The antihelminth drug rafoxanide reverses chromosomal-mediated colistin-resistance in Klebsiella pneumoniae.

The emergence and rapid spread of multi-drug-resistant (MDR) bacteria pose a serious threat to global healthcare. Although the synergistic effect of rafoxanide and colistin was reported, little is known regarding the potential mechanism of this synergy, particularly against chromosomal-mediated colistin-resistant Klebsiella pneumoniae. In the present study, we elucidated the synergistic effect of rafoxanide and colistin against chromosomal-mediated colistin-resistant Klebsiella pneumoniae isolates from human (KP-9) and swine (KP-1) infections. Treatment with 1 mg/L rafoxanide overtly reversed the MIC max to 512-fold. Time-kill assays indicated that rafoxanide acted synergistically with colistin against the growth of KP-1 and KP-9. Mechanistically, we unexpectedly found that the combination destroys the inner-membrane integrity, and ATP synthesis was also quenched, albeit, not via F1F0-ATPase; thereby also inhibiting the activity of efflux pumps. Excessive production of reactive oxygen species (ROS) was also an underlying factor contributing to the bacterial-killing effect of the combination. Transcriptomic analysis unraveled overt heterogeneous expression as treated with both administrations compared with monotherapy. Functional analysis of these differentially expressed genes (DEGs) targeted to the plasma membrane and ATP-binding corroborated phenotypic screening results. These novel findings highlight the synergistic mechanism of rafoxanide in combination with colistin which effectively eradicates chromosomal-mediated colistin-resistant Klebsiella pneumoniae. IMPORTANCE The antimicrobial resistance of Klebsiella pneumoniae caused by the abuse of colistin has increased the difficulty of clinical treatment. A promising combination (i.e., rafoxanide+ colistin) has successfully rescued the antibacterial effect of colistin. However, we still failed to know the potential effect of this combination on chromosome-mediated Klebsiella pneumoniae. Through a series of in vitro experiments, as well as transcriptomic profiling, we confirmed that the MIC of colistin was reduced by rafoxanide by destroying the inner-membrane integrity, quenching ATP synthesis, inhibiting the activity of the efflux pump, and increasing the production of reactive oxygen species. In turn, the expression of relevant colistin resistance genes was down-regulated. Collectively, our study revealed rafoxanide as a promising colistin adjuvant against chromosome-mediated Klebsiella pneumoniae.

ICEGpa1804, a novel integrative and conjugative element carrying eight resistance genes, identified in Glaesserella parasuis.

ICEGpa1804 was identified in the genome of a serovar 2, ST279 isolate EHP1804 carrying eight different resistance genes from 200 Glaesserella parasuis strains isolated from swine with lower respiratory tract infection in seven provinces of China. Susceptibility testing for EHP1804 was determined by broth microdilution, and its genetic profile was determined by whole-genome sequencing. The complete ICEGpa1804 was analysed by polymerase chain reaction, conjugation assay and bioinformatics tools. The conjugation assay was performed using EHP1804 as the donor and G. parasuis V43 (rifampicin-resistant) as the recipient. ICEGpa1804 has a size of 71,880 bp and contains 83 genes, including eight resistance genes [tet(B), blaRob-1, aphA1, strA, strB, aac(3)-IId, catA3 and sul2]. The conjugation assay showed that ICEGpa1804 could be transferred to G. parasuis V43 with frequencies of 4.3 × 10-7. To the best of the authors' knowledge, this is the first study to identify a novel integrative and conjugative element (ICE) carrying eight resistance genes and seven insertion sequence (IS) elements from a G. parasuis isolate. Tn6743, a novel transposon carrying six resistance genes, was identified. Moreover, ISGpa1, a novel IS256 family insertion element, is the first characterized example of a G. parasuis insertion element. Multiple mobile genetic elements involved in resistance genes were located in chromosomal ICEGpa1804, which showed that ICEs may serve as a vital platform for the accumulation of resistance genes.

Synergistic antibacterial activity of tetrandrine combined with colistin against MCR-mediated colistin-resistant Salmonella.

It has been recognized that colistin resistance is a growing problem that seriously impairs the clinical efficacy of colistin against bacterial infections. One strategy that has been proven to have therapeutic effect is to overcome the widespread emergence of antibiotic-resistant pathogens by combining existing antibiotics with promising non-antibiotic agents. In this work, antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of the combination. The molecular mechanisms of tetrandrine in combination with colistin were analyzed using fluorometric assay and Real-time PCR. To predict possible interactions between tetrandrine and MCR-1, molecular docking assay was taken. Finally, we evaluated the in vivo efficacy of tetrandrine in combination with colistin against MCR-positive Salmonella. Overall, the combination of tetrandrine and colistin showed significant synergistic activity. In-depth mechanistic analysis showed that the combination of tetrandrine with colistin enhances the membrane-damaging ability of colistin, undermines the functions of proton motive force (PMF) and efflux pumps in MCR-positive bacteria. The results of molecular docking and RT-PCR analyses showed that tetrandrine not only affects the expression of mcr-1 but is also an effective MCR-1 inhibitor. Compared with colistin monotherapy, the combination of tetrandrine with colistin significantly reduced the bacterial load in vivo. Our findings demonstrated that tetrandrine serves as a potential colistin adjuvant against MCR-positive Salmonella.

Molecular Characterization of a Novel Integrative Conjugative Element ICEHpa1 in Haemophilus parasuis.

ICEHpa1 was identified in the genome of a serovar 8 Haemophilus parasuis ST288 isolate YHP170504 from a case of swine lower respiratory tract infection. The aim of the present study was to characterize the integrative conjugative element ICEHpa1 and its multiresistance region. Susceptibility testing was determined by broth microdilution and the complete ICEHpa1 was identified by WGS analysis. The full sequence of ICEHpa1 was analyzed with bioinformatic tools. The presence of ICEHpa1, its circular intermediate and integration site were confirmed by PCR and sequence analysis. Transfer of ICEHpa1 was confirmed by conjugation. ICEHpa1 has a size of 68,922 bp with 37.42% GC content and harbors 81 genes responsible for replication and stabilization, transfer, integration, and accessory functions, as well as seven different resistance genes [bla Rob- 3, tet(B), aphA1, strA, strB, aac(6)'-Ie-aph(2')-Ia, and sul2]. Conjugation experiments showed that ICEHpa1 could be transferred to H. parasuis V43 with frequencies of 6.1 × 10-6. This is the first time a multidrug-resistance ICE has been reported in H. parasuis. Seven different resistance genes were located on a novel integrative conjugative element ICEHpa1, which suggests that the ICEHpa1 is capable of acquiring foreign genes and serving as a carrier for various resistance genes.