Resumo: A produção de beta-lactamase de espectro estendido (ESBL) em membros da família Enterobacteriaceae pode conferir resistência a cefalosporinas de amploespectro, aztreonam e penicilinas. Devido a esse fenômeno, a detecção exata dos produtores de ESBL é essencial para a seleção apropriada da antibioticoterapia. Para detectar a produção de beta-lactamase de espectro estendido (ESBL) em bacilos Gram-negativos, foi usado um teste de triagem com os discos de aztreonam (ATM), ceftazidima (CAZ), cefotaxima (CTX) e ceftriaxona (CRO) sobre 300 cepas, das quais trinta e cinco eram suspeitas da presença de ESBL. A produção de ESBL foi demonstrada por três métodos fenotípicos confirmatórios de fácil utilização. Os três testes fenotípicos para confirmar a produção de ESBL incluíram o teste do sinergismo (double disk), E-test? ESBL e disco combinado. Os discos utilizados no teste do sinergismo e do disco combinado foram: aztreonam (30?g-ATM), cefotaxima (30?g-CTX), ceftazidima (30?g-CAZ), cefpodoxima (10?g-CPD) ceftriaxone (30?g-CRO) e amoxicilina+ácido clavulânico(30?g-AMC), cefotaxima+ácido clavulânico (30?g-10?g), ceftazidima+ácido clavulânico (30?g- 10?g), cefpodoxima+ácido clavulânico (10?g-1? g). Para E-test foram utilizadas fitas contendo as cefalosporinas: ceftazidima versus ceftazidima/ácido clavulânico; cefotaxima versus cefotaxima/ácido clavulânico. Os testes fenotípicos confirmaram a presença de ESBL em cinco cepas de enterobactérias (1,66%). Todos os métodos são de fácil execução, contudo o método do Etest requer experiência para interpretar os resultados. Os três testes oferecem uma solução viável para confirmar a produção de ESBL no laboratório clínico. / Abstract: The production of extended spectrum beta-lactamase (ESBL) in the members of the family Enterobacteriaceae can check resistance to cephalosporins of extended-spectrum, aztreonam and penicilins. Due to this phenomenon, the exact detection of the producers of ESBL are essential for the appropriate selection of antimicrobial therapy. To detect the production of extended spectrum beta-lactamase (ESBL) in Gram-negative bacilli, a test of screening was used with the discs of aztreonam (ATM), ceftazidime (CAZ), cefotaxime (CTX) e ceftriaxone (CRO) in 300 strains, of which thirty-five were suspicious of the presence of ESBL. The production of ESBL was demonstrated by three phenotypic methods confirmed of easy utilization. The three phenotypic tests to confirm the production of ESBL included the test of sinergy (double disk), E-test? ESBL and combination disk. The disks used on the test sinergy and the combination disk were: aztreonam (30?g-ATM), cefotaxime (30?g-CTX), ceftazidime (30?g-CAZ), cefpodoxime (10?g-CPD) ceftriaxone (30?g- CRO) e amoxicillin+clavulanic acid (30?g-AMC), cefotaxime+clavulanic acid (30?g- 10? g), ceftazidime+clavulanic acid (30?g-10? g), cefpodoxime+clavulanic acid (10?g- 1? g). For E-test, were utilized strips containing the cephalosporins: ceftazidime and ceftazidime/clavulanic acid; cefotaxime and cefotaxime/clavulanic acid. The phenotypic tests confirmed the presence of ESBL in five strains Enterobacteriaceae (1,66%). All of the methods are of easy execution; however, the method of Etest requires experiment to interpret the results. The three tests offer a viable solution to confirm the production of ESBL on a clinic laboratory. / Orientador: Elisabeth Loshchagin Pizzolitto / Coorientador: Antonio Carlos Pizzolitto / Banca: Wilton Rogério Lustri / Banca: Izabel Yoko Ito / Mestre

Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-08-15Bitstream added on 2014-06-13T18:48:56Z : No. of bitstreams: 1 rodrigues_lo_me_arafcf.pdf: 364674 bytes, checksum: 25ad80987f7d2eb4d8bf537154a09922 (MD5) / Universidade Estadual Paulista (UNESP) / A produção de beta-lactamase de espectro estendido (ESBL) em membros da família Enterobacteriaceae pode conferir resistência a cefalosporinas de amploespectro, aztreonam e penicilinas. Devido a esse fenômeno, a detecção exata dos produtores de ESBL é essencial para a seleção apropriada da antibioticoterapia. Para detectar a produção de beta-lactamase de espectro estendido (ESBL) em bacilos Gram-negativos, foi usado um teste de triagem com os discos de aztreonam (ATM), ceftazidima (CAZ), cefotaxima (CTX) e ceftriaxona (CRO) sobre 300 cepas, das quais trinta e cinco eram suspeitas da presença de ESBL. A produção de ESBL foi demonstrada por três métodos fenotípicos confirmatórios de fácil utilização. Os três testes fenotípicos para confirmar a produção de ESBL incluíram o teste do sinergismo (double disk), E-test? ESBL e disco combinado. Os discos utilizados no teste do sinergismo e do disco combinado foram: aztreonam (30?g-ATM), cefotaxima (30?g-CTX), ceftazidima (30?g-CAZ), cefpodoxima (10?g-CPD) ceftriaxone (30?g-CRO) e amoxicilina+ácido clavulânico(30?g-AMC), cefotaxima+ácido clavulânico (30?g-10?g), ceftazidima+ácido clavulânico (30?g- 10?g), cefpodoxima+ácido clavulânico (10?g-1? g). Para E-test foram utilizadas fitas contendo as cefalosporinas: ceftazidima versus ceftazidima/ácido clavulânico; cefotaxima versus cefotaxima/ácido clavulânico. Os testes fenotípicos confirmaram a presença de ESBL em cinco cepas de enterobactérias (1,66%). Todos os métodos são de fácil execução, contudo o método do Etest requer experiência para interpretar os resultados. Os três testes oferecem uma solução viável para confirmar a produção de ESBL no laboratório clínico. / The production of extended spectrum beta-lactamase (ESBL) in the members of the family Enterobacteriaceae can check resistance to cephalosporins of extended-spectrum, aztreonam and penicilins. Due to this phenomenon, the exact detection of the producers of ESBL are essential for the appropriate selection of antimicrobial therapy. To detect the production of extended spectrum beta-lactamase (ESBL) in Gram-negative bacilli, a test of screening was used with the discs of aztreonam (ATM), ceftazidime (CAZ), cefotaxime (CTX) e ceftriaxone (CRO) in 300 strains, of which thirty-five were suspicious of the presence of ESBL. The production of ESBL was demonstrated by three phenotypic methods confirmed of easy utilization. The three phenotypic tests to confirm the production of ESBL included the test of sinergy (double disk), E-test? ESBL and combination disk. The disks used on the test sinergy and the combination disk were: aztreonam (30?g-ATM), cefotaxime (30?g-CTX), ceftazidime (30?g-CAZ), cefpodoxime (10?g-CPD) ceftriaxone (30?g- CRO) e amoxicillin+clavulanic acid (30?g-AMC), cefotaxime+clavulanic acid (30?g- 10? g), ceftazidime+clavulanic acid (30?g-10? g), cefpodoxime+clavulanic acid (10?g- 1? g). For E-test, were utilized strips containing the cephalosporins: ceftazidime and ceftazidime/clavulanic acid; cefotaxime and cefotaxime/clavulanic acid. The phenotypic tests confirmed the presence of ESBL in five strains Enterobacteriaceae (1,66%). All of the methods are of easy execution; however, the method of Etest requires experiment to interpret the results. The three tests offer a viable solution to confirm the production of ESBL on a clinic laboratory.

Enterobacteriaceae belong to the normal enteric flora in humans and may cause infections. Escherichia coli is the leading urinary tract pathogen with septicaemic potential, whereas Klebsiella pneumoniae causes opportunistic infections and often outbreaks in hospital settings. Beta-lactams are the first choice for treatment of infections caused by Enterobacteriaceae, and might be destroyed by extended-spectrum beta-lactamases, ESBLs. ESBLs hydrolyse all beta-lactams except cephamycin and carbapenems, and constitute a large heterogeneous group of enzymes with different origins. The phenotypic and molecular characteristics of a K. pneumoniae strain causing a major outbreak at Uppsala University Hospital between 2005 and 2008 were described. The strain was multiresistant and produced CTM-M-15, a common ESBL type in Europe. Due to the lack of obvious epidemiological links between patients, a case-control study was performed, which identified risk factors for the acquisition of the outbreak strain in urine cultures. The complex chain of transmission facilitated by patient overcrowding and the interventions applied to curb the outbreak, was revealed in the subsequent study. In the final study, the genetic background of the observed increase in ESBL-producing E. coli isolates during the K. pneumoniae outbreak was explored. The utility of six typing methods in epidemiological investigations of a local outbreak with ESBL-producing E. coli was compared. The increase of ESBL-producing E. coli isolates was not secondary to the K. pneumoniae outbreak. Twentytwo per cent belonged to the epidemic O25b-ST131 clone and only a limited number of infections were caused by nosocomial transmission. ESBL-producing Enterobacteriaceae are a challenge to clinical microbiology laboratories and infection control teams. To investigate their dissemination, typing methods need to be continuously adapted to the current situation. Proper hand disinfection and structural key problems such as over-crowding, under-staffing, lack of single rooms and bathrooms must be adressed to limit transmission.

In the first part of my thesis I focus on mapping problems associated with antibiotic therapy and subsequent development of antibiotic resistance. Tracking resistance is based primarily on data collection and evaluation of the results set sensitivity from around the world. Antibiotic resistance is a natural phenomenon that can be observed in the evolution of microbes as one of the mechanisms of adaptation to new conditions in the environment. For this work I have chosen the following research questions. Do the incidence of ESBL strains in the České Budějovice Hospital a.s. increase over time? Are these values comparable to those achieved in another region, namely in Moravian hospitals the University Hospital of Olomouc, Ostrava University Hospital and Regional University Hospital of T. Bata in Zlin? The data collection I made in collaboration with the laboratory technicians and doctors at Hospital?s Bacteriology Laboratory in České Budějovice. Bacteries tested for the detection of ESBL production originated from biological materials, witch came from patients of hospital in České Budějovice. The first objective was to compare the results achieved in the České Budějovice Hospital in the period of 2007 to 2012. If we look at the total number of ESBL strains that have been isolated since 2007, values have upward trend. While in 2007 there were only 64 strains a year later, the number more than doubled. In 2010, the value soared to 281 tribes and in the year 2012, the number was 321 tribes. The incidence of ESBL strains in 2007 increased about five times. In the long term we can say the numbers have increasing tendency and the range of each species in the production of ESBL has significantly changed. In 2007, it was K. pneumoniae strains that dominated the statistics, but over time the strains of E. coli came forefront. Values of 2012 suggest that the presence of ESBL strains of K. pneumoniae is again almost equal to the number of E. coli strains. The second objective was to compare the results of the 2012 with study of the Prevalence of ESBL-positive Enterobacteriaceae in large Moravian hospitals. In the general overview of ESBL producers values in Hospital České Budějovice (5.23%) are comparable to those in Ostrava (4.9%) and in Zlín (4.3%). Number of strains in the Hospital in Olomouc (11.8%) is about twice as high as the numbers in České Budějovice. In this comparison the České Budějovice Hospital is one of the hospitals with a lower incidence of ESBL producers. The České Budějovice Hospital is below the national average, which originate from an elaborate system of care for patients with colonization or infection with ESBL strains, and from therapy control system using antibiotic center. These results may serve to the Hospital in České Budějovice for statistical purposes, and also for proposals for improving patient care. In the discussion, I pointed out the danger of the spread of resistant strains of bacteria in the community and also the associated risks that mentioned bacteria mean for patients injured in mass accidents or disasters. In these cases, number of infections including ESBL producers can penetrate through open wounds into the affected body. Unlike conventional sensitive bacteria those strains are resistant to commonly used antibiotics and thereby endanger the lives of people affected by the accident.

The University Hospital of Liège has 955 beds in 8 intensive care units, 15 medical wards, 10 surgical wards and 1 paediatric ward. Approximately 36,000 patients are admitted each year, giving a total of 265,000 patient-days hospitalization. Extended-spectrum beta-lactamase-producing Enterobacteriaceae (E-ESBL) constitute, along with methicillin-resistant Staphylococcus aureus (MRSA), the main multi-resistant bacteria recovered in our hospital. The aims of the present study were to: - evaluate the epidemiology of E-ESBL - evaluate the impact of an infection control programme to reduce the spread of E-ESBL in the University Hospital of Liège. In order to do this, several studies were carried out between 2001 and 2007: 1. Determination of the high risk units in the CHU (2001) The high risk units were determined by comparing the incidence rates of each type of unit. Two types of high risk unit were identified in this way: the Intensive Care Units (ICUs) and the Onco-Haematology Unit. 2. Epidemiology of E-ESBL in the Onco-Haematology unit (2002-2003 and 2005-2006) Digestive tract colonization by E-ESBL was found to be relatively high (7.3%) and this explains the high incidence of E-ESBL in Onco-Haematology in comparison with the rest of the hospital. However, the clinical gravity associated with exposure to the risk factor (digestive tract colonization by E-ESBL) was found to be relatively weak. 3. Importance of digestive tract colonization by E-ESBL in General ICUs (2002-2003) Digestive tract colonization by E-ESBL was found to be relatively common (8.8%) and faecal carriage of E-ESBL was found to be a good marker for infection with E-ESBL at another body site. Even though the number of infected patients was found to be low, the risk of infection due to E-ESBL was multiplied by 14.7 in a group of digestive carriers of E-ESBL with regard to a group of non carriers. Our data also showed that Enterobacter aerogenes is the most frequent species producing extended-spectrum beta-lactamase (ESBL) and that TEM-24 is the most prevalent ESBL produced by E-ESBL species in our ICUs. No CTX-M-type genes were identified. With regard to antibiotic susceptibility, meropenem and cefepime appeared to be the most active agents against the majority of isolates. 4. Impact of an infection control programme to reduce the spread of E-ESBL (2006-2007) A surveillance programme was carried out to evaluate the implementation of infection control procedures including surveillance of ESBL-producing strains, utilization of computer alerts for E-ESBL positive patients and the application of contact precautions for colonized or infected patients. Infection control compliance observations were performed by trained referring nurses. During the 2 years of application, one or more E-ESBL were identified in 500 patients. A total of 2268 internal messages regarding the identification of E-ESBL were sent within the hospital, among which 91.84 % were received (at least 1 for every patient). An alert was associated with 406 patients, who were always hospitalized as the identification of the E-ESBL by the laboratory was obtained. A total of 257 registration forms were filled in by the referring nurses, resulting in a survey compliance of 63%. This survey showed that door signs identifying positive patients, hydro-alcoholic solution and gloves were present in 90% of the cases, but that gowns were only present in 59%. The overall incidence of nosocomial acquisition of E-ESBL between 2006 and 2007 was 0.92/1000 patient-days, more or less the same as in 2002. In relation to this research, several questions remain: - Even though the rates of digestive tract colonization with E-ESBL in the 2 types of high risk unit were found to be more or less the same (7.3 and 8.8%), the impact on infections due to E-ESBL was very different. - Are the infections due either to E-ESBL endogenous infections (owing notably to the use of broad spectrum antibiotics) or to secondary infections (resulting from cross-transmission) or to both? The implementation of an infection control programme to limit the spread of E-ESBL has been based on the limitation of the cross-transmission of these micro-organisms. An enhanced barrier precautions policy has been in place in our institution for 2 years, and we have seen no erosion in compliance. We should not however lose sight of the fact that, whatever the institutional policy for the management of multi-resistant bacteria, the correct application of standard precautions for all patients is the first measure to limit the cross-transmission of all micro-organisms.

TEM and SHV β-lactamases are the most prevalent β-lactamases among Gram-negative bacteria. The introduction and widespread use of expanded-spectrum antibiotics, particularly third generation cephalosporins, has led to the evolution of bacterial strains expressing extended spectrum β-lactamases (ESBLs). ESBLs emerge by genetic point mutation from non-extended spectrum precursors. It was found that multiple β-lactamase families within single isolates complicate the process of detecting the resistance status of isolate using non-quantitative DNA diagnostic methods. Preliminary phenotypic characterisation of probable β-lactamase enzyme family types present in 100 isolates from the Asia-Pacific and South African locales showed that single isolates frequently contained multiple β-lactamase families. SHV, TEM, AMPC and CTX-M β-lactamase families were detected in these isolates using PCR detection methods. Ninety-eight percent of all isolates tested contained as least one β-lactamase gene, with up to four to β-lactamase gene families found to co-exist in single isolates. Kinetic PCR methods for interrogating the polymorphic sites at blaSHV codons 238 & 240 and blaTEM codons 164, 238, 240 as well as promoter polymorphism were developed. A high proportion of blaSHV 238 and 240 mutant alleles was found to correlate with cefotaxime, ceftazidime and aztreonam resistance levels. In an attempt to understand the molecular basis for the co-existence of multiple blaSHV alleles within single isolates, the blaSHV promoter region was cloned from one ESBL expressing isolate. Experimental results showed that blaSHV can exist downstream of two different promoters within a single isolate. Both promoters have previously been reported, and differ by the presence or absence of IS26, which results in a change in the transcription initiation site. The blaSHV gene copy numbers in cis with the different promoters were measured, and it was found that the copy number of the IS26::blaSHV promoter was positively correlated with resistance levels. Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with promoters in individual isolates. However, mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL+ isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL+ phenotype. To further confirm the role of IS26 in resistance acquisition, ESBL negative isolates were subjected to serial passage in vitro evolution experiments and fluctuation assays. Results confirm that the insertion of the IS26 element upstream of blaSHV is positively correlated with the ability to exhibit an ESBL phenotype, when such isolates also contain the critical G238S substitution. It was also found that IS26 can catalyse the duplication and mobilisation of blaSHV within an isolate. Fluctuation experiments have shown that the frequency at which such genomic events occur resulting in ESBL phenotypes is extremely low and requires many generations of selection under sub-lethal conditions. A survey of a geographically diverse set of isolates has shown that IS26-blaSHV was found in all of the bacterial populations surveyed. However, it does not appear to be exclusively associated with SHV-mediated ESBL production.