IS6110 Insertion Research Articles

Genome analysis of tigecycline-resistant Acinetobacter baumannii reveals nosocomial lineage shifts and novel resistance mechanisms.

To investigate the characteristics and clonal dynamics of tigecycline-resistant Acinetobacter baumannii (TRAB) isolates from a Chinese hospital from 2016 to 2021. A total of 64 TRAB isolates were screened and WGS was performed. Phylogenetic analysis and non-polymorphic mutation analysis were used to analyse their clonal dynamics and tigecycline resistance-related mutations. RT-PCR was used to analyse the expression of the resistance-nodulation cell-division (RND) efflux pump genes adeB and adeJ. Gene cloning was used to explore the effect of tet(39) variants on tigecycline resistance. Most TRAB isolates were found to be MDR, with 95% (61/64) of the isolates showing resistance to carbapenems. These TRAB isolates were classified into three primary genetic clusters based on core-genome SNPs. The KL2 cluster persisted throughout the study period, whereas the KL7 cluster emerged in 2019 and became the dominant clone. The KL7 cluster carried more antimicrobial resistance genes than the other two clusters. The predominant tigecycline resistance mechanism of the KL2 cluster and KL7 cluster was IS insertion in adeN (82.1%, 23/28) and genetic alterations in adeS (76.2%, 16/21), respectively. Eleven novel AdeS mutations were identified associated with elevated AdeB expression and tigecycline resistance. Moreover, we characterized a plasmid-borne tet(39) variant with an Ala-36-Thr substitution that synergizes with the RND efflux pump to confer high-level tigecycline resistance. This work provides important insights into the diverse mechanisms associated with tigecycline resistance in A. baumannii, highlighting a pressing need for further monitoring of ST2-KL7 A. baumannii in clinical settings.

The IncC and IncX1 resistance plasmids present in multi-drug resistant Escherichia coli strains isolated from poultry manure in Poland

The study describes the whole-genome sequencing of two antibiotic-resistant representative Escherichia coli strains, isolated from poultry manure in 2020. The samples were obtained from a commercial chicken meat production facility in Poland. The antibiotic resistance profile was characterized by co-resistance to β-lactam antibiotics, aminoglycosides, and fluoroquinolones. The three identified resistance plasmids (R-plasmids), pECmdr13.2, pECmdr13.3, and pECmdr14.1, harbored various genes conferring resistance to tetracyclines (tetR[A]) for, aminoglycoside (aph, aac, and aad families), β-lactam (blaCMY-2, blaTEM-176), sulfonamide (sul1, sul2), fluoroquinolone (qnrS1), and phenicol (floR). These plasmids, which have not been previously reported in Poland, were found to carry IS26 insertion elements, the intI1-integrase gene, and conjugal transfer genes, facilitating horizontal gene transfer. Plasmids pECmdr13.2 and pECmdr14.1 also possessed a mercury resistance gene operon related to transposon Tn6196; this promotes plasmid persistence even without antibiotic selection pressure due to co-selection mechanisms such as co-resistance. The chicken manure–derived plasmids belonged to the IncX1 (narrow host range) and IncC (broad host range) incompatibility groups. Similar plasmids have been identified in various environments, clinical isolates, and farm animals, including cattle, swine, and poultry. This study holds significant importance for the One Health approach, as it highlights the potential for antibiotic-resistant bacteria from livestock and food sources, particularly E. coli, to transfer through the food chain to humans and vice versa.

Whole-genome sequencing of clinical isolates of Citrobacter Europaeus in China carrying blaOXA−48 and blaNDM−1

