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Polymyxins (polymyxin B and colistin) are last-line antibiotics against multidrug-resistant Gram-negative pathogens. Polymyxin resistance is increasing worldwide, with resistance most commonly regulated by two-component systems such as PmrAB and PhoPQ. This review discusses the regulatory mechanisms of PhoPQ and PmrAB in mediating polymyxin resistance, from receiving an external stimulus through to activation of genes responsible for lipid A modifications. By analyzing the reported nonsynonymous substitutions in each two-component system, we identified the domains that are critical for polymyxin resistance. Notably, for PmrB 71% of resistance-conferring nonsynonymous mutations occurred in the HAMP (present in histidine kinases, adenylate cyclases, methyl accepting proteins and phosphatase) linker and DHp (dimerization and histidine phosphotransfer) domains. These results enhance our understanding of the regulatory mechanisms underpinning polymyxin resistance and may assist with the development of new strategies to minimize resistance emergence.

Infections caused by multi-drug resistant (MDR) bacterial pathogens are a leading cause of mortality and morbidity across the world. Indiscriminate use of broad-spectrum antibiotics has seriously affected this situation. With the diminishing discovery of novel antibiotics, new treatment methods are urgently required to combat MDR pathogens. Polymyxins, the cationic lipopeptide antibiotics, discovered more than half a century ago, are considered to be the last-line of antibiotics available at the moment. This antibiotic shows a great bactericidal effect against Gram-negative bacteria. Polymyxins primarily target the bacterial membrane and disrupt them, causing lethality. Because of their membrane interacting mode of action, polymyxins cause nephrotoxicity and neurotoxicity in humans, limiting their usability. However, recent modifications in their chemical structure have been able to reduce the toxic effects. The development of better dosing regimens has also helped in getting better clinical outcomes in the infections caused by MDR pathogens. Since the mid-1990s the use of polymyxins has increased manifold in clinical settings, resulting in the emergence of polymyxin-resistant strains. The risk posed by the polymyxin-resistant nosocomial pathogens such as the Enterobacteriaceae group, Pseudomonas aeruginosa, and Acinetobacter baumannii, etc. is very serious considering these pathogens are resistant to almost all available antibacterial drugs. In this review article, the mode of action of the polymyxins and the genetic regulatory mechanism responsible for the emergence of resistance are discussed. Specifically, this review aims to update our current understanding in the field and suggest possible solutions that can be pursued for future antibiotic development. As polymyxins primarily target the bacterial membranes, resistance to polymyxins arises primarily by the modification of the lipopolysaccharides (LPS) in the outer membrane (OM). The LPS modification pathways are largely regulated by the bacterial two-component signal transduction (TCS) systems. Therefore, targeting or modulating the TCS signalling mechanisms can be pursued as an alternative to treat the infections caused by polymyxin-resistant MDR pathogens. In this review article, this aspect is also highlighted.

Overcoming colistin-resistant Acinetobacter baumannii (CoR-AB) has become a major concern due to the lack of effective antibiotics. This study aimed to explore the prevalence of CoR-AB clinical isolates in Thailand, their mechanisms of resistance, and test the efficacy of colistin plus sulbactam against CoR-AB isolates. The colistin resistance rate among carbapenem-resistant A. baumannii was 15.14%. The mcr gene or its variants were not detected in CoR-AB isolates by PCR screening. The lipid A mass spectra of CoR-AB isolates showed the additional [M–H] − ion peak at m / z  = 2034 that correlated to the phosphoethanolamine (pEtN) addition to lipid A (N = 27/30). The important amino acid substitutions were found at position S14P, A138T, A227V in PmrB that are associated with overexpression of the pEtN transferase (PmrC) and contributed the pEtN addition. The lipopolysacccharide production genes ( lpxACD ) were not related to lipid A mass spectra. A colistin plus sulbactam combination exhibited the synergy rate at 86.7% against CoR-AB isolates compare to sulbactam (85.89% resistance) or colistin (15.14% resistance) alone. The excellent synergistic activity of colistin plus sulbactam combination has the potential for the treatment of CoR-AB infections.

Introduction: Colistin and β-lactams are widely investigated because of their effectiveness in the treatment of human diseases. This study investigated the phenotypic and genotypic profiles of colistin- and β-lactam-resistant Escherichia coli (n = 235) obtained from bovines, swine, and workers from a mixed slaughterhouse in Brazil. Methodology: The disk diffusion method was used to test the resistance against β-lactams (amoxicillin, ampicillin, cefaclor, cefazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, imipenem, meropenem, and aztreonam). In order to test colistin resistance, the isolates were subjected to the minimum inhibitory concentration (MIC) technique using the broth microdilution method (BMD; 0.5 to 16 μg/mL) and polymerase chain reaction (PCR) assays targeting colistin- (mcr-1 to mcr-5) and β-lactam- (blaTEM, blaCTX-M, blaSHV, ampC) genes. The pmrAB mutation was further investigated. Results: The isolates presented resistance, especially to ampicillin (cattle: 14/106, swine: 62/100, humans: 10/29) and amoxicillin (cattle: 7/106, swine: 61/100, humans: 8/29). One swine isolate was characterized as extended spectrum β-lactamase (ESBL) producer. The isolates obtained from swine presented higher frequencies of colistin resistance (13/100) when compared to isolates from bovines (5/106) and humans (0/29). Molecular assays concluded that the isolates presented blaTEM (swine: 67/100, humans: 7/29), ampC (swine: 1/100), and blaCTXM (swine: 1/100). The pmrAB complex presented mutations (T31S, P42A, I128N, G144S, H2R, N358Y, D283G, K15I). Conclusions: This study highlights the presence of antimicrobial resistance and presents a method to verify these factors in the animal production chain.

