High rate of detection of OXA-23-producing Acinetobacter from two general hospitals in Brazil

Abstract INTRODUCTION In recent decades, the prevalence of carbapenem-resistant Acinetobacter isolates has increased, and the production of oxacillinase (OXA)-type carbapenemases is the main mechanism underlying resistance. We evaluated OXA production from 114 Acinetobacter isolates collected between March and December 2013 from different clinical specimens of patients in two hospitals (Hospital 1 [n = 61] and Hospital 2 [n = 53]) located in Niterói, Rio de Janeiro, Brazil. We also evaluated the genetic diversity of OXA-producing isolates. METHODS All the isolates were identified through the automated system Vitek II and matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS as belonging to the A. baumannii-A. calcoaceticuscomplex. Antimicrobial susceptibility profiles were verified through agar diffusion tests. The presence of OXA-encoding genes was confirmed by PCR. The genetic diversity of isolates positive for carbapenemase production was analyzed through pulsed-field gel electrophoresis. RESULTS There was a high rate of resistance to carbapenems in the isolates (imipenem: 96%; meropenem: 92%) from both hospitals. Moreover, a high percentage (95.6%) of OXA-23-positive isolates was observed for both hospitals, indicating that this was the main mechanism of carbapenem-resistance among the studied population. In addition, most isolates (96.5%) were positive for bla OXA-51. A high genetic diversity and a few major genotypes were found among the OXA-23-positive isolates analyzed. Only intra-hospital dissemination was observed. CONCLUSIONS The elevated dissemination of bla OXA-23-like observed among Acinetobacter isolates from both the studied hospitals highlights the need for continuous epidemiological surveillance in these institutions.

Glimpse into the genome sequence of a multidrug-resistant Acinetobacter pittii ST950 clinical isolate carrying the blaOXA-72 and blaOXA-533 genes in China

The development of carbapenem-resistant Acinetobacter species is of serious concern in the hospital settings and naturally occurring oxacillinase genes (blaOXA) have been identified in several Acinetobacter species. In this study, we report the genome sequence of A. pittii TCM178 belongs to ST950, a multidrug-resistant isolate that harbored the blaOXA-72 and blaOXA-533 genes simultaneous. The genome size was estimated to be 3,789,564 bp with 3,501 predicted coding regions, and G+C content is 37.60%. Our findings have raised awareness of the possible constitution of a reservoir for peculiar carbapenemase genes in A. pittii that may spread among other Acinetobacter species in China.

Draft genome sequence of a multidrug-resistant beta-lactamase OXA-357-producing Acinetobacter pittii ST865 clinical isolate from China

Abstract Worldwide increasing emergence of carbapenem-resistant Acinetobacter spp. has rendered the limited availability of effective antimicrobial agents and has become a major public health concern. In this study, we report the draft genome sequence of A. pittii TCM156, a multidrug-resistant isolate that harbored the blaOXA-357 gene. The genome sequence was further analyzed by various bioinformatics methods. The genome size was estimated to be 3,807,313 bp with 3508 predicted coding regions and G + C content is 38.7%. These findings have raised awareness of the possible emergence of OXA-type enzyme-producing A. pittii isolate in China.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

New Delhi metallo-beta-lactamase-1-producing Acinetobacter spp. infection: report of a survivor

Abstract: New Delhi metallo-beta-lactamase-1 (NDM-1) is a bacterial enzyme that renders the bacteria resistant to a variety of beta-lactam antibiotics. A 20-year-old man was hospitalized several times for surgical treatment and complications caused by a right-sided vestibular schwannoma. Although the patient acquired several multidrug-resistant infections, this study focuses on the NDM-1-producing Acinetobacter spp. infection. As it was resistant to all antimicrobials tested, the medical team developed a 20-day regimen of 750mg/day metronidazole, 2,000,000IU/day polymyxin B, and 100mg/day tigecycline. The treatment was effective, and the patient recovered and was discharged from the hospital.

NDM-1 (New Delhi metallo beta lactamase-1) producing Gram-negative bacilli: Emergence & clinical implications.

