Hydrolyzing 3-Lactamase from Aeromonas hydrophila - Antimicrobial ...

Vol. 27, No. 4

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1985, p. 479-484

0066-4804/85/040479-06$02.00/0 Copyright ) 1985, American Society for Microbiology

Genetic and Biochemical Properties of AER-1, a Novel CarbenicillinHydrolyzing 3-Lactamase from Aeromonas hydrophila ROBERT W. HEDGES,'t ANTONE A. MEDEIROS,2* MENASHI COHENFORD,2 AND GEORGE A. JACOBY3 Department of Bacteriology, Royal Postgraduate Medical School, London W12 OHS, United Kingdom'; The Miriam Hospital, Brown University, Providence, Rhode Island 029062; and Massachusetts General Hospital, Boston, Massachusetts 021143 Received 19 September 1984/Accepted 1 February 1985

A novel carbenicillin-hydrolyzing ,-lactamase has been discovered in a blood isolate of Aeromonas hydrophila. The enzyme resembles plasmid-determined carbenicillinases in substrate profile but differs in isoelectric point (pl 5.9) and molecular weight (22,000) and has been termed AER-1. No evidence for a plasmid location could be obtained in A. hydrophila, but the AER-1 gene and resistance to chloramphenicol, streptomycin, and sulfonamide could be transferred by mobilization with IncP plasmids to Escherichia coli, where the gene cluster inserted at a unique chromosomal site. The linked resistances are similar to those found on multiresistance 1-lactamase transposons, but since insertion of the A. hydrophila gene cluster was site specific and recA+ dependent, the cluster is not a functional transposon.

portant bacteria and to establish their substrate specificities and other characteristics. In this paper we describe a 3-lactamase with novel characteristics in a strain of Aeromonas hydrophila. The genetic determinant for this enzyme was conjugally mobilized to E. coli, where it integrated into a unique chromosomal site and there functioned as a resistance determinant.

Since the introduction of ampicillin into medical practice, there has been a dramatic increase in the proportion of isolates of enteric bacteria that are resistant by virtue of production of P-lactamases. Subsequently, other ,-lactams, notably carbenicillin, have been used, and there has been an increase in the proportion of resistant P-lactamaseproducing strains in other groups of gram-negative bacteria. The existence of plasmids capable of conjugal transfer among a wide range of bacterial families (10), the ability of resistance determinants to transpose between replicons (15), and the possibility of plasmid transformation (9) ensure that a resistance mechanism initially confined to one group of gram-negative bacteria may sooner or later be transferred to other groups and, if selective forces are favorable, may spread in these newly colonized hosts. The P-lactamase PSE-1 provides an example of this process. This enzyme seems originally to have been specific to Pseudomonas aeruginosa and may have been uniquely associated with plasmids of the P-2 incompatibility group which are confined to this genus (18, 19). Hedges and Matthew (16) showed that the determinant for this enzyme could be integrated into a broad-host-range plasmid, transferred to Escherichia coli, and P-lactam antibiotics. Medeiros et al. (30) have shown that the production of this enzyme is now well established among enteric bacteria worldwide. Recently, a strain ofAchromobacter was shown to produce a previously undescribed P-lactamase. This was encoded by a plasmid which could be transferred into E. coli by transformation (23) and which conferred P-lactam resistance in the new host. Thus, the range of 1-lactamases potentially available to, for example, the enteric bacteria may be much greater than the set of enzymes actually observed in naturally occurring strains. Considering the effort currently being expended on developing new P-lactams, both as antibiotics and as ,Blactamase inhibitors, it is important to identify the types of ,B-lactamase which can be acquired by these clinically im-

