Human Clinical Samples Research Articles

Antibiotic Resistance Profiles of Pseudomonas aeruginosa Isolates Containing Virulence Genes

Background: A most common opportunistic pathogen, Pseudomonas aeruginosa is present in both humans and animals and responsible for various nosocomial infections and healthcare settings related infections. Different virulence genes like; oprL (membrane lipoprotein L) and toxA (exotoxin A i.e. ETA) in P. aeruginosa, assist in its pathogenicity, toxicity and contribute to high antibiotic resistance. So considering the risk of zoonotic transmission of P. aeruginosa through animal-related foods, this study aimed to monitor the prevalence of oprL and toxA virulence genes and antimicrobial resistance in P. aeruginosa isolates, obtained from animal (Cow’s milk) and human clinical samples. Material and methods: Of the total 120 collected samples for this study, every 60 samples were collected from animals and humans from respective laboratories. Total 76 isolates of P. aeruginosa were isolated and identified by morphological and biochemical tests. The presence of virulence factors like oprL and toxA were evaluated by PCR analysis and antimicrobial resistance was assessed by antibiotic susceptibility test (Kirby-Bauer method). Results: From the total 76, P. aeruginosa isolates obtained from both animal and human isolates, alone presence and coexistence of both toxA and oprL genes in P. aeruginosa isolates; were detected in PCR analysis. PCR analysis results showed in P. aeruginosa isolates, alone distribution of toxA and oprL genes is, 75% and 54.16% in animals, and 84.61% and 80.76% in humans, respectively. The coexistence of both genes was 37.50% and 40.32% in animals and human isolates, along with high antibiotic resistance in most P. aeruginosa isolates. Conclusion: Therefore, this study suggested PCR analysis can be used for fast and specific detection of oprL and toxA genes in P. aeruginosa. Monitoring of these genes can help to prevent the risk of transmission of multi-drug resistant P. aeruginosa, from animals to humans.

Identification and antimicrobial susceptibility of Streptomyces and other unusual Actinobacteria clinical isolates in Spain

Two hundred and eighty-six isolates from human clinical samples were identified between 1996 and 2019 as belonging to 8 families, 19 genera and 88 species of Actinobacteria. The most identified genera were Streptomyces (182 strains from 45 species), Actinomadura (29 strains, 5 species), Nocardiopsis (21 strains, 6 species) and Dietzia (18 strains, 5 species). The rest of the identified genera (15) contained 27 species with 36 isolates. Of the species studied, only 13/88 had been documented previously as isolates from clinical samples, and in some cases, as true pathogens. In this sense, a literature review of the species found in infections or in clinical samples without clear involvement in pathology has been carried out. Finally, the susceptibility to 8 antimicrobial agents has been studied. Streptomyces showed high resistance (80.8%) against cefotaxime and cotrimoxazole (55.5%), and no isolate resistance to amikacin and linezolid have been found. Lower percentages of resistance have been found in other genera, except in Dietzia (100% against cotrimoxazole and 44.4% against erythromycin). The greatest resistance in these genera was to cotrimoxazole (29.8) and erythromycin (27,9%), and no resistance to linezolid has been found in these genera. In Microbispora, Nonomuraea and Umezawaea, no resistant isolates have been found against any antibiotic studied. Only 3/104 isolates were resistant to amikacin in Amycolatopsis, Crossiella, and Micromonosopora. One isolate of Amycolatopsis was resistant to imipenem.

Whole-Genome Analysis of a High-Risk Clone of Klebsiella pneumoniae ST147 Carrying Both mcr-1 and blaNDM-1 Genes in Peru.

The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other β-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

Chemiluminescence Measurement of Reactive Sulfur and Nitrogen Species.

