Aspirin, sodium benzoate and sodium salicylate reverse resistance to colistin in Enterobacteriaceae and Pseudomonas aeruginosa.

BACKGROUND: MDR bacterial infections are currently a serious problem for clinicians worldwide. Klebsiella pneumoniae and Enterobacter spp., among Enterobacteriaceae, and Pseudomonas aeruginosa, are part of the group of ESCAPE pathogens or bacteria that 'escape' from common antibacterial treatments. The lack of effectiveness of the first common line of antibiotics has led to the search for new therapies based on older antibiotics, such as colistin. OBJECTIVES: We searched for new enhancers of the action of colistin against MDR Gram-negative bacteria that can be easily applicable to clinical treatments. METHODS: Colistin MICs were determined alone and with the protonophores CCCP, sodium benzoate, sodium salicylate and aspirin using the broth microdilution method and FIC indexes were calculated to assess synergy between colistin and each chemical. Time-kill assays of colistin with and without protonophores were performed to determine the bactericidal action of combinations of colistin with protonophores. Likewise, the effect of sucrose, l-arginine and l-glutamic acid on the MICs of colistin alone and combined with each protonophore was assessed. RESULTS: It was found that sodium benzoate, sodium salicylate and aspirin, at concentrations allowed for human and animal use, partially or totally reversed resistance to colistin in P. aeruginosa and highly resistant enterobacterial strains. The mechanism of action could be related to their negative charge at a physiological pH along with their lipid-soluble character. CONCLUSIONS: Sodium benzoate, sodium salicylate and aspirin are good enhancers to use in antibiotic therapies that include colistin.

Antimicrobial susceptibility profiles of porcine mycoplasmas isolated from samples collected in southern Europe.

BACKGROUND: Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. hyosynoviae are significant pathogens for the porcine industry worldwide. The aim of the present study was to determine the antimicrobial susceptibility of six key antimicrobials (tylosin, tilmicosin, tylvalosin, lincomycin, tiamulin and valnemulin) routinely used for treating infections caused by these pathogens. Twenty-seven M. hyopneumoniae, 48 M. hyorhinis and 40 M. hyosynoviae field strains isolated from clinical samples from different Southern European countries between 2013 and 2018 using broth microdilution method were evaluated. RESULTS: Tylvalosin exhibited the highest in vitro activity among the macrolides assayed, with MIC90 values 4 to 5 two-fold dilutions lower than those of tylosin and tilmicosin. The pleuromutilin valnemulin showed one of the highest in vitro activities against the three mycoplasma species. On the contrary, lincomycin exhibited the highest MIC values of the antimicrobials tested. CONCLUSIONS: The data obtained in the present study supports the use of pleuromutilins and macrolides for the control of infections caused by porcine mycoplasmas. The use of lincomycin for the treatment of porcine mycoplasma infections should be carefully evaluated due to the presence of circulating field isolates with decreased susceptibility to this antimicrobial.

QnrS1 structure-activity relationships.

OBJECTIVES: Loop B is important for low-level quinolone resistance conferred by Qnr proteins. The role of individual amino acids within QnrS1 loop B in quinolone resistance and gyrase protection was assessed. METHODS: qnrS1 and 11 qnrS1 alleles with site-directed Ala mutations in loop B were expressed in Escherichia coli BL21(DE3) and proteins were purified by affinity chromatography. Ciprofloxacin MICs were determined with and without IPTG. Gyrase DNA supercoiling was measured with and without ciprofloxacin IC50 and with various concentrations of QnrS1 proteins. RESULTS: Wild-type QnrS1 and QnrS1 with Asn-110→Ala and Arg-111→Ala substitutions increased the ciprofloxacin MIC 12-fold in BL21(DE3), although QnrS1 with Gln-107→Ala replacement increased it 2-fold more than wild-type did. However, QnrS1 with Ala substitutions at His-106, Val-108, Ser-109, Met-112, Tyr-113, Phe-114, Cys-115 and Ser-116 increased ciprofloxacin MIC 1.4- to 8-fold less than wild-type QnrS1. Induction by 10-1000 µM IPTG increased ciprofloxacin MICs for all mutants, reaching values similar to those for wild-type. Purified wild-type and mutated proteins differed in protection of gyrase from ciprofloxacin action. Wild-type QnrS1 produced complete protection of gyrase supercoiling from ciprofloxacin (1.8 µM) action at 0.05 nM and half protection at 0.5 pM, whereas QnrS1 with Ala replacements that conferred the least increase in ciprofloxacin MICs also required the highest QnrS1 concentrations for protection. CONCLUSIONS: Key individual residues in QnrS1 loop B affect ciprofloxacin resistance and gyrase protection from ciprofloxacin action, supporting the concept that loop B is key for interaction with gyrase necessary for quinolone resistance.

