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.

Intereactions between doripenem and clavulanate ­ Application of minimal inhibitory concentration analysis and cytometry flow for bactericidal studies

Background: In view of the current low efficacy of bacterial infection treatment the common trend towards searching for antibiotic systems exhibiting synergistic action is well justified. Among carbapenem analogues a particularly interesting option is provided by combinations of clavulanic acid with meropenem, which have proven to be especially effective. Results: Determination of the minimal inhibitory concentration (MIC) along with the method based on flow cytometry constitutes an important tool in the identification of bacterial sensitivity to active substances. Within this study the inhibitory effect of doripenem, clavulanic acid and the doripenem-clavulanate acid system was analyzed in relation to such bacteria as Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Clostridium butyricum and Clostridium pasteurianum, Acinetobacter baumannii, Enterobacter aerogenes. The lowest MIC, amounting to 0.03 µg/mL, was observed for the doripenem-clavulanate acid system in the case of E. coli ATCC 25922. In turn, the lowest MIC for doripenem applied alone was recorded for K. pneumoniae ATCC 31488, for which it was 0.1 µg/mL. The strain which proved to be most resistant both to doripenem and the doripenem-clavulanate acid system, was A. baumannii, with MIC of 32 µg/mL (clinical isolate) and 16 µg/mL (reference strain). Cytometric analysis for P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 showed changes in cells following exposure to limiting concentrations of the active substance. Conclusions: Analysis of MIC supplies important information concerning microbial sensitivity to active substances, mainly in terms of limiting concentrations causing mortality or vitality of the tested species, which is essential when selecting appropriate antibiotic therapy.

Stability of doripenem in reconstituted solution - thermal and oxidative decomposition kinetics and degradation products by LC-MS.

A ultra-fast liquid chromatography method applied to quantitation of doripenem in powder for injection was validated. Validation parameters were assayed and a rapid analysis was established by a reversed-phase system comprising a C18 column endcapped (50 × 4.0 mm, 2.0 µm), mobile phase consisting of phosphoric acid 0.01% (pH 3.8) and acetonitrile (98:02, v/v) and a flow rate of 0.4 mL min-1 . Drug stability was studied through submission to forced conditions, allowing the major degradation products to be detected and the kinetics parameters to be established. Thermal and oxidative degradation were determined, and indicated a kinetic decomposition following first and second order, respectively. The main degradation products were identified by LC-MS analysis, and the results were evaluated in order to suggest the chemical structures corresponding to respective masses and fragmentations. Six compounds were identified, with m/z 411, 427, 437, 634, 650 and 664. All of them resulted from cleavage of ß-lactam ring and alcoholic chain and/or dimerization. These experimental results provide valuable information about the stability of doripenem reconstituted solution and procedures for its handling and storage.

Actividad comparativa in vitro de doripenem y otros carbapenemicos frente a pseudomonas aeruginosa aisladas en el laboratorio de microbiología del hospital Vargas de Caracas / Comparative in vitro activity of dorypenem and other carbapenems in pseudomonas aeruginosa isolates on Vargas Caracas Hospital´s Microbiology

Los ensayos de cuantificación de ARN plasmáticos de VIH-1 son importantes para el control de pacientes infectados, así como el monitoreo de la respuesta a la terapia antirretroviral. Por lo tanto, los ensayos comerciales empleados para este propósito deben presentar buena correlación entre si, para dar lugar al manejo terapéutico apropiado. El objetivo del estudio consistió en correlacionar los resultados obtenidos mediante el ensayo de amplificación de señal (bDNA) y PCR en tiempo real (RT-PCR), ambas casas comerciales aprobadas por la FDA y con diferente diana de detección del VIH-1. La validación se realizó con 180 muestras clínicas de pacientes referidos al INHRR. Los resultados fueron comparados con la subpoblación de linfocitos TCD4+ determinados mediante citometría de flujo. El análisis estadístico se realizó empleando el coeficiente de regresión lineal de Pearson (R2) y el valor de contraste de hipótesis con una significancia del 95 %, usando el programa SPSS Statistics v10.0. Se observó una buena correlación entre los ensayos (R2=0.961, p<0.05), siendo la RTPCR más sensible. Las diferencias cuantitativas de carga viral entre las técnicas ensayadas fue menor de 0.5 log10 copias/ml para el 89% de las muestras, y >1 log10 copias/ml solo en dos pacientes, no indicando necesariamente cambio terapéutico. Adicionalmente, se encontró una correlación inversa entre los linfocitos TCD4+ y carga viral del VIH-1 medida por bDNA (R2= 0.20, p<0.05) y RT-PCR (R2= 0.15, p<0.05). Los ensayos evaluados mostraron que ambas técnicas puedes ser empleadas indistintamente para el control de los pacientes VIH positivo.

