Symposium on Clinical Laboratory Medicine

Diagnostic Bacteriology in Small Animal Practice Robert J. Wilkins, B.V.Sc., Dip. V.P.,* and Denise (De) Vito, M.S.t

Of the many thousands of species of bacteria known, little more than a few hundred have been shown to cause disease in man and animals. The numbers are further reduced if we consider only those organisms pathogenic to the dog and cat. Of the approximately 25 genera of bacteria pathogenic to these animals, Staphylococcus, Streptococcus, Corynebacterium, Pasteurella, Escherichia coli, Klebsiella, Enterobacter, Proteus, Salmonella, Pseudomonas, and Clostridium constitute approximately 85 per cent of the pathogens causing bacterial disease. In the laboratory examination of specimens by bacterial culture, the primary aim is to isolate and distinguish these pathogens from other organisms including commensal and contaminant bacteria. In this way, the presence of an infection can be ascertained. The recognition of the common pathogens is possible by means of a reasonably simple scheme using selected biochemical and biophysical tests. Once the identity of the pathogen is established, antibiotic sensitivity testing may be carried out to determine the appropriate antibiotic to use in treatment. Thus, three basic steps in diagnostic bacteriology are essential: (1) isolation, (2) identification, and (3) sensitivity testing. This work may be sent to a diagnostic laboratory or performed by competent technical staff in the hospital laboratory. Economics, feasibility, and volume of specimens will obviously dictate this decision. Work that is sent to a reference laboratory should be done at those laboratories whose technicians are familiar with those organisms of veterinary importance and especially those isolated from the dog and cat. One should expect highly competent and efficient service (two to three days, maximum) from any private diagnostic laboratory offering to do diagnostic work for veterinarians. Those laboratories employing the services of a veterinary microbiologist or clinical pathologist are most *Staff Clinical Pathologist, The Animal Medical Center, New York, New York tStaff Microbiologist, The Animal Medical Center, New York, New York

Veterinary Clinics of North America-Vol. 6, No.4, November 1976

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helpful since many technicians trained as human medical microbiologists often tend to incorrectly identify and misinterpret the significance of bacterial isolants of veterinary importance. For those who elect to do the work "in house," Figures 1 through 4 offer a simplified and practical guide for identifying the major pathogenic genera. Some practitioners may not wish to proceed this far, and should make use of a veterinary reference laboratory. Others who wish to proceed more extensively with the classification are referred to the excellent references cited at the end of this article.

COLLECTION OF SPECIMENS Proper specimen collection is of vital importance in the entire procedure of diagnostic bacteriology. Three methods of collection are employed, depending on the specimen. In the first, a fine, cotton-tipped sterile swab is used to collect from the lesion site; at least two swabs should be collected. Prior cleansing of the lesion and surrounding area is advised to prevent gross contamination by nonpathogenic organisms. Such a technique is applicable for lesions of or discharges from the skin, ear, eye, oral cavity, throat, rectal and genital tracts, and for lesions found at surgery. The swabs may be plated directly onto primary isolation media at the time of collection or held in a transport medium such as Stuart's, modified Stuart's, charcoal transport, or Amies medium until they can be cultured in the laboratory. Several commercial swab systems which incorporate a transport medium are available.* Transport media are designed to maintain viability of the organisms without encouraging their growth or multiplication. Swabs must not be allowed to dry out between the time of collection and the time of inoculation of the culture medium. They also attempt to maintain the ratio of numbers of one organism to the others in the specimen. Obviously the longer the organisms remain in transport medium, the more likely they are to die, especially if the organisms have fastidious growth requirements. It is best for the swab to be in transport medium for as short a period as possible, ideally less than 24 hours. The second method is used for collecting fluid specimens such as pus, urine, cerebrospinal fluid, effusions, and semisolid material such as sputum. In these cases, the material is collected into a sterile tube or vial using as aseptic a technique as possible. This container is then sealed, labeled, and submitted to the laboratory. Fluid volumes should be about 3 to 5 ml, and semisolid material should weigh about 2 gm. The third collection technique is used for blood cultures, whereby blood is collected from a vein (after very carefully shaving and sterilizing *Cepti-Seal Culturette Swabs-Scientific Products, McGraw Park, Illinois; HandiSwab Culture Swab--Fischer Scientific Company, Pittsburgh, Pennsylvania.

