Detection of an integrated tetracycline-resistance plasmid in Staphylococcus aureus from a Nigerian hospital

Antimicrobial Agents International Journal of Antimicrobial Agents 6 (1995) 51-56

Detection of an integrated tetracycline-resistance plasmid in Staphylococcus aureus from a Nigerian hospital E.E. Udo”,*, W.B. Grubbb “Department ,?f Microbiology, Faculty of Medicine, Kuwait University, II 0. Box 24923, &far 13110, Kuwait bSchool of Biomedical Sciences, Curtin University of Technology, Perth, Australia

Accepted 2 June 1995

Abstract The genetics of tetracycline-resistance determinants was studied in eight methicillin-resistant and two methicillin-sensitive Staphylococcus a~lre~s isolated from a Nigerian hospital. Curing and transfer experiments demonstrated that one methicillin-sensitive S. aureus isolate WBG4762, had a 4.4 kb extrachromosomal plasmid- as well as a chromosomally-mediated tetracycline resistance. All others had chromosomal tetracycline resistance and were resistant to either tetracycline and minocycline or tetracycline only. The two methicillin-susceptible isolates were resistant to both tetracycline and minocycline. Chromosomal DNA from all the resistant isolates hybridized with a digoxigenin-1 l-dUTP labeled 4.4 kb tetracycline-resistance plasmid probe indicating that they contained tetracycline-resistance plasmisds similar to the probe integrated into their chromosomes. The results demonstrated the presence of integrated tetracycline-resistance plasmid in both methicillin-resistant and methicillin-susceptible S. aureus resistant to tetracycline and minocycline as well as those resistant only to tetracycline. This is the first demonstration of an integrated tetracycline-resistance plasmid in methicillin-sensitive S. aureus and suggests that the integrated tetracycline-resistance plasmid may be widespread in S. aureus. Keywords:

Staphylococcus

aureus; Tetracycline resistance; Integrated plasmids

1. Introduction

Tetracycline resistance has been reported among many bacterial species [l--3]. In Staphylococcus aureus tetracycline resistance is due to a decrease in the intracellular accumulation of the antibiotic [ 11.Initially, this was correlated with a decreased uptake of the antibiotic [4] but recent evidence suggests that a specific efflux mechanism [2,5,6] similar to that in Escherichia coli and ribosomal protection [3,5] also contributes to resistance in S. aureus. The genes encoding tetracycline resistance in S. aureus may reside on physically identical 4.4 kb plasmids [7-lo] or on chromosome 7, 11. Asheshov [7] identified two types of tetracycline-resistance phenotypes in S. cLureus.A plasmid-borne inducible resistance to tetra-

*Corresponding author. Fax (965) 531 8454, E-mail: Edet @HSCC.KUNIV.EDU.KW 0924-8579/95/$26.00 0 1995 Elsevier SSDI 0924-8579(95)00025-9

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cycline but not minocycline and a chromosomal resistance to both tetracycline and minocycline. These phenotypes were found to correspond to tetK and tetM resistance phenotypes [ 121. Molecular studies of methicillin-resistant Staphylococcus aureus (MRSA) isolates from eastern Australian hospitals which were resistant to tetracycline and minocycline demonstrated that their tetracycline resistance was due to the integration, into their chromosome, of a plasmid similar to pT181 [13]. A recent study by Stewart et al. [14] also found integrated tetracycline-resistance plasmids in MRSA from the USA and Greece. MRSA isolated from the Lagos University Teaching Hospital (LUTH) in Nigeria [15] have been shown to differ from the classical MRSA, the epidemic MRSA of the UK and eastern Australian MRSA (EA MRSA) in their resistance and plasmid contents. The EA MRSA [13] were resistant to tetracycline and minocycline whereas the LUTH MRSA were resistant to tetracycline but sensitive to minocycline [15]. The LUTH MRSA

