Quantitative Antibiogram as a Potential Tool for Epidemiological TypingAuthor(s): D. J. FlournoySource: Infection Control, Vol. 3, No. 5 (Sep. - Oct., 1982), pp. 384-387Published by: The University of Chicago Press on behalf of The Society for Healthcare Epidemiologyof AmericaStable URL: http://www.jstor.org/stable/30142290 .

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Ouantitative Antibiogram as a Potential Tool for Epidemiological Typing

D.J. Flournoy, Ph.D.

ABSTRACT

Forty stock, sink drain and clinical isolates of gram- negative bacilli were tested by broth microdilution against eight aminoglycoside antibiotics. Results show that quantitative antibiograms provide more information as epidemiological tools than qualitative antibiograms. [Infect Control 1982; 3(5):384-387.]

INTRODUCTION

Bacteria can be characterized, for epidemiological purposes, by resitotyping, hemagglutinin typing, bio- typing, serotyping, bacteriocin typing, phage typing and antimicrobial susceptibility testing.1-4 Each of these methods has advantages and disadvantages. An ideal method would be not only rapid and inexpensive but easy to perform, interpret, and separate organisms into many different types. Antimicrobial susceptibility test anti- biograms are perhaps one of the most commonly used methods, since this type of testing is needed for patient care, regardless of epidemiology. Although antimicrobial susceptibility testing, by disc-agar diffusion, is helpful, several organisms could have a similar resistance pattern yet vary considerably in their degree of resistance. Quantitative testing, by determining minimal inhibitory concentrations (MICs), provides a greater range of results than standard antibiograms and is therefore potentially more useful epidemiologically.

Aminoglycoside-resistant organisms have been impli- cated in hospital outbreaks8 and isolated repeatedly from sink drains at this institution.9"10 With this in mind, a quantitative antimicrobial susceptibility testing system was designed to type aminoglycoside-resistant gram- negative bacilli.

MATERIALS AND METHODS

Organisms Bacteria used in this study included stock cultures

(donated by the Schering Corporation), sink drain isolates from a previous study'0 and clinical isolates from the Veterans Administration Medical Center of Oklahoma City. Clinical isolates were consecutively-occurring gram- negative bacilli, which were classified as resistant or intermediate by disc-agar diffusion testing, performed as described elsewhere."

Antimicrobials

Antimicrobials were donated as follows: gentamicin (GM), gentamicin Cla (Cla), gentamicin C1 (C1), gentamicin C2 (C2) and netilmicin (N) by the Schering Corporation; tobramycin (TM) by Eli Lilly 8& Company; and kanamycin (K) and ainikacin (AN) by Bristol Laboratories.

Minimal Inhibitory Concentration Testing

Quantitative antimicrobial susceptibility testing was by broth microdilution as described previously.12 The range of concentrations tested was from 0.5 to 500 tg/ml. Testing conditions included: an inoculum concentration of 105 colony forming unitsiml, a final well volume of 0.1 ml, incubation at 350C for 18 to 24 hours and the diluent for the inoculum and antimicrobials was unsupple- mented Mueller-Hinton broth (Difco), which was pre- pared according to the manufacturer's instructions. MICs were performed once and interpreted as susceptible or resistant based on published literature."3

Antibiogram Pattern Classification

All MICs were interpreted qualitatively as either susceptible or resistant, based on a previous report. " The qualitative pattern for each isolate tested was then noted and classified as A-K where A was the most and K the least resistant. Pattern A was therefore used when the organ- ism was resistant to all eight antibiotics, B to seven

From the Microbiology Section, Laboratory Sernice, I 'eterans Administration Medical Center, Oklahoma City, Oklahoma.

Address reprint requests to: D.J. Flournoy, Ph.D., V'eterans Administration Medical Center (113), 921 NE 13th St., Oklahoma City, OK 73104.

