The use of cell-membrane fractions in anaerobic bacteriology An organic solution to a long-standing problem.

This innovative technique for culturing anaerobes could lighten the microbiologist's workload and save time, money, and space as well.

In today's world, the clinical microbiologist is faced with decreasing resources and an increasing workload, a situation common to many laboratories. Cultivation and identification of anaerobes is only a small fraction of the workload, yet it is one of the more costly, time-consuming, and labor-intensive jobs in the field of microbiology.

Culturing anaerobes--a necessary evil

The problems created by anaerobes in the clinical microbiology laboratory are directly related to their nature, because these organisms must be grown in an environment free of oxygen. Because ambient air contains roughly 20% oxygen, barrier apparatus designed to keep out oxygen must be constructed to house the enrichment media onto which the organisms are cultivated. The equipment and techniques used for working with anaerobes can be traced back to the early days of microbiology; they have not changed significantly over the last 100 years. The overlay of equipment and apparatus needed to establish and maintain an anoxic environment complicate the plating of a specimen to isolate and grow anaerobic bacteria.

Anaerobic bacteria are part of the normal flora found on healthy skin and mucous membranes. Ironically, these same organisms can cause serious and even life-threatening infections in humans and animals. Although it is estimated that 400 to 500 different types of anaerobic bacteria may be present as indigenous flora in and on the body, only a fraction of that number is frequently isolated from properly collected specimens taken from infectious processes. The sites of these infections typically contain polymicrobic mixtures of aerobic and anaerobic bacteria. Isolation of 5 or more microorganisms from a specimen does not imply that the specimen is contaminated. These mixtures of microorganisms frequently act synergistically to produce infection.

Respiratory enzymes replace barrier apparatus

The late Dr. Howard Adler and his colleagues at the biology division of Oak Ridge National Laboratory, Oak Ridge, TN, made a discovery that became a new method to grow anaerobic bacteria on solid surfaces without extraneous paraphernalia. Adler and his colleagues found that cell-membrane fractions containing respiratory enzymes could produce anaerobic environments.

As is often the case with novel ideas, Adler made this discovery by chance while studying the effects of ionizing radiation on Escherichia coli.[1] He and his colleagues were working with a mutant that, after exposure to radiation, retained the ability to grow (into long, nonseptate, multinucleated filaments) but could no longer divide. This was a lethal event for those radiation-damaged cells. Adler and his group were trying to explain the curious observation that such cells recovered the ability to divide if they were incubated in the presence of certain other bacteria. The phenomenon did not seem to require direct contact between the irradiated cells and the "neighbor" (nonirradiated) cells. The researchers found that a particulate fraction derived from the neighbor cells could stimulate division. The objective of their study was to characterize the particulate fraction and to understand the process of cell division.

By the late 1970s, Adler and his colleagues determined that the active particulate fraction was derived from the innermost membrane of the complex envelope that encloses the E. coli cells. This cytoplasmic membrane contains several enzymes and is important in many of the biochernical transformations carried out in bacterial cells.

Eventually, the scientists showed that the particulate fraction was promoting cell division by removing oxygen from the growth environment. They concluded that the membranes removed oxygen because they contained a respiratory enzyme system that transferred hydrogen atoms from various substrates directly to oxygen, thereby producing water. The effect on cell division was shown to be indirect, as the anaerobic environment created by the membrane fraction produced conditions for the damaged cells to continue dividing.

Although disappointed that the findings eliminated his quest for directly stimulating cell division, Adler recognized the practical importance of readily creating anaerobic environments in microbiological media. He and his team had provided a biotechnical solution to a long-standing microbiology problem--how to grow anaerobic microorganisms in an aerobic environment using simple and direct means.

Subsequent studies have shown that the E. coli enzymatic membrane fractions reduce oxygen to very low levels in microbiological environments efficiently and quickly and will continue to remove the oxygen after repeated exposure during the isolation, subculture, and identification process. It is this catalytic property that uniquely solves many of the problems of working with anaerobes.

