Effect of different drugs and drug combinations on killing stationary phase and biofilms recovered cells of Bartonella henselae in vitro.

BACKGROUND: Bartonella henselae is a Gram-negative bacterium transmitted to humans by a scratch from cat in the presence of ectoparasites. Humans infected with B. henselae can result in various clinical diseases including local lymphadenopathy and more serious systemic disease such as persistent bacteremia and endocarditis. The current treatment of persistent B. henselae infections is not very effective and remains a challenge. To find more effective treatments for persistent and biofilm Bartonella infections, in this study, we evaluated a panel of drugs and drug combinations based on the current treatment and also promising hits identified from a recent drug screen against stationary phase and biofilm recovered cells of B. henselae. RESULTS: We evaluated 14 antibiotics and 25 antibiotic combinations for activity against stationary phase B. henselae (all antibiotics were at 5 µg/ml) and found that ciprofloxacin, gentamicin, and nitrofurantoin were the most active agents, while clofazimine and miconazole had poor activity. Drug combinations azithromycin/ciprofloxacin, azithromycin/methylene blue, rifampin/ciprofloxacin, and rifampin/methylene blue could rapidly kill stationary phase B. henselae with no detectable CFU after 1-day exposure. Methylene blue and rifampin were the most active agents against the biofilm B. henselae after 6 days of drug exposure. Antibiotic combinations (azithromycin/ciprofloxacin, azithromycin/methylene blue, rifampin/ciprofloxacin, rifampin/methylene blue) completely eradicated the biofilm B. henselae after treatment for 6 days. CONCLUSIONS: These findings may facilitate development of more effective treatment of persistent Bartonella infections in the future.

Introducing RpsA Point Mutations Δ438A and D123A into the Chromosome of Mycobacterium tuberculosis Confirms Their Role in Causing Resistance to Pyrazinamide.

Pyrazinamide (PZA) is a unique frontline drug for shortening tuberculosis (TB) treatment, but its mechanisms of action are elusive. We previously found one PZA-resistant strain that harbors an alanine deletion at position 438 (Δ438A) in RpsA, a target of PZA associated with PZA resistance, but its role in causing PZA resistance has been inconclusive. Here, we introduced the RpsA Δ438A mutation along with the D123A mutation into the Mycobacterium tuberculosis chromosome and demonstrated that these RspA mutations are indeed responsible for PZA resistance.

Identification of Novel Mutations in LprG (rv1411c), rv0521, rv3630, rv0010c, ppsC, and cyp128 Associated with Pyrazinoic Acid/Pyrazinamide Resistance in Mycobacterium tuberculosis.

Despite progress, the mechanisms of action and resistance of pyrazinamide (PZA) are not well understood. We characterized 109 mutants resistant to pyrazinoic acid (POA), the active form of PZA, and found that while most (n = 101 [93%]) mutants had panD mutations and 4 (4%) had clpC1 mutations (S91G), new mutations in lprG (rv1411c) and rv0521 (n = 4 [4%]), rv3630, rv0010c, ppsC, and cyp128 (cytochrome P450 128) were identified, shedding new light on the mechanisms of action and resistance of PZA in M. tuberculosis.

Novel Mutations Associated with Clofazimine Resistance in Mycobacterium abscessus.

Mycobacterium abscessus is a major nontuberculous mycobacterial (NTM) pathogen and is responsible for about 80% of all pulmonary infections caused by rapidly growing mycobacteria. Clofazimine is an effective drug active against M. abscessus, but the mechanism of resistance to clofazimine in M. abscessus is unknown. To investigate the molecular basis of clofazimine resistance in M. abscessus, we isolated 29 M. abscessus mutants resistant to clofazimine and subjected them to whole-genome sequencing to identify possible mutations associated with clofazimine resistance. We found that mutations in the MAB_2299c gene (which encodes a possible transcriptional regulatory protein), MAB_1483, and MAB_0540 are most commonly associated with clofazimine resistance. In addition, mutations in MAB_0416c, MAB_4099c, MAB_2613, MAB_0409, and MAB_1426 were also associated with clofazimine resistance but less frequently. Two identical mutations which are likely to be polymorphisms unrelated to clofazimine resistance were found in MAB_4605c and MAB_4323 in 13 mutants. We conclude that mutations in MAB_2299c, MAB_1483, and MAB_0540 are the major mechanisms of clofazimine resistance in M. abscessus Future studies are needed to address the role of the identified mutations in clofazimine resistance in M. abscessus Our findings have implications for understanding mechanisms of resistance to clofazimine and for rapid detection of clofazimine resistance in this organism.