ObjectiveTo analyze the clinical infection characteristics and genetic environments of resistance genes in carbapenem-resistant Citrobacter europaeus using whole-genome sequencing.MethodsThe susceptibility of two clinical isolates of C. europaeus (WF0003 and WF1643) to 24 antimicrobial agents was assessed using the BD Phoenix™ M50 System and Kirby-Bauer (K-B) disk-diffusion method. Whole-genome sequencing was performed on the Illumina and Nanopore platforms, and ABRicate software was used to predict resistance and virulence genes of carbapenem-resistant C. europaeus. The characteristics of plasmids carrying carbapenem-resistance genes and their genetic environments were analyzed. Single nucleotide polymorphisms were used to construct a phylogenetic tree to analyze the homology of these two C. europaeus strains with ten strains of C. europaeus in the NCBI database.ResultsThe two strains of carbapenem-resistant C. europaeus are resistant to various antimicrobial agents, particularly carbapenems and β-lactams. WF0003 carries blaNDM− 1, which is located on an IncX3 plasmid that has high homology to the pNDM-HN380 plasmid. blaNDM− 1 is located on a truncated Tn125. It differs from Tn125 by the insertion of IS5 in the upstream ISAba125 and the deletion of the downstream ISAba125, which is replaced by IS26. WF1643 carries blaOXA− 48 in a Tn1999 transposon on the IncL/M plasmid, carrying only that single drug resistance gene. Homology analysis of these two strains of C. europaeus with ten C. europaeus strains in the NCBI database revealed that the 12 strains can be classified into three clades, with both WF0003 and WF1643 in the B clade.ConclusionTo the best of our knowledge, this is the first study to report an IncX3 plasmid carrying blaNDM− 1 in C. europaeus in China. C. europaeus strains harboring carbapenem-resistance genes are concerning in relation to the spread of antimicrobial resistance, and the presence of carbapenem-resistance genes in C. europaeus should be continuously monitored.

The effect of DNA-binding proteins on insertion sequence element transposition upstream of the bgl operon in Escherichia coli.

The bglGFB operon in Escherichia coli K-12 strain BW25113, encoding the proteins necessary for the uptake and metabolism of β-glucosides, is normally not expressed. Insertion of either IS1 or IS5 upstream of the bgl promoter activates expression of the operon only when the cell is starving in the presence of a β-glucoside, drastically increasing transcription and allowing the cell to survive and grow using this carbon source. Details surrounding the exact mechanism and regulation of the IS insertional event remain unclear. In this work, the role of several DNA-binding proteins in how they affect the rate of insertion upstream of bgl are examined via mutation assays and protocols measuring transcription. Both Crp and IHF exert a positive effect on insertional Bgl+ mutations when present, active, and functional in the cell. Our results characterize IHF's effect in conjunction with other mutations, show that IHF's effect on IS insertion into bgl also affects other operons, and indicate that it may exert its effect by binding to and altering the DNA conformation of IS1 and IS5 in their native locations, rather than by directly influencing transposase gene expression. In contrast, the cAMP-CRP complex acts directly upon the bgl operon by binding upstream of the promoter, presumably altering local DNA into a conformation that enhances IS insertion.

Accuracy of molecular diagnostic assays for detection of Mycobacterium bovis: A systematic review and meta-analysis

Bovine tuberculosis (bovine TB) is a chronic wasting disease of cattle caused primarily by Mycobacterium bovis. Controlling bovine TB requires highly sensitive, specific, quick, and reliable diagnostic methods. This systematic review and meta-analysis evaluated molecular diagnostic tests for M. bovis detection to inform the selection of the most viable assay. On a per-test basis, loop-mediated isothermal amplification (LAMP) showed the highest overall sensitivity of 99.0% [95% CI: 86.2%-99.9%] and specificity of 99.8% [95% CI: 96.2%-100.00%]. Quantitative real-time polymerase chain reaction (qPCR) outperformed conventional PCR and nested PCR (nPCR) with a diagnostic specificity of 96.6% [95% CI: 88.9%-99.0%], while the diagnostic sensitivity of 70.8% [95% CI: 58.6–80.5%] was comparable to that of nPCR at 71.4% [95% CI: 60.7–80.2%]. Test sensitivity was higher with the input of milk samples (90.9% [95% CI: 56.0%-98.7%]), while specificity improved with tests based on major M. bovis antigens (97.8% [95% CI: 92.3%-99.4%]), the IS6110 insertion sequence (95.4% [95% CI: 87.6%-98.4%]), and the RD4 gene (90.7% [95% CI: 52.2%-98.9%]). The design of the currently available molecular diagnostic assays, while mostly based on nonspecific gene targets, prevents them from being accurate enough to diagnose M. bovis infections in cattle, despite their promise. Future assay development should focus on the RD4 region since it is the only target identified by genome sequence data as being distinctive for detecting M. bovis. The availability of a sufficiently accurate diagnostic test combined with the routine screening of milk samples can decrease the risk of zoonotic transmissions of M. bovis.

