Andreza Francisco Martins

Carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme: Dissemination in Southern Brazil

Acinetobacter baumannii has emerged as an important nosocomial pathogen due to its ability of long-term survival in the hospital environment, which facilitate spreading, causing institutional outbreaks, and due to its acquired multiple mechanisms of antimicrobial resistance [1]. A number of factors such as immunosuppression, use of invasive devices, and the use of antimicrobial agents have been reported as increasing the risk of infection or colonization by this opportunistic pathogen [2]. The carbapenems used to be an effective therapeutic option for the treatment of Acinetobacter spp. infections. However, in the last few years, carbepenem-resistant Acinetobacter species have been recovered worldwide. Loss of porins, alterations in the penicillin-binding protein (PBP) affinity, and different class B (metalloenzymes) and D (OXA enzymes) b-lactamases have been associated with resistance to carbapenems in A. baumannii [3]. The OXA carbapenemases of Acinetobacter are divided into four phylogenetic subgroups: OXA-23, OXA24 and OXA-58 (acquired enzymes), and OXA-51 that is intrinsic to A. baumannii [4]. In Brazil, the first report of acquired oxacillinase has been described in Curitiba [5] in 2003, but carbapenem resistance seems to constitute an emerging problem in other regions of the country and in Latin America [6]. In Porto Alegre, a city with 1.4 million inhabitants and 7,700 beds situated in 25 hospitals, the first isolate of carbapenem-resistant A. baumannii (CRAB) was identified only in 2004, but, further, CRAB has been reported to the local health department in an unprecedented outbreak involving 16 hospitals and more than 500 detected cases from 2004 to 2008 [7]. The goal of this study was to investigate the mechanisms of emerging resistance to carbapenems in multiresistant A. baumannii isolates and to characterize the molecular type using pulsed-field gel electrophoresis (PFGE). A total of 53 CRAB isolates were obtained from patients attending two university tertiary teaching hospitals in Porto Alegre from July to December 2007 (one isolate per patient). Three isolates were obtained from the environment in hospital 2 through surveillance culture during an outbreak investigation. Isolates were identified using the GN32 card using the API 20NE system (bioMérieux, Basingstoke, United Kingdom). Antimicrobial susceptibility testing was performed by the disk diffusion method, as described by the Clinical Laboratory Standards Institute (CLSI) [8]. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were established using the E-test. The presence of genes coding for blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were detected by multiplex polymerase chain reaction (PCR) as described previously with minor modifications [9]. Positive controls included Acinetobacter spp. strains known to produce OXA-23-like, OXA-24-like, and OXA-58-like enzymes [5], and the type strain of A. baumannii ATCC 19606 (negative control) for these genes and positive for OXA-51-like (intrinsic to A. baumannii). All three isolates from the hospital environment, as well as a set of 13 clinical isolates of CRAB, randomly selected from all clinical isolates, which were stored at –80 C, were selected for molecular typing. Macrorestriction analysis of chromosomal DNA with SmaI (Invitrogen, Paisley,

Dissemination of Pseudomonas aeruginosa Producing SPM-1-like and IMP-1-like Metallo-β-lactamases in Hospitals from Southern Brazil

Background:Metallo-β-lactamase (MBL) is an emerging resistance mechanism among Pseudomonas aeruginosa. The prevalence of this mechanism is particularly high in Latin America. We aimed to describe the prevalence and molecular characteristics of SPM-1-like, IMP-1-like and VIM type MBLs among ceftazidime and/or imipenem-resistant nosocomial P. aeruginosa isolates.Methods:Pseudomonas aeruginosa isolates resistant to ceftazidime and/or imipenem recovered from hospitalized patients from two teaching hospitals from Porto Alegre, Brazil, were prospectively selected. Isolates were tested for MBL production using two phenotypic screening tests. Those isolates with positive results were further tested for the presence of MBL genes (SPM-1-like, IMP-1-like and VIM type) and submitted to molecular typing.Results:A total of 92 isolates were analyzed and 33 (35.9%) were presumptively MBL producers by phenotypic tests. The SPM-1-like gene was found in 18 isolates and IMP-1-like in 5 isolates. In ten isolates the MBL type could not be identified. Three IMP-1-like isolates were susceptible to imipenem. SPM-1-like isolates comprised a single clone, and IMP-1-like isolates another single clone.Conclusion:The prevalence of MBL production among ceftazidime-resistant P. aeruginosa isolates is relatively high in both hospitals. Infection control measures have been challenged and further improvements in such measures are required to prevent dissemination of these isolates among hospitals. This is the first report of IMP-1-like MBLs in P. aeruginosa in southern Brazil.

