Pseudomonas aeruginosa is a widespread γ-proteobacterium and an important opportunistic pathogen. The genetically diverse P. aeruginosa phylogroup 3 strains are characterized by producing the pore-forming ExlA toxin and by their lack of a type III secretion system. However, like all strains of this species, they produce several virulence-associated traits, such as elastase, rhamnolipids and pyocyanin, which are regulated by quorum sensing (QS). The P. aeruginosa QS response comprises three systems (Las, Rhl and Pqs, respectively) that hierarchically regulate these virulence factors. The Pqs QS system is composed of the PqsR transcriptional factor, which, coupled with the alkyl-quinolones HHQ or PQS, activates the transcription of the pqsABCDE operon. The products of the first four genes of this operon produce HHQ, which is then converted to PQS by PqsH, while PqsE forms a complex with RhlR and stabilizes it. In this study we report that mutations affecting the Pqs system are particularly common in phylogroup 3 strains. To better understand QS in phylogroup 3 strains we studied strain MAZ105 isolated from tomato rhizosphere and showed that it contains mutations in the central QS transcriptional regulator, LasR, and in the gene encoding the PqsA enzyme involved in the synthesis of PQS. However, it can still produce QS-regulated virulence factors and is virulent in Galleria mellonella and mildly pathogenic in the mouse abscess/necrosis model; our results show that this may be due to the expression of pqsE from a different PqsR-independent promoter than the pqsA promoter. Our results indicate that using anti-virulence therapy based on targeting the PQS system will not be effective against infections by P. aeruginosa phylogroup 3 strains.

Urinary tract infections (UTIs) are common bacterial infections caused mainly by enteric bacteria. Numerous virulence factors assist bacteria in the colonization of the bladder. Bacterial efflux pumps also contribute to bacterial communication and to biofilm formation. In this study, the phenotypic and genetic antibiotic resistance of clinical UTI pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis were determined by disk diffusion method and polymerase chain reaction (PCR). Following this, different classes of antibiotics were evaluated for their antibacterial activity at pH 5, 6, 7 and 8 by a microdilution method. Gentamicin (GEN) was the most potent antibacterial agent against E. coli strains. The effect of GEN on the relative expression of marR and sdiA genes was evaluated by quantitative PCR. The slightly acidic pH (pH 6) and GEN treatment induced the upregulation of marR antibiotic resistance and sdiA QS activator genes in both E. coli strains. Consequently, bacteria had become more susceptible to GEN. It can be concluded that antibiotic activity is pH dependent and so the artificial manipulation of urinary pH can contribute to a more effective therapy of multidrug resistant bacterial infections.

Although antibiotics have been indispensable in the advancement of modern medicine, there are downsides to their use. Growing resistance to broad-spectrum antibiotics is leading to an epidemic of infections untreatable by first-line therapies. Resistance is exacerbated by antibiotics used as growth factors in livestock, over-prescribing by doctors, and poor treatment adherence by patients. This generates populations of resistant bacteria that can then spread resistance genes horizontally to other bacterial species, including commensals. Furthermore, even when antibiotics are used appropriately, they harm commensal bacteria leading to increased secondary infection risk. Effective antibiotic treatment can induce bacterial survival tactics, such as toxin release and increasing resistance gene transfer. These problems highlight the need for new approaches to treating bacterial infection. Current solutions include combination therapies, narrow-spectrum therapeutics, and antibiotic stewardship programs. These mediate the issues but do not address their root cause. One emerging solution to these problems is anti-virulence treatment: preventing bacterial pathogenesis instead of using bactericidal agents. In this review, we discuss select examples of potential anti-virulence targets and strategies that could be developed into bacterial infection treatments: the bacterial type III secretion system, quorum sensing, and liposomes.

Hormesis is a biphasic dose-response relationship featured by low-dose stimulation and high-dose inhibition. Although the hormetic phenomenon has been extensively studied over the past decades, there is little information regarding the influence of energy source on the occurrence of hormesis, especially the time-dependent one. In this study, to explore the role of cultivation system\'s energy source in time-dependent hormesis, the toxic dose-responses of Aliivibrio fischeri (A. fischeri) bioluminescence to Sulfadoxine (SDX) during 24 h were determined in four cultivation systems with different energy source conditions. The results indicated that the time-dependent hormetic effects were induced by SDX in all cultivation systems: SDX triggered hormetic phenomenon on the bioluminescence at each growth stage over 24 h in the cultivation systems with sufficient and insufficient energy source; due to the diauxic growth of A. fischeri under multiple energy source conditions, the hormetic effects of SDX gradually disappeared after the preferred energy source was used up. It was speculated that the inhibitory action of SDX was derived from its interaction with DHPS to impede the synthesis of proteins, and SDX bound with AC to upregulate the quorum sensing (QS) system to exhibit the stimulatory action. Comparing the time-dependent hormesis in each cultivation system, it was obtained that the energy source could impact the hourly maximum stimulatory rate, the EC50 of SDX, and the time point that hormesis occurred, which might result from the influence of energy source on the stimulatory and inhibitory actions of SDX through regulating the metabolic system (individual level) and QS system (group level) of bacteria. This study clarifies the importance of energy source for hormesis occurrence, which may further promote the development of hormesis.

