococci and additional enterobacteriaceae colonize the gut - additional microbes: candida species, protoozoa

4 The impact of the intestinal flora on obesity An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006, 444, colonization of germ-free mice with obese microbiota results in a significantly greater increase in total body fat than colonization with a lean microbiota

5 Ingestion of lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve Proc. Natl. Acad. Sci. USA 2011, 108, alterations in central GABA receptor expression are implicated in in the pathogenesis of anxiety L. rhamnosus reduced stress-induced corticosterone and anxiety- and depression-related behavior effects were not found in vagotomized mice

6 Gram-negative facultative anaerobic rods: Enterobacteriaceae Habitat: gastrointestinal tract of humans and animals plants soil Grow on eosin methylene blue agar No spore formation Usually motile Metabolism: - sugars are fermented - nitrate degraded to nitrites - catalase: variable - oxidase: negative

7 Enterobacteriaceae: most important genera Escherichia: - The most abundant facultative anaerobic member of the human gastrointestinal normal flora - Produces vitamin K - Some types are enteric pathogens Klebsiella: ankylosing spondilitis (HLA-B27); Crohn disease? Enterobacter Proteus: rheumatoid arthritis (amino acid sequence homology between the urease of P. mirabilis and the joint cartilage collagen) Citrobacter Serratia

8 Enterobacteriaceae: most important features - E. coli: production of indole from tryptophan - Klebsiella: degradation of urea - Enterobacter - Proteus: motile on solid media - Citrobacter - Serratia: production of pigment; capable of colonizing the respiratory tract

9 Identification of Enterobacteriaceae first level Proteus penneri Proteums mirabilis Proteus vulgaris Proteus morganil Proteus rettgeri Providencia stuartil Edwardsiella Salmonella Citrobacter freundil Citrobacter koseri Escherichia coli Hafnia Pantoea agglomerans Serratia Enterobacter aerogenes Enterobacter cloacae Klebsiella oxytoca Klebsiella pneumoniae Motility d d H 2 S d Urease + + d - d d - - d d - - d Indole d d d d d Lysin-decarboxilase Argininedihydrol d Ornithin decarbox d d d d - Citrate d d d - Voges Proskauer Pigment d d Ferment: - Adonit + + d + d Inositol + + d + d d d Lactose d d - + d d Mannitol Saccaharose d - d d d - - d d Polymyxin-B S S S S R S S S S S S S R R R R R R

10 Identification of proteus and providencia spp. Proteus Providencia heimbachae rustigianii stuartii biocsoport 6 stuartii biocsoport 5 stuartii biocsoport 4 myxofaciens panneri inconstans rettgeri morganii ssp.sibonii Morganii ssp.morganii vulgaris mirabilis Urease Indole H 2 S d Methyl red d + Voges-Proskauer d Gelatine d Phenylalaninedeaminase Omithinedecarboxylase Prod. of gas from glucose d d d d - Fermentation - Glucose Adonit Inositol d - Maltose d - Mannitol Mannose Rhamnose d Trehalose + d d

11 Eosin-methylene blue agar Selective and differentiating medium: - Eosin and methylene blue inhibit the growth of Gram-positive bacteria - Anionactive detergent (eg. Na-laurylsulphate) inhibit the swarming of proteae - Contains lactose. Bacteria degrading lactose produce acid that precipitates eosin that will be stained by methylene blue the colonies of lactose positive bacteria are blue those of the lactose negatives are pink or grayish - Advantage: acid will be formed just under colonies

12 E. coli E. coli Eo VA Br

13 SSI Bi VA Klebsiella DC EM Br

14 Proteus growing on eosin methylene blue agar

15 Klebsiella growing on blood agar plate

16 Proteus

17 Serratia marcescens growing on blood agar plate

18 Enterobacteriaceae extraintestinal infections Most frequent nosocomial infections - Surgical wound infection (late onset) - Urogenital infection /often catheter associated/ - Pneumonia /ventilation/ - Meningitis - Sepsis (klebsiella in neonatal int. care units) Most frequent community-acquired infections - Urogenital infections /E. coli/ - Pneumonia /klebsiella/ - Otitis externa /infection of the external auditory canal: E. coli, proteus/

19 Uropathogenic E. coli Causes 90 per cent of community-acquired urethral and bladder infections Source of infection: normal GI flora Infection is more frequent in women Infection can be ascending resulting in pyelonephritis, prostatitis Uropathogenic strains harbour special virulence factors

20 Uropathogenic E. coli Characteristics of uropathogenic strains: - have P fimbriae that specifically bind to P blood group antigens which are present also on the surface of uroepithelial cells - produce haemolysins which damage also epithelial cells - capable of forming colonies inside human cells (hiding from immune response)

