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Background Multiple drug resistance (MDR) of cancer cells is the main cause of intrinsic or acquired desensitization to chemotherapy in many cancers. A number of studies have found high expression of COX‐2 to be a factor for expression of MDR gene in several cancer. Furthermore, adipose tissue derived mesenchymal stem/stromal cells (ADSC) have been found to increase cyclo‐oxygenase‐2 (COX‐2) expression in some tumour cells. The mechanism for this, however, is not yet clear and needs further study. Objective The purpose of this study was to determine whether tumour necrosis factor‐alpha stimulated gene/protein 6 (TSG‐6) secreted from ADSCs is associated with an increase in MDR genes by inducing COX‐2 gene expression in melanoma and osteosarcoma cell lines. Methods ADSCs were transfected with TSG‐6 siRNA or Control RNA respected, and cancer cell line were transfected with COX‐2 siRNA or Control RNA respected. Using trans well coculture system, the interactions of ADSCs with tumour cells were investigated. Results Increased COX‐2 expression was observed in cancer cell co‐cultured with ADSCs. Additionally, we identified that COX‐2 expression was related to drug resistance genes (P‐glycoprotein, multidrug resistance‐associated protein). Transfecting canine ADSCs with small interfering RNA, TSG‐6 secreted from ADSCs was found to be a major factor in the regulation of COX‐2 expression and drug resistance genes in osteosarcoma and melanoma cell lines. Conclusion TSG‐6 mediated COX‐2 up‐regulation is a possible mechanism of chemoresistance development induced by ADSCs. These findings provide better understanding about the mechanism associated with ADSC‐induced chemoresistance in cancer., TSG‐6 mediated COX‐2 up‐regulation could be a possible mechanism underlying the development of chemoresistance induced by mesenchymal stem cells.

International audience; ABC ("ATP-Binding Cassette") transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.

Nephrotic syndrome is a common disease of the urinary system. The aim of this study is to explore the effect of astragalus polysaccharides (APS) on multidrug resistance gene 1 (MDR1) and P-glycoprotein 170 (P-gp170) in adriamycin nephropathy rats and the underlying mechanisms.A total of 72 male Wistar rats were divided into a control group, a model group, an APS low-dose group, an APS high-dose group, an APS+micro RNA (miR)-16 antagomir group and an APS+miR-16 antagomir control group, with 12 rats in each group. Urine protein (UP) was detected by urine analyzer, and serum cholesterol (CHOL), albumin (ALB), blood urea nitrogen (BUN), and creatinine (SCr) were detected by automatic biochemical analyzer; serum interleukin-6 (IL-6), IL-1β, tumor necrosis factor α (TNF-α) levels were detected by ELISA kit; the morphological changes of kidney tissues were observed by HE staining; the levels of miR-16 and MDR1 mRNA in kidney tissues were detected by real-time RT-PCR; the expression levels of NF-κB p65, p-NF-κB p65, and P-gp170 protein in kidney tissues were detected by Western blotting; and dual luciferase was used to verify the relationship between miR-16 and NF-κB.The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration. The renal glomeruli of rats in the model group were mildly congested, capillary stenosis or occlusion, and inflammatory cell infiltration was obvious. The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion, the proliferation of mesangial cells was significantly reduced, and the inflammatory cells were reduced. Compared with the high-dose APS group and the APS+miR-16 antagomir control group, there were more severe renal tissue structure damages in the APS + miR-16 antagomir group. Compared with the control group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased (allAPS can regulate the miR-16/NF-κB signaling pathway, thereby affecting the levels of MDR1 and P-gp170, and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.

Antimicrobial resistance in Morganella morganii is increasing in recent years, which is mainly introduced via extra genetic and mobile elements. The aim of our study is to analyze the multidrug resistance (MDR) and characterize the mobile genetic elements (MGEs) in M. morganii isolates. Here, we report the characteristic of a pathogenic M. morganii isolate containing multidrug resistance genes that are mainly carried by a novel transposon Tn

Background The analysis of single nucleotide polymorphism (SNPs) in drug-resistance associated genes is a commonly used strategy for the surveillance of anti-malarial drug resistance in populations of parasites. The present study was designed and performed to provide genetic epidemiological data of the prevalence of N86Y-Y184F-D1246Y SNPs in Plasmodium falciparum multidrug resistance 1 (pfmdr1) in the malaria hotspot of Northern Nigeria. Methods Plasmodium falciparum-positive blood samples on Whatman-3MM filter papers were collected from 750 symptomatic patients from four states (Kano, Kaduna, Yobe and Adamawa) in Northern Nigeria, and genotyped via BigDye (v3.1) terminator cycle sequencing for the presence of three SNPs in pfmdr1. SNPs in pfmdr1 were used to construct NYD, NYY, NFY, NFD, YYY, YYD, YFD and YFY haplotypes, and all data were analysed using Pearson Chi square and Fisher’s exact (FE) tests. Results The prevalence of the pfmdr1 86Y allele was highest in Kaduna (12.50%, 2 = 10.50, P = 0.02), whilst the 184F allele was highest in Kano (73.10%, 2 = 13.20, P = 0.00), and the pfmdr1 1246Y allele was highest in Yobe (5.26%, 2 = 9.20, P = 0.03). The NFD haplotype had the highest prevalence of 69.81% in Kano (2 = 36.10, P = 0.00), followed by NYD with a prevalence of 49.00% in Adamawa, then YFD with prevalence of 11.46% in Kaduna. The YYY haplotype was not observed in any of the studied states. Conclusion The present study suggests that strains of P. falciparum with reduced sensitivity to the lumefantrine component of AL exist in Northern Nigeria and predominate in the North-West region.

