Multi-approach analysis of the diversity in <Emphasis Type="Italic">Colletotrichum cliviae</Emphasis> sensu lato

Colletotrichum cliviae is a fungal species reported both as pathogen and endophyte with broad geographical distribution. Some purported isolates of this species have been assigned to different taxa, including Colletotrichum aracearum, Colletotrichum orchidearum and Colletotrichum. sichuanensis, for which a preliminary analysis of extensive multilocus (ACT, GAPDH, ITS, TUB2) data in this study revealed high sequence similarity with C. cliviae. We further reassessed the species delineation by using the coalescent method of the generalized mixed Yule-coalescent (GMYC) and Poisson Tree Processes (PTP). Single and multilocus gene trees strongly supported a C. cliviae s. lat. clade including the four species. This clade unfolded eight subclades grouped into three distinct lineages, but no monophyly of any of the four species. GMYC and PTP analyses confidently supported the evolutionary independence of these lineages. C. sichuanensis and C. cliviae, except one isolate, formed the largest lineage. The second lineage was made up of isolates named C. aracearum and some of C. orchidearum sharing the haplotype and the third lineage accommodated two isolates named C. cliviae and C. orchidearum. This finding suggests the synonymization of C. sichuanensis with C. cliviae whereas the taxonomic status of C. aracearum and C. orchidearum still needs clarification. This study lays great stress upon the use of comprehensive data for sequence-based characterisation of species in the C. cliviae s. lat. It also presents the first report of C. cliviae in tropical Africa and on citrus host.     

Peculiarities of adhesion of epiphytic bacteria on leaves of the seagrass <Emphasis Type="Italic">Zostera marina</Emphasis> and on abiotic surfaces

A comparative study of the adhesion of epiphytic bacteria and marine free-living, saprophytic, and pathogenic bacteria on seagrass leaves and abiotic surfaces was performed to prove the occurrence of true epiphytes of Zostera marina and to elucidate the bacterium-plant symbiotrophic relationships. It was shown that in the course of adhesion to the seagrass leaves of two taxonomically different bacteria, Cytophaga sp. KMM 3552 and Pseudoalteromonas citrea KMM 461, isolated from the seagrass surface, the number of viable cells increased 3–7-fold after 60 h of incubation, reaching 1.0–2.0 × 10⁵ cells/cm²; however, in the case of adhesion of these bacteria to abiotic surfaces, such as glass or metal, virtually no viable cells were observed after 60 h of incubation. Such selectivity of cell adhesion was not observed in the case of three other bacterial species studied, viz., Vibrio alginolyticus KMM 3551, Bacillus subtilis KMM 430, and Pseudomonas aeruginosa KMM 433. The amount of viable cells of V. alginolyticus KMM 3551 absorbed on glass and metal surfaces increased twofold after 40 h of incubation. The cells of saprophytic B. subtilis KMM 430 and pathogenic P. aeruginosa KMM 433 adsorbed on three studied substrata remained viable for 36 h and died by the 60th hour of incubation.     

A Fatal Case of <Emphasis Type="Italic">Candida auris</Emphasis> and <Emphasis Type="Italic">Candida tropicalis</Emphasis> Candidemia in Neutropenic Patient

We report a fatal case of Candida auris that was involved in mixed candidemia with Candida tropicalis, isolated from the blood of a neutropenic patient. Identification of both isolates was confirmed by amplification and sequencing of internal transcribed spacer and D1/D2 domain of large subunit in rRNA gene. Antifungal susceptibility test by E-test method revealed that C. auris was resistant to amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole and voriconazole. On the other hand, C. tropicalis was sensitive to all antifungal tested. The use of chromogenic agar as isolation media is vital in detecting mixed candidemia.     

Biofilm and metallo beta-lactamase production among the strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> spp. at a Tertiary Care Hospital in Kathmandu,Nepal

Introduction

Pseudomonas aeruginosa and Acinetobacter spp. are found to be associated with biofilm and metallo-β-lactamase production and are the common causes of serious infections mainly in hospitalized patients. So, the main aims of this study were to determine the rates of biofilm production and metallo beta-lactamase production (MBL) among the strains of Pseudomonas aeruginosa and Acinetobacter spp. isolated from hospitalized patients.

Methods

A total of 85 P. aeruginosa isolates and 50 Acinetobacter spp. isolates isolated from different clinical specimens from patients admitted to Shree Birendra Hospital, Kathmandu, Nepal from July 2013 to May 2014 were included in this study. The bacterial isolates were identified with the help of biochemical tests. Modified Kirby-Bauer disc diffusion technique was used for antimicrobial susceptibility testing. Combined disc diffusion technique was used for the detection of MBL production, while Congo red agar method and tube adherence method were used for detection of biofilm production.

