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Candida species are highly opportunistic yeasts that are responsible for serious invasive fungal infections among immunocompromised patients worldwide. Due to the increase in drug resistance and incidence of infections, there is an urgent need to develop new antifungals and to identify co-drugs that can sensitize drug-resistant Candida to antifungals. The objective of this study was to assess the effect of saquinavir on the activity of azole antifungals against C. auris. The in vitro interaction of saquinavir and three azole antifungals (itraconazole, voriconazole, and fluconazole) was evaluated against a panel of C. auris isolates. The itraconazole/saquinavir combination exhibited a synergistic (SYN) relationship against all C. auris isolates tested with the fractional inhibitory concentration index ranging from 0.03 to 0.27. Moreover, a time-kill kinetics assay revealed that saquinavir restored the itraconazole's fungistatic activity against C. auris. Furthermore, saquinavir restored itraconazole's antifungal activity against other clinically important Candida species. The mechanistic investigation indicated that saquinavir significantly inhibited efflux pumps, glucose utilization, and ATP synthesis in Candida. Finally, a murine model of C. auris infection was used to evaluate the efficacy of the itraconazole/saquinavir combination in the presence of ritonavir (as a pharmacokinetic enhancer). The combination significantly reduced the fungal burden in the kidneys by 0.93-log10 colony-forming units (88%) compared to itraconazole alone. This study identified that saquinavir exhibits a potent SYN relationship in combination with itraconazole against Candida species, which warrants further consideration. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a global issue that threatens our progress in healthcare and life expectancy. In recent years, antimicrobial peptides (AMPs) have been considered as promising alternatives to the classic antibiotics. AMPs are potentially superior due to their lower rate of resistance development, since they primarily target the bacterial membrane ('Achilles' heel' of the bacteria). However, bacteria have developed mechanisms of AMP resistance, including the removal of AMPs to the extracellular space by efflux pumps such as the MtrCDE or AcrAB–TolC systems, and the internalization of AMPs to the cytoplasm by the Sap transporter, followed by proteolytic digestion. In this review, we focus on AMP transport as a resistance mechanism compiling all the experimental evidence for the involvement of efflux in AMP resistance in Gram-negative bacteria and combine this information with the analysis of the structures of the efflux systems involved. Finally, we expose some open questions with the aim of arousing the interest of the scientific community towards the AMPs—efflux pumps interactions. All the collected information broadens our understanding of AMP removal by efflux pumps and gives some clues to assist the rational design of AMP-derivatives as inhibitors of the efflux pumps. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1 , hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A , hmg1 , and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1 , several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro , in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity. [ABSTRACT FROM AUTHOR]

Combination therapy is a common antibiotic treatment strategy that aims at minimizing the risk of resistance evolution in several infectious diseases. Nonetheless, evidence supporting its efficacy against the nosocomial opportunistic pathogen Pseudomonas aeruginosa remains elusive. Identification of the possible evolutionary paths to resistance in multidrug environments can help to explain treatment outcome. For this purpose, we here performed whole-genome sequencing of 127 previously evolved populations of P. aeruginosa adapted to sublethal doses of distinct antibiotic combinations and corresponding single-drug treatments, and experimentally characterized several of the identified variants. We found that alterations in the regulation of efflux pumps are the most favored mechanism of resistance, regardless of the environment. Unexpectedly, we repeatedly identified intergenic variants in the adapted populations, often with no additional mutations and usually associated with genes involved in efflux pump expression, possibly indicating a regulatory function of the intergenic regions. The experimental analysis of these variants demonstrated that the intergenic changes caused similar increases in resistance against single and multidrug treatments as those seen for efflux regulatory gene mutants. Surprisingly, we could find no substantial fitness costs for a majority of these variants, most likely enhancing their competitiveness toward sensitive cells, even in antibiotic-free environments. We conclude that the regulation of efflux is a central target of antibiotic-mediated selection in P. aeruginosa and that, importantly, changes in intergenic regions may represent a usually neglected alternative process underlying bacterial resistance evolution, which clearly deserves further attention in the future. [ABSTRACT FROM AUTHOR]

Background Quinolones are broad-spectrum antibiotics, which are used for the treatment of different infectious diseases associated with Enterobacteriaceae. During recent decades, the wide use as well as overuse of quinolones against diverse infections has led to the emergence of quinolone-resistant bacterial strains. Herein, we present the development of quinolone antibiotics, their function and also the different quinolone resistance mechanisms in Enterobacteriaceae by reviewing recent literature. Methods All data were extracted from Google Scholar search engine and PubMed site, using keywords; quinolone resistance, Enterobacteriaceae, plasmid-mediated quinolone resistance, etc. Results and conclusion The acquisition of resistance to quinolones is a complex and multifactorial process. The main resistance mechanisms consist of one or a combination of target-site gene mutations altering the drug-binding affinity of target enzymes. Other mechanisms of quinolone resistance are overexpression of AcrAB-tolC multidrug-resistant efflux pumps and downexpression of porins as well as plasmid-encoded resistance proteins including Qnr protection proteins, aminoglycoside acetyltransferase (AAC(6′)-Ib-cr) and plasmid-encoded active efflux pumps such as OqxAB and QepA. The elucidation of resistance mechanisms will help researchers to explore new drugs against the resistant strains. [ABSTRACT FROM AUTHOR]

Bile salts, including sodium deoxycholate (DOC), are secreted into the intestine to aid fat digestion and contribute to antimicrobial protection. Gram-negative pathogens such as Escherichia coli , however, are highly resistant to DOC, using multiple mechanisms of which the multidrug efflux pump AcrAB-TolC is the dominant one. Given that TolC-mediated efflux masks the interaction of DOC with potential targets, we sought to identify those targets by identifying genes whose mutations cause an increase in the MIC to DOC relative to the ∆ tolC parental strain, that lacks TolC-associated functional efflux pumps. Using a mutant screen, we isolated twenty independent spontaneous mutants that had a higher MICDOC than the E. coli parental ∆tolC strain. Whole genome sequencing of these mutants mapped most mutations to the ptsI or cyaA gene. Analysis of knock-out mutants and complementation showed that elimination of PtsI, a component of the carbohydrate phosphotransferase system, or one of the two key proteins involved in cAMP synthesis and signaling, adenylate cyclase (CyaA) or cAMP receptor protein (Crp) causes low-level increased resistance of a ∆tolC E. coli strain to DOC. [ABSTRACT FROM AUTHOR]