Mining is among the human activities with widest environmental impacts, and mining-impacted environments are characterized by high levels of metals that can co-select for antibiotic resistance genes (ARGs) in microorganisms. However, ARGs in mining-impacted environments are still poorly understood. Here, we conducted a comprehensive study of ARGs in such environments worldwide, taking advantage of 272 metagenomes generated from a global-scale data collection and two national sampling efforts in China. The average total abundance of the ARGs in globally distributed studied mine sites was 1572 times per gigabase, being rivaling that of urban sewage but much higher than that of freshwater sediments. Multidrug resistance genes accounted for 40% of the total ARG abundance, tended to co-occur with multimetal resistance genes, and were highly mobile (e.g. on average 16% occurring on plasmids). Among the 1848 high-quality metagenome-assembled genomes (MAGs), 85% carried at least one multidrug resistance gene plus one multimetal resistance gene. These high-quality ARG-carrying MAGs considerably expanded the phylogenetic diversity of ARG hosts, providing the first representatives of ARG-carrying MAGs for the Archaea domain and three bacterial phyla. Moreover, 54 high-quality ARG-carrying MAGs were identified as potential pathogens. Our findings suggest that mining-impacted environments worldwide are underexplored hotspots of multidrug resistance genes.

Objectives To investigate the prevalence of the optrA, poxtA and cfr linezolid resistance genes in linezolid-resistant enterococci from Irish hospitals and to characterize associated plasmids. Methods One hundred and fifty-four linezolid-resistant isolates recovered in 14 hospitals between June 2016 and August 2019 were screened for resistance genes by PCR. All isolates harbouring resistance genes, and 20 without, underwent Illumina MiSeq WGS. Isolate relatedness was assessed using enterococcal whole-genome MLST. MinION sequencing (Oxford Nanopore) and hybrid assembly were used to resolve genetic environments/plasmids surrounding resistance genes. Results optrA and/or poxtA were identified in 35/154 (22.7%) isolates, the highest prevalence reported to date. Fifteen isolates with diverse STs harboured optrA only; one Enterococcus faecium isolate harboured optrA (chromosome) and poxtA (plasmid). Seven Enterococcus faecalis and one E. faecium harboured optrA on a 36 331 bp plasmid with 100% identity to the previously described optrA-encoding conjugative plasmid pE349. Variations around optrA were also observed, with optrA located on plasmids in five isolates and within the chromosome in three isolates. Nine E. faecium and 10 E. faecalis harboured poxtA, flanked by IS1216E, within an identical 4001 bp region on plasmids exhibiting 72.9%–100% sequence coverage to a 21 849 bp conjugative plasmid. E. faecalis isolates belonged to ST480, whereas E. faecium isolates belonged to diverse STs. Of the remaining 119 linezolid-resistant isolates without linezolid resistance genes, 20 investigated representatives all harboured the G2576T 23S RNA gene mutation associated with linezolid resistance. Conclusions This high prevalence of optrA and poxtA in diverse enterococcal lineages in Irish hospitals indicates significant selective pressure(s) for maintenance.

Multidrug efflux pumps play an important role in bacterial multidrug resistance by actively excreting antibiotics. The ATP-binding cassette-type drug efflux pump MacAB was originally reported as a macrolide-specific pump. MacAB is also known to be required for the virulence of Salmonella enterica serovar Typhimurium following oral infection in mice. Here, we performed a screening of inhibitors of Salmonella MacAB and found a compound that increased the susceptibility of a MacAB-expressing strain to macrolides. It was previously reported that MacAB is required to resist peroxide-mediated killing in vitro and that a supernatant of wild-type Salmonella rescues the growth defect of a macAB mutant in H2 O2 . In this study, we also found that the MacAB inhibitor reduced the ability of the supernatant to rescue Salmonella cells in H2 O2 . This compound could lead to a better understanding of the function of MacAB.

Introduction The multidrug resistance 1 (MDR1) gene plays an important function in carcinogens detoxification and drugs metabolism. Many authors reported that MDR1 gene influences individual susceptibility to cancers. We carried out the present case-control study to investigate the impact of MDR1 gene in the predisposition to acute myeloid leukemia (AML) in a sample of Moroccan population. In addition, we performed a meta-analysis study to discuss our results and to better highlight the influence of MDR1 gene on the susceptibility of AML. Methods The study included 187 AML patients and 206 controls. Genomic DNA was extracted from white blood cell by salting method. Polymorphisms of G2677 T and C3435 T were genotyped by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), using Mbo I and Ban I restriction enzymes. Statistical analysis was performed using the software SPSS (version 19.0; SPPS Inc., Chicago, IL, USA) and MedCalcv.11.6.1.0 software. Results No statistically significant differences in genotype and allelic distribution were found in G2677 T and C3435 T polymorphisms between AML cases and controls in the Moroccan population. On the other hand, we found that the age of onset of AML in patients with homozygous mutant genotype was statistically lower than in patients with either the heterozygous or wild type genotype for both polymorphisms (P = 0.006; P = 0.03). Meta-analysis showed a significant association of C3435 T, G2677 T polymorphisms on the susceptibility of AML when considering the recessive and the allelic models. Conclusion Our findings showed that the G2677 T and C3435 T polymorphisms of the MDR1 gene were associated with the age at onset of AML in our population. In addition, the meta-analysis showed that these polymorphisms could play a role in susceptibility to AML.

The field of synthetic biology has used the engineered assembly of synthetic gene networks to create a wide range of functions in biological systems. To date, gene-circuit-based sensors have primarily used optical proteins (for example, fluorescent, colorimetric) as reporter outputs, which has limited the potential to measure multiple distinct signals. Here we present an electrochemical interface that permits expanded multiplexed reporting for cell-free gene-circuit-based sensors. We have engineered a scalable system of reporter enzymes that cleave specific DNA sequences in solution, which results in an electrochemical signal when these newly liberated strands are captured at the surface of a nanostructured microelectrode. We describe the development of this interface and show its utility using a ligand-inducible gene circuit and toehold switch-based sensors by demonstrating the detection of multiple antibiotic resistance genes in parallel. This technology has the potential to expand the field of synthetic biology by providing an interface for materials, hardware and software.