High endemic levels of multidrug-resistant Acinetobacter baumannii among hospitals in southern Brazil

BACKGROUND Most published data on multidrug-resistant Acinetobacter baumanii (MDR Ab) are derived from outbreaks. We report incidence trends on health care-acquired infections due to MDR Ab over a 12-month period in the city of Porto Alegre in southern Brazil. METHODS Clinical and epidemiologic data were obtained from the local health care information system of the municipal health department. Polymerase chain reaction was used to detect the presence of the genes bla(OXA-23-like), bla(OXA-24-like), bla(OXA-51), and bla(OXA-58), and repetitive sequence-based polymerase chain reaction and pulsed-field gel electrophoresis were performed for molecular typing. RESULTS The highest rate of infection (9.0/1,000 inpatient-days) was identified in a trauma hospital. The gene bla(OXA-23-like) was identified in 99.0% of MDR Ab isolates. Eight main clonal groups were identified by molecular typing, and 3 of these were found in all hospitals. CONCLUSION The presence of 3 clones in all hospitals demonstrates the ability of MDR Ab to spread among hospitals. Moreover, the occurrence of one particular clone (clone 4) throughout the study period suggests its increased ability to cause outbreaks and to remain in the environment. The monitoring of epidemic strains by molecular methods is of paramount importance to prevent or reduce the spread of MDR Ab.

Emergence of NDM-1-producing Acinetobacter pittii in Brazil

Sir, The New Delhi metallo-lactamase (NDM), initially reported in Klebsiella pneumoniae and Escherichia coli, is now disseminated worldwide mostly among Enterobacteriaceae [1]. The NDM carbapenemase has also been described in Acinetobacter baumannii, but only in sporadic cases in countries such as China, India, Egypt, Germany, Israel and, more recently, Brazil [1,2]. Noteworthy, recent studies reported NDM-producers among non-baumannii Acinetobacter spp., which may also be human pathogens. Here we report the first case of NDM-1-producing Acinetobacter pittii in Brazil. A 66-year-old male patient with bladder carcinoma was admitted for radical cystectomy to a 900-bed tertiary care hospital in Porto Alegre, Southern Brazil, on 25 February 2013. Fifteen days later he presented an intestinal subocclusion and fever. Computerised tomography (CT) of the abdomen showed the presence of a collection in pelvis, which was drained surgically. This purulent secretion was cultured and a K. pneumoniae was identified (VITEK® 2 system; bioMérieux, La Balme-les-Grottes, France). Urine was also cultured and revealed the presence of Candida sp. (50 000 CFU/mL) and Acinetobacter sp. (>100 000 CFU/mL). The patient was treated with intravenous meropenem 500 mg every 12 h for 7 days, followed by cefepime 1 g every 24 h (doses adjusted to impaired renal function). Three subsequent urine cultures obtained 11, 28 and 44 days after the first culture were negative for Acinetobacter sp. The patient was therefore considered colonised by Acinetobacter sp. After 90 days the patient improved and was discharged from the hospital. The Acinetobacter sp. isolate MP was identified as A. pittii by matrix-assisted laser desorption/ionisation time-of-flight (MALDITOF) (Bruker Daltonik, Bremen, Germany), gyrB multiplex PCR and 16S rRNA gene sequencing. Minimum inhibitory concentrations (MICs) of -lactams, aminoglycosides, ciprofloxacin, fosfomycin, chloramphenicol, tigecycline, colistin and polymyxin B were determined (Etest® and microdilution method) and showed that the isolate was resistant to all -lactams (with the exception of aztreonam), including carbapenems (MICs of imipenem, ertapenem, doripenem and meropenem >32 g/mL). The isolate remained susceptible to amikacin, gentamicin, tigecycline, colistin, polymyxin B, ciprofloxacin and chloramphenicol. Carbapenemase genes were searched by real-time PCR (blaOXA-48, blaKPC, blaIMP, blaVIM and blaGES) and multiplex PCR (blaOXA-23-like, blaOXA-40-like, blaOXA-58-like and blaOXA-143). A positive signal was obtained only for the blaNDM gene, and sequencing identified the blaNDM-1 gene. To identify the location of this gene, electrotransformation assays were attempted using plasmid DNA extracts from A. pittii isolate MP using A. baumannii CIP7010 and E. coli TOP10 as recipients. Transfer of the blaNDM-1 gene by electrotransformation into these two recipient strains remained unsuccessful, suggesting that the gene might be chromosomally located in A. pittii MP, as reported in A. baumannii [3]. The genetic environment of the blaNDM-1 gene was determined by PCR mapping as described [3] and insertion sequence ISAba125 was identified upstream of the blaNDM-1 gene. However, attempts to identify another copy of ISAba125 downstream of blaNDM-1 remained unsuccessful, suggesting that the blaNDM-1 gene might be part of a truncated Tn125 transposon, as previously reported in A. baumannii [3]. Multilocus sequence typing (MLST) was performed according to the Institute Pasteur scheme (http://www.pasteur.fr) and A. pittii isolate MP was identified as ST119. Interestingly, two blaNDM-positive A. pittii isolates were recently identified in Paraguay [4], a neighbouring country of Brazil, but those isolates belonged to ST320 and ST321. The only reports of A. pittii ST119 isolates are from Japan, with isolates producing the carbapenemase IMP-19 [1]. Identification of blaNDM-positive non-baumannii Acinetobacter spp. is now increasingly reported worldwide, concomitantly with those of blaNDM-positive A. baumannii isolates. There are few reports of NDM-producing A. pittii, being from China, Turkey and recently Paraguay. This is of particular concern considering that Acinetobacter sp. may (i) act as reservoirs for blaNDM genes in non-human settings, as recently shown in several Chinese studies with identification of NDM-1-producers among Acinetobacter calcoaceticus and Acinetobacter junii from environmental samples from livestock farms [1], Acinetobacter johnsonii from hospital sewage [1] and Acinetobacter lwoffii from chickens [1], but also (ii) act as a source of blaNDM genes then horizontally transferred to enterobacterial species as evidenced [5].

