Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem- resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXYOprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Effects of the encapsulation of usnic acid into liposomes and interactions with antituberculous agents against multidrug-resistant tuberculosis clinical isolates

Mycobacterium tuberculosis (Mtb) has acquired resistance and consequently the antibiotic therapeutic options available against this microorganism are limited. In this scenario, the use of usnic acid (UA), a natural compound, encapsulated into liposomes is proposed as a new approach in multidrug-resistant tuberculosis (MDR-TB) therapy. Thus the aim of this study was to evaluate the effect of the encapsulation of UA into liposomes, as well as its combination with antituberculous agents such as rifampicin (RIF) and isoniazid (INH) against MDR-TB clinical isolates. The in vitro antimycobacterial activity of UA-loaded liposomes (UA-Lipo) against MDR-TB was assessed by the microdilution method. The in vitro interaction of UA with antituberculous agents was carried out using checkerboard method. Minimal inhibitory concentration values were 31.25 and 0.98 μg/mL for UA and UA-Lipo, respectively. The results exhibited a synergistic interaction between RIF and UA [fractional inhibitory concentration index (FICI) = 0.31] or UA-Lipo (FICI = 0.28). Regarding INH, the combination of UA or UA-Lipo revealed no marked effect (FICI = 1.30-2.50). The UA-Lipo may be used as a dosage form to improve the antimycobacterial activity of RIF, a first-line drug for the treatment of infections caused by Mtb.

Herbicides cross resistance of a multiple resistant short-awn foxtail ( Alopecurus aequalis Sobol.) population in wheat field

Alopecurus aequalis Sobol. is a common grass weed, which has become increasingly troublesome to control in China wheat fields. One A. aequalis population, collected from Anhui Province China, was suspected to be resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl. This study aimed to establish the cross-resistance pattern using the purified subpopulation and explore the potential targetsite and non-target-site based resistance mechanisms. Sequencing results showed that a single nucleotide change of ATT to AAT was present in acetyl-CoA carboxylase (ACCase) gene of the resistant (R) plants, resulting in an Ile2041Asn amino acid substitution. Besides, another single nucleotide change of CCC to CGC was present in acetolactate synthase (ALS) gene of the R plants, resulting in a Pro197Arg amino acid substitution. The homozygous resistant plants were isolated and the seeds were used in whole-plant herbicide bioassays. Compared with the susceptible (S) population, R population displayed high level resistance to fenoxaprop-P-ethyl and mesosulfuronmethyl. Cross resistance patterns showed that the R population was highly resistant to clodinafop-propargyl, moderately resistant to pyroxsulam and flucarbazoncsodium, lowly resistant to pinoxaden, and susceptible to tralkoxydim, sethoxydim, and isoproturon. The pretreatment of piperonyl butoxide reduced the 50% growth reduction (GR50) value of fenoxaprop-P-ethyl, suggesting that target-site resistance and non-target-site resistance mechanisms were both present in fenoxaprop- P-ethyl-resistance of A. aequalis. This is the first report of ACCase Ile2041Asn and ALS Pro197Arg mutation in A. aequalis.

In vitro technique for selecting onion FOR white rot disease- resistance

In vitro selection is one of the most effective and efficient techniques for plant improvement. This is due to its ability to isolate plants with the desired character(s), either by applying a selection agent on the culture media to drive the selection of somaclones with the required character(s), or by establishing particular conditions that change in the genomes of somaclones toward the required character. The objective of this study was to identify a suitable protocol for in vitro selection of Allium white rot disease ( Sclerotium cepivorum ) tolerance in commercial Egyptian onion varieties, namely Giza 20, Giza 6 and Beheri Red. Oxalic acid (OA), the phytotoxin produced by Sclerotium cepivorum, was used as the selective agent. Seeds of the three Egyptian varieties were germinated on four concentrations (0.0, 0.02, 0.2, 2 and 20 mM) of Oxalic acid. Among the tested cultivars, Beheri Red had the highest germination frequency (52%) at all concentrations tested, followed by Giza 20 (42.6%), and Giza 6 at (32%). Cotyledon explants from the varieties were cultured on toxic MSBDK medium, supplemented with 0, 3, 6 and 12 mM OA. The survival of calli on MSBDK free toxic medium was 70.7% for all tested cultivars; however, MSBDK-stressed medium, with 3 mM OA reduced the viable calli to 42.1%. The highest OA concentration (12 mM) completely inhibited calli induction from cotyledons explants. A medium supplement with 3 mM OA retarded 80% of calli growth. Among 156 tested calli of Beheri Red, only 23 calli (14.7%) survived on toxic medium for 45 days. Similarly, there was 15.6% survival for Giza 20 calli, while 40.1% of the Giza 6 calli survived. Plantlets were regenerated from surviving calli and transplanted onto ex vitro, and formed bulb after acclimatisation.

Relationship between super antigenicity, antimicrobial resistance and origin of Staphylococcus aureus isolated

Introduction: Staphylococcus aureus is a pathogen that causes food poisoning as well as hospital and community acquired infections. Objective: Establish the profile of superantigen genes among hospital isolates in relation to clinical specimen type, susceptibility to antibiotics and hospital or community acquisition. Methods: Eighty one isolates obtained from patients at Colombian hospital, were classified by antimicrobial susceptibility, specimen type and hospital or community acquired . The PCR uniplex and multiplex was used for detection of 22 superantigen genes (18 enterotoxins, tsst-1 and three exfoliative toxins). Results: Ninety five point one percent of isolates harbored one or more of the genes with an average of 5.6 genes. Prevalence of individual genes was variable and the most prevalent was seg (51.9%). Thirty nine genotypes were obtained, and the genotype gimnou (complete egc cluster) was the most prevalent alone (16.0%) and in association with other genes (13.6%). The correlation between presence of superantigens and clinical specimen or antimicrobial susceptibility showed no significant difference. But there was significant difference between presence of superantigens and the origin of the isolates, hospital or community acquired (p= 0.049). Conclusions: The results show the variability of the superantigen genes profile in hospital isolates and shows no conclusive relationship with the clinical sample type and antimicrobial susceptibility, but there was correlation with community and hospital isolates. The analysis of the interplay between virulence, epidemic and antibiotic resistance of bacterial populations is needed to predict the future of infectious diseases.