Phenotypic variation of Salmonella in host tissues delays eradication by antimicrobial chemotherapy.

Antibiotic therapy often fails to eliminate a fraction of transiently refractory bacteria, causing relapses and chronic infections. Multiple mechanisms can induce such persisters with high antimicrobial tolerance in vitro, but their in vivo relevance remains unclear. Using a fluorescent growth rate reporter, we detected extensive phenotypic variation of Salmonella in host tissues. This included slow-growing subsets as well as well-nourished fast-growing subsets driving disease progression. Monitoring of Salmonella growth and survival during chemotherapy revealed that antibiotic killing correlated with single-cell division rates. Nondividing Salmonella survived best but were rare, limiting their impact. Instead, most survivors originated from abundant moderately growing, partially tolerant Salmonella. These data demonstrate that host tissues diversify pathogen physiology, with major consequences for disease progression and control.

Tracking the progeny of bacterial persisters using a CRISPR-based genomic recorder.

The rise of antimicrobial failure is a global emergency, and causes beyond typical genetic resistance must be determined. One probable factor is the existence of subpopulations of transiently growth-arrested bacteria, persisters, that endure antibiotic treatment despite genetic susceptibility to the drug. The presence of persisters in infected hosts has been successfully established, notably through the development of fluorescent reporters. It is proposed that infection relapse is caused by persisters resuming growth after cessation of the antibiotic treatment, but to date, there is no direct evidence for this. This is because no tool or reporter currently exists to track the extent to which infection relapse is initiated by regrowth of persisters in the host. Indeed, once they have transitioned out of the persister state, the progeny of persisters are genetically and phenotypically identical to susceptible bacteria in the population, making it virtually impossible to ascertain the source of relapse. We designed pSCRATCH (plasmid for Selective CRISPR Array expansion To Check Heritage), a molecular tool that functions to record the state of antibiotic persistence in the genome of Salmonella persisters. We show that pSCRATCH successfully marks persisters by adding spacers in their CRISPR arrays and the genomic label is stable in persister progeny after exit from persistence. We further show that in a Salmonella infection model the system enables the discrimination of treatment failure originating from persistence versus resistance. Thus, pSCRATCH provides proof of principle for stable marking of persisters and a prototype for applications to more complex infection models and other pathogens.

Salmonella persisters promote the spread of antibiotic resistance plasmids in the gut.

The emergence of antibiotic-resistant bacteria through mutations or the acquisition of genetic material such as resistance plasmids represents a major public health issue1,2. Persisters are subpopulations of bacteria that survive antibiotics by reversibly adapting their physiology3-10, and can promote the emergence of antibiotic-resistant mutants11. We investigated whether persisters can also promote the spread of resistance plasmids. In contrast to mutations, the transfer of resistance plasmids requires the co-occurrence of both a donor and a recipient bacterial strain. For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli, a common member of the microbiota12. S. Typhimurium forms persisters that survive antibiotic therapy in several host tissues. Here we show that tissue-associated S. Typhimurium persisters represent long-lived reservoirs of plasmid donors or recipients. The formation of reservoirs of S. Typhimurium persisters requires Salmonella pathogenicity island (SPI)-1 and/or SPI-2 in gut-associated tissues, or SPI-2 at systemic sites. The re-seeding of these persister bacteria into the gut lumen enables the co-occurrence of donors with gut-resident recipients, and thereby favours plasmid transfer between various strains of Enterobacteriaceae. We observe up to 99% transconjugants within two to three days of re-seeding. Mathematical modelling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces the formation of reservoirs of persisters after oral infection with S. Typhimurium, as well as subsequent plasmid transfer. We conclude that-even without selection for plasmid-encoded resistance genes-small reservoirs of pathogen persisters can foster the spread of promiscuous resistance plasmids in the gut.

Salmonella Typhimurium alters galactitol metabolism under ciprofloxacin treatment to balance resistance and virulence.

