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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.

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.

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.

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.

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

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.

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 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.

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).

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.

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.

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.

Problem addressed Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), particularly of the clonal complex (CC) 398, emerged as zoonotic pathogens predominantly among humans with direct or indirect livestock contact, but also in healthcare settings. The factors contributing to the success of LA-MRSA are only poorly understood. Objective During the past years, the use of heavy metal compounds as feed-supplements was found to influence the co-selection of LA-MRSA in pig herds. This study aimed to determine the prevalence of zinc resistance among MRSA CC398 isolated from patients of a German university hospital located in a pig farming-dense area. Methods and approach In comparison to concurrent healthcare-associated MRSA (HA-MRSA), LA-MRSA CC398 comprising isolates from their first appearance in 2000 to recent isolates from 2014 were included. Results Among MRSA CC398, the overall resistance rate towards zinc chloride was 57% compared to only 3% among concurrently isolated HA-MRSA. Zinc resistance correlated with the presence of the czrC gene in 100% of the MRSA CC398 and in 67% of the HA-MRSA. Conclusions The zinc resistance rate in MRSA CC398 significantly increased from 2009 to 2014 with a maximum in 2014. Alarmingly, zinc resistance has become a frequent phenotype of human LA-MRSA in Germany potentially facilitating co-selection of antibiotic resistance genes.

The increased prevalence of the virulence factor exoU + genotype among multidrug-resistant Pseudomonas aeruginosa has been previously reported. However, the genes that are related to the multidrug resistance of the exoU + genotype strain have not been analyzed and remain to be elucidated. The objective of this study was to analyze the correlations between virulence factors and resistance genes. The exoU + genotype was frequently found in carbapenem and fluoroquinolone non-susceptible strains. The imp carbapenemase genotype, the quinolone-resistance-determining region mutation in GyrA and ParC and the defective mutation in OprD were not frequently found in the exoU + genotype and carbapenem and fluoroquinolone non-susceptible strains. On the other hand, mexY and ampC mRNA overexpressing strains were more frequently found in the exoU + genotype and carbapenem and fluoroquinolone non-susceptible strains. Moreover, sequence type 235, a high risk clone of multidrug-resistant P. aeruginosa, was prevalent among the exoU + genotype and carbapenem and fluoroquinolone non-susceptible strains. ExoU is highly virulent protein, and the overexpression of efflux pumps and AmpC β-lactamase induce a multidrug-resistant phenotype. Therefore, the increased prevalence of P. aeruginosa strains with an exoU + genotype and the overexpression of efflux pumps and AmpC β-lactamase are likely to make P. aeruginosa infections difficult to treat. An understanding of the prevalence of both the exoU + genotype and the mRNA overexpression of resistance genes may help to select empirical therapy for the treatment of nosocomial infections caused by P. aeruginosa.

RNA interference (RNAi) is considered a highly specific approach for gene silencing and holds tremendous potential for treatment of various pathologic conditions such as cardiovascular diseases, viral infections, and cancer. Although gene silencing approaches such as RNAi are widely used in preclinical models, the clinical application of RNAi is challenging primarily because of the difficulty in achieving successful systemic delivery. Effective delivery systems are essential to enable the full therapeutic potential of RNAi. An ideal nanocarrier not only addresses the challenges of delivering naked siRNA/miRNA, including its chemically unstable features, extracellular and intracellular barriers, and innate immune stimulation, but also offers "smart" targeted delivery. Over the past decade, great efforts have been undertaken to develop RNAi delivery systems that overcome these obstacles. This review presents an update on current progress in the therapeutic application of RNAi with a focus on cancer therapy and strategies for optimizing delivery systems, such as lipid-based nanoparticles.

As the first-line drug for breast cancer chemotherapy, doxorubicin (Dox) has strong cardiotoxicity. Meanwhile, prolonged Dox treatment of patients with breast cancer may result in resistance of breast cancer cells to Dox and an increased number of Dox-resistant breast cancer stem cells (BCSCs), thereby easily leading to breast cancer relapse. TanshinoneIIA (Tan IIA) has anti-tumor activity in addition to its cardiovascular protective effect. By preparing Dox resistant human breast cancer MCF-7 cells, here, we wanted to assess a new use of Tan IIA in enhancing the chemosensitivity of breast cancer cells to Dox and investigated its possible mechanisms. The results showed that Tan IIA could enhance the anti-tumor effect of Dox on MCF-7 and MCF-7/dox cells in a dose-dependent manner, especially that of on MCF-7/dox cells. Even nontoxic dose of Tan IIA could also promote intracellular Dox accumulation of MCF-7 and MCF-7/dox cells through down-regulating the expression of efflux ABC transporters including P-gp, BCRP and MRP1, which can effectively eliminated cancerous cells including BCSCs, thereby enhancing the chemosensitivity of breast cancer. Therefore, Tan IIA can be used as a new potential chemotherapeutic sensitizer for the combination treatment of breast cancer.

