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Abstract:ObjectiveMethodsResultsConclusionPrevious clinical studies have demonstrated that Wumei Pill (WP), a traditional Chinese medicine formula, can effectively alleviate nocturnal asthma-related anxiety and improve nighttime symptoms. The therapeutic mechanism of WP may involve regulation of inflammatory chemokines in peripheral blood. This mechanism is potentially linked to modulation of the circadian clock gene ARNT-like protein-1 (Bmal1), but precise pathways underlying this interaction remain unclear, requiring further investigation.Bmal1 knockout and wild-type mice were utilized to establish asthma models. Techniques such as flow cytometry, RT-PCR, and ELISA were employed to measure the levels of serum inflammatory mediators, specifically IFN-γ, CXCL16, I-TAC, and PARC. Furthermore, the pathological alterations in airway thickness were assessed. Additionally, we investigated the regulation of the Bmal1 gene and its influence on the circadian rhythm-related recruitment of leukocytes, as well as the expression patterns of downstream mediators.Compared to the wild-type (WT) group, the model group showed significantly higher levels of CXCL16, I-TAC, and PARC (p < 0.05), as well as a notable decrease in IFN-γ expression. WP treatment effectively normalized the levels of these inflammatory factors in the model group, indicating a regulatory effect of WP on inflammatory chemokines.The knockout of the Bmal1 gene, a crucial regulator of circadian rhythms, disrupts the circadian expression of inflammatory chemokines. Treatment with WP modulated Bmal1 expression, influencing the release of these mediators, offering a promising strategy for managing nocturnal asthma. Notably, wild-type nocturnal asthma mice exhibited significantly better control of airway inflammation compared to their Bmal1-deficient counterparts, highlighting the importance of circadian regulation in the pathophysiology of asthma. [ABSTRACT FROM AUTHOR]

This research delves into the evolving dynamics of antibiogram trends, the diversity of antibiotic resistance genes and antibiotic efficacy against Vibrio cholerae strains that triggered the cholera outbreak 2022 in Odisha, India. The study will provide valuable insights managing antimicrobial resistance during cholera outbreaks. Eighty V. cholerae strains isolated during the outbreak were analysed for genotypic variations in associated drug resistance genes using PCR assays. Antibiogram profiles and MIC gradient analysis were performed according CLSI guidelines to assess antibiotic effectiveness. Substitution of amino acid position in the QRDR Region was examined to understand the development of Fluoroquinolone resistance. Elevated resistances in V. cholerae strains were observed against doxycycline, azithromycin, ciprofloxacin, and chloramphenicol. The average MARI registered 0.63 value, exceeding the threshold value 0.2. PCR assays revealed higher prevalence of antibiotic resistance genes, and MIC values observed have surpassed the previously registered values during any cholera outbreaks in India. Novel mutations in the parC gene, specifically Tyr-88→Cys and Ser-85→Leu implicated Fluoroquinolone resistance in V. cholerae. This study urges moving beyond on antibiotic reliance to control cholera, emphasizing alternative strategies like OCV, rehydration therapy, probiotics and Water, Sanitation and Hygiene (WASH) interventions as effective tools to combat cholera outbreaks and mitigate antibiotic resistance. [ABSTRACT FROM AUTHOR]

Introduction: The European Partnership for the Assessment of Risks from Chemicals (PARC) is a 7-year multinational partnership aimed at consolidating and strengthening European Union's (EU) research and innovation capacity for chemical risk assessment (RA) to protect human health and the environment. It consists of nine work packages (WP) involving more than 200 participating organisations from 29 countries. PARC is currently mapping the most relevant needs in the field of European chemical RA to steer PARC's future activities in the coming years. The present study aims to gather the perspectives of WP/Task/Project Leaders of PARC to understand their experience during the first prioritisation round of PARC activities and to identify potential points of improvement for future rounds. Methods: Three online 90-min focus group discussion (FGD) sessions were conducted between the 3rd and 9th of May 2023. Each session was attended by 4-5 participants with at least one representative from each PARC WPs 4, 5 and 6 (n = 13). The sessions were recorded and transcribed, then analysed in NVivo 12 software using thematic analysis. Results: Some important aspects for the prioritisation of activities that were mentioned include: (1) having a transparent prioritisation process even though each WP might need different prioritisation criteria, (2) balancing the fulfilment of short-term regulatory needs and anticipating long-term needs in chemical RA, (3) maintaining alignment and synergy between the WPs and with other relevant EU initiatives to avoid duplication and to ensure continuity of work and (4) making sure that PARC can effectively respond to requests from different PARC stakeholders. Conclusions: The next round of PARC research activity steering process will provide an opportunity to implement the various improvements identified. PARC should utilise the advantage of having stakeholders from different backgrounds (e.g., risk assessors, policymakers, regulatory bodies, academia, etc.) within its consortium and its advising bodies to prioritise projects and activities that will support its overall objectives. These recommendations could also be of interest outside PARC in the context of prioritising research and innovation needs related to chemical RA. [ABSTRACT FROM AUTHOR]

Abstract Introduction The European Partnership for the Assessment of Risks from Chemicals (PARC) is a 7-year multinational partnership aimed at consolidating and strengthening European Union’s (EU) research and innovation capacity for chemical risk assessment (RA) to protect human health and the environment. It consists of nine work packages (WP) involving more than 200 participating organisations from 29 countries. PARC is currently mapping the most relevant needs in the field of European chemical RA to steer PARC’s future activities in the coming years. The present study aims to gather the perspectives of WP/Task/Project Leaders of PARC to understand their experience during the first prioritisation round of PARC activities and to identify potential points of improvement for future rounds. Methods Three online 90-min focus group discussion (FGD) sessions were conducted between the 3rd and 9th of May 2023. Each session was attended by 4-5 participants with at least one representative from each PARC WPs 4, 5 and 6 (n = 13). The sessions were recorded and transcribed, then analysed in NVivo 12 software using thematic analysis. Results Some important aspects for the prioritisation of activities that were mentioned include: (1) having a transparent prioritisation process even though each WP might need different prioritisation criteria, (2) balancing the fulfilment of short-term regulatory needs and anticipating long-term needs in chemical RA, (3) maintaining alignment and synergy between the WPs and with other relevant EU initiatives to avoid duplication and to ensure continuity of work and (4) making sure that PARC can effectively respond to requests from different PARC stakeholders. Conclusions The next round of PARC research activity steering process will provide an opportunity to implement the various improvements identified. PARC should utilise the advantage of having stakeholders from different backgrounds (e.g., risk assessors, policymakers, regulatory bodies, academia, etc.) within its consortium and its advising bodies to prioritise projects and activities that will support its overall objectives. These recommendations could also be of interest outside PARC in the context of prioritising research and innovation needs related to chemical RA.