Results

Around 16.4% of P. aeruginosa isolates and 22% of the strains of Acinetobacter spp. were metallo β-lactamase producers. Out of 85 P. aeruginosa isolates, 23 (27.05%) were biofilm producers according to tube adherence test while, only 13 (15.29%) were biofilm producers as per Congo red agar method. Similarly, out of 50 Acinetobacter spp. 7 (14%) isolates were biofilm producers on the basis of tube adherence test, while only 5 (10%) were positive for biofilm production by Congo red agar method. Highest rates of susceptibility of P. aeruginosa as well as Acinetobacter spp. were seen toward colistin.

Conclusion

In our study, biofilm production and metallo beta-lactamase production were observed among Pseudomonas aeruginosa and Acinetobacter spp. However, no statistically significant association could be established between biofilm production and metallo beta-lactamase production.

     

Surface analysis of nitrogen plasma-treated C<Subscript>60</Subscript>/PS nanocomposite films for antibacterial activity

The influence of N₂ plasma on the antibacterial properties of polystyrene/fullerene (C₆₀/PS) nanocomposite films with two concentrations is investigated. A comparison is made between the surface characteristics of the films before and after plasma irradiation for different time intervals. The alterations induced on the surface of the films after treatment are analyzed by contact angle and surface energy measurements, FTIR spectroscopy, and atomic force microscopy. The antibacterial properties, growth, biofilm formation, and adhesion of the nanocomposite films against two multidrug-resistant bacterial strains, Staphylococcus aureus KT337489 and Pseudomonas aeruginosa KT337488, are investigated before and after plasma irradiation. The results indicate that P. aeruginosa is more sensitive to treatment than S. aureus as well as an enhancement of the anti-adhesion of both strains to treated surfaces through exposure.     

Comparison of Killing Activity of Micafungin Against Six <Emphasis Type="Italic">Candida</Emphasis> Species Isolated from Peritoneal and Pleural Cavities in RPMI-1640, 10 and 30% Serum

Currently echinocandins are recommended in Candida peritonitis and pleuritis. We determined micafungin killing rates (k values) at therapeutic concentrations (0.25–2 mg/L) in RPMI-1640 with and without 10 and 30% serum mimicking in vivo conditions against six Candida species isolated from peritoneal and pleural fluid. In RPMI-1640, micafungin was fungicidal against C. glabrata, C. krusei and C. kefyr within 2.27?±?10.68, 2.69?±?10.29 and 3.10?±?4.41 h, respectively, while was fungistatic against C. albicans, C. tropicalis and C. parapsilosis. In 10% serum, ≥?0.25, ≥?0.5, ≥?0.5 and ≥?1 mg/L micafungin produced positive k values (killing) for all C. albicans, C. glabrata, C. kefyr and C. krusei, respectively. In 30% serum, 2 mg/L micafungin produced killing against all C. albicans, C. glabrata and C. kefyr isolates, but was ineffective against C. krusei, C. parapsilosis and 2 of 3 C. tropicalis. Micafungin exposure should be increased against non-albicans species to eradicate fungi from peritoneal and pleural cavities.     

In Vitro Susceptibility of a Large Collection of <Emphasis Type="Italic">Candida</Emphasis> Strains Against Fluconazole and Voriconazole by Using the CLSI Disk Diffusion Assay

We evaluated all Candida spp. isolates obtained from patients admitted to two tertiary care hospitals between 1999 and 2003 in the city of São Paulo, Brazil. The in vitro activities of fluconazole (FCZ) and voriconazole were determined by the agar disk diffusion test using the Clinical and Laboratory Standards Institute M44-A guidelines. The inhibition zone diameters were read and interpreted automatically by the BIOMIC^® image-analysis plate reader system. We tested a total of 4,625 strains, including 2,393 strains of C. albicans (51.7%), 658 of C. tropicalis (14.2%), 503 of C. glabrata (10.9%), 495 of C. parapsilosis (10.7%), 292 of C. rugosa (6.3%), 195 of C. guilliermondii (4.2%) and 89 of other Candida species (2.0%). Only 2.0% of the strains tested were classified as dose-dependent susceptible (DDS), and 5.8% of them were resistant to FCZ. The resistance or DDS to fluconazole was verified mainly among C. glabrata (7.8%), C. krusei (67.9%) and C. rugosa (65.1%). Voriconazole exhibited better activity in vitro than fluconazole, even in isolates fluconazole resistant. The resistance of fluconazole and voriconazole did not increase in the isolates of Candida spp. during the evaluated period.     

