Javier Sánchez-Céspedes

Prevalence of mechanisms decreasing quinolone-susceptibility among Salmonella spp. clinical isolates.

Fluoroquinolone treatment failure has been reported in patients with nalidixic acid-resistant Salmonella infections. Both chromosomal- and plasmid-mediated quinolone-resistance mechanisms have been described. The objective of this study was to identify the prevalence of these mechanisms in a collection of 41 Salmonella spp. clinical isolates causing acute gastroenteritis, obtained in the Hospital Clinic, Barcelona. The minimum inhibitory concentrations (MICs) of nalidixic acid and ciprofloxacin were determined by Etest. Mutations in the quinolone-resistance determining regions (QRDRs) of the gyrA, gyrB, and parC genes and the presence of the qnr, aac(6)-Ib-cr, and qepA genes were detected by PCR and DNA sequencing. All isolates showed constitutive expression of an efflux pump. None of the isolates were ciprofloxacin-resistant, whereas 41.5% showed nalidixic acid resistance associated with a mutation in gyrA and overexpression of an efflux pump. Although qnrS1, qnrB6, and qepA were found in four isolates, the expression of these genes was not associated with decreased quinolone susceptibility. Mutations in the gyrA gene and overexpression of an efflux pump were critical for nalidixic acid resistance and decreased susceptibility to ciprofloxacin in these isolates. However, plasmid-mediated quinolone resistance did not seem to play a major role. To our knowledge, this is the first description of qepA in Salmonella.

Characterization of the Carbapenem-Hydrolyzing Oxacillinase Oxa-58 in an Acinetobacter Genospecies 3 Clinical Isolate

ABSTRACT Based on imipenem resistance in an Acinetobacter genospecies 3 clinical isolate, we were able to identify, for the first time in this genomic species, a plasmid-encoded blaOXA-58 gene that was 100% homologous to the same gene in Acinetobacter baumannii.

Detection of dihydrofolate reductase genes by PCR and RFLP

The presence of plasmid-encoded trimethoprim resistant dfr genes is the most common mechanism responsible for the acquisition of trimethoprim resistance. The usual method to detect the presence of these genes is hybridization with specific probes. We describe an alternative, faster and easier method, based on PCR amplification and RFLP analysis, to discriminate up to sixteen different dfr genes.

Plasmid-Mediated QnrS2 Determinant from a Clinical Aeromonas veronii Isolate

The main objective of this study was to determine the prevalence of the Qnr determinants in clinical and environmental Aeromonas spp. A total of 52 Aeromonas sp. isolates identified by biochemical methods (5), 25 isolated from natural waters (1) and 27 isolated from clinical samples from hospitals in Valencia, Spain, were tested for quinolone resistance by the disk diffusion method (4) (nalidixic acid, 30 μg; oxolinic acid, 2 μg; flumequine, 30 μg; ciprofloxacin, 5 μg; and levofloxacin, 5 μg). Among the studied isolates, 27 showed resistance to nalidixic acid and susceptibility to ciprofloxacin, 24 isolates were susceptible to both nalidixic acid and ciprofloxacin, and only 1, the A. veronii A272 clinical isolate, was resistant to both nalidixic acid and ciprofloxacin. The isolates resistant to nalidixic acid were also resistant to oxolinic acid and flumequine. Moreover, A. veronii A272 was the only one resistant to levofloxacin. Screening of the qnrA, qnrB, and qnrS genes was performed by multiplex PCR using a set of specific primers for all isolates. Bacterial strains positive for each qnr gene were used as positive controls (Klebsiella pneumoniae UAB1 for qnrA, Escherichia coli J53 pMG252 for qnrB, and E. coli J53 pMG298 for qnrS) and were run in each batch of tested samples. Only an A. veronii clinical isolate (A. veronii A272) presented a qnr gene, which showed 100% homology with the qnrS2 gene previously reported in an isolate from the bacterial community of a wastewater treatment plant in Germany (2) and in a non-Typhi Salmonella clinical isolate in the United States (6).

Mechanism of Binding of Fluoroquinolones to the Quinolone Resistance-Determining Region of DNA Gyrase: Towards an Understanding of the Molecular Basis of Quinolone Resistance

We have studied the bacterial resistance to fluoroquinolones that arises as a result of mutations in the DNA gyrase target protein. Although it is known that DNA gyrase is a target of quinolone antibacterial agents, the molecular details of the quinolone–gyrase interaction remain unclear. The mode of binding of ciprofloxacin, levofloxacin, and moxifloxacin to DNA gyrase was analyzed by means of docking calculations over the surface of the QRDR of GyrA. The analysis of these binding models allows study of the resistance mechanism associated with gyrA mutations more commonly found in E. coli fluoroquinolone‐resistant strains at the atomic level. Asp87 was found to be critical in the binding of these fluoroquinolones because it interacts with the positively charged nitrogens in these bactericidal drugs. The role of the other most common mutations at amino acid codon Ser83 can be explained through the contacts that the side chain of this residue establishes with fluoroquinolone molecules. Finally, our results strongly suggest that, although Arg121 has never been found to be associated with fluoroquinolone resistance, this residue makes a pivotal contribution to the binding of the antibiotic to GyrA and to defining its position in the QRDR of the enzyme.

