Media for Detecting Fluoroquinolone Non-Susceptible Microorganisms

Cat. no. G258 MacConkey Agar with Ciprofloxacin, 10μg/ml, 15x100mm Plate, 19ml 10 plates/bag
Cat. no. K258 BHI Broth with Ciprofloxacin, 10μg/ml, 15x103mm Tube, 5ml 20 tubes/box

INTENDED USE

Hardy Diagnostics BHI Broth with Ciprofloxacin, 10μg/ml is a selective pre-enrichment broth for gram-negative microorganisms with reduced susceptibility to ciprofloxacin. Pre-enrichment with the broth is performed with a mixed population of microorganisms from a lab sample. Hardy Diagnostics MacConkey Agar with Ciprofloxacin, 10μg/ml is a companion product meant to act as a screening subculture medium for the selective isolation of non-susceptible Ciprofloxacin, gram-negative microorganisms. Concomitant cultures are necessary to recover microorganisms for identification, susceptibility testing, and epidemiological typing. A negative result does not preclude fluoroquinolone non-susceptible, gram-negative colonization. Culture results obtained from these products are not intended to guide therapy.

SUMMARY

Microbial antibiotic resistance has increased significantly among gram-negative bacilli in recent years and colonization of the gastrointestinal tract by resistant microorganisms serves as a reservoir for person-to-person transmission, as well as a likely source of potential clinical infection. The latter source is of particular importance for patients undergoing transrectal prostate biopsy in the histological diagnosis of prostate carcinoma. Approximately 800,000 biopsies are performed in the U.S. each year and complications from this procedure include urinary tract infections, prostatitis, and sepsis.(2,7-9) In general, the procedure usually follows antibiotic prophylaxes with fluoroquinolones and patients harboring fluoroquinolone-resistant microorganisms are at greater risk of contracting post-procedural infection.

The most common organism responsible for bacterial infection and clinical complication following transrectal prostate biopsy is Escherichia coli. Consequently, selective broth pre-enrichment, followed by selective culture on solid agar, is a useful tool in identifying patients colonized with resistant microorganisms prior to biopsy.(2,7-9)

Hardy Diagnostics media for detecting fluoroquinolone non-susceptible microorganisms are meant to be used for the selective pre-enrichment and isolation of fluoroquinolone non-susceptible enteric bacteria. Liquid media containing ciprofloxacin is meant to be used as a selective pre-enrichment broth from a mixed sample before subculture of the sample to solid media containing the same concentration of antibiotic. Ciprofloxacin is added to the media at 10μg/ml to aid in the selective isolation of fluoroquinolone non-susceptible microorganisms. BHI Broth with Ciprofloxacin, 10μg/ml (Cat.no. K258) should only be used as a selective pre-enrichment step with MacConkey Agar with Ciprofloxacin, 10μg/ml (Cat. no. G258) for best results.

FORMULA

Ingredients per liter of deionized water:*

MacConkey Agar with Ciprofloxacin, 10μg/ml (Cat. no. G258):
Peptone 17.0gm
Lactose 10.0gm
Sodium Chloride 5.0gm
Proteose Peptone 3.0gm
Bile Salts 1.5gm
Neutral Red 30.0mg
Ciprofloxacin** 10.0mg
Crystal Violet 1.0mg
Agar 13.5gm

Final pH 7.1 +/- 0.2 at 25ºC.

BHI Broth with Ciprofloxacin, 10μg/ml (Cat. no. K258):
Calf Brain-Beef Heart Infusion 17.5gm
Pancreatic Digestion of Gelatin 10.0gm
Sodium Chloride 5.0gm
Disodium Phosphate 2.5gm
Dextrose 2.0gm
Ciprofloxacin*** 0.01gm

Final pH 7.4 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking for agar media) or discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection and culture.(3-10) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold.

Method of Use:

1. Collect rectal swab specimen and place the swab directly into a BHI Broth with Ciprofloxacin, 10μg/ml (Cat. no. K258) tube.* If swabs are collected in the examination room, they should be placed into the BHI Broth with Ciprofloxacin tube and transported at room temperature to the laboratory without delay.

2. Incubate the swab in BHI Broth with Ciprofloxacin with slightly loose caps at 35 +/- 2ºC. for 24 hours.

3. Transfer 0.1ml of the BHI Broth with Ciprofloxacin culture to a MacConkey Agar with Ciprofloxacin plate (Cat. no. G258) and streak the specimen using a sterile loop to obtain isolated colonies.

4. Incubate plates in ambient air at 35 +/- 2ºC. for 24 to 48 hours and examine plates for colony growth. Colonies growing as gram-negative, enteric bacilli suspected of being fluoroquinolone non-susceptible Escherichia coli should be submitted for further biochemical confirmatory and susceptibility testing.

