Clonal Types Research Articles

Development of an algorithm for code clone detection in source code based on abstract syntax tree

The object of research of this work is the algorithm for searching for duplicates in the program code based on the Abstract Syntaxes Tree (AST). The main tasks solved within the framework of this study are the detection of duplicate code and the search for vulnerabilities in the program code. The obtained results showed that the proposed algorithm is resistant to type 1 and 2 clones, which means its effectiveness in detecting similar code fragments with identical or variant text. However, for type 3 and 4 clones, the algorithm may show less efficiency due to the change in the AST structure for these types of clones. Experimental studies of the proposed algorithm showed that the algorithm can detect matches between unrelated files due to the presence of typical AST chains present in many programs. This can lead to a certain level of false positives in the detection of duplicates. Testing of the algorithm in the task of finding vulnerabilities showed that: The best recognition is observed for the «SQL injection» vulnerability, but it also has the highest number of false positives. Memory leak and null pointer dereferencing vulnerabilities are detected with equal effectiveness and false positives. «Buffer overflow» has the lowest recognition rate but fewer false positives compared to «SQL injection». The study showed that the use of AST allows for the effective detection of duplicate code and vulnerabilities in the software code. The developed tool can help software developers reduce maintenance efforts, improve code quality, and ensure software product security.

Reduced Diversities and Clonally Expanded Sequences of T-Cell Receptors in Patients With Essential Hypertension and Subclinical Carotid Atherosclerosis.

It has long been hypothesized that the abnormal immune responses contribute to the essential hypertension (EH) and its subclinical target organ damage (STOD). However, the mechanism is unclear. This study aimed at exploring the potential association with abnormal T-cell responses and EH, STOD, and early atherosclerosis in patients with EH. This cross-sectional study included 146 patients with EH and 73 age-matched normotensive individuals. The expressed peripheral TCR (T-cell receptor) β repertoire was analyzed by high through-put sequencing. The TCRβ repertoires of the patients with EH were significantly different, with significantly elevated certain TCR beta variable (TRBV) and joint (TRBJ) gene usages, significantly reduced TCRβ diversity indexes (diversity 50s) and numbers of total TCRβ clonal types, significantly elevated percentages of the biggest TCRβ clones and numbers of clones accounting >0.1% sequences, compared with those in the normotensive controls. Decreased diversity 50s and increased biggest TCRβ clone percentages were independently correlated with carotid intima-media thickness and subclinical carotid atherosclerosis (SCA) in the patients with EH. Moreover, the diversity 50s were further significantly reduced and the biggest TCRβ clone percentages were significantly increased in the patients with EH with SCA (n=89) comparing to the patients with EH/patients without SCA (n=57), and in patients with EH/SCA with carotid plaque (n=22) comparing to patients with EH/SCA/patients without carotid plaque (n=67). Importantly, specific TCRβ clones were identified in different subgroups of the patients with EH. These results reveal that abnormal T-cell responses may play important roles in the progression of EH and its SCA, especially the formation of carotid plaque. URL: https://www.chictr.org.cn; Unique identifier: ChiCTR2100054414.

Microevolution of Pseudomonas aeruginosa in the airways of people with cystic fibrosis.

The chronic infections of cystic fibrosis (CF) airways with Pseudomonas aeruginosa are a paradigm of how environmental bacteria can conquer, adapt, and persist in an atypical habitat and successfully evade defense mechanisms and chemotherapy in a susceptible host. The within-host evolution of intraclonal diversity has been examined by whole-genome sequencing, phenotyping, and competitive fitness experiments of serial P. aeruginosa isolates collected from CF airways since onset of colonization for a period of up to 40 years. The spectrum of de novo mutations and the adaptation of phenotype and fitness of the bacterial progeny were more influenced by the living conditions in the CF lung than by the clone type of their ancestor and its genetic repertoire.

Whole-genome sequencing of Chlamydia psittaci from Australasian avian hosts: A genomics approach to a pathogen that still ruffles feathers.