Backgound & objectives: resistance to carbapenems in Gram-negative bacteria conferred by NDM-1 is a global health problem. We investigated the occurrence of NDM-1 in clinical isolates of gram-negative bacilli in a tertiary care hospital in Kashmir valley, India. Methods: Gram-negative bacilli from different clinical isolates were included in the study. Antimicrobial susceptibility was performed by Kirby Bauer disk diffusion method and interpreted using Clinical Laboratory Standards Institute (CLSI) guidelines. Isolates resistant to carbapenems were subjected to different phenotypic test such as modified hodge test (MHT), boronic acid and oxacillin based MHT (bA-MHT and OXA-MHT), combined disk test and minimum inhibitory concentration (MIC) with imipenem and imipenem -EDTA for determination of class B metallo enzymes. Presence of blaNDM-1 gene was established by PCR and confirmed by sequencing. Results: Of the total 1625 gram-negative isolates received, 100 were resistant to imipenem. Of the 100 isolates, 55 (55%) were positive by modified Hodge test indicating carbapenemase production. Of the 100 isolates tested by MHT, BA-MHT and OXA-MHT, 29 (29%) isolates belonged to Class A and 15 (15%) to Class B, while 56 (56%) isolates were negative. Of the 15 class B metallo beta lactamase producers, nine carried the blaNDM-1 gene. NDM-1 was found among escherichia coli (2 isolates), Klebsiella pneumoniae (2 isolates), Citrobacter freundii (3 isolates), Acinetobacter spp (1 isolate), and one isolate of Pseudomonas aeruginosa. Isolates were resistant to all antibiotic tested except polymyxin B and tigecycline. Interpretation & conclusions: Our study showed the presence of clinical isolates expressing NDM-1 in Srinagar, Jammu & Kashmir, India. These isolates harbour plasmid mediated multiple drug resistant determinants and can disseminate easily across several unrelated genera. To halt their spread, early identification of these isolates is mandatory.

Detection of blaOXA-23 in Acinetobacter spp. isolated from patients of a university hospital

INTRODUCTION: Acinetobacter spp. have emerged as notorious pathogens involved in healthcareassociated infections. Carbapenems are important antimicrobial agents for treating infections due to multidrug resistant Acinetobacter spp. Different mechanisms may confer resistance to these drugs in the genus, particularly production of class D carbapenemases. OXA-23-like family has been pointed out as one of the predominant carbapenamases among Acinetobacter. The present work aimed to investigate the occurrence of OXA-23-like carbapenemases among Acinetobacter isolates recovered from patients of a university hospital in Niterói, RJ, Brazil. METHODS: Antimicrobial susceptibility profiles were determined by disk-diffusion. Imipenem resistant isolates were submitted to Modified Hodge Test in order to screen for carbapenemase production, and later to polymerase chain reaction (PCR) to investigate the presence of blaOXA-23. RESULTS: Imipenem and meropenem resistance rates were 71.4% and 69.7%, respectively. The Modified Hodge Test revealed carbapenemase production among 76 (89.4%) of the 85 imipenem resistant isolates analyzed; according to PCR results, 81 isolates (95.4%) carried the blaOXA-23 gene. CONCLUSIONS: OXA-23-like enzymes may be an important mechanism of carbapenem resistance among isolates present in the hospital studied.

CHROMagar KPC. Comparación con el método propuesto por los Centros para el Control y Prevención de Enfermedades (CDC, EE.UU.) para el estudio de portación rectal y evaluación de falsos positivos / CHROMagar KPC. Comparison with the method proposed by the Centers for Disease Control and Prevention (CDC, USA) for rectal screening and evaluation of false positive results

Se cultivaron 81 hisopados rectales en el medio CHROMagar KPC y por el método del CDC. Fueron positivos para Klebsiella pneumoniae KPC en CHROMagar KPC, 9/81 y 6/81 con el método del CDC. El medio CHROMagar KPC tuvo dos falsos positivos: 1 K. pneumoniae y 1 Acinetobacter sp. Los falsos positivos del método CDC fueron: 25 Acinetobacter spp., 2 Escherichia coli y4K. pneumoniae. El empleo del medio CHROMagar KPC resultó ser un método con mayor recuperación de aislamientos productores de KPC y menos falsos positivos que el método del CDC. Para evaluar los falsos positivos en el medio CHROMagar KPC se cultivaron 1247 hisopados rectales. Se obtuvieron 1021 negativos, 171 K. pneumoniae KPC y 55 (4,4 %) falsos positivos. Debido al desarrollo de falsos positivos en el medio CHROMagar KPC, se debe confirmar por caracterización fenotípica la presencia de KPC en las bacterias aisladas.