The enzymatic membrane preparations are specific for oxygen and reduce it directly to water. The substrates (such as lactate and oxygen) and products (such as pyruvate and water) of this reaction are commonly found in bacteriological media, and at the concentrations used, are not toxic or inhibitory to anaerobic bacteria.

Traditional methods

Barrier and catalytic-reaction techniques. Conventional methods rely on barriers such as jars, bags, or chambers, in tandem with vacuum and purge, or on catalytic reactions to create a limited and contained environment suitable for growing the anaerobic microorganism. Most techniques for achieving an anaerobic environment use nonspecific, chemical-reducing agents or oxygen-free gases to sweep oxygen out of bacteriological media. These techniques, which are sometimes combined, are not problem-free. Because chemical agents are often toxic, they can be added only in limited amounts. Treatment with oxygen-free gases also has its limitations. If solid media are exposed to oxygen-free gases, the diffusion of oxygen from the solid into the gas phase is usually slow and inefficient.

Media prepared in the air rapidly form oxidized products, which can be toxic to anaerobic bacteria. Once formed, these oxidized products cannot be removed by subsequently reducing the medium and incubating it in an anaerobic environment. To counteract this adverse affect, media intended for anaerobes must first be prereduced and then anaerobically sterilized, poured, and sealed inside an anaerobic chamber. This method of preparation adds to the complexity and cost of working with anaerobes.

The anaerobic chamber. Many laboratory scientists consider the anaerobic chamber to be the best environment for growing anaerobes. Although well designed for growing anaerobes, it is not well suited for today's multifunctional, resource-strained, clinical microbiology laboratory. It is costly to maintain and awkward to use. It uses valuable space and disrupts the workflow in the clinical microbiology laboratory.

Microbiologists who have a low volume of anaerobic cultures or who simply choose not to invest in an anaerobic chamber may opt for anaerobic jars or bags. These, too, have their limitations. Working with anaerobes outside a chamber will expose them to oxygen, which can damage some strictly anaerobic bacteria, although most clinically important anaerobes can tolerate brief oxygen exposure. Furthermore, working with anaerobes in ambient air frustrates the use of prereduced media. Exposure during the time required to plate or examine a number of patient samples before they are bagged or jarred oxidizes the medium and reduces the benefits expected of these media.

Cell-membrane method evaluation

The microbiology department of the 300-bed Mid-Michigan Medical Center provides laboratory services to the oncological and neurosurgical specialties, 2 community hospitals, and several nursing homes and area clinics. Because of our successful outreach programs, we have substantially increased our work-load without additional manpower; consequently, we are always on the lookout for streamlined, cost-effective techniques that will lighten the burden.

Our department performs approximately 30,000 procedures annually, 2,600 of which are anaerobic cultures. Over the course of nearly 30 years, we have had extensive experience with all types of anaerobic culture methodologies, from gassed-out jars and heat-sealed bags to the anaerobic chamber. Because we were unfamiliar with cell-membrane fractions, which incorporate the mechanisms of anaerobiosis directly into the culture media, we decided to conduct our own comparison study.

We inoculated 212 infected wound specimens in duplicate. Brucella blood agar (BBA) and kanarnycin-vancomycin laked blood agar (KVL) that contained the enzymatic membrane preparation were incubated at 35[degrees]C in 6% [CO.sub.2]. BBA and KVL that did not contain the enzyme were incubated at 35[degrees]C in the anaerobic chamber. [2]

Eighty-seven culture specimens grew anaerobic bacteria, yielding 180 strains. Forty-one strains failed to grow in the anaerobic chamber but grew on the media containing the enzymatic membrane preparations. These strains were predominantly Peptostreptococcus species (28%), Eubacterium sp. (20%), and Propionibacterium sp. (20%). Fourteen strains failed to grow on the agar plates containing the enzyme. No colonial variation was noticed, and the growth rates were similar. (We dismantled and removed our anaerobic chamber at the conclusion of the study.)

