Bacteriology

·         An understanding of normal, or indigenous, microflora is essential in order to appreciate the abnormal. Usually, anatomic sites contiguous to mucous membranes are not sterile and have a characteristic normal flora.

The skin flora differs as a function of location. Skin adjacent to mucous membranes may share some of the normal flora of the gastrointestinal system. Overall, the predominant bacteria on the skin surface are S. epidermidis and Propionibacterium, an anaerobic diphtheroid.

The gastrointestinal tract is sterile at birth and soon develops a characteristic flora as a function of diet. In the adult, anaerobes such as B. fragilis and Bifidobacterium may outnumber coliforms and enterococci by a ratio of 1000:1. The colon contains 1011 to 1012 bacteria per gram of feces.

The mouth is part of the gastrointestinal tract, but its indigenous flora shows some distinct differences. While anaerobes are present in large numbers, particularly in the gingival crevice, the eruption of teeth at 6 to 9 months of age leads to colonization by organisms such as Streptococcus mutans and S. sanguis, both alpha-hemolytic streptococci. An edentulous person loses alpha-hemolytic streptococci as normal flora.

 Soon after birth, the vagina becomes colonized by lactobacilli. As the female matures, lactobacilli may still be predominant, but anaerobic cocci, diphtheroids, and anaerobic Gram-negative rods also are found as part of the indigenous flora. Changes in the chemical or microbiologic ecology of the vagina can have marked effects on normal flora and may promote infection such as vaginitis or vaginosis.

·         Two human forms of ehrlichiosis can occur:

human monocytic ehrlichiosis (HME), caused by E. chaffeensis; and

       human granulocytic ehrlichiosis (HGE), caused by an Anaplasma phagocytophilum.

Ehrlichiosis was previously recognized only as a veterinary pathogen. HME infection is transmitted by the brown dog tick and A. americanum. HGE infection is transmitted by I. scapularis, the same tick that transmits Lyme disease. Both infections cause fever and leukopenia. A rash rarely occurs. E. chaffeensis infects monocytes, and HGE infects granulocytes; both organisms produce inclusion bodies called morulae.

·         Listeria is a common inhabitant of farm animals and can be readily isolated from silage, hay, and barnyard soil. Humans at the extremes of age are most susceptible to Listeria infection but only recently has food been implicated as a vehicle. In the outbreak in Nova Scotia, it is likely that the cabbage used for the coleslaw was fertilized with animal droppings and not properly washed prior to consumption. Major Listeria outbreaks associated with cheese have been seen in the United States and most likely have originated from contaminated milk.

·         Adenosine triphosphate (ATP) is believed to be generated at three reaction points in the electron transport chain: the reductions of flavoprotein, cytochrome b, and cytochrome c. This phenomenon, demonstrated in experiments with mammalian mitochondria, can be expressed in terms of the relationship between the moles of ATP generated for each atom of oxygen consumed—the P/O ratio. In mammalian cells, the P/O ratio is 3; that is, there are three segments in the electron transfer chain in which there is a relatively large free energy drop. In bacteria, however, there appears to be only one or two of these segments. Loss of these phosphorylation sites as well as reactions that bypass these sites of ATP synthesis account for the lower P/O ratio in bacteria. Some bacteria, such as Mycobacterium phlei, have P/O ratios of 3.

·         Penicillin-resistant, non--lactamase-producing, vancomycin-resistant, Gram-positive cocci are most likely Enterococcus faecium. There are a variety of mechanisms for vancomycin resistance in E. faecium and they have been termed Van A, B, or C. These isolates have become one of the most feared nosocomial pathogens in the hospital environment. Unfortunately, no approved antibiotics can successfully treat vancomycin-resistant enterococci (VRE), only some experimental antibiotics such as Synercid.

·         Wound exudates smell bad owing to production of organic acids by such anaerobes as B. fragilis. Black exudates or a black pigment (heme) in the isolated colony is usually a characteristic of Bacteroides (Porphyromonas) melaninogenicus, not B. fragilis.

·         The structural integrity of the beta-lactam ring in penicillins is essential for their antimicrobial activity. Many resistant strains of staphylococci produce an enzyme, penicillinase, that cleaves the beta-lactam ring at the carbon-nitrogen bond. Other organisms, including certain coliform bacteria, produce an amidase enzyme that inactivates penicillin by disrupting the bond between the radical and nitrogen in the free amino group.

·         6-Phosphogluconic acid is a characteristic metabolic intermediate in the pentose-phosphate metabolic pathway. This pathway is used by heterolactic fermenters such as Leuconostoc, the organism responsible for the fermentation of cabbage in the production of sauerkraut. Leuconostoc is a Gram-positive bacterium with a dextran capsule.