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DIAGNOSTIC BACTERIOLOGY IN SMALL ANIMAL PRACTICE

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the venipuncture site) into a sterile syringe. A volume of from 2 to 10 ml of blood is transferred immediately to an enriched broth medium, such as trypticase soy broth, Columbia broth, or brain-heart infusion broth, containing sodium polyanethol sulfonate as anticoagulant. Such media are prepared in blood culture tubes and are commercially available.* When collecting specimens from necropsy material, remember that microorganisms rapidly invade the blood within a few hours after death, frequently yielding false positive cultures. When antemortem septicemia is suspected, culture at necropsy from bone marrow and blood is advised. These cultures must be taken as soon as possible after death. The bone, such as a rib or femur, is flame sterilized and broken, and a swab is used to collect directly from the bone marrow. Blood is collected directly from the heart after sterilizing the epicardium. Swab specimens from lesions may be collected by searing the surface of the lesion with a heated spatula and then incising the lining with a sterile scalpel blade. The swab is inserted through the incision, rotated several times, and withdrawn. Media may be inoculated immediately, or the swab may be maintained in a transport medium for transfer to the laboratory.

DIRECT SMEAR CYTOLOGY Before proceeding with microbial cultures, in many cases it is advisable to prepare slides from the lesion, the swabs, or the fluid collected; these are stained with Wright's and/or Gram's stain, and examined for evidence of infection. In this way definitive evidence of pathogenicity of any isolant can be established. Several criteria are essential for establishing the presence of infection in a cytology preparation: (1) the presence of inflammatory cells, especially polymorphonuclear leukocytes and monocytes; (2) the presence of bacteria, their relative numbers, morphology, and gram reaction; and (3) evidence of phagocytosis of bacteria by the inflammatory cells.

CULTURE TECHNIQUES Primary isolation is performed by inoculating the swab or fluid onto appropriate media. For most routine work, a universal medium such as 5 per cent sheep blood agar, an enteric bacteria selective medium such as eosin methylene blue agar or MacConkey agar, and an enriched broth medium that will facilitate growth of both aerobic and anaerobic bacteria (such as thioglycollate broth media) are used. *Vacutainer-Becton-Dickinson, Rutherford, New Jersey; Difco Bacto-Blood Culture Bottles-Difco Laboratories, Detroit, Michigan; Liquoid Blood Culture BottleRoche Diagnostics, Nutley, New Jersey.

748

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WILKINS AND DENISE (DE) VITO

Figure 5. Methods of inoculating agar plate media. A, For primary isolation of single colonies. B, Subculture technique to enrich growth of single colonies. C, Preparation of an antibiotic sensitivity test.

The agar plates are stroked with the swab in a fashion to isolate single colonies (Fig. 5); then the swab is broken into the broth medium. The other swab may be used to prepare smears for cytology and gram staining as described. For fluid specimens, a similar technique is used depending on the results of cytologic evaluation of the fluid. If organisms are numerous in the fluid cytology, a sterile swab or inoculating loop is dipped in the fluid and then streaked onto plates and broth. If organisms are few or not obvious in the cytology, yet infection is still suspected, prior centrifugation and culture of the sediment are advised. Urine specimens can be inoculated using a standard size (0.01 ml) platinum inoculating loop onto blood agar and Levine eosin methylene blue agar plates in order to quantitate the relative numbers of organisms. A less accurate, but frequently used, technique involves dipping a sterile swab into a fresh, aseptically collected urine specimen and inoculating the agar plates. Since the volume of urine inoculated is not constant, only relative estimates based on growth results can be made. Urine is not routinely cultured in a broth medium unless there is evidence of urinary tract infection on examination of the urine sediment and no organisms have grown on the initial plate cultures. Fecal specimens are not ordinarily cultured because pathogenic sig-

