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In this work, multicomponent trimethoprim-based pharmaceutical solid systems were developed by mechanochemistry, using coformers from the GRAS list and other active pharmaceutical ingredients. The choice of coformers took into account their potential to increase the aqueous solubility/dissolution rate of TMP or its antibacterial activity. All the binary systems were characterized by thermal analysis, powder X-ray diffraction and infrared spectroscopy, and 3 equimolar systems with FTIR pointing to salts, and 4 eutectic mixtures were identified. The intrinsic dissolution rate of TMP in combination with nicotinic acid (a salt) and with paracetamol (eutectic mixture) were 25% and 5% higher than for pure TMP, respectively. For both Gram-positive and -negative strains, the antibacterial activity of TMP with some of the coformers was improved, since the dosage used was lower than the TMP control. A significant increase in antibacterial activity against E. coli was found for the eutectic mixture with curcumin, with the best results being obtained for the eutectic and equimolar mixtures with ciprofloxacin. Combining trimethoprim with coformers offers an interesting alternative to using trimethoprim alone: multicomponent forms with enhanced TMP dissolution rates were identified, as well as combinations showing enhanced antibacterial activity relatively to the pure drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Importance: Bacteria employ a number of mechanisms to adapt to antibiotics. Mutations in transcriptional regulators alter the expression levels of genes that can change the susceptibility of bacteria to antibiotics. Two-component signaling proteins are a major class of signaling molecule used by bacteria to regulate transcription. In previous work, we found that mutations in MgrB, a feedback regulator of the PhoQP two-component system, conferred trimethoprim tolerance to Escherichia coli . Here, we elucidate how mutations in MgrB have a domino-like effect on the gene regulatory network of E. coli . As a result, pervasive perturbation of gene regulation ensues. Depending on the environmental context, this pervasive deregulation is either adaptive or maladaptive. Our study sheds light on how deregulation of gene expression can be beneficial for bacteria when challenged with antibiotics, and why regulators like MgrB may have evolved in the first place., Competing Interests: The authors declare no conflict of interest.

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR's crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4'-deoxy methyl trimethoprim (4'-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4'-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4'-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4'-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.

Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion protein's abundance. However, in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of ∼1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of fluorescence-activated cell sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as "C12", contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated a significant 2.9-fold reduction in basal levels compared to the conventional ecDHFR DD ( i.e. , R12Y/G67S/Y100I). This domain was similarly responsive to TMP with respect to dose response and maximal stabilization, indicating an overall enhanced dynamic range. Interestingly, both computational and wet-lab experiments identified the W74R and T113S mutations of C12 as the main contributors toward its basal destabilization. However, the combination of all the C12 mutations was required to maintain both its enhanced degradation and TMP stabilization. We further demonstrate the utility of C12 by fusing it to IκBα and Nrf2, two stress-responsive proteins that have previously been challenging to regulate. In both instances, C12 significantly enhanced the basal turnover of these proteins and improved the dynamic range of regulation post stabilizer addition. These advantageous features of the C12 ecDHFR DD variant highlight its potential for replacing the conventional N-terminal ecDHFR DD and improving the use of DDs overall, not only as a chemical biology tool but for gene therapy avenues as well.

Cells that cannot synthesize one of the DNA precursors, dTTP, due to thyA mutation or metabolic poisoning, undergo thymineless death (TLD), a chromosome-based phenomenon of unclear mechanisms. In Escherichia coli, thymineless death is caused either by denying thyA mutants thymidine supplementation or by treating wild-type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage in vivo comes from Fenton's reaction when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection in both thyA mutants denied thymidine, as well as in trimethoprim-treated wild-type (WT) cells, under the following three conditions: (i) intracellular iron chelation, (ii) mutational inactivation of hydrogen peroxide (HP) scavenging, and (iii) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division, which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the rffC and recBCD mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. IMPORTANCE Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via in vivo Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking in vivo Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyperthymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.

Although glutathione (GSH) has been shown to influence the antimicrobial effects of many kinds of antibiotics, little is known about its role in relation to trimethoprim (TMP), a widely used antifolate. In this study, several genes related to glutathione metabolism were deleted in different Escherichia coli strains (i.e., O157:H7 and ATCC 25922), and their effects on susceptibility to TMP were tested. The results showed that deleting gshA , gshB , grxA , and cydD caused TMP resistance, and deleting cydD also caused resistance to other drugs. Meanwhile, deleting gshA , grxA , and cydD resulted in a significant decrease of the periplasmic glutathione content. Supplementing exogenous GSH or further deleting glutathione importer genes ( gsiB and ggt ) restored TMP sensitivity to Δ cydD . Subsequently, the results of quantitative-reverse transcription PCR experiments showed that expression levels of acrA , acrB , and tolC were significantly upregulated in both Δ grxA and Δ cydD . Correspondingly, deleting cydD led to a decreased accumulation of TMP within bacterial cells, and further deleting acrA , acrB , or tolC restored TMP sensitivity to Δ cydD . Inactivation of CpxR and SoxS, two transcriptional factors that modulate the transcription of acrAB - tolC , restored TMP sensitivity to Δ cydD . Furthermore, mutations of gshA , gshB , grxA , cydC , and cydD are highly prevalent in E. coli clinical strains. Collectively, these data suggest that reducing the periplasmic glutathione content of E. coli leads to increased expression of acrAB - tolC with the involvement of CpxR and SoxS, ultimately causing drug resistance. To the best of our knowledge, this is the first report showing a linkage between periplasmic GSH and drug resistance in bacteria. IMPORTANCE After being used extensively for decades, trimethoprim still remains one of the key accessible antimicrobials recommended by the World Health Organization. A better understanding of the mechanisms of resistance would be beneficial for the future utilization of this drug. It has been shown that the AcrAB-TolC efflux pump is associated with trimethoprim resistance in E. coli clinical strains. In this study, we show that E. coli can sense the periplasmic glutathione content with the involvement of the CpxAR two-component system. As a result, reducing the periplasmic glutathione content leads to increased expression of acrA , acrB , and tolC via CpxR and SoxS, causing resistance to antimicrobials, including trimethoprim. Meanwhile, mutations in the genes responsible for periplasmic glutathione content maintenance are highly prevalent in E. coli clinical isolates, indicating a potential correlation of the periplasmic glutathione content and clinical antimicrobial resistance, which merits further investigation.