21 Enterobacteriaceae: Antibiotic resistance Most frequently used agents: - cephalosporins - carbapenems - aminoglycosides - fluoroquinolones Most important mechanisms of resistance - Production of β-lactamase - Efflux systems - Alteration of membrane proteins - Production of modifying enzymes - Mutations in target molecules

22 β-lactamase enzymes Large family of enzymes Many bacteria produces β-lactamases including free-living species Grouping: on the basis of structure or efficacy Some members of the family enterobacteriaceae constitutively produce simple β-lactamases the genes of which are usually located on the chromosome As a consequence of extensive use of antibiotics a number highly effective variants of the original β-lactamases evolved in recent decades

23 β-lactamase enzymes The most important types of β-lactamases: - extended-spectrum β-lactamases (ESBLs): klebsiella, E. coli - genes located on plasmids (easily transmit) - confers resistance against penicillins and most cephalosporins - metallo-β-lactamases (MBLs) and other carbapenemases: klebsiella, pseudomonas - genes are located on plasmids or on the chromosome - confer resistance against all β-lactam antibiotics including carbapenems

24 Significance of enteritis Estimated to cause million deaths annually all over the world (prior to the introduction of rehydration with glucoseelectrolyte solution at the beginning of the 1980s the figure could have been twice as high) Deaths occur mostly in children Geographical distribution of cases: Most affected areas: black Africa, India, Southeast Asia

25 Conditions promoting the development of enteric disease Lack of safe drinking water (a problem for 30-35% of humanity) Lack of appropriate sanitation (affects about 50% of humanity) Lack of appropriate food higyene Lack of appropriate cleaning facilities High density of population, crowded residential area

26 The consequences of recurrent enteric infections in childhood Growth retardation due to malabsorption (average: 8.2 cm until 7 years of age; later the child can catch up some growth retardation if infections seize) An intellectual retardation of about 10 IQ points (The brain and synapses develop primarily during the first two years of life) A weakened immune system ( Fitness cost associated with a predisposition to infections) Infection with particular pathogens negatively impact development even without enteric symptoms (enteroaggregative E. coli, cryptosporidium)

27 Genetic susceptibility to infection by enteropathogenic bacteria Salmonella: some IL, HLA, IFNGR genes, TNFA Helicobacter pylori: some IL genes, IFNGR1, TNFA Vibrio cholerae O1: blood group 0 Clostridium difficile: IL-8 Enteroaggregative E. coli: IL-8

28 E. coli causing enteritis Strains of E. coli can acquire pathogenicity factors carried on plasmids or other mobile genetic elements and become enteropathogenic Types of E.coli causing enteritis: - Enteropathogenic E. coli (EPEC) - Enterotoxin producing E. coli (ETEC) - Enteroinvasive E. coli (EIEC) - Enterohaemorrhagic E. coli (EHEC) - Enteroaggregative E. coli (EAEC) Types can not always be distinguished: pathogenicity factors can vary across groups Pathogenicity factors are linked to particular O serotypes in all groups

29 Enteropathogenic (dyspepsia) E. coli (EPEC) Causes disease primarily in children less than 1 year old Capable of adhering to the epithelial cells of the small intestine by its pathogenicity factor: intimine Symptoms: can be serious or mild Pathogenicity is related to certain O serotypes Earlier caused outbreaks in day care centres fro young children Therapy: fluid replacement when symptoms are serious antibiotics

30 Enterotoxin producing E. coli (ETEC) The causative agent of travellers diarrhea prevalent in developing countries symptoms: serious watery diarrhea ; affecting the small intestine Pathogenicity factors: usually located on plasmids Serotypes: diverse Identification: demonstration of pathogenicity genes with PCR underdiagnosed - adhesion factors (colonization factors) usually on fimbriae; types sepcific for particular animal species exist - toxins: LT (heat labile), ST (heat stable) Therapy: antibiotics, fluid replacement

31 Enteroinvasive E. coli (EIEC) Symptoms are same as those of shigellosis (bloody diarrhea - dysentery) EIEC strains carry the same virulence plasmid and virulence genes as shigellae EIEC strains are often lactose negative and nonmotile EIEC O antigens are also related to those of shigellas Transmission: food, contact (not as efficient as with shigellas) Most common serogroup: O124

32 Enterohaemorrhagic E. coli (EHEC) Symptoms: -serious bloody enteritis, affecting primarily the colon -haemolytic uremic syndrome (HUS): haemolytic anaemia with acute renal failure Transmission: food (beef, milk), contact Pathogenesis: - adhesion factors - toxins: Shiga-like (SLT) damaging capillaries; transmitted by phages; demonstration: PCR Most important serotype: O157 strains usually sorbitol negative Therapy: antibiotics, fluid replacement