MATERIALS AND METHODS Bacteria. A. hydrophila VL7711 was provided by J. V. Lee, Public Health Laboratory, Kent, U.K.). It was originally isolated in India from a human blood sample. It is capable of vigorous growth at 37°C, and all the transfers and antibiotic resistance determinations described in this paper were done at this temperature, although other experiments at room temperature gave similar results. The E. coli K-12 derivative strains and plasmids used are listed in Table 1. P. aeruginosa strain PU21(pMG76) encoding LCR-1 (39) was provided by I. N. Simpson and strains PU21(RPL11) encoding PSE-1 (29) and PU21(R151) encoding PSE-2 (26) came from M. Matthew. Phage. Phages P1 kc and PR4 (41) were used. Reagents. Antibiotics were gifts of the following manufacturers: cloxacillin, methicillin, and oxacillin were from Bristol Laboratories, Syracuse, N.Y.; cephaloridine, cefamandole, and moxalactam were from Eli Lilly & Co., Indianapolis, Ind.; ceftriaxone was from Hoffman-La Roche, Nutley, N.J.; Sch 29482 was from Schering-Plough Corp., Bloomfield, N.J.; cefotaxime was from Hoechst-Roussel, Somerville, N.J.; cefoxitin was from Merck Sharpe & Dohme, West Point, Pa.; carbenicillin and clavulanic acid were from Beecham Laboratories, Bristol, Tenn.; and cefoperazone and sulbactam were from Pfizer, Groton, Conn. Nitrocefin was obtained from Becton Dickinson and Co., Cockeysville, Md., and ampholines were from LKB Produketer A.B., Bromma, Sweden. Sephadex gels and standard proteins for molecular weight (MW) determinations were obtained from Pharmacia Fine Chemicals, Piscataway, N.J. Bio-Rad protein assay reagent was purchased from Bio-Rad Laboratories, Richmond, Calif. Conjugation. The technique of Clowes and Hayes (7) was used for conjugation between E. coli K-12 strains. In at-

Corresponding author. t Present address: Plant Genetic Systems, 22 Jozef Plateaustraat, B-9000 Ghent, Belgium. *

479

480

HEDGES ET AL.

tempts to transfer resistances from A. hydrophila VL7711 to E. coli K-12 derivatives, equal quantities of overnight cultures of donor and recipient were pipetted onto sterile filter paper on the surface of a nutrient agar plate. The plate was incubated for about 24 h, and then the filter was transferred to fresh broth and agitated to transfer the bacteria into the broth. The broths were centrifuged to pellet the bacteria, and these were suspended in small volumes of fresh broth and plated on appropriate selective media. Transduction. The technique of Roth (37) was used for transduction. Plasmid visualization. Plasmids were purified and their sizes were determined by agarose gel electrophoresis as previously described (30), using techniques to visualize both small and very large plasmids. Enzyme preparations. Enzymes were prepared by sonication as described previously (31) and spot tested with nitrocefin to assess their ,B-lactamase activity (25). Extracts were dialyzed against distilled deionized water. Those with low activity were then concentrated with a Micro-ProDiCon apparatus (Bio-Molecular Dynamics, Beaverton, Oreg.) on membranes with a 10,000-MW cutoff. Protein content was determined by the Bio-Rad reagent with bovine serum albumin as the standard. TEM-1, OXA-2, and PSE-1 ,Blactamases used for substrate profile determinations were prepared from E. coli K-12 strains containing plasmid R6K, R46, and pMG212, respectively. The LCR-1, PSE-1, and PSE-2 1-lactamases used for isoelectric focusing were prepared from P. aeruginosa PU21(pMG76), PU21(RPL11), and PU21(R151), respectively. Enzyme induction. A 5-ml amount of an overnight culture in brain-heart infusion broth was added to 45 ml of fresh broth and incubated in a shaker broth culture at 37°C for 2 h. Ampicillin was then added to a final concentration of 50 ,ug/ml, and the mixture was incubated for an additional 2 h. Analytical isoelectric focusing. The procedure of Matthew et al. (28), modified as described previously (30), was used for analytical isoelectric focusing. Enzyme assays. ,-Lactamase activity was assayed alkalimetrically by using a pH stat linked on-line to a microcomputer. The assays were performed by the method of Labia et al. (20) with the following modifications: the titrant was 0.02 N NaOH and the reaction mixture was 0.5 mM substrate in 3 mM NaCl. The volume of base added over time was monitored with a pH stat (Radiometer, Copenphagen) linked to an Apple II+ microcomputer (Apple Computer, Inc., Cupertino, Calif.). All readings were compared with those for control reaction mixtures to which no enzyme was added. One upit of activity was defined as the amount of enzyme catalyzing the formation of 1.0 ,umol of product in 1.0 min under the above assay conditions. MW estimations. MW was estimated by the method of Andrews (1) on a Sephadex G-100 column (1.5 by 90 cm) which was equilibrated and eluted with 50 mM sodium phosphate buffer, pH 7.0, containing 0.02% sodium azide. The column was standardized with RNase (MW 13,700), myoglobin (MW 17,800), chymotrypsinogen (MW 25,000), ovalbumin (MW 43,000), and bovine serum albumin (MW 67,000). Void volume was determined with dextran blue 2000. RESULTS A. hydrophia. A. hydrophila VL7711 was resistant to ampicillin (MIC, 100 ,ugIml), carbenicillin (MIC, 500 ,ug/ml), chloramphenicol (MIC, 20 ,ug/ml), streptomycin (MIC, 60 ,ug/ml), sulfonamide (tested by disk), and tetracycline (MIC,

ANTIMICROB. AGENTS CHEMOTHER.

