Acanthamoeba act as hosts for various microorganisms and pathogens, causing Acanthamoeba Keratitis (AK). To investigate the association between endosymbionts and AK progression, we performed a metagenomics study to characterize the intracellular microbiome from five lenses associated with AK isolates and standard strains to characterize the role of ocular flora in AK progression. The used clinical isolates were axenic cultured from lenses associated with AK patients. AK isolates and standard controls such as 16S ribosomal RNA sequencing techniques were used for analysis. The microbiome compositions and relative abundance values were compared. The orders of Clostridiales and Bacteroidales presented major populations of intracellular microbes belonging to all isolates. Comparison of the different source isolates showed that most of the abundance in keratitis isolates came from Ruminococcus gnavus (121.0 folds), Eubacterium dolichum (54.15 folds), Roseburia faecis (24.51 folds), and Blautia producta (3.15 folds). Further analysis of the relative abundance data from keratitis isolates showed that Blautia producta was positively correlated with the disease course. In contrast, Bacteroides ovatus was found to be abundant in early-stage keratitis isolates. This study reveals the abundant anaerobic Gram-positive rods present in severe keratitis isolate and characterize the association between Acanthamoeba and ocular flora in AK progression. Acanthamoeba is a free-living amoebae (FLA) that can be found in moist environments, such as in tap water and human nasopharyngeal mucosa [Acanthamoeba castellanii and Acanthamoeba polyphaga. Diseases in Acanthamebiasis contain Acanthamoeba keratitis (AK) and Granulomatous amoebic encephalitis (GAE). The protozoa obtain food sources from surrounding microorganisms via phagocytosis. Nevertheless, several bacteria can escape and latently inhabit amebae, including ocular surface flora.l mucosa ,2. The mAcanthamoeba characteristics of phagocytosis alter the surrounding microorganism distribution, thereby causing complications [Acanthamoeba can host and protect microorganisms, including pathogenic bacteria, fungi, and viruses [Acanthamoeba, these microbes could be protected from harmful environments, such as ocular immune killing [Pseudomonas aeruginosa is an important endosymbiont of Acanthamoeba and that it usually infects concurrently with AK [Mimivirus can acquire genes from the ameba host [Acanthamoeba in microorganism evolution. In addition, several studies have indicated that endosymbionts can enhance the pathogenicity of Acanthamoeba [Acanthamoeba [Acanthamoeba clinical isolates might be a potential entry point for a pathogenicity differentiation between Acanthamoeba isolates.The ocular surface harbors a variety of microbes due to continuous exposure to the environment . The ocuications ,6. Acant viruses . However killing . Previou with AK . Some paeba host . These geba host , suggestthamoeba ,13. In vthamoeba ,15. TherAcanthamoeba, which could lead to depletion of probiotics. Nevertheless, some studies have analyzed the microbiota in CLs users [Acanthamoeba involvement is less reported. As most AK isolates are related to CLs use [Most AK patients who have been wearing contact lenses (CLs) over a long period have a high risk of contracting AK. According to previous studies, the long-term wearing of CLs, not only affects the eye’s health but also interferes with the ocular surface microbiota distribution through oxygen deprivation ,17. ReceLs users ,21, wher CLs use ,23, inveAcanthamoeba and the intracellular microbiome in AK progression in a hypoxic environment.In this study, bacterial 16S ribosomal RNA sequencing techniques were performed to profile the intracellular microbiome of phagocytic microbes in various CL-associated AK isolates in order to characterize the association between Acanthamoeba spp., we collected five clinical and two standard isolates. All isolates were cultured until normalization. The clinical isolates belonged to Acanthamoeba castellanii strain T4 and contained axenic culture from Acanthamoeba keratitis (AK) patients, who were assessed at Cheng Kung University Hospital, Taiwan. All clinical isolates were collected from patients who had lenses as a risk factor [To determine the intracellular microbiome in the k factor . StandarAcanthamoeba spp. DNA was extracted from the axenic cultures and the bacterial specific 16S primer pairs were used to pre-examine the endosymbionts using RT-PCR. No contamination was detected in this study after the screening of isolate ATCC-50492 in cultured medium and NCKUH-B using PCR detection , Eubacterium dolichum (54.15 folds), Roseburia faecis (24.51 folds), and Blautia producta (3.15 folds) than those from soil sources. Although Akkermansia muciniphila was abundant in soil-source ameba (11.08 folds), the relative abundance was not different in the genus validation. These abundant intracellular microbes were also presented in genera of Ruminococcus (38.79 folds), Eubacterium (27.27 folds), Roseburia (19.16 folds), and Blautia (27.01 folds) during the validated comparison (Blautia producta (3.81 folds). This significant difference was also observed during the genus validation of Blautia (19.15 folds). The early-stage keratitis isolates harbored abundant microbes of Bacteroides ovatus (2.60 folds), as proven by genus validation (8.55 folds) (To investigate the microbiome diversity with mparison A. Furthe5 folds) B.Acanthamoeba hosts harbor various intracellular microbes, which have been known for a long time [Ruminococcus and Blautia. Of a total of seven Acanthamoeba isolates, six were obtained from lens-associated AK patients and one from a soil source which was considered as a non-disease-related strain. Of the five clinical isolates, three  were isolated from patients who received medically successful AK treatment, whereas two (NCKUH-D and H) were from patients who finally had surgery owing to poor progression of AK. The intracellular microbiome content conversion profiles in different groups showed no obvious differences. However, the intracellular microbe relative abundance profiles from gene expression databases showed interesting diversity. The clinical isolates from lens-associated keratitis had a greater abundance of intracellular microbes of R. gnavus, E. dolichum, R. faecis, and B. producta than non-disease related strains. In contrast, the NCKUH-D and H isolate from medical failure cases had more B. producta and less B. ovatus in comparison with medical success isolates. Hence, the intracellular microbe relative abundance data suggested that the anaerobic Gram-positive bacillus Blautia producta might play roles in Acanthamoeba spp. pathogenicity in lens-wearing patients.Free-living ong time . In the B. producta was described in 1941 [B. ovatus was first reported in 1933 [B. producta is Gram-positive and B. ovatus is Gram-negative. According to previous studies, human ocular microbiota was characterized to be commonly identified in Gram-positive bacteria [Acanthamoeba apparently prefer Gram-negative bacteria [P. aeruginosa and S. marcescens [S. epidermidis [Acanthamoeba. Therefore, the early-stage keratitis isolates harbored abundant Gram-negative bacteria, with B. ovatus suspected as the early food source during initial infection. Notably, the relative abundance of B. ovatus was significantly higher in patients with atopic dermatitis as an immune disorder [B. producta had demonstrated prevention of Vancomycin-resistant Enterococcus (VRE) colonization [Acanthamoeba ingestion of bacteria, which might lead to secondary inflammation or infection due to ocular surface dysbiosis. in 1941  and is c in 1941 . B. ovat in 1933  and prov in 1933 . Howeverbacteria , while Abacteria . Previourcescens , but notdermidis . It was disorder , whereasnization . These rB. producta and B. ovatus are anaerobic microbes, which were not the major populations of ocular microbiota. This was probably due to vigorous reproduction on the ocular surface, which made a hypoxic environment, leading to lens obstruction. Although several diseases can lead to ocular hypoxia, including diabetes, herpes simplex virus infection, dry eye syndrome, and limbal stem cell deficiency [Acanthamoeba pathogenicity, virulence, or susceptibility to drugs [Staphylococci and Streptococcus, can stimulate ocular immunity as a natural barrier on the corneal surface [Acanthamoeba isolates were consider as high pathogenicity. Therefore, the analysis of endosymbionts in Acanthamoeba might be beneficial for AK patient prognosis.Additionally, both ficiency , lens usficiency . Previouto drugs . However surface . Under hAcanthamoeba intracellular microbes will be necessary and may provide more information for the differential diagnostic and prognostic evaluation of AK.In conclusion, these findings indicate that the types of intracellular microbes were associated with AK progression, while the abundant anaerobic bacteria in the keratitis isolates were associated with CLs wearing, suggesting hypoxia might correlate to AK induction. Further investigation of Acanthamoeba castellanii which were isolated from soil and human cornea with keratitis and purchased from ATCC . The clinical isolates were collected from the corneal ulcers of patients who were diagnosed with AK in the Cheng Kung University Hospital [Acanthamoeba isolates were cultured in protease peptone-yeast extract-glucose (PYG) medium (pH 6.5) at 28 °C in cell culture flasks and maintained after Page’s modified Neff’s amoeba saline  washing.The standard isolates ATCC-30010 and ATCC-50492 were Hospital  and are A. castellanii, protozoan cells were grown in axenic culture and subsequently harvested. The cell pellets were suspended in PAS for genome extraction. DNA extraction was performed using a LabPrep DNA Mini kit®  according to the protocol described in the package insert.To identify the endosymbionts in Acanthamoeba were analyzed by polymerase chain reaction (PCR) . To tested intracellular symbionts presenting, all the extracted DNA of isolates were analyzed by PCR as mentioned above. With approximately 550 bp PCR product, as confirmed by gel electrophoresis, the DNA samples were further subjected to sample preparation for metagenomics sequencing. DNA samples were prepared according to the 16S Metagenomics Sequencing Library Preparation instructions . The prepared amplicons were sequenced on a MiSeq instrument  using a 600-cycle sequencing reagent and with the specified paired-end mode.The enriched DNA samples were then subjected to PCR reactions with specified forward (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCA G) and reverse (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAC TACHVG GGTATCTAATCC) primers  to ampliThe generated Next-generation sequencing (NGS) data were analyzed with the Microbial Genomics Module of CLC Genomics Workbench 9.5.4 . As shown in The comparison of intracellular bacteria gene abundance was analyzed from the NGS database. The analyzed targets were classified into keratitis, soil, early-stage AK, and late-stage AK. Keratitis includes ATCC-50492, NCKUH-A, -B, -C, -D, and –H; soil includes ATCC-30010; early-stage AK includes NCKUH-A, -B, and –C; late-stage AK includes NCKUH-D and –H. To characterize the intracellular microbiome relative abundance ratio, gene expression fold change of keratitis was divided by soil and late-stage AK was divided by early-stage AK. The significant bacteria showed consistent abundance in the comparison within family, genus, and species.


