JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1976, p. 239-244 Copyright ©) 1976 American Society for Microbiology

Vol. 4, No. 3 Printed in U.S.A.

Bacteriocin (Klebocin) Sensitivity Typing of Klebsiella CARROL L. BUFFENMYER, RUSSELL R. RYCHECK, AND ROBERT B. YEE* Departments of Microbiology* and Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received for publication 8 March 1976

High-titer preparations ofKlebsiella bacteriocins (klebocins) were obtained by using mitomycin to induce standard strains of Klebsiella. Of 296 clinical isolates of Klebsiella, 67% could be typed on the basis of their sensitivity to klebocins. The method proposed in this paper affords a standard basis for the further development of klebocin typing as a suitable procedure for hospital laboratories concerned with epidemiological investigations of hospital-associated infections. Evidence is also provided to show that high-titered klebocin typing can be used in conjunction with biochemical typing to provide a sensitive epidemiological marker for Klebsiella. The problem of hospital-associated infections is a serious one, with incidence rates of 5% and prevalence rates of 15% having been reported in various surveys (1-3, 5). The major etiological agents are various gram-negative bacilli and Staphylococcus aureus, with a trend of increased frequency of infections due to the former. Among the gram-negative bacteria, Klebsiella has been recognized as a major contributor to infections (15, 17). Epidemiological investigations on Klebsiella colonization and disease have, for the most part, relied on capsular serotyping to differentiate strains (4). Recently, biotyping combined with serotyping has been proposed as a more precise means for strain differentiation (13). We propose that another feasible system is bacteriocin typing. Although this procedure has been used successfully for other gram-negative bacteria, such as Pseudomonas, Serratia, and Shigella, its use in typing Klebsiella has been limited (9, 11, 14). Price and Sleigh (11) attempted to use antibiotic sensitivity patterns and phage and bacteriocin typing to study the epidemiological pattern of an outbreak of Klebsiella infections in a neurosurgical unit. Although many types reportedly emerged, they were unable to establish any epidemiological relationships. Specific typing methodologies and results were not stated. Hall (9) used a set of 10 klebocin-producing strains to type 630 clinical isolates of Klebsiella. Most strains fell into 16 type categories, only one of which predominated. A major problem was the lack of reproducibility among large numbers of weak reactions encountered. She felt that klebocin typing could be a useful epide-

miological tool if the method could be made more precise and sensitive. This study was done to define the conditions for obtaining higher yields of klebocins and to type clinical isolates with these preparations. MATERIALS AND METHODS Media. Nutrient broth (NB) was from the Difco Laboratories and Trypticase soy broth (TSB) was from the Baltimore Biological Laboratories. Nutrient agar (NA; Difco), when used for typing, was prepared with a final agar concentration of 2% (wt/ vol) by the addition of Bacto-agar (Difco). Klebsiella strains. Eight klebocin-producing strains, 114, 902, L4, K6, D33, D40, 823, and 525, and an indicator strain, 9C, were kindly supplied by Stefan Slopek, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland (14). The indicator strain, sensitive to all eight klebocins, was used for klebocin titration and as a positive control in typing. Klebsiella isolates were supplied by the following colleagues: Wie-Shing Lee, Presbyterian-University Hospital; Dolores Freedel, Magee-Womens Hospital; and Spyros Kominos, Mercy Hospital; all located in Pittsburgh, Pa. The 296 cultures represented isolates from outpatients, inpatients, and the hospital environment. Ten known serotypes of Klebsiella, ranging from K-1 to K-10, were obtained for us from the Center for Disease Control, Atlanta, Ga., by Joseph Sarandria of the Allegheny County Health Department Laboratories, Pittsburgh, Pa. All cultures were reconfirmed as being Klebsiella by colony morphology on MacConkey agar, reaction in triple sugar iron agar (BBL), the absence of motility in sulfide-indole motility medium (Difco), ability to grow on Simmon citrate agar (BBL), and the decarboxylation of lysine but not ornithine (4). Stock cultures of all klebsiellas were maintained on NA slants. The cultures were incubated over239

