Resistência aos antimicrobianos e diversidade genética de variantes fenotípicas de Pseudomonas aeruginosa não sensíveis aos carbapenêmicos recuperadas de pacientes com fibrose cística com infecção pulmonar crônica: uma análise temporal / Antimicrobial resistance and genetic diversity of phenotypic variants of carbapenem non susceptible Pseudomonas aeruginosa recovered from patients with cystic fibrosis with chronic lung infection: a temporal analysis

Pseudomonas aeruginosa, bactéria ubíqua e versátil, pode se comportar como um patógeno oportunista, com ampla capacidade adaptativa, por múltiplos fatores de virulência e resistência. Como agente patogênico nas infecções pulmonares em pacientes com fibrose cística (FC), é motivo de prognóstico ruim, aumento de hospitalizações e altas taxas de morbimortalidade, sendo quase impossível a sua erradicação, ao evoluírem para a cronicidade. Globalmente, é notável o aumento nos índices de amostras não sensíveis aos carbapenêmicos e a múltiplos antimicrobianos, essenciais à terapêutica. Assim, avaliamos temporalmente a susceptibilidade aos antimicrobianos e a presença de amostras hipermutáveis (HPM) em P. aeruginosa de diferentes morfotipos, não sensíveis aos carbapenêmicos (PANSC), obtidas de pacientes FC com infecção pulmonar crônica, acompanhados em dois centros de referência no Rio de Janeiro. De 2007 a 2016, a análise retrospectiva, através dos resultados obtidos no teste de disco-difusão (TDD), permitiu selecionar amostras de PANSC incluídas neste trabalho. Usando os resultados obtidos no TDD, foi definida a susceptibilidade a outros antimicrobianos, bem como os fenótipos de resistência, multi-(MDR), extensivo-(XDR) e pandroga resistentes (PDR). Adicionalmente, determinou-se a concentração inibitória mínima (CIM) para imipenem (IPM), meropenem (MEM), doripenem (DOR) e polimixina (POL). Através de teste fenotípico, foi calculada a frequência de mutação espontânea e as amostras hipermutáveis foram caracterizadas. O sequenciamento de genoma total (SGT) foi realizado em seis amostras de diferentes morfotipos, incluindo uma variante fenotípica rara, a small colony variant (SCV). Essas amostras foram recuperadas em dois episódios de exacerbação do paciente. Foram investigadas a clonalidade, resistência a antimicrobianos e virulência. Das 143 amostras, de 18 pacientes (9 pediátricos e 9 adultos), os resultados do TDD apontaram taxas de não susceptibilidade superiores a 44% para gentamicina, amicacina, tobramicina e ciprofloxacina, e maiores de 30 % para POL. Pela determinação da CIM, quase a totalidade (96%) das amostras foram não sensíveis a IMP, seguidos de 56% para MEM e 44% para DOR. Analisando-se a distribuição dos valores da CIM50 e CIM90 nos dois grupos de pacientes, os valores para IMP foram maiores entre as amostras dos pacientes pediátricos, equivalendo a 32 µg/mL e 64 µg/mL, respectivamente. Cerca de 25%, 37% e 6% eram MDR, XDR e PDR, respectivamente. Aproximadamente 12% eram HPM, e mais da metade destas foram XDR. Após o SGT, as seis amostras, recuperadas do caso clínico foram classificadas em um novo sequence type (ST2744), com a presença de genes de resistência adquiridos blaPAO, blaOXA-50, aph(3')-Iib, fosA, catB7 e crpP, apresentando mutações em genes codificadores de porinas e bombas de efluxo. Entretanto, não foram observados marcadores genéticos clássicos exclusivos para os fenótipos SCV e HPM. Este é o primeiro relato de P. aeruginosa SCV na FC, no Brasil. A vigilância epidemiológica de P. aeruginosa é crucial para a conduta terapêutica, bem como para o sucesso da resposta do paciente e erradicação da infecção pulmonar, justificando o uso de técnicas fenotípicas e moleculares na detecção dos mecanismos de resistência e virulência desse microrganismo na FC.