Introduction: O'Donnell-Luria-Rodan syndrome was recently identified as an autosomal dominant systemic disorder caused by variants in KMT2E. It is characterized by global developmental delay, some patients also exhibit autism, seizures, hypotonia, and/or feeding difficulties.Methods: Whole-exome sequencing of family trios were performed for two independent children with unexplained recurrent seizures and developmental delay. Both cases were identified as having de novo variants in KMT2E. We also collected and summarized the clinical data and diagnosed them with O'Donnell-Luria-Rodan syndrome. Structural-prediction programs were used to draw the variants' locations.Results: A 186 G>A synonymous variant [NM_182931.3:exon4: c.186G>A (p.Ala62=)] was found in one family, resulting in alternative splicing acid. A 5417 C>T transition variant [NM_182931.3:exon27: c.5417C>T (p.Pro1806Leu)] was found in another family, resulting in 1806 Pro-to-Leu substitution. Both variants were classified as likely pathogenic according to the ACMG  guidelines and verified by Sanger sequencing.Conclusion: To date, three studies of O'Donnell-Luria-Rodan syndrome have been reported with heterogeneous clinical manifestations. As a newly recognized inherited systemic disorder, O'Donnell-Luria-Rodan syndrome needs to be paid more attention, especially in gene testing. KMT2E   family  syndrome , brain magnetic resonance imaging (MRI), malformations, investigations of other organs, and gene variations were analyzed. We also combined the 43 Peripheral blood samples were collected from the probands and their families. Genomic DNA was extracted. A total amount of 1.0 μg genomic DNA per sample was used as input material for the DNA sample preparation. Sequencing libraries were generated using xGen Exome Research Panel probes  following the manufacturer's recommendations. Sequencing was performed on the Illumina Novaseq 6000 platform.de novo variants that were absent in the parents. These were further validated by Sanger sequencing. The ACMG guideline was used to judge the degree of pathogenicity of both variants to evaluate their pathogenicity.Burrows-Wheeler Aligner (BWA)  was utilTo understand the biophysical consequences of KMT2E protein sequence changes, structural-prediction programs  were applied to evaluate the presence of protein domains and major secondary structure elements .Case 1 was a full-term, 5-month-old test-tube girl with uncomplicated prenatal and neonatal courses. Her other family members, including her twin sister, were all healthy. This girl had seizure onset at 4 months, with focal to generalized tonic-clonic seizures. One small café au lait spot was found on her trunk and hypotonia of the extremities. She had microcephaly with a head circumference of only 38 cm (OFC <-3 SD). She also presented specific facial features, including a slightly large forehead, prominent cheeks, and long eyelashes . HoweverCase 2 was a 5-year-old boy whose birth was uneventful. He showed global developmental delay with mild autism. He experienced two episodes of febrile seizure at 2 years old when EEG showed slow background waves. On admission, he suffered frequent generalized tonic-clonic seizures that lasted for 30 min, which fulfilled the diagnostic criteria of convulsive status epilepticus. His occipitofrontal head circumference was 50 cm. When he was 3 years old, he could walk without support, speak simple words, and follow simple instructions. Additionally, he had difficulties with social interaction and communication without any tests for intellectual disability or autism spectrum disorder before admission. On the day of admission, he was in a coma with a Glasgow Coma Scale score of nine. He had no neck stiffness, but the Babinski sign was positive. No malformations of other organs were identified. Cytological and biochemical examinations of the cerebrospinal fluid were normal, and etiological tests including herpes simplex virus, cytomegalovirus, Epstein-Barr virus, Japanese encephalitis virus, coxsackievirus, mycoplasma pneumoniae, tubercle bacillus, and fungal culture were all negative. In addition, autoimmune encephalitis and metabolic diseases were excluded by antibody and metabolic screening. The EEG showed sharp spikes in the frontal, temporal, and parietal lobes with a slow background . Brain Mde novo variants in the KMT2E gene were identified: a 186 G>A synonymous variant (NM_182931.3:exon4: c.186G>A (p.Ala62=) resulting in alternative splicing acid and a 5417 C>T transition variant [NM_182931.3:exon27: c.5417C>T (p.Pro1806Leu)] resulting in 1806 Pro-to-Leu substitution. Sanger sequencing confirmed the variants in their families , epilepsy , and microcephaly  in the missense group were significantly higher than their overall incidence (p < 0.001) was observed in the missense group  gene (ODLURO syndrome is a recently reported genetic disease caused by heterozygous variants in the 2E) gene . To date2E) gene , 14–17. 2E) gene . Patient2E) gene .KMT2E variants with neuropathic disorder reported, only 12 had epilepsy, 5 of which were treatment-resistant epilepsy. The main forms of seizures were tonic seizures, clonic seizures, focal or generalized tonic-clonic seizures, myoclonic seizures, epileptic spasms, and absence seizures. There seem to be phenotypic differences between individuals with protein-truncating and missense variants in KMT2E. Patients carrying the missense variant might present with a more severe phenotype and are more likely to have seizures than patients with the truncating variant . Case 2 presented with global developmental delay, autism, and generalized tonic-clonic seizures. Of the 43 cases of  variant , 15. O'D variant  reportedThe MRI abnormalities of ODLURO syndrome were varied and unspecific. They included thinning or partial agenesis of the corpus callosum, widening of the subarachnoid space, ventricle enlargement, various cysts, increased white matter signal, a hyperintense signal in the basal ganglia, decreased volume, delayed myelination, small areas of heterotopia, cerebellar dysplasia, and Chiari I malformation. Among them, thinning of the corpus callosum may be common and was seen in 7 of 41 patients  , 15. In KMT2E related ODLURO syndrome cases reported had a de novo variant  falls in the terminal exon of the gene. Thus, it potentially escapes non-sense-mediated decay and extends the open reading frame. It is predicted to be functionally damaging by several bioinformatics software programs. An evolutionary analysis showed a conserved result at 62 and 1806 sites among multiple species  can be found at: NCBI BioProject, accession no: The studies involving human participants were reviewed and approved by West China Second University Hospital, Sichuan University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.YL: conceptualization, methodology, and data mining. LF, MY, and JZ: writing . RL: writing  and supervision. ZY: software, data mining, and investigation. JG: supervision and writing (reviewing and editing). All authors contributed to the article and approved the submitted version.ZY was employed by Cipher Gene LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cassia abbreviata, Cissus cactiformis, Euphorbia ingens, Ipomoea kituiensis, Synadenium cupulare, Senna petersiana, Urigenia sanguinea, Maerua angolensis, and Tabernaemontana elegans on eggs and J2 population densities of Meloidogyne incognita race 2 on tomato was examined under glasshouse conditions. These plant species have medicinal properties and are being used in South Africa by traditional healers as so-called “muti.” All plant species showed a suppressive effect. Relative to untreated control, the soil amendments consistently suppressed M. incognita population densities in tomato roots and the reproductive potential (RP) of the nematode. When compared to fenamiphos, a commercial systemic chemical nematicide, the soil amendments performed comparable or better in suppressing nematode populations in the root systems in 2008 and 2009, but fenamiphos performed better than all soil amendments in 2011. The RP of M. incognita was comparable for both soil amendment- and fenamiphos-treated plants. No consistent trend in the effect of the soil amendments on plant root and shoot bioweight was observed, except when plants were treated with T. elegans-based soil amendments and both root and shoot bioweight were consistently higher compared with untreated control plants. Our results show that the plant species examined are potential sources of phytonematicides effective against M. incognita race 2.The nematicidal activity of crudely milled powders of stems, leaves, and bulbs of  Meloidogyne spp.), are worldwide an extremely important limiting factor in vegetable production . Plant-parasitic nematodes, especially root-knot nematodes (In South Africa (SA), the majority of the rural and peri-urban populations depend for their daily dietary food needs on vegetables, grains, and leguminous crops produced mainly in household and community gardens, and on smallholder farms . In thisAliber, 2009oduction .Solanum lycopersicon L.) is one of the most common vegetables grown in SA  and wild cucumber (Cucumis myriocarpus Naudin) produce phytonematicides that can suppress root-knot nematode population densities  . The basensities .Cassia abbreviata Oliv. (sjambok pod), Cissus cactiformis Gilg. (cucumber cactus), Euphorbiaingens E. Mey. ex Boiss. , Ipomoea kituiensis (Burm.) Merr. (kitui morning glory), Maerua angolensis DC. (bead-bean tree), Senna petersiana (Bolle) Lock. , Synadenium cupulare (Boiss.) Wheeler ex A. C. White (dead-man’s tree), Tabernaemontana elegans Stapf (toad tree), and Urginea sanguinea Shinz. (rooislangkop) on growth of tomato plants and suppression of population densities of M. incognita race 2 under glasshouse conditions.So-called “muti” are African traditional medicines that make use of various natural products derived from trees and other plant species. These botanical medicines are usually prescribed by an inyanga or herbal healer and they are mostly endemic to Southern Africa. In SA, traditional healers use these mutis at controlled dosage rates to treat humans and domestic animals for various ailments . The medC. abbreviata, C. cactiformis, E. ingens, I. kituiensis, M. angolensis, S. petersiana, S. cupulare, T. elegans, and U. sanguinea were collected from traditional healers in the Mopani and Vhembe districts, Limpopo Province, SA. The botanical origin of the plant parts was identified and confirmed by a botanist from the South African National Biodiversity Institute . The plant parts were chopped into 5-cm-long pieces and oven-dried for four days at 52°C prior to grinding in a Wiley mill and passed through a 1-mm-aperture sieve  were evaluated. Based on the first trial (2006 and 2007), the soil amendments of C. abbreviata, I. kituiensis, S. petersiana, and U. sanguinea showed phytotoxicity. The three most effective plant species  were selected for further study in 2008, 2009, and 2011, along with two newly selected plant species (M. angolensis and T. elegans). Two six-weeks-old seedlings of tomato cultivar Floradade were transplanted into 5-L 150-mm-diameter plastic bags filled with a steam-pasteurized sandy loam soil , and pH 5.8. Immediately after transplanting, all seedlings were inoculated with 3,000±20 eggs and J2 in 5-cm-deep holes around the seedling stem using a 20-mL plastic syringe. In all trials, soil treatments consisted of a soil amendment of only one concentration (5 g/pot) that translates to 0.1% in w/w (0.71 metric tons/ha) of crudely milled powder. In 2006 and 2007, only untreated plants were included as controls; in 2008 and 2009, untreated plants plus fenamiphos-treated plants  were included as controls. In 2011, untreated- and fenamiphos-treated plants, and a soil amendment of 5 g dried, milled leaf meal of C. myriocarpus Naudin were included as positive controls. The addition of C. myriocarpus enable us to compare the effects of the powders used in our trials with a powder having known nematicidal activity  and Redspidercide® (tetradifon 81 g/L) alternatively every two weeks to prevent aphid and red spider mite infestation, respectively.The trials were carried out in a glasshouse at the ARC-Tropical and Subtropical Crops research institute in Mbombela, SA . In 2006 and 2007, crudely milled powder of seven plant species  transformed to remove the inherent variability prior to analysis. Treatment means were separated using Fisher’s protected least significant difference (LSD) calculated at P ≤ 0.05  using SAS/STAT statistical software . The staP ≤ 0.05 .P ≤ 0.05) suppressed the nematode population densities by 68 to 83% compared to untreated control  by 87 to 88% compared to untreated control plants while a significant difference with fenamiphos was observed in 2008. The suppressive effect of C. cactiformis, E. ingens, S. cupulare, and T. elegans were confirmed in 2011 when soil amendments based on these plant species significantly (P ≤ 0.05) suppressed nematode population densities with 78 to 94% compared to untreated control and, with results of all of the above except for M. angolensis being comparable to results with C. myriocarpus. The reproductive potential (RP) of M. incognita were comparable in both fenamiphos and soil amendment treated plants  increase in fresh root weight compared to untreated control plants was observed in the trials carried out from 2007 until 2011 in which soil amendments of C. cactiformis, E. ingens, S. cupulare, M. angolensis, and T. elegans were included (P ≤ 0.05) increase in fresh shoot weight compared to untreated control plants were observed in all the trials carried out from 2007 until 2011 for soil amendments of T. elegans and C. cactiformis (E. ingens and S. cupulare resulted in a significantly (P ≤ 0.05) higher fresh shoot weight compared to untreated control plants. Marua angolensis resulted in a significantly higher shoot weight in 2008 and 2011.There were no consistent trends in the effects of soil amendments on plant root and shoot weight. A significant ) was considered, the suppressive effect of all soil amendments was comparable to fenamiphos in 2008, 2009, and 2011. Some inconsistencies were observed but they were few and inconsistencies are not surprising in this type of trials , polyacetylenes, polyketides, terpenoids, thiophenes, and waxes . Howeverin casu M. incognita race 2) than previously reported. Products of some of these medicinal plants apparently have the potential to increase tomato growth and yield in root-knot nematode infested soil. The development of these plant materials as phytonematicides merits further investigation under microplot and field conditions. These plant materials may offer an accessible and affordable management alternative for root-knot nematode problems in household and community gardens, and smallholder fields especially in Mpumalanga, Limpopo, and KwaZulu-Natal provinces of SA where some of these medicinal plants such as C. cactiformis, M. angolensis, and T. elegans are grown abundantly. Possible human, animal, and environmental risks of their use should be determined, preferably in consultation with the traditional healers that use these materials for medicinal purposes.This study confirmed that more plant species contain nematicidal compounds against root-knot nematodes (


Burkholderia includes a wide range of Gram-negative bacterial species some of which are pathogenic to humans and other vertebrates. The most pathogenic species are Burkholderia mallei, Burkholderia pseudomallei, and the members of the Burkholderia cepacia complex (Bcc). B. mallei and B. pseudomallei, the cause of glanders and melioidosis, respectively, are considered potential bioweapons. The Bcc comprises a subset of Burkholderia species associated with respiratory infections in people with chronic granulomatous disease and cystic fibrosis. Antimicrobial treatment of Burkholderia infections is difficult due to the intrinsic multidrug antibiotic resistance of these bacteria; prophylactic vaccines provide an attractive alternative to counteract these infections. Although commercial vaccines against Burkholderia infections are still unavailable, substantial progress has been made over recent years in the development of vaccines against B. pseudomallei and B. mallei. This review critically discusses the current advances in vaccine development against B. mallei, B. pseudomallei, and the Bcc.The genus  Burkholderia comprises a wide range of environmental Gram-negative bacteria that interact with plants, insects, animals, and humans. Most of the species in the genus are plant-associated bacteria [Burkholderia mallei, Burkholderia pseudomallei, and the members of the Burkholderia cepacia complex (Bcc). All Burkholderia are intrinsically resistant to a wide range of antibiotics, which complicates the treatment of their infections; prophylactic vaccines are an attractive alternative to protect against Burkholderia infections.The genus bacteria . Howeverbacteria . The mosB. mallei and B. pseudomallei are phylogenetically similar; they cause glanders and melioidosis, respectively, and have been classified as Tier 1 select agents by the US Centers for Disease Control and Prevention (https://emergency.cdc.gov/agent/agentlist-category.asp). B. mallei is a facultative intracellular pathogen generally infecting horses, mules, and donkeys, and sporadically humans [B. pseudomallei, a saprophyte commonly present in wet soils and rice paddies, is endemic in tropical and subtropical countries [B. mallei and B. pseudomallei are resistant to broad-spectrum antibiotics including aminoglycosides, polymyxins, and β-lactams [y humans . Glandery humans . Althougy humans ,5,6,7,8.ountries ; it is aountries  and bactountries . Epidemiountries . B. mall-lactams . Both gl-lactams .Burkholderia species that cause respiratory infections in people with underlying diseases, such as cystic fibrosis and chronic granulomatous disease [B. cenocepacia, B. multivorans, B. vietnamiensis, B. dolosa, and B. cepacia [B. cenocepacia and B. multivorans are highly prevalent, as they account for approximately 85–97% of all Bcc infections in people with cystic fibrosis [The Bcc encompasses a subgroup of  disease . The Bcc disease ,16,17,18 cepacia . B. cenofibrosis . Treatmefibrosis . Developfibrosis ,22,23,24Burkholderia species. However, no ideal vaccine candidate appeared for humans, reflecting the challenges in designing safe and effective vaccines that can elicit long lasting immunity to protect against both acute and chronic infections by these bacteria. Here, we provide a critical overview of recent advances in Burkholderia vaccine development and how new technologies could drive the design of effective vaccines against Burkholderia infections.Substantial advances have been made over the past decade in understanding the pathogenesis, host–pathogen interactions, virulence factors, and host immune responses against Bordetella pertussis and Neisseria meningitidis serogroup B infections [Inactivated whole-cell vaccines contain bacteria that have lost the capacity to cause disease. Heat, chemical, and UV radiation are the most common methods to inactivate bacteria; the primary advantage of inactivated whole-cell vaccines is their ability to induce robust humoral immune responses. Nonreplicating, inactivated vaccines have been licensed against 11 different human pathogens including viruses and bacteria , such asfections .B. pseudomallei vaccines  and heat-killed (HK) zed mice . It remazed mice . A similoduction . TherefoB. mallei, heat-killed and UV-inactivated wild type and a capsule-negative mutant were evaluated in mice as protective vaccines. Immunized mice generated mixed Th1 and Th2 cytokine responses and a Th2-biased immunoglobulin response, respectively; however, these preparations did not protect mice against live challenge with B. mallei [B. mallei only conferred partial protection of mice from lethal B. mallei challenge [+, and CD8+ T cells, as well as IFN-γ and TNF-α cytokines, appear to play important roles in the protection mediated by the heat-killed B. mallei vaccine [For . mallei . Similarhallenge . B cells vaccine .B. mallei and B. pseudomallei challenge was observed in mice immunized with inactivated whole-cell vaccines, immunized mice were incompletely protected. Several factors, such as inactivation conditions, choice of adjuvants, and immunization route must be considered when evaluating the protective efficacy of an inactivated Burkholderia vaccine.Collectively, although some protection against Salmonella enterica serovar Typhi [The best example of a successful live attenuated bacterial vaccine is a licensed vaccine against ar Typhi , which iar Typhi . AttenuaBurkholderia species [ilvI) defective in the branched chain amino acid biosynthetic pathway provided significant protection in mice against semi lethal doses of wild-type B. pseudomallei [+ and CD8+ T cell depletion studies showed that the protection was mediated by CD4+ T cells [purN, ΔpurM) and additional biosynthetic pathways, were also tested as candidate attenuated vaccines [purN strains either by intranasal or intraperitoneal routes were protected against acute infection while no protection was observed against intravenous challenge. However, mice infected by intranasal or intraperitoneal routes eventually showed clinical signs of disease after 20 days or more post-challenge and succumbed from chronic infection [purN mutant making it not fully attenuated [purM mutant derived from strain 1026b, which is defective in adenine and thiamine biosynthesis, has been proven to be fully attenuated and safe for use under BSL-2 laboratory conditions [purN and ΔpurM mutants, the results using them as vaccine antigens are inconclusive [By transposon mutagenesis and genome sequencing, various essential genes have been identified in  species  and the domallei . CD4+ an T cells . Other mvaccines . Challennfection . It is ptenuated . In contnditions ,41. Despnclusive ,39.aroB and ΔaroC, have also been evaluated as attenuated vaccines [aroB strain, all mice eventually died. Further, immunization with the ΔaroC mutant protected C57Bl/6 mice but not BALB/c mice from infection challenge [aroC mutant is too attenuated to develop a protective immune response in BALB/c mice since it is rapidly cleared. A deletion mutant, Δasd, auxotrophic for diaminopimelate in rich medium and auxotrophic for diaminopimelate, lysine, methionine, and threonine in minimal medium, has been evaluated for protective efficacy as a live attenuated vaccine [relAΔspoT, which lacks (p)ppGpp-synthesizing enzymes [relAΔspoT mutant provided significantly increased survival (100% survivors) up to 30 days post-challenge; however, sterilizing immunity was not achieved.A set of two auxotrophic mutants in the biosynthesis of aromatic compounds, Δvaccines ,43. Alth vaccine . This va vaccine . Another enzymes . VaccinaB. pseudomallei infection. The B. pseudomallei ΔbipD mutant, lacking a putative protein associated with the translocation apparatus of the type 3 secretion system, was significantly attenuated in BALB/c mice [bipD partially protected mice  from infection with the wild-type B. pseudomallei challenge. However, immunization with the BipD protein alone failed to confer protection, suggesting BipD is not a protective antigen for B. pseudomallei [Due to their importance in bacterial pathogenicity, protein secretion systems have been exploited to develop attenuated vaccines against B/c mice . Howeverdomallei .B. mallei strains. The attenuated B. mallei strains ΔilvI [tssN [tonB [tonBΔhcp1 [ilvI provided partial protection in BALB/c mice against high-dose aerosol challenge with B. mallei ATCC23344. The surviving mice, however, developed chronic infection, suggesting the vaccine was not efficient to elicit enough protection to eliminate all bacteria in the inoculum or alternatively, had no effect on bacteria internalized in host cells [tssN provided some protection against aerosol challenge with high-dose ATCC23344 in BALB/c mice, but surviving mice experienced significant weight loss indicative of chronic infection [tonB conferred 100% protection to BALB/c mice against aerosol challenge with the wild type strain CSM001. However, this mutant may not have been fully attenuated, as the immunized mice developed splenomegaly and multiple splenic abscesses [tonBΔhcp1 double mutant appears to be the safest and most effective B. mallei and B. pseudomallei attenuated vaccine tested so far  demonstrating an efficiency greater than that of the live attenuated 2D2 positive control [B. cenocepacia and B. multivorans and a balanced Th1/Th2 immune response [, B. cenocepacia OmpA specific antibodies are present in sera of people with cystic fibrosis, indicating its potential to naturally stimulate humoral responses [Outer membrane proteins are surface antigens also considered for vaccine development . Immuniz species . OmpW is strains . Indeed, control . The Ompresponse . Two Ompresponse . Furtheresponses . Anotheresponses .B. pseudomallei hemolysin-coregulated protein Hcp2 provided 80% protection to challenged mice, while Hcp1, Hcp3, and Hcp6 provided 50% protection [B. pseudomallei and B. mallei [B. pseudomallei using a goat model of melioidosis identified multiple antigens. From these, the GroEL heat shock protein 60, EF-Tu, ATP synthase β chain, and the DnaK chaperone provided the strongest immune responses, suggesting they could be good candidates for further investigation [Other virulence factors, such as proteins associated with the type 6 secretion system and trimeric autotransporter adhesins have been investigated as subunit vaccine candidates. The otection . Anti-Hcotection . Trimeri. mallei ,71,72. Atigation .B. pseudomallei and B. mallei in both mice and Rhesus macaques. Immunization of mice with B. pseudomallei outer membrane vesicles elicited humoral and cellular immune responses against the bacterium [B. mallei infection, reflected by production of B. mallei-specific serum IgG in both mice and Rhesus macaques, and B. mallei-specific Th1/Th14 CD4+ and CD8+ T cell responses in mice [Outer membrane vesicles have also been tested as vaccines against acterium ,67. In R in mice ,67,74.B. pseudomallei type I fimbria subunit BPSL1626, which can recognize and bind antibodies in sera from infected patients and induce T-lymphocyte responses in vivo [B. pseudomallei (Bp1651) proteins as potential vaccine targets [Reverse vaccinology strategies based on bioinformatic pipelines have opened new possibilities to identify novel vaccine candidates. They can be used to predict new antigens, such as the  in vivo . Three e in vivo . Reverse targets .Burkholderia and the heterogeneity of the genus a multicomponent subunit vaccine is likely required to induce a balanced immune response resulting in long-lasting immunity. So far, no ideal vaccine candidate has been identified. The current candidates only offer sterilizing immunity at relatively low challenge doses, which may be insufficient for a fully protective vaccine. However, this low protection threshold could still be effective to reduce the incidence of disease and several vaccine formulations may become useful with additional optimization.Due to the intracellular survival of Polysaccharides are attractive candidate antigens for vaccine development, as they contain unusual carbohydrates that can be highly antigenic ,79,80. AB. pseudomallei and B. mallei [B. pseudomallei CPS covalently linked to diphtheria toxin mutant (CRM) induces high IgG titers and opsonizing antibodies in C57BL/6 mice [B. pseudomallei [B. pseudomallei in lungs, spleen, and liver, indicating the vaccine provides sterilizing immunity [B. pseudomallei CPS, also provided robust protection [O-acetyl-6-deoxy-β-d-manno-heptopyranose, coupled to the tetanus toxin Hc domain (TetHc), elicited IgM and IgG antibodies recognizing the native capsule and protected mice from challenge with B. pseudomallei [CPS is a recognized virulence factor for both . mallei ,83. ImmuL/6 mice . When codomallei . Furtherimmunity . Other cotection . Concernotection . Synthetdomallei .Burkholderia glycoconjugates, and they can be packed into nanoparticles to improve efficacy by promoting antigen presentation [B. mallei, using LPS from B. thailandensis, which has been tested in mice and Rhesus macaques [Lipopolysaccharide (LPS) linked to carrier proteins is another strategy used for production of glycoconjugate vaccines. TetHc, Hcp1, and FliC are commonly used as carriers for entation ,87,88. Gentation ,90,91,92macaques .N- and O-glycosylation machineries for the production of glycoconjugates has also been examined [N-glycosylation system of Campylobacter jejuni can be functionally reconstructed in Escherichia coli to express the periplasmic AcrA protein glycosylated with B. pseudomallei O-polysaccharide [O-glycosylation cluster, common to all Burkholderia species, can also be potentially exploited to produce recombinant glycoprotein-based vaccines [B. pseudomallei LPS, resulted in 100% mice survival and lower lung colonization after a lethal B. pseudomallei challenge [The potential of exploiting the examined . The N-gccharide . The O-gvaccines ,96. Glycvaccines . In silivaccines . This prhallenge .N. meningitidis, S. pneumoniae, and H. influenzae. Candidate Burkholderia glycoconjugate antigens . Viral vectors have some limitations such as the risk of cancer induced by viral genome being integrated into host genome and the potential of recombinant virus vector being rapidly eliminated by pre-existing immunity. However, viral vectors based on recombinant viruses have been used to develop new vaccines against a wide range of diseases including cancer, human immunodeficiency virus, and malaria ,107,108.t genome ,110. Numlitensis . The antB. mallei and B. pseudomallei  could aid in broadening the pool of available antigens for cross-protection. As mentioned previously, experimental data from protection studies suggest that a multicomponent approach could improve the effectiveness of a subunit vaccine. DNA vaccine and viral vector vaccines are also promising strategies that merit further exploration since they have proven to be effective in eliciting protective immune response against diverse pathogens including bacteria, viruses, and parasites.Concerning candidate antigens for vaccine development, the use of transgenic mice for immune response research could help mapping responses to antigens and thus identifying more suitable vaccine candidates for humans. Transgenic mice expressing human HLA alleles have been used to study not only the binding of antigen epitopes but also T-cell responses . HLA all


Prototheca cutis infection was diagnosed postmortem for an immunocompetent cat with respiratory signs. Pathologic examination and whole-genome sequencing identified this species of algae, and susceptibility testing determined antimicrobial resistance patterns. P. cutis infection should be a differential diagnosis for soft tissue infections of mammals.Severe nasal  Prototheca spp.  are ubiquitous algal organisms that represent emerging infectious agents of humans and animals . From June 2019 through January 2020, the nasal planum became rounded and disfigured. A biopsy sample submitted to a private diagnostic laboratory indicated a fungal infection containing organisms suggestive of Cryptococcus spp. Because of concerns over the zoonotic potential of Cryptococcus spp., the cat was euthanized and submitted for postmortem examination.In January 2020, an 11-year-old, 5.8-kg, neutered male, domestic cat was examined for sneezing, wheezing, congestion, and rhinitis. This indoor/outdoor cat was negative for feline leukemia and feline immunodeficiency viruses. The cat showed no response to treatment with steroids and cefovecin sodium . Partial sequencing of the internal transcribed spacer region and the D1/D2 region of the 28S rRNA gene yielded sequences 96% and 99% homologous to those from P. cutis available in BLAST (https://blast.ncbi.nlm.nih.gov) and CBS-KNAW  databases.Fungal culture yielded white colonies growing in the presence and absence of light at 30°C  Figure 2cytb gene potentially represents a new standard method for identifying Prototheca species (cytb as well as other genes by using Illumina MiSeq (https://www.illumina.com) (P. cutis JCM15793 strain ATCC PRA-138 (https://www.atcc.org) , chloroplast genome DNA (99.54%), 18S rRNA gene , ITS , and 28S rRNA (D1/D2 domain) , which were deposited in GenBank.Because the mitochondrial ina.com) . The nucPrototheca spp. infections in animals  to identify genes conferring resistance to β-lactams, tetracyclines, aminoglycosides, chloramphenicol, and vancomycin. Consistent with MICs, no resistance genes corresponded to pradofloxacin and trimethoprim/sulfamethoxazole (Prototheca spp.We performed susceptibility testing for antifungal and antimicrobial drugs because both have been used against  animals . Resista the cat . Oxacillhoxazole . The MICCryptococcus spp. Although Prototheca spp. are widely considered to be zoonotic agents, reports of definitive cases of zoonotic transmission are lacking in the literature. Zoonotic transmission from bovids is thought to occur via consumption of contaminated milk (P. cutis is unclear; infectivity is probably similar to that of other Prototheca spp.Our primary concern with regard to this case was determining the zoonotic potential of the agent, which was initially misdiagnosed as P. cutis isolation from a cat reinforces protothecosis as an emerging infectious disease of humans and animals. We emphasize the potential of P. cutis to infect presumably immunocompetent hosts. The veterinary and human medical communities should be aware of the unusual clinical, pathologic, and microbiological manifestations of protothecosis.Our report of Prototheca cutis infection in a cat.Detailed gross and histopathologic methods and findings associated with diagnosis of


Escherichia coli and Staphylococcus aureus are important agents of urinary tract infections that can often evolve to severe infections. The rise of antibiotic-resistant strains has driven the search for novel therapies to replace the use or act as adjuvants of antibiotics. In this context, plant-derived compounds have been widely investigated. Cuminaldehyde is suggested as the major antimicrobial compound of the cumin seed essential oil. However, this effect is not fully understood. Herein, we investigated the in silico and in vitro activities of cuminaldehyde, as well as its ability to potentiate ciprofloxacin effects against S. aureus and E. coli. In silico analyses were performed by using different computational tools. The PASS online and SwissADME programmes were used for the prediction of biological activities and oral bioavailability of cuminaldehyde. For analysis of the possible toxic effects and the theoretical pharmacokinetic parameters of the compound, the Osiris, SwissADME and PROTOX programmes were used. Estimations of cuminaldehyde gastrointestinal absorption, blood brain barrier permeability and skin permeation by using SwissADME; and drug likeness and score by using Osiris, were also evaluated The in vitro antimicrobial effects of cuminaldehyde were determined by using microdilution, biofilm formation and time-kill assays. In silico analysis indicated that cuminaldehyde may act as an antimicrobial and as a membrane permeability enhancer. It was suggested to be highly absorbable by the gastrointestinal tract and likely to cross the blood brain barrier. Also, irritative and harmful effects were predicted for cuminaldehyde if swallowed at its LD50. Good oral bioavailability and drug score were also found for this compound. Cuminaldehyde presented antimicrobial and anti-biofilm effects against S. aureus and E. coli.. When co-incubated with ciprofloxacin, it enhanced the antibiotic antimicrobial and anti-biofilm actions. We suggest that cuminaldehyde may be useful as an adjuvant therapy to ciprofloxacin in S. aureus and E. coli-induced infections. Escherichia coli and Staphylococcus aureus are important agents of urinary tract infections [E. coli is one of the major pathogens of such infections, causing bacteremia in over 6,000 per 100,000 patients per year [S. aureus is responsible for 21 to 36 cases of bacteremia per 100,000 habitants per year [Bacteria such as fections –4. Both fections , 5–7. Thper year , 9. In aper year  with morper year .S. aureus and E. coli have become resistant [E. coli and S. aureus, thus, reducing the chances of a successful treatment of infections caused by these microorganisms. The increased bacterial resistance has driven the search for novel therapeutic approaches that can either replace the use or act as adjuvants of antibiotics [The management of such infections mostly relies on antibiotic therapy, with patient survival and recovery largely depending on treatment timing and efficacy. Of note, ciprofloxacin is the most commonly prescribed fluoroquinolone antibiotic for urinary tract infections  to whichesistant , 14. Indibiotics . In thisibiotics , 17.Cuminum cyminum L. (Apiaceae), popularly known as cumin, have been largely used in the global cuisine [S. aureus and E. coli [S. aureus and E. coli strains [in silico and in vitro activities of this compound against S. aureus and E. coli. We also assessed the ability of cuminaldehyde to potentiate ciprofloxacin effects on these bacteria.The seeds of  cuisine . Its see cuisine , 19. Rec E. coli –27. This E. coli , 28, 29. strains –32. In owww.way2drug.com/passonline) composed of other organic molecules with defined biological activities [To evaluate the biological activities of cuminaldehyde, the Prediction of Activity Spectra for Substances (PASS) Online programme was used. This computational tool calculates the probability of a given organic molecule to present a biological activity