Nowadays, lung cancer, as a health problem in worldwide, has high mortality both in men and women. Despite advances in diagnosis and surgical techniques of lung cancer in recent decades, chemotherapy is still a fundamentally and extensively useful strategy. Vinca alkaloids are a class of important and widely used drugs in the treatment of lung cancer, targeting on the Vinca binding site at the exterior of microtubule plus ends. Either intrinsic or acquired resistance to chemotherapy of Vinca alkaloids has been a major obstacle to the treatment of lung cancer, which arose great interests in studies of understanding and overcoming resistance. In this review, we focused on the application and resistance mechanisms of the Vinca alkaloids such as vinblastine, vincristine, vinorelbine and vinflunine in lung cancer. We reviewed characteristic resistance mechanisms in lung cancer including over-expression of ATP-binding cassette (ABC) transporters P-glycoprotein and structural, functional or expression alterations of β-tubulin (βII, βIII, βIV) which may devote to the development of acquired resistance to the Vinca alkaloids; multidrug-resistance proteins (MRP1, MRP2, MRP3) and RLIP76 protein have also been identified that probably play a significant role in intrinsic resistance. Lung resistance-related protein (LRP) is contributed to lung cancer therapy resistance, but is not deal with the Vinca alkaloids resistance in lung cancer. Understanding the principle of the Vinca alkaloids in clinical application and mechanisms of drug resistance will support individualized lung cancer therapy and improve future therapies.

Background Selection inversion is the hypothesis for antibiotic resistant inhabitation in bacteria and collateral sensitivity is one of the proposed phenomena for achievement of this hypothesis. The presence of collateral sensitivity associated with the proton motivation pump between the aminoglycosides and beta-lactam group of antibiotics is one of the examples of collateral sensitivity in some studies. The aim of this study was to demonstrate that collateral sensitivity between aminoglycosides and beta-lactam antibiotics associated with proton motivation pump may not be true in all cases. Methods In this study, 100 Pseudomonas aeruginosa were surveyed. Gentamicin and imipenem-resistant strains were confirmed by disc diffusion method and MIC. Active proton motivation pumps were screened by pumps inhibitor. Semi-quantitative Real-Time PCR assay was used to confirm gene overexpression. Results Seventy-six and 79 out of 100 strains were resistant to gentamicin and imipenem, respectively. Seventy-five strains were resistant to both gentamicin and imipenem. The results of proton pump inhibitor test showed the involvement of active proton motivation pump in 22 of 75 imipenem- and gentamicin-resistant strains. According to Real – Time PCR assay, mexX efflux gene was overexpressed in the majority of isolates tested. Discussion The collateral sensitivity effect cannot explain the involvement of active proton motivation pumps in both imipenem and gentamicin-resistant strains simultaneously. Active and/or inactive proton pump in gentamicin-sensitive and/or resistant strains cannot be a suitable example for explanation of collateral sensitivity between aminoglycosides and beta-lactam antibiotics.

Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological malignancies. Defects in the cell growth and apoptotic pathways are responsible for both disease pathogenesis and treatment resistance. Therefore, pro-apoptotic agents are potential candidates for APL treatment. Kaempferol is a flavonoid with antioxidant and anti-tumor properties. This study was designed to investigate the cytotoxic, pro-apoptotic and differentiation-inducing effects of kaempferol on HL-60 and NB4 leukemia cells. Resazurin assay was used to determine cell viability following treatment with kaempferol (12.5-100 µM) and all-trans retinoic acid (ATRA; 10 µM; used as a positive control). Apoptosis and differentiation were also detected using propidium iodide and NBT staining techniques, respectively. Furthermore, the expression levels of genes involved in apoptosis (PI3K, AKT, BCL2, BAX, p53, p21, PTEN, CASP3, CASP8 and CASP9), differentiation (PML-RAR and HDAC1), and multi-drug resistance (ABCB1 and ABCC1) were determined using quantitative real-time PCR. The protein expressions of Bax/Bcl2 and casp3 were confirmed using western blot. The results showed that kaempferol decreased cell viability and increased subG1 population in the tested leukemic cells. This effect was associated with decreased expression of Akt, BCL2, ABCB1 and ABCC1 genes, while the expression of CASP3 and BAX/BCL-2 ratio were significantly increased at both gene and protein levels. Kaempferol promoted apoptosis and inhibited multidrug resistance in a concentration-dependent manner, without any differential effect on leukemic cells. In conclusion, this study suggested that kaempferol may be utilized as an appropriate alternative for ATRA in APL patients. This article is protected by copyright. All rights reserved

Nonfermenting Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii are widespread in the environment and are increasingly associated with nosocomial infections, often associated with multidrug-resistance phenotypes. This study aimed to evaluate epidemiological, physiological, and molecular characteristics of carbapenem resistance in P. aeruginosa and A. baumannii. In total, 63 nonreplicated strains (44 A. baumannii and 19 P. aeruginosa) were isolated from hospitalized patients. Antimicrobial resistance patterns, biocide tolerance, oxidative stress, hemolytic activity, and biofilm formation were assessed. Genetic markers related to β-lactamase synthesis, efflux systems, and porin loss were screened by PCR. Epidemiological data of patients were analyzed. Advanced age, intensive care unit admission, invasive medical devices, treatment with fluoroquinolones or β-lactams/β-lactamase inhibitor combinations, and prolonged hospital stay were predisposing factors for infection. Colistin showed to be active in vitro against these bacteria. Carbapenem-resistant P. aeruginosa strains did not show hemolytic activity and were less tolerant to oxidative stress and biocides. However, increased ability of biofilm formation was observed, comparing to the carbapenem-susceptible isolates. Genetic markers related to oxacillinases synthesis (OXA-23 and OXA-143), oprD absence, and efflux pump (adeB) were detected in carbapenem-resistant A. baumannii. Screening for OXA-51-like gene was performed as confirmatory test for A. baumannii identification. In P. aeruginosa genes encoding efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM) and SPM-1 were found; besides, oprD absence was also observed. Our results suggest that these organisms are well adapted to different environments and confirm the difficulty of therapeutic management of patients with infections associated with multidrug-resistant microorganisms, with direct impact on mortality and epidemiological control of these strains in health centers.

One mcr-1 -carrying Salmonella enterica serovar Indiana strain D90, was identified from 1320 Salmonella enterica isolates from poultry slaughterhouse in 2012 in China. The objective of this study was to verify the transferability of the mcr-1 gene and also completely characterize the sequence of the strain at the whole-genome level. Broth matting assays were carried out to detect the transferability and whole-genome sequencing (WGS) of S. enterica serovar Indiana D90 was performed using the PacBio RS II system. Open reading frames were assigned using Rapid Annotation using Subsystem Technology (RAST) and analysed by BLASTn and BLASTp. Salmonella Pathogenisity Islands (SPIs) were annotated by SPIFinder platform. The complete genome sequence of S. enterica serovar Indiana D90 contained a circular 4,779,514-bp chromosome and four plasmids. Genome analysis and sequencing revealed that 24 multi-drug resistance (MDR) genes were located on plasmids. The largest plasmid pD90-1, was found to be of an IncHI2/HI2A/Q1/N type that encoded a bla CTX-M-65 gene along with 20 additional antimicrobial resistance genes. A 60.5-kbp IncI2 plasmid pD90-2 contained a nikA - nikB - mcr-1 genetic structure, that can be successfully transferred to E. coli and S. enterica serovar Typhimurium at low transfer rates. Interestingly, comparative sequence analysis revealed the plasmids pD90-1 and pD90-2 showed considerable nucleotide similarity to pHNSHP45-2 and pHNSHP45, respectively. Moreover, the genome and the plasmid pD90-2 also showed high similarity to one carbapenem resistant S. enterica serovar Indiana strain, C629 and its plasmid pC629, respectively. This is the first report of the complete nucleotide sequence of one mcr-1 -carrying MDR S. enterica serovar Indiana strain.