DIAGNOSTIC BACTERIOLOGY IN SMALL ANIMAL PRACTICE

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nificance cannot be assigned to any isolant except perhaps Salmonella and Shigella. Specimens, however, are often taken from gastrointestinal tract lesions at surgery, necropsy, or during clinical examination (via rectal swabbing). These swabs are cultured by routine methods in addition to inoculating a Salmonella-Shigella selective broth medium, such as Selenite or GN broth (gram negative enrichment); we also culture blood agar and Levine eosin methylene blue plates anaerobically. Prereduced media can be used for this purpose. The Gas-Pak* system is the most convenient practical method for small volume anaerobic cultures. When cytology and/or clinical impressions are suggestive of systemic fungal infections, isolation can be attempted. This work is usually referred to a state diagnostic laboratory for identification and confirmation since many fungi may be pathogenic to humans when cultured on an artificial culture medium such as Sabouraud dextrose agar. For dermatophytes, Mycocel or selective Sabouraud dextrose agar, which contain chloromycetin and chlorhexidine, are preferred to inhibit growth of saprophytic fungi or bacteria. Specialized medium incorporating phenol red indicator is also commercially available.t For bacterial cultures, the plate and broth media are incubated at 37°C. for 24 hours, after which time they are checked for growth. (1) If abundant growth occurs and is reasonably pure (i.e., of one colony morphology), appropriate tests are selected to identify the organism and an antibiotic sensitivity test is prepared on Mueller-Hinton agar or 5 per cent blood agar for fastidious organisms. (2) If growth of colonies is scant or mixed, then single colonies may be subcultured onto a 5 per cent blood agar plate and streaked in a fashion as shown in Figure 5B. Culture for 12 to 24 hours and proceed with identification and sensitivity testing. (3) If no growth occurs on plate cultures, check the thioglycollate broth for growth. If no growth is obvious (turbidity) in this medium, all plate and broth media should be recultured an additional 24 to 48 hours. If still negative for growth after this period, the cultures are considered negative for bacterial growth. If growth is present in the broth, subculture the medium to 5 per cent blood agar plate and Levine eosin methylene blue agar, then culture aerobically for 24 hours. If growth occurs, identify and test for sensitivity. If no growth is present on the aerobically cultured blood plate medium and eosin methylene agar plate, reinoculate the media from the broth and culture anaerobically. Fungal cultures are generally grown at room temperature for several days to two weeks. They are checked periodically for growth during this time, and are discarded if no growth has occurred after two weeks. If growth does occur the organism can be further identified. Occasional-

* Gas-Pak, BBL.-Bioquest, Cockeysville, Maryland.

tFungassay-Pitman-Moore, Englewood, New Jersey; Dermatophyte Test Media, DTM-Schering Corporation, Kenilworth, New Jersey.

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ly, with systemic mycotic infections, growth can occur at 37°C. Generally, the organisms will grow on the initial isolating agar plates within a three-day period. ' Blood cultures are incubated for 12 to 24 hours, and first checked for growth after this period. Turbidity or change in odor of the media is usually indicative of growth. At this stage, a 5 per cent blood agar and Levine eosin methylene blue agar plate medium is inoculated, and isolated organisms identified and sensitivity tested. If no growth occurs, continue to incubate the blood culture broth and periodically check for growth. If, after 10 days, no growth has occurred, the culture may be discarded.

