Direct detection of Pseudomonas aeruginosa from patients with healthcare associated pneumonia by real time PCR.

Using oprL sequences, a TaqMan real time PCR was developed and used for quantitative detection of Pseudomonas aeruginosa from 99 broncoalveolar lavage and 11 sputum specimens collected from patients with health care associated pneumonia. All specimens were cultured on appropriate media to isolate bacteria. Twenty five specimens were positive by both methods. Polymicrobial infections were found in 13 specimens. Amplification of oprL in serial dilutions ranged from 10(9)CFU/ml to 10(2)CFU/ml. Standard curve of duplicated every dilution had slope 3.25±0.1 and R(2)>0.99 with SD 0.1. Our real time PCR assay showed high sensitivity (100%) and specificity (98.85%). This technique could detect and enumerate 100 bacteria directly from clinical specimens and showed that the threshold is 10(3)CFU/ml in cases with clinical symptoms. Our method can be used for quantitative detection of P. aeruginosa from BAL and sputum specimens in 1h and 10min.

Phenotypic characterization and plasmid analysis of Klebsiella pneumoniae strains from Iranian patients

Local knowledge of antimicrobial susceptibilities of Klebsiella pneumoniae is important for implementation of effective hospitals anti-infective policies. One hundred isolates of K. pneumoniae collected from 3 different hospitals in Iran during 2004 were screened for their susceptibilities to thirteen different antibiotics using disk diffusion test and macro broth dilution assay. Isolates were then subjected to restriction endonuclease analysis of plasmid DNA. All isolates were susceptible to imipenem. The rates of resistance to other antibiotics were in the following order: amikacin (10 por cento), piperacillin-tazobactam (por cento 2), ciprofloxacin (20 por cento), ceftizoxime (14 por cento), cefexime (31 por cento), ceftazidime (28 por cento), cefotaxime (33 por cento), nalidixic acid (32 por cento), cephalexin (32 por cento), gentamicin (30 por cento), nitrofurantoin (31 por cento) and piperacillin (66 por cento). The production of extended spectrum betalactamase (ESBL) hydrolyzing ceftazidime and cefotaxime was detected in 54 por cento of isolates. Of 100 isolates tested, 67 harbored plasmids and the remaining lacked any plasmid. Though the prevalence of ESBL phenotype in Iran is higher than western countries, it is close to figures reported from the region. Evidences for outbreaks with certain isolates of K. pneumoniae were found by restriction endonuclease analysis of plasmid DNA. This technique also showed the persistence of infections in the urinary tract of several patients.

Transposon Tn5281 is the main distributor of the aminoglycoside modifying enzyme gene among isolates of Enterococcus faecalis in Tehran hospitals.

Infections with high levels of gentamicin-resistant (HLGR) isolates of Enterococcus faecalis are common in Tehran hospitals. Genes encoding such resistance are transmissible by conjugation at high frequency. The purpose of this study was to determine the existence of Tn5281 and its flanking aminoglycoside modifying enzyme gene aac(6')-aph(2") among 102 HLGR isolates of E. faecalis cultured from patients at three hospitals in Tehran, Iran. These isolates were detected by disks containing 120 microg of gentamicin and made 65% of all E. faecalis during the study period. DNA was extracted from HLGR isolates and subjected to PCR assays targeting aac(6')-aph(2") and conjugative transposon Tn5281. The amplified aac(6')-aph(2") gene was labeled with digoxigenin and probed with Tn5281 amplicons in dot blot hybridization assays. The aac(6')-aph(2") gene was detected in 91%-92% (n = 93) of the HLGR isolates. All isolates containing aac(6')-aph(2") were positive in long-PCR targeting Tn5281 and the probe hybridized with Tn5281 amplicons. The number of HLGR isolates of E. faecalis has increased considerably in Tehran hospitals. Tn5281 is the main cause of transmission of aac(6')-aph(2") to different isolates of E. faecalis in the hospitals studied.

Phenotypic characteristics and population genetics of Enterococcus faecalis cultured from patients in Tehran during 2000-2001.

Conventional bacteriology techniques were used to identify enterococci isolates cultured from patients at different hospitals in Tehran during 2000-2001. The identification was confirmed using species-specific PCR targeting the D-alanyl-D-alanine ligase gene. A total of 59 isolates of Enterococcus faecalis were identified. The rates of resistance to different antibiotics were in the following order: penicillin 84%, ciprofloxacin 42%, high-level gentamicin 30%, nitrofurantoin 14%, imipenem 4%, and chloramphenicol 2%. Resistance to ampicillin was found to be rare among the Iranian isolates of E. faecalis. Multi-locus enzyme electrophoresis was then used to analyze the strains. Forty-five electrophoretic types were obtained when 10 enzyme loci were screened. Although the collection of bacterial isolates was limited in time and location, considerable heterogeneity was found. Analysis of strains for linkage disequilibrium demonstrated that the studied population is not clonal, since the index of association was not significantly different from zero (Ia = 0.0296). Enterococcus faecalis isolates recovered from patients in Tehran were genetically diverse and seemed to possess a high potential for genetic recombinations, though none were resistant to vancomycin.