Abstracts

TRANSFER OF CLINDAMICYN RESISTANCE BETWEEN BACTEROIDES FRAGILIS GROUP STRAINS ISOLATED FROM CLINICAL SPECIMENS

Cibele B. Mano de Carvalho^1** Corresponding author. Mailing addres. Departamento de Patologia e Medicina Legal, Universidade Federal do Ceará, Caixa Postal 3163, CEP 60441-750, Fortaleza, CE, Brasil. E-mail cbmc@sec.secrel.com.br . FAX: (+5585) 281 3454 , José Luciano B. Moreira¹, Maria Candida S. Ferreira²

¹Departamento de Patologia e Medicina Legal, Universidade Federal do Ceará, Foratleza, CE, Brasil. ²Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

Approved: July 23, 1998

SHORT COMMUNICATION

Clindamycin resistance was trasferred by a conjugation–like process from Bacteroides thetaiotaomicron 52, a multiple antibiotic–resistant strain isolated from clinical specimens, to other Bacteroides species. A possible association between a plasmid detected in the donor strain and clindamycin resistance is discussed.

Key words: Bacteroides fragilis group, antimicrobial resistance, plasmid DNA

In Brazil, studies on antimicrobial resistance of B. fragilis group strains from clinical isolates and plasmid analysis have been rarely done (2,5,6).

Considering the importance of the antimicrobial resistance analysis, the purpose of this study was to investigate the transferability of clindamycin resistance between B. fragilis group strains and to determine the plasmid content of each isolate and transconjugants.

Four multiple antibiotic-resistant strains of Bacteroides sp were used as donor strains: B. distasonis 1069 E, B. thetaiotaomicron 36, 52 and 44, all of them isolated from clinical specimens ( bile, pelvic inflamatory disease, peritonitis) at the Hospital of Federal University of Ceará during 1993/1994. To be used as the recipient strains a rifampicin resistant B. fragilis 6xRfm was obtained, using the Szybalski mutants experiments (14).

Minimum inhibitory concentrations of antibiotics were determined by the standardized agar dilution method (10). Antibiotics included clindamycin, tetracycline and rifampicin ( Sigma Chemical Co., St Louis, MO).

Matings between B. fragilis group strains were carried out for 16h on membrane filters (type HA; pore size, 0.45µm; Millipore Corp.) as described by Macrina et al. (8). Transconjugant selection was made on agar plates containing clindamycin (64µg/ml) and tetracycline (16µg/ml) plus the counterselective drug (Rifampicin at 4µg/ml). Only B. thetaiotaomicron 52 was able to transfer clindamycin resistance to 6x Rfm strain. B. thetaiotaomicron 52 was found to be resistant to clindamycin and tetracycline but susceptible to rifampicin. The minimal inhibitory concentrations for each antibiotic was as follows (in mg/ml): clindamycin > 128; tetracycline = 64 and rifampicin <0.25. B. fragilis recipient strains was found to be susceptible to clindamycin and tetracycline but resistant to rifampicin, exibiting the minimal inhibitory concentration for each antibiotic as follows: clindamycin = 4; tetracycline = 2 and rifampicin >4. The clindamycin-resistant transcipients were similar to the 6xRfm in the phenotypic characteristics. Spontaneous clindamycin – rifampicin resistant mutants were observed in the experiment. It is noteworthy that transfer of the tetracycline resistance determinant could not be detected in the mating experiments (Table 1). The frequency of transfer of clindamycin resistance was aproximately 10

From the results of the experiments done in order to characterize the mode of transferability of clindamicin resistance, it was concluded that the transfer of clindamycin resistance was not by genetic transformation since DNAse did not reduce the frequency of transfer of clindamycin resistance compared with the MgSO₄ control. Similarly, the transfer was probably not by transduction since the incubation of either cell-free filtrates of donor cells or chloroform-treated donor cells with recipient cells did not result in the production of clindamycin-resistance transconjugants. Transfer of clindamycin resistance also did not occur when the donor and recipient cells were separated by a membrane filter. It therefore appears that the transfer of clindamycin resistance was by a conjugation-like mechanism (Table 2).

