ABSTRACT
Urinary tract infections and their complications cause serious health problems affecting millions of people every year. Infections of the urinary tract are the second most common type of infection in the body. About 20% of women are especially prone to UTIs for reasons that are not yet well understood. UTIs in men are not as common as in women but can be very serious when they do occur. Accurate identification of bacterial isolates is an essential task of the clinical microbiology laboratory. Conventional identification methods of these infections lack the precision and the reproducibility, as well as they are time consuming because they rely on the growth of the bacteria. Some groups need days or even weeks to grow. Soon after the DNA becomes the worldwide common language, in this study, we compared the conventional microbiological diagnostic methods with the molecular based techniques. Our main focus was targeted to the PCR and sequencing of ribosomal markers. Our candidate in this study was 16S rRNA gene using different primer sets specifically designed to amplify different regions of our marker. We also focused on how much sequencing information is needed for blind identification of bacterial pathogens. In conclusion, the DNA sequencing based method provides a valuable tool for cheap and accurate diagnosis of Gram-negative bacteria in urinary tract infections which can be applicable in other infections
ABSTRACT
Background: Increased incidence of resistance to beta-lactams among members of the family Enterobacteriaceae has been reported worldwide. Extended spectrum beta-lactamase [ESBL] producing Gram-negative bacteria are becoming a major global concern and usually harbor plasmid-mediated enzymes of the TEM, SHV, OXA, PER, and CTX-M types. The aim of this study is to determine the prevalence of ESBL-producing Enterobacteriaceae in Mansoura hospitals and to molecularly characterize the ESBL-related bla genes, including blaTEM and blaCTX-M
Methodology: A total of 40 E. coli, 30 K. pneumoniae and 30 Proteus isolates were studied for antibiotic susceptibility pattern using different betalactam antibiotics and for the presence of ESBLs by combination of double-disc approximation test and inhibitor-potentiated disc-diffusion test. Subsequently, the hyper variable regions of beta-lactamase-encoding genes were amplified and sequenced using dye termination Sanger methodology to study the genetic variation among the clinical isolates
Results: All E. coli isolates were resistant to ampicillin, amoxicillin/clavulanate and cefadroxil. Regarding K. pneumoniae, all isolates were resistant to ampicillin, amoxicillin/clavulanate, cefadroxil, cefoxitin, cefuroxime and cefotaxime. Concerning Proteus species, all isolates were resistant to ampicillin, cefadroxil, cefotriaxone, cefuroxime and cefoperazone. In contrast 95% of E. coli isolates 80% of K. pneumoniae isolates and 90% of Proteus isolates were sensitive to imipenem. The detection of ESBLs by double-disc approximation test and inhibitor-potentiated disc-diffusion test was quiet different. Double disc approximation method lacks sensitivity. It showed false negative results in nearly 92% of the isolates that were concerned positive ESBLs producers by inhibitor-potentiated disc-diffusion test. PCR amplification and sequencing analysis revealed the presence of CTX-M and TEM type ESBLs in the tested isolates and could accurately characterize different types of blaTEM and blaCTX-M among the clinical isolates
Conclusion: Combined use of the conventional ESBLs screening methods and the molecular amplification of the ESBLs encoding genes followed by PCR based sequencing method provides a very valuable tool for identification and characterization of ESBLs producing E. coli, K. pneumoniae, and Proteus clinical isolates
ABSTRACT
Background: Gram negative bacteria are responsible for numerous infectious diseases. These diseases can occur in and harm any part of the body, the skin, eyes and the nervous, cardiovascular, respiratory, gastrointestinal and urogenital systems
Methods: In the present study, some phenotypic and molecular typing techniques were applied on 108 strains of E. coli, 88 strains of Ps. aeruginosa and 8 strains of Serratia isolated from different clinical lesions in Mansoura University Hospitals, Egypt