Evaluation of heteroresistance to polymyxin B among carbapenem-susceptible and -resistant Pseudomonas aeruginosa

One hundred and twenty-four Pseudomonas aeruginosa isolates were selected for antimicrobial susceptibility testing with anti-pseudomonal agents, MIC determination for polymyxin B and metallo-beta-lactamase detection (genes blaSPM, blaVIM-1, blaNDM-1 and blaIMP). According to the imipenem and/or meropenem susceptibility profile, a set of randomly selected isolates (12 isolates carbapenem-susceptible and 12 isolates carbapenem-resistant) were evaluated for heteroresistance to polymyxin B. Heteroresistance testing was performed by plating the isolates onto increasing concentrations of polymyxin B (from 0 to 8.0 mg l(-1)). The population analysis profile (PAP) was defined as the ratio of the number of colony-forming units on the plate with the highest concentration of polymyxin B at which bacterial growth occurred against the number of colony-forming units on the plate without antibiotic. Isolates presenting subpopulations that exhibited growth at polymyxin B concentrations ≥2 mg l(-1) were considered heteroresistant. Isolates containing subpopulations that grew at polymyxin B concentrations at least twice as high as the original MIC but <2 mg l(-1) were considered heterogeneous. Antimicrobial susceptibility testing results indicated a variable degree of susceptibility: high levels of resistance to gentamicin (30.6 %) and imipenem (29.0 %); low levels of resistance to aztreonam (1.6 %) and ciprofloxacin (4.8 %). All isolates were susceptible to polymyxin B: MIC50 and MIC90 were 1 mg l(-1) and 2 mg l(-1), respectively. Thirty-seven isolates (30 %) were carbapenem-resistant. Four isolates resistant to carbapenems were positive for blaIMP. There were no heteroresistant subpopulations in the carbapenem-susceptible group, but three isolates presented heterogeneous subpopulations. The PAP frequency ranged from 2.1×10(-4) to 6.9×10(-8). In the carbapenem-resistant group, one isolate was heteroresistant. Six isolates in this group presented heterogeneous subpopulations. In the resistant population, the PAP frequency ranged from 2.1×10(-7) to 2.6×10(-4). In this study, polymyxin B heteroresistance in P. aeruginosa was uncommon and occurred in only one carbapenem-resistant isolate, despite the fact that several isolates presented heterogeneous subpopulations with increased polymyxin B MICs.

Ocurrence of blaSPM-1 and blaIMP-1 genes of metallo-beta-lactamases in clinical isolates of Pseudomonas aeruginosa from three universitary hospitals in the city of Porto Alegre, Brazil

Descrevemos a ocorrencia dos genes de metalo-b-lactamases (MBL) blaSPM-1 e blaIMP-1 em isolados clinicos de Pseudomonas aeruginosa resistentes ao imipenem e/ou ceftazidima obtidos em tres hospitais universitarios de Porto Alegre, Brasil. A producao de MBL foi observada atraves de tecnica fenotipica e os genes foram detectados pelo metodo de PCR.