Clonal Dissemination of Yersinia enterocolitica Strains with Various Susceptibilities to Nalidixic Acid

ABSTRACT Ten epidemiologically related Yersinia enterocolitica clinical isolates were studied. Six isolates were nalidixic acid resistant (MIC > 512 μg/ml), with mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene, suggesting clonal dissemination of a nalidixic acid-susceptible Y. enterocolitica strain which has acquired different mutations generating resistance to nalidixic acid.

Perspectives for clinical use of engineered human host defense antimicrobial peptides

Abstract Infectious diseases caused by bacteria, viruses or fungi are among the leading causes of death worldwide. The emergence of drug-resistance mechanisms, especially among bacteria, threatens the efficacy of all current antimicrobial agents, some of them already ineffective. As a result, there is an urgent need for new antimicrobial drugs. Host defense antimicrobial peptides (HDPs) are natural occurring and well-conserved peptides of innate immunity, broadly active against Gram-negative and Gram-positive bacteria, viruses and fungi. They also are able to exert immunomodulatory and adjuvant functions by acting as chemotactic for immune cells, and inducing cytokines and chemokines secretion. Moreover, they show low propensity to elicit microbial adaptation, probably because of their non-specific mechanism of action, and are able to neutralize exotoxins and endotoxins. HDPs have the potential to be a great source of novel antimicrobial agents. The goal of this review is to provide an overview of the advances made in the development of human defensins as well as the cathelicidin LL-37 and their derivatives as antimicrobial agents against bacteria, viruses and fungi for clinical use.

Antiadenovirus drug discovery: potential targets and evaluation methodologies.

Human adenoviruses (HAdV) are the cause of many acute infections, mostly in the respiratory and gastrointestinal (GI) tracts, as well as conjunctivitis. HAdV diseases in immunocompetent individuals are mostly self-limiting; however, in immunocompromised individuals, especially in pediatric units, HAdV infections are the cause of high morbidity and mortality. Despite the significant clinical impact, there are currently no approved antiviral therapies for HAdV infections. Here, we provide an overview of the different targets that could be considered for the design of specific drugs against HAdV, as well as the available in vitro and in vivo tools for the screening and evaluation of candidate molecules.

Effects of a Mutation in the gyrA Gene on the Virulence of Uropathogenic Escherichia coli.

ABSTRACT Fluoroquinolones are among the drugs most extensively used for the treatment of bacterial infections in human and veterinary medicine. Resistance to quinolones can be chromosome or plasmid mediated. The chromosomal mechanism of resistance is associated with mutations in the DNA gyrase- and topoisomerase IV-encoding genes and mutations in regulatory genes affecting different efflux systems, among others. We studied the role of the acquisition of a mutation in the gyrA gene in the virulence and protein expression of uropathogenic Escherichia coli (UPEC). The HC14366M strain carrying a mutation in the gyrA gene (S83L) was found to lose the capacity to cause cystitis and pyelonephritis mainly due to a decrease in the expression of the fimA, papA, papB, and ompA genes. The levels of expression of the fimA, papB, and ompA genes were recovered on complementing the strain with a plasmid containing the gyrA wild-type gene. However, only a slight recovery was observed in the colonization of the bladder in the GyrA complement strain compared to the mutant strain in a murine model of ascending urinary tract infection. In conclusion, a mutation in the gyrA gene of uropathogenic E. coli reduced the virulence of the bacteria, likely in association with the effect of DNA supercoiling on the expression of several virulence factors and proteins, thereby decreasing their capacity to cause cystitis and pyelonephritis.

Characterization of the enzyme aac(3)-id in a clinical isolate of Salmonella enterica serovar Haifa causing traveler's diarrhea

INTRODUCTIONnThe objective of this investigation was to identify the mechanism of decreased susceptibility to gentamicin in a Salmonella clinical isolate, leading to the detection of a aminoglycoside acetyltransferase gene found in a class 1 integron.nnnMETHODSnA multidrug-resistant Salmonella strain was recovered from feces of a traveler to Egypt. The antimicrobial susceptibility test to 12 antimicrobial agents was performed with the Kirby-Bauer method. The presence of class 1 integron was determined by PCR. The amplified product was recovered and sequenced in order to establish the genes carried. In addition, susceptibility to gentamicin C1a, gentamicin C1, sisomicin, neomycin, dibekacin, kanamycin, tobramycin, amikacin, netilmicin, apramycin, dactimicin, spectinomycin, streptomycin, lividomycin and butirosin, was established. The Champion pET101 Directional TOPO Expression Kit was used to clone and express the aac(3)-I gene.nnnRESULTSnThe isolate was identified as Salmonella enterica serovar Haifa, showing resistance to nalidixic acid, tetracycline and decreased susceptibility to gentamicin. One integron with a size circa 1,500 bp, encoding an aac(3)-Id plus aadA7 genes was observed. The analysis of the susceptibility to different aminoglycosides in the E. coli TOP10F transformed with the vector carrying aac(3)-Id gene showed resistance to gentamicin C1a, gentamicin C1, and dactimicin, in accordance with the presence of this enzyme but, was susceptible to sisomicin. The homology of the amino acid and nucleotide sequences with the AAC(3)-Id enzyme was of 100%.nnnCONCLUSIONnThe presence of the AAC(3)-Id enzyme was described for the first time in a S. Haifa.