A variety of Gram-positive bacteria carry mobile mef genes

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1 Journal of Antimicrobial Chemotherapy (1999) 44, A variety of Gram-positive bacteria carry mobile mef genes JAC Vicki A. Luna a, Patricia Coates b, E. Anne Eady b, Jonathan H. Cove b, Thanh T. H. Nguyen a and Marilyn C. Roberts a * a Department of Pathobiology, Box , School of Public Health and Community Medicine, University of Washington, Seattle, WA , USA; b Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK The mefe gene codes for a membrane bound efflux protein, which confers resistance to macrolides, and has been identified in Streptococcus pneumoniae. A variety of Gram-positive organisms were examined. Twenty-six isolates of S. pneumoniae carried mefe and were resistant to erythromycin (MIC of 2 16 mg/l). Two additional isolates of Em r S. pneumoniae carried both ermb and mefe (MIC of mg/l). One Micrococcus luteus, one Corynebacterium jeikeium, three Corynebacterium spp., two viridans streptococci and seven Enterocccus spp. also carried mef genes. It was possible to move the mef gene from all 11 S. pneumoniae tested to susceptible S. pneumoniae and/or Enterococcus faecalis recipients. The addition of DNase (1 g/l) did not affect the gene transfer. It was also possible to move the mef gene from donor Enterococcus spp., viridans streptococci, M. luteus, C. jeikeium and Corynebacterium spp. to E. faecalis recipients. Transconjugant isolates were resistant to erythromycin (MIC 16 mg/l). Hybridization with a labelled mef oligonucleotide probe against Southern blots and bacterial dot blots confirmed the presence of the mef genes. This is the first time that a mobile mef gene has been identified in four different genera, from three distinct geographical locations. Introduction World-wide, the prevalence of antibiotic resistant Strepto - coccus pneumoniae has increased, 1 4 with five serogroups (6, 9, 14, 19 and 23) having developed resistance to two or more classes of antibiotic. 5 Most resistance (with the exception of that to -lactams), has been obtained through the acquisition of new genes, usually associated with conjugative transposons. 6,7 Resistance to erythromycin in the first resistant S. pneumoniae isolates described was found to be due to the presence of an rrna methylase gene, ermb, which conferred resistance to the macrolides, lincosamides and streptogramin B antibiotics. This gene is still commonly found in many streptococcal species More recently, resistance to macrolides in the absence of resistance to lincosamides or streptogramin B has been described in S. pneumoniae and -hemolytic (Lancefield Group A) streptococci. This is determined by the presence of a membrane bound efflux protein, encoded by the mef genes, mefa or mefe mefa has been cloned from Streptococcus pyogenes and has been identified in Lancefield Group C and G streptococci originating from Finland. 14,16 The mefe gene has been identified and cloned from S. pneumoniae in the USA. 13,15,17 The two genes have 90% nucleotide sequence identity, and using DNA probes for conserved regions, both mefa and mefe will be detected. In this study, probes to the conserved regions of the genes were used. The mef gene will be referred to as mefe for S. pneumoniae and the mef gene for other species. Materials and methods Thirty-four isolates of S. pneumoniae, which were erythromycin resistant (Em r ) (MIC 2 mg/l), were included in the study. Twenty-four isolates from cases of invasive disease were collected between 1995 and 1996, during a Washington State Surveillance Study; four Em r S. pneumo - niae resulted from a Paediatric Nasopharyngeal Carriage Study, conducted in 1996, in Seattle; and a further six Em r invasive isolates of S. pneumoniae were obtained in 1997 from the Washington State Health Department. Serotypes of the S. pneumoniae isolates were initially determined by the Quellung reaction 18 in this laboratory and subsequently *Corresponding author. marilynr@u.washington.edu 1999 The British Society for Antimicrobial Chemotherapy 19

