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Ralstonia metallidurans, formerly known as Alcaligenes eutrophus and thereafter as Ralstonia eutropha, is a β-Proteobacterium colonizing industrial sediments, soils or wastes with a high content of heavy metals. The type strain CH34 carries two large plasmids (pMOL28 and pMOL30) bearing a variety of genes for metal resistance. A chronological overview describes the progress made in the knowledge of the plasmid-borne metal resistance mechanisms, the genetics of R. metallidurans CH34 and its taxonomy, and the applications of this strain in the fields of environmental remediation and microbial ecology. Recently, the sequence draft of the genome of R. metallidurans has become available. This allowed a comparison of these preliminary data with the published genome data of the plant pathogen Ralstonia solanacearum, which harbors a megaplasmid (of 2.1 Mb) carrying some metal resistance genes that are similar to those found in R. metallidurans CH34. In addition, a first inventory of metal resistance genes and operons across these two organisms could be made. This inventory, which partly relied on the use of proteomic approaches, revealed the presence of numerous loci not only on the large plasmids pMOL28 and pMOL30 but also on the chromosome. It suggests that metal-resistant Ralstonia, through evolution, are particularly well adapted to the harsh environments typically created by extreme anthropogenic situations or biotopes.

Helicobacter pylori is a major pathogen associated with various gastric diseases. Despite decades of research, the treatment of H. pylori remains challenging. One of the primary mechanisms contributing to failures of therapies targeting this bacterium is genetic mutations in drug target sites, although the growing body of scientific data highlights that efflux pumps may also take part in this process. Efflux pumps are proteinaceous transporters actively expelling antimicrobial agents from the interior of the targeted cells and reducing the intracellular concentration of these compounds. Considering that efflux pumps contribute to both antimicrobial resistance and biofilm formation, an in-depth understanding of their properties may constitute a cornerstone in the development of novel therapeutics against H. pylori. In line with this, the aim of the current review is to describe the multitude of efflux pumps produced by H. pylori and present the data describing the involvement of these proteins in tolerance and/or resistance to various classes of antimicrobial substances.

Amidst the challenge posed by multiple drug-resistant (MDR) bacteria, this study investigates the efficacy of 2-Hydroxy-4-methoxybenzaldehyde (HMB) as an efflux pump inhibitor (EPI) against Proteus mirabilis . Efflux pump-mediated drug resistance stands as a major obstacle in combating MDR strains, prompting the exploration of innovative strategies. P. mirabilis , a Gram-negative bacterium renowned for urease production, forms crystalline biofilms in catheterized urinary tracts, leading to encrustation and blockage. The Bcr/cflA efflux system, which promotes P. mirabilis biofilm development, is targeted for intrinsic antibiotic resistance inhibition. HMB's antibiofilm potential against P. mirabilis crystalline biofilm stems from its efflux inhibition on the Bcr/cflA efflux system. An antibiotic susceptibility assay unveiled HMB’s ability to enhance antibiotic sensitivity in P. mirabilis . HMB demonstrated putative efflux inhibition against P. mirabilis , with a maximum efflux inhibitory concentration (MEIC) of 50 μg/ml determined through EtBr accumulation assays. Molecular modeling indicated favorable interactions between HMB and the Bcr/ClfA efflux system, suggesting its potential as an EPI. HMB treatment down-regulated efflux genes expression and enhanced antibiotic susceptibility, particularly against amikacin, chloramphenicol, kanamycin, and polymyxin-B, highlighting its therapeutic role in combating antibiotic resistance in P. mirabilis .

An ATP-binding cassette (ABC) transporter, called the PseEF efflux system, was identified at the left border of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. The PseEF efflux system was located within a 3.3-kb operon that encodes a periplasmic membrane fusion protein (PseE), and an ABC-type cytoplasmic membrane protein (PseF). The PseEF efflux system exhibited amino acid homology to a putative ABC efflux system (MacAB) of E. coli W3104 with identities of 47.2% (i.e., PseE to MacA) and 57.6% (i.e., PseF to MacB). A nonpolar mutation within the pseF gene was generated by nptII insertional mutagenesis. The resultant mutant strain showed significant reduction in secretion of syringomycin (74%) and syringopeptin (71%), as compared to parental strain B301D. Quantitative real-time RT-PCR was used to determine transcript levels of the syringomycin (syrB1) and syringopeptin (sypA) synthetase genes in strain B301D-HK7 (a pseF mutant). Expression of the sypA gene by mutant strain B301D-HK7 was approximately 6.9% as compared to that of parental strain B301D, while the syrB1 gene expression by mutant strain B301D-HK7 was nearly 14.6%. In addition, mutant strain B301D-HK7 was less virulent by approximately 67% than parental strain B301D in immature cherry fruits. Mutant strain B301D-HK7 was not reduced in resistance to any antibiotics used in this study as compared to parental strain B301D. Expression (transcript levels) of the pseF gene was induced approximately six times by strain B301D grown on syringomycin minimum medium (SRM) supplemented with the plant signal molecules arbutin and D-fructose (SRM∧AF), as compared to that of strain B301D grown on SRM (in the absence of plant signal molecules). In addition, during infection of bean plants by P. syringae pv. syringae strain B728a, expression of the pseF gene increased at 3 days after inoculation (dai). More than 180-fold induction was observed in transcript levels of the pseF gene by parental strain B728a as compared to strain B728a-SL7 (a salA mutant). Thus, the PseEF efflux system, an ABC-type efflux system, has an important role in secretion of syringomycin and syringopeptin, and is required for full virulence in P. syringae pv. syringae.