Streptococcus agalactiae is the leading cause of meningitis in newborns and a significant cause of invasive diseases in pregnant women and adults with underlying diseases. Antibiotic resistance against erythromycin and clindamycin in group B streptococcus (GBS) isolates has been increasing worldwide. GBS expresses the Srr1 and Srr2 proteins, which have important roles in bacterial infection. They have been investigated as novel vaccine candidates against GBS infection, with promising results. But a recent study detected non-srr1/2-expressing clinical isolates belonging to serotype III. Thus, we aimed to analyze the genotypes of non-srr1/2 GBS clinical isolates collected between 2013 and 2016 in South Korea. Forty-one (13.4%) of the 305 serotype III isolates were identified as non-srr1/2 strains, including sequence type 19 (ST19) (n = 16) and ST27 (n = 18) strains. The results of the comparative genomic analysis of the ST19/serotype III/non-srr1/2 strains further revealed four unique gene clusters. Site 4 in the srr1 gene locus was replaced by an lsa(E)-lnu(B)-aadK-aac-aph-aadE-carrying multidrug-resistant gene cluster flanked by two IS1216 transposases with 99% homology to the enterococcal plasmid pKUB3007-1. Despite the Srr1 and Srr2 deficiencies, which resulted in reduced fibrinogen binding, the adherence of non-srr1/2 strains to endothelial and epithelial cells was comparable to that of Srr1- or Srr2-expressing strains. Moreover, their virulence in mouse models of meningitis was not significantly affected. Furthermore, additional adhesin-encoding genes, including a gene encoding a BspA-like protein, which may contribute to colonization by non-srr1/2 strains, were identified via whole-genome analysis. Thus, our study provides important findings that can aid in the development of vaccines and antibiotics against GBS. IMPORTANCE Most previously isolated group B streptococcus (GBS) strains express either the Srr1 or Srr2 glycoprotein, which plays an important role in bacterial colonization and invasion. These glycoproteins are potential protein vaccine candidates. In this study, we first report GBS clinical isolates in which the srr1/2 gene was deleted or replaced with foreign genes. Despite Srr1/2 deficiency, in vitro adherence to mammalian cells and in vivo virulence in murine models were not affected, suggesting that the isolates might have another adherence mechanism that enhanced their virulence aside from Srr1/2-fibrinogen-mediated adherence. In addition, several non-srr1/2 isolates replaced the srr1/2 gene with the lnu(B) and lsa(E) antibiotic resistance genes flanked by IS1216, effectively causing multidrug resistance. Collectively, we believe that our study identifies the underlying genes responsible for the pathogenesis of new GBS serotype III. Furthermore, our study emphasizes the need for alternative antibiotics for patients who are allergic to β-lactams and for those who are pregnant.

Cereal-associated lactobacilli resist antimicrobial plant secondary metabolites. This study aimed to identify multi-drug-resistance (MDR) transporters in isolates from mahewu, a Zimbabwean fermented cereal beverage, and to determine whether these MDR-transporters relate to resistance against phenolic compounds and antibiotics. Comparative genomic analyses indicated that all seven mahewu isolates harbored multiple MATE and MFS MDR proteins. Strains of Lactiplantibacillus plantarum and Limosilactobacillus fermentum encoded for the same gene, termed mahewu phenolics resistance gene mprA, with more than 99% nucleotide identity, suggesting horizontal gene transfer. Strains of Lp. plantarum were more resistant than strains of Lm. fermentum to phenolic acids, other antimicrobials and antibiotics but the origins of strains were not related to resistance. The resistance of several strains exceeded EFSA thresholds for several antibiotics. Analysis of gene expression in one strain each of Lp. plantarum and Lm. fermentum revealed that at least one MDR gene in each strain was over-expressed during growth in wheat, sorghum and millet relative to growth in MRS5 broth. In addition, both strains over-expressed a phenolic acid reductase. The results suggest that diverse lactobacilli in mahewu share MDR transporters acquired by lateral gene transfer, and that these transporters mediate resistance to secondary plant metabolites and antibiotics.

Increasing resistance to antifungal therapy is an impediment to the effective treatment of fungal infections. Candida glabrata is an opportunistic human fungal pathogen that is inherently less susceptible to cost-effective azole antifungals. Gain-of-function mutations in the Zn-finger pleiotropic drug resistance transcriptional activator-encoding gene CgPDR1 are the most prevalent causes of azole resistance in clinical settings. CgPDR1 is also transcriptionally activated upon azole exposure; however, factors governing CgPDR1 gene expression are not yet fully understood. Here, we have uncovered a novel role for two FK506-binding proteins, CgFpr3 and CgFpr4, in the regulation of the CgPDR1 regulon. We show that CgFpr3 and CgFpr4 possess a peptidyl-prolyl isomerase domain and act redundantly to control CgPDR1 expression, as a Cgfpr3Δ4Δ mutant displayed elevated expression of the CgPDR1 gene along with overexpression of its target genes, CgCDR1, CgCDR2, and CgSNQ2, which code for ATP-binding cassette multidrug transporters. Furthermore, CgFpr3 and CgFpr4 are required for the maintenance of histone H3 and H4 protein levels, and fluconazole exposure leads to elevated H3 and H4 protein levels. Consistent with the role of histone proteins in azole resistance, disruption of genes coding for the histone demethylase CgRph1 and the histone H3K36-specific methyltransferase CgSet2 leads to increased and decreased susceptibility to fluconazole, respectively, with the Cgrph1Δ mutant displaying significantly lower basal expression levels of the CgPDR1 and CgCDR1 genes. These data underscore a hitherto unknown role of histone methylation in modulating the most common azole antifungal resistance mechanism. Altogether, our findings establish a link between CgFpr-mediated histone homeostasis and CgPDR1 gene expression and implicate CgFpr in the virulence of C. glabrata.