Molecular characterization of lugdunin inactivation mechanisms and their association with Staphylococcus lugdunensis genetic types

Background and purposeOur previous studies showed that lugdunin activities are associated with Staphylococcus lugdunensis genotypes, and most isolates do not exhibit lugdunin activity. As a continuation of our previous analysis, we focused on the reasons for defects in lugdunin production in S. lugdunensis clinical isolates. MethodsA comparative analysis of 36 S. lugdunensis whole genome sequencing data revealed three major mutation types, unknown deletion mechanism that caused most of lug operon genes lost, mobile genetic element (MGE) insertion, and nonsense mutations, which potentially damaged lugdunin production. A total of 152 S. lugdunensis clinical isolates belonging to lugdunin nonproducers were further examined for the above three mutation types. PCR products were sequenced to examine these variations. ResultsForty-six of the 152 isolates were CRISPR-Cas IIC isolates, including 26 ST27, 14 ST4, and 6 ST29 isolates; further investigation confirmed that all of their lug operons had lost almost all lug operon genes except lugM. An IS256 insertion in lugA was identified in 16 isolates, and most isolates (15 over 16) belonged to ST3. In addition, three nonsense mutations caused by single nucleotide substitutions (an adenine deletion in lugB at the 361th and 1219th nucleotides and an adenine deletion in lugC at the 1612nd nucleotide) that were frequently observed among 36 S. lugdunensis whole genome sequencing data were further observed in our clinical isolates. These three nonsense mutations were frequently found in most of CRISPR-Cas IIIA strains, especially in ST6 isolates. ConclusionOur findings suggest that the mechanisms affecting lugdunin production are associated with S. lugdunensis molecular types.

Genomic analyses of an Escherichia coli and Klebsiella pneumoniae urinary tract co‐infection using long‐read nanopore sequencing

AbstractEscherichia coli and Klebsiella pneumoniae isolates presenting with the same antimicrobial susceptibility profile were recovered from the same catheter sample of urine (CSU). Both strains were recovered from a patient with a long‐standing indwelling urinary catheter. Each isolate had its DNA extracted following culture. Nanopore long‐read sequencing was used to build the plasmids and chromosomes from each strain to closure to discern the potential horizontal propagation of resistance‐encoding plasmids and the relationship between resistance genes and insertion sequences. Plasmids derived from resistance strains in the urinary microbiota remain poorly characterized. The same 11 antimicrobial resistance (AMR) genes were found in plasmids from each strain. The 185,239‐bp FIB(K) pKBM1, from the K. pneumoniae strain, additionally encoded the five AMR genes: sul2, strA, strB, blaTEM‐1B, and blaCTX‐M‐15. A multimeric array of AMR genes and IS26 insertion sequences were found in the plasmids from both isolates. Both plasmids from each isolate were similar. Horizontal transfer of plasmids, followed by subsequent plasmid rearrangement, is likely to have occurred during infection. Furthermore, the resistance region in the plasmids shared similarity against the internationally prevalent plasmid, pKPN3‐307_typeA, commonly identified in K. pneumoniae ST307. Biofilm formation in catheterized patients may allow close cell contact between strains. Horizontal propagation of resistance genes may occur, leading to polymicrobial infections.

Genomic Characterization of IS6110 Insertions in Mycobacterium orygis.

Mycobacterium orygis, a subspecies of the Mycobacterium tuberculosis complex (MTBC), has emerged as a significant concern in the context of One Health, with implications for zoonosis or zooanthroponosis or both. MTBC strains are characterized by the unique insertion element IS6110, which is widely used as a diagnostic marker. IS6110 transposition drives genetic modifications in MTBC, imparting genome plasticity and profound biological consequences. While IS6110 insertions are customarily found in the MTBC genomes, the evolutionary trajectory of strains seems to correlate with the number of IS6110 copies, indicating enhanced adaptability with increasing copy numbers. Here, we present a comprehensive analysis of IS6110 insertions in the M. orygis genome, utilizing ISMapper, and elucidate their genetic consequences in promoting successful host adaptation. Our study encompasses a panel of 67 paired-end reads, comprising 11 isolates from our laboratory and 56 sequences downloaded from public databases. Among these sequences, 91% exhibited high-copy, 4.5% low-copy, and 4.5% lacked IS6110 insertions. We identified 255 insertion loci, including 141 intragenic and 114 intergenic insertions. Most of these loci were either unique or shared among a limited number of isolates, potentially influencing strain behavior. Furthermore, we conducted gene ontology and pathway analysis, using eggNOG-mapper 5.0, on the protein sequences disrupted by IS6110 insertions, revealing 63 genes involved in diverse functions of Gene Ontology and 45 genes participating in various KEGG pathways. Our findings offer novel insights into IS6110 insertions, their preferential insertion regions, and their impact on metabolic processes and pathways, providing valuable knowledge on the genetic changes underpinning IS6110 transposition in M. orygis.

