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3. The University of Szeged

4. Case of a 60-year-old alcoholic patient Clinical symptoms Diagnostics Low grade fever during the Chest radiograph past 2 weeks Computed tomography scan Foul-smelling sputum During surgery sample was Anaemia, weight loss, WBC taken from the abscess for culture

5. Microbiological finding Sputum was investigated by Gram-stain

7. Microbiological finding Sputum was investigated by Gram-stain

8. A 60-year-old alcoholic patient (cont.) Treatment Lobectomy (surgical resection was contraindicated due to the risk of spillage of the abscess contents) Prolonged antimicrobial therapy against the mixed flora and postural drainage were carried out (in the present case i.v. imipenem, followed by amoxicillin-clavulanic acid p.o.) The mortality rate of lung abscess in most recent series of publications <15% (32-34% before the antibiotic era)

9. Conditions predisposing to pleuropulmonary anaerobic infections Aspiration due to an altered level of consciousness Alcohol use Cerebrovascular accident General anaesthesia Drug overdose Seizure disorder Shock Aspiration due to dysphagia Oesophageal disease Neurological disease Oral surgery Intestinal obstruction

10. Conditions predisposing to pleuropulmonary anaerobic infections (cont.) Periodontal disease (low-level oral hygiene) Bronchiectasis Septic embolization or bacteraemia “Postobstructive” pneumonia

11. The main clinical entities in the lung where anaerobes may be involved Aspiration pneumonia (anaerobic necrotizing bronchopneumonia) Anaerobic empyema Lung abscess

12. The superior segments of the lower lobes and posterior segments of both upper lobes are frequently involved in aspiration pneumonia and lung abscess Dependent segments of the lung in the supine positionDependent segments of the lung in the supine position

13. Aspiration pneumonia following oesophagogastroduodenoscopy A before the procedure B 12 hours later Left sided infiltrates due to aspiration of gastric contens These early inflamatory events are genrally due to gastric acid and enzymes However later bacterial aspiration pneumonia occurs in >60% of the cases of chemical aspiration Acid content in the lung induces the release of proinflamatory cytokines including TNF-alfa, IL8 There are other cytokines which recruit neutrophils into the lungA before the procedure B 12 hours later Left sided infiltrates due to aspiration of gastric contens These early inflamatory events are genrally due to gastric acid and enzymes However later bacterial aspiration pneumonia occurs in >60% of the cases of chemical aspiration Acid content in the lung induces the release of proinflamatory cytokines including TNF-alfa, IL8 There are other cytokines which recruit neutrophils into the lung

14. Anaerobic empyema

15. Lung abscess Differential diagnosis of a cavitary lesion on a chest radiograph Bacteria (anaerobic oral flora members, S. aureus, Str. milleri group, Enterobacteriaceae, P. aeruginosa, Actinomyces, etc.) Mycobacteria (TB and non-TB) Fungi (endemic fungi, Aspergillus, Cryptococcus) Parasites (E. histolytica, Echinococcus ) Neoplasms (lymphoma, carcinoma) Vasculitis (Wegener granulomatosis) Cystic bronchiectasis Pulmonary sequestration Sarcoidosis Empyema with air fluid may be mistaken for lung absess on chest radiography computed thomography scen is usefull to distiguish absess from empyeamaEmpyema with air fluid may be mistaken for lung absess on chest radiography computed thomography scen is usefull to distiguish absess from empyeama

16. Anaerobes in nosocomial pneumonia

17. Prevalence of anaerobic bacteria in different infections (summarized from several publications by S. Finegold) Bacteraemia 5-20% Brain abscess 89% Subdural empyema 10% Chronic otitis 52% Aspiration pneumonia 93% Lung abscess 95% Empyema 30-50% Abdominal infection after surgery 93% Liver abscess 50-100% Appendicitis, peritonitis 96% Vulvovaginal abscess 74% Pelvic abscess 92% Endometritis 73% Bacterial vaginosis 100%