Identification of novel mutations associated with cycloserine resistance in Mycobacterium tuberculosis.

OBJECTIVES: d-Cycloserine is an important second-line drug used to treat MDR- and XDR-TB. However, the mechanisms of resistance to d-cycloserine are not well understood. Here we investigated the molecular basis of d-cycloserine resistance using in vitro-isolated resistant mutants. METHODS: Mycobacterium tuberculosis H37Rv was subjected to mutant selection on 7H11 agar plates containing varying concentrations of d-cycloserine. A total of 18 d-cycloserine-resistant mutants were isolated and subjected to WGS. The identified mutations associated with d-cycloserine resistance were confirmed by PCR and Sanger sequencing. RESULTS: We identified mutations in 16 genes that are associated with d-cycloserine resistance. Interestingly, we found mutations only in alr (rv3423c) encoding alanine racemase, but not in other known d-cycloserine resistance-associated genes such as ddl, cycA or ald. Instead, we identified 13 new genes [rv0059, betP (rv0917), rv0221, rv1403c, rv1683, rv1726, gabD2 (rv1731), rv2749, sugI (rv3331), hisC2 (rv3772), the 5' intergenic region of rv3345c and rv1435c, and the 3' region of rv0759c] that had solo mutations associated with d-cycloserine resistance. Our findings indicate that the mechanisms of d-cycloserine resistance are more complex than previously thought and involve genes participating in different cellular functions such as lipid metabolism, methyltransferase, the stress response and transport systems. CONCLUSIONS: New mutations in diverse genes associated with d-cycloserine resistance have been identified that shed new light on the mechanisms of action and resistance of d-cycloserine. Future studies are needed to verify these findings in clinical strains so that molecular detection of d-cycloserine resistance for improved treatment of MDR-TB can be developed.

Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics.

Persisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active against Escherichia coli persisters at high concentrations (25 or 50 µg/ml). At a clinically relevant lower concentration (10 µg/ml), colistin alone had no apparent effect on E. coli persisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugs in vitro The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

Mycobacterium tuberculosis Mutations Associated with Reduced Susceptibility to Linezolid.

Linezolid (LZD) has become increasingly important for the treatment of multidrug-resistant tuberculosis (MDR-TB), but its mechanisms of resistance are not well characterized. We isolated 32 mutants ofMycobacterium tuberculosiswith reduced susceptibility to LZD, which was accounted for byrrlandrplCmutations in almost equal proportions, causing lower and higher MICs, respectively. Our findings provide useful information for the rapid detection of LZD resistance for improved treatment of MDR-TB.

Conditions and mutations affecting Staphylococcus aureus L-form formation.

Staphylococcus aureus is a prominent human pathogen and is known to form L-forms in vitro and in vivo during infection. However, the conditions of L-form formation are not optimal and the mechanisms of L-form formation in this organism are unknown. Here, we optimized the conditions of S. aureus unstable L-form formation, constructed a transposon mutant library and screened for mutants defective in unstable L-form formation. Our results revealed that 20 % sucrose, 3.5 % sodium chloride, 750-1000 U penicillin and 33 °C were optimal conditions for L-form formation. Stationary phase cultures of S. aureus formed L-forms better than exponential phase cultures. The S. aureus L-form colonies showed typical 'fried-egg' morphology and the cells had deficient cell wall, showed morphological diversity, and stained Gram-negative. The mutant library screens identified 15 mutants deficient in L-form formation and sequencing analysis identified mutations in eight genes and three intergenic regions. Real-time PCR analysis indicated that, with the exception of gntK, seven genes including glpF, glpK, NWMN_0623, NWMN_0843, NWMN_0333, NWMN_0872 and NWMN_1269 were preferentially expressed in L-forms as compared with normal cell-walled form (P<0.05). The identified genes involved in L-form growth mapped in the pathways for energy production, iron homeostasis, transporters, DNA repair, membrane biogenesis, and biosynthesis. Our findings shed new insight into the molecular basis of S. aureus unstable L-form formation and may have implications for development of novel drugs targeting S. aureus L-forms for improved treatment.