Pseudomonas aeruginosa, a ubiquitous and versatile bacterium, can behave as an opportunistic pathogen, with strong adaptive capacity, due to multiple virulence and resistance factors. As a pulmonary infection pathogen in patients with cystic fibrosis (CF), it is related with poor prognosis, increased hospitalizations and high rates of morbidity and mortality, and the eradication is almost impossible, especially after chronicity. The increase rates of isolates non-susceptible to carbapenem and multiple antimicrobials, essentials to therapy, have been observed worldwide. Therefore, we assessed the antimicrobial susceptibility and the presence of hypermutability (HPM) in non-susceptible to carbapenem P. aeruginosa (PANSC) isolates from different morphotypes, obtained from CF patients with chronic pulmonary infection, followed at two reference centers in Rio de Janeiro. Using the results obtained by disk-diffusion test (DDT) between 2007 to 2016, we select 143 PANSC and susceptibility to other antimicrobials was defined, as well as the resistance phenotypes, multi- (MDR), extensive- (XDR) and pandrug resistant (PDR). Additionally, the minimum inhibitory concentration (MIC) for imipenem (IPM), meropenem (MEM), doripenem (DOR) and polymyxin (POL) was determined. Hypermutable isolates were characterized by determination of mutation frequency. Whole genome sequencing (WGS) was performed in six morphotypes isolates, including the small colony variant (SCV), a rare variant phenotype. These isolates were recovered in two exacerbation episodes. Clonality, antimicrobial resistance and virulence were investigated. Of the total (143 isolates) isolated from 18 patients (9 pediatric and 9 adults), non-susceptibility rates above than 44% for gentamicin, amikacin, tobramycin and ciprofloxacin, and more than 30% for POL were observed. Almost all (96%) of the isolates were non-susceptible to IPM by MIC determination, followed by 56% for MEM and 44% for DOR. MIC50 (32 µg/mL) and MIC90 (64 µg/mL) rates for IPM were higher among pediatric patient isolates and 25%, 37% and 6% were MDR, XDR and PDR, respectively. 12% of all isolates were classified as HPM and more than half were categorized as XDR. Using WGS, the six isolates recovered from the clinical case, were identified as a new sequence type (ST2744). Acquired resistance genes blaPAO, blaOXA-50, aph (3')-Iib, fosA, catB7 and crpP and mutations in encoding genes for porins and efflux pumps, was annotated. None exclusive classic genetic markers related to SCV and HPM phenotypes were not observed. This is the first Brazilian report of P. aeruginosa SCV in CF. Our results highlight the importance of epidemiological surveillance in P. aeruginosa. The application of phenotypic and molecular techniques to investigate resistance and virulence mechanisms, can contribute to therapeutic success in CF.

Xpert CARBA-R Assay for the Detection of Carbapenemase-Producing Organisms in Intensive Care Unit Patients of a Korean Tertiary Care Hospital

Carbapenemase-producing organisms (CPO) are rapidly disseminating worldwide, and their presence in tertiary care hospitals poses a significant threat to the management of nosocomial infections. There is a need to control CPO, especially in intensive care unit (ICU) patients, because these organisms are resistant to most beta-lactam antibiotics and are easily transmitted. At present, the identification of CPO is time-consuming; hence, this study focused on the use of the Xpert CARBA-R assay (Cepheid, USA) to determine intestinal colonization rates of CPO in patients admitted to the ICU of a tertiary care hospital in Korea. Forty clinical stool samples were collected and inoculated both in a CARBA-R cartridge and in conventional culture plates. The CARBA-R assay required only ~one hour to screen CPO, while the time required for conventional culture was over three days. We also found that the prevalences of intestinal colonization by carbapenem-resistant organisms and Enterobacteriaceae were 17.5% (7 out of 40) and 7.5% (3 out of 40), respectively. Among the colonizing strains, three that contained carbapenemase, including Klebsiella pneumonia carbapenemase (KPC), and imipenem (IMP) and Verona integron-mediated metallo-beta-lactamase (VIM) were found. With its convenience, the Xpert CARBA-R assay can be included in CPO surveillance strategies.

In Vitro Synergistic Effects of Antimicrobial Combinations on Extensively Drug-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii Isolates