52

E. E. Udo. W3. Grubbihrrrnationaf Journal of Antimicrobial Agents 6 (1995) 51-56

harboured a single 30 kb cadmium-resistance plasmid and had chromosomal determinants for penicillinase production and resistance to methicillin, kanamycin, neomycin, streptomycin and tetracycline [15]. The EA MRSA usually carried three plasmids; a plasmid of approximately 28 kb encoding resistance to nucleic acid binding compounds, a 4.5 kb chloramphenicol resistance plasmid and a 1.6-1.8 kb cryptic plasmid [9,16]. In view of the obvious differences in the tetracycline-resistance phenotypes of both the EA MRSA and the LUTH MRSA, this study was undertaken to determine the genetic nature of tetracycline-resistance determinants in the LUTH MRSA. Two methicillin-susceptible S aUreU$ isolated from the same hospital which were resistant to both tetracycline and minocycline were also studied. The results indicated that the LUTH MRSA as well as the two methicillin-sensitive S. ~UY~US contained integrated tetracycline-resistance plasmids in their chromosome similar to those found in the EA MRSA. These results suggest that chromosomally integrated tetracyclineresistance plasmids may be more widespread than presently known and raises the question of the origin of tetracycline-resistance plasmids in S. aureus.

2. Materials and methods 2.1. Bacterial strains The S. aweus isolates studied are listed in Table 1. Isolates WBG4763, WBG7208-WBG7214, WBG4761 and WBG4762 were from the Lagos University Teaching Hospital (LUTH), Lagos, Nigeria. WBG525 was an EA MRSA from an Australian hospital [16]. WBG541 and WBG1876 were fusidic acid and rifampicin-resistant recipients used for conjugation and mixed-culture transfer (MCT) experiments, respectively [17]. WBG248 was a plasmid-free and drug-sensitive isolate [ 161. WBG488 1 [18] was a WBG248 transductant and contained the tetracycline-resistance plasmid, pWBG3, used as a probe in this study. 2.2. Media Brain Heart Infusion Agar (BHIA), Brain Heart Infusion Broth, Trypticase Soy Broth (TSB) and Mueller Hinton Agar were purchased from Gibco Diagnostics (Madison, WI, USA). 2.3. Susceptibility testing Susceptibility to antimicrobial agents was tested by the disk diffusion method using Mueller Hinton Agar [16]. Minimum inhibitory concentrations (MICs) to tetracycline and minocycline were determined by the agar dilution method using an inoclllum of lo5 organisms per ml.

2.4. Loss of resistance Loss of resistance to antimicrobial agents was investigated by screening single colonies for loss of resistance after growth at 43.5”C as described previously [15]. 2.5. Plasmid isolation Plasmids were isolated using the cetyltrimethylammonium bromide method as reported previously [193. 2.6. Transfer of tetracycline resistance Plasmid transfer was attempted by mixed-culture transfer (MCT) using strain WBG1876 as recipient, and by conjugation as reported previously [20,21] using strain WBG541 as recipient. For MCT experiments 0.1 ml each of an overnight culture of donor and recipient was added to 5.0 ml of TSB containing 0.01 M CaCl, and incubated at 37°C overnight. The mixture was then centrifuged at 2000 g for 5 min, the supernatant was discarded and the deposit spread on selection media containing fusidic acid 5 agll, rifampicin 2.5 mg/l and tetracycline 5 mg/l. For mobilization experiments, the conjugative plasmid, pWBG636 [20], was transferred by conjugation to WBG4762. The resultant strain containing pWBG636 was then conjugated with strain WBG541. Tetracycline-resistant transconjugants were obtained on selection media as described for MCT experiments. 2.7. DNA-DNA

hybridization

Genomic DNA was isolated as described previously [17], digested with Hind111 restriction endonuclease and transferred to a positively charged nylon membrane (Boehringer Mannheim, GmbH, Germany) using standard conditions [22]. The tetracycline-resistance plasmid, pWBG3, was purified by elution from low melting agarose (BioRad Laboratories, Hercules CA, USA) using agarase (Boehringer Mannheim). The DNA was alcohol precipitated, digested with HindIII, labeled by the random primer method with digoxigenin-1 1-dUTP (Boehringer Mannheim) and used to probe the chromosomal DNA for the presence of homologous sequences. Hybridization, post-hybridization washes and detection with Lumi-Phos 530 (Boehringer Mannheim) were performed according to methods provided by the manufacturer. Autoradiographs were obtained with Kodak X- OMATmARfilm after 60 min incubation at room temperature.