2 V. A. Luna et al. confirmed by the University of Washington Medical Center, Seattle, and/or the Centers for Disease Control and Prevention (CDC) in Anchorage, Alaska. In addition, ten Em r Micrococcus luteus isolates and 20 Em r Corynebact - erium spp. isolates (one Corynebacterium jeikeium, one Corynebacterium Group G2, ten Corynebacterium Group ANF, three Corynebacterium Group A, three Corynebact - erium aquaticum and two Corynebacterium spp.) collected in the UK during 1997 from skin cultures of patients attending an acne clinic at the University of Leeds were examined. Thirty-two Em r Enterococcus spp. obtained from normally sterile sites, from patients at the University of Washington Medical Center Clinical Microbiology Laboratory, Seattle, were examined. Unfortunately, no patient information was available on these isolates. Twenty-nine viridans streptococcal isolates, which grew on erythromycin supplemented media (10 mg/l), collected from oral cultures of children participating in a dental study in Lisbon, Portugal, were also screened. Resistance to erythromycin was confirmed for mef positive isolates by micro-broth dilution or agar dilution as described in NCCLS guidelines. 19,20 Streptococcus spp. were grown on Brucella blood agar (BA) (Difco, Detroit, MI, USA) supplemented with 5% sheep red blood cells and incubated with 5% CO 2 at 36.5 C. M. luteus and Corynebacterium spp. were grown on Brain Heart Infusion Agar (BHIA) (Difco) supplemented with 5% NAD and 5% Vitamin K haemin. Enterococcus faecalis JH2-2 was grown on BHIA without supplements. Bacteria for DNA dot blots and DNA extractions were grown overnight at 36.5 C in BHI broth (Difco) supplemented with 0.03 M D-glucose and 0.04% DL-threonine. 21,22 Mating experiments Ten Em r S. pneumoniae from Seattle and S. pneumoniae 02J1048, known to contain the mefe gene, from Pfizer, Inc. (Groton, CT, USA), 15 Em r M. luteus 64, four Em r Corynebacterium spp. (214, 260, 274 and 388), three Em r Enterococcus spp. (102, 106 and 138) and two Em r viridans streptococci (7405B2-47 and 7405B2-48) were used as donors. The donor organisms were susceptible to fusidic acid, rifampicin and streptomycin. E. faecalis JH2-2 was used as a recipient (erythromycin susceptible), and had been previously selected for chromosomal resistance to rifampicin (25 mg/l) and fusidic acid (25 mg/l) (Em s Rif r Fus r ). 21,23 The other recipient was a clinical isolate, S. pneu - moniae 915, a 6B serotype from Alaska, which was susceptible to erythromycin and, by stepwise selection, made chromosomally resistant to streptomycin (1 g/l), fusidic acid (25 mg/l) and rifampicin (25 mg/l) (Em s Rif r Fus r Str r ). 21 Donor and recipient bacteria were grown separately at 36.5 C overnight on BA or BHIA plates. Each isolate was suspended in ml of BHI broth (Difco) to a density of approximately 10 9 cells/ml (3 McFarland). Donor and recipient at a 1:1 (donor to recipient) ratio for S. pneumoniae to S. pneumoniae matings,or a 5:1 (donor to recipient) ratio for all other matings, were mixed, plated directly on to BA plates, and incubated in CO 2 at 36.5 C for 48 h. 21 After incubation, the mating mixture was serially diluted on to antibiotic supplemented plates as previously described Transconjugants from the S. pneumoniae S. pneumoniae and S. pneumoniae viridans streptococcus matings were selected on BA plates supplemented with 2 mg/l of erythromycin and 25 mg/l of rifampicin. S. pneu - moniae transconjugants grew on BA plates supplemented with either erythromycin (2 mg/l), rifampicin (25 mg/l), fusidic acid (25 mg/l) or streptomycin (1 g/l). These isolates were identified biochemically as S. pneumoniae. 18 The E. faecalis tranconjugants were selected on BHIA supplemented with erythromycin 10 mg/l and either rifampicin 25 mg/l. E. faecalis tranconjugants grew in the presence of rifampicin (25 mg/l), erythromycin (10 mg/l) or fusidic acid (25 mg/l), as previously described, 23 and were identified biochemically as E. faecalis. 24 Plates with no growth were held for 7 days. All matings were done in duplicate. Some matings were done in the presence of DNase (1 g/l) (Sigma Chemical Co., St Louis, MO, USA) as previously described, to rule out transformation. 25 Duplicate matings were performed without DNase, and the frequencies were compared with the frequencies of the matings with DNase, and were shown to be the same. mef was detected using labelled mef oligonucleotide probe hybridization of bacterial dot blots and/or Southern blots. Labelled probes The oligonucleotide probes used were MF4 (sequence: 5 - ACC GAT TCT ATC AGC AAA-3 ), MF5 (sequence: 5 - GGT GCT GTG ATT GCA TCT ATT AC-3 ), and ErmB F (sequence: 5 -GAA AAG GTA CTC AAC CAA ATA-3 ). 