Molecular epidemiological monitoring of the tuberculosis pathogen in the Arkhangelsk region

Introduction. Against the background of improvement of the main epidemiological indicators (morbidity and mortality) for tuberculosis in the Arkhangelsk region, the proportion of newly diagnosed tuberculosis patients with multidrug-resistant pathogen (MDR-TB) increased from 18.7% in 2002 to 33.8% in 2018.
 The purpose of this study was the genotypic characterization of Mycobacterium tuberculosis strains obtained from newly diagnosed tuberculosis patients in the Arkhangelsk region in 2018.
 Materials and methods. 89 M. tuberculosis strains isolated in 2018 from newly diagnosed tuberculosis patients were studied. Beijing genotype, its clusters B0/W148 and Central Asian/Russian were determined by PCR detection of the specific markers: IS6110 insertions in the dnaA-dnaN region, mutations in codons 48 of the mutT4 gene (CGG GGG) and 58 of the mutT2 gene (GGA CGA), IS6110 insertions in the Rv2664 region-Rv2665 and Rv1359-Rv1360, substitutions G A in the sigE gene. Non-Beijing strains were spoligotyped.
 Results. Drug resistance was detected in 41.6% (37/89), MDR — in 33.7% of strains. In 90% (27/30) of MDR strains, resistance to rifampicin and isoniazid was due to rpoB Ser531Leu and katG Ser315Thr mutations. Following M. tuberculosis genotypes were identified: Beijing (67.4%), T (14.6%), Ural (4.5%), Haarlem (4.5%), LAM (2.3%) and CAS1-Delhi (1.1%). Among the Beijing strains, clusters Central-Asian/Russian (60%; 36/60) and B0/W148 (30%; 18/60) prevailed. The majority of MDR strains belonged to the Beijing family (93.3%; 28/30), of which 64.3% (18/28) and 21.4% (6/28) belonged to clusters B0/W148 and Central-Asian/Russian, respectively.
 Conclusion. In heterogeneous population of the causative agent of tuberculosis in the Arkhangelsk region, the most common strains were those of the Beijing genotype; in 2018 its share increased to 67.4% (40.4% in 1998–1999). Among MDR strains, the proportion of Beijing reached 93.3%, of which more than half (64.3%) belonged to the epidemiologically and clinically significant in Russia cluster B0/W148.

The Mechanism of Tigecycline Resistance in Acinetobacter baumannii Revealed by Proteomic and Genomic Analysis.

The mechanism of tigecycline resistance in A. baumannii remains largely unclear. In this study, we selected a tigecycline-resistant and a tigecycline-susceptible strain from a tigecycline-susceptible and a resistant strain, respectively. Proteomic and genomic analyses were performed to elucidate the variations associated with tigecycline resistance. Our study showed proteins associated with efflux pump, biofilm formation, iron acquisition, stress response, and metabolic ability are upregulated in tigecycline resistant strains, and efflux pump should be the key mechanism for tigecycline resistance. By genomic analysis, we found several changes in the genome that can explain the increased level of efflux pump, including the loss of the global negative regulator hns in the plasmid and the disruption of the hns gene and acrR gene on the chromosome by the insertion of IS5. Collectively, we not only revealed the phenomenon that the efflux pump is mainly responsible for tigecycline resistance, but also highlighted the mechanism at the genomic level, which will help in understanding the resistance mechanism in detail and provide clues for the treatment of clinical multiple drug-resistant A. baumannii.

Complete Genetic Analysis of Plasmids Carrying Multiple Resistance, Virulence, and Phage-Like Genes in Foodborne Escherichia coli Isolate.

