MacAB-TolC Contributes to the Development of Acinetobacter baumannii Biofilm at the Solid–Liquid Interface

Acinetobacter baumannii has emerged as one of the most problematic bacterial pathogens responsible for hospital-acquired and community infections worldwide. Besides its high capacity to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on inert and living surfaces leading to biofilm development. This lifestyle confers additional protection against various treatments and allows it to persist for long periods in various hospital niches. Due to their remarkable antimicrobial tolerance, A. baumannii biofilms are difficult to control and ultimately eradicate. Further insights into the mechanism of biofilm development will help to overcome this challenge and to develop novel antibiofilm strategies. To unravel critical determinants of this sessile lifestyle, the proteomic profiles of two A. baumannii strains (ATTC17978 and SDF) grown in planktonic stationary phase or in mature solid–liquid (S-L) biofilm were compared using a semiquantitative proteomic study. Of interest, among the 69 common proteins determinants accumulated in the two strains at the S-L interface, we sorted out the MacAB-TolC system. This tripartite efflux pump played a role in A. baumannii biofilm formation as demonstrated by using ΔmacAB-tolC deletion mutant. Complementary approaches allowed us to get an overview of the impact of macAB-tolC deletion in A. baumannii physiology. Indeed, this efflux pump appeared to be involved in the envelope stress response occurring in mature biofilm. It contributes to maintain wild type (WT) membrane rigidity and provides tolerance to high osmolarity conditions. In addition, this system is probably involved in the maintenance of iron and sulfur homeostasis. MacAB-TolC might help this pathogen face and adapt to deleterious conditions occurring in mature biofilms. Increasing our knowledge of A. baumannii biofilm formation will undoubtedly help us develop new therapeutic strategies to tackle this emerging threat to human health.

Colistin Resistance Mechanisms in Human Salmonella enterica Strains Isolated by the National Surveillance Enter-Net Italia (2016–2018)

Background: A collection of human-epidemiologically unrelated S. enterica strains collected over a 3-year period (2016 to 2018) in Italy by the national surveillance Enter-Net Italia was analysed. Methods: Antimicrobial susceptibility tests, including the determination of minimal inhibitory concentrations (MICs) for colistin, were performed. Colistin resistant strains were analysed by PCR to detect mobile colistin resistance (mcr) genes. In mcr-negative S. enterica serovar Enteritidis strains, chromosomal mutations potentially involved in colistin resistance were identified by a genomic approach. Results: The prevalence of colistin-resistant S. enterica strains was 7.7%, the majority (87.5%) were S. Enteritidis. mcr genes were identified only in one strain, a S. Typhimurium monophasic variant, positive for both mcr-1.1 and mcr-5.1 genes in an IncHI2 ST4 plasmid. Several chromosomal mutations were identified in the colistin-resistant mcr-negative S. Enteritidis strains in proteins involved in lipopolysaccharide and outer membrane synthesis and modification (RfbN, LolB, ZraR) and in a component of a multidrug efflux pump (MdsC). These mutated proteins were defined as possible candidates for colistin resistance in mcr-negative S. Enteritidis of our collection. Conclusions: The colistin national surveillance in Salmonella spp. in humans, implemented with genomic-based surveillance, permitted to monitor colistin resistance, determining the prevalence of mcr determinants and the study of new candidate mechanisms for colistin resistance.

Phenotypic and Molecular Characteristics of the MDR Efflux Pump Gene-Carrying Stenotrophomonas maltophilia Strains Isolated in Warsaw, Poland

An increase of nosocomial infections caused by Stenotrophomonas maltophilia strains has recently been observed all over the world. The isolation of these bacteria from the blood is of particular concern. In this study we performed the phenotypic and genotypic characterization of 94 S. maltophilia isolates, including isolates from patients hospitalized in a tertiary Warsaw hospital (n = 79) and from outpatients (n = 15). All isolates were found to be susceptible to trimethoprim-sulfamethoxazole and minocycline, while 44/94 isolates demonstrated a reduction in susceptibility to levofloxacin. A large genetic variation was observed among the isolates tested by pulsed-field gel electrophoresis. A clonal relationship with 100% similarity was observed between isolates within two sub-pulsotypes: the first included nine bloodstream isolates and the second involved six. Multilocus sequence typing showed two new sequence types (ST498 and ST499) deposited in public databases for molecular typing. Moreover, the presence of genes encoding ten different efflux pumps from the resistance-nodulation-division family and the ATP-binding cassette family was shown in the majority of the 94 isolates. The obtained knowledge about the prevalence of efflux pump genes in clinical S. maltophilia strains makes it possible to predict the scale of the risk of resistance emergence in strains as a result of gene overexpression.

Luteolin increases susceptibility to macrolides by inhibiting MsrA efflux pump in Trueperella pyogenes

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen associated with a variety of diseases in many domestic animals. Therapeutic treatment options for T. pyogenes infections are becoming limited due to antimicrobial resistance, in which efflux pumps play an important role. This study aims to evaluate the inhibitory activity of luteolin, a natural flavonoid, on the MsrA efflux pump and investigate its mechanism. The results of antimicrobial susceptibility testing indicated that the susceptibility of msrA-positive T. pyogenes isolates to six macrolides increased after luteolin treatment, while the susceptibility of msrA-negative isolates showed no change after luteolin treatment. It is suspected that luteolin may increase the susceptibility of T. pyogenes isolates by inhibiting MsrA activity. After 1/2 MIC luteolin treatment for 36 h, the transcription level of the msrA gene and the expression level of the MsrA protein decreased by 55.0–97.7% and 36.5–71.5%, respectively. The results of an affinity test showed that the equilibrium dissociation constant (KD) of luteolin and MsrA was 6.462 × 10–5 M, and hydrogen bonding was predominant in the interaction of luteolin and MsrA. Luteolin may inhibit the ATPase activity of the MsrA protein, resulting in its lack of an energy source. The current study illustrates the effect of luteolin on MsrA in T. pyogenes isolates and provides insight into the development of luteolin as an innovative agent in combating infections caused by antimicrobial-resistant bacteria.

