Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in Chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa.

Introduction: Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum ß-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Methods: Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Results: Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla PER. One isolate carried bla PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum ß-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other ß-lactamsin the in vitro evolved isolate. Discussion: PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other ß-lactams.

Mutations in the efflux pump regulator MexZ shift tissue colonization by Pseudomonas aeruginosa to a state of antibiotic tolerance.

Mutations in mexZ, encoding a negative regulator of the expression of the mexXY efflux pump genes, are frequently acquired by Pseudomonas aeruginosa at early stages of lung infection. Although traditionally related to resistance to the first-line drug tobramycin, mexZ mutations are associated with low-level aminoglycoside resistance when determined in the laboratory, suggesting that their selection during infection may not be necessarily, or only, related to tobramycin therapy. Here, we show that mexZ-mutated bacteria tend to accumulate inside the epithelial barrier of a human airway infection model, thus colonising the epithelium while being protected against diverse antibiotics. This phenotype is mediated by overexpression of lecA, a quorum sensing-controlled gene, encoding a lectin involved in P. aeruginosa tissue invasiveness. We find that lecA overexpression is caused by a disrupted equilibrium between the overproduced MexXY and another efflux pump, MexAB, which extrudes quorum sensing signals. Our results indicate that mexZ mutations affect the expression of quorum sensing-regulated pathways, thus promoting tissue invasiveness and protecting bacteria from the action of antibiotics within patients, something unnoticeable using standard laboratory tests.

A whole-cell hypersensitive biosensor for beta-lactams based on the AmpR-AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20.

Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta-lactams are lacking. To address this gap, a biosensor based on the AmpC beta-lactamase regulation system (ampR-ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR-AmpC system is well-conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta-lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta-lactam hypersensitive strain, generating a whole-cell biosensor. The biosensor demonstrated dose-dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1-10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta-lactam antibiotics, specifically carbapenems, in different matrices.

Activity of imipenem/relebactam and comparators against KPC-producing Klebsiella pneumoniae and imipenem-resistant Pseudomonas aeruginosa.

PURPOSE: Relebactam is a novel ß-lactamase inhibitor, which, when combined with imipenem/cilastatin, is active against both class A and class C ß-lactamases. To evaluate in vitro antimicrobial activity of imipenem/relebactam against a collection of recent clinical isolates of carbapenem-non-susceptible P. aeruginosa and K. pneumoniae ST258 and ST512 KPC producers belonging to different lineages from hospitals in Southern Spain. METHODS: Six hundred and seventy-eight isolates were tested: 265 K. pneumoniae (230 ST512/KPC-3 and 35 ST258/KPC-3) and 413 carbapenem-non-susceptible P. aeruginosa. Imipenem, piperacillin/tazobactam, ceftazidime, cefepime, aztreonam, ceftolozane/tazobactam, meropenem, amikacin, ciprofloxacin, colistin, and ceftazidime/avibactam were used as comparators against P. aeruginosa. Against K. pneumoniae ceftazidime, cefepime, aztreonam, and ceftolozane/tazobactam were not tested, and tigecycline was studied instead. MICs were determined in duplicate by broth microdilution according to EUCAST guidelines. RESULTS: Imipenem/relebactam displayed potent in vitro activity against both sequence types of KPC-3-producing K. pneumoniae. MIC50 and MIC90 values were 0.25 mg/L and 1 mg/L, respectively, with percent of susceptible isolates >97%. Only three K. pneumoniae ST512/KPC-3 isolates and one ST258/KPC-3 were resistant to imipenem/relebactam. Relebactam sensitized 98.5% of K. pneumoniae isolates resistant to imipenem. The activity of imipenem/relebactam against P. aeruginosa was moderate (susceptibility rate: 62.7%). Analysis of the acquired and mutational resistome of isolates with high levels of resistance to imipenem/relebactam has not shown a clear association between them. CONCLUSION: Imipenem/relebactam showed excellent activity against K. pneumoniae KPC-3. The activity of imipenem/relebactam against imipenem-resistant P. aeruginosa was moderate.

