BACTERIA MOST OFTEN SUBMITTED FOR IDENTIFICATION TESTING DURING 2010

FREQUENTLY ENCOUNTERED BACTERIAL ENVIRONMENTAL ISOLATES

Microbiological consulting professionals  are often requested to assist Clients with the identification of various environmental microorganisms to include bacteria that are isolated from their various HVAC and Utility Systems during facility commissioning and on-going operations.  These requests relate to controlling these isolates within Alert and Action Levels and never permitting them to migrate  to Out of Trend (OOT) or Out of Specification (OOS) situations. 

During environmental monitoring of ISO 5-8 controlled and classified areas, microorganisms are often isolated from airborne viable, settling, and contact plates and personnel.  Knowing the identification and sources of these bacteria becomes essential to maintaining the control of the environment and minimizing contamination of the non-sterile or sterile process and the aseptic filling of the product.  Personnel require this information to determine what sanitizer, disinfectant or sterilant to use. 

Accugenix, Inc., Newark, DE has kindly provided the enclosed list of the fifteen most frequently obtained bacterial isolates during 2010.  These bacteria represent primarily skin microflora and environmental isolates.  One of the microorganisms isolated is also an anaerobe.  Please note that no yeast, molds or streptomyces are included within this list which is presented in Table 1 below along with their frequency.

TABLE 1

RANK	GENUS/SPECIIES	PERCENTAGE
1	Micrococcus luteus	9.6%
2	Staphylococcus epidermidis	8.7%
3	Staphylococcus hominis	5.6%
4	Bacillus cereus/thuringiensis	4.3%
5	Corynebacterium tuberculostearicum	2.9%
6	Staphylococcus capitis	2.8%
7	Bacillus pumilus	2.7%
8	Staphyloccus warneri	2.5%
9	Bacillus amyloliquefaciens/subtilis	2.4%
10	Staphyloccus haemolyticus	2.2%
11	Ralstonia pickettii	1.7%
12	Paenibacillus glucanolyticus	1.5%
13	Bacillus megaterium	1.5%
14	Propionibacterium acnes	1.0%
15	Bacillus licheniformis	0.9%

 A review of these bacteria obtained by DNA Sequencing provides the following categories.  Eight of the isolates were derived from skin microflora and include Staphylococcus sp. (5), Micrococcus sp. (1), Corynebacterium sp. (1) and Propionibacterium sp. (1).  Seven of the eight were aerobic and would readily grow on Trypticase Soy Agar (TSA) or in Broth (TSB), while the Propionibacterium sp. would grow only under anaerobic conditions.  All of these bacteria should readily be killed by isopropyl alcohol.  In rare instances, a stronger disinfectant to include a Quaternary Ammonium Compound or a Phenol might be required.

Propionibacterium sp. is a microaerophilic Gram positive bacterium that will not grow under aerobic conditions and is not readily observed during daily monitoring unless provisions are made for anaerobic culturing.  It is a very slow growing bacterium that is not readily observed on TSA plates or within TSB broth and is only observed after 9 – 14 days within Fluid Thioglycollate medium (FTM).  The frequency of isolation made under these conditions suggests that if Quality Control testing labs were to use more frequent anaerobic conditions for testing, this bacterium would be more frequently observed.

The Bacillus sp. (5) and Paenibacillus sp. (1) are spore-forming Gram positive bacteria that are environmental isolates and frequently found within classified and controlled environments.  In the medical device, pharmaceutical and biotechnology industries, these bacteria are often found on corrugated (boxes), on cotton and gauze and within yeast extract.  These bacteria will not be killed as spores by either sanitizers or disinfectants and require sporicides to kill them.  Popular sporicides include hydrogen peroxide (6%), hydrogen peroxide/peracetic acid blends and bleach.  The presence of films on contact surfaces may necessitate the use of a cleaning agent to remove these films prior to the application of the sporicide.

Ralstonia sp. (1) is a Gram negative rod that is frequently associated with Purified Water (PW) water systems and will survive within these PW systems if the water is not heat treated (~80^oC) on an on-going basis.  If the system becomes heavily infected, closure of the system and its complete disinfection is required.

Microbiological consultants can provide on-site training to minimize the requirements for requiring DNA Sequencing to be performed on each isolate.  Often, knowledge of the colonial morphology and the Gram stain are sufficient to quickly initiate control measures once colonies are observed.