BACKGROUND: Extensively drug-resistant (XDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a threat to hospitalized patients. We evaluated the effects of antimicrobial combinations on XDR P. aeruginosa and A. baumannii isolates. METHODS: P. aeruginosa and A. baumannii isolates, which were resistant to all antibiotics except colistin (CL), were collected from eight hospitals in Korea. Genes encoding metallo-beta-lactamases (MBLs) and OXA carbapenemases were detected by PCR in eight P. aeruginosa and 30 A. baumannii isolates. In vitro synergy of antimicrobial combinations was tested by using the checkerboard method. RESULTS: Minimum inhibitory concentrations of beta-lactams, aminoglycosides, and fluoroquinolones were very high, while that of CL was low for majority of XDR P. aeruginosa and A. baumannii isolates. Antimicrobial combinations including Imipenem (IPM)-CL, ceftazidime (CAZ)-CL, and rifampin (RIF)-CL exerted only additive/indifferent effects on majority of XDR P. aeruginosa isolates. Proportions of XDR A. baumannii isolates that showed synergistic and additive/indifferent inhibition after treatment with antimicrobial combinations used are as follows: IPM-ampicillin-sulbactam (AMS), 17% and 80% isolates, respectively; IPM-rifampin (RIF), 13% and 81% isolates, respectively; IPM-CL, 13% and 87% isolates, respectively; and RIF-COL, 20% and 73% isolates, respectively. Significant proportion (19%) of XDR P. aeruginosa isolates produced MBLs, and majority (82%) of A. baumannii isolates produced either MBLs or OXA-23. CONCLUSIONS: Our results suggest that combinations of IPM-AMS, IPM-RIF, IPM-CL, and RIF-CL are more useful than individual drugs for treating 13-20% of XDR A. baumannii infections.

Disk Carbapenemase Test for the Rapid Detection of KPC-, NDM-, and Other Metallo-β-Lactamase-Producing Gram-Negative Bacilli

BACKGROUND: Rapid detection of carbapenemase-producing gram-negative bacilli (GNB) is required for optimal treatment of infected patients. We developed and assessed a new disk carbapenemase test (DCT). METHODS: Paper disks containing 0.3 mg of imipenem and bromothymol blue indicator were developed, and the performance of the DCT were evaluated by using 742 strains of GNB with or without carbapenemases. RESULTS: The paper disks were simple to prepare, and the dried disks were stable at -20℃ and at 4℃. The DCT detected 212 of 215 strains (98.6% sensitivity with 95% confidence interval [CI] 96.0-99.5%) of GNB with known class A (KPC and Sme) and class B (NDM, IMP, VIM, and SIM) carbapenemases within 60 min, but failed to detect GES-5 carbapenemase. The DCT also detected all two Escherichia coli isolates with OXA-48, but failed to detect GNB with OXA-232, and other OXA carbapenemases. The DCT showed 100% specificity (95% CI, 99.2-100%) in the test of 448 imipenem-nonsusceptible, but carbapenemase genes not tested, clinical isolates of GNB. CONCLUSIONS: The DCT is simple and can be easily performed, even in small laboratories, for the rapid detection of GNB with KPC, NDM and the majority of IMP, VIM, and SIM carbapenemases.

Antimicrobial Susceptibility of Clinical Isolates of Bacteroides fragilis Group Organisms Recovered from 2009 to 2012 in a Korean Hospital

BACKGROUND: Periodic monitoring of antimicrobial resistance trends of clinically important anaerobic bacteria such as Bacteroides fragilis group organisms is required. We determined the antimicrobial susceptibilities of clinical isolates of B. fragilis group organisms recovered from 2009 to 2012 in a tertiary-care hospital in Korea. METHODS: A total of 180 nonduplicate clinical isolates of B. fragilis group organisms were collected in a tertiary care hospital. The species were identified by conventional methods: the ATB 32A rapid identification system (bioMerieux, France) and the Vitek MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (bioMerieux). Antimicrobial susceptibility was determined by the CLSI agar dilution method. RESULTS: Imipenem and meropenem resistance rates were 0-6% for B. fragilis group isolates. The rate of resistance to piperacillin-tazobactam was 2% for B. fragilis and 0% for other Bacteroides species, but 17% for B. thetaiotaomicron isolates. High resistance rates to piperacillin (72% and 69%), cefotetan (89% and 58%), and clindamycin (83% and 69%) were observed for B. thetaiotaomicron and other Bacteroides spp. The moxifloxacin resistance rate was 27% for other Bacteroides spp. The MIC50 and MIC90 of tigecycline were 2-4 microg/mL and 8-16 microg/mL, respectively. No isolates were resistant to chloramphenicol or metronidazole. CONCLUSIONS: Imipenem, meropenem, chloramphenicol, and metronidazole remain active against B. fragilis group isolates. Moxifloxacin and tigecycline resistance rates are 2-27% and 8-15% for B. fragilis group isolates, respectively.