3. Results The properties

of the S. aureus isolates studied are

E. E. Udo, WB. Grubbllnternational Journal of Antimicrobial Agents 6 (1995) 51-56

summarized in Table 1. With the exception of WBG4761 and WBG4762, the isolates were methicillin-resistant. The MRSA isolates except WBG4763 were resistant to tetracycline and sensitive to minocycline. The methicillin-sensitive isolates (MSS.A), WBG4761 and WBG4762 and one MRSA (WBG4763) were resistant to tetracycline and minocycline. Their MICs for tetracycline and minocycline were as presented in Table 1. Only WBG4762 had an MIC greater than 250 mg/l for tetracycline. The LUTH MRSAs each contained a single plasmid. The resistance phenotypes of these plasmids have been reported elsewhere [15]. The MSSA isolates, WBG4761 and WBG4762, harboured two and three plasmids, respectively (Table 1). WBG4762 harboured three plasmids of 20, 4.7, and 4.4 kb (Fig. 1). 3.1. Transfer of tetracycline resistance None of the isolates transferred tetracycline resistance in MCT or conjugation experiments. They were tested in conjugation experiments to ascertain if they contained

53

conjugative transposons in view of the presence of tetracycline-resistance conjugative transposons in Enterococci [23,24]. As WBG4762 harboured a 4.4 kb plasmid which is the size of plasmids usually associated with tetracycline resistance in S. aweus [7-lo], it was studied further so that the 4.4 kb plasmid could be isolated and characterized. In curing experiments only the loss of resistance to chloramphenicol and streptomycin was observed which was accompanied by the loss of the 4.7 kb plasmid from the parental isolate (Fig. 1, lane 5). No loss of tetracycline resistance was detected in any of the of 370 colonies screened. However, since WBG4762 harboured a 4.4 kb plasmid and was resistant to both tetracycline and minocycline, it was possible that it had both plasmid-and chromosomal-borne tetracycline-resistance determinants. If this were the case, the loss of the 4.4 kb tetracycline-resistance plasmid would not result in the loss of tetracycline resistance since the chromosomal resistance would still be expressed. This possibility was investigated by screening single colonies of WBG4762 after curing at

Table 1 Properties of S. aweus strains Isolates

WBG248 WBG541 WBG1876 WBG4761 WBG4762

WBG7695 WBG6163b WBG4763 WBG7208 WBG7209 WBG7210 WBG7211 WBG7212 WBG7213 WBG7214 WBG525

WBG4881’ WBG6199* WBG6195’

Resist ante profile

None Fa,Rf’ Fa,Rf Tc,Mn,Pc,Cd,Asa, Sm,Cm Tc,Mn,Pc,Cd,Sm,Cm

Tc,Mn,Pc,Cd, Sm,Cm Tc,Fa. Rf Tc,Mn,Mc,bla,Cd,Hg, Pma,Sm,Gm,Km,Pi,Eb Tc,Mc,bla,Cd,Km,Sm Tc,Mc,bla,Cd,Km,Sm Tc,Mc,bla,Cd,Km,Sm Tc,Mc,bla,Cd,Km,Sm Tc,Mc,bla,Cd,Km,Sm Tc,Mc,bla,Cd,Km,Sm Tc,Mc,bla,Cd,Km,Sm Tc,Mn,Mc,bla,Cd,Cm, Tp,Gm,Km,Sm,Em, Hg,Pi,Eb Fa,Rf,Tc Tc,Mn,Mc,Pc,Gm,Km Tc,Mn

Plasmid kb, phenotype

None None None 36.3; Pc,Cd,Asa 4.7; Sm,Cm, 20.8; Pc,Cd 4.7; Sm,Cm 4.4;Tc 20.8;Pc,Cd 4.7;Sm,Cm 4.4;Tc 28,O;Pc,Cd,Hg,Eb,Pi 30.O;Cd 30.O;Cd 30.O;Cd 30.O;Cd 30.O;Cd 30.O;Cd 30.O;Cd 28,8;Gm,Tp,Pi,Eb 4.5;Cm 1.I;Cryptic 4.4;Tc None None

