Genomic island type IV secretion system and transposons in genomic islands involved in antimicrobial resistance in Trueperella pyogenes.

Trueperella pyogenes (T. pyogenes) is a well-known opportunistic pathogen of many animal species. It can cause a variety of suppurative infections. The objective of this research was to get insight into the gene context and the location of the antimicrobial resistance determinants in the two multi-resistant T. pyogenes isolates TP3 and TP4. Comparative analysis of key factors leading to antimicrobial resistance was performed. Both isolates were resistant to erythromycin, azithromycin and tetracycline, and susceptible to ciprofloxacin, enrofloxacin, cefazolin and florfenicol. In addition, TP4 was resistant to amikacin and gentamicin. Whole-genome analyses revealed that both TP3 and TP4 contained two different genomic islands (TP3-GI1, TP3-GI5, TP4-GI5 and TP4-GI8) involved in multi-drug resistance. There is a common region in TP3-GI1 and TP4-GI5, containing the tetracycline resistance gene tet(W) and a series of genes involved in type IV secretion systems. Several genes located on TP3-GI5 and TP4-GI8 are highly homologous. Tetracycline-resistance gene tet(33) was potentially acquired by horizontal gene transfer via IS6100 located on 57,936 bp TP3-GI5. The macrolide resistance gene erm(X) was located near the end of the TP3-GI5. The sequence analysis of TP4-GI8 showed that two copies of erm(X) and two IS1634 elements located in the same orientation may have formed a composite transposon. GI-type T4SS, transposons and multiple resistance genes located on GIs play a key role in multiple drug resistance of TP3 and TP4.

Multidrug resistance genes are associated with a 42-kb island TGI1 carrying a complex class 1 integron in Trueperella pyogenes.

OBJECTIVES: This research was conducted to ascertain the context and location of the antibiotic resistance determinants in a multiple antibiotic-resistant Trueperella pyogenes isolate TP1. METHODS: The genome was sequenced using PacBio RS II, and the filtered data were assembled using Canu. Sequences were annotated on the basis of those in GenBank, and the genomic island (GI) of the TP1 was predicted by IslandPath-DIMOB. RESULTS: TP1 as a multiple antibiotic-resistant isolate was recovered at Jilin Province (China) in 2017 from a dairy cow with pneumonia. TP1 exhibited resistance to aminoglycosides (gentamicin and amikacin), macrolides (erythromycin), lincosamides (clindamycin), sulfonamides (sulfamonomethoxine), tetracyclines (tetracycline and doxycycline) and chloramphenicols (chloramphenicol and florfenicol). An antibiotic resistance gene clustered together with the aadB, aadA1, cmlA5 and cmlA6 resistance genes located on a 7-kilobase (kb) multidrug-resistant (MDR) region, constituting a complex class 1 integron. The MDR region was located at one end of a 42-kb GI, and IS6100Δ1 mediated a genetic rearrangement with the complex class 1 integron-like SGI1 and formed a composite transposon. Furthermore, the tetW gene was located outside the four GIs consistent with tetracycline and doxycycline resistance. The ermD gene positioned in the front end of the 42-kb GI played an important role in mediating acquired erythromycin and clindamycin resistance. CONCLUSIONS: Multiple resistance genes are located in a complex class 1 integron within a 42-kb T. pyogenes genomic island (TGI1), leading to TP1 multiple drug resistance. In comparison with SG1 families, TGI1 possesses versatile gene distribution and specific gene context for it upstream and downstream, and it represents a new lineage of genomic resistance islands.

Antibacterial mode of fibrauretine and synergistic effect with kanamycin against multi-drug resistant Escherichia coli.

OBJECTIVE: The present study evaluated the antibacterial activity and mode of action of fibrauretine on Escherichia coli (E. coli) and Staphylococcus aureus, and synergistic effect with kanamycin against multi-drug resistant E. coli. RESULTS: The fibrauretine exhibited inhibitory effect on the growth of the tested bacteria with minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 2.5-5 and 5-10 mg/ml, respectively. Morphological changes of cell microstructure were observed after adding fibrauretine at MIC. The mode of action was further confirmed by measuring release of 260-nm absorbing materials and extracellular potassium ions. Checkerboard dilution test suggested that fibrauretine exhibited synergistic activity when combined with kanamycin (FICI ranging from 0.5625 to 0.625). CONCLUSIONS: Our results indicated that fibrauretine exerted synergistic effect with kanamycin and its antibacterial mode of action mainly attributed to disruption of cell membrane integrity.

Transcriptome Analysis Reveals AI-2 Relevant Genes of Multi-Drug Resistant Klebsiella pneumoniae in Response to Eugenol at Sub-MIC.

Eugenol, the major active essential oil component of clove, was reported to possess QS (quorum sensing) inhibitory activity. A previous study found that eugenol could bind to quorum sensing receptors of Pseudomonas aeruginosa and down-regulate the expression of Streptococcus mutans virulence genes at sub-MIC (minimum inhibitory concentration) without affecting the bacterial growth. However, the alterations of QS signal molecules at transcription levels was not well understood. To better understand interactions of Klebsiella pneumoniae in response to eugenol and explore molecular regulations, transcriptome sequencing was performed. A total of 5779 differentially expressed genes (DEGs) enriched in a variety of biological processes and pathways were identified. The transcriptional data was validated by qPCR and the results showed that the expression profiles of 4 major genes involved in autoinducers-2 (AI-2) synthesis, including luxS, pfs, and lsrK were consistent with transcriptome analysis except for lsrR, a transcriptional repressor gene of lsr operon, which may repress the expression of following genes responsible for AI-2 signal transmission in vivo. In vitro AI-2 synthesis assay also revealed that eugenol could inhibit AI-2 generation. The results of our study offer insights into the mechanisms of QS inhibitory activity and K. pneumoniae AI-2 alterations after eugenol treatment.

Antimicrobial resistance and presence of virulence factor genes in Trueperella pyogenes isolated from pig lungs with pneumonia.

Trueperella pyogenes (T. pyogenes) is a worldwide known pathogen of domestic ruminants and pigs causing a wide variety of infections. The objective of this study was to report the presence of major virulence genes in T. pyogenes isolated from pigs with respiratory clinical signs and determine their resistance to antibiotics at the same time. A total of 27 T. pyogenes strains were obtained from Jilin Province, and the nanH, nanP, cbpA, fimC, and fimE virulence genes were detected in 7 (25.9%), 14 (51.9%), 18 (66.7%), 8 (29.6%), and 16 (59.3%) isolates, respectively. All isolates were observed to harbor plo and fimA genes. However, 27 T. pyogenes strains tested negative for fimG gene. Antibiotic susceptibility tests revealed that the isolated strains had extensive drug resistance, all isolates were sensitive to fluoroquinolones and penicillins antibiotics, and high levels of resistance were found to gentamicin (77.8%), amikacin (74.1%), erythromycin (85.2%), and azithromycin (85.2%). These results highlights the need for prudent use of specific antimicrobial agents in veterinary clinical treatment.