Changes in Causative Pathogens of Acute Cholangitis and Their Antimicrobial Susceptibility over a Period of 6 Years / 대한소화기학회지

BACKGROUND/AIMS: We evaluated changes of causative pathogen in acute cholangitis and their antimicrobial susceptibility over six years and differences between community-acquired and hospital-acquired acute cholangitis at our institution. METHODS: Medical records of 1,596 patients with acute cholangitis and biliary drainage between August 2006 and August 2012 were reviewed retrospectively. Cases were divided according to time: period 1 (August 2006-December 2008, n=645, 40.4%), period 2 (January 2009-August 2012, n=951, 59.6%). Cases were divided according to community-acquired cholangitis (n=1,397, 87.5%) and hospital-acquired cholangitis (n=199, 12.5%). Causative pathogens and antimicrobial susceptibility were investigated in each group. RESULTS: Causative pathogen was isolated from bile culture in 1,520 out of 1,596 cases (95.2%). The three most frequently isolated Gram-negative bacteria were extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (n=485, 30.4%), E. coli (n=237, 13.2%), and Citrobacter freundii (n=110, 6.9%). Between periods 1 and 2, prevalence of ESBL-producing E. coli and Klebsiella pneumoniae did not show significant change (36.7% vs. 32.1%, p=0.073; 6.6% vs. 6.2%, p=0.732). C. freundii showed a significant increase from period 1 to period 2 (1.7% vs. 13.2%, p=0.000). In both time periods, imipenem was the antimicrobial agent showing the highest rate of susceptibility (93.3% vs. 93.9%, p=0.783). Higher prevalence of ESBL-producing E. coli and C. freundii was observed in the hospital-acquired cholangitis group (52.1% vs. 31.2%, p=0.000; 15.9% vs. 7.3%, p=0.001). CONCLUSIONS: The most common causative pathogen of acute cholangitis was ESBL-producing E. coli. Prevalence of C. freundii increased over the time period. Imipenem should be reserved as an alternative for resistant pathogens.

The First Case of Septicemia Caused by Imipenem-Susceptible, Meropenem-Resistant Klebsiella pneumoniae

No abstract available.

Molecular Epidemiology of Integron-Associated Antimicrobial Gene Cassettes in the Clinical Isolates of Acinetobacter baumannii from Northern Taiwan

BACKGROUND: The aims of this study were to understand the molecular epidemiology of integron-associated gene cassettes in Acinetobacter baumannii across four hospitals in northern Taiwan and to clarify the relationship between the presence of integrons and antibiotic-resistant phenotypes. METHODS: Sixty-five A. baumannii isolates, collected from the patients of four regional hospitals in northern Taiwan in 2009, were tested for the presence of integrons and their associated gene cassettes. The susceptibility difference between integron-positive and integron-negative A. baumannii strains was analyzed. Antibiotic-resistant phenotypes among A. baumannii with different types of gene cassette array combinations were also compared. RESULTS: Around 72% of the A. baumannii isolates carried class 1 integrase genes. Despite this, only three gene cassette arrays were found in the integrons. Integron-positive strains were significantly more resistant to all the tested antibiotics than the integrase-negative strains. All the four types of A. baumannii with different gene cassette array combinations were multidrug-resistant in nature. Gene cassette array aacA4-catB8-aadA1 existed in all the integron-positive A. baumannii isolates. Repetitive-sequence-based PCR (rep-PCR) results revealed the prevalence of one major cluster of imipenem-resistant A. baumannii strains (84%) in the four regional hospitals. CONCLUSIONS: The presence of integrons with associated antimicrobial resistance gene cassettes can be used as a representative marker of multidrug resistance in A. baumannii. Some prevalent gene cassette arrays may exist among epidemiologically unrelated A. baumannii strains.

Nosocomial Infection by Sequence Type 357 Multidrug-Resistant Acinetobacter baumannii Isolates in a Neonatal Intensive Care Unit in Daejeon, Korea

Acinetobacter baumannii is an important microorganism responsible for a number of nosocomial outbreaks, in particular, in intensive care units (ICUs). We investigated a nosocomial infection caused by multidrug-resistant (MDR) A. baumannii in a neonatal intensive care unit (NICU) in Korea. A. baumannii isolates were characterized using Etest (AB Biodisk, Sweden), two multiplex PCR assays, and multilocus sequence typing (MLST) scheme. PCR and PCR mapping experiments were performed for detecting and characterizing the determinants of antimicrobial resistance. Eight strains isolated from an NICU belonged to European (EU) clone II and revealed only one sequence type (ST), namely, ST357. All the isolates were susceptible to imipenem but were resistant to amikacin, gentamicin, ceftazidime, cefepime, and ciprofloxacin. To the best of our knowledge, this is the first report of a nosocomial infection in an NICU in Korea caused by ST357 MDR/carbapenem-susceptible A. baumannii strains. This result demonstrates that nosocomial outbreaks of MDR/carbapenem-susceptible strains as well as MDR/carbapenem-resistant isolates may occur in NICUs.