Plasmid DNA was extracted according to the alkaline lysis technique (12). 15µl of the DNA sample was submitted to horizontal electrophoresis in 0.6% agarose gel. The electrophoresis buffrer consisted of TBE plus ethidium bromide (Sigma – 0.5µg/ml). The gels were run at 24mA and 60V constant current for 12 to 18h. The sizes of the plasmids were calculated by comparing them with of l DNA – Eco R₁ fragments of known molecular weight. DNA bands were photographed over a short wave ultraviolet transiluminator (Germetec) using a Zeiss- Krow camera, a Kodak panatomic X film and a red filter.

Plasmid analysis revealed some problems. All the four donor strains showed one DNA band, correspondent to a plasmid of approximately 29kb. Nevertheless, DNA bands presented a diffuse aspect requiring a new plasmid extraction for confirmation. As regard to transconjugants, some attempts of plasmid DNA extraction were performed without satisfactory results. This segment of our study was then considered inconclusive.

The first studies about transferability of clindamycin resistance determinants among B. fragilis group strains were described in 1979 by Privitera et al. (11), Tally et al. (15) and Welch et al. (17). Two different plasmids harboring clindamycin resistance determinants were isolated in those studies and have been intensively studied: pBF4, a 41kb plasmid and pBFTM10, a 15kb plasmid isolated from B. fragilis strains.

In the present study, we performed interspecies transfer experiments using B. fragilis group strains and a good result was obtained in only one experiment with 52Bt and 6xRfm strains, donor and recipient respectively. The failure of the other experiments could be due to an unfortunate choice of strains, because it is known that not every strain is necessarily a good recipient or a good donor.

The suggested mechanism of transfer is conjugation because transformation and transduction experiments failed and also because cell-to-cell contact appears to be necessary for the transfer. Although transfer of clindamycin resistance determinants between B. fragilis group strains have been done successfully in the last years, only a few studies on this subject were done in your country (2).

The isolation of cryptic plasmids and plasmids harboring clindamicin resistance determinants from Bacteroides strains of different origins has been reported in the last ten years (9). Presuming that B. fragilis group strains are lysozime resistant, Beul et al. (3) used the hot alkaline SDS treatment, and cryptic plasmids were isolated in 9 of the 24 B. fragilis strains tested, mostly with low molecular weight. The use of lysozime treatment in combination with SDS for the isolation of plasmids of B. fragilis group strains has been recommended by Smith et al. (12) and Macrina et al. (8). Using the alkaline lysis method, we isolated plasmids of approximately 29kb from the donor strains tested. However, the presence of other plasmids of higher molecular weights cannot be discarded. Smith et al. (13) reported difficulties to detect a 82kb plasmid (pBI136) harboring clindamycin resistance determinants in a B. ovatus strain. They found later that this determinant was closely related to the determinant on the Bacteroides R plasmids pBF4 and pBFTM10.

The transconjugants obtained from the mating between Bt52 and 6xRfm were analyzed but the results are still inconclusive. It is possible that the apparent similarity in size of the plasmids isolated is not accidental, but no conclusion can be drawn until now. Elucidation of the role of these plasmids and the analysis of the transconjugants should be the next goal of our group.

ACKNOWLEDGMENTS

This work was supported by grants of the Conselho de Aperfeiçoamento e Pesquisa de Ensino Superior (CAPES). The authors wish to express their gratitude to Dr. Dalgimar Bezerra de Menezes for critical review of this manuscript.

RESUMO

Transferência de resistência à clindamicina entre cepas do grupo Bacteroides fragilis isoladas de espécimes clínicos

Resistência à clindamicina foi transferida através de processo semelhante à conjugação, de uma cepa multirresistente e isolada de espécime clínico, B. thetaiotaomicron 52, para outra espécie de Bacteroides. Uma possível associação entre um plasmídio detectado na cepa doadora e a resistência à clindamicina é discutida.

Palavras-chave: Grupo Bacteroides fragilis, resistência a antimicrobianos, DNA plasmidial.

REFERENCES

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