Candida glabrata causes difficult to treat invasive candidiasis due to its antifungal resistance, mainly to azoles. The aim of the present work was to study the role of the genes ERG11 , CDR1 , CDR2, and SNQ2 on the resistance to voriconazole (VRC) in a set of C. glabrata strains with known in vitro and in vivo susceptibility to this drug. Eighteen clinical isolates of C. glabrata were exposed in vitro to VRC, and the expression of the cited genes was quantified by real time quantitative polymerase chain reaction (q-PCR). In addition, the ERG11 gene was amplified and sequenced to detect possible mutations. Ten synonymous mutations were found in 15 strains, two of them being reported for the first time; however, no amino acid changes were detected. ERG11 and CDR1 were the most expressed genes in all the strains tested, while the expression of CDR2 and SNQ2 was modest. Our results show that gene expression does not directly correlate with the VRC MIC. In addition, the expression profiles of ERG11 and efflux pump genes did not change consistently after exposure to VRC. Although individual analysis did not result in a clear correlation between MIC and gene expression, we did observe an increase in ERG11 and CDR1 expression in resistant strains. It is of interest that considering both in vitro and in vivo results, the slight increase in such gene expression correlates with the observed resistance to VRC. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was the molecular identification of Salmonella enterica serovar Infantis (S. Infantis) isolated from poultry samples and their antimicrobial resistance and virulence profiling. Methods and Results: A total of 149 isolates, belonging to genus Salmonella, originally isolated from 340 suspected poultry post mortem specimens reported by us earlier were preliminary identified as Salmonella by biochemical methods and confirmed by polymerase chain reaction targeting genus‐specific gene invA. Targeting serovar‐specific gene fragment (fljB) resulted in confirmation of 54 isolates as S. Infantis which were further confirmed by sequencing of 16S RNA and fljB genes. Swimming and swarming motilities were detected in 98·1 and 11·1% isolates respectively. Phenotypic disc diffusion assay against 23 antimicrobial agents showed the highest resistance against pefloxacin (PEF) (94·4%), chloramphenicol (83·3%) and imipenem (77·7%) while 5·3% isolates showed extended‐spectrum β‐lactamase production. Fifty‐nine genes reported for antimicrobial resistance and 12 for conferring virulence were targeted. The most prevalent resistance gene for aminoglycosides was aadA (42·3%), for quinolone resistance determining region parE (62·5%), for penicillin's Int1 (62·9%), for chloramphenicol cat3 (66·1%) and for beta‐lactams blaTEM‐1 (44·4%). Among efflux pump coding genes, armA showed highest (74·2%) prevalence and for virulence, a high prevalence of SopE (89·2%) showed the zoonotic potential of the isolates. The activity of efflux pumps was detected through Ethidium Bromide‐agar method. Conclusions: Poultry could act as reservoirs of multidrug resistance Salmonella. Significance and Impact of the Study: We firstly report the prevalence and molecular characterization of virulence/drug resistance in S. Infantis from this region and the results may contribute to designing precisely targeted therapy. This study has also highlighted the possible emergence of S. Infantis with zoonotic potential. [ABSTRACT FROM AUTHOR]

Background Ethionamide is used to treat multidrug-resistant tuberculosis (MDR-TB). The antimicrobial pharmacokinetics/pharmacodynamics, the contribution of ethionamide to the multidrug regimen, and events that lead to acquired drug resistance (ADR) are unclear. Methods We performed a multidose hollow fiber system model of tuberculosis (HFS-TB) study to identify the 0–24 hour area under the concentration-time curve (AUC0-24) to minimum inhibitory concentration (MIC) ratios that achieved maximal kill and ADR suppression, defined as target exposures. Ethionamide-resistant isolates underwent whole-genome and targeted Sanger sequencing. We utilized Monte Carlo experiments (MCEs) to identify ethionamide doses that would achieve the target exposures in 10000 patients with pulmonary tuberculosis. We also identified predictors of time-to-sputum conversion in Tanzanian patients on ethionamide- and levofloxacin-based regimens using multivariate adaptive regression splines (MARS). Results An AUC0-24/MIC >56.2 was identified as the target exposure in the HFS-TB. Early efflux pump induction to ethionamide monotherapy led to simultaneous ethambutol and isoniazid ADR, which abrogated microbial kill of an isoniazid-ethambutol-ethionamide regimen. Genome sequencing of isolates that arose during ethionamide monotherapy revealed mutations in both ethA and embA. In MCEs, 20 mg/kg/day achieved the AUC0-24/MIC >56.2 in >95% of patients, provided the Sensititre assay MIC was <2.5 mg/L. In the clinic, MARS revealed that ethionamide Sensititre MIC had linear negative relationships with time-to-sputum conversion until an MIC of 2.5 mg/L, above which patients with MDR-TB failed combination therapy. Conclusions Ethionamide is an important contributor to MDR-TB treatment regimens, at Sensititre MIC <2.5 mg/L. Suboptimal ethionamide exposures led to efflux pump-mediated ADR. [ABSTRACT FROM AUTHOR]

Aims To understand the mode of action of thyme essential oil as an alternative biofungicide. Methods and Results The chemical composition of thyme essential oil isolated by hydrodistillation from the aerial parts of Thymus vulgaris was analyzed. The main constituents of thyme essential oil were thymol (76·96%), ρ-cymene (9·89%), γ-terpinene (1·92%) and caryophyllene oxide (1·69%). The antifungal activity of the oil and its pure major component (thymol) was assessed by the in vitro assay against Mycosphaerella graminicola. Thyme oil exhibited higher antifungal activity than thymol. The expression pattern of genes involved in fungal development and detoxification acting in M. graminicola under thyme oil and thymol treatment was analyzed. Thyme oil overexpressed, more than thymol, the genes encoding for the efflux pump ( MgMfs1, MgAtr4), the regulatory subunit of protein kinase A ( PKA) ( MgBcy1) and the MAPK MgHog1. Thyme oil repressed the expression of the genes encoding for the efflux pump MgAtr4, the MAPK ( MgSlt2) and the regulatory subunit of PKA ( MgBcy1). However, thymol repressed only MgAtr4 and MgSlt2 expression. Conclusions These data highlight the ability of thyme oil to target genes involved in fungal development and virulence of the yeast-like fungi M. graminicola, which explain its higher antifungal activity. Significance and Impact of the Study These findings will probably be useful to design an alternative biofungicide which will not lead to pathogen multidrug resistance. [ABSTRACT FROM AUTHOR]