First Detection of VIM-4 Metallo-beta-Lactamase-Producing Citrobacter freundii in China

No abstract available.

Detection and characterization of metallo-β-lactamases in Pseudomonas aeruginosa by phenotypic and molecular methods from clinical samples in a tertiary care hospital / Detección y caracterización de la metallo-betalactamasa en la Pseudomonas aeruginosa mediante métodos fenotípicos y moleculares a partir de muestras clínicas en un hospital de atención terciaria

AIMS: The aim of this study was to detect and characterize the presence of metallo-β-lactamase (MBL) production in multidrug resistant (MDR) P aeruginosa collected from clinical samples in a tertiary care hospital. METHODS AND MATERIALS: A total of 67 non-repetitive isolates of MDR P aeruginosa recovered from various clinical specimens were screened for MBL production by IPM/MEM-EDTA combined disc test. Polymerase chain reaction was performed on all isolates using blaIMP and blaVIM consensus primers to characterize them genotypically. RESULTS: Among 67 P aeruginosa isolates, 62.7% (42/67) and 70.1% (47/67) were resistant to imipenem and meropenem respectively and 47 (70.1%) were found to be MBL producers. Among this 47 MBL-producing isolates, 41 (61.1%) strains carried the blaVIM gene and 2 (3%) strains carried the blaIMP gene. Three strains were phenotypically negative but positive genotypically for blaVIM gene. One strain was resistant to both imipenem and meropenem but did not show phenotypic positivity. CONCLUSION: This study confirms the dissemination of blaVIM genes among MDR Pseudomonas aeruginosa and hence it is indispensible to identify and aptly control the threat of horizontal and vertical transfer.

OBJETIVO: El objetivo de este estudio es descubrir y caracterizar la presencia de producción de metallo-betalactamasa (MBL) en P aeruginosa resistente a los multifármacos (RMF), recogida de muestras clínicas de un hospital de atención terciaria. MÉTODO: Un total de 67 aislados no repetitivos de P aeruginosa RMF obtenidos de varios specímenes clínicos, fueron tamizados en busca de producción de MBL, mediante una prueba de disco combinado IPM/MEM-EDTA. Se efectuó una reacción en cadena de la polimerasa sobre todos los aislados, usando iniciadores de consenso blaIMP y blaVIM para la caracterización genotípica. RESULTADOS: Entre los aislados de P aeruginosa, 62.7% (42/67) y 70.1% (47/67) fueron resistentes al Imipenem y al Meropenem respectivamente, mientras que se halló que 47 (70.1%) eran productores de MBL. De los 47 aislados productores de MBL, 41 (61.1%) cepas eran portadoras del gen blaVIM en tanto que 2 (3%) cepas eran portadoras del gen blaIMP. Tres cepas fueron fenotípicamente negativas, pero genotípicamente positivas con respecto al gen blaVIM. Una cepa fue resistente tanto al Imipenem como al Meropenem, pero no mostró positividad fenotípicamente. CONCLUSIÓN: El presente estudio confirma la diseminación de los genes blaVIM entre las Pseudomonas aeruginosa RMF. Es importante identificar así como controlar adecuadamente la amenaza de la transferencia horizontal y vertical.

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

Diclofenac sodium and Imipenem action on rat intestinal mucosa: a biomechanical and histological study / Ação do Diclofenaco de sódio e Imipenem na mucosa intestinal do rato: estudo biomecânico e histológico

PURPOSE: To study diclofenac sodium induced histological and mechanical alterations and their prevention with Imipenem in rat intestine. METHODS: Male Wistar rats (n=240) were randomly assigned to four experimental groups: GI: n=60 treated with 0.9 percent saline IM; GII: n=60 treated with 6mg/kg body weight diclofenac sodium IM for four days; GIII: n=60 treated with 30mg/kg body weight Imipenem IM for four days, and GIV n=60 treated with diclofenac sodium plus Imipenem at the above doses IM for 4 days. Each group was further divided into 4 subgroups of 15 rats each and sacrificed at 4, 7, 14, and 21 days of follow-up, respectively. Abdominal cavity macroscopy and histology, and small bowel breaking strength were analyzed at each sacrifice moment. RESULTS: There were no histological or mechanical alterations in normal control rats throughout the study. Ulcerated lesions in intestinal mucosa were observed and breaking strength decreased in all diclofenac sodium treated rats. Ulcerated lesions in intestinal mucosa were prevented by Imipenem in all rats. CONCLUSION: Diclofenac sodium induced ulcerated lesions in rat intestinal mucosa can be prevented by Imipenem treatment.

