Abstract
Aquatic environments are crucial hotspots for the dissemination of antibiotic resistant microorganisms and resistance genes. Thus, the purpose of this study was to investigate the occurrence and the genetic characterization of cefotaxime-resistant (CTXR) Enterobacteriaceae at a Tunisian semi-industrial pilot plant with biological treatment (WWPP) and its receiving river (Rouriche River, downstream from WWPP) located in Tunis City, during 2017–2018. We collected 105 and 15 water samples from the WWPP and the Rouriche River, respectively. Samples were screened to recover ESBL-producing Enterobacteriaceae (ESBL-E) and isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, plasmid types and molecular typing (multilocus sequence typing, MLST). Among 120 water samples, 33 and 4 contained ESBL-producing E. coli and K. pneumoniae isolates, respectively. Most isolates were multidrug resistant and produced CTX-M-15 (28 isolates), CTX-M-1 (4 isolates), CTX-M-55 (2 isolates), CTX-M-27 (one isolate), SHV-12 (one isolate) and VEB beta-lactamases (one isolate). All K. pneumoniae were CTX-M-15-positive. Four colistin-resistant isolates were found (MIC 4–8 μg/ml), but they were negative for the mcr genes tested. Class 1 integrons were detected in 21/25 trimethoprim/sulfamethoxazole-resistant isolates, and nine of them carried the gene cassette arrays: aadA2 + dfrA12 (n = 4), aadA1 + dfrA15 (n = 2), aadA5 + dfrA17 (n = 2) and aadA1/2 (n = 1). The IncP and IncFIB plasmids were found in 30 and 16 isolates, respectively. Genetic lineages detected were as follows: E. coli (ST48-ST10 Cplx, ST2499, ST906, ST2973 and ST2142); K. pneumoniae: (ST1540 and ST661). Our findings show a high rate of CTX-M-15 and high genetic diversity of ESBL-E isolates from WWPP and receiving river water.
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References
Alcalá L, Alonso CA, Simón C, González-Esteban C, Orós J, Rezusta A, Ortega C, Torres C (2016) Wild birds, frequent carriers of extended-spectrum β-lactamase (ESBL) producing Escherichia coli of CTX-M and SHV-12 types. Microb Ecol 72:861–869. https://doi.org/10.1007/s00248-015-0718-0
Al-Marzooq F, Yusof MYM, Tay ST (2015) Molecular analysis of antibiotic resistance determinants and plasmids in Malaysian isolates of multidrug resistant Klebsiella pneumoniae. PLoS One 10:e0133654. https://doi.org/10.1371/journal.pone.0133654
Azam M, Jan AT, Haq QM (2016) blaCTX-M-152, a novel variant of CTX-M-group-25, identified in a study performed on the prevalence of multidrug resistance among natural inhabitants of river Yamuna, India. Front Microbiol 7:176. https://doi.org/10.3389/fmicb.2016.00176
Baquero F, Martínez JL, Cantón R (2008) Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 19:260–265. https://doi.org/10.1016/j.copbio.2008.05.006
Ben Sallem R, Ben Slama K, Sáenz Y, Rojo-Bezares B, Estepa V, Jouini A, Gharsa H, Klibi N, Boudabous A, Torres C (2012) Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL) – and CMY-2 – producing Escherichia coli isolates from healthy food-producing animals in Tunisia. Foodborne Pathog Dis 9:1137–1142. https://doi.org/10.1089/fpd.2012.1267
Berglund B (2015) Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with antibiotics. Infect Ecol Epidemiol 5:28564. https://doi.org/10.3402/iee.v5.28564
Bevan ER, Jones AM, Hawkey PM (2017) Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype. J Antimicrob Chemother 72:2145–2155. https://doi.org/10.1093/jac/dkx146
Bizzini A, Greub G (2010) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification. Clin Microbiol Infect 16:1614–1619. https://doi.org/10.1111/j.1469-0691.2010.03311.x
Blaak H, de Kruijf P, Hamidjaja RA, van Hoek AH, de Roda Husman AM, Schets FM (2014) Prevalence and characteristics of ESBL-producing E. coli in Dutch recreational waters influenced by wastewater treatment plants. Vet Microbiol 171:448–459. https://doi.org/10.1016/j.vetmic.2014.03.007