Chlamydia psittaci is a globally distributed veterinary pathogen with zoonotic potential. Although C. psittaci infections have been reported in various hosts, isolation and culture of Chlamydia is challenging, hampering efforts to produce contemporary global C. psittaci genomes. This is particularly evident in the lack of avian C. psittaci genomes from Australia and New Zealand. In this study, we used culture-independent probe-based whole-genome sequencing to expand the global C. psittaci genome catalogue. Here, we provide new C. psittaci genomes from two pigeons, six psittacines, and novel hosts such as the Australian bustard (Ardeotis australis) and sooty shearwater (Ardenna grisea) from Australia and New Zealand. We also evaluated C. psittaci genetic diversity using multilocus sequence typing (MLST) and major outer membrane protein (ompA) genotyping on additional C. psittaci-positive samples from various captive avian hosts and field isolates from Australasia. We showed that the first C. psittaci genomes sequenced from New Zealand parrots and pigeons belong to the clonal sequence type (ST)24 and diverse 'pigeon-type' ST27 clade, respectively. Australian parrot-derived strains also clustered in the ST24 group, whereas the novel ST332 strain from the Australian bustard clustered in a genetically diverse clade of strains from a fulmar, parrot, and livestock. MLST and ompA genotyping revealed ST24/ompA genotype A in wild and captive parrots and a sooty shearwater, whilst 'pigeon-types' (ST27/35 and ompA genotypes B/E) were found in pigeons and other atypical hosts, such as captive parrots, a little blue penguin/Kororā (Eudyptula minor) and a zebra finch (Taeniopygia guttata castanotis) from Australia and New Zealand. This study provides new insights into the global phylogenomic diversity of C. psittaci and further demonstrates the multi-host generalist capacity of this pathogen.

GENERATION OF NOVEL ANTIBODY CANDIDATES USING TRANSFORMER AND GAN-BASED DEEP LEARNING ARTIFICIAL INTELLIGENCE

Abstract Introduction Conventional library-based antibody display can only explore a small fraction of the sequences generated from animal immunization, not even to exhaust the potential sequence diversity that can be turned into antibody therapies. This is because screening for antibody is limited to sequences that can be displayed, which only constitute a subset of the entire sequences generated by B cells, whereas screening for antibody directly from single B cells can be costly. Here, we introduce a novel Artificial Intelligence-enabling tool to navigate antibody discovery from a broader range of search space with reduced cost. We trained a transformer-based model from sequences of an immunized library to cluster the clones and a generative adversarial network (GAN)-based model to generate novel sequences that can be potentially developed into antibody therapies. Background and significance One limitation in the early discovery of antibody is the number of functional candidates that can be selected. Our work provides an AI-enabling tool to discover and generate a panel of antibodies of differentiated binding strengths to a broad range of epitopes to ensure functional coverage. Methods & Results We extracted 104 sequences from the FACS-enriched yeast pool from a fully immunized alpaca (Lama pacos) using Next Generation Sequencing, from which we assembled 103 unique sdAb sequences. We fine-tuned a transformer-based deep learning model, which was previously trained from our dataset containing 100,000 antibody sequences, on such pre-processed sdAb sequences giving representation that correlates to the sequence homology for the clustering of clonal types. We postulate such representation also encodes long-range amino acid interactions in the 3D structure, making the accuracy exceeds the performance of bioinformatics-based primary sequence homology analysis. This process is fully automated and optimized to require minima computational resources. We selected 15 candidates from AI-clustered clonal groups and experimentally measured their binding activity. Kd of 12 candidates were of 10−9 affinity and 1 candidates were of 10−8 affinity, the rest one candidate was non-binding (hence a hit rate of 87%). The large sequence diversity of the CDR3 show these nanobodies are potentially good binders for a wide range of epitopes. We generated a CDR-diversifying virtual library (103) of each binding candidate by training a GAN-based models using the sequences of the same clonal group of the binder sequences. This method incorporates the probability of amino acid residues on each specific location that provides a more precise mutagenesis route than PCR-based affinity maturation. The generated sequences provided a wider CDR sequence diversity for the selection of antibodies of differentiated affinity and epitopes, which could generate candidates of different functionality. Conclusion Antibody discovery is a central step in early drug development that identification of a wide range of functional candidates could increase the success rate and reduce risks in later developments. We built an AI-enabling tool for the searching and generation of functional antibodies from animal immunization library. We believe this technology would help deliver candidates of fine-tuned affinity and functionality.

Antimicrobial resistance, molecular characteristics, virulence and pathogenicity of bla NDM-1-positive Enterobacter cloacae.