OBJETIVO: Avaliar as alterações histológicas e biomecânicas do diclofenaco de sódio na mucosa intestinal do rato e a associação com o uso de Imipenem. MÉTODOS: Foram estudados 240 ratos Wistar distribuídos aleatoriamente em quatro grupos experimentais: GI: 60 ratos tratados com injeção IM de soro fisiológico 0,9 por cento; GII: 60 ratos tratados com injeção IM de diclofenaco de sódio na dose de 6mg/kg de peso por 4 dias; GIII: 60 ratos tratados com injeção IM de Imipenem na dose de 30 mg/kg de peso por 4 dias; GIV: 60 ratos tratados com injeção IM de soro fisiológico e diclofenaco de sódio nas doses acima. Em cada grupo os animais foram posteriormente divididos em 4 momentos de 15 animais em cada um para sacrifício, respectivamente, no 4º, 7º, 14º e 21º dias após o início do tratamento. As alterações da cavidade abdominal, assim como as características histológicas e de força de ruptura do intestino delgado foram analisadas em cada momento, em cada grupo. RESULTADOS: Não foram encontradas alterações histológicas e biomecânicas nos animais do Grupo I nesse estudo. Lesões ulceradas na mucosa do intestino delgado foram observadas nos animais tratados com diclofenaco de sódio, assim como diminuição da força de ruptura. As lesões ulceradas encontradas foram prevenidas pelo uso de Imipenem. CONCLUSÃO: O diclofenaco de sódio induz lesões ulceradas na mucosa intestinal do rato que podem ser prevenidas pelo uso de Imipenem.

Resistencia a carbapenemes en aislamientos de pseudomonas aeruginosa: un ejemplo de interacción entre distintos mecanismos / Carbapenem resistance in pseudomonas aeruginosa isolates: an example of interaction between different mechanisms

Objetivo. Identificar la proteína de membrana externa ausente en los aislamientos resistentes y determinar tanto las causas de su ausencia en la membrana, como la presencia de otros mecanismos de resistencia a carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa. Métodos. Se estudió un brote de 20 aislamientos de P. aeruginosa previamente caracterizados como productores de la metalobetalactamasa IMP-13. Estos aislamientos presentaron igual expresión de la enzima IMP-13, pero solo cinco de ellos fueron resistentes acarbapenemes. En esos cinco aislamientos resistentes se confirmó la ausencia de una proteína de membrana externa. Se secuenciaron oprD y ampC; se identificaron las proteínas de membrana externa por desorción/ionización láser asistida por matriz/espectometría de masa tiempo de vuelo (MALDI-TOF); se determinó el nivel de expresión de OprD, de AmpC y de los sistemas de eflujo tipo Mex, por reacción en cadena de polimerasa en tiempo real, y por último, se determinó la contribución del déficit de OprD a la resistencia a carbapenemes. Resultados. La proteína de la membrana externa ausente en el grupo R (resistentes a ambos carbapenemes) fue identificada como OprD-TS, pero no se observaron variaciones en suexpresión. El gen oprD presentó mutaciones en los cinco aislamientos resistentes. Se observó la misma producción de la enzima tipo AmpC PDC-5 y del sistema de eflujo Mex AB-OprM entre los aislamientos sensibles y resistentes a carbapenemes. Se analizó cómo la presencia conjunta de IMP-13 y el déficit de OprD contribuyen al aumento de la resistencia.Conclusiones. Distintos mecanismos contribuyen a la resistencia de aislamientos productores de IMP-13 a carbapenemes. La posibilidad de no detectar estos aislamientos productores de IMP-13 representa un riesgo latente de selección de mutantes con mecanismos de resistencia que se suman para aumentar la resistencia a carbapenemes.

Objective. To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of othermechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. Methods. Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibitedequal expression of the IMP-13 enzyme, but only five of them were carbapenemresistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identifiedusing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. Results. The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presentedmutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and‑resistant isolates. The contribution of the combined presence of IMP-13 and reducedOprD to increased resistance was examined. Conclusions. Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistancemechanisms in order to enhance carbapenem resistance.