Significance: Reactive sulfur and nitrogen species such as hydrogen sulfide (H2S) and nitric oxide (NO•) are ubiquitous cellular signaling molecules that play central roles in physiology and pathophysiology. A deeper understanding of these signaling pathways will offer new opportunities for therapeutic treatments and disease management. Recent Advances: Chemiluminescence methods have been fundamental in detecting and measuring biological reactive sulfur and nitrogen species, and new approaches are emerging for imaging these analytes in living intact specimens. Ozone-based and luminol-based chemiluminescence methods have been optimized for quantitative analysis of hydrogen sulfide and nitric oxide in biological samples and tissue homogenates, and caged luciferin and 1,2-dioxetanes are emerging as a versatile approach for monitoring and imaging reactive sulfur and nitrogen species in living cells and animal models. Critical Issues: This review article will cover the major chemiluminescence approaches for detecting, measuring, and imaging reactive sulfur and nitrogen species in biological systems, including a brief history of the development of the most established approaches and highlights of the opportunities provided by emerging approaches. Future Directions: Emerging chemiluminescence approaches offer new opportunities for monitoring and imaging reactive sulfur and nitrogen species in living cells, animals, and human clinical samples. Widespread adoption and translation of these approaches, however, requires an emphasis on rigorous quantitative methods, reproducibility, and effective technology transfer. Antioxid. Redox Signal. 36, 337-353.

Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.

Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry

A striking feature of skin organization is that the extracellular matrix (ECM) occupies a larger volume than the cells. Skin ECM also directly contributes to aging and most cutaneous diseases. In recent years, specific ECM enrichment protocols combined with in silico approaches allowed the proteomic description of the matrisome of various organs and tumor samples. Nevertheless, the skin matrisome remains under-studied and protocols allowing the efficient recovery of the diverse ECM found in skin are still to be described. Here, we compared four protocols allowing the enrichment of ECM proteins from adult mouse back skin and found that all protocols led to a significant enrichment (up to 65%) of matrisome proteins when compared to total skin lysates. The protocols based on decellularization and solubility profiling gave the best results in terms of numbers of proteins identified and confirmed that skin matrisome proteins exhibit very diverse solubility and abundance profiles. We also report the first description of the skin matrisome of healthy adult mice that includes 236 proteins comprising 95 core matrisome proteins and 141 associated matrisome proteins. These results provide a reliable basis for future characterizations of skin ECM proteins and their dysregulations in disease-specific contexts. SignificanceExtracellular matrix proteins are key players in skin physiopathology and have been involved in several diseases such as genetic disorders, wound healing defects, scleroderma and skin carcinoma. However, skin ECM proteins are numerous, diverse and challenging to analyze by mass spectrometry due to the multiplicity of their post-translational modifications and to the heterogeneity of their solubility profiles. Here, we performed the thorough evaluation of four ECM enrichment protocols compatible with the proteomic analysis of mouse back skin and provide the first description of the adult mouse skin matrisome in homeostasis conditions. Our work will greatly facilitate the future characterization of skin ECM alterations in preclinical mouse models and will inspire new optimizations to analyze the skin matrisome of other species and of human clinical samples.

Subtype heterogeneity and epigenetic convergence in neuroendocrine prostate cancer

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.

Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples

In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.

Inclusion of double helix structural oligonucleotide (STexS) results in an enhance of SNP specificity in PCR

Genetic mutations such as single nucleotide polymorphisms (SNP) are known as one of the most common forms which related to various genetic disorders and cancers. Among of the methods developed for efficient detection of such SNP, polymerase chain reaction (PCR) methods are widely used worldwide for its cost and viable advantages. However, the technique to discriminate small amounts of SNP mixed in abundant normal DNA is incomplete due to intrinsic technical problems of PCR such as amplification occurring even in 3’mismatched cases because of high enzyme activity of DNA polymerases. To overcome the issue, specifically designed PCR platform, STexS (SNP typing with excellent specificity) using double stranded oligonucleotides was implemented as a means to emphasize the amplification of SNP templates by decreasing unwanted amplification of 3’mismatched DNA copies. In this study, the results indicate several EGFR mutations were easily detected specifically utilizing the STexS platform. Further trials show the novel method works effectively to discriminate mutations in not only general allele specific (AS)-PCRs, but also amplification refractory mutation system (ARMS)-PCR. The STexS platform will give aid in PCRs targeting potential SNPs or genetically mutated biomarkers in human clinical samples.

