Over time, “superbugs,” or bacteria that have become resistant to antibiotics, have become a great concern in modern medicine. Viable alternates are currently being looked into as effective and safe ways to prevent or treat infections caused by these superbugs. One such method is through the utilization of the second messenger molecule cyclic-di-GMP (c-di-GMP) that has been shown to regulate phenotypes within other bacteria that may control surface colonization in Acinetobacter baumannii. Through a series of experiments, the active enzymes that create c-di-GMP - diguanylate cyclases - and break down c-di- GMP - phosphodiesterases - have been inactivated in mutants to test phenotypes including biofilm formation, motility, antibiotic resistance, and desiccation survival. The research’s objective is to show that manipulation of c-di-GMP within the multi-drug resistant strain of Acinetobacter baumannii may serve as a means to control this bacteria.

Bibliography / Thesis (MMed (Pathology. Medical Microbiology))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Acinetobacter baumannii has emerged as one of the most troublesome nosocomial pathogens globally. This organism causes infections that are often extremely difficult to treat because of the widespread resistance to the major antibiotic groups. Colonization or infection with multidrugresistant A. baumannii is associated with the following risk factors: prolonged hospital stay, admission to an intensive care unit (ICU), mechanical ventilation, and exposure to broad spectrum antibiotics, recent surgery, invasive procedures, and severe underlying disease. A. baumannii has been isolated as part of the skin flora, mostly in moist regions such as axillae, groin and toe webs. It has also been isolated from the oral cavity and respiratory tract of healthy adults. Debilitated hospitalized patients have a high rate of colonization, especially during nosocomial Acinetobacter outbreaks. This organism is an opportunistic pathogen as it contains few virulence factors. Clinical manifestations of A. baumannii include nosocomial pneumonia, nosocomial bloodstream infections, traumatic battlefield and other wound infections, urinary tract infections, and post-neurological surgery meningitis. Fulminant community-acquired pneumonia has recently been reported, indicating that this organism can be highly pathogenic. The number of multidrug-resistant A. baumannii strains has been increasing worldwide in the past few years. Therefore the selection of empirical antibiotic treatment is very challenging. Antibiotic combinations are used mostly as empirical therapy in critically ill patients. One rationale for the use of combination therapy is to achieve synergy between agents. The checkerboard and time-kill methods are two traditional methods that have been used for synergy testing. These methods are labor intensive, cumbersome, costly, and time consuming. The E-test overlay method is a modification of the E-test method to determine synergy between the different antibiotics. This method is easy to perform, flexible and time efficient. The aim of this study was to assess the in vitro activity of different combinations of colistin, rifampicin, imipenem, and tobramycin against selected clinical strains of A. baumannii using the checkerboard and the E-test synergy methods. The MICs obtained with the E-test and broth microdilution method were compared. The results of the disk diffusion for imipenem and tobramycin as tested in the routine microbiology laboratory were presented for comparison. Overall good reproducibility was obtained with all three methods of sensitivity testing. The agreement of MICs between the broth dilution and E-test methods was good with not more than two dilution differences in MIC values for all isolates, except one in which the rifampicin E-test MIC differed with three dilutions from the MIC obtained with the microdilution method. However, the categorical agreement between the methods for rifampicin was poor. Although MICs did not differ with more than two dilutions in most cases, many major errors occurred because the MICs clustered around the breakpoints. The combinations of colistin + rifampicin, colistin + imipenem, colistin + tobramycin, rifampicin + tobramycin, and imipenem + tobramycin all showed indifferent or additive results by the E-test method. No results indicating synergy were obtained for all the above-mentioned combinations. There was one result indicating antagonistic effect for the combination of colistin + tobramycin. The results of the checkerboard method showed results indicating synergy in four of the six isolates for which the combination of colistin and rifampicin was tested. The other two isolates showed indifferent/additive results. All the other combinations showed indifferent/additive results for all isolates except isolate 30 (col + tob) and isolate 25 (rif + tob) which showed synergism. No antagonistic results were observed by the checkerboard method. When the results obtained with the E-test and checkerboard methods were compared, it was noted that for most antibiotic combinations an indifferent/additive result was obtained. However, for the colistin + rifampicin combination, the checkerboard method showed synergism for 4 of 6 isolates, whereas the E-test method showed indifference and an additive result in one. For the rifampicin + tobramycin, and colistin + tobramycin combinations, synergism was also shown with the checkerboard method in one isolate for each combination. The E-test method however showed an indifferent and additive result respectively. . The E-test method was found to be a rapid, reproducible, easy-to-perform, and flexible method to determine synergistic antibiotic activity. This study was however limited by low numbers of isolates. This might explain why no synergistic results were obtained with the E-test method and few synergistic results with the checkerboard method. Genotypic analysis using pulse-field gel electrophoresis (PFGE) may be considered in future studies to determine relatedness of the isolates which will facilitate the selection of different strains for synergy testing. Furthermore, clinical studies are needed to establish whether in vitro synergy testing is useful in the clinical setting and whether the results of synergy testing will have any bearing on the clinical outcome of patients infected with multidrug resistant A. baumannii. / AFRIKAANSE OPSOMMING: Acinetobacter baumannii het wêreldwyd as een van die mees problematiese nosokomiale patogene verskyn. Hierdie organisme veroorsaak infeksies wat dikwels baie moeilik is om te behandel weens wydverspreide weerstandigheid teen major antibiotikagroepe. Kolonisasie of infeksie met multi-weerstandige A. baumannii word geassosieer met die volgende riskofaktore: verlengde hospitaalverblyf, toelating tot ‘n intensiewe sorgeenheid (ICU), meganiese ventilasie, blootstelling aan breëspektrum antibiotika, onlangse chirurgie, indringende prosedures en ernstige onderliggende siekte. A. baumannii kan deel vorm van die normale velflora, veral in die axillae, inguinale area en tussen die tone. Dit is ook al vanuit die mondholte en die respiratoriese traktus van gesonde volwassenes geïsoleer. Verswakte gehospitaliseerde pasiënte word veral gekoloniseer gedurende nosokomiale Acinetobacter