Identification of novel mutations associated with clofazimine resistance in Mycobacterium tuberculosis.

OBJECTIVES: Although clofazimine has been traditionally used to treat leprosy, there is recent interest in using clofazimine for the treatment of MDR-TB and drug-susceptible TB. However, the mechanisms of resistance to clofazimine are poorly understood. Here, we investigated the molecular basis of clofazimine resistance using resistant mutants isolated in vitro. METHODS: We isolated 96 mutants of Mycobacterium tuberculosis resistant to clofazimine and performed WGS and Sanger sequencing to identify possible mutations associated with clofazimine resistance. RESULTS: We found that 97% (93/96) of clofazimine-resistant mutants had a mutation in rv0678 encoding a transcription repressor for efflux pump MmpL5. Two mutational hot spots at nucleotide positions 193 and 466 in rv0678 accounted for 43.8% (42/96) and 11.5% (11/96) of the mutations, respectively. The previously reported A202G mutation (S68G) in rv0678 occurred less frequently, in 5 of 96 mutants. The remaining 34 mutations were scattered along the entire rv0678 gene. We discovered two new genes (rv1979c and rv2535c) associated with clofazimine resistance in mutants without rv0678 mutations. CONCLUSIONS: Mutations in rv0678 are a major mechanism of clofazimine resistance. Our findings provide useful information for the design of new molecular tests for rapid detection of clofazimine resistance. Further studies are needed to address the role of rv1979c and rv2535c in clofazimine resistance and mechanisms of action.

Trans-translation mediates tolerance to multiple antibiotics and stresses in Escherichia coli.

BACKGROUND: Trans-translation mediated by SsrA (tmRNA) and its associated protein SmpB plays an important role in rescuing stalled ribosomes and detoxifying toxic protein products under stress conditions. However, the role of SsrA and SmpB in bacterial persister survival has not been studied. The recent finding that pyrazinamide as a unique persister drug inhibits trans-translation in Mycobacterium tuberculosis prompted us to examine the role of trans-translation in persister survival. METHODS: Using Escherichia coli as a model, we constructed SsrA and SmpB mutants and assessed the susceptibility of the mutants to various antibiotics and stress conditions in MIC/MBC and persister assays. RESULTS: We found that mutations in SsrA and SmpB caused a defect in persister survival as shown by their increased susceptibility to a variety of antibiotics, including gentamicin, streptomycin, amikacin, norfloxacin, trimethoprim and tetracycline, and also stresses, such as acid, weak acid salicylate, heat and peroxide. Additionally, the SsrA and SmpB mutants were 2-8-fold more susceptible than the parent strain to various antibiotics in MIC and MBC tests. The SmpB mutant was more susceptible to antibiotics and stresses than the SsrA mutant. A particularly interesting finding is the hypersusceptibility of the SmpB mutant and the SsrA mutant to trimethoprim. The defect of various SsrA and SmpB mutant phenotypes could be complemented by functional ssrA and smpB, respectively. CONCLUSIONS: We conclude that SsrA and SmpB are important for persister survival and may serve as a good target for developing new antibiotics that kill persister bacteria for improved treatment of persistent bacterial infections.

Analogs of N'-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide inhibit Mycobacterium tuberculosis methionine aminopeptidases.

Our previous target validation studies established that inhibition of methionine aminopeptidases (MtMetAP, type 1a and 1c) from Mycobacterium tuberculosis (Mtb) is an effective approach to suppress Mtb growth in culture. A novel class of MtMetAP1c inhibitors comprising of N'-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide (4c) was uncovered through a high-throughput screen (HTS). A systematic structure-activity relationship study (SAR) yielded variants of the hit, 4b, 4h, and 4k, bearing modified A- and B-rings as potent inhibitors of both MtMetAPs. Except methanimidamide 4h that showed a moderate Mtb inhibition, a desirable minimum inhibitory concentration (MIC) was not obtained with the current set of MtMetAP inhibitors. However, the SAR data generated thus far may prove valuable for further tuning of this class of inhibitors as effective anti-tuberculosis agents.

