Green Colonies Research Articles

Isolation and molecular identification of Cronobacter spp. from powdered infant formula (PIF) in Bangladesh

Cronobacter spp. formerly known as Enterobacter sakazakii is an occasional contaminant of powdered infant formula (PIF). This pathogen has been associated with out-breaks of a rare form of infant meningitis, necrotizing enterocolitis (NEC), bacteremia and neonate deaths. The organism is ranked by the International Commission for Microbiological Specifications for Foods (ICMSF) as a ‘Severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration’. Present study aimed to isolate Cronobacter spp. from PIF and clinical samples, such as blood, stool and CSF collected from 93 neonates and child patients, age ranged from 0 to 24 months. We did not detect Cronobacter spp. in any of these samples. Later 32 PIF samples collected from retail markets in Bangladesh were tested for the presence of Cronobacter spp. Of these only one was found to be contaminated with Cronobacter sp. This is the first case of Cronobacter contaminated PIF found in Bangladesh to be reported. The organism was successfully identified based on its typical culture characteristics, producing blue–green colonies on chromogenic DFI agar and also by a standardized conventional PCR assay targeting the alpha glucosidase and 16 S rRNA gene sequence of Cronobacter sp. The 16 S rRNA gene was partially sequenced to provide for the phylogenetic analysis of this isolate (DA01) and found to cluster with some other Cronobacter isolates in the phylogram.

A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.

Fatal Systemic Cladosporiosis in a Merino Sheep Flock

Systemic cladosporiosis is described in 25 merino sheep from a flock consisting 250 animals. The fungal pneumonia appeared after an intensive antibiotic treatment, because of a respiratory system disorder. The pen of the flock was humid and crowded, and animals had signs of respiratory distress, coughing, fever and anorexia. All of the ill animals died, and necropsy was performed on 10 sheep. The lesions were characterized by a multifocal pyogranulomatous pneumonia and an abomasitis. Severe hemorrhages were observed in the lungs. At the histopathological examination, severe vasculitis with thrombosis was observed in various organs, especially in the lungs and abomasums, suggestive for a hematogenous dissemination of the infection in these organs. Numerous PAS-positive fungal elements were seen in the pyogranulomatous foci. Dark green fungal colonies were seen in the blood agar and Sabouraud dextrose agar that were identified as Cladosporium cladosporioides. This report highlights that phaeohyphomycosis can cause a severe systemic and fatal disease in merino sheep under insufficient management conditions.

Photobacterium swingsii sp. nov., isolated from marine organisms

Six Gram-negative coccobacilli were isolated from Pacific oysters (Crassostrea gigas) from Mexico and haemolymph of spider crabs (Maja brachydactyla) from Spain. All of the isolates grew as small green colonies on thiosulphate-citrate-bile salts-sucrose (TCBS) agar and were facultatively anaerobic, oxidase-positive and sensitive to the vibriostatic agent O/129. Repetitive palindromic PCR analysis revealed a high degree of genomic homogeneity among the isolates. Several phenotypic traits differentiated the isolates from the type strains of species of the genus Photobacterium. DNA-DNA relatedness between two representative isolates and their closest phylogenetic neighbours by 16S rRNA gene sequence similarity, Photobacterium aplysiae CAIM 14(T) and Photobacterium frigidiphilum CAIM 20(T), was 44.01-53.85 %. We propose a novel species of the genus Photobacterium to accommodate the six isolates, with the name Photobacterium swingsii sp. nov. The type strain is CAIM 1393(T) (=CECT 7576(T)).

Understanding the cause of false negative β-d-glucuronidase reactions in culture media containing fermentable carbohydrate

To explain the basis for false negative beta-glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Escherichia coli strains were assessed for their reactions in culture media containing a beta-d-glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of beta-D-glucuronidase at pH 5.0 and pH 7.2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme beta-D-glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose-containing media expressed virtually no beta-D-glucuronidase activity at pH 5.0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. E. coli that failed to produce green colonies on MLGA produced lower levels of beta-D-glucuronidase than did strains that formed green colonies, the difference being greater at pH 5.0 than pH 7.2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme beta-D-glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7.2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).

