Colony Pigmentation Research Articles

Antimicrobial resistance pattern of Klebsiella species isolated from various clinical specimens

Objective: The present study was carried out to identify Klebsiella species and their antimicrobial resistance pattern from various clinical specimens. Methods: This cross study was conducted at a tertiary care hospital, Dhaka, Bangladesh from July2020 to June 2021. Written consent was taken from the concerned authority. A total 6739 clinical samples including urine, wound swab/ pus, sputum, blood and endotracheal aspirates were collected and processed for isolation of bacteria by following standard guidelines from adult patients. All the urine, wound swab, blood and endotracheal aspirates were inoculated in both blood agar and MacConkey agar media, for blood samples blood culture was done in BacT/ALERT 3D 60 aerobic bottles irrespective to antibiotics administration. After incubation at 370C aerobically for 24 hours incubated plates were examined. Phenotypic identification of the organisms were done by observing colony morphology, hemolytic properties, pigment production, Gram staining and biochemical tests (oxidase test, catalase test and biochemical reactions after inoculation in TSI, MIU, citrate agar media). The Kirby - Bauer disk diffusion method was used for antimicrobial susceptibility testing of the isolated organism using Mueller-Hinton agar and commercially available antibiotic discs (Oxoid Ltd, UK). The data were analyzed using SPSS software Version-20 (SPSS Inc., Chicago, IL, USA). Categorical variables were expressed as numbers (n) and percentages (%). Results: A total 6739 samples were included in the present study. Of which, 170 were urine, 162 were wound swabs, 71 were sputum, 52 were endotracheal aspirates and 45 were blood samples. Culture positivity among the samples is shown in table I. Conclusion: Most of the Klebsiella species are highly resistant to commonly used antibiotics. This higher resistance is the major cause morbidity and mortality related with infections in hospitalized patients. So, a regular surveillance of antibiotic susceptibility pattern is needed to prevent indiscriminate use of antibiotics. CME J 2024; 3(2); 12-15

Biodiversity of Actinobacteria from Kula Geopark in Türkiye

Investigating the microbial diversity of Actinobacteria inhabiting the soils of the Kula-Salihli Geopark and identifying species at the genus level using 16S rRNA gene sequences are the primary goals of this work. In the literature review of this geopark located within the borders of Manisa province, no study on actinobacterial biodiversity was found. In this study, 10 different selective isolation media were used to investigate the biodiversity of Actinobacteria in the Geopark. A total of 469 Actinobacteria strains were isolated using the dilution-plate method. From these 469 strains, 34 strains were selected based on their colony morphology and pigmentation characteristics. The isolates performed phylogenetic analysis based on sequencing of the 16S ribosomal RNA gene region. The isolates were found to belong to nine different genera, including Actinomadura, Amycolatopsis, Kribbella, Micromonospora, Nocardia, Nonomuraea, Pseudonocardia, Saccharothrix and Streptomyces, according to the results of phylogenetic analysis. Five isolates have been identified as novel species as a consequence of our current study.

Morphological Characterization of Native Isolates of Hypocreale Fungi Metarhizium rileyi (Farl.) Samson from Maize Fall Armyworm

The Present studies indicates the collection of entomopathogenic fungi from the roving survey in Northeastern region of Karnataka, India to know the diversity of M. rileyi distribution and also its growth pattern. Different morphological parameters used for describing the growth pattern which indicates diversity and uniqueness of M. rileyi i.e., colony colour which was white during initial stage and then during sporulating stage the colour changed into malachite green colour. The conidiophores were in compact patches. Conidia were aseptate, hyaline, round or ovoid in shape and elongate by forming chains. Conidiophores were short, which bears round to ovoid aseptate conidia in different isolates. The conidia size on its ratio found that, the highest size ratio was recorded in UASRBC Mr-20 2.19 then remaining isolates which has shown less than 2.0. The radial growth found only in Mr-20 and Mr-22nd Isolates, colony diameter ranged from 0.3 to 2.23 cm, concentric rings of colonies found in all the isolates except Mr-19 and Mr-20th isolates. The colony surface layer was white cottony growth to pale yellow color was found in all the isolates, the colony pigmentation in most of the isolates was light brown colored pigmentation. The growth appearance was irregular patchy growth was observed in most of the isolates. Among six different isolates the highest width was recorded in isolate UASRBC Mr-20 (6.46) followed by UASRBC Mr-22 (5.73) which was significantly superior than remaining isolates. The highest colony diameter was recorded in UASRBC Mr-18 (19 mm).