Trimethoprim and sulfamethoxazole are used commonly together as cotrimoxazole for the treatment of urinary tract and other infections. The evolution of resistance to these and other antibacterials threatens therapeutic options for clinicians. We generated and analysed a chemical-biology-whole-genome data set to predict new targets for antibacterial combinations with trimethoprim and sulfamethoxazole. For this we used a large transposon mutant library in Escherichia coli BW25113 where an outward-transcribing inducible promoter was engineered into one end of the transposon. This approach allows regulated expression of adjacent genes in addition to gene inactivation at transposon insertion sites, a methodology that has been called TraDIS- Xpress . These chemical genomic data sets identified mechanisms for both reduced and increased susceptibility to trimethoprim and sulfamethoxazole. The data identified that over-expression of FolA reduced trimethoprim susceptibility, a known mechanism for reduced susceptibility. In addition, transposon insertions into the genes tdk , deoR , ybbC , hha , ldcA , wbbK and waaS increased susceptibility to trimethoprim and likewise for rsmH , fadR , ddlB , nlpI and prc with sulfamethoxazole, while insertions in ispD , uspC , minC , minD , yebK , truD and umpG increased susceptibility to both these antibiotics. Two of these genes' products, Tdk and IspD, are inhibited by AZT and fosmidomycin respectively, antibiotics that are known to synergise with trimethoprim. Thus, the data identified two known targets and several new target candidates for the development of co-drugs that synergise with trimethoprim, sulfamethoxazole or cotrimoxazole. We demonstrate that the TraDIS- Xpress technology can be used to generate information-rich chemical-genomic data sets that can be used for antibacterial development.

Rising incidents of urinary tract infections (UTIs) among catheterized patients is a noteworthy problem in clinic due to their colonization of uropathogens on abiotic surfaces. Herein, we have examined the surface modification of urinary catheter by embedding with eco-friendly synthesized phytomolecules-capped silver nanoparticles (AgNPs) to prevent the invasion and colonization of uropathogens. The preliminary confirmation of AgNPs production in the reaction mixture was witnessed by the colour change and surface resonance plasmon (SRP) band at 410nm by UV-visible spectroscopy. The morphology, size, crystalline nature, and elemental composition of attained AgNPs were further confirmed by the transmission electron microscopy (TEM), selected area electron diffraction (SAED), X-ray diffraction (XRD) technique, Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The functional groups of AgNPs with stabilization/capped phytochemicals were detected by Fourier-transform infrared spectroscopy (FTIR). Further, antibiofilm activity of synthesized AgNPs against biofilm producers such as Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were determined by viability assays and micrographically. AgNPs coated and coating-free catheters performed to treat with bacterial pathogen to analyze the mat formation and disruption of biofilm formation. Synergistic effect of AgNPs with antibiotic reveals that it can enhance the activity of antibiotics, AgNPs coated catheter revealed that, it has potential antimicrobial activity and antibiofilm activity. In summary, C. carandas leaf extract mediated synthesized AgNPs will open a new avenue and a promising template to embed on urinary catheter to control clinical pathogens., Competing Interests: The authors have declared that no competing interests exist.

Microbial multidrug resistance (MDR) poses a huge threat to human health. Bacterial acquisition of MDR relies primarily on class 1 integron-involved horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). To date, no strategies other than the use of antibiotics can efficiently cope with MDR. Here, we report that an engineered CRISPR interference (CRISPRi) system can markedly reduce MDR by blocking a class 1 integron in Escherichia coli Using CRISPRi to block plasmid R388 class 1 integron, E. coli recombinants showed halted growth upon exposure to relevant antibiotics. A microplate alamarBlue assay showed that both subgenomic RNAs (sgRNAs) R3 and R6 led to 8- and 32-fold decreases in half-maximal inhibitory concentrations (IC 50 ) for trimethoprim and sulfamethoxazole, respectively. Reverse transcription and quantitative PCR (RT-qPCR) revealed that the strain employing sgRNA R6 exhibited 97% and 84% decreases in the transcriptional levels of the dfrB2 cassette and sul1 , two typical ARGs, respectively. RT-qPCR analysis also demonstrated that the strain recruiting sgRNA R3 showed a 96% decrease in the transcriptional level of intI1 , and a conjugation assay revealed a 1,000-fold decrease in HGT rates of ARGs. Overall, the sgRNA R3 targeting the 31 bp downstream of the Pc promoter on the intI1 nontemplate strand outperformed other sgRNAs in reducing integron activity. Furthermore, this CRISPRi system is reversible, genetically stable, and titratable by varying the concentration of the inducer. To our knowledge, this is the first report on exploiting a CRISPRi system to reduce the class 1 integron in E. coli This study provides valuable insights for future development of CRISPRi-based antimicrobial agents and cellular therapy to suppress MDR., (Copyright © 2020 Li et al.)

Evolutionary trajectories and mutational landscapes of drug-resistant bacteria are influenced by cell-intrinsic and extrinsic factors. In this study, I demonstrated that loss of the Lon protease altered susceptibility of Escherichia coli to trimethoprim and that these effects were strongly contingent on the drug concentration and genetic background. Lon, an AAA + ATPase, is a bacterial master regulator protease involved in cytokinesis, suppression of transposition events, and clearance of misfolded proteins. I show that Lon deficiency enhances intrinsic drug tolerance at sub-MIC levels of trimethoprim. As a result, loss of Lon, though disadvantageous under drug-free conditions, has a selective advantage at low concentrations of trimethoprim. At high drug concentrations, however, Lon deficiency is detrimental for E. coli I show that the former is explained by suppression of drug efflux by Lon, while the latter can be attributed to SulA-dependent hyperfilamentation. On the other hand, deletion of lon in a trimethoprim-resistant mutant E. coli strain (harboring the Trp30Gly dihydrofolate reductase [DHFR] allele) directly potentiates resistance by enhancing the in vivo stability of mutant DHFR. Using extensive mutational analysis at 3 hot spots of resistance, I show that many resistance-conferring mutations render DHFR prone to proteolysis. This trade-off between gaining resistance and losing in vivo stability limits the number of mutations in DHFR that can confer trimethoprim resistance. Loss of Lon expands the mutational capacity for acquisition of trimethoprim resistance. This paper identifies the multipronged action of Lon in trimethoprim resistance in E. coli and provides mechanistic insight into how genetic backgrounds and drug concentrations may alter the potential for antimicrobial resistance evolution. IMPORTANCE Understanding the evolutionary dynamics of antimicrobial resistance is vital to curb its emergence and spread. Being fundamentally similar to natural selection, the fitness of resistant mutants is a key parameter to consider in the evolutionary dynamics of antimicrobial resistance (AMR). Various intrinsic and extrinsic factors modulate the fitness of resistant bacteria. This study demonstrated that Lon, a bacterial master regulator protease, influences drug tolerance and resistance. Lon is a key regulator of several fundamental processes in bacteria, including cytokinesis. I demonstrated that Lon deficiency produces highly contingent phenotypes in E. coli challenged with trimethoprim and can expand the mutational repertoire available to E. coli to evolve resistance. This multipronged influence of Lon on drug resistance provides an illustrative instance of how master regulators shape the response of bacteria to antibiotics., (Copyright © 2020 American Society for Microbiology.)

