Consensus on ß-Lactamase Nomenclature.

Assigning names to ß-lactamase variants has been inconsistent and has led to confusion in the published literature. The common availability of whole genome sequencing has resulted in an exponential growth in the number of new ß-lactamase genes. In November 2021 an international group of ß-lactamase experts met virtually to develop a consensus for the way naturally-occurring ß-lactamase genes should be named. This document formalizes the process for naming novel ß-lactamases, followed by their subsequent publication.

Escherichia coli GyrA Tower Domain Interacts with QnrB1 Loop B and Plays an Important Role in QnrB1 Protection from Quinolone Inhibition.

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo-cross-linking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286 to 298 on the tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286 to 289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.

Analyzing Possible Native Functions of the Quinolone Resistance Gene qnr in Vibrio vulnificus.

The worldwide distribution of qnr genes found on plasmids and their presence on the chromosomes of aquatic bacteria, such as Vibrio vulnificus, one of the suspected sources, suggests an origin before the development of synthetic quinolones. However, their native function remains unknown. Previous work indicated that expression of qnrVv in V. vulnificus was induced by cold shock. To investigate its role further, we constructed single in-frame deletion mutants in qnrVv and cspA (the gene for cold shock protein) and a double mutant in qnrVv and cspA in V. vulnificus ATCC 17562 to evaluate the response to different environmental conditions and stresses and to exposure to various DNA-damaging agents. We found that qnrVv is involved in resistance to ciprofloxacin, levofloxacin, and mitomycin C and in the cold shock response in V. vulnificus Moreover, ΔqnrVv and ΔcspA mutants showed slower growth when they were treated with bile salts at 37°C and then shifted to 15°C (cold shock) without bile salts in the medium, with the effect being stronger in the double mutant. This transition may mimic what happens when V. vulnificus is ingested into the gastrointestinal tract and released in its natural environment. Cold shock and bile salts induced the expression of cspA and DNA gyrase and topoisomerase IV genes. However, no induction was found in the ΔqnrVv mutant, suggesting that the qnrVv gene is involved in the response to DNA damage and nucleic acid secondary structure.

Plasmids and genes contributing to high-level quinolone resistance in Escherichia coli.

INTRODUCTION: The importance of plasmid-mediated quinolone resistance (PMQR) in Enterobacterales and its high incidence has been emphasised many times. However, a clinical strain carrying more than two PMQR genes is rare. This study sequenced plasmid transconjugants from a donor strain carrying four different PMQR genes to establish their genetic locations. METHODS: An Escherichia coli clinical strain displayed remarkable quinolone resistance with a ciprofloxacin MIC of 1024 mg/L carrying four PMQR genes: qnrA1, qepA1, aac(6')1b-cr and oqxAB. When outcrossed to Escherichia coli J53 AziR, different PMQR genes were transferred and the resulting strains 7C and 8C were chosen for further characterisation. Plasmids were extracted and sequenced by the Illumina and Oxford Nanopore Technologies platforms. S1 nuclease-PFGE was used to estimate the number and size of plasmids. RESULTS: The parental strain had three plasmid bands, as determined by PFGE. Transconjugant 8C obtained three plasmids: pMG336 (162 647 bp, F18:A-:B1:C4) carrying oqxAB; pMG335 carrying qepA1 (73 874 bp, F2:A-:B-); and pMG334 (59 724 bp, IncN (ST5)) with qnrA1 and aac(6')1b-cr. Interestingly, strain 7C obtained plasmid pMG333 (134 435 bp), which was not present in the parental strain but was an IncN-IncF cointegrate of plasmids pMG334 and pMG335 linked via insertion sequence IS26. CONCLUSION: This study described the complete nucleotide sequence of three plasmids carrying four PMQR genes in a single strain and the plasmid profile obtained after outcrosses. In addition, it described a cointegrate of two plasmids formed with flanking insertion sequences.

A Standard Numbering Scheme for Class C ß-Lactamases.

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) ß-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser64, Lys67, Tyr150, and Lys315), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.