Advantages over traditional methods.

By conducting our own comparison study, we were able to see these advantages of the cell-membrane method:

* One technologist could perform total culture workup (aerobic and anaerobic) at the same time and at the same bench.

* Side-by-side comparison of the aerobic and anaerobic plates permitted recognition of the anaerobic colonies sooner and, therefore, quicker preliminary reporting.

* Removal of the anaerobic chamber freed up valuable space.

* Elimination of the chamber barrier created a paper-free work log (results were entered electronically instead of on work cards).

* Anaerobic and aerobic plates were stacked and stored together in the same incubator. Aerotolerance subcultures were stored with the primary plates.

* Enzymatic membrane additive continued to reduce oxygen in the media throughout the culture workup.

Cost analysis. We found that the cost of 2 prereduced anaerobic agar plates in tandem with the anaerobic chamber was higher than the cost of 2 of the same agar plate formulations containing the enzymatic membrane fractions. Our figures, on a cost-per-test analysis basis, showed that a wound culture using the anaerobic chamber method cost $15.64 compared to $14.37 for the cell-membrane method. For sterile fluid cultures, the cost of the anaerobic chamber method was $11.42 compared to $8.88 for the cell-membrane method. [2]

Anaerobic microbiology--a new perspective

Editor, MLO 

Plating patient specimens to isolate, identify, and characterize infectious bacteria will continue to be a basic tool of the clinical microbiologist. We discovered that the complexity associated with anaerobic microbiology was significantly reduced by using the enzymatic membrane preparations, which incorporate the mechanisms of achieving anaerobiosis directly into the growth media. This method eliminates the need for extra equipment, allows the incubation of all patient cultures in an aerobic incubator, removes the need for a separate workspace, and creates a simpler, more efficient workflow.

Colleen Gannon is the microbiology section head at MidMichigan Medical Center, Midland, Ml.

References

(1.) Adler HI, Crow W. A novel approach to the growth of anaerobic microorganisms. Biotech Bioeng. 1981;Symp.II:533-540.

(2.) Gannon CK, Thurston M. A comparison study between Oxyrase anaerobic agar plates and conventional anaerobic glove chamber for the isolation and identification of anaerobic bacteria from clinical wound infections. J Rapid Methods and Auto in Microbiol. 1997;5:13-20. Abstract C-228.

Suggested reading

Adler HI, Carroaco A, Crow W, Gill J. Cytoplasmic membrane fraction that promotes septation in an Escberichiae coli Ion mutant. J Bacteriol 1981;147:326.

Adler HI, Spady G. The use of microbial membranes to achieve anaerobiosis. J Rapid Methods and Auto in Microbiol, 1996;5:1-12.

Carlson J, Nyberg G, Wretben J. Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen. Appl Environ Microbial. 1978;36:223.

Edelstein MAC. Laboratory diagnosis of anaerobic infections in humans. In: Finegold SM, and George WL, edo. Anaerobic Infections in Humans. Orlando, FL: Academic Press; 1989.

Frolander F, Carlson J. Bactericidal effect of anaerobic broth exposed to atmospheric oxygen tested on Peptostreptococcus anaerobius. J Clin. Microbial. 1977;6:117.4.

Gill J, Adler HI. Promotion of septation in irradiated Escberichiae coli by a cytoplasmic membrane preparation. Radiation Res. 1985;102:232.

Summanen P, Baron EJ, Citron DM, et al. Wadsworth Anaerobic Bacteriology Manual. 5th ed. Belmont, CA: Star Publishing; 1993.

