Porin Gene Research Articles

Insertion sequence ISRP10 inactivation of the oprD gene in imipenem-resistant Pseudomonas aeruginosa clinical isolates

Carbapenem resistance mechanisms were investigated in 32 imipenem-resistant Pseudomonas aeruginosa clinical isolates recovered from hospitalised children. Sequence analysis revealed that 31 of the isolates had an insertion sequence element ISRP10 disrupting the porin gene oprD, demonstrating that ISRP10 inactivation of oprD conferred imipenem resistance in the majority of the isolates. Multilocus sequence typing (MLST) was used to discriminate the isolates. In total, 11 sequence types (STs) were identified including 3 novel STs, and 68.3% (28/41) of the tested strains were characterised as clone ST253. In combination with random amplified polymorphic DNA (RAPD) analysis, the imipenem-resistant isolates displayed a relatively high degree of genetic variability and were unlikely associated with nosocomial infections.

Hetero-oligomeric MspA pores in Mycobacterium smegmatis.

The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.

Reduction in horizontal transfer of conjugative plasmid by UV irradiation and low-level chlorination

The widespread presence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) in the drinking water system facilitates their horizontal gene transfer among microbiota. In this study, the conjugative gene transfer of RP4 plasmid after disinfection including ultraviolet (UV) irradiation and low-level chlorine treatment was investigated. It was found that both UV irradiation and low-level chlorine treatment reduced the conjugative gene transfer frequency. The transfer frequency gradually decreased from 2.75 × 10−3 to 2.44 × 10−5 after exposure to UV doses ranging from 5 to 20 mJ/cm2. With higher UV dose of 50 and 100 mJ/cm2, the transfer frequency was reduced to 1.77 × 10−6 and 2.44 × 10−8. The RP4 plasmid transfer frequency was not significantly affected by chlorine treatment at dosages ranging from 0.05 to 0.2 mg/l, but treatment with 0.3–0.5 mg/l chlorine induced a decrease in conjugative transfer to 4.40 × 10−5 or below the detection limit. The mechanisms underlying these phenomena were also explored, and the results demonstrated that UV irradiation and chlorine treatment (0.3 and 0.5 mg/l) significantly reduced the viability of bacteria, thereby lowering the conjugative transfer frequency. Although the lower chlorine concentrations tested (0.05–0.2 mg/l) were not sufficient to damage the cells, exposure to these concentrations may still depress the expression of a flagellar gene (FlgC), an outer membrane porin gene (ompF), and a DNA transport-related gene (TraG). Additionally, fewer pili were scattered on the bacteria after chlorine treatment. These findings are important in assessing and controlling the risk of ARG transfer and dissemination in the drinking water system.

Structural organization of porin genes (intact and disrupted by IS100) bordering the pgm locus of the Yersinia pestis and Yersinia pseudotuberculosis strains

In highly virulent strains of Yersinia pestis, the porin gene border- ing pigmentation (pgm) locus was observed to be usually broken by IS100. In this case, the pgm locus that carries virulence genes (high pathogenicity island) and biofilm formation genes (hms operon) is flanked by direct copies of IS100, which can cause its destabilization. We studied the prevalence of the intact and dis- rupted porin genes among 240 strains of Y. pestis from 39 natural centers in Russia and abroad, and 68 strains of Yersinia pseudotuberculosis from different geographical regions. The majority of the highly virulent Y. pestis strains and some phylogenetic lines of Y. pseudotuberculosis 0:1 serotype contain disrupted porin genes. At the same time, deletion of the pgm locus by flanking IS100 in Y pseudotuberculosis is impossible, because IS100 is integrated in the porin gene in the reverse orientation as compared to Y pestis. The porin genes are intact in all Y pestis strains with low epidemic importance and some phylogenetic lines of highly virulent Y pestis strains from some desert foci and Caspian sandy focus, as well as most strains of Y pseudotuberculosis 0:1 serotype. Less virulent strains of Y pseudotuberculosis 0:3 serotype revealed extensive deletion, which included the porin gene and a portion of the gene astE. The nucleotide sequence of the porin genes in Y pestis and Y pseudotuberculosis strains from different geographical regions are identical. Three alleles of the porin gene differ solely by the site of integration and orientation of IS 100 or by the lack of integration. The nucleotide sequence of IS 100, embedded in the porin gene of Yersinia, has minor differences only in two Y pestis strains isolated in America. Low frequency of Hms- mutations correlates with the intact condition of the porin gene in Y pestis. This correlation is absent in Y pseudotuberculosis.