Density of Vibrios in Hemolymph and Hepatopancreas of Diseased Pacific White Shrimp, Litopenaeus vannamei, from Northwestern Mexico

Vibriosis is one of the most widespread infectious diseases of cultured shrimp in Mexico; as a result, the Research Centre for Food and Development (CIAD, for the Spanish wording) offers a diagnostic service to the shrimp farmers where they bring samples of diseased shrimp as judged by themselves. Hemolymph (HL) and hepatopancreas (HP) of diseased Litopenaeus vannamei were analyzed from five regions of Northwestern Mexico (from 2001 to 2006) in tiosulphate citrate bile sacharose (TCBS) agar; a total of 5912 shrimp were examined. The results showed a mean vibrios density of 6.45 × 103, SD = 6.90 × 104 CFU/mL for HL and 1.40 × 105, SD = 3.20 × 105 CFU/g for HP (n = 5912). HL samples had 12% (SD = 26%) of green colonies (GCs) in TCBS, and 23% (SD = 34%) of GCs in HP samples. Of the total shrimp analyzed, 46% were not found to possess vibrios in HL, whereas 60% of the shrimp examined contained more than 105 CFU/g of HP. More than 60 and 40% of the shrimp did not have GC in HL and HP, respectively. The highest Pearson coefficient correlation was observed between vibrios HL and GCs in HL for 2006 (r = 0.611, n = 712, P < 0.001).

Evaluation of a Chromogenic Agar under Development To Screen for VRE Colonization

BBL CHROMagar VanRE (CVRE) was compared with bile esculin azide agar plus vancomycin to screen for vancomycin-resistant enterococcus (VRE) colonization. CVRE distinguishes Enterococcus faecalis (green colonies) from Enterococcus faecium (mauve colonies) on the basis of chromogenic substrate use. CVRE sensitivity and specificity were 98.6% and 99.1%. Positive and negative predictive values were 95.9% and 99.7%.

Green Colonialism and Forest Policies in South India, 1800-1900

Abstract British colonial rule and its impact on the environment of the Indian sub-continent has made for a fascinating historical research subject over the last three decades. While exploitation of forests and the socio-economic impact of the process on tribal and other forest-dependent communities are well documented, the role of scientific discourse in facilitating the hegemonic control of the colonial state over forests had not been adequately treated in the existing literature. This article looks at the history of colonial forest policies in South India to argue that initially British destroyed most the accessible forests by logging them for shipbuilding, railways and the requirements of the military and public works departments. When the sustainability of the wood supply emerged as a problem, desiccationist ideas began to be systematically propagated. The desiccationist discourse branded forest utilisation by the natives as the main culprit for deforestation and the consequent reduction of the water flow of some rivers in South India. This alarmist discourse facilitated the control of the colonial state over most of the forests in South India by establishing a rhetoric of the preservation of forests as a means to save the environment and irrigation as prerequisites for the well-being of the agrarian economy. This article illustrates how desiccationist fears were used to justify the colonial state’s monopolistic control over the forests of South India.

Identification of Candida spp. by phenotypic tests and PCR

The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgar™ Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans.

Key Residues in Mycobacterium tuberculosis Protein Kinase G Play a Role in Regulating Kinase Activity and Survival in the Host

Protein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues.

Porites randalli: a new coral species (Scleractinia, Poritidae) from American Samoa

A new species of scleractinian coral, Porites randalli spec. nov. (Scleractinia, Poritidae), previously known as Porites sp. 2, is described from American Samoa. P. randalli typically forms small pale green colonies that are usually Porites randalli spec. nov. is an example of cryptic diversity; it is a small coral that at first glance can be overlooked or mistaken for a young colony of other species.

First Report of Bacterial Leaf Spot Caused by Pseudomonas cichorii on Schefflera arboricola in Turkey.

In late winter and spring of 2006 and 2008, leaf spots with yellow halos were observed on dwarf schefflera (Schefflera arboricola cvs. Gold Capella, Trinette, and Green Gold) that were grown as potted plants in two commercial ornamental greenhouses in Adana and Mersin, Turkey. Average disease incidence was assessed as 10% during the term of the study. Isolations were made from leaf spots symptoms on King's medium B. Bacteria consistently isolated from diseased tissues formed green fluorescent colonies on the medium. Ten representative bacterial strains were examined and found to be gram negative, rod shaped, and aerobic, levan, pectolytic, and arginine dihydrolase negative, and oxidase positive. They all induced a hypersensitive response in tobacco (Nicotiana tabacum cv. Samsun). All strains were identified as Pseudomonas cichorii with similarity indices of 79 to 99% based on fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE). Pathogenicity of the strains was confirmed on five dwarf schefflera plants by leaf tissue infiltration with bacterial suspensions (107 CFU ml-1) in sterile distilled water. P. cichorii NCPPB 3802 and sterile water were used as positive and negative controls, respectively. The same symptoms as those observed in the commercial greenhouses were observed on dwarf schefflera leaves within 12 to 15 days after inoculation. The bacteria were reisolated from the inoculated plants and identified as the same as the original strain by conventional tests and FAME analysis. Negative control plants remained disease free. Occurrence of bacterial leaf spot caused by P. cichorii on vegetable crops in Turkey (1,3) and dwarf schefflera in other countries (2) has been reported previously, but to our knowledge, this is the first report of the observation of P. cichorii on dwarf schefflera in Turkey.