We previously reported on brain H1 receptor occupancy measurements of antihistamines in human brain using [11C]doxepin and positron emission tomography (PET). We proposed the use of brain H1 receptor occupancy to classify antihistamines objectively into three categories of sedating, less-sedating, and non-sedating antihistamines according to their sedative effects. Non-sedating antihistamines are recommended for the treatment of allergies such as pollinosis and atopic dermatitis because of their low penetration into the central nervous system. Physicians and pharmacists are responsible for fully educating patients about the risks of sedating antihistamines from pharmacological points of view. If a sedating antihistamine must be prescribed, its sedative effects should be thoroughly considered before choosing the drug. Non-sedating antihistamines should be preferentially used whenever possible as most antihistamines are equally efficacious, while adverse effects of sedating antihistamines can be serious. This review summarizes the pharmacological properties of clinically useful non-sedating antihistamines from the perspective of histamine function in the CNS.

The master regulator, DnrI of Streptomyces peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.

The multidrug resistance 2 knockout (Mdr2-/- ) mouse is a well-established model of cholestatic cholangiopathies. Female Mdr2-/- mice develop more severe hepatobiliary damage than male Mdr2-/- mice, which is correlated with a higher proportion of taurocholate in the bile. Although estrogen has been identified as an important player in intrahepatic cholestasis, the underlying molecular mechanisms of gender-based disparity of cholestatic injury remain unclear. The long noncoding RNA H19 is an imprinted, maternally expressed, and estrogen-targeted gene, which is significantly induced in human fibrotic/cirrhotic liver and bile duct-ligated mouse liver. However, whether aberrant expression of H19 accounts for gender-based disparity of cholestatic injury in Mdr2-/- mice remains unknown. The current study demonstrated that H19 was markedly induced (∼200-fold) in the livers of female Mdr2-/- mice at advanced stages of cholestasis (100 days old) but not in age-matched male Mdr2-/- mice. During the early stages of cholestasis, H19 expression was minimal. We further determined that hepatic H19 was mainly expressed in cholangiocytes, not hepatocytes. Both taurocholate and estrogen significantly activated the extracellular signal-regulated kinase 1/2 signaling pathway and induced H19 expression in cholangiocytes. Knocking down H19 not only significantly reduced taurocholate/estrogen-induced expression of fibrotic genes and sphingosine 1-phosphate receptor 2 in cholangiocytes but also markedly reduced cholestatic injury in female Mdr2-/- mice. Furthermore, expression of small heterodimer partner was substantially inhibited at advanced stages of liver fibrosis, which was reversed by H19 short hairpin RNA in female Mdr2-/- mice. Similar findings were obtained in human primary sclerosing cholangitis liver samples. Conclusion H19 plays a critical role in the disease progression of cholestasis and represents a key factor that causes the gender disparity of cholestatic liver injury in Mdr2-/- mice. (Hepatology 2017;66:869-884).

Patient: Male, 32 Final Diagnosis: NDM-1-producing Klebsiella pneumoniae • bacteremia Symptoms: Fever Medication: — Clinical Procedure: None Specialty: Infectious Diseases Objective: Diagnostic/therapeutic accidents Background: Infections affecting burn patients are frequently caused by Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae species. Infections with these pathogens have become increasingly difficult to treat due to evolving antibiotic resistance mechanisms, including the production of carbapenemases. Case Report: The present case report describes the evolution of a burn patient with polymicrobial healthcare-associated burn infections, including a bloodstream infection due to an emergent multidrug-resistant New Delhi metallo-beta-lactamase (NDM-1)-producing Klebsiella pneumoniae. During hospitalization, initial antibiotic treatment eradicated some of the infecting species. Newer isolates were found to be multidrug-resistant and required unique antibiotic combinations. The patient’s condition continued to deteriorate after the isolation of multidrug-resistant P. aeruginosa and NDM-1-positive K. pneumoniae from the blood. Conclusions: This case report illustrates the need for adequate antibiotic therapies in burn patients with subsequent infections due to a carbapenemase-producing multidrug-resistant bacteria. The potential danger of new bacterial pathogens should be considered in this group of susceptible patients.

