CURRENT MICROBIOLOGY Vol. 12 (1985), pp. 17-22 Current Microbiology �9 Springer-Verlag 1985

Evidence for Plasmid-mediated Resistance of Pseudomonas putida to Hexahydro- 1,3,5-triethyl-s-triazine

Elaine Hall and Robert G. Eagon

Department of Microbiology, University of Georgia, Athens, Georgia, USA

Abstract. Resistance to the industrial biocide hexahydro-1,3,5-triethyl-s-triazine (HHTT) by a strain of Pseudomonas putida was shown to be encoded by a 32.5-megadalton (Mdal) plasmid as evidenced: (a) by visualization of the plasmid DNA by agarose gel electrophoresis, (b) by the loss of HHTT resistance as well as the loss of the 32.5-Mdal plasmid upon curing with novo- biocin, and (c) by conjugal concomitant transfer of HHTT resistance and the 32.5-Mdal plas- mid by mating the novobiocin-cured HHTT-sensitive derivative with the HHTT-resistant strain. The 32.5 Mdal did not encode for heavy metal or antibiotic resistance, and it was shown not to be one of the "degradative" plasmids of Pseudomonas. The mechanism of HHTT resistance was not discerned from these studies.

The biodeterioration of many industrial products by microorganisms is a problem of great economic im- portance. Economic losses due to microbiological spoilage has become an increasingly significant problem for paper and paint industries in recent years [12]. Many components of paint formulations such as natural oils, plasticizers, and emulsifying agents can be used by bacteria and fungi as carbon sources [3]. Substances such as starch, protein, and various synthetic compounds are used as coating binders in the paper industry and these substances are also subject to microbiological attack and spoil- age [12]. Kaolin, a mineral clay used in the produc- tion of both paint and paper, has been shown to serve as a source of minerals and nutrients needed for bacterial growth [13, 17].

Industrial biocides for many years have been incorporated by paint and paper manufacturers into finished products or into components of finished products in an attempt to control bacterial and fun- gal growth and thereby reduce biodeterioration [13, 15]. In industrial parlance, the term biocide means any agent used to suppress the growth of, or to kill, microorganisms. Hexahydro-l,3,5-triethyl-s- triazine (HHTT) is an industrial biocide commonly used in kaolin slurries and in paper- and paint-mak- ing industries. This biocide has been found to be effective in protecting paper pulp, paper coating binders, paint formulations, and aqueous kaolin

Address reprint requests to: Dr. Robert G. Eagon, Department

slurries, as well as other industrial products, from biodeterioration [10, 14].

Candal and Eagon [4] studied a total of 16 iso- lates of Pseudomonas strains that were cultured from biocide-treated kaolin slurries and, in one in- stance, from a water-based latex paint with respect to their resistance to four industrial biocides. Many of the isolates showed multiple resistance patterns in that they were resistant to more than one biocide as well as to certain heavy metals and other antimi- crobial agents, and many of the isolates were resis- tant to agents to which they had no previous history of exposure. One to four plasmids were detected in each of the biocide-resistant isolates that were ex- amined. These experimental data were highly sug- gestive that resistance to the industrial biocides was due to plasmid-encoded mechanisms. Thus, the purpose of this study was to determine whether the genes that encode for resistance to HHTT were chromosomal or plasmid located.

Materials and Methods

Organisms. The HtlTT-resistant organism used in this study was one of several Pseudomonas species originally isolated from bio- cide-treated kaolin slurries showing high microbial counts [4]. This organism was subsequently identified as P. putida and it was assigned the strain designation 3-T-152. Pseudomonas aeru- ginosa PA01 and P. putida ATCC 12633 were used as control or reference organisms in these studies.

of Microbiology, University of Georgia, Athens, GA 30602, USA.

18 CURRENT MICROBIOLOGY Vol. 12 (1985)

Organisms used in plasmid screens were: P. putida 3-T-152, a novobiocin-treated (cured) derivative ofP. putida 3-T-152 (des- ignated 3-T-152 11:21), and exconjugants resulting from various conjugation experiments. Organisms used as standards in plas- mid screens were: Escherichia coli J53(RP4), E. coil J53(pSa), E. coli J53(RP4)(R300B), E. coil RRl(pBR322), and E. coil DU1040(pDU202).

