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Anaerobic Bacteriology

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Published: June 2012

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From collection of the proper specimen to selection of appropriate methods for antimicrobial susceptibility testing, working with anaerobic bacteria is challenging. Dr. Rosenblatt discusses all the steps necessary to successfully isolate and identify the pathogen from your specimen.

Presenter: Jon Rosenblatt, MD

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Transcript

Introduction

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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Jon Rosenblatt, a Professor of Medicine and Microbiology, as well as a consultant in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota. Dr. Rosenblatt discusses the process for successful culture and identification of anaerobic bacteria. Thank you, Dr. Rosenblatt.

Thank you, Sharon.

Objectives

The objectives for this session are the selection of appropriate specimens and methods of collection and transport for anaerobic culture, understanding of current methods for culture and identification of anaerobic bacteria, and the indications and methods for antimicrobial susceptibility testing of anaerobes.

Anaerobic Bacteria

It’s important to recognize that anaerobic bacteria are part of the human micro bio. They are normal flora of the skin, gastrointestinal tract, oropharynx, and female genitourinary/gynecologic tracts. They play important roles in normal physiology. Generally they are of low pathogenicitiy except when they occur in “the wrong place at the wrong time” such as a break in normal host barriers such as rupture of the intestinal mucosal barrier. Anaerobes grow in no or low oxygen concentrations in areas where there are low oxidation reduction potentials in the local environment.

Principal Anaerobic Pathogens

Principal anaerobic pathogens include the Bacteroides fragilis group, Fusobacterium, Prevotella and Porphyromonas, Bilophila, the anaerobic cocci, Clostridium, Propionibacterium and other nonspore forming gram-positive bacteria. It’s important to recognize that there have been much recent change in the taxonomy of anaerobes due to development of new molecular methods.

Specimen Selection: Avoid Contamination With Normal Flora

For specimen selection one must avoid contamination with normal flora. Appropriate specimens include blood, and we recognize that inclusion of an anaerobic bottle in the blood culture collection will not only increase the recovery of anaerobes but will also increase recovery of facultative organisms, especially Streptococci. Other appropriate specimens are normally sterile body fluids, including aspirates of pleural and synovial fluids, abscesses or cyst aspirates, surgically obtained tissues, including bone and joints, especially when there is infection of prosthetic joints suspected. One must whenever possible avoid the use of swabs in collection of specimens for anaerobic culture. This is because use of swabs, may enable or encourage collection of inappropriate or inadequate specimens and may not be amenable to suitable transport systems.

Inappropriate Specimens

Inappropriate specimens for anaerobic culture include Sputum, BAL’s, nasopharyngeal or oropharyngeal fluids. Draining sinuses to the skin except when actinomycosis is suspected. Open wounds, voided urine, cervical and vaginal samples, and any specimen which is grossly contaminated by feces including peritoneal fluid and there has been perforation of the intestinal mucosa. The only exception for culture feces is when we are trying to diagnose Clostridium difficile infection. This will not be further discussed during this session.

Collection and Transport

Collection and transport can be accomplished using needle and syringe aspirates of fluid or pus or from surgically obtained tissue. Again, avoid the use of swabs whenever possible. Fluid or pus is injected into sealed anaerobic transport vials and these should contain an EH indicator in the fluid or gel or agar in the bottom of the tube. Place tissues in upright oxygen-free tubes which contain an anaerobic gas or carbon dioxide and seal the tube. It is optimal to culture the specimen within two to three hours of collection.

Anaerobic Transport Vials

This slide is an illustration of the anaerobic transport vials which are in use in the microbiology laboratory at the Mayo Clinic. On the left is a sterile carbon dioxide filled tube for use with tissues. After the tissue is placed in the tube the cap can be resealed. On the right, is an illustration of an anaerobic vial containing peptone yeast extract with resazurin indicator to insure that not excessive oxygen has been introduced.

Anaerobic Transporters

This slide illustrates commercial versions of the same type of anaerobic transporters shown in the previous slide.

Anaerobic Culture Media

Once the specimen has reached the laboratory, it should be inoculated on fresh enriched, selective, and differential agar plates. Examples are Brucella or CDC Blood Agar, Laked Blood Agar containing Kanamycin and Vancomycin or LKV, bile-esculin agar, phenylethyl alcohol agar or PEA, and thioglycollate broth tubes. So called PRAS media that is pre-reduced anaerobically sterilized may be utilized but it is quite expensive and cumbersome to use and generally not necessary for the clinical laboratory. Inoculated plates are then placed in a holding jar until that jar is full and ready to be set up as an anaerobic jar or for use in an anaerobic chamber glove box. Initial incubation is for forty eight hours before the plates undergo examination. Plates should also be checked for new growth for up to seven days and thioglycolate broth tubes should be kept for a total of fourteen days especially if one suspects infection of the total joint arthroplasty.

Primary Culture CO2 Holding Jar with Flow Meter

This slide illustrates primary plates, inoculated and placed in a holding jar which is being flushed with carbon dioxide to maintain an oxygen free environment.

Incubation

Incubation is carried out in GasPak jars that are either mechanically flushed with an anaerobic gas or made anaerobic through the use of commercial products such as Anaero-Packs or GasPaks. All jars should have a methylene blue EH indicator to ensure that anaerobiosis has been accomplished. One may also use an anaerobic glove box either for primary incubation or for examination and work on plates which have been set up in anaerobic jars.

