Distribution and Characteristics of Bacteria Isolated from Cystic Fibrosis Patients with Pulmonary Exacerbation

Background Cystic fibrosis (CF) is an inherited recessive disorder characterized by recurrent and persistent pulmonary infections, resulting in lung function deterioration and early mortality. Methods A cross-sectional study was conducted on the bacterial profile and antibiotic resistance pattern of 103 respiratory specimens from CF patients with signs of pulmonary exacerbation. Antibiotic susceptibility testing and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa isolates were performed by the Kirby–Bauer disc diffusion method and microtiter plate assay, respectively. Molecular typing of S. aureus and P. aeruginosa isolates was carried out by spa typing and repetitive extragenic palindromic element PCR. Results In a total of 129 isolates, the most prevalent organisms were S. aureus (55.3%) and P. aeruginosa (41.7%). Other less prevalent bacterial isolates include coagulase-negative staphylococci, Escherichia coli, klebsiella spp., Enterobacter spp., and Achromobacter xylosoxidans. The highest rate of resistance for S. aureus was observed to azithromycin and erythromycin (80%), ciprofloxacin (52.3%), clindamycin (44.6%) and tetracycline (43%). Twenty percent of S. aureus isolates were methicillin-resistant S. aureus (MRSA) and 47.6% were MDR S. aureus. For P. aeruginosa isolates the highest resistance was to cefepime (38.3%) and levofloxacin (33.3%) and 20% showed MDR phenotype. Conclusion Our study demonstrated a significant decline in the prevalence of P. aeruginosa infections in comparison to previous studies. We found S. aureus to be more prevalent in younger patients, whereas mucoid P. aeruginosa showed a shift in prevalence toward older ages. Molecular typing methods showed great diversity between isolates.


Introduction
Cystic fbrosis (CF) is an inherited genetic disorder which afects 70,000 to 100,000 people around the world [1,2]. It is caused by a mutation in the transmembrane conductance regulator (CFTR) gene [3]. A defective CFTR results in thickened and viscous mucus secretions in the respiratory tract that cannot be easily cleared by the mucociliary clearance system [1,4]. Te accumulated mucus creates an appropriate niche for bacterial colonization and the development of persistent pulmonary bacterial infections [2,5]. Tis malicious cycle of chronic infection, infammation, and tissue destruction results in a progressive decline in lung function, which is the primary cause of morbidity and mortality in patients with CF [6]. In addition to chronic lung infections, CF patients also experience recurrent episodes of acute decline in pulmonary function called "pulmonary exacerbation" (PE). Te general presentations of a common PE are increased cough, change in sputum, shortness of breath, fever, decreased appetite, weight loss, and decrease in spirometric parameters [7]. PEs are often associated with the acquisition of new organisms, a change in the bacterial density, or the expansion of pre-existing strains [8]. Within the very frst months of their life, CF patients develop airway infections. Staphylococcus aureus is the most dominant bacteria during childhood, but gradually, Gram-negative bacteria, especially Pseudomonas aeruginosa, become more dominant [9]. Other prevalent bacteria recovered by conventional culture techniques that are supposed to play a role in pulmonary infections in CF patients are Burkholderia cepacia complex species, Haemophilus infuenzae, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans [9,10]. With the increasing survival of CF patients and more advanced methods for nontuberculous mycobacteria identifcation, the prevalence of NTM isolation from CF sputum samples is reported more frequently. Mycobacterium avium complex and Mycobacterium abscessus are the most common isolates [11,12].
Epidemiological studies by molecular typing methods are of great importance to determining the genetic diversity of the pathogens, determining the prevalent molecular types of the isolated bacteria, tracking the spread of infections, and assessing the potential risk of person-toperson transmission of infection. Staphylococcal protein A typing and repetitive extragenic palindromic element PCR typing methods are among the well-established typing techniques used in the study of molecular evolution and outbreak investigations of S. aureus and P. aeruginosa, respectively [13][14][15][16].
Considering the vital impact of PEs on patient health, better understanding the microbiological signals associated with PEs will help to fnd new therapeutic strategies or predictive biomarkers to reduce the frequency and/or severity of PEs [17]. Given the importance of microbiological surveillance of CF patients, this study aims to investigate the frequency, antimicrobial susceptibility pattern, and molecular typing of bacterial pathogens isolated from CF patients during the PE phase who were admitted to Te Cystic Fibrosis Center at Children's Medical Center University Hospital, Tehran, Iran, during a period of one year.