10 ,ug/ml). Derivative strains that had spontaneously lost tetracycline resistance (MIC, 2.5 ,ug/ml) were isolated, but no loss of carbenicillin or chloramphenicol resistance was ever noted (frequency less than 10-3). DNA isolated from strain VL7711 was subjected to electrophoresis, and two plasmids bands were observed, both approximately 2 megadaltons. No larger plasmids could be detected. Transfer of resistance to E. coli K-12 was attempted, but no such transfer was detected. Plasmids R772, R702, pUZ8, R388, and R792 were transferred into strain VL7711. All of these plasmids were stably maintained, and DNA species of appropriate MWs were found in these R+ strains. It was noted that in the strains carrying IncP plasmids, resistance to ampicillin, carbenicillin, and chloramphenicol (but none of the other resistances) became unstable. The MICs for the susceptible segregants were: carbenicillin, less than 12.5 ,ug/ml, and chloramphenicol, 6 ,ug/ml. The rate of loss was not consistent between experiments, but as many as 10% of R+ cells had lost these resistances after overnight growth in drug-free broth. Electrophoresis of the DNA showed no change in plasmid content associated with the loss of these resistances. Transfer of resistances to E. coli K-12. Neither R388 nor R792 could mobilize the transfer of carbenicillin or chloramphenicol resistance, but the three IncP plasmids all effected this transfer. As all three showed similar patterns of mobilization, only that for R772 will be presented. Tetracycline resistance was transferred with an efficiency of about 10-6 per donor cell (it was also transferred by R388 and R792). Usually tetracycline resistance was transferred alone (or together with the mobilizing plasmid). When transferred with the other strain VL7711 resistances, it showed no association with these (for example, it could not be cotransduced with them by phage P1), and we concluded that it had a genetic determination separate from those of the other resistances. It was not investigated further. Transfer of resistance to carbenicillin or chloramphenicol occurred at a slightly lower frequency (ca. 2 x 10-7 per donor cell). These two resistances almost always cotransferred (1 clone lacking chloramphenicol resistance was noted among about 500 carbenicillin-resistant transconjugants), and resistance to streptomycin (and spectinomycin) and sulfonamides was always cotransferred. The MICs for these E. coli transconjugants were: ampicillin, 100 ,ug/ml; carbenicillin, 200 ,ug/ml; chloramphenicol, 30 ,ug/ml; and streptomycin, 25 ,ug/ml. A proportion of the transconjugants (varying between experiments) were also resistant to kanamycin. This resistance (but not strain VL7711-derived resistances) was freely transmissible to other strains of E. coli. The kanamycin-resistant strains were susceptible to phage PR4, which lyses strains containing IncP plasmids, and selection for phage PR4 resistance produced a high proportion of kanamycinsusceptible segregants but no carbenicillin-susceptible variants (Iess than 1%). None of the kanamycin-susceptible transconjugants showed any plasmid DNA bands upon electrophoresis, whereas the kanamycin-resistant clones carried single plasmid bands indistinguishable from those formed by R772 (about 40 megadaltons). Thus, there was no evidence that resistance to carbenicillin or the other determinants from strain VL7711 was associated with R772 or transferred on an independent plasmid. Mapping resistance markers in E. coli K-12. Strain J53-2, which had received carbenicillin, chloramphenicol, streptomycin, and sulfonamide resistance (but not plasmid R772) from strain VL7711 and carried no detectable plasmid bands,

