degeneration occurring in the lower motor neurons of the spinal cord. Therefore, evaluation of remaining function and prognosis may partially be determined by evaluation of remaining muscle tonus.

One of the most diagnostic reflexes for determining neurologic function and localiza- tion of the lesion is the toepinch or flexor reflex. This reflex is performed by applying painful stimuli to the interdigital spaces. The normal animal wil l exhibit a response to pain and quickly withdraw the extremity. The peripheral nerve innervation, L4-S3 spinal segment for the rear legs, C6-TI for the front legs and an intact spinal cord are all required for normal reaction. A degenerative lesion in the spinal segment innervating the legs wil l result in loss of the flexor reflex. However, a degenerative spinal lesion.anterior to the spinal segment of the reflex arc wil l usually show flexion but no response of pain. This indicates localized loss of cord function and a poor prognosis. Inflammatory and compressive lesions usually produce an exaggerated response with cord function still remaining. Therefore, a favorable prognosis would be given to the patient showing both flexion and cortical pain perception.

The knee-jerk or patellar reflex i s utilized for determining the integrity of the L4-6 spinal cord segment. Injury and degeneration to this cord segment would result in absence of this response. However, i t must be noted that most injuries to the cord are well above this cord segment and that this reflex i s usually normal or hyperreflexic in these cases.

Perhaps the greatest diagnostic aid in the spinal examination is that of neuroradiography. However, radiographic examination is a supple- ment and not a substitute for basic neurologic examination procedures. Since many spinal lesions are minute and lack radiographic density, spinal radiography requires the best of techniques and equipment. The patient must be handled very carefully and moved only as a single unit to prevent any flexion of the spine. Both ventrodorsal and lateral views are required to determine the degree of spinal canal displace- ment and fragmentation of bone. The neurologic examination should indicate the spinal segment involved so that films may be taken accordingly. The most common sites of spinal fractures in order of occurrence include the lower thoracic and upper lumbar (Tg-L l ) , lower lumbar and sacral (L4-S3) and the upper cervical (Cl-2) spines.

A tragic error in the neurologic evaluation of the patient with spinal injury i s that frequent assumption that the spinal cord i s severed just because the patient cannot move the legs. This

54

assumption i s not valid in many cases. It i s only following careful neurologic and radiographic examinations that a clinician makes the most accurate assessment of the neurologic damage. Time is a major factor in these cases. Therefore, it i s imperative that a careful evaluation be made early so that treatment procedures may be initiated before irreversible neuronal degenera- tion occurs.

REFERENCES

1 . Gage, E.D.: Atlas of Neurologic Diagnosis and Neurosurgery in the Dog. 2nd. ed Texas A B M University Press, College Station, Tex , (1974): 24-38. 2. Gage, E.D.. Surgical Reapir of Spinal Fractures in Small-Breed Dogs. VMISAC. 66.1, (Nov. 1971): 1095-1101. 3. Gage, E.D.: Surgical Repair of Fractured Cervical Spine in the Dog. J.A.V.M.A., 153, 11 . (Dec. 1968): 1407-1411. 4. Hoerlein, B.F.: Canine Neurology. 2nd ed. W.B. Saunders Co., Philadelphia, Pa., (1971)' 303-412. 5. Jackson, F.E.: Pathophysiology of Head Injuries. Clinical Symposia, 18, 3, (1966): 67-68. 6. Jackson, F.E.. Treatment of Head Injuries. Clinical Symposia, 19, 1, (1967): 6-1 4

ANTIMICROBIAL SUSCEPTIBILITY TESTING IN SURGICAL MICROBIOLOGY

Mary McCarthy, B.S., M.Sc.t

Bacter ia l in fect ions are no t in f requent problems in the surgical patient. Until recently, it was not economically feasible for most practicing veterinarians to determine the anti- microbial susceptibility of the organisms causing infections The treatment regimen of such intections was based on experience and the use of antibiotic combinations or broad-spectrum antibiotics. A number of recent reports have shown the multiple resistance of bacterial isolates from animals, especially to Strepto- mycin, Tetracycline and Penicillin. l5, 17, 20, 21

Since these antimicrobials are some ot the most liberally used in veterinary medicine, i t appears that the incorporation of susceptibility testing into the evaluation and treatment of the infected patient would be helpful.