Ciprofloxacin-resistant Salmonella Typhimurium (S. Typhimurium) causes a significant health burden worldwide. A wealth of studies has been published on the contributions of different mechanisms to ciprofloxacin resistance in Salmonella spp. But we still lack a deep understanding of the physiological responses and genetic changes that underlie ciprofloxacin exposure. This study aims to know how phenotypic and genotypic characteristics are impacted by ciprofloxacin exposure, from ciprofloxacin-susceptible to ciprofloxacin-resistant strains in vitro. Here, we investigated the multistep evolution of resistance in replicate populations of S. Typhimurium during 24 days of continuously increasing ciprofloxacin exposure and assessed how ciprofloxacin impacts physiology and genetics. Numerous studies have demonstrated that RamA is a global transcriptional regulator that prominently perturbs the transcriptional landscape of S. Typhimurium, resulting in a ciprofloxacin-resistant phenotype appearing first; the quinolone resistance-determining region mutation site can only be detected later. Comparing the microbial physiological changes and RNA sequencing (RNA-Seq) results of ancestral and selectable mutant strains, the selectable mutant strains had some fitness costs, such as decreased virulence, an increase of biofilm-forming ability, a change of "collateral" sensitivity to other drugs, and inability to utilize galactitol. Importantly, in the ciprofloxacin induced, RamA directly binds and activates the gatR gene responsible for the utilization of galactitol, but RamA deletion strains could not activate gatR. The elevated levels of RamA, which inhibit the galactitol metabolic pathway through the activation of gatR, can lead to a reduction in the growth rate, adhesion, and colonization resistance of S. Typhimurium. This finding is supported by studies conducted in M9 medium as well as in vivo infection models. IMPORTANCE: Treatment of antibiotic resistance can significantly benefit from a deeper understanding of the interactions between drugs and genetics. The physiological responses and genetic mechanisms in antibiotic-exposed bacteria are not well understood. Traditional resistance studies, often retrospective, fail to capture the entire resistance development process and typically exhibit unpredictable dynamics. To explore how clinical isolates of S. Typhimurium respond to ciprofloxacin, we analyzed their adaptive responses. We found that S. Typhimurium RamA-mediated regulation disrupts microbial metabolism under ciprofloxacin exposure, affecting genes in the galactitol metabolic pathways. This disruption facilitates adaptive responses to drug therapy and enhances the efficiency of intracellular survival. A more comprehensive and integrated understanding of these physiological and genetic changes is crucial for improving treatment outcomes.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Clonal spread of blaNDM-1-carrying Salmonella enterica serovar Typhimurium clone ST34 and wide spread of IncHI2/ST3-blaNDM-5 plasmid in China.

OBJECTIVES: To characterize blaNDM-carrying Salmonella recovered from a pig slaughterhouse. METHODS: In this study, 46 environment samples were collected from a slaughterhouse in China, and screened for carbapenem-resistant Enterobacterales. WGS, antimicrobial susceptibility testing and conjugation experiments were carried out to identify the isolates' resistance phenotypes and genetic characteristics. The phylogenetic relatedness of the Salmonella isolates obtained in this study and Salmonella (ST34 and ST29) in GenBank was determined. RESULTS: Two ST34 Salmonella Typhimurium and one ST29 Salmonella Stanley, recovered from three environmental samples (6.52%), were positive for blaNDM-1 and blaNDM-5, respectively. The two ST34 S. Typhimurium strains exhibited a close relationship (10-36 SNPs) with two human-derived blaNDM-1-bearing isolates from China (Hong Kong and Guangxi Province) and two blaNDM-negative ST34 Salmonella strains from the UK. The blaNDM-1 genes were located on IncHI2/ST3 plasmids. The capture of blaNDM-1 by the IncHI2/ST3 plasmid seems to be due to homologous recombination mediated by circular structures, as the genetic arrangements of the blaNDM-1 gene contain two IS26 elements of the same orientation. The blaNDM-5 gene was also carried by the IncHI2/ST3 plasmid, which shares highly similar structures with other blaNDM-5-bearing IncHI2/ST3 plasmids from other sources (fish, chicken, duck, human). CONCLUSIONS: This is the first report of a blaNDM-5-carrying IncHI2/ST3 plasmid in Salmonella. The clonal spread of NDM-1-producing ST34 S. Typhimurium across human and animal-associated environments, and the widespread dissemination of epidemic blaNDM-5-carrying IncHI2/ST3 plasmids among Enterobacteriaceae in China indicate the potential of further dissemination of blaNDM among Salmonella, which poses a threat to public health.