26 The probes were labelled using the Genius Oligonucleotide Labeling Kit (Boehringer Mannheim, Indianapolis, IN, USA), following the manufacturer s procedures for purified Southern blots and whole cell DNA dot blots only. 32 P-labelled probes were used for whole bacterial cell dots using T4 polynucleotide kinase (10 U) (Promega, Madison, WI, USA) as previously described. 22 Whole cell DNA extraction Whole cell DNA was prepared from isolates and transconjugants, using cells grown overnight, in 100 ml of supplemented BHI broth, as previously described. 27 After extractions with Tris-saturated phenol, ph 8.0, and chloroform, the DNA was precipitated with ethanol, resuspended in nanopure sterile water and stored at 20 C until needed. The whole cell DNA was run on a 0.7% agarose gel and Southern blots were prepared

3 Gram-positive bacteria carrying mobile mef genes Dot blots Overnight bacterial growth (1 ml) in supplemented BHI broth was placed into 1.5 ml sterile Eppendorf tubes, centrifuged at 10,000 rpm for 2 min, and the supernatant decanted. The bacterial pellet was resuspended with 1 ml BHI broth to create a turbid suspension (corresponding to 3 McFarland; 10 9 bacteria/ml). Suspension (200 L) was spotted onto GeneScreenPlus membrane (NEN Research, Boston, MA, USA), dried, treated with 0.5 M NaOH for 10 min, 1 M Tris HCl for 3 min and 1 M Tris HCl with 1.5% NaCl, ph 7.5, for 10 min. The membrane was dried, washed twice in chloroform isoamylalcohol (24:1), rinsed in water twice, then washed in 1 M Tris HCl, and 1 M Tris HCl with 1.5% NaCl, and baked at 80 C for 1 h. The filters were stored at room temperature until labelled. 28 Polymerase chain reaction (PCR) A PCR assay was used as a second method for detection of the mef genes in donors and transconjugants. The PCR assay used 40 ng of genomic DNA from S. pneumoniae 02J1048 as a positive control, and 40 ng genomic DNA as a template from S. pneumoniae, Micrococcus, Corynebac - terium spp., viridans streptococci and Enterococcus spp. The primers used were: MF4a (5 -ACC GAT TCT ATC AGC AAA G-3 ) and MF6 (5 -GGA CCT GCC ATT GGT GTG-3 ). 17 Both are in the conserved regions of mefa and mefe genes. Each reaction contained 2 units of Taq polymerase (Perkin Elmer Cetus, Norwalk, CT, USA), 200 M deoxynucleoside triphosphates, 1 PCR buffer (1.5 mm MgCl 2 ) and 100 ng of each primer. Using a Perkin Elmer Cetus thermal cycler, the reactions were carried out by denaturing at 94 C for 1 min, annealing at 37 C for 1 min and elongation at 72 C for 2 min for 35 cycles. The PCR products were lyophilized, resuspended in 1/10 volume sterile water, and run on a 1.5% agarose gel with 0.5 TBE running buffer. Ethidium bromide staining allowing visualization of PCR bands and Southern blots were prepared. The 940 bp PCR product was confirmed by hybridization with a labelled internal mef probe, MF5 (5 -GGT GCT GTG ATT GCA TCT ATT AC-3 ). Negative and positive controls were included in each run. DNA DNA hybridization Southern blots of the uncut whole cell DNA were prepared on Magnagraph nylon (Micron Separation Inc., Westboro, MA, USA) and hybridized with nonradiolabelled oligonucleotide probe MF4 or ermb F following the manufacturer s directions (Boehringer). Southern blots of the PCR assay were prepared using the Magnagraph nylon membrane (Micron Separation, Inc.) and hybridized with labelled MF5. Detection of the probe was performed using the CDP-Star reagent at a 1:1000 dilution, following the manufacturer s instructions (Boehringer Mannheim Biochemica). DNA dot blots containing g of purified whole cell DNA were placed on GeneScreenPlus membrane (NEN Research Products) and hybridized with the nonradiolabelled probe. The whole bacterial cell dot blots were placed on GeneScreenPlus membrane and hybridized with a 32 P-labelled oligonucleotide probe. 22 Results Detection of the mef and ermb genes in four genera of bacteria Sixty-three (50%) of the total 125 bacterial isolates tested hybridized with the mef and/or ermb probes (Table I). Twenty-six (76%) of the 34 S. pneumoniae isolates tested hybridized with the mef probe only, two isolates hybridized with mef and ermb probes, and four isolates hybridized with the ermb probe only (Table I). One of the M. luteus, four Corynebacterium spp. (one C. jeikeium, one Coryne - bacterium Group A, and two Corynebacterium spp.), seven of the Enterococcus spp. and two viridans streptococci hybridized with the mef probe. Six Enterococcus spp., one M. luteus, one Corynebacterium (Group A) and nine viridans streptococci hybridized with the ermb probe (Table I). The PCR assay with hybridization