Phenotypic Characteristics, Pathogenicity, and Molecular Identification of Hypomyces perniciosus Causing Wet Bubble Disease of Edible Mushrooms

Abstract The research aimed to determine the phenotypic characteristic, pathogenicity and molecular characterization of Hypomyces perniciosus isolates responsible for wet bubble disease of the white button mushroom Agaricus bisporus. Phenotypic characteristics such as colony appearance, mycelium texture, and pigmentation were studied on four different agar media, i.e., potato dextrose agar (PDA), malt extract agar (MEA), Czapek-Dox yeast agar (CYA), and Sabouraud dextrose agar (SDA), after eight days of incubation. Additionally, the growth rate of the tested isolates was studied depending on the pH of the medium. Fungal isolates showed the highest mycelial growth on MEA and SDA at pH 6.0. However, on CYA at pH 7.0, the mycelium exhibited the worst growth. Isolate identification and genetic relationship analysis were carried out using internal transcribed spacer region sequencing and the random amplified polymorphic DNA method. The research confirmed that all Polish isolates belong to the species H. perniciosus, and the genetic diversity is relatively low. Phylogenetic analyses revealed three subgroups of H. perniciosus isolates. The first group included three genetically distinct isolates with a similarity coefficient in the range of 0.76–0.85 to isolates CBS 815.73 and CBS 322.52. The second group was divided into two subgroups and included 16 isolates with a genetic similarity range of 0.91 to 1.0 to CBS 815.73 and CBS 322.52 isolates. Furthermore, the eight genetically similar isolates exhibited the greatest pathogenicity towards A. bisporus.

Pisang Nangka (Musa paradisiaca) As An Alternative Media For Aspergillus niger Growth

Nangka banana (Musa paradisiaca) is one of the most popular bananas in Indonesia. Therefore, the production of waste will increase along with the use of bananas. Nangka banana peel and flesh can be used as an alternative media for mushroom growth because of its high carbohydrate content. The purpose of this study was to observe the growth of Aspergillus niger on alternative media made from nangka banana with concentrations of 8%, 9%, 10%, 11% and 12% and to determine the optimum variation and concentration of alernative media. Aspergillus niger was grown on nangka banana flesh media, nangka banana peel media and mix of nangka banana peel and flesh as well as PDA (Potato Dextrose Agar) media as a positive control using the single dot method. Colony diameter and pigmentation were observed for five days. The variation of nangka banana mix media with a concentration of 12% is the optimum variation and concentration to grow Aspergillus niger. Keywords: Alternative media, Aspergillus niger, nangka banana

Isolation and identification of microorganisms from bitumen, and soil around bitumen deposit site

Isolation of microorganisms from hydrocarbon and oil polluted environment has been reported over the years. This study focused on the isolation and identification of microorganisms from bitumen, and soil around bitumen deposit site. Microorganisms were isolated from soil sample collected around bitumen deposit and bitumen itself using pour plate method. The cultural and morphological characteristics of each bacteria isolates such as colony shape, elevation, pigmentation, colour, cell shape, gram reaction, motility, and endospore test were carried out. From the result, the following bacteria were identified; Acinetobacter sp (soil and bitumen), Pseudomonas Aeruginosa (soil), Bacillus subtilis (soil and bitumen), Staphylococcus aureus (soil). Mould isolation was carried out by cutting a small portion of mycelium, stained it with cotton blue in lactophenol and the examined carefully under light microscope with x40 0bjective. The result revealed the following mould isolates; Aspergillus niger and Arthrobotrys oligospora both isolated from the soil. The result showed the isolation of microorganisms from bitumen and soil around bitumen deposit, thus their potential usefulness in remediation of bitumen polluted environment if the exploitation of the resource commenced.