Eighty one rectal swabs (RS) were cultured on CHROMagar KPC and the CDC method. Of the 81 samples, 9 were positive for KPC-producing Klebsiella pneumoniae on CHROMagar KPC, and 6 for the CDC method. CHROMagar KPC had two false positive (FP) results: 1 K. pneumoniae and 1 Acinetobacter sp. FP results on the CDC method were: 25 Acinetobacter spp., 2 Escherichia coli and 4 K. pneumoniae. CHROMagar KPC yielded a better recovery of KPC-producing bacteria and less FP results than CDC method. In order to evaluate FP results on CHROMagar KPC, 1247 RS were cultured and yielded 1021 negatives, 171 KPC-producing K. pneumoniae and 55 FP (4.4 %). Because of the FP results growing on CHROMagar KPC, KPC must be phenotypically confirmed in the bacteria isolated.

Resistencia a los antibacterianos en América Latina: consecuencias para la infectología / Antibacterial drug resistance in Latin America: consequences for infectiousdisease control

Antibacterial drug resistance is a particularly significant issue in Latin America. This article explores antimicrobial resistance in three classes of clinically important bacteria: gram-positive bacteria, enterobacteria, and nonfermenting gram-negativebacilli. The gram-positive bacteria frequently responsible for infections in humans are for the most part cocci: staphylococci, streptococci (including pneumococci), and enterococci,in both community and hospital settings. This situation is no different in theRegion of the Americas. Among the gram-positive bacteria, the causative agents of bacteremia are most commonly strains of coagulase-negative Staphylococcus, followed by enterococci. This report explores the resistance of these species to different antimicrobial drugs, resistance mechanisms in community and hospital strains, and new drugs for treating infections caused by these bacteria. In Latin America, antimicrobialresistance in Enterococcus strains is still a minor problem compared to the situation in the United States. The strains of the genus Streptococcus isolated from respiratory infections are still sensitive to penicillin. Furthermore, the resistance of enterobacteriais extremely important in the Region, particularly because of the broad dissemination of CTX-M extended-spectrum beta-lactamases (ESBL), some of which originated in Latin America. This article analyzes the resistance of Streptococcus pneumoniae, betahemolytic streptococci, and viridans group streptococci. Among the nonfermentinggram-negative bacilli, while Pseudomonas aeruginosa strains remain the leading cause of bacteremia, infections caused by strains of Acinetobacter spp. have proliferatedextensively in some areas. With regard to antibiotics, several options are available for treating gram-positive bacterial infections...

La resistencia a los fármacos antibacterianos tiene particular importancia en América Latina. En este artículo se analiza la resistencia a los antimicrobianos de tres clases de bacterias de importancia clínica: bacterias grampositivas, enterobacterias y bacilos gramnegativos no fermentadores.Las bacterias grampositivas que producen infecciones humanas frecuentes son, en su mayoría, cocos: estafilococos, estreptococos (incluidos neumococos) y enterococos, tanto en elmedio comunitario como en el nosocomial. Esta situación no es diferente en la Región de las Américas. Entre las bacterias grampositivas, las que causan bacteriemia con mayor frecuencia corresponden a cepas de estafilococos coagulasa negativos, seguidas de las de enterococos. Eneste informe se analiza la resistencia de estas especies a distintos antimicrobianos, los mecanismosde resistencia para las cepas de origen hospitalario y comunitario y los nuevos medicamentos para tratar las infecciones por estas bacterias. La resistencia a los antimicrobianos delas cepas de Enterococcus en América Latina todavía es un problema menor en relación con la situación en los Estados Unidos de América. Las cepas del género Streptococcus aisladasde infecciones respiratorias aún son sensibles a penicilina. Por otra parte, la resistencia de las enterobacterias es de gran importancia en la Región, particularmente por la gran difusión debetalactamasas de espectro extendido (BLEE) de tipo CTX-M, algunas de las cuales se originaron en América Latina. En el presente artículo se analizan la situación de la resistencia de las cepas de Streptococcus pneumoniae, y de los estreptococos betahemolítico y del grupo viridans. Entre los bacilos gramnegativos no fermentadores, si bien las cepas de Pseudomonasaeruginosa siguen siendo la causa principal de bacteriemias, la proliferación de infecciones por cepas de Acinetobacter spp. tiene en algunas partes gran magnitud...