Brown spot needle blight associated with Mycosphaerella dearnessii occurs on Pinus rotundata in the Czech Republic

Lecanosticta acicola is the anamorph of Mycosphaerella dearnessii, the cause of brown spot needle blight on pines. The disease has been recorded in several European countries including Austria, Bulgaria, France, Germany, Spain, Italy, Switzerland and former Yugoslavia (e.g. Holdenrieder & Sieber, 1995; La Porta & Capretti, 2000). There are much earlier records of brown spot needle blight in Europe although it is unclear how these were distinguished from Dothiostroma septospora infections producing similar symptoms. Lecanosticta acicola was confirmed in the Czech Republic from samples of bog pine, Pinus rotundata, collected on the 10 June, 2007 from Červené Blato Nature Reserve, South Bohemia (48°51′37″N, 14°48′44″E); 430 metres above sea level. Bog pines 10–40 years-old had severe needle defoliation inside the nature reserve which were absent in pines (P. rotundata, P. sylvestris) in surrounding managed forests. Symptoms begin with the appearance of yellow and brown spots, later becoming bands on first-year needles which then decline from the tip. Acervuli erupt from needles incubated for two to three days in a moisture chamber. Conidia are subhyaline, thick-walled, straight to curved, fusiform to cylindrical, with rounded apex and truncate base, 1–5 septate, occasionally slightly constricted at the septa, 3–5 × 21–44 µm. The surface of conidia have small warts or spines. After three to four weeks incubation of conidia at 21°C on 2% malt extract agar, dark green olive stromatic mycelial colonies produced conidia identical to those detected on incubated needles. DNA sequences of the ITS region of mycelium from the isolated strain (GenBank Accession. No. EU117117) showed 99–100% similarity with other sequences of M. dearnessii held in GenBank. A dried specimen of infected needles is preserved in BRNL herbarium (specimen no. 1010), and the fungus has been deposited in the CCF Culture Collection (strain no. 3743). We appreciate the financial support of the project NAZV QH81039.

Ochratoxin- and aflatoxin-producing fungi associated with green and roasted coffee samples consumed in Argentina

The aims of this work were to identify the Aspergillus sections Nigri and Flavi, and to evaluate the natural occurrence of ochratoxin A (OTA) and aflatoxins in green and roasted coffee bean samples. The capacity to produce these toxins by Aspergillus species was also studied. Fifty samples of Colombian coffee beans (25 green and 25 roasted) were obtained from a processor plant located in the south of Córdoba province (Argentina). OTA and aflatoxin analysis were performed by high performance liquid chromatography (HPLC). OTA production by strains belonging to Aspergillus niger aggregate were cultivated using YES medium and detected by HPLC. Aflatoxin production was tested in strains belonging to section Flavi on malt extract agar and was detected by thin liquid chromatography (TLC). From green coffee samples, the predominant species isolated belonged to A. niger aggregate, 60 and 55%, in dichloran rose bengal chloramphenicol agar (DRBC) and dichloran 18% glycerol agar (DG18) respectively. While A. flavus strains were isolated in 14% from DRBC and A. parasiticus strains in 12% and 28% in DRBC and DG18, respectively. From roasted coffee samples, A. flavus was the most predominant fungi, isolated in similar percentages from both media (28%); followed by A. niger aggregate isolated in 28 and 14% in DRBC and DG18, respectively. In green and roasted coffee samples mean colony counts ranged from 2×103 to 3.5×104 colony forming units per gram of sample (cfu/g). OTA and aflatoxins were not detected in any sample analyses (<1 and 0.5 ng/g for OTA and aflatoxins, respectively). Twenty-five percent of black Aspergillus strains were OTA producers. The total of A. flavus strains assayed produced aflotoxin B1 (AFB1) and 80% of the A. parasiticus strains were AFB1 and aflotoxin G1 producers. The high percentage of A. flavus and A. parasiticus aflatoxin-producing strains suggest a potential risk for contamination in coffee with aflatoxins.

