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Antibiotic Sensitivities of Alken-Murray Bacterial Strains
S = SUSCEPTIBLEI = INTERMEDIATER = RESISTANT ORGANISM
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Additional Antibiotics tested for some strains:
S = SUSCEPTIBLEI = I INTERMEDIATER = RESISTANT ORGANISM
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NOTES PERTAINING TO ANTIOBIC SENSITIVITY TABLES
1. B. pumilus AMH109 is also susceptible to Nalidixic acid.
2. B. pumilus AMH 111 is also sensitive to Polymyxin B. 3. M. hydrocarbonoclasticus 840 is also susceptible to Nalidixic acid and resistant to Oleandomycin, Staphylomicinand Vibriostatic agent 4. B. subtilis 633 is also sensitive to Nalidixic acid, Oxytetracycline and Sulbactam and is resistant to Ofloxacin,results reported in Comparative Study of Probiotic Cultures to Control Growth of Escherichia coli O156:H7 andSalmonella typhimurium by Moustafa Y. M. El-Naggar, Biotechnology V 2, Issue 3 2004, pp. 174 - 180 Page 8 of 11 Antibiotic Sensitivity Tests INTERNAL IDENTIFICATION CODES AND NUMBERS
When Alken-Murray began isolating and maintainig live bacterial cultures, for research and future production of
commercial bioremediation and probiotic products, each new isolate was assigned a 3 digit identification NUMBER
for better record-keeping, with NO repetition of numbers within the same genus-species combination. More recently
isolated strains are assigned an Alken-Murray three-letter prefix, that begins with the abbreviation for Alken-Murray
itself “AM”. A third letter is then selected, either to indicate something about the site of isolation or about one of more
of the strains belonging to that group. As of December 2006, six (6) prefixes beginning with AM have been assigned,
as follows:
AMC= Two unrelated strains got assigned to AMC & stayed there - NOT related, they are AMC 300 & AMC 872.
AMH= Alken-Murray strains isolated from Humified peat-soil in Georgia, USA
AMN= Alken-Murray strains isolated from environment in New York, USA
AMP= Alken-Murray strains isolated from Pine forest in Houston, Texas, USA
AMT= Alken-Murray strains isolated from refinery wastewater in Houston, Texas, USA
AMV = Alken-Murray strains isolated from various locations in Virginia, USA
Public culture collections take on the massive responsibility of accepting, growing, protecting, storing and providing
small vials of valuable cultures from researchers worldwide. Such deposits are REQUIRED to allow a patent
examiner to verify the claims asserted about a strain in a patent application. When a researcher believes that he/she
has isolated a new genus or species, the authorities that grant approval for such naming policies, generally require
that small freeze-dried plugs of the “new” strain be placed on deposit with three different culture collections, with at
least one of those OUTSIDE the country of origin, all designed to make it easier for the scientific community to
expand their knowledge, while preserving biodiversity. When a public culture collection accepts the responsibility of
protection, growth and preservation of a strain, they first verify that it was received alive, then grow it up, freeze-drying
small vials for future distribution. The freeze-dried vials can then be ordered by the original depositor, as needed, or if
the strain is patented, it may be ordered by patent examiners to verify new patents. Alken-Murray’s most valuable
strains ARE accessioned at either the ARS (Agricultural Research Service, the first USA culture collection) or at
ATCC, the world-famous American Type Culture Collection. Since public culture collections verify both viability
(survival) of strains they accession and sufficient information about the safety of the genus and species involved to
assure themselves that the submitted culture is safe for their staff to handle,, some import authorities are reassured
that commercial cultures known to be accessioned by a public culture collection can simply be accepted as “safe”
when they submit accession numbers for those cultures, along with normal import application documentation
requested by that authority. To help our distributors and “private-label” direct clients obtain the fastest import
licensing, Alken-Murray is willing to share our “confidential specifications sheets”, which provide in-depth profiling of
each strain, including primary and secondary enzymes, antibiotic sensitivity (taken from this table). An accession list
for the specific product will be placed on its last page of the “Confidential Specifications Sheet”, once the import
authorities sign and return a copy of our “trade-secret agreement”, agreeing that anyone requiring to study our
confidential information will use that information ONLY to verify safety of our products and further to protect such
information from any potential competitor or potential counterfeiter. Below are three examples of the proper reporting
of bacterial strains that are accessioned to public culture collections.