Bacterial antimicrobial resistance, especially phenotypic resistance to multiple drugs (MDR), has posed a serious threat to public health worldwide. To clarify the mechanism of transmission of multidrug resistance encoding plasmids in Enterobacterales, all seven plasmids of an Escherichia coli (E. coli) strain 1108 obtained from a chicken meat sample were extracted and sequenced by Illumina Nextseq 500 and MinION platforms. Plasmids in strain 1108 possessed 16 known antimicrobial resistance genes, with p1108-NDM (~97K) being the most variable plasmid. The multidrug resistance region of p1108-NDM was punctuated by eight IS26 insertion sequences; thus, four MDR regions were found in the backbone of this plasmid. The plasmid p1108-MCR (~65K) was found to lack the ISApl1 element and harbor the blaCTX-M-64-ISEcp1 transposition unit. Moreover, the ISEcp1-blaCMY-2 transposition unit was found in plasmid p1108-CMY2 (~98K), whereas plasmid p1108-emrB (~102K) was associated with resistance to erythromycin (emrB) and streptomycin (aadA22). p1108-IncY (96K) was a phage P1-like plasmid, while p1108-IncFIB (~194K) was found to harbor a virulence region similar to ColV plasmids, and they were found to encode a conserved conjugative transfer protein but harbor no resistance genes. Finally, no mobile element and resistant genes were found in p1108-ColV (~2K). Carriage of mcr-1-encoding elements in carbapenemase-producing Escherichia coli will potentially render all antimicrobial treatment regimens ineffective. Enhanced surveillance and effective intervention strategies are urgently needed to control the transmission of such multidrug resistance plasmids. IMPORTANCE Antimicrobial resistance (AMR) has been increasingly prevalent in agricultural and clinical fields. Understanding the genetic environment involved in AMR genes is important for preventing transmission and developing mitigation strategies. In this study, we investigated the genetic features of an E. coli strain (1108) isolated from food product and harboring 16 AMR genes, including blaNDM-1 and mcr-1 genes encoding resistance to last line antibiotics, meropenem, and colistin. Moreover, this strain also carried virulence genes such as iroBCDEN, iucABCD, and iutA. Our findings confirmed that multiple conjugative plasmids that were formed through active recombination and translocation events were associated with transmission of AMR determinants. Our data warrant the continuous monitoring of emergence and further transmission of these important MDR pathogens.

Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism.

Due to the high specificity in targeting DNA and highly convenient programmability, CRISPR-Cas-based antimicrobials applied for eliminating specific strains such as antibiotic-resistant bacteria in the microbiome were gradually developed. However, the generation of escapers makes the elimination efficiency far lower than the acceptable rate (10-8) recommended by the National Institutes of Health. Here, a systematic study was carried out providing insight into the escaping mechanisms in Escherichia coli, and strategies for reducing the escapers were devised accordingly. We first showed an escape rate of 10-5-10-3 in E. coli MG1655 under the editing of pEcCas/pEcgRNA established previously. Detailed analysis of the escapers obtained at ligA site in E. coli MG1655 uncovered that the disruption of cas9 was the main cause of the generation of survivors, notably the frequent insertion of IS5. Hence, the sgRNA was next designed to target the "perpetrator" IS5, and subsequently the killing efficiency was improved 4-fold. Additionally, the escape rate in IS-free E. coli MDS42 was also tested at the ligA site, ∼10-fold decrease compared with MG1655, but the disruption of cas9 was still observed in all survivors manifested in the form of frameshifts or point mutations. Thus, we optimized the tool itself by increasing the copy number of cas9 to retain some cas9 that still has the correct DNA sequence. Fortunately, the escape rates dropped below 10-8 at 9 of the 16 tested genes. Furthermore, the λ-Red recombination system was added to generate the pEcCas-2.0, and a 100% gene deletion efficiency was achieved at genes cadA, maeB, and gntT in MG1655, whereas those genes were edited with low efficiency previously. Last, the application of pEcCas-2.0 was then expanded to the E. coli B strain BL21(DE3) and W strain ATCC9637. This study reveals the mechanism of E. coli surviving Cas9-mediated death, and a highly efficient editing tool is established based on the mechanism, which will accelerate the further application of CRISPR-Cas.

Impact of ompk36 genotype and KPC subtype on the in vitro activity of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam against KPC-producing K. pneumoniae clinical isolates.

