Mariana Domínguez

Extended-spectrum β-lactamases belonging to CTX-M group produced by Escherichia coli strains isolated from companion animals treated with enrofloxacin

We studied the antibiotic resistance among Escherichia coli isolates obtained from fecal samples of dogs and cats treated and untreated with enrofloxacin in veterinary clinics. Resistant patterns of 70 strains and the presence of extended-spectrum beta-lactamase (ESBL) were studied. The genes encoding the following families of beta-lactamases: CTX-M, GES, PER, TEM and SHV, were investigated by PCR-RFLP and sequencing. The strains isolated from enrofloxacin-treated animals were multi-drug-resistant exhibiting resistant patterns including fluorquinolones, beta-lactams, aminoglycosides, tetracycline and phenicols. On the contrary, the strains obtained from the untreated group of animals exhibited narrower antibiotic resistant profiles. The synthesis of ESBL was detected in 14 strains (20%) isolated from treated animals. The ESBL encoded by genes bla CTX-M-1, bla CTX-M-9 group and bla PER-2 were detected by PCR. We believe that this is the first report on the presence of ESBL in E. coli strains isolated from small animals in Chile, and the first report of beta-lactamase belonging to the CTX-M-9 group (CTX-M-14). The presence of these genes in bacteria isolated from pets is an important fact that constitutes a public health concern.

Principales factores de virulencia de Listeria monocytogenesy su regulación

Listeria monocytogeneses un patogeno intracelular facultativo, ubicuo y agente etiologico de listeriosis. La principal via de adquisicion es el consumo de alimentos contaminados, pudiendo ocasionar cuadros clinicos muy graves como septicemia, meningitis y gastroenteritis, especialmente en ninos, individuos inmunocomprometidos y de la tercera edad, y aborto en mujeres embarazadas. Se ha informado un aumento en los casos de listeriosis a escala mundial y se estima que su frecuencia en los paises desarrollados esta en un rango de 2 a 15 casos por millon de habitantes. Este microorganismo se caracteriza por realizar una transicion desde el medio ambiente hacia la celula eucariota. Para este proceso se han descrito varios factores de virulencia, los cuales estan involucrados en el ciclo intracelular y estan regulados, principalmente, por la proteina PrfA, la cual a su vez esta regulada por diferentes mecanismos que actuan a nivel transcripcional, traduccional y post-traduccional. Ademas, se han descrito otros mecanismos regulatorios como: factor Sigma, sistema VirR/S y ARN sin sentido. No obstante, PrfA es el mecanismo de control mas importante y el cual es requerido para la expresion de los factores de virulencia esenciales para el ciclo intracelular.

Molecular typing and genetic environment of the blaKPC gene in Chilean isolates of Klebsiella pneumoniae

The aim of this work was to determine the genetic environment and transferability of blaKPC as well as the pulsotypes of KPC-producing Klebsiella pneumoniae strains isolated from clinical samples in Chilean hospitals. Seventeen strains, principally isolated in Santiago (the capital of Chile) during the years 2012 and 2013, were included. The genetic environment of blaKPC was elucidated by PCR mapping and sequencing. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Curing and conjugation experiments were performed with six strains of different sequence types (STs) and pulsotypes. Thirteen pulsotypes and six STs, mainly belonging to clonal complex 258, were found. In addition, seven strains belonged to a new ST assigned ST1161. The blaKPC sequence indicated that 16 strains had the KPC-2 variant; in only one strain (UC331) an amino acid change (R6P) was detected, corresponding to a new KPC variant designated KPC-24. Molecular characterisation of the blaKPC genetic environment revealed two distinct platforms, namely variant 1a and the Tn4401a isoform, with the first being the most common (11/17 strains). Mating experiments failed to produce transconjugants; however, loss of blaKPC was achieved by plasmid curing in all assayed strains. In conclusion, in Chilean strains of K. pneumoniae, blaKPC is primarily found associated with the variant 1a and is located in non-transferable plasmids. In addition, this study highlights the description of the new ST1161 and the new KPC-24 variant.