MIC (mti) Tc

Mn

0.5 0.5 ND 64

0.25 0.25

> 250

8

64

8

> 250

8

64 64 64 64 64 64 64 64 64

16 0.5 0.5 0.5 0.5 0.5 0.5 0.5 16

> 250 64 64

0.25 8 8

8

Asa, arsenate; Cd, cadmium; Cm, chloramphenicol; Eb, ethidium bromide; Em, erythromycin; Fa, fusidic acid; Gm, gentamicin; Hg, mercuric chloride; Km, kanamycin; MC, methicillin; Mn, minocycline; PC, benzyl penicillin; Pma, phenyl mercuric acetate; Pi, propamidine isethionate; Rf, rifampicin; Sm, streptomycin; Tc, tetracycline; Tra +, conjugative plasmid; bla, beta-lactamase; kb, kilobase, MJC, minimum inhibitory concentration; ND, not determined. a WBG4762 cured of the 4.4 kb plasmid. bA WBG4762 transconjugant coniaining the 4.4 kb plasmid pWBG641. ’ WBG248 containing the tetracycline-resistance plasmid pWBG3. dWBG4763 cured of the 30 kb plasmid and resistance to Cd, Hg, Pma, Pi, Eb. e WBG4761 cured of the 36.6 kb plasmid and resistance to PC, Asa.

54

E.E. Udo. WB. Grubbllnternational Journal of Antimicrobial Agents 6 (1995) 51-56

MRSA isolates, WBG4761, WBG7695, WBG248 and the EA MRSA, WBG525, digested with Hind111 and probed with a tetracycline-resistance plasmid, pWBG3, probe labeled with DIG-l I-dUTP in Southern blot experiments. Chromosomal DNA from WBG7695 (WBG4762 cured of the extrachromosomal4.4 kb plasmid) was used instead of WBG4762 to eliminate the chance of the chromosomal plasmid being masked by the extrachromosomal4.4 kb plasmid. The labeled pWBG3 probe hybridized to chromosomal fragments of all the isolates except WBG248 (Fig. 2). The probe hybridized to three fragments which corresponded to the 2.4, 1.5 and 0.5 kb Hind111 fragments of pWBG3 in all the isolates and to additional chromosomal fragments, some of which were larger than the 2.4 kb Hind111 fragments of pWBG3, in WBG525, WBG4761, WBG7211, WBG7695 and WBG4763 (Fig. 2). In another experiment using chromosomal DNA from derivatives of WBG7208, WBG7211, WBG476 1 and WBG4763 cured of their cadmium-resistance plasmids the probe gave similar hybridizing patterns to their parental isolates (not shown). This experiment was conducted to ascertain if their plasmids contributed to the extra bands which hybridized to the probe.

4. Discussion

Fig. 1. Plasmids of WBG4762 and its derivatives. Sizes are in kb. Only the CCC forms are labeled. Lane 1, WBG4762 parental isolate containing three plasmids. Lane 2, WBG6163 (WBG541 transconjugant carrying the 4.4 kb pWBG641). Lane 3, a transconjugant of WBG541 carrying the 4.7 kb Sm-Cm plasmid. The plasmid is present largely in the relaxed OC form. Lane 4, WBG7695 (WBG4762 cured of the 4.4 kb plasmid. pWBG641). Lane 5, WBG4762 cured of the 4.7 kb plasmid.

43.5”C for plasmid content. One of 37 colonies screened was found to have lost the 4.4 kb plasmid (Fig. 1, lane 4) but was still resistant to tetracycline and minocycline. This strain was designated WBG7695. Mobilization experiments were then performed using the conjugative plasmid, pWBG636 [20], which resulted in the transfer of the 4.4 kb plasmid to WBG541. The transfer of the 4.4 kb plasmid conferred tetracycline, only on WBG541 indicating that it encoded tetracycline resistance. One of the tetracycline-resistant transconjugants was designated WBG6163 (Fig. 1, lane 2). The MICs for tetracycline were determined for WBG6163 and WBG7695 (WBG4762 cured of the 4.4 kb plasmid) and were > 250 and 64 mg/l, respectively. 3.2. DNA-DNA hybridization Chromosomal