The assay for quantification of plasma HIV-1 RNA are important for the control of patients infected, as well as the monitoring of the response to antiretroviral therapy. Therefore, the commercial assays used for this purpose must submit good correlation between to give place to the appropriate therapeutic management. In this study, we correlate the results obtained through the testing of signal amplification (bDNA) and real-time PCR (RT-PCR), two comercial technical approved by the FDA and with different targets of detection HIV-1. The validation was carried out with 180 clinical samples of patients referred to the INHRR. The results were compared with the subpopulation of lymphocytes TCD4+ determined by flow cytometry. The statistical analysis was performed using the program SPSS Statistics v10. It was observed good correlation between the tests studied (R2=0.961, p<0.05), with RT-PCR more sensitive. The quantitative differences in viral load between the techniques tested was less than 0.5 log10 copies/ml for the 89% of the samples, and >1 log10 copies/ml in only two patients. Additionally, it was found an inverse correlation between lymphocytes TCD4+ and viral load of HIV-1, measured by bDNA (R2= 0.20, p<0.05) and RT-PCR (R2= 0.15, p<0.05). Therefore, these assays can be employed for the patient control HIV.

A meta-analysis of efficacy and safety of doripenem for treating bacterial infections.

OBJECTIVE: The aim of this article is to compare the efficacy and safety of doripenem for bacterial infections. METHODS: We included six randomized clinical trials identified from PubMed and Embase up to July 31, 2014. The included trials compared efficacy and safety of doripenem for complicated intra-abdominal infections, complicated urinary tract infection, nosocomial pneumonia, and acute biliary tract infection. The meta-analysis was carried on by the statistical software of Review Manager, version 5.2. RESULTS: Compared with empirical antimicrobial agents on overall treatment efficacy, doripenem was associated with similar clinical and microbiological treatment success rates (for the clinical evaluable population, odds ratio [OR]=1.26, 95% confidence interval [CI] 0.93-1.69, p=0.13; for clinical modified intent-to-treatment population, OR=0.88, 95% CI 0.55-1.41, p=0.60; for microbiology evaluable population, OR=1.16, 95% CI 0.90-1.50, p=0.26; for microbiological modified intent-to-treatment (m-mITT), OR=0.98, 95% CI 0.81-1.20, p=0.87). We compared incidence of adverse events and all-cause mortality to analyze treatment safety. The outcomes suggested that doripenem was similar to comparators in terms of incidence of adverse events and all-cause mortality on modified intent-to-treatment population (for incidence of AEs, OR=1.10, 95% CI 0.90-1.35, p=0.33; for all-cause mortality, OR=1.08, 95% CI 0.77-1.51, p=0.67). In nosocomial pneumonia and ventilator-associated pneumonia treatment, doripenem was not inferior to other antibacterial agents in terms of efficacy and safety. CONCLUSION: From this meta-analysis, we can conclude that doripenem is as valuable and well-tolerated than empirical antimicrobial agents for complicated intra-abdominal infections, complicated urinary tract infection, acute biliary tract infection and nosocomial pneumonia treatment.

Actividad in vitro de doripenem comparado con meropenem frente a aislamientos de Pseudomonas aeruginosa en un centro asistencial de tercer nivel de Bogotá (Colombia) / Activity of Doripenem compared to Meropenem against Pseudomonas aeruginosa isolates at Tertiary Care Teaching Hospital in Bogotá (Colombia)

Objetivo: El objetivo de este estudio fue evaluar la actividad in vitro de doripenem comparado con meropenem frente a aislamientos locales de Pseudomonas aeruginosa (P. aeruginosa) . Materiales y métodos: En el Hospital San José de Bogotá (Colombia), un hospital universitario de 300 camas, se recolectaron entre 2009 y 2011, 83 aislamientos no repetidos de P. aeruginosa , en los cuales se determinó la concentración inhibitoria mínima frente a meropenem y doripenem por el método de E-test. Se aplicaron los puntos de corte del Clinical and Laboratory Standars Institute para carbapenémicos vigentes para 2012 Resultados: La sensibilidad global de los aislamientos de P. aeruginosa a meropenem fue del 69% y a doripenem del 75%, siendo la concentración inhibitoria mínima 50 y 90 (µg/mL) de 0,75 y 28,8 para meropenem y de 0,64 y 15,2 para doripenem. Conclusiones: La concentración inhibitoria mínima frente a doripenem fue menor que la correspondiente a meropenem. Se requiere realizar en el ámbito local estudios de este tipo con el fin de llevar a cabo los ajustes a los protocolos de antibióticos institucionales.

objective: The aim of this study was to evaluate the in vitro activity of doripenem compared to meropemen against local isolates of Pseudomonas aeruginosa (P. aeruginosa) . Materials and methods: At the San José Hospital in Bogotá-Colombia, a 300-bed teaching hospital, 83 nonduplicate isolates of P. aeruginosa were collected between 2009 and 2011.Meropenem and Doripenem Minimum Inhibitory Concentration (MIC) were determined by the E test method. The 2012 Clinical and Laboratory Standards Institute breakpoints for carbapenems were applied. Results: The global susceptibility of P. aeruginosa isolates to meropemen was 69% and to doripenem was 75%, being the CIM 50 and 90 (ug/mL) of 0.75 and 28.8 for meropenem and of 0.64 and 15.2 for doripenem. Conclusions: The MIC of doripenem was lower than the corresponding MIC of meropenem. It is required to carry out local studies of this type in order to make adjustments to the institutional antibiotics protocol.