Activity of cefepime, cefotaxime, ceftazidime, and aztreonam against extended-spectrum-producing isolates of Klebsiella pneumoniae and Escherichia coli from Chilean hospitals

Resistance of Gram-negative bacilli to third-generation cephalosporins has been increasing due to the production of extended-spectrum beta-lactamases. In this work, the activities of cefepime, cefotaxime, ceftazidime, and aztreonam, alone and in association with clavulanic acid, against isolates of Klebsiella pneumoniae and Escherichia coli are compared. These isolates produce extended-spectrum beta-lactamases as shown by the synergy tests and by the decrease in the MICs of cephalosporins in the presence of clavulanic acid. Cefepime was the most active compound against these microorganisms. In addition, the microorganisms exhibited lower frequencies of resistance to this cephalosporin.

The efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) increases resistance to carbapenems in Chilean clinical isolates of KPC-producing Klebsiella pneumoniae

OBJECTIVES KPC-producing strains present a wide range of carbapenem minimum inhibitory concentrations (MICs). This variation may be due to differential expression of blaKPC and porin genes, efflux pump activity and the production of extended-spectrum β-lactamases and/or AmpC β-lactamases. The aim of this study was to determine the role of efflux pumps inhibited by phenylalanine-arginine β-naphthylamide (PAβN) in resistance to carbapenems in Chilean clinical isolates of blaKPC-harbouring Klebsiella pneumoniae. METHODS MICs were determined by the agar dilution method for imipenem, meropenem, ertapenem and ciprofloxacin in the presence and absence of PAβN (25mg/L) in 17 carbapenem-resistant KPC-producing K. pneumoniae strains. Outer protein membrane (OMP) profiles were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Expression levels of the ompK35 and ompK36 genes were also determined by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS No contribution of PAβN-inhibited efflux pumps to carbapenem resistance was found, unlike ciprofloxacin resistance. However, a ≥4-fold increase in the MIC of at least one carbapenem was observed in 13 isolates in the presence of PAβN. Additionally, decreased gene expression of ompK35 and ompK36 in the presence of PAβN was detected, however no obvious differences in porin band intensity were observed by SDS-PAGE. CONCLUSIONS The presence of PAβN resulted in an increase in carbapenem MICs unrelated to efflux pump inhibition, and a decrease in the expression of ompK35 and ompK36 genes without an obvious difference in OMP profiles observed by SDS-PAGE. Therefore, additional factors are responsible for the increase in carbapenem MIC in the presence of PAβN.

Colistín en la era post-antibiótica

One of the most important features of the post-antibiotic era in the late 20th century is the resurgence of colistin for the treatment of extensively drug resistant gram-negative bacteria (XDR). Colistin is a narrow spectrum anti-biotic, active against microorganisms with clinical significance such as Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. Nowadays its toxicity is lower, partly explained by better pharmaceuticals and management of the critically ill patients. However, there has been much confusion regarding the dosage of the drug, its name and labeling, therefore, experts have recommended using a common language about this polymyxin. The lack of PK/PD studies for colistin is perhaps the main weakness of this area of knowledge, even though the before mentioned approach has contributed with new ways to manage and calculate the dose of this antimicrobial. Indeed, the efficiency of colistin in association with a second agent in reducing mortality has not been demonstrated.

[Isolation of Staphylococcus aureus hetero-resistant to vancomycin (hVISA) in the Regional Hospital of Concepción, Chile].

Methicillin-resistant Staphylococcus aureus (MRSA) is widely distributed in hospital environments, causing serious infections, mainly the bloodstream, surgical site infection and pneumonia. Vancomycin (VAN) is the antibiotic of choice for treating severe MRSA infections; however, nowadays worldwide resistant strains (VRSA), with intermediate susceptibility (VISA) and decreased susceptibility or hetero-resistance to VAN (hVISA) have been reported, related to treatment failure and increased mortality. This report describes the first confirmed isolation of MRSA with hVISA phenotype in a public hospital in Chile.