384 Quantitatize A:ntibiogram Flournov

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TABLE 1

QUALITATIVE ANTIBIOGRAM PATTERNS OF 40 BACTERIAL STRAINS

Organism (no. tested) GM Clia C1 C2 N TM AN K Pattern

P. aeruginosa (4) R R R R R R R R A Pseudomonas sp. (3) P. cepacia (1) P. maltophilia (1) S. marcescens (1) E. agglomerans (1) E. coi (1)

S. marcescens (6) R R R R R R S R B P. aeruginosa (3)

P. maltophilia (1)

K. pneumoniae (1) R R R R S R S R C Pr. rettgeri (1) P. aeruginosa (1)

S. marcescens (1) S R S S R R R R D P. aeruginosa (1)

P. aeruginosa (1) S S R R R S R R E

P. aeruginosa (1) R R R R S S S S F

S. marcescens (1) S S S S R R S R G

P. aeruginosa (1) S S R S R S S R H

P. aeruginosa (1) S S R S S S S R I

P. aeruginosa (1) S S S S R S S R J

P. aeruginosa (1) S S S S S S S R K

R-resistant. S-susceptible.

of eight, C to six of eight, etc. Each of the quali- tative patterns was then further separated quanti- tatively by numerical designations. The most resis- tant quantitative designation was 1, with higher con- secutive numbers representing different, less resistant patterns, respectively. An organism was considered more resistant when it was resistant to several antibiotics as opposed to having a high MIC against only one antibiotic. Quantitatively, in the twofold broth microdilution test, a deviation of one well from the mean was not considered significant. Therefore an MIC of 1.0 gg/ml would be grouped in the same type as MICs of 0.5 and 2.0 .tg/ml. The overall type was represented by a qualitative and quantitative designation. Al was the most resistant pattern qualitatively and quantitatively and K4 was the least resistant.

RESULTS

Minimal inhibitory concentrations were done against stock cultures, sink drain and clinical isolates of Serratia marcescens, Pseudomonas sp., P. aeruginosa, P. malto- philia, P. cepacia, Klebsiella pneumoniae, Providencia rettgeri, Enterobacter agglomerans and Escherichia coli. They were then interpreted as susceptible or resistant and categorized into 11 distinct qualitative patterns (Table 1),

each with a different code letter. A was the most resistant type and K the least resistant. Each of these 11 qualitative antibiogram patterns was then broken down quantita- tively from most to least resistant. Lower numbers represented greater resistance than higher numbers. The qualitative and quantitative code for each strain are noted in the last column of Tables 2 and 3. P. aeruginosa isolate MICs are shown in Table 2 and other organisms in Table 3. Therefore in Table 2, P. aeruginosa (782) belonged to type K1 which was one of the least resistant; however, it was more resistant than P. aeruginosa (674) which was type K3. There were only two types which had more than one species, A3 with P. cepacia, P. maltophilia and P. aeruginosa and C1 with P. aeruginosa and K. pneumoniae. Type B3 had five strains of S. marcescens and was the commonest type followed by K1 with four strains of P. aeruginosa. Organisms from different sources represented different types except in one case. Three P. aeruginosa type K1 were from sink drains and one from a clinical specimen.

Gentamicin Cla and C2 were most active against P. aeruginosa sink drain isolates, whereas amikacin and netilmicin showed the greatest overall activity versus the clinical isolates. The least active gentamicin component was C1.

INFECTION CONTROL 1982 VI'ol. 3, No. 5 385

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TABLE 2

MICs (jg/ml) OF P. AERUGINOSA ISOLATES

Organism (strain/source) GM Cia C1 C2 N TM AN K Type

P aeruginosa (B-3a/b) 125 125 250 125 125 31 62 31 A3 P. aeruginosa (1-18b/b) 31 125 31 31 125 125 62 125 A6

P. aeruginosa (74100301/a) 31 31 62 31 31 8 31 125 A8 P aeruginosa (g-19a/b) 16 31 16 16 62 31 31 31 A9 P. aeruginosa (24/c) 500 500 >500 500 16 125 8 250 B2 P aeruginosa (6/c) 125 125 125 125 125 125 8 125 B4 P aeruginosa (11652/c) 31 62 8 125 >500 62 1 500 B5 P. aeruginosa (11/c) 62 125 125 125 4 125 8 125 C1 P aeruginosa (73101501/a) 2 31 4 4 500 62 62 250 D1 P. aeruginosa (2-52b/b) 4 4 8 8 8 4 31 125 El