Resistance to 16-Membered Macrolides, Tiamulin and Lincomycin in a Swine Isolate of Acholeplasma laidlawii.

Acholeplasma (A.) laidlawii is an opportunistic pathogen with the ability to disseminate resistance determinants to antibiotics; however, its resistance to macrolides has been less studied. The aim of the present study was to characterize the mechanisms responsible for the resistance to macrolides, tiamulin and lincomycin found in a strain of A. laidlawii isolated from a pig with pneumonia. MICs of erythromycin, 15- and 16-membered macrolides, tiamulin and lincomycin were determined by microdilution method with and without reserpine, an inhibitor of ABC efflux pumps and regions of the genome were sequenced. Reserpine only decreased lincomycin MIC but it did not change the MICs of macrolides and tiamulin. The analysis of the DNA sequence of 23S rRNA showed nucleotide substitutions at eight different positions, although none of them were at positions previously related to macrolide resistance. Five mutations were found in the L22 protein, one of them at the stop codon. In addition, two mutations were found in the amino acid sequence of L4. The combination of multiple mutations in the ribosomal proteins L22 and L4 together with substitutions in 23S rRNA DNA sequence was associated with the resistance to macrolides, the pleuromutilin and lincomycin in the studied A. laidlawii strain.

Quorum-sensing regulator sdiA and marA overexpression is involved in in vitro-selected multidrug resistance of Escherichia coli.

OBJECTIVES: The role of sdiA in the acquisition of low-level multidrug resistance (MDR) was analysed and compared with that of marA and soxS in two Escherichia coli clinical isolates and two in vitro-selected mutants. METHODS: The mutants were developed by growth in lomefloxacin and ceftazidime. The sdiA, marA, soxS, ftsI, tolC and acrB gene transcript levels were determined by RT-PCR. Analyses of 2,4-dinitrophenol susceptibility, the effect of an active efflux inhibitor on antibiotic and mitomycin C susceptibility, beta-lactamase hydrolytic activity, outer and inner membrane proteins and acrR gene sequencing were also performed. RESULTS: Both mutants showed elevated marA and sdiA gene transcript levels, which were associated with increased susceptibility to 2,4-dinitrophenol; soxS overexpression was only seen in the mutant selected with ceftazidime. The two mutants showed MDR phenotypes in which ceftazidime, cefpirome and aztreonam MICs increased 4- to 128-fold, in addition to decreased susceptibility to quinolones, chloramphenicol and mitomycin C. The highest ceftazidime MIC in one of the mutants coincided with a frameshift mutation in acrR and the highest transcript level of ftsI (penicillin-binding protein 3), but not with a higher beta-lactamase activity. Likewise, active efflux associated with increased levels of acrB and tolC and decreased OmpF expression contributed to low-level MDR in both mutants. CONCLUSIONS: marA and sdiA overexpression was a common feature of multidrug-resistant mutants selected by growth in lomefloxacin and ceftazidime. To our knowledge, this report is the first to describe in vitro selection with a fluoroquinolone or ceftazidime triggering sdiA overexpression in E. coli isolates.

Biofilm Formation among Stenotrophomonas maltophilia Isolates Has Clinical Relevance: The ANSELM Prospective Multicenter Study.