The flexibility of microbes to undergo or adapt to the changes in their physiology and genotypical traits has enabled the microbes acquiring resistance to latest or recently discovered drugs which have consequently led to the menace of multidrug resistance (MDR). There is a surge in the discovery of novel antibiotics to counter the rising MDR phenomena, and in such a quest, for investigating an efficient alternative mechanism or compound to combat MDR, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has piqued the interests of the researchers across the globe. CRISPR-Cas9 technology is a genome-editing tool with successful widespread applications in cell lines, plants, animals, and even in human clinical trials, and it is seriously being considered as a potential candidate for countering MDR. This review encompasses the broad scope of CRISPR-Cas9 along with its various variations, underlying principles, mechanisms, as well as applications. Furthermore, the implications of recent advancements in various disciplines are highlighted to enhance the applicability of this technique. Consequently, its research gaps and challenges are also identified so that they can be addressed in the possible future thereby further expanding the lore of CRISPR-Cas9 technique.

Objectives In this study, 77 Enterobacter spp. isolates from a collection of 175 Gram-negative bacilli isolated from Tlemcen University Hospital Center (North-West of Algeria) were tested for antibiotic resistance, biocide tolerance and genetic determinants of antimicrobial resistance. Methods The isolates were identified by 16S rDNA gene sequencing. Biocide tolerance was determined by broth microdilution, and antibiotic resistance was determined by disk diffusion. Genetic determinants of resistance were studied by PCR amplification using suitable primers. Results The most common Enterobacter species was Enterobacter cloacae (58.4%), followed by Enterobacter hormaechei (24.7%). The most common antibiotic resistance was to ticarcillin either alone or in combination with clavulanic acid (70.1%), followed by cefepime (68.8%), cefotaxime (63.6%), ceftazidime (54.5%) and gentamicin (54.5%). Tobramycin was active against 87.0% of the isolates. Levels of biocide tolerance were high for hexachlorophene and to a lesser extent for benzalkonium chloride. The extended-spectrum β-lactamase genes blaTEM and blaCTX-M were detected in 44.2% and 36.4% of isolates, respectively. Other antimicrobial resistance genes (ARGs) frequently detected were aac(6ʹ)-Ib (57.1%) and sul2 (50.6%). Multidrug-resistant isolates carrying several ARGs were common. Significant positive correlations were detected for efflux pump genes with ARGs and also between ARGs. Conclusion The results of this study reveal thatEnterobacter spp. isolates from hospital settings are both resistant to clinically-used antibiotics and tolerant to biocides. Biocide tolerance could be an advantage for antibiotic-resistant strains in hospitals.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are global challenges due to the limited number of effective drugs for treatment. Treatment with less than 4–5 effective drugs might lead to the further emergence of drug resistance and poor clinical outcomes. For better prediction of treatment outcomes, we compared drug-resistance profiles of consecutive clinical MDR Mycobacterium tuberculosis isolates from high- and low-burden settings. This was a retrospective cohort study. We analysed 225 and 229 MDR isolates from Moscow (Russia) and Taiwan, respectively, obtained between 2014 and 2015. Drug susceptibility testing was performed by the Bactec MGIT 960 automated system and the agar proportion method. Detection of resistance-associated mutations in the M. tuberculosis genome was carried out by an array and/or sequencing of selected loci. The principal differences between resistance profiles of MDR isolates in the two countries were the percentages of pre-XDR (40.9% vs. 14.8%) and XDR (34.7% vs. 1.7%) isolates, both of which were significantly higher in Moscow isolates. Forty-eight (33%) of 147 MDR and pre-XDR Russian isolates fall into a group with less than four effective drugs, which accounts for 40% (N = 120) of these isolates. The other 60% in this group were XDR strains (N = 72). Consequently, the average number of effective anti-tuberculosis drugs for MDR-TB treatment was lower for Russian isolates (3 vs. 7). Furthermore, a notable percentage (9%) of isolates resistant to kanamycin harboured mutations in the whiB7 locus, which was not detected by molecular tests targeting common mutations in the rrs and eis loci. We found that 98.2% and 45.9% of MDR isolates from Moscow and Taiwan, respectively, were resistant to streptomycin. Molecular tests for detecting resistance to drugs other than rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs are needed for individualized therapy. The conventional MDR treatment schemes most probably fail in these cases due to the limited number of effective drugs.

Acute myeloid leukemia is an aggressive disease that arises from clonal expansion of malignant hematopoietic precursor cells of the bone marrow. Deletions on the long arm of chromosome 9 (del(9q)) are observed in 2% of acute myeloid leukemia patients. Our deletion analysis in a cohort of 31 del(9q) acute myeloid leukemia patients further supports the importance of a minimally deleted region composed of seven genes potentially involved in leukemogenesis: GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3 and NTRK2. Importantly, among them HNRNPK, encoding heterogeneous nuclear ribonucleoprotein K is proposed to function in leukemogenesis. We show that expression of HNRNPK and the other genes of the minimally deleted region is significantly reduced in patients with del(9q) compared with normal karyotype acute myeloid leukemia. Also, two mRNAs interacting with heterogeneous nuclear ribonucleoprotein K, namely CDKN1A and CEBPA are significantly downregulated. While the deletion size is not correlated with outcome, associated genetic aberrations are important. Patients with an additional t(8;21) show a good prognosis. RUNX1-RUNX1T1, which emerges from the t(8;21) leads to transcriptional down-regulation of CEBPA. Acute myeloid leukemia patients with mutations in CEBPA have a good prognosis as well. Interestingly, in del(9q) patients with CEBPA mutation mRNA levels of HNRNPK and the other genes located in the minimally deleted region is restored to normal karyotype level. Our data indicate that a link between CEBPA and the genes of the minimally deleted region, among them HNRNPK contributes to leukemogenesis in acute myeloid leukemia with del(9q).