18. Anaerobes and pleuropulmonary infections (according to recent data ) Kato et al.: Incidence of anaerobic infections among patients with pulmonary diseases CID (1996): Anaerobes were isolated in 50%, 100% and 33% of cases of pneumonia, lung abscess and AE chronic lower respiratory tract infection, respectively, if no prior antibiotic usage was present. (56 episodes) Majon P.: Oral health and respiratory infection. J. Can. Dent. Assoc. (2002): Anaerobes may facilitate the overreaction of the inflammatory process that leads to destruction of the connective tissues, which is present both in periodontal disease and in emphysema. Okuda K at al.: Involvement of periodontopathic anaerobes in aspiration pneumonia. J. Periodontol. (2005): In mice model it was confirmed that P. gingivalis and T. denticola has a synergistic effect in causing sever bronchopneumonia and high mortality with a parallel increase of cytokines including TNF-a in the BALF of the mice Porphyromonas gingivalis, Treponema denticola BALF bronchoalveolar lavage fluidPorphyromonas gingivalis, Treponema denticola BALF bronchoalveolar lavage fluid

19. Anaerobes and pleuropulmonary infections (according to recent data ) Wang J-L., et al.: Changing bacteriology of adult C-A lung abscess in Taiwan: K. pneumoniae versus anaerobes. CID (2oo5) Percutenious trasthoracic aspiration, surgical specimens, pleural effusion sample, blood culture From 9o patients 118 bacteria were isolated 41 anaerobes (12 in pure culture) and 77 aerobes (59 in pure culture). K. pneumoniae was isolated from 3o patients (28 in pure culture) Bartlett JG.: The role of anaerobicc bacteria in lung abscess (editorial commentary) CID (2oo5) Out of 336 cases only 9o (27%) were evaluated (selection bias) 25% of the patients had recived antibiotics for >3 days prior bact. studies For anaerobic bacteria, the burden of proof is the lab. background

20. Finegold (1977): An anaerobe is a bacterium that requires a reduced oxygen tension for growth and fails to grow on the surface of solid media in 10% CO2 in air (18% oxygen).

21. Why are anaerobic bacteria important in general and in pulmonary infections? They were among the first living organisms on the Earth They dominate our normal flora on mucosal surfaces (1011-1012 CFU/gram of dental plaque; aerobe:anaerobe 1:1000) They can cause severe, life-threatening infections, also in the lung They are definitely underdiagnosed We do not follow the development of resistance as much as in aerobes

22. Few requests from the clinicians - Few CM laboratories carry out high-level anaerobic diagnostics - Due to available potent drugs, therapy is often empirical - Time and work-consuming methods to isolate and identify anaerobic bacteria from mixed infections

23. Oxygen is toxic for anaerobic bacteria Az elektronhiány elöször a z anaerob baktériumok életfontosságú funkcióinak lelassulását jelentik (Bacterio staticus hatás A toxikus anyagok felszaporodása az anaerobok pusztulásához vezet. A folyamat gyorsasága és hatékonysága függ az adott species enzimtermelésétol (Szuperoxid dizmutáz, kataláz, peroxidáz) Eloször superoxid anion keletkezikAz elektronhiány elöször a z anaerob baktériumok életfontosságú funkcióinak lelassulását jelentik (Bacterio staticus hatás A toxikus anyagok felszaporodása az anaerobok pusztulásához vezet. A folyamat gyorsasága és hatékonysága függ az adott species enzimtermelésétol (Szuperoxid dizmutáz, kataláz, peroxidáz) Eloször superoxid anion keletkezik

24. Anaerobes commonly encountered in pleuropulmonary infections

25. Prevotella melaninogenica

26. Fusobacterium nucleatum

27. Peptostreptococcus anaerobius

28. Virulence factors associated with anaerobes frequently found in lung infections Bacteroides fragilis Capsular polysaccharides LPS Proteases Enterotoxin Hemagglutinin Neuraminidase Porphyromonas gingivalis Proteases LPS Capsule Haemolysin Prevotella spp. LPS Proteases Fusobacterium necrophorum Leukotoxin Haemolysin LPS Phospholipase Protease Fusobacterium nucleatum LPS Adhesins Proteases Leukotoxin

29. Gingival crevice- rés hasadékGingival crevice- rés hasadék

30. Fogazat = dentitionFogazat = dentition

31. What is needed to be successful in the diagnosis and treatment of anaerobic lung infections From the side of the clinician To think of the possibility of an anaerobic infection To avoid contamination of the sample by the normal oral flora To send the sample to the laboratory in an anaerobic environment as soon as possible From the side of the laboratory To have proper facilities to culture strict anaerobic bacteria To use special media To have special interest in anaerobes