Introduction. The bla NDM-1 -positive Enterobacter cloacae has led to limited therapeutic options for clinical treatment.Hypothesis/Gap Statement. Analysing the antimicrobial resistance and molecular typing of bla NDM-1-positive E. cloacae is of great significance. Meanwhile, the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae remains unclear and should be assessed.Aim. To understand bla NDM-1-positive E. cloacae from different perspectives.Methodology. The PCR was used to screen bla NDM-1-positive E. cloacae, then, antimicrobial susceptibility tests and multilocus sequence typing (MLST) were performed on them; sixty-nine strains of bla NDM-1-negative E. cloacae were collected as the controls, 28 pairs of virulence-related genes' carriage and biofilm-forming ability were detected for preliminary evaluation of the virulence phenotype of the strains; to gain insight into the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae, the bla NDM-1-positive E. cloacae T2 (NDM-1), the T2 bla NDM-1 knockout strain (ΔNDM-1) and ATCC13047 (ST) were studied, compared the motility, anti-serum killing ability, and virulence to cells. Then, the mice intraperitoneal infection model was established, the survival curve, histopathological characteristics, bacterial load in spleen and the contents of cytokines were compared.Results. (1) Thirty-five bla NDM-1-positive E. cloacae exhibited multidrug resistance. MLST distinguished 12 STs, ST74 was the most common clonal type (11/35), followed by ST114 (10/35). (2) The detection rates of virulence genes clpB, icmf, VasD/Lip and acrA in the bla NDM-1-positive E. cloacae were significantly higher than those in bla NDM-1-negative E. cloacae (P<0.05), while there was no significant difference in the amount of biofilm formation between two groups. (3) The presence of bla NDM-1 gene attenuated the motility diameter of E. cloacae, but had no significant effect on their ability to resist serum killing, and the virulence to cells. The survival rate, histopathological changes, bacterial load in spleen and inflammatory cytokines were not significantly affected.Conclusions. (1) The bla NDM-1-positive E. cloacae exhibited multidrug resistance, and the MLST typing was mainly ST74 and ST114, with a small-scale clonal spread of the ST114 strain in the hospital NICU ward. (2) The bla NDM-1 gene did not affect the virulence and pathogenicity of E. cloacae.

Association of Virulence, Biofilm, and Antimicrobial Resistance Genes with Specific Clonal Complex Types of Listeria monocytogenes.

Precise classification of foodborne pathogen Listeria monocytogenes is a necessity in efficient foodborne disease surveillance, outbreak detection, and source tracking throughout the food chain. In this study, a total of 150 L. monocytogenes isolates from various food products, food processing environments, and clinical sources were investigated for variations in virulence, biofilm formation, and the presence of antimicrobial resistance genes based on their Whole-Genome Sequences. Clonal complex (CC) determination based on Multi-Locus Sequence Typing (MLST) revealed twenty-eight CC-types including eight isolates representing novel CC-types. The eight isolates comprising the novel CC-types share the majority of the known (cold and acid) stress tolerance genes and are all genetic lineage II, serogroup 1/2a-3a. Pan-genome-wide association analysis by Scoary using Fisher's exact test identified eleven genes specifically associated with clinical isolates. Screening for the presence of antimicrobial and virulence genes using the ABRicate tool uncovered variations in the presence of Listeria Pathogenicity Islands (LIPIs) and other known virulence genes. Specifically, the distributions of actA, ecbA, inlF, inlJ, lapB, LIPI-3, and vip genes across isolates were found to be significantly CC-dependent while the presence of ami, inlF, inlJ, and LIPI-3 was associated with clinical isolates specifically. In addition, Roary-derived phylogenetic grouping based on Antimicrobial-Resistant Genes (AMRs) revealed that the thiol transferase (FosX) gene was present in all lineage I isolates, and the presence of the lincomycin resistance ABC-F-type ribosomal protection protein (lmo0919_fam) was also genetic-lineage-dependent. More importantly, the genes found to be specific to CC-type were consistent when a validation analysis was performed with fully assembled, high-quality complete L. monocytogenes genome sequences (n = 247) extracted from the National Centre for Biotechnology Information (NCBI) microbial genomes database. This work highlights the usefulness of MLST-based CC typing using the Whole-Genome Sequence as a tool in classifying isolates.

The Slaughterhouse as Hotspot of CC1 and CC6 Listeria monocytogenes Strains with Hypervirulent Profiles in an Integrated Poultry Chain of Italy.