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BUFFENMYER, RYCHECK, AND YEE

night at 37°C, overlaid with sterile paraffin oil, Saybolt viscosity 335/350 (Fisher Scientific Co.), and stored at room temperature. Induction of klebocin production. Inductions by ultraviolet (UV) irradiation (10) or with mitomycin C (7, 8, 16) were compared by varying the environmental conditions, e.g., type of medium, length of treatment, and incubation temperature. The methods given below, using UV irradiation or mitomycin C, were found most effective. All cultures were incubated at 37°C. For UV irradiation, an 18-h TSB culture of a klebocin producer was distributed in 7-ml amounts in sterile borosilicate petri dishes (100 by 15 mm). The dishes were placed on a rotary shaker (Eberbach) at a distance of 35 cm from two Sylvania germicidal 15W lamps (G15T8), which produced a UV intensity of 460 ,jW/cm2 at the level of the dishes. The cultures were exposed to the UV light with shaking for 20 s. They were then transferred to sterile culture tubes (18 by 150 mm) and incubated for 24 h. Subsequently each culture was sterilized by the addition of 0.3 ml of reagent grade chloroform (Fisher Scientific Co.) followed by vigorous mixing for 10 s on a Vortex mixer. Most of the chloroform was removed by centrifugation at 1,500 rpm (International Refrigerated Centrifuge model PR-2, head no. 823A) for 5 min. The supernatant was transferred to loosely capped culture tubes (13 by 100 mm), and the residual chloroform was allowed to evaporate. For the mitomycin C method, each producer strain was cultured in 5 ml of TSB in a 50-ml Erlenmeyer flask. After incubation with shaking (Eberbach variable speed shaker) for 18 h, 0.7 ml of a culture was transferred to 3.3 ml of TSB in a 50-ml Erlenmeyer flask and incubated at 37'C for 3 h with shaking. Then 1.0 ml of mitomycin C (Sigma Chemical Co.) (5 ,ug/ml) was added to each culture (final concentration, 1 Ag/ml), and incubation was continued with shaking for an additional 5 h. The remaining viable cells in the culture were killed by the addition of 0.25 ml of chloroform followed by vigorous mixing and centrifugation as described above. The klebocin preparations were maintained at 4°C until they were titrated and used for typing. Klebocin titration. The klebocins were serially diluted eightfold in NB, ranging from 1:8 to 1:512, and twofold thereon to 1:65,536, except for D33 which was diluted twofold from 1:8 to 1:256. A Precision pipette (Medical Laboratory Automation, Inc., Mount Vernon, N.Y.) was used to deliver a 0.025-ml drop of each dilution onto an NA (2% agar) plate (15 by 100 mm). After the spots had diffused into the agar, the plates were streaked with a cotton-tipped applicator that had been moistened in a culture of the 9C indicator strain. This culture was prepared by subculturing from a working stock culture into 2 ml of NB and incubating at 37°C for 6 h. The plates were incubated overnight at 22°C. The highest dilution of klebocin yielding completely clear inhibition of the indicator strain was defined as the inhibitory dilution (ID). Control spots of chloroform-treated TSB containing 1 4g of mitomycin C per ml showed no inhibition of the indicator strain. Klebocin typing. Unknown Klebsiella strains

inoculated by a straight needle from NA slants into 2 ml of NB and incubated at 37°C for 6 h. These cultures were streaked with cotton-tipped applicators onto NA plates (2% agar) that had been spotted with 256x ID of the klebocin preparations. (Initially, typings were done by first spotting plates with klebocin and then streaking with the test strain and conversely by first streaking with the test strain and then spotting with klebocin. The former appeared to yield slightly clearer zones of inhibition.) A positive control plate streaked with a 6-h culture of the 9C indicator strain was included with every typing. The klebocin preparations were titrated before every typing. Readings were made by grading the inhibition seen, using a scale of no reaction to 4+. A reaction was typified by just barely discernible inhibition. A 1 + reaction was defined as a partial inhibition with confluent growth. A 2+ reaction was partial inhibition showing patches of semiconfluent growth or more than 10 colonies. A 3+ reaction was a clear zone containing no more than 10 distinct colonies. A 4 + reaction was a completely clear zone of inhibition. All reactions that were 1 + or greater were designated as being positive, and those that were less than 1+ were designated as negative. The negative and positive reactions to the eight klebocins were recorded, using a mnemonics system (Tables 1 and 2) similar to that of Farmer (6). The eight klebocins were divided into four pairs. Each combination of were

