To investigate the mechanisms of multiple resistance and the horizontal transfer of resistance genes in animal pathogens, we characterized the molecular structures of the resistance gene-related sequences in a multidrug-resistant Klebsiella pneumoniae strain R46 isolated from a rabbit. Molecular cloning was performed to clone the resistance genes, and minimum inhibitory concentrations (MICs) were measured to determine the resistance characteristics of the cloned genes and related strains. A conjugation experiment was conducted to assess the transferability of the resistance plasmids. Sequencing and comparative genomic methods were used to analyze the structures of the resistance gene-related sequences. The K. pneumoniae R46 genome consisted of a chromosome and three resistance plasmids named pR46-27, pR46-42, and pR46-270, respectively. The whole genome encoded 34 antibiotic resistance genes including a newly identified chromosome-encoded florfenicol resistance gene named mdfA2. pR46-270, besides encoding 26 antibiotic resistance genes, carried four clusters of heavy metal resistance genes and several virulence-related genes or gene clusters. The plasmid-encoded resistance genes were mostly associated with mobile genetic elements. The plasmid with the most similarity to the floR gene-harboring plasmid pR46-27 was pCTXM-2271, a plasmid from Escherichia coli. The results of this work demonstrated that the plasmids with multidrug resistance genes were present in animal-derived bacteria and more florfenicol resistance genes such as mdfA2 could be present in bacterial populations. The resistance genes encoded on the plasmids may spread between the bacteria of different species or genera and cause the resistance dissemination.

Evidence was obtained to indicate that equine strains of organisms previously described as Actinobacillus suis or hemolytic variants of Actinobacillus equuli might constitute a separate group of organisms provisionally designated taxon 11. Four biovars were noticed within taxon 11. Selected DNA:DNA hybridizations support the classification of the mannitol positive biovar 2 of taxon 11 distinct from porcine A. suis. The final taxonomical position of taxon 11, however, has to await more detailed genetic studies including all biovars of taxon 11. A species name has not been suggested for the same reasons. The present observations also indicate that strains identified as taxon 11 apparently constitute a part of the normal bacterial flora in the oral cavity of horses.

Due to inappropriate use, florfenicol resistance is becoming increasingly serious among animal respiratory tract and gut bacteria. To detect the florfenicol resistance mechanism among Enterobacteriaceae bacteria, 292 isolates from animal feces were examined. The agar dilution method was conducted to determine the minimum inhibitory concentration (MIC) for florfenicol, and polymerase chain reaction (PCR) was performed to detect florfenicol resistance genes. To further explore the molecular mechanism of florfenicol resistance, the whole-genome Leclercia adecarboxylata R25 was sequenced. Of the strains tested, 61.6% (180/292) were resistant to florfenicol, 64.4% (188/292) were positive for floR, and 1.0% (3/292) for cfr. The whole-genome sequence analysis of L. adecarboxylata R25 revealed that the floR gene is carried by a transposon and located on a plasmid (pLA-64). Seven other resistance genes are also encoded on pLA-64, all of which were found to be related to mobile genetic elements. The sequences sharing the greatest similarities to pLA-64 are the plasmids p02085-tetA of Citrobacter freundii and p234 and p388, both from Enterobacter cloacae. The resistance gene-related mobile genetic elements also share homologous sequences from different species or genera of bacteria. These findings indicate that floR mainly contributes to the high rate of florfenicol resistance among Enterobacteriaceae. The resistance gene-related mobile genetic elements encoded by pLA-64 may be transferred among bacteria of different species or genera, resulting in resistance dissemination.

Due to the inappropriate use of florfenicol in agricultural practice, florfenicol resistance has become increasingly serious. In this work, we studied the novel florfenicol resistance mechanism of an animal-derived Leclercia adecarboxylata strain R25 with high-level florfenicol resistance. A random genomic DNA library was constructed to screen the novel florfenicol resistance gene. Gene cloning, gene knockout, and complementation combined with the minimum inhibitory concentration (MIC) detection were conducted to determine the function of the resistance-related gene. Sequencing and bioinformatics methods were applied to analyze the structure of the resistance gene-related sequences. Finally, we obtained a regulatory gene of an RND (resistancenodulation-cell division) system, ramA, that confers resistance to florfenicol and other antibiotics. The ramA-deleted variant (LA-R25ΔramA) decreased the level of resistance against florfenicol and several other antibiotics, while a ramA-complemented strain (pUCP24-prom-ramA/LA-R25ΔramA) restored the drug resistance. The whole-genome sequencing revealed that there were five RND efflux pump genes (mdtABC, acrAB, acrD, acrEF, and acrAB-like) encoded over the chromosome, and ramA located upstream of the acrAB-like genes. The results of this work suggest that ramA confers resistance to florfenicol and other structurally unrelated antibiotics, presumably by regulating the RND efflux pump genes in L. adecarboxylata R25.

PurposeThe aim of this work was to investigate the molecular characterization of a clinical Enterococcus casseliflavus strain with a resistance plasmid.Materials and methodsEn. casseliflavus EC369 was isolated from a patient in a hospital in southern China. The minimum inhibitory concentration was found by means of the agar dilution method to determine the antimicrobial susceptibilities of the strains. Whole-genome sequencing and comparative genomics analysis were performed to analyze the mechanism of antibiotic resistance and the horizontal gene transfer of the resistance gene-related mobile genetic elements.ResultsEn. casseliflavus EC369 showed resistance to erythromycin, kanamycin, and streptomycin, but was susceptible to vancomycin, ampicillin, and streptothricin and other antimicrobials. There were six resistance genes (aph3′, ant6, bla, sat4, and two ermBs) carried by a transposon identified on the plasmid pEC369 and a complete resistance gene cluster of vancomycin and a tet (M) gene encoded on the chromosome. This is the first complete plasmid sequence reported in clinically isolated En. casseliflavus. The plasmid with the greatest sequence identity with pEC369 was the plasmid of Enterococcus sp. FDAARGOS_375, followed by the plasmids of Enterococcus faecium strains F12085 and pRE25, whereas the sequence with the greatest identity to the resistance genes carrying a transposon of pEC369 was on the chromosome of Staphylococcus aureus strain GD1677.ConclusionThe resistance profiles of En. casseliflavus EC369 might contribute to the resistance genes encoded on the plasmid. The fact that the most similar sequence to the transposon carrying resistance genes of pEC369 was encoded in the chromosome of a S. aureus strain provides insights into the mechanism of dissemination of multidrug resistance between bacteria of different species or genera through horizontal gene transfer.