of the internal MF5 probe confirmed the presence of the mef gene in the isolates. Correlations were found between low-level erythromycin resistance (MIC of 2 4 mg/l) and the presence of the mefe gene in S. pneumoniae, or high-level erythromycin resistance (MIC of 16 to 128 mg/l) with ermb, or mefe and ermb in S. pneumoniae (Table II). In Enterococcus spp. there were similar correlations between low-level erythromycin resistance (MIC of 2 16 mg/l) and the presence of the mef gene and high-level resistance (MIC 32 mg/l) and the presence of ermb. In viridans streptococci, low-level erythromycin resistance (MIC of 2 4 mg/l) was associated with the mef gene, but both low- and high-level resistance was seen in the isolates with the ermb gene (Table II). MIC in M. luteus and Coryne - bacterium spp. did not correlate with the presence of the mef gene, but the numbers tested were small (Table II). Other erm genes (erma, ermc, etc.) were not examined. Conjugal transfer of the mef gene Erythromycin resistance erm genes are often associated with mobile elements. Therefore, it was of interest to see whether the mef gene was associated with mobile elements in these isolates. It was possible to move the mef gene from three of four S. pneumoniae donors to the S. pneumoniae recipient, at frequencies ranging from 10 6 to 10 7 per recipient (Table III). To selected matings, DNase (1 g/l) was added to the mating mixtures and the frequencies were compared with matings without DNase. The frequencies of transfer were within 0.5 log 10 with DNase, compared with 21

4 V. A. Luna et al. Table I. Results of DNA probe hybridization (values in parentheses are percentages) Number tested ermb mef Genus (n 125) (n 23) (n 42) Number negative a S. pneumoniae b 34 6 (18) 28 (82) 2 Enterococcus spp (19) 7 (22) 19 M. luteus 10 1 (10) 1 (10) 8 C. aquaticum 3 0 (0) 0 (0) 3 C. jeikeium 1 0 (0) 1 (100) 0 Corynebacterium Group ANF 10 0 (0) 0 (0) 10 Corynebacterium Group G2 1 0 (0) 0 (0) 1 Corynebacterium Group A 3 1 (33) 1 (33) 1 Corynebacterium spp. 2 0 (0) 2 (100) 0 viridans streptococci 29 9 (31) 2 (7) 18 The Micrococcus and Corynebacterium were from the UK, viridans streptococci were from Portugal and all other species were from the USA. a Did not hybridize with either ermb or mef, but could carry other erm genes such as erma or ermc, which were not examined in this study. b Two isolates carried both ermb and mefe. Table II. Correlation of MIC (mg/l) of erythromycin with presence of mef or ermb gene Isolate serogroup erythromycin clindamycin DNA probe S. pneumoniae F mefe S. pneumoniae F mefe S. pneumoniae F mefe S. pneumoniae n mefe S. pneumoniae w mefe S. pneumoniae F mefe S. pneumoniae F mefe S. pneumoniae ermb S. pneumoniae ermb S. pneumoniae ermb/mefe S. pneumoniae ermb S. pneumoniae ermb S. pneumoniae 54 19F a ermb/mefe M. luteus 64 NA mef C. jeikeium 388 NA mef Corynebacterium spp. 274 NA 16 4 mef MIC a Clindamycin resistance was induced by exposure to 0.5 mg/l erythromycin to give a clindamycin MIC of 128 mg/l. the same matings without DNase, suggesting that conjugation rather than transformation was taking place in S. pneumoniae S. pneumoniae matings. Two mating pairs are shown in Table III. In these three S. pneumoniae donors and eight other (total 11) S. pneumoniae donors, transfer of the mefe gene to the E. faecalis recipient could be demonstrated, at frequencies ranging from 10 6 to 10 8 per recipient (Table III). The m e f gene could also be moved from M. luteus 64, C. jeikeium 388 and Corynebacterium spp. (214, 274) to E. faecalis JH2-2, at frequencies of 10 6 to 10 8 per recipient (Table III). The same transfer of mef genes was effected from Enterococcus spp. (102, 106, 138) and viridans streptococci donors (7405B2-47, 7405B2-48) to E. faecalis JH2-2, at frequencies of 10 8 and 22