TABLE 1. Mnemonic system for reporting klebocin sensitivlty patterns Mnemonic notation

Reactions to a pair of klebocins

+ +"1 2 + -b

_+

3 4

__

"Inhibition of isolate by both klebocins. Inhibition of isolate by first klebocin and no inhibition by second klebocin. TABLE 2. Examples of klebocin sensitivity pattern using mnemonic system Example 1 (type 1331)

Example 2 (type 4244)

Producer MneMnestrainMn-ne Reaction monic monic ti rveactlon Reaction noa nota-

nota-

tion

114 902 L4 K6 D33 D40 823 525

+ +

1I

+

3

+

3

+

tion

+

4 2

4

_

+ 1 4 +, Inhibition of isolate by klebocin; -, no inhibition.

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KLEBOCIN TYPING OF KLEBSIELLA

241

using the procedure of Traub et al. (16). A comparison of this agent and UV irradiation demonstrated that the mitomycin C was a better inducer (Table 3). Subsequently we found that even higher activity could be obtained by shaking the broth cultures during treatment RESULTS Induction of klebocin production. Prelimi- with the drug (Table 4). Only strains L4 and D33 failed to produce exceptionally high connary work indicated that induction with UV irradiation or mitomycin C would be needed to centrations of klebocins. Storage. Typing of Klebsiella would be faciliobtain high yields of klebocin. Complete inhibition of the 9C indicator strain was obtained, if tated if the klebocin preparations could be at all, only with undiluted supernatants of stored with no marked decrease in activity. We spontaneously induced 18-h TSB cultures of the found storage at 5°C was not feasible since there was four- to eightfold reduction in titer klebocin-producing strains. Eighteen-hour TSB cultures of the klebocin- after 10 days. Storage at either -20 or -68°C producing Klebsiella were irradiated for vary- was suitable, with -68°C being better (Table ing time periods ranging from 5 to 60 s. The 5). With the exception of L4 klebocin preparasupernatants were tested after incubation at 2, tions, no appreciable decreases in titer were 4, 6, 8, and 24 h. The highest yields of klebocin observed after 4 weeks of storage at -68°C. Laboratories that do not have access to -68°C were obtained after 10 or 20 s of irradiation and freezers can store their preparations at -20°C, a postinduction time of 24 h. The ID of these but should recognize that klebocin of strains preparations was 10-1 or 10-2. Mitomycin C also was tested as an inducer, 902, 823, and 525 are less stable at this temperareactions

for a given pair was assigned a mnemonic

notation. Thus, the klebocin type for a given isolate is reported as a mnemonic composed of four numbers. The pairing of the klebocins, the sequence of the pairs, and two examples are given in Table 2.

ture.

TABLE 3. UV irradiation and mitomycin C (Mito) as inducers of klebocin production Klebocin producer strain

Inducer

114

UV

902

Mito UV Mito

L4

Klebocin dilution (reciprocal of log0() 1 2 3 4

UV Mito K6 UV Mito UV D33 Mito D40 UV Mito 823 UV Mito 525 UV Mito a Intensity of inhibition; done.

4+a 4+ 4+ 4+ 1+ 4+ 4+ 4+

4+ 4+ 4+ 4+ NR 4+ 4+ 4+

2+ 4+ 2+ 4+ NR 1+ 1+ 3+

NR

NR NR

NR NR

4+ 4+ 4+ 4+ 4+ 4+

2+ 4+ 2+ 4+

1+ 4+ 4+ 4+ 4+ 4+ 4+

2+ 3+

ND 2+ ND 3+ ND ± ND 2+ ND NR ND 3+ ND 3+ ND 3+

NR, no reaction; ND, not

TABLE 4. IDa of klebocin preparations obtained by the mitomycin C method of Traub et al. and by modified mitomycin C method Klebocin pro- Mitomycin C inducducer strain tion method

