Ismini Nakouti

A new approach to isolating siderophore‐producing actinobacteria

Aims: This study was conducted to investigate the application of 2,2′‐dipyridyl as a new approach to isolating siderophore‐producing actinobacteria.

Characterisation of five siderophore producing actinomycetes from soil samples and the use of antibiotic resistance to differentiate the isolates.

Organisms were isolated on their basis of survival in an iron-limited environment. The survivors of this treatment were largely actinomycetes. Of the viable cultures, most were found to produce siderophore like compounds. The most prolific producers as assessed by the Chromo Azuerol Sulphate assay were further characterised and found to belong to the genus Streptomyces. Attempts to taxonomically characterise these organisms illustrated the conserved nature of the 16S rRNA gene in this group of organisms. Physiological characterisa- tion was undertaken and it was found that the resistance of these organisms to a range of antibiotics proved to be a useful discriminating factor . Keywords- Actinomycetes, iron chelation, siderophores, soil, streptomycetes, classification, resistance

Real-Time Monitoring of Pseudomonas Aeruginosa Concentration Using a Novel Electromagnetic Sensors Microfluidic Cell Structure

This study demonstrates an electromagnetic wave-based sensor embedded within a fluidic cell for the purposes of quantifying Pseudomonas aeruginosa in real time, which implies it could be applied for provision of point-of-care diagnostics. The sensors operates through the interaction of the electromagnetic field with the analyte flowing through the fluidic system, and via the sensor head which has a specifically designed planar pattern to maximize the sensor sensitivity for the given bacteria type. The sensor is demonstrated to respond linearly (R2 = 0.9942) to OD550 25 × 10-3 - 1.0 bacteria concentration through changing resonant frequency and peak quality factor. This innovative approach is expected to contribute to better provision of healthcare services, minimizing the need for hospital visits through real-time point-of-care diagnostics as opposed to lengthy laboratory assays.

A new approach to studying ion uptake by actinomycetes

A streptomycete that had the ability to avidly sequester iron via siderophores was previously isolated from environmental soil samples. The chelating agent expressed by this organism is confirmed by HPLC as desferrioxamine E. Although the traditional chromo azuerol sulphate (CAS) assay for detection of siderophores is based upon the chelation of iron we were interested to examine the relationship of these iron‐capturing molecules with other ions. Consequently, a new approach was employed that enabled the assessment of the affinity of the siderophore moieties for other ions by adapting the CAS assay. The present study reveals that the isolate produced a siderophore that was capable of sequestering a range of ions including Mn, Co, Cd, Ni, Al, Li, Cu, Zn and Mg. On the basis of the assay described it would appear that the organism sequesters copper more readily than iron. This raises an age‐old debate surrounding the replacement of copper as a fundamentally essential element with iron as a consequence of the evolution of the di‐oxygen environment.

Phenotypic detection of AmpC β-lactamases, extended-spectrum β-lactamases and metallo-β-lactamases in Enterobacteriaceae using a resazurin microtitre assay with inhibitor-based methods

Dissemination of antibiotic resistance in Enterobacteriaceae mediated by AmpC β-lactamase, extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) is clinically significant. A simple and relatively quick method for the detection of these resistance phenotypes would greatly improve chemotherapeutic recommendation. This technology would provide valuable input in our surveillance of resistance on a global stage, particularly if the methodology could be applicable to resource-poor settings. A resazurin microtitre plate (RMP) assay incorporating cloxacillin, clavulanic acid and EDTA for the rapid phenotypic identification of AmpC, ESBL and MBL and the co-existence of β-lactamases has been developed. A total of 47 molecularly characterized Enterobacteriaceae clinical isolates producing AmpCs, ESBLs, co-producers of ESBL and AmpC, MBLs and co-producers of ESBL and MBL were phenotypically examined using the RMP assay. The ceftazidime- and cefotaxime-based RMP assays successfully detected all 16 AmpC, 14 ESBL and 9 MBL producers, 6 ESBL-AmpC co-producers and 2 ESBL-MBL co-producers without false-positive results. The ceftazidime-based assay was more reliable in detecting AmpC alone, while the cefotaxime-based assay performed better in identifying co-producers of ESBL and AmpC. There was no difference in the detection of ESBL and MBL producers. The findings of the present study suggest that use of the RMP assay with particular β-lactamase inhibitors explicitly detects three different β-lactamases, as well as co-existence of β-lactamases, within 6 h of initial isolation of the pathogen. This assay is applicable to carry out in any laboratory, is cost-effective and is easy to interpret. It could be implemented in screening patients and controlling infection and for surveillance purposes.