Activity of Pyrazinamide against Mycobacterium tuberculosis at Neutral pH in PZA-S1 Minimal Medium.

Susceptibility testing of tuberculosis (TB) drugs on Mycobacterium tuberculosis is essential for the rapid detection of strains resistant to the drugs, providing the patient with effective treatment, and preventing the spread of drug-resistant TB strains. Pyrazinamide (PZA) is one of the first-line agents used for the treatment of TB. However, current phenotypic PZA susceptibility testing is unreliable due to its performance in acidic pH conditions. The aims of this study were to develop minimal media to determine the activity of PZA at a neutral pH at 37 °C to avoid problems caused by an acidic pH, which is currently used in PZA susceptibility tests, and to identify PZA-resistant M. tuberculosis in media with reproducibility and accuracy. Different minimal media were used to determine the activity of PZA using the broth microdilution method with M. tuberculosis H37Ra as the reference strain. The PZA-S1 minimal medium was proposed as the most suitable medium. PZA inhibited the growth of M. tuberculosis in PZA-S1 at a neutral pH of 6.8, which is the optimal pH for M. tuberculosis growth. Moreover, PZA showed activity at a neutral pH on a PZA-S1 agar plate when using the disk diffusion method. PZA-resistant M. tuberculosis could be identified at a neutral pH in PZA-S1 minimal medium. This study establishes valuable information regarding the testing of PZA's susceptibility in relation to M. tuberculosis at a neutral pH of 6.8 with reliability and accuracy in clinical settings.

PhoY2 but not PhoY1 is the PhoU homologue involved in persisters in Mycobacterium tuberculosis.

OBJECTIVES: Mycobacterial persistence is thought to be the underlying cause of the current lengthy tuberculosis therapy and latent infection. Despite some recent progress, the mechanisms of bacterial persistence are poorly understood. We have recently identified a new persister gene phoU from Escherichia coli and have shown that the phoU mutant has a defect in persisters. The objective of this study is to evaluate the role of two phoU homologues phoY1 and phoY2 from Mycobacterium tuberculosis in mycobacterial persistence. METHODS: M. tuberculosis phoY1 and phoY2 mutant strains were constructed. The persister-related phenotypes of the phoY1 and phoY2 mutants were assessed in vitro by MIC testing, drug exposure assays and also by survival in the mouse model of tuberculosis infection. RESULTS: We demonstrated that M. tuberculosis PhoY2 is the equivalent of E. coli PhoU in that inactivation of phoY2 but not phoY1 caused a defect in persistence phenotype as shown by increased susceptibility to rifampicin and pyrazinamide in both MIC testing and drug exposure assays and also reduced persistence in the mouse model. CONCLUSIONS: This study provides further validation that PhoU is involved in persistence not only in E. coli but also in M. tuberculosis and has implications for the development of new drugs targeting persisters for improved treatment.

Identification of essential oils with activity against stationary phase Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is the most dominant human pathogen, responsible for a variety of chronic and severe infections. There is mounting evidence that persisters are associated with treatment failure and relapse of persistent infections. While some essential oils were reported to have antimicrobial activity against growing S. aureus, activity of essential oils against the stationary phase S. aureus enriched in persisters has not been investigated. METHODS: In this study, we evaluated the activity of 143 essential oils against both growing and stationary phase S. aureus by minimum inhibitory concentration (MIC) testing and by colony forming unit assay. RESULTS: We identified 39 essential oils (Oregano, Cinnamon bark, Thyme white, Bandit "Thieves", Lemongrass (Cymbopogon flexuosus), Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa, Cinnamon leaf, Clove bud, Citronella, Geranium bourbon, Marjoram, Peppermint, Lemongrass, Cornmint, Elemi, Ho wood, Head ease, Lemon eucalyptus, Litsea cubeba, Myrrh, Parsley seed, Coriander oil, Dillweed, Hyssop, Neroli, Rosewood oil, Tea tree, Cajeput, Clove bud, Lavender, Sleep tight, Vetiver, Palo santo, Sage oil, Yarrow) at 0.5% (v/v) concentration, 10 essential oils (Cinnamon bark, Oregano, Thyme white, Bandit "Thieves", Lemongrass, Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa at 0.25% (v/v) concentration, and 7 essential oils (Oregano, Cinnamon bark, Thyme white, Lemongrass, Allspice, Amyris, Palmarosa at 0.125% (v/v) concentration to have high activity against stationary phase S. aureus with no visible growth on agar plates after five-day exposure. Among the 10 essential oils which showed high activity at 0.25% (v/v) concentration, 9 (Oregano, Cinnamon bark, Thyme white, Bandit "Thieves", Lemongrass, Health shield, Allspice, Palmarosa, Amyris showed higher activity than the known persister drug tosufloxacin, while Sandalwood oil had activity at a higher concentration. In Oregano essential oil combination studies with antibiotics, Oregano plus tosufloxacin (or levofloxacin, ciprofloxacin) and rifampin completely eradicated stationary phase S. aureus cells, but had no apparent enhancement for linezolid, vancomycin, sulfamethoxazole, trimethoprim, azithromycin or gentamicin. CONCLUSIONS: Our findings indicate that some essential oils have excellent activity against both growing and stationary phase S. aureus. Further studies are needed to identify the active components, evaluate safety, pharmacokinetics, and their activity to eradicate S. aureus infections in vivo.