Integrative analysis of potential biomarkers and immune cell infiltration in Parkinson’s disease

BackgroundParkinson’s disease (PD) is a common neurodegenerative disease in the elderly population. However, there are no reliable diagnostic biomarkers for PD, and the pathogenesis of PD still needs further study. The aim of the current study was to identify potential biomarkers and explore the pathogenesis of PD. MethodsWe conducted an integrative analysis of messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA) expression profiles of PD using data from the Gene Expression Omnibus (GEO). The GSE110720, GSE110719 and GSE133347 data sets were selected and analysed. Gene ontology (GO) enrichment and gene set variation analysis (GSVA) were performed for annotation, visualization, and integrated discovery. Protein–protein interaction (PPI) and competing endogenous RNA (ceRNA) networks were constructed, and hub genes were identified. Meanwhile, the immune infiltration analysis of hub genes was analysed. Moreover, receiver operating characteristic (ROC) curves were generated to verify the diagnostic value of the differentially expressed genes (DEGs). Finally, the genes with high area under the curve (AUC) values were verified by human samples. ResultsWe identified 464 DEGs closely related to PD, including 154 mRNAs, 134 miRNAs, and 176 lncRNAs. The GO analyses indicated that changes in PD were mainly enriched in receptor ligand activity and cytokine receptor binding. The KEGG enrichment analysis showed that these DEGs were significantly involved in cytokine-cytokine receptor interactions, signalling pathways regulating the pluripotency of stem cells and Th17 cell differentiation. GSVA suggested that growth factor binding, IL2-stat5 signalling, and IL6-jak-stat3 signalling were crucial in the development of PD. A total of five hub genes (NPBWR2, CXCL10, CXCL5, S1PR5, and GALR1) were selected via the PPI network. A ceRNA network of the CXCL5, CXCL10 and S1PR5 genes was constructed, and target genes of the three genes were screened. The immune infiltration analysis showed that there were significant differences in a variety of immune cells between the hub genes. The expression of DEGs was validated in clinical human samples by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression levels of hsa-miR6895–5p, hsa-miR6791–5p, hsa-miR518f-5p, hsa-miR455–3p and TEKT4P2 were decreased, while the levels of TPTE2P6 were increased in human samples. These findings are consistent with the bioinformatics analysis results. ConclusionWe found that the immune inflammatory response and immune cell regulation were involved in the pathogenesis of PD. Five hub genes involved in the immune infiltration biological processes of PD based on bioinformatics. We verified the DEGs with significant differences by qRT-PCR. These findings might provide new insight into the pathogenesis of PD and the development of diagnostic and therapeutic strategies for PD.

Detection of Antibiotic Resistance Genes Among Multiple Drug Resistant Pseudomonas Aeruginosa Isolated from Clinical Sources in Selected Health Institutions in Kwara State.

Pseudomonas aeruginosa (P. aeruginosa) is a frequent nosocomial pathogen that causes severe diseases in many clinical and community settings. Strains of P. aeruginosa are associated with increased morbidity, mortality and healthcare costs. The rapid emergence of antimicrobial resistance among these strains is a public health crisis. Moreover, there is a paucity of data on the characterization of P. aeruginosa isolates from human clinical samples in Kwara State. The objectives of this study are to investigate the occurrence of metallo β-lactamase enzyme, multiple antibiotic resistant P. aeruginosa among clinical samples and detection of antibiotic resistance genes among them. Two hundred and thirty-five samples comprising of 145 males and 90 females human clinical specimens were collected aseptically from five selected health institutions within Kwara State, Nigeria. The samples were cultured immediately using standard microbiological procedures. Multiple drug resistance patterns of the micro-organisms to different antibiotics were determined using the Bauer Kirby disc diffusion technique. Metallo β-lactamase production was determined using E - test strip and the DNA samples of the multiple resistant P. aeruginosa strains were extracted and subjected to Polymerase Chain Reaction (PCR) for resistant genes determination. Data were subjected to descriptive statistics using Statistical Package for Social Sciences (SPSS) software. A total of 145 isolates were identified for P. aeruginosa from the clinical samples. Thirty were positive for metallo β-lactamase production; 11 (8%) males and 19 (13%) females. Absolute resistance to ceftazidime (100%), gentamicin (100%), ceftriaxone (100%) were observed while low resistance to ciprofloxacin (12.4%), piperacillin (6.9%) and imipenem (6.9%). All isolates were sensitive to colistin. The prevalence of various encoding genes blaVIM, , blaCTX-M and blaTEM were 34.4%, 46.7%, 16.7% and 37.7% respectively. This study has shown that there is a high occurrence of metallo β-lactamase enzyme producing and antibiotic-resistant strains of P. aeruginosa in clinical specimens from the studied area. Necessary measures must, therefore be implemented to stop the problems of this antibiotic resistance.

EDCs and female fertility - what can we learn from human clinical samples?

EDCs and female fertility - what can we learn from human clinical samples?

