Phosphorylation-Driven Epichaperome Assembly: A Critical Regulator of Cellular Adaptability and Proliferation.

The intricate protein-chaperone network is vital for cellular function. Recent discoveries have unveiled the existence of specialized chaperone complexes called epichaperomes, protein assemblies orchestrating the reconfiguration of protein-protein interaction networks, enhancing cellular adaptability and proliferation. This study delves into the structural and regulatory aspects of epichaperomes, with a particular emphasis on the significance of post-translational modifications in shaping their formation and function. A central finding of this investigation is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 situated within an intrinsically disordered region, as critical determinants in epichaperome assembly. Our data demonstrate that the phosphorylation of these serine residues enhances HSP90's interaction with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Furthermore, this study establishes a direct link between epichaperome function and cellular physiology, especially in contexts where robust proliferation and adaptive behavior are essential, such as cancer and stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone complexes in diseases characterized by epichaperome dysregulation, bridging the gap between fundamental research and precision medicine.

Unraveling the Mechanism of Epichaperome Modulation by Zelavespib: Biochemical Insights on Target Occupancy and Extended Residence Time at the Site of Action.

Drugs with a long residence time at their target sites are often more efficacious in disease treatment. The mechanism, however, behind prolonged retention at the site of action is often difficult to understand for non-covalent agents. In this context, we focus on epichaperome agents, such as zelavespib and icapamespib, which maintain target binding for days despite rapid plasma clearance, minimal retention in non-diseased tissues, and rapid metabolism. They have shown significant therapeutic value in cancer and neurodegenerative diseases by disassembling epichaperomes, which are assemblies of tightly bound chaperones and other factors that serve as scaffolding platforms to pathologically rewire protein-protein interactions. To investigate their impact on epichaperomes in vivo, we conducted pharmacokinetic and target occupancy measurements for zelavespib and monitored epichaperome assemblies biochemically in a mouse model. Our findings provide evidence of the intricate mechanism through which zelavespib modulates epichaperomes in vivo. Initially, zelavespib becomes trapped when epichaperomes bound, a mechanism that results in epichaperome disassembly, with no change in the expression level of epichaperome constituents. We propose that the initial trapping stage of epichaperomes is a main contributing factor to the extended on-target residence time observed for this agent in clinical settings. Zelavespib's residence time in tumors seems to be dictated by target disassembly kinetics rather than by frank drug-target unbinding kinetics. The off-rate of zelavespib from epichaperomes is, therefore, much slower than anticipated from the recorded tumor pharmacokinetic profile or as determined in vitro using diluted systems. This research sheds light on the underlying processes that make epichaperome agents effective in the treatment of certain diseases.

Structural and functional complexity of HSP90 in cellular homeostasis and disease.

Heat shock protein 90 (HSP90) is a chaperone with vital roles in regulating proteostasis, long recognized for its function in protein folding and maturation. A view is emerging that identifies HSP90 not as one protein that is structurally and functionally homogeneous but, rather, as a protein that is shaped by its environment. In this Review, we discuss evidence of multiple structural forms of HSP90 in health and disease, including homo-oligomers and hetero-oligomers, also termed epichaperomes, and examine the impact of stress, post-translational modifications and co-chaperones on their formation. We describe how these variations influence context-dependent functions of HSP90 as well as its interaction with other chaperones, co-chaperones and proteins, and how this structural complexity of HSP90 impacts and is impacted by its interaction with small molecule modulators. We close by discussing recent developments regarding the use of HSP90 inhibitors in cancer and how our new appreciation of the structural and functional heterogeneity of HSP90 invites a re-evaluation of how we discover and implement HSP90 therapeutics for disease treatment.

How aberrant N-glycosylation can alter protein functionality and ligand binding: An atomistic view.

Protein-assembly defects due to an enrichment of aberrant conformational protein variants are emerging as a new frontier in therapeutics design. Understanding the structural elements that rewire the conformational dynamics of proteins and pathologically perturb functionally oriented ensembles is important for inhibitor development. Chaperones are hub proteins for the assembly of multiprotein complexes and an enrichment of aberrant conformers can affect the cellular proteome, and in turn, phenotypes. Here, we integrate computational and experimental tools to investigte how N-glycosylation of specific residues in glucose-regulated protein 94 (GRP94) modulates internal dynamics and alters the conformational fitness of regions fundamental for the interaction with ATP and synthetic ligands and impacts substructures important for the recognition of interacting proteins. N-glycosylation plays an active role in modulating the energy landscape of GRP94, and we provide support for leveraging the knowledge on distinct glycosylation variants to design molecules targeting GRP94 disease-associated conformational states and assemblies.

