Franciéli Pedrotti Rozales

Emergence of NDM-1-producing Enterobacteriaceae in Porto Alegre, Brazil

OBJECTIVES To evaluate the emergence of New Delhi metallo-β-lactamase 1 (NDM-1)-producing Enterobacteriaceae isolates in Brazil. METHODS From April to October 2013, following the detection of the first NDM-1-producing isolate, a surveillance study was performed for the detection of blaNDM-1 among Enterobacteriaceae isolates with reduced susceptibility to carbapenems in 17 hospitals of Porto Alegre, Brazil. Real-time PCR was used to determine the presence of carbapenemase genes, which were further sequenced. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE). RESULTS A total of 1134 isolates were evaluated. blaNDM-1 was detected in 11 (0.97%) isolates: nine Enterobacter cloacae complex (eight belonging to a single clone recovered from two distinct hospitals and the other strain from a third hospital) and two Morganella morganii (belonging to a single clone recovered from one hospital). Most isolates presented high-level resistance to carbapenems. CONCLUSIONS NDM-1-producing Enterobacteriaceae have emerged rapidly in the hospitals of the Brazilian city where they were first detected. The emergence of NDM-1 in Brazil is of great concern, since it is a severe threat to antimicrobial therapy against Enterobacteriaceae in this country.

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].

Detection of OXA-370, an OXA-48-related class D β-lactamase, in Enterobacter hormaechei from Brazil

Jorge L. M. Sampaio, Vanessa B. Ribeiro, Juliana Coutinho Campos, Franciéli P. Rozales, Cibele M. Magagnin, Diego R. Falci, Renato Cassol F. da Silva, Micheline G. Dalarosa, Daniela I. Luz, Fabiane J. Vieira, Laura C. Antochevis, Afonso Luis Barth, Alexandre P. Zavascki Departamento de Análises Clínicas e Toxicológicas—Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil; Microbiologia—Fleury Medicina e Saúde, São Paulo, Brazil; Laboratório de Pesquisa em Resistência Bacteriana—LABRESIS, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; Programa de PósGraduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; Infection Control Service, Hospital Nossa Senhora da Conceição, Porto Alegre, Brazil; Infectious Diseases Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; Department of Internal Medicine, Medical School, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

Detection of blaGES-5 in Carbapenem-Resistant Kluyvera intermedia Isolates Recovered from the Hospital Environment

Vanessa B. Ribeiro, Alexandre P. Zavascki, Franciéli P. Rozales, Mariana Pagano, Cibele M. Magagnin, Carolina S. Nodari, Renato Cassol Ferreira da Silva, Micheline G. Dalarosa, Diego R. Falci, Afonso L. Barth ‹Laboratório de Pesquisa em Resistência Bacteriana, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Brazil; Infectious Diseases Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas; Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil; Programa de Pós-Graduação em Ciências Médicas, UFRGS, Porto Alegre, Brazil; Hospital Nossa Senhora da Conceição, Porto Alegre, Brazil

Carbapenem-resistant GES-5-producing Klebsiella pneumoniae in Southern Brazil

GES-type enzymes belong to Ambler Class A plasmidencoded -lactamases. Although GES enzymes usually exhibit extended-spectrum -lactamases (ESBL) properties, changes in its active site enhance its activity conferring the ability to hydrolyze carbapenems.1 GES-type carbapenemases (GES2, GES-4, GES-5, GES-8, GES-11, GES-18, GES-20) have already been described in several countries.2 Although it has been reported in a Klebsiella pneumoniae from Brazil in 2010, in a patient admitted in 2008,3 no other isolate has ever since been reported in this country. Here, we described another carbapenem-resistant K. pneumoniae (CRKP) isolate carrying the blaGES-5 gene in Brazil. The CRKP, identified by VITEK2 system (bioMeriéux, France), was recovered from the blood of an adult patient admitted to a university hospital in Porto Alegre, Brazil, in 2011. The patient had acute myeloid leukemia with recent exposure to many antibiotics including piperacillin–tazobactam, polymyxin B, meropenem, vancomycin, and cefepime. Despite treatment with polymyxin B and tigecycline, the patient ultimately died six days after CRKP recovering. Minimum inhibitory concentrations (MICs), performed by Etest®, and the phenotypic tests for carbapenemases detection are shown in Table 1. The presence of blaGES was demonstrated by multiplex real-time PCR that also included blaKPC, blaVIM, blaNDM, blaOXA-48 and blaIMP genes.4 Sequencing of blaGES gene was performed using the primers previously described.5 GenBank was used to access the GES sequences deposited to date and BioEdit program was used to compare the similarity between sequences, and revealed the presence of a GES-5. Plasmidial DNA from CRKP was obtained from alkaline lysis and it was electroporated into an Escherichia coli Top10 (Invitrogen). Transformants were selected on Luria-Bertani agar containing 0.5 g/mL of ceftazidime. The plasmid electroporation resulted in transformants, which confirmed the presence of blaGES by PCR (Table 1). We reported the second case of a CRKP carrying the blaGES-5 gene in Brazil. Although the first GES-5-producing CRKP was reported in São Paulo in 2008,3 there has been no other report of this enzyme in Enterobacteriaceae since then in Brazil. Table 1 – Phenotypic characterization of Klebsiella pneumoniae and Escherichia coli transformant carrying blaGES-5. MIC ( g/mL)a

PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data

Tuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission in patients. The aims of this study were to evaluate two in-house molecular tests: nested PCR (nPCR) and real-time PCR (rtPCR) to detect M. tuberculosis complex directly from clinical samples. The results were compared to the culture results and to the culture results plus clinical data of patients. The rtPCR and nPCR presented high sensitivity (Se) and specificity (Sp) (rtPCR 97·6% and 91·5%, nPCR 85·7% and 92·7%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the Se and Sp were 90·2% and 97·3% for rtPCR and 80·4% and 98·6% for the nPCR, respectively. The results demonstrated that molecular assays of M. tuberculosis can provide a sensitive and rapid diagnostic of TB, and when used in addition to the clinical data of TB patients will help to improve the Sp of the diagnosis of pulmonary TB.

Performance of rapid tests for carbapenemase detection among Brazilian Enterobacteriaceae isolates.

The global emergence of carbapenemases led to the need of developing new methods for their rapid detection. The aim of this study was to evaluate the performance of the rapid tests for carbapenemase-producing and non-producing Enterobacteriaceae. Carbapenem non-susceptible Enterobacteriaceae from a surveillance study submitted to a multiplex real time PCR for carbapenemase detection were included in this study. The isolates were subjected to the rapid phenotypic tests Carba NP, Blue-Carba and Carbapenem Inactivation Method (CIM). A total of 83 carbapenemase-producing (43) and non-producing (40) isolates were included in the study. The sensitivity/specificity were 62.7%/97.5%, 95.3%/100%, and 74.4%/97.5% for Carba NP, Blue-Carba and CIM, respectively. Both Carba NP and Blue-Carba presented their final results after 75 min of incubation; the final results for CIM were obtained only after 8 h. Failure to detect OXA-370 carbapenemase was the main problem for Carba NP and CIM assays. As the Blue-Carba presented the highest sensitivity, it can be considered the best screening test. Conversely, CIM might be the easiest to perform, as it does not require special reagents. The early detection of carbapenemases aids to establish infection control measures and prevent carbapenemases to spread reducing the risk of healthcare associated infections and therapeutic failure.

Characteristics of Enterobacteriaceae Isolates Coharboring Distinct Carbapenemase Genes