by comparing the molecule structure to a database  [2, ii) LogP ≤ 5, iii) molecular weight <500 daltons, iv) number of acceptor hydrogen bonds (nALH) ≤ 10, and v) number of donor hydrogen bonds (nDLH) ≤ 5.The SwissADME programme (ex.php#)  was usedex.php#) , 36. By www.organic-chemistry.org/prog/peo/drugScore.html) [http://www.swissadme.ch/index.php#) [For analysis of the possible toxic effects and the theoretical pharmacokinetic parameters  of cuminaldehyde, the Osiris (re.html)  and Swisex.php#)  programmex.php#) .http://tox.charite.de/protox_II/index.php?site=compound_input) [50 ≤ 5, class ii- fatal if swallowed when 5<LD50 ≤ 50, class iii- toxic if swallowed when 50 <LD50 ≤ 300, class iv- harmful if swallowed when 300 <LD50 ≤ 2000, class v- harmful if swallowed when 2000 <LD50 ≤ 5000, and class vi- non-toxic when LD50> 5000. Estimations of cuminaldehyde gastrointestinal absorption, permeability through the blood brain barrier and skin permeation (log Kp in centimeters (cm)/s) were assessed by the SwissADME programme (http://www.swissadme.ch/index.php#) [LD50 values in milligram per kilogram (mg/kg) were estimated by using the PROTOX programme (d_input)  and usedex.php#) . Of notewww.organic-chemistry.org/prog/peo/drugScore.html), by combining the values obtained for iLogP, drug-likeness, solubility  [Additionally, the probability of cuminaldehyde to becoming a commercial drug ("drug-score") was calculated by the Osiris programme (ubility) , molar mubility) .S. aureus ATCC 6538 and enteroaggregative E. coli 042 (EAEC 042), and the clinical isolates from urinary tract infections (S. aureus-A (Saa), S. aureus-V (Sav) and E. coli-R (Ecr)) were kindly provided by the bacterial collection sector of the Universidade CEUMA. The bacteria were cultured on Müeller-Hinton  agar, at 37°C for 24 h as previously described [The standard strains of escribed .8 colony forming units (CFU)/milliliter (ml)). For determining the MICs, 10 μl of the bacterial suspension were incubated in MH broth  containing different concentrations of cuminaldehyde (0.0234–24 mg/ml). Serial dilutions of ciprofloxacin (0.0009–200 μg/ml) were used as positive controls. Sterile dimethyl sulfoxide  was used to increase cuminaldehyde solubility and as negative control. Samples were incubated for 24 h at 37°C, and the MICs  were evaluated. For this, the absorbances were read at 600 nm.Cuminaldehyde  antimicrobial effects were investigated by the microdilution method . For thiIn parallel, the effects of the sub-inhibitory concentrations (MIC/2-MIC/8) of cuminaldehyde and ciprofloxacin on bacterial viability were assessed and calculated by addition of the PrestoBlue® reagent , according to the manufacturer’s instructions. The absorbance was read at 570 nm and 600 nm and cell viability expressed as Δ absorbance in nm.Biofilm formation was quantified as previously described . For thi10 CFU/ml was calculated. The bactericidal combinatory effects were assessed by variation on Log10 CFU/ml (ΔLC). Synergy was defined as a decrease of ≥ 2 log10 CFU/ml and antagonism as an increase of #x2265; 2 log10 CFU/ml. If ΔLC was between 1 and 2 log10 CFU/ml, the effects were recorded as additive, and as indifferent if ΔLC = ± 1 log10 CFU/ml [An aliquot (200 μl) of each bacterial suspension was added to 2 ml of MH broth (Merck Millipore) containing ciprofloxacin (MIC/2) or cuminaldehyde (MIC/2-MIC/4) alone, or in combination. Vehicle -treated bacteria were used as negative control. Cell growth was monitored by plating 10 μl of 10-fold-diluted suspensions from each sample at different time-points (0.15–8 h) in MH agar (Merck Millipore) plates. After 8 h of incubation at 37°C, the colonies were counted and then, the Log0 CFU/ml .Effects on biofilm formation). For this, 10 μl of each bacterial suspension were added per well into a 96-well cell culture plate containing cuminaldehyde (MIC/8) and ciprofloxacin (MIC/2) in 190 μl of MH broth (Merck Millipore). Vehicle -treated bacteria and broth without bacteria were used as negative controls.The potential of cuminaldehyde to interact with ciprofloxacin was assessed on biofilm formation. Biofilm mass formation was quantified  probability to present 726 activities. Of those, 198 have moderate probability (Pa>0.5) of occurrence and 69, high probability of occurrence (Pa>0.7). Of the total identified activities, 15 were antimicrobial .For comparison, the biological activities of ciprofloxacin were also evaluated. Forty-seven activities with > 30% probability of occurrence were identified for ciprofloxacin. Of those, only 04 had high probability of occurrence (Pa>0.7). Analysis of all 47 biological activities indicated that 12 of them were antimicrobial .For predicting the oral bioavailibility of cuminaldehyde, its TPSA, iLogP, molecular weight, nALH and nDLH values were analyzed. Information on the estimations of gastrointestinal absorption, permeability through the blood brain barrier and skin permeation (log Kp in centimeters (cm)/s) are shown in S. aureus ATCC 6538 and EAEC 042, and the clinical isolates Saa, Sav and Ecr. Cuminaldehyde presented antimicrobial activity against all tested bacteria, being more effective against EAEC 042 (1.5 mg/ml). All bacteria, except the clinical isolate Ecr, were susceptible to ciprofloxacin . However, it enhanced the amount of Saa-dependent biofilm formation  and ciprofloxacin (MIC/2) sub-inhibitory concentrations were analysed . Data deormation , withoutormation .C. cyminum seeds indicated its antimicrobial potential against a series of bacteria including S. aureus and E. coli [C. cyminum seeds [Studies with the essential oil of  E. coli –27. Cumium seeds , 28.in silico evaluation of the biological activities of cuminaldehyde confirmed its antimicrobial potential. We aimed at gaining further knowledge of the anti-S. aureus and anti-E. coli actions of cuminaldehyde. Therefore, its properties and effects were compared to those of ciprofloxacin which has been commonly used for the treatment of urinary tract infections.In our study, the In silico analysis also revealed that, as observed for ciprofloxacin, cuminaldehyde is likely to be absorbed by the gastrointestinal tract. Of note, this compound was suggested to be able to cross the blood brain barrier, meaning its dosing for systemic use should be carefully considered as it may result in unwanted effects in the nervous system.Evaluation of cuminaldehyde oral bioavailability estimated the compound is a good candidate for oral intake with a drug score of 0.55 , similar to the one observed for ciprofloxacin. However, doses >1,320 mg/kg of the compound would be lethal to mammalians, as cuminaldehyde was classified as harmful if swallowed at this LD50 . S. aureus is a common antibiotic-resistant pathogen colonizing skin wounds [Prediction of skin permeation (log Kp) indicated that cuminaldehyde is more likely to be absorbable by skin layers than ciprofloxacin. Of note, n wounds , 42. ThuE. coli and S. aureus; respectively [E. coli. Interestingly, the compound was antimicrobial against the ciprofloxacin-resistant clinical isolate Ecr. Analysis of sub-inhibitory concentrations of cuminaldehyde on bacterial viability reinforced its effectiveness against E. coli. Ciprofloxacin sub-inhibitory concentrations also reduced, although in a smaller degree, Ecr viability ,Recent studies demonstrated cuminaldehyde has antimicrobial effects, presenting a MIC of 0.311 mg/ml and 0.650–4.98 mg/ml for ectively ,32,43. IC. cyminum seeds reduces Streptococcus mutans and S. pyogenes biofilm formation [Klebsiella pneumoniae [E. coli [S. aureus biofilm mass formation. By comparison, the sub-inhibitory concentrations of ciprofloxacin were only able to diminish Saa biofilm viability, in addition to Saa and Sav biofilm mass formation. These results indicate that cuminaldehyde presents a broader anti-biofilm activity than ciprofloxacin at the tested concentrations.Of importance, compounds with anti-biofilm actions have been investigated as alternative therapies to treat bacterial infections –46. It wormation . Similareumoniae . Recentl[E. coli . Herein,C. cyminum seed essential oil to potentiate ciprofloxacin antimicrobial effects was shown against K. pneumoniae [in silico prediction of the biological activities of cuminaldehyde indicated this compound may act as a membrane permeability enhancer. Therefore, we hypothesized on whether this compound enhances the antimicrobial effects of ciprofloxacin. Our results demonstrate that cuminaldehyde (MIC/2-MIC/4) is synergistic to ciprofloxacin (MIC/2), as their association effectively reduces S. aureus and E. coli survival. A similar effect was seen in S. aureus (ATCC 6538 and Sav) biofilm formation. On the other hand, cuminaldehyde potentiated biofilm mass formation in ciprofloxacin-treated Saa, suggesting the combinatory effects of these drugs on biofilm mass formation may be strain-dependent.Many compounds have been investigated as adjuvants to antibiotic therapy to overcome bacterial resistance , 17. Theeumoniae . Herein,S. aureus and E. coli-induced infections, lower pressure on bacterial resistance would probably result from this combined therapy; a hypothesis which remains to be investigated.Overall, the evidences gathered herein, suggest that cuminaldehyde may be useful as an adjuvant to ciprofloxacin therapy. As reduced doses of this commercial antibiotic would be used in conjunction with cuminaldehyde to treating S1 Rawdata(XLS)Click here for additional data file.