Abstract Background The emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing. Results Out of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6’)-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene. Conclusions Our results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

Based on toxicological evidence, children's exposure to phthalates may contribute to altered neurodevelopment and abnormal regulation of brain-derived neurotrophic factor (BDNF). We analyzed data from five aligned studies of the Human Biomonitoring for Europe (HBM4EU) project. Ten phthalate metabolites and protein BDNF levels were measured in the urine samples of 1148 children aged 6–12 years from Italy (NACII-IT cohort), Slovakia (PCB-SK cohort), Hungary (InAirQ-HU cohort) and Norway (NEBII-NO). Serum BDNF was also available in 124 Slovenian children (CRP-SLO cohort). Children's total, externalizing and internalizing behavioral problems were assessed using the Child Behavior Checklist at 7 years of age (only available in the NACII-IT cohort). Adjusted linear and negative binomial regression models were fitted, together with weighted quantile sum (WQS) regression models to assess phthalate mixture associations. Results showed that, in boys but not girls of the NACII-IT cohort, each natural-log-unit increase in mono-n-butyl phthalate (MnBP) and Mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) was cross-sectionally associated with higher externalizing problems [incidence rate ratio (IRR): 1.20; 95% CI: 1.02, 1.42 and 1.26; 95% CI: 1.03, 1.55, respectively]. A suggestive mixture association with externalizing problems was also observed per each tertile mixture increase in the whole population (WQS—IRR = 1.15; 95% CI: 0.97, 1.36) and boys (IRR = 1.20; 95% CI: 0.96, 1.49). In NACII-IT, PCB-SK, InAirQ-HU and NEBII-NO cohorts together, urinary phthalate metabolites were strongly associated with higher urinary BDNF levels, with WQS regression confirming a mixture association in the whole population (percent change (PC) = 25.9%; 95% CI: 17.6, 34.7), in girls (PC = 18.6%; 95% CI: 7.92, 30.5) and mainly among boys (PC = 36.0%; 95% CI: 24.3, 48.9). Among CRP-SLO boys, each natural-log-unit increase in ∑DINCH concentration was associated with lower serum BDNF levels (PC: −8.8%; 95% CI: −16.7, −0.3). In the NACII-IT cohort, each natural-log-unit increase in urinary BDNF levels predicted worse internalizing scores among all children (IRR: 1.15; 95% CI: 1.00, 1.32). Results suggest that (1) children's exposure to di-n-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP) metabolites is associated with more externalizing problems in boys, (2) higher exposure to DINCH may associate with lower systemic BDNF levels in boys, (3) higher phthalate exposure is associated with higher urinary BDNF concentrations (although caution is needed since the possibility of a "urine concentration bias" that could also explain these associations in noncausal terms was identified) and (4) higher urinary BDNF concentrations may predict internalizing problems. Given this is the first study to examine the relationship between phthalate metabolite exposure and BDNF biomarkers, future studies are needed to validate the observed associations. [ABSTRACT FROM AUTHOR]

Background: The emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing. Results: Out of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6')-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene. Conclusions: Our results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region. [ABSTRACT FROM AUTHOR]

High-level delafloxacin-resistant (H-L DLX-R) Staphylococcus aureus isolates (minimum inhibitory concentration ≥1 mg/L) associated with mutations affecting position 84 of ParC have emerged. We aimed to elucidate the role of these mutations as a mechanism of H-L DLX resistance in methicillin-resistant S. aureus (MRSA) isolates recovered from blood cultures. Susceptibility to DLX was determined in 75 MRSA isolates by E-test, and an rt-PCR was developed to detect mutations affecting position 84 of ParC to screen a further 185 MRSA isolates. The genomes of 48 isolates, including all DLX-R isolates or with alterations at position 84, and also a subset of DLX-susceptible isolates were analyzed. Among the 75 isolates studied, 77.34% were DLX-susceptible and only 4 H-L DLX-R isolates were found. Seven (3.8%) isolates with alterations at position 84 of ParC were detected by rt-PCR. Genomic analysis showed that 89.9% (8/9) of isolates with the substitution E84K/G in ParC, together with other mutations in gyrA and parC, were H-L DLX-R. However, the E84K substitution in ParC alone or with other alterations was found in two isolates without H-L DLX-R. Alterations at position 84 of ParC are rare but play a key role in H-L DLX resistance in MRSA but only when other alterations in GyrA are present. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance in Neisseria gonorrhoeae (NG) is increasing worldwide. Second-line treatments with macrolides or fluoroquinolones are an option for NG infections in some cases following the STI guideline recommendations. In our study, we compared the gradient diffusion test using EUCAST 2024 breakpoints with a new molecular method using the Allplex™ NG&DR assay (Seegene®) including A2059G/C2611 mutations (23S rRNA) associated with high/moderate-level macrolide resistance and S91F mutation (gyrA) relationship with fluoroquinolone resistance in NG isolates (n = 100). We calculated the sensitivity, specificity, and correlation of the molecular test for fluoroquinolone using the gradient diffusion as the reference method. In twenty-three strains was not detected any mutation associated with macrolides or fluoroquinolone resistance. No A2059G/C2611T mutations were detected, and the S91F mutations were detected in 77 out of the 100 isolates screened. Twenty-three NG isolates were reported to be resistant to azithromycin (ECOFF: >1 mg/L), and 78 NG isolates were resistant to ciprofloxacin (MIC: >0.06 mg/L). The molecular method showed a sensitivity of 96.1% and, a specificity of 90.9% for fluoroquinolone susceptibility, but the statistical analysis between the molecular test and gradient diffusion test was not statistically significant for fluoroquinolone resistance (p = 1). Statistical analysis was not performed for macrolides because of the absence of positive RT-PCR results. According to our data, Allplex™ assay cannot replace the gradient diffusion test for macrolide resistance. However, the assay could be used to test fluoroquinolone resistance in NG isolates as a replacement for phenotypic methods. [ABSTRACT FROM AUTHOR]

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC). [ABSTRACT FROM AUTHOR]