·         Dipicolinic acid, formed in the synthesis of diaminopimelate (DAP), is a prominent component of bacterial spores but is not found in vegetative cells or eukaryotic appendages or fimbrial structures. The calcium salt of dipicolinic acid apparently plays an important role in stabilizing spore proteins, but its mechanism of action is unknown. Dipicolinic acid synthetase is an enzyme unique to bacterial spores.

·         ADP-ribosylates G_s = Turn the On, On

Vibrio

ETEC

·         ADP-ribosylates G_i = Turns the Off, Off

Bordetella pertussis

·         Ribosylation of EF₂

Pseudomonas

Diptheria

·         Common bacteria in the back of the throat

o  Strep. Pyogenes

o  S. pneumonia - encapsulated

o  H. influenza - encapsulated

o  Neisseria Cataralis - encapsulated

§  These bacteria can live in the back of throat because they contain IgA Protease to protect against IgA activity

§  Therefore, they are the MCC of sinusitis, otitis, bronchitis, and pneumonia

·         Enterococcus is the only nitrite negative UTI.

·         Cultures of Actinomyces grow as white masses with a domed surface, which is called a “molar tooth” appearance.

·         Nocardiae are aerobic and acid-fast, in contrast to Actinomyces species, which are strict anaerobes and not acid-fast.

Actinomyces israelli is treated with penicillin and Nocardia asteroides is sensitive to trimethoprim-sulfamethoxazole.

·         L. monocytogenes is a short, gram-positive, non-spore-forming bacillus that can produce neonatal disease or can result in stillbirth. Characteristics that are unique to Listeria include a tumbling motility on hanging drop and an umbrella-shaped motility pattern when a specimen is stabbed into a test tube agar slant.

Listeria may be mistaken for diphtheroids on Gram stain; both are gram-positive bacilli. Listerial infection often occurs in immunocompromised patients. Risk factors include cirrhosis, neoplastic disease, renal failure, pregnancy, chronic steroid therapy, and extremes of age (i.e., very young and elderly).

·         Bartonella infections are also characterized by proliferations of blood vessels. Examples of Bartonella include B. quintana, B. henselae, and B. bacilliformis,

B.bacilliformis, the causative agent of Oroya fever.

B. quintana is spread by the human body louse and is the causative agent of trench fever (seen in the trenches of World War I) and bacillary angiomatosis. This latter term refers to a lesion seen in patients with AIDS consisting of a lobular proliferation of capillaries with abundant leukocytoclastic debris.

 B. henselae is the causative agent of cat-scratch fever. Histologically, this disease is characterized by the formation of stellate microabscesses with necrotizing granulomas.

·         Persons with acute and chronic alcoholism become infected with Klebsiella pneumoniae, most often by aspiration. K. pneumoniae pneumonia often localizes in the upper lobe, sometimes the minor fissures bows downward, reflecting the bogginess of the upper lobe involved with this infection.

·         The process of transduction involves the transfer of a portion of DNA from one bacterium to the chromosome of another bacterium by means of a viral infection.

Conjugation is the transfer of a so-called male chromosomal DNA to the DNA of an acceptor, or female, bacterial cell.

Colinearity defines the relationship between genes and proteins in that the sequence of amino acids in proteins is a result of the sequence of base triplets in template genes.

 Recombination is simply the exchange of sequences between two molecules of DNA.

 Transformation results when exogenous DNA fragments are incorporated into the chromosome of another organism, as in the transformation of pneumococcal bacteria that led Avery and McLeod to recognize the genetic significance of DNA.

·         ENTEROBACTERIACEAE ANTIGENIC MARKERS:

1.       O-Antigen: Carbohydrate in the cell wall.

2.       K-Antigen: Capsular antigen, when present.

3.       H-Antigen: Flagellar antigen, when present: Campylobacter, Helicobacter, Escherichia.

4.       F-Antigen: Pili antigen.

·         ENTEROTOXINS A, B, C1, C2, D: Affects the GI-tract in S. AURES food poisoning.

They are heat stable -- heating (or reheating) food does not denature the toxin.

·         Virulence of S.saprophyticus:

Urease: Enable survival in urinary tract.

Tropic for urinary transitional epithelium.

Lysostaphin is lethal to S. AURES and other bugs.

·         B. anthracis species is identified by a String of Pearls formation in penicillin. The colonies are susceptible to penicillin, whereas other Bacillus species are not.

