February 2020 • Microbe Online

Anaerobic infections range from local abscesses to life-threatening infections. In most cases, our own normal flora (i.e., endogenous origin) are involved, but in some cases, exogenous sources play a role. Characteristics of Anaerobic Infections Anaerobic infections usually are polymicrobial; these may include obligate aerobes, facultative anaerobes, or microaerophiles as concomitant microorganisms. In polymicrobial infections, these different types of bacteria can coexist: for example, facultative anaerobes can deplete the amount of oxygen present, making the environment conducive for strict anaerobe for growth. The site of the infection may be non-sterile body parts or the sterile site, when the normal skin or mucous flora reaches there via local trauma, surgery, or viscus perforation (for example appendicitis if not diagnosed and treated timely, may lead to perforation of appendix thus giving enteric anaerobes access to the peritoneal cavity). Tissue necrosis and impaired clearance of a sterile site (chronic sinusitis, pneumonia) also predispose to anaerobic infections. Common locations of infections involving anaerobic bacteria (Image source: Koneman, Ref-1) Common anaerobic infections Brain abscess Dental infections (dental abscesses, gingivitis, and periodontitis) Head and neck infections ( suppurative infections of retropharyngeal abscess, peritonsillar abscess, cervical lymphadenitis, deep neck abscesses, and parotitis) Abdominal abscesses, Aspiration pneumonia, Bite infections (animal/human), Brain abscesses, Lung abscesses, and Necrotizing infections of soft tissue. Etiological agents of anaerobic infections Name of the Organism Gram characteristics Anaerobic infection Clostridium difficile Gram-Positive, spore-forming bacilli C. difficile hospital-acquired (nosocomial) diarrhea Clostridium perfringens Gram-Positive, spore-forming bacilli Gas gangrene (myonecrosis) or soft tissue infections Clostridium botulinum Gram-Positive, spore-forming bacilli Infant botulism, food-borne botulism, and wound botulism. Actinomyces israelii Gram-positive, non-spore-forming bacilli Actinomycosis Propionibacterium acne Gram-positive, non-spore-forming bacilli Acne vulgaris Bifidobacterium Gram-positive, non-spore-forming bacilli Usually non-pathogenic but it may cause chronic otitis media, abdominal abscesses, and peritonitis in children. Lactobacillus Gram-positive, non-spore-forming bacilli Organism with low pathogenic potential but its involvement has been seen in abdominal abscesses, aspiration pneumonia, and bacteremia, particularly in neonates. Peptococcus and Peptostreptococcus Gram-positive, cocci Chronic otitis media, chronic sinusitis, aspiration pneumonia, and pelvic inflammatory disease Bacteroides fragilis Gram-negative, non-spore-forming bacilli Intra-abdominal abscesses, aspiration pneumonia, brain abscesses etc. Fusobacterium spp Gram-negative, non-spore-forming bacilli Peritonsillar abscesses Campylobacter spp Gram-negative, non-spore-forming bacilli Acute gastroenteritis Prevotella spp Gram-negative, non-spore-forming bacilli Peritonsillar abscesses, retropharyngeal abscesses, and perineal or perianal infections such as pilonidal abscess; mostly in children. Veillonella spp Gram-negative, diplococci Abdominal abscesses and aspiration pneumonia in children Common clues to anaerobic infections are: foul-smelling discharge, presence of gas in the tissues, necrotic tissue, abscess formation, the unique morphology of certain anaerobes on Gram's Stain, and failure to obtain growth on aerobic culture despite the presence of organisms on Gram-stained direct smear. Acceptable Specimens for Anaerobic Culture Specimens for anaerobic culture should be properly collected and transported. Indigenous anaerobes are often present in large numbers as normal flora on mucosal surfaces (e.g., mouth). So the sample from sites known to have anaerobes as part of the normal flora is unacceptable for anaerobic culture. Specimen for anaerobic culture is best obtained by tissue biopsy or by aspiration using a needle and syringe. Generally, the following clinical samples are suitable for anaerobic culture. Bile Biopsy of endometrial tissue obtained with an endometrial suction curette Blood Bone marrow Branchial washings obtained with double lumen plugged catheter Cerebrospinal fluid Culdocentesis aspirate Decubitus ulcer, if obtained from the base of the lesion after thorough debridement of surface debris Fluid from normally sterile sites (e.g. joint) Material aspirated from abscesses (the best specimens are from loculated or walled-off lesions) Percutaneous (direct) lung aspirate or biopsy Peritoneal (ascitic) fluid Sulfur granules from draining fistula Suprapubic bladder aspirate Thoracentesis (pleural) fluid Tissue obtained at biopsy or autopsy Transtrancheal aspirate Uterine contents, if collected using a protected swab Laboratory diagnosis The culture and isolation of anaerobic organisms are not accessible due to their fastidious nature. Proper collection and transport of the sample are of utmost importance to increase their recovery whenever samples are shipped to a reference laboratory. Gram stain is an essential rapid tool for anaerobic bacteriology. All the specimens submitted for anaerobic culture should be examined by Gram staining prior to culture. Gram stain reveals the types and relative numbers of microorganisms and host cells present in the sample. Gram stain also serves as a quality control measure for the adequacy of anaerobic techniques. After the bacteria's isolation, the isolate's clinical relevance is determined by correlating it with the initial Gram stain. Is there any modification of the Gram staining technique for the anaerobes? Yes. We use the same standard gram stain procedure and reagents but the safranin counterstain is left on for 3 to 5 minutes. Alternatively, 0.5% aqueous basic fuchsin can be used as the counterstain. Commonly used methods for anaerobic bacterial cultures are: McIntosh-Fildes jar GasPak Modified candle-jar system Anaerobic glove box Coy anaerobic chambers Pre-reduced anaerobically sterilized medium Sodium azide selective medium and Anoxomat Presumptive identification of anaerobic bacteria can be made using macroscopic examination, examination of Gram-stained smears, cultural characteristics in differential agar media, and results of spot tests. Earlier, microbiologists used to rely on the phenotypic characteristics of the anaerobes for their identification but nowadays, various commercial microsystems (RapID ANA II (Remel), the RapID CB-Plus (Remel), the BBL Crystal ANR ID (BD Microbiology Systems) and the RapID 32A (bioMérieux, Inc.) are available for the identification of anaerobes. Other methods of identification are; the determination of metabolic products by Gas-Liquid Chromatography, the use of molecular and mass spectrometric methods (e.g., MALDI-TOF), etc. What do positive gram stain and negative culture indicate? Positive grams stain with a negative culture report gives information regarding the adequacy of sample collection, transport, and culture methods used. This situation may come in the following mentioned conditions: Poor transport methods Excessive exposure to air during sample processing Inadequate types of media or old media, or The anaerobic system (jar, pouch, and chamber) failed to achieve an anaerobic atmosphere. That microorganisms have been killed by antimicrobial therapy Treatment Treatment of anaerobic infections usually requires a combination of surgical (debridement of necrotic tissue or amputation of a limb) and medical measures. Antibiotic therapy for anaerobic infections is different from that used for many infections involving aerobes or facultative anaerobes. Frequently used drugs for anaerobic infections are penicillin G, chloramphenicol, clindamycin, metronidazole, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate, meropenem, doripenem, and vancomycin. The choice of antimicrobial agents varies with the bacterial species involved. References and further reading Koneman's Color Atlas And Textbook Of Diagnostic Microbiology (check the latest edition) Noor A, Khetarpal S. Anaerobic Infections.