Objectives: The objective of this study was to evaluate the proficiency of Spanish laboratories with respect to accurate susceptibility testing and the detection and interpretation of quinolone resistance phenotypes in Enterobacteriaceae. Methods: Thirteen strains of Enterobacteriaceae were sent to 62 participating centres throughout Spain; strains harboured GyrA/ParC modifications, reduced permeability and/or plasmid-mediated quinolone resistance genes. The centres were requested to evaluate nalidixic acid and five quinolones, provide raw/interpreted clinical categories and to detect/infer resistance mechanisms. Consensus results from reference centres were used to assign minor, major and very major errors (mEs, MEs and VMEs, respectively). Results: Susceptibility testing in the participating centres was frequently performed using the MicroScan WalkAway, Vitek 2 and Wider systems (48%, 30% and 8%, respectively). CLSI/EUCAST breakpoints were used in 71%/29% of the determinations. The percentage of VMEs for all quinolones was well below 2%. Only ofloxacin and moxifloxacin showed higher values for raw VMEs (6.6%), which decreased to 0% and 2.9%, respectively, in the interpreted VMEs. These errors were particularly associated with the CC-03 strain [qnrS2+aac(6')-Ib-cr]. For MEs, percentages were always ,10%, except in the case of ofloxacin and nalidixic acid. There was a significantly higher percentage of all types of errors for strains whose MICs were at the border of clinical breakpoints. Conclusions: The use of different breakpoints and methods, the complexity of mutation-driven and transferable resistance mechanisms and the absence of specific tests for detecting low-level resistance lead to high variability and represent a challenge to accuracy in susceptibility testing, particularly in strains with MICs on the border of clinical breakpoints. [ABSTRACT FROM AUTHOR]

A number of microorganisms have the ability to thrive in the presence of a range of toxic solvents. Tolerance to these chemicals is a multifactorial process, meaning that bacterial cells use a set of physiological and gene expression changes to overcome the damage imparted by these chemicals. This review focuses mainly on issues related to tolerance to aromatic hydrocarbons and butanol in Pseudomonas, although other microorganisms are also discussed. Pseudomonas putida strains contain a circular chromosome of approximately 6 Mbp which encodes about 5300 genes. A combination of physiological and biochemical assays, a genome-wide collection of mutants and several omics approaches have provided useful information to help identify functions involved in solvent tolerance in P. putida. The solvent response involves fine-tuning of lipid fluidity to adjust membrane functions including impermeabilization, activation of a general stress-response system, increased energy generation and induction of specific efflux pumps that extrude solvents to the medium. These responses are modulated at the transcriptional level by local and global regulators as well as by a number of sRNAs whose levels fluctuate with the presence of solvents in the environment. Taken as a whole these regulatory inputs orchestrate the complex network of metabolic responses observed after solvent addition. [ABSTRACT FROM AUTHOR]

Objectives:We report the synthesis, antibacterial activity and toxicity of 24 bis-indolic derivatives obtained during the development of new ways of synthesis of marine bis-indole alkaloids from the spongotine, topsentin and hamacanthin classes. Methods: Innovative ways of synthesis and further structural optimizations led to bis-indoles presenting either the 1-(1H-indol-3'-yl)-1,2-diaminoethane unit or the 1-(1H-indol-3-yl)ethanamine unit. MIC determination was performed for reference and clinical strains of Staphylococcus aureus and CoNS species. MBC, time-kill kinetics, solubility, hydrophobicity index, plasma protein-binding and cytotoxicity assays were performed for lead compounds. Inhibition of the S. aureus NorA efflux pump was also tested for bis-indoles with no antistaphylococcal activity. Results: Lead compounds were active against both S. aureus and CoNS species, with MICs between 1 and 4 mg/L. Importantly, the same MICs were found for MRSA and vancomycin-intermediate S. aureus strains. Early concentration- dependent bactericidal activity was observed for lead derivatives. Compounds with no intrinsic antibacterial activity could inhibit the S. aureus NorA efflux pump, which is involved in resistance to fluoroquinolones. At 0.5 mg/L, the most effective compound led to an 8-fold reduction of the ciprofloxacin MIC for the SA-1199B S. aureus strain, which overexpresses NorA. However, the bis-indole compounds displayed a high hydrophobicity index and high plasma protein binding, which significantly reduced antibacterial activity. Conclusions:We have synthesized and characterized novel bis-indole derivatives as promising candidates for the development of new antistaphylococcal treatments, with preserved activity against MDR S. aureus strains. [ABSTRACT FROM AUTHOR]

Objectives To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. Methods The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. Results Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. Conclusions This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance. [ABSTRACT FROM PUBLISHER]

Objectives The components of the AcrAB–TolC efflux pump function as a tripartite efflux system conferring resistance to multiple antibiotics and the individual components can also function in conjunction with other efflux pumps. This study aimed to establish whether redundancy exists between the homologous periplasmic adaptor proteins (PAPs) AcrA and AcrE and to measure the impact of this redundancy on antimicrobial resistance and the potential efficacy of inhibitor molecules. Methods The acrE gene was inactivated in Salmonella enterica serovar Typhimurium SL1344 and a ΔacrA mutant by insertion of the aph gene. The mutants were complemented with plasmids carrying acrA or acrE. The antimicrobial susceptibility of the mutants to various antimicrobials was determined and the accumulation or efflux of various substrates was measured. Results Inactivation of acrE alone had no phenotypic effect. However, the effect of inactivation of PAPs was additive; the acrA acrE mutant was more susceptible to certain antimicrobials and accumulated more Hoechst dye than single acrA, acrE or acrB mutants. In addition, the double mutant invaded human intestinal epithelial cells poorly. The phenotypic defects of the acrA acrE mutant were ameliorated by expression of either acrA or acrE, but the proteins exhibited some substrate specificity. Conclusions These data show for the first time the level of redundancy between the PAPs AcrA and AcrE, and highlight the PAPs as excellent targets for inhibitor molecules that could be used to potentiate the action of clinical antimicrobials. However, the redundancy that exists between AcrA and AcrE means potential inhibitors must act on both targets to be effective. [ABSTRACT FROM PUBLISHER]