An allosteric inhibitor of the PhoQ histidine kinase with therapeutic potential against Salmonella infection.

BACKGROUND: The upsurge of antimicrobial resistance demands innovative strategies to fight bacterial infections. With traditional antibiotics becoming less effective, anti-virulence agents or pathoblockers, arise as an alternative approach that seeks to disarm pathogens without affecting their viability, thereby reducing selective pressure for the emergence of resistance mechanisms. OBJECTIVES: To elucidate the mechanism of action of compound N'-(thiophen-2-ylmethylene)benzohydrazide (A16B1), a potent synthetic hydrazone inhibitor against the Salmonella PhoP/PhoQ system, essential for virulence. MATERIALS AND METHODS: The measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes was used to evaluate the specificity of A16B1 to the PhoP regulon. Autokinase activity assays with either the native or truncated versions of PhoQ were used to dissect the A16B1 mechanism of action. The effect of A16B1 on Salmonella intramacrophage replication was assessed using the gentamicin protection assay. The checkerboard assay approach was used to analyse potentiation effects of colistin with the hydrazone. The Galleria mellonella infection model was chosen to evaluate A16B1 as an in vivo therapy against Salmonella. RESULTS: A16B1 repressed the Salmonella PhoP/PhoQ system activity, specifically targeting PhoQ within the second transmembrane region. A16B1 demonstrates synergy with the antimicrobial peptide colistin, reduces the intramacrophage proliferation of Salmonella without being cytotoxic and enhances the survival of G. mellonella larvae systemically infected with Salmonella. CONCLUSIONS: A16B1 selectively inhibits the activity of the Salmonella PhoP/PhoQ system through a novel inhibitory mechanism, representing a promising synthetic hydrazone compound with the potential to function as a Salmonella pathoblocker. This offers innovative prospects for combating Salmonella infections while mitigating the risk of antimicrobial resistance emergence.

Sanitization of hydroponic farming facilities in Singapore: what, why, and how.

This study performed microbial analysis of nutrient film technique (NFT) hydroponic systems on three indoor farms in Singapore (the "what"). To justify the necessity of sanitizing hydroponic systems, strong biofilm-forming bacteria were isolated from the facility and investigated for their influence on Salmonella colonization on polyvinyl chloride (PVC) coupons in hydroponic nutrient solutions (the "why"). Finally, sanitization solutions were evaluated with both laboratory-scale and field-scale tests (the "how"). As a result, the microbiome composition in NFT systems was found to be highly farm specific. The strong biofilm formers Corynebacterium tuberculostearicum C2 and Pseudoxanthomonas mexicana C3 were found to facilitate the attachment and colonization of Salmonella on PVC coupons. When forming dual-species biofilms, the presence of C2 and C3 also significantly promoted the growth of Salmonella (P < 0.05). Compared with hydrogen peroxide (H2O2) and sodium percarbonate (SPC), sodium hypochlorite (NaOCl) exhibited superior efficacy in biofilm removal. At 50 ppm, NaOCl reduced the Salmonella Typhimurium, C2, and C3 counts to <1 log CFU/cm2 within 12 h, whereas neither 3% H2O2 nor 1% SPC achieved this effect. In operational hydroponic systems, the concentration of NaOCl needed to achieve biofilm elimination increased to 500 ppm, likely due to the presence of organic matter accumulated during crop cultivation and the greater persistence of naturally formed multispecies biofilms. Sanitization using 500 ppm NaOCl for 12 h did not impede subsequent plant growth, but chlorination byproduct chlorate was detected at high levels in the hydroponic solution and in plants in the sanitized systems without rinsing. IMPORTANCE: This study's significance lies first in its elucidation of the necessity of sanitizing hydroponic farming systems. The microbiome in hydroponic systems, although mostly nonpathogenic, might serve as a hotbed for pathogen colonization and thus pose a risk for food safety. We thus explored sanitization solutions with both laboratory-scale and field-scale tests. Of the three tested sanitizers, NaOCl was the most effective and economical option, whereas one must note the vital importance of rinsing the hydroponic systems after sanitization with NaOCl.