Letter to the editor: Escherichia coli harbouring mcr-1 gene isolated from poultry not exposed to polymyxins in Brazil

SA Lentz 1 2 , D de Lima-Morales 2 3 , VM Cuppertino 1 , LdS Nunes 3 , AS da Motta 1 , AP Zavascki 4 , AL Barth 3 , AF Martins 1 3 1. 1. ICBS – Instituto de Ciências Básicas da Saúde, UFRGS Univ. Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 2. These authors contributed equally to this work. 3. LABRESIS – Lab. de Pesquisa em Resistência Bacteriana, HCPA Hosp. de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil 4. Infectious Diseases Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil

Mobile genetic elements related to carbapenem resistance in Acinetobacter baumannii.

Acinetobacter baumannii is widely recognized as an important pathogen associated with nosocomial infections. The treatment of these infections is often difficult due to the acquisition of resistance genes. A. baumannii presents a high genetic plasticity which allows the accumulation of these resistance determinants leading to multidrug resistance. It is highlighted the importance of the horizontal transfer of resistance genes, through mobile genetic elements and its relationship with increased incidence of multidrug resistant A. baumannii in hospitals. Considering that resistance to carbapenems is very important from the clinical and epidemiological point of view, the aim of this article is to present an overview of the current knowledge about genetic elements related to carbapenem resistance in A. baumannii such as integrons, transposons, resistance islands and insertion sequences.

Acinetobacter multirresistente ? um desafio para a saúde pública

OBJETIVOS: Discutir os diferentes aspectos associados a emergencia da resistencia aos antibioticos carbapenemicos em Acinetobacter baumannii e a disseminacao de clones epidemicos. FONTE DE DADOS: Foram consultadas as bases de dados SciELO, PubMed e Science Direct. Foram incluidos artigos publicados em ingles, prioritariamente no periodo de 2000 a 2011. Os descritores utilizados foram Acinetobacter , multidrug resistance (multirresistencia), outbreaks (surtos) infection control measures (medidas de controle de infeccoes), e risk factors (fatores de risco). SINTESE DOS DADOS: Acinetobacter baumannii e um patogeno oportunista envolvido em um amplo espectro de infeccoes hospitalares, incluindo bacteremia, meningite e infeccao do trato urinario. Sua maior prevalencia e como agente de pneumonia hospitalar, particularmente pneumonia associada a ventilacao mecânica em unidades de terapia intensiva. Os antimicrobianos carbapenemicos ja foram utilizados como tratamento das infeccoes causadas por Acinetobacter, mas atualmente as elevadas taxas de resistencia a essa classe de antimicrobianos tem limitado as opcoes terapeuticas. A resistencia aos carbapenemicos pode estar relacionada a perda de porinas, a producao de beta-lactamases da classe B (metalo-beta-lactamases) e, de forma mais significativa, a producao de beta-lactamases da classe D (OXA-carbapenemases). Surtos associados a producao de OXA-carbapenemases vem sendo descritos em diferentes paises desde o final da decada de 1990, e a disseminacao de clones epidemicos tem sido documentada. CONCLUSOES: O elevado numero de surtos ja relatados na literatura, a facilidade de disseminacao de clones epidemicos e a dificuldade no tratamento tornam as infeccoes por Acinetobacter um grave problema de saude publica.

Genetic similarity of Burkholderia cenocepacia from cystic fibrosis patients

Burkholderia cenocepacia may cause serious infections in patients with cystic fibrosis, and this microorganism can be highly transmissible. Pulsed-field gel electrophoresis is widely used to study the dynamics of strain spread in cystic fibrosis patients. The aim of this work was to perform pulsed-field gel electrophoresis-based molecular typing of B. cenocepacia isolates to evaluate the epidemiology of this species at our hospital. A total of 28 isolates from 23 cystic fibrosis patients were analyzed. Initially, we compared isolates obtained from the same patient at different periods of time. We then compared the pulsed-field gel electrophoresis profiles of 15 IIIA isolates, and in a third analysis, evaluated the genetic profile of 8 IIIB isolates from different patients. The pulsed-field gel electrophoresis profiles of isolates from the same patient indicated that they are genetically indistinguishable. Analysis of isolates from different patients revealed the presence of multiple clonal groups. These results do not indicate cross-transmission of a unique clone of B. cenocepacia among cystic fibrosis patients, although this has been observed in some patients. Our findings highlight the importance of adequate patient follow-up at cystic fibrosis centers and adherence to management and segregation measures in cystic fibrosis patients colonized with B. cenocepacia.