Objectives Acinetobacter baumannii is an important opportunistic pathogen and multidrug-resistant isolate. Although tigecycline is a potent antibiotic for treating infections with multidrug-resistant isolates, resistance is becoming a problem. This study aimed to explore the mechanism of tigecycline resistance in A. baumannii. Methods A serial passage experiment was performed to collect isolates selected by tigecycline. The expression of efflux pumps was quantified in the final selected isolate, 19606-T8. The whole genome of 19606-T8 was sequenced and the putative mutations were confirmed using PCR and Sanger sequencing. A complementation experiment was performed to evaluate the contribution of the mutations to decreased susceptibility to tigecycline. The significance of a deletion mutation was further investigated in terms of growth rate and antibiotic susceptibilities. Results We collected serial isolates by selective pressure of tigecycline, and designated them 19606-T1 to 19606-T8. The efflux pumps AdeABC, AdeFGH and AdeIJK were not overexpressed in 19606-T8, which had decreased susceptibility to tigecycline. Isolate 19606-T8 carried one deletion mutation in trm and three non-synonymous substitutions in msbA (A84V), lolA (P91L) and filC (N168K). The deletion mutation in trm (encoding S-adenosyl-l-methionine-dependent methyltransferase) resulted in decreased susceptibility to tigecycline as well as to minocycline and doxycycline. In complementation experiments, the MICs of tigecycline, minocycline and doxycycline in a tigecycline-resistant isolate were restored by complementation with wild-type trm. Conclusions Given that the deletion mutation in trm was associated with decreased susceptibility to tigecycline and that a wild-type trm plasmid could restore the susceptibility, trm is considered to play an important role in decreased susceptibility to tigecycline in A. baumannii. [ABSTRACT FROM PUBLISHER]

Objectives Although the prevalence of fluoroquinolone resistance among methicillin-resistant Staphylococcus aureus (MRSA) is known to be higher than in methicillin-susceptible S. aureus (MSSA), the reasons have never been identified. Methods We randomly selected 115 isolates of S. aureus collected from 10 different hospitals in Korea between June 2009 and May 2011. To investigate the difference in fluoroquinolone resistance mechanisms between MRSA and MSSA, we evaluated gyrA and parC mutations and the relative expression of the multidrug efflux pump genes norA, norB and norC. Results All 62 ciprofloxacin-resistant S. aureus had either gyrA or parC mutations. The S84L mutation of gyrA (59/62, 95.2%) and the S80F mutation of parC (61/62, 98.4%) were the most common. Fifty-eight (93.6%) strains had both the S84L mutation of gyrA and the S80F mutation of parC. Among the 115 isolates, norB overexpression was the most common, occurring in 49 (42.6%) strains. There were only two (1.7%) strains with norA overexpression and none with norC overexpression. Strains overexpressing norB were more common among ciprofloxacin-resistant S. aureus (33/62, 53.2%) than ciprofloxacin-susceptible S. aureus (16/53, 30.2%) (P = 0.013). When we analysed 62 ciprofloxacin-resistant S. aureus strains, those overexpressing norB were more common in ciprofloxacin-resistant MRSA (28/46, 60.9%) than in ciprofloxacin-resistant MSSA (5/16, 31.3%) (P = 0.041). Conclusions Increased expression of norB can be a factor that contributes to ciprofloxacin resistance in MRSA strains. [ABSTRACT FROM PUBLISHER]

Background Enteropathogens have shown a high level of resistance against commonly used antibacterial drugs in Peru and it is necessary to explore alternative treatments. The aim of this study was to analyse the in vitro activity of rifaximin against diarrhoeagenic and commensal Escherichia coli in children less than 2 years of age. Methods The minimal inhibitory concentration (MIC) to rifampicin and rifaximin was determined for 210 strains in the presence and absence of phenyl-arginine-β-naphthylamide (PAβN) and the mechanisms of resistance were investigated. Results The MIC levels ranged between 8 and >256 mg/litre and the predominant mechanism of resistance to rifaximin was the efflux pumps inhibited by PAβN in 95.2% of the isolates. Conclusions The present MIC values are higher than those observed in other studies. Efflux pumps inhibited by PAβN were the cause of the rifaximin resistance in the majority of cases and suggest the presence of an environmental selective pressure. Consequently, rifaximin should be used with caution in the treatment of diarrhoea in Peru. [ABSTRACT FROM AUTHOR]

Objectives Recent increases in triazole resistance in Aspergillus fumigatus have been attributed primarily to target site (cyp51A) mutations. A recent survey of resistant isolates in Manchester showed that >50% of resistant isolates had no mutation in cyp51A or its promoter. We investigated the mechanisms of resistance in clinical azole-resistant isolates without cyp51A mutations. Methods Twelve azole-resistant isolates, 10 of which were itraconazole resistant, were studied. Bioinformatic comparisons between Candida albicans efflux genes and A. fumigatus genome data identified 20 putative azole transporter genes. Basal and azole-induced expression of these genes and cyp51A was quantified using RT–PCR with comparison with clinical azole-susceptible isolates. Function of high basal or itraconazole-induced expression transporters was tested by gene knockout in azole-susceptible and azole-resistant isolates. Results All susceptible strains showed minimal basal expression of cdr1B compared with 8 of 10 azole-resistant strains with high basal expression of this gene (>5-fold), 3 of which showed >30-fold increased expression. Knockout of this gene resulted in a 4-fold reduction in itraconazole, posaconazole and voriconazole MICs for a susceptible clinical isolate and a 4-fold reduction in itraconazole susceptibility in a clinical resistant isolate. One strain showed a >500-fold induction of cyp51A. No increase in basal expression or expression after induction was seen for the 18 remaining putative transporters. Conclusions The reasons behind the shift away from target site mutation in azole-resistant isolates from Manchester are unknown. The modest change in expression of cdr1B in azole-susceptible strains implies that only study of resistant isolates will lead to further understanding of resistance mechanisms in A. fumigatus. [ABSTRACT FROM PUBLISHER]

