1.5 Rapidly Growing
Mycobacteria (RGM)
RGM are common in the environment and include saprophytic
species as well as opportunistic pathogens, which may be a particular concern in immunocompromised individuals. They are currently
divided into six groups: Mycolicibacterium fortuitum group, Mycobacteroides chelonae/Mycobacteroides abscessus complex, Mycolicibacterium smegmatis group, Mycolicibacterium mucogenicum group,
Mycolicibacterium mageritense/Mycolicibacterium wolinskyi group,
and pigmented RGM. Approximately 50% of known mycobacterial
species are RGM with three species comprising approximately 80%
of clinically known RGM pathogens: M. fortuitum, M. chelonae,
and M. abscessus [12]. RGM lack pigmentation and produce a
positive arylsulfatase at 3 days [13]. High Performance Liquid
Chromatography (HPLC) of mycolic acids, RFLP analysis of
genes including hsp65, and sequencing could be used to identify
isolates down to the species level [13]. Many NTM can be found in
the environment, and therefore, it is important to establish the
isolate’s clinical significance which is based on clinical, radiologic,
and microbiologic criteria [11, 14].
1.6 Methods
for Continuously
Monitored Broth
Systems
There are several commercial platforms designed to help the clinical
mycobacteriologists to automate mycobacterial cultures. Here we
will describe three of these platforms. For all others, we encourage
users to refer to the specific manufacturer’s instructions and guidelines. The first platform to be described is the BACTEC MGITautomated mycobacterial systems (BD Diagnostic Systems, Sparks,
MD). This system automatically detects tube placement, i.e., scanand-load technology; uses MGIT medium containing patented
sensors that detect oxygen consumption, indicating mycobacterial
growth; PANTA antibiotic mixture is added to the MGIT tube
prior to inoculation; and a fluorescent compound, embedded in
silicone in the tube bottom, is quenched by the presence of oxygen.
When the oxygen is metabolized by mycobacteria or another
microorganism, the compound fluoresces [15].
A second platform is the BacT/Alert Mycobacteria detection
system (bioMe ´rieux, Durham, NC). It utilizes a colorimetric sensor
and reflected light to monitor the presence and production of CO 2.
The sensor changes color because of CO 2 produced by the metabolic activity of mycobacteria and other microorganisms; enrichment fluid containing bovine serum albumin, sodium chloride,
oleic acid, and saponin is added; bottles contain antibiotic mixture
of amphotericin B, azlocillin, nalidixic acid, polymyxin B, trimethoprim, and additional growth factors.
A third commonly used platform is the VersaTREK (Thermo
Fisher Scientific, Waltham, MA). In this system, the bottles contain
antibiotic mixture of polymyxin B, vancomycin, nalidixic acid, and
amphotericin B. After inoculation, each bottle is continuously
monitored for changes in gas pressure, due to consumption of
oxygen by mycobacteria and production of gas by their metabolic
activity [15].
Culturing Mycobacteria
7
Mycobacteria (RGM)
RGM are common in the environment and include saprophytic
species as well as opportunistic pathogens, which may be a particular concern in immunocompromised individuals. They are currently
divided into six groups: Mycolicibacterium fortuitum group, Mycobacteroides chelonae/Mycobacteroides abscessus complex, Mycolicibacterium smegmatis group, Mycolicibacterium mucogenicum group,
Mycolicibacterium mageritense/Mycolicibacterium wolinskyi group,
and pigmented RGM. Approximately 50% of known mycobacterial
species are RGM with three species comprising approximately 80%
of clinically known RGM pathogens: M. fortuitum, M. chelonae,
and M. abscessus [12]. RGM lack pigmentation and produce a
positive arylsulfatase at 3 days [13]. High Performance Liquid
Chromatography (HPLC) of mycolic acids, RFLP analysis of
genes including hsp65, and sequencing could be used to identify
isolates down to the species level [13]. Many NTM can be found in
the environment, and therefore, it is important to establish the
isolate’s clinical significance which is based on clinical, radiologic,
and microbiologic criteria [11, 14].
1.6 Methods
for Continuously
Monitored Broth
Systems
There are several commercial platforms designed to help the clinical
mycobacteriologists to automate mycobacterial cultures. Here we
will describe three of these platforms. For all others, we encourage
users to refer to the specific manufacturer’s instructions and guidelines. The first platform to be described is the BACTEC MGITautomated mycobacterial systems (BD Diagnostic Systems, Sparks,
MD). This system automatically detects tube placement, i.e., scanand-load technology; uses MGIT medium containing patented
sensors that detect oxygen consumption, indicating mycobacterial
growth; PANTA antibiotic mixture is added to the MGIT tube
prior to inoculation; and a fluorescent compound, embedded in
silicone in the tube bottom, is quenched by the presence of oxygen.
When the oxygen is metabolized by mycobacteria or another
microorganism, the compound fluoresces [15].
A second platform is the BacT/Alert Mycobacteria detection
system (bioMe ´rieux, Durham, NC). It utilizes a colorimetric sensor
and reflected light to monitor the presence and production of CO 2.
The sensor changes color because of CO 2 produced by the metabolic activity of mycobacteria and other microorganisms; enrichment fluid containing bovine serum albumin, sodium chloride,
oleic acid, and saponin is added; bottles contain antibiotic mixture
of amphotericin B, azlocillin, nalidixic acid, polymyxin B, trimethoprim, and additional growth factors.
A third commonly used platform is the VersaTREK (Thermo
Fisher Scientific, Waltham, MA). In this system, the bottles contain
antibiotic mixture of polymyxin B, vancomycin, nalidixic acid, and
amphotericin B. After inoculation, each bottle is continuously
monitored for changes in gas pressure, due to consumption of
oxygen by mycobacteria and production of gas by their metabolic
activity [15].
Culturing Mycobacteria
7