Cold shock induces chromosomal qnr in Vibrio species and plasmid-mediated qnrS1 in Escherichia coli.

qnr genes are found in aquatic bacteria and preceded the development of synthetic quinolones. Their natural functions are unknown. We evaluated the expression of chromosomal qnr in Vibrio species in response to environmental stresses and DNA damaging agents. Sub-inhibitory concentrations of quinolones, but not other DNA damaging agents, induced the expression of chromosomal qnr by more than five times in Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio mytili Cold shock also induced the expression of qnr in V. parahaemolyticus, V. vulnificus, and V. mytili, as well as qnrS1 in Escherichia coli qnrS1 induction by cold shock was not altered in ΔihfA or ΔihfB mutants or in a strain over-expressing dnaA, that otherwise directly modulate qnrS1 induction by ciprofloxacin. In contrast, qnrS1 induction by cold shock was reduced in a ΔcspA mutant in the cold shock regulon compared to the wild type. In conclusion, cold shock as well as quinolones induce chromosomal qnr in Vibrio species, and the related qnrS1 in E. coli.

Multiple Copies of qnrA1 on an IncA/C2 Plasmid Explain Enhanced Quinolone Resistance in an Escherichia coli Mutant.

In a previous study, mutants with enhanced ciprofloxacin resistance (Cipr) were selected from Escherichia coli J53/pMG252 carrying qnrA1 Strain J53 Cipr 8-2 showed an increase in the copy number and transcription level of qnrA1 We sequenced the plasmids on Illumina and MinION platforms. Parental plasmid pMG252 and plasmid pMG252A from strain J53 Cipr 8-2 were almost identical, except for the region containing qnrA1 that in pMG252A contained 4 additional copies of the qnrA1-qacEΔ1-sul1-ISCR1 region.

Genes and Proteins Involved in qnrS1 Induction.

Expression of the quinolone resistance gene qnrS1 is increased by quinolones, but unlike induction of some other qnr genes, the bacterial SOS system is not involved and no lexA box is found upstream. Nonetheless, at least 205 bp of upstream sequence is required for induction to take place. An upstream sequence bound to beads trapped potential binding proteins from cell extracts that were identified by mass spectrometry as Dps, Fis, Ihf, Lrp, CysB, and YjhU. To further elucidate their role, a reporter plasmid linking the qnrS1 upstream sequence to lacZ was introduced into cells of the Keio collection with single-gene deletions and screened for lacZ expression. Mutants in ihfA and ihfB had decreased lacZ induction, while induction in a cysB mutant was increased and dps, fis, lrp, yjhU, and other mutants showed no change. The essential upstream sequence contains potential binding sites for Ihf and DnaA. A dnaA deletion could not be tested because it provides essential functions in cell replication; however, increased dnaA expression decreased qnrS1 induction while decreased dnaA expression enhanced it, implying a role for DnaA as a repressor. In a mobility shift assay, purified IhfA, IhfB, and DnaA proteins (but not CysB) were shown to bind to the upstream segment. Induction decreased in a gyrA quinolone-resistant mutant, indicating that GyrA also has a role. Thus, quinolones acting through proteins DnaA, GyrA, IhfA, and IhfB regulate expression of qnrS1.

Chromosomal mutations that accompany qnr in clinical isolates of Escherichia coli.

We examined 13 qnr-positive and 14 qnr-negative clinical isolates of Escherichia coli for mutations previously seen in a qnr-containing laboratory strain exposed to supra minimum inhibitory concentrations (MICs) of ciprofloxacin. Among the qnr-positive strains, those with ciprofloxacin MICs of ≥ 2 µg/mL had at least one mutation in gyrA. Mutations in parC were present in strains with a ciprofloxacin MIC of ≥ 128 µg/mL. The 6 most ciprofloxacin-resistant strains contained additional plasmid-mediated quinolone resistance determinants. aac(6')-Ib-cr was found in 5 of the 6 strains. Eleven of the 13 strains had alterations in MarR, 9 in SoxR, and 5 had mutations in AcrR. All had elevated expression of at least one efflux pump gene, predominantly acrA (92% of the strains), followed by mdtE (54%) and ydhE (46%). Nine had functionally silent alterations in rfa, two had mutations in gmhB, and one of these also had a mutation in surA. An E. coli with ciprofloxacin MIC of 1024 µg/mL contained 4 different plasmid-mediated quinolone resistance determinants as well as gyrA, parC, parE and pump overexpression mutations. Nine of the 14 qnr-negative strains had mutations in topoisomerase genes with a ciprofloxacin MIC of 0.25 to 256 µg/mL. The three most resistant strains also had mutations in parE. Twelve had alterations in MarR, 10 in SoxR and 5 in AcrR. Ten of the 14 strains had elevated expression of efflux pumps with acrA (71.4%), followed by ydhE (50%) and mdtE (14.3%). A diversity of resistance mechanisms occurs in clinical isolates with and without qnr genes.