This study aimed at evaluating the in vitro antifungal susceptibility of Candida albicans isolates obtained during necropsy of a wild Brazilian porcupine and the mechanism of azole resistance. Initially, we investigated the in vitro susceptibility of the three isolates to amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole. Afterwards, three sub-inhibitory concentrations (47, 21 and 12 mg/l) of promethazine, an efflux pump inhibitor, were tested in combination with the antifungal drugs in order to evaluate the role of these pumps in the development of antifungal resistance. In addition, the three isolates were submitted to RAPD-PCR and M13-fingerprinting analyses. The minimum inhibitory concentrations (MICs) obtained with the isolates were 1, 0.03125, 250, 125, 8 and 250 mg/l for amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole, respectively, and the isolates were found to be resistant to all tested azoles. The addition of the three subinhibitory concentrations of promethazine resulted in statistically significant ( P < 0.05) reductions in the MICs for all tested drugs, with decreases to azoles being statistically greater than those for amphotericin B and caspofungin ( P < 0.05). The molecular analyses showed a genetic similarity among the three tested isolates, suggesting the occurrence of candidemia in the studied animal. These findings highlight the importance of monitoring antifungal susceptibility of Candida spp. from veterinary sources, especially as they may indicate the occurrence of primary azole resistance even in wild animals. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa are known to have a wide physiological potential allowing them to constantly populate diverse environments leading to severe infections of humans such as septicemia, leg ulcers, and burn wounds. We set out to probe physiological characteristics of P. aeruginosa isolates from diabetic leg ulcers collected from Helsinki metropolitan area. A total of 61 clinical isolates were obtained. Detailed phenotypic (physiological) characteristics [outer membrane ( OM) permeability, membrane voltage, and activity of multidrug resistance pumps] were determined in several growth phases leading to the division of the analyzed set of P. aeruginosa strains into five distinct clusters including cells with similar physiological properties. In addition, their antibiotic resistance patterns and genetic heterogeneity were determined. Multiple isolates from the same patient were genetically very closely related and belonged to the same phenotypic cluster. However, genetically close isolates from different patients expressed very different phenotypic properties. The characteristics of infected patients seem to determine the growth environments for microorganisms that adapt by changing their physiological and/or genetic properties. [ABSTRACT FROM AUTHOR]

Objectives To evaluate the proficiency of Spanish laboratories regarding accurate susceptibility testing, detection and interpretation of Pseudomonas aeruginosa β-lactam resistance phenotypes. Methods Thirteen characterized strains were sent to 54 participating centres: clinical strains producing horizontally acquired β-lactamases [extended-spectrum β-lactamases (ESBLs; PER-1 and OXA-161) and class A (GES-5) and B (VIM-2) carbapenemases] and mutants with combinations of chromosomal mechanisms (AmpC, OprD and/or efflux). The centres were requested to evaluate six antipseudomonal β-lactams, provide raw/interpreted clinical categories and detect/infer the resistance mechanisms. Consensus results from reference centres were used to assign minor, major or very major errors (mEs, MEs or VMEs). Results Vitek2, MicroScan WalkAway and Wider were the most used devices (25%–30% each). CLSI/EUCAST breakpoints were used in 86%/14% of the determinations. Discrepancies exclusively due to the differential application of breakpoints were highest for aztreonam, followed by piperacillin/tazobactam. The lowest percentage of VMEs was for Vitek2, followed by Wider. The highest percentages of VMEs (6%) were for the AmpC-hyperproducing OprD− strain and for the GES-5 producer, while among antibiotics the highest percentage of VMEs (22%) involved piperacillin/tazobactam. Appropriate inference of resistance mechanisms was high for the VIM-2-producing strain (83%), but low (<40%) for strains producing ESBLs or non-metallo-β-lactamase carbapenemases. Conclusions The use of different breakpoints and devices, the complexity of mutation-driven resistance mechanisms and the lack of unequivocal tests to detect ESBLs or carbapenemases in P. aeruginosa leads to extraordinary variability and low accuracy in susceptibility testing, which may have consequences for the treatment and control of nosocomial infections. [ABSTRACT FROM PUBLISHER]

Objectives To investigate the epidemiology and genetic elements of the efflux pump gene, oqxAB, in clinical isolates of Escherichia coli and Klebsiella pneumoniae. Methods The prevalence of the oqxAB gene in a total of 308 consecutive clinical isolates of E. coli, K. pneumoniae and other Klebsiella spp. was investigated by PCR amplification. The full-length oqxAB gene was sequenced. Conjugation experiments were carried out in oqxAB-positive E. coli strains. Results Of 136 E. coli strains, 71.3% and 70.6% were non-susceptible to ciprofloxacin and levofloxacin, respectively, while 66.9% and 50.7% of the 154 K. pneumoniae were non-susceptible to these two quinolones. oqxA and oqxB were present in 6.6% of E. coli and in all K. pneumoniae strains. The oqxA and oqxB sequences of E. coli and randomly selected K. pneumoniae strains showed high similarity to the original oqxAB sequence of pOLA52. One or two amino acid substitutions were detected in the OqxA or OqxB sequences from two E. coli strains and were designated oqxA2, oqxB2 and oqxB3. oqxAB was transferred in seven of nine oqxAB-positive E. coli strains, and transconjugants showed 2- to 256-fold increases in ciprofloxacin and levofloxacin MICs relative to that for the recipient. Conclusions Variants of the oqxAB gene, oqxA2, oqxB2 and oqxB3, were identified in two E. coli strains. The oqxAB gene was present in all K. pneumoniae strains studied and is likely located on the chromosome, thus identifying the genome of K. pneumoniae as a possible reservoir of oqxAB. [ABSTRACT FROM PUBLISHER]

Objectives Bacterial drug resistance is a worrying public health problem and there is an urgent need for research and development to provide new antibacterial molecules. Peptide deformylase (PDF) is now a well-described intracellular target selected for the design of a new antibiotic group, PDF inhibitors (PDFIs). The initial bacterial susceptibility to an inhibitor of a cytoplasmic target is directly associated with the diffusion of the compound through the membrane barrier of Gram-negative bacteria and with its cytosolic accumulation at the required concentration. Methods We have recently demonstrated that the activity of different PDFIs is strongly dependent on the accumulation of the active molecules by using permeabilizing agents, efflux inhibitors or efflux-mutated strains. In this work we assessed various combination protocols using different putative inhibitors (PDFIs, methionine aminopeptidase inhibitors etc.) to improve antibacterial activity against various resistant Gram-negative bacteria. Results The maximum effect was observed when combining actinonin with a dual inhibitor of methionine aminopeptidase and PDF, this molecule being also able to interact with the target while actinonin is bound to the PDF active site. Conclusions Such a combination of inhibitors acting on two tightly associated metabolic steps results in a cooperative effect on bacterial cells and opens an original way to combat multidrug-resistant bacteria. [ABSTRACT FROM PUBLISHER]

