Genotypic and phenotypic characteristics of Cronobacter species, with particular attention to the newly reclassified species C. helveticus, C. pulveris, and C. zurichensis [forthcoming]

In 2013, Enterobacter helveticus, E. pulveris and E. turicensis, were reclassified as Cronobacter helveticus, C. pulveris and C. zurichensis, respectively. Previously these species had been used as negative controls for some Cronobacter detection assays. This study examined cultural, biochemical and molecular Cronobacter detection and identification assays, with emphasis on the new species. Additionally, 32 Cronobacter genomes were examined for the presence of PCR target genes using the BLAST function of the online Cronobacter BIGSdb facility. The results of the cultural methods varied and no single medium was able to correctly detect all Cronobacter spp. Since the supporting databases have not been updated to include the Cronobacter genus, Enterobacter sakazakii was returned for four strains of the newly reclassified species with ID32E and none with API 20E. PCR probes targeting rpoB and ompA could not correctly identify the new Cronobacter spp., due to primer specificity or absent target genes. As neonates have been identified as a high-risk group for infection, international standards require the absence of all Cronobacter species in powdered infant formula. However, many conventional detection methods cannot correctly identify the newly recognized species. Conversely, DNA sequence-based methods can adapt to taxonomic revisions and will likely become more common

Insights Into Halophilic Microbial Adaptation: Analysis of Integrons and Associated Genomic Structures and Characterization of a Nitrilase in Hypersaline Environments

Hypersaline environments are extreme habitats that can be exploited as biotechnological resources. Here, we characterized a nitrilase (NitraS-ATII) isolated from Atlantis II Deep brine pool. It showed higher thermal stability and heavy metal tolerance compared to a closely related nitrilase. We also studied integrons in halophiles and hypersaline environments. Integrons are genetic platforms in which an integron integrase (IntI) mediates the excision and integration of gene cassettes at specific recombination sites. In order to search for integrons in halophiles and hypersaline metagenomes, we used a PCR-based approach, in addition to different bioinformatics tools, mainly IntegronFinder. We found that integrons and clusters of attC sites lacking a neighboring integron-integrase (CALINs) are abundant in halophilic bacterial genomes; and we detected archaeal integrons for the first time. Moreover, we found different classes of insertion sequences (ISs) within and nearby integrons and CALINs in halophilic genomes. We detected Group IIB introns for the first time associated with different integrons and CALINs. Finally, using 16S rRNA-based analysis, we assessed the prokaryotic diversity in two Egyptian hypersaline environments: Aghormy Lake in Siwa Oasis and Sebeaka saltern at the vicinity of Bardawil Lagoon. Both sites showed an abundance of family Rhodothermaceae, Bacteroidetes. Aghormy Lake showed abundance in Deinococcus-Thermus phylum known by its extremophilic members. The observed dissimilarities between both brines may reflect the differences in the biogeographical and physicochemical nature of the two sites. Our study may shed light towards a possible interplay of integrons with different associated MGEs in the adaptation of prokaryotes in hypersaline environments

Cloning, expression and preliminary characterization of a novel nitrilase from the Red Sea, Atlantis II Deep brine pool using a metagenomic approach

With the abundance of hazardous nitrile compounds in nature and with the extensive use of them in industries of fine chemicals, the attention towards nitrilases has profoundly increased as biocatalysts in different industries and in bioremediation. The use of nitrilases, which hydrolyze nitriles into carboxylic acids and ammonia, was introduced as a biological method superior to conventional chemical means. In contrast to conventional microbiological techniques using cultured microorganisms, and with the loads of genomic sequences available in the databases, identification of novel nitrilases using metagenomic approaches seems more promising. In this study, a nitrilase was isolated from the microbial metagenomic DNA obtained from the Lower Convective Layer (LCL) of Atlantis II Deep Brine Pool in the Red Sea. Sequences of putative nitrilases were retrieved from the LCL database, followed by PCR amplification and gene synthesis of the chosen sequences. Amplified genes were cloned into pET SUMO expression vector, whereas a synthesized gene, with optimized codons for E. coli, was cloned into pET-28b+ vector flanked by SacI and HindIII restriction sites. The recombinant proteins were then expressed in Escherichia coli BL21 (DE3) cells. The His-tagged protein (Nitra-S), from the recombinant pET-28b+ transformed cells, was successfully purified using Ni-NTA affinity chromatography columns under native conditions. Preliminary studies on the induced cells, crude extracts and the purified enzyme showed that Nitra-S exhibited nitrilase activity towards dinitriles (succinonitrile and gluteronitrile) rather than other nitrile classes. The used quantitative activity assay was based on measuring the amounts of released ammonia upon the action of nitrilases on their nitrile substrates. Full characterization of the purified nitrilase is still to be done; however, the isolation of this protein from the LCL with its unique characteristics, increase the odds towards finding exceptional properties of the newly identified nitrilase

Multilocus sequence typing of Cronobacter spp. from powdered infant formula and milk powder production factories

This study applied the Cronobacter spp. multilocus sequence typing (MLST) scheme to three strain collections, then known as Enterobacter sakazakii, which had been isolated between 1988 and 2009 from 14 countries. The results revealed the predominance (85%) of C. 29 sakazakii (72 strains) in all three collections. The remaining strains were C. turicensis (10%), C. malonaticus (4%), and C. muytjensii (1%). No strains of C. dublinensis, C. universalis or C. condimenti were identified. Twenty-one out of seventy two C. sakazakii strains were in the clinically significant ST4 clonal complex, and were found in all three strain collections. These results confirm C. sakazakii ST4 is one of the predominant clonal complexes over the past 20 years in several parts of the world. Further understanding of the ecosystem and sources of the organism may be used for the development of improved intervention strategies in the dairy industry

Shari`ah and State Formation: Historical Perspective

This Article focuses on issues of Islamic discrimination against women and asks how centuries of legal practice in Shari\u27ah courts illustrate Muslim societies\u27 regard of the witness of women, women\u27s work, women\u27s seclusion, and the existence of or the need for a private/public divide in a woman\u27s role in society. Furthermore, this Article explores the legal system when Shari\u27ah courts practiced Shari\u27ah law before the coming of the West or the modernization of law in Muslim countries. Were presumptions, such as the nature of women, at the heart of the system, or were rules of evidence the determinants of justice? How closely followed were the conclusions of jurists and mufti\u27s (jurisconsults) when it came to decisions made by qadis (judges)? Finally, how can we define justice in Islamic courts? I deal with these questions through two main inquiries using Shari\u27ah archival records from Egypt and Palestine during the Ottoman period and into the nineteenth century. The first inquiry deals with the public/private divide, which is the usual justification for giving less credibility to the witness of women. The second inquiry will deal with archival evidence regarding the witness of women including their role as expert witnesses

Investigation of biofilm formation on contact eye lenses caused by methicillin resistant Staphylococcus aureus

Objective: The objective was to investigate the biofilm‑forming capacity of methicillin resistant Staphylococcus aureus (MRSA) isolated from eye lenses of infected patients.Materials and Methods: A total of 32 MRSA isolated from contact lenses of patients with ocular infections were screened for their biofilm‑forming capacity using tube method (TM), Congo red agar (CRA), and microtiter plate (MtP) methods. The effect of some stress factor on the biofilm formation was studied. The biofilm‑forming related genes, icaA, icaD and 10 microbial surface components that recognize adhesive matrix molecule (MSCRAMM), of the selected MRSA were also detected using polymerase chain reaction.Results: Of 32 MRSA isolates, 34.37%, 59.37%, and 81.25% showed positive results using CRA, TM or MtP, respectively. Biofilm production was found to be reduced in the presence of ethanol or ethylenediaminetetraacetic acid and at extreme pH values. On the other hand, glucose or heparin leads to a concentration dependent increase of biofilm production by the isolates. The selected biofilm producing MRSA isolate was found to harbor the icaA, icaD and up to nine of 10 tested MSCRAMM genes, whereas the selected non biofilm producing MRSA isolate did not carry any of the tested genes.Conclusions: The MtP method was found to be the most effective phenotypic screening method for detection of biofilm formation by MRSA. Furthermore, the molecular approach should be taken into consideration for the rapid and correct diagnosis of virulent bacteria associated with contact eye lenses.Key words: Biofilm formation, eye lenses, methicillin resistant Staphylococcus aureus, microtiter plate, polymerase chain reactio

Comparative evaluation of dexmedetomidine versus magnesium sulphate on the adequacy of hypotensive anesthesia and post-operative recovery for patients undergoing endoscopic transnasal transsphenoidal pituitary tumor resection

Background: Perioperative use of magnesium sulfate (MgSO4), dexmedetomidine, have been tried in order to provide beneficial clinical effects during general anesthesia (GA). However, few literature discussed it with varying results. Several clinical researches have showed that usage of MgSO4 infusion was associated with a reduction in anesthetic requirement and postsurgical analgesic consumption during GA.Objective: This study aimed to assess the pharmacologic effects of the use of dexmedetomidine and MgSO4 on anesthetic requirement, intra operative haemodynamics stability and postsurgical analgesic effects on the adequacy of hypotensive anesthesia during transsphenoidal resection of pituitary tumours.Patients and methods: A total of 110 cases were enrolled in this prospective study. They were randomized into 2 groups: Group D (55 cases) that was commenced on dexmedetomidine, and group M (55 cases) which received MgSO4Results: The mean values of Boezaart score were significantly decreased in Group D in comparison to group M. In addition, isoflurane and propranolol consumption showed a significant decrease in group D. However, blood loss showed no significant difference when comparing the same groups. Group D expressed significantly longer emergence and extubation times compared to Group M.Conclusion: Dexmedetomidine appears to be superior compared to magnesium sulphate in achieving hypotensive anesthesia during pituitary surgery

Anaphora Resolution in Business Process Requirement Engineering

Anaphora resolution (AR) is one of the most important tasks in natural language processing which focuses on the problem of resolving what a pronoun, or a noun phrase refers to. Moreover, AR plays an essential role when dealing with business process textual description, either when trying to discover the process model from the text, or when validating an existing model. It helps these systems in discovering the core components in any process model (actors and objects).In this paper, we propose a domain specific AR system. The approach starts by automatically generating the concept map of the text, then the system uses this map to resolve references using the syntactic and semantic relations in the concept map. The approach outperforms the state-of-the art performance in the domain of business process texts with more than 73% accuracy. In addition, this approach could be easily adopted to resolve references in other domains

Physiological characterisation of the neonatal meningitic bacterium Cronobacter sakazakii

The Cronobacter genus is a member of the Enterobacteriaceae family that comprises 7 species. A multiple locus sequence typing (MLST) scheme was designed for Cronobacter and was used to identify over 400 sequence types (STs). Notably, C. sakazakii ST4 and its single/double loci variants, which together form clonal complex 4 (CC4), are predominantly associated with neonatal meningitis. The present PhD study aimed to investigate the diversity of C. sakazakii especially CC4 using MLST analysis, physiological characterisation of stress responses along with genomic analysis associated genes. In the first part of the study, MLST was used to analyse three collections of environmental bacterial isolates. The isolates were from powdered infant formula, milk powder factory environments, and milk powder processing equipment; they had not been profiled prior to this study and had previously been identified as E. sakazakii. These environmental strains were obtained from geographically diverse countries. A total of 39 STs were identified across the studied isolates. The analysis revealed that despite their geographical and temporal spread, only 4 Cronobacter species were isolated from these three collections of strains, namely C. sakazakii, C. malonaticus, C. turicensis, and C. muytjensii. Interestingly, this study demonstrated that C. sakazakii CC4 isolates represent a predominantly stable lineage within the Cronobacter genus in these environments. This result led to further investigation to study the physiological factors in the CC4 clone associated with its persistence in the environment. This included desiccation stress, heat tolerance, acid resistance and serum resistance assays. The desiccation and serum resistance assays indicated that all of the tested C. sakazakii CC4 and non-CC4 strains showed equal resistance against desiccation and serum. The experiments also indicated that C. sakazakii CC4 strains were more heat tolerant than non-CC4 strains to 100°C heat. The most interesting observation was that C. sakazakii CC4 strains were significantly more acid-resistant than non-CC4 strains to pH 3.5, which is the pH of the neonatal stomach. The results were supported by the outer membrane protein (OMP) profiling where C. sakazakii strains revealed more OMPs than non-CC4 at pH 3.5. Capsule production assays showed that most of the C. sakazakii CC4 and non-CC4 strains produced mucoid capsules when cultured on milk agar. The motility of the tested strains was associated with the presence of the fliRQPON flagellar genes. An exhaustive BLAST search of sequenced strains to identify genes associated with virulence or with environmental fitness found no significant difference between the virulence potential of C. sakazakii CC4 and C. sakazakii non-CC4 strains. The present study provides important insights into neonatal meningitis C. sakazakii CC4. Further studies are warranted to characterise the OMPs that are predominant in C. sakazakii CC4 and elucidate their significance towards virulence of this important neonatal health-associated pathogen