BACKGROUND Ultrasound/microbubble (USMB)-mediated sonoporation is a new strategy with minimal procedural invasiveness for targeted and site-specific drug delivery to tumors. The purpose of this study was to explore the effect of different breast cancer cell lines on sonoporation efficiency, and then to identify an optimal combination of USMB parameters to maximize the sonoporation efficiency for each tumor cell line. MATERIAL AND METHODS Three drug-sensitive breast cell lines - MCF-7, MDA-MB-231, and MDA-MB-468 - and 1 multidrug resistance (MDR) cell line - MCF-7/ADR - were chosen. An orthogonal array experimental design approach based on 3 levels of 3 parameters (A: microbubble concentration, 10%, 20%, and 30%, B: sound intensity, 0.5, 1.0, and 1.5 W/cm², C: irradiation time, 30, 60, and 90 s) was employed to optimize the sonoporation efficiency. RESULTS The optimal USMB parameter combinations for different cell lines were diverse. Under optimal parameter combinations, the maximum sonoporation efficiency differences between different breast tumor cell lines were statistically significant (MDA-MB-231: 46.70±5.79%, MDA-MB-468: 53.44±5.69%, MCF-7: 59.88±5.53%, MCF-7/ADR: 65.39±4.01%, P

RND (Resistance-Nodulation-Division) family transporters have a vital role in both intrinsic and acquired multi-drug resistance in Gram-negative bacteria. It is important to find a conserved domain in the RND family between different pathogenic bacteria for diagnostic and therapeutic purpose. Total sequences of three-component system RND efflux pumps were retrieved from NCBI nucleotide and protein database and were subjected to conservation and variation analysis using the multiple sequence alignment feature of the CLC workbench. The phylogenetic tree for main transporters was drawn and the three-dimensional structure was also evaluated. From the sequence conservation analysis, highly conserved residues with 282 base pair (94 amino acid) long were identified. The location of the highly conserved domain is positioned in the domain 1 crystallographic structure of AcrB Escherichia coli and MexB Pseudomonas aeruginosa. The main transporter component phylogenetic tree shows the clusters of different genotypes and their evolutionary association. Each of three components of RND proteins is crucial for drug efflux, and the absence of even one component makes the entire complex totally nonfunctional. Therefore, this highly conserved region can be used to disable the RND multidrug efflux pumps. In addition, this highly conserved can also be used for diagnostic aspects.

Multidrug resistance gene (MDR1a) and P-glycoprotein (P-gp) play an important role in the development of ulcerative colitis (UC) and influence the therapeutic effect of glucocorticoids, which may lead to drug resistance mechanically. UC may be related to miR-145 to some extent, and the relationship still needs further exploration. In this study we found that the expression of miR-145 was downregulated in the colonic tissues of rats with Dextran sodium sulfate (DSS)-induced UC. Also, the expression of MDR1a in colon tissues of each group negatively correlated with the expression of miR-145 in rats. The change in the plasma peak concentration (Cmax) in each group positively related to the miR-145 level. Mechanistically, miR-145 negatively regulated the expression and function of P-gp via acting directly on the 3'-UTR of MDR1 mRNA. Overall, these results indicated that miR-145 had a protective effect on the colorectal mucosa, and its downregulation may enhance the expression and function of MDR1a and P-gp, promoting the occurrence and development of UC. The downregulation of miR-145 reduced the drug sensitivity of 5-aminosalicylic acid (5-ASA) and glucocorticoids in treating UC, indicating that miR-145 might be a potential therapeutic target for UC.

Bacterial multidrug resistance causes many therapeutic failures, making it more difficult to fight against bacterial diseases. This study aimed to investigate the antibacterial activity of extract, fractions, and phytochemicals from Plectranthus glandulosus (Lamiaceae) against multidrug-resistant (MDR) Gram-negative phenotypes expressing efflux pumps. The crude extract after extraction was subjected to column chromatography, and the structures of the isolated compounds were determined using spectrometric and spectroscopic techniques. Antibacterial assays of samples alone and in the presence of an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide, PAβN) were carried out using the broth microdilution method. The phytochemical study of P. glandulosus plant extract afforded seven major fractions (A–G) which lead to the isolation of seventeen known compounds. The ethanol extract of P. glandulosus was not active at up to 1024 μg/mL, whereas its fractions showed MICs varying from 32 to 512 μg/mL on the studied bacteria. Fraction C of P. glandulosus showed the lowest MIC (32 μg/mL) on E. coli ATCC8739 strain. Fraction D presented the highest activity spectrum by inhibiting the growth of 90% (9/10) of the studied bacteria. The presence of PAβN has improved the activity of extract and all fractions. Overall, the tested phytochemicals showed low activity against the studied bacteria. The overall results obtained in this study show that some fractions from P. glandulosus, mainly fractions C and D, should be investigated more for their possible use to fight against MDR bacteria.

The persistence of plasmids in bacterial populations represents a puzzling evolutionary problem with serious clinical implications due to their role in the ongoing antibiotic resistance crisis. Recently, major advancements have been made toward resolving this “plasmid paradox” but mainly in a nonclinical context. Here, we propose an additional explanation for the maintenance of multidrug-resistance plasmids in clinical Escherichia coli strains. After coevolving two multidrug-resistance plasmids encoding resistance to last resort carbapenems with an extraintestinal pathogenic E. coli strain, we observed that chromosomal media adaptive mutations in the global regulatory systems CCR (carbon catabolite repression) and ArcAB (aerobic respiration control) pleiotropically improved the maintenance of both plasmids. Mechanistically, a net downregulation of plasmid gene expression reduced the fitness cost. Our results suggest that global chromosomal transcriptional rewiring during bacterial niche adaptation may facilitate plasmid maintenance.