English

Phoenix dactylifera L. (Date palm) is one of the leading perennial fruit crops regarding its nutritional profile, medicinal significance, excellent yields, long life, and high adaptability to diverse climatic conditions of semi-arid and arid regions globally. Eleven major palm-growing districts of Punjab, Sindh, and Khyber Pakhtunkhwa were surveyed for wilt disease assessment on date palms. The date palm plantation was highly affected with Fusarium wilt diseases with 55%, the incidence in Khairpur (Sindh province), followed by Bahawalpur 47.4% (Punjab Province) and 45% in Dera Ismail Khan (KPK province). Disease severity was recorded as 65% in Khairpur, 55% in Bahawalpur, 50% in Dera Ismail Khan (Sindh, Punjab, and KPK, respectively). Sixty-three Fusarium isolates were obtained from three hundred and eighty (380) diseased samples, and thirty isolates randomly selected out of 63 were studied for morpho-cultural and microscopic characterization. Morpho-cultural characters i.e colony pigmentation, growth pattern, conidia (macro and microconidia), shape, apical cell shape, and phalidies studied were coinciding with typical characters of Fusarium oxysporum. Twenty isolates of Fusarium population was subjected for the ITS region sequence data-based phylogenetic network. The Neighbor net algorithm computed an unrooted phylogenetic network displayed as four branched genetic-network that depicted the division of twenty fungal isolates into four groups. F. oxysporum (FMB-FO-PD-011) inoculated onto healthy symptomless detached date palm leaves, seedlings and suckers under controlled as well as field conditions showed typical wilt disease symptoms. Hence this study proved that F. oxysporum is the potential cause of date palm wilt in Pakistan.

Distribution and correlation of Soil bacterial population and chemical properties in salt affected landuses of Kaithal and Fatehabad district of Haryana

This study attempts to examine the relationship between soil bacterial population and physico-chemical properties of soil in Kaithal and Fatehabad district of Haryana. Soil pH in the study ranged from 7.7 to 10.32 in the selected sites. EC in the study ranged from 200 to 1780 μS/m at 25 °C. Organic carbon ranged from 0.10 to 1.32% in the present study. Majority of soil samples showed medium status of available nitrogen which ranged from 0.0005 to 0.02%. Soil phosphorus is most available for plant use at pH values of 6 to 7. Potassium content ranged from 0.001 to 0.05%. The present study revealed that bacteria isolated from bottom segment showed higher growth rate in anaerobic condition. A decreasing trend of total microbial population and organic carbon content of soil were found with increase in depth. The maximum number of bacterial colonies was observed at the depth of 0-30 cm. Gram’s staining Revealed that 0-30 cm depth was occupied by majority of Gram negative bacteria in Haryana. 30-60 cm depth was analyzed, there was a slight but significant difference in the bacterial population and majority were Gram positive. Simultaneously there was less variation with respect to colony morphology and pigment production as compared to the depth of 0-30 cm.

Mycoviral gene integration converts a plant pathogenic fungus into a biocontrol agent.

Mycovirus-infected fungi can suffer from poor growth, attenuated pigmentation, and virulence. However, the molecular mechanisms of how mycoviruses confer these symptoms remain poorly understood. Here, we report a mycovirus Stemphylium lycopersici alternavirus 1 (SlAV1) isolated from a necrotrophic plant pathogen Stemphylium lycopersici that causes altered colony pigmentation and hypovirulence by specifically interfering host biosynthesis of Altersolanol A, a polyketide phytotoxin. SlAV1 significantly down-regulates a fungal polyketide synthase (PKS1), the core enzyme of Altersolanol A biosynthesis. PKS1 deletion mutants do not accumulate Altersolanol A and lose pathogenicity to tomato and lettuce. Transgenic expression of SlAV1 open-reading frame 3 (ORF3) in S. lycopersici inhibits fungal PKS1 expression and Altersolanol A accumulation, leading to symptoms like SlAV1-infected fungal strains. Multiple plant species sprayed with mycelial suspension of S. lycopersici or S. vesicarium strains integrating and expressing ORF3 display enhanced resistance against virulent strains, converting the pathogenic fungi into biocontrol agents. Hence, our study not only proves inhibiting a key enzyme of host phytotoxin biosynthesis as a molecular mechanism underlying SlAV1-mediated hypovirulence of Stemphylium spp., but also demonstrates the potential of mycovirus-gene integrated fungi as a potential biocontrol agent to protect plants from fungal diseases.

Phenotypic and genomic assessment of the potential threat of human spaceflight-relevant Staphylococcus capitis isolates under stress conditions.

