Clinical and lab aspect of anaerobic infection Ali Somily MD, FRCPC,ABMM

Classification 1. Anaerobic spore forming bacilli (Clostridia) 2. Gram negative bacilli non-sporing forming (Bacteroides) 3. Anaerobic streptococci (Peptostreptococcus) 4. Anaerobic staphylococcus (Peptococcus) 5. Gram negative diplococci (Veillonella) 6. Gram positive bacilli (Actinomyces)

Anaerobiosis Lack cytochrome-cannot use oxygen as hydrogen acceptor Lack cytochrome-cannot use oxygen as hydrogen acceptor Most Lack Most Lack – Catalase – Peroxidase Contain flavoprotein so in the presence of oxygen produce H2O2 which is toxic Contain flavoprotein so in the presence of oxygen produce H2O2 which is toxic Some lack enzyme superoxide dismutase so many killed, peroxide and toxic radicales enzyme like fumarate reductase must be in reduced form to work Some lack enzyme superoxide dismutase so many killed, peroxide and toxic radicales enzyme like fumarate reductase must be in reduced form to work

HABITAT I : These organism are normal flora in: These organism are normal flora in: A.Oropharynx A.Oropharynx eg. 1. Bacteroides melaninogenicus eg. 1. Bacteroides melaninogenicus  Now called provetella melaninogenicus – 2. Fusobacteria – 3. Veillonella

HABITAT II: B.Gastrointestinal tract B.Gastrointestinal tract – Found mainly in the large colon in large numbers – Total number of anaerobes = – While all aerobes (including E. coli) = 10 4 – examples are  (1) B acteroides fragilis  (2) Bifidobacterium species C.Female genital tract (mainly in the vagina) C.Female genital tract (mainly in the vagina)

INFECTIONS CAUSED BY,NONSPORING ANAEROBES A.The head, neck and respiratory tract A.The head, neck and respiratory tract B. The lower abdomen and the pelvis B. The lower abdomen and the pelvis

FEATURES OF ANAEROBIC INFECTIONS Characterized by Infections are always near to the site of the body which are habitat. Infections are always near to the site of the body which are habitat. Infection from animal bites. Infection from animal bites. Deep abscesses Deep abscesses The infections are also polymicrobial foul smell The infections are also polymicrobial foul smell Gas formation Gas formation Detection of "Sulphur granules"' due to actinomycosis Detection of "Sulphur granules"' due to actinomycosis Failure to grow organism from pus if not culture anaerobically. Failure to grow organism from pus if not culture anaerobically. Failure to respond to usual antibiotics. Failure to respond to usual antibiotics.

INFECTIONS BEGIN DISRUPTION OF BARRIERS – – TRAUMA – – OPERATIONS – – CANCEROUS INVASION OF TISSUES DISRUPTION OF BLOOD SUPPLY – – DROPS OXYGEN CONTENT OF TISSUE – – DECREASE IN Eh POTENTIAL – – TISSUE NECROSIS

WHAT ARE THE INFECTION CAUSED BY THESE ANAEROBIC ORGANISMS I 1. Post operative wound infection 2. Brain abscess 3. Dental abscesses 4. Lung abscess 5. Intra abdominal abscess, appendicitis, diverculitis 6. All these infection can cause bacteriaemia

WHAT ARE THE INFECTION CAUSED BY THESE ANAEROBIC ORGANISMS II 1. Infection of the female genital tract 2. Septic abortion 3. Puerperal infection or sepsis 4. Endometritis 5. Pelvic abscess Other infections – a) Breast abscess in puerperal sepsis – b) Infection of diabetic patients (diabetic foot infections). – c) Infection of pilonidal sinus

ORAL & DENTAL > 400 SPECIES OF ANO2 IN MOUTH MOST INFECTIONS = POLYMICROBIC – – MIXED ORGANISMS – – ENTER AS A GROUP ANO2 NOT INITIAL INVADER – – USUALLY SECONDARY – – 1 ST ORGANISM DECREASES [O2] & Eh

ORAL & DENTAL COMMONLY ASSOCIATED WITH 1. 1.DENTAL ABSCESSES 2. 2.ROOT CANALS 3. 3.JUVENILE PERIODONTITIS 4. 4.ADULT PERIODONTITIS 5. 5.CLENCHED FIST INJURIES

ENT – HEAD & NECK CHRONIC OTITIS MEDIA CO-PATHOGENS WITH CHRONIC STREP TONSILLITIS ACUTE SINUSITIS – – POST-DENTAL EXTRACTIONS OR TRAUMA – – 2 o INVADER