Staphylococcus (S) equorum and S. saprophyticus were the species most frequently found in P production, followed by S. succinis and S. warneri; a wider species variability was observed in A factory being S. equorum, S. capitis, S. xylosus,S. pasteuri, S. epidermidis and S. saprophyticus as the main identified species. The technological characterization of 28 CNS strains showed their ability to hydrolyze gelatin and tributyrin together with a relevant nitrate reductase activity. Phenotypic and genotypic approaches were conducted to investigate the main safety traits. Llama’s CNS strains exhibited weak decarboxylase and hemolytic activity and low biofilm production; additionally, no enterotoxin genes were detected. Correlation analysis between phenotypic and genotypic data showed low values for the biofilm parameters, while high correlation was observed for oxacillin, ampicillin, tetracycline and aminoglycosides resistance and their genetic determinants. Data obtained may contribute to broaden knowledge about the autochthonous strains of this poorly studied fermented product, thus helping to select an appropriate combination of potential starter cultures to improve llama sausage safety and quality.Llama sausage is still produced following artisanal procedures, with the autochthonous microbiota being mainly responsible for the fermentation process. In this work, the taxonomical identification and technological-safety criteria of coagulase-negative staphylococci (CNS) isolated from two different productions of llama sausages  were investigated.  Lama guanicoe) and vicuna (Vicugna vicugna) are wild, while llama (Lama glama) and alpaca (Lama pacos) are domesticated [Lactobacillus sakei, constituting this native microbiota a source of biodiversity with potential technological and beneficial properties [The Andean region in Northwestern Argentina exhibits singular environmental conditions that limit agricultural and breeding activities in several areas. Here, high altitudes allow the survival of native camelids of south America, of which guanaco  were enumerated and isolated at different ripening times  on Mannitol Salt Agar  during 48 h at 30 °C, from naturally fermented llama sausages produced in Northwest Argentina as reported by Fontana et al. [®, Microzone Ltd., Haywards Heath, Sussex, UK), following the manufacturer’s instructions. To achieve the strain typing, the isolates were screened by Random Amplification of Polymorphic DNA (RAPD) according to Fontana et al. [® ExoSAP-IT® PCR Cleanup  following the manufacturer’s instruction and sequenced at the BMR Genomics . The taxonomical assessment was performed using BLAST . All isolated strains were stored in Brain Hearth Infusion broth (BHI)  plus 20% (v/v) glycerol at –20 °C and deposited at Università Cattolica del Sacro Cuore collection with UC code ranged from UC7541 to UC7569.One-hundred and twenty colonies were screened by PCR (Polimerase Chain Reaction) using specific primers  for consa et al. , using aThe proteolytic activity on sarcoplasmic and myofibrillar pork proteins was assayed according to Mauriello et al. . ProteolTributyrin agar , and BHI agar medium supplemented with 1% of Tween 20 were employed for the assessment of the lipolytic activities . Plates 3 as previously described [Nitrate reductase activity was determined by the agar plate method on Yeast Tryptone Agar  supplemented with 1 g/l KNOescribed . The redThe ability of the CNS strains to produce biogenic amines  was phenotypically evaluated following the protocol described by Bover-Cid and Holzapfel . The strThe α-β-and δ-hemolytic activity was determined following the protocol of Jeong et al. . For the600) was <0.1, moderate if OD600 was between >0.1– <1.0, and positive if OD600 was ≥ 1.0.Biofilm production was assessed in microtiter using the method reported by Kwasny et al.  and AsaiThe phenotypic AR profile was investigated by the disc diffusion test , accordiPCR amplification of well-known structural genes associated with AR, staphylococcal enterotoxins and biofilm-related genes were performed using the primers and conditions reported in Staphylococcus genus . Plate count performed by Fontana et al. [In this study, 95 out of 120 colonies isolated from llama meat sausages (P and A productions) were taxonomically identified by molecular techniques as belonging to the a et al. , on P anS. equorum and S. saprophyticus were the species most frequently found in P production together with one strain belonging to S. succinus and S. warneri species. In contrast, a wider species variability was observed in the artisanal manufacturing where identified strains belonged to S. equorum, S. capitis, S. xylosus, S. pasteuri, S. epidermidis and S. saprophyticus species. A correspondence analysis showed the influence of the type of production on the CNS species development during the fermentation process , S. epidemidis (2), S succinis (1) and the second cluster grouping all S. saprophyticus (7 strains), S. capitis (3), S xylosus (2), S. pasteuri (2). The solely S. warneri strain constitute a singleton. The predominance of S. equorum and S. saprophyticus species has also been described in other fermented dry sausage manufactured in Italy and in other Mediterranean regions [S. equorum was observed in Spanish dry-cured salami [S. saprophyticus was the dominant species in artisanal Argentinean sausage [To investigate their genetic relatedness, 28 biotypes were clustered by UPGMA; dendogram from  regions ,20. The d salami ,21,22 and salami , whereas strains , S. epidS. capitis strains. Only the 18% and 25% of strains showed weak proteolysis against sarcoplasmic or myofibrillar proteins, respectively. Contrary to our results, Cachaldora et al. [S. equorum the less lipolytic strains , while the embP gene was only found in the negative biofilm producer S. pasteuri UC7551  and sea (50%) determinants among CNS strains isolated from commercial and artisanal salami.The enterotoxigenic potential of CNS has always been a subject of controversy ,31. The S. equorum strains and S. pasteuri UC7551 were sensitive to all antibiotics considered in this study. Moreover, multidrug resistance (MDR) was observed in more than half (53.6%) of CNS analyzed, with at least one strain of each species showing resistance to two or three antibiotics  was found between phenotypic AR for oxacillin, ampicillin, tetracycline and aminoglycosides with the presence of their respective genetic determinant h et al.  and Fijah et al. , who didy et al. , Landetay et al. , and mory et al. , reportedetected ,38. On tas found . Althougl origin , Rebecchl origin  reportednorA gene was observed; in fact, this genetic determinant was detected only in S. epidermidis UC7564. Chajecka-Wierzchowska et al. [norA gene in S. aureus codifies for the efflux pumps, providing resistance to a variety of fluoroquinolones [mecA and norA genes was found . Based on the technological and safety profile obtained from the autochthonous CNS, the most relevant technological performance was observed in


Leishmania major Yakimoff & Shokhor and transmitted by Phlebotomus papatasi (Scopoli) is a public health concern in Morocco. The disease is endemic mainly in pre-Saharan regions on the southern slope of the High Atlas Mountains. The northern slope of the High Atlas Mountains and the arid plains of central Morocco remain non-endemic and are currently considered high risk for ZCL. Here we investigate and compare the population genetic structure of P. papatasi populations sampled in various habitats in historical foci and non-endemic ZCL areas. A fragment of the mtDNA cytochrome oxidase I (COI) gene was amplified and sequenced in 59 individuals from 10 P. papatasi populations. Haplotype diversity was probed, a median-joining network was generated (FST) and molecular variance (AMOVA) were analyzed. Overall, we identified 28 haplotypes with 32 distinct segregating sites, of which seven are parsimony informative. The rate of private haplotypes was high; 20 haplotypes (71.4%) are private ones and exclusive to a single population. The phylogenetic tree and the network reconstructed highlight a genetic structuration of these populations in two well defined groups: Ouarzazate (or endemic areas) and Non-Ouarzazate (or nonendemic areas). These groups are separated by the High Atlas Mountains. Overall, our study highlights differences in terms of population genetics between ZCL endemic and non-endemic areas. To what extent such differences would impact the transmission of L. major by natural P. papatasi population remains to be investigated.Zoonotic cutaneous leishmaniasis (ZCL) caused by  Phlebotomus papatasi  is the proven vector of L. major Yakimoff & Shokhor 1914, which causes zoonotic cutaneous leishmaniasis (ZCL) in North Africa and other geographic areas, with rodents acting as reservoir hosts [L. major zymodeme MON 25 is transmitted by P. papatasi [Meriones shawi grandis is the reservoir host [L. major as well as favorable eco-climatic conditions. These areas should therefore be classified as at high risk for ZCL [The Leishmaniases are a group of parasitic diseases caused by various species of protozoa belonging to the genus Leishmania  and transmitted by several species of phlebotomine sand fly vectors  ,2,3. Phlir hosts . In Moro for ZCL ,11.Phlebotomus papatasi present a wide geographical distribution [P. papatasi exceeds that of L. major and its reservoir hosts, extending to southern Europe and the eastern regions of the Indian subcontinent, where it is often abundant in domestic and peri-domestic biotopes far from rodent colonies [L. major between rodents and from rodents to humans [cytochrome b mtDNA sequences showed that Phlebotomus papatasi populations are relatively homogeneous [P. papatasi is also demonstrated with another molecular marker, the second internal transcribed spacer ribosomal DNA (rDNA ITS2) [P. papatasi populations from the North African and the Mediterranean subregions have provided evidence for genetic differentiation [P. papatasi populations at a microgeographic scale [ribution  and adapribution ,14,15,16colonies . It is ho humans . The undogeneous  despite ogeneous ,21,22,23NA ITS2) . Microsantiation ,26 and hic scale .P. papatasi are one of the most frequently trapped sandfly species [P. papatasi are recorded in urban areas and in unhealthy suburbs of large cities such as Marrakech and Agadir, particularly in wild dumps and areas