Occurrence and the susceptibility to antimicrobial agents in Pseudomonas aeruginosa and Acinetobacter sp. at a tertiary hospital in southern Brazil / Ocorrência e perfil de sensibilidade a antimicrobianos em Pseudomonas aeruginosa e Acinetobacter sp. em um hospital terciário, no sul do Brasil

INTRODUCTION: The main mechanism of emerging resistance in Pseudomonas aeruginosa and Acinetobacter sp. isolates is the production of metallo-β-lactamases (MβLs). MβLs are enzymes capable of hydrolyzing cephalosporins, penicillins and carbapenems, but not monobactams (aztreonam), which are often used as antimicrobial therapy to treat nosocomial infections. METHODS: An observational descriptive and retrospective study was designed to assess the frequency of MβLs among strains of P. aeruginosa and Acinetobacter sp. obtained from a tertiary hospital in southern Brazil. RESULTS: MβL production was observed in 77.6 percent (n = 173/223) for P. aeruginosa isolates and 22.4 percent (n = 50/223) of Acinetobacter sp. isolates. The Acinetobacter sp. isolates showed 92.8 percent sensitivity to amikacin and P. aeruginosa isolates showed 58.9 percent sensitivity to aztreonam. CONCLUSIONS: The MβL indices determined confirm the global concern with this mechanism of resistance.

INTRODUÇÃO: O principal mecanismo de resistência entre isolados de Pseudomonas aeruginosa e Acinetobacter sp. é a produção de metalo-β-lactamases (MβLs). As MβLs são enzimas capazes de hidrolisar cefalosporinas, penicilinas e carbapenêmicos, mas não monobactâmicos (aztreonam) antibióticos que se encontram entre as principais opções terapêuticas para o tratamento de infecções causadas por bactérias não fermentadoras de glicose. MÉTODOS: Um estudo observacional, transversal, descritivo e retrospectivo foi desenvolvido para avaliar a frequência e o perfil de susceptibilidade cepas de P. aeruginosa e Acinetobacter sp. produtoras de MβLs isoladas no Hospital São Vicente de Paulo, Passo Fundo, Brasil. RESULTADOS: A produção de MβLs foi observada em 77,6 por cento (n = 173/223) dos isolados de P. aeruginosa e em 22,4 por cento (n = 50/223) dos isolados de Acinetobacter sp. Dentre as cepas produtoras de MβL, a maioria apresentou mais de 90 por cento de resistência a seis antimicrobianos dos 12 testados, enfatizando a resistência a ceftazidima, gentamicina, aztreonam, piperaciclina/tazobactam, cefepime, ciprofloxacina, meropenem e tobramicina. CONCLUSÕES: Os índices de MβL encontrados confirmam a preocupação mundial com a disseminação desse mecanismo de resistência.

Pesquisa de Acinetobacter sp e Pseudomonas aeruginosa produtores de metalo-β-lactamase em hospital de emergência de Porto Alegre, Estado do Rio Grande do Sul, Brasil / Investigation of metallo-β-lactamase-producing Acinetobacter sp and Pseudomonas aeruginosa at an emergency hospital in Porto Alegre, State of Rio Grande do Sul, Brazil

INTRODUÇÃO: O aparecimento de Pseudomonas aeruginosa e Acinetobacter sp produtores de metalo-β-lactamases (MBLs) é um desafio para os hospitais. MÉTODOS: Verificou-se a produção de MBL em cepas clínicas de Pseudomonas aeruginosa e Acinetobacter sp de um hospital de emergência de Porto Alegre pelo método de aproximação de disco e E-test MBL. Os genes bla foram pesquisados pela PCR. RESULTADOS: Duas cepas de Pseudomonas aeruginosa e oito Acinetobacter sp demonstraram fenótipo de MBLs. A amplificação do gene blaSPM-1 confirmou a enzima em P. aeruginosa.. CONCLUSÕES: Deve-se ter cautela ao avaliar testes fenotípicos utilizados na detecção rotineira de metalo-enzima.

INTRODUCTION: The appearance of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa and Acinetobacter sp. is a challenge for hospitals. METHODS: The production of MBL in clinical isolates of Pseudomonas aeruginosa and Acinetobacter sp. From an emergency hospital in Porto Alegre was investigated using the disk approximation test and MBL E-test. The bla genes were determined using PCR. RESULTS: Two strains of Pseudomonas aeruginosa and eight of Acinetobacter sp were shown to be MBL phenotypes. Amplification of the blaSPM-1 gene confirmed the presence of the enzyme in P. aeruginosa. CONCLUSIONS: Caution is needed in evaluating phenotype tests used for routine detection of metallo-β-lactamases.

Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative Gram-negative bacteria.