ENT – HEAD & NECK VINCENT’S ANGINA – – COMBINATION OF FUSOBACTERIUM & SPIROCHETE SPECIES OVERGROWTH – – ANAEROBIC PHARYNGITIS – – GRAY MEMBRANE – – FOUL ODOR

Vincent’s disease Trench mouth Trench mouth Sudden onset of pain in the gingiva (mastication) Sudden onset of pain in the gingiva (mastication) Necrosis of the gingiva Necrosis of the gingiva – interdental papilla – a marginated, punched-out, and eroded appearance A superficial grayish pseudomembrane A superficial grayish pseudomembrane altered taste sensation is present altered taste sensation is present Fever, malaise, and regional lymphadenopathy Fever, malaise, and regional lymphadenopathy

Ludwig’s Angina

Lemierre Syndrome

Expansion of the retropharyngeal soft tissues

PLELRO PULMONARY I FECTION ASPIRATION LUNG ABSCESS ASPIRATION LUNG ABSCESS ASPIRATION PNEUMONIA ASPIRATION PNEUMONIA M ETASTATIC LUNG ABSCESS M ETASTATIC LUNG ABSCESS BRONCHIACTSIS BRONCHIACTSIS ALL OF ABOVE CAN CAUSE EMPYEMA ALL OF ABOVE CAN CAUSE EMPYEMA

LUNG & PLEURAL ASPIRATION PNEUMONIA EMPHYSEMA LUNG ABSCESSES MALIGNANCIES LEUKOPENIA

THORACIC ACTINOMYCOSIS

ACTINOMYCOSIS

Molar tooth appearance of Actinomyces israeIii

Macroscopic colony (left) Gram stain (right) of Actinomyces

SKIN & SOFT TISSUE TRAUMATIZED & DEVITALIZED TISSUE 1. 1.TRAUMATIC WOUNDS 2. 2.HUMAN/ANIMAL BITES 3. 3.ISCHEMIA OF EXTREMITIES DIABETES ATHEROSCLEROSIS

CLENCHED FIST INJURIES

DIABETIC FOOT

HUMAN BITE

NECROTIZING CELLULITIS

PUERPERAL INFECTION SEPTIC ABORTION PUERPERAL ABSCESS PUERPERAL ABSCESS SEPTIC ABORTION SEPTIC ABORTION BACTERAEMIA BACTERAEMIA PELVIC ABSCESS PELVIC ABSCESS ADENXAL ABSCESS ADENXAL ABSCESS PERITONITIS PERITONITIS ENDOMETRITIS ENDOMETRITIS

ABDOMINAL INFECTIONS MANIPULATION, INVASION OR TRAUMA TO GI TRACT 1. 1.TRAUMA 2. 2.SURGERY 3. 3.APPENDICITIS 4. 4.MALIGNANCIES COLON CANCER

CNS HEAD TRAUMA HEMATOGENOUS SPREAD – – FROM ANY INFECTED BODY SITE GEOGRAPHIC SPREAD – – SINUS INFECTIONS – – DENTAL ABSCESSES

BONE & JOINT HEMATOGENOUS SPREAD TRAUMA PERIVASCULAR DISEASE JUVENILE PERIODONTITIS

OTHER INFECTIONS GRAM NEGATIVE BACTREMIA GRAM NEGATIVE BACTREMIA BREAST ABSCESS BREAST ABSCESS AXILLARY ABSCESS AXILLARY ABSCESS INFECTION OF DIABETIS EG.DIABETIC ULCERS INFECTION OF DIABETIS EG.DIABETIC ULCERS INFECTION OF PILONIDAL SINUS INFECTION OF PILONIDAL SINUS PARONYCHIA PARONYCHIA

LABORATORY DIAGNOSIS: When anaerobic infection is suspected; When anaerobic infection is suspected; – a) Specimens have to be collected from the site containing necrotic tissue. – b) Pus is better than swabs. – c) Specimens has to be send to the laboratory within 1/2 hour why? – d) Fluid media like cooked meat broth are the best culture media. – e) Specimens have to incubated anaerobically for 48 hours.

Anaerobic chamber

TREATMENT: Bacteroides fragilis is always resistant to penicillin. Bacteroides fragilis is always resistant to penicillin. But penicillin can he used for other anaerobes But penicillin can he used for other anaerobes Flagyl (metronidazole) is the drug of choice. Flagyl (metronidazole) is the drug of choice. Clindamycin can also be used. Clindamycin can also be used.