Previous studies have reported that spaceflight specific conditions such as microgravity lead to changes in bacterial physiology and resistance behavior including increased expression of virulence factors, enhanced biofilm formation and decreased susceptibility to antibiotics. To assess if spaceflight induced physiological changes can manifest in human-associated bacteria, we compared three spaceflight relevant Staphylococcus capitis isolates (DSM 111179, ISS; DSM 31028, clean room; DSM 113836; artificial gravity bedrest study) with the type strain (DSM 20326T). We tested the three strains regarding growth, colony morphology, metabolism, fatty acid and polar lipid pattern, biofilm formation, susceptibility to antibiotics and survival in different stress conditions such as treatment with hydrogen peroxide, exposure to desiccation, and irradiation with X-rays and UV-C. Moreover, we sequenced, assembled, and analyzed the genomes of all four strains. Potential genetic determinants for phenotypic differences were investigated by comparative genomics. We found that all four strains show similar metabolic patterns and the same susceptibility to antibiotics. All four strains were considered resistant to fosfomycin. Physiological differences were mainly observed compared to the type strain and minor differences among the other three strains. The ISS isolate and the bedrest study isolate exhibit a strong delayed yellow pigmentation, which is absent in the other two strains. Pigments were extracted and analyzed by UV/Vis spectroscopy showing characteristic carotenoid spectra. The ISS isolate showed the highest growth rate as well as weighted average melting temperature (WAMT) of fatty acids (41.8°C) of all strains. The clean room isolate showed strongest biofilm formation and a high tolerance to desiccation. In general, all strains survived desiccation better in absence of oxygen. There were no differences among the strains regarding radiation tolerance. Phenotypic and genomic differences among the strains observed in this study are not inevitably indicating an increased virulence of the spaceflight isolate. However, the increased growth rate, higher WAMT and colony pigmentation of the spaceflight isolate are relevant phenotypes that require further research within the human spaceflight context. We conclude that combining genetic analysis with classical microbiological methods allows the detailed assessment of the potential threat of bacteria in highly regulated and extreme environments such as spaceflight environments.

Characterization of Fusarium spp. causal agent of wilt in pecan

One of the limiting factors for pecan production is the incidence of disease caused by Fusarium spp. which are a threat to orchards in the south of the country. Therefore, the study aimed to confirm the pathogenicity and evaluate the morphophysiological and molecular characteristics of Fusarium spp. associated with pecan in Rio Grande do Sul and Paraná. For this, samples of symptomatic plant material were collected, and potentially pathogenic isolates were evaluated for pathogenicity, by immersing the roots of the plants in a spore suspension of Fusarium spp. In addition, the severity of the disease was assessed using a rating scale according to the symptoms expressed by the plants. The isolates were also analyzed for morphophysiological variability, through evaluations of mycelial growth, colony and aerial mycelium pigmentation, and characteristics of reproductive structures. Molecular characterization was performed which amplify the region of the 1-alpha elongation factor and sequencing. Nineteen isolates were obtained, which were considered pathogenic, however, there was variability in their aggressiveness and morphophysiological characteristics. In addition, the sequencing was used to identify different species. Therefore, species of Fusarium are responsible for the occurrence of wilt in pecan in southern Brazil.

Characterization of Fungal Melanins from Black Stains on Paper Artefacts

Melanins play a fundamental role in the biology and ecology of several fungal species. Unfortunately, this group of amorphous macromolecules also severely (and most times irreversibly) stains cultural heritage objects. Despite efforts made throughout the years, knowledge of the chemical composition and structure of melanins is still insufficient, which hampers the task of safely cleaning these colourants from cultural heritage materials in a targeted way without causing further deterioration. This work aimed therefore to contribute towards enlightening the characteristics of fungal melanins from three fungi that are common paper colonizers: Aspergillus niger, Chaetomium globosum and Cladosporium cladosporioides. The extracted melanins were characterized by FTIR, Raman, UV-vis, Solid-State NMR and MALDI-TOF MS spectroscopies and the effect of inhibitors of DHN-melanin and DOPA-melanin pathways on colony pigmentation and growth was evaluated. Although all the extracted colourants show a predominantly aromatic structure with carbonyl and phenolic groups, some differences between the melanins can be highlighted. Melanins obtained from Ch. globosum and Cl. cladosporioides exhibited similar structures and composition and both presented DHN-melanin characteristics, while A. niger’s melanins revealed a more complex and ordered structure, with a higher prevalence of highly conjugated carbonyls than the others, besides the additional presence of a yellow/green component. These conclusions cannot be overlooked while selecting targeted cleaning methodologies for melanin stains on cultural heritage materials.