Background & objectives: The production of carbapenemases is an important mechanism responsible for the carbapenem resistance. A simple and inexpensive testing method for screening of carbapenemase producers is essential. A prospective study was undertaken to detect metallo-β-lactamases (MBLs) and AmpC β-lactamases in nonfermentative Gram negative bacteria and to evaluate the various methods for detection of carbapenemases and MBLs. Methods: A total of 100 Acinetobacter spp. (78 A. baumannii and 22 A. lwoffi i) and 140 Pseudomonas spp. (103 P. aeruginosa and 37 other Pseudomonas spp.) were screened for meropenem resistance by Kirby- Bauer disc diffusion method. Modifi ed Hodge test, EDTA disk synergy (EDS) test and AmpC disk test were used for the detection of carbapenemases, MBLs and AmpC β-lactamases, respectively. Results: Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffi i, 32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonas spp. were resistant to meropenem. Among the 32 meropenem resistant P. aeruginosa, 15 (46.9%) were AmpC β-lactamase producers, 16 (50.0%) MBL producers by EDS test, but only 9 (28.1%) found positive for carbapenemases by modifi ed Hodge test. Among the 46 meropenem resistant A. baumannii, 31 (67.4%) were AmpC β-lactamase producers, 3 (6.5%) MBL producers, but only 1 (14.3%) was positive for carbapenemases by modifi ed Hodge test. One P. aeruginosa was positive for carbapenemase by modifi ed Hodge test, but was negative for MBL and AmpC β-lactamase. Interpretation & conclusions: MBL production is an important mechanism of carbapenem resistance among Pseudomonas species but not among Acinetobacter species. EDS is more sensitive for detection of MBLs than modifi ed Hodge test. Both EDTA-meropenem and EDTA-ceftazidime combination must be used to detect all the MBL producers. Carbapenemases other than MBL may also be responsible for carbapenem resistance. AmpC β-lactamase is also a contributory factor for carbapenem resistance among the isolates in the hospital.

Metallo-beta-lactamase-producing clinical isolates of Acinetobacter species and Pseudomonas aeruginosa from intensive care unit patients of a tertiary care hospital.

Prompt detection of metallo-beta-lactamase (MBL) producing isolates is necessary to prevent their dissemination. Frequency of MBLs producing strains among multidrug resistant (MDR) Acinetobacter species and Pseudomonas aeruginosa was evaluated in critical care patients using imipenem-EDTA disk method. One hundred MDR Acinetobacter spp. and 42 Pseudomonas aeruginosa were checked for MBL production, from January to June 2001. MBL was produced by 96.6 % of imipenem-resistant Acinetobacter isolates, whereas 100% imipenem-resistant Pseudomonas aeroginosa isolates were MBL producers. Carbapenem resistance in MDR Acinetobacter spp. and Pseudomonas aeruginosa isolates in this study was due to MBLs. This calls for strict infection control measures to prevent further dissemination.

E-test para determinar concentraciones inhibitorias mínimas, estimar la diversidad bacteriana e identificar presuntivamente beta-lactamasas en cepas de pseudomonas aeruginosa y acinetobacter baumanii asociadas a infecciones intrahospitalarias / E-test to study minimal inhibitory concentrations, bacterial diversity and to presumably identify beta-lactamases in pseudomonas aeruginosa and acinetobacter baumanii strains associated to intrahospitaly infections

Sixty eight strains of Pseudomonas Aeruginosa and acinetobacter baumanii isolated on a teaching hospital were analized with E-test strips to determine their minimal inhibitory concentration for different antimicrobials. More than 75 percent of Acinetobacter baumanii were resistent to Piperacillin, Cefpirome, Cefepime, Gentamicin or Amikacin, 40 percent of strains were resistant to Ceftazidime, 27 and 53 percent of isolates had a decreased susceptibility to Meropenem and Piperacillin-tazobactam respectively. Twenty eight to 54 percent of Pseudomonas aeruginosa strains were resistant to Cefpirome, Cefepime, Ciprefloxacin and Gentamicin. 18 and 10 percent of were resistant to Meropenem and Imipenem respectively. Less than 10 percent of strains were resistant to Amikacin, Aztreonam, Piperacillin-tazobactam or Ceftazidime. Most of beta-lactam resistance of Pseudomonas aeruginosa was associated to dicreased susceptibility or resistance to Cefpirome, Cefepime or to Meropenem-Imipenem and did not match clearly with known beta-lactamase profiles. The knowledge of susceptibility of these bacteria responsible for nosocomial infections, will help to plan the appropiate use of antimicrobials