In an effort to find new repurposed antibacterial compounds, we performed the screening of an FDA-approved compounds library against Staphylococcus aureus American Type Culture Collection (ATCC) 25923. Compounds were evaluated for their capacity to prevent both planktonic growth and biofilm formation as well as to disrupt pre-formed biofilms. One of the identified initial hits was fingolimod (FTY720), an immunomodulator approved for the treatment of multiple sclerosis, which was then selected for follow-up studies. Fingolimod displayed a potent activity against S. aureus and S. epidermidis with a minimum inhibitory concentration (MIC) within the range of 12-15 µM at which concentration killing of all the bacteria was confirmed. A time-kill kinetic study revealed that fingolimod started to drastically reduce the viable bacterial count within two hours and we showed that no resistance developed against this compound for up to 20 days. Fingolimod also displayed a high activity against Acinetobacter baumannii (MIC 25 µM) as well as a modest activity against Escherichia coli and Pseudomonas aeruginosa. In addition, fingolimod inhibited quorum sensing in Chromobacterium violaceum and might therefore target this signaling pathway in certain Gram-negative bacteria. In conclusion, we present the identification of fingolimod from a compound library and its evaluation as a potential repurposed antibacterial compound.

Rehmannia glutinosa, a perennial medicinal plant, suffers from severe replant disease under consecutive monoculture. The rhizosphere microbiome is vital for soil suppressiveness to diseases and for plant health. Moreover, N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS) regulates diverse behavior in rhizosphere-inhabiting and plant pathogenic bacteria. The dynamics of short-chain AHL-mediated QS bacteria driven by consecutive monoculture and its relationships with R. glutinosa replant disease were explored in this study. The screening of QS bacteria showed that 65 out of 200 strains (32.5%) randomly selected from newly planted soil of R. glutinosa were detected as QS bacteria, mainly consisting of Pseudomonas spp. (55.4%). By contrast, 34 out of 200 (17%) strains from the diseased replant soil were detected as QS bacteria, mainly consisting of Enterobacteriaceae (73.5%). Functional analysis showed most of the QS bacteria belonging to the Pseudomonas genus showed strong antagonistic activities against Fusarium oxysporum or Aspergillus flavus, two main causal agents of R. glutinosa root rot disease. However, the QS strains dominant in the replant soil caused severe wilt disease in the tissue culture seedlings of R. glutinosa. Microbial growth assays demonstrated a concentration-dependent inhibitory effect on the growth of beneficial QS bacteria (i.e., Pseudomonas brassicacearum) by a phenolic acid mixture identified in the root exudates of R. glutinosa, but the opposite was true for harmful QS bacteria (i.e., Enterobacter spp.). Furthermore, it was found that the population of quorum quenching (QQ) bacteria that could disrupt the beneficial P. brassicacearum SZ50 QS system was significantly higher in the replant soil than in the newly planted soil. Most of these QQ bacteria in the replant soil were detected as Acinetobacter spp. The growth of specific QQ bacteria could be promoted by a phenolic acid mixture at a ratio similar to that found in the R. glutinosa rhizosphere. Moreover, these quorum-quenching bacteria showed strong pathogenicity toward the tissue culture seedlings of R. glutinosa. In conclusion, consecutive monoculture of R. glutinosa contributed to the imbalance between beneficial and harmful short-chain AHL-mediated QS bacteria in the rhizosphere, which was mediated not only by specific root exudates but also by the QQ bacterial community.

Time-dependent cross-phenomenon in which the cross between the actual concentration-response curve (CRC) for mixture crosses the CRCs for reference model varies with time has been frequently reported in previous studies, expressed as a heterogeneous pattern of joint toxic action. However, the variation tendency of time-dependent cross-phenomenon is rarely addressed. In this study, the joint toxic actions of binary antibacterial mixtures (i.e., two quorum sensing inhibitors, tetracycline hydrochloride, erythromycin, and chloramphenicol with sulfonamides) were judged using independent action (IA) model to find the variation tendency of time-dependent cross-phenomenon. The results show that the time-dependent cross-phenomena of the test binary antibacterial mixtures follow a unified variation tendency and the corresponding joint toxic actions change regularly with an increase of both concentration and time. Through investigating the relationship between the stimulatory and inhibitory modes of action for the single agents and the time-dependent cross-phenomena of binary mixtures, the regular time-dependent cross-phenomena is speculated to be derived from the hormetic effects of the components in the mixtures. This study offers an advance for the variation tendency and mechanistic explanation of time-dependent cross-phenomenon, which will provide a support for the future development in the exploration of time-dependent cross-phenomenon and environmental risk assessment of pollutant mixtures.