The availability of new β-lactam/β-lactamase inhibitors ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam have redefined contemporary treatment of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) infections. We aimed to characterize and contrast the in vitro activity of these agents against genetically diverse KPC-Kp clinical isolates. We analysed genomes of 104 non-consecutive KPC-Kp isolates and compared the in vitro antibiotic activity by KPC subtype and ompK36 genotype. MICs were determined in triplicate by CLSI methods. Twenty representative isolates were selected for time-kill analyses against physiological steady-state and trough concentrations, as well as 4× MIC for each agent. Fifty-eight percent and 42% of isolates harboured KPC-2 and KPC-3, respectively. OmpK36 mutations were more common among KPC-2- compared with KPC-3-producing Kp (P < 0.0001); mutations were classified as IS5 insertion, glycine-aspartic acid insertion at position 134 (GD duplication) and other mutations. Compared to isolates with WT ompK36, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam MICs were elevated for isolates with IS5 by 2-, 4- and 16-fold, respectively (P < 0.05 for each). Against isolates with GD duplication, imipenem/relebactam and meropenem/vaborbactam MICs were increased, but ceftazidime/avibactam MICs were not. In time-kill studies, ceftazidime/avibactam-mediated killing correlated with ceftazidime/avibactam MICs, and did not vary across ompK36 genotypes. Imipenem/relebactam was not bactericidal against any isolate at trough concentrations. At steady-state imipenem/relebactam concentrations, regrowth occurred more commonly for isolates with IS5 mutations. Log-kills were lower in the presence of meropenem/vaborbactam for isolates with GD duplication compared with IS5 mutations. Our investigation identified key genotypes that attenuate, to varying degrees, the in vitro activity for each of the new β-lactam/β-lactamase inhibitors. Additional studies are needed to translate the importance of these observations into clinical practice.

Selection of IS6110 conserved regions for the detection of Mycobacterium tuberculosis using qPCR and LAMP.

IS6110 insertion sequence is a frequently used target for Mycobacterium tuberculosis detection. However, its sequence variability is studied insufficiently. We aimed to identify the most conservative and variable regions in IS6110 sequences and develop qPCR and LAMP oligonucleotide sets for the conservative regions. Using in-house Python scripts, 3609M. tuberculosis genome sequences from the NCBI database were aligned; conservative regions were identified to design oligonucleotide sets. IS6110 fragments located within the 31-231bp region were the most conservative and represented in genomes and were used to design qPCR and LAMP oligonucleotides. The in silico sensitivity of the qPCR oligonucleotides on the whole genome set was 99.1% and 98.4%. For the LAMP primers developed, the sensitivity was 96.9%. For qPCR, the limit of detection with 95% confidence (LoD95%) was four IS6110 copies per reaction, with LoD90% being 200 BCG cells per ml of artificial sputum. For LAMP, LoD95% was 16 copies per reaction, with LoD90% being 400 Mycobacterium bovis Bacille Calmette-Guerin (BCG) cells per ml of artificial sputum. We have demonstrated the IS6110 sequence variability and designed highly sensitive qPCR and LAMP oligonucleotides to detect M. tuberculosis.

THE PECULARITIES OF TUBERCULOSIS, CAUSED BY BEIJING STRAINS

East Asian family Beijing M. tuberculosis is a family characterized by active distribution. The article analyzes the association between the features of the course of newly diagnosed tuberculosis in patients infected with pathogens of the Beijing family in comparison with the non-Beijing group. Cultures of M.tuberculosis for molecular genetic research were obtained from 87 patients with first diagnosed pulmonary tuberculosis. According to the presence of the IS6110 insertion fragment in the dnaA-dnaN region, 26,4% of the isolated cultures were reffered to the Beijing family. The medical records of 87 patients with pulmonary tuberculosis were analyzed. Patients infected with pathogens of the Beijing family more often has disseminated form of tuberculosis then infiltrative (60,9% to 30,5%, RR 2,0 CI 1,0 – 4,03), longer period of positive bacterioscopic and cultural tests for M.tuberculosis (RR 1, 8 CI 1,04 – 3,01), more common treatment failure. Higher frequency of mutations of the katG, inhA, rpoB genes, which cause resistance to isoniazid and rifampicin, and multiresistance was detected more often compared to non-Beijing pathogens. In the group of Beijing strains, the level of multiresistance detected by cultural method 31,6% versus 8.5% in non-Beijing, i.e. strains of the Beijing family were characterized by multiresistance 3.7 times more often according to the cultural study than isolates of the non-Beijing group (р&lt;0.05; χ2=8.22) The obtained results permit to speculate that M. tuberculosis infection of the Beijing family is a risk factor for more severe corse of the disease. Key words: tuberculosis, effectiveness of treatment, Beijing family, drug resistance, M.tuberculosis.

