PmrD Is Required for Modifications to Escherichia coli Endotoxin That Promote Antimicrobial Resistance

In Salmonella enterica, PmrD is a connector protein that links the two-component systems PhoP-PhoQ and PmrA-PmrB. While Escherichia coli encodes a PmrD homolog, it is thought to be incapable of connecting PhoPQ and PmrAB in this organism due to functional divergence from the S. enterica protein. However, our laboratory previously observed that low concentrations of Mg²⁺, a PhoPQ-activating signal, leads to the induction of PmrAB-dependent lipid A modifications in wild-type E. coli (C. M. Herrera, J. V. Hankins, and M. S. Trent, Mol Microbiol 76:1444–1460, 2010, http://dx.doi.org/10.1111/j.1365-2958.2010.07150.x). These modifications include phosphoethanolamine (pEtN) and 4-amino-4-deoxy-l-arabinose (l-Ara4N), which promote bacterial resistance to cationic antimicrobial peptides (CAMPs) when affixed to lipid A. Here, we demonstrate that pmrD is required for modification of the lipid A domain of E. coli lipopolysaccharide (LPS) under low-Mg²⁺ growth conditions. Further, RNA sequencing shows that E. coli pmrD influences the expression of pmrA and its downstream targets, including genes coding for the modification enzymes that transfer pEtN and l-Ara4N to the lipid A molecule. In line with these findings, a pmrD mutant is dramatically impaired in survival compared with the wild-type strain when exposed to the CAMP polymyxin B. Notably, we also reveal the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system. These results illuminate a more complex network of protein interactions surrounding activation of PhoPQ and PmrAB in E. coli than previously understood.     

Characterization of Mutations Contributing to Sulfathiazole Resistance in Escherichia coli

A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia coli repeatedly selected for sulfathiazole resistance was mapped to folP by P1 transduction. The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis. A single C→T transition resulted in a Pro→Ser substitution at amino acid position 64. Replacement of the mutant folP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains. Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E. coli. Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters. An additional mutation contributing to sulfathiazole resistance, sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by these purU strains.     

Pseudomonas aeruginosa Ceftolozane-Tazobactam Resistance Development Requires Multiple Mutations Leading to Overexpression and Structural Modification of AmpC

We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ΔmutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 μg/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 μg/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.     

Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

Amdinocillin (mecillinam) is a β-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 × 10⁻⁸ to 2 × 10⁻⁵ per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli.     

Correlation of Resistance to Proflavine and Penicillin in Escherichia coli

A number of proflavine (PF)-resistant mutants of Escherichia coli B were also resistant to penicillin and cephalothin. Mutants resistant to 1.0 mM PF were 10 times more penicillin resistant than were the PF-susceptible, wild-type cells. Single-step mutants selected for resistance to either PF or penicillin were also resistant to the other drug. None of the resistant mutants tested possessed β-lactamase activity. These results suggest that resistance to PF and penicillin in E. coli B may be due to permeability changes in the cell envelope.     

Interplay between Drug Efflux and Antioxidants in Escherichia coli Resistance to Antibiotics

