[Identification of Burkholderia cepacia in patients with cystic fibrosis by pulsed-field gel electrophoresis]

Background: Complex of Burkholderia cepacia is one of the main and serious causes of infections in cystic fibrosis patients that can be highly transmissible

Small hospital outbreaks are frequent and are usually due to a single contaminated environmental source. The pulsed-field gel electrophoresis [PFGE] is widely used to identify the strain emission sources in cystic fibrosis patients

The aim of this research was to study geno-typing of Burkholderia cepacia using PFGE method, and to evaluate diversity complex of clinical strains isolated from cystic fibrosis patients

Methods: This is a descriptive study, in which 100 pulmonary secretion specimens of cystic fibrosis patients admitted in Masih Daneshvari Hospital, Tehran Iran in period of 12 months 2012 to 2013 were collected

The specimens were cultured on BCSA plate's. After incubation suspected colonies were isolated and identified by biochemical and phenotypic method. All samples were checked by API system [API20NE] and by specific PCR method for genus Bulkhorderia and Bcc as well. DNA was extracted by alkaline lysis method and confirmed by PCR analysis of recA genes. Genetic diversity of isolate was performed by PFGE analysis according to Pulsenet guideline by using Xbal, Spel as restriction enzyme which digests infrequently among the Burkholderia cepacia genome

Results: Out of 100 samples five were identified as Burkholderia cepacia. It is obviously different at variously reports. The electrophoresis data of PCR products and comparison of band in samples from patients with standard strain ATCC 25416 Burkholderia cepacia and compare and analyse the PFGE size marker bands of Salmonella choleransuis serotype Braenderup H9812 strain, were the same

Conclusion: Application of PFGE and identification of pulse-type is a potential tool to enhance the investigation of apparent nosocomial outbreaks of B.cepacia. Similar type of pulse patterns was observed in this study means that all of infection has been from one source; therefore the hypothesis of transferring person to person will be rejected. Base on these results environmental sources sampling should be considered in future investigation

Detection of Brucella abortus by alkB and IS711 based primers

Brucellosis is a zoonotic disease, which involves both animals and human. Although the conventional methods have been widely used for its laboratory diagnosis, the PCR techniques have proved to be useful due to specificity, sensitivity and the rapidness. Various target sequences of brucella bacterium such as OMP2, 16s RNA and IS711 have been used for the primer designing. All primer sets have shown different sensitivities and specificities. In present investigation, PCR protocol and primer designated based on IS711 and a fragment of chromosomal DNA all were optimized with standard genome and clinical samples. Numerous tissue samples [liver, kidney, lymph node, and uterus] were prepared and were cultured by the bacteriological standard methods along with the serology positive human samples. PCR protocol was optimized and the primer's sensitivity and the specificity were checked using pure genome of B. abortus. All samples were tested by the standard bacteriological methods. The samples were then subject to PCR amplification and the PCR product was confirmed using the RFLP technique. The culture results indicated a poor sensitivity as it was previously reported. The PCR product 157 bp was observed on the agarose gel indicating that significant number of clinical samples [human brucellosis cases] were positive by PCR but not by the culture method. Although B. abortus DNA was detected in all the culture positive veterinary specimens, some cross-reactions with close related bacteria were observed that might influence the interpretation of the results. The sensitivity of the present PCR protocol was significantly higher when alk B and IS711 based primers were used in compare to each of the alkB and IS711 based primers alone. More research will be needed to improve the specificity and sensitivity of the PCR protocol before recommending for routine laboratory works

Isolation of Brucella from blood culture of hospitalized brucellosis patients

Brucellosis is a zoonotic disease that is endemic in Iran. Appropriate and rapid diagnosis has a vital role in public health improvement. Low isolation rate of the organism has reported frequently in various reports. The present study was conducted to determine the isolation rate of organism in culture from collected specimens of hospitalized patients who were not under antibiotic therapy. Meanwhile, comparing the direct inoculation to biphasic media with lysis method was also determined. Twenty-five hospitalized brucellosis patients diagnosed on the basis of clinical manifestations and positive serologic tests were included. Blood samples were provided and cultured either as direct inoculation into biphasic media or lysis method by washing with distilled water before culture on solid media. Brucella was isolated in 4 samples [16%]. Further studies revealed all these four cases to be B. melitensis. Washing method did not differ in isolation rate with direct inoculation; however, Brucella was isolated in a shorter period in washing method. Higher isolation rate when compared with prior studies indicates an appropriate sampling time and technique, rapid inoculation to the media, and the lack of antibiotic therapy before sampling. Washing method has the preference of shorter isolation time to direct inoculation; however, it is faced with a higher risk of contamination