Patients and Medical Records. Between March 2018 and
February 2019, all confrmed CF patients (sweat test and clinical presentations) with signs of pulmonary exacerbation according to Goss and Burns criteria [18], referred to the cystic fbrosis center at Children's medical center University Hospital, Tehran, Iran, were included in the study. Patients who had multiple referrals over the sampling period were also included. Spontaneous sputum samples were obtained from patients involved during the study period. Te patient was asked to rinse her/his mouth, cough, and expectorate the sputum into a sterile container. Sputum samples with high saliva contamination were excluded. When it was not possible to obtain spontaneous sputum samples, an oropharyngeal (OP) swab was used as a sample. All participants included in the study and/or parents (in the case of children) were provided with oral consent before enrollment in the study. All sputum specimens were produced voluntarily. All the patients' information was kept confdential.

Bacterial Identifcation.
Homogenized sputum or throat swab samples were inoculated on the following culture media for primary screening: general media (blood agar and chocolate agar) and selective/diferential media (MacConkey agar, mannitol salt agar, Burkholderia cepacia selective agar (BCSA), and stenotrophomonas maltophilia agar). Chocolate agar plates were incubated in 5% CO 2 atmosphere. Plates were incubated at 37°C for 48 hours [19]. BCSA plates were incubated for a further 5 days at room temperature. Suspected colonies were isolated and subcultured. Te isolates were identifed to the species level using standard biochemical methods [20]. For the detection of mycobacteria, sputum samples were processed by the modifed Petrof's method [21] and inoculated on LJ (Löwenstein-Jensen) medium. All slopes were observed for any signs of growth daily for the frst week and then at weekly intervals for 8 weeks. Te absence of growth at the end of 8 weeks was regarded as a negative culture.
S. aureus and P. aeruginosa as the two most prevalent pathogens of CF patient's respiratory tract, were investigated in more details to illustrate a more precise picture of their phenotypic and molecular features.

Antimicrobial Susceptibility
Testing. Antimicrobial susceptibility tests were performed by Kirby-Bauer's disc difusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2019) [22]. To determine the susceptibility pattern of S. aureus isolates the following antibiotics were tested: cefoxitin (FOX: 30  All the antibiotic discs were purchased from MAST Company (Mast Group Ltd, UK). E. coli ATCC 25922 was used as the control strain [23]. Detection of methicillinresistant Staphylococcus aureus (MRSA) isolates was performed by 30 μg cefoxitin antibiotic discs (Mast Group Ltd, UK) according to recommendations of CLSI 2019 [22]. Te molecular confrmation of MRSA was accomplished by amplifcation of the mecA gene. Resistance to at least one agent in three or more antimicrobial classes was considered as multidrug resistance (MDR) [24].

Bioflm Formation Assay for S.aureus and P. aeruginosa
Isolates. Bioflm formation was evaluated according to the method of O'Toole and Kolter [25] with some modifcations. Briefy, 100 μL of each bacterial suspension, adjusted to McFarland standard 0.5 was inoculated into each well of fatbottomed 96-well polystyrene microtiter plate (SPL Plastic Labware, Korea). After overnight incubation at 37°C the medium was removed and washed twice with 0.9% NaCl. Te formed bioflms were fxed by methanol and stained with 1% (w/v) crystal violet solution; then 33% glacial acetic acid (Merck, Germany) was added to the wells. After 10 minutes the absorbance of solubilized crystal violet was measured at 550 nm. Te experiments were performed in triplicate. Uninoculated medium was considered a negative control sample. According to the results of microtiter plate test, bioflm producer isolates were characterized as follows based on the optical density: nonbioflm producers (OD test < ODc), weak bioflm producers (ODc < OD < 2 × ODc), moderate bioflm producers (2 × ODc < OD < 4 × ODc), and strong bioflm producers (4 × ODc < OD) [26].
2.6. Spa Typing for S. aureus Isolates. Te Spa typing was performed according to Harmsen et al. [27]. Briefy, the polymorphic X region of the spa gene was amplifed by specifc primers. Te PCR reaction was performed in 12.5 μL fnal volumes containing 5 μL of PCR Master Mix (Bio-FACT, South Korea), 4.5 μL of distilled water, 0.25 μL of each primer, and 2.5 μL of extracted DNA. Te PCR amplifcation conditions were as follows: the initial denaturation at 94°C for fve minutes, and the next 35 cycles consisting of a denaturation step at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, and a fnal extension step at 72°C for ten minutes. Te spa gene PCR products were sequenced at Pishgam Biotech Company (Tehran, Iran). Isolates were assigned to particular spa types according to the guidelines described by the spa typing website (https://www.spaserver.ridom.de) [23].