In recent years, high frequencies of trimethoprim resistance in urinary tract infections (UTIs) caused by E. coli are have been reported. Co-resistance to other antimicrobial drugs may play a role in this increase. Therefore, we investigated whether previous use of other antimicrobial drugs was associated with trimethoprim resistance. We conducted a nested case-control study with urinary cultures with E. coli from participants of the Rotterdam Study sent in by general practitioners to the regional laboratory between 1 January 2000 and 1 April 2016. Multivariable logistic regression analysis was performed to study the association between prior prescriptions of several antimicrobial drug groups and trimethoprim resistance using individual participant data. Urinary cultures of 1264 individuals with a UTI caused by E. coli were included. When adjusted for previous other antimicrobial drug use, a history of > 3 prescriptions of extended-spectrum penicillins (OR 1.68; 95% CI 1.10-2.55) was significantly associated with trimethoprim resistance of E. coli as was the use of > 3 prescriptions of sulfonamides and trimethoprim (OR 2.22; 95% CI 1.51-3.26). The use of > 3 prescriptions of nitrofuran derivatives was associated with a lower frequency of trimethoprim resistance (OR 0.60; 95% CI 0.39-0.92), after adjustment for other antimicrobial drug prescriptions. We found that previous use of extended-spectrum penicillins is associated with trimethoprim resistance. On the contrary, previous nitrofurantoin use was associated with a lower frequency of trimethoprim resistance. Especially in individuals with recurrent UTI, co-resistance should be taken into account and susceptibility testing before starting trimethoprim should be considered.

Objective: The objective of this study was to assess the level of resistance of trimethoprim alone (TMP) with respect to E. coli strains isolated from the urines of women with simple acute cystitis in community., Patients and Methods: Prospective study realized for 9 months in 2017-18. A total of 351 urine samples were analyzed. Culture has been made according to the usual techniques and antibiogram was carried out according to the recommendations of the CA-SFM., Results: The rate of resistance to TMP was 16.5% (58/351). Only 11 strains of E. coli (3%) producing ESBL were found, 5 of which were sensitive to TMP., Conclusion: The resistance rate of E. coli to TMP remains below 20%, the threshold for choosing a probabilistic treatment of a non-serious infection. Considering the good tolerance of TMP and its weak effect on the microbiota during a short treatment, one can propose TMP alone in the probabilistic treatment of simple acute cystitis., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

There are substantial limitations in understanding of the distribution of antimicrobial resistance (AMR) in humans and livestock in developing countries. This papers present the results of an epidemiological study examining patterns of AMR in Escherichia coli isolates circulating in sympatric human (n = 321) and livestock (n = 633) samples from 99 households across Nairobi, Kenya. E. coli isolates were tested for susceptibility to 13 antimicrobial drugs representing nine antibiotic classes. High rates of AMR were detected, with 47.6% and 21.1% of isolates displaying resistance to three or more and five or more antibiotic classes, respectively. Human isolates showed higher levels of resistance to sulfonamides, trimethoprim, aminoglycosides and penicillins compared with livestock (P<0.01), while poultry isolates were more resistant to tetracyclines (P = 0.01) compared with humans. The most common co-resistant phenotype observed was to tetracyclines, streptomycin and trimethoprim (30.5%). At the household level, AMR carriage in humans was associated with human density (P<0.01) and the presence of livestock manure (P = 0.03), but keeping livestock had no influence on human AMR carriage (P>0.05). These findings revealed a high prevalence of AMR E. coli circulating in healthy humans and livestock in Nairobi, with no evidence to suggest that keeping livestock, when treated as a single risk factor, contributed significantly to the burden of AMR in humans, although the presence of livestock waste was significant. These results provide an understanding of the broader epidemiology of AMR in complex and interconnected urban environments., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Whole-genome sequencing of trimethoprim-resistant Escherichia coli strains MF2165 and PF9285 from healthy Swiss fattening calves revealed a so far uncharacterized dihydrofolate reductase gene, dfrA35 Functionality and association with trimethoprim resistance were demonstrated by cloning and expressing dfrA35 in E. coli The DfrA35 protein showed the closest amino acid identity (49.4%) to DfrA20 from Pasteurella multocida and to the Dfr determinants DfrG (41.2%), DfrD (40.8%), and DfrK (40.0%) found in Gram-positive bacteria. The dfrA35 gene was integrated within a florfenicol/chloramphenicol-sulfonamide resistance IS CR2 element ( floR -IS CR2 - dfrA35 - sul2 ) next to a Tn 21 -like transposon that contained genes with resistance to sulfonamides ( sul1 ), streptomycin ( aadA1 ), gentamicin/tobramycin/kanamycin ( aadB ), and quaternary ammonium compounds ( qacE Δ 1 ). A search of GenBank databases revealed that dfrA35 was present in 26 other E. coli strains from different origins as well as in Acinetobacter IMPORTANCE The presence of dfrA35 associated with IS CR2 in Escherichia coli from animals, as well as its presence in other E. coli strains from different sources and countries and in Acinetobacter , highlights the global spread of this gene and its potential for further dissemination. The genetic link of IS CR2 - dfrA35 with other antibiotic and disinfectant resistance genes showed that multidrug-resistant E. coli may be selected and maintained by the use of either one of several antimicrobials., (Copyright © 2019 Wüthrich et al.)

We show that trimethoprim (TMP), an antibiotic in current use, displays a strong synergistic effect on mutagenesis in Escherichia coli when paired with the base analog 2-aminopurine (2AP), resulting in a 35-fold increase in mutation frequencies in the rpoB -Rif r system. Combination therapies are often employed both as antibiotic treatments and in cancer chemotherapy. However, mutagenic effects of these combinations are rarely examined. An analysis of the mutational spectra of TMP, 2AP, and their combination indicates that together they trigger a response via an alteration in deoxynucleoside triphosphate (dNTP) ratios that neither compound alone can trigger. A similar, although less strong, response is seen with the frameshift mutagen ICR191 and 2AP. These results underscore the need for testing the effects on mutagenesis of combinations of antibiotics and chemotherapeutics., (Copyright © 2019 American Society for Microbiology.)

The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity. For example, daptomycin requires medium supplemented with Ca 2+ , and the lipoglycopeptides dalbavancin and oritavancin require Tween 80 to be added to the growth medium to prevent the depletion of free drug via adsorption to the plastic microplate. In this report, we examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in medium alone and in medium supplemented with the commonly used additives Tween 80, lysed horse blood, and 50% human serum. We observed very significant differences in measured MICs (up to 100-fold) for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, and found that nonspecific binding plates can replace the need for surfactant additives. Microtiter plate types and any additives should be specified when reporting broth dilution MIC values, as results can vary dramatically for some classes of antibiotics., (Copyright © 2018 Kavanagh et al.)

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, might enable selection against resistance during treatment. However, the success of this approach would depend on the conservation of CS networks across genetically diverse bacterial strains. Here, we examine CS conservation across diverse Escherichia coli strains isolated from urinary tract infections. We determine collateral susceptibilities of mutants resistant to relevant antimicrobials against 16 antibiotics. Multivariate statistical analyses show that resistance mechanisms, in particular efflux-related mutations, as well as the relative fitness of resistant strains, are principal contributors to collateral responses. Moreover, collateral responses shift the mutant selection window, suggesting that CS-informed therapies may affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that rapid detection of resistance mechanisms is important to accurately predict collateral responses.