Dynamics of the MRSA Population in a Chilean Hospital: a Phylogenomic Analysis (2000-2016).

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P < 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or more limited typing techniques that lack the resolution to represent an accurate description of the genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000 and 2016 in Chile providing the largest and most detailed study of clonal dynamics of MRSA in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing in frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Latin America.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine ß-naphthylamide (PAßN). In the absence of PAßN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAßN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAßN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.

Isolation of an Extensively Drug-Resistant Pseudomonas aeruginosa exoS+/O4 Strain Belonging to the "High-Risk" Clone ST654 and Coproducer of NDM-1 and the Novel VIM-80.

The aim of this study was to investigate the genomic features of an extensively drug-resistant (XDR) Pseudomonas aeruginosa isolate (P-469) emerging in Chile. Antibiotic susceptibility was determined by disk diffusion and "colistin agar" test. Whole-genome sequencing (WGS) was performed by the Illumina NextSeq 2000 platform, and epidemiologically and clinically relevant data (i.e., sequence-type, serotype, mobile genetic elements, virulome, resistome, plasmidome, prophages, and CRISPR-Cas systems) were retrieved using multiple bioinformatic tools. The P-469 strain displayed an XDR profile, remaining susceptible to colistin. Genomic analysis revealed that this isolate belonged to the "high-risk" clone ST654 (CC654), serotype O4, and genotype exoS+. Strikingly, two CRISPR-Cas systems, five intact prophages sequences, and a broad resistome that included blaNDM-1 and the novel blaVIM-80 carbapenemase genes were predicted. Our results revealed the genomic characteristics of P. aeruginosa belonging to the high-risk clone ST654/O4 coproducing NDM-1 and VIM-80 in Chile, supporting that genomic surveillance is necessary to track the emergence and spread of epidemiologically successful WHO's critical priority pathogens in order to prevent their rapid dissemination.

Acquisition of resistance to ceftazidime-avibactam during infection treatment in Pseudomonas aeruginosa through D179Y mutation in one of two blaKPC-2 gene copies without losing carbapenem resistance.

Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing Pseudomonas aeruginosa (KPC-PA) and Enterobacterales. CAZ/AVI resistance is driven by several mechanisms. In P. aeruginosa this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in Enterobacterales it frequently occurs through D179Y substitution in the active site of KPC enzyme. This aminoacid change abolishes AVI binding to the KPC active site, hence inhibition is impaired. However, this substitution also decreases KPC-mediated resistance to carbapenems ("see-saw" effect). The goal of this work was to characterize the in vivo acquisition of CAZ/AVI resistance through D179Y substitution in a KPC-PA isolated from a hospitalized patient after CAZ/AVI treatment. Two KPC-PA isolates were obtained. The first isolate, PA-1, was obtained before CAZ/AVI treatment and was susceptible to CAZ/AVI. The second isolate, PA-2, was obtained after CAZ/AVI treatment and exhibited high-level CAZ/AVI resistance. Characterization of isolates PA-1 and PA-2 was performed through short and long-read whole genome sequencing analysis. The hybrid assembly showed that PA-1 and PA-2A had a single plasmid of 54,030 bp, named pPA-1 and pPA-2 respectively. Each plasmid harbored two copies of the bla KPC-containing Tn4401b transposon. However, while pPA-1 carried two copies of bla KPC-2, pPA-2 had one copy of bla KPC-2 and one copy of bla KPC-33, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the "see-saw" effect. The bla KPC-33 allele was detected only through hybrid assembly using a long-read-first approach. The present work describes a KPC-PA isolate harboring a plasmid-borne CAZ/AVI resistance mechanism based on two copies of bla KPC-2-Tn4401b and D179Y mutation in one of them, that is not associated with loss of resistance to carbapenems. These findings highlight the usefulness of a fine-tuned combined analysis of short and long-read data to detect similar emerging resistance mechanisms.