Repetitive Extragenic Palindromic Element PCR (Rep-PCR)
Genotyping for P.aeruginosa Isolates. Te molecular typing of P. aeruginosa isolates was performed by rep-PCR as previously described with some modifcations [16]. DNA amplifcation was performed in a fnal volume of 25 μl containing 16 μl of 2X Multi-Star PCR Master Mix (Bio-FACT, South Korea), 1 μl of each primer (rep-F: 5′-ICGICTTATCIGGCCTAC-3′ and rep-R: 5′-IIIICGICGI-CATCIGGC-3′), 5 μl of distilled water, and 2 μl of the template DNA. Te cycling conditions were as follows: initial denaturation for 2 minutes at 95°C, followed by 35 cycles for 1 minute at 95°C, 1 minute at 42°C, 4 minutes at 72°C, and a fnal extension for 16 minutes at 72°C. Te rep-PCR products were loaded on a 1.5% (w/vol) agarose gel and were analyzed by gel electrophoresis at 80 V for 2 h. A 1 kilobase DNA ladder (Termo Fisher scientifc, USA) was used as a molecular size standard. To monitor the reproducibility of the method, a P. aeruginosa ATCC 27853 reference strain was used as a control in each PCR reaction. Te rep-PCR fngerprints of P. aeruginosa isolates were analyzed using GelCompar II software, version 4.0 (Applied Maths, Belgium), on the basis of the number and weight of band diferences. Te relatedness among isolates was deduced as previously described: linked isolates (similarity above 95%) and diferent (similarity less than 95%) [28].

Statistical
Analysis. Data were analyzed in SPSS software (version 23; IBM Corp, USA). Nominal variables have been described with frequencies and percentages. Continuous variables were described as the mean ± SD. In addition, a binary logistic regression analysis was utilized to establish the association between the outcome variable (presence or absence of diferent bacteria) and the explanatory variable (age).
A P value less than 0.05 was considered statistically signifcant.

Study Population.
In this descriptive cross-sectional study, 103 samples from 85 CF patients between 8 months and 30 years of age with pulmonary exacerbation signs were collected. Nine patients had two referrals, and four patients had three referrals over the sampling period that were eligible for inclusion in our study. A summary of basic patient demographics and types of samples included in the study are provided in Table 1. Most cases (28.2%) were in age group of 2 to 5 years and the least cases (5.9%) were in 20 to 30 years age group. Te most common symptoms observed in patients were increased coughing (74.7%) and reduced FEV1 (59.2%).

Prevalence of Microbial Isolates.
A total of 120 bacteria and 9 yeasts were isolated from 103 respiratory samples. Te most prevalent isolated species were S. aureus and P. aeruginosa. Other less prevalent bacterial isolates include coagulase-negative staphylococci, Escherichia coli, klebsiella spp., Enterobacter spp., and Achromobacter xylosoxidans. In 14.5% (15/103) of the samples, no pathogenic bacteria were isolated. Te distribution pattern of each species within diferent age groups is presented in Table 2.
Te prevalence of mono-microbial and polymicrobial infections was 52.4% (54/103) and 33.0% (34/103), respectively. S. aureus and P. aeruginosa coinfections were detected in 22.3% (23/103) of the samples that were mostly from patients in the 6 to 10 age group. Other double, triple, and quadruple coinfection patterns that were detected from patients are presented by age group in Figure 1.  Figure 2(b). A history of antibiotic use at the time of sampling in patients with MDR S. aureus can be found in Table 2 of the supplementary files.   Results of the microtiter plate assay for bioflm production of S. aureus isolates showed that all 65 S. aureus isolates were bioflm producers in which 8 isolates (12.3%) were strong bioflm producers, 32 (49.2%) moderate, and 25 (38.4%) were weak bioflm producers. Te bioflm formation status of resistant strains is illustrated in Figure 2(a). No relation between bioflm formation status and antibiotic resistance was evident.
As presented in Figure 2(c), S. aureus isolates fall into three clusters according to their respective resistance patterns. Isolates from the frst and third clusters demonstrate similarities in the relative frequencies of resistance combinations and types of samples from which they have been isolated. MRSA isolates mostly ft into frst and third cluster and MDR isolates mostly grouped into the frst and to a lesser extent to the third cluster. Te second cluster mostly included isolates with low relative frequencies of resistance most of them were methicillin susceptible and were mostly isolated from throat swabs.
Spa typing of S. aureus isolates revealed that the isolates came from a variety of genotypes and are not from a CFspecifc clade. While most spa types were unique, t037 was found in 4 patients (all were MDR and grouped in the frst cluster), t701 (all were MDR/MSSA and grouped in the second cluster), and t021 were found in 3 patients and t1149, t14870, t325, and t084 in 2 patients. Te detailed result for each isolate is shown in Figure 2(c) and supplementary Table 1.