FIRST IS A SINGLE STRAIN DEPOSITED AT ATCC
Bacillus thuringiensis, subspecies israelensis, strain AMC 872
Accession # ATCC 700872
THE NEXT ARETWO STRAINS DEPOSITED AT ARS
Bacillus amyloliquefaciens, strain AMV 923
Accession # NRRL B-30741
Bacillus pumilus, strain AMH 115
Accession # NRRL B-30732
If the above-listed trio of strains represented a complete formula, ANTIBIOTIC SENSITIVITY TESTING PROTOCOLS USED:
KIRBY-BAUER
Zones of inhibition were read and compared with the values of susceptibility interpretive breakpoints issued by the
National Committee for Clinical Laboratory Standards (NCCLS) to determine the degree of sensitivity to each
antibiotic tested on each strain tested, following18 hours of incubation at 35EC on BBL Mueller Hinton or Mueller-
Hinton II Agar, plated 4 mm deep on 100 or 150 mm agar plates, following the Kirby-Bauer test protocol, using aseptic
techniques. Plated Mueller-Hinton agar was obtained from Culture Media & Supplies, Oswego, IL, USA or was
prepared by Alken-Murray laboratory technicians, according to directions on the bottle of BBL Mueller-Hinton II agar,
using aseptic techniques and autoclaving at 121EC at 15 psi for 15 minutes, cooled to 50EC and plated aseptically.
BBL Sensi-discs (antibiotics) were purchased from Hardy Diagnostics in California, USA or Culture Media & Supplies
in IL, USA. Strains were brought to a uniform density by aseptically diluting 24 hour colonies grown on TSA or Nutrient
Agar in 5 ml bioMerieux sterile 0.85% saline ampules until they matched McFarland Standard 0.5 (purchased as part
of bioMerieux McFarland Standard set or else prepared fresh by an AMC laboratory technician, with accuracy verified
with a Perkin-Elmer Lambda 2 dual beam spectrophotometer, with a reading at 625 nm, of 0.08 to 0.1 and/or a
reading at 550 nm of 0.125, prior to inoculation. Mueller-Hinton agar plates were inoculated using sterile cotton swabs
in the confluent pattern, prescribed in the Kirby-Bauer procedure, included with the BBL Sensi-Disks and further
detailed in Alken-Murray QC 117. Zones of inhibition were measured using a metric caliper or transparent ruler, while
viewing the plates with a Quebec Darkfield colony counter. A positive control of inoculation procedure, with no
antibiotic discs applied was used for each round of testing to verify proper inoculation protocol. Control strains
Page 9 of 11 Antibiotic Sensitivity Tests suggested for each vial of antibiotic discs, by manufacturer BBL, (50 discs per vial) were used to verify performanceof antibiotic discs. Results were rounded to the nearest whole number, using standard conventions for roundingnumbers (any number below ½ higher than the lower number was rounded down and any number above ½ wasrounded up).
KIRBY-BAUER MODIFICATIONS FOR TESTING NITRIFIERS
Since nitrifier species (Nitrosomonas, Nitrobacter, Nitrosococcus and Nitrospira) are unable to utilize an organic
source of carbon, Mueller-Hinton or Mueller-Hinton II agars cannot be used as a growth substrate for them. This also
makes it impossible for them to infect any species of living organism. AMC’s QC 2 (named STP broth) was modified
by adding 17 g agar to 500 ml distilled water and label Solution E. All solutions were autoclaved for 15 minutes at
121EC (15 lbs. pressure. After cooling to 50EC, components were combined, altering the recipe by adding 3 ml/L of
Solution D, the phenol red indicator dye solution, to make it easier to visualize results. Before pouring, pH was
adjusted to 7.75 ± 0.15 and was then plated 4 mm deep in 100 mm sterile disposable polystyrene agar plates from
Weber Scientific. Nitrobacter winogradskyi 391 and Nitrosomonas europaea 978 were diluted in a 5 ml of bioMerieux
sterile saline solution ampule, matching density to bioMerieux McFarland Standards 0.5. Sterile cotton swabs were
used to apply strains in the confluent pattern, prescribed in the Kirby-Bauer protocol. Four antibiotic discs were
applied per inoculated STP agar. To compensate for lower incubation temperatures, required by nitrifier species, and
slow germination and growth times, inoculated plates with antibiotic discs were incubated at 25E - 28EC for 48 hours.
Results were determined colorimetrically. If agar under and around an antibiotic disc maintained a bright fuschia pink
color, strain was reported as “Sensitive”. If agar under and around an antibiotic disc turned yellow, result was reported
as “Resistant” to the antibiotic tested. No intermediate state was verifiable using this procedure. Although nitrifiers
are incapable of causing infection or growing inside any living organism, they were tested to comply with requests
from a few international clients.
MINIMUM INHIBITORY CONCENTRATION (MIC)
Minimum Inhibitory Concentration (MIC) tests were performed on some strains by an independent laboratory. Levels
of sensitivity were interpreted using National Committee for Clinical Laboratory Standards (NCCLS) standards to allow
addition of these strains to these tables. To verify correlation, some strains originally tested using MIC protocol were
retested using the Kirby-Bauer agar diffusion antibiotic sensitivity protocol.