The MexJK Multidrug Efflux Pump Is Not Involved in Acquired or Intrinsic Antibiotic Resistance in Pseudomonas aeruginosa, but Modulates the Bacterial Quorum Sensing Response.

Multidrug efflux pumps are critical elements in both intrinsic and acquired antibiotic resistance of bacterial populations. Consequently, most studies regarding these protein machineries focus on this specific phenotype. Nevertheless, different works show that efflux pumps participate in other aspects of bacterial physiology too. Herein, we study the Pseudomonas aeruginosa multidrug efflux pump MexJK. Previous studies, using model strains lacking MexAB-OprM and MexCD-OprJ efflux pumps, support that MexJK can extrude erythromycin, tetracycline, and triclosan. However, the results here reported indicate that this potential increased extrusion, in a mutant overexpressing mexJK, does not alter the antibiotics susceptibility in a wild-type genetic background where all intrinsic multidrug efflux pumps remain functional. Nevertheless, a clear impact on the quorum sensing (QS) response, mainly in the Pqs-dependent QS regulation network and in the expression of Pqs-regulated virulence factors, was observed linked to mexJK overexpression. The production of the siderophore pyoverdine strongly depended on the level of mexJK expression, suggesting that MexJK might participate in P. aeruginosa pyoverdine-dependent iron homeostasis. All in all, the results presented in the current article support that the functions of multidrug efflux pumps, as MexJK, go beyond antibiotic resistance and can modulate other relevant aspects of bacterial physiology.

Genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria (MDRB) isolated from the gut microbiota of highly antibiotic treated Salmo salar.

OBJECTIVES: The main objective of this study was the genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria isolated from the intestinal microbiota of aquaculture salmon treated with high concentrations of antibiotics. METHODS: In 82 multidrug resistant bacterial isolates, the prevalence of both the conserved elements of the integrons, qacEΔ1 and sul1 genes, and the variable region (VR) was determined. Further, whole genome sequencing and complete genetic analysis was performed in VR-positive isolates. RESULTS: Despite the fact that 100% of the bacterial isolates presented the intI1 gene, only 12.3% carried the qacEΔ1 and sul1 genes and only two (2.4%) presented a VR with gene cassettes. In the Pseudomonas baetica 25P2F9 isolate, a VR carrying aac(6')31, qacH, and blaOXA-2 gene cassettes was described, whereas the VR of Aeromonas salmonicida 30PB8 isolate showed a dfrA14 gene cassette. The array of gene cassettes found in the Pseudomonas isolate appears with high frequency in clinically relevant pathogens such as Pseudomonas aeruginosa or Escherichia coli. Additionally, it was possible to determine that these integrons are contained in plasmids and coul be easily transferred. Resistome analysis demonstrated that both isolates carried a great diversity of antibiotic resistance genes, including many ß-lactamases. Even in the Aeromonas isolate a new oxacillin-hydrolyzing beta-lactamase gene was described (blaOXA-956). CONCLUSION: The presence of multidrug resistant bacteria and clinically relevant genetic elements in the salmon intestinal microbiota make the aquaculture a hotspot in the phenomenon of antibiotic resistance; therefore, the control of antibiotics used in this activity is a key point to avoid its escalation.

Real-World Performance of Susceptibility Testing for Ceftolozane/Tazobactam against Non-Carbapenemase-Producing Carbapenem-Resistant Pseudomonas aeruginosa.