Adaptation to a Commercial Quaternary Ammonium Compound Sanitizer Leads to Cross-Resistance to Select Antibiotics in Listeria monocytogenes Isolated From Fresh Produce Environments

The effective elimination of Listeria monocytogenes through cleaning and sanitation is of great importance to the food processing industry. Specifically in fresh produce operations, the lack of a kill step requires effective cleaning and sanitation to mitigate the risk of cross-contamination from the environment. As facilities rely on sanitizers to control L. monocytogenes, reports of the development of tolerance to sanitizers and other antimicrobials through cross-resistance is of particular concern. We investigated the potential for six L. monocytogenes isolates from fresh produce handling and processing facilities and packinghouses to develop cross-resistance between a commercial sanitizer and antibiotics. Experimental adaptation of isolates belonging to hypervirulent clonal complexes (CC2, CC4, and CC6) to a commercial quaternary ammonium compound sanitizer (cQAC) resulted in elevated minimum inhibitory concentrations (2–3 ppm) and minimum bactericidal concentrations (3–4 ppm). Susceptibility to cQAC was restored for all adapted (qAD) isolates in the presence of reserpine, a known efflux pump inhibitor. Reduced sensitivity to 7/17 tested antibiotics (chloramphenicol, ciprofloxacin, clindamycin, kanamycin, novobiocin, penicillin, and streptomycin) was observed in all tested isolates. qAD isolates remained susceptible to antibiotics commonly used in the treatment of listeriosis (i.e., ampicillin and gentamicin). The whole genome sequencing of qAD strains, followed by comparative genomic analysis, revealed several mutations in fepR, the regulator for FepA fluoroquinolone efflux pump. The results suggest that mutations in fepR play a role in the reduction in antibiotic susceptibility following low level adaptation to cQAC. Further investigation into the cross-resistance mechanisms and pressures leading to the development of this phenomenon among L. monocytogenes isolates recovered from different sources is needed to better understand the likelihood of cross-resistance development in food chain isolates and the implications for the food industry.

Nanocluster Mediated Photothermia Improves Eradication Efficiency and Antibiotic Sensitivity of Helicobacter Pylori

Background: Helicobacter pylori (H. pylori) eradication plays a crucial role in gastric cancer prevention, but the antimicrobial resistance of H. pylori is obstructing this elimination process. In this study, we developed nanoclusters (NCs) from Zn0.3Fe2.7O4 nanoparticles using a poly(ethylene glycol)-b-poly(ε-caprolactone)-based nanocarrier as an innovative antibiotic-independent H. pylori management.Results: The nanocluster showed minimal toxicity and maximal biocompatibility. With a low concentration (50 µg/mL) of NCs under a short time period (～2 min) of near-infrared (808nm) irradiation, we kept the culture medium temperature to 41 °C for 20 minutes with continuous irradiation. The heated NCs exhibited efficient photothermal effects and resulted in an excellent inhibition of H. pylori growth, adhesion ability and cell vacuolization ability in in vitro investigation. Transmission electron microscopy showed a dramatic morphologic change after NCs photothermia on H. pylori, including cell wall and membrane rupture, as well as ribosome damage. Besides, levofloxacin and clarithromycin resistance were improved after photothermal treatment in H. pylori NCTC 11637 and/or clinical strains, however metronidazole resistance was unchanged. We also discovered a significant decrease in the biofilm formation of H. pylori under the NCs-based photothermal application, while efflux pump function was unchanged.Conclusions: Based on this novel NCs-based photothermal approach, we were able to demonstrate in vitro a significant inhibition of both H. pylori growth and molecular toxicity, and its improvement in antibiotic resistance alone with the eradication of H. pylori biofilms previously believed to be resistant to conventional antibiotics.

Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

A Novel Tigecycline Adjuvant ML-7 Reverses the Susceptibility of Tigecycline-Resistant Klebsiella pneumoniae

The increasing incidence of tigecycline resistance undoubtedly constitutes a serious threat to global public health. The combination therapies had become the indispensable strategy against this threat. Herein, 11 clinical tigecycline-resistant Klebsiella pneumoniae which mainly has mutations in ramR, acrR, or macB were collected for tigecycline adjuvant screening. Interestingly, ML-7 hydrochloride (ML-7) dramatically potentiated tigecycline activity. We further picked up five analogs of ML-7 and evaluated their synergistic activities with tigecycline by using checkerboard assay. The results revealed that ML-7 showed certain synergy with tigecycline, while other analogs exerted attenuated synergistic effects among tigecycline-resistant isolates. Thus, ML-7 was selected for further investigation. The results from growth curves showed that ML-7 combined with tigecycline could completely inhibit the growth of bacteria, and the time-kill analysis revealed that the combination exhibited synergistic bactericidal activities for tigecycline-resistant isolates during 24 h. The ethidium bromide (EtBr) efflux assay demonstrated that ML-7 could inhibit the functions of efflux pump. Besides, ML-7 disrupted the proton motive force (PMF) via increasing ΔpH, which in turn lead to the inhibition of the functions of efflux pump, reduction of intracellular ATP levels, as well as accumulation of ROS. All of which promoted the death of bacteria. And further transcriptomic analysis revealed that genes related to the mechanism of ML-7 mainly enriched in ABC transporters. Taken together, these results revealed the potential of ML-7 as a novel tigecycline adjuvant to circumvent tigecycline-resistant Klebsiella pneumoniae.