Several methods are available for suscepti- bility testing. The most common include 1) the Broth Dilution Technique of determining the Minimum Inhibitory Concentration (MIC) of the antimicrobial for the organism in question, 2) the agar Dilution Technique for determining the MIC, and 3) the Standardized Single Disk Method commonly called the Kirby-Bauer Met hod,

Broth Dilution Method: lo. 12, 25

This method employs Mueller-Hinton broth for serial dilutions of the antimicrobial to be

tMicrobiologist, Clinical Research Service, Madigan Army Medical Center, Tacoma, Washington 98431

tested. Fastidious organisms including some streptococci, may not grow adequately i n this medium. I n such cases a casein soy peptone (Trypticase of Tryptic soy) broth or Levinthal medium is recommended. Such dilutions might include 64, 32, 16, 8, 4, 2, 1, and 0.5 mcg/ml respectively. Each tube is inoculated wi th approximately 105 to 106 bacteria per m l and incubated overnight at 35OC. The tubes are examined visually for signs of bacterial growth. The M I C is read as the lowest concentration of a n t i m i c r o b i a l i n h i b i t i n g v i s ib le g r o w t h . Subculturing those tubes wi th n o visible growth onto antibiotic free agar medium wi l l determine the M in imum Bacterial Concentration (MBC). After overnight incubation of the agar medium, the MBC is read as the iowest concentration of antimicrobial that yielded n o growth o n subculture. The difference between the MIC and MBC is usually zero to two di lut ion intervals for bactericidal antibiotic5. Bacteriostatic anti- microbials may have greater differences in MIC and MBC values. This method may be used t o test aerobic or anaerobic organisms. Agar Dilution Method: l o . ’*. 25

The agar di lut ion method is very similar to the broth di lut ion technique except that the d i f f e re n t a n t i b i o t i c con c e n t r at i o n s are i n c or por - ated into a Mueller-Hinton agar medium in a petri dish. Blood may be added for growth of more fastidious organisms. An example of a typical assay may include 8 agar petri dishes each containing a different concentration of antimicrobial: 64, 32, 16, 8, 4, 2, 1, or 0 . 5 mcg/ml respectively. A drop of bacterial inoculum containing 3 t o 6x105 organisms is dispensed onto each agar plate, incubated at 3 5 O C overnight, and observed for growth. The lowest di lut ion showing no growth is the M I C . The MBC cannot be determined by this method.

The Standardized Single Disk Method of Kirby- Bauer:

The di lut ion methods give accurate quantita- tive data regarding the susceptibility of a bacterial strain, bu t are t ime consuming and not practical for most cl inical laboratories. Discs impregnated wi th antibiot ic have been used in diffusion susceptibility testing for some t ime. The disc i s placed on the surface of an agar medium seeded wi th bacteria and the antibiot ic diffuses through the medium. After overnight incubation at 3 5 O , the plate is observed for a zone of inhibit ion around the disk. It was not unti l 1966 that a standardized method was introduced to control al l the variables in this test. Bauer, Kirby, Sherris, and Turck described their standardized single disk method of susceptibility testing a t this t ime. 4 I t is

teriaceae family, Staphylococci, and Pseudo- monas sp., and the most common pathogenic Streptococci. The National Committee for Clinical Laboratory Standards (NCCLS) and the FDA make periodic recommendations for changing and improving the method.23

The test is based on the principle that the size of the zone of growth inhibi t ion around the antibiot ic disc is inversely related t o the M I C of the organism (Figure 1) as determined by the di lut ion test described above. The zone site is a measure of the approximate MIC value, which can be read from the regression l ine shown in Figure 1

R = RESISTANT I = INTERMEDIATE S = SENSITIVE

\

-I-

7 6 10 14 18 22 26 30 34 38

ZONE DIAMETER (mm.) Courtesy of Eli Lil ly & Company

FIGURE 1

Suggested interpretation of zone size for cephalexin (30-ug. disc) determined by the Bauer-Kirby method.

From the M I C value the cl inician can determine whether or no t his patient wi l l respond to the antibiot ic b y f inding what the attainable blood levels are for that antibiot ic. To facilitate this decision making process, the categories o f Resistant, Intermediate, and Susceptible (Sensitive) have been established for the Kirby-Bauer Method. These categories are based o n the zone size - M I C value relationship and the fol lowing information: 22

a. Relationship of the M I C of the organism to the blood level (or i n some cases urine level) obtained on usual dosage schedules.

b. Relationship of the susceptibility of the tested strain to other members of the same species.

55

c. Clinical effectiveness as indicated by experience with the agent under consideration.

The zone size interpretive standards are given in Table 1. Each antimicrobial i s listed with the appropriate disc potency to use and the zone diameters ( in mm) that categorize the organism as Resistant, Intermediate or Susceptible.

Susceptible implies that an infection due to the tested strain wi l l respond to the usual anti- microbial dosage recommended for that strain and type of infection. The Intermediate category includes strains which may respond to blood

levels attained by high dosages, or strains which mav be eliminated from the urinary tract, where the an t im ic rob ia l is concentrated. Few organisms fall in the intermediate zone. Resistant strains are not inhibited by the concentrations of antimicrobial attainable in the blood with the usual therapeutic dosages. Certain antimicrobials are used only for urinary tract infections and the M I C zone diameters are based on urine levels rather than blood levels. These include Nitrofurantoin and Nalidixic Acid (Table 1).

TABLE 1

ZONE-SIZE INTERPRETATIVE CHART

Antibiotic of Disc Inhibit ion Zone Diameter in m m chemotherapeutic Agent Potency Resistant Intermediate Sensitive

Ampicillin Gram negative 10 mcg 11 or less

Ampicillin Staphylococci and 10 mcg 20 or less

8 or less Baci traci n 10 u Carbenicillin Proteus species

and E. coli 50 mcg 17 or less Carbenicill in 50 mcg 12 or less

end enterococci

highly Penicillin- sensitive organisms

1 2-1 3

21 -28

14 or more

29 or more

9-1 2 13 or more

18-22 23 or more 13-1 4 15 or more

Pseudomonas aeruginosa Cephalorid i ne

Cephaloth i n Chloramphenicol Clindamycin Colistin Erythromycin Gentamicin Kanamycin Lincomycin Methicillin Nalidixic Acid' Neomycin N itrof u rantoi n * Novobiocin Oleandomycin Pen ici I I in-G Staphylococci Pencicillin-G Other organisms Polymyxin-B Streptomycin Triple Sulfa"' Tetracycline Vancomycin

30 mcg 30 mcg 30 mcg

2 rncg 10 mcg 15 mcg 10 mcg 30 mcg 2 mcg 5 mcg

30 mcg 30 mcg

300 mcg 30 mcg 15 mcg

10 u 10 u

300 U 10 mcg

250 mcg 30 mcg 30 mcg

1 or less 4 or less 2 or less 4 or less 8 or less 3 or less 2 or less 3 or less 9 or less 9 or less

13 or less 12 or less 14 or less 17 or less 11 or less 20 or less 11 or less

8 or less 11 or less 12 or less 14 or less

9 or less

2-1 5 16 or more 5-1 7 18 or more 3-1 7 18 or more 5-1 6 17 or more 9-1 0 11 or more 4-1 7 18 or more

13 or more 4-1 7 18 or more

10-1 4 15 or more 10-1 3 14 or more 1 4-1 8 13-1 6 1 5-1 6 18-21 12-1 6 21 -28 12-21 9-1 2

1 2-1 4 1 3-1 6 15-18 10-1 1

19 or more 17 or more 17 or more 22 or more 17 or more 29 or more 22 or more 12 or more 15 or more 17 or more 19 or more 12 or more

*Urinary Tract infections only * *Not applicable to blood containing media

"'Any of the commercially available 300 or 250 mcg sulfonamide discs can be used with the same standards of zone inter-

""This category includes some organisms such as enterococci which may cause systemic infections treatable by high doses pretation

of Penicillin-G.

