Biofilm formation in clinical isolates of nosocomial Acinetobacter baumannii and its relationship with multidrug resistance

Objective: To check biofilm formation by Acinetobacter baumannii (A. baumannii) clinical isolates and show their susceptibility to different antibiotics and investigate a possible link between establishment of biofilm and multidrug resistance. Methods: This study was performed on clinical samples collected from patients with nosocomial infections in three hospitals of Tehran. Samples were initially screened by culture and biochemical tests for the presence of different species of Acinetobacter. Identifications were further confirmed by PCR assays. Their susceptibilities to 11 antibiotics of different classes were determined by disc diffusion method according to Clinical and Laboratory Standards Institute guidelines. The ability to produce biofilm was investigated using methods: culture on Congo red agar, microtiter plate, and test tube method. Results: From the overall clinical samples, 156 specimens were confirmed to contain A. baumannii. The bacteria were highly resistant to most antibiotics except polymyxin B. Of these isolates, 10.26% were able to produce biofilms as shown on Congo red agar. However, the percentage of bacteria with positive biofilm in test tube, standard microtiter plate, and modified microtiter plate assays were 48.72%, 66.66%, and 73.72%, respectively. At least 92% of the biofilm forming isolates were multidrug resistant. Conclusions: Since most of the multidrug resistant strains produce biofilm, it seems necessary to provide continuous monitoring and determination of antibiotic susceptibility of clinical A. baumannii. This would help to select the most appropriate antibiotic for treatment.

Molecular cloning of virB12 gene of Brucella melitensis 16M strain in pET28a vector

OBJECTIVE@#To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.@*METHODS@#Brucella melitensis (B. melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep® Genomic DNA Extraction Kit. Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5' end of the forward and reverse primers, respectively. DNA amplification was performed using PrimSTAR® HS DNA polymerase and the PCR product was purified by DNA AccuPrep®Gel Purification Kit. Purified DNA was cloned into pJET1.2 cloning vector. VirB12 gene fragment was excised from pJET1.2 using BamHI/HindIII and subsequently subcloned into pET28a (+).@*RESULTS@#Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a (+) plasmids. PCR and restriction enzyme digestion confirms the procedure.@*CONCLUSION@#We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.

Comparison of polymerase chain reaction and culture for detection of genital mycoplasma in clinical samples from patients with genital infections

Comparison of polymerase chain reaction [PCR] and culture for detection of genital mycoplasma [Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma urealyticum] in clinical samples from patients with genital infections. Duplicate genital swabs were taken from 210 patients, referred to the gynecology clinic of Rasool Hospital, Tehran, Iran between December 2007 and June 2008. They were transported to the laboratory in a selective mycoplasma transport medium and in phosphate buffer solution. The specimens were inoculated into specific broth and solid medium for culture. Characteristic mycoplasma colonies were determined with Diennes' stain and examined microscopically. For PCR, samples were analyzed with genus specific primers. The primer sets, which were originally designed in our laboratory, amplified a 465 bp fragment [Mycoplasma genitalium], 559 bp fragment [Ureaplasma urealyticum], and 630bp fragment [Mycoplasma hominis]. Samples containing a band of the expected sizes for mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme. Of the 210 samples, mycoplasma strains were isolated from 83 patients [39.5%], [23 mycoplasma isolates, 11%; and 69 ureaplasma isolates, 32.9%] by using a selective mycoplasma isolation media. Using PCR, a total of 120 [57.1%] samples were found to be positive for mycoplasmas [28 mycoplasma spp., 13.3%; and 67 ureaplasma spp., 31.9%] and co-infections with both species were detected in 25 samples [11.9%]. The PCR was found to be highly sensitive when genus specific primers were used for diagnosis of genital mycoplasmas in comparison with culture

Isolation and identification of anionic surfactant degrading bacteria from activated sludge

Linear alkylbenzene sulfonate [LABS] is an anionic surfactant widely used all over the world. They will eventually end-up and accumulate in household or industrial sewage. Due to their high foaming capabilities which can cause numerous problems in sewage treatment facilities as well as direct toxic effects on many different organisms in ecosystem; they are generally considered as serious pollutants. Many reports have indicated that common bacteria can readily degrade LABS. In this survey, two different bacteria were isolated from Tehran municipal active sludge that showed the ability to degrade LABS rapidly and actively upon using it as their sole source of carbon. Biochemical tests as well as 16S rRNA gene sequencing performed. Results have indicated the two isolates to be Acinetobacter johnsoni and Pseudomonas beteli. After experiments to optimize the pH and temperature for growth of the two bacterial isolates, the extent of LABS, utilization was evaluated by HPLC method. The Pseudomonas beteli and Acinetobacter johnsoni isolates were able to degrade 96.4% and 97.2% of the original LABS levels after 10 days of growth, respectively. Mixed culture of the two isolates did not significantly increase LABS utilization [97.6%]. Our study showed the ability of two isolated steains to rapidly biodegrade LABS under aerobic conditions