We aimed to investigate the association between the polymorphism and expression patterns of multiple drug resistance genes (MDR) in breast cancer (BC). The MDR gene expression levels were measured in tumor tissues of 106 breast cancer patients using quantitative real-time PCR. Affymetrix CytoScan™ HD Array chips were used to assess genotypes. Pairwise correlation analysis for ABCB1, ABCC1, ABCC2 and ABCG2 gene expression levels was carried out to reveal co-expression clusters. Associations between SNPs of MDR genes and their preoperative expression levels were assessed using analysis of covariance adjusting for covariates. The SNPs associated with the expression of the ABCB1, ABCC1, ABCC2 and ABCG2 genes before NAC were detected. In addition, 21 SNPs associated with the expression of four ABC-transporter genes and involved in the expression regulation were identified. Validation in an independent sample confirmed the association between the MDR cluster genes and 11 SNPs. Four MDR genes: ABCB1, ABCC1, ABCC2 and ABCG2 were shown to form the functional expression cluster in breast tumor. Further studies are required to discover precise mechanisms of the cluster regulation, thereby providing new approaches and targets to combat the development of the MDR phenotype during chemotherapy.

The discovery of penicillin followed by streptomycin, tetracycline, cephalosporins and other natural, semi-synthetic and synthetic antimicrobials completely revolutionized medicine by reducing human morbidity and mortality from most of the common infections. However, shortly after they were introduced to clinical practice, the development of resistance was emerged. The decreasing interest from antibiotic industry in spite of rapid global emergence of antibiotic resistance is a tough dilemma from the pointview of public health. The efficiency of antimicrobial treatment is determined by both pharmacokinetics and pharmacodynamics. In spite of their selective toxicity, antibiotics still cause severe, life-threatening adverse reactions in host body mostly due to defective drug metabolism or excessive dosing regimen. The present article aims at updating current knowledge on pharmacokinetics/pharmacodynamics concepts and models, toxicity of antibiotics as well as antibiotic resistance mechanisms, resistome analyses and search for novel antibiotic resistance determinants with special emphasis given to the-state-of-the-art regarding multidrug efflux pumps and their additional physiological functions in stress adaptation and virulence of bacteria. All these issues are highly linked to each other and not only important for most efficient and prolonged use of current antibiotics, but also for discovery and development of new antibiotics and novel inhibitors of antibiotic resistance determinants of pathogens.

Emerging antibiotic resistance in the oropharyngeal microbiota, of which Streptococcus salivarius is a prominent species, represents a challenge for treating paediatric populations. In this study, we investigated the role of Streptococcus salivarius as a reservoir for antibiotic resistance genes (ARG) in the oral microbiota by analysing 95 Streptococcus salivarius isolates from 22 healthy infants (2–16 months of age). MICs of penicillin G, amoxicillin, erythromycin, tetracycline, doxycycline and streptomycin were determined. ARG profiles were assessed in a subset of 21 strains by next-generation sequencing of genomes, followed by searches of assembled reads against the Comprehensive Antibiotic Resistance Database. Strains resistant to erythromycin, penicillins and tetracyclines were isolated from 83.3, 33.3 and 16.6 %, respectively, of infants aged 2 to 8 months with no prior antibiotic treatment. These percentages were100.0, 66.6 and 50.0 %, by 13 to 16 months of age. ARG or polymorphisms associated with antibiotic resistance were the most prevalent and involved genes for macrolide efflux (mel, mefA/E and macB), ribosomal protection [erm(B), tet(M) and tet(O)] and β-lactamase-like proteins. Phylogenetically related strains showing multidrug-resistant phenotypes harboured multidrug efflux ARG. Polymorphic genes associated with antibiotic resistance to drugs affecting DNA replication, folate synthesis, RNA/protein synthesis and regulators of antibiotic stress responses were detected. These data imply that Streptococcus salivarius strains established during maturation of the oral microbiota harbour a diverse array of functional ARG, even in the absence of antibiotic selective pressures, highlighting a potential role for this species in shaping antibiotic susceptibility profiles of oropharyngeal communities.

