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ANTIMICROBIAL agents are often used to prevent or treat chlamydial infections. The widespread use of tetracycline antibiotics in this way has encouraged selection for resistant organisms. The aim of this study was to evaluate the sensitivity to tetracycline of some strains of Chlamydia suis isolated in Italy.
In the period from 2004 to 2007, 14 chlamydial isolates were collected from pigs with conjunctival and/or reproductive disorders reared in four different farms in northern and southern Italy. dna from the isolates grown in llc-mk2 cells (a continuous cell line derived from rhesus monkey kidney tissue) was extracted for molecular analysis using a commercially available kit (Tissue Kit; Qiagen) and used as a template for amplification of a 1050 base pair (bp) fragment of the chlamydial ompA gene (Sayada and others 1995). The amplicons were purified using a commercially available kit (qiaquick pcr Purification Kit; Qiagen) and sequenced. Nucleotide sequences were compared with the same regions of the C suis type strain s45 available in the GenBank database, using blast software. The ompA amplification showed the expected products. Alignment of the deduced amino acid sequences of the momp protein of the 14 isolates with the same sequence of the reference C suis s45 strain revealed an amino acid homology of 81 to 91 per cent.
The sensitivity of the 14 isolates to doxycycline was tested and compared with the susceptibility to doxycycline of the urethral Italian isolate go86 of Chlamydia trachomatis serovar D, the reference strain 6bc of Chlamydophila psittaci, and isolates of Chlamydophila, abortus, Chlamydophila pecorum and Chlamydophila felis. The susceptibility tests were performed in llc-mk2 cells grown in 24-well plates, as previously described by Donati and others (2002). After incubation of the cell monolayers with chlamydiae and centrifugation, the medium was removed and replaced with a medium containing serial twofold dilutions of doxycycline. All the tests were run in triplicate. After incubation at 35°C for 48 hours, the cultures were fixed and stained for the detection of chlamydial inclusions by immunofluorescence using a fluorescein-conjugated monoclonal antibody specific for the chlamydial lipopolysaccharide genus-specific antigen.
The minimum inhibitory concentration (mic) of doxycycline was defined as the lowest concentration preventing the detection of more than 90 per cent of the chlamydial inclusions compared with the drug-free control. The minimum bactericidal concentration (mbc) was determined by aspirating the antimicrobial-containing medium, washing the wells twice with phosphate-buffered saline, adding antimicrobial-free medium, and reincubating the plates for 48 hours at 35°C. The mbc was the lowest antimicrobial concentration resulting in more than a 90 per cent reduction of inclusions. mic values of 4 μg/ml or greater and mbc values of 8 μg/ml or greater were defined as indicating resistance of the isolates (Lenart and others 2001).
Dugan and others (2004) demonstrated that the C suis tetracycline resistance (Tcr) phenotype is associated with a resistance gene, tet(C), integrated into the chlamydial chromosome. Therefore, the C suis isolates were tested for the presence of the tet(C) resistance gene by a pcr assay amplifying a 525 bp product of the tet(C) gene-coding region. The reaction was performed according to Dugan and others (2004), using the following forward and reverse oligo-nucleotide primers: cs43 5′-agcactgtccgaccgctttg-3′ and cs47 5′-tcctcgccgaaaatgaccc-3′. Each dna preparation (5 μl) was added to a pcr mixture (final volume 50 μl) containing 1·5 U of AmpliTaq Gold dna polymerase (Applied Biosystems/Roche), 2·0mM magnesium chloride, 200μM of each deoxynucleoside triphosphate, 1 × Gold Buffer and 20 pmol of each primer. The cycling conditions were as follows: four minutes of denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for one minute, annealing at 55°C for one minute and chain elongation at 72°C for one minute. A step at 72°C for seven minutes completed the reaction. The same reaction was applied to tetracycline-sensitive strains of C trachomatis, Cp psittaci, Cp abortus, Cp pecorum, Cp felis. The amplicons were purified and sequenced.
In comparison to the strains of C trachomatis, Cp psittaci, Cp abortus, Cp pecorum and Cp felis, which were sensitive to doxycycline with mics and mbcs ranging from 0·03 to 0·125 μg/ml, the mic and mbc values of doxycycline for most of the 14 isolates of C suis ranged from 4 to 8 μg/ml (Table 1). All of the C suis isolates carried an identical nucleotide sequence, which showed 100 per cent homology with that of the tet(C) resistance gene described by Dugan and others (2000) (GenBank accession number ay428551). None of the five tetracycline-sensitive chlamydial strains carried the tet(C) gene.
These results concerning tetracycline resistance of C suis isolates in Italy were consistent with data reported from the usa (Andersen and Rogers 1998, Lenart and others 2001, Dugan and others 2004). At present, data about other geographical areas are lacking. It was of interest that all the Italian C suis isolates carried the tet(C) gene associated with tetracycline resistance, and all but two of the isolates were tetracycline resistant. In Italy, as in the usa, tetracycline is routinely used to ward off infections in pig herds, therefore the widespread use of this antibiotic could explain the selection for resistant organisms. However, epidemiological studies performed in other regions where the use of tetracycline is less or absent could give more insight and could help in better defining the mechanisms that favour tetracycline resistance in C suis.
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