Isolation and identification of Pandoraea spp. From bronchoalveolar lavage of cystic fibrosis patients in Iran

From November 2017 to August 2018, following the sedation of CF patients in Namazi hospital (Shiraz, Iran), 31 samples of bronchoalveolar lavage and sputum were collected. Then the samples were transferred to the bacteriology laboratory, faculty of veterinary medicine of Shiraz University, in cold condition. The study was approved by the Ethics Committee of Shiraz University of Medical Sciences.

To obtain strains and to compare culture and PCR results, cultures were performed initially as enrichment cultures. Upon arrival, bronchoalveolar lavage and sputum were cultured in BHI broth and incubated aerobically at 37 °C for 24 h. Initial enriched cultures were transferred to the enrichment culture plates by serial streaking the cultures on sheep blood agar and incubated aerobically at 37 °C for 24–48 h.

Then, a loop full of the identified colonies were streaked on a new blood agar and incubated aerobically at 37 °C for 24 h. Next, from culture positive plates, typical colonies were subjected to gram’s staining to study staining reactions and cellular morphology under light microscope. Mixed and gram-negative bacteria were further sub cultured with due care, on both blood agar and MacConkey agar plates for final identification. The growth of typical colonies on both blood agar and MacConkey agar was characterized using blood agar for the presence and type of haemolysis, and the general appearance of colonies (morphology, color, shape, size and consistency) and the MacConkey agar for the ability to ferment lactose. Pure cultures of single colony type were further analyzed by catalase and oxidase tests. Confirmation of bacteria to species level was aided by using the biochemical tests including, metabolism of sugars such as glucose, fructose, lactose and tests for metabolic end products such as nitrate reduction, unease and citrate activity, growth on cetrimide agarat 42 °C and O/F medium following standard procedures. For final confirmation, isolates evaluated by two PCR reactions. A first PCR assay was designed to identify all Pandoraea spp. Subsequent PCR assays were designed to identify individual Pandoraea species.

A few colonies from the phenotypically characterized pure cultures of Pandoraea from 24 to 48 h growth on blood agar plates were transferred into 1.5 ml Eppendorf tubes. Total DNA was prepared using the Gram negative DNA extraction kit (Cinagen, Iran). The protocol for Gram negative bacteria as described in the kit was followed for extractions. The extracted DNA were determined to be of good quality and DNA concentration was measured using Nanodrop (10,000 V 3.52). DNA concentrations were adjusted to 15 ng μL^− 1 before PCR amplification. Finally, extracted DNA stored at −20 °C for further use.

The oligonucleotide primers used in this study were synthesized by Cinagen Company (Iran). The forward and the reverse primers sequences are as PanF (5´GGGCTYAACCTGGGAACTGCATTC3´), and PanR (5´CGRYTTGGCRRCCCTCTGTACCG3´) [26]. Also PCR evaluations have been done with two universal primers for amplification of 16S rRNA gene. The reaction mixture solution for PCR was prepared using 2 μl of 10x PCR buffer, 1.5 μl (1.5 mM) MgCl₂, 1.2 μl (200 mM) dNTPs, 1.2 μl (100 nmol) each primer of Pandoraea, 0.2 U of Taq DNA polymerase, 14.7 μl H₂O, and 3 μl of DNA template to have a final volume of 25 μl. Using 0.2 ml thin wall microtubes. After initial denaturation (at 94 °C for 5 min), amplification conditions were, denaturation at 94 °C for 1 min, annealing at 60 °C for 45 s, and extension at 72 °C for 1 min. This was repeated for 30 cycles in a Block assembly 96G thermocycler with a hot top assembly (Analytic Jena, Germany), with a final extension of 72 °C for 10 min and the PCR products remained in the thermal cycler at 4 °C until they were collected. All DNA isolation procedures were carried out in a Class II biological safety cabinet in a room geographically separate from that used to set up reaction mixes and also from the ‘post PCR’ room, in order to prevent crossover contamination by extraneous nucleic acids and in accordance with good molecular diagnostic practice. All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA, and from the amplification and post-PCR room, in order to minimize contamination. A negative control consisting of all component of reaction mixture except the DNA template was included in all PCR. Positive controls were included in the PCR from colonies that were confirmed according to NCBI gene bank sequencing. In order to evaluate the PCR results electrophoresis in agarose gel was carried out. PCR amplicons were assessed by loading 8 μl of the PCR product plus 2 μl of loading buffer into separate wells of a 1% (w/v) agarose gel (Agarose I; Cinnagen, Iran) containing safe stain. A molecular weight marker (50 bp; Cinnagen, Iran) was loaded into the first well to determine the size of the amplified fragments. The gel was immersed in TBE buffer and subjected to a voltage difference of 100 V that led to separation of the fragments. Visualization was undertaken using an ultraviolet transilluminator (BTS-20, Japan), and the resulting image was captured by a computer software program (Alpha Ease; Alpha Innotech).

For the final confirmation of isolates of Pandoraea, PCR products of Pan primers for some samples according to the Macrogene Company’s instructions were prepared at 50 μl volume and were transferred for sequencing. In addition, 16S rRNA gene sequencing for species identification was applied for some of the Pandoraea isolates. The sequences were compared with previously published 16SrRNA (GenBank accession no.NR-152002), Pandoraea gene sequences.

Antimicrobial susceptibility testing for Pandoraea spp. isolates was performed by disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) [27]. Disk diffusion method was performed on Muller-Hinton agar (Merck, Germany), using an inoculum of 10⁵ CFU/ml. Antibiotic disks (Padtan teb, Iran) including amikacin, ciprofloxacin, trimethoprim- sulfumethoxazole, gentamicin, piperacillin tetracycline and imipenem. These antibiotic disks were then placed on agar plates and incubated at 37 °C for 24 h. Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used as quality controls strain in each susceptibility determination. The diameter of inhibition zone was measured in millimeters and isolates were scored as susceptible or resistant by comparing results with values recommended on standard charts.

In order to evaluate the ability to create biofilms in Pandoraea spp. isolates used the method of Merritt et al. and Wakimoto model [28, 29]. Briefly, 100 μl of each diluted culture was relocate into individual wells of microtiter dish and grown at 37 °C overnight. All wells were stained with 125 μl of 0.2% (w/v) crystal violet solution for 10 min at room temperature. The excess stain was washed twice with distilled water and the plate was allowed to air dried. 200 μl of 95% ethanol was added to solubilize the stained biofilm and incubated for 10–15 min at room temperature. 125 μl of each well was transferred into a new microtiter dish. Finally, the optical density (OD) of each sample was measured at 570 nm by a spectrophotometer. The isolates with the optical density higher than 0.2 were considered as biofilm former isolates.

In this study susceptibility to antibiotics was detected using 7 usual antibiotics prescribed for treatment of chronic lung infection including amikacin, ciprofloxacin, trimethoprim-sulfumethoxazole, gentamicin, piperacillin, tetracyline and imipenem. Antibiotic susceptibility test were indicated all isolates were resistant to gentamicin, amikacin and imipenem and susceptible to ciprofloxacin, trimethoprim-sulfumethoxazole, piperacillin and tetracycline (Fig. 3).