Media. Pseudomonas putida 3-T-152 and HHTT-resistant trans- conjugants were maintained on Luria (L) agar slants (10 g tryp- tone, 5 g yeast extract, 10 g NaCI, 15 g agar, and deionized water to 1 liter) containing 500 ppm (final concentration) of HHTT. Pseudomonas aeruginosa PA01 and P. putida ATTCC 12633 were maintained on slants of nutrient agar (Difco Labora- tories, Detroit, MI). All cultures were kept at room temperature and transferred every 2-3 weeks.

For conjugation experiments, L agar plates supplemented with the appropriate selective agents were used to recover trans- conjugants.

Minimal inhibitory concentration (MIC) determinations. MIC de- terminations were made in Antibiotic Medium 3 (Difco Labora- tories, Detroit, MI). MIC values were determined by the broth dilution test tube method as described by Anderson [2]. The MIC was defined as the lowest concentration of biocide preventing visible growth. MIC values were recorded after 24 h of incuba- tion at 32~

Curing experiments. Pseudomonas putida 3-T-152 was subjected to curing treatments utilizing the antibiotic novobiocin (Sigma Chemical Company, Saint Louis, MO) as the curing agent. Or- ganisms were inoculated into Erlenmeyer flasks containing 30 ml of L broth and incubated overnight, with shaking, at 32~ Cul- tures were then diluted I : 1000 in L broth and 1.5-ml samples were inoculated into a series of tubes containing L broth and nine different concentrations of novobiocin. The concentrations of novobiocin used were: 1000/~g/ml, 800/~g/ml, 600/~g/ml, 400 /~g/ml, 200/~g/ml, 100 p~g/ml, 50/~g/ml, and 0/~g/ml. All samples were incubated overnight at 32~ From each series of tubes, the culture was selected that showed a just detectable increase in turbidity. From this culture, dilutions were made and 0.1-ml samples from these dilutions were plated onto nutrient agar. Plates were incubated overnight at 32~ Individual colonies were "picked and patched" (using sterile toothpicks) onto L agar master plates. After 24 h of incubation at 32~ the master plates were replica-plated onto L agar containing 250 ppm and 500 ppm of HHTT to test for loss of resistance.

The organism 3-T-152 and its cured derivative, 3-T-152 11:21, were used as donor and recipient, respectively, in conju- gation experiments. Escherichia coli J53(RP4) was also mated with the HHTT-resistant isolate 3-T-152.

Isolation and characterization of plasmid DNA. Plasmid DNA was extracted from the organisms by the method of Kado and Liu 11 l] modified slightly as previously described [4].

Conjugation experiments. To determine whether the R plasmid encoding HHTT resistance was conjugative (self-transmissible), mating experiments were performed using the 3-T-152 isolate as the donor and its HHTT-sensitive, cured derivative, 3-T-152 11:21, as the recipient. Spontaneous mutants (resistant to differ- ent antibiotics) of each of these organisms were obtained in the laboratory. The donor was resistant to rifampicin (Rif) (125/~g/ ml), while the recipient was streptomycin (Sin) resistant (250/~g/

ml). Donor and recipient organisms were inoculated into two different 250-ml Erlenmeyer flasks each containing 50 ml of L broth. Cultures were incubated overnight with shaking at 32~ Equal volumes (approximately 0.05 ml) of each parent culture were mixed together on an L agar plate. This mixture was incu- bated for 12 h at 32~ After incubation, the agar was washed with 5 ml of sterile saline and cells were scraped from the agar surface with a sterile toothpick. Cells were collected with a pi- pet, transferred to a sterile test tube and mixed by vortexing. Dilutions were made of the resultant suspension and 0.2-ml sam- ples were spread onto L agar plates containing 250/~g/ml Sm and 500 ppm HHTT to select for exconjugants. Exconjugants that appeared on the selective medium were retested for their resis- tance or sensitivity to Sin, Rif, and HHTT by replica plating.

Because results obtained from the experiments described above indicated that the transfer frequency of the HHTT resis- tance plasmid was low, this plasmid was mobilized using plasmid RP4. Escherichia coil J53(RP4) (Ap r, Kn r, Tc r, Rift, HHTT ~) was used as the donor organism and the 3-T-152 isolate (Rif', HHTT r, Kn ~) served as the recipient (abbreviations: Ap, ampi- cillin; Kn, kananycin; Tc, tetracycline). Matings were carried out as described above, and exconjugants were selected for using L agar plates supplemented with Rif (125/zg/ml) and Kn (50/zg/ ml). Colonies that grew on this medium were then tested for HHTT resistance. The exconjugant, 3-T-152(RP4) (Kn r, Rift, HHTTL Sm s) was then used as a donor and mated with 3-T-152 11:21 (Sin r, KnL RifL HHTT~). Plates of L agar containing Sm (250/zg/ml), Kn (50/zg/ml), and HHTT (500 ppm) were used to select for exconjugants.