Molecular Genotyping of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> Species Complex

Background

The Candida parapsilosis complex species has emerged as an important cause of human disease. The molecular identification of C. parapsilosis isolates at the species level can be helpful for epidemiological studies and then for the establishment of appropriate therapies and prophylactic measures.

Methods

The present study was undertaken to analyze 13 short tandem repeat (STR) markers (7 minisatellites and 6 microsatellites) in a global set of 182 C. parapsilosis complex isolates from different origins including invasive and superficial clinical sites.

Results

Upon the analysis of 182 strains of C. parapsilosis complex species, 10–17 haplotypes were detected for each minisatellite marker. The combination of 7 minisatellite markers yielded 121 different genotypes with a 0.995 D value. Upon the analysis of 114 isolates (68 from invasive infections and 46 from superficial infections), 21–32 genotypes were detected for each microsatellite marker. The combination of all 13 markers yielded 96 different genotypes among 114 isolates with a high degree of discrimination (0.997 D value).The same multilocus genotype was shared by isolates recovered from some patients and from the hand of theirs correspondent healthcare worker. For another patient, the same multilocus genotype of C. metapsilosis was detected in blood and skin confirming that candidemia usually arises as an endogenous infection following prior colonization.

Conclusions

These STR markers are a valuable tool for the differentiation of C. parapsilosis complex strains, to support epidemiological investigations especially studies of strain relatedness and pathways of transmission.

     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.     

Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous <Emphasis Type="Italic">GFP</Emphasis> genes in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura⁺ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10^?8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.     

Coating polypropylene surfaces with protease weakens the adhesion and increases the dispersion of <Emphasis Type="Italic">Candida albicans</Emphasis> cells

Objectives

To investigate the ability of the proteases, subtilisin and α-chymotrypsin (aCT), to inhibit the adhesion of Candida albicans biofilm to a polypropylene surface.

Results

The proteases were immobilized on plasma-treated polypropylene by covalently linking them with either glutaraldehyde (GA) or N′-diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS). The immobilization did not negatively affect the enzyme activity and in the case of subtilisin, the activity was up to 640% higher than that of the free enzyme when using N-acetyl phenylalanine ethyl ester as the substrate. The efficacies against biofilm dispersal for the GA-linked SubC and aCT coatings were 41 and 55% higher than the control (polypropylene coated with only GA), respectively, whereas no effect was observed with enzymes immobilized with DIC and NHS. The higher dispersion efficacy observed for the proteases immobilized with GA could be both steric (proper orientation of the active site) and dynamic (higher protein mobility/flexibility).

Conclusions

Proteases immobilized on a polypropylene surface reduced the adhesion of C. albicans biofilms and therefore may be useful in developing anti-biofilm surfaces based on non-toxic molecules and sustainable strategies.

     

Induction of two cyclotide-like genes <Emphasis Type="Italic">Zmcyc1</Emphasis> and <Emphasis Type="Italic">Zmcyc5</Emphasis> by abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis>

Cyclotides are small plant disulfide-rich and cyclic proteins with a diverse range of biological activities. Cyclotide-like genes show key sequence features of cyclotides and are present in the Poaceae. In this study the cDNA of the nine cyclotide-like genes were cloned and sequenced using 3′RACE from Zea mays. The gene expression of two of these genes (Zmcyc1 and Zmcyc5) were analyzed by real-time PCR in response to biotic (Fusarium graminearum, Ustilago maydis and Rhopalosiphum maydis) and abiotic (mechanical wounding, water deficit and salinity) stresses, as well as in response to salicylic acid and methyl jasmonate elicitors to mimic biotic stresses. All isolated genes showed significant similarity to other cyclotide-like genes and were classified in two separate clusters. Both Zmcyc1 and Zmcyc5 were expressed in all studied tissues with the highest expression in leaves and lowest expression in roots. Wounding, methyl jasmonate and salicylic acid significantly induced the expression of Zmcyc1 and Zmcyc5 genes, but the higher expression was observed for Zmcyc1 as compared with Zmcyc5. Expression levels of these two genes were also induced in inoculated leaves with F. graminearum, U. maydis and also in response to insect infestation. In addition, the 1000-base-pairs (bp) upstream of the promoter of Zmcyc1 and Zmcyc5 genes were identified and analyzed using the PlantCARE database and consequently a large number of similar biotic and abiotic cis-regulatory elements were identified for these two genes.     