32. Basic rules of sample-taking in lung infections where anaerobic bacteria are assumed Samples should be taken by (transtracheal aspiration) transthoracic needle aspiration in a protected way through bronchoscopy (protected brush) protected BAL during surgery Sample taken by swab versus sample taken by syringe Transport : normally in transport medium in a plastic tube; if >2 h required for transport, then in a glass tube

33. A suger plug protects the contamination of the lumen the plug is expelled from near the turget area The same is true for the protected specimen brushingA suger plug protects the contamination of the lumen the plug is expelled from near the turget area The same is true for the protected specimen brushing

34. How to send samples to the laboratory to detect anaerobes

36. Incubation in an anaerobic environment Átmenet = transitionÁtmenet = transition

38. Processing of the specimen Direct examination: Gram staining (or native ) - preliminary diagnosis Direct GLC examination – preliminary examination Aerobic and CO2 primary culture result (24-48 h) Anaerobic primary culture result (48-96 h) (Aerobic/anaerobic liquid cultures – as a back-up) Isolation of colonies from mixed cultures Species and resistance determination – final diagnosis

39. Identification levels Level I.: isolation presumptive identification of the most common anaerobic bacteria maintain anaerobes in pure culture send to reference laboratory

40. Identification levels Level II: simple tests to identify the anaerobic bacteria miniaturized biochemical systems (API ANA, REMEL) rapid enzyme detection panels (RapID ANA, Crystal, VITEK) disks (ROSCO tablets) gas-liquid chromatography susceptibility testing Level III: Reference laboratories – several classical biochemical tests Modern molecular biological methods (PCR, LCR, ARDRA, typing methods…)

41. Primary plate incubated anaerobically

43. Special potency disk patterns Vanco Kana Colistin SPS 5 µg 1000 µg 10 µg B. fragilis group R R R Campylobacter/B. ureolyticus R S S Fusobacterium spp. R S S Bilophila/Suttorella R S S Desulfomonas/Desulfovibrio R S R Porphyromonas spp. S R R Veillonella spp. R S S Capnocytophaga spp. R S R Prevotella spp. R R S Selenomonas spp. R S R Pept. anaerobius S S R S

44. Antibiotic resistance determination in the case of anaerobic bacteria

45. Therapy of anaerobic lung infections Antibiotics for empirical therapy for lung abscess and aspiration pneumonia

46. Antibiotics which may be used in the treatment of anaerobic lung infections (Resistance to antibiotics in anaerobes) Ampicillin – amoxicillin (>90% R) ß-Lactam + ß-lactamase inhibitor (3-5% R) Clindamycin (5-35% R) Erythromycin – tetracyclin (>80% R) 4th -generation quinolones (moxifloxacin, gatifloxacin) (???) Carbapenems (<1%) Metronidazole (in combination) Chloramphemicol!!!Chloramphemicol!!!

47. Resistance trends in Bacteroides isolates in the USA and Europe 1Cuchural et al. 1990; Antimicrob Agents Chemother 34: 479-480. 2Snydman et al. 2007; Antimicrob Agents Chemother 51: 1649-1655. 3Hedberg at al. 2003; Clinical Microbiology and Infection 8: 475-488

48. Prevalence of antibiotic resistances among other anaerobes

49. Prevalence of cfiA gene and carbapenem resistance among Bacteroides strain, in different countries Country (no.) cfiA + Resistance to IMP France (500) 2.4% 0.8% (Podglajen 1995) UK (175) 6.9% 0.6% (Edwards 1999) Hungary (345) 5.4% 0.8% (Soki 2000; Terhes 2005) Japan (286) 1.9-4.1% 1.2-0.8% (Yamazoe 1999)

50. Therapy of anaerobic lung infections (cont.) Antibiotics (mono or combination therapy active against anaerobes) Surgery (if needed) Lung abscesses usually drain themselves through communication with large airways Drainage of lung abscess to contralateral lung with patient positioning CT-guided percutaneous drainage (if necessary)

51. To summarize Anaerobic bacteria are frequently involved in lower respiratory tract infections following aspiration Sample-taking should avoid contamination with the normal flora Isolation and identification of anaerobic bacteria in such infections need special requirements Anaerobic bacteria can also acquire antibiotic resistance; empirical treatment may fail in such cases

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