DNA was isolated from all of the

This study investigated the genetic nature of tetracycline resistance in methicillin-resistant and methicillinsusceptible S. aureus isolated from a Nigerian hospital. The results have provided evidence that they contained chromosomally integrated tetracycline-resistance plasmids. With the exception of one MSSA, WBG4762 that harboured a 4.4 kb plasmid, none of the isolates carried extrachromosomal tetracycline-resistance plasmid. This lack of an association of extrachromosomal plasmids with tetracycline resistance in the isolates clearly established that their tetracycline-resistance determinants were chromosomal and was consistent with the criteria used to establish the chromosomal location of ,&lactamase plasmids [25,26] and tetracycline-resistance determinants [7,11,27,28] in S. aureus. None of the isolates transferred chromosomal tetracycline resistance by conjugation, suggesting that they lack tetracycline-resistance conjugative transposons which have been found in some strains of Enterococci [23,24]. Probing the isolates with a labeled pWBG3 probe demonstrated that the chromosomal DNA fragments of MRSA and MSSA from Nigeria and WBG525, an EA MRSA, which hybridized to the probe corresponded to the 2.4, 1.5 and 0.5 kb Hind111 fragments of the probe providing strong evidence that complete plasmids similar to pWBG3 were integrated into their chromosomes. The additional chromosomal bands which hybridized with the probe in some of the isolates are most likely due to

E E. Vdo, WB. Grubbllnternational Journal

A abcdefghi

of

Antimicrobial Agents 6 (1995) 51-56

iklm

55

ilm

2.4

2.4

l-5

l-5 0.5

0,5

Fig. 2. Probing of S. aureus genomic DNA with a tetracycline-resistance plasmid probe. Autoradiograph of Hind111 digested chromosomal DNA after hybridization with DIG-11-dUTP labeled pWBG3 probe. Lane a, pWBG3 used as probe. Lane b, WBG7208; Lane c, WBG7209; Lane d, WBG7210; Lane e, WBG7211; Lane f, WBG7212; Lane g, WBG7213; Lane h, WBG7214; Lane i, WBG4761; Lane j, WBG7695; Lane k. WBG4763; Lane 1, WBG525; Lane m, WBG248. Note that there was no hybridization with chromosomal DNA of WBG248.

incompletely digested DNAs or to the plasmid integrating into different sites relative to the Hind111 sites. Obviously, the additional bands hybridizing to the probe were not due to cross hybridization with their large plasmids since derivatives cured of l.hese plasmids gave patterns similar to those of the uncured parental isolates. The results obtained with WBG525, an EA MRSA, confirm the study by Gillespie et al. [13] who first reported the presence of an integrated tetracycline-resistance plasmid, similar to pT181, in the chromosome of MRSA isolated from Australian hospitals. Two types of chromosomal tetracycline-resistance phenotypes were observed in the LUTH isolates. One type was resistant to tetracycline only and was detected in the MRSAs, WBG7208-‘WBG7214. The second type was resistant to tetracycline and minocycline and was methicillin-susceptible isolates, detected in the WBG4761 and WBG4762, and an MRSA, WBG4763. These phenotypes correspond to the t&K and tetM phenotypes, respectively [12]. From these results only WBG4763 had a similar tetracycline-resistance phenotype to the EA MRSA, W7BG525 (tetM). WBG7208WBG7214 were different, possessing the tetK phenotype. Some MRSA isolated in Europe [l l] and Western Australia [27] also carry chromosomal tetracycline without minocycline resistance. It is interesting that although WBG7208-WBG7214 differed from the EA MRSA in their tetracycline-resistance phenotype, they all expressed similar levels of tetracycline resistance (MIC 64 mg/l) and gave similar hybridization patterns with the

