Development Of Cross-resistance Research Articles

The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is severely constrained by random peptide mixtures.

The prevalence of antibiotic-resistant pathogens has become a major threat to public health, requiring swift initiatives for discovering new strategies to control bacterial infections. Hence, antibiotic stewardship and rapid diagnostics, but also the development, and prudent use, of novel effective antimicrobial agents are paramount. Ideally, these agents should be less likely to select for resistance in pathogens than currently available conventional antimicrobials. The usage of antimicrobial peptides (AMPs), key components of the innate immune response, and combination therapies, have been proposed as strategies to diminish the emergence of resistance. Herein, we investigated whether newly developed random antimicrobial peptide mixtures (RPMs) can significantly reduce the risk of resistance evolution in vitro to that of single sequence AMPs, using the ESKAPE pathogen Pseudomonas aeruginosa (P. aeruginosa) as a model gram-negative bacterium. Infections of this pathogen are difficult to treat due the inherent resistance to many drug classes, enhanced by the capacity to form biofilms. P. aeruginosa was experimentally evolved in the presence of AMPs or RPMs, subsequentially assessing the extent of resistance evolution and cross-resistance/collateral sensitivity between treatments. Furthermore, the fitness costs of resistance on bacterial growth were studied and whole-genome sequencing used to investigate which mutations could be candidates for causing resistant phenotypes. Lastly, changes in the pharmacodynamics of the evolved bacterial strains were examined. Our findings suggest that using RPMs bears a much lower risk of resistance evolution compared to AMPs and mostly prevents cross-resistance development to other treatments, while maintaining (or even improving) drug sensitivity. This strengthens the case for using random cocktails of AMPs in favour of single AMPs, against which resistance evolved in vitro, providing an alternative to classic antibiotics worth pursuing.

Drying-wetting cycle enhances stress resistance of Escherichia coli O157:H7 in a model soil

Outbreaks of Escherichia coli (E. coli) O157:H7 in farms are often triggered by heavy rains and flooding. Most cells die with the decreasing of soil moisture, while few cells enter a dormant state and then resuscitate after rewetting. The resistance of dormant cells to stress has been extensively studied, whereas the molecular mechanisms of the cross-resistance development of the resuscitated cells are poorly known. We performed a comparative proteomic analysis on O157:H7 before and after undergoing soil dry-wet alternation. A differential expression of 820 proteins was identified in resuscitated cells compared to exponential-phase cells, as determined by proteomics analysis. The GO and KEGG pathway enrichment analyses revealed that up-regulated proteins were associated with oxidative phosphorylation, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, ribosome activity, and transmembrane transporters, indicating increased energy production and protein synthesis in resuscitated O157:H7. Moreover, proteins related to acid, osmotic, heat, oxidative, antibiotic stress and horizontal gene transfer efficiency were up-regulated, suggesting a potential improvement in stress resistance. Subsequent validation experiments demonstrated that the survival rates of the resuscitated cells were 476.54 and 7786.34 times higher than the exponential-phase cells, with pH levels of 1.5 and 2.5, respectively. Similarly, resuscitated cells showed higher survival rates under osmotic stress, with 7.5%, 15%, and 30% NaCl resulting in survival rates that were 460.58, 1974.55, and 3475.31 times higher. Resuscitated cells also exhibited increased resistance to heat stress, with survival rates 69.64 and 139.72 times higher at 55 °C and 90 °C, respectively. Furthermore, the horizontal gene transfer (HGT) efficiency of resuscitated cells was significantly higher (153.12-fold) compared to exponential phase cells. This study provides new insights into bacteria behavior under changing soil moisture and this may explain O157:H7 outbreaks following rainfall and flooding, as the dry-wet cycle promotes stress cross-resistance development.

Роль D-маннозы и проантоцианидинов клюквы в профилактике рецидивов инфекции мочевыводящих путей.

Introduction. Prevention of urinary tract infections (UTI) is an urgent problem due to their high prevalence. Regular use of antibiotics, especially when recurrent urinary tract infections occur, contributes to the formation of resistant strains of microorganisms and the development of antibiotic cross-resistance. Thus, the relevance of finding alternative approaches for the treatment and prevention of such diseases is increasing. Materials and methods. Literature review from eLibrary.ru and PubMed revealed insufficient study of the use of D-mannose and proanthocyanidins of cranberry. Results. D-mannose is a simple sugar that has structural similarity to the mannose residues of uroplakin-1a, which coats the surface of urothelial umbrella epithelial cells. D-mannose is able to saturate FimH-adhesins, which prevents E. coli attachment mediated by FimH and type 1 fimbriae through a competitive inhibition mechanism, and excrete pathogens with urine. Cranberry consumption also inhibits bacterial adhesion. Although roanthocyanidins contained in cranberries do not have their own antibacterial activity, they prevent the attachment of uropathogenic E. coli by blocking the P-type fimbriae. Knowledge of the mechanisms of action of D-mannose and proanthocyanidins of cranberry, which exert their anti-adhesive effect by interacting with different types of fimbriae, suggests that the combination of these substances can bring much greater benefit and maximally block the adhesion of pathogenic bacteria. Conclusion. The published results of clinical studies have confirmed the efficacy of prescribing D-mannose and cranberry proanthocyanidins for the prevention of UTIs, but further reliably designed clinical trials of the combined use of different prophylactic methods to control urinary tract infection are needed.

Abstract 5834: The GR-LEDGF/p75 transcriptional network promotes therapy cross-resistance in prostate cancer cells

Abstract Prostate cancer (PCa) is the second leading cause of cancer mortality for men in the U.S. Patients who develop advanced PCa ultimately acquire therapy resistance and succumb to the disease. An emerging challenge in the treatment of advanced PCa is the development of therapy cross-resistance, in which molecular pathways promoting resistance to a particular drug or treatment also promote resistance to subsequent treatments. Although the mechanisms driving PCa therapy cross-resistance are not clearly understood, recent studies from our group and others implicate the glucocorticoid receptor (GR), a transcription factor, in resistance to both androgen receptor signaling inhibitors such enzalutamide (ENZ), and taxane drugs such as docetaxel (DTX). Our studies also showed that GR upregulates and interacts with the lens epithelium derived growth factor of 75 kD (LEDGF/p75) in a large panel of PCa cell lines. LEDGF/p75 is a stress transcription co-activator and oncoprotein that promotes cancer cell resistance to DTX and DNA-damaging drugs. GR and LEDGF/p75 are part of a large transcriptional network that include other oncogenic transcription factors linked to cancer chemoresistance such as β-catenin, Menin, MLL, JPO2, and c-Myc. We hypothesized that this transcriptional network plays a role in PCa therapy cross-resistance and could be a promising therapeutic target. We observed by Western blotting (WB) that both GR and LEDGF/p75 proteins are upregulated in ENZ-resistant and DTX-resistant PCa cell lines, and that GR silencing in both contexts abrogated LEDGF/p75 expression. MTT viability assays showed that ENZ-resistant PCa cells also exhibited cross-resistance to DTX. Bioinformatics studies, using publicly available RNA-seq datasets, identified overlapping differentially expressed genes (DEGs) between ENZ-resistant and DTX-resistant PCa cells. Individual silencing of GR and LEDGF/p75 in two DTX-resistant PCa cell lines followed by RNA-seq analysis also identified an overlap between DEGs regulated by GR and LEDGF/p75 with DEGs associated with both ENZ and DTX resistance in PCa. GSEA analysis of DEGs targeted by both GR and LEDGF/p75 in DTX-resistant PCa cells revealed enriched pathways associated with stress responses, androgen responses, and regulation of cell death and cell cycle. Further, we initiated studies to evaluate the efficacy of small molecule inhibitors targeting multiple components of the GR-LEDGF/p75 transcriptional network in sensitizing therapy-resistant PCa cells to DTX and ENZ. In conclusion, our results implicate the GR-LEDGF/p75 axis in promoting resistance to both ENZ and DTX in PCa cells. Further mining of RNA-seq data will identify potential molecular targets associated with PCa therapy cross-resistance, leading to the design of novel therapeutic strategies to attenuate this resistance. Citation Format: Pedro T. Ochoa, Evelyn S. Sanchez-Hernandez, Kai Wen Cheng, Isaac Kremsky, Charles Wang, Carlos A. Casiano. The GR-LEDGF/p75 transcriptional network promotes therapy cross-resistance in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5834.

Acquired resistance or tolerance? – in search of mechanisms underlying changes in the resistance profile of Candida albicans and Candida parapsilosis as a result of exposure to methotrexate

The increasing prevalence of fungal strains showing acquired resistance and multidrug resistance is an increasing therapeutic problem, especially in patients with a severely weakened immune system and undergoing chemotherapy. What is also extremely disturbing is the similarity of the resistance mechanisms of fungal cells and other eukaryotic cells, including human cells, which may contribute to the development of cross-resistance in fungi in response to substances used in e.g. anticancer treatment. An example of such a drug is methotrexate, which is pumped out of eukaryotic cells by ABC transmembrane transporters - in fungi, used to remove azoles from fungal cells.For this reason, the aim of the study was to analyze the expression levels of genes: ERG11, MDR1 and CDR1, potentially responsible for the occurrence of cross-resistance in Candida albicans and Candida parapsilosis as a result of fungal exposure to methotrexate (MTX).In vitro exposure of C. albicans and C. parapsilosis strains to methotrexate showed a high increase in resistance to fluconazole and a partial increase in resistance to voriconazole. Analysis of the expression of resistance genes showed varied responses of the tested strains depending on the species. In the case of C. albicans, an increase in the expression of the MDR1 gene was observed, and a decrease in ERG11 and CDR1. However, for C. parapsilosis there was an increase in the expression of the CDR1 gene and a decrease in ERG11 and MDR1.We noted the relationship between the level of resistance to voriconazole and the level of ERG11 gene expression in C. albicans. This indicates that this type of relationship is different for each species. Our research confirms that the mechanisms by which fungi acquire resistance and develop cross-resistance are highly complex and most likely involve several pathways simultaneously. The emergence of multidrug resistance may be related to the possibility of developing tolerance to antimycotics by fungi.

In Vitro Microevolution and Co-Selection Assessment of Amoxicillin and Cefotaxime Impact on Escherichia coli Resistance Development.

The global spread of antimicrobial resistance has become a prominent issue in both veterinary and public health in the 21st century. The extensive use of amoxicillin, a beta-lactam antibiotic, and consequent resistance development are particularly alarming in food-producing animals, with a focus on the swine and poultry sectors. Another beta-lactam, cefotaxime, is widely utilized in human medicine, where the escalating resistance to third- and fourth-generation cephalosporins is a major concern. The aim of this study was to simulate the development of phenotypic and genotypic resistance to beta-lactam antibiotics, focusing on amoxicillin and cefotaxime. The investigation of the minimal inhibitory concentrations (MIC) of antibiotics was performed at 1×, 10×, 100×, and 1000× concentrations using the modified microbial evolution and growth arena (MEGA-plate) method. Our results indicate that amoxicillin significantly increased the MIC values of several tested antibiotics, except for oxytetracycline and florfenicol. In the case of cefotaxime, this increase was observed in all classes. A total of 44 antimicrobial resistance genes were identified in all samples. Chromosomal point mutations, particularly concerning cefotaxime, revealed numerous complex mutations, deletions, insertions, and single nucleotide polymorphisms (SNPs) that were not experienced in the case of amoxicillin. The findings suggest that, regarding amoxicillin, the point mutation of the acrB gene could explain the observed MIC value increases due to the heightened activity of the acrAB-tolC efflux pump system. However, under the influence of cefotaxime, more intricate processes occurred, including complex amino acid substitutions in the ampC gene promoter region, increased enzyme production induced by amino acid substitutions and SNPs, as well as mutations in the acrR and robA repressor genes that heightened the activity of the acrAB-tolC efflux pump system. These changes may contribute to the significant MIC increases observed for all tested antibiotics. The results underscore the importance of understanding cross-resistance development between individual drugs when choosing clinical alternative drugs. The point mutations in the mdtB and emrR genes may also contribute to the increased activity of the mdtABC-tolC and emrAB-tolC pump systems against all tested antibiotics. The exceptionally high mutation rate induced by cephalosporins justifies further investigations to clarify the exact mechanism behind.

Trans-kingdom fungal pathogens infecting both plants and humans, and the problem of azole fungicide resistance.

Azole antifungals are abundantly used in the environment and play an important role in managing fungal diseases in clinics. Due to the widespread use, azole resistance is an emerging global problem for all applications in several fungal species, including trans-kingdom pathogens, capable of infecting plants and humans. Azoles used in agriculture and clinics share the mode of action and facilitating cross-resistance development. The extensive use of azoles in the environment, e.g., for plant protection and wood preservation, contributes to the spread of resistant populations and challenges using these antifungals in medical treatments. The target of azoles is the cytochrome p450 lanosterol 14-α demethylase encoded by the CYP51 (called also as ERG11 in the case of yeasts) gene. Resistance mechanisms involve mainly the mutations in the coding region in the CYP51 gene, resulting in the inadequate binding of azoles to the encoded Cyp51 protein, or mutations in the promoter region causing overexpression of the protein. The World Health Organization (WHO) has issued the first fungal priority pathogens list (FPPL) to raise awareness of the risk of fungal infections and the increasingly rapid spread of antifungal resistance. Here, we review the main issues about the azole antifungal resistance of trans-kingdom pathogenic fungi with the ability to cause serious human infections and included in the WHO FPPL. Methods for the identification of these species and detection of resistance are summarized, highlighting the importance of these issues to apply the proper treatment.

Hypochlorous acid-the future of wound care?

Dear Editor, Wounds, especially chronic wounds and associated infections, impose a substantial burden on individuals and healthcare systems, leading to escalated morbidity rates and a significant ris in healthcare costs. Chronic wounds create ideal conditions for infection and the formation of biofilms, making it essential to implement comprehensive wound care techniques to promote wound healing1. Antiseptics are substances that impede or eradicate pathogens found within wounds or on the skin. Iodine-based agents such as povidone-iodine (PVP-I) have been used to treat wounds for decades. PVP-I is widely used and accepted due to its broad activity spectrum against gram-positive and negative bacteria, no development of cross-resistance upon use and its ability to penetrate biofilms. While being widely utilized worldwide, certain concerns have been raised regarding hypersensitivity, inadequate penetration, and potential cytotoxicity on host cells associated with its use. Reports have emerged documenting instances of both immediate and delayed allergic reactions to PVP-I. There have also been reports of iodine toxicity in patients when PVP-I was used as a constant wound irrigant2. Hypochlorous acid (HOCl) is a potent antimicrobial compound naturally synthesized by the immune system. It exhibits remarkable efficacy against a wide range of bacterial, viral, and fungal pathogens, effectively neutralizing their presence. A study conducted by Sakarya et al. concluded that stabilized HOCl solution is minimally allergenic as it’s produced naturally in the body and is also an ideal wound care solution3. A few comparative studies have been conducted between HOCl and PVP-I. In very recent research, it was concluded that in neonatal circumcision, the group of patients that used PVP-I had a greater occurrence of edema and delayed wound healing as compared to patients that used HOCl4. Moreover, PVP-I has been demonstrated to be cytotoxic to cellular components of wound healing while HOCl controls bacterial spread without disrupting the wound healing process as it is non-cytotoxic to cells at concentrations that achieve antimicrobial action. The amount of exudate also decreases when Hypochlorous acid is used instead of Povidone iodine5. To sum it up, Physicians and nurses face a formidable hurdle in identifying a topical antiseptic agent that is both safe and efficacious. PVP-I is being widely used as an antiseptic agent for wound healing but patients do experience some side effects. Research suggests that hypochlorous acid is also an effective option to treat lesions with less adverse effects, although more research should be conducted to reach absolute conclusive results.

Phenomenon of suppression of estrogen signaling in breast cancer cells under ultraviolet irradiation: role of Snail proteins

Introduction. The study of the effect of irradiation or any other DNA-damaging agents on the sensitivity of tumors to conservative therapy, drug or hormonal, is among the most imporant tasks that determine the efficiency of combined therapy of cancer patients.Aim. To investigate the effect of irradiation on the activity of key signaling proteins and the level of hormone dependence of breast cancer cells.Materials and methods. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells. Ultraviolet (UV) irradiation in the range of 254 nm with the intensity of 25–50 J/m2 was used as an experimental model to study the response of tumor cells to DNA damage. Cell growth rate was determined using the MTT test, cell survival after irradiation was analyzed using the colony-forming test. Estrogen receptor transcriptional activity was determined by reporter assay; cellular protein expression was determined by immunoblotting.Results. Single UV irradiation of MCF-7 cells leads to a marked increase in the level of apoptotic markers: p53, cPARP, suppression of expression of growth signaling proteins: CDK4/6 and estrogen receptor α (ERα). The above changes are accompanied with an increase in phosphorylation of Akt protein kinase and a marked increase in the expression of Snail1, one of the key proteins of epithelial-mesenchymal transition. In UV-resistant MCF-7/UVR cell subline obtained under repeated irradiation cycles, the levels of apoptotic and growth signaling proteins (p53, cPARP, CDK4/6) return to control levels, except for the phosphorylated form of Akt and Snail1, whose content remains high. Transfection of Snail1-expressing plasmid into MCF-7 cells is accompanied by activation of apoptotic signaling, suppression of ERα activity, and development of partial hormone resistance; however, the sensitivity of cells to irradiation is practically unchanged. Transfection of microRNA-181a-2, one of the microRNAs associated with cell resistance, simultaneously activates Akt and Snail1 and leads to the development of cross-resistance of cells to irradiation and hormonal drugs.Conclusion. The obtained data allow us to consider irradiation-induced Snail1 activation as one of the factors involved in deregulation of estrogen signaling and formation of cell resistance to hormonal drugs, while simultaneous activation of Akt and Snail1 is accompanied by the development of cross-resistance to irradiation and hormonal drugs.

Composition of Conditioned Media from Radioresistant and Chemoresistant Cancer Cells Reveals miRNA and Other Secretory Factors Implicated in the Development of Resistance.

Resistance to chemo- or radiotherapy is the main obstacle to consistent treatment outcomes in oncology patients. A deeper understanding of the mechanisms driving the development of resistance is required. This review focuses on secretory factors derived from chemo- and radioresistant cancer cells, cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and cancer stem cells (CSCs) that mediate the development of resistance in unexposed cells. The first line of evidence considers the experiments with conditioned media (CM) from chemo- and radioresistant cells, CAFs, MSCs, and CSCs that elevate resistance upon the ionizing radiation or anti-cancer drug exposure of previously untreated cells. The composition of CM revealed factors such as circular RNAs; interleukins; plasminogen activator inhibitor; and oncosome-shuttled lncRNAs, mRNAs, and miRNAs that aid in cellular communication and transmit signals inducing the chemo- and radioresistance of sensitive cancer cells. Data, demonstrating that radioresistant cancer cells become resistant to anti-neoplastic drug exposure and vice versa, are also discussed. The mechanisms driving the development of cross-resistance between chemotherapy and radiotherapy are highlighted. The secretion of resistance-mediating factors to intercellular fluid and blood brings attention to its diagnostic potential. Highly stable serum miRNA candidates were proposed by several studies as prognostic markers of radioresistance; however, clinical studies are needed to validate their utility. The ability to predict a treatment response with the help of the miRNA resistance status database will help with the selection of an effective therapeutic strategy. The possibility of miRNA-based therapy is currently being investigated with ongoing clinical studies, and such approaches can be used to alleviate resistance in oncology patients.

Adaptive laboratory evolution of Salmonella enterica in acid stress.

Adaptive laboratory evolution (ALE) studies play a crucial role in understanding the adaptation and evolution of different bacterial species. In this study, we have investigated the adaptation and evolution of Salmonella enterica serovar Enteritidis to acetic acid using ALE. Acetic acid concentrations below the minimum inhibitory concentration (sub-MIC) were used. Four evolutionary lineages (EL), namely, EL1, EL2, EL3, and EL4, of S. Enteritidis were developed, each demonstrating varying levels of resistance to acetic acid. The acetic acid MIC of EL1 remained constant at 27 mM throughout 70 days, while the MIC of EL2, EL3, and EL4 increased throughout the 70 days. EL4 was adapted to the highest concentration of acetic acid (30 mM) and demonstrated the highest increase in its MIC against acetic acid throughout the study, reaching an MIC of 35 mM on day 70. The growth rates of the evolved lineages increased over time and were dependent on the concentration of acetic acid used during the evolutionary process. EL4 had the greatest increase in growth rate, reaching 0.33 (h-1) after 70 days in the presence of 30 mM acetic acid as compared to EL1, which had a growth rate of 0.2 (h-1) after 70 days with no exposure to acetic acid. Long-term exposure to acetic acid led to an increased MIC of human antibiotics such as ciprofloxacin and meropenem against the S. enterica evolutionary lineages. The MIC of ciprofloxacin for EL1 stayed constant at 0.016 throughout the 70 days while that of EL4 increased to 0.047. Bacterial whole genome sequencing revealed single-nucleotide polymorphisms in the ELs in various genes known to be involved in S. enterica virulence, pathogenesis, and stress response including phoP, phoQ, and fhuA. We also observed genome deletions in some of the ELs as compared to the wild-type S. Enteritidis which may have contributed to the bacterial acid adaptation. This study highlights the potential for bacterial adaptation and evolution under environmental stress and underscores the importance of understanding the development of cross resistance to antibiotics in S. enterica populations. This study serves to enhance our understanding of the pathogenicity and survival strategies of S. enterica under acetic acid stress.

Therapeutic Resistance Models and Treatment Sequencing in Advanced Prostate Cancer.

Current common treatments for castration-resistant prostate cancer (CRPC) typically belong to one of three major categories: next-generation anti-androgen therapies (NGAT) including enzalutamide, abiraterone acetate, apalutamide, and darolutamide; taxane therapy represented by docetaxel; and PARP inhibitors (PARPi) like olaparib. Although these treatments have shown efficacy and have improved outcomes for many patients, some do not survive due to the emergence of therapeutic resistance. The clinical landscape is further complicated by limited knowledge about how the sequence of treatments impacts the development of therapeutic cross-resistance in CRPC. We have developed multiple CRPC models of acquired therapeutic resistance cell sublines from C4-2B cells. These include C4-2B MDVR, C4-2B AbiR, C4-2B ApaR, C4-2B DaroR, TaxR, and 2B-olapR, which are resistant to enzalutamide, abiraterone, apalutamide, darolutamide, docetaxel, and olaparib, respectively. These models are instrumental for analyzing gene expression and assessing responses to various treatments. Our findings reveal distinct cross-resistance characteristics among NGAT-resistant cell sublines. Specifically, resistance to enzalutamide induces resistance to abiraterone and vice versa, while maintaining sensitivity to taxanes and olaparib. Conversely, cells with acquired resistance to docetaxel exhibit cross-resistance to both cabazitaxel and olaparib but retain sensitivity to NGATs like enzalutamide and abiraterone. OlapR cells, significantly resistant to olaparib compared to parental cells, are still responsive to NGATs and docetaxel. Moreover, OlapR models display cross-resistance to other clinically relevant PARP inhibitors, including rucaparib, niraparib, and talazoparib. RNA-sequencing analyses have revealed a complex network of altered gene expressions that influence signaling pathways, energy metabolism, and apoptotic signaling, pivotal to cancer's evolution and progression. The data indicate that resistance mechanisms are distinct among different drug classes. Notably, NGAT-resistant sublines exhibited a significant downregulation of androgen-regulated genes, contrasting to the stable expression noted in olaparib and docetaxel-resistant sublines. These results may have clinical implications by showing that treatments of one class can be sequenced with those from another class, but caution should be taken when sequencing drugs of the same class.

Repeat-exposure in vitro protocol to assess the risk of antimicrobial resistance (AMR) development from use of personal care products: Case study using an antibacterial liquid handwash

The global crisis we are facing with regard to antibiotic resistance has been largely attributed to the overuse and misuse of antibiotics in healthcare and agriculture. However, there is also growing global concern about cross-resistance between biocides and antibiotics. This has made clear the need for more research in this area along with easy-to-perform, but realistic, methods to characterise the potential risk associated with cross-resistance to antibiotics due to biocide use. The primary aim of this work was to develop a repeat-exposure method for predicting bacterial resistance to microbicides, including their cross-resistance to antibiotics. Realism is incorporated in the presented protocol through the use of relevant concentrations and contact times, validated neutralisers, appropriate test organisms and repeat-exposures. The protocol can be applied to formulated microbicides, as shown in the liquid handwash case study presented here. Five bacterial strains were included in the study: Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, Staphylococcus epidermidis ATCC 14990, Escherichia coli ATCC 10536 and Enterococcus hirae ATCC 10541. The protocol parameters used in the case study reflected a worst-case exposure scenario (in terms of contact time and concentration). The results demonstrated that repeated exposure to the liquid handwash would not be expected to lead to development of bacterial resistance or cross-resistance to antibiotics. It is envisaged that this protocol could be used by manufacturers of microbicidal formulations to assess whether repeated use of the test products would contribute to bacterial resistance development or cross-resistance to antibiotics.

Helicoverpa armigera (Hubner) Associated Cross Resistance Patterns in South Indian Crop Ecosystem

Aim: To study the cross resistance patterns associated with Mahaboobnagar, Raichur, Nagpur populations of Helicoverpa armigera (Hubner).
 Study design: Bioassay
 Place and Duration of Study: The experiment was carried out from February 2010 to May 2011 at Department of Entomology, College of Agriculture, Rajendranagar, Hyderabad, Telangana.
 Methodology: Helicoverpa armigera was selected for indoxacarb in F1 and F2 continuously then the population subjected to different selected insecticides to know the cross resistance patterns associated.
 Results: Mahaboobnagar population recorded 1.109 and 0.816 fold resistance at LD50 and LD90, respectively, while Raichur population has developed still higher levels of relative resistance by 1.591 and 0.846 fold when compared with the Nagpur population at LD50 and LD90, respectively. Similarly, the Raichur population has developed 1.435 and 1.037 folds relative resistance at LD50­ and LD90, respectively as compared with the Mahaboobnagar population.
 The Mahaboobnagar population resistant to indoxacarb at F3, when subjected to selected insecticides like cypermethrin, methomyl, spinosad showed a negative cross resistance ratio of 0.665, 0.830, 0.916 to cypermethrin, methomyl, spinosad respectively, and a positive cross resistance ratio of 1.019 to indoxacarb, while similar trend was displayed by Raichur population showing a negative cross resistance ratio of 0.932, 0.565, 0.803 to cypermethrin, methomyl, spinosad respectively and positive cross resistance of 1.036 indoxacarb further, same trend was shown by Nagpur population by displaying a negative cross resistance ratio of 0.610, 0.735, 0.519 to cypermethrin, methomyl, spinosad and positive cross resistance ratio of 1.026 to indoxacarb.
 Conclusion: Continuous application of single insecticide belonging to a specific group across the generations increases the resistance from F1 to F3. Alternating the new chemistries with old conventional chemicals resulted in no cross resistance development as it was observed in all test populations.

Salicylic acids and pathogenic bacteria: new perspectives on an old compound.

Salicylic acids have been used in human and veterinary medicine for their anti-pyretic, anti-inflammatory, and analgesic properties for centuries. A key role of salicylic acid-immune modulation in response to microbial infection-was first recognized during studies of their botanical origin. The effects of salicylic acid on bacterial physiology are diverse. In many cases, they impose selective pressures leading to development of cross-resistance to antimicrobial compounds. Initial characterization of these interactions was in Escherichia coli, where salicylic acid activates the multiple antibiotic resistance (mar) operon, resulting in decreased antibiotic susceptibility. Studies suggest that stimulation of the mar phenotype presents similarly in closely related Enterobacteriaceae. Salicylic acids also affect virulence in many opportunistic pathogens by decreasing their ability to form biofilms and increasing persister cell populations. It is imperative to understand the effects of salicylic acid on bacteria of various origins to illuminate potential links between environmental microbes and their clinically relevant antimicrobial-resistant counterparts. This review provides an update on known effects of salicylic acid and key derivatives on a variety of bacterial pathogens, offers insights to possible potentiation of current treatment options, and highlights cellular regulatory networks that have been established during the study of this important class of medicines.

Current Understanding of Potential Linkages between Biocide Tolerance and Antibiotic Cross-Resistance.

Antimicrobials (e.g., antibiotics and biocides) are invaluable chemicals used to control microbes in numerous contexts. Because of the simultaneous use of antibiotics and biocides, questions have arisen as to whether environments commonly treated with biocides (e.g., hospitals, food processing, wastewater, agriculture, etc.) could act as a reservoir for the development of antibiotic cross-resistance. Theoretically, cross-resistance could occur if the mechanism of bacterial tolerance to biocides also resulted in antibiotic resistance. On the other hand, biocides would likely present a higher evolutionary barrier to the development of resistance given the different modes of action between biocides and antibiotics and the broad-based physicochemical effects associated with most biocides. Published studies have shown that the induction of biocide tolerance in a laboratory can result in cross-resistance to some antibiotics, most commonly hypothesized to be due to efflux pump upregulation. However, testing of environmental isolates for biocide tolerance and antibiotic cross-resistance has yielded conflicting results, potentially due to the lack of standardized testing. In this review, we aim to describe the state of the science on the potential linkage between biocide tolerance and antibiotic cross-resistance. Questions still remain about whether the directed evolution of biocide tolerance and the associated antibiotic cross-resistance in a laboratory are or are not representative of real-world settings. Thus, research should continue to generate informative data to guide policies and preserve these tools' utility and availability.

Exposure of Salmonella enterica serovar 1,4, [5],12:i:- to benzalkonium chloride leads to acquired resistance to this disinfectant and antibiotics.

Disinfectants such as benzalkonium chloride (BC) extensively used in animal farms and food-processing industries contribute to the development of adaptive- and cross-resistance in foodborne pathogens, posing a serious threat to food safety and human health. The purpose of this study is to explore whether continuous exposure of Salmonella enterica serovar 1,4, [5],12: i:- (S. 1,4, [5],12: i:-) to sublethal concentrations of BC could result in acquired resistance to this agent and other environmental stresses (e.g. antibiotics, heat, and acid). BC tolerance increased in all tested strains after exposure to gradually increasing concentrations of BC, with increases in minimum inhibitory concentrations between two and six-fold. The survival rate of BC-adapted strains was significantly (P<0.05) higher than that of their wild-type (non-adapted) counterparts in lethal concentrations of BC. In addition, significant reductions (P<0.05) in zeta potential were observed in BC-adapted strains compared to wild-type ones, indicating that reduction in cell surface charge was a cause of adaptative resistance. More importantly, two BC-adapted strains exhibited increased antibiotic resistance to levofloxacin, ceftazidime, and tigecycline, while gene mutations (gyrA, parC) and antibiotic efflux-related genes (acrB, mdsA, mdsB) were detected by genomic sequencing analysis. Moreover, the tolerance of BC-adapted strains to heat (50, 55, and 60°C) and acid (pH 2.0, 2.5) was strain-dependent and condition-dependent. Repeated exposure to sublethal concentrations of BC could result in the emergence of BC- and antibiotic-resistant S. 1,4, [5],12: i:- strains.

Sublethal Exposure to Common Benzalkonium Chloride Leads to Antimicrobial Tolerance and Antibiotic Cross-Resistance in Commensal and Opportunistic Bacterial Species

The production and consumer use of disinfectants has substantially increased during the COVID-19 pandemic. Benzalkonium chloride (BAC) is a mixture of alkyl benzyl dimethyl ammonium chloride compounds and is the most common active ingredient in surface cleaning and disinfecting products. Accordingly, BAC compounds are routinely in contact with microorganisms in indoor environments, which may contribute to the development of antimicrobial tolerance and cross-resistance. To investigate the impact of BAC exposure on commensal and opportunistic bacteria of public health importance, we exposed Staphylococcus epidermidis, Corynebacterium xerosis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa to a standard BAC mixture (BAC12–14), as well as purified BAC16. Minimum inhibitory concentrations (MICs) and antibiotic susceptibilities were determined before and after repeated exposure to sublethal BAC concentrations. MICs for Gram-negative bacteria were significantly higher than Gram-positive bacteria. Additionally, BAC12–14 MICs were significantly higher for opportunistic pathogens and BAC-tolerance was associated with antibiotic cross-resistance. These results suggest that common Gram-negative opportunistic pathogens are less sensitive to BAC-inhibition than commensal species and may preferentially develop antimicrobial tolerance upon repeated or prolonged exposure to BAC12–14. Reevaluating the formulation and concentration of BAC-containing products in efforts to limit the development of antimicrobial tolerance and antibiotic co-resistance is warranted.

Cross Resistance Patterns Associated with Spinosad Resistant Helicoverpa armigera (Hubner) in South India

Aim: To study the cross resistance patterns associated with Helicoverpa armigera (Hubner) in south India.&#x0D; Study Design: Bioassay.&#x0D; Place and Duration of Study: The experiment was carried out in the Department of Entomology, College of Agriculture, Rajendranagar, Hyderabad,Telangana from February 2010 to May 2011.&#x0D; Methodology: Spinosad resistant Helicoverpa armigera population in F1 and F2 subjected to different insecticides to know the cross resistance patterns associated.&#x0D; Results: American bollworm population of Mahaboobnagar has developed 0.308 and 0.646 folds and 0.284 and 0.624 folds in Raichur population as compared with the Nagpur baseline population at F1. Mahaboobnagar population displayed a negative cross resistance ratio of 0.677 fold to cypermethrin, 0.806 fold to methomyl, 0.935 fold to indoxacarb and positive cross resistance of 1.039 fold to spinosad, similar trend was followed in Raichur population with a negative cross resistance ratio of 0.918 fold to cypermethrin, 0.543 fold to methomyl, 0.642 fold to indoxacarb and 1.060 fold to spinosad. Further, the Nagpur population exihibited a similar trend with a negative cross resistance ratio of 0.604 fold to cypermethrin, 0.690 fold to methomyl, 0.570 fold to indoxacarb and positive cross resistance ratio of 1.077 fold to spinosad at F3.&#x0D; Conclusion: The present study revealed that the continuous application of same insecticide across the generations increases the resistance from F1 to F3. Alternating the new chemistries with old conventional chemicals results in no cross resistance development as it was observed in all three populations studied.

Chromosome 1 trisomy confers resistance to aureobasidin A in Candida albicans.

Candida albicans is a prevalent opportunistic human fungal pathogen. However, there are currently very few antifungal treatments available. Inositol phosphoryl ceramide synthase is an essential and fungal-specific protein that also provides a novel and promising antifungal target. Aureobasidin A is a widely used inhibitor of inositol phosphoryl ceramide synthase, however the mechanism of resistance to aureobasidin A is largely unknown in pathogenic fungi. Here we investigated how C. albicans adapted to low and high concentrations of aureobasidin A. We identified trisomy of chromosome 1 as the predominant mechanism of rapid adaptation. Resistance to aureobasidin A was unstable because of the inherent instability of aneuploids. Importantly, chromosome 1 trisomy simultaneously regulated genes which were associated with aureobasidin A resistance that are on this aneuploid chromosome as well as on other chromosomes. Furthermore, the pleiotropic effect of aneuploidy caused altered resistance not only to aureobasidin A but also to other antifungal drugs including caspofungin and 5-flucytosine. We posit aneuploidy provides a rapid and reversible mechanism of development of drug resistance and cross resistance in C. albicans.