Iranian Journal of Medical Microbiology

Pseudomonas is a Gram-negative, aerobic, non-spore forming, oxidase positive, catalase positive bacteria belonging to the family Pseudomonadaceae (1). Pseudomonas aeruginosa is an important opportunistic pathogen among cystic fibrosis and burnt patients and can be colonized in hospital sewage systems and enter the environment (2). The pathogenesis of P. aeruginosa is due to the production of several cellular and extracellular virulence factors. The cellular factors include flagellum, pyocyanin, fimbria and lipopolysaccharide, and pili, among which bacterial type IV pilus (Tfp) is one of the most important and necessary factors for multiple cellular functions, including biofilm formation, surface motility, host-cell binding, adhesion, cell signaling, DNA uptake by natural transformation (3). Two models of O-glycosylation systems have been found in P. aeruginosa. In the first model, glycosylation happens through TfpO (Pil O). The Pil O is an Oligo-saccharyl-transferase that transfers an antigen-O unit to the serine amino acid placed at the C-terminal of the group I pilins. The second pilin glycosylation system in P. aeruginosa was found in strains IV, Pa5196 and Pa7, which lacked TfpO (6). P. aeruginosa has been separated into five groups according to the presence or absence of varying downstream accessory genes adjacent the pilin gene, pilA. The PilA pilins have different lengths, amino acid sequences and posttranslational modifications.
The first recognized groups are groups I and II.  Strains PA01, PAK, and PA103 express group II pilins. Spangenberg and colleagues showed that Type III polymorphism has 173 amino acids, which is taller than the prior pilins, and an open-ended read-only template with undefined function, now called TfpY, which lies under the PilA gene. More isolates with group III alleles, as well as isolates with 2 new pilin alleles (groups III and IV), were found in more than 300 strains of P. aeruginosa. The strains of the group V pilin express the auxiliary pilin and protein (Tfp Z) as group III. The group IV pilin consists of two isolates (Pa97, Pa5196) that code the TfpW and TfpX auxiliary proteins at the downstream of the PilA4 (9).
Considering that type IV pilin is one of the most important structures of P. aeruginosa, its presence and variation may contribute to the pathogenesis of the isolates possessing it. This pilin may also act as a new target for antibiotic therapy which has been recently very challenging worldwide (10-12). Hence we aimed to study the presence of type IV pilin subtypes among P. aeruginosa isolates from three different sources; hospital waste water, burnt and cystic fibrosis patients.
 
Material and Methods
Totally, 90 isolates of P. aeruginosa strains; 35 from environmental samples, 30 from burnt patients and 25 from cystic fibrosis children referred to Mofid Hospital of Tehran, Iran, were isolated from November 2015 to December 2016. Sampling was done under sterile conditions and the samples were immediately transferred to the laboratory of Shahid Beheshti University of Medical Sciences at the Faculty of Medicine. Sputum specimens of patients with cystic fibrosis were inoculated into McCanky agar and blood agar medium and incubated at 37°C for 24 hours. Finally, the isolates were identified as P. aeruginosa using microbial and biochemical methods such as oxidase and catalase, TSI and OF, and growth at temperatures of 4 and 41°C, as well :as char:acteristics such a pigment and odor production . The standard strain of P. aeruginosa PAO1 was used as control. P. aeruginosa samples isolated from burnt and cystic fibrosis patients and the environment isolates were stored in TSB containing 30% glycerol at -70°C until further investigation.
DNA extraction and polymerase chain reaction
For DNA extraction of the isolates, the boiling technique was applied. For this purpose, 200 ml of sterilized water was added into the micro-tube and 3-4 fresh colonies (24 hour growth) were dissolved in water and the suspensions were kept at 100°C for 10 minutes, after which centrifugation at 10,000 rpm was done for 5 minutes, and the supernatant containing DNA was transferred to the new microtubule for PCR. In this study, the presence of the genes encoding two subtypes of type IV pilin including TfpO and TfpZ genes were investigated by PCR method using specific primers listed in Table 1. The PCR reaction was done in a volume of 25 μL and performed as (Amplicon, Iran) 12.5 μL Master Mix 1x buffer, 0.5 μL of each primer with concentration of 10 mM, 300 ng/L DNA, and 4 μL deionized distilled water. After preparation of PCR reaction reagents, the microtubules were placed in a Thermocycler machine (Bio Intellectica, Canada). The program for each gene amplification is summarized in Table 2.

Reference	Product length (bp)	Oligonucleotide sequence ('3-'5)	Primer
in this Study	363	Forward 5′-  CGTAGGGCTTGCACTGCTAT-3′   Reverse 5′- AAGAACCCTCCCAACACGAC-3′	TfpO
in this Study	464	Forward 5′-  AGATTAGGGCGTTCGCTGTT-3′ Reverse 5′- CCGCAGGACATCCATTAGCA-3′	TfpZ

	TfpZ			TfpO			Gene
time		Temperature	time		Temperature	Stages
15 m		95	15 m		95	Initial denaturation
30 s		95	30 s		95	denaturation
1 m		72	30 s		72	Connection
45 s		60	30 s		58	Elongation
7 m		72	7 m		72	Final elongation
35		35	35		35	Cycle

After the PCR reaction, electrophoresis of the PCR products was performed on a 1.5% agarose gel containing safe staining dye and recorded by Gel Documentation (Figures 1 and 2). PCR products were sequenced bi-directionally (the Bioneer Co., Korea) and results were analyzed using the NCBI database. Statistical analysis was performed using SPSS 23 (SPSS Inc. Chicago, Illinois, USA).
 