CLASSIFICATION 1. Anaerobic spore forming bacilli (Clostridia) 2. Gram negative bacilli nonsporing (Bacteroides) 3. Anaerobic streptococci (Peptostreptococcus) 4. Anaerobic staphylococcus (Peptococcus) 5. Gram negative diplococci (Veillonella) 6. Gram positive bacilli (Actinomyces)

ORGANISM GROUPS GRAM NEGATIVE RODS – – BACTEROIDES – – PREVOTELLA – – PORPHYROMONAS – – FUSOBACTERIUM – – BUTYRIVIBRIO – – SUCCINOMONAS

Bacteroides fragilis

Propionibacterium

Fusobacterium nucleatum

BACTEROIDES STRICT ANAEROBE STRICT ANAEROBE PLEOMORPHIC PLEOMORPHIC GRAM NEGATIVE BACILLI (COCCO BACILLI) GRAM NEGATIVE BACILLI (COCCO BACILLI) NORMAL FLORA IN NORMAL FLORA IN – OROPHARYNX – GASTROINTESTINAL TRACT – VAGINA

BACTEROIDES FRAGILIS GP GROUP = B. FRAGILIS, B. VULGARIS, B.THETAIOTAMICRON, B. UNIFORMIS – – ACCOUNT FOR 1/3 OF ALL ISOLATES – – RESISTANT TO 20% BILE – – RESISTANT TO MANY ANTIBIOTICS PENICILLIN, KANAMYCIN, VANCOMYCIN, COLISTIN – AND MANY MORE

BACTEROIDES FRAGILIS GP GLC = MAJOR ACETIC & SUCCINIC, LACTIC & PROPIONIC ACIDS NO PIGMENTATION OF COLONIES OR FLUORESCENCE

BACTEROIDES OTHER SP BACTEROIDES SPECIES OTHER THAN B. FRAGILIS GROUP – – GLC = MAJOR ACETIC & SUCCINIC ONLY – – BILE SENSITIVE – – RESISTANT TO KANAMYCIN ONLY – – SOME PIGMENTED

BACTEROIDES B. FRAGILIS IN THE GUT AND VAGINA B. FRAGILIS IN THE GUT AND VAGINA B.MELANINOGESUS AND B.ORALIS IN THE MOUTH AND OROPHARYNX B.MELANINOGESUS AND B.ORALIS IN THE MOUTH AND OROPHARYNX B. FRAGILIS PENICILLIN RESISTANT, B. FRAGILIS PENICILLIN RESISTANT, OTHER ARE SENSITIVE, OTHER ARE SENSITIVE, IT IS THE COMMONEST ORGANISM IN THE GUT ORGANISM /GRAM OF FAECES IT IS THE COMMONEST ORGANISM IN THE GUT ORGANISM /GRAM OF FAECES

Bacteroides and other anaerobic bacilli

BACTEROIDES AND FUSOBCTERIUM B.FRAG B.NECROPH ORUS B.MELANINO GENICUS B.CORRODE NS FUSOBACTE RIUM BLACKPIG PITTING---+- INDOLE LYSINE++ BILE GROWTH +

Growth of Bacteroides fragilis on Bacteroides bile-esculin agar

PEPTOCOCCUS NIGER GRAM POSITIVE COCCI GLC = ACETIC, BUTYRIC, ISOBUTYRIC, ISOVALERIC, CAPROIC BLACK PIGMENT

PEPTOSTREPTOCOCCUS GRAM POSITIVE COCCI GLC = ACETIC, SOME BUTYRIC Ps. ASACCHAROLYTICUS INDOLE + Ps. ANAEROBIUS, Ps. MAGNUS, Ps.PREVOTI, Ps. INDOLECUS

STREP & STAPH ANAEROBIC SPECIES OF STAPH AND STREP STREPTOCOCCUS INTERMEDIUS STAPHYLOCOCCUS SACCHAROLYTICUS

VEILLONELLA PARVULA GRAM NEGATIVE COCCI GLC = ACETIC & PROPIONIC NITRATE + HEAD AND NECK INFECTIONS DENTAL ABSCESSES

CLOSTRIDIUM SPECIES LARGE GRAM POSITIVE RODS SPORE FORMATION SPECIFIC DISEASES – – PSEUDOMEMBRANOUS COLITIS – – TETANUS – – BOTULISM – – GANGRENE - MYONECROSIS

C. difficile

CLOSTRIDIA

CLOSTRIDIA Causative Agents For Causative Agents For – 1.Gas gangrene : Cl. perfringens and other e.g septicum – 2.Tetanus : Cl. tetani – 3.Botulism : Cl. botulinum – 4.Toxic enterocolitis : Cl. difficile (Pseudomembernous colitis)