Study of Plasmid-Mediated Quinolone Resistance in Bacteria.

Plasmid-mediated quinolone resistance (PMQR) involves genes for proteins that protect the quinolone targets, an enzyme that inactivates certain quinolones as well as aminoglycosides, and pumps that efflux quinolones. Quinolone susceptibility is reduced by these mechanisms but not to the level of clinical resistance unless chromosomal mutations are also present. PCR primers and conditions for PMQR gene detection are described as well as how to establish a plasmid location.

Topoisomerase Inhibitors: Fluoroquinolone Mechanisms of Action and Resistance.

Quinolone antimicrobials are widely used in clinical medicine and are the only current class of agents that directly inhibit bacterial DNA synthesis. Quinolones dually target DNA gyrase and topoisomerase IV binding to specific domains and conformations so as to block DNA strand passage catalysis and stabilize DNA-enzyme complexes that block the DNA replication apparatus and generate double breaks in DNA that underlie their bactericidal activity. Resistance has emerged with clinical use of these agents and is common in some bacterial pathogens. Mechanisms of resistance include mutational alterations in drug target affinity and efflux pump expression and acquisition of resistance-conferring genes. Resistance mutations in one or both of the two drug target enzymes are commonly in a localized domain of the GyrA and ParC subunits of gyrase and topoisomerase IV, respectively, and reduce drug binding to the enzyme-DNA complex. Other resistance mutations occur in regulatory genes that control the expression of native efflux pumps localized in the bacterial membrane(s). These pumps have broad substrate profiles that include other antimicrobials as well as quinolones. Mutations of both types can accumulate with selection pressure and produce highly resistant strains. Resistance genes acquired on plasmids confer low-level resistance that promotes the selection of mutational high-level resistance. Plasmid-encoded resistance is because of Qnr proteins that protect the target enzymes from quinolone action, a mutant aminoglycoside-modifying enzyme that also modifies certain quinolones, and mobile efflux pumps. Plasmids with these mechanisms often encode additional antimicrobial resistances and can transfer multidrug resistance that includes quinolones.

Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1.

Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6')-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6')-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate.

Functions of a GyrBA fusion protein and its interaction with QnrB and quinolones.

In order to study the interactions between Escherichia coli DNA gyrase and the gyrase interacting protein QnrB in vivo, we constructed a gyrB-gyrA fusion and validated its ability to correct the temperature-sensitive growth of gyrA and gyrB mutants. Like wild-type gyrA, the gyrB-gyrA fusion complemented a quinolone-resistant gyrA mutant to increase susceptibility. It functioned as an active type II topoisomerase, catalyzed negative supercoiling of DNA, was inhibited by quinolone, and was protected by QnrB.

Protective effect of Qnr on agents other than quinolones that target DNA gyrase.

