NOTES

Isolation of auxotrophs of Bacteroides fragilis ROGERL. VANTASSEL.L A N D TRACY D. W I L K I N S ' A~ltrcroheLoDortrro~:\,,Vi~.gi~ricc Polyrcclrtric Itr.\tirrerc, colt1 Sttrre Ut~i~.or.\iry, Bltrt~X.\0111;~, VA , U.S.A. 24060

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/10/14 For personal use only.

Accepted September 21. 1978 VAN TASSEL.^., R. L., i ~ n dT. D. WIL.KINS. 1978. Isolation o f auxotrophs ofBtrc~rooittes fiiceIqiIiis. Can. J . Microbiol. 24: 1619-1621. Techniques employing ethane methanesulfonate and modified penicillin enrichment were l d readily isolated. Several a~rxodeveloped whereby auxotrophs o f Boc,reroitles f i ~ t e g i l ic. ~o ~ ~ be trophic phenotypes are described. VAN TASSELL,R. L.. et T. D. WILKINS. 1978. Isolation o f auxotrophs o f Bc~creroitlesfktrgili.~. Can. J . Microbiol. 24: 1619-1621. Nous avons ~ ~ t i l i slee methane-sulfon;~tecl'ethane et une technique modifiee de I'enrichissement par la penicilline pourisoler lesBtrcrc~c~oitl~s.fkt~giIi.~ auxotropheset nousdecrivons plusieurs phenotypes a~~xotrophes. [Trxduit par lejourn;~l]

Until recently, genetic studies of strict anaerobes have been restricted to studies on the transfer of antibiotic resistance (Sebald and Brefort 1975; Burt and Woods 1976; Mancini and Behme 1977). Only with CloLstricli~rm pet:fi.it~go~;\ has a group of specific auxotrophic mutants been isolated and used to examine the potential for exchange of chromosomal markers in anaerobes (Sebald and Costilow 1975). For reasons similar to those that made C . pet:fiitlgetl.s a good choice among gl-am-positive anerobes, we felt Brrcrcroicles .fkcrgilis was an appropriate choice for beginning genetic studies of the gl-am-negative anaerobes. For example, it can tolerate prolonged periods of exposure to air and is easily cultured on complex and defined (minimal) agar media. We have also isolated several phages specific for many B. fk~gilisstrains, making it possible to study the potential of this organism for transduction. In the early stages of any genetic work, it is necessary to isolate mutants containing stable chromosomal markers. Several researchers have attempted the isolation of auxotrophic mutants of Btrcreroitles species; however, the techniques employed have been unsuccessfi~l(B. Woods: personal communication). After trying a variety of mutagens (e.g. ultl-aviolet light, nitrosoguanidine and hydroxylamine) we developed methods using ethane methane sulfonate (EMS) for isolating auxotrophs of B. fi.agilis. This article presents these methods and includes descriptions of several of the auxotrophic mutants. The bacterial strains used were B. fkagilis, VPI strains 12256 (ATCC 29768) and 2044 (ATCC 'Author to whom reprint requests should be sent.

29771). Both strains were chosen because of their ability to propagate a variety of bacteriophages previously isolated in our laboratory. The range of phage sensitivity of each strain could be used to ensure that each auxotroph was actually derived from its respective wild-type parent. Strain 12256 was naturally resistant to clindamycin, tetracycline, and penicillin. The minimal inhibitory concentrations (MICs) for these antibiotics. as determined by the broth-dilution method (Sutter et crl. 1975) were 512pg/ml, 16 to 32pg/ml, and 1028 unitslml respectively. Strain 2044 which was not unusually resistant to antibiotics was screened for isolates resistant to rifampicin. A spontaneous rifampicin-resistant mutant was isolated by streakings cu1tu1.eonto a brain heart infusion (BHI) agar plate containing a gradient (0. I to 5 pg/ml) of rifampicin (Szybalski and BI-yson 1952). The MIC of strain 2044 was increased from 0.1 pg/ml to greater than 25 pg/ml in the stable mutant. We designated this mutant 2044R3 (ATCC 29762) and used it in all subsequent mutation experiments. Prel-educed supplemented BHI (Holdeman and Moore 1975) was used in all experiments involving complex media. The prereduced minimal broth was adapted from the medium of Varel and Bryant (1974) by substituting 0.1 M phosphate buffer for the sodium bicarbonate. Minimal agar medium was prepared by adding the essential components of Varel and BI-yant's medium to Eschericlzicr coli minimal A medium (J. Kowalski and J . L. Johnson, personal communication). It contained per litre: (NH4),S04, 2g; sodium citrate, 0.5 g; vitamin B I Z , 5 p g ; KH2P04, 7 g ; K2HP04, 8 g ; MnCI2.4H20, 10 mg; MgCI,. 6 H 2 0 , 20 mg; FeCl,. 6 H z 0 , 0.3 mg; CaCI,.2H20, 30 mg; NaHCO,, 4 g; cysteine HCl,

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/10/14 For personal use only.