uitbrake. Hierdie organisme is ‘n opportunistiese patogeen en bevat min virulensie faktore. Kliniese manifestasies van A. baumannii sluit nosokomiale pneumonie, nosokomiale bloedstroom infeksies, troumatiese slagveld- en ander wondinfeksies, urienweginfeksies en meningitis wat volg op neurologiese chirurgie in. Fulminerende gemeenskapsverworwe pneumonie is onlangs beskryf en dui aan dat hierdie organisme hoogs patogenies kan wees. Die aantal multi-weerstandige A. baumannii stamme het wêreldwyd toegeneem oor die laaste paar jare. Daarom is die seleksie van empiriese antibiotiese behandeling ‘n uitdaging. Antibiotika kombinasies word meestal as empiriese behandeling in ernstige siek pasiënte gebruik. Die beginsel hiervan is om sinergistiese werking tussen agente te verkry. Die “checkerboard” en “time-kill” metodes is twee tradisionele metodes van sinergisme toetsing. Hierdie metodes is werksintensief, duur en tydrowend. Die E-toets sinergisme metode is gebaseer op die E-toets metode. Hierdie metode is maklik, buigbaar en tydseffektief. Die doel van hierdie studie was om die in vitro aktiwiteit tussen verskillende antibiotika kombinasies van colistin, rifampisien, imipenem, en tobramisien teen geselekteerde kliniese A. baumannii isolate te toets met die “checkerboard” en E-toets sinergisme toetsing metodes. Die minimum inhibitoriese konsentrasies (MIKs) verkry met die E-toets en “broth microdilution” metode is ook vergelyk. Die resultate van die skyfie diffusie metode (die metode wat in die roetiene mikrobiologie laboratorium gebruik word) vir imipenem en tobramisien word ook verskaf vir vergelyking van die resultate van verskillende sensitiwiteitsmetodes. In oorsig is goeie herhaalbaarheid van resultate verkry met al drie metodes van sensitiwiteitstoetsing. Die ooreenstemming van MIKs tussen die “broth dilution” en E-toets metodes was goed en resultate het met nie meer as twee verdunnings in MIK waardes verskil nie. Daar is een uitsondering waar die rifampisien E-toets MIK waarde met drie verdunnings van die MIK waarde verkry met die “microdilution” metode verskil. Die ooreenstemming tussen die sensitiwiteitskategorie resultate tussen die twee metodes was egter swak vir rifampisien. Alhoewel die MIKs in die meeste gevalle met nie meer as twee verdunnings in waarde verskil het nie, was daar baie major foute aangetoon omdat die MIKs rondom die breekpunte geval het. Die kombinasies van colistin + rifampisien, colistin + imipenem, colistin + tobramisien, rifampisien + tobramisien, en imipenem + tobramisien het oorwegend slegs matige interaksie met die E-toets metode getoon. Geen sinergisme is verkry met enige van die antibiotika kombinasies met hierdie metode nie. Daar was egter een resultaat wat antagonisme getoon het vir die kombinasie van colistin + tobramycin. Die resultate van die “checkerboard” metode het sinergisme getoon in vier van die ses isolate wat vir die kombinasie van colistin en rifampisien getoets was. Die ander twee isolate het slegs matige interaksie getoon. Al die ander kombinasies het ook slegs matige interaksie getoon, behalwe in isolaat 30 (col + tob) en isolaat 25 (rif + tob) waar die spesifieke kombinasies sinergisme getoon het. Geen antagonisme is waargeneem met die “checkerboard” metode nie. Met vergelyking van die E-toets en “checkerboard” metodes, is dit opmerklik dat vir die meeste van die antibiotika kombinasies slegs matige interaksie verkry is. Vir die colistin + rifampisien kombinasie toon die “checkerboard” metode egter sinergisme vir 4 uit 6 isolate, terwyl die E-toets metode slegs matige interaksie toon. Vir rifampisien + tobramisien, en colistin + tobramisien kombinasies is sinergisme getoon met die “checkerboard” metode in een isolaat vir elke kombinasie. Die E-toets metode het slegs matige interaksie getoon. Die E-toets sinergisme metode was vinnig, herhaalbaar en maklik om uit te voer. Hierdie studie word egter beperk deur lae getalle van isolate. Dit mag verklaar waarom geen sinergistiese resultate met die E-toets metode verkry is nie en die min sinergistiese resultate met die “checkerboard” metode. Genotipiese analiese met “pulse-field gel electrophoresis” mag in aanmerking geneem word in toekomstige studies om die verwantskap tussen isolate te bepaal wat die seleksie van verskillende stamme vir sinergisme toetsing sal vergemaklik. Verder, kliniese studies is nodig om te bepaal of in vitro sinergisme toetsing van waarde is en of die resultate van sinergisme toetsing ‘n rol speel in die kliniese uitkoms van pasënte geïnfekteer met multiweerstandige A. baumannii. / The National Health Laboratory Serivice

Contexto: O Acinetobacter spp. é um cocobacilo gram-negativo, considerado patógeno oportunista e de grande importância nas infecções hospitalares. Estão envolvidos em amplo espectro de infecções nosocomiais, incluindo bacteremia, meningite secundária e infecção do trato urinário, mas sua maior prevalência é como agente de pneumonia associada à ventilação mecânica em pacientes internados em unidades de terapia intensiva (UTIs); podendo ocasionar um agravamento do quadro clínico e o óbito desses pacientes. Considera-se como um patógeno de baixa virulência, podendo permanecer sobre a pele ou dentro do corpo humano sem causar doença. A disseminação pelas mãos dos profissionais de saúde geralmente não é detectada e quando as infecções pelo Acinetobacter tornam-se aparentes o número de pacientes colonizados é, provavelmente, muito elevado. Assim sendo, as precauções para prevenir um surto tornam-se tardias. Estudos prévios indicaram como fatores de risco para aquisição de infecção por Acinetobacter a gravidade da doença dos pacientes, uso prévio de antimicrobiano, número de dias com procedimento invasivo, tempo de permanência no hospital, contaminação ambiental. Os fatores de risco associados à mortalidade de pacientes com A. baumannii ainda não foram totalmente elucidados pela literatura, mas a idade, colonização prévia por esta bactéria, neutropenia, escore de gravidade APACHE II (Acute Physiology and Chronic Health Evaluation) elevado, procedimentos como ventilação mecânica, terapia antimicrobiana inapropriada são apontados como alguns dos fatores relacionados. Objetivos: Caracterizar a mortalidade atribuível a infecções causadas por Acinetobacter baumannii resistente à carbapenêmicos (CRAB) em um surto no 13 Centro de Terapia Intensiva adulto de um hospital universitário. Métodos: Foi realizado um estudo de coorte retrospectivo pareado como parte da investigação do surto de pacientes no Centro de Tratamento Intensivo (CTI) Adulto infectados com a bactéria Acinetobacter baumannii apresentando resistência à carbapenêmicos. Os pacientes foram selecionados entre 01/01/2007 a 31/07/2008 e foram considerados como casos os pacientes com cultura positiva para CRAB. Os controles foram pacientes internados no CTI no mesmo período que os casos, mas que não apresentaram infecção na qual foi isolada a presença da bactéria em questão. Os fatores avaliados como possível associação com o risco de mortalidade foram avaliados. Determinou-se a mortalidade atribuível a infecções causadas por CRAB e através da curva de sobrevivência avaliou-se essa distribuição entre casos e controles. Resultados: Foram selecionados 90 pacientes como casos e 179 pacientes pareados como controles. A média de idade, as proporções de pacientes com Escore de Chalson ³ 2, de pacientes internados não eletivamente, as reinternações e a freqüência de realização de cirurgias foram muito semelhantes entre os grupos estudados. Entre os casos, houve maior proporção de pacientes transferidos de outro hospital (P<0,001), internados em área contígua à presença de casos de colonização ou infecção por CRAB (P<0,001), de pacientes submetidos a alimentação parenteral (P<0,001); ventilação mecânica (P<0,001), cateteres urinários (P=0,031), cateteres para acesso vascular central (P=0,006) e cateteres para hemodiálise (P<0,001) comparativamente aos controles. Da mesma maneira, casos apresentaram maior freqüência de exposição prévia a antimicrobianos, comparativamente aos controles: penicilinas (P<0,001), cefalosporinas de 1ª e/ou 2ª gerações (P<0,001), carbapenêmicos (P<0,001), aminoglicosídeos (P=0,046), quinolonas (P=0,004) e 14 glicopeptídeos (P=0,001). Os casos apresentaram tempo médio de internação superior aos controles, incluindo duração total da internação (P=0,002), permanência na CTI (P<0,001) e permanência na CTI antes da infecção por CRAB (P=0,03). O escore de APACHE II por ocasião da admissão no CTI também teve média significativamente maior entre os casos comparativamente aos controles (P<0,001). Houve diferença na taxa de mortalidade bruta intra-hospitalar entre casos e controles, respectivamente, 58,9% (53/90) e 36,9% (66/179) (P=0,001). A mortalidade atribuível foi 22% (IC 95%; 8,8%-35,2%) e as curvas de sobrevivência cumulativa para casos e controles não apresentaram diferença significativa entre os grupos (P=0,207; log rank test) A análise multivariável indica que pacientes com escore de APACHE II maiores e que mais freqüentemente foram submetidos a procedimentos invasivos como ventilação mecânica, suporte nutricional (dieta parenteral) e que permaneceram um período maior no hospital estiveram mais propensos a risco de mortalidade associada à infecção por CRAB. Conclusões: Nesse estudo os fatores associados com a mortalidade e a taxa de mortalidade atribuível identificados vão ao encontro da literatura e indica que pacientes mais graves estão mais propensos a risco de morte associada à infecção por CRAB. A literatura enfatiza também a necessidade de consistentes estratégias de controle de infecção para prevenir infecções por Acinetobacter multirresistente. A investigação da mortalidade atribuível ao A. baumannii apresenta muitas limitações e ainda não é conclusiva. / Context: Acinetobacter spp. is a bacilli gram-negative considered an opportunistic pathogen and of great importance in nosocomial infections. They are involved in a wide spectrum of nosocomial infections, including bacteremia, secondary meningitis and urinary tract infection, but is prevalent as an agent of mechanical ventilatorassociated pneumonia in patients admitted to intensive care units (ICUs); this factor can lead to an increase morbidity and mortality of these patients. It is considered as a pathogen of low virulence and may remain on or within the human body without causing disease. The spread by the hands of clinical staff is often not detected and when Acinetobacter infections become apparent, the number of colonized patients is probably very high, therefore, precautions to prevent an outbreak are late. Previous studies have observed as risk factors for acquisition of Acinetobacter infection by the disease severity of patients, prior use of antimicrobials, number of days with invasive procedures, length of stay in hospital environmental contamination. Risk factors associated with mortality of patients with A. baumannii have not been fully elucidated in the literature, but showed that age, previous colonization by this bacterium, neutropenia, high severity score APACHE II (Acute Physiology and Chronic Health Evaluation), procedures such as mechanical ventilation, inappropriate antimicrobial therapy as some of the factors related to mortality. Objectives: To characterize attributable mortality to infections caused by Acinetobacter baumannii resistant to carbapenem (CRAB) in an outbreak in the adult intensive care unit of a university hospital. 16 Methods: We performed a matched retrospective cohort as part of outbreak investigation of patients in the ICU adult infected with the bacteria Acinetobacter baumannii exhibiting resistance to carbapenems. Patients were selected from 01/01/2007 to 31/07/2008 and the cases were considered patients with positive culture for CRAB. Controls were patients admitted to the ICU during the same period as cases, but showed no infection in which was isolated the presence of the bacterium in question. Factors evaluated as possible association with the risk of mortality were evaluated. Determined the attributable mortality to infections caused by CRAB and through the survival curve was evaluated this distribution between cases and controls. Results: 90 patients were selected as cases and 179 patients matched as controls. The average age, the proportions of patients with a Chalson score ³ 2 from inpatients not elective, the frequency of hospitalizations and surgeries were similar among studied groups. Among the cases, a greater proportion of patients transferred from another hospital (P <0.001), admitted in an area contiguous to the presence of cases of colonization or infection by CRAB (P <0.001) in patients undergoing parenteral nutrition (P <0.001) ; mechanical ventilation (P <0.001), urinary catheters (P = 0.031), central catheters for vascular access (P = 0.006) and catheters for hemodialysis (P <0.001) compared to controls. Likewise, cases had higher frequency of prior exposure to antimicrobials, compared with controls: penicillin (P <0.001), cephalosporins of 1st and / or 2nd generation (P <0.001), carbapenems (P <0.001), aminoglycosides (P = 0.046), quinolones (P = 0.004) and glycopeptides (P = 0.001). The cases presented mean length of stay higher than controls, including total duration of hospitalization (P = 0.002), stay in ICU (P <0.001) and stay in the ICU before 17 infection by CRAB (P = 0.03). The APACHE II score on admission to the ICU was also significantly higher average among cases compared with controls (P <0.001). There was a difference in the rate of in-hospital crude mortality among cases and controls, respectively, 58,9% (53/90) e 36,9% (66/179) (P = 0.001). The attributable mortality was 22% (95% CI 8.8% -35.2%) and cumulative survival curves for cases and controls showed no significant difference between groups (P = 0.207, log rank test) Multivariate analysis indicates that patients with APACHE II score higher and more frequently underwent invasive procedures such as mechanical ventilation, nutritional support (parenteral nutrition) and remained a longer period in hospital were more likely to risk of mortality associated with infection by CRAB. Conclusions: In this study the factors associated with mortality and the attributable mortality rate identified are in line with the literature and indicates that more severe patients are more prone to risk of mortality associated with infection by CRAB. The literature also emphasizes the need for consistent infection control strategies to prevent infection by multidrug resistant Acinetobacter. The investigation of attributable mortality to A. baumannii has many limitations and is not conclusive yet.

Introdução: As polimixinas são consideradas as opções terapêuticas de resgate para o tratamento das infecções de corrente sanguínea (ICS) por bacilos Gram-negativos (BGN) com resistência aos carbapenens (CR) e a combinação com outro antimicrobiano tem sido utilizada apesar da falta de evidência clínica para esta prática. Objetivo: Avaliar o uso de polimixina B intravenosa em monoterapia e em combinação com outro antimicrobiano para ICS por BGN CR. Pacientes e Métodos: Foi um estudo de coorte retrospectivo em um hospital terciário, incluindo 99 pacientes. A comparação dos tipos terapias foi através do propensity score. Resultados: A mortalidade global em 30 dias foi de 43,4%: 40,7% (24 de 59) e 47,5% (19 de 40), p=0,51, em pacientes que receberam combinação e monoterapia, respectivamente. A sepse grave/choque séptico no dia da ICS, alta pontuação do escore de bacteremia de Pitt e a presença de neoplasia como doença de base foram, independentemente, associados a maior mortalidade em 30 dias no modelo de regressão de Cox. A terapia combinada não foi significativamente associada a este resultado (hazard ratio, 0,70; intervalo de confiança de 95%, 0,36-1,36); p=0,29). Apesar de não ser significativa, houve uma tendência a um efeito benéfico da associação em pacientes com ICS por BGN CR da família Enterobacteriaceae. Não houve diferença no desenvolvimento de insuficiência renal aguda em pacientes que receberam terapia de combinação comparado com os que receberam monoterapia. Conclusão: Não houve diferença na mortalidade em 30 dias em pacientes com ICS por BGN CR tratados com polimixina B em combinação com outro antimicrobiano ou em monoterapia. A prática rotineira de combinar um segundo antibiótico em esquemas baseados em polimixinas, especialmente, se a bactéria apresenta resistência in vitro ao carbapenem, ainda necessita estudos clínicos adicionais. / Background: Polymyxins are usually the last resort therapy for carbapenem-resistant Gram-negative bacteria (CR GNB) bloodstream infections (BSIs), combination with another antimicrobial has been used despite the lack of clinical evidence supporting such practice. Objetive: We aimed to assess the use of intravenous polymyxin B in combination with another antimicrobial in comparison with polymyxin B as a single drug for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. Patient and methods: We compared combination versus monotherapy with polymyxin B for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. It was a retrospective cohort study at a tertiary-hospital including 99 patients. Results: The overall 30-day mortality was 43.4%: 40.7% (24 of 59) and 47.5% (19 of 40), P=0.51, in patients receiving combination and monotherapy, respectively. Severe sepsis/ septic shock at BSI onset higher Pitt bacteremia score and neoplasia were independently associated with higher 30-day mortality in a Cox-regression model. Combination therapy was not significantly associated with this outcome (Hazard Ratio, 0.70; 95% confidence interval, 0.36-1.36); P=0.29). Although not significant, there was a tendency to a beneficial effect of combination in patients with Enterobacteriaceae CR GNB BSIs. There was no difference in development of AKI in patients receiving combination therapy compared to those receiving monotherapy. Conclusions: There was no difference in 30-day mortality in patients with CR GNB BSIs treated with polymyxin B in combination with another antimicrobial compared with polymyxin B alone. The routine practice of combining a second antibiotic in polymyxins-based regimes, especially if the bacteria present in vitro resistance to the agent, still lacks support from clinical studies.

Contexto: O Acinetobacter spp. é um cocobacilo gram-negativo, considerado patógeno oportunista e de grande importância nas infecções hospitalares. Estão envolvidos em amplo espectro de infecções nosocomiais, incluindo bacteremia, meningite secundária e infecção do trato urinário, mas sua maior prevalência é como agente de pneumonia associada à ventilação mecânica em pacientes internados em unidades de terapia intensiva (UTIs); podendo ocasionar um agravamento do quadro clínico e o óbito desses pacientes. Considera-se como um patógeno de baixa virulência, podendo permanecer sobre a pele ou dentro do corpo humano sem causar doença. A disseminação pelas mãos dos profissionais de saúde geralmente não é detectada e quando as infecções pelo Acinetobacter tornam-se aparentes o número de pacientes colonizados é, provavelmente, muito elevado. Assim sendo, as precauções para prevenir um surto tornam-se tardias. Estudos prévios indicaram como fatores de risco para aquisição de infecção por Acinetobacter a gravidade da doença dos pacientes, uso prévio de antimicrobiano, número de dias com procedimento invasivo, tempo de permanência no hospital, contaminação ambiental. Os fatores de risco associados à mortalidade de pacientes com A. baumannii ainda não foram totalmente elucidados pela literatura, mas a idade, colonização prévia por esta bactéria, neutropenia, escore de gravidade APACHE II (Acute Physiology and Chronic Health Evaluation) elevado, procedimentos como ventilação mecânica, terapia antimicrobiana inapropriada são apontados como alguns dos fatores relacionados. Objetivos: Caracterizar a mortalidade atribuível a infecções causadas por Acinetobacter baumannii resistente à carbapenêmicos (CRAB) em um surto no 13 Centro de Terapia Intensiva adulto de um hospital universitário. Métodos: Foi realizado um estudo de coorte retrospectivo pareado como parte da investigação do surto de pacientes no Centro de Tratamento Intensivo (CTI) Adulto infectados com a bactéria Acinetobacter baumannii apresentando resistência à carbapenêmicos. Os pacientes foram selecionados entre 01/01/2007 a 31/07/2008 e foram considerados como casos os pacientes com cultura positiva para CRAB. Os controles foram pacientes internados no CTI no mesmo período que os casos, mas que não apresentaram infecção na qual foi isolada a presença da bactéria em questão. Os fatores avaliados como possível associação com o risco de mortalidade foram avaliados. Determinou-se a mortalidade atribuível a infecções causadas por CRAB e através da curva de sobrevivência avaliou-se essa distribuição entre casos e controles. Resultados: Foram selecionados 90 pacientes como casos e 179 pacientes pareados como controles. A média de idade, as proporções de pacientes com Escore de Chalson ³ 2, de pacientes internados não eletivamente, as reinternações e a freqüência de realização de cirurgias foram muito semelhantes entre os grupos estudados. Entre os casos, houve maior proporção de pacientes transferidos de outro hospital (P<0,001), internados em área contígua à presença de casos de colonização ou infecção por CRAB (P<0,001), de pacientes submetidos a alimentação parenteral (P<0,001); ventilação mecânica (P<0,001), cateteres urinários (P=0,031), cateteres para acesso vascular central (P=0,006) e cateteres para hemodiálise (P<0,001) comparativamente aos controles. Da mesma maneira, casos apresentaram maior freqüência de exposição prévia a antimicrobianos, comparativamente aos controles: penicilinas (P<0,001), cefalosporinas de 1ª e/ou 2ª gerações (P<0,001), carbapenêmicos (P<0,001), aminoglicosídeos (P=0,046), quinolonas (P=0,004) e 14 glicopeptídeos (P=0,001). Os casos apresentaram tempo médio de internação superior aos controles, incluindo duração total da internação (P=0,002), permanência na CTI (P<0,001) e permanência na CTI antes da infecção por CRAB (P=0,03). O escore de APACHE II por ocasião da admissão no CTI também teve média significativamente maior entre os casos comparativamente aos controles (P<0,001). Houve diferença na taxa de mortalidade bruta intra-hospitalar entre casos e controles, respectivamente, 58,9% (53/90) e 36,9% (66/179) (P=0,001). A mortalidade atribuível foi 22% (IC 95%; 8,8%-35,2%) e as curvas de sobrevivência cumulativa para casos e controles não apresentaram diferença significativa entre os grupos (P=0,207; log rank test) A análise multivariável indica que pacientes com escore de APACHE II maiores e que mais freqüentemente foram submetidos a procedimentos invasivos como ventilação mecânica, suporte nutricional (dieta parenteral) e que permaneceram um período maior no hospital estiveram mais propensos a risco de mortalidade associada à infecção por CRAB. Conclusões: Nesse estudo os fatores associados com a mortalidade e a taxa de mortalidade atribuível identificados vão ao encontro da literatura e indica que pacientes mais graves estão mais propensos a risco de morte associada à infecção por CRAB. A literatura enfatiza também a necessidade de consistentes estratégias de controle de infecção para prevenir infecções por Acinetobacter multirresistente. A investigação da mortalidade atribuível ao A. baumannii apresenta muitas limitações e ainda não é conclusiva. / Context: Acinetobacter spp. is a bacilli gram-negative considered an opportunistic pathogen and of great importance in nosocomial infections. They are involved in a wide spectrum of nosocomial infections, including bacteremia, secondary meningitis and urinary tract infection, but is prevalent as an agent of mechanical ventilatorassociated pneumonia in patients admitted to intensive care units (ICUs); this factor can lead to an increase morbidity and mortality of these patients. It is considered as a pathogen of low virulence and may remain on or within the human body without causing disease. The spread by the hands of clinical staff is often not detected and when Acinetobacter infections become apparent, the number of colonized patients is probably very high, therefore, precautions to prevent an outbreak are late. Previous studies have observed as risk factors for acquisition of Acinetobacter infection by the disease severity of patients, prior use of antimicrobials, number of days with invasive procedures, length of stay in hospital environmental contamination. Risk factors associated with mortality of patients with A. baumannii have not been fully elucidated in the literature, but showed that age, previous colonization by this bacterium, neutropenia, high severity score APACHE II (Acute Physiology and Chronic Health Evaluation), procedures such as mechanical ventilation, inappropriate antimicrobial therapy as some of the factors related to mortality. Objectives: To characterize attributable mortality to infections caused by Acinetobacter baumannii resistant to carbapenem (CRAB) in an outbreak in the adult intensive care unit of a university hospital. 16 Methods: We performed a matched retrospective cohort as part of outbreak investigation of patients in the ICU adult infected with the bacteria Acinetobacter baumannii exhibiting resistance to carbapenems. Patients were selected from 01/01/2007 to 31/07/2008 and the cases were considered patients with positive culture for CRAB. Controls were patients admitted to the ICU during the same period as cases, but showed no infection in which was isolated the presence of the bacterium in question. Factors evaluated as possible association with the risk of mortality were evaluated. Determined the attributable mortality to infections caused by CRAB and through the survival curve was evaluated this distribution between cases and controls. Results: 90 patients were selected as cases and 179 patients matched as controls. The average age, the proportions of patients with a Chalson score ³ 2 from inpatients not elective, the frequency of hospitalizations and surgeries were similar among studied groups. Among the cases, a greater proportion of patients transferred from another hospital (P <0.001), admitted in an area contiguous to the presence of cases of colonization or infection by CRAB (P <0.001) in patients undergoing parenteral nutrition (P <0.001) ; mechanical ventilation (P <0.001), urinary catheters (P = 0.031), central catheters for vascular access (P = 0.006) and catheters for hemodialysis (P <0.001) compared to controls. Likewise, cases had higher frequency of prior exposure to antimicrobials, compared with controls: penicillin (P <0.001), cephalosporins of 1st and / or 2nd generation (P <0.001), carbapenems (P <0.001), aminoglycosides (P = 0.046), quinolones (P = 0.004) and glycopeptides (P = 0.001). The cases presented mean length of stay higher than controls, including total duration of hospitalization (P = 0.002), stay in ICU (P <0.001) and stay in the ICU before 17 infection by CRAB (P = 0.03). The APACHE II score on admission to the ICU was also significantly higher average among cases compared with controls (P <0.001). There was a difference in the rate of in-hospital crude mortality among cases and controls, respectively, 58,9% (53/90) e 36,9% (66/179) (P = 0.001). The attributable mortality was 22% (95% CI 8.8% -35.2%) and cumulative survival curves for cases and controls showed no significant difference between groups (P = 0.207, log rank test) Multivariate analysis indicates that patients with APACHE II score higher and more frequently underwent invasive procedures such as mechanical ventilation, nutritional support (parenteral nutrition) and remained a longer period in hospital were more likely to risk of mortality associated with infection by CRAB. Conclusions: In this study the factors associated with mortality and the attributable mortality rate identified are in line with the literature and indicates that more severe patients are more prone to risk of mortality associated with infection by CRAB. The literature also emphasizes the need for consistent infection control strategies to prevent infection by multidrug resistant Acinetobacter. The investigation of attributable mortality to A. baumannii has many limitations and is not conclusive yet.

Contexto: O Acinetobacter spp. é um cocobacilo gram-negativo, considerado patógeno oportunista e de grande importância nas infecções hospitalares. Estão envolvidos em amplo espectro de infecções nosocomiais, incluindo bacteremia, meningite secundária e infecção do trato urinário, mas sua maior prevalência é como agente de pneumonia associada à ventilação mecânica em pacientes internados em unidades de terapia intensiva (UTIs); podendo ocasionar um agravamento do quadro clínico e o óbito desses pacientes. Considera-se como um patógeno de baixa virulência, podendo permanecer sobre a pele ou dentro do corpo humano sem causar doença. A disseminação pelas mãos dos profissionais de saúde geralmente não é detectada e quando as infecções pelo Acinetobacter tornam-se aparentes o número de pacientes colonizados é, provavelmente, muito elevado. Assim sendo, as precauções para prevenir um surto tornam-se tardias. Estudos prévios indicaram como fatores de risco para aquisição de infecção por Acinetobacter a gravidade da doença dos pacientes, uso prévio de antimicrobiano, número de dias com procedimento invasivo, tempo de permanência no hospital, contaminação ambiental. Os fatores de risco associados à mortalidade de pacientes com A. baumannii ainda não foram totalmente elucidados pela literatura, mas a idade, colonização prévia por esta bactéria, neutropenia, escore de gravidade APACHE II (Acute Physiology and Chronic Health Evaluation) elevado, procedimentos como ventilação mecânica, terapia antimicrobiana inapropriada são apontados como alguns dos fatores relacionados. Objetivos: Caracterizar a mortalidade atribuível a infecções causadas por Acinetobacter baumannii resistente à carbapenêmicos (CRAB) em um surto no 13 Centro de Terapia Intensiva adulto de um hospital universitário. Métodos: Foi realizado um estudo de coorte retrospectivo pareado como parte da investigação do surto de pacientes no Centro de Tratamento Intensivo (CTI) Adulto infectados com a bactéria Acinetobacter baumannii apresentando resistência à carbapenêmicos. Os pacientes foram selecionados entre 01/01/2007 a 31/07/2008 e foram considerados como casos os pacientes com cultura positiva para CRAB. Os controles foram pacientes internados no CTI no mesmo período que os casos, mas que não apresentaram infecção na qual foi isolada a presença da bactéria em questão. Os fatores avaliados como possível associação com o risco de mortalidade foram avaliados. Determinou-se a mortalidade atribuível a infecções causadas por CRAB e através da curva de sobrevivência avaliou-se essa distribuição entre casos e controles. Resultados: Foram selecionados 90 pacientes como casos e 179 pacientes pareados como controles. A média de idade, as proporções de pacientes com Escore de Chalson ³ 2, de pacientes internados não eletivamente, as reinternações e a freqüência de realização de cirurgias foram muito semelhantes entre os grupos estudados. Entre os casos, houve maior proporção de pacientes transferidos de outro hospital (P<0,001), internados em área contígua à presença de casos de colonização ou infecção por CRAB (P<0,001), de pacientes submetidos a alimentação parenteral (P<0,001); ventilação mecânica (P<0,001), cateteres urinários (P=0,031), cateteres para acesso vascular central (P=0,006) e cateteres para hemodiálise (P<0,001) comparativamente aos controles. Da mesma maneira, casos apresentaram maior freqüência de exposição prévia a antimicrobianos, comparativamente aos controles: penicilinas (P<0,001), cefalosporinas de 1ª e/ou 2ª gerações (P<0,001), carbapenêmicos (P<0,001), aminoglicosídeos (P=0,046), quinolonas (P=0,004) e 14 glicopeptídeos (P=0,001). Os casos apresentaram tempo médio de internação superior aos controles, incluindo duração total da internação (P=0,002), permanência na CTI (P<0,001) e permanência na CTI antes da infecção por CRAB (P=0,03). O escore de APACHE II por ocasião da admissão no CTI também teve média significativamente maior entre os casos comparativamente aos controles (P<0,001). Houve diferença na taxa de mortalidade bruta intra-hospitalar entre casos e controles, respectivamente, 58,9% (53/90) e 36,9% (66/179) (P=0,001). A mortalidade atribuível foi 22% (IC 95%; 8,8%-35,2%) e as curvas de sobrevivência cumulativa para casos e controles não apresentaram diferença significativa entre os grupos (P=0,207; log rank test) A análise multivariável indica que pacientes com escore de APACHE II maiores e que mais freqüentemente foram submetidos a procedimentos invasivos como ventilação mecânica, suporte nutricional (dieta parenteral) e que permaneceram um período maior no hospital estiveram mais propensos a risco de mortalidade associada à infecção por CRAB. Conclusões: Nesse estudo os fatores associados com a mortalidade e a taxa de mortalidade atribuível identificados vão ao encontro da literatura e indica que pacientes mais graves estão mais propensos a risco de morte associada à infecção por CRAB. A literatura enfatiza também a necessidade de consistentes estratégias de controle de infecção para prevenir infecções por Acinetobacter multirresistente. A investigação da mortalidade atribuível ao A. baumannii apresenta muitas limitações e ainda não é conclusiva. / Context: Acinetobacter spp. is a bacilli gram-negative considered an opportunistic pathogen and of great importance in nosocomial infections. They are involved in a wide spectrum of nosocomial infections, including bacteremia, secondary meningitis and urinary tract infection, but is prevalent as an agent of mechanical ventilatorassociated pneumonia in patients admitted to intensive care units (ICUs); this factor can lead to an increase morbidity and mortality of these patients. It is considered as a pathogen of low virulence and may remain on or within the human body without causing disease. The spread by the hands of clinical staff is often not detected and when Acinetobacter infections become apparent, the number of colonized patients is probably very high, therefore, precautions to prevent an outbreak are late. Previous studies have observed as risk factors for acquisition of Acinetobacter infection by the disease severity of patients, prior use of antimicrobials, number of days with invasive procedures, length of stay in hospital environmental contamination. Risk factors associated with mortality of patients with A. baumannii have not been fully elucidated in the literature, but showed that age, previous colonization by this bacterium, neutropenia, high severity score APACHE II (Acute Physiology and Chronic Health Evaluation), procedures such as mechanical ventilation, inappropriate antimicrobial therapy as some of the factors related to mortality. Objectives: To characterize attributable mortality to infections caused by Acinetobacter baumannii resistant to carbapenem (CRAB) in an outbreak in the adult intensive care unit of a university hospital. 16 Methods: We performed a matched retrospective cohort as part of outbreak investigation of patients in the ICU adult infected with the bacteria Acinetobacter baumannii exhibiting resistance to carbapenems. Patients were selected from 01/01/2007 to 31/07/2008 and the cases were considered patients with positive culture for CRAB. Controls were patients admitted to the ICU during the same period as cases, but showed no infection in which was isolated the presence of the bacterium in question. Factors evaluated as possible association with the risk of mortality were evaluated. Determined the attributable mortality to infections caused by CRAB and through the survival curve was evaluated this distribution between cases and controls. Results: 90 patients were selected as cases and 179 patients matched as controls. The average age, the proportions of patients with a Chalson score ³ 2 from inpatients not elective, the frequency of hospitalizations and surgeries were similar among studied groups. Among the cases, a greater proportion of patients transferred from another hospital (P <0.001), admitted in an area contiguous to the presence of cases of colonization or infection by CRAB (P <0.001) in patients undergoing parenteral nutrition (P <0.001) ; mechanical ventilation (P <0.001), urinary catheters (P = 0.031), central catheters for vascular access (P = 0.006) and catheters for hemodialysis (P <0.001) compared to controls. Likewise, cases had higher frequency of prior exposure to antimicrobials, compared with controls: penicillin (P <0.001), cephalosporins of 1st and / or 2nd generation (P <0.001), carbapenems (P <0.001), aminoglycosides (P = 0.046), quinolones (P = 0.004) and glycopeptides (P = 0.001). The cases presented mean length of stay higher than controls, including total duration of hospitalization (P = 0.002), stay in ICU (P <0.001) and stay in the ICU before 17 infection by CRAB (P = 0.03). The APACHE II score on admission to the ICU was also significantly higher average among cases compared with controls (P <0.001). There was a difference in the rate of in-hospital crude mortality among cases and controls, respectively, 58,9% (53/90) e 36,9% (66/179) (P = 0.001). The attributable mortality was 22% (95% CI 8.8% -35.2%) and cumulative survival curves for cases and controls showed no significant difference between groups (P = 0.207, log rank test) Multivariate analysis indicates that patients with APACHE II score higher and more frequently underwent invasive procedures such as mechanical ventilation, nutritional support (parenteral nutrition) and remained a longer period in hospital were more likely to risk of mortality associated with infection by CRAB. Conclusions: In this study the factors associated with mortality and the attributable mortality rate identified are in line with the literature and indicates that more severe patients are more prone to risk of mortality associated with infection by CRAB. The literature also emphasizes the need for consistent infection control strategies to prevent infection by multidrug resistant Acinetobacter. The investigation of attributable mortality to A. baumannii has many limitations and is not conclusive yet.

Introdução: As polimixinas são consideradas as opções terapêuticas de resgate para o tratamento das infecções de corrente sanguínea (ICS) por bacilos Gram-negativos (BGN) com resistência aos carbapenens (CR) e a combinação com outro antimicrobiano tem sido utilizada apesar da falta de evidência clínica para esta prática. Objetivo: Avaliar o uso de polimixina B intravenosa em monoterapia e em combinação com outro antimicrobiano para ICS por BGN CR. Pacientes e Métodos: Foi um estudo de coorte retrospectivo em um hospital terciário, incluindo 99 pacientes. A comparação dos tipos terapias foi através do propensity score. Resultados: A mortalidade global em 30 dias foi de 43,4%: 40,7% (24 de 59) e 47,5% (19 de 40), p=0,51, em pacientes que receberam combinação e monoterapia, respectivamente. A sepse grave/choque séptico no dia da ICS, alta pontuação do escore de bacteremia de Pitt e a presença de neoplasia como doença de base foram, independentemente, associados a maior mortalidade em 30 dias no modelo de regressão de Cox. A terapia combinada não foi significativamente associada a este resultado (hazard ratio, 0,70; intervalo de confiança de 95%, 0,36-1,36); p=0,29). Apesar de não ser significativa, houve uma tendência a um efeito benéfico da associação em pacientes com ICS por BGN CR da família Enterobacteriaceae. Não houve diferença no desenvolvimento de insuficiência renal aguda em pacientes que receberam terapia de combinação comparado com os que receberam monoterapia. Conclusão: Não houve diferença na mortalidade em 30 dias em pacientes com ICS por BGN CR tratados com polimixina B em combinação com outro antimicrobiano ou em monoterapia. A prática rotineira de combinar um segundo antibiótico em esquemas baseados em polimixinas, especialmente, se a bactéria apresenta resistência in vitro ao carbapenem, ainda necessita estudos clínicos adicionais. / Background: Polymyxins are usually the last resort therapy for carbapenem-resistant Gram-negative bacteria (CR GNB) bloodstream infections (BSIs), combination with another antimicrobial has been used despite the lack of clinical evidence supporting such practice. Objetive: We aimed to assess the use of intravenous polymyxin B in combination with another antimicrobial in comparison with polymyxin B as a single drug for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. Patient and methods: We compared combination versus monotherapy with polymyxin B for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. It was a retrospective cohort study at a tertiary-hospital including 99 patients. Results: The overall 30-day mortality was 43.4%: 40.7% (24 of 59) and 47.5% (19 of 40), P=0.51, in patients receiving combination and monotherapy, respectively. Severe sepsis/ septic shock at BSI onset higher Pitt bacteremia score and neoplasia were independently associated with higher 30-day mortality in a Cox-regression model. Combination therapy was not significantly associated with this outcome (Hazard Ratio, 0.70; 95% confidence interval, 0.36-1.36); P=0.29). Although not significant, there was a tendency to a beneficial effect of combination in patients with Enterobacteriaceae CR GNB BSIs. There was no difference in development of AKI in patients receiving combination therapy compared to those receiving monotherapy. Conclusions: There was no difference in 30-day mortality in patients with CR GNB BSIs treated with polymyxin B in combination with another antimicrobial compared with polymyxin B alone. The routine practice of combining a second antibiotic in polymyxins-based regimes, especially if the bacteria present in vitro resistance to the agent, still lacks support from clinical studies.

Introdução: As polimixinas são consideradas as opções terapêuticas de resgate para o tratamento das infecções de corrente sanguínea (ICS) por bacilos Gram-negativos (BGN) com resistência aos carbapenens (CR) e a combinação com outro antimicrobiano tem sido utilizada apesar da falta de evidência clínica para esta prática. Objetivo: Avaliar o uso de polimixina B intravenosa em monoterapia e em combinação com outro antimicrobiano para ICS por BGN CR. Pacientes e Métodos: Foi um estudo de coorte retrospectivo em um hospital terciário, incluindo 99 pacientes. A comparação dos tipos terapias foi através do propensity score. Resultados: A mortalidade global em 30 dias foi de 43,4%: 40,7% (24 de 59) e 47,5% (19 de 40), p=0,51, em pacientes que receberam combinação e monoterapia, respectivamente. A sepse grave/choque séptico no dia da ICS, alta pontuação do escore de bacteremia de Pitt e a presença de neoplasia como doença de base foram, independentemente, associados a maior mortalidade em 30 dias no modelo de regressão de Cox. A terapia combinada não foi significativamente associada a este resultado (hazard ratio, 0,70; intervalo de confiança de 95%, 0,36-1,36); p=0,29). Apesar de não ser significativa, houve uma tendência a um efeito benéfico da associação em pacientes com ICS por BGN CR da família Enterobacteriaceae. Não houve diferença no desenvolvimento de insuficiência renal aguda em pacientes que receberam terapia de combinação comparado com os que receberam monoterapia. Conclusão: Não houve diferença na mortalidade em 30 dias em pacientes com ICS por BGN CR tratados com polimixina B em combinação com outro antimicrobiano ou em monoterapia. A prática rotineira de combinar um segundo antibiótico em esquemas baseados em polimixinas, especialmente, se a bactéria apresenta resistência in vitro ao carbapenem, ainda necessita estudos clínicos adicionais. / Background: Polymyxins are usually the last resort therapy for carbapenem-resistant Gram-negative bacteria (CR GNB) bloodstream infections (BSIs), combination with another antimicrobial has been used despite the lack of clinical evidence supporting such practice. Objetive: We aimed to assess the use of intravenous polymyxin B in combination with another antimicrobial in comparison with polymyxin B as a single drug for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. Patient and methods: We compared combination versus monotherapy with polymyxin B for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. It was a retrospective cohort study at a tertiary-hospital including 99 patients. Results: The overall 30-day mortality was 43.4%: 40.7% (24 of 59) and 47.5% (19 of 40), P=0.51, in patients receiving combination and monotherapy, respectively. Severe sepsis/ septic shock at BSI onset higher Pitt bacteremia score and neoplasia were independently associated with higher 30-day mortality in a Cox-regression model. Combination therapy was not significantly associated with this outcome (Hazard Ratio, 0.70; 95% confidence interval, 0.36-1.36); P=0.29). Although not significant, there was a tendency to a beneficial effect of combination in patients with Enterobacteriaceae CR GNB BSIs. There was no difference in development of AKI in patients receiving combination therapy compared to those receiving monotherapy. Conclusions: There was no difference in 30-day mortality in patients with CR GNB BSIs treated with polymyxin B in combination with another antimicrobial compared with polymyxin B alone. The routine practice of combining a second antibiotic in polymyxins-based regimes, especially if the bacteria present in vitro resistance to the agent, still lacks support from clinical studies.