In Brazil, the production of KPC-type carbapenemases in Enterobacteriales is endemic, leading to widespread use of polymyxins. In the present study, 502 Klebsiella pneumoniae isolates were evaluated for resistance to polymyxins, their genetic determinants and clonality, in addition to the presence of carbapenem resistance genes and evaluation of antimicrobial resistance. Resistance to colistin (polymyxin E) was evaluated through initial selection on EMB agar containing 4% colistin sulfate, followed by Minimal Inhibitory Concentration (MIC) determination by broth microdilution. The susceptibility to 17 antimicrobials was assessed by disk diffusion. The presence of bla KPC , bla NDM and bla OXA-48-like carbapenemases was investigated by phenotypic methods and conventional PCR. Molecular typing was performed by PFGE and MLST. Allelic variants of the mcr gene were screened by PCR and chromosomal mutations in the pmrA , pmrB , phoP , phoQ and mgrB genes were investigated by sequencing. Our work showed a colistin resistance frequency of 29.5% (n = 148/502) in K. pneumoniae isolates. Colistin MICs from 4 to >128 µg/mL were identified (MIC 50 = 64 µg/mL; MIC 90 >128 µg/mL). All isolates were considered MDR, with the lowest resistance rates observed for amikacin (34.4%), and 19.6% of the isolates were resistant to all tested antimicrobials. The bla KPC gene was identified in 77% of the isolates, in consonance with the high rate of resistance to polymyxins related to its use as a therapeutic alternative. Through XbaI -PFGE, 51 pulsotypes were identified. MLST showed 21 STs, with ST437, ST258 and ST11 (CC11) being the most prevalent, and two new STs were determined: ST4868 and ST4869. The mcr-1 gene was identified in 3  K. pneumoniae isolates. Missense mutations in chromosomal genes were identified, as well as insertion sequences in mgrB . Furthermore, the identification of chromosomal mutations in K. pneumoniae isolates belonging from CC11 ensures its success as a high-risk epidemic clone in Brazil and worldwide.

Heteroresistance may pose a threat to the prognosis of patients following colistin treatment. We investigated colistin heteroresistance in Klebsiella pneumoniae isolates from South Korea. Among 252 K. pneumoniae blood isolates, 231 were susceptible to polymyxins. Heteroresistance to colistin was determined using population analysis profiles, disk diffusion assays, and E-test strip tests for the susceptible isolates. As a result, we identified three colistin-heteroresistant K. pneumoniae isolates belonging to separate clones (ST11, ST461, and ST3217) by multilocus sequence typing analysis. Two colistin-resistant subpopulations were selected from each heteroresistant isolate in either disk diffusion testing or E-testing. Two resistant subpopulations from the same isolate exhibited different amino acid substitutions in the two-component regulatory systems PmrAB and PhoPQ. An in vitro time–kill assay showed that meropenem combined with colistin had a 1× minimum inhibitory concentration bactericidal effect against a multidrug-resistant, colistin-heteroresistant isolate.

Plasmid-mediated colistin resistance (mcr) determinants are challenging the efficacy of polymyxins against Gram-negative pathogens. Among 10 mcr genes described so far, the major determinants mcr-1 and mcr-3 are found closely linked to hpap2 or dgkA genes, encoding a hypothetical phosphatidic acid phosphatase of type 2 (PAP2) and a diacylglycerol kinase, respectively, whose functions are still unknown. In this study, mcr-1, mcr-1–hpap2, mcr-3, and mcr-3–dgkA were expressed in Escherichia coli, and recombinant strains were analyzed to detect antimicrobial susceptibility and changes in the expression of genes involved in phospholipid metabolism. The mcr-1 or mcr-3 single genes were enough to drive growth on colistin selective media, although co-expression of linked genes conferred maximal antibiotic resistance. Expression of mcr determinants downregulated endogenous genes involved in lipopolysaccharide (LPS) modification or phospholipid recycling, although to different extents of repression: strong for arnB, ybjG, and pmrR; medium for eptA, lpxT, and dgkA; small for bacA and pgpB. Four of these genes (bacA, lpxT, pgpB, and ybjG) encode undecaprenyl pyrophosphate (UPP) phosphatases. In these conditions, cells presented resistance against bacitracin, an antibiotic that sequesters UPP from PAP2 enzymes. The hpap2 and dgkA genes might play a role in colistin resistance by compensating for phospholipid metabolism functions altered during LPS modification by colistin resistance determinants.