Ceftolozane/tazbactam (C/T) is a potent anti-pseudomonal agent that has clinical utility against infections caused by non-carbapenemase, producing-carbapenem-resistant Pseudomonas aeruginosa (non-CP-CR-PA). Accurate, precise, and reliable antimicrobial susceptibility testing (AST) is crucial to guide clinical decisions. However, studies assessing the performance of different AST methods against non-CP-CR-PA (the main clinical niche for C/T), are lacking. Here, we evaluated performance of gradient strips (Etest and MIC test strip [MTS], and disk diffusion [DD]) using CLSI breakpoints. Additionally, we assessed the performance of DD using EUCAST breakpoints. For all susceptibility tests, we used a collection of 97 non-CP-CR-PA clinical isolates recovered from 11 Chilean hospitals. Both gradient strips and DD had acceptable performance when using CLSI breakpoints, yielding a categorical agreement (CA) of >90% and 92%, respectively. In contrast, DD using EUCAST breakpoints performed suboptimally (CA 81%). MTS yielded a higher essential agreement (EA, >90%) than Etest (84%). Importantly, the performance of all methods varied significantly when the isolates were stratified by their degree of susceptibility to other anti-pseudomonal ß-lactams. All methods had 100% CA when testing isolates that were pan-susceptible to all ß-lactams (Pan-ß-S). However, the CA markedly decreased when testing isolates resistant to all ß-lactams (Pan-ß-R). Indeed, the CA was 81% for Etest (six errors), 78% for MTS (seven errors), and 78% and 56% for DD when using CLSI (seven errors) or EUCAST breakpoints (14 errors), respectively. Our results suggest that all manual AST methods have strikingly decreased performance in the context of Pan-ß-R P. aeruginosa with potentially major clinical implications.

Primer aislado de Pseudomonas aeruginosa productora de Sao Paulo metalo-beta-lactamasa (SPM-1) en un paciente chileno / First isolate of SPM-1- producing Pseudomonas aeruginosa (Sao Paulo metallo-beta-lactamase) in a Chilean patient

Resumen Las enzimas VIM, IMP, y NDM son carbapenemasas de tipo metalo-beta-lactamasas (MBLs) que se encuentran ampliamente distribuidas en el mundo. La SPM-1 (Sao Paulo metalo-beta-lactamasa) es una MBL que fue descrita en Pseudomonas aeruginosa en Sao Paulo (Brasil) en 2002. Comunicamos por primera vez la presencia de SPM-1 en Chile, en un aislado de P aeruginosa resistente a meropenem e imipenem, detectado en un cultivo rectal de vigilancia de carbapenemasas desde un paciente internado en nuestra institución. La secuencia del producto de la RPC fue 100% idéntica a la secuencia de SPM-1 reportada en Brasil. El paciente tenía antecedentes de una angioplastía realizada en Brasil en 2004-2005. Como consecuencia de este hallazgo, la detección de SPM mediante RPC será incorporada a la búsqueda de rutina de carbapenemasas en P aeruginosa.

Abstract VIM, IMP, and NDM carbapenemases are metallo-beta-lactamases (MBLs) that are widely distributed throughout the world. SPM-1 (Sao Paulo metallo-beta-lactamase) is an MBL that was described in Pseudomonas aeruginosa in Sao Paulo (Brazil) in 2002. We report for the first time the presence of SPM-1 in Chile, in an isolate of P aeruginosa resistant to meropenem and imipenem, detected in a carbapenemase surveillance rectal swab culture, in a patient admitted to our institution. The sequence of the PCR product was 100% identical to the sequence of SPM-1 reported in Brazil. The patient had a history of an angioplasty performed in Brazil in 2004-2005. As a consequence of this finding, the detection of SPM by PCR will be incorporated into the routine screening for carbapenemases in P aeruginosa.

Discovery of inhibitors of Pseudomonas aeruginosa virulence through the search for natural-like compounds with a dual role as inducers and substrates of efflux pumps.

Multidrug efflux pumps are ancient elements encoded in every genome, from bacteria to humans. In bacteria, in addition to antibiotics, efflux pumps extrude a wide range of substrates, including quorum sensing signals, bacterial metabolites, or plant-produced compounds. This indicates that their original functions may differ from their recently acquired role in the extrusion of antibiotics during human infection. Concerning plant-produced compounds, some of them are substrates and inducers of the same efflux pump, suggesting a coordinated plant/bacteria coevolution. Herein we analyse the ability of 1243 compounds from a Natural Product-Like library to induce the expression of P. aeruginosa mexCD-oprJ or mexAB-oprM efflux pumps' encoding genes. We further characterized natural-like compounds that do not trigger antibiotic resistance in P. aeruginosa and that act as virulence inhibitors, choosing those that were not only inducers but substrates of the same efflux pump. Four compounds impair swarming motility, exotoxin secretion through the Type 3 Secretion System (T3SS) and the ability to kill Caenorhabditis elegans, which might be explained by the downregulation of genes encoding flagellum and T3SS. Our results emphasize the possibility of discovering new anti-virulence drugs by screening natural or natural-like libraries for compounds that behave as both, inducers and substrates of efflux pumps.