Evaluation of Disulfiram Drug Combinations and Identification of Other More Effective Combinations against Stationary Phase Borrelia burgdorferi.

Lyme disease, caused by Borrelia burgdorferi, is the most common vector-borne disease in USA, and 10-20% of patients will develop persistent symptoms despite treatment ("post-treatment Lyme disease syndrome"). B. burgdorferi persisters, which are not killed by the current antibiotics for Lyme disease, are considered one possible cause. Disulfiram has shown to be active against B. burgdorferi, but its activity against persistent forms is not well characterized. We assessed disulfiram as single drug and in combinations against stationary-phase B. burgdorferi culture enriched with persisters. Disulfiram was not very effective in the drug exposure experiment (survival rate (SR) 46.3%) or in combinations. Clarithromycin (SR 41.1%) and nitroxoline (SR 37.5%) were equally effective when compared to the current Lyme antibiotic cefuroxime (SR 36.8%) and more active than disulfiram. Cefuroxime + clarithromycin (SR 25.9%) and cefuroxime + nitroxoline (SR 27.5%) were significantly more active than cefuroxime + disulfiram (SR 41.7%). When replacing disulfiram with clarithromycin or nitroxoline in three-drug combinations, bacterial viability decreased significantly and subculture studies showed that combinations with these two drugs (cefuroxime + clarithromycin/nitroxoline + furazolidone/nitazoxanide) inhibited the regrowth, while disulfiram combinations did not (cefuroxime + disulfiram + furazolidone/nitazoxanide). Thus, clarithromycin and nitroxoline should be further assessed to determine their role as potential treatment alternatives in the future.

Identification of Essential Oils Including Garlic Oil and Black Pepper Oil with High Activity against Babesia duncani.

Some evidence indicated that human babesiosis caused by Babesia duncani has spread widely in North America. However, current therapeutic regimens (atovaquone + azithromycin) for human babesiosis are suboptimal with frequent recrudescence and side effects, and furthermore, there is no specific treatment for human babesiosis caused by B. duncani. Here, we screened 97 essential oils and identified 10 essential oils (garlic, black pepper, tarragon, palo santo, coconut, pine, meditation, cajeput, moringa, and stress relief) at a low concentration (0.001%; v/v) that showed good inhibitory activity against B. duncani in the hamster red blood cell culture model. Among them, garlic oil and black pepper oil performed best, as well as their potential active ingredients diallyl disulfide (DADS) and ß-caryophyllene (BCP), respectively. Interestingly, further subculture study indicated that B. duncani could relapse after treatment with current therapeutic drugs atovaquone or azithromycin even at high concentrations. In contrast, the combination of garlic oil or DADS and azithromycin showed eradication of B. duncani at low concentrations without regrowth. These results are encouraging and suggest that the garlic-derived sulfur compound DADS and ß-caryophyllene (BCP) may be promising drug candidates for evaluation of their ability to cure persistent B. duncani infections in the future.

The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD.