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying welldefined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation). Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN). Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research. Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development. [ABSTRACT FROM AUTHOR]

High-level delafloxacin-resistant (H-L DLX-R) Staphylococcus aureus isolates (minimum inhibitory concentration ≥1 mg/L) associated with mutations affecting position 84 of ParC have emerged. We aimed to elucidate the role of these mutations as a mechanism of H-L DLX resistance in methicillin-resistant S. aureus (MRSA) isolates recovered from blood cultures. Susceptibility to DLX was determined in 75 MRSA isolates by E-test, and an rt-PCR was developed to detect mutations affecting position 84 of ParC to screen a further 185 MRSA isolates. The genomes of 48 isolates, including all DLX-R isolates or with alterations at position 84, and also a subset of DLX-susceptible isolates were analyzed. Among the 75 isolates studied, 77.34% were DLX-susceptible and only 4 H-L DLX-R isolates were found. Seven (3.8%) isolates with alterations at position 84 of ParC were detected by rt-PCR. Genomic analysis showed that 89.9% (8/9) of isolates with the substitution E84K/G in ParC, together with other mutations in gyrA and parC, were H-L DLX-R. However, the E84K substitution in ParC alone or with other alterations was found in two isolates without H-L DLX-R. Alterations at position 84 of ParC are rare but play a key role in H-L DLX resistance in MRSA but only when other alterations in GyrA are present.

Background: Traditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of Mycoplasma genitalium when cultured in vitro. Sanger sequencing is the standard method for detecting drug resistance-associated mutations. It has been used in some laboratories to guide the choice of macrolide antibiotics for Mycoplasma genitalium infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge. Objective: Sequencing analysis can detect unknown mutations, but it is timeconsuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance. Results: 105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001). Conclusions: The results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice. [ABSTRACT FROM AUTHOR]

Introduction: Dupilumab is approved for a variety of type 2 inflammatory diseases. Changes in chemokine levels during treatment require further analysis. Aim: We evaluated changes in eotaxin-3 and PARC levels after dupilumab treatment through a meta-analysis, aiming to provide more comprehensive results. Material and methods: Databases were searched to select eligible publications. The study quality was assessed after inclusion. The standardized mean difference (SMD) was used for evaluation. Results: Four studies were included. Eotaxin-3 levels were not seen significantly decreased at weeks 1 and 12, with SMD = -0.39 (95% CI: -1.78, 0.99) and -2.60 (95% CI: -5.77, 0.57), respectively (p > 0.05). Eotaxin-3 levels decreased significantly at weeks 2, 4, 8, 16, 24, 36, and 52, with SMD = -0.94 (95% CI: -1.61, -0.27); -1.17 (95% CI: -1.49, -0.84); -1.20 (95% CI: -1.52, -0.88); -1.31 (95% CI: -1.83, -0.79); -4.57 (95% CI: -6.90, -2.33); -5.28 (95% CI: -5.52, -5.04); and -4.03 (95% CI: -4.22, -3.85) (p < 0.05), respectively. PARC levels decreased significantly at weeks 4, 8, 12, and 16, with SMD = -1.08 (95% CI: -1.59, -0.58); -1.17 (95% CI: -1.68, -0.66); -1.11 (95% CI: -1.61, -0.60); and -1.15 (95% CI: -1.66, -0.64) (p < 0.05), respectively. Conclusions: Eotaxin-3 and PARC levels can be significantly reduced in patients treated with dupil [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is an important cause of nosocomial infections and displays increasing resistance to fluoroquinolones (FQ). This study surveyed the mechanisms of FQ resistance and molecular typing of K. pneumoniae isolates from intensive care units patients in Tehran, Iran. A total of 48 ciprofloxacin (CIP) resistant K. pneumoniae isolates from urine samples were included in this study. Broth microdilution assays revealed high-level CIP resistance (MIC > 32 μg/mL) in 31.25% of the isolates. Plasmid-mediated quinolone resistance genes were detected in 41 (85.4%) isolates. Among which, qnrS (41.67%) was the most prevalent followed by qnrD (35.42%), qnrB (27.1%), qnrA (25%), qepA (22.9%), aac(6ʹ)-Ib-cr (20.83%), and qnrC (6.25%). Target site mutations (gyrA and parC) were assessed using PCR and sequencing on all isolates. A single mutation in gyrA (S83I) was found in 13 (27.1%) isolates and two isolates harbored six simultaneous mutations. Fourteen isolates (29.2%) had mutations in parC and S129A and A141V mutations were the most prevalent. Real time PCR showed an increase in the expression level of acrB and oqxB efflux genes in 68.75 and 29.16% isolates, respectively. Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed 14 genotypes and 11 of them were classified by multilocus sequence typing (MLST) into 11 different sequence types belonging to seven clonal complexes and two singletons, most of them have not been reported in Iran yet. We are concerned about the spread of these clones throughout our country. Most FQ resistance mechanisms were detected among our isolates. However, target site mutation had the greatest effect on CIP resistance among our isolates. [ABSTRACT FROM AUTHOR]

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

New approach methodologies (NAMs) have the potential to become a major component of regulatory risk assessment, however, their actual implementation is challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) was designed to address many of the challenges that exist for the development and implementation of NAMs in modern chemical risk assessment. PARC’s proximity to national and European regulatory agencies is envisioned to ensure that all the research and innovation projects that are initiated within PARC agree with actual regulatory needs. One of the main aims of PARC is to develop innovative methodologies that will directly aid chemical hazard identification, risk assessment, and regulation/policy. This will facilitate the development of NAMs for use in risk assessment, as well as the transition from an endpoint-based animal testing strategy to a more mechanistic-based NAMs testing strategy, as foreseen by the Tox21 and the EU Chemical’s Strategy for Sustainability. This work falls under work package 5 (WP5) of the PARC initiative. There are three different tasks within WP5, and this paper is a general overview of the five main projects in the Task 5.2 ‘Innovative Tools and methods for Toxicity Testing,’ with a focus on Human Health. This task will bridge essential regulatory data gaps pertaining to the assessment of toxicological prioritized endpoints such as non-genotoxic carcinogenicity, immunotoxicity, endocrine disruption (mainly thyroid), metabolic disruption, and (developmental and adult) neurotoxicity, thereby leveraging OECD’s and PARC’s AOP frameworks. This is intended to provide regulatory risk assessors and industry stakeholders with relevant, affordable and reliable assessment tools that will ultimately contribute to the application of next-generation risk assessment (NGRA) in Europe and worldwide.