Objectives: Trimethoprim has been recently included in the French guidelines for the treatment of urinary tract infections, but no epidemiological data supports its use. We aimed to determine the trimethoprim susceptibility of Escherichia coli isolates responsible for community-acquired urinary tract infections in women of childbearing age., Materials and Methods: We conducted a national prospective survey. A total of 350 strains of E. coli isolated from urines in 35 laboratories were included. Antibiotic susceptibility testing was performed in each laboratory., Results: We reported a susceptibility rate of 78%, and a similar clinical categorization between trimethoprim and cotrimoxazole for 97.4% of isolates. We pointed out an association between resistance to trimethoprim and other antibiotic classes., Conclusion: The results support trimethoprim as a second-line therapy based on antibiotic susceptibility testing results. We confirm that trimethoprim and cotrimoxazole susceptibility rates are very close., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.

Pig feed may contain various levels of antimicrobial residues due to cross-contamination. A previous study showed that a 3% carry-over level of doxycycline (DOX) in the feed results in porcine faecal concentrations of approximately 4 mg/L. The aim of this study was to determine the effect of residual DOX concentrations (1 and 4 mg/L) in vitro on selection of DOX-resistant porcine commensal Escherichia coli and transfer of their resistance plasmids. Three different DOX-resistant porcine commensal E. coli strains and their plasmids were characterised. These strains were each brought in competition with a susceptible strain in a medium containing 0, 1 and 4 mg/L DOX. Resistant bacteria, susceptible bacteria and transconjugants were enumerated after 24 h and 48 h. The tet(A)-carrying plasmids showed genetic backbones that are also present among human E. coli isolates. Ratios of resistant to susceptible bacteria were significantly higher at 1 and 4 mg/L DOX compared with the blank control, but there was no significant difference between 1 and 4 mg/L. Plasmid transfer frequencies were affected by 1 or 4 mg/L DOX in the medium for only one of the resistance plasmids. In conclusion, DOX concentrations of 1 and 4 mg/L can select for resistant E. coli in vitro. Further research is needed to determine the effect of these concentrations in the complex environment of the porcine intestinal microbiota., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Objective: Several studies showed that a substantial decline in the use of co-trimoxazole did not result in a decline in resistance rates among Escherichia coli isolates. Since mathematical models have shown that it may take decades before resistance rates start to decline to relevant levels, we performed a new analysis using more recently collected data., Methods: Data were extracted from Guy's and St Thomas' Hospitals Transmission and Antimicrobial Record database which contains microbiological test results from all specimens tested between 2002 and 2014. We selected all blood samples positive for E. coli which were tested for resistance against co-trimoxazole. Prevalence of co-trimoxazole resistance among the tested samples by year was modelled by a Poisson model., Results: Almost all (96%) of E. coli blood isolates were tested for co-trimoxazole resistance. In total, 2070 E. coli isolates were available for analyses. Resistance to co-trimoxazole fluctuated over the years, but there was no clear increasing or decreasing trend; the annual percentage change in the prevalence of co-trimoxazole resistance was 0.52 (95% confidence interval -0.75% to 1.81%). Including co-trimoxazole or trimethoprim use in the year before the sample was taken did not improve the model., Conclusion: The prevalence of co-trimoxazole resistance among E. coli blood isolates remained high, almost three decades after a substantial decline in co-trimoxazole use. Our results further emphasize the importance of prudent antibiotics use, as antibiotic resistance may not always be easily reversible., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

There is often overlap in the diagnostic features of common pathologic processes such as infection, sterile inflammation, and cancer both clinically and using conventional imaging techniques. Here, we report the development of a positron emission tomography probe for live bacterial infection based on the small-molecule antibiotic trimethoprim (TMP). [ 18 F]fluoropropyl-trimethoprim, or [ 18 F]FPTMP, shows a greater than 100-fold increased uptake in vitro in live bacteria ( Staphylococcus aureus , Escherichia coli , and Pseudomonas aeruginosa ) relative to controls. In a rodent myositis model, [ 18 F]FPTMP identified live bacterial infection without demonstrating confounding increased signal in the same animal from other etiologies including chemical inflammation (turpentine) and cancer (breast carcinoma). Additionally, the biodistribution of [ 18 F]FPTMP in a nonhuman primate shows low background in many important tissues that may be sites of infection such as the lungs and soft tissues. These results suggest that [ 18 F]FPTMP could be a broadly useful agent for the sensitive and specific imaging of bacterial infection with strong translational potential., Competing Interests: The authors declare no conflict of interest.

Antibiotic-resistant infections kill approximately 23,000 people and cost $20,000,000,000 each year in the United States alone despite the widespread use of small-molecule antimicrobial combination therapy. Antibiotic combinations typically have an additive effect: the efficacy of the combination matches the sum of the efficacies of each antibiotic when used alone. Small molecules can also act synergistically when the efficacy of the combination is greater than the additive efficacy. However, synergistic combinations are rare and have been historically difficult to identify. High-throughput identification of synergistic pairs is limited by the scale of potential combinations: a modest collection of 1,000 small molecules involves 1 million pairwise combinations. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, the overlap2 method (O2M). O2M extracts patterns from chemical-genetic datasets, which are created when a collection of mutants is grown in the presence of hundreds of different small molecules, producing a precise set of phenotypes induced by each small molecule across the mutant set. The identification of mutants that show the same phenotype when treated with known synergistic molecules allows us to pinpoint additional molecule combinations that also act synergistically. As a proof of concept, we focus on combinations with the antibiotics trimethoprim and sulfamethizole, which had been standard treatment against urinary tract infections until widespread resistance decreased efficacy. Using O2M, we screened a library of 2,000 small molecules and identified several that synergize with the antibiotic trimethoprim and/or sulfamethizole. The most potent of these synergistic interactions is with the antiviral drug azidothymidine (AZT). We then demonstrate that understanding the molecular mechanism underlying small-molecule synergistic interactions allows the rational design of additional combinations that bypass drug resistance. Trimethoprim and sulfamethizole are both folate biosynthesis inhibitors. We find that this activity disrupts nucleotide homeostasis, which blocks DNA replication in the presence of AZT. Building on these data, we show that other small molecules that disrupt nucleotide homeostasis through other mechanisms (hydroxyurea and floxuridine) also act synergistically with AZT. These novel combinations inhibit the growth and virulence of trimethoprim-resistant clinical Escherichia coli and Klebsiella pneumoniae isolates, suggesting that they may be able to be rapidly advanced into clinical use. In sum, we present a generalizable method to screen for novel synergistic combinations, to identify particular mechanisms resulting in synergy, and to use the mechanistic knowledge to rationally design new combinations that bypass drug resistance.

In recent years the emergence of multiple drug resistance microbes has become a global public health problem. The aim of the present investigation was to evaluate possible antibiofilm efficacy of a gallotannin 1,2,6-tri-O-galloyl-β-D-glucopyranose from Terminalia chebula fruits alone and in combination with gentamicin and trimethoprim against preformed biofilms of multidrug-resistant (MDR) uropathogenic E. coli isolates using microbroth dilution, checkerboard titration and kill kinetics methods. Test gallotannin showed > 50% antibiofilm efficacy after 24 h when administered alone whereas gentamicin and trimethoprim failed to do so. But in combination, test gallotannin/gentamicin and test gallotannin/trimethoprim showed 71.24±6.75% and 93.4±8.46% antibiofilm activity respectively. On the basis of FICI values, test gallotannin/gentamicin showed synergistic interactions against 71.42% and test gallotannin/trimethoprim against 85.71% biofilm forming test bacterial isolates. Kill-kinetics study confirmed their synergistic interactions. Thus, gentamicin and trimethoprim in combination with test gallotannin may have potential for treatment of urinary tract infections caused by biofilm forming MDR uropathogenic E. coli.

Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family ( dfrB ). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a >2,000-fold increased trimethoprim resistance on overexpression in E. coli Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance., (Copyright © 2017 American Society for Microbiology.)

Purpose: The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals., Methodology: In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic-resistant and virulent factors was analysed by PCR., Results: A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3')-IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole-resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %)., Conclusion: To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichiacoli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic-resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

In present study, nineteen novel trimethoprim (TMP) derivatives were designed, synthesized and evaluated for their antibacterial potential. Hydroxy trimethoprim 2 (HTMP) was synthesized by following the demethylation of 4-methoxy group at trimethoxy benzyl ring of TMP. Structure-activity relationship (SAR) studies were explored on HTMP by incorporating various substituents leading to the identification of some new compounds with improved antibacterial activities. The results revealed that the introduction of benzyloxy (4a-e) and phenyl ethanone (5a-e) group at 4-position of dimethoxy benzyl ring leads to overall increase in the antibacterial activity. The most potent antibacterial compound discovered is benzyloxy derivative 4b with MIC value of 5.0μM against Staphylococcus aureus and 4.0μM against Escherichia coli strains higher than the standard TMP (22.7μM against S. aureus and 55.1μM against E. coli). Substitution at 4-NH 2 group was not tolerated and the resulting Schiff base derivatives 3a-h demonstrated very little or no antibacterial activity in the tested concentration domain. We further performed exploratory docking studies on dihydrofolate reductase (DHFR) to rationalize the in vitro biological data and to demonstrate the mechanism of antibacterial activity. For the ability to cross lipophilic outer membrane, logP was computed. It was found that the compounds possessing high hydrophobicity have high activity against E. coli., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Culture-based approaches are used to monitor, e.g., drinking water or bathing water quality and to investigate species diversity and antibiotic resistance levels in environmental samples. For health risk assessment, it is important to know whether the growing cultures display the actual abundance of, e.g., clinically relevant antibiotic resistance phenotypes such as vancomycin-resistant Enterococcus faecium/Enterococcus faecalis (VRE) or methicillin-resistant Staphylococcus aureus. In addition, it is important to know whether sub-inhibitory antibiotic concentrations, which are present in surface waters, favor the growth of antibiotic-resistant strains. Therefore, clinically relevant bacteria were isolated from different water sources and the growth behavior of 58 Escherichia coli, 71 Enterococcus, and 120 Staphylococcus isolates, belonging to different species and revealing different antibiotic resistance patterns, was studied with respect to "environmental" antibiotic concentrations. The finding that VRE could only be detected after specific enrichment can be explained by their slow growth compared to non-resistant strains. Interpreting their absence in standardized culture-based methods as nonexistent might be a fallacy. Sub-inhibitory antibiotic concentrations that were detected in sewage and receiving river water did not specifically promote antibiotic-resistant strains. Generally, those antibiotics that influenced cell metabolism directly led to slightly reduced growth rates and less than maximal optical densities after 48 h of incubation.

This study describes the antimicrobial resistance temporal trends and seasonal variation of Escherichia coli (E. coli) urinary tract infections (UTIs) over five years, from 2009 to 2013, and compares prevalence of resistance in hospital- and community-acquired E. coli UTI. A cross sectional study of E. coli UTIs from patients attending a tertiary referral hospital in Canberra, Australia was undertaken. Time series analysis was performed to illustrate resistance trends. Only the first positive E. coli UTI per patient per year was included in the analysis. A total of 15,022 positive cultures from 8724 patients were identified. Results are based on 5333 first E. coli UTIs, from 4732 patients, of which 84.2% were community-acquired. Five-year hospital and community resistance rates were highest for ampicillin (41.9%) and trimethoprim (20.7%). Resistance was lowest for meropenem (0.0%), nitrofurantoin (2.7%), piperacillin-tazobactam (2.9%) and ciprofloxacin (6.5%). Resistance to amoxycillin-clavulanate, cefazolin, gentamicin and piperacillin-tazobactam were significantly higher in hospital- compared to community-acquired UTIs (9.3% versus 6.2%; 15.4% versus 9.7%; 5.2% versus 3.7% and 5.2% versus 2.5%, respectively). Trend analysis showed significant increases in resistance over five years for amoxycillin-clavulanate, trimethoprim, ciprofloxacin, nitrofurantoin, trimethoprim-sulphamethoxazole, cefazolin, ceftriaxone and gentamicin (P<0.05, for all) with seasonal pattern observed for trimethoprim resistance (augmented Dickey-Fuller statistic = 4.136; P = 0.006). An association between ciprofloxacin resistance, cefazolin resistance and ceftriaxone resistance with older age was noted. Given the relatively high resistance rates for ampicillin and trimethoprim, these antimicrobials should be reconsidered for empirical treatment of UTIs in this patient population. Our findings have important implications for UTI treatment based on setting of acquisition., Competing Interests: The authors have declared that no competing interests exist.

In view of continued high clinical prevalence of infections involving extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, this study sought to characterise the blaCTX-M genes, their associated mobile genetic elements and the integrons present in 100 ESBL-producing E. coli isolates collected in a Dublin hospital and associated community healthcare facilities. Polymerase chain reaction (PCR) mapping and sequencing was used to detect blaCTX-M alleles, their associated insertion sequences (ISs) and class 1 and 2 integrons in the collection. ESBL plasmids were characterised by PCR-based replicon typing and replicon sequence typing (RST). Cefotaximases were harboured by 94% of isolates (66 blaCTX-M-15, 8 blaCTX-M-14, 7 blaCTX-M-1, 4 blaCTX-M-3, 3 blaCTX-M-9, 2 blaCTX-M-27, 2 blaCTX-M-55, 1 blaCTX-M-32 and 1 blaCTX-M-2). An ISEcp1 promoter was linked to a group 1 blaCTX-M gene in 45% of isolates. A further 34% of isolates contained blaCTX-M-15 downstream of IS26, an arrangement typical of epidemic UK strain A. Class 1 integrons were found in 66% of isolates, most carrying trimethoprim/aminoglycoside resistance genes. CTX-M plasmids were primarily of multireplicon IncF or IncI1 type, but IncN and unidentified types were also found. Novel IncF RSTs F1:A-:B-, F45:A1:B-, F45:A4:B- and a novel IncI1 sequence type, ST159, were identified. CTX-M plasmids and integrons resembled those identified recently in animal isolates from Ireland and Western Europe. The molecular epidemiology of CTX-M-producing E. coli in Dublin suggests that horizontal spread of mobile genetic elements contributes to antimicrobial resistant human infections. Further investigations into whether animals or animal products represent an important local reservoir for these elements are warranted.