1620

C A N . J. MICROBIOL. VOL. 24. 1978

0.5 g; glucose, log; agar (BBL purified agar. lot No. 100 GWS), 20g: hemin, 5 mg; and resazurin, 1 mg. The medium was prepared by dissolving the first seven components in 350 ml distilled water and adjusting the final pH to 6.65. The agar, FeCI,.6Hz0 and CaClz .2H,O were dissolved in 500 ml of distilled water and boiled. The two mixtures were autoclaved at 121°C for 15 min. After sterilization, the molten agar was cooled to 60°C and aseptically added to the salts broth. Sterile autoclaved solutions of glucose (10% wt./vol.), cysteine HCI (5% wt./vol.), and sodium carbonate (10% wt./vol.) were aseptically added to yield final concentrations of I%, 0.05'3, and 0.4% I-espectively. Plates were poured and placed in an anaerobic glove box (85% N 2 , 10% Hz, 5% CO?) for at least 24 h before being used (Aranki and Freter 1972). Essentially all of the B. fi.rrgi1i.s strains we tested showed moderate to excellent growth on this medium. Mutagenesis of bacterial strains was carried out as follows. Ten-millilitre volumes of BHI 'broth were inoculated with 0.2 to 0.5 ml of an overnight BHI culture and incubated at 37OC for 4 to 6 h. When midlog phase was reached (OD = 0.3 to 0.5, Bausch and Lomb Spectronic-20, 18-mm light path, 650 nm), EMS was aseptically added to the cultures which were then incubated for30min at 37°C. Final concentrations of EMS were 2% (vol./vol.) for strain 2044R3 and 2.5% (vol./vol.) for strain 12256. These concentrations consistently yielded 5 to 10% cell survival. Aftel incubation with the mutagen, the cultures were centrifuged for IOmin at I0000 x g (4°C). The pellet was resuspended in BHI broth at an optical density of 0.2 (1-2 x lo8 CFU/ml) and incubated at 37°C for 14 to 16 h (overnight) to allow for cell division and subseqitent segregation of mutant traits. The next stage in the mutation scheme, semiselective enrichment, involved the use of clavulanic acid (Beecham Labs, BI-istol, TN.) in conjunction with penicillin-G (Sigma, St. Louis, MO). Clavulanic acid increases the susceptibility of B. .fi.ngilis strains to penicillin (Wiist and Wilkins 1978) by competitively inhibiting p-lactamase activity. I t lowered the MIC of strain 12256 from 1025 to 32 units/ml, making penicillin enrichment with this strain practical. The MIC for 2044R3 was 16 to 32 units/ml and the use of clavulanic acid during enrichment was not necessary. For both strains the MIC in BHI was similar to the MIC in minimal broth. The overnight BHI cultures were washed twice with minimal broth (10000 x g , l0min) and the cells were suspended in lOml minimal broth. After allowing for initiation of growth of the prototrophs

and depletion of endogenous nutrients of the auxotrophs (incubation of 4 to 6 h at 37"C), the optical density ofeach cu1tu1.ewas adjusted to 0.03 to0.05. Filter-sterilized penicillin was added to the cultures of both 2044R3 and 12256 to give a final concentration of 128 ~lnits/nil.To the 12256 culture, sterile clavulanic acid solution also was added yielding a final concentration of 10pg/ml. The cultures were incubated for 12 to 18h at 37°C. The cells were centrifuged at 15 000 X g for 20min and resuspended in an equal volume (10ml) of BHI broth. Each cell suspension and 10-fold serial dilutions thereof were plated (0.1 mi) on BHI agar and incubated for 24 to 48 h at 37°C in the anaerobic glove box. Individual colonies from the plates were replicated to both BHI agar and minimal agar plates and incubated an additional 48 to 72 h. All clones which grew on BHI and not on the minimal medium were transferred into chopped meat broth (Holdeman rt rrl. 1977) and incubated for 18 to 20 h at 37°C. These cultures were presumed to be auxotrophic and were used in final screening. The frequency of such mutant clones, isolated following penicillin enrichment, was between 15 and 30% of the total number of replicated clones for both strains. No auxotl-ophs were ever isolated from nonmutagenized cultures, non-mutagenized penicillinenriched cultures. or cultures treated with various concentrations of nitrosoguanidine under the conditions described herein. Each presumed auxott-ophic culture was spotted using a Steer's I-eplicator (Steers el rrl. 1959) onto BHI agar, minimal agar, and minimal agar supplemented with a single nutritional substance (amino acid, vitamin, purine. or pyrimidine) (Sebald and Costilow 1975). Cultures which grew on the supplemented minimal agar and not on ~lnsupplemented agar were streaked on BHI and isolated clones were stocked in chopped meat broth. Of the total mutant population, 10 to 20% were identified as to their specific single requirement. The growth requirements and stability were confirmed by repeated streaking onto both si~pplementedand unsupplemented minimal agar. Each mutant strain was tested for sensitivity to rifampicin, clindamycin, and bacteriophages specific fo~.B..fi.~gili~ and it was confirmed that each isolate was a mutant derived from its parental strain. Brrcteroides .fi.crgilis strains 12256 and 2044R3 were sensitive to a total of 17 and 16 of our B. Jktrgilis specific bacteriophage respectively. No variation of phage sensitivity was seen with any mutant strain. Estimations of reversion frequencies were performed by counting the number of colonies that arose when 5 x los cells from a 24-h BHI culture of each strain was spread

NOTES

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/10/14 For personal use only.