P aeruginosa (2-48/b) 8 8 16 8 .0.5 .0.5 .0.5 .0.5 F1 P aeruginosa (F/c) 2 2 8 2 16 4 16 125 H1 P. aeruginosa (C/c) 2 2 8 2 4 - 16 125 11 P aeruginosa (1-34b/b) 2 .0.5 4 .0.5 8 2 16 62 J1 P aeruginosa (2-40a/b) 1 .0.5 2 .0.5 2 2 4 62 K1 P aeruginosa (G-33a/b) 2 1 4 1 2 .0.5 8 62 K1 P aeruginosa (B-21b/b) 2 .0.5 4 1 2 1 8 62 K1 P aeruginosa (782/c) 2 2 4 2 2 1 8 16 K1 P aeruginosa (B-14b/b) 4 1 4 2 4 4 8 125 K2 P aeruginosa (674/C) .0.5 .0.5 1 1 2 .0.5 4 125 K3 P aeruginosa (1-27/b) .0.5 .0.5 4 .0.5 .0.5 .0.5 2 62 K4

Source. a-stock, b-sink drain, c-clinical

TABLE 3

MICs (jg/ml) OF OTHER ORGANISMS

Organism (strain/source) GM Clia C1 C2 N TM AN K Type

Pseudomonas sp. (J/c) >500 >500 >500 >500 >500 >500 500 >500 Al Pseudomonas sp. (736/c) >500 500 >500 >500 250 500 250 125 A2 P. cepacia (505/c) 125 125 250 125 125 62 125 62 A3 P maltophilia (B/c) 125 62 125 62 62 62 125 125 A3 S. marcescens (8/c) 250 250 250 250 16 250 31 250 A4 E. agglomerans (C/c) 31 125 31 31 125 250 62 500 A5 Pseudomonas sp. (450/c) 31 31 125 31 31 31 62 62 A7 E. coli(L/c) 8 8 8 8 8 8 31 62 A10 P maltophilia (11591/c) 500 500 >500 500 125 62 16 250 B1 S. marcescens (10/c 125 125 250 125 16 125 16 250 B3 S. marcescens (12/c) 125 125 250 125 8 125 8 250 B3 S. marcescens (19/c) 125 125 125 125 16 125 8 250 83 S. marcescens (1/c) 125 125 125 125 8 125 16 250 83 S. marcescens (7/c) 125 125 250 125 8 125 16 250 B3 S. marcescens (6/c) 62 62 125 62 8 125 4 125 B6 K. pneumoniae (18/c) 125 125 125 125 4 125 8 250 C1 Pr. rettgeri (275/c) 8 16 16 16 4 8 .0.5 16 C2 S. marcescens (76032303/a) 1 16 2 4 62 31 31 500 D2 S. marcescens (90/c) 2 4 2 2 16 16 16 32 G1

Source: a-stock, b-sink drain, c-clinical

DISCUSSION

Many clinical microbiology laboratories are performing quantitative antimicrobial susceptibility tests routinely. With the trend toward quantitation, this type of epidemiological tool could prove useful. For those laboratories which routinely do disc-agar diffusion

testing, the MIC typing approach could be used selectively to characterize mu!tiple-antimicrobial-resistant organ- isms which are considered to be potentially dangerous in causing outbreaks. Aminoglycoside-resistant P. aeruginosa strains were studied because they have been isolated repeatedly from sink drains at this hospital and

386 Quantitative Antibiogram/Flournoy

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represent a potential threat to debilitated patients.10 The potential epidemiological value of the quantitative

antibiogram can readily be seen in Table 2 when P. aeruginosa strains 24 and 11652 are compared. These organisms would have identical qualitative patterns, B, both resistant to all antimicrobials except amikacin, even though their quantitative antibiograms differ drastically. Crichton and Old' also noted that qualitative antibio- grams were of limited value because of too few types.

Commercially-prepared gentamicin consists of three components, C1, Cla and C2, which are usually present in near equal proportions. Gentamicin and its components are good epidemiological markers since they can provide different MICs (Table 2, strain B-21b).