The ability to form biofilms is a recognized trait of Stenotrophomonas maltophilia, but the extent of its clinical relevance is still unclear. The present multicenter prospective study (ANSELM) aims at investigating the association between biofilm formation and clinical outcomes of S. maltophilia infections. One hundred and nine isolates were collected from various geographical origins and stratified according to their clinical relevance. Biofilm formation was evaluated by the microtiter plate assay and correlated with microbiological and clinical data from the associated strains. Antibiotic susceptibility of the planktonic cells was tested by the disk diffusion technique, while antibiotic activity against mature biofilms was spectrophotometrically assessed. Most strains (91.7%) were able to form biofilm, although bloodborne strains produced biofilm amounts significantly higher than strains causing hospital- rather than community-acquired infections, and those recognized as "definite" pathogens. Biofilm formation efficiency was positively correlated with mechanical ventilation (p = 0.032), whereas a negative relationship was found with antibiotic resistance (r2 = 0.107; p < 0.001), specifically in the case of the pathogenic strains. Mature S. maltophilia biofilms were markedly more resistant (up to 128 times) to cotrimoxazole and levofloxacin compared with their planktonic counterparts, especially in the case of bloodborne strains. Our findings indicate that biofilm formation by S. maltophilia is obviously a contributing factor in the pathogenesis of infections, especially in deep ones, thus warranting additional studies with larger cohort of patients and isolates.

Review on bacterial stress topics.

A complex network of regulatory systems ensures a coordinated and effective response to different types of stress that can act on a bacterium. Bacterial stress response generates changes that influence efflux system and virulence factor expression. Thus, partial or total loss of pathogenicity islands in uropathogenic Escherichia coli can be induced by SOS-dependent or SOS-independent pathways related to selection of quinolone-resistant mutants. Likewise, hyperosmolarity and some chemicals, including fluoroquinolones, salicylate, nonantimicrobial medicaments like diazepam and anti-inflammatory drugs are all able to induce an increased active efflux, cyclohexane tolerance, loss of porins, and decreased susceptibility to multiple antimicrobials in enterobacterial strains, suggesting that bacterial response to the stress caused by an increase in osmolarity might be linked to the development of the multidrug-resistant phenotypes. Finally, a sudden downshift of the growth temperature (cold-shock) triggers a drastic reprogramming of bacterial gene expression to allow cell survival under the new unfavorable conditions. The strategy developed by E. coli to reach this goal consists in the induction of a set of (cold-shock) genes whose expression is regulated at both transcriptional and posttranscriptional levels.

Interaction of carbapenems and ß-lactamase inhibitors towards CTX-M-15 and CTX-M-15G238C mutant.

OBJECTIVES: The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various ß-lactamase inhibitors. METHODS: A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. RESULTS: In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. CONCLUSIONS: Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.

Ciprofloxacin, salicylate, and 2,4-dinitrophenol decrease production of AmpC-type beta-lactamase in two Citrobacter freundii clinical isolates.

The effect of ciprofloxacin and two mar RAB inducers on the susceptibility to beta-lactam antibiotics in two AmpC beta-lactamase semi-constitutive producer Citrobacter freundii clinical isolates (the DM 1 and DM 2 strains) was studied. Possible changes in outer membrane protein expression, permeability to cephaloridine, active efflux, and hydrolytic activity of beta-lactamase-crude extracts were evaluated under the influence of ciprofloxacin, sodium salicylate, and 2,4-dinitrophenol. Results were compared with those of the effect of the same three chemicals on a normally beta-lactamase-inducible wild-type C. freundii strain. The three assayed compounds decreased beta-lactamase hydrolysis on cephaloridine in both the two clinical isolates as well as in the wild-type strain. However, only the DM 1 and DM 2 strains showed increased susceptibility to beta-lactams. Sodium salicylate and 2,4-dinitrophenol, but not ciprofloxacin, reduced the expression of a 45-kDa outer membrane protein in the three studied strains, which was accompanied by a 4- to 20-fold diminution in permeability to cephaloridine. In conclusion, two mar RAB inducers and ciprofloxacin induced the Mar phenotype and repressed AmpC beta-lactamase synthesis in the DM 1 and DM 2 clinical isolates.