Hit, Lead & Candidate Discovery Drug repurposing has become a recent trend in drug development programs, where previously developed drugs are explored for hit and redeveloped into potential therapeutic agents for new diseases. Globally, in any drug development program, a series of molecules are synthesized and evaluated for the hypothesized activity. Hits are developed into lead molecules or drugs, whereas the negative ones are shelved in the lab with no immediate use. We in this project took the previously sidelined small chemical molecules to the next level of utility, where previously developed in-house small molecules library are tested for the unexplored biological relevant activity. As biofilm formation and quorum sensing play a vital role in bacterial pathogenesis, we believe that they could be one of the most effective targets for antimicrobial agents. In this study, we report the evaluation of 50 different compounds for anti-biofilm and anti-quorum sensing activity against Pseudomonas aeruginosa. Out of the screened compounds, three hydrazine-carboxamide hybrid derivatives showed promising anti-biofilm property and inhibition of pyocyanin production without any direct antimicrobial activity and cytotoxicity issues. Hydrazine-carboxamide hybrids can be a new class and promising leads for further anti-biofilm and anti-virulence development against microbial infections.

Hormesis occurs frequently in broadly ranging biological areas (e.g. plant biology, microbiology, biogerontology), toxicology, pharmacology and medicine. While numerous mechanisms (e.g. receptor and pathway mediated pathway responses) account for stimulatory and inhibitory features of hormetic dose responses, the vast majority emphasizes the inclusion of many doses but only one timepoint or use of a single optimized dose that is assessed over a broad range of timepoints. In this paper, a toxicity study was designed using a large number of properly spaced doses with responses determined over a large number of timepoints, which could help us reveal the underlying mechanism of hormesis. We present the results of a dose-time-response study on hormesis using five antibacterial chemicals on the bioluminescence of Aliivibrio fischeri, measuring expression of protein mRNA based on quorum sensing, simulating bioluminescent reaction and analyzing toxic actions of test chemicals. The findings show dose-time-dependent responses conforming to the hormetic dose-response model, while revealing unique response dynamics between agent induced stimulatory and inhibitory effects within bacterial growth phase dynamics. These dynamic dose-time features reveal a type of biological seesaw model that integrates stimulatory and inhibitory responses within unique growth phase, dose and time features, which has faultlessly explained the time-dependent hormetic phenomenon induced by five antibacterial chemicals (characterized by low-dose stimulation and high-dose inhibition). This study offers advances in understanding cellular dynamics, the biological integration of diverse and opposing responses and their role in evolutionary adaptive strategies to chemicals, which can provide new insight into the mechanistic investigation of hormesis.

Cross-phenomenon in which the concentration-response curve (CRC) for a mixture crosses the CRC for the reference model has been identified in many studies, expressed as a heterogeneous pattern of joint toxic action. However, a mechanistic explanation of the cross-phenomenon has thus far been extremely insufficient. In this study, a time-dependent cross-phenomenon was observed, in which the cross-concentration range between the CRC for the mixture of sulfamethoxypyridazine (SMP) and (Z-)-4-Bromo-5-(bromomethylene)-2(5H)-furanone (C30) to the bioluminescence of Aliivibrio fischeri (A. fischeri) and the CRC for independent action model with 95% confidence bands varied from low-concentration to higher-concentration regions in a timely manner expressed the joint toxic action of the mixture changing with an increase of both concentration and time. Through investigating the time-dependent hormetic effects of SMP and C30 (by measuring the expression of protein mRNA, simulating the bioluminescent reaction and analyzing the toxic action), the underlying mechanism was as follows: SMP and C30 acted on the quorum sensing (QS) system of A. fischeri, which induced low-concentration stimulatory effects and high-concentration inhibitory effects; in the low-concentration region, the stimulatory effects of SMP and C30 made the mixture produce a synergistic stimulation on the bioluminescence; thus, the joint toxic action exhibited antagonism. In the high-concentration region, the inhibitory effects of SMP and C30 in the mixture caused a double block in the loop circuit of the QS system; thus, the joint toxic action exhibited synergism. With the increase of time, these stimulatory and inhibitory effects of SMP and C30 were changed by the variation of the QS system at different growth phases, resulting in the time-dependent cross-phenomenon. This study proposes an induced mechanism for time-dependent cross-phenomenon based on QS, which may provide new insight into the mechanistic investigation of time-dependent cross-phenomenon, benefitting the environmental risk assessment of mixtures.