Molecular Typing of Enterococcus faecalis from Persistent Endodontic Infections

Molecular techniques that provide valuable information about the epidemiology of oral strains. The purpose of this study was to determine the genetic relatedness of 83 Enterococcus faecalis strains isolated from treated root canals. These strains were obtained from patients who were treated for persistent endodontic infections. E. faecalis isolates were molecular typed by Pulsed Field Gel Electrophoresis using SmaI. Ten clonal groups and 13 pulse types with 38.7 % similarity for the less related strains were identified. Genetic heterogeneity among strains from different patients and a high level of genetic homogeneity among intrapatient strains were observed. Therefore, restriction endonuclease fingerprinting of genomic DNA from E. faecalis strains confirmed the polyclonality of the isolates obtained from the root canals of patients diagnosed with persistent endodontic infections, compared with other reports. These results provide additional data for a better understanding of the epidemiological aspects of root canal infections by E. faecalis.

Genetic Platforms of blaCTX-M in Carbapenemase-Producing Strains of K. pneumoniae Isolated in Chile

Objective: To elucidate whether the genetic platforms of blaCTX-M contribute to the phenotypes of multi-drug-resistance (MDR) in the first carbapenemase-producing K. pneumoniae strains isolated in Chile. Method: Twenty-two carbapenemase-producing K. pneumoniae strains isolated from different Chilean patients and hospitals were studied. Their genetic relatedness was assessed by PFGE and MLST. The levels of antibiotic resistance were evaluated by determining the minimum inhibitory concentration of various antimicrobials. In addition, several antibiotic resistance genes of clinical relevance in Chile were investigated. The prevalence, allelic variants, and genetic platforms of blaCTX-M were determined by PCR and sequencing. Results: Out of the 22 strains studied, 20 carry KPC, one carries NDM-1, and one carries OXA-370. The PFGE analysis showed three clades with a genetic relatedness >85%, two formed by four strains and one by eight strains. The other strains are not genetically related, and a total of 17 different pulse types were detected. Ten different STs were identified, the main ones being ST258 (five strains) and ST1161 (seven strains). The isolates presented different percentages of resistance, and 82% were resistant to all the β-lactams tested, 91% to ciprofloxacin, 73% to colistin, 59% to gentamicin, 50% to amikacin, and only 9% to tigecycline. All isolates carried blaTEM and blaSHV, whereas 71% carried aac(6′)Ib-cr, and 57% one qnr gene (A, B, C, D, or S). The blaCTX-M gene was found in 10 of the isolates (4 blaCTX-M−15 and 6 blaCTX-M−2). The characterization of the platform, in seven selected strains, revealed that the gene is associated with unusual class 1 integrons and insertion sequences such as ISCR1, ISECp1, and IS26. Conclusion: In the first carbapenemase-producing K. pneumoniae strains isolated in Chile the genetic platform of blaCTX-M−2 corresponds to an unusual class 1 integron that can be responsible for the MDR phenotype, whereas the genetic platforms of blaCTX-M−15 are associated with different IS and do not contribute to multi-drug resistance.

Se debe revisar la determinación de la CIM de colistín en Acinetobacter baumannii

Currently, there is a controversy in how to determine the minimal inhibitory concentration (MIC) of colistin against Acinetobacter baumannii. We compared three methods, concluding that the addition of Tween-80 (0.002%) to Müller-Hinton broth in the microdilution method could improve MIC determination and it could reduce false resistance.Currently, there is a controversy in how to determine the minimal inhibitory concentration (MIC) of colistin against Acinetobacter baumannii. We compared three methods, concluding that the addition of Tween-80 (0.002%) to Müller-Hinton broth in the microdilution method could improve MIC determination and it could reduce false resistance.