Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

Do viscous forces affect survival of marine fish larvae? Revisiting the ‘safe harbour’ hypothesis

Aquatic organisms physically interact with the water that surrounds them, and this interaction is fundamental in shaping many aspects of their biology. General characteristics of the hydrodynamic interactions between organisms and the flow around them can be captured by the dimensionless Reynolds number (Re), depicting the ratio between inertial and viscous forces operating on the organism. The characteristic flow regime of larval fish that cruise at slow speeds is a regime of low Re, where viscous forces dominate. In this study, we experimentally test the ‘safe harbour’ hypothesis, which proposes that increasing larval body size facilitates an ‘escape’ from the detrimental effects of low Re. Larval gilthead seabream (Sparus aurata) were reared during early ontogeny under artificially manipulated water viscosities to expose larvae to low Re regimes. Larval survival decreased significantly with increasing water viscosity, and increased with increasing standard length. Surviving larvae exceeded the mean length of mortalities by ~1 mm, on average. Our findings provide direct experimental support for the ‘safe harbour’ hypothesis, indicating that marine larvae incur a fitness cost when operating under low Re conditions. Moreover, the results highlight the need to recognize the hydrodynamic environment when considering the a-biotic characteristics that may influence organismal performance and fitness.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

Binuclear rhenium carbonyl nitrosyls related to dicobalt octacarbonyl and their decarbonylation products

The geometries and thermochemistry of Re₂(NO)₄(CO) _n (n?=?4, 3, 2, 1, 0) structures isovalent with the binuclear cobalt carbonyls Co₂(CO) _n+4 have been examined using density functional theory. Eight low-energy Re₂(NO)₄(CO)₄ structures, all with formal Re–Re single bonds, lie within 6 kcal mol^?1 of the global minimum. These eight structures include unbridged structures as well as structures with two bridging NO groups but no structures with bridging CO groups. Similarly, five low-energy Re₂(NO)₄(CO)₃ structures, all with formal Re=Re double bonds, lie within 6 kcal mol^?1 of the global minimum. Again these five structures include unbridged structures as well as structures with one or two bridging NO groups but no structures with bridging CO groups. The Re₂(NO)₄(CO) _n (n?=?4, 3) appear to be fluxional systems similar to the well-known Co₂(CO)₈ for which doubly bridged and unbridged structures have approximately the same energies. The lowest energy Re₂(NO)₄(CO)₂ structures have formal Re=Re double bonds including a structure with a five-electron donor bridging η²-μ-NO group. Isomeric Re₂(NO)₄(CO)₂ structures with formal Re≡Re triple bonds lie approximately ～10 kcal mol^?1 above the global minimum. For the more highly unsaturated Re₂(NO)₄(CO) and Re₂(NO)₄ systems, the lowest energy structures have formal Re≡Re triple bonds of length ～2.6 Å. Higher energy Re₂(NO)₄(CO) structures have shorter Re–Re distances of length ～2.5 Å suggesting formal quadruple bonds.

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Graphical Abstract The geometries and thermochemistry of Re₂(NO)₄(CO) _n (n?=?4, 3, 2, 1, 0) structures isovalent with the binuclear cobalt carbonyls Co₂(CO) _n+4 have been examined using density functional theory. A number of energetically closely spaced Re₂(NO)₄(CO)₄ and Re₂(NO)₄(CO)₃ structures are found, including unbridged and NO-bridged structures but no CO-bridged structures. The Re₂(NO)₄(CO) _n (n?=?2, 1, 0) systems provide examples of Re–Re multiple bonds of orders ranging from 2 to 4.

     

Expression of extracellular polysaccharides and proteins by clinical isolates of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in response to environmental conditions

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.     