Systems-level analyses of protein-protein interaction network dysfunctions via epichaperomics identify cancer-specific mechanisms of stress adaptation.

Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.

Synthesis, biological evaluation and mechanistic studies of 4-(1,3-thiazol-2-yl)morpholine-benzimidazole hybrids as a new structural class of antimicrobials.

In spite of several attempts to develop newer pharmacophores as potential antimicrobial agents, the benzimidazole scaffold is still considered as one of the most sought after structural component towards the design of compounds that act against a wide spectrum of microbes. Herein, we report the design and synthesis of a new structural class of 4-(1,3-thiazol-2-yl)morpholine-benzimidazole hybrids as antimicrobial agents. The most potent analog, 6g shows IC50 of 1.3 µM, 2.7 µM, 10.8 µM, 5.4 µM and 10.8 µM against Cryptococcus neoformans, Candida albicans, Candida parapsilosis, Escherichia coli and Staphylococcus aureus, respectively. Interestingly 6g exhibits selectivity towards the cryptococcal cells with fungicidal behavior. Propidium iodide uptake study shows permeabilization of pathogenic cells in the presence of 6g. Flow cytometric analysis confirms that cell death is predominantly due to apoptosis. Moreover, electron microscopic analysis specifies that it shrinks, disrupts and initiate pore(s) formation in the cell membrane leading to cell lysis.

Cryoconservation of in vitro grown shoot tips of Cicer microphyllum: A crop wild relative of chickpea.

BACKGROUND: Cicer microphyllum Benth. is a crop wild relative (CWR) of chickpea (C. arietinum L.), that possess useful genes for cold and drought tolerance. The species is being conserved in the In Vitro Active Genebank for short- to medium-term conservation. Cryopreservation would be a useful complementary approach for its long-term conservation. OBJECTIVE: The current work aimed to develop an efficient cryoconservation protocol for cryobanking of C. microphyllum shoot tips. MATERIALS AND METHODS: In vitro shoot tips excised from 4-month old shoot cultures grown on B5 + 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO3 medium were cryoconserved using a droplet-vitrification technique. Post-thaw regrowth was evaluated after: (i) preculture medium (B5 basal, B5 + 3, 4, 6 and 10% sucrose), (ii) preculture incubation temperature (25 ± 2, 10, 8 and 22/5 degree C), (iii) PVS2 duration (10, 20, 30. 40, 50 and 60 min) and (iv) regrowth medium (B5) supplemented with 0.5 mg/L KIN + 0.1 NAA mg/L; 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO3; 0.2 mg/L BAP + 10 mg/L AgNO3; 0.2 mg/L BAP + 20 mg/L AgNO3 and 0.2 mg/L BAP + 30 mg/L AgNO3. RESULTS: In vitro shoot tips grown on B5 + 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO3, precultured on B5 + 6% sucrose at 10 degree C for 3 days, followed by PVS2 treatment for 20 min, unloading solution for 60 min and regrowth on B5 + 0.2 mg/L BAP + 20 mg/L AgNO3 resulted in highest survival (57%) and regrowth (40%) after cryoconservation. CONCLUSION: The standardized protocol was successfully used for cryobanking of in vitro shoot tips of C. microphyllum in the In Vitro Base Genebank of ICAR-NBPGR, New Delhi. Doi.org/10.54680/fr23610110412.

Synthesis of 124I-labeled epichaperome probes and assessment in visualizing pathologic protein-protein interaction networks in tumor bearing mice.

Epichaperomes are disease-associated pathologic scaffolds composed of tightly bound chaperones and co-chaperones. They provide opportunities for precision medicine where aberrant protein-protein interaction networks, rather than a single protein, are detected and targeted. This protocol describes the synthesis and characterization of two 124I-labeled epichaperome probes, [124I]-PU-H71 and [124I]-PU-AD, both which have translated to clinical studies. It shows specific steps in the use of these reagents to image and quantify epichaperome-positivity in tumor bearing mice through positron emission tomography. For complete details on the use and execution of this protocol, please refer to Bolaender et al. (2021), Inda et al. (2020), and Pillarsetty et al. (2019).

Disease-specific interactome alterations via epichaperomics: the case for Alzheimer's disease.

