Comparison of methods for the detection of in vitro synergy in multidrug-resistant gram-negative bacteria.

BACKGROUND: The use of combined antibiotic therapy has become an option for infections caused by multidrug-resistant (MDR) bacteria. The time-kill (TK) assay is considered the gold standard method for the evaluation of in vitro synergy, but it is a time-consuming and expensive method. The purpose of this study was to evaluate two methods for testing in vitro antimicrobial combinations: the disk diffusion method through disk approximation (DA) and the agar gradient diffusion method via the MIC:MIC ratio. The TK assay was included as the gold standard. MDR Gram-negative clinical isolates (n = 62; 28 Pseudomonas aeruginosa, 20 Acinetobacter baumannii, and 14 Serratia marcescens) were submitted to TK, DA, and MIC:MIC ratio synergy methods. RESULTS: Overall, the agreement between the DA and TK assays ranged from 20 to 93%. The isolates of A. baumannii showed variable results of synergism according to TK, and the calculated agreement was statistically significant in this species against fosfomycin with meropenem including colistin-resistant isolates. The MIC:MIC ratiometric agreed from 35 to 71% with TK assays. The kappa test showed good agreement for the combination of colistin with amikacin (K = 0.58; P = 0.04) among the colistin-resistant A. baumannii isolates. CONCLUSIONS: The DA and MIC:MIC ratiometric methods are easier to perform and might be a more viable tool for clinical microbiology laboratories.

Synergistic Effect of Ceftazidime-Avibactam with Meropenem against Panresistant, Carbapenemase-Harboring Acinetobacter baumannii and Serratia marcescens Investigated Using Time-Kill and Disk Approximation Assays.

Susceptibility of ceftazidime-avibactam and in vitro synergy with meropenem were investigated using disk approximation and time-kill assays against 11 multiresistant Acinetobacter baumannii isolates harboring oxacillinases and 5 Serratia marcescens isolates carrying blaKPC-2 Ceftazidime-avibactam was very active and synergistic with meropenem against multiresistant S. marcescens isolates. On the other hand, only the A. baumannii isolates coharboring blaOXA-23 and blaOXA-117 displayed synergy. The disk approximation technique presented good sensitivity for synergism in S. marcescens infection.

Methicillin-resistant Staphylococcus aureus DNA electrophoretic pattern: temporal changes in an endemic hospital environment.

OBJECTIVE: To describe the analysis of geographical and temporal distribution of DNA profiles determined by pulsed-field gel electrophoresis (PFGE) of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from hospitalized patients in a tertiary care university hospital in Brazil. METHODS: Ninetynine samples of MRSA obtained from 89 patients in the period 1999-2004 were studied. MRSA strains were isolated from central venous catheters (33 isolates) and bloodstream infections (66 strains). PFGE with 20 units of SmaI restriction endonuclease was used for genomic typing. RESULTS: Analysis of DNA PFGE of 99 strains of MRSA revealed 26 profiles and their respective related profiles. The mean time interval for detecting MRSA infection was 26 days from hospital admission. Forty-nine patients (57.6%) had a recent hospitalization. The DNA PFGE MRSA profiles were distributed in three clonal groups-I, II, and III-according to the period of time when the MRSA strains were isolated. DNA PFGE MRSA profiles were spread homogeneously through all hospital wards. CONCLUSIONS: Changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospital ward or any specific area of the hospital.

Alternative drugs against multiresistant Gram-negative bacteria.

OBJECTIVES: Enterobacterales and other non-fermenting Gram-negative bacteria have become a threat worldwide owing to the frequency of multidrug resistance in these pathogens. On the other hand, efficacious therapeutic options are quickly diminishing. The aims of this study were to describe the susceptibility of 50 multiresistant Gram-negative bacteria, mostly pan-resistant, against old and less-used antimicrobial drugs and to investigate the presence of antimicrobial resistance genes. METHODS: A total of 50 genetically distinct isolates were included in this study, including 14 Acinetobacter baumannii (belonging to ST79, ST317, ST835 and ST836), 1 Pseudomonas aeruginosa (ST245), 8 Serratia marcescens and 27 Klebsiella pneumoniae (belonging to ST11, ST340, ST258, ST16, ST23, ST25, ST101, ST234, ST437 and ST442). The isolates were submitted to antimicrobial susceptibility testing and whole-genome sequencing to evaluate lineages and resistance genes. RESULTS: Our results showed that some strains harboured carbapenemase genes, e.g. blaKPC-2 (28/50; 56%) and blaOXA-23 (11/50; 22%), and other resistance genes encoding aminoglycoside-modifying enzymes (49/50; 98%). Susceptibility rates to tigecycline (96%) in all species (except P. aeruginosa), to minocycline (100%) and doxycycline (93%) in A. baumannii, to ceftazidime/avibactam in S. marcescens (100%) and K. pneumoniae (96%), and to fosfomycin in S. marcescens (88%) were high. Chloramphenicol and quinolones (6% susceptibility each) did not perform well, making their use in an empirical scenario unlikely. CONCLUSIONS: This study involving genetically distinct bacteria showed promising results for tigecycline for all Gram-negative bacteria (except P. aeruginosa), and there was good activity of minocycline against A. baumannii, ceftazidime/avibactam against Enterobacterales, and fosfomycin against S. marcescens.

Genetic and virulence characterization of colistin-resistant and colistin-sensitive A. baumannii clinical isolates.

Treatment of infections caused by A. baumannii is becoming a challenge due to the ability to develop multidrug-resistance, virulence, and high mortality. We described the colistin resistance and virulence genes present in sixA. baumannii clinical isolates using WGS, expression by qPCR, and virulence in the Galleria mellonella model. The colistin-resistant isolates were assigned as ST233 and the colistin-susceptible isolates as ST236 and ST407. The colistin-resistant isolates contained mutations within PmrA/PmrB, and the pmrA showed up-regulation in all of them. Only one colistin-resistant isolate indicating virulence in G. mellonella. This particular isolate belonged to a different clone, and it was the only isolate that presented non-synonymous mutations in pmrB. Colistinresistance in A. baumannii isolates seems to be caused by up-regulation of pmrA gene. Only one isolate appeared to be virulent in the G. mellonella model. This finding indicating low virulence in isolates belonging to emerging clones circulating in our hospital.

High mortality of bloodstream infection outbreak caused by carbapenem-resistant P. aeruginosa producing SPM-1 in a bone marrow transplant unit.

PURPOSE: Carbapenem resistance in P. aeruginosa is increasing worldwide. In Brazil, SPM-1 is the main P. aeruginosa carbapenemase identified. Little is known about the virulence factor in SPM-1 clones.Methodolgy. We describe a carbapenem-resistant P. aeruginosa bloodstream infection (CRPa-BSI) outbreak in a bone marrow transplant Unit (BMT). Twenty-nine CRPa-BSI cases were compared to 58 controls. Microbiological characteristics of isolates, such as sensitivity, carbapenemase gene PCR for P. aeruginosa, and PFGE are described, as well as the whole-genome sequence (WGS) of three strains.Results/Key findings. The cultures from environmental and healthcare workers were negative. Some isolates harboured KPC and SPM. The WGS showed that the 03 strains belonged to ST277, presented the same mutations in outer membrane protein, efflux pump, and virulence genes such as those involved in adhesion, biofilm, quorum-sensing and the type III secretion system, but differ regarding the carbapenemase profile. A predominant clone-producing SPM harbouring Tn 4371 was identified and showed cross-transmission; no common source was found. Overall mortality rate among cases was 79 %. The first multivariate analysis model showed that neutropenia (P=0.018), GVHD prophylaxis (P=0.016) and prior use of carbapenems (P=0.0089) were associated with CRPa-BSI. However, when MASCC>21 points and platelets were added in the final multivariate analysis, only prior use of carbapenems remained as an independent risk factor for CRPa-BSI (P=0.043). CONCLUSIONS: The predominant clone belonging to ST277 showed high mortality. Carbapenem use was the only risk factor associated with CRPa-BSI. This finding is a wake-up call for the need to improve management in BMT units.

Antimicrobial Combinations against Pan-Resistant Acinetobacter baumannii Isolates with Different Resistance Mechanisms.