This table was assembled using results reported by various laboratory technicians in bound laboratory notebooks. Toimprove clarity of the chart, the category “moderately susceptible” was eliminated. All old results classified as“moderately susceptible” were changed to “intermediate” Official genus and species reassignments were made as they appeared in peer-reviewed microbiological journals.
Old name = new name, as follows: Bacillus pasteurii = Sporosarcina pasteurii, Bacillus polymyxa = Paenibacilluspolymyxa, Thiobacillus novellus = Starkeya novella, Micrococcus denitrificans 367 = Paracoccus denitrificans 367,Thiosphaera pantotropha 512 = Paracoccus pantotrophus 512, Pseudomonas testosteroni = Comomonastestosteroni, Corynebacterium nitrophilus 419 = Rhodococcus pyridinovorans 419 UPDATES
Initial update and alphabetical order indexing 07/07/2001
Update - 4/21/2002 - B. megaterium 112 and P. putida 633 added, 16S rRNA & FAME identification of species.
Update - 5/22/2002 - Bacillus cereus AMH 121, Bacillus pumilus. AMH 111, Bacillus cereus AMV 200 & Bacillus
thuringiensis, subsp. israelensis AMC 872 were added to the tables. Oxoid Bacillus cereus Enterotoxin test performed
for all B. thuringiensis and B. cereus strains. Positive results: B. cereus AMH 101, AMH 116; B .thuringiensis 367,679
and 866. Negative results: B. cereus AMH 121 and AMV 200; B .thuringiensis AMC 872
Update - 6/21/2002 - Bacillus mojavensis AMH 118, B. pumilus AMH 114, and B. pumilus AMH 119 were added to
these tables. Bacillus pumilus AMH 115 was tested for antibiotic sensitivity to Trimethoprim/Sulfoxazole, with
Sensitive response.
Update - 10/22/2002 - Starkeya novella 093 was added to the tables.
Update - 11/18/2002 - Bacillus amyloliquefaciens AMP100 and AMP104, Bacillus subtilis AMV359, Brevibacillus
parabrevis AMV306-H, Bacillus pumilus AMV308-H and AMV 310-H were added to the tables.
Update - 12/2/2002 - Bacillus amyloliquefaciens 842, B. licheniformis AMV134-S, B. megaterium 254, B. pumilus
AMV119-S and AMV 121-S, and B. amyloliquefaciens AMV 923 were added to tables.
Update - 1/5/2003 - Streptomyces viridosporus 479, Marinobacter hydrocarbonoclasticus 132, 189 and 840 were
added to tables. Lactobacillus delbruckeii was removed and dropped from AMC collection for shelf-life and
commercial fermentation issues.
Update - 03/17/2004 - Bacillus megaterium AMC 300 added. May be retested, since antibiotic discs were old when
Page 10 of 11 Antibiotic Sensitivity Tests Update 05/01/2004 - Certain Bacillus genus and species names changed according to new 16S rRNA test results
from Accugenix Laboratories. Paenibacillus polymyxa AMV 407 = Bacillus amyloliquefaciens AMV 407, Bacillus
subtilis AMV 923 = Bacillus amyloliquefaciens AMV 923
Update 12/4/2004 - Added B. megaterium 581 and Marinobacter hydrocarbonoclasticus 189.
Update 10/20/2005 - Second table was redone to include Neomycin, Novobiocin, Azithromycin, Enrofloxacin
antibiotics (Zithromax & Baytril)
Update 11/15/2005 - Added Paracoccus denitrificans 367 and added tests for Bacillus megaterium AMC 300 and
Bacillus pumilus AMH 119.
Update 11/17/2005 - Added Paracoccus pantotrophus 512 antibiotic sensitivity test results.
Update 12/1/2005 - Added retest of Bacillus megaterium AMC 300, Bacillus pumilus, strain AMH 119, Paracoccus
pantotrophus 512 & Paracoccus denitrificans 367 for Clindamycin, Azithromycin. Oxacillin. Confirmed Cephalothin &
GentamIcin. Updated Bacillus subtilis, spizizenii 633 for Penicillin G., Tetracycline, Streptomycin, Erythromycin &
Neomycin.
Update 05/37/2006 - Added Bacillus licheniformis 713 and Bacillus pumilus, strain AMT 304.
Update 12/11/2006 - At one time, Alken-Murray had two antibiotic sensitivity tables, one for Active, Commercial
Strains and one for Recent Isolates, Research Cultures and Abandoned/Lost Strains. Gradually, as new strains
graduated through research to commercial status, the present single table emerged, containing antibiotic sensitivity
data for all strains that an Alken-Murray researcher could wish to screen for undiscovered talents in the future. Only a
single strain managed to evade inclusion in this table, Acinetobacter calcoaceticus, subspecies anitratus, strain 305,
which was reinserted into the table today.
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Source: http://alkenmurray.hypermart.net/antibio-strain.pdf