Cynthia Rodríguez

Resistencia enzimática a betalactámicos en el género Proteus y evaluación de los fenotipos y genotipos de resistencia a cefalosporinas de tercera y cuarta generación en Proteus mirabilis

Introduccion. El objetivo de este trabajo fue evaluar la resistencia a betalactamicos en el genero Proteus y caracterizar las betalactamasas responsables de dicha resistencia. Metodos. Se analizaron 99 cepas (87 P. mirabilis; 10 P. vulgaris, y 2, P. penneri) aisladas de pacientes atendidos en un Hospital Universitario. Los ensayos de susceptibilidad a antibioticos se realizaron de acuerdo con las recomendaciones del National Committee for Clinical Laboratory Standards. La presencia de betalactamasas de espectro extendido (BLEE) fue inferida por el metodo de difusion de doble disco y por la concentracion inhibitoria minima (CIM) de cefalosporinas de tercera y cuarta generacion solas y en presencia de acido clavulanico. Se estimo el punto isoelectrico (pI) por isoelectroenfoque y la presencia de los genes codificantes se confirmo por reaccion en cadena de la polimerasa (PCR). Resultados. Una betalactamasa de amplio espectro fue detectada en aquellos aislamientos resistentes a penicilinas y cefalosporinas de primera generacion (28%), mientras que la enzima CTX-M-2 fue detectada en los aislamientos de P. mirabilis resistentes a cefalosporinas de tercera y cuarta generacion (18%). Uno de los P. vulgaris presento sensibilidad disminuida a cefotaxima debido a una enzima de pI 7,4, mientras que la resistencia a cefotaxima en un P. penneri fue relacionada con una enzima de pI 6,8. Ambas enzimas fueron activas sobre cefotaxima (1.000 mg/l) en el ensayo iodometrico. Conclusion. La betalactamasa de amplio espectro en el genero Proteus fue TEM-1 mientras que CTX-M-2 fue la BLEE responsable de la resistencia a cefalosporinas de tercera y cuarta generacion en P. mirabilis. En P. vulgaris y P. penneri esta resistencia se asocio a la hiperproduccion de la betalactamasa cromosomica.

Hyperendemic clone of KPC producing Klebsiella pneumoniae ST 258 in Buenos Aires hospitals

Since KPC-type carbapenemases were first reported in Klebsiella pneumoniae in 2001 in the USA (Yigit et al., 2001), they have become a frequent resistant marker encountered also in other Enterobacteriaceae and Pseudomonads from the Americas, Europe, Asia and Middle East (Villegas et al., 2007; Nordmann et al., 2009; Walsh, 2010). Their wide spread across multiple continents and species has been associated with a mobile genetic element, Tn4401 (Naas et al., 2008). More recently, the molecular epidemiology of KPCproducing K. pneumoniae isolates has revealed the successful dissemination of a single sequence type 258 clone (Kitchel et al., 2009). Although in Argentina KPC-2 producing K. pneumoniae were first detected in 2006 (Pasterán et al., 2008), a substantial increase was observed in 2010, in Buenos Aires (http://www.ine. gov.ar/publi_pdfs/Carbapenemasas.pdf). To characterize this occurrence, single, non-repetitive K. pneumoniae isolates obtained from 57 patients from May 2009 through April 2010 in six hospitals in Buenos Aires were included (Hosp 1:3, Hosp 2:5, Hosp 3:30, Hosp 4:3, Hosp 5: 2, Hosp 6:14). Antimicrobial susceptibility tests were conducted by disk diffusion according to CLSI recommendations (CLSI, 2010). Taking into account the local resistance prevalence in hospital-acquired K. pneumoniae infections, the selected antibiotic disks were placed on two primary 90 mm Mueller Hinton agar plates as follows: plate 1: ampicillin, cephalotin, gentamicin, amikacin, ciprofloxacin and trimethoprime/sulfamethoxazole; plate 2: amoxicillin/clavulanic acid, piperacillin/tazobactam, cefotaxime, ceftazidime, imipenem, and meropenem. As it has been previously reported, phenyl boronic acid is a good inhibitor of both AmpC and KPC b-lactamases (Yagi et al., 2005; Pasteran et al., 2009), we included a disk containing 300 lg phenyl boronic acid in the center of the second plate (distance between disks was 25 mm center to center). An enhancement in the inhibition zone of the carbapenemcontaining disks adjacent to the one containing the inhibitor was considered a positive screening for KPC. All the isolates displayed inhibition zones for imipenem and/or meropenem 621 mm and a positive synergy with phenyl boronic acid. Minimal inhibitory concentrations (MICs) were determined according to CLSI. The isolates showed a multidrug-resistant phenotype with varying levels of carbapenem resistance (Table 1). MIC50 and MIC90 values were as follows (lg/ml) ampicillin: >256 and >256, cephalotin: >256 and >256, ceftazidime: 256 and >256, cefotaxime: 128 and >256, imipenem: 2 and 16, meropenem: 2 and 32, colistin: 4 and 8, amikacin: >128 and >128 and gentamicin 2 and 4, respectively. Three isolates displayed MICs of colistin of 128 lg/ml. The presence of blaKPC was confirmed by PCR amplification using the following primers: KPC-F: 50ATGTCAC TGTATCGCCGTCT 30 and KPC-R: 50TTTT CAGAGCCTTACTGCCC 30 on heat-extracted DNA as template