<Emphasis Type="Italic">Burkholderia xenovorans</Emphasis> LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the <Emphasis Type="Italic">pha</Emphasis> genes and synthesizes poly(3-hydroxybutyrate) under nitrogen-limiting conditions

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that are synthesized by diverse bacteria. In this study, the synthesis of PHAs by the model aromatic-degrading strain Burkholderia xenovorans LB400 was analyzed. Twelve pha genes including three copies of phaC and five copies of the phasin-coding phaP genes are distributed among the three LB400 replicons. The phaC1ABR gene cluster that encodes the enzymes of the PHA anabolic pathway is located at chromosome 1 of strain LB400. During the growth of strain LB400 on glucose under nitrogen limitation, the expression of the phaC1, phaA, phaP1, phaR, and phaZ genes was induced. Under nitrogen limitation, PHA accumulation in LB400 cells was observed by fluorescence microscopy after Nile Red staining. GC-MS analyses revealed that the PHA accumulated under nitrogen limitation was poly(3-hydroxybutyrate) (PHB). LB400 cells grown on glucose as the sole carbon source under nitrogen limitation accumulated 40?±?0.96% PHB of the cell dry weight, whereas no PHA was observed in cells grown in control medium. The functionality of the phaC1 gene from strain LB400 was further studied using heterologous expression in a Pseudomonas putida KT40C1ZC2 mutant strain derived from P. putida KT2440 that is unable to synthesize PHAs. Interestingly, KT40C1ZC2[pVNC1] cells that express the phaC1 gene from strain LB400 were able to synthesize PHB (33.5% dry weight). This study indicates that B. xenovorans LB400 possesses a functional PHA synthetic pathway that is encoded by the pha genes and is capable of synthesizing PHB.     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

Centromeric and non-centromeric satellite DNA organisation differs in holocentric <Emphasis Type="Italic">Rhynchospora</Emphasis> species

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.     

In Vitro Activity of Berberine Alone and in Combination with Antifungal Drugs Against Planktonic Forms and Biofilms of <Emphasis Type="Italic">Trichosporon Asahii</Emphasis>

Trichosporon asahii (T. asahii) is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients, with high mortality rates up to 80% despite treated with antifungal drugs. The biofilms-forming ability of T. asahii on indwelling medical devices may account for the resistance to antifungal drugs. Berberine (BBR) has been demonstrated to have antifungal activity and synergistic effects in combination with antifungal drugs against pathogenic fungi. In the present study, the in vitro activities of BBR alone or combined with fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against planktonic forms and biofilms of 21 clinical T. asahii isolates were evaluated using checkerboard microdilution method and XTT reduction assay, respectively. The fractional inhibitory concentration index (FICI) was used to interpret drug interactions. BBR alone did not exhibit significant antifungal activities against both T. asahii planktonic cells (MICs, 32 → 128 μg/ml) and T. asahii biofilms (SMICs, >128 μg/ml). However, BBR exhibited synergistic effects against T. asahii planktonic cells in combination with AMB, FLC and CAS (FICI ≤ 0.5) and exhibited synergistic effects against T. asahii biofilms in combination with AMB and CAS (FICI ≤ 0.5). BBR/ITC and BBR/VRC combinations yielded mainly indifferent interactions against T. asahii planktonic cells. BBR/FLC, BBR/ITC and BBR/VRC combinations also yielded indifferent interactions against T. asahii biofilms. Our study highlights the therapeutic potential of BBR to be used as an antifungal synergist in combination with antifungal drugs against T. asahii infections, especially BBR/AMB combination. Further in vivo studies are needed to validate our findings.     

Human milk and mucosal lacto- and galacto-<Emphasis Type="Italic">N</Emphasis>-biose synthesis by transgalactosylation and their prebiotic potential in <Emphasis Type="Italic">Lactobacillus</Emphasis> species

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-d-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.     

Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress.

Results

Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g^-1 dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain.

Conclusion

The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

     

<Emphasis Type="Italic">Novosphingobium profundi</Emphasis> sp. nov. isolated from a deep-sea seamount

A marine bacterial strain, F72^T, was isolated from a solitary scleractinian coral, collected in Yap seamounts in the Pacific Ocean. Strain F72^T is a Gram-negative, light-yellow-pigmented, motile, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain F72^T is related to the genus Novosphingobium and has high 16S rRNA gene sequence similarities with the type strains of Novosphingobium pentaromativorans US6-1^T (97.7 %), Novosphingobium panipatense SM16^T (97.6 %), Novosphingobium mathurense SM117^T (97.2 %) and Novosphingobium barchaimii LL02^T (97.1 %). Ubiquinone Q-10 was detected as the dominant quinone. The predominant cellular fatty acids were C_18:1ω7c and C_17:1ω6c. The genomic DNA G+C content of strain F72^T was 63.4 mol %. The polar lipids profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylcholine, sphingoglycolipid and one uncharacterized lipid. Strain F72^T shared DNA relatedness of 25 % with N. pentaromativorans JCM 12182^T, 31 % with N. panipatense DSM 22890^T, 21 % with N. mathurense DSM 23374^T and 26 % with N. barchaimii DSM 25411^T. Combined data from phenotypic, phylogenetic and DNA–DNA relatedness studies demonstrated that the strain F72^T is a representative of a novel species of the genus Novosphingobium, for which we propose the name Novosphingobium profundi sp. nov. (type strain F72^T = KACC 18566^T = CGMCC 1.15390^T).     