of sewage application, far away from human dwellings [P. papatasi are frequently recorded, with symmetrical anomalies being observed in the lateral lobes, along with abnormal styles, which correlate with environmental human-caused disturbances [cytochrome c oxidase I gene as a molecular marker, we investigated the genetic structure of wild P. papatasi populations sampled in endemic and non-endemic areas for ZCL in Morocco.In Morocco,  species ,28,29,30 species . High dewellings . Phenotyurbances . Here, uL. major in Ouarzazate, southern Morocco. The second one, in central and southwestern Morocco, is a non-endemic area for L. major. The two areas are separated by the High Atlas Mountains (Sand fly specimens were collected between April and November from 2006 to 2008. They were captured using CDC-miniature light traps placed in houses and sticky traps (castor oil papers) placed in intra-domestic, peri-domestic, and sylvatic habitats . Collectountains .The head and the abdominal Terminalia of each specimen were dissected with sterilized microneedles and mounted on a slide in Canada balsam . MorpholTotal DNA was extracted using the QIAmp kit , according to the manufacturer’s protocol.COI gene [Polymerase chain reaction (PCR) was performed in a total volume of 50 µL. The primers LepF (5′-ATTCAACCAATCATAAAGATATTGG-3′) and LepR (5′-TAAACTTCTGGATGTCCAAAAAATCA-3′) were used to amplify a 681 bp fragment of the COI gene . Each amhttp://ribosome.toulouse.inra.fr/multalin/multalin) using the cytochrome c oxidase I gene as a molecular marker. Gaps were treated as missing data. Sequence analysis and Kimura’s two-parameter genetic distance calculation were performed using MEGA v. 4.0 [FST) [Fst values > 0.25 indicate strong genetic differentiation. The median-joining algorithm, implemented in the software package NETWORK v. 3.1 [https://www.phylogeny.fr/) [P. papatasi COI gene sequences from Morocco, or from Spain (LC090048.1), Serbia (KY848828.1), Turkey (MH780862.1), Cyprus (MF968974.1), and of P. ariasi (KJ1481169.1), P. sergenti (KC755398.1), or P. perniciosus (KJ1481136.1).Sequences (681 bp) were manually edited and aligned with the online service Multalin   . SignifiK v. 3.1 ,42, was eny.fr/) . AlignmeCOI gene produced a band of approximately 700 bp in a 1% agarose gel. The 681 bp sequences were manually trimmed, resulting in good quality sequences with a length of 654 bp. Sequence alignment of the 654 bp COI sequences did not identify any nucleotide insertion/deletions (indels), and sequences can be fully translated with the Drosophila mitochondrial code. Seven parsimony-informative sites were recorded in more than one haplotype. All sequences are available in GenBank under accession numbers MT074053-054, MT075056-57, MT075059-61, MT075064-75, MT075057, and MT075078-85. Sequence analysis delineated the presence of 28 haplotypes with 32 distinct segregating sites  are high. Finally, Tajima’s D and Fu’s Fs neutrality values are negative and significant in all sampled populations. These pinpoint probable episodes of population expansion . See The PCR amplification of the 3′ end of the mitochondrial ng sites . A relatP. papatasi samples are presented in P. papatasi sampled in endemic and non-endemic areas. The other 24 of the 28 identified haplotypes (85.7%) are private ones. Thirty private haplotypes  are associated with Ouarzazate, five  with Nfifa, one (H9) with Labrouj, and one with Taferiat (H5). The rate of private haplotypes is high in populations from the ZCL-endemic areas of Ouarzazate. Here, 13 of the 15 identified haplotypes (86.6%) are unique. In non-endemic areas, the corresponding rate is only of 42.8% (6 out of 14 haplotypes).The 28 haplotypes recorded within the 59 Phlebotomus papatasi belongs to the Phlebotomus subgenus. The consensus tree  ariasi, P. (Laroussius) perniciosus or P. (Transphlebotomus) sergenti which belong to the Laroussius and Transphebotomus subgenus. Overall, within P. papatasi samples collected in Morocco, the presence of at least two P. papatasi populations that fall into two groups which show some genetic relatedness is depicted . Most specimens sampled in the endemic area of Ouarzazate are grouped together and constitute the “Ouarzazate group”, with a bootstrap value of 56%. This group is interrupted by two individuals from Labrouj in the central Morocco area. The other specimens from non-endemic areas, or “Non-Ouarzazate” areas, constitute a geographically mixed group.sus tree A recoverThe median-joining analysis displays a highly structured network. Within each major clade, the internal and terminal nodes can be interpreted as old and recently derived haplotypes. The network display places all haplotypes into two groups: “Ouarzazate” or endemic; and “Non-Ouarzazate” or non-endemic. Within the Non-Ouarzazate group, two groups (named Mix1 and Mix2) are distinguishable . These tP. papatasi was defined by F statistics. AMOVA results presented in The genetic differentiation among populations of FST values were calculated in a pairwise manner for the main populations . With a STF value of 0.31026, the matrix of significance for 110 permutations reached p = 0.05, which highlights some genetic differentiation between the two groups that were analyzed using our 59 samples.The L. major is largely widespread. The central Moroccan plateaus overlooking the northern slope of the High Atlas, notably the Haouz plain and Chichaoua, remain non-endemic but, according to entomological data, is considered high risk.Natural barriers like high mountains play an important role for limiting dispersal and isolating populations. The Atlas, a natural barrier, may have played a role in limiting the diffusion of zoonotic cutaneous leishmaniasis to the Saharan regions of Morocco. This Atlas consists of three parallel ranges: the Anti-Atlas in the southwest, which gives way to the High Atlas, where the country’s highest peak rises to 4165 m; the Middle Atlas is located northeast of the High Atlas. Rolling plateaus east of the mountains gradually lead into the Sahara, located in southeastern and southern Morocco. Here, in sub-Saharan areas, cytochrome c oxidase subunit I (Cox1 or COI) as molecular marker to gather clues on the influence of the Atlas Mountains on the evolutionary history of P. papatasi populations in Morocco. The cytochrome c oxidase gene is used as a DNA barcode to guide the identification of new animal species and to delineate cryptic taxon and the association between their life stages [Lutzomyia longipalpis [L. umbratilis sibling species [COI in combination with the cytochrome b (Cytb) gene can differentiate two closely related species, P. chabaudi and P. riouxi [COI gene in Morocco, reminiscent of a genetic structuration in P. papatasi populations. Samples collected were indistinguishable by morphological observation and genomic analysis of the 18srRNA (data not shown). The haplotype distributions displayed in the median-joining network as well as the phylogenetic analysis supported the notion of a geographic clustering of P. papatasi, in Morocco. Two groups, with distinct genetic structures, can be differentiated. The first one encompasses P. papatasi samples from the ZCL endemic areas of Ouarzazate. The second one, designated the “Non-Ouarzazate” group, includes a mix of individuals from non-endemic areas sampled on the northern slope of the High Atlas Mountains and the arid plains of central Morocco, as well as samples from endemic areas.We have investigated the genetic structure of 10 populations collected from both endemic and non-endemic areas for zoonotic cutaneous leishmaniasis using the e stages ,45,46. Tgipalpis , and to  species . In Old . riouxi . Here, wP. papatasi populations. A complex scenario of P. papatasi populations might have taken place in Morocco, with expansion events allowing the appearance of multiple haplotype subgroups. The Ouarzazate population of P. papatasi, which derives from the populations of the southern slope of the High Atlas Mountains, is expected to have experienced a period of isolation due to the physical barrier of the Atlas Mountains. Then, population expansion and genetic diversification might have occurred, maybe following a climatic shift. These assumptions are supported by the high rate of private haplotypes recorded in this group (87%), which could reflect some local adaptation. The diversity of the ecological conditions may affect the distribution and density of P. papatasi, including the bioclimate, anthropization, urbanization, and elevation [P. papatasi density is high and in mountainous localities where P. papatasi is less frequent [P. papatasi from endemic areas, and the more distant Mix2. The Mix1 and Mix2 populations are not geographically isolated by the High Atlas Mountains. It therefore appears that ecological habitats do not play a key role in shaping the genetic variation of P. papatasi populations. This finding agrees with other reports from Hamarsheh et al. [P. papatasi linked to latitude rather than to ecological environment.The median-joining network, as well as nucleotide analyses, provide additional evidence of a probable demographic expansion of levation . The biofrequent . Despiteh et al. ,25,26 thP. papatasi populations and therefore have had an impact on the current and future ZCL incidence in Morocco. To this aim, the use of microsatellite markers may help to more precisely define the demographic history of Moroccoan P. papatasi populations.It will be now of interest to gather more precise information on the evolutionary scenarios that have governed the dispersal of COI gene), highlights clear differences in the genetic structure between P. papatasi populations living the L. major-endemic area of Ouarzazate and those of non-endemic areas of central Morocco. In addition, morphological differences have been reported in P. papatasi populations in Morocco [L. major by its specific vector P. papatasi [PpGalec) with a mutation affecting an amino acid involved in substrate recognition. Although performed on a limited number of samples, this further highlights an unequal distribution of this mutation in populations from endemic and non-endemic areas for zoonotic cutaneous leishmaniasis [P. papatasi populations from the southern and the northern slope of the High Atlas Mountains in Morocco.This work, based on a maternally inherited marker (the  Morocco . Indeed, Morocco , which ppapatasi . This stmaniasis . All the