Background Although single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs and their contribution to fluoroquinolone failure. Methods Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019–February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n = 194), the association between SNPs and microbiologic treatment outcome was analyzed. Results The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC / gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA cooccurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/ gyrA dual SNPs when compared with infections with single S83I SNP alone from analysis of (1) 194 cases in this study (81.2% vs 45.8%, P =.047), and (2) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; P =.0027), indicating a strong additive effect. Conclusions Compared with parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC / gyrA had twice the likelihood of failing moxifloxacin. Although antimicrobial resistance varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing. [ABSTRACT FROM AUTHOR]

Salmonella is one of the most important foodborne zoonotic pathogens, causing global morbidity and mortality in both humans and animals. Due to the extensive use of antimicrobials in food-producing animals, the antimicrobial resistance of Salmonella has attracted increasing attention globally. There have been many reports concerning the antimicrobial resistance of Salmonella from food-producing animals, meats and the environment. However, few studies on Salmonella from food-producing animals have been reported in Chongqing municipality, China. The aim of the present study was to determine the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella isolated from livestock and poultry in Chongqing. Meanwhile, we also want to know the presence of β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance-determining region (QRDR) mutations of Salmonella isolates. A total of 129 Salmonella strains were recovered from 2,500 fecal samples at 41 farms from pigs, goats, beef cattle, rabbits, chickens, and ducks. Fourteen serovars were identified, with S. Agona and S. Derby being the dominant serovars. The 129 isolates had high resistance to doxycycline (87.6%), ampicillin (80.6%), tetracycline (79.8%), trimethoprim (77.5%), florfenicol (76.7%) chloramphenicol (72.9%), and trimethoprim-sulfamethoxazole (71.3%), but were susceptible to cefepime. A total of 114 (88.4%) isolates showed multidrug resistant phenotypes. The prevalence of β-lactamase genes in Salmonella isolates was 89.9% (116/129), and among these isolates, 107 (82.9%) harbored blaTEM, followed by blaOXA (26, 20.2%), blaCTX-M (8, 6.2%), and blaCMY (3, 2.3%). In addition, qnrB, qnrD, qnrS, oqxA, oqxB, and aac(6′)-Ib-cr were detected in 11, 2, 34, 34, 43, and 72 PMQR-producing isolates, respectively. Moreover, QRDR mutations were very common in PMQR-positive Salmonella isolates (97.2%, 70/72) with mutation(s) in parC or combinative mutations in gyrA and parC. More significantly, 32 extended spectrum beta-lactamase (ESBL)-producing isolates were identified, and 62.5% of them were found to harbor one to four PMQR genes. Furthermore, 11 sequence types were identified from the isolates, and most of ESBL-producing isolates were attributed to ST34 (15.6%) and ST40 (62.5%). The coexistence of PMQR genes with β-lactamase genes and the extensive mutations in QRDR present in Salmonella isolates from food-producing animals suggest a potential threat to public health. Reasonable utilization and strict control strategies for antimicrobials in animal husbandry and animal treatment are necessary to reduce the emergence and dissemination of drug-resistant Salmonella isolates. [ABSTRACT FROM AUTHOR]

The currently accepted methods for neurotoxicity (NT) testing rely on animal studies. However, high costs and low testing throughput hinder their application for large numbers of chemicals. To overcome these limitations, in vitro methods are currently being developed based on human-induced pluripotent stem cells (hiPSC) that allow higher testing throughput at lower costs. We applied six different protocols to generate 3D BrainSphere models for acute NT evaluation. These include three different media for 2D neural induction and two media for subsequent 3D differentiation resulting in self-organized, organotypic neuron/astrocyte microtissues. All induction protocols yielded nearly 100% NESTIN-positive hiPSC-derived neural progenitor cells (hiNPCs), though with different gene expression profiles concerning regional patterning. Moreover, gene expression and immunocytochemistry analyses revealed that the choice of media determines neural differentiation patterns. On the functional level, BrainSpheres exhibited different levels of electrical activity on microelectrode arrays (MEA). Spike sorting allowed BrainSphere functional characterization with the mixed cultures consisting of GABAergic, glutamatergic, dopaminergic, serotonergic, and cholinergic neurons. A test method for acute NT testing, the human multi-neurotransmitter receptor (hMNR) assay, was proposed to apply such MEA-based spike sorting. These models are promising tools not only in toxicology but also for drug development and disease modeling. [ABSTRACT FROM AUTHOR]

Background: Acinetobacter baumannii, are involved in hospital-acquired infections and are increasingly developing resistance to fluoroquinolones, such as ciprofloxacin. The most common method of fluoroquinolone resistance is alteration in genes that encode DNA gyrase (gyrA) and topoisomerase IV (parC).Methods: We sought to isolate fluoroquinolone-resistant A. baumannii and search for changes in gyrA (Ser83Leu) and parC (Ser80Leu) loci by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Results: We found that all 68 A. baumannii isolates that were part of this research were multidrug resistant and harbored gyrA and parC loci. Most isolates had ciprofloxacin minimal inhibitory concentrations of >128 µg/mL (50.8%). Mutations in gyrA were the most prevalent (47.45%), followed by parC mutations (33.9%) and combined mutations in both genes (23.7%).Conclusions: Single mutations in either gyrA (Ser83Leu) or parC (Ser80Leu) genes may be attributed to fluroquinolone resistance in A. baumannii. [ABSTRACT FROM AUTHOR]