Borsa BA, Demirci M, Gungordu-Dalar Z, Karabiyik G, Aygun G, Kucukbasmaci O (2019) Molecular mechanisms of colistin resistance among Klebsiella pneumoniae strains. Clin Lab 65(7). https://doi.org/10.7754/Clin.Lab.2019.180705
Bouki C, Venieri D, Diamadopoulos E (2013) Detection and the fate of antibiotic resistant bacteria in wastewater treatment plants: a review. Ecotoxicol Environ Saf 91:1–9. https://doi.org/10.1016/j.ecoenv.2013.01.016
Bungau S, Mirela Tit D, Fodor K, Cioca G, Agop M, Iovan C, Nistor Cseppento DC, Bumbu A, Cristiana Bustea C (2018) Aspects regarding the pharmaceutical waste management in Romania. Sustainability 10: 2788; https://doi.org/10.3390/su10082788
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ (2005) Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63:219–228. https://doi.org/10.1016/j.mimet.2005.03.018
Clermont O, Bonacorsi S, Bingen E (2000) Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 66:4555–4558. https://doi.org/10.1128/aem.66.10.4555-4558.2000
Clinical and Laboratory Standards Institute (2017) Performance standards for antimicrobial susceptibility testing. 27th ed. Wayne, PA. Clinical and Laboratory Standards Institute, CLSI Supplement M100
Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74:417–433. https://doi.org/10.1128/MMBR.00016-10
Dropa M, Lincopan N, Balsalobre LC, Oliveira DE, Moura RA, Fernandes MR, da Silva QM, Matté GR, Sato MI, Matté MH (2016) Genetic background of novel sequence types of CTX-M-8-and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae from public wastewater treatment plants in São Paulo, Brazil. Environ Sci Poll Res Int 23:4953–4958. https://doi.org/10.1007/s11356-016-6079-5
Elbediwi M, Li Y, Paudyal N, Pan H, Li X, Xie S, Rajkovic A, Feng Y, Fang W, Rankin SC, Yue M (2019) Global burden of colistin-resistant bacteria: mobilized colistin resistance genes study (1980–2018). Microorganisms 7:461. https://doi.org/10.3390/microorganisms7100461
Elghaieb H, Freitas AR, Abbassi MS, Novais C, Zouari M, Hassen A, Peixe L (2019) Dispersal of linezolid-resistant enterococci carrying poxtA or optrA in retail meat and food-producing animals from Tunisia. J Antimicrob Chemother 74:2865–2869. https://doi.org/10.1093/jac/dkz263
El-Sayed Ahmed MAEG, Zhong LL, Shen C, Yang Y, Doi Y, Tian GB (2020) Colistin and its role in the era of antibiotic resistance: an extended review (2000–2019). Emerg Microbes Infect 9:868–885. https://doi.org/10.1080/22221751.2020.1754133
Freitas AR, Elghaieb H, León-Sampedro R, Abbassi MS, Novais C, Coque TM, Hassen A, Peixe L (2017) Detection of optrA in the African continent (Tunisia) within a mosaic Enterococcus faecalis plasmid from urban wastewaters. J Antimicrob Chemother 72:3245–3251. https://doi.org/10.1093/jac/dkx321
Furlan JPR, Stehling EG (2019) Draft genome sequence of a multidrug-resistant tetA/IncF-harbouring Escherichia coli ST906 obtained from a soil cultivated with jaboticaba (Plinia cauliflora). J Glob Antimicrob Resist 16:181–182. https://doi.org/10.1016/j.jgar.2019.01.026
Gatica J, Cytryn E (2013) Impact of treated wastewater irrigation on antibiotic resistance in the soil microbiome. Environ Sci Pollut Res Int 20:3529–3538. https://doi.org/10.1007/s11356-013-1505-4
Hassen B, Saloua B, Abbassi MS, Ruiz-Ripa L, Mama OM, Hassen A, Hammami S, Torres C (2019) mcr-1 encoding colistin resistance in CTX-M-1/CTX-M-15-producing Escherichia coli isolates of bovine and caprine origins in Tunisia. First report of CTX-M-15-ST394/D E. coli from goats. Comp Immunol Microbiol Infect Dis 67:101366. https://doi.org/10.1016/j.cimid.2019.101366
Ibrahim C, Hammami S, Chérif N, Mejri S, Pothier P, Hassen A (2019) Detection of Sapoviruses in two biological lines of Tunisian hospital wastewater treatment. Int J Environ Health Res 29:400–413. https://doi.org/10.1080/09603123.2018.1546835
Izdebski R, Baraniak A, Herda M, Fiett J, Bonten MJ, Carmeli Y, Goossens H, Hryniewicz W, Brun-Buisson C, Gniadkowski M, Muzlovic I, Nardi G, Petrikkos G, Stammet P, Salomon J, Lawrence C, Legrand P, Rossini A, Salvia A, Samso J, Fierro J, Paul M, Lerman Y (2014) MLST reveals potentially high-risk international clones of Enterobacter cloacae. J Antimicrob Chemother 70:48–56. https://doi.org/10.1093/jac/dku359
Jouini A, Vinué L, Slama KB, Saenz Y, Klibi N, Hammami S, Boudabous A, Torres C (2007) Characterisation of CTX-M and SHV extended-spectrum β-lactamases and associated resistance genes in Escherichia coli strains of food samples in Tunisia. J Antimicrob Chemother 60:1137–1141. https://doi.org/10.1093/jac/dkm316
Kittinger C, Lipp M, Folli B, Kirschner A, Baumert R, Galler H, Grisold AJ, Luxner J, Weissenbacher M, Farnleitner AH, Zarfel G (2016) Enterobacteriaceae isolated from the river Danube: antibiotic resistance, with a focus on the presence of ESBL and carbapenemases. PLoS One 11:e0165820. https://doi.org/10.1371/journal.pone.0165820
Lepuschitz S, Schill S, Stoeger A, Pekard-Amenitsch S, Huhulescu S, Inreiter N, Hartl R, Kerschner H, Sorschag S, Springer B, Brisse S, Allerberger F, Mach RL, Ruppitsch W (2019) Whole genome sequencing reveals resemblance between ESBL-producing and carbapenem resistant Klebsiella pneumoniae isolates from Austrian rivers and clinical isolates from hospitals. Sci Total Environ 662:227–235. https://doi.org/10.1016/j.scitotenv.2019.01.179
Long SW, Olsen RJ, Eagar TN, Beres SB, Zhao P, Davis JJ, Brettin T, Xia F, Musser JM (2017) Population genomic analysis of 1,777 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates, Houston, Texas: an unexpected abundance of clonal group 307. MBio 8:e00489–e00417. https://doi.org/10.1128/mBio.00489-17
Lupo A, Coyne S, Berendonk TU (2012) Origin and evolution of antibiotic resistance: the common mechanisms of emergence and spread in water bodies. Front Microbiol 3:18. https://doi.org/10.3389/fmicb.2012.00018
Manges AR, Geum HM, Guo A, Edens TJ, Fibke CD, Pitout JD (2019) Global extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Clin Microbiol Rev 32:e00135–e00118. https://doi.org/10.1128/CMR.00135-18
Mathers AJ, Peirano G, Pitout JD (2015) The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae. Clin Microbiol Rev 28:565–591. https://doi.org/10.1128/CMR.00116-14
Mughini-Gras L, Dorado-García A, van Duijkeren E, van den Bunt G, Dierikx CM, Bonten MJ, Schmitt H, Hald T, Evers EG, de Koeijer A, van Pelt W, Franz E, Mevius DJ, Heederik DJJ (2019) Attributable sources of community-acquired carriage of Escherichia coli containing β-lactam antibiotic resistance genes: a population-based modelling study. Lancet Planet Health 3:e357–e369. https://doi.org/10.1016/S2542-5196(19)30130-5
Naas T, Coignard B, Carbonne A, Blanckaert K, Bajolet O, Bernet C, Verdeil X, Astagneau P, Desenclos JC, Nordmann P (2006) VEB-1 extended-spectrum β-lactamase-producing Acinetobacter baumannii, France. Emerg Infect Dis 12:1214–1222. https://doi.org/10.3201/eid1708.051547
Nasreen M, Sarker A, Malek MA, Ansaruzzaman MD, Rahman M (2015) Prevalence and resistance pattern of Pseudomonas aeruginosa isolated from surface water. Adv Microbiol 5:74–81. https://doi.org/10.4236/aim.2015.51008
Olaitan AO, Morand S, Rolain JM (2016) Emergence of colistin-resistant bacteria in humans without colistin usage: a new worry and cause for vigilance. Int J Antimicrob Agents 47:1–3. https://doi.org/10.1016/j.ijantimicag.2015.11.009
Poirel L, Naas T, Guibert M, Chaibi EB, Labia R, Nordmann P (1999) Molecular and biochemical characterisation of VEB-1, a novel class A extended-spectrum β-lactamase encoded by an Escherichia coli integron gene. Antimicrob Agents Chemother 43:573–581
Poirel L, Mugnier PD, Toleman MA, Walsh TR, Rapoport MJ, Petroni A, Nordmann P (2009) ISCR2, another vehicle for blaVEB gene acquisition. Antimicrob Agents Chemother 53:4940–4943. https://doi.org/10.1128/AAC.00414-09
Poirel L, Jayol A, Nordmann P (2017) Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. Clin Microbiol Rev 30:557–596. https://doi.org/10.1128/CMR.00064-16
Raddaoui A, Chebbi Y, Tanfous FB, Mabrouk A, Achour W (2020) First description of clinical linezolid resistant Enterococcus sp. in North Africa. J Glob Antimicrob Resist 21:169–170. https://doi.org/10.1016/j.jgar.2020.04.012
Ruiz E, Sáenz Y, Zarazaga M, Rocha-Gracia R, Martínez-Martínez L, Arlet G, Torres C (2012) qnr, aac (6′)-Ib-cr and qepA genes in Escherichia coli and Klebsiella spp.: genetic environments and plasmids and chromosomal location. J Antimicrob Chemother 67:886–897. https://doi.org/10.1093/jac/dkr548
Sáenz Y, Briñas L, Domínguez E, Ruiz J, Zarazaga M, Vila J, Torres C (2004) Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal and food origins. Antimicrob Agents Chemother 48:3996–4001. https://doi.org/10.1128/AAC.48.10.3996-4001.2004
Sghaier S, Abbassi MS, Pascual A, Serrano L, Díaz-De-Alba P, Said MB, Hassen B, Ibrahim C, Hassen A, López-Cerero L (2019) Extended-spectrum β-lactamase-producing Enterobacteriaceae from animal origin and wastewater in Tunisia: the first detection of O25b-B23-CTX-M-27-ST131 Escherichia coli and CTX-M-15/OXA-204-producing Citrobacter freundii from wastewater. J Glob Antimicrob Resist 17:189–194. https://doi.org/10.1016/j.jgar.2019.01.002
Shahin K, Bouzari M, Wang R, Khorasgani MR (2019) Distribution of antibiotic resistance genes and integrons among Shigella spp. isolated from water sources. J Glob Antimicrob Resist 19:122–128. https://doi.org/10.1016/j.jgar.2019.04.020
Shivakumaraswamy SK, Deekshit VK, Vittal R, Akhila DS, Mundanda DM, Raj JRM, Chakraborty A, Karunasagar I (2019) Phenotypic & genotypic study of the antimicrobial profile of bacteria isolates from environmental samples. Indian J Med Res 149:232–239. https://doi.org/10.4103/ijmr.IJMR_2097_17
Tacão M, Moura A, Correia A, Henriques I (2014) Co-resistance to different classes of antibiotics among ESBL-producers from aquatic systems. Water Res 48:100–107. https://doi.org/10.1016/j.watres.2013.09.021
Wang X, Wang Y, Zhou Y, Li J, Yin W, Wang S, Zhang S, Shen J, Shen Z, Wang Y (2018) Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae. Emerg Microbes Infect 7:122–129. https://doi.org/10.1038/s41426-018-0124-z
WHO (2019) Ten threats to global health in 2019, https://www.who.int/emergencies/ten-threats-to-global-health-in-2019
Zurfluh K, Jakobi G, Stephan R, Hächler H, Nüesch-Inderbinen M (2014) Replicon typing of plasmids carrying blaCTX-M-1 in Enterobacteriaceae of animal, environmental and human origin. Front Microbiol 5:555. https://doi.org/10.3389/fmicb.2014.00555
Acknowledgements
We would like to acknowledge all the workers of the WWPP of El Menzeh 1, Tunis, Tunisia, who have kindly participated in the collection and transport of the water samples.
Funding
The work performed at the University of La Rioja was financed by project SAF2016-76571-R of the Agencia Estatal de Investigation (AEI) of Spain and FEDER of the EU. B. Hassen has a fellowship from the Tunisian Ministry of Higher Education and Scientific Research (Tunisia). L. Ruiz-Ripa has a predoctoral fellowship at the University of La Rioja (Spain). O. M. Mama has a predoctoral fellowship of Mujeres por África-Universidad de La Rioja.
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Hassen, B., Abbassi, M.S., Benlabidi, S. et al. Genetic characterization of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from wastewater and river water in Tunisia: predominance of CTX-M-15 and high genetic diversity. Environ Sci Pollut Res 27, 44368–44377 (2020). https://doi.org/10.1007/s11356-020-10326-w
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DOI: https://doi.org/10.1007/s11356-020-10326-w