[First isolate of SPM-1- producing Pseudomonas aeruginosa (Sao Paulo metallo-ß-lactamase) in a Chilean patient]. / Primer aislado de Pseudomonas aeruginosa productora de Sao Paulo metalo-ß-lactamasa (SPM-1) en un paciente chileno.

VIM, IMP, and NDM carbapenemases are metallo-ß-lactamases (MBLs) that are widely distributed throughout the world. SPM-1 (Sao Paulo metallo-ß-lactamase) is an MBL that was described in Pseudomonas aeruginosa in Sao Paulo (Brazil) in 2002. We report for the first time the presence of SPM-1 in Chile, in an isolate of P aeruginosa resistant to meropenem and imipenem, detected in a carbapenemase surveillance rectal swab culture, in a patient admitted to our institution. The sequence of the PCR product was 100% identical to the sequence of SPM-1 reported in Brazil. The patient had a history of an angioplasty performed in Brazil in 2004-2005. As a consequence of this finding, the detection of SPM by PCR will be incorporated into the routine screening for carbapenemases in P aeruginosa.

The impaired quorum sensing response of Pseudomonas aeruginosa MexAB-OprM efflux pump overexpressing mutants is not due to non-physiological efflux of 3-oxo-C12-HSL.

Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analysed efflux pump. Herein, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This 'apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.

Naringenin Inhibition of the Pseudomonas aeruginosa Quorum Sensing Response Is Based on Its Time-Dependent Competition With N-(3-Oxo-dodecanoyl)-L-homoserine Lactone for LasR Binding.

Bacterial quorum sensing (QS) is a cell-to-cell communication system that governs the expression of a large set of genes involved in bacterial-host interactions, including the production of virulence factors. Conversely, the hosts can produce anti-QS compounds to impair virulence of bacterial pathogens. One of these inhibitors is the plant flavonoid naringenin, which impairs the production of QS-regulated Pseudomonas aeruginosa virulence factors. In the present work, we analyze the molecular basis for such inhibition. Our data indicate that naringenin produces its effect by directly binding the QS regulator LasR, hence competing with its physiological activator, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12-HSL). The in vitro analysis of LasR binding to its cognate target DNA showed that the capacity of naringenin to outcompete 3OC12-HSL, when the latter is previously bound to LasR, is low. By using an E. coli LasR-based biosensor strain, which does not produce 3OC12-HSL, we determined that the inhibition of LasR is more efficient when naringenin binds to nascent LasR than when this regulator is already activated through 3OC12-HSL binding. According to these findings, at early exponential growth phase, when the amount of 3OC12-HSL is low, naringenin should proficiently inhibit the P. aeruginosa QS response, whereas at later stages of growth, once 3OC12-HSL concentration reaches a threshold enough for binding LasR, naringenin would not efficiently inhibit the QS response. To test this hypothesis, we analyze the potential effect of naringenin over the QS response by adding naringenin to P. aeruginosa cultures at either time zero (early inhibition) or at stationary growth phase (late inhibition). In early inhibitory conditions, naringenin inhibited the expression of QS-regulated genes, as well as the production of the QS-regulated virulence factors, pyocyanin and elastase. Nevertheless, in late inhibitory conditions, the P. aeruginosa QS response was not inhibited by naringenin. Therefore, this time-dependent inhibition may compromise the efficiency of this flavonoid, which will be effective just when used against bacterial populations presenting low cellular densities, and highlight the importance of searching for QS inhibitors whose mechanism of action does not depend on the QS status of the population.