VOL. 27, 1985

A. HYDROPHILA P-LACTAMASE

chosen for study. To describe such a strain, the designation J53-2 fQ7711 will be used, where fQ7711 represents the DNA inserted from strain VL7711 (33). The chromosomemobilizing (17) plasmid Rl drdl6 was introduced, and the R+ derivative was used as a donor to various F- recipients. Crosses with strain X195 showed that the resistances were linked to the tyrA gene and lay between the his operon and the tyrA gene. More precise localization of the resistance genes was attempted by performing P1 transduction. These transductions were used to prove that all strain VL7711-derived resistances were inherited as a group. When selection was made for transfer of chloramphenicol resistance, all (of more than 100) transductants also received the other resistances. When carbenicillin resistance was selected, cotransduction of streptomycin and sulfonamide resistance was always observed, and in all but 2 of 500 cases tested, chloramphenicol resistance was also cotransduced. Lysates were prepared on strain J53-2 fQ7711 and used to transform various strains carrying mutations in the his-tyr chromosome segment. Transduction to strain RKS1703 showed close linkage of resistance to the guaB gene. When selection was made for

was

TABLE 1. Strains of E. coli K-12 and plasmids used Strain or

Relevant

or

Source

phenotype

or

reference

plasmid

Strain AT2457

42 HfrH glyA6 relAl spoTI thi-l AT2465 42 HfrH guaA6 relAl spoTI thi-J C600 recAa F- lacYl /euB6 recA 3 thi-l thr-l J53-2 F- met pro Rifr 14 HfrH gal-att X-bio K. Shimada KS1703

guaB

(personal communication)

PCO111

F- leuB6 metBI

RKS1703

purC rpsL (strA)126 thr-J F- guaB Rifr

KS1703

W3110

F- recA

3

B. Bachmann

(personal communication) x W3110 Thy recombinant

recAa

W3110

F-

thy Rifr

2

ThyX195

F- ga/K35 his68 /ac6 35; B. Bachman

pro3 purB51 pyrF287 rpsL (strA)125 thi-J trpC60 tyrA2 Plasmids Rl drdl6b R6K

pUZ8 R46

pMG212 R388 R702 R772 R792

Kmr IncFII Apr (TEM-1) Smr IncX Kmr Tcr Hgr IncP Apr (OXA-2) Smr Sur Tcr IncN Apr (PSE-1) Cmr Smr Sur Tcr IncFII Sur Tpr IncW Kmr Smr Sur Tcr Hgr IncP Kmr IncP Kmr Sur IncC

(personal communication)

32 12

16 12

31 11 15 8 14

aRifampin-resistant mutants of these strains recipients. bA derivative having lost Apr Cmr Smr Sur.

were

isolated and used

as

481

TABLE 2. Influence of Q7711 on cotransduction of guiaB and

purC No. of Gua+ Donor strain

PCO111 PCO111 PCO111 Q7711 PCO111 Q7711

Recipient strain

RSK1703 RSK1703 Q7711 RSK1703 RSK1703 Q7711

transductantsa PurC+

PurC-

286 245 361 525

45 36

X Cotransduction of PurC

14

13 4 4 a Gua+ transductants were selected on minimal agar plates containing adenine and then scored for an adenine requirement. 17 20

carbenicillin resistance, 99% of the transductants (320 of 323) were gua +, whereas when selection was made for prototrophy, ca. 90% (207 of 239) of the transductants were resistant to carbenicillin. (Similar linkage was observed when the guaA mutation of strain AT2465 was studied.) Analysis of the three-point transduction events implied that the resistance marker had been inserted between purC and guaB rather than between guaB and glyA. This finding was confirmed by measuring the influence of the Q7711 insertion on cotransduction in this region. No influence was found on cotransduction of glyA and guaB (about 5%; data not shown), but whereas guaB and purC were 14% cotransduced from strain pC0111, this value fell to 4% when the donor carried the insert (Table 2). Transfer of carbenicillin resistance from A. hydrophila VL7711 to E. coli RKS1703 was made by four separate conjugations. In each case the transfer of carbenicillin resistance was accompanied by transfer of resistance to chloramphenicol, streptomycin, and sulfonamides. All transconjugants retained the gua character of the recipient, and in all cases this auxotrophic marker was shown to be cotransducible with the resistance determinants. Thus, these resistance determinants must be inserted at or very close to the site of insertion of the original transconjugant. Transfer of carbenicillin resistance from strain VL7711 (R772) to recA E. coli K-12 strains (C600 recA or W3110 recA) was attempted but failed consistently. Similarly, strain J53-2 Q7711 carrying plasmid R772 failed to transmit resistance to carbenicillin, chloramphenicol, or sulfonamide to several recA E. coli recipients, although kanamycin resistance (determined by R772) transferred readily. A. hydrophila VL771 ,B-lactamase. Isoelectric focusing in polyacrylamide gels showed that sonic extracts of A. hydrophila VL7711 had 3-lactamases which appeared as three distinct, equally dense bands at isoelectric points 7.1, 6.6, and 5.9. The transconjugant E. coli, however, produced only the 3-lactamase of pl 5.9, which appeared as a single band nearly equidistant from the main bands of the PSE-1 and PSE-2 3-lactamases, in addition to its chromosomal 3-lactamase (pl 8.0) (Fig. 1). The isoelectric focusing band of the A. hydrophila enzyme was very close to that of the LCR-1 ,B-lactamase but slightly higher. The two were easily distinguished by loading the enzyme samples at both the anode and the cathode (Fig. 2). The amount of ,B-lactamase produced by A. hydrophila VL7711 increased with induction by ampicillin. Isoelectric focusing of extracts of induced and noninduced cells showed that with induction, the pl 7.1 and 6.6 bands became much denser than the pI 5.9 band (cf. Fig. 1), suggesting that only the nontransferable P-lactamases had been induced. The sonic extract prepared from the E. coli transconjugant had weak 1-lactamase activity, 0.2 ,umol of penicillin G hydrolyzed per min per mg of protein. It hydrolyzed carben-