Screening for additional plasmid-specified characteristics. The re- sistance of 3-T-152 and 3-T-15 z 11:21 to silver, mercury, tellu- rium, arsenate, and arsenite were tested. Approximately l07 cells from overnight cultures of each organism were inoculated onto L agar plates containing, in final concentration, 1.0 mM AgCI2, 50/~M HgCl2, 0.02 mM TeO, 80 mM Na2HAsO4 - 7 H20, and 10 mM NaAsO2. The plates were incubated at 32~ and observed for growth after 24, 48, and 72 h.

The ability of the two strains to utilize various organic com- pounds as sole carbon and energy sources was also tested. The following compounds were used: salicylate, naphthalene, cam- phor, toluene, n-octane, n-hexadecane, n-dodecane, and m-xy- lene. Salicylate (0.05%, final concentration) was added to the basal salts (EPBS) agar described previously [7]. Crystals of ei- ther naphthalene or camphor were added to the lids of inverted EPBS agar plates. Filter-paper circles were placed in the lids of inverted EPBS agar plates and saturated with approximately 0.4 ml of either toluene, n-octane, n-hexadecane, n-dodecane, or m- xylene. All plates were inoculated with approximately 107 cells from overnight cultures. Salicylate plates were incubated at 32~ All other plates were incubated at ambient temperature. Plates were observed for growth after 24, 48, and 72 h.

R e s u l t s

MIC. The MIC of HHTT for the HHTT-resistant isolate, P. putida 3-T-15 2, its novobiocin-cured de- rivative, 3-T-152 11:21, P. putida ATCC 12633, and P. aeruginosa PA01 is shown in Table 1. It can be seen that resistance to HHTT was readily elimi- nated from 3-T-152 by novobiocin as a curing agent. Although not shown in Table 1, out of 100 colonies

E. Hall and R. G. Eagon: Plasmid-mediated Resistance to a s-Triazine-type Biocide 19

Table 1. MIC of HHTT for HHTT-resistant Pseudomonas putida 3-T-152, its novobiocin-cured HHTT-sensitive derivative, 3-T- 152 11:21, and wild-types P. putida ATCC 12633 and P. aeruginosa PA01

Organism HHTT (ppm)

3-T-15 z 1000 3-T-15 z 11:21 32 12633 125 PA01 125

Fig. 1. Agarose gel electrophoresis of plasmid DNA from the HHTT-resistant Pseudomonas putida 3-T-15 ~ (lane 7), the novo- biocin-cured, HHTT-sensitive recipient 3-T-15 z 11:21 (lane 6), and an exconjugant (lane 5) recovered from the mating of these partners. Lane 3 shows plasmid DNA from 3-T-152(RP4) (HHTTq bearing both plasmid RP4 and the HHTT resistance plasmid. In lane 1, plasmid DNA from 3-T-15 z 11:21 after mating with 3-T-152(RP4)(HHTT r) containing both plasmid RP4 and the HHTT resistance plasmid is seen. Lanes 4 and 2 depict plasmid DNA from 3-T-15 z 11:21 after mating with 3-T-15z(RP4)(HHTT r) containing only RP4 and the HHTT resistance plasmid, respec- tively. The molecular weight standards are as follows: lane 8, pDU202, (60 Mdal); lane 9, pBR322 (3.3 Mdal); lane 10, pSa (24 Mdal); and lane 11, RP4 and R300B (38 and 5 Mdal, respec- tively). The arrow indicates the 32.5-Mdal plasmid encoding for HHTT resistance.

tes ted f rom the novob ioc in - t rea ted cul ture, only seven grew on L agar containing 250 ppm of H H T T and none grew on L agar containing 500 ppm of H H T T .

Fig. 2. Agarose gel electrophoresis of plasmid DNA from HHTT-resistant Pseudomonas putida 3-T-152 (lane 2) and its novobiocin-cured, HHTT-sensitive derivative, 3-T-152 11:21 (lane 1). The molecular weight standards are as follows: lane 3, RP4 and R300B (38 and 5 Mdal, respectively); lane 4, pSa (24 Mdal); lane 5, RP4 (38 Mdal); and lane 6, pDU202 (60 Mdal). The arrow indicates the 32.5-Mdal plasmid encoding for HHTT resis- tance.