TABLEI. Auxotrophic niutants of Bcrcleroidesfingilis 2044R3 and 12256 Strain designation

Phenotype

2044R3 A2 (ATCC 29763) A40 H6 (ATCC 29764) HI6

argarghishis-

H25 H40 H76 M27 M32 (ATCC 29765) M 47 M 80 Th18 Th34 Th64 T23 T55 (ATCC 29767) P I2 (ATCC 29766) 12256 A8 A1 2 (ATCC 29769) A15 H4 H7 P8 (ATCC 29770) PI9

hishishisniet met met thrthrthrtrptrpade- and aua-

argargarghishisade- and guaade- and a u a -

Revertants per 5 x lo8 cells

162 1

we are now concentrating on this problem. Several of the most stable mutant strains as well as the parental strains have been deposited with the American Type Culture Collection in hope that s will these strains and the t e c h n i q ~ ~ edescribed prove useful to other investigators in this area.

0

Acknowledgments We acknowledge David K. MacDonald for his excellent technical assistance and thank Betty Bond for supplying 11s with Bcrcter.oicies fi.trgilis strain 12256. This research was supported by Public Health Service grant AI- 12 137-03 from the National Institute of Allergy and Infectious Diseases.

0 0 52

35 0

-77 5 250

-3 3

on unsupplemented minimal plates. For each phenotype, only one mutant of each strain from a single mutation experiment was chosen assuring that each auxotrophic trait originated from a different mutational event. The phenotypic characteristics of several auxotrophs are listed in Table I. T o the best of our knowledge, the mutants described in this study are the first examples of auxotrophs isolated from the Bncter.oicic~group. Using the phage to which these strains are sensitive, we have not yet been able to demonstrate any potential for transduction. Similarly, attempts in our laboratory to exchange chi-omosomal markers via conjugation have also been unsuccessful, although

A R A N K A,. I . and R. FRETER.1972. Use ofanaerobicglove boxes for the cultivation of strictly anaerobic bacteria. Am. J . Clin. N ~ r t r25: . 1324-1329. BURT. S . J . , and D. R . WOODS. 1976. R filctor transfer to obligate anaerobes from E.cc~hericlriircoli. J . Gen. Microbiol. 93: 405-409. HOLDEMAN L.. V.. E. P. CA.IO.i ~ n dW. E. C . MOORE.(W~/OI..S.) 1977. Anaerobe laboratory manual. 4th etl. Vil.ginia Polytechnic Insritute and State University, Blacksbu~.g. MANC.INI. C., and R. J . B E H M I ~1977. . Transfer of multiple antibiotic resistance from Bnc~teroitle.s.fii~~yiIi.s to E.sclre~~iclritr coli. J . Infect. Dis. 136: 597-600. S E B A L DM., . and M. B. BREFOR-I. 1975. T ~ x n s f e rdu plasniide tet~.acycline-chlol-:lniphenicol chez Clostr.iclirrr>rperfiirrgerrs. C . R . Acad. Sci. Paris. 281: 317-319. S E B A L DM., , and R. N. COSTILOW.1975. Minimal growth requirements of Clo.c~ritlii~rri per:fiirryerrs and isolation of ~ I L I X O trophic mutants. Appl. Microbiol. 29: 1-6. STEERS, E . . E. L. FOLTZ,ilnd B. S . GRAVES.1959. An inocula replica~ing;:lpparatus for routine testing of bacterial susceptibility t o a n t ~ b ~ o t i c Antibiot. s. Chernother. 9: 307-31 1. SUTTER,V. L., V. L. VARGO,i ~ n dS. M. FINECOLD.1975. Wadsworth anaerobic bacteriology manual. 2nd ed. Wadsworth Hospital Center, Los Angeles. S Z Y B A L S KW., I , and V. S. BRYSON.1952. Genetic studies on microbial cross resist;~nceto toxic agents I. Cross resistance o f E . coli to fifteen antibiotics. J . Bacterial. 64: 489-499. VAREL.V. H.. ilnd M . P. BRYANT.1974. Nutritional f e a t ~ ~ r e s o f Btrcteroit1e.s tkirgilis subsp. fkirgilis. Appl. Microbiol. 28: 25 1-257. WfjST. J . . and T . D. W I L K I N S1978. . Effect ofcl;~vulnnicacidon anaerobic bacteria resist;~ntto beta-lactam antibiotics. Antirnicrob. Agents Chernother. 13: 130- 133.

Isolation of auxotrophs of Bacteroides fragilis.

NOTES Isolation of auxotrophs of Bacteroides fragilis ROGERL. VANTASSEL.L A N D TRACY D. W I L K I N S ' A~ltrcroheLoDortrro~:\,,Vi~.gi~ricc Polyrccl...
204KB Sizes 0 Downloads 0 Views