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available–and are currently in the process of regulatory validation–there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Salmonella is one of the most important foodborne zoonotic pathogens, causing global morbidity and mortality in both humans and animals. Due to the extensive use of antimicrobials in food-producing animals, the antimicrobial resistance of Salmonella has attracted increasing attention globally. There have been many reports concerning the antimicrobial resistance of Salmonella from food-producing animals, meats and the environment. However, few studies on Salmonella from food-producing animals have been reported in Chongqing municipality, China. The aim of the present study was to determine the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella isolated from livestock and poultry in Chongqing. Meanwhile, we also want to know the presence of β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance-determining region (QRDR) mutations of Salmonella isolates. A total of 129 Salmonella strains were recovered from 2,500 fecal samples at 41 farms from pigs, goats, beef cattle, rabbits, chickens, and ducks. Fourteen serovars were identified, with S. Agona and S. Derby being the dominant serovars. The 129 isolates had high resistance to doxycycline (87.6%), ampicillin (80.6%), tetracycline (79.8%), trimethoprim (77.5%), florfenicol (76.7%) chloramphenicol (72.9%), and trimethoprim-sulfamethoxazole (71.3%), but were susceptible to cefepime. A total of 114 (88.4%) isolates showed multidrug resistant phenotypes. The prevalence of β-lactamase genes in Salmonella isolates was 89.9% (116/129), and among these isolates, 107 (82.9%) harbored blaTEM, followed by blaOXA (26, 20.2%), blaCTX-M (8, 6.2%), and blaCMY (3, 2.3%). In addition, qnrB, qnrD, qnrS, oqxA, oqxB, and aac(6′)-Ib-cr were detected in 11, 2, 34, 34, 43, and 72 PMQR-producing isolates, respectively. Moreover, QRDR mutations were very common in PMQR-positive Salmonella isolates (97.2%, 70/72) with mutation(s) in parC or combinative mutations in gyrA and parC. More significantly, 32 extended spectrum beta-lactamase (ESBL)-producing isolates were identified, and 62.5% of them were found to harbor one to four PMQR genes. Furthermore, 11 sequence types were identified from the isolates, and most of ESBL-producing isolates were attributed to ST34 (15.6%) and ST40 (62.5%). The coexistence of PMQR genes with β-lactamase genes and the extensive mutations in QRDR present in Salmonella isolates from food-producing animals suggest a potential threat to public health. Reasonable utilization and strict control strategies for antimicrobials in animal husbandry and animal treatment are necessary to reduce the emergence and dissemination of drug-resistant Salmonella isolates.

Copyright of Al-Mustansiriyah Journal of Science is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

100 isolates of Acinetobacter baumannii were collected from different clinical sources including (blood, sputum and burns) from hospitals in Baghdad - Iraq. In order to investigate its resistance to fluoroquinolones. MIC assay for ciprofloxacin was performed using E-test, and PCR assay for gry A and parC genes. The results of the MIC showed that A. baumannii was sensitive to ciprofloxacin at concentration=

Background & Objective: Pseudomonas aeruginosa is an opportunistic pathogen and one of the most common causes of nosocomial infections. This bacterium's antibiotic resistance to the common fluoroquinolone antibiotics, especially ciprofloxacin, is due to mutations in the gyrA and parC genes. This study aimed to investigate the effect of the mutation in (gyrA, parC) on ciprofloxacin resistance in clinical isolates of Pseudomonas aeruginosa.Methods: A total of 140 clinical samples were collected from hospitals. The samples were identified by standard biochemical tests, and the antibiotic resistance was investigated by the disk diffusion method. DNA was extracted from 30 isolates, and PCR was performed. PCR-sequencing was carried out to assess gyrA and parC mutations in drug-resistant isolates. NCBI-Blast and MEGA7 software was used to analyze the nucleotide sequences.Results: 30 clinical isolates were 80% resistant to ciprofloxacin; meanwhile, in 21 samples, mutations were observed. 87/5% of mutations were related to gyrA (Thr83 → Ile), 79/16 % parC (Ser87 → Leu), and 4/18% (Glu91 → Lys). The antibiotic resistance to ciprofloxacin and mutations in gyrA and parC genes in resistant isolates are significantly related to each other (P

Simple Summary: Bovine Mycoplasma species, particularly antimicrobial resistant Mycoplasma bovis are important causes of bovine respiratory disease (BRD) in cattle, which causes major economic losses worldwide. Thus, the current study aimed to determine the prevalence and antimicrobial resistance profiles of bovine Mycoplasma spp. isolated from cattle's respiratory tracts, in addition to evaluating the fluoroquinolone resistance in the recovered isolates using broth microdilution and conventional PCR techniques in Egypt. Our result showed that M. bovis was the most common spp. (61%), followed by M. bovirhinis (15%). In total, mycoplasma isolates were more prevalent among all examined lung tissues (38%), followed by nasal swabs (35%), tracheal tissues (28%), and tracheal swabs (27%). All the examined mycoplasma isolates (n = 76) were 100% susceptible to spectinomycin, tulathromycin, spiramycin, and tylosin, but high doxycycline and enrofloxacin minimum inhibitory concentrations (MICs) values were observed among 43.4% and 60.5% of the tested isolates, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%), meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, understanding antimicrobial resistance mechanisms is a significant tool for the future development of genetic-based diagnostic techniques for the rapid detection of resistant mycoplasma strains. Among many bovine Mycoplasma species (spp.), Mycoplasma bovis is recognized as a significant causative agent of respiratory diseases in cattle. In recent years, resistant M. bovis isolates, especially to fluoroquinolones, have been reported globally as a result of the extensive usage of antimicrobials in the treatment of bovine pneumonia. Therefore, the aim of this study is to investigate the prevalence and antimicrobial susceptibility patterns of bovine Mycoplasma spp. isolated from the respiratory tracts of cattle in Egypt and to assess the fluoroquinolones resistance in the recovered mycoplasma isolates via broth microdilution and conventional PCR techniques. Conventional phenotypic methods identified 128 mycoplasma isolates (32%) from 400 different samples, with M. bovis being the predominant spp. (61%), followed by M. bovirhinis (15%). Of note, mycoplasma isolates were rarely isolated from total healthy lung tissues (7/55, 12.7%), but they were frequently isolated from pneumonic lungs (31/45, 68.9%). All the examined mycoplasma isolates (n = 76) were sensitive to tilmicosin, tylosin, tulathromycin, spiramycin, and spectinomycin (100% each), while 60.5% and 43.4% of the examined isolates had high minimum inhibitory concentration (MIC) values to enrofloxacin and doxycycline, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%); meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, our findings revealed alarming resistance to enrofloxacin and doxycycline antibiotics; thus, antimicrobial usage must be restricted and molecular techniques can help in the rapid detection of the resistant strains. [ABSTRACT FROM AUTHOR]