Characterization of plasmid DNA. Plasmid D N A isolated f rom bo th the HHTT- re s i s t a n t 3-T-152 iso- lated and its HHTT-sens i t i ve derivat ive, 3-T-152 11:21, was subjec ted to agarose gel e lec t rophores is . Four E. coli strains conta in ing plasmids o f k n o w n molecular weights were used as s tandards . Figure 1 shows the e lec t rophore t ic profiles o f plasmid D N A f rom 3-T-152 (lane 2) and 3-T-152 11:21 (lane 1). A plasmid can be seen in 3-T-152 that is absent f rom 3- T-152 11:21. The molecu la r weight o f this p lasmid was approx ima te ly 32.5 megada l tons (Mdal). The e lec t rophore t ic profiles o f the two organisms were o therwise identical; each conta ined at least three small p lasmids , all having molecular weights o f less than 5 Mdal.

Conjugation exper iments . In o rder to s tudy the con- jugal t ransfer o f the H H T T res is tance plasmid, sev- eral con juga t ion exper imen t s were pe r fo rmed . To de te rmine whe the r the H H T T res is tance plasmid was self- t ransmissible, initial mat ing exper iments were pe r fo rmed using 3-T-152 ( H H T T r, Rif r, Sm S) as the d o n o r and 3-T-152 11:21 (Sin r, H H T T s, Rif s) as the rec ip ient (Table 2, line 1). Excon jugan t s were r ecove red , bu t the t ransfer f r equency was low (4.4 • 10-8). F igure 2 shows plasmid D N A f rom the 3-T- 152 d o n o r (lane 7), the 3-T-152 11:21 recipient (lane 6), and an excon jugan t (lane 5). The same plasmid

2 0 CURRENT MICROBIOLOGY VOI. 12 (1985)

Table 2. Conjugal t ransfer o f the HHTT-res i s tan t plasmid with and without plasmid RP4

Transfer f requency of res is tance to"

Donor Recipient Selective agents H H T T Kn

1) 3-T-15 ~ 3-T-152 11:21 (HHTT ~, Rif r, Sm ~) (Sm r, H H T T ~, Rif ~) Sm + H H T T 4.4 x 10 8 _

2) Escherichia coli J53 3-T-152 (RP4) (Kn r, H H T T S, Rif s) (HHTT r, Rif r, Kn s) Rif + Kn - - 5.3 x 10 i

3) 3-T-15 z 3-T-15 z 11:21 (RP4) (HHTT r, KnL Sm s) (Sm r, H H T T L Kn ~) Sm, Kn + H H T T 2.6 • 10 5 3.1 x 10 -2~

a Frequency of t ransfer Was calculated as the number of exconjugants divided by the number of donor cells present at the commenc e - ment o f mating. b Selection was on L agar containing Sm (250/zg/ml) and Kn (50 tzg/ml). Of 150 exconjugants tested, none grew on 500 ppm H H T T , indicating that the plasmid RP4 had transferred but the H H T T resis tance plasmid had not.

seen in the donor was also seen in the exconjugant, but this plasmid was absent from the recipient. Ex- conjugants had MIC values of 1000 ppm when tested with HHTT (data not shown).

Mobilization of the HHTT resistance plasmid was accomplished using plasmid RP4, which is known to mobilize certain nontransmissible plas- mids [5]. Plasmid RP4 was first moved into the 3-T- 152 isolate by using E. coli J53(RP4) (Kn r, HHTT s, Rif s) as the donor and mating it with 3 - T - 1 5 2

(HHTT r, Rif r, Kn S) (Table 2, line 2). Plasmid RP4 transferred to the 3-T-152 recipient with a frequency of 5.3 x 10 -1. Plasmid DNA from an exconjugant resulting from this mating can be seen in Fig. 2, lane 3. Both plasmid RP4 and the slightly smaller HHTT resistance plasmid can be discerned.