Outbreak of severe disseminated aspergillosis in a flock of ostrich (Struthio camelus)

This study was undertaken to describe clinical, mycological and histopathological findings in black neck ostriches affected with severe aspergillosis in a flock including 80 birds, near Tehran, Iran. The signs included anorexia, depression, notable weight loss, diarrhoea, severe respiratory distress and death. Grossly, the lungs showed numerous white to yellow caseous nodules and the walls of the thoracic and abdominal air sacs were thickened with inflammatory exudates containing cellular debris, necrotic masses and green mold colonies. Multiple nodules were observed in the liver, spleen and gastrointestinal tract as well. Histopathologically, there were conidial heads and fungal hyphae in the air sacs and multifocal necrotic and granulomatous lesions with septated and dichotomously branched hyphae in various tissues, which were stained with haematoxylin and eosin and Grocott's methenamine silver nitrate. Aspergillus fumigatus was isolated in various tissues taken from affected ostriches.

Response to “Another look at green Trichodesmium colonies”

Response to “Another look at green <i>Trichodesmium</i> colonies”

Another look at green Trichodesmium colonies

Another look at green <i>Trichodesmium</i> colonies

Distribution and photobiology of Symbiodinium types in different light environments for three colour morphs of the coral Madracis pharensis: is there more to it than total irradiance?

The role of symbiont variation in the photobiology of reef corals was addressed by investigating the links among symbiont genetic diversity, function and ecological distribution in a single host species, Madracis pharensis. Symbiont distribution was studied for two depths (10 and 25 m), two different light habitats (exposed and shaded) and three host colour morphs (brown, purple and green). Two Symbiodinium genotypes were present, as defined by nuclear internal transcribed spacer 2 ribosomal DNA (ITS2-rDNA) variation. Symbiont distribution was depth- and colour morph-dependent. Type B15 occurred predominantly on the deeper reef and in green and purple colonies, while type B7 was present in shallow environments and brown colonies. Different light microhabitats at fixed depths had no effect on symbiont presence. This ecological distribution suggests that symbiont presence is potentially driven by light spectral niches. A reciprocal depth transplantation experiment indicated steady symbiont populations under environment change. Functional parameters such as pigment composition, chlorophyll a fluorescence and cell densities were measured for 25 m and included in multivariate analyses. Most functional variation was explained by two photobiological assemblages that relate to either symbiont identity or light microhabitat, suggesting adaptation and acclimation, respectively. Type B15 occurs with lower cell densities and larger sizes, higher cellular pigment concentrations and higher peridinin to chlorophyll a ratio than type B7. Type B7 relates to a larger xanthophyll-pool size. These unambiguous differences between symbionts can explain their distributional patterns, with type B15 being potentially more adapted to darker or deeper environments than B7. Symbiont cell size may play a central role in the adaptation of coral holobionts to the deeper reef. The existence of functional differences between B-types shows that the clade classification does not necessarily correspond to functional identity. This study supports the use of ITS2 as an ecological and functionally meaningful marker in Symbiodinium.

Utilization of the chromogenic agar MRSA ID in the surveillance of methicillin-resistant Staphylococcus aureus in a low endemic unit for MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogen responsible for nosocomial infections.Approximately 20% of patients undergoing surgical operations acquires at least one nosocomial infection. Nasal carriers of S. aureus have been identified as one of the populations at major risk to develop this type of infection, particularly, after operations, dialysis, transplants and admission in intensive care unit. Our study was conducted on patients who came from a Neurosurgery Intensive Care Unit, where the incidence of MRSA is very low (8%) and where it is very important to identify MRSA carriers in order to adopt measures to avoid transmission.The use of chromogenic agar MRSA ID allowed us to identify green colonies of MRSA within 24 hours and to make a timely and careful diagnosis.

Holographic assembly workstation for optical manipulation

We report a holographic assembler workstation for optical trapping and micro-manipulation. The workstation is based on a titanium sapphire laser, making it particularly suited for biomaterials and incorporates a choice of user interfaces for different applications. The system is designed around a commercial inverted microscope and is configured such that it can be easily used by the non-specialist. We demonstrate the bio-capabilities of our system by manipulating a group of yeast cells, a single red blood cell and a single cell of the green algae colony Volvox.