In Europe, very few studies are available regarding the diversity of Listeria monocytogenes (L. monocytogenes) clonal complexes (CCs) and sequence types (ST) in poultry and on the related typing of isolates using whole genome sequencing (WGS). In this study, we used a WGS approach to type 122 L. monocytogenes strains isolated from chicken neck skin samples collected in two different slaughterhouses of an integrated Italian poultry company. The studied strains were classified into five CCs: CC1-ST1 (21.3%), CC6-ST6 (22.9%), CC9-ST9 (44.2%), CC121-ST121 (10.6%) and CC193-ST193 (0.8%). CC1 and CC6 strains presented a virulence gene profile composed of 60 virulence genes and including the Listeria Pathogenicity Island 3, aut_IVb, gltA and gltB. According to cgMLST and SNPs analysis, long-term persistent clusters belonging to CC1 and CC6 were found in one of the two slaughterhouses. The reasons mediating the persistence of these CCs (up to 20 months) remain to be elucidated, and may involve the presence and the expression of stress response and environmental adaptation genes including heavy metals resistance genes (cadAC, arsBC, CsoR-copA-copZ), multidrug efflux pumps (mrpABCEF, EmrB, mepA, bmrA, bmr3, norm), cold-shock tolerance (cspD) and biofilm-formation determinants (lmo0673, lmo2504, luxS, recO). These findings indicated a serious risk of poultry finished products contamination with hypervirulent L. monocytogenes clones and raised concern for the consumer health. In addition to the AMR genes norB, mprF, lin and fosX, ubiquitous in L. monocytogenes strains, we also identified parC for quinolones, msrA for macrolides and tetA for tetracyclines. Although the phenotypical expression of these AMR genes was not tested, none of them is known to confer resistance to the primary antibiotics used to treat listeriosis The obtained results increase the data on the L. monocytogenes clones circulating in Italy and in particular in the poultry chain.

Escherichia coli ST2797 Is Abundant in Wastewater and Might Be a Novel Emerging Extended-Spectrum Beta-Lactamase E. coli.

The increasing prevalence of antibiotic-resistant bacteria is an emerging threat to global health. The analysis of antibiotic-resistant enterobacteria in wastewater can indicate the prevalence and spread of certain clonal groups of multiresistant bacteria. In a previous study of Escherichia coli that were isolated from a pump station in Norway over 15 months, we found a recurring E. coli clone that was resistant to trimethoprim, ampicillin, and tetracycline in 201 of 3,123 analyzed isolates (6.1%). 11 representative isolates were subjected to whole-genome sequencing and were found to belong to the MLST ST2797 E. coli clone with plasmids carrying resistance genes, including blaTEM-1B, sul2, dfrA7, and tetB. A phenotypic comparison of the ST2797 isolates with the uropathogenic ST131 and ST648 that were repeatedly identified in the same wastewater samples revealed that the ST2797 isolates exhibited a comparable capacity for temporal survival in wastewater, greater biofilm formation, and similar potential for the colonization of mammalian epithelial cells. ST2797 has been isolated from humans and has been found to carry extended spectrum β-lactamase (ESBL) genes in other studies, suggesting that this clonal type is an emerging ESBL E. coli. Collectively, these findings show that ST2797 was more ubiquitous in the studied wastewater than were the infamous ST131 and ST648 and that ST2797 may have similar abilities to survive in the environment and cause infections in humans. IMPORTANCE The incidence of drug-resistant bacteria found in the environment is increasing together with the levels of antibiotic-resistant bacteria that cause infections. The COVID-19 pandemic has shed new light on the importance of monitoring emerging threats and finding early warning systems. Therefore, to mitigate the antimicrobial resistance burden, the monitoring and early identification of antibiotic-resistant bacteria in hot spots, such as wastewater treatment plants, are required to combat the occurrence and spread of antibiotic-resistant bacteria. Here, we applied a PhenePlate system as a phenotypic screening method for genomic surveillance and discovered a dominant and persistent E. coli clone ST2797 with a multidrug resistance pattern and equivalent phenotypic characteristics to those of the major pandemic lineages, namely, ST131 and ST648, which frequently carry ESBL genes. This study highlights the continuous surveillance and report of multidrug resistant bacteria with the potential to spread in One Health settings.

First Isolation of Methicillin-Resistant Livestock-Associated Staphylococcus aureus CC398 and CC1 in Intensive Pig Production Farms in Argentina.