Chart courtesy of Bioquest, Division of Becton, Dickinson and Company

56

A brief description of the Kirby-Bauer Method would be most appropriate at this point. 4- 1 1 , 22

Several colonies of the organism to be tested are transferred to 4 m l of a suitable broth such as trypticase soy broth or soybean casein digest broth, and incubated for 2 to 5 hours at 35 to 37 C. The suspension is diluted, i f necessary, with sterile saline or broth to give a turbidity comparable to a Ba CI2 standard (Prepared by adding 0.5 m l of 0.048 M Ba CI2 to 99.5 m l of 0.36 N H2504). Within 15 minutes of adjusting the density of the bacterial suspension, a sterile cotton swab is saturated with the suspension and rotated on the side of the tube to remove excess i nocu lum .

The dried surface of a Mueller-Hinton agar plate Is inoculated by streaking the swab over the whole surface. The plate is rotated approxi- mately 60° and the streaking repeated. The plate is rotated and streaked a third time to ensure an even distribution of the bacterial inoculuin, resulting in a confluent growth of bacteria after incubation. The plate i s allowed to stand 3 to 5 minutes to dry (not longer than 15 minutes) and the antimicrobial discs are applied.

A n alternate method of inoculating the Mueller-Hinton plate is described by A . L . Barry. 2 This i s commonly referred to as the agar overlay method. Select 4 to 5 colonies of the organism to be tested and prepare a suspension in 0.5 m l of brain-heart infusion broth. Incubate in a 35 to 37 C waterbath for 4 to 8 hours. Mix the culture and transfer 0.001 ml (using a calibrated loop) to 9.0 ml of a melted agar solution held at 45 to 50 C . Mix and pour the seeded agar evenly over a 4mm depth Mueller-Hinton Agar plate. Let the plate stand 3 to 5 minutes and apply the susceptibility discs. The discs may be applied with sterile forceps, gently pressing down to make contact with the agar. Commercial dispensers are available that insure the proper number of discs per plate and the proper distances between discs.

The plate is inverted and incubated at 35OC within 15 minutes after discs are applied. After 16 to 18 hours incubation, the plate is examined and the zones of inhibition measured with a sliding caliper, a ruler, or a template especially prepared to read millimeter zone diameters. The zone for each drug tested are recorded in millimeters and the susceptibility determined from the chart shown in Table 1. Swarming Proteus may form a veil inside the zone of inhibition. This i s ignored in measuring the zone diameter. Sulfonamides may not inhibit growth for at least several generations. Therefore, the zone of inhibition is measured at the margin of heavy growth.

I t is critical to follow the Kirby-Bauer or the

agar overlay procedure exactly as described. Factors such as the pH of the Mueller-Hinton media, concentration of the organisms in the inoculum, depth of the agar, storage of discs, temperature of incubation, and periods of incubat ion are a l l c r i t i ca l for proper performance of the test. These factors are discussed further in a number of important references. ’. 9 . 1 0 . 1 1 , 2 6 It should be emphasized that the use of a mixed culture in the procedure can result in erroneous zone diameters and should NEVER be used. If a mixed culture i s mistakenly used, the test should be repeated on a pure culture.

To assure accuracy of the disc susceptibility testing method, i t is recommended that stock cultures of Staphyloccoccus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) be obtained and tested with each batch of tests performed. Zone sizes are recorded and compar- ed with the acceptable range of zone sizes de- termined previously. ” If there is an unacceptable discrepancy in zone sizes, the laboratory technician should be aware of an existing error in the test procedure and take the necessary measures to correct i t .

Other methods of antimicrobial susceptibility testing are available. These include automated methods based on an optical counting system and a semiautomated microcalor imetr ic method. 6, l 6 The disc diffusion methods without the proper standardization of the Kirby-Bauer Method have been and s t i l l are being used. These methods lack the excellent reproducibil- ity of the Kirby-Bauer Method.

Choice of Antimicrobials in Treatment of Infec- tions:

A number of factors should be considered in choosing the proper antibiotic for treatment of an infection. The in vitro antimicrobial suscep- tibility of the organism(s) is only one factor. Other important concerns are the site of infection, the immunological status of the patient, and how the patient wi l l tolerate the toxicity of certain drugs. It i s recommended that a narrow-spectrum antibiotic be utilized when possible to avoid unnecessary alteration of the animal’s normal flora. I f a Combination of antibiotics must be used in infections caused by multiple organisms, they should be chosen with care to avoid any possible antagonistic effects. This problem is discussed in greater detail by Aronson.