Novel Inducers of the Expression of Multidrug Efflux Pumps That Trigger Pseudomonas aeruginosa Transient Antibiotic Resistance.

The study of the acquisition of antibiotic resistance (AR) has mainly focused on inherited processes, namely, mutations and acquisition of AR genes. However, inducible, noninheritable 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 nonantibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tight regulation of their expression. Their contribution to acquired AR relies in their overexpression. Here, 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 to modify 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 the mexCD-oprJ operon. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine, or atropine are present. This study emphasizes the need to study compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests.

Role of the Multidrug Resistance Efflux Pump MexCD-OprJ in the Pseudomonas aeruginosa Quorum Sensing Response.

Multidrug efflux pumps constitute a category of antibiotic resistance determinants that are a part of the core bacterial genomes. Given their conservation, it is conceivable that they present functions beyond the extrusion of antibiotics currently used for therapy. Pseudomonas aeruginosa stands as a relevant respiratory pathogen, with a high prevalence at hospitals and in cystic fibrosis patients. Part of its success relies on its low susceptibility to antibiotics and on the production of virulence factors, whose expression is regulated in several cases by quorum sensing (QS). We found that overexpression of the MexCD-OprJ multidrug efflux pump shuts down the P. aeruginosa QS response. Our results support that MexCD-OprJ extrudes kynurenine, a precursor of the alkyl-quinolone signal (AQS) molecules. Anthranilate and octanoate, also AQS precursors, do not seem to be extruded by MexCD-OprJ. Kynurenine extrusion is not sufficient to reduce the QS response in a mutant overexpressing this efflux pump. Impaired QS response is mainly due to the extrusion of 4-hydroxy-2-heptylquinoline (HHQ), the precursor of the Pseudomonas Quinolone Signal (PQS), leading to low PQS intracellular levels and reduced production of QS signal molecules. As the consequence, the expression of QS-regulated genes is impaired and the production of QS-regulated virulence factors strongly decreases in a MexCD-OprN P. aeruginosa overexpressing mutant. Previous work showed that MexEF-OprJ, another P. aeruginosa efflux pump, is also able of extruding kynurenine and HHQ. However, opposite to our findings, the QS defect in a MexEF-OprN overproducer is due to kynurenine extrusion. These results indicate that, although efflux pumps can share some substrates, the affinity for each of them can be different. Although the QS response is triggered by population density, information on additional elements able of modulating such response is still scarce. This is particularly important in the case of P. aeruginosa lung chronic infections, a situation in which QS-defective mutants are accumulated. If MexCD-OprJ overexpression alleviates the cost associated to triggering the QS response when un-needed, it could be possible that MexCD-OprJ antibiotic resistant overproducer strains might be selected even in the absence of antibiotic selective pressure, acting as antibiotic resistant cheaters in heterogeneous P. aeruginosa populations.

The development of efflux pump inhibitors to treat Gram-negative infections.

INTRODUCTION: One of the possibilities for reducing the emergence and spread of antibiotic resistance is the use of anti-resistance compounds capable of resensitizing resistant microorganisms to current antimicrobials. For this purpose, multidrug efflux pumps, whose inhibition may increase bacterial susceptibility to several antibiotics, including macrolides to which Gram-negatives are considered intrinsically resistant, have emerged as suitable targets. Areas covered: In the current review, the authors discuss different mechanisms that can be exploited for inhibiting multidrug efflux pumps and describe the properties and the potential therapeutic value of already studied efflux pumps inhibitors. Although efforts have already been made to develop these inhibitors, there are currently no good candidates for treating infectious diseases. Consequently, the authors also discuss potential approaches for their development. Expert opinion: Classical anti-resistance drugs such as beta-lactamases inhibitors, while useful, are only purposeful for treating infections caused by beta-lactamase producers. However, inhibitors of multidrug efflux pumps, which are present on all organisms, can sensitize both susceptible and resistant bacteria to antibiotics belonging to several different structural families. Since some efflux pumps are involved in bacterial infections, their inhibition may also reduce the infectivity of Gram-negative bacterial pathogens.