One problem with currently used epidemiological typing systems is that there are so many and they vary from organism to organism. These various systems are cumber- some and impractical for most clinical microbiology laboratories. Emphasis should be placed on fewer systems to type more organisms. A combination quantitative antibiogram/resistogram might be the best overall system, since it could possibly provide antibiotic susceptibility and physiological clues to the type of organism involved in an outbreak. Numerous metals and disinfectants have also been studied.14'"15 Buck et a1'6 have utilized a combination of antimicrobials and other compounds to identify bacteria with the Autobac I (Pfizer Diagnostics). This automated system generated quantitative data. Miller et al'7 have also surveyed aminoglycoside-resistant patterns and characterized their enzymatic inactivation mechanisms on the basis of MICs of aminoglycosides. An automated quantitative typing system which could provide information on enzymatic inactivation mechan- isms of antimicrobials and resistance to disinfectants and other compounds against many different types of clinical isolates would be a great epidemiological tool. Until more sophisticated typing systems are developed, however, the quantitative antibiogram could prove useful for typing aminoglycoside-resistant gram-negative bacilli and maybe other organisms. This system could provide epidemiological information for any hospital, and especially those where funds, personnel and time are severely limited, as in this case.

REFERENCES

1. Crichton PB, Old DC: Differentiation of strains of Escherichia coli: Multiple typing approach. J Clin Microbiol 1980; 11:635-640.

2. Govan JRW, Gillies RR: Further studies in the pyocine typing of Pseudomonas pyocyanea. J Med Microbiol 1969; 2:17-25.

3. Kocka FE, Srinivasan S, Mowjood M, Kantor HS: Nosocomial multiply resistant Providencia stuartii: A long-term outbreak with multiple biotypes and serotypes at one hospital. J Clin Microbiol 1980; 11:167-169.

4. Lewis SA, Altemeier WA: Emergence of clinical isolates of Staphylococcus aureus resistant to gentamicin and correlation of resistance with bacteriophage type. J Infect Dis 1978; 137:314-317.

5. Craven PC, Jorgensen JH, Kaspar RL, et al: Amikacin therapy of patients with multiply antibiotic-resistant Serratia marcescens infections. Am J Med 1977; 62:902-910.

6. Guerrant RL, Strausbaugh RP, Wenzel RP, et al: Nosocomial bloodstream infections caused by gentamicin-resistant gram- negative bacilli. Am J Med 1977; 62:894-901.

7. Jaurequi L, Cushing RD, Lerner AM: Gentamicin/amikacin- resistant gram-negative bacilli at Detroit General Hospital, 1975- 1976. Am J Med 1977; 62:882-888.

8. Rennie RP, Duncan IBR: Emergence of gentamicin-resistant Klebsiella in a general hospital. Antimicrob Agents Chemother 1977; 11:179-184.

9. Flournoy DJ, Muchmore HG, Francis EB: Noscomial infection linked to handwashing. Hospitals 1979; 53:105-107.

10. Perryman FA, Flournoy DJ: Prevalence of gentamicin- and amikacin-resistant bacteria in sink drains. J Clin Microbiol 1980; 12:79-83.

11. Performance standards for antimicrobial disc susceptibility tests, M2-A2S. Villanova, Pennsylvania, National Committee on Clinical Laboratory Standards, 1981.

12. Flournoy DJ, Perryman FA: LY127935, a new Beta-lactam antibiotic, versus Proteus, Klebsiella, Serratia and Pseudomonas. Antimicrob Agents Chemother 1979; 16:641-643.

13. Barry AL, Thornsberry C: Susceptibility testing: Diffusion test procedures, in Lennette EH, Balows A, Hausler WJ, et al (eds): Manual of Clinical Microbiology, ed 3. Washington, American Society for Microbiology, 1980, p 464.

14. Summers AO; Jacoby GA: Plasmid-determined resistance to boron and chromium compounds in Pseudomonas aeruginosa. Anti- microb Agents Chemother 1978; 13:637-640.

15. Sutton L, Jacoby GA: Plasmid-determined resistance to hexa- chlorophene in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1978; 13:634-636.

16. Buck GE, Sielaff BH, Boshard R, et al: Automated, rapid identification of bacteria by pattern analysis of growth inhibition profiles obtained with Autobac I. J Clin Microbiol 1977; 6:46-49.

17. Miller GH, Sabatelli FJ, Hare RS, et al: Survey of aminoglycoside resistance patterns. Developments in Industrial Microbiology 1980; 21:91-104.

INFECTION CONTROL 1982 1ol. 3, No. 5 387

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