ABSTRACT Plasmids that encode the same replication machinery are generally unable to coexist in the same bacterial cell. However, Clostridium perfringens strains often carry multiple conjugative toxin or antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many bacteria, plasmid partitioning upon cell division involves a ParMRC system; in C. perfringens plasmids, there are approximately 10 different ParMRC families, with significant differences in amino acid sequences between each ParM family (15% to 54% identity). Since plasmids carrying genes belonging to the same ParMRC family are not observed in the same strain, these families appear to represent the basis for plasmid compatibility in C. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-binding proteins and their parC binding sites. The ParR proteins bound to sequences within a parC site from the same ParMRC family but could not interact with a parC site from a different ParMRC family. These data provide evidence that compatibility of the conjugative toxin plasmids of C. perfringens is mediated by their parMRC-like partitioning systems. This process provides a selective advantage by enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for different host targets. IMPORTANCE Toxins produced by the Gram-positive pathogen Clostridium perfringens are primarily encoded by genes found on different conjugative plasmids. These plasmids encode highly similar replication proteins and therefore should be incompatible, but they are often found to coexist within the same isolate. In this study, we showed that a series of phylogenetically related ParMRC plasmid partitioning systems, structures that are normally responsible for ensuring that plasmids segregate correctly at cell division, dictate which toxin plasmid combinations can coexist within the same bacterial cell. We dissected the recognition steps between the DNA-binding ParMRC component, ParR, and the plasmid-derived centromere, parC. Our data suggested a mechanism by which plasmids encoding ParMRC systems from the same family are incompatible, whereas plasmids encoding ParMRC systems from distinct families are compatible. This work provides insight into how these cells can maintain multiple highly similar toxin plasmids, which is a critical first step in understanding how to limit the disease-causing potential of C. perfringens.

Type II topoisomerases, including DNA gyrase (GyrA) and topoisomerase IV (ParC), contribute to fluoroquinolone resistance in Enterococcus spp. This study investigated the mutational status of the quinolone resistance-determining regions (QRDRs) of GyrA and ParC in the clinical isolates of enterococci from a hospital in Baotou, China. We analyzed 110 enterococcal isolates, including 57 Enterococcus faecalis and 53 Enterococcus faecalis faecium. The resistance rates of E. faecalis and E. faecium to ciprofloxacin were 63.16% and 84.91%, respectively. We found that 32 samples of E. faecalis and 42 of E. faecium had single or combined mutations in gyrA and/or parC, which were all resistant to ciprofloxacin. Only two ciprofloxacin-resistant E. faecalis isolates had no mutation. No mutations in gyrA and parC genes in all ciprofloxacinsusceptible isolates were found. Ciprofloxacin minimal inhibitory concentrations (MICs) in the mutation group were significantly higher than those of the non-mutation group, indicating that mutations in the QRDRs of gyrA and parC were correlated with MIC elevation. Two novel substitutions (GyrA Ser83Phe and ParC Ser80Leu) of E. faecalis were identified herein. Three-dimensional modeling revealed that these novel amino acid substitutions could disrupt the water/metal-ion bridge and decrease the interaction between the enzymes and ciprofloxacin. The data showed a diversity of mutation types in QRDRs of type II topoisomerases whose association with fluoroquinolone resistance in clinical isolates of enterococci warrants further investigation.

Abstract Background Fluoroquinolone agents, such as enrofloxacin and marbofloxacin, are commonly used for pseudomonal infection in veterinary medicine. However, the rate of resistance to fluoroquinolones is rapidly increasing, according to multiple studies in various countries. Point mutations in the quinolone resistance-determining region (QRDR) are closely related to the increased fluoroquinolone resistance of Pseudomonas aeruginosa. The aim of this study was to investigate current antimicrobial susceptibility and fluoroquinolone resistance in P. aeruginosa strains isolated from dogs. The presence of point mutations in the QRDR was confirmed by gyrA and parC polymerase chain reaction and nucleotide sequencing analysis. Results A total of 84 nonduplicated P. aeruginosa strains were obtained from 228 healthy dogs (healthy group) and 260 dogs with clinical signs (infected group). Among these isolates, 38 strains from the healthy group were detected in several sample types, whereas 46 strains from the infected group were obtained mostly from dogs’ ears with otitis externa (41/260, 15.8%). All strains were resistant to nalidixic acid, while some were also resistant to enrofloxacin (23/84, 27.4%), marbofloxacin (17/84, 20.2%), levofloxacin (12/84, 14.3%), or ciprofloxacin (11/84, 13.1%). Enrofloxacin resistance was significantly higher in strains from the infected group than in those from the healthy group (p

Antimicrobial resistance in Neisseria gonorrhoeae is an important global health concern. The genetically related commensal Neisseria act as a reservoir of resistance genes, and horizontal gene transfer (HGT) has been shown to play an important role in the genesis of resistance to cephalosporins and macrolides in N. gonorrhoeae. In this study, we evaluated if there was evidence of HGT in the genes gyrA/gyrB and parC/parE responsible for fluoroquinolone resistance. Even though the role of gyrB and parE in quinolone resistance is unclear, the subunits gyrB and parE were included as zoliflodacin, a promising new drug to treat N. gonorrhoeae targets the gyrB subunit. We analyzed a collection of 20,047 isolates; 18,800 N. gonorrhoeae, 1,238 commensal Neisseria spp., and nine Neisseria meningitidis. Comparative genomic analyses identified HGT events in genes, gyrA, gyrB, parC, and parE. Recombination events were predicted in N. gonorrhoeae and Neisseria commensals. Neisseria lactamica, Neisseria macacae, and Neisseria mucosa were identified as likely progenitors of the HGT events in gyrA, gyrB, and parE, respectively.

Antimicrobial resistance in Neisseria gonorrhoeae is an important global health concern. The genetically related commensal Neisseria act as a reservoir of resistance genes, and horizontal gene transfer (HGT) has been shown to play an important role in the genesis of resistance to cephalosporins and macrolides in N. gonorrhoeae. In this study, we evaluated if there was evidence of HGT in the genes gyrA/gyrB and parC/parE responsible for fluoroquinolone resistance. Even though the role of gyrB and parE in quinolone resistance is unclear, the subunits gyrB and parE were included as zoliflodacin, a promising new drug to treat N. gonorrhoeae targets the gyrB subunit. We analyzed a collection of 20,047 isolates; 18,800 N. gonorrhoeae , 1,238 commensal Neisseria spp., and nine Neisseria meningitidis. Comparative genomic analyses identified HGT events in genes, gyrA , gyrB , parC , and parE. Recombination events were predicted in N. gonorrhoeae and Neisseria commensals. Neisseria lactamica , Neisseria macacae , and Neisseria mucosa were identified as likely progenitors of the HGT events in gyrA , gyrB , and parE , respectively. [ABSTRACT FROM AUTHOR]

