Lisa M Hodges, Ashley Cooper, Adam Koziol, Catherine D Carrillo
Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.
{"title":"Characterization of MLST-99 <i>Salmonella</i> Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.","authors":"Lisa M Hodges, Ashley Cooper, Adam Koziol, Catherine D Carrillo","doi":"10.1099/mic.0.001456","DOIUrl":"10.1099/mic.0.001456","url":null,"abstract":"<p><p><i>Salmonella enterica</i> subsp. <i>enterica</i> Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated <i>S</i>. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of <i>S</i>. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as <i>S</i>. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other <i>Salmonella</i> and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other <i>Salmonella</i> evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes <i>umuC</i> and <i>umuD</i>, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11256474/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140946285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Solenne Ithurbide, Roshali T de Silva, Hannah J Brown, Vinaya Shinde, Iain G Duggin
Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.
考古细胞生物学是一个新兴领域,有望确定基本的细胞过程,帮助解析细胞生命的深层进化史,并为生物技术和合成生物学贡献新的成分和功能。为了促进这些研究,我们开发了质粒载体,可以在模式古菌 Haloferax volcanii 中方便地克隆和生产带有柔性、刚性或半刚性连接体的蛋白质和融合蛋白。为了利用荧光蛋白(FP)标签进行蛋白质亚细胞定位研究,我们创建了包含一系列密码子优化荧光蛋白的载体,用于 N 端或 C 端标记,包括 GFP、mNeonGreen、mCherry、YPet、mTurquoise2 和 mScarlet-I。对于涉及多种相互作用的蛋白质来说,获得功能性融合蛋白是一项挑战,主要原因是立体干扰。我们展示了使用新载体系统筛选细胞骨架蛋白 FP 融合蛋白功能的改进,并鉴定出在细胞分裂中起作用的 FtsZ1-FPs 和在运动和杆状细胞发育中起作用的 CetZ1-FPs。连接体的类型和FP的类型都会影响融合产物的功能。这种载体设计还便于克隆和串联表达两个基因或融合基因,并由改良的色氨酸诱导启动子控制。这些工具将促进古生物分子和细胞生物学及生物技术的进一步发展和应用。
{"title":"A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in <i>Haloferax volcanii</i>.","authors":"Solenne Ithurbide, Roshali T de Silva, Hannah J Brown, Vinaya Shinde, Iain G Duggin","doi":"10.1099/mic.0.001461","DOIUrl":"10.1099/mic.0.001461","url":null,"abstract":"<p><p>Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon <i>Haloferax volcanii</i>. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in <i>H. volcanii</i> cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11165654/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141087743","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Katarzyna Okurowska, Peter N Monk, Esther Karunakaran
Introduction. Bacterial keratitis, particularly caused by Pseudomonas aeruginosa, is challenging to treat because of multi-drug tolerance, often associated with the formation of biofilms. Antibiotics in development are typically evaluated against planktonic bacteria in a culture medium, which may not accurately represent the complexity of infections in vivo.Hypothesis/Gap Statement. Developing a reliable, economic ex vivo keratitis model that replicates some complexity of tissue infections could facilitate a deeper understanding of antibiotic efficacy, thus aiding in the optimization of treatment strategies for bacterial keratitis.Methodology. Here we investigated the efficacy of three commonly used antibiotics (gentamicin, ciprofloxacin and meropenem) against Pseudomonas aeruginosa cytotoxic strain PA14 and invasive strain PA01 using an ex vivo porcine keratitis model.Results. Both strains of P. aeruginosa were susceptible to the MIC of the three tested antibiotics. However, significantly higher concentrations were necessary to inhibit bacterial growth in the minimum biofilm eradication concentration (MBEC) assay, with both strains tolerating concentrations greater than 512 mg l-1 of meropenem. When MIC and higher concentrations than MBEC (1024 mg l-1) of antibiotics were applied, ciprofloxacin exhibited the highest potency against both P. aeruginosa strains, followed by meropenem, while gentamicin showed the least potency. Despite this, none of the antibiotic concentrations used effectively cleared the infection, even after 18 h of continuous exposure.Conclusions. Further exploration of antibiotic concentrations and aligning dosing with clinical studies to validate the model is needed. Nonetheless, our ex vivo porcine keratitis model could be a valuable tool for assessing antibiotic efficacy.
导言。细菌性角膜炎,尤其是由铜绿假单胞菌引起的细菌性角膜炎,由于对多种药物的耐受性,往往与生物膜的形成有关,因此治疗具有挑战性。开发中的抗生素通常是针对培养基中的浮游细菌进行评估,这可能无法准确反映体内感染的复杂性。开发一种可靠、经济的体外角膜炎模型,复制组织感染的某些复杂性,有助于深入了解抗生素的疗效,从而帮助优化细菌性角膜炎的治疗策略。在此,我们利用猪角膜炎体外模型研究了三种常用抗生素(庆大霉素、环丙沙星和美罗培南)对铜绿假单胞菌细胞毒性菌株 PA14 和侵袭性菌株 PA01 的疗效。两株铜绿假单胞菌对三种测试抗生素的 MIC 均敏感。然而,在最小生物膜根除浓度(MBEC)试验中,抑制细菌生长所需的浓度明显更高,两株菌株都能耐受浓度高于 512 毫克升/升的美罗培南。当抗生素的 MIC 和浓度高于 MBEC(1024 毫克/升)时,环丙沙星对两种铜绿假单胞菌菌株的效力最高,其次是美罗培南,而庆大霉素的效力最低。尽管如此,所使用的抗生素浓度都不能有效清除感染,即使在连续接触 18 小时后也是如此。需要进一步探讨抗生素浓度,并根据临床研究调整剂量,以验证模型。尽管如此,我们的体外猪角膜炎模型仍可作为评估抗生素疗效的重要工具。
{"title":"Increased tolerance to commonly used antibiotics in a <i>Pseudomonas aeruginosa ex vivo</i> porcine keratitis model.","authors":"Katarzyna Okurowska, Peter N Monk, Esther Karunakaran","doi":"10.1099/mic.0.001459","DOIUrl":"10.1099/mic.0.001459","url":null,"abstract":"<p><p><b>Introduction</b>. Bacterial keratitis, particularly caused by <i>Pseudomonas aeruginosa</i>, is challenging to treat because of multi-drug tolerance, often associated with the formation of biofilms. Antibiotics in development are typically evaluated against planktonic bacteria in a culture medium, which may not accurately represent the complexity of infections <i>in vivo</i>.<b>Hypothesis/Gap Statement.</b> Developing a reliable, economic <i>ex vivo</i> keratitis model that replicates some complexity of tissue infections could facilitate a deeper understanding of antibiotic efficacy, thus aiding in the optimization of treatment strategies for bacterial keratitis.<b>Methodology</b>. Here we investigated the efficacy of three commonly used antibiotics (gentamicin, ciprofloxacin and meropenem) against <i>Pseudomonas aeruginosa</i> cytotoxic strain PA14 and invasive strain PA01 using an <i>ex vivo</i> porcine keratitis model.<b>Results</b>. Both strains of <i>P. aeruginosa</i> were susceptible to the MIC of the three tested antibiotics. However, significantly higher concentrations were necessary to inhibit bacterial growth in the minimum biofilm eradication concentration (MBEC) assay, with both strains tolerating concentrations greater than 512 mg l<sup>-1</sup> of meropenem. When MIC and higher concentrations than MBEC (1024 mg l<sup>-1</sup>) of antibiotics were applied, ciprofloxacin exhibited the highest potency against both <i>P. aeruginosa</i> strains, followed by meropenem, while gentamicin showed the least potency. Despite this, none of the antibiotic concentrations used effectively cleared the infection, even after 18 h of continuous exposure.<b>Conclusions.</b> Further exploration of antibiotic concentrations and aligning dosing with clinical studies to validate the model is needed. Nonetheless, our <i>ex vivo</i> porcine keratitis model could be a valuable tool for assessing antibiotic efficacy.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11165664/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140913218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Response to comments on the tolerance to <i>Clostridioides difficile</i> spores to sodium hypochlorite disinfection.","authors":"Humaira Ahmed, Lovleen Tina Joshi","doi":"10.1099/mic.0.001463","DOIUrl":"10.1099/mic.0.001463","url":null,"abstract":"","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11165626/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141072139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mycobacterium tuberculosis (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.
{"title":"Targeting <i>Mycobacterium tuberculosis</i> pH-driven adaptation.","authors":"Shelby J Dechow, Robert B Abramovitch","doi":"10.1099/mic.0.001458","DOIUrl":"10.1099/mic.0.001458","url":null,"abstract":"<p><p><i>Mycobacterium tuberculosis</i> (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11165653/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140877820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jennifer L Cadnum, Claire E Kaple, William A Rutala, Curtis J Donskey
{"title":"Comment on the effectiveness of sodium hypochlorite against <i>Clostridioides difficile</i> spores.","authors":"Jennifer L Cadnum, Claire E Kaple, William A Rutala, Curtis J Donskey","doi":"10.1099/mic.0.001436","DOIUrl":"10.1099/mic.0.001436","url":null,"abstract":"","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11165594/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141072129","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
J W Decousser, F Barbut, R Baron, P Parneix, T Lavigne, S Romano-Bertrand, For The Scientific Committee Of The French Society For Hospital Hygiene And The National Reference Laboratory For
{"title":"Comments on the tolerance of <i>Clostridioides difficile</i> spores to disinfection with sodium hypochlorite disinfectant.","authors":"J W Decousser, F Barbut, R Baron, P Parneix, T Lavigne, S Romano-Bertrand, For The Scientific Committee Of The French Society For Hospital Hygiene And The National Reference Laboratory For","doi":"10.1099/mic.0.001435","DOIUrl":"10.1099/mic.0.001435","url":null,"abstract":"","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11165617/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141072135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Marco Gontijo, Mateus Pereira Teles, Hugo Martins Correia, Genesy Pérez Jorge, Isabella Carolina Rodrigues Santos Goes, Anthony Jhoao Fasabi Flores, Márcia Braz, Lucas de Moraes Ceseti, Priscila Zonzini Ramos, Ivan Rosa E Silva, Pedro Marcus Pereira Vidigal, Jörg Kobarg, Rafael Miguez Couñago, Cristina Elisa Alvarez-Martinez, Carla Pereira, Carmen S R Freire, Adelaide Almeida, Marcelo Brocchi
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.
{"title":"Combined effect of SAR-endolysin LysKpV475 with polymyxin B and <i>Salmonella</i> bacteriophage phSE-5.","authors":"Marco Gontijo, Mateus Pereira Teles, Hugo Martins Correia, Genesy Pérez Jorge, Isabella Carolina Rodrigues Santos Goes, Anthony Jhoao Fasabi Flores, Márcia Braz, Lucas de Moraes Ceseti, Priscila Zonzini Ramos, Ivan Rosa E Silva, Pedro Marcus Pereira Vidigal, Jörg Kobarg, Rafael Miguez Couñago, Cristina Elisa Alvarez-Martinez, Carla Pereira, Carmen S R Freire, Adelaide Almeida, Marcelo Brocchi","doi":"10.1099/mic.0.001462","DOIUrl":"10.1099/mic.0.001462","url":null,"abstract":"<p><p>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 <i>in vitro</i> and <i>in vivo</i> 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 <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 μg ml<sup>-1</sup> against <i>Pseudomonas aeruginosa</i> ATCC 27853, 16.25 μg ml<sup>-1</sup> against <i>S</i>. <i>enterica</i> Typhimurium ATCC 13311, and 32.50 μg ml<sup>-1</sup> against <i>Klebsiella pneumoniae</i> ATCC BAA-2146 and <i>Enterobacter cloacae</i> P2224. LysKpV475 showed bactericidal activity only for <i>P. aeruginosa</i> ATCC 27853 (32.50 μg ml<sup>-1</sup>) and <i>P. aeruginosa</i> P2307 (65.00 μg ml<sup>-1</sup>) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against <i>K. pneumoniae</i> ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and <i>S</i>. <i>enterica</i> Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against <i>S</i>. <i>enterica</i> 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 S<i>almonella</i> 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.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11170124/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140913205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation of ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.
{"title":"A historical sequence deletion in a commonly used <i>Bacillus subtilis</i> chromosome integration vector generates undetected loss-of-function mutations.","authors":"K Julia Dierksheide, Gene-Wei Li","doi":"10.1099/mic.0.001455","DOIUrl":"10.1099/mic.0.001455","url":null,"abstract":"<p><p>Since the 1980s, chromosome-integration vectors have been used as a core method of engineering <i>Bacillus subtilis</i>. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the <i>amyE</i> locus. Here, we report a gap in the homology region inherited from the original <i>amyE</i> integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of <i>amyE</i> and the downstream gene <i>ldh</i>, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation of <i>ldh</i>, which encodes lactate dehydrogenase. Although both types of colonies test positive for <i>amyE</i> disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11084560/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140873093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Danna R Gifford, Anish Bhattacharyya, Alexandra Geim, Eleanor Marshall, Rok Krašovec, Christopher G Knight
Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterized the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a ΔluxS deletant strain - a gene with roles in both quorum sensing and the recycling of methylation products used in E. coli's DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive ΔluxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.
{"title":"Environmental and genetic influence on the rate and spectrum of spontaneous mutations in <i>Escherichia coli</i>.","authors":"Danna R Gifford, Anish Bhattacharyya, Alexandra Geim, Eleanor Marshall, Rok Krašovec, Christopher G Knight","doi":"10.1099/mic.0.001452","DOIUrl":"10.1099/mic.0.001452","url":null,"abstract":"<p><p>Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterized the spectrum of spontaneous mutations in <i>Escherichia coli</i> grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a Δ<i>luxS</i> deletant strain - a gene with roles in both quorum sensing and the recycling of methylation products used in <i>E. coli</i>'s DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive Δ<i>luxS</i> deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11084559/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140868621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}