Qnr is a plasmid-encoded and chromosomally determined protein that protects DNA gyrase and topoisomerase IV from inhibition by quinolones. Despite its prevalence worldwide and existence prior to the discovery of quinolones, its native function is not known. Other synthetic compounds and natural products also target bacterial topoisomerases. A number were studied as molecular probes to gain insight into how Qnr acts. Qnr blocked inhibition by synthetic compounds with somewhat quinolone-like structure that target the GyrA subunit, such as the 2-pyridone ABT-719, the quinazoline-2,4-dione PD 0305970, and the spiropyrimidinetrione pyrazinyl-alkynyl-tetrahydroquinoline (PAT), indicating that Qnr is not strictly quinolone specific, but Qnr did not protect against GyrA-targeting simocyclinone D8 despite evidence that both simocyclinone D8 and Qnr affect DNA binding to gyrase. Qnr did not affect the activity of tricyclic pyrimidoindole or pyrazolopyridones, synthetic inhibitors of the GyrB subunit, or nonsynthetic GyrB inhibitors, such as coumermycin A1, novobiocin, gyramide A, or microcin B17.Thus, in this set of compounds the protective activity of Qnr was confined to those that, like quinolones, trap gyrase on DNA in cleaved complexes.

Mechanisms of drug resistance: quinolone resistance.

Quinolone antimicrobials are synthetic and widely used in clinical medicine. Resistance emerged with clinical use and became common in some bacterial pathogens. Mechanisms of resistance include two categories of mutation and acquisition of resistance-conferring genes. Resistance mutations in one or both of the two drug target enzymes, DNA gyrase and DNA topoisomerase IV, are commonly in a localized domain of the GyrA and ParE subunits of the respective enzymes and reduce drug binding to the enzyme-DNA complex. Other resistance mutations occur in regulatory genes that control the expression of native efflux pumps localized in the bacterial membrane(s). These pumps have broad substrate profiles that include quinolones as well as other antimicrobials, disinfectants, and dyes. Mutations of both types can accumulate with selection pressure and produce highly resistant strains. Resistance genes acquired on plasmids can confer low-level resistance that promotes the selection of mutational high-level resistance. Plasmid-encoded resistance is due to Qnr proteins that protect the target enzymes from quinolone action, one mutant aminoglycoside-modifying enzyme that also modifies certain quinolones, and mobile efflux pumps. Plasmids with these mechanisms often encode additional antimicrobial resistances and can transfer multidrug resistance that includes quinolones. Thus, the bacterial quinolone resistance armamentarium is large.

Interactions between QnrB, QnrB mutants, and DNA gyrase.

Plasmid-encoded protein QnrB1 protects DNA gyrase from ciprofloxacin inhibition. Using a bacterial two-hybrid system, we evaluated the physical interactions between wild-type and mutant QnrB1, the GyrA and GyrB gyrase subunits, and a GyrBA fusion protein. The interaction of QnrB1 with GyrB and GyrBA was approximately 10-fold higher than that with GyrA, suggesting that domains of GyrB are important for stabilizing QnrB1 interaction with the holoenzyme. Sub-MICs of ciprofloxacin or nalidixic acid reduced the interactions between QnrB1 and GyrA or GyrBA but produced no reduction in the interaction with GyrB or a quinolone-resistant GyrA:S83L (GyrA with S83L substitution) mutant, suggesting that quinolones and QnrB1 compete for binding to gyrase. Of QnrB1 mutants that reduced quinolone resistance, deletions in the C or N terminus of QnrB1 resulted in a marked decrease in interactions with GyrA but limited or no effect on interactions with GyrB and an intermediate effect on interactions with GyrBA. While deletion of loop B and both loops moderately reduced the interaction signal with GyrA, deletion of loop A resulted in only a small reduction in the interaction with GyrB. The loop A deletion also caused a substantial reduction in interaction with GyrBA, with little effect of loop B and dual-loop deletions. Single-amino-acid loop mutations had little effect on physical interactions except for a Δ105I mutant. Therefore, loops A and B may play key roles in the proper positioning of QnrB1 rather than as determinants of the physical interaction of QnrB1 with gyrase.

Effect of Qnr on Plasmid Gyrase Toxins CcdB and ParE.

Plasmid toxins CcdB and ParE are part of addiction systems promoting plasmid maintenance. Both target host DNA gyrase, as do quinolones and plasmid-determined Qnr proteins that protect gyrase from quinolone inhibition. We cloned qnrB4, qnrS1, ccdB, parE, and the antitoxin-encoding genes ccdA and parD on compatible plasmids and tested them in combination. CcdB and ParE had no specific effect on quinolone susceptibility or Qnr protection, and Qnr did not act as a CcdB or ParE antitoxin.