Introducción: Durante los XX últimos años, Acinetobacter baumannii se ha posicionado como una de las principales infecciones intrahospitalarias resistentes a antibióticos. A. baumannii multidrogoresistente (MDR) está considerado por la OMS dentro del grupo más crítico de resistencia. Uno de los mecanismos de resistencia identificados en dicho patógeno son las bombas de eflujo, por lo que, se han desarrollado inhibidores de las mismas, generando así menos resistencia por parte de las bacterias hacia los antibióticos. Objetivos: En nuestro estudio, el objetivo fue evaluar el efecto de la adición del inhibidor de bomba de eflujo CCCP sobre la actividad bactericida de imipenem y cefepime en cepas de A. baumannii Métodos: 49 cepas aisladas como A. baumannii fueron obtenidas en el Hospital Regional de Cajamarca. Mediante técnicas moleculares en el laboratorio de Biología Molecular de la Universidad Peruana de Ciencias Aplicadas (UPC) se confirmaron 47 cepas positivas para A. baumannii. Se utilizó PCR- en tiempo real para identificar el gen blaOXA-51-like y para la determinación de la CMI el método de microdilución en caldo. Finalmente se añadió el inhibidor CCCP para poder evaluar si efecto sobre los antibióticos Resultados: Un total de 49 cepas aisladas de A. baumannii fueron obtenidas en el Hospital Regional de Cajamarca. Se determinó la susceptibilidad antimicrobiana mediante la concentración mínima inhibitoria y la actividad de la bomba de eflujo fue evaluada usando CCCP. Conclusiones: Las bombas de eflujo puede jugar un rol importante en la resistencia antibiótica de A. baumannii. El inhibidor CCCP junto a imipenem y cefepime, mejora la sensibilidad antibiótica. Asimismo, nuevas estrategias terapéuticas son requeridas para eliminar el transporte de eflujo de las cepas resistentes que causan infecciones nosocomiales / Introduction: In the last years the rapid expansion of multidrug-resistant A. baumannii strains have become a major health problem. Efflux pumps are a group of transport proteins that contribute to the development of antibiotic resistance. The aim of this study was to evaluate the effect of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the antimicrobial action of imipenem and cefepime on clinical strains of A. baumannii. Materials and methods: A total of 49 non-duplicate clinical samples were collected during January through December of 2018 from patients hospitalized in the Hospital Regional Docente de Cajamarca. Of the 49 samples obtained, the confirmatory identification of A. baumannii was performed on 47 samples by molecular methods. The amplification of the blaOXA-51-like gene was carried out by polymerase chain reaction (PCR). The determination of the minimum inhibitory concentration (MIC) was calculated using the microdilution method in culture broth. The susceptibility to both antibiotics (cefepime and imipenem) was evaluated in the presence and absence of the inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Results: A total of 47 strains of A. baumannii were isolated: 97.87% (46/47) were resistant to Imipenem, 2.13% (1/47) of them were classified as intermediate and none of these strains were susceptible. On the other hand, 51.06% (24/47) of isolates were resistant to cefepime; 19.15% (9/47) intermediate and 29.79% (14/47) susceptible. We considered a significant difference in antibiotic susceptibility if the MIC changed at least 4 dilutions, after the addition of the inhibitor. In the case of CCCP in addition to imipenem, 2.1% (1/47) had a significant change of 4 or more reductions in MIC, 59.6% (28/47) achieved a change equal or less than 3 dilutions and 17.0% (8/47) did not have any change. In the case of CCCP with cefepime the percentage of strains with the significant change of MIC was 8.5% (4/47). On the other hand, 53.2% (24/47) presented a reduction equal or less than 3 dilutions and 12.8% (6/47) did not show changes. Conclusion: In conclusion, our results demonstrate that the use of CCCP may improve the antibiotic effect of imipenem and cefepime on clinical strains of A. baumannii. The relevance of this study is that it provides evidence that this efflux pump inhibitor may be an alternative treatment against multidrug-resistant A. baumannii. / Tesis

As espécies do Complexo Acinetobacter calcoaceticus-A. baumannii (ACB) são importantes causadoras de Infecções Relacionadas à Assistência à Saúde em todo o mundo. Detêm maior relevância os isolados com resistência aos antimicrobianos, os quais impactam negativamente no prognóstico, na mortalidade e custos associados ao cuidado com o paciente. O objetivo deste estudo foi avaliar a diversidade genética e o perfil de susceptibilidade antimicrobiana de 134 isolados multirresistentes de A. baumannii presentes no Hospital das Clínicas da Faculdade de Medicina de Botucatu, entre 2007 e 2014. A identificação de A. baumannii deu-se pela pesquisa dos genes blaOXA-51-like e gltA, detectados em 85% (n=114) dos isolados Os isolados de Acinetobacter não-baumannii foram identificados por sequenciamento gênico como A. nosocomialis (n=4; 3,1%), A. pittii, A. bereziniae (n=2; 1,7%, cada), A. ursingii, A. variabilis, A. gyllenbergii (n=1; 0,9% cada) e Acinetobacter spp (n=2; 1,7%). Os isolados de A. baumannii foram submetidos às técnicas de PCR multiplex para detecção de outras oxacilinases, pesquisa de ISAba1, teste de susceptibilidade antimicrobiana, tipagem molecular por eletroforese em campo pulsado (PFGE), por sequência trilocus (3LST) e por sequência multilocus (MLST). Detectou-se o gene blaOXA-23-like em 105 isolados (92,1%), estando 100% associados a ISAba1; blaOXA-72 em um isolado (0,9%) e blaOXA-231 em dois isolados (1,7%). A maior parte (n=66; 57,9%) dos isolados foi classificada como extensivamente resistentes (XDR). O PFGE agrupou os isolados em 11 clusters (A-K) e o MLST identificou os isolados pertencentes majoritariamente aos clones CC79 (42,4%), CC1 (16,6%), CC15 (12,1%) e ao ST317 (18,2%). Os resultados do MLST e 3LST concordaram em 95,6%. Foi verificada a ocorrência de diferentes perfis de PFGE em A. baumannii MDR e XDR, predominando cepas carreadoras de ISAba1/OXA-23-like e pertencentes aos CC1, CC15, CC79, ST317. Predominaram o ST317 nos anos iniciais e o CC79 (ST730) de 2011 a 2014. Estes resultados fornecem subsídios que ressaltam a necessidade de monitoramento e controle de patógenos multirresistentes / Species of Acinetobacter calcoaceticus-A. baumannii Complex (ACB) are important causes of Healthcare Associated Infections worldwide. More relevant are isolates with antimicrobial resistance, which have a negative impact on the outcome, mortality and costs associated with patient care. The aim of this study was to evaluate the genetic diversity and the antimicrobial susceptibility profile of 134 multiresistant ACB spcies strains present at Botucatu Medical School Teaching Hospital between 2007 and 2014. Identification of A. baumannii species was by detection of blaOXA-51-like and gltA genes, detected in 85% (n=114) of the isolates. Non-baumannii Acinetobacter species were identified by gene sequencing as A. nosocomialis (n=4, 3.1%), A. ursingii, A. variabilis, A. gyllenbergii (n=1, 0.9% each) and Acinetobacter spp (n=2; 1.7%). A. baumannii isolates were submitted to multiplex PCR for other oxacillinases and ISAba1 detection, antimicrobial susceptibility testing, molecular typing by pulsed field gel electrophoresis (PFGE), trilocus sequence typing (3LST) and multilocus sequence typing (MLST). blaOXA-23-like gene was detected in 105 isolates (92.1%), of which 100% were associated with ISAba1; blaOXA-72 was present in one isolate (0.9%) and blaOXA-231, in two isolates (1.7%). The majority (n=66; 57.9%) of isolates were classified as extensively resistant (XDR). The PFGE grouped the isolates into 11 clusters (A-K) and MLST identified the isolates belonging mainly to CC79 (42.4%), CC1 (16.6%), CC15 (12.1%) and ST317 (18.2%). MLST and 3LST results were 95.6% concordant. We verified the occurrence of different PFGE profiles in multidrug-resistant (MDR) and extensively drug-resistant (XDR) A. baumannii, predominantly presenting ISAba1/OXA-23-like genes and belonging to CC1, CC15, CC79 and ST317. There was a prevalence of ST317 in the early years and CC79 (ST730) from 2011 to 2014. Our results highlight the importance of surveillance and control of multiresistant pathogens