10-26-09 Introduction to Anaerobic Bacteria of Medical Relevance 1.

Understand the basis of anaerobiosis and the pathogenic mechanisms of anaerobic bacteria. The basic definition of anaerobes is the ability to grow with little or no oxygen requirement. The biochemical basis of anaerobiosis is the ability to use catabolic pathways other than respiration for energy, namely fermentation. Anaerobes are also characterized by their lack of catalase (decomposition of peroxide) or superoxide dismutase (decomposition of superoxide). Obligate anaerobes in particular lack these enzymes and therefore are subject to ROS accumulation and subsequent death in the presence of oxygen. Pathogenic anaerobes may have these enzymes, which allow for their survival. There are four types of anerobes: 1. obligate anaerobes: must be in an environment without oxygen (oxygen causes ROS formation) e.g. Clostridia 2. aerotolerant anaerobes: will tolerate oxygen, but always use fermentative embolism 3. facultative anaerobes: these anaerobes are normally found in oxygen environments; during situations where oxygen is not available, facultative anaerobes switch to fermentation (facultative is flexible) 4. microaerophiles: requires low levels of oxygen at low levels, but are inhibited by high levels of oxygen (micro air) PATHOGENICITY Anaerobes are known for their pathogenicity when they are moved from normal anatomic sites to aseptic tissues i.e. aspiration of GI/mouth fluids, pre-existing periodontal disease (inflammation of tissues that support and surround the teeth), and trauma. Many anaerobes are commensals, and GI is the major source/reservoir for anaerobes. Clostridia is also commonly found in soil. Pathogenicity results from the various virulence factors that anaerobic bacteria possess: tissuedegrading exotoxins, polysaccharide capsules, adhesins, binary toxins, and occasional peroxidase or superoxide dismutase expression that provides resistance to ROS. CULTURE CHARACTERISTICS Anaerobe infections are usually polymicrobic, with aerobic species mixed in. Staph. aureus and Strep. pyogenes are both facultative anaerobes and are associated with deep wounds. Anaerobic infections often occur grossly as localized collections of pus or abscesses, since low partial pressure of oxygen in necrotic areas favor growth of anaerobes. Gas and foul-smelling discharges are often produced. TREATMENT

Penicillin G is the treatment of choice, especially for aerobics arising from oropharyngeal sources. Recall how most anaerobic infections are polymicrobic. Consequently, one or more antibiotics with wide spectrums are often used. For example, Bacteriodes and Prevotella geni are known for their beta lacatamase (and consequently penicillin resistance) activity. They must be treated with penicllin + beta-lactamase inhibitor. The most active drugs against anaerobics are clindamycin and metronidazole, because relatively few anaerobes are resistant to either of them. 2. Know the major groups of pathogenic anaerobic bacteria. Bacilli, Gram Positive, Spore Formers Clostridium: C. tetani, C. perfringens, C. botulinum, C. difficle Bacilli, Gram Positive, non-spore formers Actinomyces Mobiluncus Lactobacillus Propionibacterium Bacilli, Gram Negative Bacteroides (penicillin resistant) Biolophilia Desulfomonas Fusobacterium Mitsuokella Mobiluncus Prevotella Tissierella Cocci, Gram Positive Peptostreptococcus Cocci, Gram Negative Megasphaera Veillonella 3. Understand the modes of action of Clostridial toxins (i.e. potential terror agents). There are two major Clostridial toxins: botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT). Both are binary toxins originating from a single toxin gene. Post-translational modification allows for the expression of A and B domains. The A domains are protein-specific endopeptidases: TeNT A domain cleaves VAMP/synaptobrevin BoNT A domain cleaves syntaxin 1A or SNAP-25, depending on toxin serotype The B domains are target different nerve terminals: TeNT B domain targets GABAergic or Glycinergic synapses afferent to motoneurons BoNT B domain targets neuromuscular junctions