The study investigated the effect of antibiotic combinations against 20 clinical isolates of A. baumannii (seven colistin-resistant and 13 colistin-susceptible) with different resistance mechanisms. Clinical data, treatment, and patient mortality were evaluated. The following methods were used: MIC, PCRs, and outer membrane protein (OMP) analysis. Synergy was investigated using the checkerboard and time-kill methods. Clonality was evaluated by PFGE. Based on clonality, the whole genome sequence of six A. baumannii isolates was analyzed. All isolates were resistant to meropenem, rifampicin, and fosfomycin. OXA-23 and OXA-143 were the most frequent carbapenemases found. Four isolates showed loss of a 43kDa OMP. The colistin-susceptible isolates belonged to different clones and showed the highest synergistic effect with fosfomycin-amikacin. Among colistin-resistant isolates, the highest synergistic effect was observed with the combinations of colistin-rifampicin followed by colistin-vancomycin. All colistin-resistant isolates harbored blaOXA-23-like and belonged to CC113. Clinical and demographic data were available for 18 of 20 patients. Fourteen received treatment and eight patients died during treatment. The most frequent site of infection was the blood in 13 of 14 patients. Seven patients received vancomycin plus an active drug against A. baumannii; however, mortality did not differ in this group. The synergistic effect was similar for colistin-susceptible isolates of distinct clonal origin presenting with the same resistance mechanism. Overall mortality and death during treatment was high, and despite the high synergism in vitro with vancomycin, death did not differ comparing the use or not of vancomycin plus an active drug against A. baumannii.

Methicillin-resistant Staphylococcus aureus DNA electrophoretic pattern: temporal changes in an endemic hospital environment / Patrón electroforético del ADN de Staphylococcus aureus resistente a la meticilina: cambios temporales en unmedio hospitalario endémico

Objective. To describe the analysis of geographical and temporal distribution of DNA profiles determined by pulsed-field gel electrophoresis (PFGE) of methicillin-resistant Staphylococcusaureus (MRSA) strains isolated from hospitalized patients in a tertiary care university hospital in Brazil. Methods. Ninety-nine samples of MRSA obtained from 89 patients in the period 1999–2004 were studied. MRSA strains were isolated from central venous catheters (33 isolates) and bloodstream infections (66 strains). PFGE with 20 units of SmaI restriction endonucleasewas used for genomic typing. Results. Analysis of DNA PFGE of 99 strains of MRSA revealed 26 profiles and theirrespective related profiles. The mean time interval for detecting MRSA infection was 26 days from hospital admission. Forty-nine patients (57.6%) had a recent hospitalization. The DNAPFGE MRSA profiles were distributed in three clonal groups—I, II, and III—according to the period of time when the MRSA strains were isolated. DNA PFGE MRSA profiles were spreadhomogeneously through all hospital wards. Conclusions. Changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospitalward or any specific area of the hospital.

Objetivo. Analizar la distribución geográfica y temporal de los perfiles de ADN determinados mediante electroforesis en gel de campo pulsado (PFGE) de cepas de Staphylococcus aureus resistente a la meticilina (SARM) aisladas de pacientes internados en un hospital universitario de atención terciaria en el Brasil. Métodos. Se estudiaron 99 muestras de SARM obtenidas 89 de pacientes en el período1999–2004. Las cepas de SARM se aislaron de infecciones de catéteres venosos centrales (33 aislados) y del torrente sanguíneo (66 cepas). Para la tipificación genómica se empleó PFGE con 20 unidades de endonucleasa de restricción SmaI. Resultados. El análisis del ADN de 99 cepas de SARM mediante PFGE reveló 26 perfiles, con sus respectivos perfiles relacionados. El intervalo medio de detección de la infección por SARM fue de 26 días desde el ingreso al hospital. En 49 pacientes (57,6%) había habido una hospitalización previa reciente. Los perfiles de ADN de SARM determinados mediante PFGE se distribuyeron en tres grupos clonales —I, II y III— según el período en el que se aislaron las cepas de SARM. Estos perfiles de ADN se encontraban distribuidos de manera homogénea en todos los servicios del hospital. Conclusiones. Los cambios en la distribución de los perfiles de ADN de SARM determinados mediante PFGE fueron en gran medida temporales, con reemplazo de los grupos clonales con el transcurso del tiempo, y sin predominio en ningún servicio ni área específica del hospital.