Antibiotics used for the treatment of Gram-positive cocci include the penicillins, the semisynthetic penicillinase-resistant penicillins, the ceph alospor i ns, eryt h romyc i n, I i ncomyc i n- clindamycin, and vancomycin. In addition, some broad spectrum agents used chiefly in

57

treatment of Gram-negative infections can also be used in treatment of Cram-positive cocci infections. These include the tetracyclines, chloramphenicol, the sulfonamides, and the am i noglycosides (gentam ic in, kanamycin, streptomycin). The more specific narrow spectrum antibiotics are recommended for use in treatment of Gram-positive infections. A possible exception is the useful synnergistic effect against the enterococci achieved by a combination of penicillin or ampicillin with streptomycin. 1 3 1 3

The aerobic Gram-negative bacilli may respond t o the fo l low ing ant imicrobia ls : ampicillin, carbenicillin, the cephalosporins, c hloram phen icol, gentam ici n, kanamyci n , the polymyxins, the tetracyclines, and the sulfona- mides. Antimicrobial susceptibility testing i s important in infections caused by the aerobic Gram-negative bacilli due to their variable sensitivity patterns.

Anaerobic infect ions present a fur ther problem. The standarized disc susceptibility testing of Kirby and Bauer cannot be applied to the anaerobes without certain changes currently in progress. The agar and broth dilutions for determining MIC values are very t ime consuming and impractical in a clinical laboratory. Thus, the choice of antimicrobial used in these infections i s based on suscep- tibility patterns currently available. The most f requent ly isolated c l in ica l l y impor tan t anaerobic i s Bacteroides fragilis. Clindamycin and chloramphenicol are currently the drugs of choice in its treatment. Antimicrobials which may be effective in treatment of anaerobic infections (excluding 6. fragilis) include the tetracyclines, erythromycin, the penicillins and the cephalosporins. The aminoglycosides and polymyxins are, in general, inactive against all the major anaerobic pathogens. This should be remembered when treating infections with these antibiotics where anaerobes may have been overlooked. 19

Susceptibility Testing in Veterinary Practice:

The decision to incorporate antimicrobial susceptibility testing into a private veterinary practice wil l depend on various factors. The cost of the bacteriological media and sensitivity discs i s relatively minimal, but the time involved to perform the culture and sensitivity may discourage clinicians from routinely performing such tests. It is relatively expensive ($18 to $25) to have a culture and sensitivity test performed in a commercial clinical laboratory, bu t in cases where the client's finances are not in question this would be an alternative to the clinician

performing the test in his office. If susceptibility testing i s not employed, due

to financial or other reasons, the clinician should make every attempt to choose the correct antimicrobial based on the current suscepti- bility pattern of the infectious organism. With the increasing interest in the Kirby-Bauer method of susceptibility testing among veter- inarians, i t i s probable that additional reliable data wil l become available on the antimicrobial sensitivity patterns of the common Enterobac- teriaceae, staphylococci, and enterococci of animal origin. 5. 14. 15. 17. 18, 20. 21

