First report of isolation and identification of Brevundimonase ( Pseudomonas ) diminuta from collected nasopharyngeal specimens in suspected patients to pertussis

Brevundimonase diminuta as an opportunistic environmental bacterium, due to the increase in isolation rate from clinical specimens and its antibiotic resistance is considered as a new threat to human health care. After performing conventional phenotypic methods and molecular approaches, antibiotics resistance pattern on the identified Brevundimonase isolates was performed as minimum bactericidal concentration by disk diffusion method. Out of 1084 nasopharyngeal specimens, 2/1084 (%0.18) Brevundimonase diminuta, and 1/1084 (%0.09) Brevundimonase subvibrioides were isolated. Evaluation of the resistance pattern from isolated Brevundimonase strains indicated that: levofloxacin, ciprofloxacin, ampicillin, amikacin (Excluding B. subvibrioides), chloroamphenicol and ceftazidime had poor susceptibility results. Also azythromycin, gentamycin, sulfamethoxazole/trimethoprim (Excluding B. subvibrioides), ciprofloxacin, kanamycin showed good susceptibility results against Brevundimonase isolated strains. In conclusion, Although, Brevundimonase diminuta rarely has been isolated from other clinical specimens, but, due to reports of resistance to some of antimicrobial agents in this genus all laboratories should be equipped for the identification and evaluation of susceptibility patterns of this species. It is first the report of isolated Brevundimonase diminuta from nasopharyngeal specimen from suspected patients to pertussis in Iran.

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Brevundimonase diminuta previously described as an opportunistic environmental bacterium and assigned in the genus of Pseudomonas (lefson and Hugh, 1954;Segers et al., 1994;Gilligan et al., 2003;Han and Andrade, 2005).Recently, due to the presence of unique 16srRNA gene, Brevundimonase spp have been reclassified (Lee et al., 2011).Han and Andrade (2005) reported that, B. diminuta has been isolated and identified from water and clinical specimens.Pathogenecity of B. diminuta has not been determined yet and due to small size of the bacterium routinely is used as an indicator of the efficiency of water filters (Holt et al., 1994).Segers et al. (1994) reported that, B. diminuta strains could be isolated from human specimens such as human urine (Goteborg, Sweden), blood plasma (Slagelse, Denmark), human ear (Boras, Sweden), hemoculture (Hospital, Brussel, Belgium), pleura liquid (Hospital, Brussel, Belgium) and blood from patients with endocardities (Culture Collection of the University of Goteborg, Department of Clinical Bacteriology, University of Goteborg, Goteborg, Swede (CCUG 1797).Brevundimonase vesicularis from environmental (United States) and human specimens including pleura liquid (Hospital, Brussel, Belgium), Vaginal swab (Hospital, Brussels, Belgium), hemoculture (Hospital, Brussel, Belgium) and urinary bladder (Hirudu sp) has been isolated.This study generally designed for identification and confirmation of the presence of Brevundimonase diminuta from nasopharyngeal samples in suspected patients to pertussis based on the conventional biochemical tests and molecular approaches and further analysis of antibiotic resistance which housed them.

Routine procedure for isolation and identification of Bordetella pertussis in Pasteur Institute of Iran
Nasopharyngeal specimens from suspected patients were collected by sterile Dacron swabs.Subsequently, samples immediately emulsified in Riegan Lowe Medium (Difco) as transport medium.All samples were suspended in phosphate buffer solution (PBS).DNA extracting process directly performed (in nasopharyngeal samples from suspected patients to pertussis) by high pure DNA extraction kit (Roche, Diagnostics GmbH, Mannheim, Germany).Taq man real time PCR assay optimized for rapid detection of B. pertussis was used.For precise determination of B. pertussis in clinical samples, insertion sequence 481(IS 481) and sequence BP 283 (Codon gene between tyolase region) were targeted by using of specific primers and probes (Reischl et al., 2001, Probert et al., 2008).Positive strains for pertussis were affirmed by conventional phenotypic methods including grow in Bordet gengou agar with and without 40µg/ml cephalexin.As well, colony morphology, biochemical activity tests consisting of oxidase and catalase activity, gram stain, utilizing scheme for amino acids and carbohydrate fermentation and other substrate by API 20 E (BioMerieux, Inc., Hazelwood, MO) were performed.All isolated B. pertussis strains reconfirmed by serological test using antiserum kit (BioMerieux, Inc., Hazelwood, MO).

