Real-time monitoring of translocation of selected type-III effectors from Xanthomonas oryzae pv. oryzae into rice cells

Type-III (T3) effectors PthXo1 and AvrXa10 of Xanthomonas oryzae pv. oryzae are translocated into rice cells to inducevirulence and avirulence on susceptible- and resistant-rice varieties Nipponbare and IRBB10, respectively. The translocation needs the bacterial T3 translocator Hpa1 and rice Oryza sativa plasma membrane protein OsPIP1;3. Here, weemployed the b-lactamase (BlaM) reporter system to observe PthXo1 and AvrXa10 translocation. The system wasestablished to monitor effectors of animal-pathogenic bacteria by quantifying the BlaM hydrolysis product [P] and fluorescence resonance energy transfer (FRET) of the substrate. The feasibility of the BlaM reporter in rice protoplasts wasevaluated by three criteria. The first criterion indicated differences between both [P] and FRET levels among wild types andOsPIP1;3-overexpressing and OsPIP1;3-silenced lines of both Nipponbare and IRBB10. The second criterion indicateddifferences between [P] and FRET levels in the presence and absence of Hpa1. The last criterion elucidated the coincidenceof PthXo1 translocation with induced expression of the PthXo1 target gene in protoplasts of Nipponbare and the coincidence of AvrXa10 translocation with induced expression of the AvrXa10 target gene in protoplasts of IRBB10. Theseresults provide an experimental avenue for real-time monitoring of bacterial T3 effector translocation into plant cells with apathological consequence.

Isolation of blaNDM-producing Enterobacteriaceae in a public hospital in Salvador, Bahia, Brazil

ABSTRACT Carbapenemases have great importance in the global epidemiological scenario since infections with carbapenemase-producing bacteria are associated with high mortality, especially in hospitalized patients in intensive care units. This study describes two microorganisms producers of the New Delhi Metallo-b-lactamase, Klebsiella pneumoniae and Citrobacter freundii, from two patients admitted to a public hospital in Salvador, Bahia. These are the first clinical cases of New Delhi Metallo-b-lactamase described in microorganisms in the north and northeast Brazil. The isolates were characterized by antimicrobial susceptibility test, with resistance to all β-lactams including carbapenems, negative Modified Hodge Test and the synergy test with Ethylenediaminetetraacetic acid, Phenylboronic Acid and Cloxacillin was positive only with Ethylenediaminetetraacetic acid (difference of >5 mm in the inhibition zone between the disk without and with the inhibitor). Analysis by multiplex PCR for blaIMP, blaVIM, blaNDM, blaKPC and blaOXA-48 enzymes confirmed the presence of blaNDM gene. This report of two different New Delhi Metallo-b-lactamase-producing microorganisms in a different region of Brazil confirms the risk of spreading resistance genes between different species and emphasizes the need for prevention and control of infections caused by these pathogens, which have limited treatment options and have been linked to high mortality rates.

Molecular Detection Of The New Delhi Metallo-B-Lactamase-1 Gene In Enterobacteriaceae Isolates In A Tertiary Medical Centre

Background: New Delhi metallo-b-lactamase-1 (NDM-1) is a relatively recent carbapenemase enzyme that inactivates all b-lactam antibiotics with the exception of aztreonam. This study aims to ascertain the baseline prevalence and antibiotic susceptibility patterns of NDM-1-producing Enterobacteriaceae in a tertiary medical center in Malaysia. Methods: Over a period of one year, all Enterobacteriaceae isolates from all clinical specimens with reduced susceptibility to at least one carbapenem and resistance to at least one third generation cephalosporin were subjected to antibiotic susceptibility testing by disk diffusion and molecular detection of the NDM-1 gene by single-target PCR followed by gel electrophoresis. Results: A total of 13,098 Enterobacteriaceae isolates were screened and 63 (0.48%) had reduced susceptibility to at least one carbapenem. Of this 63, 18 (29%) were NDM-1-positive. Of this 18, 16 (89.0%) were Klebsiella pneumoniae, one (5.5%) was Escherichia coli and one (5.5%) was Klebsiella ornithinolytica. Reduced susceptibility to at least one aminoglycoside was seen in 17 (94%) of the NDM-1-positive isolates. All 18 (100%) had reduced susceptibility to ertapenem and were resistant to all the second and third generation cephalosporin antibiotics tested. Conclusion: The prevalence of NDM-1-producing Enterobacteriaceae among all the Enterobacteriaceae isolates in our institution is low (0.14%) and screening for the NDM-1 gene is best performed using ertapenem-impregnated disks.

Purification of metallo-B-lactamase CcrA from Bacteroides fragilis with salting-out method

A simple and effective salting-out method was developed for the purification of the metallo-β-lactamase CcrA from Bacteroides fragilis based on the plasmid pMSZ02, in which the crude protein secreted into growth medium was precipitated by 80% sulfate saturation of the medium and purified with Q-Sepharose to offer pure CcrA with yield of 20.1 mg per litter medium. The dependence of the amount of protein precipitation on sulfate saturation was investigated, which showed that more than 80% sulfate saturation resulted the maximum protein precipitated. The purified CcrA was evaluated by steady-state kinetics using penicillin G and cephalothin V as substrates, which showed the Km values of 68±2 and 17±2 µM and Kcat values of 63±1 and 102±3 S-1, respectively. The comparison with the data of the protein from literature method showed that the salting-out method was viable, and it could be useful for the purification of other proteins secreted into growth medium.

