![]() Purified plasmid DNA from strain HS1543 was used as a template in subsequent sequencing reactions. Oligonucleotides were designed at the ends of the cloned fragments. When required, a liquid medium version of TM/SN, termed TMB, was prepared by omitting the agar from TM/SN.ĭNA sequencing, analysis, and annotation of plasmids.ĭNA fragments from a midi-prep (QIAGEN) isolation of plasmids derived from strain HS1543 digested with either Sau3A (cloned fragment sizes, 0.3 and 0.6 kb) or MspI (cloned fragment sizes, 1.0 and 1.6 kb) were cloned into pUC19 digested with BamHI or AccI, respectively, and sequenced using universal M13 forward and reverse primers. ![]() Immediately before pouring, TM/SN medium is supplemented with the following sterile additives: 0.0025% NADH, 0.005% thiamine, 1% heat-inactivated horse serum, and 5% oleic acid bovine serum albumin complex consisting of 4.75% bovine serum albumin (fraction V) in normal saline (with the normal saline containing 0.06% oleic acid and 5% 0.05N NaOH). Briefly, TM/SN is prepared from a basal medium known as TM which contains 1% biosate peptone, 1% NaCl, 0.1% starch, 0.05% glucose, and 1.5% agar. All Pasteurellaceae strains used in this study were grown on TM/SN medium prepared as described previously ( 20). parasuis strains surveyed for tetracycline resistance, including additional outbreak strains, are listed in Table Table2. paragallinarum strains 221 and HP31, and Haemophilus influenzae strain Rd were used in electroporations using plasmid preparations containing both plasmids pHS-Tet and pHS-Rec. pleuropneumoniae (HS1861) strains used in this study were obtained from the culture collection at the Animal Research Institute, Brisbane, Australia. parasuis, Haemophilus paragallinarum (HP31 and 221), and A. coli Genetic Stock Center, Yale University) was used to make the tetR probe. Strain XL1-Blue MR (Stratagene) was used in electroporations using plasmid pHS-Tet, and strain TST-1 ( E. parasuis isolate from this outbreak.Įscherichia coli strains were cultured in Luria-Bertani medium (LB) with appropriate antibiotics. Tetracycline resistance determinant tet(B) was identified in an A. parasuis and Actinobacillus pleuropneumoniae. One of these tetracycline-resistant field isolates was isolated from a disease outbreak involving both H. parasuis strains, identifying tet(B) plasmid-mediated tetracycline resistance in two additional H. A survey of tetracycline resistance was undertaken in 45 H. parasuis plasmids, one of which encoded the Tet B tetracycline resistance determinant. In this study, we have identified and sequenced two native H. parasuis have previously been reported in Austria ( 15) and Denmark ( 1) although the mechanism of this resistance has not been elucidated. Tetracycline resistance determinants Tet B, Tet H, and Tet M have previously been found in other members of the Pasteurellaceae ( 7, 9). Tetracycline has a long history of use in the swine industry ( 17), and its use generates a strong selective pressure that has resulted in the exchange of tetracycline resistance genes associated with plasmids or transposons within and between bacterial species ( 13). The use of antibiotics in animal production as treatment or prophylaxis against common infections or at subtherapeutic levels in feed to promote growth is under increasing scrutiny ( 11). ![]() Common symptoms of this disease include anorexia, pyrexia, and lameness, though some pigs may die suddenly during acute outbreaks ( 19). Haemophilus parasuis is the causative agent of Glässer's disease in swine. parasuis plasmids and Tet B-mediated tetracycline resistance in this microorganism. This is believed to be the first report of native H. Southern hybridization analysis of these plasmids showed that the Tet B determinant was present, and restriction digest comparisons suggest that these plasmids are related. An approximately 10.6-kb plasmid was identified in field isolate HS226 and outbreak strains HS1857, HS1859, and HS1861. parasuis strain (HS1857, serovar 8) and a tetracycline-resistant Actinobacillus pleuropneumoniae strain (HS1861). Analysis of three additional isolates from the same disease outbreak as strain HS1859 revealed a further tetracycline-resistant H. parasuis isolates surveyed (15 international reference strains, 15 field isolates selected for their genetic diversity, and 15 recent Australian field isolates), 2 tetracycline-resistant field isolates (HS226 and HS1859) were identified. Plasmid pHS-Tet contains four open reading frames including a tet(B) tetracycline resistance gene which unusually did not have an associated tetR repressor gene. The complete sequence of two plasmids, pHS-Tet (5.1 kb) and pHS-Rec (9.5 kb), isolated from Haemophilus parasuis strain HS1543 has been obtained. ![]()
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