*Alternatively, direct plating of rectal swabs to MacConkey Agar with Ciprofloxacin has been described in the literature.(12)

INTERPRETATION OF RESULTS

Positive growth of BHI Broth with Ciprofloxacin cultures subbed to MacConkey Agar with Ciprofloxacin plates showing the growth of gram-negative enteric bacilli shall be suspected of presenting reduced susceptibility to fluoroquinolone antibiotics. Concomitant cultures are necessary to recover organisms for identification, susceptibility testing, and epidemiological typing. A negative result does not preclude fluoroquinolone non-susceptible, gram-negative colonization. Culture results obtained from these products are not intended to guide therapy.

LIMITATIONS

MacConkey Agar with Ciprofloxacin is recommended for selective isolation of gram-negative bacilli with reduced susceptibility to fluoroquinolone antibiotics. The formulation is not intended for use as a method of antimicrobial susceptibility testing or to guide therapy.

Confirmation of resistance by an approved method is necessary, as some organisms on initial isolation may overcome the inhibitory effecs of this medium.

The absence of suspect colonies on MacConkey Agar with Ciprofloxacin medium does not rule out the presence of fluoroquinolone non-susceptible microorganisms.

If no growth of positive BHI Broth with Ciprofloxacin cultures subbed to MacConkey Agar with Ciprofloxacin plates is observed after 24 hours, allow plates to incubate for up to 48 hours before determining negative cultures.(6)

Greater sensitivity of low-level fluoroquinolone-resistance has been shown by direct plating of stool and perirectal cultures to MacConkey Agar supplemented with greater than or equal to 0.125μg/ml levofloxacin and incubated in 5 to 10% CO2; consequently, culture of perirectal or rectal swabs alone may fail to identify fluoroquinolone-resistant organisms in patients with low-level colonization.(6)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, pipets, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
MacConkey Agar with Ciprofloxacin, 10μg/ml (Cat. no. G258):
Acinetobacter baumannii
ATCC ® BAA-1605**
A 24hr 35°C Aerobic Growth; colorless to slightly pink colonies
Escherichia coli
ATCC ® 25922**
B 24hr 35°C Aerobic Inhibited
Enterococcus faecalis
ATCC ® 29212
B 24hr 35°C Aerobic Partial to complete inhibition
BHI Broth with Ciprofloxacin, 10μg/ml (Cat. no. K258):
Acinetobacter baumannii
ATCC ® BAA-1605**
A 24hr 35°C Aerobic Growth; broth becomes turbid
Escherichia coli
ATCC ® 25922**
B 24hr 35°C Aerobic No growth; broth remains the same

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

Physical Appearance

MacConkey Agar with Ciprofloxacin, 10μg/ml should appear clear, slightly opalescent, and reddish-purple in color.
BHI Broth with Ciprofloxacin, 10μg/ml should appear clear, and medium amber in color.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Batura, D., G.G. Rao, and P.B. Nielsen. 2010. Prevalence of Antimicrobial Resistance in Intestinal Flora of Patients Undergoing Prostatic Biopsy; Implications for Prophylaxis and Treatment of Infections After Biopsy. BJU Int.; 106:1017-1020.

3. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

6. Lautenbach, E., A.D. Harris, E.N. Perencevich, I. Nachamkin, P. Tolomeo, and J.P. Metlay. 2005. Test Characteristics of Perirectal and Rectal Swab Compared to Stool Sample for Detection of Fluoroquinolone-Resistant Escherichia coli in the Gastrointestinal Tract. Antimicro. Agents and Chemo.; 49(2):798-800.

7. Liss, M.A., A.N. Peeples, L. Pham, and E.M. Peterson. 2010. Detection of Fluoroquinolone Resistant Organisms from Rectal Swabs. Presented at the 2010 American Society for Microbiology General Meeting. San Diego, CA.

8. Liss, M.A., A.N. Peeples, and E.M. Peterson. 2011. Detection of Fluoroquinolone-Resistant Organisms from Rectal Swabs by Use of Selective Media Prior to a Transrectal Prostate Biopsy. J. Clin. Micro.; 49(3):1116-1118.

9. Liss, M.A., A. Chang, R. Santos, A.N. Peeples, E.M. Peterson, K. Osann, J. Billimek, R.J. Szabo, and A. Dash. 2011. Prevalence and Significance of Fluoroquinolone Resistant Escherichia coli in Patients Undergoing Transrectal Ultrasound Guided Prostate Needle Biopsy. J. Urol.; 185(4):1283-1288.

10. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

11. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

12. Liss, M.A., K.K. Nakamura, and E.M. Peterson. 2013. Comparison of Broth Enrichment to Direct Plating for Screening of Rectal Cultures for Ciprofloxacin-Resistant Escherichia coli. J. Clin. Microbiol. 51(1):249.

13. Bryan, L.G., K.C. Carroll, J. Miller, A. Thompson. 2013:D-1146 ICAAC Poster. Evaluation of Selective Media to Survey for Ciprofloxacin-Resistant Gram negative Bacterial Colonization in Patients Undergoing Prostate Biopsy. www.icaaconline.com accessed 26-May-2015.


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