Gerardo González

Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

ABSTRACT Transcriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes. Klebsiella pneumoniae is a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription of ramA is associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the available Klebsiella genome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded in K. pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568, and Enterobacter cloacae. We show that the overexpression of rarA results in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show that rarA (MGH 78578 KPN_02968) and its neighboring efflux pump operon oqxAB (KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest that rarA overexpression upregulates the oqxAB efflux pump. Additionally, it appears that oqxR, encoding a GntR-type regulator adjacent to the oqxAB operon, is able to downregulate the expression of the oqxAB efflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

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.

Evaluating the effects of chlortetracycline on the proliferation of antibiotic-resistant bacteria in a simulated river water ecosystem.

ABSTRACT Antibiotics and antibiotic metabolites have been found in the environment, but the biological activities of these compounds are uncertain, especially given the low levels that are typically detected in the environment. The objective of this study was to estimate the selection potential of chlortetracycline (CTC) on the antibiotic resistance of aerobic bacterial populations in a simulated river water ecosystem. Six replicates of a 10-day experiment using river water in continuous flow chemostat systems were conducted. Each replicate used three chemostats, one serving as a control to which no antibiotic was added and the other two receiving low and high doses of CTC (8 μg/liter and 800 μg/liter, respectively). The addition of CTC to the chemostats did not impact the overall level of cultivable aerobic bacteria (P = 0.51). The high-CTC chemostat had significantly higher tetracycline-resistant bacterial colony counts than both the low-CTC and the control chemostats (P < 0.035). The differences in resistance between the low-CTC and control chemostats were highly nonsignificant (P = 0.779). In general a greater diversity of tet resistance genes was detected in the high-CTC chemostat and with a greater frequency than in the low-CTC and control chemostats. Low levels of CTC in this in vitro experiment did not select for increased levels of tetracycline resistance among cultivable aerobic bacteria. This finding should not be equated with the absence of environmental risk, however. Low concentrations of antibiotics in the environment may select for resistant bacterial populations once they are concentrated in sediments or other locations.

Role of shellfish hatchery as a reservoir of antimicrobial resistant bacteria.

The main aim of this study was to determine the occurrence of resistant bacteria in florfenicol-treated and untreated scallop larval cultures from a commercial hatchery and to characterize some selected florfenicol-resistant strains. Larval cultures from untreated and treated rearing tanks exhibited percentages of copiotrophic bacteria resistant to florfenicol ranging from 0.03% to 10.67% and 0.49-18.34%, respectively, whereas florfenicol resistance among oligotrophic bacteria varied from 1.44% to 35.50% and 3.62-95.71%, from untreated and treated larvae, respectively. Florfenicol resistant microbiota from reared scallop larvae mainly belonged to the Pseudomonas and Pseudoalteromonas genus and were mainly resistant to florfenicol, chloramphenicol, streptomycin and co-trimoxazole. This is the first study reporting antimicrobial resistant bacteria associated to a shellfish hatchery and the results suggest that a continuous surveillance of antimicrobial resistance even in absence of antibacterial therapy is urgently required to evaluate potential undesirable consequences on the surrounding environments.

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.

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.

Inhibition of Flavobacterium psychrophilum biofilm formation using a biofilm of the antagonist Pseudomonas fluorescens FF48

The most important bacterial pathology currently occurring in Chilean freshwater salmon farming is the cold-water disease produced by the psychrotrophic bacteria Flavobacterium psychrophilum. The main aim of this study was to characterize the inhibitory activity of an antagonist strain on the formation of biofilms of a F. psychrophilum strain. The antagonistic strain Pseudomonas fluorescens FF48 was isolated from the sediment beneath the salmon cages of a freshwater Chilean salmon farm and was identified by using the 16S rRNA gene sequence analysis. The production of siderophores, mainly during the stationary phase of growth of the antagonist strain was demonstrated using the Chrome Azurol S method and through F. psychrophilum inhibition under iron saturation conditions. Subsequently, the effect of the antagonist supernatant on the formation of F. psychrophilum biofilm was tested using the crystal violet staining method observing an inhibition of the growth of F. psychrophilum, but no effect was observed when iron saturation concentrations were used. Furthermore, when the antagonist strain was previously deposited on the support, it completely inhibited the formation of F. psychrophilum biofilms, but when both bacteria were inoculated simultaneously no inhibitory effect was detected. In conclusion, it was demonstrated that FF48 strain is able to inhibit the formation of F. psychrophilum biofilms in vitro probably mediated by the siderophore production, suggesting its potential use as a biocontrol biofilm in freshwater fish rearing systems to prevent the persistence of biofilms of the fish pathogenic species F. psychrophilum.