Gram Negative Anaerobic Rods Cerrahpasa Medical Faculty ANAEROBIC BACTERIA Gram Negative Anaerobic Rods & Anaerobic cocci Doç.Dr.Hrisi BAHAR Istanbul University Cerrahpasa Medical Faculty

Lıfe without air…. ●Anaerobic means "life without air." ● Anaerobic bacteria grow in places which completely, or almost completely, lack oxygen. ● They are normally found in the mouth, gastrointestinal tract, and vagina, and on the skin.

Anaerobic bacteria ● Obligate anaerobes replicate at sites with low oxidation-reduction potential (eg, necrotic, devascularized tissue). ●Obligate anaerobes have been categorized based on their O2 tolerance: ● Strict anaerobes grow in ≤ 0.4% O2; ● Moderate anaerobes grow in 0.8 to 2.5% O2; ● Aerotolerant anaerobes grow in ≥ 2.5% O2.

Anaerobic Infection ● An “anaerobic infection” is an infection caused by bacteria called anaerobes which can not grow in the presence of oxygen. ● Anaerobic bacteria can infect deep wounds, deep tissues, and internal organs where there is little oxygen. ● These infections are characterized by abscess formation, foul-smelling pus, and tissue destruction

Anaerobic Infection ● Anaerobic bacteria can cause an infection when a normal barrier is damaged due to surgery, injury, trauma or disease. ● The low oxygen condition can result from blood vessel disease, shock, injury, and surgery.

Anaerobic bacteria can cause infection anywhere in the body -1- ● Mouth, head, and neck. ● Lung. ● Intraabdominal. ● Female genital tract

Anaerobic bacteria can cause infection anywhere in the body -3- ● Skin and soft tissue. ● Central nervous system ● Bloodstream

Specimen collection from the site of infection -1- The keys to effective anaerobic bacteria cultures include ● Collecting a contamination-free specimen ● Protecting it from oxygen exposure

Specimen collection from the site of infection -2- ● Swabs should be avoided when collecting specimens for anaerobic culture because cotton fibers may be detrimental to anaerobes. ● Abscesses or fluids can be aspirated using a sterile syringe that is then tightly capped to prevent entry of air.

Specimen collection from the site of infection -3- ● Tissue samples should be placed into a degassed bag and sealed, or into a gassed out screw top vial that may contain oxygen-free prereduced culture medium and tightly capped.

Specimen collection from the site of infection -4- ● All procedures must be performed aseptically. ● The physician who collects the specimen should be prepared to take two samples, One for anaerobic culture and One for aerobic culture, since it is unknown whether the pathogen can grow with or without oxygen. ● In addition, health care professionals should document any antibiotics that the patient is currently taking and any medical conditions that could influence growth of bacteria.

Condition predisposing to anaerobic infections 1-Exposure of a sterile body location to a high inoculum of indigenous bacteria of mucous membrane flora . 2- Inadequate blood supply and tissue necrosis which lower the oxidation and reduction potential which support the growth of anaerobes.

Condition which can lowers the blood supply and can predispose to anaerobic infection are: ● Splenectomy, ● Neutropenia, ● Immunosuppression, ● Hypogammaglobinemia, ● Leukemia, ● Collagen vascular disease ● Cytotoxic drugs ● Diabetes mellitus ● Trauma, ● Foreign body ● Malignancy ● Surgery ● Edema ● Shock ● Colitis ● Vascular disease

Pathophysiology-1- ● A preexisting infection caused by aerobic or facultative organisms can alter the local tissue conditions and make them more favorable for the growth of anaerobes. ● Impairment in defense mechanisms due to anaerobic conditions can favor anaerobic infection.

Pathophysiology-2- These include > Production of leukotoxins > Phagocytosis intracellular killing impairments > Chemotaxis inhibition > Proteases degradation of serum proteins

Clues to anaerobic infection ● Polymicrobial results on Gram stain or culture. ● Gas in pus or infected tissues. ● Foul odor of pus or infected tissues. ● Sulfur granules in pus of infected tissues. ● Necrotic infected tissues. ● Site of infection near mucosa where anaerobic microflora normally reside.

Infections more likely caused by anaerobic bacteria and suspected as anaerobic infections -1- ● Brain abscess ● Human or animal bites, ● Aspiration pneumonia and lung abscesses, ● Amnionitis, endometritis, septic abortions, tubo-ovarian abscess, peritonitis and abdominal abscesses

● Pus-forming necrotizing infections of soft tissue or muscle Infections more likely caused by anaerobic bacteria and suspected as anaerobic infections -2- ● Pus-forming necrotizing infections of soft tissue or muscle ● Some solid malignant tumors,

Management of anaerobic infection -1- The main principles of managing anaerobic infections are ● Neutralizing the toxins produced by anaerobic bacteria, ● Preventing the local proliferation of these organisms by altering the environment and preventing their dissemination and spread to healthy tissues.