Introduction: Increasing resistance in Escherichia coli and Klebsiella pneumoniae to firstline antibiotics makes therapeutic options for urinary tract infections (UTIs) challenging. This study investigated the in vitro efficacies of 6 antibiotics against multidrug resistant (MDR) uropathogens., Materials and Methods: Minimum inhibitory concentrations to ceftibuten, cefpodoxime, fosfomycin, mecillinam, temocillin, and trimethoprim were determined against 155 MDR-isolates of E. coli and K. pneumoniae. The presence of extended-spectrum beta-lactamases (ESBL) and plasmid-borne AmpC enzymes was determined by phenotypic testing with genotyping performed by multiplex polymerase chain reaction., Results: Temocillin demonstrated highest susceptibility rates for both E. coli (95%) and K. pneumoniae (95%) when breakpoints for uncomplicated UTIs were applied; however, temocillin susceptibility was substantially lower when "systemic infection" breakpoints were used. Fosfomycin demonstrated the best in vitro efficacy of the orally available agents, with 78% and 69% of E. coli and K. pneumoniae isolates susceptible, respectively. The next most effective antibiotics were ceftibuten (45%) and mecillinam (32%). ESBL and ampC genes were present in 47 (30%) and 59 (38%) isolates., Conclusion: This study demonstrated few oral therapeutic options for MDR-uropathogens, with fosfomycin demonstrating the best in vitro activity.

Antimicrobial use and resistance in animal and food production are of concern to public health. The primary aims of this study were to determine the frequency of resistance to 12 antimicrobials in Escherichia coli isolates from 39 pig farms and to identify patterns of antimicrobial use on these farms. Further aims were to determine whether a categorization of farms based on the duration of in-feed antimicrobial use (long-term versus short-term) could predict the occurrence of resistance on these farms and to identify the usage of specific antimicrobial drugs associated with the occurrence of resistance. Escherichia coli were isolated from all production stages on these farms; susceptibility testing was carried out against a panel of antimicrobials. Antimicrobial prescribing data were collected, and farms were categorized as long term or short term based on these. Resistance frequencies and antimicrobial use were tabulated. Logistic regression models of resistance to each antimicrobial were constructed with stage of production, duration of antimicrobial use and the use of 5 antimicrobial classes included as explanatory variables in each model. The greatest frequencies of resistance were observed to tetracycline, trimethoprim/sulphamethoxazole and streptomycin with the highest levels of resistance observed in isolates from first-stage weaned pigs. Differences in the types of antimicrobial drugs used were noted between long-term and short-term use farms. Categorization of farms as long- or short-term use was sufficient to predict the likely occurrence of resistance to 3 antimicrobial classes and could provide an aid in the control of resistance in the food chain. Stage of production was a significant predictor variable in all models of resistance constructed and did not solely reflect antimicrobial use at each stage. Cross-selection and co-selection for resistance was evident in the models constructed, and the use of trimethoprim/sulphonamide drugs in particular was associated with the occurrence of resistance to other antimicrobials., (© 2015 Blackwell Verlag GmbH.)

Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we developed a predictive biophysics-based fitness landscape of trimethoprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency. Protein stability is concurrently affected by the resistant mutants, which precludes a precise description of fitness from a single molecular trait. Application of the kinetic flux theory provided an accurate model to predict resistance phenotypes (IC50) quantitatively from a unique combination of the in vitro protein molecular properties. Further, we found that a controlled modulation of the GroEL/ES chaperonins and Lon protease levels affects the intracellular steady-state concentration of DHFR in a mutation-specific manner, whereas IC50 is changed proportionally, as indeed predicted by the model. This unveils a molecular rationale for the pleiotropic role of the protein quality control machinery on the evolution of antibiotic resistance, which, as we illustrate here, may drastically confound the evolutionary outcome. These results provide a comprehensive quantitative genotype-phenotype map for the essential enzyme that serves as an important target of antibiotic and anticancer therapies.

The rare sulphonamide resistance gene sul3 was found in the commensal Escherichia coli ST95 strain 22.1-R1 that was isolated in 2010 from the faeces of a healthy Australian adult. The genome of 22.1-R1 was sequenced and a 144,344bp RepFII/FIB plasmid, pCERC3, carrying sul3 was assembled. The sul3 gene is part of a class 1 integron featuring a sul3-containing conserved segment (sul3-CS) that replaced the classic sul1-containing 3'-conserved segment (3'-CS) usually seen in class 1 integrons. The integron contained the cassette array dfrA12-orfF-aadA2-cmlA1-aadA1-qacH, conferring resistance to trimethoprim, streptomycin, spectinomycin, chloramphenicol and quaternary ammonium compound. Two additional antibiotic resistance genes, blaTEM (ampicillin resistance) and tetA(B) (tetracycline) were adjacent to the integron, forming a single resistance region. In pCERC3, the sul3-type class 1 integron was flanked by sequence derived from the tnp and mer modules of Tn21 and was in the same location as In2, the sul1-containing In5-type class 1 integron of Tn21. At one end the sequence extends into Tn2670-derived sequence and then into sequence derived from the plasmid NR1 (R100). Examination of the sequences of eleven more complete sul3-containing plasmids in GenBank confirmed the relationship between sul3-associated integrons and Tn21/Tn2670/NR1. This suggests that the events that formed sul3-associated class 1 integrons occurred within the Tn21/Tn2670 context, most likely in NR1 or a related plasmid. The backbone of pCERC3 is most closely related to the backbones of ColV virulence plasmids and contains a complete ColV operon as well as several virulence associated genes and gene clusters. Hence, pCERC3 is both an antibiotic resistance and virulence plasmid., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We used classical mutagens in Gram-negative Escherichia coli to study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. We used 4-nitroquinoline 1-oxide (4NQO), zebularine (ZEB), 5-azacytidine (5AZ), 2-aminopurine (2AP), and 5-bromodeoxyuridine (5BrdU) as mutagens (with bactericidal potency of 4NQO > ZEB > 5AZ > 2AP > 5BrdU) and vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL) as antibiotics. We detected the strongest synergies with 4NQO, an agent that oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics VAN, TMP, CPR, and GEN, but no synergies with the bacteriostatic antibiotics TET, ERY, and CHL. Each of the other mutagens displays synergies with the bactericidal antibiotics to various degrees that reflect their potencies, as well as with some of the other mutagens. The results support recent models showing that bactericidal antibiotics kill bacteria principally by ultimately generating more double-strand breaks than can be repaired. We discuss the synergies seen here and elsewhere as representing dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. We also used the results of pairwise tests to place the classic mutagens into functional antibacterial categories within a previously defined drug interaction network., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Topoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) of Escherichia coli topoisomerase I interact directly with RNA polymerase to remove transcription-driven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study, Escherichia coli with a topA66 single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by this topA66 mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in the topA66 mutant. The increased antibiotic sensitivity for the topA66 mutant can be reversed by the expression of recombinant E. coli topoisomerase I but not by the expression of recombinant Mycobacterium tuberculosis topoisomerase I that has a nonhomologous CTD even though the recombinant M. tuberculosis topoisomerase I can restore most of the plasmid DNA linking number deficiency caused by the topA66 mutation. Direct interactions of E. coli topoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Administration of antimicrobials to food-producing animals increases the risk of higher antimicrobial resistance in the normal intestinal flora of these animals. The present cross-sectional study was conducted to investigate antimicrobial susceptibility and extended-spectrum β-lactamase (ESBL)-producing strains and to characterize class 1 integrons in Escherichia coli in healthy swine in Thailand. All 122 of the tested isolates had drug-resistant phenotypes. High resistance was found to ampicillin (98.4% of isolates), chloramphenicol (95.9%), gentamicin (78.7%), streptomycin (77.9%), tetracycline (74.6%), and cefotaxime (72.1%). Fifty-four (44.3%) of the E. coli isolates were confirmed as ESBL-producing strains. Among them, blaCTX-M (45 isolates) and blaTEM (41 isolates) were detected. Of the blaCTX-M-positive E. coli isolates, 37 carried the blaCTX-M-1 cluster, 12 carried the blaCTX-M-9 cluster, and 5 carried both clusters. Sequence analysis revealed blaTEM-1, blaTEM-135, and blaTEM-175 in 38, 2, and 1 isolate, respectively. Eighty-seven (71%) of the 122isolates carried class 1 integrons, and eight distinct drug-resistance gene cassettes with seven different integron profiles were identified in 43 of these isolates. Gene cassettes were associated with resistance to aminoglycosides (aadA1, aadA2, aadA22, or aadA23), trimethoprim (dfrA5, dfrA12, or dfrA17), and lincosamide (linF). Genes encoding β-lactamases were not found in class 1 integrons. This study is the first to report ESBL-producing E. coli with a class 1 integron carrying the linF gene cassette in swine in Thailand. Our findings confirm that swine can be a reservoir of ESBL-producing E. coli harboring class 1 integrons, which may become a potential health risk if these integrons are transmitted to humans. Intensive analyses of animal, human, and environmental isolates are needed to control the spread of ESBL-producing E. coli strains.