β-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: A new blaTEM gene

Resistance to β-lactam/β-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second- and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a (1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV β-lactamases (K. pneumoniae and P. mirabilis). A new blaTEM variant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8μg/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrum β-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.

Reliability of Gradient Diffusion Methods for Detection of Acquired Colistin Resistance.

We read with interest the study published by Dafopoulou et al. (1). We agree that the emergence and spread of multidrugresistant Gram-negative bacilli (MDRGNB) have renewed the necessity of using old antibiotics, such as colistin (COL). We want to discuss some results of the mentioned study and ask the authors about some key points which arose from the results exposed. First, the authors (1) reported unacceptable very major errors (VME), namely, 24 (39.3%) and 19 (31%) VME after using Etest and another gradient diffusion strip (MIC test strips [MTS]), respectively. However, 5/24 isolates tested by Etest and 10/19 isolates tested with MTS showed a MIC difference of only 1 log2 dilution from results of broth dilution (BD). After reviewing the literature, we noted that although there are few studies that have evaluated COL susceptibility testing methods, there is a great dispersion of results concerning correlations among MIC-based methodologies. There is little agreement regarding the accuracy of automated systems and Etest methods; the rates of occurrence of VME with the Etest varied from 50% to 0% in a comparison with results of the reference method, BD. Hindler and Humphries reported VME rates of up to 50% with 19 COL-resistant GNB (2), and Tan and Ng also recorded 4% VME with specimens of Enterobacteriaceae (3). Conversely, many other authors reported VME rates within acceptable limits; Behera et al. in a study comprising 24 COL-resistant GNB recorded 1% VME (4), Arroyo et al. recorded 1.7% VME with Acinetobacter baumannii (5), and some others did not observe VME and reported good correlation between the Etest and BD results (6, 7, 8). Etest results also showed good concordance with BD results for Klebsiella pneumoniae and A. baumannii clinical isolates studied in the last few years in our hospital. Second, it is noteworthy that 20/58 (35%) isolates exhibited low levels of resistance to COL, with the MIC corresponding to the current resistance breakpoint (4 g/ml). This number is significantly great; in other studies, the authors detected few isolates (3 or fewer) for which the MIC was 4 g/ml (2, 8). Have the authors considered the possibility of evidencing a phenomenon of dissemination of a single clone among these isolates? In case of clonal dissemination, VME would be overrated. The third issue that arose when we read the work by Dafopoulou et al. (1) was that they did not detect heteroresistance despite having a large number of COL-resistant isolates; the recommended methodology is the population analysis profile, but there are reported studies using Etest strips (6, 9). Reading the inhibition of growth after 24 h of incubation might improve the detection of small colonies and, thus, decrease the disagreement between the results of BD and gradient diffusion techniques. We believe that this methodology is advisable in regions with high rates of acquired resistance to COL in K. pneumoniae and A. baumannii. In conclusion, we would not be so strict as to prevent the use of gradient diffusion methods, as was suggested by the authors (1). Further studies using this methodology should be conducted by following strict recording and interpretative criteria, considering that COL has become one of the options of last resort in the treatment of infections by MDRGNB, which is a high-impact subject worldwide, and that use of a reference method is not always possible for all laboratories.