Mechanisms involved in quinolone resistance in Mycoplasma mycoides subsp. capri.

Mycoplasma mycoides subsp. capri is a causative agent of contagious agalactia in goats. In this study, M. mycoides subsp. capri mutants were selected for resistance to fluoroquinolones (norfloxacin, enrofloxacin and ciprofloxacin) by serial passes in broth with increasing concentrations of antibiotic. Mutations conferring cross-resistance to the three fluoroquinolones were found in the quinolone resistance determining regions of the four genes encoding DNA gyrase and topoisomerase IV. Different mutations in the DNA gyrase GyrA subunit suggest a different mechanism of inhibition between norfloxacin and the other tested fluoroquinolones. The presence of an adenosine triphosphate-dependent efflux system was suggested through the use of the inhibitor orthovanadate.

Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli.

An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase. The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme. The N-terminal amino acid sequence was determined to be NH2-QTADVQ... Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates. Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.

Salicylate decreases production of AmpC type beta-lactamases and increases susceptibility to beta-lactams in a Morganella morganii clinical isolate.

The effect of salicylate, a marRAB inducer, on the resistance to beta-lactams was characterized in an AmpC beta-lactamase hyperproducer Morganella morganii clinical isolate (the M1 strain). Results were compared with those of the effect of salicylate in a wild-type M. morganii strain. Salicylate induced a decreased susceptibility to nalidixic acid, norfloxacin and tetracycline and simultaneously increased the susceptibility to beta-lactams apparently due to the repression of AmpC beta-lactamase synthesis in the M1 strain. Likewise, salicylate only repressed 46 kDa outer membrane protein expression in the wild-type strain, since the clinical isolate M1 did not express it.

Enhanced active efflux, repression of porin synthesis and development of Mar phenotype by diazepam in two enterobacteria strains.

The aim of this work was to determine whether diazepam could induce the multiple antibiotic resistance (Mar) phenotype in Klebsiella pneumoniae and Escherichia coli strains. The Mar phenotype is characterized by decreased susceptibility to multiple antibiotics due to the loss of porins and/or increased expression of active efflux systems. The effect of subinhibitory concentrations of diazepam on the susceptibility of different antimicrobial agents, outer-membrane protein expression and norfloxacin intracellular accumulation was studied. The results revealed that diazepam concentrations equal or twice adult dosage induced the same Mar phenotype as two well known E. coli marRAB inducers, sodium salicylate and sodium benzoate. Susceptibility to norfloxacin in a K. pneumoniae clinical isolate and E. coli strain Ag100 decreased due to enhanced active efflux and loss of porin expression. A decreased susceptibility to chloramphenicol, tetracycline, nalidixic acid and beta-lactam antibiotics was also observed. In conclusion, like sodium salicylate or sodium benzoate, diazepam may induce the Mar phenotype.

Resistance to ceftazidime in Escherichia coli associated with AcrR, MarR and PBP3 mutations and overexpression of sdiA.

The mechanisms responsible for the increase in ceftazidime MIC in two Escherichia coli in vitro selected mutants, Caz/20-1 and Caz/20-2, were studied. OmpF loss and overexpression of acrB, acrD and acrF that were associated with acrR and marR mutations and sdiA overexpression, together with mutations A233T and I332V in FtSI (PBP3) resulted in ceftazidime resistance in Caz/20-2, multiplying by 128-fold the ceftazidime MIC in the parental clinical isolate PS/20. Absence of detectable ß-lactamase hydrolytic activity in the crude extract of Caz/20-2 was observed, and coincided with Q191K and P209S mutations in AmpC and a nucleotide substitution at -28 in the ampC promoter, whereas ß-lactamase hydrolytic activity in crude extracts of PS/20 and Caz/20-1 strains was detected. Nevertheless, a fourfold increase in ceftazidime MIC in Caz/20-1 compared with that in PS/20 was due to the increased transcript level of acrB derived from acrR mutation. The two Caz mutants and PS/20 showed the same mutations in AmpG and ParE.