REFERENCES

1 . Aronson. A L.. Kirk, R.W.. Antibiotic Therapy in Kirk, R.W. (ed): Current Veterinary Therapy V . W.B. Saunders Co., Philadelphia, 1974. 2. Barry. A.L , Garcia, F.. Thrupp, L.D : An Improved Single-disk Method for Testing the Antibiotic Susceptibility of Rapidly Growing Pathogens. Amer J Ciin Pathol 53.149-158. 1970. 3. Barry, A.L., Joyce, L.J.. Adams, H.P., Benner. E.J.: Rapid Determination of Antimicrobial Susceptibility for Urgent Clinical Situations. Amer J Clin Pathol 59.693-699, 1973. 4. Bauer. A.W., Kirby, W.M.M., Sherris, J.C., Turck, M.: Antibiotic Susceptibility Testing by a Standarized Single Disk Method. Amer J Clin Pathol 45 493-496, 1966. 5. Biberstein, E.L.. Franti. C.E., Jang, S.S., Ruby, A.: Antimicrobial Sensitivity Patterns in Sfaphyloceccus aureus from Animals. JAVMA 164.1183-1 186, 1974 6. Binford. J.S.. Binford, L.F.. Adler. P.: A Semiautomated Microcalorimetric Method of Antibiotic Sensitivity Testing. Amer J Clin Pathol 59236-94, 1973. 7. Blazevic, D.J., Koepcke. M.H.. Mstsen, J.M.: Quality Control Testing with the Disk Antibiotic Susceptibility Test of Bauer-Kirby-Sherris-Turck. Amer J Clin Pathol 57:592-597. 1972 8 Boxerbaum, B.. Antimicrobial Drugs for Treatment of Infections Caused by Aerobic Gram-negative Bacilli. Med Clin N Amer 58:519-532. 1974. 9. Drew, W.L., Barry, A.L., O'Toole, R., Sherris. J.C.: Reliability of the Kirby- Bauer Disc Diffusion Method for Detecting Methicillin-resistant Strains of Staphylococcus aureus. Appl Microbiol 24:240-247, 1972. 10 Ericsson. H.M.. Sherris, J.C.: Antibiotic Sensitivity Testing. Report of International Collaborative Study. Acta Pathol Microbiol Scand, Suppl 217(B):90, 1971 11. Gavan, T.L.: Antimicrobial Susceptibility Testing, ASCP Commission on Continuing Education Check Sample Program. Microbiology No. MB-64:l-37, 1973. 12. Gavan. T.L.: In vitro Antimicrobial Susceptibility Testing. Clinical Implica- tions and Limitations. Med Clin N Amer 58:493-503, 1974. 13. Graham, R.C.: Antibiotics for Treatment of Infections Caused by Gram- positive Cocci. Med Clin N Amer 58:505-517, 1974. 14. Hariharan. H , Barnum, D.A.: Antimicrobial Drug Susceptibility of Certain Bacterial Pathogens from Dogs and Cats. Can Vet Jour 15:108-113. 1974. 15. Hirsch, D.C: Multiple Antimicrobial Resistance in Escherichia coli Isolated from the Urine of Dogs and Cats with Cystitis. JAVMA 162:885-887, 1973. 16. Isenberg, H.D., Reischler, A,, Wiseman, D.: Prototype of a Fully Automated Device for Determination of Bacterial Antibiotic Susceptibility in the Clinical Laboratory. Appl Microbiol 22:980-986, 1971. 17 Jasper, D.E.: Antimicrobial Susceptibility of Staphylococcus Isolated from Bovine Mastitis. California Vet 26:12-15, 1972. 18. Ladiges, W.C.: Standardized Procedure for Test ing Ant ib io t i c Susceptibility of Bacterial Pathogens. JAAHA 10:407-411, 1974. 19. Lerner, P.I.: Antimicrobial Considerations in Anaerobic Infections. Med Clin N Amer 58:533-543. 1974. 20. Loken, K.I., Wagner, W. , Henke. C.L.: Transmissible Drug Resistance in Enterobacteriaceae Isolated from Calves Given Antibiotics. Amer J Vet Res 323207-1212, 1971 21. Mercer, H.D.. Prcurull. D.. Gaines, S., Wilson, S., Bennett, J.U.: Characteristics of Antimicrobial Resistance of Escherichia Coli from Animals: Relationship to Veterinary and Management Uses of Antimicrobial Agents. Appl Microbial 22:700-705, 1971. 22. Ryan, J.J., Schoenknecht, F.D., Kirby, W.M.: Disc Sensitivity Testing. HOSD Practice 5:91-100. 1970. 23. National Committee on Clinical Laboratory Standards: Performance Standards for Antimicrobial Disc Susceptibility Tests, as Used in Clinical Laboratories, Revised Tentative Standards. Philadelphia, PA, 1974. 24. Shahidi, A., Ellner, P.D.: Effect of Mixed Cultures on Antibiotic Susceptibility Testing. Appl Microbiol 18:766-770, 1969. 25. Washington, J.A., Barry, A.L.: Dilution Test Procedures, in Lennette, E.H., Spaulding. E.H., Truant, J.P. (eds) Manual of Clinical Microbiology, American Society for Microbiology, Bethesda, 1974, pp. 410-417. 26. Wick, W.E.: Influence of Antibiotic Stability on the Results of the In vitro Testing Procedures. J Bacteriol 873162-1170. 1964.

58