The increasingly appreciated prevalence of complicated stressor-to-phenotype associations in human disease requires a greater understanding of how specific stressors affect systems or interactome properties. Many currently untreatable diseases arise due to variations in, and through a combination of, multiple stressors of genetic, epigenetic, and environmental nature. Unfortunately, how such stressors lead to a specific disease phenotype or inflict a vulnerability to some cells and tissues but not others remains largely unknown and unsatisfactorily addressed. Analysis of cell- and tissue-specific interactome networks may shed light on organization of biological systems and subsequently to disease vulnerabilities. However, deriving human interactomes across different cell and disease contexts remains a challenge. To this end, this opinion article links stressor-induced protein interactome network perturbations to the formation of pathologic scaffolds termed epichaperomes, revealing a viable and reproducible experimental solution to obtaining rigorous context-dependent interactomes. This article presents our views on how a specialized 'omics platform called epichaperomics may complement and enhance the currently available conventional approaches and aid the scientific community in defining, understanding, and ultimately controlling interactome networks of complex diseases such as Alzheimer's disease. Ultimately, this approach may aid the transition from a limited single-alteration perspective in disease to a comprehensive network-based mindset, which we posit will result in precision medicine paradigms for disease diagnosis and treatment.

Pharmacologically controlling protein-protein interactions through epichaperomes for therapeutic vulnerability in cancer.

Cancer cell plasticity due to the dynamic architecture of interactome networks provides a vexing outlet for therapy evasion. Here, through chemical biology approaches for systems level exploration of protein connectivity changes applied to pancreatic cancer cell lines, patient biospecimens, and cell- and patient-derived xenografts in mice, we demonstrate interactomes can be re-engineered for vulnerability. By manipulating epichaperomes pharmacologically, we control and anticipate how thousands of proteins interact in real-time within tumours. Further, we can essentially force tumours into interactome hyperconnectivity and maximal protein-protein interaction capacity, a state whereby no rebound pathways can be deployed and where alternative signalling is supressed. This approach therefore primes interactomes to enhance vulnerability and improve treatment efficacy, enabling therapeutics with traditionally poor performance to become highly efficacious. These findings provide proof-of-principle for a paradigm to overcome drug resistance through pharmacologic manipulation of proteome-wide protein-protein interaction networks.

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

Low-Level Tolerance to Fluoroquinolone Antibiotic Ciprofloxacin in QAC-Adapted Subpopulations of Listeria monocytogenes.

There was a development of low-level tolerance to fluoroquinolone antibiotic ciprofloxacin in Listeria monocytogenes after sublethal adaptation to quaternary ammonium compound (QAC). Using eight L. monocytogenes strains, we determined the changes in short-range MIC, growth rate, and survival for heterologous stress response to ciprofloxacin, after sublethal exposure to daily cycles of fixed or gradually increasing concentration of QAC. Three main findings were observed. (1) MIC increase-QAC-adapted subpopulations exhibited a significant increase in short-range MIC of ciprofloxacin, by 1.5 to 2.9 fold, as compared to non-adapted control for 4/8 strains (p < 0.05). (2) Growth rate increase-QAC-adapted subpopulations exhibited significant 2.1- to 6.8- fold increase in growth rate (OD600 at 10 h) in ciprofloxacin-containing broth, as compared to non-adapted control for 5/8 strains (p < 0.05). (3) Survival increase-QAC-adapted subpopulations of L. monocytogenes yielded significantly higher survival in ciprofloxacin-containing agar by 2.2 to 4.3 log CFU/mL for 4/8 strains, as compared to non-adapted control (p ˂ 0.05). However, for other 4/8 strains of L. monocytogenes, there was no increase in survival of QAC-adapted subpopulations, as compared to non-adapted control in ciprofloxacin. These findings suggest the potential formation of low-level ciprofloxacin-tolerant subpopulations in some L. monocytogenes strains when exposed to residual QAC concentrations (where QAC might be used widely) and such cells if not inactivated might create food safety risk.

Decreased biofilm formation by planktonic cells of Listeria monocytogenes in the presence of sodium hypochlorite.

The objective of this study was to determine if the adaptation at planktonic stage to subinhibitory concentrations (SIC) of sodium hypochlorite (NaOCl) could modulate the biofilm forming ability of five Listeria monocytogenes strains V7, Scott A, FSL-N1-227, FSL F6-154 and ATCC 19116 representing serotypes 1/2a, 4b and 4c. Biofilm formation by NaOCl nonadapted and adapted L. monocytogenes planktonic cells was measured in the presence or absence of SIC of NaOCl. The biofilm formation ability of NaOCl nonadapted and adapted L. monocyotgenes planktonic cells was reduced only in the presence of NaOCl (P < 0.05). Scanning electron microscopy revealed that the continuous exposure of NaOCl induced morphological changes in the L. monocytogenes biofilm structure and reduced its attachment to polystyrene surface. The qRT-PCR results also showed that the subinhibitory NaOCl reduced biofilm formation related gene expression such as motility and quorum sensing signals (P < 0.05). These findings indicate that subinhibitory NaOCl can reduce the ability of L. monocytogenes planktonic cells to form biofilms on polystyrene surface.

Clinical summary guide: reproduction in women with previous abdominopelvic radiotherapy or total body irradiation.

STUDY QUESTION: What is the evidence to guide the management of women who wish to conceive following abdominopelvic radiotherapy (AP RT) or total body irradiation (TBI)? SUMMARY ANSWER: Pregnancy is possible, even following higher doses of post-pubertal uterine radiation exposure; however, it is associated with adverse reproductive sequelae and pregnancies must be managed in a high-risk obstetric unit. WHAT IS KNOWN ALREADY: In addition to primary ovarian insufficiency, female survivors who are treated with AP RT and TBI are at risk of damage to the uterus. This may impact on its function and manifest as adverse reproductive sequelae. STUDY DESIGN SIZE DURATION: A review of the literature was carried out and a multidisciplinary working group provided expert opinion regarding assessment of the uterus and obstetric management. PARTICIPANTS/MATERIALS SETTING METHODS: Reproductive outcomes for postpubertal women with uterine radiation exposure in the form of AP RT or TBI were reviewed. This included Pubmed listed peer-reviewed publications from 1990 to 2019, and limited to English language.. MAIN RESULTS AND THE ROLE OF CHANCE: The prepubertal uterus is much more vulnerable to the effects of radiation than after puberty. Almost all available information about the impact of radiation on the uterus comes from studies of radiation exposure during childhood or adolescence.An uncomplicated pregnancy is possible, even with doses as high as 54 Gy. Therefore, tumour treatment doses alone cannot at present be used to accurately predict uterine damage. LIMITATIONS REASONS FOR CAUTION: Much of the data cannot be readily extrapolated to adult women who have had uterine radiation and the publications concerning adult women treated with AP RT are largely limited to case reports. WIDER IMPLICATIONS OF THE FINDINGS: This analysis offers clinical guidance and assists with patient counselling. It is important to include patients who have undergone AP RT or TBI in prospective studies to provide further evidence regarding uterine function, pregnancy outcomes and correlation of imaging with clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: This study received no funding and there are no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Aneurysmal bone cyst of thoracic spine with neurological deficit and its recurrence treated with multimodal intervention - A case report.

BACKGROUND: Aneurysmal bone cysts (ABCs) are rare, representing about 1% of primary bone tumors, and 15% of all primary spine/sacral tumors. Notably, when they are located in poorly accessible regions such as the spine and pelvis, their management may be challenging. Treatment options include selective arterial embolization (SAE), curettage, en bloc excision with reconstruction, and radiotherapy. CASE DESCRIPTION: A 16-year-old male presented with 2 months of mid back pain, left-sided thoracic radiculopathy, and left lower limb weakness (MRC - 3/5). MR imaging revealed an expansile, lytic lesion involving the T9 vertebral body, and the left-sided posterior elements resulting in cord compression. He underwent SAE followed by intralesional excision, bone grafting, and a cage - instrumented fusion. ABC was diagnosed from the biopsy sample. Postoperatively, the pain was reduced, and he was neurologically intact. Five months later, he presented with a new lesion that was treated with repeated SAE and three doses of zoledronic acid. At the end of 2 years, the subsequent, MRI and CT studies documented new bone formation in the lytic areas, with healing of lesion; additionally, he clinically demonstrated sustained pain relief. CONCLUSION: Here, we emphasized the importance of surgery for patients with ABC who develop focal neurological deficits. Treatment options should include SAE with bisphosphonate therapy for lesions that recur without neurological involvement.

A Chemical Biology Approach to the Chaperome in Cancer-HSP90 and Beyond.

Cancer is often associated with alterations in the chaperome, a collection of chaperones, cochaperones, and other cofactors. Changes in the expression levels of components of the chaperome, in the interaction strength among chaperome components, alterations in chaperome constituency, and in the cellular location of chaperome members, are all hallmarks of cancer. Here we aim to provide an overview on how chemical biology has played a role in deciphering such complexity in the biology of the chaperome in cancer and in other diseases. The focus here is narrow and on pathologic changes in the chaperome executed by enhancing the interaction strength between components of distinct chaperome pathways, specifically between those of HSP90 and HSP70 pathways. We will review chemical tools and chemical probe-based assays, with a focus on HSP90. We will discuss how kinetic binding, not classical equilibrium binding, is most appropriate in the development of drugs and probes for the chaperome in disease. We will then present our view on how chaperome inhibitors may become potential drugs and diagnostics in cancer.

Rugose Morphotype in Salmonella Typhimurium and Salmonella Heidelberg Induced by Sequential Exposure to Subinhibitory Sodium Hypochlorite Aids in Biofilm Tolerance to Lethal Sodium Hypochlorite on Polystyrene and Stainless Steel Surfaces.

Salmonella biofilms act as a continuous source for cross-contamination in the food processing environments. In this study, a stable rugose morphotype of Salmonella was first induced by sequential exposure to subinhibitory concentrations (SICs) of sodium hypochlorite (NaOCl) (ranging from 50 to 300 ppm over 18-day period) in tryptic soy broth. Then, rugose and smooth morphotypes of Salmonella Typhimurium ATCC 14028 and Salmonella Heidelberg ATCC 8326 were characterized for biofilm forming abilities on polystyrene and stainless steel surfaces. Rugose morphotype of both ATCC 14028 and ATCC 8326 exhibited higher Exopolysaccharide (EPS) formation than smooth morphotype (p ≤ 0.05). Also, the SICs of NaOCl (200 or 300 ppm in broth model) increased the biofilm formation ability of rugose morphotype of ATCC 8326 (p ≤ 0.05) but decreased that of ATCC 14028. The 2-day-old Salmonella biofilms were treated with biocidal concentrations of 50, 100, or 200 ppm NaOCl (pH 6.15) in water for 5, 10, or 20 min at room temperature. The biofilm reduction in CFU/cm2 for the rugose was lower than the smooth morphotype on both surfaces (p ≤ 0.05) by lethal NaOCl in water. Scanning electron micrographs on both polystyrene and stainless steel surfaces demonstrated that the rugose morphotype produced a denser biofilm than the smooth morphotype. Transmission electron micrographs revealed the cell wall roughness in rugose morphotype, which may help in tolerance to NaOCl. The gene expression data indicate that the expression of biofilm regulator (csgD), curli (csgA, csgB, and csgC), and cellulose (bcsE) was significantly increased in rugose morphotype when induced by sequential exposure of NaOCl SICs. These findings reveal that the rugose morphotype of S. Typhimurium and S. Heidelberg produced significantly denser biofilm on food contact surfaces, which also increased with sequential exposure to SICs of NaOCl in the case of S. Heidelberg, and these biofilms were more tolerant to biocidal NaOCl concentrations commonly used in the food processing plants.

Effect of Chlorine-Induced Sublethal Oxidative Stress on the Biofilm-Forming Ability of Salmonella at Different Temperatures, Nutrient Conditions, and Substrates.

The present study was conducted to evaluate the effect of chlorine-induced oxidative stress on biofilm formation by various Salmonella strains on polystyrene and stainless steel (SS) surfaces at three temperatures (30, 25 [room temperature], and 4°C) in tryptic soy broth (TSB) and 1/10 TSB. Fifteen Salmonella strains (six serotypes) were exposed to a sublethal chlorine concentration (150 ppm of total chlorine) in TSB for 2 h at the predetermined temperatures. The biofilm-forming ability of the Salmonella strains was determined in 96-well polystyrene microtiter plates by using a crystal violet staining method and on SS coupons in 24-well tissue culture plates. All tested strains of Salmonella produced biofilms on both surfaces tested at room temperature and at 30°C. Of the 15 strains tested, none (chlorine stressed and nonstressed) formed biofilm at 4°C. At 30°C, Salmonella Heidelberg (ID 72), Salmonella Newport (ID 107), and Salmonella Typhimurium (ATCC 14028) formed more biofilm than did their respective nonstressed controls on polystyrene ( P ≤ 0.05). At room temperature, only stressed Salmonella Reading (ID 115) in 1/10 TSB had significantly more biofilm formation than did the nonstressed control cells ( P ≤ 0.05). Salmonella strains formed more biofilm in nutrient-deficient medium (1/10 TSB) than in full-strength TSB. At 25°C, chlorine-stressed Salmonella Heidelberg (ATCC 8326) and Salmonella Enteritidis (ATCC 4931) formed stronger biofilms on SS coupons ( P ≤ 0.05) than did the nonstressed cells. These findings suggest that certain strains of Salmonella can produce significantly stronger biofilms on plastic and SS upon exposure to sublethal chlorine.

