Antimicrobial Drug Efflux Systems as Components of Bacterial Stress Responses

Antimicrob Agents Chemother

Corona

Martínez

2018

Multidrug resistance efflux pumps frequently present low levels of basal expression. However, antibiotic-resistant mutants that overexpress these resistance determinants are selected during infection. In addition, increased expression of efflux pumps can be induced by environmental signals/cues, which can lead to situations of transient antibiotic resistance. In this study, we have applied a novel high-throughput methodology in order to identify inducers able to trigger the expression of the SmeVWX and SmeYZ efflux pumps. To that end, bioreporters in which the expression of the yellow fluorescent protein (YFP) is linked to the activity of either or promoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. YFP production was also measured by flow cytometry, and the levels of expression of and in the presence of a set of selected compounds were also determined by real-time reverse transcription-PCR (RT-PCR). The expression of was induced by iodoacetate, clioquinol, and selenite, while boric acid, erythromycin, chloramphenicol, and lincomycin triggered expression. The susceptibility to antibiotics that are known substrates of the efflux pumps decreased in the presence of the inducers. However, the analyzed multidrug efflux systems did not contribute to resistance to the studied inducers. To sum up, the use of fluorescent bioreporters in combination with Biolog plates is a valuable tool for identifying inducers of the expression of bacterial multidrug resistance efflux pumps, and likely of other bacterial systems whose expression is regulated in response to signals/cues.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Biolog Phenotype Microarray Is a Tool for the Identification of Multidrug Resistance Efflux Pump Inducers

Antimicrob Agents Chemother

Corona

Martínez

2018

“…MDR efflux pumps can also take part of general mechanisms of response to cellular stress that contribute to ameliorate the adverse effects caused by stressors agents or stress by-products (e.g. oxidative stress or envelope stress) (18, 40). We hypothesize that when MDR efflux systems are participating in this global cellular stress response, a general common inducer mechanism of stress could be identified through the analysis of their inducer compounds, despite their structural diversity.…”

Section: Discussionmentioning

confidence: 99%

“…Besides the assigned specific function of RND efflux pumps, these systems are in occasions considered stress defense determinants (e.g. oxidative and nitrosative stress), acting as scape valves for the accumulation of toxic compounds or stress by-products (18).…”

Section: Introductionmentioning

confidence: 99%

Biolog phenotype microarray: a tool for the identification of multidrug resistance efflux pumps inducers

Corona

Martínez

2018

Preprint

1Overexpression of multidrug resistance efflux pumps is a relevant mechanism of antibiotic 2 resistance for bacterial pathogens. These systems use to present low levels of basal 3 expression. However, they can be induced by environmental signals or stresses which can 4 lead to situations of phenotypic induced resistance. In contrast to efflux pumps substrates, 5 inducers of these systems have not been thoroughly studied. In this work, we have applied a 6 novel high-throughput methodology in order to identify inducer molecules of the 7Stenotrophomonas maltophilia SmeVWX and SmeYZ efflux pumps. To that goal, 8 bioreporters in which the expression of the yellow fluorescent protein is linked to the activity 9 of either the smeVWX or the smeYZ promoters were developed and used for the screening of 10 potential inducers of the expression of these efflux pumps using Biolog phenotype 11 microarrays. Confirmation of induction was carried out measuring YFP production along the 12 bacterial growth and by flow cytometry; mRNA levels of smeV and smeY were also 13 determined by real-time RT-PCR after exposure to the selected compounds. Among the 144 14 tested compounds, iodoacetate, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and sodium 15 selenite were found to be smeVWX inducers, while boric acid, erythromycin, chloramphenicol 16 and lincomycin are able to trigger the expression of smeYZ. While the presence of the 17 inducers allowed a decrease in the susceptibility to antibiotics that are known substrates of 18 the efflux pumps, our results indicate that these efflux pumps did not contribute to S. 19 maltophilia resistance to the analyzed inducers. 20

“…It is worth mentioning that, besides being AR determinants (16, 17), efflux pumps present other physiological functions important for bacterial behavior, such as modulation of Quorum Sensing (QS) signaling (18–20), response to stress situations (21) and to host defenses (22–24), or plant/bacteria interactions (25, 26). Although the basal level of expression of each efflux pump can vary, it is well established that their expression may increase in the presence of some compounds or situations (27).…”

Section: Introductionmentioning

confidence: 99%

Novel inducers of the expression of multidrug efflux pumps that triggerPseudomonas aeruginosatransient antibiotic resistance

Laborda

Alcalde-Rico

et al. 2019

Preprint

The study of the acquisition of antibiotic resistance (AR) has mainly focused in inherited processes, namely mutations and acquisition of AR genes. However, inducible, non-inheritable AR has received less attention and most information in this field derives from the study of antibiotics as inducers of their associated resistance mechanisms. Less is known about non-antibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tightly regulation of their expression. Their contribution to acquired AR relies in their overexpression. Herein we analyzed potential inducers of the expression of the chromosomally-encoded Pseudomonas aeruginosa clinically-relevant efflux pumps, MexCD-OprJ and MexAB-OprM. For this purpose, we developed a set of luxCDABE-based P. aeruginosa biosensor strains, which allows the high-throughput analysis of compounds able of modifying the expression of these efflux pumps. Using these strains, we analyzed a set of 240 compounds present in Biolog Phenotype Microarrays. Several inducers of the expression of the genes that encode these efflux pumps were found. The study focused in dequalinium chloride, procaine and atropine, compounds that can be found in clinical settings. Using real-time PCR, we confirmed that these compounds indeed induce the expression of mexCD-oprJ. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine or atropine are present. This work emphasizes the need of studying compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests.