Antibacterial efficacy of cinnamon bark essential oil against multi-resistant Enterococci using LEGO® MINDSTORMS®-based automated sterilization equipment

Natural antimicrobials are currently being tested by many researchers for active packaging applications as a response to consumer demands for safer food products. In previous work, several packaging materials consisting of ethylene vinyl alcohol (EVOH)‐coated polypropylene (PP) films containing essential oils or their constituents as active agents were successfully developed and tested for antimicrobial activity. In this work, selected films from those materials, namely EVOH coatings with carvacrol, citral, marjoram essential oil, or cinnamon bark essential oil, on PP and polyethylene terephthalate (PET) substrates, were subjected to diverse physicochemical analyses in order to assess their suitability for food packaging applications. Concretely, the investigated properties were the stability of EVOH coatings on PP and PET substrates, the retainability of EVOH matrices for active compounds, the mechanical, optical, surface and barrier properties of the final active films and the effects of a matrix modification based on the addition of bentonite nanoclay on the performance of PP/EVOH active packages studied in actual working conditions.Results showed that the application of corona discharge followed by a polyethyleneimine‐based primer was the best anchorage treatment available to stabilize EVOH coatings on PP and PET substrates. Furthermore, they demonstrated that the retention of active agents into EVOH matrices ranged from low to moderate, depending on the embedded substance, and that their presence into an EVOH coating in the final multilayer films did not noticeably affect their mechanical, optical or barrier properties, although it considerably improved their wettability. They also indicated that the inclusion of bentonite nanoparticles into their carrier layers substantially enhanced the performance of the final packages, while maintaining or slightly improving their other physical properties. Hence, as a conclusion, all the assayed multilayer films were considered perfectly valid for food packaging applications, and the incorporation of bentonite nanoclay to their carrier layers was also highly recommended. Copyright © 2014 John Wiley & Sons, Ltd.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Data availability References Decision letter Author response Article and author information Metrics Abstract Total RNA sequencing (RNA-seq) is an important tool in the study of mosquitoes and the RNA viruses they vector as it allows assessment of both host and viral RNA in specimens. However, there are two main constraints. First, as with many other species, abundant mosquito ribosomal RNA (rRNA) serves as the predominant template from which sequences are generated, meaning that the desired host and viral templates are sequenced far less. Second, mosquito specimens captured in the field must be correctly identified, in some cases to the sub-species level. Here, we generate mosquito rRNA datasets which will substantially mitigate both of these problems. We describe a strategy to assemble novel rRNA sequences from mosquito specimens and produce an unprecedented dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African Republic, Madagascar, and French Guiana). These sequences will allow both physical and computational removal of rRNA from specimens during RNA-seq protocols. We also assess the utility of rRNA sequences for molecular taxonomy and compare phylogenies constructed using rRNA sequences versus those created using the gold standard for molecular species identification of specimens—the mitochondrial cytochrome c oxidase I (COI) gene. We find that rRNA- and COI-derived phylogenetic trees are incongruent and that 28S and concatenated 28S+18S rRNA phylogenies reflect evolutionary relationships that are more aligned with contemporary mosquito systematics. This significant expansion to the current rRNA reference library for mosquitoes will improve mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA depletion protocols for a broader range of species and streamlining species identification by rRNA sequence and phylogenetics. Editor's evaluation Mosquitoes are an important vector for viruses and other pathogens worldwide. However, significant genomic resources are scarce for the study of these species. In this work, the authors create a significant genomic resource that will enable the study of mosquitoes and the pathogens that they carry. https://doi.org/10.7554/eLife.82762.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Mosquitoes top the list of vectors for arthropod-borne diseases, being implicated in the transmission of many human pathogens responsible for arboviral diseases, malaria, and lymphatic filariasis (WHO, 2017). Mosquito-borne viruses circulate in sylvatic (between wild animals) or urban (between humans) transmission cycles driven by different mosquito species with their own distinct host preferences. Although urban mosquito species are chiefly responsible for amplifying epidemics in dense human populations, sylvatic mosquitoes maintain the transmission of these viruses among forest-dwelling animal reservoir hosts and are involved in spillover events when humans enter their ecological niches (Valentine et al., 2019). Given that mosquito-borne virus emergence is preceded by such spillover events, continuous surveillance and virus discovery in sylvatic mosquitoes is integral to designing effective public health measures to pre-empt or respond to mosquito-borne viral epidemics. Metagenomics on field specimens is a powerful method in our toolkit to understand mosquito-borne disease ecology through the One Health lens (Webster et al., 2016). With next-generation sequencing becoming more accessible, such studies have provided unprecedented insights into the interfaces among mosquitoes, their environment, and their animal and human hosts. As mosquito-associated viruses are mostly RNA viruses, RNA sequencing (RNA-seq) is especially informative for surveillance and virus discovery. However, working with lesser studied mosquito species poses several problems. First, metagenomics studies based on RNA-seq are bedevilled by overabundant ribosomal RNAs (rRNAs). These non-coding RNA molecules comprise at least 80% of the total cellular RNA population (Gale and Crampton, 1989). Due to their length and their abundance, they are a sink for precious next-generation sequencing reads, decreasing the sensitivity of pathogen detection unless depleted during library preparation. Yet the most common rRNA depletion protocols require prior knowledge of rRNA sequences of the species of interest as they involve hybridizing antisense oligos to the rRNA molecules prior to removal by ribonucleases (Fauver et al., 2019; Phelps et al., 2021) or by bead capture (Kukutla et al., 2013). Presently, reference sequences for rRNAs are limited to only a handful of species from three genera: Aedes, Culex, and Anopheles (Ruzzante et al., 2019). The lack of reliable rRNA depletion methods could deter mosquito metagenomics studies from expanding their sampling diversity, resulting in a gap in our knowledge of mosquito vector ecology. The inclusion of lesser studied yet medically relevant sylvatic species is therefore imperative. Second, species identification based on morphology is notoriously complicated for members of certain species subgroups. This is especially the case among Culex subgroups. Sister species are often sympatric and show at least some competence for a number of viruses, such as Japanese encephalitis virus, St Louis encephalitic virus, and Usutu virus (Nchoutpouen et al., 2019). Although they share many morphological traits, each of these species have distinct ecologies and host preferences, thus the challenge of correctly identifying vector species can affect epidemiological risk estimation for these diseases (Farajollahi et al., 2011). DNA molecular markers are often employed to a limited degree of success to distinguish between sister species (Batovska et al., 2017; Zittra et al., 2016). To address the lack of full-length rRNA sequences in public databases, we sought to determine the 28S and 18S rRNA sequences of a diverse set of Old and New World sylvatic mosquito species from four countries representing three continents: Cambodia, the Central African Republic, Madagascar, and French Guiana. These countries, due to their proximity to the equator, contain high mosquito biodiversity (Foley et al., 2007) and have had long histories of mosquito-borne virus circulation (Desdouits et al., 2015; Halstead, 2019; Héraud et al., 2022; Jacobi and Serie, 1972; Ratsitorahina et al., 2008; Saluzzo et al., 2017; Zeller et al., 2016). Increased and continued surveillance of local mosquito species could lead to valuable insights on mosquito virus biogeography. Using a unique score-based read filtration strategy to remove interfering non-mosquito rRNA reads for accurate de novo assembly, we produced a dataset of 234 novel full-length 28S and 18S rRNA sequences from 33 mosquito species, 30 of which have never been recorded before. We also explored the functionality of 28S and 18S rRNA sequences as molecular markers by comparing their performance to that of the mitochondrial cytochrome c oxidase subunit I (COI) gene for molecular taxonomic and phylogenetic investigations. The COI gene is the most widely used DNA marker for molecular species identification and forms the basis of the Barcode of Life Data System (BOLD) (Hebert et al., 2003; Ratnasingham and Hebert, 2007). Presently, full-length rRNA sequences are much less represented compared to other molecular markers. However, given the availability of relevant reference sequences, 28S and concatenated 28S+18S rRNA sequences can be the better approach for molecular taxonomy and phylogenetic studies. We hope that our sequence dataset, with its species diversity and eco-geographical breadth, and the assembly strategy we describe would further facilitate the use of rRNA as markers. In addition, this dataset enables the design of species-specific oligos for cost-effective rRNA depletion for a broader range of mosquito species and streamlined molecular species identification during RNA-seq. Results Poor rRNA depletion using a non-specific depletion method During library preparations of mosquito samples for RNA-seq, routinely used methods for depleting rRNA are commercial kits optimised for human or mice samples (Belda et al., 2019; Bishop-Lilly et al., 2010; Chandler et al., 2015; Kumar et al., 2012; Weedall et al., 2015; Zakrzewski et al., 2018) or through 80–100 base pair antisense probe hybridisation followed by ribonuclease digestion (Fauver et al., 2019; Phelps et al., 2021). In cases where the complete reference rRNA sequence of the target species is not known, oligos would be designed based on the rRNA sequence of the closest related species (25, this study). These methods should deplete reads from the conserved regions of rRNA sequences. However, reads from the variable regions remain at abundances high enough to compromise RNA-seq output. In our hands, we have found that using probes designed for the Ae. aegypti rRNA sequence followed by RNase H digestion according to the protocol published by Morlan et al., 2012, produced poor depletion in Aedes albopictus, and in Culicine and Anopheline species (Figure 1), in which between 46% and 94% of reads post-depletion were ribosomal. Additionally, the lack of full-length reference rRNA sequences compromises the in silico clean-up of remaining rRNA reads from sequencing data, as reads belonging to variable regions would not be removed. To solve this and to enable RNA-seq metagenomics on a broader range of mosquito species, we performed RNA-seq to generate reference rRNA sequences for 33 mosquito species representing 10 genera from Cambodia, the Central African Republic, Madagascar, and French Guiana. Most of these species are associated with vector activity for various pathogens in their respective ecologies (Table 1). In parallel, we sequenced the mitochondrial COI gene to perform molecular species identification of our samples and to comparatively evaluate the use of rRNA as a molecular marker (Figure 2). Figure 1 Download asset Open asset Percentage of rRNA reads in mosquito total RNA sequencing (RNA-seq) data after depletion using probes antisense to Aedes aegypti sequences. Pools of five individual mosquitoes from genera Aedes (Ae), Culex (Cx), Mansonia (Ma), and Anopheles (An) were ribodepleted by probe hybridisation followed by RNase H digestion according to the protocol by Morlan et al., 2012. Y-axis depicts percentages of remaining rRNA reads calculated as the number of rRNA reads over total reads per sample pool. Depletion efficiency decreases with taxonomic distance from Ae. aegypti underlining the need for reference sequences for species of interest. Table 1 Mosquito species represented in this study and their vector status. Mosquito taxonomy‡Origin*Collection site (ecosystem type)Vector for†ReferenceAedes (Fredardsius) vittatusCFRural (village)ZIKV, CHIKV, YFVDiallo et al., 2020Aedes (Ochlerotatus) scapularisGFRural (village)YFVVasconcelos et al., 2001Aedes (Ochlerotatus) serratusGFRural (village)YFV, OROVCardoso et al., 2010; Romero-Alvarez and Escobar, 2018Aedes (Stegomyia) aegyptiCFUrbanDENV, ZIKV, CHIKV, YFVKraemer et al., 2019Aedes (Stegomyia) albopictusCF, KHRural (village, nature reserve)DENV, ZIKV, CHIKV, YFV, JEVAuerswald et al., 2021; Kraemer et al., 2019Aedes (Stegomyia) simpsoniCFRural (village)YFVMukwaya et al., 2000Anopheles (Anopheles) baezaiKHRural (nature reserve)Unreported–Anopheles (Anopheles) coustaniMG, CFRural (village)RVFV, malariaMwangangi et al., 2013; Nepomichene et al., 2018; Ratovonjato et al., 2011Anopheles (Cellia) funestusMG, CFRural (village)ONNV, malariaLutomiah et al., 2013; Tabue et al., 2017Anopheles (Cellia) gambiaeMG, CFRural (village)ONNV, malariaBrault et al., 2004Anopheles (Cellia) squamosusMGRural (village)RVFV, malariaRatovonjato et al., 2011; Stevenson et al., 2016Coquillettidia (Rhynchotaenia) venezuelensisGFRural (village)OROVTravassos da Rosa et al., 2017Culex (Culex) antennatusMGRural (village)RVFVNepomichene et al., 2018; Ratovonjato et al., 2011Culex (Culex) duttoniCFRural (village)Unreported–Culex (Culex) neaveiMGRural (village)USUVNikolay et al., 2011Culex (Culex) orientalisKHRural (nature reserve)JEVKim et al., 2015Culex (Culex) perexiguusMGRural (village)WNV, USUVVezenegho et al., 2022Culex (Culex) pseudovishnuiKHRural (nature reserve)JEVAuerswald et al., 2021Culex (Culex) quinquefasciatusMG, CF, KHRural (village, nature reserve)ZIKV, JEV, WNV, DENV, SLEV, RVFV, Wuchereria bancroftiBhattacharya and Basu, 2016; Maquart et al., 2021; Ndiaye et al., 2016; Serra et al., 2016Culex (Culex) tritaeniorhynchusMG, KHRural (village, nature reserve)JEV, WNV, RVFVAuerswald et al., 2021; Hayes et al., 1980; Jupp et al., 2002Culex (Melanoconion) spissipesGFRural (village)VEEVWeaver et al., 2004Culex (Melanoconion) portesiGFRural (village)VEEV, TONVTalaga et al., 2021; Weaver et al., 2004Culex (Melanoconion) pedroiGFRural (village)EEEV, VEEV, MADVTalaga et al., 2021; Turell et al., 2008Culex (Oculeomyia) bitaeniorhynchusMG, KHRural (village, nature reserve)JEVAuerswald et al., 2021Culex (Oculeomyia) poicilipesMGRural (village)RVFVNdiaye et al., 2016Eretmapodites intermediusCFRural (village)Unreported–Limatus durhamiiGFRural (village)ZIKVBarrio-Nuevo et al., 2020Mansonia (Mansonia) titillansGFRural (village)VEEV, SLEVHoyos-López et al., 2015; Turell, 1999Mansonia (Mansonioides) indianaKHRural (nature reserve)JEVArunachalam et al., 2004Mansonia (Mansonioides) uniformisMG, CF, KHRural (village, nature reserve)RVFV, Wuchereria bancroftiLutomiah et al., 2013; Ughasi et al., 2012Mimomyia (Etorleptiomyia) mediolineataMGRural (village)Unreported–Psorophora (Janthinosoma) feroxGFRural (village)ROCVMitchell et al., 1986Uranotaenia (Uranotaenia) geometricaGFRural (village)Unreported– * Dengue virus, DENV; Zika virus, ZIKV; chikungunya virus, CHIKV; Yellow Fever virus, YFV; Oropouche virus, OROV; Japanese encephalitis virus, JEV; Rift Valley Fever virus, RVFV; O’Nyong Nyong virus, ONNV; Usutu virus, USUV; West Nile virus, WNV; St Louis encephalitis virus, SLEV; Venezuelan equine encephalitis virus, VEEV; Tonate virus, TONV; Eastern equine encephalitis virus, EEEV; Madariaga virus, MADV; Rocio virus, ROCV. † Origin countries are listed as their ISO alpha-2 codes: Central African Republic, CF; Cambodia, KH; Madagascar, MG; French Guiana, GF. ‡ Subgenus indicated in brackets. Figure 2 Download asset Open asset Novel mosquito rRNA sequences were obtained using a unique reads filtering method. (A) Schematic of sequencing and bioinformatics analyses performed in this study to obtain full-length 18S and 28S rRNA sequences as well as cytochrome c oxidase I (COI) DNA sequences. Nucleic acids were isolated from mosquito specimens for next-generation (for rRNA) or Sanger (for COI) sequencing. Two in-house libraries were created from the SILVA rRNA gene database: Insecta and Non-Insecta, which comprises 8,585 sequences and 558,185 sequences, respectively. Following BLASTn analyses against these two libraries, each RNA-sequencing (RNA-seq) read is assigned a ratio of BLASTn scores to describe their relative nucleotide similarity to insect rRNA sequences. Based on these ratios of scores, RNA-seq reads can then be filtered to remove non-mosquito reads prior to assembly with SPAdes to give full-length 18S and 28S rRNA sequences. Image created with https://biorender.com/. (B) Based on their ratio of scores, reads can be segregated into four categories, as shown on this ratio of scores versus number of reads plot for the representative specimen ‘CF S27’: (i) reads with hits only in the Insecta library (shaded in green), (ii) reads with a higher score against the Insecta library (shaded in blue), (iii) reads with a higher score against the Non-Insecta library (shaded in yellow), and (iv) reads with no hits in the Insecta library (shaded in red). We applied a conservative threshold at 0.8, indicated by the black horizontal line, where only reads above this threshold are used in the assembly with SPAdes. For this given specimen, 175,671 reads (96.3% of total reads) passed the ≥0.8 cut-off, 325 reads (0.18% of total reads) had ratios of scores <0.8, while 6,423 reads (3.52%) did not have hits against the Insecta library. rRNA reads filtering and sequence assembly Assembling Illumina reads to reconstruct rRNA sequences from total mosquito RNA is not a straightforward task. Apart from host rRNA, total RNA samples also contain rRNA from other organisms associated with the host (microbiota, external parasites, or ingested diet). As rRNA sequences share high homology in conserved regions, Illumina reads (150 bp) from non-host rRNA can interfere with the contig assembly of host 28S and 18S rRNA. Our score-based filtration strategy, described in detail in the Materials and methods section, allowed us to bioinformatically remove interfering rRNA reads and achieve successful de novo assembly of 28S and 18S rRNA sequences for all our specimens. Briefly, for each Illumina read, we computed a ratio of BLAST scores against an Insecta library over scores against a Non-Insecta library (Figure 2A). Based on their ratio of scores, reads could be segregated into four categories (Figure 2B): (i) reads mapping only to the Insecta library, (ii) reads mapping better to the Insecta relative to Non-Insecta library, (iii) reads mapping better to the Non-Insecta relative to the Insecta library, and (iv) reads mapping only to the Non-Insecta library. By applying a conservative threshold at 0.8 to account for the non-exhaustiveness of the SILVA database, we removed reads that likely do not originate from mosquito rRNA. Notably, 15 of our specimens were engorged with vertebrate blood, a rich source of non-mosquito rRNA (Appendix 1—table 1). The successful assembly of complete 28S and 18S rRNA sequences for these specimens demonstrates that this strategy performs as expected even with high amounts of non-host rRNA reads. This is particularly important in studies on field-captured mosquitoes as females are often sampled already having imbibed a blood meal or captured using the human landing catch technique. We encountered challenges for three specimens morphologically identified as Mansonia africana (Specimen ID S33–S35) (Appendix 1—table 1). COI amplification by PCR did not produce any product, hence COI sequencing could not be used to confirm species identity. In addition, the genome assembler SPAdes (Bankevich et al., 2012) was only able to assemble partial length rRNA contigs, despite the high number of reads with high scores against the Insecta library. Among other Mansonia specimens, these partial length contigs shared the highest similarity with contigs obtained from sample ‘Ma uniformis CF S51’. We then performed a guided assembly using the 28S and 18S sequences of this specimen as references, which successfully produced full-length contigs. In two of these specimens (Specimen ID S34 and S35), our assembly initially produced two sets of 28S and 18S rRNA sequences, one of which was similar to mosquito rRNA with low coverage and another with 10-fold higher coverage and 95% nucleotide sequence similarity to a water mite of genus Horreolanus known to parasitize mosquitoes. Our success in obtaining rRNA sequences for mosquito and water mite shows that our strategy can be applied to metabarcoding studies where the input material comprises multiple insect species, provided that appropriate reference sequences of the target species or of a close relative are available. Altogether, we were able to assemble 122 28S and 114 18S full-length rRNA sequences for 33 mosquito species representing 10 genera sampled from four countries across three continents. This dataset contains, to our knowledge, the first records for 30 mosquito species and for seven genera: Coquillettidia, Mansonia, Limatus, Mimomyia, Uranotaenia, Psorophora, and Eretmapodites. Individual GenBank accession numbers for these sequences and specimen information are listed in Appendix 1—table 1. Comparative phylogeny of novel rRNA sequences relative to existing records To verify the assembly accuracy of our rRNA sequences, we constructed a comprehensive phylogenetic tree from the full-length 28S rRNA sequences from our study and relevant rRNA sequences from GenBank (Figure We applied a for GenBank sequences with at least 95% coverage of our sequence to as many species or genera as Although we found records for the species in our the resulting tree that our 28S sequences according to their respective species and by to at with the of and 28S rRNA sequences a with related sequences from Anopheles Anopheles and Anopheles high homology for or other members of (Figure in 28S rRNA sequences a to sister species Culex (Figure in Figure with 2 all Download asset Open asset 28S sequences from this study with or from existing GenBank phylogenetic tree based on full-length 28S sequences bp) from this study and from GenBank was using the method and constructed to in et al., 2018) using an Horreolanus species found among our samples as an at each from from GenBank are with and their accession numbers are For sequences from this each specimen information on and specimen ID specimens produced to two 28S this is indicated by the numbers 1 or 2 at the of the specimen genera are indicated by Culex in Anopheles in Aedes in Mansonia in in in in in in in and in at is Figure data 1 sequence of 28S rRNA sequences from this study and from GenBank Download 28S rRNA phylogenetic (Figure with GenBank Figure this study to that of 18S rRNA sequences (Figure 2). Although all rRNA trees show the of into in and other the phylogenetic relationships between the 28S and 18S rRNA trees and are The 18S rRNA tree also several taxonomic (i) the lack of by species the Culex (ii) the lack of between 18S rRNA sequences of and (iii) the of CF a Culex and (iv) the lack of a (Figure 2). However, 28S and 18S rRNA sequences are by in and should not be when concatenated 28S+18S rRNA sequences were from the specimens (Figure the phylogenetic tree resulting from these sequences more the 28S tree (Figure with to the of the the with in tree in 28S rRNA in concatenated 28S+18S rRNA For were higher in the concatenated tree compared to the 28S the 28S+18S rRNA tree an from genera yellow), Aedes blue), and driven by the inclusion of 18S rRNA sequences. also the found in the 18S rRNA tree and to the close between Culex and Mansonia relative to the 28S tree (Figure the Culex and Mansonia genera are no in the concatenated 28S+18S rRNA tree (Figure Culex is with to of genus Mansonia (Figure and which we to be Mansonia a distinct in 28S or 18S rRNA thus representing a of Figure with 2 all Download asset Open asset 28S and 18S rRNA sequences phylogenetic relationships that are with with higher 28S sequences This phylogenetic tree based on concatenated 28S+18S rRNA sequences bp) from this study was using the method and constructed to using et al., 2018) using an Horreolanus species found among our samples as an at each from specimen information on indicated in and specimen ID specimens produced to two 28S+18S rRNA this is indicated by the numbers 1 or 2 at the of the specimen genera are indicated by Culex in Anopheles in Aedes in Mansonia in in in in in in and in at is Figure data 1 sequence of 122 28S rRNA sequences, two sequences from Horreolanus Download Figure data 2 sequence of 114 18S rRNA sequences, two sequences from Horreolanus Download The concatenated 28S+18S rRNA tree (Figure is known the of our specimens, (i) the of from (ii) the of genus Anopheles into two Anopheles and (iii) the of genus Aedes into and (iv) the of the the Culex genus and 2016). rRNA as a molecular marker for taxonomy and phylogeny We sequenced a of the COI gene to confirm morphological species identification of our specimens and to compare the functionality of rRNA and COI sequences as molecular markers for taxonomic and phylogenetic investigations. COI sequences were able to determine the species in most specimens for the COI sequences from our of specimen shared high nucleotide similarity with several other Anopheles species such as the most and closest In the case of Ae. three specimens had been morphologically identified as Ae. their COI sequences similarity to that of Ae. As GenBank no records of Ae. COI at the of this we aligned the Ae. COI sequences against two sister species of Ae. Ae. and Ae. We found they shared only and respectively. Given this significant we these specimens to be Ae. were especially among Culex specimens belonging to the or where the sequence with of the top two hits by a For between and of the and between and of the Among our three specimens of two to to a species that is different from related to We that these specimens could be based on morphological similarity were not able to verify this by molecular as no COI reference sequence is for this species. These specimens are hence as ‘Ma

A score-based read selection strategy enables the assembly of novel full-length ribosomal RNA sequences for mosquitoes, which improves the physical and computational removal of interfering ribosomal RNA reads in RNA-seq and provides another molecular marker for taxonomic and phylogenetic inquiries.

A score-based read selection strategy enables the assembly of novel full-length ribosomal RNA sequences for mosquitoes, which improves the physical and computational removal of interfering ribosomal RNA reads in RNA-seq and provides another molecular marker for taxonomic and phylogenetic inquiries.

Conventionally, the initialization of new prototypes and concepts in laboratory automation and life science software applications has required close collaboration between hardware and software experts, as well as lab personnel such as biologists, chemists, biotechnologists, or process engineers. This setup - still common today - often means that the ideas and requests of lab personnel must be translated into software applications by software developers, which frequently results in long development times. Low-Code Development Platforms (LCDPs) seek to address this challenge by providing a way to accelerate application development by reducing dependence on traditional software development methods, empowering lab personnel to build applications without writing extensive amount of code. By offering a visual, drag-and-drop interface, lab personnel can actively participate in the software development process. This helps democratize application creation and can lead to the quick setup of software solutions tailored to laboratory needs. This study demonstrates the implementation of four different use cases in a bioprocessing laboratory environment using an open-source LCDP and commercially available upstream and downstream equipment. The LCDP facilitated the integration and control of different device types with varying communication protocols also enabling dashboarding, monitoring and data processing capabilities. This methodology highlights the suitability of LCDPs for rapidly prototyping and evaluating laboratory and bioprocess automation pipelines, potentially expediting the development of biotechnological production processes and products. All developed components are made available through a publicly accessible repository, facilitating reuse and further development by the scientific community.

Antibacterial effects of cinnamon (Cinnamomum zeylanicum) bark essential oil on Porphyromonas gingivalis

ABSTRACT: The consumption of food contaminated with bacteria or their toxins can result in foodborne infections or illnesses. This study investigated the antibacterial and antibiofilm activity of essential oils against bacteria isolated from milk samples collected in Pune, Maharashtra, India. Twenty-five raw milk samples were collected from local vendors and examined for the presence of biofilm-forming microorganisms. The isolated bacteria were identified and characterized using morphological and biochemical tests, revealing that 20% of the samples were contaminated with Acinetobacter spp. and 28% with E. coli. The antibacterial activity of the essential oils was evaluated using the disc diffusion method, and the minimum inhibitory concentration (MIC) was determined using 96-well plates. The minimum bactericidal concentration (MBC) was also assessed by inoculating assay mixtures from wells exhibiting no microbial growth onto sterile nutrient agar medium. Biofilm formation and disruption were evaluated using crystal violet assay and biofilm disruption assay, respectively. The results demonstrated that the Cinnamon Bark and Oregano essential oils exhibited significant antibacterial and antibiofilm activity at the lowest MIC value of 0.02-0.04 μg/ml against the isolated bacteria. The findings suggest that essential oils could be potential natural alternatives to conventional antibiotics for controlling bacterial contamination and biofilm formation in milk and dairy products. Further research is needed to explore the practical applications of essential oils in the dairy industry and to ensure their safety and efficacy as natural antimicrobial agents.

Dear Editor Coryneform bacteria are gram-positive, catalase-positive, usually non-motile, glucose fermentative or non-fermentative, aerobic or facultatively anaerobic rods [1]. Several pathogenic Corynebacterium spp. have been identified, and nosocomial Corynebacterium infection has garnered substantial attention [2,3]. For example, 281 cases of Corynebacterium infections associated with prosthetic devices, including central venous catheters (CVCs), have been reported [4]. Here, we present the first case in Korea of CVC-related bloodstream infection by C. striatum identified by 16S rRNA and rpoB gene sequencing. A 51-yr-old man was admitted to our hospital with altered mental status. He had a history of cerebral infarction resulting from hypertension and diabetes mellitus. Initial complete blood count revealed: WBC count, 10.3×109/L; hemoglobin level, 11.6 g/dL; and platelet count, 692×109/L. C-reactive protein (CRP) level was 4.15 mg/dL. He became confused and developed recurrent seizures. Under the diagnosis of status epilepticus, he was treated with infusions of carbamazepine, levetiracetam, and quetiapine. A CVC was inserted into his jugular vein to ensure venous access. Since rashes were observed around the CVC and the patient developed a fever (37.5℃) at day 17, the CVC was changed. Cultures from blood and CVC tip (collected simultaneously) yielded no isolates. The patient's white blood cell count decreased to 0.8×109/L, and absolute neutrophil count was <0.1×109/L. Owing to potential drug-induced neutropenia, he was placed in reverse isolation and all drugs were stopped. Three days later, his temperature and CRP level increased to 39.0℃ and 14.74 mg/dL, respectively. Three sets of blood cultures, including one from the CVC, yielded gram-positive bacilli identified as Corynebacterium spp. on days 36 and 38 (Fig. 1). Cultures repeated two days later yielded coryneforms, identified as C. striatum by API Coryne (bioMerieux, Durham, NC, USA). The isolates were positive for nitrate/nitrite reduction, pyrazinamidase, catalase, and glucose and sucrose fermentation at 42℃, and were negative for the Christie, Atkins, and Munch-Peterson test and urease, maltose and galactose fermentation. They were non-motile and exhibited growth at 20℃. Similar results were obtained at day 40 as well. Fig. 1 Peripheral blood culture on blood agar showing pure isolates as white, opaque, and smooth-edged colonies. Although empirical treatment for neutropenic fever, ceftriaxone and meropenem, was administered, patient showed no change in temperature and CRP levels. The CVC via left subclavian approach was exchanged for one via right subclavian approach at day 42. Blood and catheter tip cultures (sampled at the time of exchange) yielded Corynebacterium spp. (>15 colony forming unit [CFU]). Susceptibility of the isolates to ciprofloxacin, clindamycin, erythromycin, linezolid, cefoxitin, penicillin, rifampin, trimethoprim/sulfamethoxazole, teicoplanin, and vancomycin was tested by the disk diffusion method on 5% sheep blood agar plates. Results were interpreted by using the criteria for staphylococci. As C. striatum lacks approved breakpoints for ciprofloxacin, penicillin, and vancomycin, these results were interpreted using the British Society for Antimicrobial Chemotherapy (BSAC) guidelines [5]. The strains were susceptible to linezolid, rifampin, teicoplanin, and vancomycin and resistant to ciprofloxacin, clindamycin, erythromycin, cefoxitin, penicillin, and trimethoprim/sulfamethoxazole. For more robust phenotypic data and species identification, the 16S rRNA gene of the first isolates was sequenced by capillary sequencing (Macrogen Inc., Seoul, Korea) using the primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5'-TACCAGGGTATCTAATCC-3'). The sequence showed 99.86% similarity to that of C. striatum (strain: BRRJ 1954, GenBank: {type:entrez-nucleotide,attrs:{text:JF342700.1,term_id:327239535,term_text:JF342700.1}}JF342700.1). Since a prior study reported that 16S rRNA sequences are not polymorphic enough for phylogenetic studies among all Corynebacterium species, rpoB was further sequenced by using the primers C2700F (5'-CGWATGAACATYGGBCAGGT-3') and C3130R (5'-TCCATYTCRCCRAARCGCTG-3') [6]; it showed 99.49% similarity with C. striatum sequence, thereby confirming the identification. The antibiotic regimen was changed to vancomycin, which decreased the fever. Bacterial growth was not observed in subsequent cultures. The patient had fever and elevated CRP levels with no apparent source of bloodstream infection except for the catheter. The same species was isolated from peripheral venous blood and CVC tip. Blood cultures subsequent to the CVC removal did not show bacterial growth. Additionally, all isolates were susceptible to vancomycin, which effectively managed the symptoms. Our finding is consistent with the previous report on coryneform bacteria being susceptible to vancomycin [7]. Vancomycin should therefore be considered the first line of treatment for CVC-related bloodstream infections. C. striatum can cause bloodstream infections in immunocompromised patients, especially in those implanted with medical devices such as intravenous catheters. Catheters are a major route by which natural skin flora, such as Corynebacterium, enter the bloodstream. Appropriate guidelines are needed for management of corynebacterial catheter-related bloodstream infection. This is the first report of catheter-related bloodstream infection with C. striatum in Korea identified by repeated blood culture and confirmed by 16S rRNA and rpoB gene sequencing.

Study question Can quantitative PCR (qPCR) serve as a reliable, cost-efficient alternative to sequencing-based methods for accurately analysing microbial composition and relative abundance in the vagina? Summary answer qPCR outperformed sequencing in species detection and quantification, demonstrating high accuracy and providing a reliable alternative for vaginal microbiome analysis at a fraction of cost. What is known already The vaginal microbiome is increasingly recognised as a key factor in reproductive health, with emerging research linking its imbalance to ART failure and adverse pregnancy outcomes. This has driven efforts to characterise its composition and role in fertility. 16S rRNA and whole-genome sequencing are the gold standard for microbiome analysis but are very costly, labour-intensive, and require extensive computational analysis, limiting feasibility for large-scale studies and clinical application. As only a limited number of species are known to inhabit the vagina, sequencing may not always be necessary, and qPCR may serve as a cost-effective and rapid alternative for microbiome analysis. Study design, size, duration First, a qPCR assay was developed for vaginal microbiome profiling, with target species selected through literature review. Accuracy in species detection and quantification was assessed using 10 artificial genomic DNA mixtures (3-6 species per mixture; mean: 3.9) with known bacterial DNA proportions (1–88%), representing 39 individual species measurements. qPCR results were compared with 16S rRNA and whole-genome sequencing. Subsequently, all three methods were applied to vaginal swabs from 56 ART patients, to compare their performance. Participants/materials, setting, methods qPCR employed specific assays targeting 22 bacterial species, including commensal Lactobacillus spp. and dysbiosis-associated species linked to bacterial vaginosis and aerobic vaginitis. Additionally, four pan-genus assays covering the most common vaginal genera (Lactobacillus, Bifidobacterium, Streptococcus and Gardnerella) ensured a comprehensive microbiome assessment. 16S rRNA sequencing analysed all nine hypervariable regions, unlike conventional approaches targeting selected regions, enabling a comparison of sequencing variability. Whole-genome sequencing captured entire microbial genomes, allowing for high-resolution species identification and quantification. Main results and the role of chance For artificial gDNA mixtures, qPCR demonstrated high accuracy in species detection and quantification. Results closely matched theoretical values, showing a strong correlation (r = 0.98, p &amp;lt; 0.0001) and minimal deviation (2.1%±2.0%) across 39 species measurements, confirming its reliability in reflecting known bacterial DNA proportions.16S rRNA sequencing showed lower accuracy in species-level detection, with relative quantification also being unreliable and varying widely across hypervariable regions (r = 0.43-0.79, p &amp;lt; 0.0001). It frequently misclassified both beneficial Lactobacillus spp. and dysbiotic species. Additionally, species resolution depended on the hypervariable region analysed, often failing to distinguish closely related species and sometimes lacking genus-level classification. On average, 23.0%±9.1% of sequencing output (range: 14.2-34.6%) was either misclassified or unassignable at the species level. Whole-genome sequencing was more reliable than 16S rRNA sequencing for species identification, providing improved species-level resolution. However, 23.4%±22.9% of sequencing reads remained unassigned at species level. Additionally, species quantification accuracy was only moderate (r = 0.53, p &amp;lt; 0.0001).In vaginal swabs from ART patients, 16S sequencing showed substantial variability between hypervariable regions, particularly at the species level. Such misclassification is problematic, as bacterial species, not just genera, impact vaginal health and ART success. qPCR and whole-genome sequencing showed greater concordance, with whole-genome sequencing detecting few additional species, though not at clinically relevant proportions. Limitations, reasons for caution qPCR can only detect preselected species, potentially missing novel or unexpected microbes that sequencing might identify. Additionally, its targeted nature may overlook low-abundance species. However, given the well-defined composition of the vaginal microbiome, these limitations are unlikely to significantly affect its effectiveness for clinical and research use. Wider implications of the findings Besides its accuracy in vaginal microbiome analysis, qPCR costs three to four times less than sequencing and avoids complex bioinformatics and high-performance computing. With a 4-hour processing time versus 40–50 hours for sequencing, its cost-effectiveness and speed make it ideal for large-scale vaginal microbiome studies and clinical use. Trial registration number No

In recent years, enterococci have emerged as important pathogens. These organisms are now the third most commonly encountered nosocomial bloodstream infection pathogens in many part of the world. Due to increasing frequency with which multi-drug resistant enterococci are isolated from clinical specimens, there is a need for rapid reporting of results of identification tests and tests for susceptibility testing to antimicrobial agents. In 1999, we published an update of regarding the identification of the different species of enterococci as well as the recommendations for accurately detecting resistance among these isolates. In the current report, we will discuss additional tests for identification of enterococcal species as well as, the current recommendations for susceptibility testing

During the last 10 years, the introduction of PCR-based DNA typing technologies in forensic applications has been highly successful. This technology has become pervasive throughout forensic laboratories and it continues to grow in prevalence. For many criminal cases, it provides the most probative evidence. Criminal genotype data banking and victim identification initiatives that follow mass-fatality incidents have benefited the most from the introduction of automation for sample processing and data analysis. Attributes of offender specimens including large numbers, high quality and identical collection and processing are ideal for the application of laboratory automation. The magnitude of kinship analysis required by mass-fatality incidents necessitates the application of computing solutions to automate the task. More recently, the development activities of many forensic laboratories are focused on leveraging experience from these two applications to casework sample processing. The trend toward increased prevalence of forensic genetic analysis will continue to drive additional innovations in high-throughput laboratory automation and information systems.

Hospital-acquired infections are caused by multidrug-resistant Enterococcus (E.) species, especially E. faecalis and E. faecium, which are zoonotic bacteria and pose a significant public health challenge. This study aimed to identify multidrug-resistant Enterococcus isolates and evaluate the antibacterial activity of Rosmarinus officinalis (R. officinalis) extract against E. faecalis and E. faecium. Fifty urine and stool samples were collected from hospitalized patients. Four multidrug-resistant strains—E. faecalis 6E, 7B, 10D, and E. faecium 15E—were identified by polymerase chain reaction (PCR) with universal primers (8F and 1492R) targeting the 16S rDNA gene to confirm their identity as Enterococcus species. The antibacterial efficacy of R. officinalis extract was assessed using disk diffusion and minimum inhibitory concentration (MIC) determination. High-Performance Liquid Chromatography (HPLC) analysis was conducted to identify phenolic bioactive compounds in R. officinalis. The extract demonstrated significant antibacterial activity, with MICs of 25 mg/mL and 30 mg/mL for E. faecalis and E. faecium, respectively. HPLC revealed phenolic compounds such as rosmarinic acid, caffeic acid, and ferulic acid, likely contributing to the antimicrobial properties. The R. officinalis extract could be a promising natural antimicrobial agent against multidrug-resistant Enterococcus species. Compounds such as rosmarinic acid, caffeic acid, ferulic acid, apigenin-7-glucoside, syringic acid, and p-hydroxybenzoic acid show potential as safer, environmentally friendly alternatives to traditional antibiotics, aiding in the fight against rising antibiotic resistance.

Comparing four mitochondrial genes in earthworms – Implications for identification, phylogenetics, and discovery of cryptic species

Enterococci were frequently considered to be commensal organism and were ignored when isolated in clinical laboratory. But recently due to its capability of causing variety of infections, especially in hospitalized patients and difference in antimicrobial sensitivity of each species to varying antibiotics the present study wasundertaken with a view at characterizing the clinical isolates of enterococcus, recovered from the cases of urinary tract infections (UTI). A total of 59 Enterococci were isolated from UTI patient for a period of 1 year. In the present study it was the 3rd leading organism causing UTI. They were speciated by an identification system based on the phenotypic characteristics of enterococcus species and their antimicrobial sensitivity pattern was determined. Most of the isolates were E.faecalis 42(71.18%) followed by E.faecium accounted for 10(16.94%),E.avium 1(2.43%), E.raff 1(2.43%) and 5(8.47%) remained unidentified. E.faecium showed increased resistance to amoxicillin (90%), co-trimoxazole (80%), ciprofloxacin (70%), gentamycin (80%), ceftriaxone (90%), and cefuroxime (80%). Most of the E.faecalis was resistant to amoxicillin (66.66%), cotrimoxazole (71.42%), ciprofloxacin (76.19%), gentamycin (71.42%), ceftriaxone (64.28%) and cefuroxime (80.95%). Multidrug-resistant enterococci are emerging as a leading nosocomial uropathogen. Identification of species along with knowledge of the antimicrobial resistance profile may ultimately contribute to development of strategies for prevention and to formulate treatment guidelines for infections caused by enterococci.Bangladesh J Med Microbiol 2014; 08 (01): 16-20

Isolation, characterization, and application of a lytic bacteriophage SSP49 to control Staphylococcus aureus contamination on baby spinach leaves