GRAM POSITIVE BACILLI

DANILO D. DEVEZA JR., M.D.

Spore-Forming Gram Positive Bacilli Bacilli species Clostridium species Bacillus species Bacillus anthracis Bacillus cereus Large aerobic, gram positive rods in chains Most are saprophylic Most do not cause disease Important diseases: Anthrax, Food Poisoning Spores located in center Non-motile Cut glass appearance in culture Use nitrogen & carbon for energy Spores are resistant to environmental changes, dry heat, certain disinfectants

Cutaneous anthrax 1-7 days: pruritic rash Papule vesicle necrotic ulcer Lesion has central black eschar Edema, lymphangitis, lymphadenopathy Healing by granulation & leaves a scar Incubation period: 6 weeks Inhalation (Wool sorters disease) Hemorrhagic necrosis & edema of mediastinum Pleural effusion Sepsis GI: bowel ulceration Brain: hemorrhagic menigitis

DIAGNOSTICS

Bacillus anthracis Anthrax: primarily disease of herbivores Humans are infected incidentally Acquired by the entry of spores Injured skin (cutaneous anthrax) Mucous membrane (gastrointestinal anthrax) Inhalation (inhalation anthrax) PATHOLOGY Spores germinate in tissues at the site of entry Formation of edema & congestion Spread via lymphatics Three Anthrax toxin Protective antigen (PA) Edema factor (EF) Lethal factor (LF) PA binds to specific cell receptors: entry of EF & LF EF + PA = edema toxin LF + PA = lethal toxin (virulence factor) CLINICAL FINDINGS Humans: 95% (cutaneous), 5% (inhalation)

Specimem: fluid from local lesion, blood & sputum Chains of large gram positive rods Blood agar: non-hemolytic gray to white colonies, with comma shaped outgrowths (Medusa head) TREATMENT Ciprofloxacin: recommended Penicillin G, Gentamycin, Streptomycin PREVENTION Proper disposal of animal carcasses Decontamination of animal products Protective handling of potentially infected materials Active immunization of domestic animals Bacillus cereus Produce toxins that cause disease Spores germinate, vegetative cells produce toxins

CLINICAL FINDINGS Emetic type (rice) Nausea, vomiting, abdominal cramps Self limiting Diarrheal type (meat dishes & sauces) Diarrhea w/ abdominal pain & cramps Enterotoxin : pre-formed or produced in the intestine Eye infections Organisms are introduced by foreign bodies Local & systemic infections Endocarditis, meningitis, osteomyelitis Presence of medical device (IV lines) & IV drugs Presence of B. cereus in stool is not diagnostic 105 bacteria or more per gram of food is diagnostic Culture: exhibit motility swarming

Large, anaerobic, gram (+), motile rods Decompose proteins or form toxins or both Spore is place centrally, sub-terminally or terminally Produce large raised colonies (C. perfringens) Small colonies (C. tetani) Many produce hemolysis on blood agar (C. perfringens: double zone) Ferment a variety of sugars Many digest proteins

Clostridium botulinium CHARACTERISTICS Causes Botulism Found in soil Produce toxins: Type A, B, E, occasionally F: human illness Among the most toxic substances Destroyed by heating for 20 mins at 100C PATHOGENESIS Illness is not infection INTOXICATION Toxins acts by blocking the release of acetylcholine at synapses & neuromuscular junctions FLACCID PARALYSIS CLINICAL FINDINGS Visual disturbances Dysphagia Speech difficulty Signs of bulbar paralysis Cause of death: respiratory paralysis or cardiac arrest TREATMENT Antitoxins (A, B and E) Supportive Ventilation

TREATMENT Drug of choice: vancomycin or clindamycin Resistant to penicillins & cephalosporins Clostridium Species Clostridium botulinium Clostridium tetani Clostridium perfringens Clostridium difficile

Clostridium tetani Characteristics Gram (+) bacilli, terminal spores Obligate anaerobes, motile Reservoir Soil/ feces of animals Transmission Puncture wounds/trauma Requires low tissue oxygenation (Eh) LOCALIZED

PATHOGENESIS Spores germinate in the tissues: tetanospasmin A fragment: blocks NT release at inhibitory synapses B fragment: mediates binding to neuron and cell penetration of A fragment Carried intra-axonally to CNS Binds to ganglioside receptors Blocks release of inhibitory mediators (glycine and GABA) at spinal synapses Excitatory neurons are unopposed extreme muscle spasm

CLINICAL FINDINGS

Incubation period: 4 5 days (up to weeks) Tonic contraction of voluntary muscles Spasm first in area of injury, then the muscles of the jaw External stimuli may precipitate muscle spasm Death: spasm of respiratory muscles DIAGNOSIS Clinical TREATMENT Hyperimmune human globulin (TIG) to neutralize toxin + Metronidazole or Penicillin Spasmolytic drugs (diazepam), debride, delay course PREVENTION