To the Editor—The emergence of carbapenemase-producing Enterobacteriaceae (CPE) isolates is an important public health problem; the treatment of carbapenem-resistant isolates is extremely difficult because few options remain available for clinical use. Usually, CPE harbors only 1 carbapenemase gene, although other resistance mechanisms (ESBL, porin loss, eflux pumps) may also be present. However, relatively few studies have reported Enterobacteriaceae isolates producing more than 1 carbapenemase. In the present study, we describe the characteristics of 10 Enterobacteriaceae coharboring carbapenemase genes. The isolates were selected from an epidemiologic study evaluating Enterobacteriaceae with reduced susceptibility to carbapenems in several hospitals in the southernmost state of Brazil. The methods of this epidemiologic study are detailed elsewhere. Briefly, those isolates harboring more than 1 of the following genes were selected for further evaluation in this report: blaKPC, blaVIM, blaGES, blaNDM, blaOXA-48, and blaIMP (detected by a multiplex real-time polymerase chain reaction [PCR]). These isolates were initially identified in the original institution by the VITEK2 system (bioMeriéux, France), and isolates coharboring more than 1 carbapenemase gene were confirmed by 16S rRNA sequencing. The presence of the carbapenemase genes was confirmed by conventional PCR, and the amplicons were purified and sequenced using a BigDie Terminator kit (version 3.1) and an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA). GenBank was used to access the sequences deposited to date, and the BioEdit program was used to compare similarities among sequences. Plasmids were extracted by alkaline lysis and were transformed into cells of an Escherichia coli TOP10 eletrocompetent by electroporation. Transformants were selected on LuriaBertani agar containing 2mg/L ceftazidime. Estimation of plasmid size was performed after 0.7% agarose gel electrophoresis, using a curve obtained by plotting the distance (mm), compared to E. coli 39R861. Minimum inhibitory concentrations (MIC) of carbapenems were evaluated using the broth microdilution method and were interpreted according to the Clinical and Laboratory Standards Institute (CLSI). Overall, 10 isolates coproducing carbapenemases were identified: 5 Enterobacter cloacae complexes with blaNDM-1 and blaOXA-370 genes, 3 Klebsiella pneumoniae, 1 E. cloacae complex with blaNDM-1 and blaKPC-2 genes, and 1 K. pneumoniae with blaKPC-2 and blaOXA-370 genes. We detected multiple plasmids in 8 clinical isolates (Table 1); 2 isolates presented only one plasmid. One Providencia rettgeri presented blaGES and blaIMP genes in the multiplex PCR but the sequencing of the amplicon did not yield the specific variant of these genes. Notably, the P. rettgeri presented a peculiar result: it was positive for both blaIMP and blaGES, which are supposed to be in a plasmid, but the plasmid was not identified in either the clinical isolate or the transformant (data not show). The MICs of transformants were much higher than that of E. coli TOP10, which indicates that the plasmids are enough to confer resistance to antibiotics. The analysis of the antimicrobial susceptibility profile of the transformants compared to the wild-type isolates showed that most isolates present lower MICs for carbapenems. The transformants that received only the OXA-370 gene present very lowMICs to both meropenem and imipenem, which may indicate that this OXA-48 variant lacks carbapenemase activity. Plasmid analysis demonstrated a heterogeneous pattern of plasmid sizes: 92, 110, 128, 130, and 154 kbp. Moreover, we observed that carbapenemases were inserted in different plasmids, which was also observed in other studies. Balm et al reported a K. pneumoniae isolate coharboring blaNDM and blaOXA-181 genes on ~160 kb and ~280 kb plasmids, respectively. Another study demonstrated coproduction of blaNDM-1 and blaOXA-232 in E. coli: the blaNDM-1 gene was located on a plasmid of 129,085 bp and the blaOXA-232 gene was located on a small plasmid of 6,141 bp. A study of K. pneumoniae coharboring blaVIM and blaKPC revealed 2 plasmids of 70 and 150 kb, while the blaVIM transconjugants had a single plasmid of 150 kb and the blaKPC-bearing transconjugant had a single plasmid of 70 kb. We were able to transfer at least 1 carbapenemase gene to the E. coli TOP10 receptor, with the exception of 1 (4517F) E. cloacae that transferred both carbapenemase genes (blaNDM-1 and blaKPC-2). The most frequently reported Enterobacteriaceae species carrying 2 or more carbapenemases is Klebsiella pneumoniae, although other species with this property have also been reported sporadically. Moreover, a few studies have indicated that the number of blaNDM-1 and blaOXA-48 is constantly increasing, and this combination has been the most frequently described. Relatively few reports of Enterobacteriaceae coproducing carbapenemases are available; here, we describe 2 additional species harboring 2 carbapenemases, and we observed other combinations such as a New Delhi metallo (NDM)-codifying gene with an OXA-48 variant carbapenemase. In the present study, we observed the occurrence of 10 clinical isolates coproducing different carbapenemases located in a variety of plasmids, demonstrating the plasticity of these mobile genetic elements. The dissemination infection control & hospital epidemiology

Emergence of Acinetobacter baumannii ST730 carrying the blaOXA-72 gene in Brazil

Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and has been commonly associated with high morbimortality. In Brazil, this resistance is mainly attributed to the spread of OXA-23-producing clones and, to a lesser extent, to OXA-143-producing clones. Here, we describe, for the first time, two OXA-72-producing A. baumannii isolates in southern Brazil to a broad spectrum of antibiotics, except polymyxin B and tigecycline. Molecular typing by multilocus sequence typing (MLST) demonstrated that both OXA-72-producing isolates belong to a new sequence type (ST), ST730, which was recently identified in OXA-23-producing A. baumannii isolates in São Paulo, Brazil. We demonstrate that the two A. baumannii ST730 isolates carrying blaOXA-72share a common ancestral origin with the blaOXA-23producers in Brazil. This observation reinforces the importance of strain-typing methods in order to clarify the dynamics of the emergence of new clones in a geographic region.