Plant Growth-Promoting (PGP) Traits of Endophytic Bacteria from In Vitro Cultivated Tectona grandis L.f

Tectona grandis L.f. (teak) is a tropical tree cultivated mainly due to its resistance, valuable wood and tolerance to biotic and abiotic factors. An abundant bacteria community exists in teak tissues and knowledge of the functional roles of teak endophytic bacteria, from in vitro tissue culture, is essential for improving micropropagation techniques. In this study, we isolated endophytic bacteria with plant growth-promoting (PGP) traits from two teak clones (Proteca® A3 and E4) in calli, leaves, and stems segments in a culture medium. We analyzed colony pigmentation, gram reaction, and evaluated PGP traits (phosphorous solubilization, nitrogen fixation and indol-acetic acid production) of 54 colonies from clone A3 and 50 colonies from clone E4. A total of 35 colonies of clone A3 and 42 colonies of clone E4 were capable of fixing nitrogen. Four isolated bacteria from clone A3 were capable of solubilizing phosphorous (P-Ca3(PO4)2), while no strain of E4 clone showed that capacity. Furthermore, 49 endophytic bacteria from clone A3 showed capacity to synthesize indol-acetic acid, while only 4 bacteria from the E4 clone presented that characteristic. We also identified six teak endophytic bacteria, by analysis of the 16S-23S rDNA intergenic spacer region, showing high identity with the genera Curtobacterium sp., Ochrobactrum sp., and Bacillus spp. Therefore, we demonstrate here that the abundant bacterial community existing in teak tissues, including those of in vitro cultivated plants, have PGP traits that can be further harnessed for preparation of bioformulations, for example. Our findings open the possibility for studying these isolated endophytic bacteria more closely in order to understand their association with teak growth.

First Report of Maize Seedling Blight Caused by Fusarium pseudograminearum in China.

Maize (Zea mays L.) is one of the most important food and feed crops in China, with a cultivation area of more than 40 million hectares (http://www.fao.org/faostat/en/#data/QC). In July 2021, a serious maize seeding blight occurred in Changjia Town, Gaoqing Country, Zibo City, Shandong Province, China, and the disease incidence was up to 50% in some fields. The root system of infected plants displayed poor development. The primary roots were brown and rotted. The leaves at the base of the plants were drying up, then the whole plant withered. To determine the cause agent of the disease, symptomatic roots of diseased seedlings were collected and surface-sterilized (70% ethanol for 30 s and 3% sodium hypochlorite (NaClO) for 90 s), subsequently rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), then incubated at 25°C for 2 days. Two cultures with similar morphological characteristics were purified through single-spore isolation technique and identified by morphology and molecular methods as Fusarium pseudograminearum O'Donnell & T. Aoki 1999. Plentiful macroconidia formed in 5-day-old carboxymethyl cellulose (CMC) cultures; microconidia were absent. Macroconidia were thick-walled and curved, usually 3- to 5- septa, 31.6 ± 0.6 μm × 4.8 ± 0.1 μm (n = 50). Colony pigmentation on PDA was pink to red, with white to pink aerial mycelia on PDA cultures was abundant and filled the petri dishes. For molecular identification, the rDNA internal transcribed spacer (ITS) gene and translation elongation factor 1 alpha (TEF-1α) gene of two isolates (SAIA41B and SAIA41C) were amplified with ITS1/ITS4 (White et al., 1990) and EF-1/EF-2 (O'Donnell et al., 1998), respectively. Blastn analysis of both the ITS sequence (accession numbers OM108101 and OM108102) and TEF-1α sequence (accession numbers OM142205 and OM142206) revealed 100% (481/481 bp for ITS and 637/637 bp for TEF-1α) sequence identity with the sequences of F. pseudograminearum reported in GenBank (MW699613 for ITS and JN862232 for TEF-1α). The molecular identification was further confirmed by the F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell 1999). The expected 523-bp fragments were obtained for isolates SAIA41B and SAIA41C. In the pathogenicity test, healthy germinating maize roots (Zhengdan958) were inoculated with PDA culture blocks of isolate SAIA41C. Plants inoculated only with PDA culture blocks served as controls. Maize plants were put in petri dishes and placed in an incubator with a 12-h photoperiod at 25 oC and 100% relative humidity. Seven days later, roots of the plants inoculated with isolate SAIA41C were poorly developed and became brown necrotic and rotted, which were identical to the symptoms observed in the fields, whereas the roots of control plants were developed normally. The pathogen was re-isolated from the necrotic tissue of the inoculated roots but not from the control plants, and its identity was confirmed by PCR with the primes Fp1-1/Fp1-2 described above, fulfilling Koch's Postulates. To our knowledge, this is the first report of maize seedling blight caused by F. pseudograminearum in China. Our finding indicates the potential spread of F. pseudograminearum on maize, and more attention should be paid to prevention and control of maize seedling blight caused by F. pseudograminearum. The author(s) declare no conflict of interest. Acknowledgements: This research was supported by National Natural Science Foundation of China (No. 32102181), Shandong Provincial Natural Science Foundation (No. ZR2021QC059), Wheat Industry Technology System of Shandong Province (No. SDAIT-01-10), and Agricultural Science and Technology Innovation Project of SAAS (No. CXGC2021A38 and CXGC2021A33).