Identification of Brevundimonase diminuta
Conventional biochemical tests were done according to the Bergeys Manual of Determinative Bacteriology recipes and further isolates were confirmed using API 20 E technology (BioMerieux, Inc., Hazelwood, MO) (Holt et al., 1994).Clinical characteristics of patients and Biochemical characterization of B. diminuta and B. subvibrioides isolated strains are listed in Tables 1 and 2.

Molecular techniques
Genomic DNA was extracted from each isolates using a comer-cial kit.Briefly, pure identified colonies were emulsified in 500µl PBS.Cells harvested by centrifugation at 8000 × g for 5 min.Subsequently, pellets consisting bacterial cells were used for DNA extraction process.Total DNA extraction was completed using a high pure DNA extraction kit (Roche, Diagnostics GmbH Mannheim, Germany).Purified DNA was dissolved in 50µl distilled water.Quantity of extracted DNA was measured by NANO-DROP ®ND-1000 instrument (spectrophotometer 1000, USA) and adjusted to 500 ng/µL -1 .

Amplification of rrs gene and sequencing process
Purified DNA was evaluated by polymerase chain reaction (PCR) utilizing universal rrs gene (16srRNA).Sequences of forward and reverse primer for 1475 bp PCR product were 5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-GGTTACCTTGTTACGACTT-3′ (Tsai et al., 2004).Amplification protocol originally described by previously published study (Tsai et al., 2004).PCR was performed using 2 µl genomic DNA as template in total volume 25 µl containing; 12 µl master amplicon (Biolab, New England, UK), Primer Forward 1µm, Primer Reverse 1 µm and 9 µl mineral oil.PCR process was optimized with purified DNA of E.coli ATCC 25922 as positive genotype.Amplification was performed on Gene Amp PCR system (Applied Biosystem, USA) using a program as follow: an initial cycle of denaturation 95°C for 5 min followed by 35 cycles of 95°C for 1 min, 50°C for 1 min and 72°C 1 min, with final extension step of 72°C for 10 min.Amplified products (1475 bp) were visualized on 1.5% agarose gel (Max pure agarose, Spain) stained by etidium bromide using Gel logic 212pro.Figure 1 showed amplified products of rrs gene.

Sequencing
PCR amplification from DNA preparations of rrs yielded corresponding products 1475 bp (Figure 2) were sequenced following the purification of PCR products using of purifying kit (QIAquick PCR Purification Kit, Qiagen).Sequencing process was accomplished by the dye terminal method in an ABI 377 sequencer and sequencing results interpreted through a query to the Gen Bank local alignment search tool (BLAST) (blast.ncbi.nlm.nih.gov/Blast.cgi?).
After PCR process and sequencing of generated amplicon, using online software and comparison with a data base identification of isolated B. diminuta and B. subvibrioides strains with match point ≥95, were confirmed.Subsequent comparison with the results obtained by biochemical and molecular methods two Brevundimonase diminuta isolates, the similar results were obtained which indicates that the results of both biochemical and molecular tests were correct.Unrooted phylogenic tree are shown in Figure 3.After performed disk diffusion method, azythromycin, gentamicin and sulfamethoxazole/trimethoprim have shown an appropriate inhibitory effect against B. diminuta isolated strains.On the contrary, antibacterial agents such as levofloxacin, ciprofloxacin, ceftazidime, ampicillin and amikacin due to low activity against B. diminuta strains have not detected for suitable drug treatment process.
Susceptibility pattern of the mentioned antibiotics against B. subvibrioides strain indicated that chloroamphenicol, azythromycin, ampicillin, sulfamethoxazole/ trimethoprim, levofloxacin, ceftazidime and ciprofloxacin were not sufficient inhibitory effect against isolated strain.Also B. subvibrioides isolated strain has good susceptibility results against some of antibiotics such as gentamycin, kanamycin, cefepime and ceftriaxone.Detailed data are listed in Table 3.