Management of anaerobic infection -2- ● Toxin can be neutralized by specific antitoxins, mainly in infections caused by Clostridia (tetanus and botulism). ● Controlling the environment can be attained by: > Draining the pus, > Surgical debriding of necrotic tissue, > Improving blood circulation, > Improving tissue oxygenation. > Therapy with hyperbaric oxygen may also be useful.

Management of anaerobic infection -3- The main goal of antimicrobials is in restricting the local and systemic spread of the microorganisms. ● The available parenteral antimicrobials for most infections are metronidazole, clindamycin, chloramphenicol, cefoxitin, a penicillin (i.e. ticarcillin, ampicillin, piperacillin) and a beta-lactamase inhibitor (i.e. clavulanic acid, sulbactam, tazobactam), and a carbapenem (imipenem, meropenem, doripenem, ertapenem).

Management of anaerobic infection -4- ● An antimicrobial effective against Gram-negative enteric bacilli (i.e. aminoglycoside) or an anti-pseudomonal cephalosporin (i.e. cefepime ) are generally added to metronidazole, and occasionally cefoxitin when treating intra-abdominal infections to provide coverage for these organisms. ● Clindamycin should not be used as a single agent as empiric therapy for abdominal infections.

Management of anaerobic infection -5- ● Penicillin can be added to metronidazole in treating of intracranial, pulmonary and dental infections to provide coverage against microaerophilic streptococci, and Actinomyces. ● Oral agents adequate for polymicrobial oral infections include the combinations of , clindamycin and metronidazole plus a macrolide

Management of anaerobic infection -6- ● A macrolide can be added to metronidazole in treating upper respiratory infections to cover S. aureus and aerobic streptococci. ● Penicillin can be added to clindamycin to supplement its coverage against Peptostreptococcus spp. and other Gram-positive anaerobic organisms

Antimicrobials that are ineffective Anaerobic bacteria are very commonly recovered in chronic infections, and are often found following the failure of therapy with them, such as trimethoprim-sulfamethoxazole (co-trimoxazole), aminoglycosides and the earlier quinolones

Gram Negative Anaerobic Rods ● The anaerobic Gram negative bacilli constitute the predominant bacterial flora of the gastrointestinal tract. ● These organisms outnumber the facultative bacteria approximately 1000:1. ● They are also significant flora on the urogenital and oropharyngeal mucosa.

Gram Negative Anaerobic Rods ● The anaerobic Gram negative bacilli are isolated in over one half of infections involving anaerobes. They can be placed into 5 groups ● 1)Bacteroides fragilis group (bile resistant, VanR, KanR, ColR) ● 2)Pigmented, saccharolytic Prevotella (bile sensitive, VanR, KanR, ColV)

● 3)Pigmented, asaccharolytic Porphyromonas (bile sensitive, VanS, KanR, ColR) ● 4)Non‑pigmented Bacteroides and non-pigmented Prevotella (bile sensitive, VanR, KanR/S, ColV) ● 5)Fusobacterium species (bile variable, VanR, KanS, ColS)

Bacteroides fragilis (Bile Resistant) Group ● Seventy percent of the isolates from this group are B. fragilis and B. thetaiotaomicron; other members include B. ovatus, B. vulgatus, B. distasonis. ● Gram Stain-pale irregularly staining pleomorphic cells with rounded ends. Colonies are usually non-hemolytic, grey to white, smooth entire and convex 1-3 mm in diameter; The indole reaction can used to separate the most common isolates of this group; B. fragilis is indole negative while B.thetaiotaomicron is indole positive; both are catalase positive.

Bacteroides fragilis (Bile Resistant) Group ● It is important to identify this group because it is significantly more resistant to antimicrobic therapy than other anaerobes; most resistance is mediated, in part, through the production of β-lactamases. ● The B. fragilis group is found predominantly associated with abdominal or peritoneal anaerobic infections; they are GI tract commensals, but may be recovered on occasion from the female genital tract. ● They are not considered commensal flora of the oropharynx.

Pigmented, saccharolytic, bile sensitive Gram negative rods ● Gram Stain-pale staining coccobacilli, may be pleomorphic; young cultures may appear gram variable. Pigment production may take up to two weeks, faster on laked rabbit blood. Pigmentation may vary from light tan to black; intensity of pigment is useful for identification of members within the group. ● Prevotella melaninogenica, ● P. denticola, ● P. corporis Indole negative; ColR/s, KanR, VanR; light pigment (may require prolonged incubation; >5 days) ● Prevotella intermedia, P. nigrescens Indole positive, ColS, KanR, VanR, black pigment,

Pigmented, asaccharolytic, bile sensitive Gram negative rods Gram Stain-pale staining coccobacilli, may be pleomorphic; colonies are often mucoid. Porphyromonas asaccharolyticus. P. gingivalis. P. endodontalis. They are indole positive, ColR, VanS, KanR, dark pigment, lipase negative.