482

HEDGES ET AL.


0

70

II~~~~~~0

-5.0 Bs

A

C

D

FIG. 1. Isoelectric

focusing gel showing band pattemns of1lactamases from strain PU21(RPL11) (PSE-1 J3-lactamase prototype) (lane A), A. hydrophila VL7711 (lane B), strain J53-2 fl7711 (lane C), and strain PU21(R151) (PSE-2 13-lactamase prototype) (lane D). 13-Lactamase activity was detected with the chromogenic substrate nitrocefin (28).

icillin as rapidly as it did penicillin G, similar to PSE-1 (Table 3). However, it hydrolyzed cefazolin, cephalothin, and cefotaxime relatively more rapidly than PSE-1. The A. hydrophila 13-lactamase had an MW of 22,000 as determined by Sephadex gel filtration. DISCUSSION

The

resistance

of A.

hydrophila VL7711 to 13-lactam by several 13-lactamases, one of which was distinct from those previously described. This enzyme hydrolyzed carbenicillin rapidly and formed a band at pI 5.9, nearly equidistant from those of the PSE-1 and PSE-2 13-lactamases after isoelectric focusing in polyacrylamide. This pattern differed from those reported for other antibiotics

was

mediated

carbenicillinases (13, 22, 27) but resembled that of the plasmid-determined enzyme LCR-1 (39). However, by differential isoelectric focusing (6), the two 13-lactamases differed, and their substrate profiles were quite distinct as well (39). The A. hydrophila enzyme also appeared to differ from previously described carbenicillinases in hydrolyzing cephalothin relatively rapidly. This finding may, however, be due to residual E. coli chromosomal cephalosporinase (21, 24) present in the sonic extracts tested. The low level of the novel 13-lactamase produced by the transconjugant E. coli made it difficult to purify the enzyme for more definitive substrate profile determination. The estimated MW (22,000) of the A. hydrophila enzyme was similar to those of TEM-1 and TEM-2 but different from those of other carbenicillin-

FIG. 2. Isoelectric focusing gel showing band patterns of ,Blactamases from strain J53-2 fQ7711 (lanes B, D, and F) and P. aeruginosa PU21(pMG76) (LCR-1 1-lactamase prototype) (lanes A, C, and E). Enzyme samples were loaded at the cathode (lanes A and B) and at the anode (lanes C through F).

hydrolyzing enzymes (22, 27). This 3-lactamase also differed from the one extracted by Sawai et al. (38) from a fish strain of A. hydrophila in not hydrolyzing methicillin, oxacillin, or cloxacillin. We suggest that this enzyme of A. hydrophila VL7711 be designated ,-lactamase AER-1. The gene encoding the enzyme was part of a cluster of resistance determinants that were normally cotransferred in conjugation or by P1 transduction. Since no suitable plasmid could be detected in strain VL7711 and no difference in plasmid content could be observed after loss of the resistances, we suppose that this cluster of genes was chromosomally located. The presence of an IncP plasmid in strain VL7711 leads to destabilization of carbenicillin and chloramphenicol resistance. The rate of loss was variable but markedly less than that associated with plasmid incompatibility; there was no evidence for alteration in plasmid content, and the introduced IncP plasmid was stably inherited. Thus, the instability is thought not to be TABLE 3. Substrate profiles of the A. hydrophila enzyme and some plasmid-mediated P-lactamases produced by E. coli K-12 Relative rate of hydrolysisa

TEM-1 72 12 E type

AER-1 38 98 TEM-1 72 12 PSE-1 84 100

OXA-2 236

1.3

4

U0 ~ 0.9 0.3 4 1.3 9 1.1

271581

31

01

u

0

u

20

4

4

uUZ

0 2677 44 1817 b 3 20 47 0 60 13 1120 0 28 0.7 4 46 0 27 0 0 10 0 00 0 31

157108161167132 0118

0

0-

a Activity relative to that with penicillin G (set at 100). With penicillin G as the substrate, 13-lactamase activity ranged from 0.2 to 1.9 ~Lmolmin per mg of protein. No hydrolysis was observed for any of the enzymes with moxalactam or Sch 29482. b-, Not tested.

A. HYDROPHILA

VOL. 27, 1985

related to incompatibility functions but may be some sort of excision effected by a plasmid-determined enzyme. It may be significant that plasmids which destabilized these resistances were able to mobilize their transfer to E. coli. When transferred to E. coli, the cluster of resistance genes integrated into the E. coli chromosome. In no case has insertion into any mobilizing plasmid been demonstrated, and so far as our evidence goes, it seems that the f17711 insertion always occurred in the same region of the chromosome, between purC and guaAB. In some respects the f17711 gene cluster was like a transposon. Tn7, for example, integrates at a unique site in the E. coli K-12 chromosome (4) but at many different sites in plasmid replicons (5). Similarly, Tn2521 occurs naturally at a unique chromosomal site in P. aeruginosa and, like the A. hydrophila gene cluster, determines a carbenicillin-hydrolyzing ,-lactamase (PSE-4) and resistance to streptomycin and sulfonamide, but it, too, can integrate into various plasmids (40). Indeed, linkage of P-lactamase production with resistance to chloramphenicol, streptomycin, sulfonamide, and other antibiotics is characteristic of a number of multiresistance ,-lactamase transposons (30, 36, 43; Levesque and Jacoby, unpublished data). However, an essential attribute of a transposon is its ability to integrate at a variety of nonhomologous sites. Since integrations of Q17711 appeared to be both site specific and RecA+ dependent, it cannot be considered a typical transposon. Perhaps the unit has lost a site or function essential for transposition and can now be transferred only by plasmids capable of chromosome mobilization (17). That such a process may be important in the genesis of P-lactam resistance in clinical isolates is illustrated by the recent demonstration that chromosomal P-lactamase hyperproduction has spread from Shigella sonnei to E. coli (34). ACKNOWLEDGMENTS We thank J. V. Lee and B. Bachmann for supplying strains and Elaine Papa, Frank Prevost, Lorraine Sutton, and Jennifer Zollner for expert technical assistance. This work was supported in part by the Miriam Hospital Research Trust and by National Science Foundation grant PCM 8110808 to G.A.J. LITERATURE CITED 1. Andrews, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91:222-233. 2. Bachmann, B. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 3. Barth, P. T., and N. Datta. 1977. Two naturally occurring transposons indistinguishable from Tn7. J. Gen. Microbiol. 102:129-134. 4. Barth, P. T., N. Datta, R. W. Hedges, and N. J. Grinter. 1976. Transposition of a DNA sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J. Bacteriol. 125:800-810. 5. Barth, P. T., and N. J. Grinter. 1977. Map of plasmid RP4 derived by insertion of transposon C. J. Mol. Biol. 113:455-474. 6. Barthelemy, M., M. Guionie, and R. Labia. 1978. Beta-lactamases: determination of their isoelectric points. Antimicrob. Agents Chemother. 13:695-698. 7. Clowes, R. C., and W. Hayes. 1968. Experiments in microbial genetics. Blackwell Scientific Publications, Oxford. 8. Coetzee, J. N., G. Lecatsas, W. F. Coetzee, and R. W. Hedges. 1979. Properties of R plasmid R772 and the corresponding pilus-specific phage PR772. J. Gen. Microbiol. 110:263-272. 9. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic resistance in bacteria: genetic transfor-