Enterococcus faecalis is a commensal and nosocomial pathogen, which is also ubiquitous in animals and insects, representing a classical generalist microorganism. Here, we study E. faecalis isolates ranging from the pre-antibiotic era in 1936 up to 2018, covering a large set of host species including wild birds, mammals, healthy humans, and hospitalised patients. We sequence the bacterial genomes using short- and long-read techniques, and identify multiple extant hospital-associated lineages, with last common ancestors dating back as far as the 19th century. We find a population cohesively connected through homologous recombination, a metabolic flexibility despite a small genome size, and a stable large core genome. Our findings indicate that the apparent hospital adaptations found in hospital-associated E. faecalis lineages likely predate the “modern hospital” era, suggesting selection in another niche, and underlining the generalist nature of this nosocomial pathogen., Enterococcus faecalis is a commensal microorganism of animals, insects and humans, but also a nosocomial pathogen. Here, the authors analyse genomic sequences from E. faecalis isolates from animals and humans, and find that the last common ancestors of multiple hospital-associated lineages date to the pre-antibiotic era.

Patient: Male, 39-year-old Final Diagnosis: Hepatotoxicity Symptoms: Jaundice Medication: — Clinical Procedure: Cholecystectomy Specialty: Gastroenterology and Hepatology • Surgery Objective: Unknown ethiology Background: Although many cases of unusual liver discoloration exist, such as blue liver syndrome which is linked to oxaliplatin-based chemotherapy, our finding was seen in a patient who was not on chemotherapy. A 39-year-old male who presented with jaundice was found to have blue liver discoloration. Case Report: A 39-year-old male presented with jaundice of one-month’s duration evidenced by elevated total and direct bilirubin. An ultrasound and magnetic resonance cholangiopancreatography (MRCP) demonstrated thickened gall bladder wall but no common bile duct stones. A robotic-assisted laparoscopic cholecystectomy with liver biopsy was performed. Intraoperatively, the liver was noted to be unusually blue in color. During his postoperative course, the patient developed excessive incisional bleeding associated with an increase in international normalized ratio (INR) and increasing direct hyperbilirubinemia. This was managed with blood transfusions, and ursodeoxycholic acid was begun, which resulted in improvement of his bilirubin levels and overall recovery. Conclusions: Drug induced cholestasis and liver injury is a common cause of elevated liver enzymes. However, the unusual blue appearance of the liver should prompt an evaluation for other unusual and rare causes of obstructive jaundice.

Antibiotics are used to treat or prevent some types of bacterial infection. The inappropriate use of antibiotics unnecessarily promotes antibiotic resistance and increases resistant bacteria, and controlling these bacteria is difficult. While the emergence of drug-resistant bacteria is a serious problem, the behavior of drug-resistant bacteria is not fully understood. In this study, we investigated the behavior of Streptococcus mutans, a major etiological agent of dental caries that is resistant to bacitracin, which is a cell wall-targeting antibiotic, and focused on biofilm formation in the presence of bacitracin. S. mutans UA159 most strongly induced extracellular DNA (eDNA)-dependent biofilm formation in the presence of bacitracin at 1/8× MIC. The ΔmbrC and ΔmbrD mutant strains, which lack bacitracin resistance, also formed biofilms in the presence of bacitracin at 1/2× MIC. This difference between the wild type and the mutants was caused by the induction of atlA expression in the mid-log phase. We also revealed that certain rgp genes involved in the synthesis of rhamnose-glucose polysaccharide related to cell wall synthesis were downregulated by bacitracin. In addition, glucosyltransferase-I was also involved in eDNA-dependent biofilm formation. The biofilm led to increased transformation efficiencies and promoted horizontal gene transfer. Biofilms were also induced by ampicillin and vancomycin, antibiotics targeting cell wall synthesis, suggesting that cell envelope stress triggers biofilm formation. Therefore, the expression of the atlA and rgp genes is regulated by S. mutans, which forms eDNA-dependent biofilms, promoting horizontal gene transfer in response to cell envelope stress induced by sub-MICs of antibiotics. IMPORTANCE Antibiotics have been reported to induce biofilm formation in many bacteria at subinhibitory concentrations. Accordingly, it is conceivable that the MIC against drug-sensitive bacteria may promote biofilm formation of resistant bacteria. Since drug-resistant bacteria have spread, it is important to understand the behavior of resistant bacteria. Streptococcus mutans is bacitracin resistant, and the 1/8× MIC of bacitracin, which is a cell wall-targeted antibiotic, induced eDNA-dependent biofilm formation. The ΔmbrC and ΔmbrD strains, which are not resistant to bacitracin, also formed biofilms in the presence of bacitracin at 1/2× MIC, and biofilms of both the wild type and mutants promoted horizontal gene transfer. Another cell wall-targeted antibiotic, vancomycin, showed effects on biofilms and gene transfer similar to those of bacitracin. Thus, treatment with cell wall-targeted antibiotics may promote the spread of drug-resistant genes in biofilms. Therefore, the behavior of resistant bacteria in the presence of antibiotics at sub-MICs should be investigated when using antibiotics.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M.tb), causes highest number of deaths globally for any bacterial disease necessitating novel diagnosis and treatment strategies. High-throughput sequencing methods generate a large amount of data which could be exploited in determining multi-drug resistant (MDR-TB) associated mutations. The present work is a computational framework that uses artificial intelligence (AI) based machine learning (ML) approaches for predicting resistance in the genes rpoB, inhA, katG, pncA, gyrA and gyrB for the drugs rifampicin, isoniazid, pyrazinamide and fluoroquinolones. The single nucleotide variations were represented by several sequence and structural features that indicate the influence of mutations on the target protein coded by each gene. We used ML algorithms - naïve bayes, k nearest neighbor, support vector machine, and artificial neural network, to build the prediction models. The classification models had an average accuracy of 85% across all examined genes and were evaluated on an external unseen dataset to demonstrate their application. Further, molecular docking and molecular dynamics simulations were performed for wild type and predicted resistance causing mutant protein and anti-TB drug complexes to study their impact on the conformation of proteins to confirm the observed phenotype.