             Enzymatic membrane method vs. traditional methods
      Traditional methods
          (2 benches)
      Aerobic plates
Day 1 1. Culture specimen: inoculate media in biological safety cabinet
      2. Incubate 35[degrees]C [CO.sub.2]
      3. Enter aerobic workup into
         computer; reincubate plates
Day 2 4. Enter aerobic workup into
         computer; reincubate plates
Day 3 5. Enter aerobic workup into
         computer; reincubate plates
Day 4 6. Enter final physician information
         into computer
      Anaerobic plates
Day 1
      2. Anaerobic chamber
      3. Enter anaerobic workup onto
         written work cards; reincubate
         plates
Day 2 4. Enter anaerobic workup onto
         written work cards; reincubate
         plates
      5. Subculture anaerobic plates
         for aerotolerance; incubate
         35[degrees]C [CO.sub.2]
      6. Enter preliminary physician
         information into computer
Day 3 7. Enter anaerobic workup onto
         written work cards; reincubate
         plates
Day 4 8. Enter all workup from written
         work cards into computer to
         generate final physician report
      Cell-membrane method
             (1 bench)
       Enzymatic membrane plates
      (anaerobic) and aerobic plates
Day 1
      2. Incubate 35[degrees]C [CO.sub.2]
      3. Enter aerobic and anaerobic
         workup into computer; reincubate
         all plates
Day 2 4. Enter aerobic and anaerobic
         workup into computer; subculture
         anaerobic plates for aerotolerance;
         reincubate
Day 3 5. Enter aerobic and anaerobic
         workup into computer; reincubate
         all plates
Day 4 6. Enter final physician information
         into computer
                    Total anaerobic isolates recovered
Isolate                             No.
Actinomyces israeli                   1
Actinomyces meyeri                    2
Actinomyces viscosis                  1
Bacteroides caccae                    2
Bacteroides capillosus                4
Bacteroides fragilis                  5
Bacteroides fragilis group            4
Bacteroides gracilis                  1
Bacteroides ovatus                    4
Bacteroides ruminicola                1
Bacternides thetaiotaomicron          8
Bacteroides uniformis                 5
Bacteroides urealyticus               6
Bifidobacterium ssp.                  1
Clostridium clostridiforme            4
Clostridium innocuum                  1
Clostridium perfringens               1
Clostridium ramosum                   3
Eubacterium lentum                    4
Eubacterium ssp.                      4
Fusobacterium ssp.                    1
Fusobacterium symbiosum               1
Fusobacterium varium                  2
Mobiluncus                            2
Peptostreptococcus anaerobius         4
Peptostreptococcus asaccharolyticus   7
Peptostreptococcus magnus             8
Peptostreptococcus prevotii           8
Peptostreptococcus ssp.              21
Peptostreptococcus tetradius          1
Porphyromonas asaccharolyticus        3
Porphyromonas endodontalis            1
Porphyromonas gingivalis              1
Pre votella bivia                     8
Prevotella corporis                   1
Prevotella disiens                    1
Prevotella loeschii                   1
Prevotella oralis group               3
Prevotella melaninogenicus            4
Prevotella nonpigmented group         2
Prevotella pigmented group            1
Prevotella ssp.                       6
Propionibacterium acnes              15
Propionibacterium avidum              4
Propionibacterium granulosum          1
Propionibacterium ssp.                2
Streptococcus intermedius             7
Veillonella ssp.                      2
          Isolates recovered only from media with enzyme additive
Isolate                      No.
Actinomyces meyeri             1
Bacteroides fragilis           1
Bacteroides ovatus             1
Bacteroides thetaiotaomicron   1
Bacteroides urealyticus        3
Bifidobacterium ssp.           1
Clostridium clostridiforme     2
Eubacterium ssp.               8
Peptostreptococcus ssp.       11
Porphyromonas ssp.             1
Prevotella ssp.                2
Propionibacterium ssp.         8
Streptococcus intermedius      1
                    Isolates recovered only in chamber
Isolate                 No.
Bacteroides fragilis      2
Bacteroides urealyticus   1
Clostridium perfringens   1
Eubacterium ssp.          1
Fusobacterium ssp.        2
Peptostreptococcus ssp.   2
Prevotalla ssp.           2
Propionibacterium ssp.    3