Cationic polymer contributes to broaden the spectrum of vancomycin activity achieving eradication of Pseudomonas aeruginosa.

Vancomycin (VAN) is unable to penetrate the outer membrane of Gram-negative bacteria and reach the target site. One approach to overcome this limitation is to associate it with compounds with permeabilizing or antimicrobial properties. EudragitE100® (Eu) is a cationic polymer insufficiently characterized for its potential antimicrobial action. Eu-VAN combinations were characterized, the antimicrobial efficacy against Pseudomonas aeruginosa was evaluated and previous studies on the effects of Eu on bacterial envelopes were extended. Time-kill assays showed eradication of P. aeruginosa within 3-6h exposure to Eu-VAN, whilst VAN was ineffective. Eu showed regrowth in 24h and delayed colony pigmentation. Although permeabilization of bacterial envelopes or morphological alterations observed by TEM and flow cytometry after exposure to Eu were insufficient to cause bacterial death, they allowed access of VAN to the target site, since Eu-VAN/Van-FL-treated cultures showed fluorescent staining in all bacterial cells, indicating Van-FL internalization. Consequently, Eu potentiated the activity of an otherwise inactive antibiotic against P. aeruginosa. Moreover, Eu-VAN combinations exhibited improved physicochemical properties and could be used in the development of therapeutic alternatives in the treatment of bacterial keratitis.

A retrospective study to analyse epidemiological profile of community acquired infectious keratitis in a tertiary care hospital located in Northern Mumbai

Background: Epidemiology of infectious keratitis varies with geographical location. The aim of the study was to review the etiology of infectious keratitis in community that will help in management strategy.Methods: A retrospective study was conducted at tertiary care hospital from November 2017 to December 2020. Corneal scrapings and corneal button were processed as per microbiological standards. Bacterial identification and antimicrobial sensitivity testing was done by automated Vitek-2 compact system. Significant fungal growth was confirmed by its colony morphology, pigment production and lacto phenol cotton blue (LPCB) mount examination.Results: Of total 130 patients, culture positivity was 63 (48%), of these 59 (53%) were from corneal scrapping and 4 (21%) from corneal button. The most common bacterial isolates were Streptococcus pneumoniae 12 (38%), followed by Pseudomonas aeruginosa 6 (19%) and Staphylococcus aureus 4 (12%). The most common fungi isolated was Fusarium spp 14 (47%) followed by Aspergillus spp 9 (30%). One specimen showed Acanthamoba spp in wet mount preparation. Major associated factors were injury with vegetative matter 30 (48%) followed by contact lens use 12 (19%) and diabetes mellitus 11 (17%). Antibiotic susceptibility tests showed that all gram-positive cocci were susceptible to vancomycin (100%) and fluoroquinolones (100%) followed by third generation cephalosporins (80%). Whereas all gram-negative bacilli were susceptible to aminoglycosides (100%) followed by fluoroquinolones (90%). Conclusions: Epidemiology of infectious keratitis vary with geographical location. Understanding local microbiological profile assist in empirical therapy when diagnostic facilities are not readily available.

Phenotypic and genotypic changes of Staphylococcus aureus in the presence of the inappropriate concentration of chlorhexidine gluconate