Two-Color Duplex Platform for Point-of-Care Differential Detection of Malaria and Typhoid Fever.

Malaria and typhoid fever are two febrile illnesses prevalent in the tropics that often present overlapping symptoms. In this work, we demonstrate an optical reader-based diagnostics platform for rapid codetection and quantification of two antigen targets: lipopolysaccharide (LPS) for typhoid fever and plasmodium lactate dehydrogenase (pLDH) for malaria infections. We report a limit of detection (LoD) of 5 ng/mL for LPS and 10 ng/mL for pLDH in a spiked serum test. We also validated the duplex test's performance of differentiating malaria infection, typhoid fever infection, and coinfection by testing clinical samples in human serum. Our platform provides the potential for further multiplexing by encoding different color codes to various detection targets. The rapid result (∼15 min), low cost (∼$2), and minimal volume requirement for human serum clinical samples (4 μL) of our diagnostic platform offer great potential for deployment in resource-limited settings to help distinguish common causes for acute febrile illnesses at the point-of-need.

An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.

BSCI-03. Adaptation of colorectal cancer cells to the brain microenvironment: The role of IRS2

Abstract Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM). Yet, molecular mechanisms supporting it are unknown. We aimed to explore drivers enabling adaptation of CRC cells to the brain and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of IRS2 gene amplification was observed in 13% of BM, compared to only 3% of primary tumors or other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BM. We constructed an in-vitro system mimicking the brain microenvironment using cultured human astrocytes or their conditioned media. Under these conditions, IRS2-overexpressed CRC cells survived better and formed larger 3D spheres. IRS2-silenced CRC cells showed a mirror image. Moreover, in an intracranial CRC BM mouse model, IRS2-overexpressed cells generated larger brain lesions, while silencing IRS2 dramatically decreased tumor outgrowth and extended survival. Interestingly, transcriptomic analysis revealed enrichment of oxidative phosphorylation (OXPHOS) and Wnt/β-catenin pathways by IRS2. Indeed, IRS2-expressing cells showed increased mitochondrial activity and glycolysis-independent viability. Furthermore, IRS2-expressing cells had increased β-catenin transcriptional activity. Interestingly, β-catenin or IRS2 inhibition (using NT219) in IRS2-expressing cells decreased their viability, β-catenin transcriptional activity, and OXPHOS gene expression, suggesting involvement of IRS2 in modulating OXPHOS through β-catenin. β-catenin is known to confer 5-FU resistance; consequently, we showed that combination of 5-FU and NT219 worked in synergy, inhibited the formation of BM, and extended animal survival. These data reveal the unique genomic profile of CRC BM and suggest IRS2 inhibition as a novel target for treatment of these patients.

Modeling Neuroimmune Interactions in Human Subjects and Animal Models to Predict Subtype-Specific Multidrug Treatments for Gulf War Illness.

Gulf War Illness (GWI) is a persistent chronic neuroinflammatory illness exacerbated by external stressors and characterized by fatigue, musculoskeletal pain, cognitive, and neurological problems linked to underlying immunological dysfunction for which there is no known treatment. As the immune system and the brain communicate through several signaling pathways, including the hypothalamic–pituitary–adrenal (HPA) axis, it underlies many of the behavioral and physiological responses to stressors via blood-borne mediators, such as cytokines, chemokines, and hormones. Signaling by these molecules is mediated by the semipermeable blood–brain barrier (BBB) made up of a monocellular layer forming an integral part of the neuroimmune axis. BBB permeability can be altered and even diminished by both external factors (e.g., chemical agents) and internal conditions (e.g., acute or chronic stress, or cross-signaling from the hypothalamic–pituitary–gonadal (HPG) axis). Such a complex network of regulatory interactions that possess feed-forward and feedback connections can have multiple response dynamics that may include several stable homeostatic states beyond normal health. Here we compare immune and hormone measures in the blood of human clinical samples and mouse models of Gulf War Illness (GWI) subtyped by exposure to traumatic stress for subtyping this complex illness. We do this via constructing a detailed logic model of HPA–HPG–Immune regulatory behavior that also considers signaling pathways across the BBB to neuronal–glial interactions within the brain. We apply conditional interactions to model the effects of changes in BBB permeability. Several stable states are identified in the system beyond typical health. Following alignment of the human and mouse blood profiles in the context of the model, mouse brain sample measures were used to infer the neuroinflammatory state in human GWI and perform treatment simulations using a genetic algorithm to optimize the Monte Carlo simulations of the putative treatment strategies aimed at returning the ill system back to health. We identify several ideal multi-intervention strategies and potential drug candidates that may be used to treat chronic neuroinflammation in GWI.

