First Identification of ß-Lactamases in Antibiotic-Resistant Escherichia coli, Citrobacter freundii, and Aeromonas spp. Isolated in Stream Macroinvertebrates in a Central Italian Region.

Antibiotic-resistant bacteria (ARB) are widespread in nature and represent a serious public and environmental problem. In the present study, we report for the first time the presence of bacterial ß-lactamases in two macroinvertebrate species with different feeding traits. The class A ß-lactamases, SHV-1 and TEM-1, were found in Citrobacter freundii isolated from Gammarus elvirae and Escherichia coli from water samples, respectively. The metallo-ß-lactamase CphA was found in Aeromonas veronii and Aeromonas hydrophila strains isolated from the predator Dina lineata. The presence of a large plasmid was ascertained only in E. coli strains isolated from water. In all strains studied, an integrase I typical of class I integrin was found. In contaminated freshwater habitats, ARB and antibiotic resistance genes could be disseminated through trophic links with important ecological implications. Transmission through the food chain may contribute to spreading and transferring antibiotic resistance not only in freshwater ecosystems but also outside the aquatic habitat.

Curcumin inhibits the SOS response induced by levofloxacin in Escherichia coli.

The role of RecA protein in bacterial resistance to antibiotics makes this protein attractive from a pharmacological point of view. In this study we demonstrate that curcumin is able to inhibit the SOS response in Escherichia coli induced by levofloxacin. The blaTEM-1 gene has been placed under the control of the LexA-binding box and used as reporter gene. The expression of TEM-1 ß-lactamase enzyme was increased in the presence of ssDNA induced by levofloxacin, while, the presence of curcumin at 8µg/ml, reduced dramatically the expression of the reporter gene. Moreover a simple microplate assay suitable for high-throughput screening has been developed.

Emergence of blaKPC-3-Tn4401a in Klebsiella pneumoniae ST512 in the municipal wastewater treatment plant and in the university hospital of a town in central Italy.

In this study, 20 carbapenem-resistant environmental Klebsiella pneumoniae strains were found to correlate with 18 clinical K. pneumoniae isolates from the teaching hospital of L'Aquila city, Italy. All strains analysed by multilocus sequence typing (MLST) were included in the same clone (ST512), and pulsed-field gel electrophoresis demonstrated a genetic relationship between the clinical isolates and most environmental strains. Both environmental and clinical strains harboured the same mobile genetic elements: transposon Tn4401a including a blaKPC-3 determinant; and a class 1 integron with the gene cassette aadA2.

Antibacterial activity of selected metabolites from Chilean lichen species against methicillin-resistant staphylococci.

The in vitro antibacterial activities of eight compounds isolated from lichens, collected in several Southern regions of Chile (including Antarctica), were evaluated against methicillin-resistant clinical isolates strains of Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus warneri. The minimum inhibitory concentrations, calculated in microdilution, were ranging from 8 µg mL(-1) for sphaerophorin to 1024 µg mL(-1) for fumarprotocetraric acid. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

In vitro antimicrobial activity of pannarin alone and in combination with antibiotics against methicillin-resistant Staphylococcus aureus clinical isolates.

The in vitro antimicrobial activities of pannarin, a depsidone isolated from lichens, collected in several Southern regions of Chile (including Antarctica), was evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC(90), MIC(50), as well as MBC(90) and MBC(50), were evaluated. A moderate synergistic action was observed in combination with gentamicin, whilst antagonism was observed in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. In order to asses cellular lysis after exposure to pannarin, cell membrane permeability assay was performed. The treatment with pannarin produces bactericidal activity without significant calcein release, consistent with lack of lysis or even significant structural damage to the cytoplasmic membrane. Furthermore, pannarin shows low hemolytic activity and moderate cytotoxic effect on peripheral blood mononuclear cells. These findings suggest that the natural compound pannarin might be a good candidate for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus aureus clinical isolates determined by FICI and ΔE model methods.

The in vitro antimicrobial activities of usnic acid were evaluated in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, were evaluated. A synergistic action was observed in combination with gentamicin, while antagonism was observed with levofloxacin. The combination with erythromycin showed indifference, while variability was observed for clindamycin and oxacillin. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index (FICI) and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings, beside confirm the well known antimicrobial activity of usnic acid, suggest, however, that this substance might be a good candidate for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Occurrence of class 1 and 2 integrons in resistant Enterobacteriaceae collected from a urban wastewater treatment plant: first report from central Italy.