In summary, the two neurotoxins have nearly identical A domains (that inhibit SNARE neurotransmitter exocytosis), but different B domains that produce entirely opposite symptoms (by targeting different neurons): TeNT inhibits release of GABA and Gly inhibitory neurotransmitters at synapses afferent to motoneurons, resulting in spastic paralysis (muscular rigidity or tetany). BoNT inhibits acetylcholine release at skeletal neuromuscular junctions resulting in flaccid paralysis. Why can both of these toxins be used as potential terror agents? Firstly, both TeNT and BoNT can be produced in large quantities via recombinant DNA techniques Secondly, both have low LD50s, making them very toxic So why havent they been successfully used by terrorists? Chlorination of water effectively denatures the toxins; heat will also cause denaturation Water heavily dilutes the toxins, thus requiring truckloads to achieve effective toxicity in water supply Treatment and recovery from TeNT and BoNT Treatment is focused on support measures The biological effects of the toxins diminish steadily but slowly over time, since the toxin proteins are eventually degraded in the nerve cell. Recovery is lengthy because functional synapses must be regenerated. Additional notes on Clostridium perfringens Classification: gram positive bacilli, subterminal spore Location: soil, human/animal GI tract Secretions: produces digestive exotoxins typed A to E (lecithinase, collagenase, hyaluronidase, DNAase, neuraminidase, fibrinolysin, proteases, ADP ribosylase binary toxin) Pathogenesis: major cause of gas gangrene causes self-limiting gastroenteritis symptoms: intense abdominal cramps and diarrhea 8-22 hours after eating infected food (associated with institutional food) self-limits by 24 hours without antibiotics causes soft tissue infections such as cellulitis, myositis, myonecrosis, and septicemia prognosis of myositis and myonecrosis treated with debridement and high-dose penicillin (penicillin G); antitoxins have questionable efficacy Clostridium botulinum Classification: gram positive bacilli, subterminal spore; types A, B, E, F pathogenic Location: soil, meat/animal products, produce Secretions: BoNT binary exotoxin with a very low LD50=0.001 micrograms/mg

BoNT is specific for peripheral cholinergic nerves and prevents release of Ach, causing flaccid paralysis Pathogenesis: Food poisoning does not require live C. botulinum, only the toxin need be present. Foodborne illness: symptoms appear 1-2 days after ingestiondizziness, blurred vision, fixed dilated pupils, dry mouth, abdominal pain; no fever Weakness of peripheral muscles; possible death due to respiratory paralysis Infant botulism: fewer than 100 cases per year, often due to honey; may be cause of sudden infant death syndrome (SIDS) Wound botulism: no GI tract involvement, but symptoms are similar to the foodborne illness with a longer incubation period Diagnosis: detection of bacteria in food or patient serum/feces Treatment: metronidazole or penicillin, botulinum antitox, ventilatory support Recovery is long term due to need for regeneration of neuron junctions

Clostridium tetani Classification: gram positive bacilli, terminal spore, clubbed/racket shaped due to large spores distending the bacteria Location: soil Pathogenesis: acquired by soil contamination of wounds; neonate and cephalic tetanus forms are also observed Secretions: secretes TeNT, a binary exotoxin that blocks release of inhibitory GABA and Gly at presynaptic terminals Symptoms include muscle spasms and rigidity known as generalized tetanus Diagnosis: early symptoms of lockjaw/trismus/sardonic smile, drooling, sweating, irritability, and back spasms late symptoms of cardiac arrhythmia, BP fluctuations, profound sweating, dehydration bacteria may be cultured from wound, though absence does not negate diagnosis Treatment: routine immunization with tetanus toxoid and boosters, plus immediate immunization of patients with soil-contaminated wounds Antitoxin is available, but it only neutralizes toxin before cell binding and is ineffective once TeNT enters the cells Clostridium difficile Classification: gram positive bacilli, spore former (subterminal, spherical or ovoid); obligate anaerobe, motile Location: normal GI flora Pathogenesis: outgrowth of C. difficile from normal GI bacteria occurs with antibiotic treatment; such outgrowth is associated with Secretions: enterotoxin (toxA) and cytotoxin (toxB) Causes antibiotic associated colitis: colitis and hemorrhagic necrosis Toxb may also be associated with cardiac edema

Diagnosis: isolation of C. difficile from feces, or detection of toxA or toxB from immunoassay/cytotoxicity assay. Treatment: discontinuance of offending antibiotic (usually ampicillin or clindamycin) For serious relapsing disease, treat with metronidazole or vancomycin. Relapsing disease is due to death of sporulating vegetative cells, spore survival, and germination of new vegetative cells.