B. Cereus is penicillin-resistant. They will form chains of rods rather than string of pearls in penicillin suspension.

BACILLUS SUBTILIS at Risk: Heroin preparations can be contaminated with it.

·         FRANCISELLA TULARENSIS: CYSTEINE-AGAR must be used.

·         In Legionella, Dieterle Silver Stain shows rod shaped bacteria within macrophages. NO STAIN with Gram Stain, Acid-Fact Stain, or H&E. CYSTEINE is absolutely required for growth.

·         Y. pestis, Bacteria reproduce in flea to the point where they clog the proventriculus. Human bite ------> inoculation of bacteria into host.

·         Pyoderma of Streptococci:

IMPETIGO: Cutaneous lesions in kids, usually on face. Very similar to the Staph bullous lesions, except that these are vesicular lesions because these are actually full of bugs.

ERYSIPELAS: Cutaneous lesions in adults. Painful. Again contains organisms.

·         Todd-Hewitt Broth: Done when few organisms were recovered.

Strep: Granular precipitates on sides and bottom of tube.

Staph: Diffuse turbidity.

·         CAMP TEST: Mix normally non-hemolytic Staph Aureus with specimen, and hemolytic zone is augmented. Staph Aureus is normally only hemolytic at 4C.

·         ENTEROCOCCUS FAECALIS (GROUP D)

Virulence: LIPOTECHOIC acid, very lipid rich, leads to gram-variable appearance.

Treatment: Penicillin resistant, strongly, due to altered Penicillin-binding proteins.

Also have acquired vancomycin and gentamycin resistance.

·         Vincent's Disease can be confused with diphtheria.

 Vincent's Disease is pseudomembranous pharyngitis, and is caused by poor oral hygiene.

Vincent's will show halitosis and signs of gingivitis (swelling, bleeding).Stain with Vincent's disease will show a whole mess of bacteria -- both Gram (+) and Gram (-), while Diphtheria will show only slender Gram (+) rods.

·         P. Vulgaris: Positive for metabolizing tryptophan ------> indole

Ampicillin-resistant. Use aminoglycoside and then wait for sensitivity tests.

P. Mirabilis: Negative for metabolizing tryptophan ------> indole

Treated with Ampicillin

·         NEISSERIA MENINGIDITIS (MENINGEOCOCCUS)

Type-B is the most virulent because it is not immunogenic -- we cannot make protective antibodies against it.It is made out of alpha-2,8-n-acetylneuraminic acid. It is rapidly degraded and thus not immunogenic.

LPS: Induces the Schwartzman Reaction. Causes the rash and can lead to necrosis.

·         BRUCELLOSIS:

Brucella Agar = Selective Agar containing Erythritol, which the bugs subsist on.

Erythritol is found in reproductive tract of cattle, hence these bugs cause abortion in cattle. Humans don't have erythritol so no abortion happens.

·         BORRELIA BURGDORFERI:

Enriched Selected Kelly's Medium. Enriched with fatty acids, but it's still difficult to grow.

·         The 16S rRNA of each species of bacteria has stable (conserved) portions of the sequence. Many copies are present in each organism. Labeled probes specific for the 16S rRNA of a species are added, and the amount of label on the double-stranded hybrid is measured. This technique is widely used for the rapid identification of many organisms. Examples include the most common and important Mycobacterium species, Coccidioides immitis, Histoplasma capsulatum, and others.

Portions of the 16S rRNA are conserved across many species of microorganisms. Amplifying the 16S rRNA using primers to these conserved regions allows isolation and sequencing of the variable regions of the molecules. These variable sequences are genus- or species-specific markers that allow identification of microorganisms. Pathogens that are difficult or impossible to culture in the laboratory have been identified using this technique. One example is Tropheryma whipplei, the cause of Whipple's disease.

·         CHOLERA:

HOLDING MEDIA:  VR Medium, Cary-Blair Medium, Autoclaved sea water

ENRICHMENT MEDIA: Allkaline Peptone water at pH 8.2, Monsur's Taurocholate tellurite peptone water at pH 9.2,

PLATING MEDIA: Alkaline Bile salt agar, Monsur's Gelatin taurocholate trypicase tellurite agar(GTTA) Medium, TCBS Medium.

V. cholerae O139 Bengal is in fact virtually identical to V. cholerae O1 El Tor except for two important differences: production of the novel O139 LPS and of an immunologically related O-antigen polysaccharide capsule. Encapsulation is not a feature of O1 strains and may explain the resistance of O139 strains to human serum in vitro as well as the occasional development of O139 bacteremia.