Co-exposure to polyethylene fiber and Salmonella enterica serovar Typhimurium alters microbiome and metabolome of in vitro chicken cecal mesocosms.

Humans and animals encounter a summation of exposures during their lifetime (the exposome). In recent years, the scope of the exposome has begun to include microplastics. Microplastics (MPs) have increasingly been found in locations, including in animal gastrointestinal tracts, where there could be an interaction with Salmonella enterica serovar Typhimurium, one of the commonly isolated serovars from processed chicken. However, there is limited knowledge on how gut microbiomes are affected by microplastics and if an effect would be exacerbated by the presence of a pathogen. In this study, we aimed to determine if acute exposure to microplastics in vitro altered the gut microbiome membership and activity. The microbiota response to a 24 h co-exposure to Salmonella enterica serovar Typhimurium and/or low-density polyethylene (PE) microplastics in an in vitro broiler cecal model was determined using 16S rRNA amplicon sequencing (Illumina) and untargeted metabolomics. Community sequencing results indicated that PE fiber with and without S. Typhimurium yielded a lower Firmicutes/Bacteroides ratio compared with other treatment groups, which is associated with poor gut health, and overall had greater changes to the cecal microbial community composition. However, changes in the total metabolome were primarily driven by the presence of S. Typhimurium. Additionally, the co-exposure to PE fiber and S. Typhimurium caused greater cecal microbial community and metabolome changes than either exposure alone. Our results indicate that polymer shape is an important factor in effects resulting from exposure. It also demonstrates that microplastic-pathogen interactions cause metabolic alterations to the chicken cecal microbiome in an in vitro chicken cecal mesocosm. IMPORTANCE: Researching the exposome, a summation of exposure to one's lifespan, will aid in determining the environmental factors that contribute to disease states. There is an emerging concern that microplastic-pathogen interactions in the gastrointestinal tract of broiler chickens may lead to an increase in Salmonella infection across flocks and eventually increased incidence of human salmonellosis cases. In this research article, we elucidated the effects of acute co-exposure to polyethylene microplastics and Salmonella enterica serovar Typhimurium on the ceca microbial community in vitro. Salmonella presence caused strong shifts in the cecal metabolome but not the microbiome. The inverse was true for polyethylene fiber. Polyethylene powder had almost no effect. The co-exposure had worse effects than either alone. This demonstrates that exposure effects to the gut microbial community are contaminant-specific. When combined, the interactions between exposures exacerbate changes to the gut environment, necessitating future experiments studying low-dose chronic exposure effects with in vivo model systems.

Accumulation of resistance genes in Salmonella Typhimurium transmitted between poultry and dairy farms increases the risk to public health.

Salmonella Typhimurium is a zoonotic pathogen that poses a major threat to public health. This generalist serotype can be found in many hosts and the environment where varying selection pressures may result in the accumulation of antimicrobial resistance determinants. However, the transmission of this serotype between food-producing hosts, specifically between poultry layer flocks and nearby dairy herds, was never demonstrated. We investigated an outbreak at a dairy in Israel to determine the role of nearby poultry houses to be sources of infection. The 2-month outbreak resulted in a 47% mortality rate among 15 calves born in that period. Routine treatment of fluid therapy, a nonsteroidal anti-inflammatory, and cefquinome was ineffective, and control was achieved by the introduction of vaccination of dry cows against Salmonella (Bovivac S, MSD Animal Health) and a strict colostrum regime. Whole genome sequencing and antimicrobial sensitivity tests were performed on S. Typhimurium strains isolated from the dairy (n = 4) and strains recovered from poultry layer farms (n = 10). We identified acquired antimicrobial-resistant genes, including the blaCTX-M-55 gene, conferring resistance to extended-spectrum cephalosporins, which was exclusive to dairy isolates. Genetic similarity with less than five single nucleotide polymorphism differences between dairy and poultry strains suggested a transmission link. This investigation highlights the severe impact of S. Typhimurium on dairy farms and the transmission risk from nearby poultry farms. The accumulation of potentially transferable genes conferring resistance to critically important antimicrobials underscores the increased public health risk associated with S. Typhimurium circulation between animal hosts.IMPORTANCESalmonella Typhimurium is one of the major causes of food-borne illness globally. Infections may result in severe invasive disease, in which antimicrobial treatment is warranted. Therefore, the emergence of multi-drug-resistant strains poses a significant challenge to successful treatment and is considered one of the major threats to global health. S. Typhimurium can be found in a variety of animal hosts and environments; however, its transmission between food-producing animals, specifically poultry layers flocks and dairy herds, was never studied. Here, we demonstrate the transmission of the pathogen from poultry to a nearby dairy farm. Alarmingly, the multi-drug-resistant strains collected during the outbreak in the dairy had acquired resistance to extended-spectrum cephalosporins, antibiotics critically important in treating Salmonellosis in humans. The findings of the study emphasize the increased risk to public health posed by zoonotic pathogens' circulation between animal hosts.

A novel family of defensin-like peptides from Hermetia illucens with antibacterial properties.

BACKGROUND: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections. RESULTS: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa. CONCLUSIONS: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa.

Synergistic antimicrobial activity of alpha-linolenic acid in combination with tetracycline or florfenicol against multidrug-resistant Salmonella typhimurium.

Salmonella is a major foodborne pathogen that can be transmitted from livestock and poultry to humans through the food chain. Due to the widespread use of antibiotics, antibiotic resistance Salmonella has become an important factor threatening food safety. Combining antibiotic and non-antibiotic agents is a promising approach to address the widespread emergence of antibiotic-resistant pathogens. In this study, we investigated the antibiotic resistance profile and molecular characterization of different serotypes of Salmonella isolated from large-scale egg farms using drug susceptibility testing and whole genome sequencing. The synergistic effect of alpha-linolenic acid (ALA) with antibiotics was evaluated using the checkerboard test and time-kill curve. The molecular mechanism of α-linolenic acid synergism was explored using biochemical assays, pull-down assays, and molecular docking. In vivo efficacy of ALA in combination with florfenicol (FFC) or tetracycline (TET) against multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar typhimurium was also investigated using a mouse model. We found that ALA reduced the minimum inhibitory concentration (MIC) of tetracycline and florfenicol in all strains tested. When ALA (512 mg/L) was combined with florfenicol (32 mg/L) or tetracycline (16 mg/L), we observed disruption of cell membrane integrity, increased outer membrane permeability, lowered cell membrane potential, and inhibition of proton-drive-dependent efflux pumps. The synergistic treatment also inhibited biofilm production and promoted oxidative damage. These changes together led to an increase in bacterial antibiotic susceptibility. The improved efficacy of ALA combination treatment with antibiotics was validated in the mouse model. Molecular docking results indicate that ALA can bind to membrane proteins via hydrogen bonding. Our findings demonstrated that combined treatment using ALA and antibiotics is effective in preventing infections involving MDR bacteria. Our results are of great significance for the scientific and effective prevention and control of antibiotic resistance Salmonella, as well as ensuring food safety.

Sensitivity to antimicrobial peptide A (SapA) contributes to the survival of Salmonella typhimurium against antimicrobial peptides, neutrophils and virulence in mice.

Host-generated antimicrobial peptides (AMPs) play a pivotal role in defense against bacterial pathogens. AMPs kill invading bacteria majorly by disrupting the bacterial cell walls. AMPs are actively synthesized and released into the lumen of the gastrointestinal tract to limit colonization of enteric pathogens like Salmonella typhimurium (S. typhimurium). However, S. typhimurium has evolved several resistance mechanisms to defend AMPs. The multicomponent SapABCDF uptake transporter is one such system that helps in resisting AMPs. In the current study, we analyzed the role of S. typhimurium SapA against stress survival and virulence of this bacterium. ∆sapA mutant strain showed hypersensitivity to AMPs, like melittin and mastoparan. Further, ∆sapA mutant showed more than 22 folds (p = 0.019) hypersensitivity to neutrophils as compared to the WT strain of S. typhimurium. In addition, ∆sapA strain showed defective survival in mice. In conclusion, the results of the current study suggest that the SapA is essential for survival against AMPs and virulence of S. typhimurium.

Multiplex PCR-based genotyping of Salmonella Enteritidis and Salmonella Typhimurium from food sources and assessment of their antimicrobial resistance profiles in Egypt.

BACKGROUND: Salmonellosis is a widespread zoonotic disease that poses a significant threat to livestock and public health. This study aimed to serotype 20 Salmonella isolates obtained from sixty retail chicken meats, assess Salmonella contamination from eggs, and evaluate antibiotic resistance profiles. METHODS AND RESULTS: Twenty eggs were randomly collected in the new Borg El Arab market. Bacterial isolation was carried out utilizing both traditional culture, biochemical, and PCR methods. Among the twenty eggs analyzed, three (15%) tested positive for Salmonella, while the remaining seventeen (85%) were confirmed as negative. Genotyping through multiplex PCR revealed the presence of two S. Enteritidis and other serovar, with the use of three specific gene sets: a random sequence for Salmonella spp., sdfI gene for S. Enteritidis, and flagellin (fliC gene) for S. Typhimurium. Out of the 20 isolates obtained from chicken meat, five (25%) were identified as S. Typhimurium, and three (15%) were classified as S. Enteritidis. All isolates sourced from chicken meat exhibited resistance to Rifampicin and Amoxicillin, with 90% displaying sensitivity to cefotaxime, gemifloxacin, and Erythromycin. Importantly, S. Blegdam, identified via serological methods, displayed resistance to all tested antibiotics. For the three isolates obtained from eggs, 66.6% showed sensitivity to cefotaxime, erythromycin, cefuraxime, and cefaclor, while displaying complete resistance (100%) to Amoxicillin, rifampicin, clarithromycin, and cefadroxil. Notably, one serovar exhibited absolute resistance to all tested drugs. CONCLUSION: Stakeholders must implement strict control measures and rationalize antibiotic use in veterinary and human medicine due to the rise of antibiotic-resistant strains.

Species-specific activity of antibacterial drug combinations.

The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine1,2. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.

Reducing Ribosome Biosynthesis Promotes Translation during Low Mg2+ Stress.

The synthesis of ribosomes is regulated by both amino acid abundance and the availability of ATP, which regenerates guanosine triphosphate (GTP), powers ribosomes, and promotes transcription of rRNA genes. We now report that bacteria supersede both of these controls when experiencing low cytosolic magnesium (Mg2+), a divalent cation essential for ribosome stabilization and for neutralization of ATP's negative charge. We uncover a regulatory circuit that responds to low cytosolic Mg2+ by promoting expression of proteins that import Mg2+ and lower ATP amounts. This response reduces the levels of ATP and ribosomes, making Mg2+ ions available for translation. Mutants defective in Mg2+ uptake and unable to reduce ATP levels accumulate non-functional ribosomal components and undergo translational arrest. Our findings establish a paradigm whereby cells reduce the amounts of translating ribosomes to carry out protein synthesis.

Escaping the cohort of concern: in vitro experimental evidence supports non-mutagenicity of N-nitroso-hydrochlorothiazide.

In recent years, nitrosamine impurities in pharmaceuticals have been subject to intense regulatory scrutiny, with nitrosamine drug substance-related impurities (NDSRIs) treated as cohort of concern impurities, regardless of predicted mutagenic potential. Here, we describe a case study of the NDSRI N-nitroso-hydrochlorothiazide (NO-HCTZ), which was positive in the bacterial reverse mutation (Ames) test but is unstable under the test conditions, generating formaldehyde among other products. The mutagenic profile of NO-HCTZ was inconsistent with that expected of a mutagenic nitrosamine, exhibiting mutagenicity in the absence of metabolic activation, and instead aligned well with that of formaldehyde. To assess further, a modified Ames system including glutathione (3.3 mg/plate) to remove formaldehyde was developed. Strains used were S. typhimurium TA98, TA100, TA1535, and TA1537, and E. coli WP2 uvrA/pKM101. In this system, formaldehyde levels were considerably lower, with a concomitant increase in levels of S-(hydroxymethyl)glutathione (the adduct formed between glutathione and formaldehyde). Upon retesting NO-HCTZ in the modified system (1.6-5000 µg/plate), a clear decrease in the mutagenic response was observed in the strains in which NO-HCTZ was mutagenic in the original system (TA98, TA100, and WP2 uvrA/pKM101), indicating that formaldehyde drives the response, not NO-HCTZ. In strain TA1535, an increase in revertant colonies was observed in the modified system, likely due to a thiatriazine degradation product formed from NO-HCTZ under Ames test conditions. Overall, these data support a non-mutagenic designation for NO-HCTZ and demonstrate the value of further investigation when a positive Ames result does not align with the expected profile.

In Vivo and in Vitro Mitigation of Salmonella Typhimurium Isolates by Fortunella Japonica Fruit Extract.

The vast dissemination of resistance to different antibiotics among bacterial pathogens, especially foodborne pathogens, has drawn major research attention. Thus, many attempts have been made to reveal novel alternatives to the current antibiotics. Due to their variable pharmacologically active phytochemicals, plants represent a good solution for this issue. This study investigated the antibacterial potential of Kumquat or Fortunella japonica methanol extract (FJME) against Salmonella typhimurium clinical isolates. Gas chromatography coupled with mass spectrometry (GC/MS) characterized 39 compounds in FJME. Palmitic acid (15.386%) and cis-vaccenic acid (15.012%) are the major active constituents detected by GC/MS. Remarkably, FJME had minimum inhibitory concentrations from 128 to 512 µg/mL in vitro. In addition, a systemic infection model revealed the in vivo antibacterial action of FJME. The antibacterial therapeutic activity of FJME was noticed by improving the histological features of the liver and spleen. Moreover, there was a perceptible lessening (p < 0.05) of the levels of the oxidative stress markers (nitric oxide and malondialdehyde) using ELISA. In addition, the gene expression of the proinflammatory cytokine (interleukin 6) was downregulated. On the other hand, there was an upregulation of the anti-inflammatory cytokine (interleukin 10). Accordingly, future clinical investigations should be done to reveal the potential antibacterial action of FJME on other food pathogens.

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test.

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity. In addition, preincubations were conducted for both 60 min and 30 min. These test variables were evaluated by testing 12 small-molecule N-nitrosamines and 17 NDSRIs for mutagenicity in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA (pKM101). Eighteen of the 29 N-nitrosamine test substances tested positive under one or more of the testing conditions and all 18 positives could be detected by using tester strains TA1535 and WP2 uvrA (pKM101), preincubations of 30 min, and S9 mixes containing 30% hamster liver S9. In general, the conditions under which NDSRIs were mutagenic were similar to those found for small-molecule N-nitrosamines.

In vitro genotoxicological evaluation of protein-rich powder derived from Xanthobacter sp. SoF1.

One way of limiting the environmental impact of food production and improving food security is to replace part of the animal- or plant-based protein in the human diet with protein sourced from microorganisms. The recently discovered bacterium Xanthobacter sp. SoF1 (VTT-E-193585) grows autotrophically using carbon dioxide gas as the only carbon source, yielding protein-rich biomass that can be processed further into a powder and incorporated into various food products. Since the safety of this microbial protein powder for human consumption had not been previously assessed, its genotoxic potential was evaluated employing three internationally recognized and standardized studies: a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes. No biologically relevant evidence of genotoxicity or mutagenicity was found.