In Gram-negative bacteria, a synergistic relationship between slow passive uptake of antibiotics across the outer membrane and active efflux transporters creates a permeability barrier, which efficiently reduces the effective concentrations of antibiotics in cells and, hence, their activities. To analyze the relative contributions of active efflux and the passive barrier to the activities of antibiotics, we constructed Escherichia coli strains with controllable permeability of the outer membrane. The strains expressed a large pore that does not discriminate between compounds on the basis of their hydrophilicity and sensitizes cells to a variety of antibacterial agents. We found that the efficacies of antibiotics in these strains were specifically affected by either active efflux or slow uptake, or both, and reflect differences in the properties of the outer membrane barrier, the repertoire of efflux pumps, and the inhibitory activities of antibiotics. Our results identify antibiotics which are the best candidates for the potentiation of activities through efflux inhibition and permeabilization of the outer membrane.

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.

Riemerella anatipestifer is a Gram-negative bacterium, which is an important pathogen infecting ducks and resistant to various antibiotics. The efflux pump is an important resistance mechanism of Gram-negative bacteria, but little research has been done in R. anatipestifer. In this study, the drug resistance mediated by RIA_1614 gene of R. anatipestifer RA-GD strain was studied, because the gene was presumed to be an efflux pump component of ABC. Firstly, the deletion strain RA-GD△RIA_1614 and complemented strain RA-GD△RIA_1614 pCPRA::RIA_1614 were constructed. Then, MICs of various antimicrobial agents to parent and deletion strains and the tolerance of the strains to organic solvents were detected to screen the substrates for RIA_1614 gene. Moreover, the transcription levels of RIA_1614 gene in the parent and the complemented strains exposed to the substrates were detected by quantitative real-time RT-PCR. Furthermore, the efflux abilities of parent, deletion and complemented strains to substrates were determined by antibiotic accumulation test. In addition, in vitro competition ability and virulence of the strains were also detected. The results showed that the deletion strain was more sensitive to aminoglycosides and organic solvents than parental strain RA-GD. When RA-GD and complemented strain were exposed to sub-repression levels of aminoglycosides and organic solvents, the transcription levels of RIA_1614 gene were significantly up-regulated. Sodium o-vanadate inhibitor assay confirmed that RIA_1614 protein contributed to amikacin and streptomycin resistance and organic solvent tolerance. Streptomycin accumulation test showed that the RIA_1614 protein was able to export streptomycin, and the addition of ATPase inhibitor sodium o-vanadate increased the accumulation of streptomycin, indicating that RIA_1614 protein was an ATP-dependent efflux transporter. Growth and competition experiments revealed that RIA_1614 protein had no significant effect on growth of RA-GD, but decreased in vitro competition ability of the strain. Furthermore, pathogenicity tests showed that RIA_1614 protein involved in the virulence of the strain. Based on the results and amino acid sequence analysis, it was determined that RIA_1614 protein was a member of ABC efflux pumps, and the protein was named RanB.

The presence of antimicrobial-resistant bacteria and resistance genes in aquatic environments is a serious public health concern. This study focused on Escherichia coli possessing bla(CTX-M) genes in wastewater inflows. Twelve crude inflow water samples from wastewater treatment plant (WWTP) A and two samples each from three other WWTPs were collected in 2017 and 2018. A total of 73 E. coli isolates with 31 different sequence types (STs) harboring distinctive bla(CTX-M) gene repertoires were detected. In WWTP A influents, bla(CTX-M-14) (14 isolates) was dominant, followed by bla(CTX-M-15 )(12 isolates) and bla(CTX-M-27) (10 isolates). The chimeric bla(CTX-M-)(64) and bla(CTX-M-123) genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The bla(CTX-M-27) gene was associated with five of seven B2-ST131 isolates, including three isolates of the B2-O25b-ST131-H30R/non-Rx lineage. One of the remaining two isolates belonged to the B2-O25b-ST131-H30R/Rx lineage harboring the bla(CTX-M-15) gene. As for the B2-025b-ST131-H30R/non-Rx lineage, two isolates with bla(CTX-M-27) were recovered from each of the WWTP B and D influents, and one isolate with bla(CTX-M-174) was also recovered from WWTP B influent. Whole-genome sequencing of chimeric bla(CTX-M) -harboring E. coli isolates revealed that the bla(CTX-M-64) gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The bla(CTX-M-123)-carrying Incl1 plasmid pB64 was 109,169 bp in length with pST108. The overall findings suggest that wastewater may act as a probable reservoir of clinically significant clonal lineages mediating antimicrobial resistance genes and chimeric genes that have not yet been identified from human isolates of domestic origin in Japan., Article, Applied and Environmental Microbiology 85(22) : e01740-19-(2019)