The 3-T-152 isolate bearing both plasmid RP4 and the HHTT resistance plasmid was then used as the donor and mated with the cured 3-T-152 11:21 (Sin r, HHTT s, Kn ~) (Table 2, line 3). Plates of L agar containing Sm, Kn, and HHTT were used to select for exconjugants that had received both plas- mid RP4 and the HHTT resistance plasmid. In these experiments, HHTT resistance was transferred with a frequency of 2.6 x 10 -5. Figure 2, lane 1, shows an exconjugant of 3-T-152 11:21 after mating with 3-T-152(RP4)(HHTT9 that bears both the RP4 plasmid and the HHTT resistance plasmid. Excon- jugants that had received only plasmid RP4 were also recovered. These exconjugants grew on L agar containing 50/zg/ml Kn but did not grow on L agar containing 500 ppm HHTT. Plasmid DNA from one of these exconjugants is seen in Fig. 2, lane 4. A few Sm r exconjugants were recovered that had appar- ently received the HHTT resistance plasmid but no plasmid RP4 since they grew on L agar containing

500 ppm HHTT but did not grow on L agar contain- ing 50/xg/ml Kn. In Fig. 2, lane 2, plasmid DNA from one of these exconjugants, 3-T-152 11:21 (HHTT r, Sm S, Rifs), can be seen.

Screening for additional plasmid-specified character- istics. The HHTT-resistant 3-T-152 isolate and its cured, HHTT-sensitive derivative, 3-T-152 11:21, were screened for differences in their resistance to the following toxic metallic elements: silver, mer- cury, tellurium, arsenate, and arsenite. Both strains were sensitive to silver, tellurium, and arsenite, but both were resistant to mercury. The 3-T-152 strain was resistant to arsenate, while 3-T-152 I1:21 was sensitive to arsenate.

In addition, both strains were screened for their ability to utilize salicylate, naphthalene, camphor, toluene, n-octane, n-hexadecane, n-dodecane, and m-xylene as sole sources of carbon and energy. Neither strain was capable of growth using any of these compounds as the sole carbon and energy s o u r c e .

D i s c u s s i o n

Resistance to many toxic metal ions and to other antimicrobial agents is known to be plasmid-medi- ated in Pseudomonas species (for review, see Cha- krabarty [5]). It was suspected, therefore, that resistance to industrial biocides may be plas- mid-mediated as well, especially in view of the fact that all biocide-resistant isolates of Pseudo- monas that were previously studied were shown to contain 1-4 plasmids [4].

The following results indicate that HHTT resis- tance was plasmid-associated in the P. putida 3-T-

E. Hall and R. G. Eagon: Plasmid-mediated Resistance to a s-Triazine-type Biocide 21

152 isolate. First, curing experiments using novo- biocin as the curing agent resulted in the loss of H H T T resistance in approximate ly 93% of the sin- gle colonies examined. Second, agarose gel electro- phoresis of plasmid D N A isolated f rom the H H T T - resistant 3-T-15 z isolate revealed a plasmid with a molecular weight of approximately 32.5 Mdal. This particular plasmid was not observed when plasmid D N A from 3-T-152 11:21, the novobiocin-cured HHTT-sens i t ive derivative of the 3-T-152 isolate, was analyzed.

Conjugal t ransfer of H H T T resistance using the 3-T-152 isolate as a donor and its cured derivative, 3-T-152 11:21, as the recipient was demonstrated. Exconjugants were recovered from these matings, but the t ransfer f requency was low (approximately 4.4 • 10-8).

Plasmid RP4 has been shown to be transferable to many Gram-negat ive bacter ia [6, 19]. This plas- mid was originally isolated f rom P. aeruginosa and confers resistance to ampicillin, kanamycin/neomy- cin, and tetracycline [9]. Plasmid RP4 was used in conjugation exper iments , therefore, to mobilize the H H T T resistance plasmid. In these matings, H H T T resistance was t ransferred to the HHTT-sens i t ive 3- T-152 11:21 organism at a f requency of about 2.6 • 10-5.

Both 3-T-152 and its cured derivative, 3-T-152 11:21, were resistant to mercury. Mercury resis- tance is known to be plasmid-mediated in bacter ia [1, 18]. This suggested that mercury resistance was probably not specified by the 32.5-Mdal plasmid seen in 3-T-152 but absent f rom 3-T-152 11:21. Ex- cept for this 32.5-Mdal plasmid, the plasmid profiles of the two organisms were identical, i.e., each or- ganism contained three small (<5 Mdal) plasmids.

Both 3-T- 152 and 3-T- 152 11:21 were sensitive to silver, tellurium, and arsenite. However , 3-T-152 was resistant to arsenate while 3-T-152 11:21 was sensitive to this agent. In E. coli and S taphy lococ - cus aureus , the genes coding for resistance to arse- nate and arsenite have been shown to be closely linked on the same plasmids [8, 16]. I f these genes are also closely linked on plasmids in P s e u d o n o - mas , the observat ion described above is hard to ex- plain, but mutant plasmids of S. aureus have been isolated that were arsenate-resis tant and arsenite- sensitive [16].