Pigmented, asaccharolytic, bile sensitive Gram negative rods

Non‑pigmented, bile‑sensitive species ● This group includes B. ureolyticus and B. gracilis Both species can produce a flat colony that pits the agar and both are sensitive to kanamycin. ● Both are recovered from a wide range of infections including, pulmonary, abdominal, urogenital and soft tissue.

Non‑pigmented, bile‑sensitive species Several species of non-pigmented Prevotella are placed in this group. P. buccae and P. oralis are found in the oral cavity and are recovered from pulmonary infections. P. bivia and P. disiens are recovered from female genital infections.

F. nucleatum ,F.necrophorum, Fusobacteria are usually bile sensitive, Fusobacterium Fusobacterium species are found as commensal flora of the oropharynx as well as the gastrointestinal tract and female urogenital tract.3 of the most common isolates are F. nucleatum ,F.necrophorum, F.mortiferum. Fusobacteria are usually bile sensitive, Van R, Col S, Kan S

Fusobacterium The fusobacteria are generally involved in the same types of infections as the pigmented Prevotella and Porphyromonas species. F. necrophorum infections are often refractile to standard therapy for anaerobic infections. This species has been reported to produce a leukocidin that may enhance its potential for virulence.

FUSOBACTERIUM F.necrophorum F.nucleatum F.necrophorum

F. nucleatum Group F. nucleatum colonies often fluoresce chartreuse under UV light. Gram stain usually demonstrates pale staining rods with pointed ends (F. nucleatum ss. nucleatum is always fusiform). F. nucleatum is indole positive. This group is usually involved with infections of the head and neck as well as lower respiratory tract infections.

F. necrophorum F. necrophorum is considered to be a particularly virulent anaerobic pathogen. It has been isolated from severe infections in children and young adults. These infections often begin as pharyngotonsillitis and metastasize to produce multiple abscesses. F. necrophorum infections are often refractile to standard therapy for anaerobic infections. This species has been reported to produce a leukocidin that may enhance its potential for virulence.

F. mortiferum-varium Group ●This group differs from other members of the genus in that they are resistant to 20% bile. Members of this group often demonstrate bizarre forms when Gram stained. Most members are also indole negative and positive for esculin hydrolysis. ● Colonies of F. mortiferum-varium do not fluoresce.

Lemierre's syndrome Lemierre's syndrome (or “Lemierre's disease”, also known as “postanginal sepsis” and “human necrobacillosis”) is a form of thrombophlebitis usually caused by the bacterium Fusobacterium necrophorum and occasionally by other members of the genus Fusobacterium (F. nucleatum, F. mortiferum and F. varium etc.) and usually affects young, healthy adults

Lemierre's syndrome Pathophysiology ●Lemierre's syndrome is initiated by an infection of the head and neck usually with pharyngitis but it can also be initiated by an otitis, a mastoiditis, a sinusitis or a parotitis. ● During the primary infection, F. necrophorum colonizes the infection site and the infection spreads to the parapharyngeal space. The bacteria then invade the peritonsillar blood vessels where they can spread to the internal jugular vein. In this vein, the bacteria cause the formation of a thrombus containing these bacteria .

Lemierre's syndrome

Anaerobic cocci ● They live in the normal flora of skin, mouth, intestinal tract, and genitourinary tract ●Anaerobic Gram-positive cocci of clinical significance are found in one genera Peptostreptococcus ● There is one Gram-negative genus Veillonella

Anaerobic cocci Clinical Manifestation ●Anaerobic cocci are not involved in any single specific disease process; rather, they may be present in a great variety of infections involving all areas of the human body ●They cause a great variety of infections including abscesses, gangrene, cellulitis, bacteremia, pneumonia, peritonitis, bite wounds, and pelvic inflammatory disease

Anaerobic cocci Pathogenesis ● Infection usually results from invasion of damaged tissue by normal microbial flora. ● Most infections are polymicrobic. however, approximately 10%–15% of all clinical isolates come from pure culture infections. Treatment Antibiotic therapy (e.g., penicillin, clindamycin), abscess drainage, debridement of necrotic tissue

Anaerobic gram-positive cocci ● The species of anaerobic gram-positive cocci isolated most commonly include *Finegoldia magna (Peptostreptococcus magnus) *Peptostreptococcus asaccharolyticus, *Peptostreptococcus anaerobius, *Peptostreptococcus prevotii, and *Peptostreptococcus micros ● The species of anaerobic gram-negative cocci isolated most commonly include *Veillonelaa parvula