We investigated possible cross talk between endogenous antioxidants glutathione, spermidine, and glutathionylspermidine and drug efflux in Escherichia coli. We found that cells lacking either spermidine or glutathione are less susceptible than the wild type to novobiocin and certain aminoglycosides. In contrast, exogenous glutathione protects against both bactericidal and bacteriostatic antibiotics. The glutathione protection does not require the AcrAB efflux pump but fails in cells lacking TolC because exogenous glutathione is toxic to these cells.In Escherichia coli and other Gram-negative bacteria, the major mechanism responsible for high levels of intrinsic resistance to antibiotics is active efflux, which decreases effective concentrations of drugs in the cytoplasm and periplasm (14). This efflux is carried out by multidrug efflux pumps comprising an inner membrane transporter from the resistance-nodulation-cell division superfamily, a periplasmic membrane fusion protein (MFP), and an outer membrane channel. The best-characterized example of such pumps is AcrAB-TolC from E. coli, which forms a protein conduit spanning the entire two-membrane E. coli envelope (15). Among substrates of AcrAB-TolC are a broad range of antibiotics, detergents, dyes, organic solvents, and hormones.Results of recent studies suggested that in addition to inhibiting specific intracellular targets, bactericidal antibiotics induce production of hydroxyl radicals, which are eventually responsible for cell death (6). Thus, besides drug efflux, the intracellular processes responsible for protection against oxidative damage might contribute to antibiotic resistance. E. coli cells produce two highly abundant compounds implicated in protection against reactive oxygen species: glutathione (GSH) and spermidine (SPE) (3, 13, 18). In addition, glutathionylspermidine (GSP), a conjugate of GSH and SPE, was also proposed previously to play a role in protection against oxidative stress (17). Whether these antioxidants protect E. coli cells against bactericidal antibiotics and how this protection is related to drug efflux remain unclear.To investigate the possible contribution of endogenous antioxidants to protection against bactericidal antibiotics, we measured MICs of antibiotics and detergents for strains lacking GSP synthetase (ΔgspS), GSH synthetase (ΔgshB), and SPE synthetase (ΔspeE). In addition to gspS, the E. coli chromosome contains ygiC and yjfC genes, which encode proteins with significant homology to the C-terminal GSP synthetase domain of GspS (2). The high level of conservation of catalytically important residues in YgiC and YjfM suggested that these proteins could be GSP synthetases (5). We therefore constructed a mutant lacking ygiBC, yjfMC, and gspS and investigated its antibiotic susceptibility as well.The mutants were either obtained from the Keio collection or constructed using the λ Red recombination approach (4). The MICs were determined by using Luria-Bertani (LB) medium supplemented with 2-fold-increasing concentrations of antibiotic as described before (19). As shown in Table ​Table1,1, E. coli cells that do not produce GSH and SPE did not become hypersusceptible to any of the tested compounds. Furthermore, MICs of amikacin and kanamycin increased 16-fold for ΔspeE cells and 4-fold for ΔgshB cells. In addition, ΔgshB cells were 8-fold less susceptible to novobiocin. Thus, endogenous SPE and GSH do not protect against antibiotics and, in some cases, are synergistic with them. The synergistic interactions between polyamines and aminoglycosides were reported before and attributed to interactions at the binding sites on ribosomes (8, 9, 12). It is surprising that similar interactions between GSH and certain antibiotics exist. Interestingly, although the ΔgspS mutant showed the same level of susceptibility as the wild type (WT), GD108 cells, lacking all three GSP enzymes, were 4-fold more susceptible than the WT to norfloxacin but not to other tested antibiotics. This result suggested that GSP enzymes function in the same, norfloxacin-sensitive pathway.

TABLE 1.

Susceptibilities of E. coli strains to antibiotics

Resistance to various tetracyclines mediated by transposon Tn10 in Escherichia coli K-12.

Levels of resistance to tetracycline, chlortetracycline, demethylchlortetracycline, doxycycline, oxytetracycline, methacycline, pyrrolidinotetracycline, minocycline, and beta-chelocardin of Escherichia coli K-12 carrying transposon Tn10 or defined DNA segments of Tn10 were determined. In all cases, tetA was the only gene required for resistance. Doxycycline was the most effective inducer of tetA gene expression.     

Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

Streptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Str^r) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Str^r mutants of Escherichia coli O157:H7 have been generated for in vivo studies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) of E. coli O157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB in E. coli O157:H7 Str^r restrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions in E. coli O157:H7. Therefore, we suggest that E. coli O157:H7 strains selected for Str^r should be thoroughly characterized before in vivo and in vitro experiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.     

Host Mutations (miaA and rpsL) Reduce Tetracycline Resistance Mediated by Tet(O) and Tet(M)

The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. and Streptococcus spp., respectively, were investigated by using mutants of Salmonella typhimurium and Escherichia coli. The miaA, miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. In S. typhimurium, this involves biosynthesis of N⁶-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms²io⁶A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i⁶) group or to a combination of the methylthioadenosine (ms²) and i⁶ groups but not to the ms² group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in the E. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.     

Resistance among Escherichia coli to sulphonamides and other antimicrobials now little used in man

OBJECTIVES: We investigated whether sulphonamide resistance in Escherichia coli remained prevalent in 2004, 9 years since the formal introduction of a UK prescribing restriction on co-trimoxazole. Resistance to other agents no longer in common use was also examined. METHODS: Consecutive urinary E. coli isolates were obtained at the diagnostic microbiology laboratory of the Royal London Hospital from January to March 2004. The presence of the sulphonamide resistance genes, sul1, sul2 and sul3, and the class I integrase gene, int1, were determined by PCR. RESULTS: Of the 391 E. coli isolates recovered in 2004, 45.5% were sulphonamide-resistant compared with 46.0% in 1999 and 39.7% in 1991. The sul2 gene remained the most prevalent sulphonamide resistance determinant, present in 81% of resistant isolates in 2004 compared with 79% and 67% in 1999 and 1991, respectively; 28% of resistant isolates carried both sul1 and sul2 genes; sul3 was not found. Resistance to streptomycin also remained common, whereas resistance to chloramphenicol and kanamycin had decreased since 1999. CONCLUSION: Sulphonamide resistance in E. coli persists undiminished despite the prolonged withdrawal of this antibiotic in the UK; resistance to streptomycin also seems stable whilst that to chloramphenicol and kanamycin is declining.