5 Gram-positive bacteria carrying mobile mef genes Table III. Conjugal transfer of mef gene Donor Recipient Frequency (per recipient) S. pneumoniae 02J1048 S. pneumoniae S. pneumoniae 915 a E. faecalis JH S. pneumoniae w002 S. pneumoniae E. faecalis JH S. pneumoniae w017 S. pneumoniae E. faecalis JH S. pneumoniae E. faecalis JH S. pneumoniae E. faecalis JH S. pneumoniae 196 S. pneumoniae S. pneumoniae 915 a viridans streptococci 7405B2-48 S. pneumoniae E. faecalis JH Enterococcus spp. 102 E. faecalis JH Enterococcus spp. 138 E. faecalis JH C. jeikeium 388 E. faecalis JH Corynebacterium spp. 214 E. faecalis JH Corynebacterium spp. 274 E. faecalis JH M. luteus 64 E. faecalis JH a Matings were performed with DNase (1 g/l) present per recipient, respectively (Table III). The MIC of erythromycin in the transconjugants from each mating with the various donors was five-fold higher than the MIC of the parental recipient (0.5 mg/l vs 16 mg/l). Whole cell DNA from donors, recipients and transconjugants were run on 0.7% agarose gel and Southern blots were prepared. Transconjugants hybridized with the labelled mef probe, whereas the susceptible recipients did not. The probe hybridized with the chromosomal fraction, suggesting a chromosomal location (data not shown). Discussion The most important finding of this study was that the mef genes examined were associated with DNase resistant mobility, suggesting a conjugative element in four Grampositive genera, from three distinct geographical locations (USA, UK and Portugal) (Table III). Transfer occurred between S. pneumoniae S. pneumoniae, as well as between unrelated genera. Plasmids were not detected in the donors or transconjugants tested. The mef probe hybridized with the chromosomal fraction in Southern blots of uncut DNA, suggesting a chromosomal location. Although the mef genes were transferred (Table III), the nature of the putative conjugative elements is unknown, but could be related to previously described conjugative elements found in streptococci. 7 As this paper was being reviewed, a paper was published which demonstrated the plasmid-free conjugal transfer of the mef gene from S. pyogenes to JH2-2 E. faecalis recipient. 29 In earlier studies, the mef genes have been found in a limited number of hosts (S. pneumoniae, S. pyogenes and Lancefield Groups C and G streptococci) ,29 This study has demonstrated that the mef genes are present in three new Em r Gram-positive genera (Corynebacterium, Entero - coccus and Micrococcus) (Table I) as well as in Em r viridans streptococci. Recently sequencing of the PCR fragments from M. luteus and Corynebacterium spp. 388 and 214 showed 93%, 93% and 95% DNA homology with the mefe GeneBank sequence, respectively. Twenty-six S. pneumoniae carried the mefe gene, and two additional isolates carried both the mefe and ermb genes. A correlation was found between lower erythromycin MIC (2 4 mg/l) for isolates carrying the mefe gene in S. pneumoniae and for enterococci (2 16 mg/l) carrying the mef gene. In contrast, isolates with higher MICs ( 16 mg/l) carried the ermb gene, either alone or in addition to the mef gene (Table II). More examples from different locations will need to be studied to determine whether the presence of these genes would predict the therapeutic usefulness of clindamycin. What impact, if any, the differences seen between carriage of mef versus erm genes will have on treatment of S. pneumoniae or other Gram-positive disease has not yet been examined. It 23

6 V. A. Luna et al. is clear from this study that the host range of the mef genes in Gram-positive bacteria needs to be more fully examined. Acknowledgements This work was presented in part at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, September This study was supported in part by the NIH National Institute of Dental Health (Grant U01-DE and Contract N01-DE-72623). We wish to thank Dr T. Fritsche and Ms S. Swanzy from the Department of Laboratory Medicine, University of Washington Medical Center, Seattle, for the Enterococcus spp., and Mr S. Deliganis from Northwest Pharmaceutical Research Network for S. pneumoniae isolates. We also wish to thank Dr A. Parkinson at the Arctic Investigations Program of the Centers for Disease Control and Prevention, Anchorage, Alaska, for confirmation of S. pneumoniae serotypes. References 1. Jacobs, M. R., Koornhof, H. J., Robins-Browne, R. M., Stevenson, C. M., Vermaak, Z. A., Freiman, I. et al. (1978). 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7 Gram-positive bacteria carrying mobile mef genes 27. Anderson, D. G. & McKay, L. L. (1983). Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Applied and Environmental Microbiology 46, Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn, pp Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 29. Kataja, J., Huovinen, P., Skurnik, M. & Seppala, H. (1999). Erythromycin resistance genes in group A streptococci in Finland. Antimicrobial Agents and Chemotherapy 43, Received 21 October 1998; returned 5 February 1999; revised 17 February 1999; accepted 12 April

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