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Sci. U.S.A. 69:2110-2114. 10. Datta, N., and R. W. Hedges. 1972. Host ranges of R factors. J. Gen. Microbiol. 70:453-460. 11. Datta, N., and R. W. Hedges. 1972. Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae. J. Gen. Microbiol. 72:349-355. 12. Datta, N., and P. Kontomichalou. 1965. Penicillinase synthesis controlled by factors in enterobacteriaceae. Nature (London) 208:239-241. 13. Furth, A. V. 1975. Purification and properties of a constitutive beta-lactamase from Pseudomonas aeruginosa Dalgleish. Biochim. Biophys. Acta 377:431-433. 14. Hedges, R. W. 1974. R factors from Providence. J. Gen. Microbiol. 81:171-181. 15. Hedges, R. W., and A. E. Jacob. 1974. Transposition of ampicillin resistance from RP4 to other replicons. Mol. Gen. Genet. 132:31-40. 16. Hedges, R. W., and M. Matthew. 1979. Acquisition by Escherichia coli of plasmid-borne 13-lactamases normally confined to Pseudomonas spp. Plasmid 2:269-278. 17. Holloway, B. W. 1979. Plasmids that mobilize bacterial chromosome. Plasmid 2:1-19. 18. Jacoby, G. A., A. E. Jacob, and R. W. Hedges. 1976. Recombination between plasmids of incompatibility groups P-1 and P-2. J. Bacteriol. 127:1278-1285. 19. Jacoby, G. A., L. Sutton, L. Knobel, and P. Mammen. 1983. Properties of IncP-2 plasmids of Pseudomonas spp. Antimicrob. Agents Chemother. 24:168-175. 20. Labia, R., J. Andrillon, and F. LeGoffic. 1973. Computerized microacidimetric determination of ,-lactamase Michallis-Menten constants. FEBS Lett. 33:42-44. 21. Labia, R., G. Brunet, M. Guionie, A. Phillipon, M. Heitz, and V. S. Pitton. 1976. Cephalosporinases constitutives de Escherichia coli. Ann. Microbiol. Inst. Pasteur 127B:453-461. 22. Labia, R., M. Guionie, M. Barthelemy, and A. Philippon. 1981. Properties of three carbencillin-hydrolyzing P-lactamases from Pseudomonas aeruginosa. J. Antimicrob. Chemother. 7:49-56. 23. Levesque, R., P. H. Roy, R. Letarte, and J. C. Pechere. 1982. A plasmid-mediated cephalosporinase from Achromobacter species. J. Infect. Dis. 145:753-761. 24. Lindstrom, E. B., H. G. Boman, and B. B. Steele. 1970. Resistance of Escherichia coli to penicillin. VI. Purification and characterization of the chromosomally mediated penicillinase present in ampA-containing strains. J. Bacteriol. 101:218-231. 25. Marre, R., A. A. Medeiros, and A. W. Pasculle. 1982. Characterization of the beta-lactamases of six species of Legionella. J. Bacteriol. 151:216-221. 26. Matthew, M. 1978. Properties of the ,B-lactamase specified by the pseudomonas plasmid R151. FEMS Microbiol. Lett. 4:241-244. 27. Matthew, M. 1979. Plasmid-mediated 1-lactamases of gram-negative bacteria: properties and distribution. J. Antimicrob. Chemother. 5:349-358. 28. Matthew, M., A. M. Harris, M. J. Marshall, and G. W. Ross. 1975. The use of analytical isoelectric focusing for detection and identification of P-lactamases. J. Gen. Microbiol. 88:169-178. 29. Matthew, M., and R. B. Sykes. 1977. Properties of the beta-lactamase specified by the Pseudomonas plasmid RPL11. J. Bacteriol. 132:341-345. 30. Medeiros, A. A., R. W. Hedges, and G. A. Jacoby. 1982. Spread of a "Pseudomonas-specific" ,-lactamase to plasmids of enterobacteria. J. Bacteriol. 149:700-707. 31. Medeiros, A. A., and T. F. O'Brien. 1975. Ampicillin-resistant Haemophilus influenzae type b processing a TEM-type ,Blactamase but little permeability barrier to ampicillin. Lancet

ii:716-718. 32. Meynell, E., and N. Datta. 1967. Mutant drug resistant factors of high transmissibility. Nature (London) 214:885-887. 33. Novick, R. P., R. C. Clowes, S. N. Cohen, R. Curtiss III, N. Datta, and S. Falkow. 1976. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol. Rev. 40:168-189. 34. Olsson, O., S. Bergstrom, F. P. Lindberg, and S. Normark. 1983. ampC P-lactamase hyperproduction in Escherichia coli: natural

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35.

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