BackgroundChlorhexidine gluconate (CHG) is a disinfectant agent with different applications in health care. Improper use of CHG causes antimicrobial resistance in bacteria as a public health threat. Since Staphylococcus aureus is a common bacteria, it is expected usually exposed to CHG in the hospital and community. The present study aimed to correlate the phenotypic and genotypic changes in a S. aureus strain upon serial adaptation with supra-inhibitory CHG concentration for 50 days.ResultsAfter in vitro serial culture of 5 × 105 CFU/ml of a clinical vancomycin-susceptible S. aureus strain (VAN-S) into brain heart infusion (BHI) broth containing CHG 1/4, 1/2, 1, and 2 × minimal inhibitory concentration (MIC) values of VAN-S in 37 °C during 50 days, we isolated a S. aureus strain (CHGVan-I) with a ≥ twofold decrease in susceptibility to CHG and vancomycin. CHG-induced CHGVan-I strain was considered as a vancomycin-intermediate S. aureus (VISA) strain with a VAN MIC of 4 μg/ml using the broth macro dilution method. However, reduced resistance was observed to tetracycline family antibiotics (doxycycline and tetracycline) using a modified Kirby-Bauer disk diffusion test. Moreover, a remarkable reduction was detected in growth rate, hemolysis activity (the lysis of human red blood cells by alpha-hemolysin), and colony pigmentation (on BHI agar plates). Biofilm formation (using the Microtiter plate method and crystal violet staining) was significantly increased upon CHG treatment. Adaptive changes in the expression of a set of common genes related to the development of VISA phenotype (graTSR, vraTSR, walKR, agr RNAIII, sceD, pbpB, and fmtA) were analyzed by Reverse Transcription quantitative PCR (RT-qPCR) experiment. Significant changes in vraTSR, agr RNAIII, sceD, and pbpB expression were observed. However, gene sequencing of the two-component system vraTSR using the Sanger sequencing method did not detect any non-synonymous substitution in CHGVan-I compared to wild-type. The clonality of VAN-S and CHGVan-I strains was verified using the pulsed-field gel electrophoresis (PFGE) method.ConclusionsThe importance of the present study should be stated in new detected mechanisms underlying VISA development. We found a link between the improper CHX use and the development of phenotypic and genotypic features, typical of VISA clinical isolates, in a CHG-induced strain.Since disruption of the cell wall biosynthesis occurs in VISA isolates, our CHG-induced VISA strain proved new insights into the role of CHG in the stimulation of the S. aureus cell wall.

First Report of Crown Rot of Banana Caused by Fusarium proliferatum in Georgia, U.S.A.

Crown rot is one of the most damaging disease of banana fruit characterized by rot and necrosis of crown tissues. In severe cases, the disease can spread to the pedicel and banana pulp. Crown rot can be infected by several common fungi, including Lasiodiplodia theobromae, Musicillium theobromae, Colletotrichum musae, and a complex of Fusarium spp. and lead to softening and blackening of tissues (Lassois et al., 2010; Kamel et al., 2016; Triest et al., 2016; Snowdon, 1990). In November 2020, typical crown rot of banana fruits (cv. Pisang Awak, belonging to the tetraploid AABB genome) were observed from UGA Banana Research 12 Plots, Tifton, GA, with incidence rates of 15%. Initial symptoms appeared in the infected crown of green banana fruits. As the infection progressed, the crown tissues became blackened and softened, followed by an internal development of infection affecting the peduncle and the fruit, triggered early ripening of bananas. At last, the development of necrosis on the pedicels and fruits appeared and caused the fingers to fall off. To identify the pathogen, tissue pieces (~0.25 cm2) from the infected crown and pedicles were surface-sterilized in a 10% bleach solution for 1 min, followed by 30 s in 70% EtOH. The disinfected tissues were rinsed in sterile water 3 times and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 25°C in the dark for 5-10 days. Isolates of the pathogen were purified using the single-spore isolation method (Leslie and Summerell 2006). Colonies on PDA produced fluffy aerial mycelium and developed an intense purple pigment when viewed from the underside. A range of colony pigmentation and growth rates were observed among the isolates. The microconidia were ovoid, hyaline, or ellipse in shape. The morphological features of the isolates were identified as Fusarium proliferatum (Leslie and Summerell, 2006). To further identify the isolates, genomic DNA was extracted from a representative isolate. And the internal transcribed spacer (ITS) region, the partial elongation factor (TEF1-α) gene and the β-tubulin gene (TUB2)were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012), EF-1 /EF-2 (O'Donnell et al. 1998) and B-tub1 /B-tub2 (O'Donnell and Cigelnik, 1997), respectively. The amplicons were sequenced and deposited in NCBI (accessions no. MZ292989, MZ293071 for ITS: MZ346602, MZ346603 for TEF1-α and MZ346600 and MZ346601 for B-tub). The ITS, TEF1-α, and B-tub sequences of the isolates showed 100% sequence similarity with Fusarium proliferatum isolates (accessions no. MT560212, LS42312, and LT575130, respectively) using BLASTn in Genbank. For pathogenicity testing, three whole bunched bananas sterilized with 10% bleach solutions and washed by sterilized water, were cut into 5 bananas per brunch. The cut surface of the banana crown was inoculated with conidial suspension (1.0 × 107 cfu/ml) of the pathogen with pipette tips. Equal number of bananas were treated with sterilized water in the same volume as a control. All bananas were sealed in a plastic bag and incubated at 25°C. After 7 days post inoculation, all inoculated bananas showed initial crown rot symptoms while no symptoms were observed on the control bananas. The fungus was re-isolated from the symptomatic tissues of infected bananas and confirmed to be genetically identical to F. proliferatum of the original inoculated strains according to morphological characteristics and molecular identification, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F. proliferatum causing crown rot on bananas in Georgia, USA.