Anaerobic Jars Set Up Using the Anaero-Pack

This is an illustration of anaerobic jars which have been set up through the use of the Anaero-Pack. Note that using this system; the plating catalyst is not necessary to be placed in the lid of the jar.

Anaerobic Glove Box

This slide illustrates the anaerobic glove box in use in the clinical laboratory at the Mayo Clinic. Other commercial products may utilize a rigid frame or the use of sleeveless hand portals or other convenience devices.

Anaerobe Culture Triage

After forty eight hours of incubation, the cultures are examined to determine the extent of identification to be performed. In our laboratory, we use a system called anaerobic culture triage. That is, we fully speciate anaerobes if one or two anaerobes are present by morphology, however, no more than three total morphologies should be present. When there are more than two anaerobes present or more than three total morphologies, we use Partial Plus in which we speciate anaerobic gram-negative bacilli and probable clostridia.

We use a system called partial identification in which we report morphologies present by gram stain and aerotolerant when there are four or more total morphologies or three or more anaerobic morphologies. And finally, we simply report the presence of mixed flora when there are five or more anaerobic morphologies present.

Identification

Identification initially is determined on the basis of growth on selective or differential media, the gram stain and colonial morphology, present of pigmentation on colonies, hydrolysis of esculin and fluorescence under UV light. Rapid test which can be initially helpful are zones of inhibition around Kanamycin, Vancomycin and colistin disks or rapid indole, urease, nitrate and catalase test. We also employ a beta-lactamase or Cefinase disk on gram-negative anaerobic bacteria. Egg yolk agar may also be utilized to determine the production of lecithinase and lipase.

Colonial Morphology

This slide illustrates the colonial morphology of various anaerobes growing on blood agar plates. In the upper left hand corner, we see the spreading colonies of Clostridium barati. In the upper right hand corner, we see the large mucoid colonies of Bacteroides fragilis. And in the lower right hand corner, we see the pigmented colonies of Prevotella melaninogenica.

Pigmented Colonies

On this slide in the upper right hand column, we also see pigmented colonies of Prevotella denticola and in the lower right hand column we see the so called “fried egg appearance” of colonies of Fusobacterium necrophorum.

Rapid Identification Using Gram Stain

Rapid identification of Propionibacterium acnes can be accomplished when seen in an appropriate gram stain and having positive catalase and quick indole test results.

Identification

Identification can be further accomplished through the use of rapid commercial biochemical kits such as the Rapid ID-ANA II, API 20A, Vitek, and others. All have variable accuracy. Generally, they can identify to a species level approximately sixty percent of clinically significant anaerobes. Again, one may choose to use PRAS biochemical’s but they are expensive, cumbersome, and generally, not necessary in the clinical laboratory. Gas liquid chromatography for the detection of all fatty acids may also be employed. Currently the new standard for identification and taxonomy of anaerobes is the use of 16S ribosomal RNA sequencing, which unfortunately is not available in many clinical laboratories. In the future, MALDI-TOF mass spectrometry will probably be employed because it is such a rapid and simple methodology. However, it is expensive equipment wise and at the present time because of limited database, it will not achieve complete identification of anaerobes.

16S Ribosomal RNA Sequence of Bacteroides fragilis

This is a graphic representation of the 16S ribosomal RNA sequence of Bacteroides fragilis.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing of anaerobic bacteria can be utilized for individual patient care on isolates from blood, the brain, bone and joints, when there is critical or long-term treatment considerations or when there is an unpredictable antimicrobial susceptibility pattern anticipated for the isolated organism. It can also be employed when there has been a treatment failure. Another option for performing susceptibility testing is to establish a hospital antibiogram by testing annually batches of organisms and posting of the results to serve as a guide for local physicians and pharmacists.

AST Methods

Methods for anaerobic susceptibility testing include the CLSI reference agar dilution method which is primarily used as a standard for comparison of other methods. However, it is generally not suitable for clinical laboratory use. Broth micro dilution panel plates are commercially available. They are somewhat time consuming and cumbersome and one is restricted to use of the antibiotics which are included in the panel on that particular plate. The E-test method is a concentration gradient strip agar diffusion method which is probably most practical for the average clinical laboratory. There is also a commercially available method called the MIC evaluator. However, currently, it is available only for purchase in Europe. The E-test method requires forty eight hours of incubation and will provide both an MIC and categorical value result.

Illustration of the Components of the E-test Method

This is an illustration of the components of the E-test method. There is available an automated mechanical dispenser for placing the strips on agar plates. However, they can also be placed manually. The MIC is read where the growth of bacteria and the inhibition of that growth intersects with the E-test strip.

Susceptibility Testing by E-test

Susceptibility testing of anaerobic bacteria using the E-test at the Mayo Clinic include routinely the following antimicrobials.

Anaerobe Antimicrobial Panels

However, we do utilize the antimicrobials in various panels for different organisms. For instance, Anaerobic gram-negative bacilli which are beta-lactamase positive are tested using Pipericillin/tazobactam, clindamycin, metronidazole, and ertapenem. Clostridia other than Clostridium perfringens receive testing with Pepericillin/tazobactam, clindamycin, metronidazole, ertapenem, and penicillin. Anaerobic Cocci and nonsporing gram-positive bacilli with the exception of actinomyces are tested using clindamyciin, metronidazole, and penicillin. Propionibacteria are tested with moxifloxacin, minocycline, and penicillin.


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