The hydrophobe/amphiphile efflux-1 (HAE-1) family, part of the Resistance-Nodulation-Division (RND) superfamily, plays a critical role in the development of multidrug resistance (MDR) in bacteria. Known for its broad substrate transport capacity, this family of efflux pumps can actively expel a wide range of molecules, including antibiotics, salts, and dyes, thereby reducing the intracellular concentration of toxic substances. These transporters, which form efflux systems, are primarily found in bacteria within the phylum Pseudomonadota (Proteobacteria), where they are strongly associated with increased resistance and enhanced virulence, thus contributing to bacterial survival in hostile environments. In addition, efflux systems are composed of two other protein components: Membrane Fusion Proteins (MFPs) and Outer Membrane Factors (OMFs). Notably, several bacterial species identified by the World Health Organization (WHO) as urgent priorities for new antibiotic development, such as Escherichia coli and Pseudomonas aeruginosa , have well-studied HAE-1 efflux systems, such as AcrAB-TolC and MexAB-OprM. These systems efficiently transport molecules from the periplasm to the extracellular space, facilitating bacterial persistence. In this review, we examined the current knowledge of HAE-1 efflux transporters and their roles in the physiology and survival of bacteria in the Pseudomonadota phylum.

Levofloxacin is considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of efflux pumps. In this study, the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical isolates was demonstrated using phenotypic (minimal inhibitory concentrations, MICs, of antibiotics determination ± efflux pump inhibitors, EPIs) and molecular (real-time polymerase-chain-reaction and sequencing) methods. Previously, the occurrence of genes encoding ten efflux pumps was shown in 94 studied isolates. Additionally, 44/94 isolates demonstrated reduction in susceptibility to levofloxacin. Only 5 of 13 isolates (with ≥4-fold reduction in levofloxacin MIC) in the presence of EPIs showed an increased susceptibility to levofloxacin and other antibiotics. The overexpression of smeD and smeV genes (in five and one isolate, respectively) of 5 tested efflux pump operons was demonstrated. Sequencing analysis revealed 20–35 nucleotide mutations in local regulatory genes such as smeT and smeRv. However, mutations leading to an amino acid change were shown only in smeT (Arg123Lys, Asp182Glu, Asp204Glu) for one isolate and in smeRv (Gly266Ser) for the other isolate. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance.

In Malaysia, an increase in non-carbapenemase-producing carbapenem-resistant (NC-CRKP) has been observed over the years. Previously, four NC-CRKP with increased susceptibility to ciprofloxacin in the presence of phenylalanine-arginine β-naphthylamide (PAβN) were identified. However, no contribution of the PAβN-inhibited efflux pump to carbapenem resistance was observed. All four NC-CRKP harboured non-carbapenemase β-lactamase, with two also exhibiting porin loss. In this study, we further investigated the genomic features and resistance mechanisms of these four isolates. All four NC-CRKP were subjected to whole-genome sequencing, followed by comparative genomic and phylogenetic analyses. Multi-locus sequence typing (MLST) analysis divided the four NC-CRKP into different sequence types: ST392, ST45, ST14, and ST5947. Neither major nor rare carbapenemase genes were detected. Given the presence of non-carbapenemase β-lactamase in all isolates, we further investigated the potential mechanisms of resistance by identifying related chromosomal mutations. Deletion mutation was detected in the cation efflux system protein CusF. Insertion mutation was identified in the nickel/cobalt efflux protein RcnA. Missense mutation of ompK36 porin was detected in two isolates, while the loss of ompK36 porin was observed in another two isolates. This study revealed that NC-CRKP may confer carbapenem resistance through a combination of non-carbapenemase β-lactamase and potential chromosomal mutations including missense mutation or loss of ompK36 porin and/or a frameshift missense mutation in efflux pump systems, such as cation efflux system protein CusF and nickel/cobalt efflux protein RcnA. Our findings highlighted the significance of implementing whole-genome sequencing into clinical practice to promote the surveillance of carbapenem resistance mechanisms among NC-CRKP.