Clostridium perfringens (CI. welchii) Morphology large rods gram +ve Morphology large rods gram +ve With bulging endospores With bulging endospores Not motile Not motile Capsulated Capsulated

Clostridium perfringens

C. perfringens

Culture: A)Blood agar with haemolytic colonies (double zone of haemolysis A)Blood agar with haemolytic colonies (double zone of haemolysis B)Cooked meat medium B)Cooked meat medium Gives the NAGLAR'S Reaction & toxin neutralization on Egg yolk medium & toxin is a phospholipase Gives the NAGLAR'S Reaction & toxin neutralization on Egg yolk medium & toxin is a phospholipase

C. perfringens

NAGLAR'S Reaction

Lipase and/or lecithinase (EYA),

Diseases Caused by C. perfringens 1) Wound Contamination 1) Wound Contamination 2) Wound infection 2) Wound infection 3) Gas Gangrene - most important disease 3) Gas Gangrene - most important disease 4) Gas Gangrene of the uterus in criminal abortion 4) Gas Gangrene of the uterus in criminal abortion 5) Food Poisoning 5) Food Poisoning Spores are swallowed Germinate in gut after 18 hours Spores are swallowed Germinate in gut after 18 hours Toxin Toxin abdominal pain and diarrhoea abdominal pain and diarrhoea

GAS GANGRENE Causes mainly Causes mainly – (Cl perfringens) (Cl. welchil) – CI. novyl, – CI. Septicum – CI oedemaritians Pathogenesis: Pathogenesis: – Traumatic open wounds – Compound fractures – Muscle damages – Contamination with dirt etc, Mainly in war wounds, Mainly in war wounds, Old age, Old age, – Low blood supply Amputation of thigh Amputation of thigh – Prophylaxis with penicillin

NECROTIZING FASCIATITIS

MYOSITIS

Gram Stain of vaginal aspirate 1.Clostridiae necrotizing (myonecrosis)

Prevention and Treatment Remove dead tissue Remove dead tissue Remove debris Remove debris Foreign bodies Foreign bodies Penicillin Penicillin Hyperbaric oxygen Hyperbaric oxygen

TETANUS

Cl.tetani Causative organism Cl.tetani Causative organism Cl.tetani Morphology gram +ve anaerobic with terminal spore Drum Stick appearance Morphology gram +ve anaerobic with terminal spore Drum Stick appearance Lives in soil and animal feaces. e,g horse Lives in soil and animal feaces. e,g horse Any wound can infected if contaminated by spores Any wound can infected if contaminated by spores Face & neck wounds are more dangerous why ? Face & neck wounds are more dangerous why ?

C. tetani

Clinical Features Incubation period 1-2 weeks Incubation period 1-2 weeks Symptoms: Painful muscle spasm around infected wound Symptoms: Painful muscle spasm around infected wound Contraction of muscles Contraction of muscles of face= – Trismus ( Lockjaw) – Risus Sardonicus strychnine Back Back – Araching of Back

Opisthotonus opistho meaning "behind" and tonos meaning "tension", opistho meaning "behind" and tonos meaning "tension", Extrapyramidal effect and is caused by spasm of the axial along the spinal column. Extrapyramidal effect and is caused by spasm of the axial along the spinal column. Caused by Caused by – Tetanus. – Cerebral palsy – Traumatic brain injury

Pathogenesis 1 )Tetanospasmin most important powerful exotoxin 1 )Tetanospasmin most important powerful exotoxin 2)Totanolysin 2)Totanolysin No invasion or Bacteraernia No invasion or Bacteraernia Toxin  is a protein Toxin  is a protein It inhibits transmission of normal inhibitory messages from central nervous system at anterior horn cells of cord It inhibits transmission of normal inhibitory messages from central nervous system at anterior horn cells of cord

Pathogenesis

Diagnosis Mainly by clinical Mainly by clinical Laboratory not important Laboratory not important Lab Lab – Organism strict anaerobe – Very motile, spread on agar.