Antibiotic resistance genes (ARGs) are emerging contaminants that pose a potential threat to human health worldwide. Urban wastewater treatment plants (WWTPs) are a main source of both antibiotic-resistant bacteria and ARGs released into the environment. Nevertheless, the propagation of ARGs and their underlying mechanisms and the dynamics of mobile genetic elements (MGEs) in WWTPs have rarely been investigated in South Korea. In this study, shotgun metagenomic analysis was used to identify comprehensive ARGs and their mechanisms, bacterial communities, and MGEs from 4 configurations with 2 activated sludge (AS) and 2 anaerobic digestion sludge (ADS) samples. A total of 181 ARG subtypes belonging to 22 ARG types were broadly detected, and the ARG abundances in the AS samples were 1.3-2.0 orders of magnitude higher than in the ADS samples. Multidrug and bacitracin resistance genes were the predominant ARG types in AS samples, followed by ARGs against sulfonamide, tetracycline, and β-lactam. However, the composition of ARG types in ADS samples was significantly changed. The abundance of multidrug and β-lactam resistance genes was drastically reduced in the ADS samples. The resistance genes of MLS were the predominant, followed by ARGs against sulfonamide and tetracycline in the ADS samples. In addition, plasmids were the dominant MGEs in the AS samples, while integrons (intI1) were the dominant MGEs in the ADS samples. These results provide valuable information regarding the prevalence of ARG types and MGEs and the difference patterns between the AS and ADS systems.

Even though Escherichia coli strains may share nearly identical phenotypic AMR profiles and AMR genes and overlap in space and time, the diversity of clones and plasmids challenges research that aims to identify sources of AMR. Horizontal gene transfer appears to play a more significant role than clonal expansion in the spread of AMR in this community., The increased prevalence of antimicrobial resistance (AMR) among Enterobacteriaceae has had major clinical and economic impacts on human medicine. Many of the multidrug-resistant (multiresistant) Enterobacteriaceae found in humans are community acquired, and some of them are possibly linked to food animals (i.e., livestock raised for meat and dairy products). In this study, we examined whether numerically dominant commensal Escherichia coli strains from humans (n = 63 isolates) and domestic animals (n = 174 isolates) in the same community and with matching phenotypic AMR patterns were clonally related or shared the same plasmids. We identified 25 multiresistant isolates (i.e., isolates resistant to more than one antimicrobial) that shared identical phenotypic resistance patterns. We then investigated the diversity of E. coli clones, AMR genes, and plasmids carrying the AMR genes using conjugation, replicon typing, and whole-genome sequencing. All of the multiresistant E. coli isolates (from children and domestic animals) analyzed had at least 90 or more whole-genome SNP differences between one another, suggesting that none of the strains was recently transferred. While the majority of isolates shared the same antimicrobial resistance genes and replicons, DNA sequencing indicated that these genes and replicons were found on different plasmid structures. We did not find evidence of the clonal spread of AMR in this community: instead, AMR genes were carried on diverse clones and plasmids. This presents a significant challenge for understanding the movement of AMR in a community. IMPORTANCE Even though Escherichia coli strains may share nearly identical phenotypic AMR profiles and AMR genes and overlap in space and time, the diversity of clones and plasmids challenges research that aims to identify sources of AMR. Horizontal gene transfer appears to play a more significant role than clonal expansion in the spread of AMR in this community.

Many laboratories are unable to perform colistin susceptibility testing. Diffusion-based antimicrobial susceptibility testing methods are not recommended, and not all laboratories have the capacity to perform broth microdilution (BMD). Using a multistep tiered approach, we investigated whether the adapted use of the MicroScan colistin well (4 μg/ml) could enhance laboratory capacity for the detection and subsequent molecular characterization of colistin-resistant Enterobacteriaceae. For the MicroScan colistin well, categorical agreement with BMD was 92.7%, and the very major error rate was 10.7%. For gradient diffusion strips, the categorical agreement was 86.4%, and the very major error rate was 53.6%. The MicroScan colistin well detected all isolates carrying mcr-1 or mcr-2 genes (n = 16), but gradient diffusion strips identified an MIC of ≥4 for colistin for only 62.5% of these isolates. A 6-month prospective phenotypic and genotypic study performed at a single clinical microbiology laboratory assessed isolates growing in the MicroScan colistin well for concordance. While 37 of 39 isolates growing in the MicroScan colistin well displayed a colistin MIC of ≥4 by BMD, all were determined to be negative for the mcr-1 and mcr-2 genes by PCR. A retrospective review of all Escherichia coli, Klebsiella spp., and Enterobacter spp. tested by MicroScan at this laboratory in 2016 identified 260 of 7,894 (3.3%) isolates that grew in the MicroScan colistin well. Based on the data presented, clinical and public health laboratories could use the MicroScan colistin well as a first screen for the detection of isolates displaying elevated colistin MICs, which could then undergo further characterization.