The ability to form biofilm on different surfaces is typical of most Candida species. Microscopic structure and genetic aspects of fungal biofilms have been the object of many studies because of very high resistance to antimycotic agents because of the scarce permeability of the external matrix and to the alterations in cell metabolism. In our study, 31 isolates of Candida parapsilosis, isolated from bloodstream infections, were tested for their ability to produce biofilm and were found to be good producers. The susceptibility to voriconazole, assayed by colorimetrical XTT assay, revealed a very elevated minimum inhibitory concentrations for sessile cells in comparison with planktonic ones. The addition of ambroxol, a mucolytic agent, increased the susceptibility of biofilm forming cells to voriconazole. Expression of the efflux pump genes CDR and MDR was analyzed in biofilms alone or treated with ambroxol, evidencing a role of ambroxol in the expression of genes involved in azole resistance mechanisms of C. parapsilosis biofilms. In conclusion, our data seem to encourage the use of different substances in combination with classical antimycotics, with the aim of finding a solution to the increasing problem of the resistance of biofilms formed on medical devices by nonalbicans Candida species. [ABSTRACT FROM AUTHOR]

Objectives The initial aim of this study was to use a systems biology approach to analyse a ciprofloxacin-selected multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium, L664. Methods The whole genome sequence and transcriptome of L664 were analysed. Site-directed mutagenesis to recreate each mutation was carried out, followed by phenotypic characterization and mutation frequency analysis. As a mutation in the TCA cycle was detected we tested the controversial hypothesis regarding the bacterial response to bactericidal antibiotics, put forward by Kohanski et al. (Cell 2007; 130: 797–810 and Mol Cell 2010; 37: 311–20), that exposure of bacteria to agents such as ciprofloxacin produces reactive oxygen species (ROS), which transiently increase the mutation rate giving rise to MDR bacteria. Results L664 contained a mutation in ramR that conferred MDR. A mutation in tctA affected the TCA cycle and conferred the inability to grow on minimal agar. The virulence of L664 was not attenuated. Ciprofloxacin exposure produced ROS in L664 and SL1344 (tctA::aph), but it was reduced and occurred later. There were no significant differences in the rates of killing or mutations per generation to antibiotic resistance between the strains. Conclusions Whilst we confirm production of ROS in response to ciprofloxacin, we have no data to support the hypothesis that this leads to selection of MDR strains. Our results indicate that the mutations in tctA and glgA were random as they did not pre-exist in the parental strain, and that the mutation in tctA did not provide a survival advantage or disadvantage in the presence of antibiotic. [ABSTRACT FROM PUBLISHER]

Objectives To determine the fluoroquinolone resistance determinants in Salmonella enterica serovar Schwarzengrund from imported foods. Methods Antibiotic susceptibility of Salmonella Schwarzengrund to 16 antibiotics was examined using disc agar diffusion and Etest. Quinolone resistance determinants were examined by sequence analysis of gyrA, gyrB, parC and parE, PCR amplification of qnrA, qnrB and qnrS, and expression of acrB, ramA, marA, soxS and rob using quantitative RT–PCR. The contribution of efflux pump activities to antibiotic resistance was determined by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP). The effect of ramR deletion on ciprofloxacin resistance was determined by complementing with wild-type ramR. Results Salmonella strains 30 and 487 were susceptible to ciprofloxacin and had a single mutation in gyrA as compared with strain 75, which was highly resistant to ciprofloxacin and had a double mutation in gyrA. Increased expression of ramA was associated with high resistance to ciprofloxacin. Strain 75 had a deletion of 315 bp in the ramR gene, which regulates ramA expression. Overexpression of ramA was possibly related to a loss of ramR. Introduction of ramR decreased the MIC of ciprofloxacin from 48 to 24 mg/L. The addition of CCCP did not reduce antibiotic resistance. To our knowledge, this study reports for the first time the natural deletion of ramR in Salmonella Schwarzengrund. Conclusions This study indicates that fluoroquinolone-resistant Salmonella are prevalent in imported food. Double mutations in gyrA and a loss of ramR were associated with high-level quinolone resistance in multidrug-resistant Salmonella Schwarzengrund strain 75. [ABSTRACT FROM PUBLISHER]

The primary objective of this work was to evaluate the capability of curcumin, a natural compound found in the Curcuma longa plant, to sensitize a clinical isolate of Candida albicans, which was found to have a high resistance to fluconazole. In addition, we assessed whether the resistance of this isolate was the result of the existence of efflux pumps, which could confer a multiple drug resistance phenotype. To evaluate azole resistance, we used the Clinical Laboratory Standard Institute (CLSI) MIC assays procedures with minor modifications. For evaluation of synergistic interaction of curcumin and fluconazole, checkerboard experiments were employed. Nile red and Rhodamine 6G accumulation assays were used to evaluate efflux pump activity. Curcumin was found to have a great capability to inhibit fluconazole resistance of the isolate of C. albicans. It was capable of restoring its sensitivity to this azole when used at 11 μM. Analysis with different azoles and the two indicated dyes showed that an efflux pump could be acting and contributing to the resistance of this isolate to fluconazole. The results suggest that a major facilitator superfamily (MFS) transporter might be involved in this process. [ABSTRACT FROM PUBLISHER]

Objectives To investigate the mechanisms of carbapenem resistance in the 175 Pseudomonas aeruginosa isolates (39%; 175/448) showing non-susceptibility (European Committee on Antimicrobial Susceptibility Testing breakpoints) to imipenem (35%), meropenem (33%) and/or doripenem (33%) recovered in 2008–09 from 16 Spanish hospitals during the Comparative Activity of Carbapenem Testing (COMPACT) surveillance study. Methods MICs (Etest), clonal relatedness (PFGE) and metallo-β-lactamase (MBL) production (Etest-MBL, PCR and sequencing) were determined. Mutation-driven resistance was studied in 60 non-MBL producers according to the doripenem MICs (15 isolates from each of four MIC groups: ≤1, 2–4, 8–16 and ≥32 mg/L). The expression of ampC, mexB, mexY, mexD and mexF was determined by real-time reverse transcription–PCR and the presence of mutations in oprD by PCR and sequencing. Isogenic mutants expressing combinations of mutation-driven carbapenem resistance were constructed. Results Twelve (6.9%) isolates were MBL (VIM-20, VIM-2 or VIM-13) producers and all showed high-level resistance (MIC 32 mg/L) to all three carbapenems. Regarding mutation-driven resistance, all but 1 of the 60 isolates were non-susceptible (MIC >32 mg/L) to imipenem, linked to oprD inactivation. In addition, 50% of the isolates overexpressed ampC, 33% mexY, 32% mexB and 15% mexF, while none overexpressed mexD. Increasing prevalence of ampC overexpression correlated with increasing doripenem MICs (≤1, 13%; 2–4, 53%; 8–16, 60%; and ≥32, 73%) while overexpression of efflux pumps correlated only with moderate resistance. Doripenem showed slightly higher activity than meropenem against isolates overexpressing ampC, especially mexB or mexY. The analysis of a collection of isogenic laboratory mutants supported this finding. Conclusions Although the prevalence of MBL producers is increasing, mutation-driven resistance is still more frequent in Spain. Imipenem resistance was driven by OprD inactivation, while additional AmpC and particularly efflux pump hyperproduction had a lower impact on the activity of doripenem compared with meropenem. [ABSTRACT FROM PUBLISHER]