The exper imental evidence presented herein strongly suggested that the genes that encode for resistance to H H T T were located on 32.5-Mdal plasmid rather than on the bacterial chromosome. This evidence does not provide information, how-

ever, on the biochemical mechanism of this plas- mid-mediated resistance. Further experiments need to be conducted in order to determine the actual resistance mechanism involved (e.g., whether H H T T is being degraded by the microorganisms, whether access of H H T T to a particular sensitive target within the cell is being blocked, or whether resistance is due to some other mechanism).

P s e u d o m o n a s pu t ida 3-T-152 was unable to use various alkanes and aromatic compounds . Thus, this indicated that the 32.5-Mdal plasmid encoding for H H T T resis tance was not one of the known "deg rada t ive" plasmids of P s e u d o m o n a s species (for review, see Chakrabar ty [5]).

We believe that this is the first report of a R- plasmid encoding resistance to an industrial biocide of this type. It is likely that plasmid-mediated resis- tance to industrial biocides may be as diverse, per- plexing, and interesting as that of plasmid-mediated antibiotic and heavy-meta l resistance.

ACKNOWLEDGMENT

This work was supported in part by a contract from the Freeport Kaolin Company.

Literature Cited

1. Ahonkhai I, Russell AD (1979) Response of RP1 + and RP1 strains of Escherichia coli to antibacterial agents and trans- fer of resistance to Pseudomonas aeruginosa. Curr Micro- biol 3:89-94

2. Anderson TG (1970) Testing of susceptibility of antimicro- bial agents in body fluids. In: Blair JE, Lennette EH, Truant JP (eds) Manual of clinical microbiology. Bethesda MD: American Society for Microbiology, pp 299-310

3. Block SS (1977) Preservatives for industrial products. In: Block SS (ed) Disinfection, sterilization, and preservation, 2nd edn. Philadelphia: Lea and Febiger, pp 788-833

4. Candal FJ, Eagon RG (1985) Evidence for plasmid-mediated bacterial resistance to industrial biocides. Int Biodeteriora- tion 20:221-224

5. Chakrabarty AM (1976) Plasmids of Pseudomonas. Annu Rev Genet 10:7-30

6. Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH (1971) Properties of an R factor from Pseudomonas aeru- ginosa. J Bacteriol 108:1244-1249

7. Eagon RG, Phibbs PV Jr (1971) Kinetics of transport of glu- cose, fructose, and mannitol by Pseudomonas aeruginosa. Can J Biochem 49:1031-1041

8. Foster RJ (1983) Plasmid-determined resistance to antimi- crobial drugs and toxic metal ions in bacteria. Microbiol Rev 47:361-409

9. Holloway BW, Richmond MH (1973) R-Factors used for genetic studies in strains of Pseudomonas aeruginosa and their origin. Genet Res 21:103-105

10. Izzat IN, Bennett EO (1979) The potentiation of cutting fluid preservatives by diethylene triamine pentaacetic acid. Int Biodeterioration Bull 15:1-6

22 CURRENT MICROBIOLOGY Vol. 12 (1985)

11. Kado CI, Liu S-T (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365- 1373

12. Lutey RW, May OW (1978) Preservatives. In: Landers BG, Knoll L (eds) Paper coating additives. New York: TAPP1 Press, pp 106-125

13. Onyekwelu IU, Bennett EO (1979) The effects of filtering agents upon the activity of preservatives in cutting fluids. Int Biodeterioration Bull 15:88-95

14. Rossmore HW (1979) Heterocyclic compounds as industrial biocides. Dev Indust Microbiol 20:41-71

15. Sharpell F (1980) Industrial uses of biocides in processes and products. Dev Indust Microbiol 21:133-140

16. Silver S, Budd K, Leahy KM, Shaw WV, Hammond D, Novick RP, Willsky GR, Malamy MH, Rosenberg H (1981) Inducible plasmid-determined resistance to arsenate, arse- nite, and antimony(Ill) in Escherichia coli and Staphylococ- cus aureus. J Bacteriol 146:983-996

17. Stotzky G, Rein LT (1966) Influence of clay minerals on microorganisms. Can J Microbiol 12:547-563

18. Summers AO, Lewis E (1972) Volatilization of mercuric chloride by mercury resistant plasmid-bearing strains of Es- cherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. J Bacteriol 113:1070-1072

19. Sykes RB, Richmond MH (1970) lntergeneric transfer of a/3- lactamase gene between Ps. aeruginosa and E. coli. Nature 226:952-954