Results: The distribution of antibiotic resistance among the isolated strains showed high incidence of resistance and imipenem was the most active antibiotic. Using the active pyocin typing, 72 strains of Ps. Aeruginosa could be typed into 35 pyotypes. Using PCR technique it was found that 84% of the 50 tested isolates were found to have at least one of the tested ESBLs. Also TolC and AcrA genes were present in all tested E. coli except 4 strains and did not present in Ps. aeruginosa except 4 strains. Plasmid profiles of 23 tested E. coli appear to be diverse. Also the prevalence of plasmids in 22 tested Ps. aeruginosa strains was lower than in tested E. coli therefore 59.1% of tested Ps. aeruginosa strains harbored plasmids. Using Pyrosequencing technique, the sequenced region of gyrA, gyrB and ParC were able to differentiate between the tested strains and neighbor-joining tree was constructed to determine relatedness between the isolated strains. Moreover, Molecular cloning of the whole sequence of bla-TEM, bla-SHV and bla-CTX-M was carried out experimentally to study the expression of these genes and determine which genes of them responsible for the resistance
Conclusion: Molecular-based typing methods of are more advantageous compared with phenotypic typing methods in terms of better discrimination and reproducibility. Significant genetic variation was observed among different strains represented by the diversity of their plasmid profiles. All molecular genetics methods for distinguishing organism subtypes are based on differences in the DNA sequence
ABSTRACT
Background: Pseudomonas aeruginosa plays an important role in opportunistic and nosocomial infections affecting individuals with predisposing conditions
Methods: In this respect, we evaluated the production of MBLs among 100 clinical isolates of Pseudomonas aeruginosa sources in Mansoura University Hospitals
Results: Most isolates were highly resistant to penicillins and cephalosporins. On the other hand, most strains were sensitive to imipenem as 91% of the isolates were susceptible. The effect of MBL inhibitors using a simple disk diffusion test revealed the efficiency of EDTA and 2-mercaptoethanol as Metalo-beta-lactamase inhibitors due the observed expansion of the growth-inhibitory zone of the two inhibitors. Cupric chloride also gave a clear result, but with a weak inhibitory effect. IMP and VIM genes were amplified from both genomic and plasmid DNA extracted from 26 isolates. The amplification primers of both markers were specifically designed to amplify 600 bp for IMP and 500 bp for VIM. On the other hand, two isolates did not harbor IMP gene on their plasmids while six isolates did not contain VIM on their plasmids. Subsequent sequencing of the generated amplicons showed different types of mutation in the sequenced regions of the tested resistance genetic markers. They include insertion, deletion and substitution mutations. Moreover, the region 178-822 of IMP and the region 111-627 of VIM, commonly found in resistant Pseudomonas aeruginosa, are the most predominant variable regions
Conclusion: particular PCR and subsequent sequencing provide a useful tool for accurate and cost efficient characterization of MBLs producing Pseudomonas aeruginosa isolates. The accuracy of the sequencing method can answer the epidemiological questions that can't be answered by the traditional microbiological methods. This approach will help in choosing the best effective antibiotic to overcome the nosocomial infection
ABSTRACT
Background: Klebsiella species cause 3-7% of all nosocomial infections, placing them in the top 10 of nosocomial bacterial pathogens
Materials: In this respect, we evaluated the differences in some quinolone resistance determinants among 70 clinical isolates of Klebsiella pneumoniae collected from Mansoura University Hospitals
Results: In the present investigation, some molecular typing techniques were applied on 70 isolates of K. pneumoniae isolated from Mansoura University Hospitals from different clinical lesions. The distribution of antibiotic resistance among the isolated strains showed high incidence of resistance to extended-spectrum cephalosporins [70 to 94.29%] and to quinolones [38.57 to 55.7 %] was also observed. Imipenem was the most active antibiotic so; it could be considered the drug of choice for treatment of infections caused by multi-resistant K. pneumoniae. Plasmid profiles of the tested strains appear to be diverse, although some similarities were found among tested strains. Sixty seven out of 70 strains contained plasmid DNA. PCR amplification was used to detect some quinolone resistance determinant genes such as gyrA, gyrB and Onr in the collected Klebsiella pneumoniae isolates. Using pyrosequencing technique, the sequenced region of gyrA gene was able to differentiate between resistant and sensitive strains however, the sequenced region of gyrB gene failed to differentiate between resistant and sensitive strains. Qnr gene was detected in all tested strains except strains No. 24 and 28
Conclusion: Using PCR and DNA sequencing of the target region of gyrA gene, we were able to differentiate between resistant and sensitive strains. While, amplification of another region of gyrB or Qnr genes failed to differentiate between the isolates. But, it could detect different types of mutations between the clinical isolates