Objectives: As an initial step in developing novel antibacterials against Burkholderia pseudomallei, we have characterized the FabI enoyl-ACP reductase homologues in the type II fatty acid biosynthesis pathway from this organism and performed an initial enzyme inhibition study.Methods: A BLAST analysis identified two FabI enoyl-ACP reductase homologues, bpmFabI-1 and bpmFabI-2, in the B. pseudomallei genome, which were cloned, overexpressed in Escherichia coli and purified. Steady-state kinetics was used to determine the reaction mechanism and the sensitivity of bpmFabI-1 to four diphenyl ether FabI inhibitors. The antibacterial activity of the inhibitors was assessed using a wild-type strain of Burkholderia thailandensis (E264) and an efflux pump mutant (Bt38).Results: Consistent with its annotation as an enoyl-ACP reductase, bpmFabI-1 catalysed the NADH-dependent reduction of 2-trans-dodecenoyl-CoA via a sequential Bi Bi mechanism. In contrast, bpmFabI-2 was inactive with all substrates tested and only bpmfabI-1 was transcriptionally active under the growth conditions employed. The sensitivity of bpmFabI-1 to four diphenyl ethers was evaluated and in each case the compounds were slow-onset inhibitors with K(i) values of 0.5-2 nM. In addition, triclosan and PT01 had MIC values of 30 and 70 mg/L for B. pseudomallei as well as a wild-type strain of B. thailandensis (E264), but MIC values of <1 mg/L for the efflux pump mutant Bt38. A reduction in MIC values was also observed for the pump mutant strain with the other diphenyl ethers.Conclusions: Provided that efflux can be circumvented, bpmFabI-1 is a suitable target for drug discovery. [ABSTRACT FROM AUTHOR]

Objectives Campylobacter jejuni is a leading foodborne pathogen worldwide and its resistance to antimicrobials is a major concern for public health. The cmeG (Cj1375) gene in C. jejuni encodes a putative efflux transporter of the major facilitator family, but its function in antimicrobial resistance has not been determined. This study aimed to characterize the function of CmeG in conferring resistance to antibiotics and oxidative stress. Methods The cmeG gene (Cj1375) in C. jejuni was inactivated by insertional mutagenesis and overexpressed by cloning with a shuttle vector. These constructs were compared with the wild-type strain using antimicrobial susceptibility tests and drug accumulation assays. Results The cmeG mutation reduced bacterial growth and rendered C. jejuni more susceptible to ciprofloxacin, erythromycin, gentamicin, tetracycline, rifampicin, ethidium bromide and cholic acid as well as hydrogen peroxide, and in trans complementation restored the susceptibility to near wild-type level. RT–PCR showed that cmeG is co-transcribed with its downstream gene cmeH (Cj1376) encoding a putative periplasmic protein, but mutation of cmeH alone did not affect the susceptibility to antibiotics. Notably, overexpression of the cmeGH operon in C. jejuni NCTC 11168 significantly increased its resistance to fluoroquinolones. In addition, the cmeG mutant accumulated more EtBr and ciprofloxacin than the wild-type strain. Conclusions These results indicate that CmeG functions as a multidrug efflux transporter contributing to antibiotic resistance and oxidative defence in Campylobacter. [ABSTRACT FROM PUBLISHER]

The antifungal activity of purpurin (1,2,4-trihydroxy-9,10-anthraquinone), a natural red anthraquinone pigment in madder root ( Rubia tinctorum L.), was evaluated by a broth microdilution assay against a total of 24 Candida isolates representing six species. The minimum inhibitory concentration (MIC) range of purpurin was 1.28-5.12 μg/ml. Mechanistic studies using the rhodamine 6G extrusion assay indicated that purpurin inhibited the energy-dependent efflux pumps of the Candida isolates in a dose-dependent manner. Furthermore, purpurin demonstrated a dose-dependent depolarization of mitochondrial membrane potential, one of the biochemical checkpoints regulating cell death in eukaryotic cells, suggesting a possible linkage between purpurin antifungal mechanism and Candida apoptosis. [ABSTRACT FROM AUTHOR]

Objectives: A possible mechanism for HIV therapy failure is the efflux of HIV drugs from viral target cells or certain body compartments by ATP-binding cassette (ABC) transporters, allowing ongoing viral replication. Here, we investigated the interaction between protease inhibitors (PIs) and ABC transporters. [ABSTRACT FROM PUBLISHER]

Objectives: Mutants of five Salmonella enterica serovars were investigated for structural changes in regulatory regions known to be involved in the up-regulation of efflux pumps. [ABSTRACT FROM PUBLISHER]

Many studies have shown that Candida biofilms are highly resistant to fluconazole. The mechanisms of acquired fluconazole resistance of Candida glabrata in non-biofilms have been elucidated, but the relevance of them relative to C. glabrata in biofilms are unknown. We examined by real-time PCR the expression of CgCDR1, CgCDR2, and CgERG11 of four bloodstream isolates of C. glabrata during the early (6 h), intermediate (15 h), and mature (48 h) phases of biofilm development. We found high levels of biofilm formation in vitro. At 6 and 15 h, the biofilms exhibited, in comparison to planktonic cells, approximately 1.5- and 3.3-fold upregulation of CgCDR1 and 0.5- and 3.1-fold upregulation of CgCDR2, respectively. However, at 48 h, neither gene was upregulated. In comparison, the expression of CgERG11 did not significantly increase during any of the three phases. Our results indicate a temporary increase in the expression of both CgCDR1 and CgCDR2 during the intermediate phase of C. glabrata biofilm development. In addition, the data collected at 48 h suggest that CgCDR1, CgCDR2, and CgERG11 may not play a role in the azole resistance of C. glabrata in mature biofilms. [ABSTRACT FROM AUTHOR]