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% 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% 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% 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% 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.

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% of Enterobacter spp. isolates were resistant to ceftazidime but 85.7% 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%) and meropenem (44.5%) 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%) at MIC values of < 4 µg/mL than did meropenem (0.0%). 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.

[In vitro activity of doripenem and other carbapenems against Pseudomonas aeruginosa]. / Actividad comparativa in vitro de doripenem y de otros carbapenemes frente a Pseudomonas aeruginosa.

Doripenem, a new carbapenem, has shown to be more active against Pseudomonas aeruginosa than other carbapenems. The activity of doripenem, imipenem and meropenem was evaluated against 93 P. aeruginosa isolates, by agar dilution and disk diffusion methods. MIC50 and MIC90, were as follows (microg/ml): doripenem, 2 and 4; meropenem, 2 and 8; and imipenem, 4 and 8, respectively. Doripenem MICs were 1 to 3 dilutions lower (i.e. more active) than those for imipenem in 82% of the isolates. In comparison with meropenem, doripenem was 1 to 3 dilutions more active in 50% of the isolates. Forty-nine percent of isolates showed the same MIC for both antibiotics. Resistance percentages for both methods were (dilution/diffusion): imipenem = 7.5%/49.5% and meropenem = 3.2%/9.7%. As the CLSI has not established cut off values for doripenem yet, resistance rates for this antibiotic were estimated by considering (a) the same cut off values for imipenem/meropenem set up by the CLSI, and (b) those suggested by Brown et al. In case (a), resistance rates would be 1.1%/2.2% whereas in case (b) 1.1%/17.2% for agar dilution and disk diffusion, respectively. In scenarios where resistance to carbapenem is based on mechanisms other than carbapenemases, doripenem has a promising future for treating P. aeruginosa infections.

Efficacy and safety of doripenem versus piperacillin/tazobactam in nosocomial pneumonia: a randomized, open-label, multicenter study.

OBJECTIVE: Doripenem is a new carbapenem that has broad-spectrum activity against bacterial pathogens commonly responsible for nosocomial pneumonia (NP). It has several advantages over currently available carbapenems and other classes of drugs used in this indication. This prospective, randomized, open-label, multicenter study was designed to establish whether doripenem was noninferior to piperacillin/tazobactam in NP. METHODS: Adults (n=448) with signs and symptoms of NP, including non-ventilated patients and those ventilated for <5 days, were stratified by ventilation mode, illness severity (Acute Physiology and Chronic Health Evaluation II score), and geographic region and then randomly allocated to treatment with doripenem 500 mg every 8 h by a 1-h intravenous (IV) infusion or piperacillin/tazobactam 4.5 g every 6 h by 30-min IV infusion. After receiving IV study drug for at least 72 h, eligible patients could be switched to oral levofloxacin 750 mg once daily. Antibiotic therapy was continued for a total of 7-14 days. The primary endpoint was the clinical cure rate, assessed 7-14 days after treatment completion, in clinically evaluable patients and in the clinical modified intent-to-treat population (cMITT). TRIAL REGISTRATION: ClinicalTrials.gov, NCT00211003. RESULTS: Doripenem was noninferior to piperacillin/tazobactam. Clinical cure rates in clinically evaluable patients (n=253) were 81.3% in the doripenem arm and 79.8% in the piperacillin/tazobactam arm (between-treatment difference: 1.5%; 95% confidence interval [CI], -9.1 to 12.1%) and in the cMITT population 69.5% and 64.1%, respectively, (between-treatment difference: 5.4%; 95% CI, -4.1 to 14.8%). Baseline resistance of Klebsiella pneumoniae and Pseudomonas aeruginosa to piperacillin/tazobactam was 44% and 26.9%, respectively; a doripenem minimum inhibitory concentration (MIC) >8 mug/mL occurred in 0% and 7.7%, respectively. Favorable microbiological outcome rates against Gram-negative pathogens were numerically higher with doripenem than with piperacillin/tazobactam, but the difference was not statistically significant. Both study drugs were generally well tolerated, as only 16.1% and 17.6% of patients receiving doripenem and piperacillin/tazobactam, respectively, had a drug-related adverse event. Study limitations included the open-label design, the low rate of monotherapy (adjunctive use of aminoglycoside was required when P. aeruginosa was suspected), and the exclusion of the most critically ill and immunocompromized patients. CONCLUSIONS: Doripenem was clinically and microbiologically effective in patents with NP, including those with early-onset ventilator-associated pneumonia, and was therapeutically noninferior to piperacillin/tazobactam.