same probe. The plasmid, pWBG3, used as probe is similar to pT181, [21] and therefore carries the tetK determinant [12]. This may be due to the fact that the EA MRSA, WBG525, WBG4763 and the MSSA isolates WBG4761 and WBG4762 carry both the tetK and tetM determinants. MRSA resistant to both tetracycline and minocycline have been shown to hybridize to both tetK and tetM probes [12]. Bismuth et al. [12] found that the level of tetracycline resistance was similar in S. aureus isolates from France whether they expressed the tetK or tetM phenotypes. They also found that the levels were increased in isolates harbouring both resistance determinants. In our study only WBG4762 carrying the extrachromosomal tetracycline-resistance plasmid expressed higher levels of resistance (MIC >250 mg/l). The higher MIC observed here may be because the extrachromosomal plasmids are present in more copies than the chromosomal determinant, thereby exhibiting a gene dosage effect, The finding that WBG4762 harboured an extrachromosomal as well as a chromosomally integrated tetracycline-resistance plasmid is very interesting as it raises the question of incompatibility between the two plasmids. Normally, plasmids belonging to the same incompatibility group do not co-exist within the cytoplasm. As the 4.4 kb tetracycline-resistance plasmids in S. aureus belong to the same incompatibility group [lo] the presence of two tetracycline-resistance plasmids in WBG4762 would suggests that either the plasmids belong to different incompatibility groups or that the integrated plasmid is defective

E.E. Udo. WB. Grubbllnternational

56

Journal of Antimicrobial Agents 6 (1995) 51-56

for incompatibility. However, incompatibility has been demonstrated between a chromosomally integrated and an extrachromosomal plasmid in Salmonella d&in [29]. The detection of integrated plasmids in the chromosomes of S. aureus isolates from LUTH is significant because it demonstrated not only the presence of integrated tetracycline-resistance plasmids in MRSA isolates other than the EA MRSA and MRSA from USA and Greece [14], but also their presence in MSSA isolates not previously studied. It also demonstrated the presence of integrated tetracycline-resistance plasmids in isolates carrying the tetracycline-resistance as well as the tetracycline-minocycline-resistance phenotypes and suggests that these elements are probably more widespread than has hitherto been known. Finally, these findings together with the association of insertion sequences IS257 with the chromosomal integration of tetracycline-resistance determinants into the chromosome [14,30,31] raise the following questions. Is the chromosomal integration of tetracycline-resistance plasmid a recent event in S. aureus or did it exist originally in the integrated form and has it only recently been excised? What was responsible for its integration or excision? Answers to these questions will enhance our understanding of the origin and evolution of tetracycline-resistance plasmids in S. aureus. Acknowledgments We thank Prof. V.O. Rotimi, for providing the isolates. This work was supported by grants from the National Health and Medical Research Council to WBG. References 111Chopra I, Howe TGB. Bacterial resistance to the tetracyclines. Microbial Rev 1978;42:707-724.

121Salyers AA, Speer BS, Shoemaker NB. New perspectives in tetracycline resistance. Mol Microbial 1990;4:151-156. t31Speer BS, Shoemaker NB, Salyers AA. Bacterial resistance to

tetracycline: mechanisms, transfer and clinical significance. Clin Microbial Rev 1992;5:389-399. D, Zaidenzaig Y, Ziegher-Schlomowitz R, [41Sompolinsky Abramova N. Mechanism of tetracycline resistance in Staphylococcus aureus. J Gen Microbial 1970;62:351-362. PI Chopra I, Hawkey PM, Hinton M. Tetracyclines, molecular and clinical aspects. J Antimicrob Chemother 1992;29:245-277. VI McMurry LM, Petrucci RE, Levy SB. Active efIIux of tetracycline encoded by four genetically different determinants. Proc Nat1 Acad Sci USA 1980;77:3974-3977. t71Asheshov EA. The genetics of tetracycline resistance in Staphylococcus aureus. J Gen Microbial 1975;88:32-140. [8] Lacey RW. Antibiotic resistance plasmids of Staphylococcus aureus and their clinical significance. Bacterial Rev 1975;39:1-32. [9] Lyon BR, Skurray R. Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbial Rev 1987;51:88-134. [lo] Iordanescu S, Surdeanu M, Latta PD, Novick R. Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker. Plasmid 1987;1:468-479. [l l] Kayser, F.H., Wust, J. and Santanam, P. Genetic and molecular

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