Zoonotic Microorganisms and Spread of Acquired Polymyxin Resistance Determinants

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Alberto Quesada
Faculty of Veterinary Medicine, University of Extremadura
Maria Jorge Campos
Polytechnic Institute of Leiria
Azucena Mora Gutiérrez
University of Santiago de Compostela
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hemotherapy"},"guid":624194,"images":[{"height":400,"url":"https://www.frontiersin.org/files/myhome article library/624194/624194_Thumb_400.jpg","width":342,"caption":null},{"height":1582,"url":"https://www.frontiersin.org/files/Articles/624194/fmicb-12-624194-HTML/image_m/fmicb-12-624194-g001.jpg","width":1350,"caption":"Map of the districts where sampling was performed."},{"height":3756,"url":"https://www.frontiersin.org/files/Articles/624194/fmicb-12-624194-HTML/image_m/fmicb-12-624194-g002.jpg","width":2917,"caption":"Maximum-likelihood phylogeny of E. coli isolates from poultry. The phylogenetic tree was constructed based on nucleotide sequence alignments of the core genes. Metadata columns include farm district, STs, and presence/absence of the mcr-1 gene and resistance genes."}],"journal":{"guid":310,"name":"Frontiers in Microbiology","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fmicb.2021.624194","pubDate":"2021-03-08","score":52.64340461571051,"title":"WGS Analysis of Clonal and Plasmidic Epidemiology of Colistin-Resistance Mediated by mcr Genes in the Poultry Sector in Lebanon","topics":["Lebanon","Poultry","IncX4","MCR-1","IncI2"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fmicb.2021.624194/pdf"},{"__typename":"Feed_Article","_id":"6809bb5d968ae81840c99b48","abstract":"Background: The resistance to colistin and carbapenems in Klebsiella pneumoniae infections have been associated with increased morbidity and mortality worldwide. A retrospective observational study was conducted to determine the prevalence and molecular events contributing to colistin resistance.Methods: Clinical samples were screened for colistin resistance and underlying mechanisms were studied by PCR-based amplification and sequence analysis of genes of two-component regulatory system (phoPQ and pmrAB), regulatory transmembrane protein-coding mgrB, and mobilized colistin resistance genes (mcr-1-8). Gene expression of pmrC and pmrK was analyzed by qRT-PCR, and the genetic relationship was assessed by MLST. The putative effect of amino-acid substitutions was predicted by a combination of bioinformatics tools.Results: Of 335 Klebsiella spp. screened, 11 (3.2%) were identified as colistin-resistant (MIC range, 8 to \u003e128 μg/ml). K. pneumoniae isolates belonged to clonal complex-11 (CC11) with sequence types (STs): 14, 16, 43, 54, 147 and 395, whereby four isolates conferred three novel STs (3986, 3987 and 3988) profiles. Sequence analysis revealed non-synonymous potentially deleterious mutations in phoP (T151A), phoQ (del87–90, del263–264, L30Q, and A351D), pmrA (G53S), pmrB (D150V, T157P, L237R, G250C, A252G, R315P, and Q331H), and mgrB (C28G) genes. The mgrB gene in three strains was disrupted by insertion sequences encoding IS1-like and IS5/IS1182 family-like transposase genes. All 11 isolates showed an elevation in the transcription level of pmrC gene. Mobilized colistin-resistance (mcr) genes were not detected. All but one of the colistin-resistant isolates was also resistant to carbapenems; β-lactamase genes blaNDM-1-like, blaOXA-48-like, and blaCTX-M-like were detected in eight, five, and nine isolates, respectively.Conclusion: All the studied colistin- and carbapenem-resistant K. pneumoniae isolates were genetically distinct, and various mechanisms of colistin resistance were detected, indicating its spontaneous emergence in this bacterial species.","htmlAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The resistance to colistin and carbapenems in \u003ci\u003eKlebsiella pneumoniae\u003c/i\u003e infections have been associated with increased morbidity and mortality worldwide. A retrospective observational study was conducted to determine the prevalence and molecular events contributing to colistin resistance.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Clinical samples were screened for colistin resistance and underlying mechanisms were studied by PCR-based amplification and sequence analysis of genes of two-component regulatory system (\u003ci\u003ephoPQ\u003c/i\u003e and \u003ci\u003epmrAB\u003c/i\u003e), regulatory transmembrane protein-coding \u003ci\u003emgrB\u003c/i\u003e, and mobilized colistin resistance genes (\u003ci\u003emcr-1-8\u003c/i\u003e). Gene expression of \u003ci\u003epmrC\u003c/i\u003e and \u003ci\u003epmrK\u003c/i\u003e was analyzed by qRT-PCR, and the genetic relationship was assessed by MLST. The putative effect of amino-acid substitutions was predicted by a combination of bioinformatics tools.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Of 335 \u003ci\u003eKlebsiella\u003c/i\u003e spp. screened, 11 (3.2%) were identified as colistin-resistant (MIC range, 8 to \u0026gt;128 μg/ml). \u003ci\u003eK. pneumoniae\u003c/i\u003e isolates belonged to clonal complex-11 (CC11) with sequence types (STs): 14, 16, 43, 54, 147 and 395, whereby four isolates conferred three novel STs (3986, 3987 and 3988) profiles. Sequence analysis revealed non-synonymous potentially deleterious mutations in \u003ci\u003ephoP\u003c/i\u003e (T151A), \u003ci\u003ephoQ\u003c/i\u003e (del87–90, del263–264, L30Q, and A351D), \u003ci\u003epmrA\u003c/i\u003e (G53S), \u003ci\u003epmrB\u003c/i\u003e (D150V, T157P, L237R, G250C, A252G, R315P, and Q331H), and \u003ci\u003emgrB\u003c/i\u003e (C28G) genes. The \u003ci\u003emgrB\u003c/i\u003e gene in three strains was disrupted by insertion sequences encoding IS\u003ci\u003e1\u003c/i\u003e-like and IS\u003ci\u003e5\u003c/i\u003e/IS\u003ci\u003e1182\u003c/i\u003e family-like transposase genes. All 11 isolates showed an elevation in the transcription level of \u003ci\u003epmrC\u003c/i\u003e gene. Mobilized colistin-resistance (\u003ci\u003emcr\u003c/i\u003e) genes were not detected. All but one of the colistin-resistant isolates was also resistant to carbapenems; β-lactamase genes \u003ci\u003ebla\u003csub\u003eNDM-1-like\u003c/sub\u003e\u003c/i\u003e, \u003ci\u003ebla\u003csub\u003eOXA-48-like\u003c/sub\u003e\u003c/i\u003e, and \u003ci\u003ebla\u003csub\u003eCTX-M-like\u003c/sub\u003e\u003c/i\u003e were detected in eight, five, and nine isolates, respectively.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e All the studied colistin- and carbapenem-resistant \u003ci\u003eK. pneumoniae\u003c/i\u003e isolates were genetically distinct, and various mechanisms of colistin resistance were detected, indicating its spontaneous emergence in this bacterial species.