TETANUS Trismus, risus sardonicus, opisthotonus

DTP, DTaP, Td TIG Proper wound care Wound Management Clostridium perfringens CHARACTERISTICS Encapsulated Non motile Double hemolysis Ferment CHO Reservoir Soil and human colon INVASIVE TOXINS Alpha toxin (Lecithinase) Necrotizing & hemolytic effect Theta toxin Necrotizing & hemolytic effect DNase & Hyaluronidaes Digest collagen Enterotoxin Alters cell membrane, disrupting ion transport PATHOGENESIS Spores germinate under anaerobic conditions in tissues Distention of tissues &interference of blood supply, presence of toxins Spread of infection Tissue necrosis

Hemolytic anemia Severe toxemia Gas gangrene (clostridial myonecrosis) Mixed infection Toxigenic & proteolytic clostridia Various cocci & gram negative bacteria

CLINICAL FINDINGS (Gas Gangrene) Spreads in 1-3 days Crepitation & subcutaneous tissues & muscles Foul smelling discharge Necrosis, fever Toxemia shock DEATH (Food poisoning) Enterotoxin forms in GUT Diarrhea without vomiting or fever in 6-18 hours Resolves in 1-2 days DIAGNOSIS Clinical Laboratory Culture Chopped meat Growth + gas BAP

 Target or double zone of hemolysis DISEASES Gas gangrene (myonecrosis) Anaerobic cellulitis Food poisoning (alpha toxin) Necrotic enteritis (fire in the bowel) Clostridial endometritis TREATMENT Surgical debridement: most important Antibiotics: Penicillin Food poisoning: supportive Clostridium difficile PSEUDOMEMBRANOUS COLITIS Drug resistant C. difficile produce toxins Toxin A: enterotoxin Toxin B: cytotoxin Pseudomembranes/ microabscesses in bowels Watery/ bloody diarrhea Clindamycin, Cephalosporins, Amoxicillin, Ampicillin TREATMENT Discontinue offending antibiotic Metronidazole or vancomycin ANTIBIOTIC-ASSOCIATED DIARRHEA Administration of antibiotics leads to mild to moderate diarrhea Less severe than pseudomembranous colitis 25% associated with C. difficile DIAGNOSIS Toxins (stools) Endoscopic exam

Non-Spore Forming Gram Positive Bacilli Listeria Erysipelothrix Actinomycetes Listeria monocytogenes Gram positive Short rod Catalase positive Beta hemolysis on BA Tumbling motility at 22 28oC Diffrentiates from diptheroids Widespread of disease in human & animals Important food-borne pathogen Survive in refrigerator temperature (4oC) Survive low pH Survive high salt conditions Overcome food preservation and safety barriers Antigenic Classifications 1/2a 1/2b 4b: causes most food-borne outbreaks

PATHOGENESIS Ami, Fbp A, Flagellin protein Bind to host cells & virulence Internalin A Interacts with E-cadherin: cell well protein in epithelial cells Promote phagocytosis Listeriolysin O: enzyme Lyses membrane, bacteria escape to cytoplasm Act A: surface protein Induces host cell actin polymerization Propels bacteria to cell membrane Cause formation of Filipods Filipods Ingested by epithelial cells, macrophages & hepatocytes Lifecycle begins abain

RESERVOIR Widespread: animals (gastrointestinal and genital tracts), Unpasteurized milk products Plants and soil Cold growth: soft cheeses, deli meats, cabbages (coleslaw) PERINATAL HUMAN LISTERIOSIS Early onset syndrome Granulomatosis infantseptica Infection in utero Neonatal sepsis, pustular & granulomas Death; before / after delivery Late onset syndrome Meningitis: birth to 3rd week of life Caused by serotype 4b High mortality rate ADULTS Listeria meningoencephalitis Immunocompromised Insidious to fulminant Bacteremia Focal infections TREATMENT Ampicillin, Erythromycin, IV Trimethoprom-sulfamethoxazole Ampicillin + Gentamycin recommended PREVENTION Precautions with food may reduce incidence Erysipelothrix rhusopathiae Distinguishing Characteristics: Gram positive bacilli, non-branching Catalase (-), Oxidase (-), Indole (-) Alpha hemolysis on BA Produced H2S on TSI (Butt Black) Differentiated from L. monocytogenes Distributed in land & sea animals Transmitted by direct inoculation Greatest risk: fishermen, fish handlers, butchers, those in contact with animal products ERYSIPELOID/ SEA FINGER/ WHALE FINGER

Icubation Period: 2-7 days Raised lesion w/ violaceous color Pain & swelling No pus