Listeria monocytogenes Response to Sublethal Chlorine Induced Oxidative Stress on Homologous and Heterologous Stress Adaptation.

The objective of this study was to determine the effect of chlorine induced sublethal oxidative stress against homologous and heterologous stress adaptations in five Listeria monocytogenes (Lm) strains. Lm cells were exposed to gradually increasing sublethal concentrations of total chlorine/day: 250 ppm (day 1), 270 ppm (day 2), 290 ppm (day 3), 310 ppm (day 4), 330 ppm (day 5), 350 ppm (day 6), and 375 ppm (day 7) in tryptic soy broth (TSB). Changes in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Lm cells exposed to chlorine and control (non-adapted cells) were determined by the macro-dilution method. Chlorine-adapted Lm cells were also evaluated for changes in antibiotic resistance using the Kirby-Bauer disk diffusion and MIC double dilution assay as per the Clinical and Laboratory Standards Institute (CLSI, 2016) guidelines. In four Lm strains (Scott A, V7, FSL-N1-227 and FSL-F6-154) after adapted to sublethal chlorine, the MIC (600 ppm) and MBC (700 ppm) values of chlorine were slightly higher as compared to control (500 ppm MIC, and 600 ppm MBC). The Kirby-Bauer and MIC double dilution assays showed some significant changes in antibiotic susceptibility patterns for antibiotics such as streptomycin, gentamicin and ceftriaxone (p < 0.05). However, the changes in zones of inhibition and MIC values to all antibiotics tested for the chlorine-adapted and non-adapted (control) Lm cells were still within the susceptible range. Transmission electron microscopy studies showed that changes in cell wall and membrane integrity resulting, from the elongation of cells, may contribute to the possible routes of its increase in tolerance to chlorine and selective antibiotics. These findings indicate that the continuous exposure of Lm cells to chlorine may lead to significant changes in homologs and heterologous stress adaptation.

Salmonella enterica growth and biofilm formation in flesh and peel cantaloupe extracts on four food-contact surfaces.

Salmonella enterica is responsible for the highest number of foodborne disease outbreaks pertaining to cantaloupe industry. The objective of this study was to examine the growth and biofilm formation by outbreak strains of S. enterica ser. Poona (S. Poona), S. enterica ser. Stanley (S. Stanley) and S. enterica ser. Montevideo (S. Montevideo) on different food-contact processing surfaces in cantaloupe flesh and peel extracts at 22 °C and 10 °C. The generation time of all S. enterica strains tested was shorter in the high concentration (50 mg/ml) of cantaloupe extract and high temperature. In 50 mg/ml of cantaloupe flesh or peel extract, the populations of S. enterica were increased by 5 log CFU/ml in 24 h at 22 °C and 1 log CFU/ml in 72 h at 10 °C. In 2 mg/ml of cantaloupe flesh or peel extracts, the populations of S. enterica were increased by 3.5 log CFU/ml in 56 h at 22 °C, but there were no changes in 72 h at 10 °C. The biofilm production of S. enterica was greater at 50 mg/ml of cantaloupe extract and 22 °C, but no major differences (P ≥ 0.05) were found among the strains tested. In 50 mg/ml cantaloupe extract, S. enterica produced 5-6 log CFU/cm2 biofilm in 4-7 d at 22 °C and approximately 3.5-4 log CFU/cm2 in 7 d at 10 °C. In 2 mg/ml of cantaloupe extract, S. enterica produced 4-4.5 log CFU/cm2 biofilms in 4-7 d at 22 °C and 3 log CFU/cm2 in 7 d at 10 °C. Biofilm formation by S. Poona (01A4754) was lowest on buna-n rubber compared to stainless steel, polyethylene and polyurethane surfaces under the majority of conditions tested. Overall, these findings show that S. enterica strains can grow rapidly and form biofilms on different cantaloupe processing surfaces in the presence of low concentrations of cantaloupe flesh or peel extracts.