Diversity of Molecular Mechanisms Conferring Carbapenem Resistance toPseudomonas aeruginosaIsolates from Saudi Arabia

Background. This study described various molecular and epidemiological characters determining antibiotic resistance patterns in Pseudomonas aeruginosa isolates. Methods. A total of 34 carbapenem-resistant P. aeruginosa clinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE) were performed. Results. All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC) > 256 mg/L). Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrum β-lactamase (VEB-1) (n = 16/34) and oxacillinase (OXA-10) (n = 14/34) were the most prevalent extended-spectrum β-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28) and imipenemase (IMP-7) variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD) expression was seen in nine isolates, and an increase in efflux pump gene (MexAB) expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15) were found among the 34 isolates. The predominant serotype was O:11 (16 isolates), followed by O:15 (nine isolates). PFGE analysis of the 34 carbapenem-resistant P. aeruginosa isolates revealed 14 different pulsotypes. Conclusions. These results revealed diverse mechanisms conferring carbapenem resistance to P. aeruginosa isolates from Saudi Arabia.

Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC).

The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system.

Genetic Characterization of Carbapenem-Resistant Enterobacteriaceae and the Spread of Carbapenem-Resistant Klebsiella pneumonia ST340 at a University Hospital in Thailand.

Carbapenem-resistant Enterobacteriaceae (CRE) has increasingly spread worldwide in the past decade. The prevalence and characteristics of CRE in Thailand are unknown. In this study, we conducted a 2-year surveillance of CRE among 12,741 clinical isolates of Enterobacteriaceae at the largest university hospital in Thailand with molecular characterization of beta-lactamase (bla) genes, including carbapenemase genes. The CRE prevalence was 1.4%. bla KPC-13 and bla IMP-14a were the only carbapenemase genes detected among these CRE isolates. bla KPC-13 gene was found in a single isolate of Escherichia coli, Enterobacter cloacae and Citrobacter freundii, and bla IMP-14a was found in four isolates of Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) isolates were resistant to multiple carbapenems at a higher ratio than other CRE species, and thus were further characterized for resistance phenotypes, bla genotypes and molecular epidemiology. Most CRKP isolates harboured multiple bla genes, especially those related to extended-spectrum beta-lactamases. Seven CRKP isolates were resistant to all tested carbapenems, and showed decreased ompK35 and/or ompK36 porin gene expression. Molecular typing of CRKP based on pulsed-field gel electrophoresis (PFGE) demonstrated several unrelated clones. Multilocus sequence typing (MLST) was partially concordant with PFGE results and revealed that ST340, a member of drug-resistant K. pneumoniae clonal complex 258, was the most predominant clone, followed by ST48, ST11 and ST273. The novel ST1645 was identified from this study. ST340 has neither been shown to be predominated among CRKP from other studies, nor been reported in Thailand. Therefore, it emphases a critical concern to monitor and control the spread of CRKP.

Effects of Klebsiella pneumoniae carbapenemase subtypes, extended-spectrum β-lactamases, and porin mutations on the in vitro activity of ceftazidime-avibactam against carbapenem-resistant K. pneumoniae.