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended-spectrum-β-lactamases, AmpC β-lactamases and co-β-lactamases in Enterobacteriaceae

A promising means of rapid screening of extended‐spectrum‐β‐lactamase (ESBL), AmpC β‐lactamase, and co‐production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed β‐lactamase‐producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, nine co‐producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co‐existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co‐β‐lactamase‐producing Enterobacteriaceae are endemic.

A combined disc method with resazurin agar plate assay for early phenotypic screening of KPC, MBL and OXA-48 carbapenemases among Enterobacteriaceae.

To validate a combined disc method along with resazurin chromogenic agar for early screening and differentiation of Klebsiella pneumoniae carbapenemase, metallo‐β‐lactamase and OXA‐48 carbapenemase‐producing Enterobacteriaceae.

Electromagnetic wave sensing of euglena gracilis viability and quantification

Euglena is a naturally occurring algae which can be found in any fresh water source.It is non-toxic, easy to handle, visualize and relatively resilient to variation in environment.This, along with the relatively large size of Euglena, means it can be readily used as a modelfor environmental monitoring of other smaller pathogenic micro-organisms (e.g. Escherichiacoli ). Currently the behavior of Euglena is observed through the use of an optical microscopefor sensing purposes. However, this method su ers from following major pitfalls: (1) the sizeand expense of the microscope; (2) the small observation volume (approx. 1 L); (3) the imageprocessing requirements and (4) need for a skilled human operator to acquire those images. Byusing electromagnetic (EM) wave technology in the GHz frequency range we seek to overcomethese challenges, since it has been demonstrated by the authors to be cost e ective, have alarge sensing volume (> 100L) and produce comparatively simple output data. Furthermoreit is possible to use simple software algorithms to process the sensor output data, and providereal-time information on Euglena gracilis viability and quantity. This paper shows proof ofconcept work to verify the feasibility of the proposed EM wave technology as an alternative tothe current optical microscopy methods.

A nitrocefin disc supplemented with ertapenem for rapid screening of carbapenemase-producing Enterobacteriaceae

Reliable, simple and rapid methods for laboratory detection of carbapenemases are important for an appropriate antibiotic administration. A nitrocefin disc containing ertapenem for rapid screening of carbapenemase production among Enterobacteriaceae is developed in the present study. A total of 87 molecularly-confirmed Enterobacteriaceae including 31 carbapenemase producers and 56 non-carbapenemase producers, were tested with nitrocefin discs supplemented with and without ertapenem (20 μg/disc). Nitrocefin discs with ertapenem successfully discriminated all 31 carbapenemase and all non-carbapenemase producers within 30 minutes. The sensitivity and specificity of the method were 100%. The minimum inhibitory concentrations (MICs) of ertapenem against all carbapenemase-producing isolates ranged from 1 to ≥256 μg/mL. This simple test could help to minimize the treatment failure and control the dissemination of infections caused by carbapenemase-associated resistant bacteria. It is a promising approach that could be performed routinely in any laboratory.

Antimicrobial activity of kojic acid from endophytic fungus Colletotrichum gloeosporioides isolated from Sonneratia apetala, a mangrove plant of the Sundarbans

Objective: To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate (EtOAc) extract of endophytic fungus Colletotrichum gloeosporioides (C. gloeosporioides) isolated from Sonneratia apetala. Methods: Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Gram-positive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay. Results: The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites. Kojic acid (1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1D, 2D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms. Whilst the minimum inhibitory concentration (MIC) values from the EtOAc extract ranged between 2.4× 10-4 mg/mL and 2.5 mg/mL, those of kojic acid (1) were between 0.125 mg/mL and 1 mg/mL. The EtOAc extract and kojic acid (1) were most active against Pseudomonas aeruginosa (MIC = 2.4×10-4 mg/mL) and Micrococcus luteus (MIC = 0.125 mg/mL), respectively. Conclusions: The results revealed that the endophytic fungus C. gloeosporioides could be a good source of commercially important kojic acid, which exhibited antimicrobial properties.