33 Enteroaggregative E. coli (EAEC) Attachment to epithelial cells is associated with the aggregation of the bacteria Symptoms: serious watery diarrhea chronic diarrhea Pathogenicity factors: pili, fimbriae The fimbriae aggregate human collagen, fibronectin, laminin Identification: demonstration of pathogenicity factors by PCR underdiagnosed

34 Yersinia enterocolitica Motile, lactose negative coccobacilli Many serotypes: mainly the O3 and O9 strains are widespread in Europe Symptoms: diarrhea, swelling of lymphatic glands, ileitis terminalis Reservoir: animal, human Transmission: contact, food Pathogenicity: adhesion proteins Secondary autoimmune sequel: arthritis Diagnosis: culture of Y. enterocolitica on selective media Therapy: antibiotics

35 Vibrionaceae family Most important group: Vibrio genus Curved Gram-negative rods, motile, oxidase positive Natural habitat: saline-, sweet waters Strains can be grouped on the basis of salt requirement: halophilic, non-halophilic strains Vibrios are usually susceptible to acidic environment but tolerate alkaline ph well (characteristic used for isolation)

36 Vibrio cholerae Most important species in the genus Vibrio On the basis of O antigen more than 100 serogroups are recognized; all carry the same flagella antigen Large epidemics are caused by serogroups O1 and O139 The O1 strains have two biotypes: - classic - El Tor Both groups are subdivided according to the structure of the O antigen for serotypes: Ogawa, Inaba, Hikojima

37 Vibrio cholerae Natural habitat: sea (Banghlades), lives in copepods (crustacean); dormant form survives for months in the sediment of estuaries Transmission: water, food (many bacteria are required for infection) Epidemics: often pandemics (prevalent in India, black Afrika, South-America)

38 Cholera (1) Incubation: from a few hours to a couple of days depending on the number of infecting bacteria Symptoms: serious watery diarrhea, desiccation occurs within hours often causing hypovolaemic shock Pathogenesis: - the pathogen attaches to the epithelial cells of the small intestine with its adhesins. - produces cholera toxin (coded for by a phage inserted in the chromosome) similar to the heat labile toxin of the ETEC strains. Toxin s pathomechanism: activation of adenylate cyclase, camp accumulated within cells escape of ions, water

39 Cholera (2) Diagnosis: isolation of the pathogen Medium: TCBS tiosulphate-citrate-bilesaccharose Enrichment: alkaline peptone water Therapy: quick fluid and salt replacement antibiotics (tetracycline) Vaccine: of limited value

40 Vibrio colonies on TCBS agar

41

42 Other vibrios Halophile vibrios require salt for growth Important species: V. parahaemolyticus V. vulnificus Transmission: not sufficiently heat treated sea food swimming in sea water Symptoms: usually not serious Skin/wound infection does occur

43 Campylobacter genus Gram-negative curved rods; motile Require microaerophilic atmosphere Oxidase positive Relevant species: C. jejuni, C. coli, C. fetus, C. lari Habitat: animals Transmission: contact, food Symptoms: variable (can be serious) Culture: on selective medium in microaerophilic atmosphere at 42 C Therapy: macrolide, fluoroquinolone antibiotics Secondary autoimmune sequel: Guillain-Barre syndrome

44 Campylobacter - Gram stain

45 Colonies of campylobacter on selective medium

46 Recorded cases of campylobacterosis in the United Kingdom Annual number of cases in Hungary: about 6000

47 Helicobacter pylori Gram-negative curved rods, sometimes assume non-culturable coccoid forms Microaerophilic Motile Oxidase and catalase positives Strongly urease positives ensures survival in acidic gastric environment Strains harbouring the Cag pathogenicity island are the most virulent (type IV secretion system; CagA protein)

48 Helicobacter pylori About 50% of humanity carries H. pylori Carriage is symptomless in about 80% of cases Conditions caused by H. pylori - gastritis - ulcer - gastric carcinoma - MALT (mucosa-associated lymphoid tissue) lymphoma

49 Helicobacter pylori Culture: sample to be taken into transport medium Isolation: on selective medium (similar to campylobacter agar), at 37 C microaerophilic atmosphere Diagnosis: urease breath test (UBT) labeled urea swallowed; exhaled labeled carbondioxide detected detection of antigen in feces (ELISA, latex) detection of antibody from urine or blood (ELISA) isolation of pathogen from gastric biopsy sample when determination of antibiotic resistance is necessary demonstration of H. pylori DNA in gastric biopsy sample by PCR

50 Helicobacter pylori Therapy: - cephalosporins - macrolides Resistence is not rare! Proton pump inhibitors Extended, repeated treatment

51 Helicobacter pylori

52 Helicobacter pylori: pathogenicity

53 Helicobacter pylori on gastric epithelial cells

54 Thank you for your attention