TREATMENT Penicillin G

Actinomycetes Aerobic Large diverse group of gram positive bacteria Form chains or filaments Categories: Acid fast positive: Mycobacteria Weakly positive: Nocardia & Rhodococcus Acid fast negative: Streptomyces & Actinimadura Nocardia Human infection: N. nova complex N. farcinica N. asteroides type IV N. brasiliensis DISTINGUISHING CHARACTERISTICS Aerobic Gram-positive branching rods Catalase positive Partially acid-fast Produce urease Can digest paraffin Inhalation of bacteria Not transmitted from person to person Opportunistic infection Corticosteroid treatment, immunosuppression, organ transplantation, AIDS, TB Begins with lobar pneumonia Mimic TB Granulona & caseation are rare Abscess formation: usual pathologic process Spread to CNS, skin, kidney & eyes

TREATMENT Trimethoprim-Sulfamethoxazole Treatment of choice Amikacin, imipenem, minocycline, linezolide & ceftaxime Surgical drainage Actinomycetoma Mycetoma (Madura Foot) Most Common cause: N. asteroides, N. brasiliensis, Streptomyces somaliensis & Actinomadura madurae Localized, slowly progressive chronic infection Begins in subcutaneous tissues & spreads to adjacent tissues Destructive & painless ANAEROBIC BACTERIOLOGY Definition of Terms Aerobic Bacteria require oxygen, and will not grow in the absence of oxygen Anaerobic Bacteria do not use oxygen for growth & metabolism but obtain their energy from fermentation reactions Capnophilic Bacteria require CO2 for growth Facultative Anaerobes can grow either oxidatively or use fermentation reactions to obtain energy FACTORS THAT INHIBIT GROWTH OF ANAEROBES BY OXYGEN Toxic compounds are produced e.g. H2O2 , Superoxides Absence of catalase & superoxide dismutase Oxidation of essential sulfhydyl groups in enzymes without sufficient reducing power to regenerate them ANAEROBIC BACTERIA OF CLINICAL IMPORTANCE Bacilli Gram-negative

Bacteriodes fragilis Prevotella melaninogenica Fusobacterium Gram-positive Actinomyces Lactobacillus Clostridium Cocci Gram-positive Peptostreptococcus Peptococcus Gram-negative Veilonella PATHOGENESIS OF ANAEROBIC INFECTIONS Polysaccharide capsule Ability to induce abscess formation Lipopolysaccharide Endotoxin: lack lipopolysaccaride structures with endotoxic activity Not directly produce clinical signs of sepsis Enzymes Proteases, Neuraminidases, Cyclolysins Cause hemolysis of erythrocytes Damage and destroys tissues Superoxide dismutase (some bacteria) Can survive in the presence of oxygen for days

DIAGNOSIS OF ANAEROBIC INFECTIONS Foul-smelling discharge (pus) Short-chain fatty acid products of anaerobic metabolism Infection in proximity to a mucosal surface Anaerobes are part of normal flora Gas in tissues Production of CO2 and H2 Negative aerobic cultures Involve mixture of organisms Form closed spaced infections or burrowing through tissues (Lungs, brain, pleura, pelvis) Most are susceptible to penicillin G Except: Bacteroides, some Prevotella species

Favored by reduce blood supply, necrotic tissues, low Eh Interfere with delivery of antibiotics Uses special collection methods & transport media

for the Presumptive Identification of Anaerobes

METHODS USED TO PRODUCE ANAEROBIOSIS Gas Pak Jar, Brewer Jar, Torbal Jar Cooked meat medium / Chopped cooked meat medium sealed with petrolatum Anaerobic glove box & chamber METHODS USED TO PRODUCE ANAEROBIOSIS PRAS Medium Pre-Reduced Anaerobically Sterilized medium e.g. Roll Tube of Hungate

KVC PATTERN RRR- B. fragilis SRS Fusobacterium B. ureolyticus Veillonella SSR- Clostridium, gram (+) cocci RSR- Porphyromonas, P. anaerobius RRS- Prevotella RESPIRATORY TRACT Prevotella melaninogenica, Fusobacterium & Peptostreptoccocus Periodeontal infections, perioral abscess, sinusitis, mastoiditis Saliva aspiration: necrotizing pneumonia, lung abscess & empyema CENTRAL NERVOUS SYSTEM Brain abscess, subdural empyema, septic thrombophlebitis Originate from respiratory tract, spread hematogenously INTRA-ABDOMINAL & PELVIC INFECTIONS Flora of colon: B. fragilis, Clostridia, Peptostreptococcus Infection due to perforated bowel Prevotella originate from female genital organs

Thioglycollate medium Aerobic Anaerobic Microaerophilic Resazurin Kanamycin-VancomycinColistin (KVC) test Antibi otic Disks

SKIN & SOFT TISSUES Anaerobes & aerobes: synergistic infections Gangrene, necrotizing fasciitis, cellulitis Anaerobic Infections TREATMENT Surgical drainage: most important Antimicrobial Therapy Penicillin G Clindamycin

Preferred for infections above diaphragm Metronidazole

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