The mechanisms of fluoroquinolone-resistance of Mycoplasma genitalium were analysed by a new method. M. genitalium strains from urinary sediments of patients with urethritis were isolated and examined antimicrobial susceptibilities and the mutations in ParC, GyrA and 23S rRNA. Docking models between gyrase and topoisomerase IV with sitafloxacin showed that two binding modes in which the amine moiety at the C-7 position rotated could be constructed. Among 18 strains, 13 strains had mutations with amino-acid changes at Serine 83 in ParC. The MICs of moxifloxacin or sitafloxacin for three strains with only S83I in ParC were 2, 1 and 8 mg/L (moxifloxacin) or 0.13, 0.13 and 1 mg/L (sitafloxacin), respectively. In contrast, the MICs of moxifloxacin or sitafloxacin for 3 strain with S83N in ParC were 0.25, 0.13 and 0.25 mg/L (moxifloxacin) or 0.06, 0.03, and 0,03 mg/L (sitafloxacin), respectively, not significantly different from wild-type isolates. The docking model of sitafloxacin and topoisomerase IV showed that the oxygen atom at the gamma position of Serine 83 of ParC interacted with the sitafloxacin carboxylate moiety. When the S83I substitution occurs, the isoleucine side chain is lipophilic and the residue hydropathy changes from hydrophilicity to hydrophobicity and important H-bond interactions between serine and the carboxylate moiety are lost. When the serine 83 to asparagine substitution (S83N) occurred, the asparagine side chain is hydrophilic and the residue hydropathy does not change. The docking model suggests that Ser83 replacements causes attenuation or loss of activity of fluoroquinolones such as sitafloxacin. [ABSTRACT FROM AUTHOR]

Introduction: Resistance to fluoroquinolones is mainly due to point mutations that gave rise to amino acid substitutions in the quinolone resistance-determining regions of either gyrA or parC genes, which may be augmented by plasmid mediated resistance. Accordingly, the main aim of the study was to investigate the mutations in gyrA and parC genes as well as the qnrA and qnrB genes acquisition. Methodology: 193 Klebsiella pneumoniae and Escherichia coli isolates were collected, identified and MICs for ciprofloxacin, levofloxacin and moxifloxacin were determined. Polymerase Chain Reaction to investigate qnrA, qnrB, gyrA and parC genes followed by DNA sequencing analysis to identify mutations in gyrA and parC genes. Results: The most prominent mutation in gyrA gene was ser83leu, followed by asp87asn, and lys154arg. Regarding parC mutations, ser80ile was the most detected. Other mutations val141ala and glu84ala were also noted. In addition to a substitution mutation at codon 157 of leucine to tyrosin. To the best of our knowledge this mutation was not previously reported. qnrB was the most detected gene, as 64.7% Klebsiella pneumoniae and 57.1% Escherichia coli were positive. qnrA gene was detected in 11% Klebsiella pneumoniae and 4% of Escherichia coli isolates tested. Conclusions: This study suggests that the indiscriminate use of fluoroquinolones resulted in the increase of development of resistance either through mutations in the quinolone resistance-determining regions of either gyrA or parC genes augmented by plasmid mediated resistance. The irrational use of new fluoroquinolones such as moxifloxacin has created selective pressure for the appearance of new mutation. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the occurrence of mutations in the quinolone resistance-determining regions (QRDRs) of the genes gyrA and parC in M. gallisepticum positive field samples from broiler flocks in Ecuador. DNA was extracted from 24 M. gallisepticum PCR-positive samples from 22 commercial broiler flocks. The genes gyrA and parC were amplified by PCR. PCR products were sequenced by Sanger technology to analyze the genetic characteristics. To identify the mutations involved in fluoroquinolone resistance (FQR), the sequences obtained were processed and analyzed using the tools Geneious R11, BLASTn, MAFFT, ExPASy MBWS, and BioEdit. All samples had mutations in both gyrA and parC genes, resulting in changes at amino acid positions Ser-83→Ile and Ile-157→Val in GyrA, and Ser-80→Trp in ParC. In addition, a change at position His-59→Tyr in GyrA was also found in one sample. The results showed that alterations in both genes have been commonly linked to FQR in mutants of other Mycoplasma species, including M. gallisepticum. This is the first study on M. gallisepticum positive samples from chickens in Ecuador which revealed the occurrence of mutations resulting in amino acid changes previously linked to FQR. [ABSTRACT FROM AUTHOR]

Expanded-spectrum quinolones (ciprofloxacin) are highly effective against gram-negative bacteria, but significant resistance to quinolones has been increasingly reported. We sought to evaluate the prevalence of gram-negative ciprofloxacin-resistant isolates (CRIs) from our hospital and their mechanism of action. Gram-negative CRIs were identified as per standard procedures and confirmed using the Ezy MICTM Strip (HiMedia). DNA from 67 CRIs was amplified for the quinolone resistance–determining region (QRDR) and plasmid-mediated quinolone resistance genes. Thirty isolates positive for QRDR DNA were sequenced by Sanger's method to detect mutation. Of the isolates, 42.5% were found to be CRIs, the majority (74.42%) from inpatient departments, and E scherichia coli (64.19%) was the predominant isolate. Among the CRIs, 24.55% were ESBL producers and 35.29% were multidrug resistant. The polymerase chain reaction results showed the majority were amplified by QRDR target regions of gyr A (35.4%) while 4.61% were amplified for the plasmid-mediated fluoroquinolone resistance region of the qnrB gene. Further sequencing of QRDR-positive genes showed point mutations with amino acid changes at codons Ser83 and Asp87 in the gyr A gene and Ser80, Glu84, and Leu88 positions in the par C gene. Ciprofloxacin resistance observed in our study was mostly due to point mutations. Hence, strategies for rational use of ciprofloxacin and adherence to the dose and duration of treatment could be helpful to prevent selection and spread of mutant CRIs/strains. [ABSTRACT FROM AUTHOR]

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objectives: Given the paucity of literature, this study investigated whether a prevention and recovery care (PARC) service supported recovery in patients with borderline personality disorder (BPD).Method: This retrospective study included patients with BPD who had their first (index) admission to North West PARC between 2011 and 2016. Patient medical records and the state-wide database were the sources of information.Results: Of the 67 patients included, over 70% attended group activities. All patients achieved their recovery goals, either fully or partially. Compared to admission, the frequency of substance use and the Health of the Nation Outcome Scale (HoNOS) scores at discharge were significantly less. A significantly smaller number of patients needed inpatient treatment during the 12 months following their PARC admission.Conclusion: The PARC service appears to promote clinical and psychosocial recovery in patients with BPD. [ABSTRACT FROM AUTHOR]