Background A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). Methods We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. Results Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). Conclusions Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.

A putative multidrug efflux gene

A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows targeted depletion at a higher degree of specificity than has been previously achieved. We have applied this technique to study trogocytosis, the intercellular transfer of cell surface molecules, but this principle could also be adapted using technology already available for use in other fields of study.

Staphylococcus aureus is an important food-related pathogen associated with bacterial poisoning that is difficult to treat due to its multidrug resistance. The cfr and lsa(E) genes both cause multiple drug resistance and have been identified in numerous Staphylococcus species, respectively. In this study, we found that a methicillin-resistant S. aureus (MRSA) strain, 2868B2, which was isolated from a sample of frozen dumplings in Hangzhou in 2015, co-carried these two different multidrug resistance genes. Further analysis showed that this strain was resistant to more than 18 antibiotics and expressed high-level resistance to florfenicol, chloramphenicol, clindamycin, tiamulin, erythromycin, ampicillin, cefepime, ceftazidime, kanamycin, streptomycin, tetracycline, trimethoprim-sulfamethoxazole and linezolid (MIC = 8 μg/mL). Whole genome sequencing was performed to characterize the genetic environment of these resistance genes and other genomic features. The cfr gene was located on the single plasmid p2868B2 (39,159 bp), which demonstrated considerable similarity to many plasmids previously identified in humans and animals. p2868B2 contained the insertion sequence (IS) element IS21-558, which allowed the insertion of cfr into Tn558 and played an important role in the mobility of cfr. Additionally, a novel multidrug resistance region (36.9 kb) harbouring lsa(E) along with nine additional antibiotic resistance genes (ARGs) (aadD, aadE, aacA-aphD, spc, lnu(B), lsa(E), tetL, ermC and blaZ) was identified. The multidrug resistance region harboured four copies of IS257 that were active and can mediate the formation of four circular structures containing ARGs and ISs. In addition, genes encoding various virulence factors and affecting multiple cell adhesion properties were identified in the genome of MRSA 2868B2. This study confirmed that the cfr and lsa(E) genes coexist in one MRSA strain and the presence of plasmid and IS257 in the multi-ARG cluster can promote both ARG transfer and dissemination. Furthermore, the presence of so many ARGs and virulence genes in food-related pathogens may seriously compromise the effectiveness of clinical therapy and threaten public health, its occurrence should pay public attention and the traceability of these genes in food-related samples needs further surveillance.

The development of resistance to multiple antimicrobial agents in pathogenic bacteria has become a threat to public health. Multidrug-resistant strains that are particularly dangerous include MDR, XDR and PDR strains.Aspirate material from paranasal sinuses, obtained from patients with chronic sinusitis undergoing functional endoscopic sinus surgery (FESS) in Medical Center MML in Warsaw, was subjected to bacteriologic analysis. The isolated strains were identified to the species level and tested for antibiotic resistance. Then, minimal inhibitory concentration (MIC) was determined. R esults: The isolated strains of coagulase-negative staphylococci were resistant mainly to macrolides, aminoglycosides and tetracycline. Nine of the isolated strains exhibited multidrug-resistance.Bacteria causing chronic sinusitis are becoming increasingly resistant to antimicrobial agents. The diagnostic process for coagulase-negative staphylococci (CNS) is often limited to the identification of species, or even genus of the bacteria. The CNS strains are considered to be non-pathogenic and they are not subject to eradication. This may lead to erroneous therapeutic decisions and, consequently, to the development of antibiotic resistance. CNS infections are classified as nosocomial and therefore, appropriate epidemiological procedures have to be followed. The authors highlight the necessity to determine MIC values for antibiotics and to introduce personalized treatment.

The capacity of bacterial pathogens to infect their hosts depends on the tight spatiotemporal regulation of virulence genes. The Listeria monocytogenes (Lm) metal efflux pump repressor CadC is highly expressed during late infection stages, modulating lipoprotein processing and host immune response. Here we investigate the potential of CadC as broad repressor of virulence genes. We show that CadC represses the expression of the bile salt hydrolase impairing Lm resistance to bile. During late infection, in absence of CadC-dependent repression, the constitutive bile salt hydrolase expression induces the overexpression of the cholic acid efflux pump MdrT that is unfavorable to Lm virulence. We establish the CadC regulon and show that CadC represses additional virulence factors activated by sB during colonization of the intestinal lumen. CadC is thus a general repressor that promotes Lm virulence by down-regulating, at late infection stages, genes required for survival in the gastrointestinal tract. This demonstrates for the first time how bacterial pathogens can repurpose regulators to spatiotemporally repress virulence genes and optimize their infectious capacity. This work was supported by grants to DC (FEDER - Fundo Europeu de Desenvolvimento Regional funds through the NORTE 2020 - Norte Portugal Regional Operational Programme, Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project NORTE-01-0145-FEDER-030020 PTDC/SAU-INF/30020/2017); to PC (European Research Council - ERC - Advanced Grant BacCellEpi 670823). R.P. and A.C. received an FCT Doctoral Fellowship (SFRH/BD/89542/2012, SFRH/BD/29314/2006) through FCT/MEC co-funded by QREN and POPH (Programa Operacional Potencial Humano). SS was supported by FCT Investigator program (COMPETE, POPH and FCT).