The <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> efficacy of fluconazole in combination with farnesol against <Emphasis Type="Italic">Candida albicans</Emphasis> isolates using a murine vulvovaginitis model

Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150–300 μM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 μM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16–0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 μM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole-resistant isolate, but not the other one.     

Abiotic and biotic factors influence the habitat use of four species of <Emphasis Type="Italic">Gymnogobius</Emphasis> (Gobiidae) in riverine estuaries in the Seto Inland Sea

Gobies that are phylogenetically related or coexist in the same marine and estuarine systems often exhibit abiotic and/or biotic habitat segregation. Thus, it is possible that species of Gymnogobius inhabiting the same riverine estuaries also exhibit abiotic and/or biotic habitat segregation. The goal of this study was to determine the differences in abiotic and biotic habitat use between these species by sampling goby and host shrimps, and by examining the physical environments of the rivers where these species are found. The surveys of goby and host shrimps were conducted in the estuaries of the Saba and Ibo rivers, which drain into the Seto Inland Sea, a body of water that separates three of the four main islands of Japan. We used hand nets and shovels to collect goby and host shrimps, and measured median sediment particle size, elevation, and salinity at each site. Generalized linear models (GLMs) were used to assess the preferences in abiotic and biotic habitat use by the goby species. Median particle size, salinity, and elevation were used as the abiotic environmental predictors, whereas the presence/absence of host shrimps were re-organized into four categories consisting of “Upogebia major” only, “Nihonotrypaea japonica” only, “Upogebia major & Nihonotrypaea japonica,” and “Upogebia yokoyai,” which were used as the biotic environmental predictors. The GLMs demonstrated that median particle size had the largest influence of the abiotic variables, with goby species segregating according to differences in sediments; moreover, there was some evidence suggesting that the host and symbiont do not always correlate at the species level. Our results indicated that although there is some overlap in abiotic and biotic habitat use among the four species of Gymnogobius, the differences were broad enough to provide an explanatory mechanism as to how these species can coexist in the same river systems.     

Development of <Emphasis Type="Italic">mazF</Emphasis>-based markerless genome editing system and metabolic pathway engineering in <Emphasis Type="Italic">Candida tropicalis</Emphasis> for producing long-chain dicarboxylic acids

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the β-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH–cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.     

Recent perspectives on the molecular basis of biofilm formation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and approaches for treatment and biofilm dispersal

Pseudomonas aeruginosa, a Gram-negative, rod-shaped bacterium causes widespread diseases in humans. This bacterium is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteriaemia especially in immunocompromised patients. The current review focuses on the recent perspectives on biofilms formation by these bacteria. Biofilms are communities of microorganisms in which cells stick to each other and often adhere to a surface. These adherent cells are usually embedded within a self-produced matrix of extracellular polymeric substance (EPS). Pel, psl and alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell surface interactions during biofilm formation. Recent studies suggested that cAMP signalling pathway, quorum-sensing pathway, Gac/Rsm pathway and c-di-GMP signalling pathway are the main mechanism that leads to the biofilm formation. Understanding the bacterial virulence depends on a number of cell-associated and extracellular factors and is very essential for the development of potential drug targets. Thus, the review focuses on the major genes involved in the biofilm formation, the state of art update on the biofilm treatment and the dispersal approaches such as targeting adhesion and maturation, targeting virulence factors and other strategies such as small molecule-based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides and natural therapies and vaccines to curtail the biofilm formation by P. aeruginosa.     

Resveratrol,pterostilbene, and baicalein: plant-derived anti-biofilm agents

Microbial adhesion to surfaces and the subsequent biofilm formation may result in contamination in food industry and in healthcare-associated infections and may significantly affect postoperative care. Some plants produce substances with antioxidant and antimicrobial properties that are able to inhibit the growth of food-borne pathogens. The aim of our study was to evaluate antimicrobial and anti-biofilm effect of baicalein, resveratrol, and pterostilbene on Candida albicans, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. We determined the minimum inhibitory concentrations (MIC), the minimum adhesion inhibitory concentration (MAIC), and the minimum biofilm eradication concentration (MBEC) by crystal violet and XTT determination. Resveratrol and pterostilbene have been shown to inhibit the formation of biofilms as well as to disrupt preformed biofilms. Our results suggest that resveratrol and pterostilbene appear potentially very useful to control and inhibit biofilm contaminations by Candida albicans, Staphylococcus epidermidis, and Escherichia coli in the food industry.