Avibactam is a novel β-lactamase inhibitor with affinity for Klebsiella pneumoniae carbapenemases (KPCs). In combination with ceftazidime, the agent demonstrates activity against KPC-producing K. pneumoniae (KPC-Kp). KPC-Kp strains are genetically diverse and harbor multiple resistance determinants, including defects in outer membrane proteins and extended-spectrum β-lactamases (ESBLs). Mutations in porin gene ompK36 confer high-level carbapenem resistance to KPC-Kp strains. Whether specific mechanisms of antimicrobial resistance also influence the activity of ceftazidime-avibactam is unknown. We defined the effects of ceftazidime-avibactam against 72 KPC-Kp strains with diverse mechanisms of resistance, including various combinations of KPC subtypes and ESBL and ompK36 mutations. Ceftazidime MICs ranged from 64 to 4,096 μg/ml and were lowered by a median of 512-fold with the addition of avibactam. All strains exhibited ceftazidime-avibactam MICs at or below the CLSI breakpoint for ceftazidime (≤4 μg/ml; range, 0.25 to 4). However, the MICs were within two 2-fold dilutions of the CLSI breakpoint against 24% of the strains, and those strains would be classified as nonsusceptible to ceftazidime by EUCAST criteria (MIC > 1 μg/ml). Median ceftazidime-avibactam MICs were higher against KPC-3 than KPC-2 variants (P = 0.02). Among KPC-2-Kp strains, the presence of both ESBL and porin mutations was associated with higher drug MICs compared to those seen with either factor alone (P = 0.003 and P = 0.02, respectively). In conclusion, ceftazidime-avibactam displays activity against genetically diverse KPC-Kp strains. Strains with higher-level drug MICs provide a reason for caution. Judicious use of ceftazidime-avibactam alone or in combination with other agents will be important to prevent the emergence of resistance.

Single cell super-resolution imaging of E. coli OmpR during environmental stress.

Two-component signaling systems are a major strategy employed by bacteria, and to some extent, yeast and plants, to respond to environmental stress. The EnvZ/OmpR system in E. coli responds to osmotic and acid stress and is responsible for regulating the protein composition of the outer membrane. EnvZ is a histidine kinase located in the inner membrane. Upon activation, it is autophosphorylated by ATP and subsequently, it activates OmpR. Phosphorylated OmpR binds with high affinity to the regulatory regions of the ompF and ompC porin genes to regulate their transcription. We set out to visualize these two-components in single bacterial cells during different environmental stress conditions and to examine the subsequent modifications to the bacterial nucleoid as a result. We created a chromosomally-encoded, active, fluorescent OmpR-PAmCherry fusion protein and compared its expression levels with RNA polymerase. Quantitative western blotting had indicated that these two proteins were expressed at similar levels. From our images, it is evident that OmpR is significantly less abundant compared to RNA polymerase. In cross-sectional axial images, we observed OmpR molecules closely juxtaposed near the inner membrane during acidic and hyposomotic growth. In acidic conditions, the chromosome was compacted. Surprisingly, under acidic conditions, we also observed evidence of a spatial correlation between the DNA and the inner membrane, suggesting a mechanical link through an active DNA-OmpR-EnvZ complex. This work represents the first direct visualization of a response regulator with respect to the bacterial chromosome.

Three Dimensional Checkerboard Synergy Analysis of Colistin, Meropenem, Tigecycline against Multidrug-Resistant Clinical Klebsiella pneumonia Isolates.

The spread of carbapenem-non-susceptible Klebsiella pneumoniae strains bearing different resistance determinants is a rising problem worldwide. Especially infections with KPC (Klebsiella pneumoniae carbapenemase) - producers are associated with high mortality rates due to limited treatment options. Recent clinical studies of KPC-blood stream infections revealed that colistin-based combination therapy with a carbapenem and/or tigecycline was associated with significantly decreased mortality rates when compared to colistin monotherapy. However, it remains unclear if these observations can be transferred to K. pneumoniae harboring other mechanisms of carbapenem resistance. A three-dimensional synergy analysis was performed to evaluate the benefits of a triple combination with meropenem, tigecycline and colistin against 20 K. pneumoniae isolates harboring different β-lactamases. To examine the mechanism behind the clinically observed synergistic effect, efflux properties and outer membrane porin (Omp) genes (ompK35 and ompK36) were also analyzed. Synergism was found for colistin-based double combinations for strains exhibiting high minimal inhibition concentrations against all of the three antibiotics. Adding a third antibiotic did not result in further increased synergistic effect in these strains. Antagonism did not occur. These results support the idea that colistin-based double combinations might be sufficient and the most effective combination partner for colistin should be chosen according to its MIC.

A putative porin gene of Burkholderia sp. NK8 involved in chemotaxis toward β-ketoadipate.