Toxin Gene Contents and Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strains Against Two Sugarcane Borer Species, <Emphasis Type="Italic">Diatraea saccharalis</Emphasis> (F.) and <Emphasis Type="Italic">D. flavipennella</Emphasis> (Box)

Bacillus thuringiensis (Berliner) bears essential characteristics in the control of insect pests, such as its unique mode of action, which confers specificity and selectivity. This study assessed cry gene contents from Bt strains and their entomotoxicity against Diatraea saccharalis (F.) and Diatraea flavipennella (Box) (Lepidoptera: Crambidae). Bioassays with Bt strains were performed against neonates to evaluate their lethal and sublethal activities and were further analyzed by PCR, using primers to identify toxin genes. For D. saccharalis and D. flavipennella, 16 and 18 strains showed over 30% larval mortality in the 7th day, respectively. The LC₅₀ values of strains for D. saccharalis varied from 0.08 × 10⁵ (LIIT-0105) to 4104 × 10⁵ (LIIT-2707) spores + crystals mL^?1. For D. flavipennella, the LC₅₀ values of strains varied from 0.40 × 10⁵ (LIIT-2707) to 542 × 10⁵ (LIIT-2109) spores + crystals mL^?1. For the LIIT-0105 strain, which was the most toxic to D. saccharalis, the genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, cry8, and cry9C were detected, whereas for the strain LIIT-2707, which was the most toxic to D. flavipennella, detected genes were cry1Aa, cry1Ab, cry1Ac, cry1B, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, and cry9. The toxicity data and toxin gene content in these strains of Bt suggest a great variability of activity with potential to be used in the development of novel biopesticides or as source of resistance genes that can be expressed in plants to control pests.     

In vitro activity of micafungin against biofilms of <Emphasis Type="Italic">Candida albicans</Emphasis>, <Emphasis Type="Italic">Candida glabrata</Emphasis>, and <Emphasis Type="Italic">Candida parapsilosis</Emphasis> at different stages of maturation

Candida spp. is able to form a biofilm, which is considered resistant to the majority of antifungals used in medicine. The aim of this study was to evaluate the in vitro activity of micafungin against Candida spp. biofilms at different stages of their maturation (2, 6, and 24 h). We assessed the inhibitory effect of micafungin against 78 clinical isolates of Candida spp., growing as planktonic or sessile cells, by widely recommended broth microdilution method. The in vitro effect on sessile cells viability was evaluated by colorimetric reduction assay. All examined strains were susceptible or intermediate to micafungin when growing as planktonic cells. At the early stages of biofilm maturation, from 11 (39.3%) to 20 (100%), tested strains, depending on the species, exhibited sessile minimal inhibitory concentrations (SMICs) of micafungin at ≤ 2 mg/L. For 24-h-old Candida spp. biofilms, from 3 (10.7%) to 20 (100%) of the tested strains displayed SMICs of micafungin at ≤ 2 mg/L. Our findings confirm that micafungin exhibits high potential anti-Candida-biofilm activity. However, this effect does not comprise all Candida species and strains. All strains were susceptible or intermediate to micafungin when growing as planktonic cells, but for biofilms, micafungin displays species- and strain-specific activity. Paradoxical growth of C. albicans and C. parapsilosis was observed. Antifungal susceptibility testing of Candida spp. biofilms would be the best solution, but to date, no reference method is available. The strongest antibiofilm activity of micafungin is observed at early stages of biofilm formation. Possibly, micafungin could be considered as an effective agent for prevention of biofilm-associated candidiasis, especially catheter-related candidaemia.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.