Since the mid-2000s, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been identified among pigs worldwide, CC398 being the most relevant LA-MRSA clone. In the present work, nasal swabs were taken from healthy pigs of different age categories (25 to 154 days) from 2019 to 2021 in four intensive farms located in three provinces of Argentina. The aim of the present study was to characterize the first LA-MRSA isolates that colonized healthy fattening pigs in Argentina in terms of their resistance phenotype and genotype and to know the circulating clones in the country. Antimicrobial susceptibility, presence of the mecA gene and PCR screening of CC398 were evaluated in all the isolates. They were resistant to cefoxitin, penicillin, tetracycline, chloramphenicol and ciprofloxacin but susceptible to nitrofurantoin, rifampicin, vancomycin and linezolid. Furthermore, 79% were resistant to clindamycin and lincomycin, 68% to erythromycin, 58% to gentamicin and 37% to trimethoprim/sulfamethoxazole. All the isolates were multidrug resistant. The clonal relation was assessed by SmaI-PFGE (pulsed-field gel electrophoresis) and a representative isolate of each PFGE type was whole genome sequenced by Illumina. MLST (multilocus sequence typing), resistance and virulence genes and SCCmec typing were performed on sequenced isolates. The isolates were differentiated in three clonal types by PFGE, and they belonged to sequence-type ST398 (58%) and ST9, CC1 (42%) by MLST. SCCmec typeV and several resistance genes detected showed complete correlation with resistance phenotypes. The present study revealed that LA-MRSA colonizing healthy pigs in Argentina belongs to CC398 and CC1, two MRSA lineages frequently associated to pigs in other countries.

Identification of a Specific Biomarker of Acinetobacter baumannii Global Clone 1 by Machine Learning and PCR Related to Metabolic Fitness of ESKAPE Pathogens.

Since the emergence of high-risk clones worldwide, constant investigations have been undertaken to comprehend the molecular basis that led to their prevalent dissemination in nosocomial settings over time. So far, the complex and multifactorial genetic traits of this type of epidemic clones have allowed only the identification of biomarkers with low specificity. A machine learning algorithm was able to recognize unequivocally a biomarker for early and accurate detection of Acinetobacter baumannii global clone 1 (GC1), one of the most disseminated high-risk clones. A support vector machine model identified the U1 sequence with a length of 367 nucleotides that matched a fragment of the moaCB gene, which encodes the molybdenum cofactor biosynthesis C and B proteins. U1 differentiates specifically between A. baumannii GC1 and non-GC1 strains, becoming a suitable biomarker capable of being translated into clinical settings as a molecular typing method for early diagnosis based on PCR as shown here. Since the metabolic pathways of Mo enzymes have been recognized as putative therapeutic targets for ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, our findings highlight that machine learning can also be useful in knowledge gaps of high-risk clones and provides noteworthy support to the literature to identify relevant nosocomial biomarkers for other multidrug-resistant high-risk clones. IMPORTANCE A. baumannii GC1 is an important high-risk clone that rapidly develops extreme drug resistance in the nosocomial niche. Furthermore, several strains have been identified worldwide in environmental samples, exacerbating the risk of human interactions. Early diagnosis is mandatory to limit its dissemination and to outline appropriate antibiotic stewardship schedules. A region with a length of 367 bp (U1) within the moaCB gene that is not subjected to lateral genetic transfer or to antibiotic pressures was successfully found by a support vector machine model that predicts A. baumannii GC1 strains. At the same time, research on the group of Mo enzymes proposed this metabolic pathway related to the superbug's metabolism as a potential future drug target site for ESKAPE pathogens due to its central role in bacterial fitness during infection. These findings confirm that machine learning used for the identification of biomarkers of high-risk lineages can also serve to identify putative novel therapeutic target sites.

Molecular characterisation of the Australian and New Zealand livestock Chlamydia pecorum strains confirms novel but clonal ST23 in association with ovine foetal loss

Chlamydia pecorum is a veterinary pathogen associated with abortions and perinatal mortality in sheep. Recent studies investigating foetal and perinatal lamb mortality in sheep from Australia and New Zealand identified C. pecorum clonal sequence type (ST)23 strains in aborted and stillborn lambs. Presently, there is limited genotypic information on C. pecorum strains associated with reproductive disease, although whole genome sequencing (WGS) of one abortigenic ST23 C. pecorum strain identified unique features, including a deletion in the CDS1 locus of the chlamydial plasmid. We applied WGS on two ST23 strains detected in aborted and stillborn lambs from Australia and used phylogenetic and comparative analyses to compare these to the other available C. pecorum genomes. To re-evaluate the genetic diversity of contemporary strains, we applied C. pecorum genotyping, and chlamydial plasmid sequencing to a range of C. pecorum positive samples and isolates from ewes, aborted foetuses and stillborn lambs, cattle and a goat from diverse geographical regions across Australia and New Zealand.The two new C. pecorum genomes are nearly identical to the genome of the Australian abortigenic strain including the unique deletion in the chlamydial plasmid. Genotyping revealed that these novel C. pecorum ST23 strains are widespread and associated with sheep abortions on Australian and New Zealand farms. In addition, a goat C. pecorum strain (denoted ST 304) from New Zealand was also characterised. This study expands the C. pecorum genome catalogue and describes a comprehensive molecular characterisation of the novel livestock ST23 strains associated with foetal and lamb mortality.