Reciprocal of klebocin ID

Traub 1,024 Modified 16,384 902 Traub 4,096 Modified 32,768 L4 Traub 128 Modified 4,096 K6 Traub 1,024 Modified 16,384 D33 Traub 2 Modified 64 D40 Traub 2,048 Modified 16,384 823 Traub 2,048 Modified 16,384 525 Traub 2,048 Modified 16,384 " Highest dilution totally inhibiting 9C indicator strain. 114

TABLE 5. Titers of klebocins after storage at -20 and -68°C for 1 and 4 weeks Storage temp (°C)

-20 -68

Reciprocal of klebocin ID

Weeks of storage

114

902

L4

K6

D33

D40

823

525

0 1 4 0 1 4

16,384 8,912 8,192 16,384 8,192 8,192

32,768 16,384 8,192 32,768 32,768 16,384

4,096 1,024

16,384 8,192 8,192 16,384 16,384 8,192

64 32 32 64

32,768 16,384 16,384 32,768 32,768 16,384

32,768

4,096 8,192 32,768

32,768 8,192 8,192 32,768

16,384 16,384

32,768 16,384

1,024

4,096 2,048 1,024

64 64

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One of the local hospitals submitted an addiKlebocin typing. The feasibility of using the above klebocin preparations for typing was ini- tional six cultures for typing. All of them were tially tested by using the ID of the prepara- found to be the same type, 1131, with the intentions. The results with the first 28 clinical iso- sities of inhibition being the same. Upon relates were encouraging since 54% of them were porting the results to the hospital, it was typable. However, only 40% of a second set of 19 learned that the isolates were from four sepaclinical isolates could be typed. This suggested rate blood cultures of a premature infant. Later that we were either dealing with a high per- the same hospital submitted two cultures that centage of untypable strains or needed to use we knew before typing to be a cerebrospinal fluid isolate and a blood isolate from another higher concentrations of klebocins for typing. Therefore, all of the 296 clinical isolates were infant. Both cultures were type 1131. The reproducibility of the typing procedure typed, using test dilutions of 4, 16, 32, and 256 x ID of the klebocins. The highest percentage of was examined by typing 43 of the clinical isotypable Klebsiella, 67%, was obtained using 256x ID of each klebocin, with the exception of TABLE 7. Percentage of klebocin types of clinical Klebsiella isolated from each of three hospitals the D33 preparation (Table 6). This latter klebocin could only be obtained at a lower titer, Klebocin types (%) from hospital: and its maximum concentration was 128x ID. Klebocin Total B C A type' Subsequent typing was done using D33 at a (n = 133) (n = 103) (n = 60) (n = 296) concentration of 128 x ID and the klebocins at 24 18 256 x ID. The distribution of the klebocin types 21 29 133 lb 7 16 24 13 1131 within each of three hospitals is given in Table 14 1111 16 6 19 7. Sixteen klebocin types were identified over6 16 6 0 3331 all, with two or three types predominant in 2 2 2 1 4244 each institution. 2 2 2 0 4331 Known serotypes, obtained from the Center 1 0 2 0 3131 for Disease Control, were also typed with our 1 2 0 1 4224 set of klebocins. Five of the nine strains were 0 1 0 1431 * 1 0 typable (Table 8). The results with this limited 0 2224 * 0 1 0 2331 number of strains suggest that a given klebocin * 1 0 0 2431 type may be found among more than one sero* 0 0 1 4333 type. 4334 4344 4432 Untypa-

TABLE 6. Percentage of typable Klebsiella isolates using different test dilutions of klebocins No. of

Hospital clinicial isolates A B C Total

133 103 60 296

%

test dilution (x ID)

32

256

20 21 22 21

37 40 38 38

43 39 41 41

70 64 66 67

Klebocin type

K-1 K-2 K-3 K-5 K-6 K-7 K-8 K-9 K-10

131 la 4444 4444 1111 1331 4444 1331 1111 4444

114

b

33

Total

100

100

100

100

n Obtained using D33 pneumocin at 128 x ID and other klebocins at 256x ID. b Mnemonic; see Table 2. d