Plasmid-mediated colistin resistance (PMCR), a consequence of the mcr genes, is a significant public health concern given its potential to easily spread among clinical pathogens. Recently, it was discovered that MCR enzymes require zinc for activity. Thus, we modified the colistin broth-disk elution (CBDE) test to screen for plasmid-mediated colistin resistance (PMCR) genes based on any reduction of colistin MIC in the presence of EDTA. Eighty-five isolates of the order Enterobacteriales (12 mcr positive) were tested by CBDE ± EDTA. The sensitivity and specificity of the EDTA-CBDE method to detect PMCR compared to the molecular genotype results were 100% and 95.8%, respectively. Isolates positive by the EDTA-CBDE test should be further evaluated to confirm the presence of mcr genes.

Objective:The smr and qacA/B genes in Staphylococcus aureus confer tolerance to antiseptics and are associated with nosocomial acquisition of infection and underlying medical conditions. Such antiseptic tolerance (AT) genes have also been reported in coagulase-negative staphylococci (CoNS) and enterococci, however, few data are available regarding their prevalence. We sought to describe the frequency of AT genes among bloodstream isolates of S. aureus, CoNS and enterococci at Texas Children’s Hospital (TCH).Methods:Banked CoNS, S. aureus and enterococci isolated from blood cultures collected bewteen October 1, 2016, and October 1, 2017, were obtained from the TCH clinical microbiology laboratory. All isolates underwent polymerase chain reaction (PCR) assay for the qacA/B and smr genes. Medical records were reviewed for all cases.Results:In total, 103 CoNS, 19 Enterococcus spp, and 119 S. aureus isolates were included in the study, and 80.6% of the CoNS possessed at least 1 AT gene compared to 37% of S. aureus and 43.8% of E. faecalis isolates (P < .001). Among CoNS bloodstream isolates, the presence of either AT gene was strongly associated with nosocomial infection (P < .001). The AT genes in S. aureus were associated with nosocomial infection (P = .025) as well as the diagnosis of central-line–associated bloodstream infection (CLABSI; P = .04) and recent hospitalizations (P < .001). We found no correlation with genotypic AT in E. faecalis and any clinical variable we examined.Conclusions:Antiseptic tolerance is common among bloodstream staphylococci and E. faecalis isolates at TCH. Among CoNS, the presence of AT genes is strongly correlated with nosocomial acquisition of infection, consistent with previous studies in S. aureus. These data suggest that the healthcare environment contributes to AT among staphylococci.

Summary Objectives The heavy use of antibiotics in farm animals contributes to the enrichment and spread of antibiotic resistance genes (ARGs) in “one-health” settings. Numerous ARGs have been identified in livestock-associated environments but not in Chinese live poultry markets (LPMs). Methods We collected 753 poultry fecal samples from LPMs of 18 provinces and municipalities in China and sequenced the metagenomes of 130 samples. Bioinformatic tools were used to construct the gene catalog and analyze the ARG content. PCR amplification and Sanger sequencing were used to survey the distribution of mcr-1 gene in all 753 fecal samples. Results We found that a low number of genes but a high percentage of gene functions were shared among the poultry, human and pig gut gene catalogs. The poultry gut possessed 539 ARGs which were classified into 235 types. Both the ARG number and abundance were significantly higher in poultry than that in either pigs or humans. Fourteen ARG types were found present in all 130 samples, and tetracycline resistance (TcR) genes were the most abundant ARGs in both animals and humans. Moreover, 59.63% LPM samples harbored the colistin resistance gene mcr-1, and other mcr gene variants were also found. Conclusions We demonstrated that the Chinese LPMs is a repository for ARGs, posing a high risk for ARG dissemination from food animals to humans under such a trade system, which has not been addressed before.