DISCUSSION
Since 1994, according to the presence of unique 16srRNA gene Brevundimonase spp have been reclassified (Han and Andrade, 2005).Brevundimonase diminuta and B. versicularis as environmental opportunistic bacteria commonly were isolated from immunocompromised patients (Holt et al., 1994;Han and Andrade, 2005).
Previously, B. diminuta was isolated from human specimen such as sputum in cystic fibrosis patients, urine, pleura effusion and blood (Chi et al., 2004;Han and Andrade, 2005;Menuet et al., 2008).According to the previously published study by Shayegani (1978), API 20 E technology was evaluated and presented as an efficient method for the identification of non-fermentative gram negative bacteria (Shayegani et al., 1978).In this study, using API 20 E technology two strains of B. diminuta and one strain of B. subvibrioides were identified according to the data included in the API 20 E data base.Although pathogenecity relationship of B. diminuta to pertussis not confirmed, and this bacterium frequently be isolated from mouth cavity but observation of mentioned bacterium from nasopharyngeal specimen in suspected patients to pertussis is considerable (Paster et al., 2002;Davis et al., 1997).
Recently, molecular techniques such as PCR and DNA sequencing have been utilized for fast and accurate detection of a wide range of microorganisms (Han and Andrade, 2005;Lee et al., 2011).In this study, generated amplicon by universal rrs primers was sequenced using sequencer instrument (ABI 377 sequencer) and following the sequence analysis through a query to the GenBank Basic Local Alignment Search Tool™ (BLAST), based on the www.NCBI.org,our phenotypic methods were affirmed and isolation and identification of B. diminuta were justifiable.
Because of multiple antimicrobial resistance characteristics in many isolates of Brevundimonase spp, selection of efficient antibiotics for treatment of related infections caused by these bacteria is difficult (Han and Andrade, 2005).Although, Brevundimonase species previously classified in the genus Pseudomonas, but routine antibacterial agents for the treatment of Pseudomonas infections (fluoroquinolones and ampicillin) have no detrimen-tal effects on the this genera of bacteria (Davis et al., 1997;Lee et al., 2011).
Although mutations in GyrB and ParC subunit, have related to quinolones resistance, but in P.aeruginosa efflux pumps have been shown to be important for the quinolones resistance (Han and Andrade, 2005).In current study, azythromycin (exception one B. subvibrioides), gentamycin exhibited high affect in antibiogram process and could be considered as a treatment option.Detection of responsible genes or other mechanisms in our B. diminuta isolates need to more study (Almuzara et al., 2012).
In the present study, levofloxacin, ciprofloxacin, ampicillin, amikacin, chloroamphenicol and ceftazidime due to poor results at the antibiogram were described as an inappropriate treatment for related infections caused by Brevundimonase strains.Moreover, azythromycin (excluding B. subvibrioides), sulfamethoxazole/trimethoprim (excluding B. subvibrioides) and gentamicin, regarding good results in antibiogram process, were introduced as suitable candidates for the treatment of associated infec-tions with isolated strains.

Conclusion
Due to the isolation rate of B. diminuta as the infectious agent, and attribution of human infections to this bacterium, bacterial identification and detection of antimicrobial susceptibility testing should be considered in the laboratories.Although most species in this genus are less pathogenic, but isolated strains from clinical specimens can be the alarm for the health system.

Table 1 .
Clinical characteristics of patients infected with B. diminuta strains.

Table 2 .
Biochemical characterization of B. diminuta and B. subvibrioides isolated strains from patients.

Table 3 .
Evaluation of antibiotic resistance pattern on B. diminuta and B. subvibrioides isolated strains.