Effects of Global and Specific DNA-Binding Proteins on Transcriptional Regulation of the E. coli bgl Operon.

Using reporter gene (lacZ) transcriptional fusions, we examined the transcriptional dependencies of the bgl promoter (Pbgl) and the entire operon regulatory region (Pbgl-bglG) on eight transcription factors as well as the inducer, salicin, and an IS5 insertion upstream of Pbgl. Crp-cAMP is the primary activator of both Pbgl and the bgl operon, while H-NS is a strong dominant operon repressor but only a weak repressor of Pbgl. H-NS may exert its repressive effect by looping the DNA at two binding sites. StpA is a relatively weak repressor in the absence of H-NS, while Fis also has a weak repressive effect. Salicin has no effect on Pbgl activity but causes a 30-fold induction of bgl operon expression. Induction depends on the activity of the BglF transporter/kinase. IS5 insertion has only a moderate effect on Pbgl but causes a much greater activation of the bgl operon expression by preventing the full repressive effects of H-NS and StpA. While several other transcription factors (BglJ, RcsB, and LeuO) have been reported to influence bgl operon transcription when overexpressed, they had little or no effect when present at wild type levels. These results indicate the important transcriptional regulatory mechanisms operative on the bgl operon in E. coli.

Comparison of Automated Ribotyping, spa Typing, and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods.

Frequency, composition and mobility of Escherichia coli-derived transposable elements in holdings of plasmid repositories.

SummaryBy providing the scientific community with uniform and standardized resources of consistent quality, plasmid repositories play an important role in enabling scientific reproducibility. Plasmids containing insertion sequence elements (IS elements) represent a challenge from this perspective, as they can change the plasmid structure and function. In this study, we conducted a systematic analysis of a subset of plasmid stocks distributed by plasmid repositories (The Arabidopsis Biological Resource Center and Addgene) which carry unintended integrations of bacterial mobile genetic elements. The integration of insertion sequences was most often found in, but not limited to, pBR322‐derived vectors, and did not affect the function of the specific plasmids. In certain cases, the entire stock was affected, but the majority of the stocks tested contained a mixture of the wild‐type and the mutated plasmids, suggesting that the acquisition of IS elements likely occurred after the plasmids were acquired by the repositories. However, comparison of the sequencing results of the original samples revealed that some plasmids already carried insertion mutations at the time of donation. While an extensive BLAST analysis of 47 877 plasmids sequenced from the Addgene repository uncovered IS elements in only 1.12%, suggesting that IS contamination is not widespread, further tests showed that plasmid integration of IS elements can propagate in conventional Escherichia coli hosts over a few tens of generations. Use of IS‐free E. coli hosts prevented the emergence of IS insertions as well as that of small indels, suggesting that the use of IS‐free hosts by donors and repositories could help limit unexpected and unwanted IS integrations into plasmids.

Prevalence of non-tuberculosis mycobacteria among samples deposited from the National Tuberculous Reference Laboratory of Iran (2011-2018)

Objective: To investigate the prevalence of non-tuberculosis mycobacteria (NTM) among the samples deposited from the National Tuberculosis Reference Laboratory of Iran between 2011 and 2018. Methods: The study evaluated the prevalence of NTM among specimens from patients with pulmonary tuberculosis symptoms (n=15 771) deposited at the National Tuberculosis Reference Laboratory of Iran from 2011 to 2018. Detection of Mycobacterium (M.) tuberculosis was based on presence of a 190-bp amplicon from IS6110 insertion sequence using Tb1 and Tb2 primers, and amplicon-negative specimens were tested for NTM and M. tuberculosis (refractory to IS6110 amplification) using restriction fragment length polymorphism PCR of hsp65 amplicon fragment. Results: A total of 7 307 (46.33%) M. tuberculosis and 658 (4.17%) NTM specimens were found, the latter mainly comprising M. abscessus (10.18%), M. avium (2.28%), M. chelonae (8.97%), M. intracellulare (10.49%), M. kansasii (4.71%), and M. simiae (56.08%). Conclusions: As treatment for NTM differs from that for M. tuberculosis, accurate detection of Mycobacterium sp. is of public health significance.

Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks.

Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3′LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.