IDENTIFICATION OF MICROORGANISMS At this stage, several important criteria have been established with which preliminary identification can be assumed. One knows: (1) whether the organism grows aerobically or is strictly anaerobic; (2) if it will grow only on blood agar or will also grow on enteric selective media, (3) the approximate size of single individual colonies after 24 hours of growth (i.e., either smaller or greater than 1 mm); (4) colony morphology; and (5) the ability of the organism to hemolyze blood. A smear is prepared from the blood agar plate, heat fixed, and stained by the Gram method.* This will indicate the gram reaction and illustrate the organism's morphology. Some further biochemical or culture tests may be carried out according to Figures 1 through 4 in order to further differentiate genus and/or species. One convenient test is the triple sugar iron agar slant which tests for the ability of the organism to grow on nutrient agar in the absence of blood factors, to oxidize or ferment any of three sugars (glucose, lactose, and sucrose), and to produce hydrogen sulfide from sulfates. The catalase test is also useful and is performed by adding one or two drops of hydrogen peroxide to isolated colonies on plate medium or to the TSI agar slant. If the test is performed on blood agar colonies, a weak reaction may be observed due to liberated erythrocyte catalase and may be mistaken for a positive test. Most catalase positive organisms give a strong catalase reaction which is unlikely to be confused with the slower false reaction. Reagents and media for carrying out the other tests cited in Figures 1 through 4 are available either as prepared or raw materials. Formulation and methods for carrying out these tests are available in the references and will not be discussed here.

*Gram Stain, Difco Gram Stain Pak-Difco Laboratories, Detroit, Michigan.

DIAGNOSTIC BACTERIOLOGY IN SMALL ANIMAL PRACTICE

751

SPECIAL CULTURING TECHNIQUES Salmonella This organism can be isolated from heavily contaminated material (e.g., intestinal or fecal swabs) by culturing such swabs for 12 hours in Selenite or GN gram-negative enrichment broth and subculturing the broth to bismuth sulfite agar. Clear or black colonies should be tested biochemically. Most Salmonella are lactose, sucrose, and urea negative, citrate and hydrogen sulfide positive, although the latter can be negative. Confirmation of any isolant may be made using Salmonella polyvalent 0 antigen* in a slide agglutination test. Speciation requires the use of special 0 and H antisera and is best carried out by a state or federal reference laboratory.

Brucella Brucella canis may require an enriched broth medium and reduced atmospheric conditions (10 per cent carbon dioxide) for enhanced primary isolation.

Nocardia Nocardia affects dogs and cats chiefly as a wound infection or as an empyema particularly involving the pleura. Primary isolation is often difficult and is attempted when cytology is suggestive of Nocardia infection (i.e., the finding of long, often beaded, filamentous gram-positive bacilli in smears). Primarily, growth on enriched blood agar under 10 per cent carbon dioxide (Gas-Pak) can be attempted along with anaerobic cultures. Note that one of the features of differentiating Nocardia from Actinomyces israelii is based on the strict anaerobiasis of the latter.

Leptospira The culture and isolation of Leptospira organisms is difficult and potentially dangerous, and for this reason should be referred to state or federal diagnostic laboratories. In most cases demonstration of the organism in blood or urine and serologic testing is all that is necessary to establish the diagnosis of leptospirosis.

Common Bacterial Isolants from Dog and Cat Table 1 summarizes some of the common pathogens isolated in a one-year period at The Animal Medical Center from various sites of the body on dogs and cats. This table is intended to be used as a guide for establishing the significance of bacterial isolants from these sites. *Salmonella Polyvalent 0 Antigen-Difco Laboratories, Detroit, Michigan.

Table I. Common Microorganisms Isolated from VarioU5 Sites in Dogs and Cats

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Abscess Wound

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Ear

Genital Tract

Nasal Cavity l

Trachea or Urine or Respiratory Tract Urinary Tract I

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REFERENCES 1. Bailey, R. W., and Scott, E. G.: Diagnostic Microbiology. Edition 4. St. Louis, C. V. Mosby Co., 1974. 2. Carter, G. R.: Diagnostic Procedures in Veterinary Bacteriology and Mycology. Edition 2. Springfield, III., Charles C Thomas, 1975 3. Coles, E. H.: Veterinary Clinical Pathology. Edition 2. Philadelphia, W. B. Saunders Co., 1974. 4. Lennette, E. H., Spaulding, E. H., and Truant,]. P.: Manual of Clinical Microbiology. Washington, D. C., American Society for Microbiology, 1974. 5. Osbaldiston, G. W.: Laboratory Procedures in Clinical Veterinary Bacteriology. Baltimore, University Park Press, 1973. The Animal Medical Center 510 East 62nd Street New York, New York 10021