High prevalence of clonally related ST182 NDM-1-producing Enterobacter cloacae complex clinical isolates in Greece

NDM-type metallo-β-lactamase (MBL)-producing Enterobacterales remain uncommon in the European region, especially among species other than Klebsiella pneumoniae and Escherichia coli. The aim of this study was to describe epidemiological and molecular characteristics of a widespread NDM-1-producing Enterobacter cloacae complex outbreak in Greece. Over a 6-year period (March 2016-March 2022), a retrospective study was conducted in a tertiary care Greek hospital. Ninety single-patient carbapenem-non-susceptible E. cloacae complex clinical isolates were recovered consecutively. The isolates were subjected to further investigation, including antimicrobial susceptibility testing and combined disc tests for carbapenemase production, polymerase chain reaction and sequencing for resistance genes, molecular fingerprinting by pulsed-field gel electrophoresis (PFGE), plasmid profiling, replicon typing, conjugation experiments, genotyping by multi-locus sequence typing (MLST), whole-genome sequencing and phylogenetic analysis. Phenotypic and molecular testing confirmed the presence of blaNDM-1 in 47 (52.2%) of the E. cloacae complex isolates. MLST analysis clustered all but four of the NDM-1 producers into a single MLST sequence type (ST182), whereas single isolates belonged to different sequence types (ST190, ST269, ST443 and ST743). PFGE analysis revealed that ST182 isolates were clustered into a single clonal type, with three subtypes, which differed from the clonal types detected among the remaining carbapenem non-susceptible E. cloacae complex isolates identified during the study period. All ST182 blaNDM-1-carrying isolates also harboured the blaACT-16 AmpC gene, while the blaESBL, blaOXA-1 and blaTEM-1 genes were detected in most cases. In all clonal isolates, the blaNDM-1 gene was located on an IncA/C-type plasmid, and flanked upstream by an ISAba125 element and downstream by bleMBL. Conjugation experiments failed to produce carbapenem-resistant transconjugants, indicating a low dynamic for horizontal gene transfer. Application of enforced infection control measures led to the absence of new NDM-positive cases for periods of time during the survey. This study represents the largest clonal outbreak of NDM-producing E. cloacae complex in Europe.

Yield and yield components of superior cocoa (Theobroma cocoa L.) clones rejuvenated by ring budding technique

The ring budding technique gave a higher suitability for rejuvenation of cocoa plants compared to side cleft grafting it can be combined with the use of superior clones for optimal production. However, information related to this is still limited. The research aimed to determine the effects of ring budding technique on yield components and yields of three types of superior cocoa clones. The experiment was arranged in a Nested Design. The first factor is the type of vegetative propagation technique, consisting of side cleft grafting and ring budding. The second factor was clones, which consisted of clones KKM 22, RCC 70 and RCC 71. Clones were nested in vegetative propagation. The research was conducted from September 2020 to January 2021 at the Pagilaran Company. Observations were made on several yield component variables. The data were analyzed using Analysis of Variance (ANOVA) and continued with the Least Significant Difference Test (LSD) at the 5%. The results showed that the number of pods and the weight of beans per cocoa stand were significantly higher in the ring budding technique than the side cleft grafting. This condition caused the dry bean weight per hectare much higher as well. The three cacao clones, which were rejuvenated by ring budding, gave a good response and high yields. Meanwhile, only KKM 2 gave good response and high yield after treated with side cleft grafting technique, while RCC 70 and RCC 71 clones gave low yields. The KKM 22 clone is the best option in terms of rehabilitation with ring budding and side cleft grafting.

Whole-genome sequencing reveals high-risk clones of Pseudomonas aeruginosa in Guangdong, China.