Antimicrobial activity of doripenem against Gram-negative pathogens: results from INVITA-A-DORI Brazilian study

In vitro activity of doripenem and comparator antimicrobial agents was evaluated against Gram-negative bacilli recently isolated from Brazilian private hospitals that were enrolled in the INVITA-A-DORI Brazilian Study. A total of 805 unique Gram-negative bacilli were collected from patients hospitalized at 18 medical centers between May/08 and March/09. Each hospital was asked to submit 50 single Gram-negative bacilli isolated from blood, lower respiratory tract or intraabdominal secretions. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using Clinical Laboratory Standards Institute (CLSI) microdilution method at a central laboratory. CLSI M100-S21 (2011) or US-FDA package insert criteria (tigecycline) was used for interpretation of the antimicrobial susceptibility results. Doripenem was as active as meropenem and more active than imipenem against E. coli and K. pneumoniae isolates. A total of 50.0 percent of Enterobacter spp. isolates were resistant to ceftazidime but 85.7 percent of them were inhibited at doripenem MICs < 1 µg/mL. Polymyxin B was the only agent to show potent activity against Acinetobacter spp. (MIC50/90, < 0.5/1 µg/mL) and P. aeruginosa (MIC50/90, 1/2 µg/mL). Although high rates of imipenem (53.1 percent) and meropenem (44.5 percent) resistance were detected among P. aeruginosa, doripenem showed MIC50 of 16 µg/mL against imipenem-resistant P. aeruginosa and inhibited a greater number of imipenem-resistant P. aeruginosa (10.5 percent) at MIC values of < 4 µg/mL than did meropenem (0.0 percent). In this study, doripenem showed similar in vitro activity to that of meropenem and retained some activity against imipenem-resistant P. aeruginosa isolated from Brazilian medical centers.

Assessing the pharmacodynamic profile of intravenous antibiotics against prevalent Gram-negative organisms collected in Colombia

OBJECTIVES: This study was designed to simulate standard and optimized dosing regimens for intravenous antibiotics against contemporary populations of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa using MIC distribution data to determine which of the tested carbapenem regimens provided the greatest opportunity for obtaining maximal pharmacodynamic (PD) activity. METHODS: The isolates studied were obtained from the COMPACT-COLOMBIA surveillance program conducted between February and November 2009. Antimicrobial susceptibility testing was conducted by broth microdilution method according to the CLSI guidelines. Doripenem, imipenem-cilastatin, and meropenem, were the modeled antibiotics. A 5,000 patient Monte Carlo simulation was performed for each regimen and PD targets were defined as free drug concentrations above the MIC for at least 40 percent of the dosing interval. RESULTS: All carbapenem regimens obtained optimal exposures against E. coli, unlike the other Enterobacteriaceae tested. Against P. aeruginosa, only a prolonged infusion of doripenem exceeded the 90 percent cumulative fraction of response (CFR) threshold. Worrisomely, no regimens for any of the drugs tested obtained optimal CFR against A. baumannii. For P. aeruginosa intensive care unit (ICU) isolates, CFR was approximately 20 percent lower for isolates collected in the respiratory tract compared with bloodstream or intra-abdominal for imipenem and meropenem. Noteworthy, all doripenem and meropenem regimens achieved greater than 90 percent CFR against bloodstream and respiratory isolates of K. pneumoniae. CONCLUSIONS: Our data suggests that higher dosing and prolonged infusion of doripenem or meropenem may be suitable for empirically treating ICU P. aeruginosa, while none of the carbapenems achieved optimal cumulative fraction of response against A. baumannii. Standard dosing regimens of all the carbapenems tested achieved optimal CFR against E. coli isolates, but higher carbapenem dosages might be required for empiric treatment of K. pneumoniae, particularly from an intra-abdominal source. Non-standard dosage regimens studied in this modeling should be proven effective in prospective clinical trials.

Detection of SPM and IMP metallo-β-lactamases in clinical specimens of Pseudomonas aeruginosa from a Brazilian public tertiary hospital

Phenotypic and genotypic SPM and IMP metallo-β-lactamases (MBL) detection and also the determination of minimal inhibitory concentrations (MIC) to imipenem, meropenem and ceftazidime were evaluated in 47 multidrug-resistant Pseudomonas aeruginosa isolates from clinical specimens. Polymerase chain reaction detected 14 positive samples to either blaSPM or blaIMP genes, while the best phenotypic assay (ceftazidime substrate and mercaptopropionic acid inhibitor) detected 13 of these samples. Imipenem, meropenem and ceftazidime MICs were higher for MBL positive compared to MBL negative isolates. We describe here the SPM and IMP MBL findings in clinical specimens of P. aeruginosa from the University Hospital of Botucatu Medical School, São Paulo, Brazil, that reinforce local studies showing the high spreading of blaSPM and blaIMP genes among brazilian clinical isolates.