Study of antimicrobial susceptibility of clinically significant microorganisms isolated from selected areas of Dhaka, Bangladesh.

Objective: Pathogenic resistance against antibiotics is substantially mounting in the developing countries including Bangladesh. Present study thus attempted to obtain the baseline information on such resistance among the community people coming to the local dispensaries around the city of Dhaka for treatment. Materials and Methods: A total of 2,700 clinical specimens were examined for the presence of Gram positive and Gram negative pathogens. Antibiotic susceptibility tests of the isolates were carried out. Extended spectrum b- lactamase (ESBL) activity, and the presence of methicillin resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE) were also detected. Results: Escherichia coli were most prevalent (45.5%) among 1044 pathogenic bacteria isolated from 2,700 samples. E. coli predominated urine, pus, wound swab, blood, high vaginal swab (HVS) and sputum specimens, and exhibited the highest frequency of ESBL activity (35%). Prevalence of Klebsiella spp. and S. aureus among the clinical specimens were 11.5% and 9.86%, respectively. Most of the Gram negative bacilli were found resistant against ciprofloxacin (5 mg), tetracycline (30 mg) and cotrimoxazole (25 mg). Majority of Pseudomonas spp. were found resistant against most of the commonly used antibiotics. Interestingly, around half of the S. aureus isolates were observed to be methicillin resistant, but not vancomycin resistant. Conclusion: Overall, such a revelation of increased antibiotic resistance demands for restrictive and appropriate antibiotic usage in accordance with the updated antibiotic prescribing policy in Bangladesh.

Metaloenzimas tipo VIM detectadas en aislamientos clínicos en Pseudomonas aeruginosa en cuatro hospitales en Venezuela / VIM- type metalloenzymes detected in clinical isolates of Pseudomonas aeruginosa in four Venezuelan hospitals

En los últimos años se ha observado mundialmente un incremento del aislamiento de cepas de Pseudomonas aeruginosa resistentes a múltiples antibióticos, siendo la producción de β-lactamasas la principal causa de resistencia a los antibióticos β-lactámicos, los cuales constituyen la única opción terapéutica en muchos casos. Las metalo-β-lactamasas (MBLs) son una familia de enzimas degradadoras de β-lactámicos que recientemente han emergido como determinantes de resistencia de importancia clínica y que son activas contra los carbapenem; no hidrolizan a los monobactámicos; son inhibidas por agentes quelantes de iones metálicos como el EDTA y el ácido dipicolínico; no son inhibidas por el ácido clavulánico, sulbactan ni tazobactan y presentan uno o dos iones de zinc en su sitio activo. Este reporte describe la detección de MBLs tipo VIM mediante ensayos fenotípicos y moleculares en nueve cepas de Pseudomonas aeruginosa resistentes a carbapenems aisladas de muestras clínicas de cuatro hospitales de Venezuela. Por métodos fenotípicos se evidenció que 100% de las cepas eran productoras de MBLs, y por PCR todas las MBLs resultaron ser de la familia VIM, las cuales confieren alto nivel de resistencia a los antibióticos β-lactámicos, con excepción del Aztreonam, único antibiótico al cual se observó sensibilidad, complicando así las opciones terapéuticas.

In recent years there has been a worldwide raise in the isolation of Pseudomonas aeruginosa strains with resistance to multiple antibiotics, being β-lactamase production the main cause of resistance to β-lactamic antibiotic (which are the only therapeutic option in many cases). The MBLs are an β-lactamic- degrading enzymatic family that have emerged as clinically relevant resistance determinants and are active against carbapemens, don´t hydrolise monobactams, have one or two zinc ions in its active site and are inhibited by metallic ions chelating agents such as EDTA and dipicolinic acid, but aren´t inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam or tazobactam. This report describes the detection of VIM-type MBLs by phenotypic and molecular methods in 9 carbapenemsresistant Pseudomonas aeruginosa strains isolated from clinical samples of four Venezuelan hospitals. By phenotypic methods it was evidenced that 100% of the strains were MBLs producers, and by final- point PCR it was determined that all the MBLs were from the VIM family, which confer high- level resistance to the β-lactamic antibiotics (except Aztreonam), and because they are carried by plasmids with the ability to transfer horizontally to other bacterial families, they can be responsible for therapeutic complications in an individual or the patients collective.

Plasmid Profile and B - Lactamase Type of Multidrug - Resistant Salmonella typhi Isolated from Korea, 1997

Eight strains of multidrug-resistant (MDR) Salmonella typhi were isolated from Kyonggi area during January-February,1997. They were resistant to ampiciUin, amoxicillin, carbeniciillin, tetracycline, chloramphenicol, trimethoprim/sulfamethoxazole, trimethoprim. Eight strains had one plasmid respectively which size was approximately M.W 220 kb and showed same restriction pattern by endonuclease HindIII. The plasmid was similar to the plasmid in size that was related to multidrug resistant S. typhi isolated from southeast Asia. It were transferred by conjugation to recipient E, coli K-12 in frequency of 2.43 x10-4 - 1.73 x 10-2 and transconjugant showed same drug-resistant pattem with donor cells. All of 8 strains produced B-lactamase that was assummed to TEM-1 type by isoelectric focusing and PCR.