This genus contains both phototrophs and nonphototrophic members. Here, we present a high-quality complete genome of the strain CHu59-6-5T, isolated from a freshwater sediment. The circular chromosome (4.39 Mbp) of the strain CHu59-6-5T has 64.4% G+C content and contains 4240 genes, of which a total of 3918 genes (92.4%) were functionally assigned to the COG (clusters of orthologous groups) database. Functional genes for denitrification (narGHJI, nirK and qnor) were identified on the genomes of the strain CHu59-6-5T, except for N2O reductase (nos) genes for the final step of denitrification. Genes (soxBXAZY) for encoding sulfur oxidation proteins were identified, and the FSD and soxF genes encoding the monomeric flavoproteins which have sulfide dehydrogenase activities were also detected. Lastly, genes for the assembly of two different RND (resistance-nodulation division) type efflux systems and one ABC (ATP-binding cassette) type efflux system were identified in the Rhodoferax sediminis CHu59-6-5T. Phylogenetic analysis based on 16S rRNA sequences and Average Nucleotide Identities (ANI) support the idea that the strain CHu59-6-5T has a close relationship to the genus Rhodoferax. A polyphasic study was done to establish the taxonomic status of the strain CHu59-6-5T. Based on these data, we proposed that the isolate be classified to the genus Rhodoferax as Rhodoferax sediminis sp. nov. with isolate CHu59-6-5T.

To better understand the function of transporter in heavy metal detoxification of bacteria, the transporters associated with heavy metal detoxification in S. rhizophila JC1 were analyzed, among which four members were verified by RT-qPCR. In addition, the removal rates of four single metal ions (Cr 6+ , Cu 2+ , Zn 2+ , Pb 2+ ) and polymetallic ions by strain JC1 were studied, respectively. We also researched the physiological response of strain JC1 to different metal stress via morphological observation, elemental composition, functional group and membrane permeability analysis. The results showed that in the single metal ion solution, removal capacities of Cu 2+ (120 mg/L) and Cr 6+ (80 mg/L) of S. rhizophila JC1 reached to 79.9% and 89.3%, respectively, while in polymetallic ions solution, the removal capacity of each metal ion all decreased, and in detail, the adsorption capacity was determined Cr 6 +>Cu 2+ >Zn 2+ >Pb 2+ under the same condition. The physiological response analyses results showed that extracellular adsorption phenomena occurred, and the change of membrane permeability hindered the uptake of metal ions by bacteria. The analysis of transporters in strain JC1 genome illustrated that a total of 323 transporters were predicted. Among them, two, six and five proteins of the cation diffusion facilitator, resistance–nodulation–division efflux and P-type ATPase families were, respectively, predicted. The expression of corresponding genes showed that the synergistic action of correlative transporters played important roles in the process of adsorption. The comparative genomics analysis revealed that S. rhizophila JC1 has long-distance evolutionary relationships with other strains, but the efflux system of S. rhizophila JC1 contained the same types of metal transporters as other metal-resistant bacteria. Graphical abstract

Thirty Acinetobacter baumannii from animal origins were obtained from 210 animal carcasses submitted for postmortem examination, including dogs (n=112), cats (n=45), rabbits (n=17), birds (n=15), pigs (n=7) and exotic animals (n=14). All the isolates were evaluated for antimicrobial susceptibility with the absence and presence of efflux inhibitors, i.e., reserpine and carbonyl cyanide ra-chlorophenylhydrazone (CCCP) and expression profile of important six multidrug efflux systems (i.e., AdeABC, AdeDE, AdeFGH, AdelJK, AbeM and AmvA). Fifteen isolates expressing different Ade multidrug efflux pumps were selected to determine transcription levels and regulatory mutations. A continuous in vitro exposure experiment with benzalkonium chloride, chlorhexidine, and triclosan was performed in the isolates susceptible to most antibiotics and biocides tested (n=12). The results showed that 70% of the isolates (n=21) were multidrug-resistant, and all expressed AdelJK The inhibitory effect of reserpine and CCCP suggested that active efflux pumps using a different type of energy (i.e., ATP and proton-motif force) contribute to the reduced susceptibility to antimicrobials in the isolates. Exposure to triclosan resulted in a spontaneous-mutant derivative, ABJ302-1, exhibiting cross-resistance to 10 of 15 antibiotics tested and resulted in downregulated expression of AdeG and AdeJ in almost all the isolates, which may be affected by the stress response of triclosan.