The ever-increasing prevalence of infections produced by multidrug-resistant or extensively drug-resistant Pseudomonas aeruginosa is commonly linked to a limited number of aptly-named epidemical 'high-risk clones' that are widespread among and within hospitals worldwide. The emergence of new potential high-risk clone strains in hospitals highlights the need to better and further understand the underlying genetic mechanisms for their emergence and success. P. aeruginosa related high-risk clones have been sporadically found in China, their genome sequences have rarely been described. Therefore, the large-scale sequencing of multidrug-resistance high-risk clone strains will help us to understand the emergence and transmission of antibiotic resistances in P. aeruginosa high-risk clones. In this study, 212 P. aeruginosa strains were isolated from 2 tertiary hospitals within 3 years (2018-2020) in Guangdong Province, China. Whole-genome sequencing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing were applied to analyze the genomic epidemiology of P. aeruginosa in this region. We found that up to 130 (61.32%) of the isolates were shown to be multidrug resistant, and 196 (92.45%) isolates were Carbapenem-Resistant Pseudomonas aeruginosa. MLST analysis demonstrated high diversity of sequence types, and 18 reported international high-risk clones were identified. Furthermore, we discovered the co-presence of exoU and exoS genes in 5 collected strains. This study enhances insight into the regional research of molecular epidemiology and antimicrobial resistance of P. aeruginosa in China. The high diversity of clone types and regional genome characteristics can serve as a theoretical reference for public health policies and help guide measures for the prevention and control of P. aeruginosa resistance.

Phylogroup-specific variation shapes the clustering of antimicrobial resistance genes and defence systems across regions of genome plasticity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C). Here, we assessed phylogroup-specific evolutionary dynamics across available and also new P.aeruginosa genomes. In this genomic analysis, we first generated new genome assemblies for 18 strains of the major P.aeruginosa clone type (mPact) panel, comprising a phylogenetically diverse collection of clinical and environmental isolates for this species. Thereafter, we combined these new genomes with 1991 publicly available P.aeruginosa genomes for a phylogenomic and comparative analysis. We specifically explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups. We found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of pangenome families, and show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these cargo genes is often significantly correlated. Moreover, inter- and intra-phylogroup interactions occur at the accessory genome level, suggesting frequent recombination events. Finally, we provide here the mPact panel of diverse P.aeruginosa strains that may serve as a valuable reference for functional analyses. Altogether, our results highlight distinct pangenome characteristics of the P.aeruginosa phylogroups, which are possibly influenced by variation in the abundance of CRISPR-Cas systems and are shaped by the differential distribution of other defence systems and AMR genes. German Science Foundation, Max-Planck Society, Leibniz ScienceCampus Evolutionary Medicine of the Lung, BMBF program Medical Infection Genomics, Kiel Life Science Postdoc Award.

Code Cloning in Smart Contracts on the Ethereum Platform: An Extended Replication Study

Smart contracts are programs deployed on blockchains that run upon meeting predetermined conditions. Once deployed, smart contracts are immutable, thus, defects in the deployed code cannot be fixed. As a consequence, software engineering anti-patterns, such as code cloning, pose a threat to code quality and security if unnoticed before deployment. In this paper, we report on the cloning practices of the Ethereum blockchain platform by analyzing 33,073 smart contracts amounting to over 4MLOC. Prior work reported an unusually high 79.2% of code clones in Ethereum smart contracts. We replicate this study at the conceptual level, i.e., we answer the same research questions by employing different methods. In particular, we analyze clones at the granularity of functions instead of code files, thereby providing a more fine-grained estimate of the clone ratio. Furthermore, we analyze more complex clone types, allowing for a richer analysis of cloning cases. To achieve this finer granularity of cloning analysis, we rely on the NiCad clone detection tool and extend it with support for Solidity, the programming language of the Ethereum platform. Our analysis shows that most findings of the original study hold at the finer granularity of our study as well; but also sheds light on some differences, and contributes new findings. Most notably, we report a 30.13% overall clone ratio, out of which 27.03% are exact duplicates. Our findings motivate improving the reuse mechanisms of Solidity, and in a broader context, of programming languages used for the development of smart contracts. Tool builders and language engineers can use this paper in the design and development of such reuse mechanisms. Business stakeholders can use this paper to better assess the security risks and technical outlooks of blockchain platforms.

Occult Vancomycin-Resistant Enterococcus faecium ST117 Displaying a Highly Mutated vanB2 Operon