Pyrazinamide has been a mainstay in the multidrug regimens used to treat tuberculosis. It is active against the persistent, non-replicating mycobacteria responsible for the protracted therapy required to cure tuberculosis. Pyrazinamide is a pro-drug that is converted into pyrazinoic acid (POA) by pyrazinamidase, however, the exact target of the drug has been difficult to determine. Here we show the enzyme PanD binds POA in its active site in a manner consistent with competitive inhibition. The active site is not directly accessible to the inhibitor, suggesting the protein must undergo a conformational change to bind the inhibitor. This is consistent with the slow binding kinetics we determined for POA. Drug-resistant mutations cluster near loops that lay on top of the active site. These resistant mutants show reduced affinity and residence time of POA consistent with a model where resistance occurs by destabilizing the closed conformation of the active site.

Identification of a novel gene argJ involved in arginine biosynthesis critical for persister formation in Staphylococcus aureus.

Staphylococcus aureus can cause both acute and recurrent persistent infections such as peritonitis, endocarditis, abscesses, osteomyelitis, and chronic wound infections. Effective therapies to treat persistent disease are paramount. However, the mechanisms of S. aureus persistence are poorly understood. In this study, we performed a comprehensive and unbiased high-throughput mutant screen against a transposon-insertion mutant library of S. aureus USA300 and focused on the role of argJ encoding an acetyltransferase in the arginine biosynthesis pathway, whose transposon insertion caused a significant defect in persister formation using multiple drugs and stresses. Genetic complementation and arginine supplementation restored persistence in the argJ transposon insertion mutant while generation of mutations on the active site of the ArgJ protein caused a defect in persistence. Quantitative RT-PCR analysis showed that the genes encoded in the arg operon were over-expressed under drug stressed conditions and in stationary phase cultures. In addition, the argJ mutant had attenuated virulence in both mouse and C. elegans. Our studies identify a new mechanism of persistence mediated by arginine metabolism in S. aureus. These findings provide not only novel insights about the mechanisms of S. aureus persistence but also offer novel therapeutic targets that may help to develop more effective treatment of persistent S. aureus infections.

Identification and Ranking of Clinical Compounds with Activity Against Log-phase Growing Uropathogenic Escherichia coli.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a major cause of Urinary Tract Infections (UTIs). Due to increasing antibiotic-resistance among UPEC bacteria, new treatment options for UTIs are urgently needed. OBJECTIVE: To identify new agents targeting growing bacteria that may be used for the treatment of antibiotic-resistant UTIs. METHODS: We screened a clinical compound library consisting of 1,524 compounds using a high throughput 96-well plate assay and ranked the activities of the selected agents according to their MICs against the UPEC strain UTI89. RESULTS: We identified 33 antibiotics which were active against log-phase clinical UPEC strain UTI89. Among the selected antibiotics, there were 12 fluoroquinolone antibiotics (tosufloxacin, levofloxacin, sparfloxacin, clinafloxacin, pazufloxacin, gatifloxacin, enrofloxacin, lomefloxacin, norfloxacin, fleroxacin, flumequine, ciprofloxacin), 15 beta-lactam or cephalosporin antibiotics (cefmenoxime, cefotaxime, ceftizoxime, cefotiam, cefdinir, cefoperazone, cefpiramide, cefamandole, cefixime, ceftibuten, cefmetazole, cephalosporin C, aztreonam, piperacillintazobactam, mezlocillin), 3 tetracycline antibiotics (meclocycline, doxycycline, tetracycline), 2 membrane-acting agents (colistin and clofoctol), and 1 protein synthesis inhibitor (amikacin). Among them, the top 7 hits were colistin, tosufloxacin, levofloxacin, sparfloxacin, clinafloxacin, cefmenoxime and pazufloxacin, where clinafloxacin and pazufloxacin were the newly identified agents active against UPEC strain UTI89. We validated the key results obtained with UTI89 on two other UTI strains CFT073 and KTE181 and found that they all had comparable MICs for fluoroquinolones while CFT073 and KTE181 were more susceptible to cephalosporin antibiotics and tetracycline antibiotics but were less susceptible to colistin than UTI89. CONCLUSION: Our findings provide possible effective drug candidates for the more effective treatment of antibiotic-resistant UTIs.