\u003c/p\u003e","authors":[{"fullName":"Mudsser Azam","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/283340/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/283340/overview","affiliation":{"name":"ICMR-National Institute of Pathology","address":null},"affiliations":[{"name":"ICMR-National Institute of Pathology","address":null}],"nessieId":"352187964625"},{"fullName":"Rajni Gaind","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1213643/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1213643/overview","affiliation":{"name":"Department of Microbiology, VMMC and Safdarjung Hospital","address":null},"affiliations":[{"name":"Department of Microbiology, VMMC and Safdarjung Hospital","address":null}],"nessieId":"231928882755"},{"fullName":"Gulshan Yadav","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1134467/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1134467/overview","affiliation":{"name":"ICMR-National Institute of Pathology","address":null},"affiliations":[{"name":"ICMR-National Institute of Pathology","address":null}],"nessieId":"257698687802"},{"fullName":"Amit Sharma","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1155166/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1155166/overview","affiliation":{"name":"Department of Microbiology, VMMC and Safdarjung Hospital","address":null},"affiliations":[{"name":"Department of Microbiology, VMMC and Safdarjung Hospital","address":null}],"nessieId":"257698561683"},{"fullName":"Kirti Upmanyu","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1138858/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1138858/overview","affiliation":{"name":"ICMR-National Institute of Pathology","address":null},"affiliations":[{"name":"ICMR-National Institute of Pathology","address":null}],"nessieId":"85899994952"},{"fullName":"Manisha Jain","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1226609/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1226609/overview","affiliation":{"name":"Department of Microbiology, VMMC and Safdarjung Hospital","address":null},"affiliations":[{"name":"Department of Microbiology, VMMC and Safdarjung Hospital","address":null}],"nessieId":"111669672667"},{"fullName":"Ruchi Singh","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/119053/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/119053/overview","affiliation":{"name":"ICMR-National Institute of Pathology","address":null},"affiliations":[{"name":"ICMR-National Institute of Pathology","address":null}],"nessieId":"661425612773"}],"dates":{"acceptedDate":"2021-01-22","recentDate":"2021-02-22"},"doi":"10.3389/fmicb.2021.609840","frontiersExtra":{"articleType":"Original Research","impact":{"citations":47,"crossrefCitations":0,"downloads":387,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":10285},"isPartOfResearchTopic":true,"isPublished":true,"section":"Antimicrobials, Resistance and Chemotherapy"},"guid":609840,"images":[{"height":188,"url":"https://www.frontiersin.org/files/myhome article library/609840/609840_Thumb_400.jpg","width":400,"caption":null},{"height":953,"url":"https://www.frontiersin.org/files/Articles/609840/fmicb-12-609840-HTML/image_m/fmicb-12-609840-g001.jpg","width":2032,"caption":"Clonal complex CC11 with 1,562 sequence types. Green halos represents novel sequence types found in this study and submitted to curator of Klebsiella pneumoniae MLST database Pasteur institute (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html), pink halos represent isolates with defined sequence types. K. pneumoniae ATCC 700603 is ST489 and designated as singleton by eBURST v3 and hence not shown here."},{"height":792,"url":"https://www.frontiersin.org/files/Articles/609840/fmicb-12-609840-HTML/image_m/fmicb-12-609840-g002.jpg","width":2033,"caption":"Fold changes ±SD in the expression of pmrC and pmrK genes among colistin-resistant K. pneumoniae isolates (n = 11) with respect to colistin-susceptible K. pneumoniae ATCC 700603 isolate are represented here. The rpsl gene was used as internal control. All the reactions (both under colistin treated and untreated conditions) were normalized using colistin-susceptible K. pneumoniae ATCC 700603 (colistin untreated- wild type). (A) relative fold-change of pmrC gene expression under colistin treated (pmrC T) and untreated conditions (pmrC UT); (B) relative fold-change of pmrK gene expression under colistin treated (pmrK T) and untreated conditions (pmrK UT). Values given are mean ± SD of three different experiments with qRT-PCR reactions performed in triplicate."}],"journal":{"guid":310,"name":"Frontiers in Microbiology","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fmicb.2021.609840","pubDate":"2021-02-22","score":92.95502858352775,"title":"Colistin Resistance Among Multiple Sequence Types of Klebsiella pneumoniae Is Associated With Diverse Resistance Mechanisms: A Report From India","topics":["Klebsiella pneumoniae","PhoPQ","PmrAB","Carbapenem resistance","Colistin resistance"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fmicb.2021.609840/pdf"},{"__typename":"Feed_Article","_id":"6809bb5d968ae81840c99b55","abstract":"ObjectivesThe performance of mainstream commercial antimicrobial susceptibility testing systems on polymyxins has not been well evaluated in China. In this study, three antimicrobial susceptibility testing systems were evaluated for polymyxin B and colistin.\u003c/sec\u003eMethodsThe MICs of 257 Gram-negative strains collected from clinical cases and livestock were determined and analyzed. Using Broth Microdilution as the gold standard, the performance of VITEK 2® COMPACT, PhoenixTM M50, and Bio-kont AST System were evaluated. Essential agreement (EA), category agreement (CA), very major error (VME), and major error (ME) were calculated for comparison. The results of mcr-1 positive strains were separately discussed.\u003c/sec\u003eResultsThe EA, CA, VME, and ME to polymyxin B for Bio-kont were 83.5, 95.6, 13.1, and 0.6%, respectively. The EAs, CAs, VMEs, and MEs to colistin were as follows: Bio-kont, 86.7%/96.5%/7.2%/1.7%; Vitek 2, 64.2%/86.8%/41.0%/0%, and Phoenix M50, 92.9%/92.9%/21.7%/0%. The performance of Bio-kont to polymyxin B and colistin for Pseudomonas spp. (EA, CA \u003c 90%, VME \u003e 1.5%, ME = 5.6%/10%) and Enterobacter spp. (EA, CA \u003c 90%, VME \u003e 1.5% and ME = 0%), Vitek to colistin for most genera, and Phoenix to colistin for Enterobacter spp. (EA, CA \u003c 90%, VME \u003e 1.5%, ME = 0%) were unsatisfactory compared with other genera. The performance of Bio-kont to polymyxins for Escherichia spp. and Phoenix to colistin for Citrobacter spp., Escherichia spp., and Klebsiella spp., which all met the CLSI standard, were satisfactory. When the susceptibility of mcr-1 positive E. coli was tested, Bio-kont and Phoenix M50 presented excellent performance with no category errors, while Vitek 2 performed a high VME (25.5%).\u003c/sec\u003eConclusionWith relatively more accurate results for polymyxin B and colistin and lower VME, Bio-kont has an advantage in polymyxin antimicrobial susceptibility testing, especially for Escherichia spp., Klebsiella spp., Citrobacter spp. and Acinetobacter spp.