Background The impact of human activities on the environmental resistome has been documented in many studies, but there remains the controversial question of whether the increased antibiotic resistance observed in anthropogenically impacted environments is just a result of contamination by resistant fecal microbes or is mediated by indigenous environmental organisms. Here, to determine exactly how anthropogenic influences shape the environmental resistome, we resolved the microbiome, resistome, and mobilome of the planktonic microbial communities along a single river, the Han, which spans a gradient of human activities. Results The bloom of antibiotic resistance genes (ARGs) was evident in the downstream regions and distinct successional dynamics of the river resistome occurred across the spatial continuum. We identified a number of widespread ARG sequences shared between the river, human gut, and pathogenic bacteria. These human-related ARGs were largely associated with mobile genetic elements rather than particular gut taxa and mainly responsible for anthropogenically driven bloom of the downstream river resistome. Furthermore, both sequence- and phenotype-based analyses revealed environmental relatives of clinically important proteobacteria as major carriers of these ARGs. Conclusions Our results demonstrate a more nuanced view of the impact of anthropogenic activities on the river resistome: fecal contamination is present and allows the transmission of ARGs to the environmental resistome, but these mobile genes rather than resistant fecal bacteria proliferate in environmental relatives of their original hosts.

The aim of this study was to investigate mechanisms of nitrofurantoin resistance and epidemiological characteristics in Escherichia coli clinical isolates. From a total of 1444 E. coli clinical isolates collected from our hospital in 2015, 18 (1.2%) nitrofurantoin-resistant E. coli isolates were identified with nitrofurantoin minimum inhibitory concentrations (MICs) ranging from 128 µg/mL to ≥512 µg/mL. The prevalence of the nfsA gene in nitrofurantoin-resistant, -intermediate and -susceptible isolates was 88.9%, 88.9% and 100%, respectively, and the prevalence of the nfsB gene was 66.7%, 61.1% and 100%, respectively. Eight nitrofurantoin-resistant isolates and two nitrofurantoin-intermediate isolates possessed oqxAB genes. In nitrofurantoin-resistant isolates, mutations in NfsA (the majority of mutated sites were I117T and G187D, accounting for 38.9%) and/or NfsB were detected, whereas only NfsA mutations were found in intermediate isolates and no sequence changes were detected in susceptible isolates. A ≥4-fold decrease in MIC was observed in eight nitrofurantoin-resistant isolates following addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mean expression level of oqxB in nitrofurantoin-resistant isolates increased ca. 7-fold compared with intermediate isolates. Multilocus sequence typing (MLST) categorised the 18 nitrofurantoin-resistant isolates into 11 different sequence types. Pulsed-field gel electrophoresis (PFGE) analysis revealed that homology among the nitrofurantoin-resistant isolates was low and sporadic. In conclusion, mutations in nfsA and nfsB were the main mechanisms leading to nitrofurantoin resistance, and overexpression of the oqxAB gene might help to further increase the MIC of nitrofurantoin.

The present study examines the trend in distribution of Candida species and their antifungal resistance patterns in hospitals across Haryana, a North Indian state with poorly addressed epidemiology of fungal infections. In our collection of 228 Candida isolates, Candida albicans dominated in both high vaginal swab (HVS) and urine samples while Candida glabrata and Candida tropicalis were the second-highest non-albicans Candida species (NAC), respectively. Of note, in blood samples, C. tropicalis and C. albicans were present in equal numbers. All 228 isolates were subjected to antifungal susceptibility tests, whereby 51% of C. albicans recovered from HVS samples displayed fluconazole resistance. To understand its mechanistic basis, expression profiling of efflux pump genes CDR1, CDR2, MDR1 and azole drug target, ERG11 was performed in 20 randomly selected resistant isolates, wherein many isolates elicited higher expression. Further, ERG11 gene sequencing suggested that most of the isolates harbored mutations, which are not reported with azole resistance. However, one isolate, RPCA9 (MIC 64 μg/mL) harbored triple mutation (Y132C, F145L, A114V), wherein Y132 and F145 sites were previously implicated in azole resistance. Interestingly, one isolate, (RPCA61) having MIC > 128 μg/mL harbored a novel mutation, G129R. Of note, HVS isolates RPCA 21, RPCA 22, and RPCA 44 (MICs 64 to > 128 μg/mL) did not show any change in alteration in ERG11 or overexpression of efflux pump genes. Together, this study presents a first report of Candida infections in selected hospitals of Haryana State.

Staphylococcus aureus has been reported as one of the most difficult to treat. In the search for new treatment alternatives, isolated plant substances such as phenolic compounds, have demonstrated the ability to reverse bacterial resistance. The present study aims to evaluate the inhibitory action of caffeic acid and gallic acid on efflux pumps from S. aureus resistant strains. The broth microdilution assay was carried out to obtain the MICs of caffeic acid and gallic acid while the efflux pump inhibition test was assessed through the reduction of the minimum inhibitory concentration of the antibiotic and ethidium bromide. In addition, in silico theoretical parameters were analyzed to determine the theoretical efficacy of the compound and its free energy of interaction. In the results, the inhibition concentration of the two compounds did not certify clinical relevance with 1024 μg/mL for all strains. In the efflux pump inhibition effect, caffeic acid inhibited the MrsA pumps of the strain RN-4220 and NorA of the strain 1199B. Caffeic acid showed greater efficacy in the docking model, in agreement with the demonstrated experimental efficacy. Isolated compounds can be indicated as efficient options in the inhibition of resistance mechanisms.