The spread of antibiotic resistant bacteria is a worldwide problem. Due to the importance of P. aeruginosa as a multidrug resistant bacterium, this study aimed, through molecular techniques, to detect point mutations in chromosomal genes responsible for the quinolones class of antibiotics resistance. A total of 52 isolates from burn infections were identified using specific primers for P. aeruginosa 16S rDNA. Ciprofloxacin minimum inhibitory concentrations (MIC) were estimated using the agar dilution assay. DNA sequences of the quinolone resistancedetermining regions of gyrA and parC were determined for detecting the mutations found in these genes and the relations among the isolates by constructing phylogenetic trees. The results revealed that only 43 (82.7%) of isolates were P. aeruginosa, of which 31 (72.06%) were resistant to different concentrations of ciprofloxacin, ranging between 4 and >32 μg/ml. Twenty six isolates were selected for sequencing, including sensitive, intermediately resistant, and highly resistant to ciprofloxacin. The ciprofloxacin sensitive isolates did not exert any amino acid alterations in gyrA or parC genes; however, a single intermediately resistant isolate had a single mutation at each gene. Of the total resistant isolates (20), 6 isolates had no mutations at different MIC levels, While 14 isolates had Thr-83-Ile substitution in gyrA and Ser-87-Leu substitution in parC, only five isolates had a second mutation, namely Asp-87-Asn, in gyrA. The phylogenetic analysis of the studied groups showed divergence from the P. aeruginosa PAO1 and PAO1OR reference strains due to increased mutations and polymorphisms in studied isolates. In conclusion, P. aeruginosa occurrence was increased in burn infections and the fluoroquinolones in current use are not as effective as before; the main resistance mechanism in local clinical isolates of P. aeruginosa is mutations, where the main target of fluoroquinolones is gyrA gene. [ABSTRACT FROM AUTHOR]

Sarvenaz Esfahani, Roya Ahmadrajabi, Hamidreza Mollaei, Fereshteh Saffari Department of Microbiology and Virology, School of Medicine, Kerman University of Medical Sciences, Kerman, IranCorrespondence: Fereshteh Saffari Email fsafari@kmu.ac.irIntroduction: Enterococcus faecalis is one of the most common pathogens in urinary tract infections (UTIs). Fluoroquinolones have been frequently used to treat E. faecalis UTIs, and the emergence of fluoroquinolone-resistant E. faecalis strains has recently been reported in several countries. This study aimed to elucidate the mechanisms involved in fluoroquinolone resistance in clinical E. faecalis isolates by analyzing mutations in quinolone- resistance-determining regions (QRDRs) of gyrA and parC and investigating the role of some efflux pumps.Methods: In total, 70 clinical E. faecalis isolates collected from UTIs were identified by phenotypic and genotypic methods. Antimicrobial susceptibility testing was performed and multidrug-resistant (including ciprofloxacin resistant) isolates were studied for minimum inhibitory concentrations to ciprofloxacin, levofloxacin, and ofloxacin. In the following, mutations in QRDRs of gyrA and parC and expression of EfrA, EfrB, and EmeA efflux pumps were investigated in 20 high-level ciprofloxacin resistant and two ciprofloxacin susceptible isolates.Results: High-level resistance to ciprofloxacin was detected in 97.5% of isolates. Sequencing of QRDRs revealed that 65% and 75% of isolates carried mutations in gyrA and parC, respectively. The presence of efflux genes was detected in all studied isolates, but expression of efrA, emeA, and efrB was demonstrated in 50%, 40%, and 30% of resistant isolates, respectively. Neither QRDR mutation nor the expression of efflux genes showed any significant association with MIC.Conclusion: Co-incidence of mutation and efflux gene expression in more than half of isolates (13/20) suggests that both mechanisms may play a role in fluoroquinolone resistance. The other unknown mechanisms including different efflux pumps and probably other QRDRs mutations may contribute to fluoroquinolone resistance in E. faecalis.Keywords: E. faecalis, UTI, gyrA, parC, efflux pump

Type II topoisomerases, including DNA gyrase (GyrA) and topoisomerase IV (ParC), contribute to fluoroquinolone resistance in Enterococcus spp. This study investigated the mutational status of the quinolone resistance-determining regions (QRDRs) of GyrA and ParC in the clinical isolates of enterococci from a hospital in Baotou, China. We analyzed 110 enterococcal isolates, including 57 Enterococcus faecalis and 53 Enterococcus faecalis faecium. The resistance rates of E. faecalis and E. faecium to ciprofloxacin were 63.16% and 84.91%, respectively. We found that 32 samples of E. faecalis and 42 of E. faecium had single or combined mutations in gyrA and/or parC, which were all resistant to ciprofloxacin. Only two ciprofloxacin-resistant E. faecalis isolates had no mutation. No mutations in gyrA and parC genes in all ciprofloxacinsusceptible isolates were found. Ciprofloxacin minimal inhibitory concentrations (MICs) in the mutation group were significantly higher than those of the non-mutation group, indicating that mutations in the QRDRs of gyrA and parC were correlated with MIC elevation. Two novel substitutions (GyrA Ser83Phe and ParC Ser80Leu) of E. faecalis were identified herein. Three-dimensional modeling revealed that these novel amino acid substitutions could disrupt the water/metal-ion bridge and decrease the interaction between the enzymes and ciprofloxacin. The data showed a diversity of mutation types in QRDRs of type II topoisomerases whose association with fluoroquinolone resistance in clinical isolates of enterococci warrants further investigation. [ABSTRACT FROM AUTHOR]

Abstract Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of β-lactamases were detected by phenotypic methods, while presence of β-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different β-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajima’s D test and Fu’s Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, β-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them had integrase I gene and had high level of mutations in QRDR regions in gyrA, parC and parE genes. All these factors may play an important role in the invasiveness of these strains and make them difficult to treat. Isolation of these strains from different medical centers, indicate the need for a strict application of infection control measures in Medical centers in the North West Bank-Palestine that aim to reduce expense and damage caused by P. aeruginosa infections. Molecular analyses showed that Palestinian fluoroquinolone resistant P. aeruginosa haplotypes are not genetically differentiated; however, more mutations may exist in these strains.