CprA is a short‐chain dehydrogenase/reductase (SDR) that contributes to resistance against colistin and antimicrobial peptides. The cprA gene is conserved across Pseudomonas aeruginosa clades and its expression is directly regulated by the two‐component system PmrAB. We have shown that cprA expression leads to the production of outer membrane vesicles (OMVs) that block autophagic flux and have a greater capacity to activate the non‐canonical inflammasome pathway. In a murine model of sepsis, a P. aeruginosa strain deleted for cprA was less virulent than the wild‐type (WT) strain. These results demonstrate the important role of CprA in the pathogenicity of P. aeruginosa. It is worth noting that CprA is also a functional ortholog of hemolysin F (HlyF), which is encoded by virulence plasmids of Escherichia coli. We have shown that other cryptic SDRs encoded by mammalian and plant pathogens, such as Yersinia pestis and Ralstonia solanacearum are functional orthologs of CprA and HlyF. These SDRs also induce the production of OMVs which block autophagic flux. This study uncovers a new family of virulence determinants in Gram‐negative bacteria, offering potential for innovative therapeutic interventions and deeper insights into bacterial pathogenesis.

Background The Salmonella enterica serovar Typhimurium PhoPQ two component system (TCS) is activated by low Mg 2+ levels, low pH and by antimicrobial peptides (AP). Under Mg 2+ limitation, the PhoPQ system induces pmrD expression, which post-translationally activates the PmrAB TCS. PhoPQ and PmrAB control many genes required for intracellular survival and pathogenesis. These include the polymyxin resistance ( pmr) operon, which is required for aminoarabinose modification of LPS and protecting the outer membrane from antimicrobial peptide disruption and killing. Extracellular DNA is a ubiquitous polymer in the matrix of biofilms and accumulates in some infection sites. Extracellular DNA chelates cations and thus activates the Pseudomonas aeruginosa PhoPQ/PmrAB systems, leading to expression of the orthologous arn ( pmr ) operon. Results Here we show that extracellular DNA induces expression of the S . Typhimurium pmr antimicrobial peptide resistance operon in a PhoPQ and PmrAB-dependent manner. Induction of the pmr genes by DNA was blocked when present with excess Mg 2+ . Exogenous DNA led to increased resistance of planktonic cultures to aminoglycosides, antimicrobial peptides (AP) and ciprofloxacin, but only AP resistance was PhoPQ/PmrAB-dependent. Extracellular DNA was shown to be a matrix component of S . Typhimurium biofilms cultivated in flow chambers and on glass surfaces. A pmrH-gfp fusion was highly expressed in flow chamber biofilms cultivated in medium with repressing levels of 10 mM Mg 2+ and co-localized with eDNA. Expression of pmrH-lux was monitored in plastic peg biofilms and shown to require PhoPQ and PmrAB. Biofilms had higher levels of pmrH expression compared to planktonic cultures. We propose that DNA accumulation in biofilms contributes to the increased pmrH-lux expression in biofilms. Conclusions The Salmonella PhoPQ/PmrAB systems and antimicrobial peptide resistance are activated by the cation chelating properties of extracellular DNA. DNA-induced AP resistance may allow immune evasion and increased survival of S. Typhimurium biofilms formed during extracellular growth stages of an infection or outside the host.

Colistin is considered a last-hope antibiotic against extensively drug-resistant isolates of Acinetobacter baumannii . Resistance to colistin has been rarely reported for A. baumannii . Genetic alterations in the PmrA-PmrB two-component system and lipid A biosynthesis genes may be associated with colistin resistance. We investigated molecular mechanisms of colistin resistance in three clinical colistin-resistant (ColR) and two colistin-susceptible (ColS) A. baumannii isolates. A ColR mutant was generated in vitro by repetitive drug exposure. The pmrA , pmrB , lpxA , lpxC , and lpxD genes were amplified and sequenced. To evaluate association between colistin resistance and upregulation of pmrCAB operon, transcriptional level of the pmrC gene encoding for lipid A phosphoethanolamine (PEtN) transferase was quantified by reverse transcription quantitative PCR (RT-qPCR) analysis. All clinical and in vitro- selected ColR isolates harbored at least one point mutation in the pmrB gene, including A142V, P233S, T235I, and A227V substitutions as well as duplication of H325. No alteration was found in the pmrA and other amino acid substitutions identified in the pmrB as well as lpx genes did not seem to be involved in colistin resistance as they were found in both ColS and ColR isolates. RT-qPCR analysis revealed a correlation between colistin resistance and pmrC overexpression. Specific alterations in the PmrB, linked to overproduction of PEtN transferase, triggered colistin resistance in the studied A. baumannii isolates.