Results
Out of the 90 P. aerosinosa isolates, 72 (80%) possessed the TfpO gene. The results of electrophoresis are shown in Figures 1 and 2. Based on the differentiation of the studied groups, the frequency of the TfpO gene in each group was as follows;
Among the 35 environmental samples, 31 strains (88.57%) had TfpO gene and 4 samples (11.42%) were recognized as negative for this gene. Out of the 25 samples from cystic fibrosis, 17 strains (68%) had TfpO gene and 8 samples (32%) did not possess this gene. Among the 30 isolates from burnt patients, 24 (80%) had TfpO gene, whilst 6 (20%) isolates did not have this gene (Figure 1).
The TfpZ gene was positive in 2 (2.2%) out of 90 isolates, regardless of the source of isolates and 88 (97.8%) isolates lacked this gene. The TfpZ gene was negative in isolates from environmental samples as well as patients with cystic fibrosis. Two (6.66%) positive strain for TfpZ belonged to isolates from burnt patients (P>0.05) (Figure 2).

 

Figure 1. Electrophoresis results of the TfpO gene among P. aeruginosa isolates (363bp). Lane 1, negative control; lane 2, positive control; M: size marker of 50 bp, lanes 3-16 PCR products of the TfpO.


 

Figure 2. Electrophoresis results of the TfpZ gene of our P. aeruginosa isolates (364bp). Lane 1, positive control; lane 2, negative control; M: size marker of 50 bp, lanes 3-16 PCR products of TfpZ gene.



Discussion
High adaptability, colonization and survival of P. aeruginosa is due to its massive sets of chrosomal and non-chromosome DNA fragments and genome-modifying factors such as bacteriophages, integrons, transposons, and other elements (13). P. aeruginosa is colonized on different surfaces and the type IV pilin is one of the important factors for colonization. It has now been accepted that P. aeruginosa is able to express three distinct types of pilin subtypes IVa, IVb and Tad. (14). The IVa type is the most common pilin type in bacteria that grow in laboratories.
 To our knowledge, no study has so far been conducted on the comparison of the frequency of type IV pilin in strains and populations of P. aeruginosa, and only limited studies have been done on genomic diversity and frequency of IVa pilin subtypes (15-17).
Therefore, the present study was conducted to investigate the presence of IV pilin sub types by PCR among the P. aeruginosa community isolated from burnt and cystic fibrosis patients, as well as in the sewage system of the burn centers. According to the results, a high percentage of P. aeruginosa strains isolated from different sources in this study were group I of type IV (TfpO). Subsequently, 68% of the samples isolated from patients with cystic fibrosis had this type of type IV. This finding suggests that strains from environment and burnt patients are more closely related to strains isolated from cystic fibrosis patients. In 2004, Kus et al. found that the prevalence of TfpO in P. aeruginosa strains was more than other groups of type IV pilin (7). According to Kus et al. in 2008, the prevalence of TfpO among cystic fibrosis patients, other clinical samples, and the environmental samples were 69.7%, 30.5%, and 58.3% respectively; which compared to our results, showed a higher prevalence among clinical samples (18). Also Deligiann et al. in 2010 observed that the prevalence of TfpO in cystic fibrosis isolated P. aeroginosa was more than other groups and that it was associated with bacterial colonization (19). Based on the study of Pirnay et al. in 2009, the frequency of TfpO in cystic fibrosis isolated P. aeroginosa was 55.8%, which is somewhat above the average (48.2%) (20), but contrary to our study an association between TfpO and cystic fibrosis was not observed. In this study, group V pilin type IV (TfpZ) was only observed in 6.66% of the samples from burnt patients, and the other isolates did not possess this group of type IV pilin.
 
Conclusion
Overall, our study showed that the prevalence of group I type IV pilin (TfpO) was high in the population of Pseudomonas aeruginosa strains isolated from burnt and cystic firbosis patients, as well as environmental samples. Since type IV pilin is involved in a variety of bacterial processes, especially in post-translational modification (pili glycosylation) and biofilm formation, their relatively high prevalence indicates a high level of virulence among the Iranian strains of P. aeruginosa studied. Based on the studies and the results obtained, we recommend that further studies with an increased number of samples, a more diverse society, involving more genotypic and phenotypic characteristics of type IV pilin subtypes.
 
Acknowledgements
Great thanks to the Department of Microbiology of Shahid Beheshti University of Medical Sciences, for the support and providing the facilities for this work.
 
Conflict of Interest
The authors reported no conflict of interest.