Genomic Comparisons and Phenotypic Diversity of Dickeya zeae Strains Causing Bacterial Soft Rot of Banana in China.

Bacterial soft rot of banana, caused by Dickeya zeae, is spreading rapidly in important banana growing areas in China and seriously threatens banana production. In this study, we sequenced the high-quality complete genomes of three typical banana strains, MS1 (size: 4,831,702-bp; genome coverages: 538x), MS_2014 (size: 4,740,000-bp; genome coverages: 586x) and MS_2018 (size: 4,787,201-bp; genome coverages: 583x), isolated in 2009, 2014, and 2018, respectively. To determine their genomic and phenotypic diversity with respect to their hosts of origin, they were compared with other D. zeae strains, including another representative banana strain MS2 from China. The sequenced strains were similar in utilization of carbon source and chemical substrates, and general genomic features of GC content, and tRNA and rRNA regions. They were also conserved in most virulence determinants, including gene-encoding secretion systems, plant cell wall degrading enzymes, and exopolysaccharides. We further explored their genomic diversity in the predicted genomic islands (GIs). These GIs were rich in integrases and transposases, where some genomic dissimilarity was observed in the flagellar gene cluster and several secondary metabolite gene clusters. Different constituents of core biosynthetic modules were found within the bacteriocin and aryl polyene (APE) pigment gene clusters, and the strains from banana showed different phenotypes with respect to antibiosis effects and colony pigmentation. Additionally, clustered regularly interspaced short palindromic repeat (CRISPR) and prophage elements, such as type I-F and III-A CRISPR arrays and an intact prophage of MS1-P5, contributed to bacterial diversity. Phylogenetic tree analysis and genome-genome nucleotide comparison confirmed the genomic divergence among the strains isolated from banana. Considering these characteristics, MS2 and MS_2014 probably diverged later than MS1, while MS_2018 was different and more similar to foreign strains isolated from other hosts in several characteristics. Strain MS_2018 caused severe symptoms on banana varieties previously considered moderately resistant or moderately susceptible, including varieties of Cavendish (Musa AAA) and Plantain (Musa ABB). Our study of genomic and phenotypic diversity raises public attention to the risk of spreading new pathogenic variants within banana growing regions and supports development of predictive strategies for disease control.

Generalised mutagenesis with transposon Tn5. A laboratory procedure for the identification of genes responsible for a bacterial phenotype and its regulation, illustrated with phenazine production in Pseudomonas chlororaphis

ABSTRACT Forward genetics involves the identification of an unknown DNA sequence (genotype) associated with the expression of that sequence (phenotype). This is often done by generating variants (mutants) of that feature. One of the most practical methods is transposon mutagenesis, which facilitates the genotype–phenotype association through a sequence tag. This six-session laboratory practical illustrates the concept of forward genetics in bacteria using the random DNA target selection of the Tn5 mini-transposon to generate a mutant library. The target bacterium is Pseudomonas chlororaphis subsp. aurantiaca strain SMMP3, whose broad-spectrum fungal antagonism is largely due to a diffusible, orange-pigmented antibiotic of the phenazine class. The library is screened on appropriate media to select clones with reduced colony pigmentation. Upon qualitative and quantitative phenotype confirmation, a subset of mutants is subjected to an arbitrary PCR to identify the interrupted genes through an in-silico comparative analysis of the sequencing products in public genomic databases. Students thus identify previously described or novel biosynthetic and regulatory genes involved in phenazine production. Along with the objective of illustrating a forward genetics approach, the exercise emulates a real scientific activity in which supervised students pursue a path to interpret the phenotypic consequences of gene knockouts.