WD40 repeat 43 mediates cell survival, proliferation, migration and invasion via vimentin in colorectal cancer

BackgroundWD40 repeat (WDR)43 is an RNA-binding protein that belongs to the WDR domain protein family. Its biological function is largely unclear, particularly in colorectal cancer (CRC).MethodsIn the present study, we searched the TCGA database and found the correlation between WDR43 and CRC. Subsequently, the high expression of WDR43 in human clinical samples of CRC was validated and we further examined the biological functions of it in CRC cells. Finally, we explored potential downstream proteins or pathways and established subcutaneous xenograft model to verify our findings.ResultsImmunohistochemistry of 16 patient specimens confirmed that the expression of WDR43 was elevated in CRC. WDR43 knockdown was shown to increase apoptosis and inhibit the proliferation, migration and invasion of CRC cells in vitro and reduce tumorigenesis in animal models. In addition, it was found that WDR43 knockdown inhibited vimentin (VIM) expression in CRC cells and overexpression of VIM can partially reverse the effects of WDR43 both in vitro and in vivo.ConclusionIn conclusion, the role of WDR43 in the occurrence and development of CRC was investigated in the present study. WDR43 may serve as a valuable biomarker and provide new options for the diagnosis and treatment of colorectal cancer.

Reduction trend of mcr-1 circulation in Emilia-Romagna Region, Italy.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018-2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018-2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Comparative genomics of Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) reveals lineage-specific host adaptation of ST432.

Unlike most Salmonella enterica subsp. diarizonae , which are predominantly associated with cold-blooded animals such as reptiles, the serovar IIIb 61:k:1,5,(7) (termed SASd) is regarded as host-adapted to sheep. The bacterium is rarely associated with disease in humans but, nevertheless, SASd isolates are sporadically obtained from human clinical samples. It is unclear whether these transmissions are directly linked to sheep or whether transmissions may, for example, occur through other domestic animals also carrying SASd. For this reason, we utilized whole-genome sequencing to investigate a set of 119 diverse SASd isolates, including sheep-associated and human-associated isolates, as well as isolates obtained from other matrices. We discovered that serovar IIIb 61:k:1,5,(7) is composed of two distinct lineages defined by their sequence types ST432 and ST439. These two lineages are distinguished by a number of genetic features, as well as their prevalence and reservoir. ST432 appears to be the more prevalent sequence type, with the majority of isolates investigated in this study belonging to ST432. In contrast, only a small number of isolates were attributed to ST439. While ST432 isolates were of sheep, human or other origin, all ST439 isolates with source information available, were obtained from human clinical samples. Regarding their genetic features, lineage ST432 shows increased pseudogenization, harbours a virB/D4 plasmid that encodes a type IV secretion system (T4SS) and does not possess the iro gene cluster, which encodes a salmochelin siderophore for iron acquisition. These characteristics likely contribute to the ability of ST432 to persistently colonize the intestines of sheep. Furthermore, we found isolates of the lineage ST432 to be highly clonal, with little variation over the sampling period of almost 20 years. We conclude from the genomic comparisons that SASd underlies a microevolutionary process and that it is specifically lineage ST432 that should be considered as host-adapted to sheep.

Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species.

BackgroundDipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. In Leishmania, DPPIII, was reported with other peptidases to play a significant role in parasites’ growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene for Leishmania species identification and for phylogeny, and thus for diagnostic and molecular epidemiology studies of Old World Leishmania species.MethodologyOrthologous DDPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assays was developed and seventy-two Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples.FindingsSequence analysis showed 478 variable sites (302 being parsimony informative). Test of natural selection (dN-dS) (-0.164, SE = 0.013) inferred a negative selection, characteristic of essential genes, corroborating the DPPIII importance for parasite survival. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment. Sequence analyses and phylogenies confirmed occurrence of 6 clusters congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and one to the L. infantum and L. donovani species complex.A PCR-RFLP algorithm for Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica and L. turanica and the L. (Sauroleishmania) L. tarentolae. The species L. donovani, and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples.ConclusionThe study demonstrates that DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. Thus, it can contribute to evidence-based disease control and surveillance.