Objectives To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3′)-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. Methods Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. Results APH(3′)-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3′)-IIb. Expression of aph(3′)-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3′)-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3′)-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3′)-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M−1 s−1. Genetic environment and comparative genomic analyses suggested that aph(3′)-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. Conclusions APH(3′)-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3′)-IId represents the fourth characterized example of an APH(3′)-II enzyme.

AmpG is a transmembrane protein with permease activity that transports meuropeptide from the periplasm to the cytoplasm, which is essential for the induction of the ampC encoding β-lactamase. To obtain new insights into the relationship between AmpG structure and function, comparative genomics analysis, secondary and tertiary structure modeling, site-directed mutational analyses and genetic complementation experiments were performed in this study. AmpGs from different genera of bacteria (Escherichia coli, Vibrio cholerae and Acinetobacter baumannii) could complement AmpG function in Pseudomonas aeruginosa. The minimal inhibitory concentration (MIC) to ampicillin is 512 μg/ml for wild type strain PAO1, while it is 32 μg/ml for an ampG deletion mutant strain (PAO1ΔampG) with a corresponding decrease in the activity of the ampC-encoded β-lactamase. Site-directed mutagenesis of conserved AmpG residues (G29, A129, Q131 and A197) resulted in a loss of function, resulting in a loss of resistance to ampicillin in PAO1ΔampG. The G29A, G29V, A129T, A129V, A129D, A197S and A197D mutants had lower resistance to ampicillin and significantly decreased activity of the AmpC β-lactamase. The G29A, G29V, A129V, A197S and A197D mutants had decreased ampG mRNA transcript levels. The A129T and A129D mutants had normal ampG mRNA transcript levels, but the function of the protein was drastically reduced. Our experimental results demonstrate that the conserved amino acids played essential roles in maintaining the function of AmpG. Combined with the AmpG structural information, these critical amino acids can be targeted for the development of new anti-bacterial agents.

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.

In analyzing the drug resistance phenotype and mechanism of resistance to macrolide antibiotics of clinical Pseudomonas aeruginosa isolates, the agar dilution method was used to determine the minimum inhibitory concentrations (MICs), and PCR (polymerase chain reaction) was applied to screen for macrolide antibiotics resistance genes. The macrolide antibiotics resistance genes were cloned, and their functions were identified. Of the 13 antibiotics tested, P. aeruginosa strains showed high resistance rates (ranging from 69.5–82.1%), and MIC levels (MIC90 > 256 μg/ml) to macrolide antibiotics. Of the 131 known macrolide resistance genes, only two genes, mphE and msrE, were identified in 262 clinical P. aeruginosa isolates. Four strains (1.53%, 4/262) carried both the msrE and mphE genes, and an additional three strains (1.15%, 3/262) harbored the mphE gene alone. The cloned msrE and mphE genes conferred higher resistance levels to three second-generation macrolides compared to two first-generation ones. Analysis of MsrE and MphE protein polymorphisms revealed that they are highly conserved, with only 1–3 amino acids differences between the proteins of the same type. It can be concluded that even though the strains showed high resistance levels to macrolides, known macrolide resistance genes are seldom present in clinical P. aeruginosa strains, demonstrating that a mechanism other than this warranted by the mphE and msrE genes may play a more critical role in the bacteria’s resistance to macrolides.

Rhodococcus equi, a member of the Rhodococcus genus, is a gram-positive pathogenic bacterium. Rhodococcus possesses an open pan-genome that constitutes the basis of its high genomic diversity and allows for adaptation to specific niche conditions and the changing host environments. Our analysis further showed that the core genome of R. equi contributes to the pathogenicity and niche adaptation of R. equi. Comparative genomic analysis revealed that the genomes of R. equi shared identical collinearity relationship, and heterogeneity was mainly acquired by means of genomic islands and prophages. Moreover, genomic islands in R. equi were always involved in virulence, resistance, or niche adaptation and possibly working with prophages to cause the majority of genome expansion. These findings provide an insight into the genomic diversity, evolution, and structural variation of R. equi and a valuable resource for functional genomic studies.

Florfenicol is widely used to control respiratory diseases and intestinal infections in food animals. However, there are increasing reports about florfenicol resistance of various clinical pathogens. floR is a key resistance gene that mediates resistance to florfenicol and could spread among different bacteria. Here, we investigated the prevalence of floR in 430 Pseudomonas aeruginosa isolates from human clinical samples and identified three types of floR genes (designated floR, floR-T1 and floR-T2) in these isolates, with floR-T1 the most prevalent (5.3%, 23/430). FloR-T2 was a novel floR variant identified in this study, and exhibited less identity with other FloR proteins than FloRv. Moreover, floR-T1 and floR-T2 identified in P. aeruginosa strain TL1285 were functionally active and located on multi-drug resistance region of a novel incomplete Tn4371-like integrative and conjugative elements (ICE) in the chromosome. The expression of the two floR variants could be induced by florfenicol or chloramphenicol. These results indicated that the two floR variants played an essential role in the host’s resistance to amphenicol and the spreading of these floR variants might be related with the Tn4371 family ICE.

The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance.

Background: Florfenicol is widely used to control respiratory diseases and intestinal infections in food animals. However, dramatic and serious florfenicol resistance in various clinical strains was reported. As a key resistance gene for florfenicol, floR has often been associated with mobile genetic elements. To analyze the potential transmission of floR, we investigated floR gene in Pseudomonas aeruginosa isolates from human clinical samples and characterize two floR variants, floR-T1 and floR-T2.Methods: Pooled genomic DNA sequencing and PCR were used to analyze the floR gene in P. aeruginosa. The floR variants were cloned, and the minimum inhibitory concentrations (MICs) were determined. Quantitative RT-PCR was used to compare the gene expression of the two floR variants in TL1285 with or without florfenicol/chloramphenicol. Whole-genome sequencing was used to identify the genetic context of the floR variants in TL1285.Results: Three types of floR variants (designated floR, floR-T1 and floR-T2) were identified in the clinical P. aeruginosa isolates, and floR-T1 was the most prevalent variant. The positive rates of the floR-T1 gene in the P. aeruginosa strains collected in 2008-2009 and 2015-2017 were 3.00% (6/200) and 7.39% (17/230), respectively. FloR-T2 exhibited less identity with other FloR proteins than FloRv. The two floR variants, floR-T1 and floR-T2, in P. aeruginosa TL1285 were functionally active and located on a novel incomplete Tn4371 family integrative and conjugative element (ICE). The expression of the two floR variants could be induced by florfenicol and chloramphenicol at different levels.Conclusions: Two floR variants, floR-T1 and floR-T2, were identified in a clinical P. aeruginosa strain. Tn4371 family ICEs contribute to the dissemination of resistance genes among P. aeruginosa. Antimicrobial resistance could be transmitted from animal bacteria to human pathogens, posing a severe threat to public health.