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      Hospitalized Pets as a Source of Carbapenem-Resistance

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          Abstract

          The massive and irrational use of antibiotics in livestock productions has fostered the occurrence and spread of resistance to “old class antimicrobials.” To cope with that phenomenon, some regulations have been already enforced in the member states of the European Union. However, a role of livestock animals in the relatively recent alerts on the rapid worldwide increase of resistance to last-choice antimicrobials as carbapenems is very unlikely. Conversely, these antimicrobials are increasingly administered in veterinary hospitals whose role in spreading bacteria or mobile genetic elements has not adequately been addressed so far. A cross-sectional study was carried out on 105 hospitalized and 100 non-hospitalized pets with the aim of measuring the prevalence of carbapenem-resistant Gram-negative bacteria (GNB) colonizing dogs and cats, either hospitalized or not hospitalized and estimating the relative odds. Stool samples were inoculated on MacConkey agar plates containing 1 mg/L imipenem which were then incubated aerobically at 37°C ± 1 for 48 h. Isolated bacteria were identified first by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and were confirmed by 16S rRNA sequencing. The genetic basis of resistance was investigated using PCR methods, gene or whole genome sequencing (WGS). The prevalence of pets harboring carbapenem-resistant bacteria was 11.4 and 1.0% in hospitalized and not-hospitalized animals, respectively, with an odds ratio of 12.8 ( p < 0.01). One pet carried two diverse isolates. Overall, 14 gram-negative non-fermenting bacteria, specifically, one Acinetobacter radioresistens, five Acinetobacter baumannii, six Pseudomonas aeruginosa and two Stenotrophomonas maltophilia were isolated. The Acinetobacter species carried acquired carbapenemases genes encoded by bla NDM-1 and bla OXA-23. In contrast, Pseudomonas phenotypic resistance was associated with the presence of mutations in the oprD gene. Notably, inherent carbapenem-resistant isolates of S. maltophilia were also resistant to the first-line recommended chemotherapeutic trimethoprim/sulfamethoxazole. This study estimates the risk of colonization by carbapenem-resistant non-fermenting GNB in pets hospitalized in veterinary tertiary care centers and highlights their potential role in spreading resistance genes among the animal and human community. Public health authorities should consider extending surveillance systems and putting the release of critical antibiotics under more strict control in order to manage the infection/colonization of pets in veterinary settings.

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          Most cited references60

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          Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR.

          Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC beta-lactamases are limited because these organisms are usually resistant to all the beta-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC beta-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC beta-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC beta-lactamase expression in organisms with or without a chromosomal AmpC beta-lactamase gene.
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            Global spread of carbapenem-resistant Acinetobacter baumannii.

            We have investigated the molecular epidemiology and distribution of carbapenemase genes in 492 imipenem-non-susceptible Acinetobacter baumannii worldwide isolates (North and Latin America, Europe, Asia, South Africa and Australia). MICs were determined by broth microdilution and Etest. The presence of carbapenemase-encoding genes was investigated by PCR. Molecular epidemiology was performed by repetitive sequence-based PCR (rep-PCR; DiversiLab), sequence-type multiplex PCR and PFGE. Imipenem non-susceptibility was associated with ISAba1 upstream of the intrinsic bla(OXA-51-like) or the acquired carbapenemase bla(OXA-23-like), bla(OXA-40-like) or bla(OXA-58-like). Isolates were grouped into eight distinct clusters including European clones I, II and III. European clone II was the largest (246 isolates) and most widespread group (USA, pan-Europe, Israel, Asia, Australia and South Africa). The global dissemination of eight carbapenem-resistant lineages illustrates the success this organism has had in epidemic spread. The acquired OXA enzymes are widely distributed but are not the sole carbapenem resistance determinant in A. baumannii.
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              The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii.

              ISAba1 was found in all widespread clones of Acinetobacter baumannii in the United Kingdom. All isolates studied had a blaOXA-51-like carbapenemase gene; some also had blaOXA-23-like and/or blaOXA-58-like. Among isolates with blaOXA-51-like as sole carbapenemase gene, only those with ISAba1 adjacent to blaOXA-51-like were carbapenem resistant. Minor differences in blaOXA-51-like sequence were observed in resistant and susceptible isolates. Isolates with blaOXA-23-like in addition were consistently resistant to carbapenems; in all of these ISAba1 lay upstream of blaOXA-23-like, but was not associated with blaOXA-51-like. These results suggest that ISAba1 is providing the promoter for blaOXA-51-like and, probably, for blaOXA-23-like.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                06 December 2018
                2018
                : 9
                : 2872
                Affiliations
                [1] 1Department of Veterinary Medical Sciences, University of Bologna , Bologna, Italy
                [2] 2Genefast Srl , Forlì, Italy
                [3] 3Department of Agricultural and Food Sciences, University of Bologna , Bologna, Italy
                [4] 4Ospedale: Veterinario I Portoni Rossi s.r.l. , Bologna, Italy
                [5] 5Unit of Microbiology, University of Calgary and Calgary Laboratory Services , Calgary, AB, Canada
                [6] 6Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata , Foggia, Italy
                [7] 7The Great Romagna Hub Laboratory , Pievesestina, Italy
                [8] 8Department of Experimental, Diagnostic and Specialty Medicine - DIMES, University of Bologna , Bologna, Italy
                Author notes

                Edited by: Rustam Aminov, University of Aberdeen, United Kingdom

                Reviewed by: Nilton Lincopan, Universidade de São Paulo, Brazil; Kristina Kadlec, Friedrich Loeffler Institut, Germany

                *Correspondence: Renato Giulio Zanoni, renatogiulio.zanoni@ 123456unibo.it

                This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2018.02872
                6291488
                30574124
                a9601050-57a6-458b-b11f-a0b4360eb548
                Copyright © 2018 Gentilini, Turba, Pasquali, Mion, Romagnoli, Zambon, Terni, Peirano, Pitout, Parisi, Sambri and Zanoni.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 30 November 2017
                : 08 November 2018
                Page count
                Figures: 2, Tables: 2, Equations: 0, References: 68, Pages: 9, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                one health,carbapenem resistance,acinetobacter radioresistens,dogs and cats,hospitalization,veterinary tertiary care facilities,blandm-1blaoxa-23

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