\u003c/sec\u003e","htmlAbstract":"\u003cp\u003e\u003cb\u003eObjectives:\u003c/b\u003e The performance of mainstream commercial antimicrobial susceptibility testing systems on polymyxins has not been well evaluated in China. In this study, three antimicrobial susceptibility testing systems were evaluated for polymyxin B and colistin.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods:\u003c/b\u003e The MICs of 257 Gram-negative strains collected from clinical cases and livestock were determined and analyzed. Using Broth Microdilution as the gold standard, the performance of VITEK 2\u003csup\u003e®\u003c/sup\u003e COMPACT, Phoenix\u003csup\u003eTM\u003c/sup\u003e M50, and Bio-kont AST System were evaluated. Essential agreement (EA), category agreement (CA), very major error (VME), and major error (ME) were calculated for comparison. The results of \u003ci\u003emcr-1\u003c/i\u003e positive strains were separately discussed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults:\u003c/b\u003e The EA, CA, VME, and ME to polymyxin B for Bio-kont were 83.5, 95.6, 13.1, and 0.6%, respectively. The EAs, CAs, VMEs, and MEs to colistin were as follows: Bio-kont, 86.7%/96.5%/7.2%/1.7%; Vitek 2, 64.2%/86.8%/41.0%/0%, and Phoenix M50, 92.9%/92.9%/21.7%/0%. The performance of Bio-kont to polymyxin B and colistin for \u003ci\u003ePseudomonas\u003c/i\u003e spp. (EA, CA \u0026lt; 90%, VME \u0026gt; 1.5%, ME = 5.6%/10%) and \u003ci\u003eEnterobacter\u003c/i\u003e spp. (EA, CA \u0026lt; 90%, VME \u0026gt; 1.5% and ME = 0%), Vitek to colistin for most genera, and Phoenix to colistin for \u003ci\u003eEnterobacter\u003c/i\u003e spp. (EA, CA \u0026lt; 90%, VME \u0026gt; 1.5%, ME = 0%) were unsatisfactory compared with other genera. The performance of Bio-kont to polymyxins for \u003ci\u003eEscherichia\u003c/i\u003e spp. and Phoenix to colistin for \u003ci\u003eCitrobacter\u003c/i\u003e spp., \u003ci\u003eEscherichia\u003c/i\u003e spp., and \u003ci\u003eKlebsiella\u003c/i\u003e spp., which all met the CLSI standard, were satisfactory. When the susceptibility of \u003ci\u003emcr-1\u003c/i\u003e positive \u003ci\u003eE. coli\u003c/i\u003e was tested, Bio-kont and Phoenix M50 presented excellent performance with no category errors, while Vitek 2 performed a high VME (25.5%).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion:\u003c/b\u003e With relatively more accurate results for polymyxin B and colistin and lower VME, Bio-kont has an advantage in polymyxin antimicrobial susceptibility testing, especially for \u003ci\u003eEscherichia\u003c/i\u003e spp., \u003ci\u003eKlebsiella\u003c/i\u003e spp., \u003ci\u003eCitrobacter\u003c/i\u003e spp. and \u003ci\u003eAcinetobacter\u003c/i\u003e spp.\u003c/p\u003e","authors":[{"fullName":"Ying Zhu","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1094581/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1094581/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},{"name":"Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences","address":null}],"nessieId":"412317510079"},{"fullName":"Peiyao Jia","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/919226/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/919226/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},{"name":"Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences","address":null}],"nessieId":"111669815672"},{"fullName":"Menglan Zhou","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/349653/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/349653/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":"180389267721"},{"fullName":"Jingjia Zhang","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/858447/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/858447/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":"17180518917"},{"fullName":"Ge Zhang","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1055850/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1055850/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":"34360387799"},{"fullName":"Wei Kang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":null},{"fullName":"Simeng Duan","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1055860/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1055860/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":"884763878724"},{"fullName":"Tong Wang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":null},{"fullName":"Yingchun Xu","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/429471/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/429471/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":"8590585355"},{"fullName":"Qiwen Yang","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/370084/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/370084/overview","affiliation":{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null},"affiliations":[{"name":"Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","address":null}],"nessieId":"60130204545"}],"dates":{"acceptedDate":"2020-12-15","recentDate":"2021-02-22"},"doi":"10.3389/fmicb.2020.610604","frontiersExtra":{"articleType":"Methods","impact":{"citations":6,"crossrefCitations":0,"downloads":1,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":4582},"isPartOfResearchTopic":true,"isPublished":true,"section":"Antimicrobials, Resistance and Chemotherapy"},"guid":610604,"images":[{"height":219,"url":"https://www.frontiersin.org/files/myhome article library/610604/610604_Thumb_400.jpg","width":400,"caption":null},{"height":1065,"url":"https://www.frontiersin.org/files/Articles/610604/fmicb-11-610604-HTML/image_m/fmicb-11-610604-g001.jpg","width":1950,"caption":null}],"journal":{"guid":310,"name":"Frontiers in Microbiology","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.610604","pubDate":"2021-02-22","score":26.540165149269683,"title":"Evaluation of the Clinical Systems for Polymyxin Susceptibility Testing of Clinical Gram-Negative Bacteria in China","topics":["polymyxin","Antimicrobial susceptibility testing","major error","very major error","Essential agreement","category agreement"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.610604/pdf"},{"__typename":"Feed_Article","_id":"6809bb5d968ae81840c99b49","abstract":"The DedA family is a conserved membrane protein family found in most organisms. A Burkholderia thailandensis DedA family protein, named DbcA, is required for high-level colistin (polymyxin E) resistance, but the mechanism awaits elucidation. Modification of lipopolysaccharide lipid A with the cationic sugar aminoarabinose (Ara4N) is required for colistin resistance and is dependent upon protonmotive force (PMF) dependent transporters. B. thailandensis ΔdbcA lipid A contains only small amounts of Ara4N, likely leading to colistin sensitivity. Two B. thailandensis operons are required for lipid A modification with Ara4N, one needed for biosynthesis of undecaprenyl-P-Ara4N and one for transport of the lipid linked sugar and subsequent lipid A modification. Here, we directed overexpression of each arn operon by genomic insertion of inducible promoters. We found that overexpression of arn operons in ΔdbcA can partially, but not completely, restore Ara4N modification of lipid A and colistin resistance. Artificially increasing the PMF by lowering the pH of the growth media also increased membrane potential, amounts of Ara4N, and colistin resistance of ΔdbcA. In addition, the products of arn operons are essential for acid tolerance, suggesting a physiological function of Ara4N modification. Finally, we show that ΔdbcA is sensitive to bacitracin and expression of a B. thailandensis UppP/BacA homolog (BTH_I1512) can partially restore resistance to bacitracin. Expression of a different UppP/BacA homolog (BTH_I2750) can partially restore colistin resistance, without changing the lipid A profile. This work suggests that maintaining optimal membrane potential at slightly alkaline pH media by DbcA is responsible for proper modification of lipid A by Ara4N and provides evidence of lipid A modification-dependent and -independent mechanisms of colistin resistance in B. thailandensis.","htmlAbstract":"\u003cp\u003eThe DedA family is a conserved membrane protein family found in most organisms. A \u003ci\u003eBurkholderia thailandensis\u003c/i\u003e DedA family protein, named DbcA, is required for high-level colistin (polymyxin E) resistance, but the mechanism awaits elucidation. Modification of lipopolysaccharide lipid A with the cationic sugar aminoarabinose (Ara4N) is required for colistin resistance and is dependent upon protonmotive force (PMF) dependent transporters. \u003ci\u003eB. thailandensis\u003c/i\u003e Δ\u003ci\u003edbcA\u003c/i\u003e lipid A contains only small amounts of Ara4N, likely leading to colistin sensitivity. Two \u003ci\u003eB. thailandensis\u003c/i\u003e operons are required for lipid A modification with Ara4N, one needed for biosynthesis of undecaprenyl-P-Ara4N and one for transport of the lipid linked sugar and subsequent lipid A modification. Here, we directed overexpression of each \u003ci\u003earn\u003c/i\u003e operon by genomic insertion of inducible promoters. We found that overexpression of \u003ci\u003earn\u003c/i\u003e operons in Δ\u003ci\u003edbcA\u003c/i\u003e can partially, but not completely, restore Ara4N modification of lipid A and colistin resistance. Artificially increasing the PMF by lowering the pH of the growth media also increased membrane potential, amounts of Ara4N, and colistin resistance of Δ\u003ci\u003edbcA\u003c/i\u003e. In addition, the products of \u003ci\u003earn\u003c/i\u003e operons are essential for acid tolerance, suggesting a physiological function of Ara4N modification. Finally, we show that Δ\u003ci\u003edbcA\u003c/i\u003e is sensitive to bacitracin and expression of a \u003ci\u003eB. thailandensis\u003c/i\u003e UppP/BacA homolog (\u003ci\u003eBTH_I1512)\u003c/i\u003e can partially restore resistance to bacitracin. Expression of a different UppP/BacA homolog (\u003ci\u003eBTH_I2750)\u003c/i\u003e can partially restore colistin resistance, without changing the lipid A profile. This work suggests that maintaining optimal membrane potential at slightly alkaline pH media by DbcA is responsible for proper modification of lipid A by Ara4N and provides evidence of lipid A modification-dependent and -independent mechanisms of colistin resistance in \u003ci\u003eB. thailandensis.\u003c/i\u003e\u003c/p\u003e","authors":[{"fullName":"Pradip R. Panta","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1005955/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1005955/overview","affiliation":{"name":"Department of Biological Sciences, Louisiana State University","address":null},"affiliations":[{"name":"Department of Biological Sciences, Louisiana State University","address":null}],"nessieId":"653252"},{"fullName":"William T. Doerrler","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/38987/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/38987/overview","affiliation":{"name":"Department of Biological Sciences, Louisiana State University","address":null},"affiliations":[{"name":"Department of Biological Sciences, Louisiana State University","address":null}],"nessieId":"223338946986"}],"dates":{"acceptedDate":"2020-12-17","recentDate":"2021-01-12"},"doi":"10.3389/fmicb.2020.618389","frontiersExtra":{"articleType":"Original Research","impact":{"citations":21,"crossrefCitations":0,"downloads":2,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":4068},"isPartOfResearchTopic":true,"isPublished":true,"section":"Antimicrobials, Resistance and Chemotherapy"},"guid":618389,"images":[{"height":370,"url":"https://www.frontiersin.org/files/myhome article library/618389/618389_Thumb_400.jpg","width":400,"caption":null},{"height":1576,"url":"https://www.frontiersin.org/files/Articles/618389/fmicb-11-618389-HTML/image_m/fmicb-11-618389-g001.jpg","width":1707,"caption":"Generation and characterization of Ara4N synthesis and transport conditional mutants. (A) Genetic organization of arn gene clusters and flanking regions of B. thailandensis E264. The gray box represents the plasmid, pSC200 with an inducible rhamnose promoter (PrhaB). E264B/ΔdbcAB (B for biosynthesis) represents a strain of E264 or ΔdbcA:FRT with pSC200 insertion in front of the putative arn biosynthesis transcriptional unit. E264T/ΔdbcAT (T for transport) represents a strain of E264 or ΔdbcA:FRT with pSC200 insertion in front of the putative arn transport transcriptional unit. (B) Growth rate of E264; vec, ΔdbcA; vec, E264B, E264T, ΔdbcAB, and ΔdbcAT at times 0, 4, 8, 12, and 24 h in LB liquid media containing the indicated concentrations of rhamnose. The graph was created through GraphPad Prism 8.4.3. The error bars indicate standard deviations of three biological replicates. (C) Spot assay of E264; vec, ΔdbcA; vec, E264B, E264T, ΔdbcAB, and ΔdbcAT on LB agar media. 1:10 dilutions of indicated strains from 2nd overnight culture grown without any rhamnose were spotted on LB, 100 Tmp, and different amounts of rhamnose as indicated. Plates were analyzed after 24 h of growth at 37°C."},{"height":1189,"url":"https://www.frontiersin.org/files/Articles/618389/fmicb-11-618389-HTML/image_m/fmicb-11-618389-g002.jpg","width":2023,"caption":"Lipid A analysis by TLC. (A) Lower abundance of lipid A modified with two Ara4N (species 2) in ΔdbcA. Lipid A was extracted from the indicated strains following labeling with 32P and resolved using thin layer chromatography. Analysis was conducted using a Phosphorimager equipped with IQMac software. Indicated structures were ascertained from comparison with our MS data (Panta et al., 2019). Species “2”, “1,” and “0” correspond to pentaacylated lipid A modified with two, one, or zero Ara4N, respectively. Numbers below each spot correspond to the percentage of that species of the total signal of species “2”, “1,” and “0.” (B) Analysis of species 2 (pentaacylated lipid A modified with two Ara4N) with different concentrations of rhamnose in E264B, E264T, ΔdbcAB, and ΔdbcAT strains. E264; vec and ΔdbcA; vec were included as controls. A bar graph is also included for clarity."