The Co-occurrence of NDM-5, MCR-1, and FosA3-Encoding Plasmids Contributed to the Generation of Extensively Drug-Resistant Klebsiella pneumoniae

The rise and global dissemination of extensively drug-resistant (XDR) bacteria are often related to plasmid-borne mobile antimicrobial resistance genes. Notably, isolates having multiple plasmids are often highly resistant to almost all the antibiotics available. In this study, we characterized an extensively drug-resistant Klebsiella pneumoniae 1678, which exhibited high-level resistance to almost all the available antibiotics. Through whole-genome sequencing (WGS), more than 20 resistant elements and 5 resistant plasmids were observed. Notably, the tigecycline resistance of K. pneumoniae 1678 was not related to the plasmid-borne tetA gene but associated with the overexpression of AcrAB and OqxAB efflux pumps, according to the susceptibility results of tetA-transformant and the related mRNA quantification of RND efflux pumps. Except for tigecycline resistance, three plasmids, mediating resistance to colistin, Fosfomycin, and ceftazidime–avibactam, respectively, were focused. Detailed comparative genetic analysis showed that all these plasmids belonged to dominated epidemic plasmids, and harbored completed conjugation systems. Results of conjugation assay indicated that these three plasmids not only could transfer to E. coli J53 with high conjugation frequencies, respectively, but also could co-transfer to E. coli J53 effectively, which was additionally confirmed by the S1-PFGE plasmids profile. Moreover, multiple insertion sequences (IS) and transposons (Tn) were also found surrounding the vital resistant genes, which may form several novel mechanisms involved in the resistant determinants’ mobilization. Overall, we characterized and reported the uncommon co-existence and co-transferring of FosA3-, NDM-5, and MCR-1-encoding plasmids in a K. pneumoniae isolate, which may increase the risk of spread of these resistant phenotypes and needing great concern.

In-vitro Anti-Tuberculosis, Anti-Efflux Pumps and Anti-Biofilm Effects of Crinum Asiaticum Bulbs

Drug resistant tuberculosis remains one of the major challenges associated with treatment and management of tuberculosis (TB) in the public health system and in clinical settings. In 2020, the World Health Organization (WHO) estimated that about 186,772 people died from drug-resistant tuberculosis out of the 500000 reported cases and this is alarming. There is a pressing need from every angle in drug discovery to develop novel compounds that could possess diverse mechanisms of action to tackle drug-resistant tuberculosis. The Crinum asiaticum bulbs extract are used ethno medicinally to treat upper respiratory tract infections and as well as wound healing agent. The aim of this work is to investigate the in-vitro anti-tuberculosis effect of Crinum asiaticum bulbs extracts and to assess the inhibitory properties against bacteria efflux pumps expression and biofilm formation. The results obtained showed that the Crinum asiaticum bulbs extracts (CAE) were effective in inhibiting Mycobacterium smegmatis (NCTC 8159) and Mycobacterium aurum (NCTC 10437) with minimum inhibitory concentration (MIC) of 125 μg/ml and 250 μg/ml against M. smegmatis and M. aurum respectively. The CAE markedly inhibited the efflux pumps of both M. smegmatis and M. aurum from expressing with the chloroform extract producing the greatest inhibition. The CAE (ethanol, methanol, chloroform and hexane) significantly (***ρ˂0.005) inhibited M. smegmatis’ and M. aurum’s biofilm formation in-vitro. Among the various extracts of Crinum asiaticum, the chloroform extract exhibited the greatest inhibition against M. smegmatis and M. aurum biofilm formation with significance levels of ***ρ˂0.005 and ***ρ˂0.005. In conclusion the CAE has anti-tuberculosis effect and could tackle drug resistant TB as exhibited through the anti-efflux and anti-biofilm forming properties of the extract against the selected Mycobacterium species.

Staphylococcus aureus Efflux Pumps and Tolerance to Ciprofloxacin and Chlorhexidine following Induction by Mupirocin

Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a YFP fluorescence reporter of NorA. Mupirocin exposure also produced reduced susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact norA and mepA , respectively.

Farnesol abrogates biofilm- and efflux pump- associated genes in drug resistant Candida auris strains

Background: Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and over expression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. Methods: In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. Rhodamine-6-G extracellular efflux and intracellular accumulation assays were used to study active drug efflux mechanism. We further studied the role of farnesol in modulating development of biofilms and drug efflux in C. auris. Down-regulation of biofilm- and efflux pump- associated genes by farnesol was also investigated. CLSM analysis for examining C. auris biofilm architecture among treated and untreated isolates. Results: Most of the isolates (twenty-two) were found resistant to FLZ whereas five were resistant to AmB. All the isolates were found capable of biofilm formation and ornamented with active drug efflux mechanism. The MIC for planktonic cells ranged from 62.5-125 mM and for sessile cells was 125 mM (0 h and 4 h biofilm) and 500 mM (12 h and 24 h biofilm), CLSM studies also confirmed these findings. Farnesol also blocked efflux pumps and down-regulated biofilm- and efflux pump- associated genes. Conclusion: Modulation of biofilm- and efflux pump- associated genes by farnesol represent a promising approach in combating C. auris infection.

A glyphosate-based herbicide cross-selects for antibiotic resistance genes in bacterioplankton communities

Agrochemicals often contaminate freshwater bodies, affecting microbial communities that underlie aquatic food webs. For example, Roundup, a widely-used glyphosate-based herbicide (GBH), has the potential to indirectly select for antibiotic resistant bacteria. Such cross-selection could occur, for example, if the same genes (e.g. encoding efflux pumps) confer resistance to both glyphosate and antibiotics. To test for cross-resistance in natural aquatic bacterial communities, we added Roundup to 1,000-L mesocosms filled with water from a pristine lake. Over 57 days, we tracked changes in bacterial communities with shotgun metagenomic sequencing, and annotated metagenome-assembled genomes (MAGs) for the presence of known antibiotic resistance genes (ARGs), plasmids, and resistance mutations in the enzyme targeted by glyphosate (enolpyruvyl-shikimate-3-phosphate synthase; EPSPS). We found that high doses of GBH significantly increased ARG frequency and selected for multidrug efflux pumps in particular. The relative abundance of MAGs after a high dose of GBH was predictable based on the number of ARGs encoded in their genomes (17% of variation explained) and, to a lesser extent, by resistance mutations in EPSPS (2% of variation explained). Together, these results indicate that GBHs have the potential to cross-select for antibiotic resistance in natural freshwater bacteria.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus

Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections.