Integrons; Resistència antimicrobiana; Infeccions nosocomials i comunitàries adquiridesIntegrons; Antimicrobial resistance; Acquired nosocomial and community infectionsIntegrones; Resistencia antimicrobiana; Infecciones nosocomiales y comunitarias adquiridasAquesta comunicació presenta l’estudi de diferents qüestions microbiològiques relacionades amb la resistència antimicrobiana en bacteris gramnegatius a l'àrea de les Terres de l'Ebre

Pérez-Moreno, Mar Olga

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Scientia, Dipòsit d’Informació Digital del Departament de Salut

  INTEGRON-RELATED ANTIMICROBIAL RESISTANCE IN  NOSOCOMIAL AND COMMUNITY ACQUIRED  INFECTIONS  Mar Olga Pérez Moreno Servei d’Anàlisis Clíniques Institut per a la Investigació en Ciències de la Salut Dr. Ferran  Hospital de Tortosa Verge de la Cinta  3ª  JORNADA DE RECERCA A L’INSTITUT CATALÀ DE LA SALUT (OCTUBRE 2010) OBJECTIVES  The focus of interest of our group is the study of different microbiological issues related to antimicrobial resistance in gram-negative bacteria in our geographical area.  ANTIBIOTIC RESISTANCE: AN EMERGING PROBLEM  One of the most pressing causes of health concern from individual (clinical), ecological (public health) and social perspective, aggravated by the lack of new antimicrobials development.  1928 80 years latter Resistant Strains Rare Resistant Strains  Dominant Antimicrobial         Exposure   Selection for antimicrobial-resistant Strains Campaign to Prevent Antimicrobial Resistance in Healthcare Settings  Emergence of antibiotic resistance is an inevitable consequence of antimicrobials use in any setting (humans, animals, agriculture). 12 Steps to Prevent Antimicrobial Resistance: Hospitalized Adults    12      Break the chain               11   Isolate the pathogen            10 Stop treatment when cured        9   Know when to say “no” to vanco    8  Treat infection, not colonization               7  Treat infection, not contamination            6         5  Practice antimicrobial control     4  Access the experts               3 Target the pathogen            2  Get the catheters out        1  Vaccinate                Prevent Transmission   Use Antimicrobials Wisely  Diagnose & Treat Effectively Prevent Infections Campaign to Prevent Antimicrobial Resistance in Healthcare Settings INVESTIGATION LINES  6  Characterization of mechanisms involved in antimicrobial resistance in gram-negative bacteria.    Particularly resistance to β-lactams (amoxicillin-clavulanate) and Integron-associated resistance.   Optimization of antibiotic therapy: improvement in effectiveness and prevention of emergence of resistances.    Molecular epidemiology studies of isolates displaying troublesome or uncommon resistance mechanisms aimed at establishing the reasons for their spread and designing strategies to bring it under control  Horizontal transfer of genetic resistance elements (antibiotic policy)   Spread of a limited number of clones (infection control measures) INTEGRONS  Integrons   ● widespread, potentially mobile, rather small, genetic elements.  ● generally located on plasmids or transposons.  ● able to incorporate and mobilize resistance gene cassettes throughout a site-specific recombination system directed by an integrase.   Class 1 integrons are the commonest.  Two highly conserved regions (5’CS and 3’CS ends) flanking a variable region where gene cassettes encoding resistance to a wide range of antibiotics are inserted.           Important and efficient (low biological cost) source for the acquisition, and spread of resistance and cross-resistance in gram-negative bacteria.  Promotor PROJECTS (I)   Characterization of mechanisms involved in resistance to associations of B-lactams plus -lactamase inhibitors in Escherichia coli isolates recovered in Terres de l’Ebre 8 METHODS: Isoelectrophocusing  (characterization of β-lactamases according to pI) and  spectophotometry (β-lactamase specific activity  and IC50 of clavulanate) RESULTS (I) 9 RESULTS (I)  RESULTS (I)            A number of mechanisms responsible for resistance to amoxicillin-clavulanate in Escherichia coli  ● TEM-1 hyperproduction ● AMPc hyperproduction or plasmidic cephamycinases. ● IRT β-lactamases production  ● OXA-1 production    Only one previous surveillance study in Spain to know the prevalence of IRT enzymes in E. coli (specific resistance mechanism against β-lactamase inhibitors).  ● Mechanism difficult to recognize by conventional antibiogram. ● Inclusion of low concentrations of cefepime in microdilution panels is a helpful tool for distinguishing OXA-1 from IRT B-lactamases.   ● Low but on the increase prevalence specially in community acquired UTI (related to the wide use of amoxicillin-clavulanate?).       11 PROJECTS (II)  Molecular epidemiology and characterization of resistance mechanisms involved in reduced susceptibility to amoxicillin-clavulanate in Klebsiella pneumoniae isolates from a chronic-care center.    Striking rate of decreased susceptibility to amoxicillin-clavulanate among cephalosporin-susceptible K. pneumoniae isolates recovered from patients from a chronic-care hospital, where amoxicillin-clavulanate was the most prescribed antibiotic followed by fluorquinolones,  since 2003 to 2009.         12 METHODS  PCR amplification or PCR-mapping assays and nucleotide sequencing:   tracking of genes encoding β-lactamases and other antibiotic resistance determinants,   chromosomal mutations in gyrA and parC   presence of class 1 integrons and identification of resistance cassettes within the integron variable region.  ERIC-PCR: DNA fingerprinting to assess epidemiological relationship between isolates.   Conjugation experiments:  To ascertain the eventual location of resistance genetic elements in conjugative plasmids.  13 RESULTS (II) 14 RESULTS (II)    Mechanisms involved: Coexistence of isolates displaying two different -lactamases, quite uncommon in this species, conferring specific resistance to β-lactamase inhibitors (IRT-11and OXA-1 -lactamase).    In almost all cases gene encoding OXA-1 located within a class 1 integron together with other antibiotic resistance determinants (aminoglycosides,  quinolones, chloramphenicol and rifampicin) and associated to qnrS2 (transferible resistance to quinolones) in a conjugative plasmid (Estructure only reported in an aquatic isolate of Aeromonas allosacharophila).      An identical integron, although in a different genetic environment – associated to genes encoding plasmidic cephamycinases - has been described in some sporadic human Enterobacterial isolates.   Three isolates carried an atypical class 1 integron lacking qacE1 and sul (typical of environmental isolates).             15 16 E3a      E4       E5        E5        E3                    E5         E5       E3        E2        E1   E3a    E4    E5      E5       E3              E5     E5         E3      E2     E1    ERIC-PCR Patterns Lanes 1-5 and 7-8: OXA-1 producing isolates Lanes 9-11: IRT-11 producing isolates Lane 6: amoxicillin/clavulanate-susceptible isolate   IRT-11 producing isolates ascribed to three clones (E1 to E3)  Clonal dissemination and horizontal transfer.   OXA-1 producing isolates carrying typical class integron ascribed to  two closely related clones (E3 and E3a)  Clonal dissemination.    OXA-1 producing isolates carrying defective class 1 integron ascribed to one clone (E4)  Clonal dissemination.   But in 2009 two qnrS2 Escherichia coli isolates harbouring the same atypical integron.  RESULTS (II)  CONCLUSIONS (II)  Second description reporting an outbreak involving IRT enzymes (the first one occurred in a chronic-care facility in France).  Importance of diversifying antibiotics used in an institution in order to avoid selective pressure which favours emergence of resistances.  • High consumption of ACL in the hospital lead to the emergence and selection of a resistance mechanism typical of community-acquired isolates.   Outstanding: adherence to standard infection control precautions. • Cross-infection between patients lead to dissemination of resistant clones.  Role played by integrons in co-selection (use of a single antimicrobial may select for resistance to other antibiotics) and horizontal transfer of resistance.     17 18    Role of integrons in antibiotic resistance in a typical zoonotic community-acquired infection: Food-borne disease caused by Non-typhoidal Salmonella.    PROJECTS (III) RESULTS (III)   Class 1 integron dissemination among multiresistant Salmonella enterica human isolates from Terres de l’Ebre  19 RESULTS (III)   Molecular epidemiology of infections  caused by multiresistant integron-carrying human Salmonella enterica serotype Typhimurium isolates from the health Region of Terres de l’Ebre 20 RESULTS AND CONCLUSIONS (III) 0%10%20%30%40%50%60%70%80%90%100%Figura 3: Freqüència de resistència als  diferents antimicrobians Total (n=522) Aïllats amb integrons (n=140) Aïllats sense integrons (n=382)  Multiresistance linked to integrons is widespread among human Salmonella enterica  isolates from our geographical region  ● 59% of MR isolates carried integrons. ● 10 different types of integrons in 14 different serotypes of Salmonella.   Resistance genes born by or linked to integrons  usually confer resistance to antibiotics employed in livestock and veterinary medicine but also to some that are the first choice in treatment of systemic human salmonellosis (amoxicillin-clavulanate, broad-range cephalosporins).   ● Need of wise antibiotic use in animals: selective pressure posed by antibiotic use in food animals may have negative impact on the treatment of human infections.   21 •dfrA1- aadA1 •dfrA12-orf-aadA2 •dfrA12-orf-aadA2-  cmlA1-aadA1 •bla oxa-1 –aadA1 •aadA2/ bla pse-1  RESULTS AND CONCLUSIONS (III)  Dramatic increase in the prevalence of serotype Typhimurium, usually more resistant and in some cases more virulent, to the detriment of serotype Enteritidis.  ● Efforts to minimize the transmission of all food-borne pathogens by insistence on good hygiene practices on farms, in abattoirs, during distribution and marketing of food and in food preparation.    First description of ● The presence of integrons in some unusual serotypes (Kapemba,  Goettingen  and S. Sub. I [9,12: lv:-] ● The emergence of S. enterica isolates harbouring atypical sul3-associated integrons, not previously reported in Spain,  that are not detected by conventional methods.  ● The emergence of S. Typhimurium isolates possessing a particular plasmid-borne integron (2 Kb/ bla oxa-1 –aadA1) which nowadays have became endemic in Spain and have  displaced the pandemic SGI1 isolates. ● Most isolates of both types belonged to a predominant clone and shared the same resistance phenotype except for resistance to nitrofurantoin (related with fraudulent use of nitrofurans in animal husbandry?).  22 FUTURE PROJECTS  Going on with surveillance of local antimicrobial resistance  and underlying mechanisms and  epidemiology ● Follow of resistance trends ● Alert for new resistance mechanisms  ● Identify risk factors for antimicrobial       resistance  ● Design and evaluate interventions to     control antimicrobial resistance  Extending the scope of our research on resistance mechanisms ● Transferable quinolone resistance ● Characterization of an SHV-derived β-lactamase conferring an atypical resistance phenotype.    23 THE TEAM 24 Facultatius Servei Anàlisis Clíniques (laboratori microbiogia) Hospital Verge de la Cinta: Mª Isabel Llovet, Anna Jardí, Mar Olga Pérez.  Residents: Isabel Fort, Ester Picó, Jesús Grande. DUES i TEL: Pilar Cid, Carmen López,Rosa Moreno. Assessorament i suport tècnic: Joaquim Ruiz (CRESIB. IDIBAPS). Carmen Torres (CIBIR. Universidad La Rioja). Ex-colaboradors: Gloria Pous, Guillermina Romera, Manel Panisello, Conchita Rubio, Marta Pérez, Mireia Carulla, Xavier Tejedor, Mª José Centelles, María Cortell, Mercé Escobedo, Rosa Gisbert, Joan Zaragoza.  25 Moltes gràcies 

Integron-Related Antimicrobial Resistance in Nosocomial and Community Acquired Infections

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Integron-Related Antimicrobial Resistance in Nosocomial and Community Acquired Infections

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Scientia, Dipòsit d’Informació Digital del Departament de Salut