Burkholderia sp. NK8 can utilize 3-chlorobenzoate (3CB) as a sole source of carbon because it has a megaplasmid (pNK8) that carries the gene cluster (tfdT-CDEF) encoding chlorocatechol-degrading enzymes. The expression of tfdT-CDEF is induced by 3CB. In this study, we found that NK8 cells were attracted to 3CB and its degradation products, 3- and 4-chlorocatechol, and β-ketoadipate. Capillary assays revealed that a pNK8-eliminated strain (NK82) was defective in chemotaxis toward β-ketoadipate. The introduction of a plasmid carrying a putative outer membrane porin gene, which we name ompNK8, into strain NK82 restored chemotaxis toward β-ketoadipate. RT-PCR analyses demonstrated that the transcription of the ompNK8 gene was enhanced in the presence of 3CB.

First detection of insertion sequence element ISPa1328 in theoprD porin gene of an imipenem-resistant Pseudomonas aeruginosa isolate from an idiopathic pulmonary fibrosis patient in Marseille, France.

We report here the first case of a carbapenem-resistant Pseudomonas aeruginosa clinical isolate harboring the insertion sequence (IS) element ISPa1328 in the oprD gene in an idiopathic pulmonary fibrosis patient in France previously treated with imipenem.

Quantifying Ligand Binding and Signal Transduction in Escherichia coli Using the Taz Chimera

The purpose of this project was to alter the EnvZ signal transduction process in Escherichia coli through replacement of its osmotic sensor with the aspartate binding region of the chemotaxis sensor protein Tar as in Utsumi et al.1 Aspartate binds Tar and signals nonrandom movement of the bacteria toward increasing aspartate concentration. EnvZ is normally an osmotic sensor that activates the protein OmpR. OmpR inactivates the OmpF gene promoter, which produces large membrane pores and it also activates the OmpC gene promoter which produces small membrane pores. Activity of the Tar / EnvZ hybrid, Taz will be determined by measuring light output from bacterial luciferase under control of the OmpC promoter. We hypothesize that light output will be proportional to aspartate concentration. A Tar knockout background will be used to prevent competition with the Taz chimera. This research was funded by NSF award #0966085 provided to Hartwick College and the Hartwick College Chemistry Department. 1Utsumi R, Brissette RE, Rampersaud A, Forst SA, Oosawa K, Inouye M. Activation of bacterial porin gene expression by a chimeric signal. Science. 1989.

A response regulator from a soil metagenome enhances resistance to the β-lactam antibiotic carbenicillin in Escherichia coli.

Functional metagenomic analysis of soil metagenomes is a method for uncovering as-yet unidentified mechanisms for antibiotic resistance. Here we report an unconventional mode by which a response regulator derived from a soil metagenome confers resistance to the β-lactam antibiotic carbenicillin in Escherichia coli. A recombinant clone (βlr16) harboring a 5,169 bp DNA insert was selected from a metagenomic library previously constructed from a remote Alaskan soil. The βlr16 clone conferred specific resistance to carbenicillin, with limited increases in resistance to other tested antibiotics, including other β-lactams (penicillins and cephalosporins), rifampin, ciprofloxacin, erythromycin, chloramphenicol, nalidixic acid, fusidic acid, and gentamicin. Resistance was more pronounced at 24°C than at 37°C. Zone-of-inhibition assays suggested that the mechanism of carbenicillin resistance was not due to antibiotic inactivation. The DNA insert did not encode any genes known to confer antibiotic resistance, but did have two putative open reading frames (ORFs) that were annotated as a metallopeptidase and a two-component response regulator. Transposon mutagenesis and subcloning of the two ORFs followed by phenotypic assays showed that the response regulator gene was necessary and sufficient to confer the resistance phenotype. Quantitative reverse transcriptase PCR showed that the response regulator suppressed expression of the ompF porin gene, independently of the small RNA regulator micF, and enhanced expression of the acrD, mdtA, and mdtB efflux pump genes. This work demonstrates that antibiotic resistance can be achieved by the modulation of gene regulation by heterologous DNA. Functional analyses such as these can be important for making discoveries in antibiotic resistance gene biology and ecology.