This study aimed to identify changing antimicrobial resistance patterns in isolates commonly obtained from equine clinical submissions. Laboratory records from 1999 to 2012 were searched for equine samples from which Escherichia coli or Streptococcus species was isolated. Susceptibility to enrofloxacin, ceftiofur, gentamicin, penicillin G, trimethoprim sulfamethoxazole (TMPS) and tetracyclines was noted. Isolates were divided into those identified between 1999 and 2004 (Early) and between 2007 and 2012 (Late). The proportion of isolates resistant to each antimicrobial and multiple drug-resistant (MDR) isolates (≥3 antimicrobial classes) was compared between time periods. There were 464 isolates identified (242 Early; 222 Late). A significant increase in the percentage of E coli isolates resistant to ceftiofur (7.3-22.7 per cent, P=0.002), gentamicin (28.5-53.9 per cent, P<0.001), tetracyclines (48.4-74.2 per cent, P=0.002) and MDR (26.6-49.4 per cent, P=0.007) was identified. There was a significant increase over time in the percentage of all streptococcal species resistant to enrofloxacin, ranging from 0 per cent (Early) up to 63 per cent (Late) depending on species. For Streptococcus zooepidemicus, resistance over time to tetracyclines and MDR increased. There was also a decrease in the proportion of S zooepidemicus resistant to TMPS over time. An increase in resistance over time of common equine pathogens to a number of commonly used antimicrobials supports the responsible use of antimicrobials., (British Veterinary Association.)

Toxin/antitoxin (TA) systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone; i.e., whether a toxin which lacks a corresponding antitoxin (orphan toxin) is physiologically relevant. By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate) is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein), nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis.

Gram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We used Escherichia coli mutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-type E. coli at concentrations of VAN as low as 6.25 μg/ml. This suggests that a very small percentage of exogenous VAN does enter E. coli but normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489-494, 2006, http://dx.doi.org/10.1038/ng1755)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Prevalence and genetic relatedness were determined for third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) detected in Swiss beef, veal, pork, and poultry retail meat. Samples from meat-packing plants (MPPs) processing 70% of the slaughtered animals in Switzerland were purchased at different intervals between April and June 2013 and analyzed. Sixty-nine 3GC-R-Ec isolates were obtained and characterized by microarray, PCR/DNA sequencing, Multi Locus Sequence Typing (MLST), and plasmid replicon typing. Plasmids of selected strains were transformed by electroporation into E. coli TOP10 cells and analyzed by plasmid MLST. The prevalence of 3GC-R-Ec was 73.3% in chicken and 2% in beef meat. No 3GC-R-Ec were found in pork and veal. Overall, the bla(CTX-M-1) (79.4%), bla(CMY-2) (17.6%), bla(CMY-4) (1.5%), and bla(SHV-12) (1.5%) β-lactamase genes were detected, as well as other genes conferring resistance to chloramphenicol (cmlA1-like), sulfonamides (sul), tetracycline (tet), and trimethoprim (dfrA). The 3GC-R-Ec from chicken meat often harbored virulence genes associated with avian pathogens. Plasmid incompatibility (Inc) groups IncI1, IncFIB, IncFII, and IncB/O were the most frequent. A high rate of clonality (e.g., ST1304, ST38, and ST93) among isolates from the same MPPs suggests that strains persist at the plant and spread to meat at the carcass-processing stage. Additionally, the presence of the blaCTX-M-1 gene on an IncI1 plasmid sequence type 3 (IncI1/pST3) in genetically diverse strains indicates interstrain spread of an epidemic plasmid. The bla(CMY-2) and bla(CMY-4) genes were located on IncB/O plasmids. This study represents the first comprehensive assessment of 3GC-R-Ec in meat in Switzerland. It demonstrates the need for monitoring contaminants and for the adaptation of the Hazard Analysis and Critical Control Point concept to avoid the spread of multidrug-resistant bacteria through the food chain.

This study demonstrates a low-cost, portable diagnostic system for rapid antimicrobial susceptibility testing in resource-limited settings. To determine the antimicrobial resistance phenotypically, the growth of pathogens in microwell arrays is detected under different antibiotic conditions. The use of a colorimetric cell viability reagent is shown to significantly improve the sensitivity of the assay compared with standard absorbance spectroscopy. Gas-permeable microwell arrays are incorporated for facilitating rapid bacterial growth and eliminating the requirement of bulky supporting equipment. Antibiotics can also be precoated in the microwell array to simplify the assay protocol toward point-of-care applications. Furthermore, a low-cost cell phone-based microphotometric system is developed for detecting the bacterial growth in the microwell array. By optimizing the operating conditions, the system allows antimicrobial susceptibility testing for samples with initial concentrations from 10(1) to 10(6) cfu/mL. Using urinary tract infection as the model system, we demonstrate rapid antimicrobial resistance profiling for uropathogens in both culture media and urine. With its simplicity and cost-effectiveness, the cell phone-based microphotometric system is anticipated to have broad applicability in resource-limited settings toward the management of infectious diseases caused by multidrug-resistant pathogens., (© 2013 Society for Laboratory Automation and Screening.)