Prevalence and clinical outcomes of episodes of ventilator-associated pneumonia caused by SPM-1-producing and non-producing imipenem-resistant Pseudomonas aeruginosa / Prevalência e evolução clínica de episódios de pneumonia associada à ventilação mecânica causada por Pseudomonas aeruginosa resistente a imipenem produtoras e não-produtoras de SPM-1

INTRODUCTION: Pseudomonas aeruginosa is a leading cause of ventilator-associated pneumonia (VAP) and exhibits high rates of resistance to several antimicrobial drugs. The carbapenens are usually the drugs of choice against this microorganism. However, the carbapenem resistance has increased among these strains worldwide. The presence of metallo-β-lactamases (MBL) has been pointed out as a major mechanism of resistance among these strains. No previous study addressed outcomes of respiratory infections caused by these strains. METHODS: Our group sought to analyze the epidemiology and clinical outcomes of patients with VAP caused by imipenem-resistant P. aeruginosa. A total of 29 clinical isolates of carbapenem-resistant Pseudomonas aeruginosa were screened for metallo-β-lactamase (MBL) genes. RESULTS: Demographic and clinical variables were similar between the SPM-1-producing and non-SPM-1-producing group. Five (17.2 percent) isolates were positive for blaSPM-1. No other MBL gene was found. All patients were treated with polymyxin B. The infection-related mortality was 40 percent and 54.2 percent for SPM-1-producing and -non-producing isolates, respectively. CONCLUSIONS: There were no differences in epidemiological and clinical outcomes between the two groups.

INTRODUÇÃO: Pseudomonas aeruginosa é uma importante causa de pneumonia associada à ventilação mecânica (PAV) e exibe altas taxas de resistência a vários antimicrobianos. Os carbapenens são usualmente as drogas de escolha para esse microorganismo. Contudo, a resistência a carbapenens tem crescido entre essas amostras em todo o mundo. A presença de metalo- β-lactamase (MBL) tem sido apontado como um importante mecanismo de resistência nessas cepas. Nenhum estudo prévio avaliou desfechos clínicos de infecções respiratórias causadas por essas amostras MÉTODOS: Nosso grupo analisou a epidemiologia e evolução clínica de episódios de PAV causada por P. aeruginosa resistente a imipenem. Um total de vinte e nove isolados clínicos de Pseudomonas aeruginosa resistente a carbapenem foram avaliados quanto à presença de genes para metalo-β-lactamase (MBL). RESULTADOS: Variáveis clínicas e demográficas foram similares entre o grupo produtor de SPM-1 e o não-produtor. Cinco (17,2 por cento) isolados foram positivos para blaSPM-1. Nenhum outro gene para MBL foi encontrado. Todos os pacientes foram tratados com polimixina B. A mortalidade relacionada à infecção foi de 40 por cento e 50 por cento respectivamente para os isolados produtores de SPM-1 e não-produtores de SPM-1. CONCLUSÕES: Nao houve diferença entre os dados epidemiológicos e a evolução clínica entre os dois grupos.

Antimicrobial resistance and prevalence of resistance genes in intestinal Bacteroidales strains

OBJECTIVE: This study examined the antimicrobial resistance profile and the prevalence of resistance genes in Bacteroides spp. and Parabacteroides distasonis strains isolated from children's intestinal microbiota. METHODS: The susceptibility of these bacteria to 10 antimicrobials was determined using an agar dilution method. β-lactamase activity was assessed by hydrolysis of the chromogenic cephalosporin of 114 Bacteriodales strains isolated from the fecal samples of 39 children, and the presence of resistance genes was tested using a PCR assay. RESULTS: All strains were susceptible to imipenem and metronidazole. The following resistance rates were observed: amoxicillin (93 percent), amoxicillin/clavulanic acid (47.3 percent), ampicillin (96.4 percent), cephalexin (99 percent), cefoxitin (23 percent), penicillin (99 percent), clindamycin (34.2 percent) and tetracycline (53.5 percent). P-lactamase production was verified in 92 percent of the evaluated strains. The presence of the cfiA, cepA, ermF, tetQ and nim genes was observed in 62.3 percent, 76.3 percent, 27 percent, 79.8 percent and 7.8 percent of the strains, respectively. CONCLUSIONS: Our results indicate an increase in the resistance to several antibiotics in intestinal Bacteroides spp. and Parabacteroides distasonis and demonstrate that these microorganisms harbor antimicrobial resistance genes that may be transferred to other susceptible intestinal strains.