The role of porins in copper acquisition by mycobacteria

Aims and objectivesCopper poisoning in macrophages plays an important role in immunity against invading pathogens. Many bacteria, including Mycobacterium tuberculosis (Mtb), have evolved mechanisms to combat copper-derived innate immune responses. Copper homeostasis in Mtb consists of several components, including a multi-copper oxidase, copper efflux pump, cytoplasmic metallothionein and copper-sensing transcriptional regulators. However, components involved in copper uptake are unknown, which prompted this study to investigate the possible role of porins in copper uptake in mycobacteria. MethodsMycobacterium smegmatis porin mutants were created and tested for their ability to grow under copper-reduced or copper-rich conditions. The M. smegmatis porin gene mspA was expressed in Mtb, and its copper susceptibility profile was investigated in the presence of different copper concentrations. The expression level of a copper detoxifying protein, mycobacterial multi-copper oxidase (MmcO), was monitored by western blot to assess intracellular copper content. ResultsDeletion of porin genes from M. smegmatis caused a severe growth defect on trace copper medium. Copper supplementation alleviated this phenotype. The inability to acquire copper in sufficient amounts due to lack of porins can explain this phenomenon. Moreover, porin mutants showed elevated tolerance to copper at concentrations that were toxic for wild-type strains, indicating that the lack of porins protects these strains from copper poisoning. On the other hand, heterologous expression of mspA in Mtb significantly impaired growth at 2.5μM copper and eliminated growth at 15μM, while wild-type Mtb eventually reached its normal cell density at this copper concentration. Consistent with a role of porins in copper uptake, expression levels of MmcO in Mtb expressing the M. smegmatis porin mspA was above wild-type levels, indicating that cytoplasmic copper-sensing transcriptional regulators respond by derepressing the expression of copper resistance genes. Moreover, the polyamine spermine, a known inhibitor of porin activity in gram-negative bacteria, increased the tolerance of wild-type Mtb for copper suggesting that endogenous outer membrane proteins with channel-forming activity exist and contribute to copper acquisition and toxicity in Mtb. ConclusionsIt was concluded from these results that porins are involved in copper uptake in mycobacteria. Moreover, the outer membrane of Mtb was found to be an important barrier against copper intoxication so that permeabilization of this barrier (e.g., by porins) renders Mtb extremely vulnerable to copper. Consequently, copper homeostasis of Mtb provides a promising drug target for the development of a new class of anti-tuberculosis compounds that can induce a copper hypersensitivity phenotype in Mtb.

In Vitro Activity of Imipenem and Colistin against a Carbapenem-Resistant Klebsiella pneumoniae Isolate Coproducing SHV-31, CMY-2, and DHA-1.

We investigated the synergism of colistin and imipenem against a multidrug-resistant K. pneumoniae isolate which was recovered from a severe hip infection. PCR and DNA sequencing were used to characterize the outer membrane porin genes and the resistance genes mediating the common β-lactamases and carbapenemases. Synergism was evaluated by time-kill studies. The bla SHV-31, bla CMY-2, and bla DHA-1 were detected. Outer membrane porin genes analysis revealed loss of ompK36 and frame-shift mutation of ompK35. The common carbapenemase genes were not found. Time-kill studies demonstrated that a combination of 1x MIC of colistin (2 mg/L) and 1x MIC of imipenem (8 mg/L) was synergistic and bactericidal but with inoculum effect. Bactericidal activity without inoculum effect was observed by concentration of 2x MIC of colistin alone or plus 2x MIC of imipenem. In conclusion, colistin plus imipenem could be an alternative option to treat carbapenem-resistant K. pneumoniae infections.

MspA-Mycobacterium tuberculosis-transformant with reduced virulence: The “unbirthday paradigm”

Expressing mspA porin gene from Mycobacterium smegmatis in Mycobacterium tuberculosis attenuated this pathogen. Intracellular growth of the transformants into free-living amoeba and murine and human macrophages decreased. Furthermore, transformants decreased the microbicidal program of human monocyte-derived macrophages. BALB/c mice inoculated with transformants exhibited higher weights, lower histological lesions and lower M. tuberculosis inoculum in the liver, spleen and lungs than control mice challenged with wild-type M. tuberculosis. Preliminary evaluation indicated that mice inoculated with this transformant showed higher weights and lower numbers of lung nodules and tissular mycobacteria than control mice when challenged with wild-type M. tuberculosis. Similar to the paradoxical “unbirthday” gift coined by Lewis Carroll in Alice's Adventures in Wonderland, adding mspA gene reduced the virulence of M. tuberculosis and yielded a protective effect. Lost of non-virulence genes is a mechanism for virulence in mycobacteria. Engineering non-virulence genes in M. tuberculosis may yield strains with decreased virulence and increased immunogenicity.

Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.

Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established.

Polymorphism in gyrA is associated to quinolones resistance in Chilean Piscirickettsia salmonis field isolates.

Keywords: antibiotic resistance, gyrA, Piscirickettsiasalmonis, quinolones.Piscirickettsia salmonis is a gram-negative, pleomor-phic coccoid-shaped and facultative intracellularfish pathogenic bacterium, that causes piscirickettsi-osis, a disease also known as salmonid rickettsialsepticaemia (SRS) (Fryer et al. 1990). In Chile,P. salmonis was isolated for the first time in 1989,but probably has been present since 1983 (Bravo C Rozas &Enriquez 2013). SRS has also been reported inimportant salmon producing countries such asCanada, Ireland, Scotland and Norway, but with alower economic impact (Rozas & Enriquez 2013).P. salmonis cells are slow growing and hard to culti-vate in free-cell media, which has hindered its isola-tion and further characterization. However, recentlydescribed supplemented media (Mikalsen et al.2008) facilitate their growth and the antibiotictesting assays to evaluate effective antimicrobial ther-apy. In this regard, quinolones were the former mostwidely used antimicrobial compounds in Chileanaquaculture, especially as a first line drug againstSRS.In Chile, a large proportion of antibiotics is usedfor prophylaxis rather than for chemotherapy. Forinstance, in comparison with Norway and Canada,1500 and eight times more antimicrobials wereneeded to obtain similar amounts of salmon inChile in 2007 (Cabello et al. 2013). The same year,930 metric tons of antibiotics were mostly used insalmon production. Of those antibiotics, 24% werequinolones (Cabello et al. 2013). This practice hasalready altered the microbiota of sediments in closeproximity to aquaculture sites, where a significantfraction of isolated bacteria is antibiotic resistant(Buschmann et al. 2012). Therefore, the emer-gence and potential spread of antibiotic-resistantP. salmonis isolates cannot be ruled out.Twenty P. salmonis isolates cultured from kid-ney samples of diseased fish collected from diversegeographic origin (Fig. 1) between 2010 and2012 were phenotypically characterized for quinol-ones susceptibility. The isolates were grown onagar (Mikalsen et al. 2008), at 18 °C for 10 daysand kept frozen in liquid nitrogen. For antibioticsensitivity testing, the minimal inhibitory concen-tration (MIC) for enrofloxacin, flumequine andoxolinic acid were assessed according to theinstructions given by the Clinical and LaboratoryStandards Institute (CLSI), guide M49-A. 96-wellmicroplates containing twofold serial dilutions(0.03–64 lgmL

ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production

The opportunistic pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilms of this bacterium are much more resistant to antibiotics than planktonic cells. Thirty-six metal ions have been investigated to identify antivirulence and antibiofilm metal ions. Zinc ions and ZnO nanoparticles were found to markedly inhibit biofilm formation and the production of pyocyanin, Pseudomonas quinolone signal (PQS), pyochelin, and hemolytic activity of P. aeruginosa without affecting the growth of planktonic cells. Transcriptome analyses showed that ZnO nanoparticles induce the zinc cation efflux pump czc operon and several important transcriptional regulators (porin gene opdT and type III repressor ptrA), but repress the pyocyanin-related phz operon, which explains observed phenotypic changes. A mutant study showed that the effects of ZnO nanoparticles on the control of pyocyanin production and biofilm formation require the czc regulator CzcR. In addition, ZnO nanoparticles markedly increased the cellular hydrophilicity of P. aeruginosa cells. Our results support that ZnO nanoparticles are potential antivirulence materials against recalcitrant P. aeruginosa infections and possibly other important pathogens.