Fluoroquinolones are frequently used to treat infectious disease that is caused by Escherichia coli in dairy cattle. However, fluoroquinolone resistance occurs and is due either to chromosomal mutations in the bacterial topoisomerase genes and/or to plasmid-mediated resistance genes. The purpose of this study was to determine the prevalence and molecular characteristics of fluoroquinolone resistance determinants in E. coli strains (n=148) isolated from dairy cattle with bovine endometritis in Inner Mongolia (China). Analysis of the mutations in the quinolone resistance-determining regions of resistant E. coli isolates confirmed previously reported substitutions in the GyrA and ParE. However, we identified additional substitutions in the ParC and GyrB that have not been reported earlier. No plasmid-mediated quinolone resistance genes in any of the isolates were found. The number of point mutations found per isolate correlated with an increase in the minimum inhibitory concentration of ciprofloxacin. Overall, 45.5% of the isolates were positive for the class I integrase gene along with four gene cassettes that were responsible for resistance to trimethoprim (dfr1 and dfrA17) and aminoglycosides (aadA1 and aadA5), respectively. The prevalence of extended-spectrum β-lactamases (ESBLs) was 100%, and the blaTEM gene was predominant in all of the isolates. In conclusion, our results identify the mechanism of quinolone resistance for the first time and reveal the prevalence of integron and ESBLs in E. coli isolates from dairy cattle with bovine endometritis in China after 20 years of quinolone usage in cattle.

We report here the nucleotide sequence of a novel blaKPC-2-harboring incompatibility group N (IncN) plasmid, pECN580, from a multidrug-resistant Escherichia coli sequence type 131 (ST131) isolate recovered from Beijing, China. pECN580 harbors β-lactam resistance genes blaKPC-2, blaCTX-M-3, and blaTEM-1; aminoglycoside acetyltransferase gene aac(6')-Ib-cr; quinolone resistance gene qnrS1; rifampin resistance gene arr-3; and trimethoprim resistance gene dfrA14. The emergence of a blaKPC-2-harboring multidrug-resistant plasmid in an epidemic E. coli ST131 clone poses a significant potential threat in community and hospital settings.

The phenotypic detection of plasmid-acquired AmpC (pAmpC) in Escherichia coli is challenging, and molecular methods are required for confirmation. In addition to cefoxitin resistance, multiresistance and high-level resistance to cephalosporins have both been suggested as criteria for targeting isolates with pAmpC, but data to support these proposed criteria are lacking. A Swedish collection of 378 isolates with either putative chromosomal hyperproduction of AmpC (cAmpC) or pAmpC were subjected to disk diffusion and minimum inhibitory concentration (MIC) determination with the Etest. The frequency of resistance to gentamicin, ciprofloxacin, and trimethoprim among cAmpC and pAmpC was compared to elucidate the issue of multidrug resistance. Lastly, methods for the phenotypic confirmation of pAmpC were compared. One in-house disk diffusion method, one method employing NeoSensitabs (Rosco), and one Etest method (bioMérieux) were compared. The analysis of histograms based on both disk diffusion and the Etest showed that resistance [according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)] to cefotaxime and/or ceftazidime occurred in almost all isolates. By coining resistance instead of non-susceptibility, the number of isolates required to subject to phenotypic testing/genotypic confirmation dropped by more than 40 %, without compromising the sensitivity substantially. Further, almost 70 % of isolates with pAmpC were non-multidrug resistant, clearly indicating that this is an inappropriate criterion for further investigation. The phenotypic tests all had more than 90 % sensitivity, and the best sensitivities were obtained with the in-house method and with the ceftazidime ± cloxacillin NeoSensitab. In conclusion, clinical resistance to cefotaxime and/or ceftazidime seems to be an appropriate criterion for pAmpC screening, and several phenotypic methods perform well for the phenotypic confirmation of AmpC production prior to genotypic confirmation.

Low protein solubility is a problem in many areas of protein science. Although chemical methods have been developed to solubilize proteins these are not always effective and add to the cost of producing the protein. One way of overcoming these difficulties is to evolve the protein to be more soluble. A major hurdle in this process is the ability to select mutant proteins with enhanced solubility from a large library of randomly mutated proteins. In this article, we describe such a method. The method relies on the fact that increasing the expression of dihydrofolate reductase (DHFR) makes Escherichia coli resistant to Trimethoprim (TMP). Proteins fused to DHFR will produce chimeras with altered levels of resistance to TMP. This variation in TMP resistance can be used to identify mutant proteins with enhanced solubility.

Objectives: Individual and group level factors associated with the probability of antimicrobial resistance of uropathogenic Escherichia coli were analysed in a multilevel model., Methods: Adult patients consulting with a suspected urinary tract infection (UTI) in 22 general practices over a 9 month period supplied a urine sample for laboratory analysis. Cases were patients with a UTI associated with a resistant E. coli. Previous antimicrobial exposure and other patient characteristics were recorded from the medical files., Results: Six hundred and thirty-three patients with an E. coli UTI and a full record for all variables were included. Of the E. coli isolates, 36% were resistant to trimethoprim and 12% to ciprofloxacin. A multilevel logistic regression model was fitted. The odds that E. coli was resistant increased with increasing number of prescriptions over the previous year for trimethoprim from 1.4 (0.8-2.2) for one previous prescription to 4.7 (1.9-12.4) for two and 6.4 (2.0-25.4) for three or more. For ciprofloxacin the ORs were 2.7 (1.2-5.6) for one and 6.5 (2.9-14.8) for two or more. The probability that uropathogenic E. coli was resistant showed important variation between practices and a difference of 17% for trimethoprim and 33% for ciprofloxacin was observed for an imaginary patient moving from a practice with low to a practice with high probability. This difference could not be explained by practice prescribing or practice resistance levels., Conclusions: Previous antimicrobial use and the practice visited affect the risk that a patient with a UTI will be diagnosed with an E. coli resistant to this agent, which was particularly important for ciprofloxacin.

Objectives: To explore the role of topoisomerase I in gene activation and increased RecA levels during the bacterial SOS response, as well as the effect of antibiotic treatment and stress challenge on cell killing initiated by trapped topoisomerase I cleavage complex., Methods: A mutant Escherichia coli strain with a ΔtopA mutation was used to investigate the role of topoisomerase I function in the SOS response to trimethoprim and mitomycin C. Induction of the recA and dinD1 promoters was measured using luciferase reporters of these promoters fused to luxCDABE. An increase in the RecA level following trimethoprim treatment was quantified directly by western blotting. The effect of stress challenge from trimethoprim and acidified nitrite treatments on cell killing by topoisomerase I cleavage complex accumulation was measured by the decrease in viability following induction of recombinant mutant topoisomerase I that forms a stabilized cleavage complex., Results: Topoisomerase I function was found to be required for efficient transcriptional activation of the recA and dinD1 promoters during the E. coli SOS response to trimethoprim and mitomycin C. The role of topoisomerase I in the SOS response was confirmed with quantitative western blot analysis of RecA following trimethoprim treatment. The bactericidal effect from topoisomerase I cleavage complex accumulation was shown to be enhanced by stress challenge from trimethoprim and acidified nitrite., Conclusions: Bacterial topoisomerase I function is actively involved in the SOS response to antibiotics and stress challenge. Cell killing initiated by the topoisomerase I cleavage complex would be enhanced by antibiotics and the host response. These findings provide further support for bacterial topoisomerase I as a therapeutic target.