The purpose of this study was to investigate the presence and distribution of integron-carrying isolates among Enterobacteriaceae resistant to ß-lactam antibiotics collected from a wastewater effluent of the city of L'Aquila (Italy). A total of 471 Enterobacteriaceae were collected during a period of 2 years (2005-2006). The presence and distribution of class 1 and 2 integrons was investigated by colony blot hybridization using specific probes labelled with dUTP-fluoresceine kit. The variable region of class 1 and 2 integrons was analyzed by polymerase chain reaction and DNA sequencing in 24 isolates with different random amplified polymorphic DNA profile. The characterization of class 1 and 2 integrons gene cassettes of 24 nonrelated strains showed the presence of four different arrays: dfr17-aadA5; aadA10; dfr1-sat1; dfr1-sat1-aadA1. This is the first report from Italy in which the authors confirm the presence of Enterobacteriaceae carrying class 1 and 2 integrons in a wastewater treatment plant that collects the urban and hospital discharges.

Natural D240G Toho-1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43.

OBJECTIVES: The aim of this article is biochemical and kinetic characterization of CTX-M-43, a natural Asp-240-->Gly mutant of CTX-M-44 (ex Toho-1), from a clinical isolate of Acinetobacter baumannii isolated in a Bolivian hospital. METHODS: Steady-state kinetic parameters (K(m) and k(cat)) were determined for a large pattern of substrates. Analysis of inactivators and transient inactivators was performed to determine the efficiency of acylation (k(+2)/K) and the deacylation constant (k(+3)). Molecular modelling of Michaelis complex of ceftazidime, cefotaxime and ceftibuten, obtained from molecular mechanics calculations, was carried out. RESULTS: CTX-M-43 showed a general increase in affinity towards all cephalosporins tested, with respect to CTX-M-44. Carbapenems acted as inactivators with a good acylation efficiency for meropenem and ertapenem and significant deacylation constant for imipenem. MICs of imipenem obtained at a higher bacterial inoculum of recombinant Escherichia coli were increased. CONCLUSIONS: Kinetic data and molecular modelling of Michaelis complex confirmed that Asp-240-->Gly allows a better accommodation of the bulky C7beta aminothiazol-oxyimino substituent, resulting in a general increase in the enzyme affinity towards oxyimino cephalosporins. The ascertained potentialities of CTX-M-type enzymes, supported by the kinetic data and the behaviour of the recombinant E. coli at different bacterial inocula towards carbapenems, make a possible evolution of those enzymes towards a carbapenemase activity plausible.

Antimicrobial susceptibility of clinical isolates of Enterobacteriaceae producing complex beta-lactamase patterns including extended-spectrum enzymes.

The antimicrobial susceptibility of 103 clinical isolates of Enterobacteriaceae to 11 antibiotics, was investigated, using a conventional inoculum size (5 x 10(5) CFU) and a higher inoculum size (5 x 10(8) CFU). All the isolates produced complex beta-lactamase patterns, including an extended-spectrum beta-lactamase (ESBL) of the TEM- or SHV-type plus other enzymes (a TEM-type or an SHV-type non-ESBL and/or a class C enzyme). The following repertoire of ESBLs was produced by the isolates: TEM-15, TEM-19, TEM-26, TEM-52, TEM-72, TEM-87, TEM-92, SHV-2a, SHV-5 and SHV-12, as assessed by sequencing. Production of the other enzymes was showed by analytical isoelectric focusing. Overall, meropenem was the most active agent and less influenced by inoculum size, while other beta-lactams showed a lower activity and a significant inoculum size effect. In conclusion, from its in vitro performance, meropenem could be considered as the last resource drug against strains producing complex beta-lactamase patterns including an ESBL.

Cefotaxime, des-Cefotaxime and Ceftazidime: in vitro activity and stability to hydrolysis from TEM-derived extended spectrum beta-lactamases.

Beta-lactams represent one of the most important class of antibiotics for the treatment of infectious diseases due to pathogenic bacteria. The selective pressure exerted from the wide spread use of third generation cephalosporins generated mutant beta-lactamases belonging mainly to the TEM or SHV family that are able to extend the activity spectrum of hydrolysis. Moreover, extended spectrum cephalosporins are often a good choice in clinical practice towards Enterobacteriaceae. Here we report a comparative analysis of stability between cefotaxime, desacetyl-cefotaxime and ceftazidime with some common TEM-derivatives extended spectrum beta-lactamases.