C. tetani

Prevention Toxoid vaccine: Toxoid vaccine: Vaccination D P T Vaccination D P T 2, 4, 6, 18 months &5 Year 2, 4, 6, 18 months &5 Year Booster every 10 years Booster every 10 years

Treatment. Cleaning of wound Cleaning of wound Removal of Foreign body Removal of Foreign body Specific by antitoxin Specific by antitoxin – Horse serum can caused anaphylaxis & shock must be tested first – Human immunoglobulin – Antibiotics. Penicillin Supportive treatment Supportive treatment – 2. Dark pace, fluids – 3. Sedative valium

CLOSTRIDIUM BOTULINUIM

Habitat Soil,Ponds AND Lakes Soil,Ponds AND Lakes

Toxin Exotoxin Exotoxin Protein Protein Heat labile at 100 O C Heat labile at 100 O C – The most powerful toxin known Lethal dose 1 µg human 3 kg kill all population of the world 3 kg kill all population of the world Dictated for by lysogenic phage Dictated for by lysogenic phage Resist gastrointestinal enzymes Resist gastrointestinal enzymes

Botulism From canned food., sea food e_g. salmon From canned food., sea food e_g. salmon Not well cooked Not well cooked Spores resist heat at 100 o C Spores resist heat at 100 o C  then multiply and produce toxin  then multiply and produce toxin

ENFANTILE BOTULISM Ingestion of Spores  germination in the gut  Botulism Ingestion of Spores  germination in the gut  Botulism Week child Week child Cranial nerve Cranial nerve Constipation Constipation Other Other

Botulism Patogenesis Ingested - incubation period hour Ingested - incubation period hour 7 Types 7 Types Mainly types A, B, E, F Mainly types A, B, E, F Attacks neuromuscular junctions Attacks neuromuscular junctions Prevents release of acetylcholine Prevents release of acetylcholine

Symptoms Funny eye movement as if cranial nerve affected when bulbar area of the brain affected Funny eye movement as if cranial nerve affected when bulbar area of the brain affected Respiratory and circulatory collapse Respiratory and circulatory collapse

SPECIMENS Suspected food Suspected food From the patient From the patient – Faeces growth – Serum Toxin detection by mouse Toxin detection by mouse – incubation paralysis and death

INFANTILE BOTULISM Week lethargic child Week lethargic child Constipation Constipation Respiratory and cardiac arrest Respiratory and cardiac arrest Due to colonization of intestine by CI. botulinum Due to colonization of intestine by CI. botulinum Diagnosis by -Culture of stools Diagnosis by -Culture of stools Detection of toxin in feaces Detection of toxin in feaces

Treatment Treatment 1) Supportive 1) Supportive 2) Horse antitoxin 2) Horse antitoxin Prevention Prevention 1)Adequate pressure cooking autoclaving 1)Adequate pressure cooking autoclaving 2)Heating of food for 10 minutes at 100 O C 2)Heating of food for 10 minutes at 100 O C

Botox

C. DIFFICILE PSEUDOMEMBRANOUS COLITIS – – 90% OF CASES CAUSED BY C. DIFF – – LONG TERM TREATMENT WITH BROAD SPECTRUM ANTIBIOTICS OR CHEMO NOSOCOMIAL DISEASE KNOCK DOWN NORMAL FLORA CLINDAMYCIN, AMPICILLIN, CEPHALOSPORINS CHEMOTHERAPEUTIC AGENTS

C. DIFFICILE OVERGROWTH OF C. DIFFICILE – – TOXIN THEN PRODUCED A -FRAGMENT = ENTEROTOXIN B -FRAGMENT = CYTOLYTIC TOXIN PSEUDOMEMBRANE SIMILAR TO THAT OF C. DIPHTHERIAE – – BACTERIA, FIBRIN, WBC, DEAD – – TISSUE CELLS - TOUGH

C. DIFFICILE DIARRHEA FIRST – – ELECTROLYTE & FLUID LOSS – – LEADS TO DEHYDRATION INTESTINAL BLOCKAGE – – CONTENTS BLOCKED – – COLON BULGES PERFORATION, RUPTURE  SEPSIS

Clinical pictures

C. DIFFICILE RAPID AGGRESSIVE COURSE IN YOUNG CHILDREN DIFFICULT TO SELECTIVELY CULTURE – – 5-10% CULTURE + EVEN WITH CONFIRMED DISEASE – – TOO MANY NORMAL ANO2 PRESENT

C. DIFFICILE SPECIALIZED ISOLATION MEDIA – – CCFA – CYCLOSERINE, CEFOXITIN,FRUCTOSE, EGG YOLK AGAR – – CCMA – CCFA BUT MANNITOL FOR FRUCTOSE – – CDMN – CYSTEINE HYDROCHLORIDE, MOXALACTAM, NORFLOXACIN AGAR

C. difficile

C. DIFFICILE C. DIFFICILE IS NORMAL FLORA – – ISOLATION NOT ENOUGH NEED TOXIN ASSAY TO CONFIRM CELL-FREE STOOL EXTRACT – – LATEX AGGLUTINATION SCREEN SOME CROSS-REACTIVITY – – EIA TO CONFIRM

Major Clostridial Diseases