Aims: Proton motive force (PMF) inhibition enhances the intracellular accumulation of autoinducers possibly interfering with biofilm formation. We evaluated the effect of the PMF inhibitor carbonyl cyanide- m-chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development. Methods and Results: Four epidemiologically unrelated P. aeruginosa isolates were studied. A MexAB-oprM overproducing strain was used as control. Expression of gene mexB was examined and biofilm formation after incubation with 0, 12·5 and 25 μmol l−1 of CCCP was investigated. Mean values of optical density were analysed with one-way analysis of variance and t-test. Two isolates subexpressed mexB gene and only 25 μmol l−1 of CCCP affected biofilm formation. Biofilms of the other two isolates and control strain PA140 exhibited significantly lower absorbance ( P ranging from <0·01 to <0·05) with either 12·5 or 25 μmol l−1 of CCCP. Conclusions: The PMF inhibitor CCCP effect was correlated with the expression of MexAB-OprM efflux system and found to compromise biofilm formation in P. aeruginosa. Significance and Impact of the Study: These data suggest that inhibition of PMF-dependent trasporters might decrease biofilm formation in P. aeruginosa. [ABSTRACT FROM AUTHOR]

The genus Xanthomonas contains plant pathogens exhibiting innate resistance to a range of antimicrobial agents. In other genera, multidrug resistance is mediated by a synergy between a low-permeability outer membrane and expression of a number of multidrug efflux systems. This report describes the isolation of a novel gene cluster xmeRSA from Xanthomonas strain IG-8 that mediates copper chloride resistance. Subsequent analysis of these genes showed that they were responsible for the high level of multiple resistance in this strain and were homologues of the sme system of Stenotrophomonas maltophilia. Knock-out mutants of this gene cluster indicate that these genes are required for the copper resistance phenotype of strain IG-8. Expression analysis using lacZ fusions indicates that the genes are regulated by copper and other antimicrobials. Bioinformatic analysis suggests that these genes were acquired by horizontal gene transfer. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa carries several multidrug efflux operons, including mexEF-oprN, that contribute to its resistance to multiple antibiotics. mvaT affects the expression of several P. aeruginosa genes. In this study, we show that the mvaT mutant PAOΔ mvaT is more resistant than its parent PAO1 strain to chloramphenicol and norfloxacin but more sensitive to imipenem; yet both were less resistant to chloramphenicol, norfloxacin, and imipenem than ‘typical’ nfxC-type mutants. Neither strain carries the deletion described for nfxC-type mutants in mexT, the mexEF-oprN regulatory gene. Expression of mexEF-oprN is increased by five- to sixfold in PAOΔ mvaT, while the expression of oprD is reduced by approximately twofold. mvaT mutation had no effect on the expression of other multidrug resistance operons, although it increased the expression of several ATP-binding cassette transporter genes. We show that mvaT mutation does not affect mexEF-oprN expression through mexT or mexS. We also explored several other potential mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: The contribution of target gene mutations and active efflux to varying levels of quinolone resistance in Irish Campylobacter isolates was studied. The Thr-86-Ile modification of GyrA did not correlate with the level of quinolone resistance. The efflux pump inhibitor Phe-Arg-β-Naphthylamide (PAβN) had no effect on the MICs to ciprofloxacin. In contrast, a PAβN sensitive efflux system contributed to the low-level nalixidic acid resistance phenotype. The lack of effect of PAβN in high-level resistant nalidixic isolates may be attributable to mutations identified in the CmeB efflux pump of these isolates. PAβN may have limited diagnostic value in the assessment of the contribution of efflux pump activity to ciprofloxacin resistance in Campylobacter. [Copyright &y& Elsevier]

The Pseudomonas genus, which includes environmental and pathogenic species, is known to present antibiotic resistances, and can receive resistance genes from multi-resistant enteric bacteria released into the environment via faecal rejects. This study was aimed to investigate the resistome of Pseudomonas populations that have been in contact with these faecal bacteria. Thus, faecal discharges originating from human or cattle were sampled (from 12 points and two sampling campaigns) and 41 Pseudomonas species identified (316 isolates studied). The resistance phenotype to 25 antibiotics was determined in all isolates, and we propose a specific antibiotic resistance pattern for 14 species (from 2 to 9 resistances). None showed resistance to aminoglycosides, tetracycline, or polymyxins. Four species carried a very low number of resistances, with none to β-lactams. Interestingly, we observed the absence of the transcriptional activator soxR gene in these four species. No plasmid transfer was highlighted by conjugation assays, and a few class 1 but no class 2 integrons were detected in strains that may have received resistance genes from Enterobacteria. These results imply that the contribution of the Pseudomonas genus to the resistome of an ecosystem first depends on the structure of the Pseudomonas populations, as they may have very different resistance profiles. [ABSTRACT FROM AUTHOR]

Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented. [ABSTRACT FROM AUTHOR]

The article discusses the isolation of a tigecycline-resistant Enterococcus faecalis from the blood cultures of a patient with complicated intra-abdominal sepsis. It mentions the case of a patient who was admitted to the hospital for an elective laparotomy and a small bowel resection with primary anastomosis, removal of a common bile duct stent and a choledochoduodenostomy. It notes the possibility of selecting tigecycline-resistant enterococci in the treatment of intra-abdominal infections.

A letter to the editor is presented about a study of fluoroquinolone resistance in Pseudomonas stutzeri isolated at the Vrije Universiteit (VU) Medical Center in Amsterdam, the Netherlands in 2007.

Summary: Burkholderia pseudomallei is the aetiological agent of melioidosis. Therapy for this disease is lengthy and limited to only a few antibiotics because of this bacterium''s intrinsic antibiotic resistance to many clinically useful antibiotics. These properties of B. pseudomallei may partially be due to expression of efflux pumps of the resistance-nodulation-cell-division (RND) family. The patterns and magnitude of RND efflux pump expression in commonly used strains and clinical isolates of B. pseudomallei from the Royal Darwin Hospital, Darwin, Australia, were assessed in cells grown to late exponential phase using quantitative real-time PCR (qRT-PCR). Expression of the three previously identified RND efflux pumps AmrAB-OprA, BpeAB-OprB and BpeEF-OprC, as well as four other yet uncharacterized pumps, was found to be widespread in the clinical isolates. In 45 of 50 isolates (90%), mRNA was detected for at least one of the seven RND pumps. Of these 45 isolates, 41 (82%) expressed multiple pumps with nine strains expressing all seven pumps tested. While these studies revealed no striking correlation between RND efflux pump expression and clinically significant antibiotic resistance, the data support the notion that RND pumps probably play important roles in this bacterium''s physiology, defence against toxic compounds, and perhaps virulence. [Copyright &y& Elsevier]