Rare information is available on clinical Enterococcus faecium encountered in Sardinia, Italy. This study investigated the antimicrobial susceptibility profiles and genotypic characteristics of E. faecium isolated at the University Hospital of Sassari, Italy, using the Vitek2 system and PCR, MLST, or WGS. Vitek2 revealed two VanB-type vancomycin-resistant Enterococcus faecium (VREfm) isolates (MICs mg/L = 8 and ≥32) but failed to detect vancomycin resistance in one isolate (MIC mg/L ≤ 1) despite positive genotypic confirmation of vanB gene, which proved to be vancomycin resistant by additional phenotypic methods (MICs mg/L = 8). This vanB isolate was able to increase its vancomycin MIC after exposure to vancomycin, unlike the "classic" occult vanB-carrying E. faecium, becoming detectable by Vitek 2 (MICs mg/L ≥ 32). All three E. faecium had highly mutated vanB2 operons, as part of a chromosomally integrated Tn1549 transposon, with common missense mutations in VanH and VanB2 resistance proteins and specific missense mutations in the VanW accessory protein. There were additional missense mutations in VanS, VanH, and VanB proteins in the vanB2-carrying VREfm isolates compared to Vitek2. The molecular typing revealed a polyclonal hospital-associated E. faecium population from Clade A1, and that vanB2-VREfm, and nearly half of vancomycin-susceptible E. faecium (VSEfm) analyzed, belonged to ST117. Based on core genome-MLST, ST117 strains had different clonal types (CT), excluding nosocomial transmission of specific CT. Detecting vanB2-carrying VREfm isolates by Vitek2 may be problematic, and alternative methods are needed to prevent therapeutic failure and spread.

The first genomic insight into Chlamydia psittaci sequence type (ST)24 from a healthy captive psittacine host in Australia demonstrates evolutionary proximity with strains from psittacine, human, and equine hosts

Chlamydia psittaci is a zoonotic pathogen that infects birds, humans, and other mammals. Notably, recent studies suggested the human-to-human transmission of C. psittaci, and this pathogen also causes equine reproductive loss in Australia. Molecular studies in Australia to date have focused on and described clonal sequence type (ST)24 strains infecting horses, wild psittacine, and humans. In contrast, the genetic identity of C. psittaci strains from captive psittacine hosts is scarce. In 2022, C. psittaci was detected in the faeces of a healthy captive blue-fronted parrot (Amazona aestiva). Genomic DNA was extracted and underwent whole-genome sequencing. Here we report the 1,160,701 bp circular chromosome of C. psittaci strain BF_amazon_parrot13 and the 7,553 bp circular plasmid pCpsBF_amazon_parrot13. Initial in silico multi-locus sequence typing and ompA genotyping revealed that BF_amazon_parrot13 belongs to the clonal ST24 lineage and has an ompA genotype A. Further context involved the genomes of 31 published ST24 strains, utilising a single-nucleotide variant (SNV) based clustering approach. Despite temporal, host, and biogeographical separation, a core-genome SNV-based phylogeny revealed that BF_amazon_parrot13 clustered in a distinct subcluster with seven C. psittaci strains from equines in Australia (maximum pairwise distance of 13 SNVs). BF_amazon_parrot13 represents the first complete C. psittaci ST24 genome from a captive psittacine in Australia. Furthermore, by using whole-genome sequencing to coordinate surveillance, we can also learn more about the possible health risks and routes of chlamydia transmission among people, livestock, wild animals, and domesticated animals.

Emergence of Pandemic Clonal Lineage Sequence Types 131 and 69 of Extraintestinal Escherichia coli as a Cause of Meningitis: Is It Time To Revise Molecular Assays?

Two Escherichia coli strains, respectively responsible for neonatal and adult meningitis, were isolated and their phenotypic antibiotic susceptibility and genomic features characterized by whole-genome sequencing (WGS). Multiplex real-time PCR targeting the principal microorganisms involved in meningitis etiology failed to identify either isolate. Afterwards, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify the isolates as E. coli strains. Genomic analysis showed that they belonged to sequence types 131 and 69 (ST131 and ST69). Neither of the isolates harbored the K1 capsular antigen or belonged to other capsular serotypes, but they shared different virulence factors, including ibe genes, responsible for invasion of brain endothelial cells. IMPORTANCE The extraintestinal pathogenic Escherichia coli group is characterized by the presence of uropathogenic E. coli (UPEC), sepsis-associated E. coli (SEPEC), and neonatal meningitis E. coli (NMEC). All of these members exhibit many virulence factors, such as lipopolysaccharides, toxins, iron acquisition factors, invasins, fimbriae, and capsules. Urinary infections are the most common infections caused by this group, followed by globally increasing numbers of both community- and nosocomially acquired bloodstream infections, associated with considerable patient morbidity and mortality. Some lineages tend to become dominant; in addition to enhanced fitness, this epidemiological success stems from increased virulence, antibiotic resistance, gut colonization, and greater host-to-host transmission. Our results underline the importance of continuous surveillance of these new emerging lineages and the need to develop new meningitis molecular assay panels able to identify them.