},{"height":1359,"url":"https://www.frontiersin.org/files/Articles/618389/fmicb-11-618389-HTML/image_m/fmicb-11-618389-g003.jpg","width":1931,"caption":"Partial complementation of colistin sensitivity in ΔdbcAB and ΔdbcAT. All the strains were grown similarly as described in Figure 1B. 1:10 dilutions were spotted on LB, 100 Tmp plates with different amount of colistin and rhamnose as indicated. Plates were analyzed after 72 h of incubation at 37°C. Higher concentrations of rhamnose are used for this experiment because the rhamnose promoter is integrated in the chromosome and arn genes are transcribed through this promoter as a single copy in compared to multicopy expression plasmids used elsewhere."},{"height":572,"url":"https://www.frontiersin.org/files/Articles/618389/fmicb-11-618389-HTML/image_m/fmicb-11-618389-g004.jpg","width":1925,"caption":"pH dependent colistin sensitivity, lipid A modification and membrane potential in B. thailandensis. (A) Minimal inhibitory concentration (MIC) was determined for indicated strains using colistin E-test strips on LB, 100 Tmp plates buffered with 100 mM of MES for pH 5.5 and 6.5, and 70 mM BIS-TRIS propane (BTP) buffer for pH 7.5 and 8.0. Approximate MIC is denoted by white arrows. Plates were analyzed after 48 h. (B) Analysis of lipid A at different pH media adjusted with either MES or BTP as described above. Lipid A was analyzed as previously described in Figure 2. Numbers below each spot correspond to the percentage of that species of the total signal of species “2”, “1,” and “0.” A “percentage of the total lipid A” bar graph for this figure is shown in Supplementary Figure S3. (C) Assessment of membrane potential (Δψ) of E264; vec and ΔdbcA; vec at different pH media using JC-1 dye represented as the red (595 nm)/green (530 nm) ratio. 2.5 × 107 cells from an overnight culture were inoculated in 25 ml LB, 100 Tmp, pH 5.5 (MES adjusted) or pH 7.5 (BTP adjusted). Each experiment was repeated three times. Bars represent mean ± SD of three independent determinations and statistical significance was calculated by unpaired Student’s t-test using GraphPad Prism 8.4.3 ***p \u003c 0.001, **p \u003c 0.01."},{"height":1231,"url":"https://www.frontiersin.org/files/Articles/618389/fmicb-11-618389-HTML/image_m/fmicb-11-618389-g005.jpg","width":1869,"caption":"A link between low pH and lipid A modification with Ara4N. (A) Lipid A modification with Ara4N is essential for acid tolerance. 1:10 dilutions of indicated strains were spotted on different pH media, LB and 100 Tmp agar plates with indicated concentration of rhamnose. Plates were buffered with either MES or BTP. The plates were analyzed after 72 h of incubation at 37°C. (B) Low pH complementation of colistin sensitivity of ΔdbcA depends on the expression of arn operons. 1:10 dilutions of indicated strains were spotted on LB, 100 Tmp, pH 5.5 with 0.05% of rhamnose and colistin as indicated. The plates were analyzed after 72 h of incubation at 37°C. (C) Burkholderia cenocepacia ArnT suppressor mutant strain, MH-55 is sensitive to acid stress. 10-fold dilution of 3 × 108 cells from overnight cultures of K-56 and MH-55 strains were spotted on LB plates at different pH media plates adjusted with either MES or BTP."},{"height":1387,"url":"https://www.frontiersin.org/files/Articles/618389/fmicb-11-618389-HTML/image_m/fmicb-11-618389-g006.jpg","width":2023,"caption":"Complementation of bacitracin and colistin sensitivity of ΔdbcA; vec by two B. thailandensis uppP homologs (Bth_I1512 and Bth_I2750) (A) ΔdbcA is sensitive to bacitracin. 10-fold dilution of cells from overnight cultures of indicated strains were spotted on LB, 100 Tmp, 0.002% rha and different amount of bacitracin. (B) Complementation of bacitracin and colistin sensitivity of ΔdbcA by Bth_I1512 and Bth_I2750, respectively. (C) Partial complementation of ΔdbcA colistin sensitivity by Bth_I2750. Here, we used a multicopy expression plasmid that requires a lower concentration of rhamnose for adequate expression, compared to the integrated construct used in Figure 3. (D) Lipid A analysis by TLC with overexpressed Bth_I2750 in E264 and ΔdbcA. Lipid A was extracted and analyzed for indicated strains. Species 2 was compared for all the strains. A “percentage of the total lipid A” bar graph is also shown on the right-hand side for more clarity."}],"journal":{"guid":310,"name":"Frontiers in Microbiology","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.618389","pubDate":"2021-01-12","score":39.165657421130774,"title":"A Burkholderia thailandensis DedA Family Membrane Protein Is Required for Proton Motive Force Dependent Lipid A Modification","topics":["Colistin","lipopolysaccharide","antibiotic resistance","membrane protein","proton motive force","Lipid A modification"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.618389/pdf"},{"__typename":"Feed_Article","_id":"6809bb5d968ae81840c99b4d","abstract":"Retrospective studies involving the screening of frozen stored collections of samples are commonplace when a new threat emerges, but it has been demonstrated that the freeze-thaw process can affect bacterial viability. The study of colistin-resistant bacteria in human and animal samples is an example of this issue. In this study, we compared culture-based and PCR-based methods for analyzing relative occurrence and diversity of colistin-resistant bacteria in caecal samples to determine the most appropriate method for frozen samples. Thus, 272 samples from the caecal contents of healthy pigs were tested before and after a 6-month freezing period. A selective medium was used when traditional isolation of colistin-resistant bacteria was tested, while a real-time SYBR® Green I PCR assay was applied for mcr-1 quantification. The number of samples with colistin-resistant isolates was higher in fresh samples (247/272) than in frozen ones (67/272) and showed a higher diversity of colistin-resistant genera. PCR identification of mcr colistin resistance genes evidenced that mcr-1 was the most prevalent mcr gene and mcr-2 was detected for the first time in pigs from Spanish animal production. The number of samples with mcr-1-carrying bacteria after a freezing period decreased, while real-time quantitation of the mcr-1 gene showed similar values in frozen and fresh samples. Therefore, when frozen cecal samples need to be analyzed, molecular detection of DNA could be the best option to provide a highly representative frame of the initial population present in the sample, and culture-based methods might be a useful complement to study colistin resistance levels.","htmlAbstract":"\u003cp\u003eRetrospective studies involving the screening of frozen stored collections of samples are commonplace when a new threat emerges, but it has been demonstrated that the freeze-thaw process can affect bacterial viability. The study of colistin-resistant bacteria in human and animal samples is an example of this issue. In this study, we compared culture-based and PCR-based methods for analyzing relative occurrence and diversity of colistin-resistant bacteria in caecal samples to determine the most appropriate method for frozen samples. Thus, 272 samples from the caecal contents of healthy pigs were tested before and after a 6-month freezing period. A selective medium was used when traditional isolation of colistin-resistant bacteria was tested, while a real-time SYBR\u003csup\u003e®\u003c/sup\u003e Green I PCR assay was applied for \u003ci\u003emcr-1\u003c/i\u003e quantification. The number of samples with colistin-resistant isolates was higher in fresh samples (247/272) than in frozen ones (67/272) and showed a higher diversity of colistin-resistant genera. PCR identification of \u003ci\u003emcr\u003c/i\u003e colistin resistance genes evidenced that \u003ci\u003emcr-1\u003c/i\u003e was the most prevalent \u003ci\u003emcr\u003c/i\u003e gene and \u003ci\u003emcr-2\u003c/i\u003e was detected for the first time in pigs from Spanish animal production. The number of samples with \u003ci\u003emcr-1\u003c/i\u003e-carrying bacteria after a freezing period decreased, while real-time quantitation of the \u003ci\u003emcr-1\u003c/i\u003e gene showed similar values in frozen and fresh samples. Therefore, when frozen cecal samples need to be analyzed, molecular detection of DNA could be the best option to provide a highly representative frame of the initial population present in the sample, and culture-based methods might be a useful complement to study colistin resistance levels.\u003c/p\u003e","authors":[{"fullName":"Pedro Miguela-Villoldo","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1000482/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1000482/overview","affiliation":{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null},"affiliations":[{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null},{"name":"Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense","address":null}],"nessieId":"481036971813"},{"fullName":"Miguel A. Moreno","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/133594/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/133594/overview","affiliation":{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null},"affiliations":[{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null},{"name":"Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense","address":null}],"nessieId":"498216854729"},{"fullName":"Marta Hernández","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/379990/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/379990/overview","affiliation":{"name":"Laboratorio de Biología Molecular y Microbiología, Instituto Tecnológico Agrario de Castilla y León","address":null},"affiliations":[{"name":"Laboratorio de Biología Molecular y Microbiología, Instituto Tecnológico Agrario de Castilla y León","address":null}],"nessieId":"317828225952"},{"fullName":"David Rodríguez-Lázaro","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/114703/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/114703/overview","affiliation":{"name":"Área de Microbiología, Departamento de Biotecnología y Ciencia de los Alimentos, Facultad de Ciencias, Universidad de Burgos","address":null},"affiliations":[{"name":"Área de Microbiología, Departamento de Biotecnología y Ciencia de los Alimentos, Facultad de Ciencias, Universidad de Burgos","address":null}],"nessieId":"266288624051"},{"fullName":"Alejandro Gallardo","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1089202/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1089202/overview","affiliation":{"name":"Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura","address":null},"affiliations":[{"name":"Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura","address":null}],"nessieId":"17180525791"},{"fullName":"Carmen Borge","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/569181/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/569181/overview","affiliation":{"name":"Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Córdoba","address":null},"affiliations":[{"name":"Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Córdoba","address":null}],"nessieId":"532576595206"},{"fullName":"Alberto Quesada","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/405293/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/405293/overview","affiliation":{"name":"Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura","address":null},"affiliations":[{"name":"Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura","address":null},{"name":"INBIO G+C, Universidad de Extremadura","address":null}],"nessieId":"85899992334"},{"fullName":"Lucas Domínguez","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/410288/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/410288/overview","affiliation":{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null},"affiliations":[{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null},{"name":"Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense","address":null}],"nessieId":"644207"},{"fullName":"María Ugarte-Ruiz","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/405440/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/405440/overview","affiliation":{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null},"affiliations":[{"name":"VISAVET Health Surveillance Centre, Universidad Complutense","address":null}],"nessieId":"137439602803"}],"dates":{"acceptedDate":"2020-10-15","recentDate":"2020-11-09"},"doi":"10.3389/fmicb.2020.572712","frontiersExtra":{"articleType":"Original Research","impact":{"citations":5,"crossrefCitations":0,"downloads":2,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":2431},"isPartOfResearchTopic":true,"isPublished":true,"section":"Antimicrobials, Resistance and Chemotherapy"},"guid":572712,"images":[{"height":364,"url":"https://www.frontiersin.org/files/myhome article library/572712/572712_Thumb_400.jpg","width":400,"caption":null},{"height":2052,"url":"https://www.frontiersin.org/files/Articles/572712/fmicb-11-572712-HTML/image_m/fmicb-11-572712-g001.jpg","width":2256,"caption":"Quantitative real-time PCR results for quantitation of the mcr-1 gene in both fresh and frozen samples."}],"journal":{"guid":310,"name":"Frontiers in Microbiology","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.572712","pubDate":"2020-11-09","score":15.889519373279775,"title":"Complementarity of Selective Culture and qPCR for Colistin Resistance Screening in Fresh and Frozen Pig Cecum Samples","topics":["Swine","antimicrobial resistance","Freeze-thaw process","MCR-1","mcr-2","caecal samples"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.572712/pdf"},{"__typename":"Feed_Article","_id":"6809bb5d968ae81840c99b50","abstract":"Due to the high prevalence of colistin-resistant Enterobacteriaceae in poultry and pigs, process waters and wastewater from slaughterhouses were considered as a hotspot for isolates carrying plasmid-encoded, mobilizable colistin resistances (mcr genes). Thus, questions on the effectiveness of wastewater treatment in in-house and municipal wastewater treatment plants (WWTPs) as well as on the diversity of the prevailing isolates, plasmid types, and their transmissibility arise. Process waters and wastewater accruing in the delivery and unclean areas of two poultry and two pig slaughterhouses were screened for the presence of target colistin-resistant bacteria (i.e., Escherichia coli, Klebsiella spp., Enterobacter cloacae complex). In-house and municipal WWTPs (mWWTPs) including receiving waterbodies were investigated as well. Samples taken in the poultry slaughterhouses yielded the highest occurrence of target colistin-resistant Enterobacteriaceae (40.2%, 33/82), followed by mWWTPs (25.0%, 9/36) and pig slaughterhouses (14.9%, 10/67). Recovered isolates exhibited various resistance patterns. The resistance rates using epidemiological cut-off values were higher in comparison to those obtained with clinical breakpoints. Noteworthy, MCR-1-producing Klebsiella pneumoniae and E. coli were detected in scalding waters and preflooders of mWWTPs. A total of 70.8% (46/65) of E. coli and 20.6% (7/34) of K. pneumoniae isolates carried mcr-1 on a variety of transferable plasmids with incompatibility groups IncI1, IncHI2, IncX4, IncF, and IncI2 ranging between 30 and 360 kb. The analyzed isolates carrying mcr-1 on transferable plasmids (n = 53) exhibited a broad diversity, as they were assigned to 25 different XbaI profiles. Interestingly, in the majority of colistin-resistant mcr-negative E. coli and K. pneumoniae isolates non-synonymous polymorphisms in pmrAB were detected. Our findings demonstrated high occurrence of colistin-resistant E. coli and K. pneumoniae carrying mcr-1 on transferrable plasmids in poultry and pig slaughterhouses and indicate their dissemination into surface water.","htmlAbstract":"\u003cp\u003eDue to the high prevalence of colistin-resistant \u003ci\u003eEnterobacteriaceae\u003c/i\u003e in poultry and pigs, process waters and wastewater from slaughterhouses were considered as a hotspot for isolates carrying plasmid-encoded, mobilizable colistin resistances (\u003ci\u003emcr\u003c/i\u003e genes). Thus, questions on the effectiveness of wastewater treatment in in-house and municipal wastewater treatment plants (WWTPs) as well as on the diversity of the prevailing isolates, plasmid types, and their transmissibility arise. Process waters and wastewater accruing in the delivery and unclean areas of two poultry and two pig slaughterhouses were screened for the presence of target colistin-resistant bacteria (i.e., \u003ci\u003eEscherichia coli\u003c/i\u003e, \u003ci\u003eKlebsiella\u003c/i\u003e spp., \u003ci\u003eEnterobacter cloacae\u003c/i\u003e complex). In-house and municipal WWTPs (mWWTPs) including receiving waterbodies were investigated as well. Samples taken in the poultry slaughterhouses yielded the highest occurrence of target colistin-resistant \u003ci\u003eEnterobacteriaceae\u003c/i\u003e (40.2%, 33/82), followed by mWWTPs (25.0%, 9/36) and pig slaughterhouses (14.9%, 10/67). Recovered isolates exhibited various resistance patterns. The resistance rates using epidemiological cut-off values were higher in comparison to those obtained with clinical breakpoints. Noteworthy, MCR-1-producing \u003ci\u003eKlebsiella pneumoniae\u003c/i\u003e and \u003ci\u003eE. coli\u003c/i\u003e were detected in scalding waters and preflooders of mWWTPs. A total of 70.8% (46/65) of \u003ci\u003eE. coli\u003c/i\u003e and 20.6% (7/34) of \u003ci\u003eK. pneumoniae\u003c/i\u003e isolates carried \u003ci\u003emcr-1\u003c/i\u003e on a variety of transferable plasmids with incompatibility groups IncI1, IncHI2, IncX4, IncF, and IncI2 ranging between 30 and 360 kb. The analyzed isolates carrying \u003ci\u003emcr-1\u003c/i\u003e on transferable plasmids (\u003ci\u003en\u003c/i\u003e = 53) exhibited a broad diversity, as they were assigned to 25 different \u003ci\u003eXba\u003c/i\u003eI profiles. Interestingly, in the majority of colistin-resistant \u003ci\u003emcr\u003c/i\u003e-negative \u003ci\u003eE. coli\u003c/i\u003e and \u003ci\u003eK. pneumoniae\u003c/i\u003e isolates non-synonymous polymorphisms in \u003ci\u003epmrAB\u003c/i\u003e were detected. Our findings demonstrated high occurrence of colistin-resistant \u003ci\u003eE. coli\u003c/i\u003e and \u003ci\u003eK. pneumoniae\u003c/i\u003e carrying \u003ci\u003emcr-1\u003c/i\u003e on transferrable plasmids in poultry and pig slaughterhouses and indicate their dissemination into surface water.\u003c/p\u003e","authors":[{"fullName":"Mykhailo Savin","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Institute of Animal Sciences, University of Bonn","address":null},"affiliations":[{"name":"Institute of Animal Sciences, University of Bonn","address":null},{"name":"Institute for Hygiene and Public Health, Medical Faculty, University of Bonn","address":null}],"nessieId":null},{"fullName":"Gabriele Bierbaum","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Institute for Medical Microbiology, Immunology and Parasitology, Medical Faculty, University of Bonn","address":null},"affiliations":[{"name":"Institute for Medical Microbiology, Immunology and Parasitology, Medical Faculty, University of Bonn","address":null}],"nessieId":null},{"fullName":"Khald Blau","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Julius Kühn-Institut, Federal Research Centre for Cultivated Plants","address":null},"affiliations":[{"name":"Julius Kühn-Institut, Federal Research Centre for Cultivated Plants","address":null}],"nessieId":null},{"fullName":"Marijo Parcina","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Institute for Medical Microbiology, Immunology and Parasitology, Medical Faculty, University of Bonn","address":null},"affiliations":[{"name":"Institute for Medical Microbiology, Immunology and Parasitology, Medical Faculty, University of Bonn","address":null}],"nessieId":null},{"fullName":"Esther Sib","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Institute for Hygiene and Public Health, Medical Faculty, University of Bonn","address":null},"affiliations":[{"name":"Institute for Hygiene and Public Health, Medical Faculty, University of Bonn","address":null}],"nessieId":null},{"fullName":"Kornelia Smalla","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Julius Kühn-Institut, Federal Research Centre for Cultivated Plants","address":null},"affiliations":[{"name":"Julius Kühn-Institut, Federal Research Centre for Cultivated Plants","address":null}],"nessieId":null},{"fullName":"Ricarda Schmithausen","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Institute for Hygiene and Public Health, Medical Faculty, University of Bonn","address":null},"affiliations":[{"name":"Institute for Hygiene and Public Health, Medical Faculty, University of Bonn","address":null}],"nessieId":null},{"fullName":"Céline Heinemann","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1090490/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1090490/overview","affiliation":{"name":"Institute of Animal Sciences, University of Bonn","address":null},"affiliations":[{"name":"Institute of Animal Sciences, University of Bonn","address":null}],"nessieId":"738734917892"},{"fullName":"Jens A. Hammerl","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Department for Biological Safety, German Federal Institute for Risk Assessment","address":null},"affiliations":[{"name":"Department for Biological Safety, German Federal Institute for Risk Assessment","address":null}],"nessieId":null},{"fullName":"Judith Kreyenschmidt","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1121029/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1121029/overview","affiliation":{"name":"Institute of Animal Sciences, University of Bonn","address":null},"affiliations":[{"name":"Institute of Animal Sciences, University of Bonn","address":null},{"name":"Department of Fresh Produce Logistics, Hochschule Geisenheim University","address":null}],"nessieId":"609886006192"}],"dates":{"acceptedDate":"2020-10-07","recentDate":"2020-10-30"},"doi":"10.3389/fmicb.2020.575391","frontiersExtra":{"articleType":"Original Research","impact":{"citations":36,"crossrefCitations":0,"downloads":1,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":8063},"isPartOfResearchTopic":true,"isPublished":true,"section":"Antimicrobials, Resistance and Chemotherapy"},"guid":575391,"images":[{"height":375,"url":"https://www.frontiersin.org/files/myhome article library/575391/575391_Thumb_400.jpg","width":400,"caption":null},{"height":2093,"url":"https://www.frontiersin.org/files/Articles/575391/fmicb-11-575391-HTML/image_m/fmicb-11-575391-g001.jpg","width":2237,"caption":"Percentage of samples containing colistin-resistant target bacteria taken in poultry and pig slaughterhouses as well as in the municipal WWTPs."},{"height":1693,"url":"https://www.frontiersin.org/files/Articles/575391/fmicb-11-575391-HTML/image_m/fmicb-11-575391-g002.jpg","width":2917,"caption":"Occurrence of target bacteria tested as colistin-resistant across the sampling points in poultry slaughterhouses (n = 82). Number of samples taken at each sampling point is stated."},{"height":1773,"url":"https://www.frontiersin.org/files/Articles/575391/fmicb-11-575391-HTML/image_m/fmicb-11-575391-g003.jpg","width":2821,"caption":"Occurrence of target bacteria tested as colistin-resistant across the sampling points in pig slaughterhouses (n = 67) and in the municipal WWTPs receiving wastewater from the investigated pig slaughterhouses (n = 36). Number of samples taken at each sampling point is stated."},{"height":2037,"url":"https://www.frontiersin.org/files/Articles/575391/fmicb-11-575391-HTML/image_m/fmicb-11-575391-g004.jpg","width":2933,"caption":"Resistance to antimicrobial agents detected among target colistin-resistant isolates of (A) E. coli, (B) K. pneumonia, and (C) E. cloacae complex with MICs interpreted according to the epidemiological cut-off values (ECOFFs) of EUCAST (scheme A). MICs (mg/L) of antimicrobials with undefined epidemiological cut-offs for E. cloacae complex isolates are shown in Table 1. AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; CST, colistin; GEN, gentamicin; NAL, nalidixic acid; SMX, sulfamethoxazole; TET, tetracycline; TGC, tigecycline; TMP, trimethoprim; FEP, cefepime; ETP, ertapenem; CTX, cefotaxime; FOX, cefoxitin; IMI, imipenem; MEM, meropenem; CAZ, ceftazidime. []* – antimicrobials with undefined ECOFFs."},{"height":2045,"url":"https://www.frontiersin.org/files/Articles/575391/fmicb-11-575391-HTML/image_m/fmicb-11-575391-g005.jpg","width":2933,"caption":"Resistance to antimicrobial agents detected among target colistin-resistant isolates of (A) E. coli, (B) K. pneumonia, and (C) E. cloacae complex with MICs interpreted according to the clinical breakpoints of EUCAST (scheme B). TEM, temocillin; PIP, piperacillin; TZP, piperacillin-tazobactam; CTX, cefotaxime; CAZ, ceftazidime; CZA, ceftazidime-avibactam; C/T, ceftolozane-tazobactam; IMP, imipenem; MEM, meropenem; AMK, amikacin; TGC, tigecycline; CIP, ciprofloxacin; LVX, levofloxacin; CHL, chloramphenicol; SXT, sulfamethoxazole-trimethoprim; FOF, fosfomycin; CST, colistin."}],"journal":{"guid":310,"name":"Frontiers in Microbiology","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.575391","pubDate":"2020-10-30","score":72.03057378784698,"title":"Colistin-Resistant Enterobacteriaceae Isolated From Process Waters and Wastewater From German Poultry and Pig Slaughterhouses","topics":["Escherichia coli","Klebsiella pneumoniae","wastewater","Colistin resistance","Slaughterhouse","Enterobacter cloacae complex","zoonotic microorganisms","mcr genes"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fmicb.2020.575391/pdf"}]],"pageParams":[null]},"dataUpdateCount":1,"dataUpdatedAt":1756916612302,"error":null,"errorUpdateCount":0,"errorUpdatedAt":0,"fetchFailureCount":0,"fetchFailureReason":null,"fetchMeta":null,"isInvalidated":false,"status":"success","fetchStatus":"idle"},"queryKey":["research-topic-articles",14542,1],"queryHash":"[\"research-topic-articles\",14542,1]"},{"state":{"data":{"researchTopicId":14542,"articleViews":51963,"articleDownloads":19073,"topicViews":1813,"summary":72849},"dataUpdateCount":1,"dataUpdatedAt":1756916612259,"error":null,"errorUpdateCount":0,"errorUpdatedAt":0,"fetchFailureCount":0,"fetchFailureReason":null,"fetchMeta":null,"isInvalidated":false,"status":"success","fetchStatus":"idle"},"queryKey":["research-topic-impact",14542],"queryHash":"[\"research-topic-impact\",14542]"}]}},"__N_SSG":true},"page":"/research-topics/[id]/[slug]/mag","query":{"id":"14542","slug":"zoonotic-microorganisms-and-spread-of-acquired-polymyxin-resistance-determinants"},"buildId":"ZvyQJ1c6REyZAR__c3437","assetPrefix":"/_rtmag","isFallback":false,"gsp":true,"scriptLoader":[{"id":"google-analytics","strategy":"afterInteractive","children":"(function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':\n new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],\n j=d.createElement(s),dl=l!='dataLayer'?'\u0026l='+l:'';j.async=true;j.src=\n 'https://tag-manager.frontiersin.org/gtm.js?id='+i+dl+ '\u0026gtm_auth=PYjuAXuPWCihEq8Nf7ErrA\u0026gtm_preview=env-1\u0026gtm_cookies_win=x';f.parentNode.insertBefore(j,f);\n })(window,document,'script','dataLayer','GTM-PT9D93K');"}]}