Pseudomonas aeruginosa.
Te second most prevalent species was P. aeruginosa which was positive in 41.7% (43/103) of the samples. Tere was a signifcant positive correlation between increased patients age and P. aeruginosa infection (p > 0.00); however, we failed to fnd any correlation between age and other bacteria. Among the P. aeruginosa isolates, 71.6% (43/60) showed a mucoid phenotype and were mostly isolated from patients older than 16 years of age (Figures 3(c) and 3(d)). Fourteen patients (patient numbers 4,6,10,14,19,20,23,30,43,85,86,96, and 98) were infected with two or three morphotypes of P. aeruginosa with a diference in being mucoid/nonmucoid, pigment production, and/or diferent antibiotic susceptibility pattern (supplementary Table 3). Accordingly, 60 morphotypes of P. aeruginosa were evaluated for further investigations.
Te highest resistance rate was for cefepime 38.3% (23/ 60) and levofoxacin 33.3% (20/60), and the lowest resistance was for ceftazidime 6.6% (4/60), imipenem 5.0% (3/60), and tobramycin 3.3% (2/60) (Figure 3(a)). Prevalence of MDR P. aeruginosa was 20% (12/60) which is represented by age group in Figure 3(b). Te highest and lowest prevalences of MDR P. aeruginosa were observed in the 2 to 5 and less than two age groups, respectively. Additional data on antibiotic consumption at the time of sampling in patients with MDR P. aeruginosa can be found in Table 4 of the supplementary fles.
Using a similarity cut-of of 95%, rep-PCR typing allowed the diferentiation of 53 P. aeruginosa isolates into 41 types (supplementary Figure 1). Rep-types are presented in numbers for each isolate in Figure 3(d) and supplementary Table 3.
As it is evident in Figure 3(d), P. aeruginosa isolates fall into four clusters. Isolates within the frst cluster show a high level of resistance to amikacin, are more likely to express a mucoid phenotype, and are isolated mostly from adolescent patients. Isolates in the second cluster showed the lowest individual and resistance combination levels of all populations. Tey are mostly isolated from throat swabs, and the relative prevalence of the mucoid phenotype is the lowest in this group. A relatively high level of resistance is apparent in the third and fourth cluster. MDR P. aeruginosa isolates with a prevalence of 20% are grouped in these two clusters.

Discussion
Cystic fbrosis patients are predisposed to bacterial colonization and infections throughout their life. Tis study provides an overview of the bacterial profle and their antibiotic resistance pattern from the CF patients visiting the main referral children hospital in Iran. Te most prevalent isolate in our study was S. aureus 55.3% (57/103) which is roughly comparable with Ukraine (40.8%) [29], but more than studies conducted in India (15.7%) [30], Canada (24%)   Canadian Journal of Infectious Diseases and Medical Microbiology [31], and Iran on 2012 (9.3%) [32] and Germany (63.3%) [33]. In the latest report from Iran on 2021, the prevalence of S. aureus was 15.6% [34]. Tis inconsistency may be due to dissimilar demographic composition, diferent identifcation methods, and longer incubation times in our study which improve the chance of slow-growing strains isolation.     identical to our study [35,36]. Several reports have suggested that chronic MRSA infection is associated with a high decline rate of lung function, failure to recover lung function after a pulmonary exacerbation and decreased survival [37,38]. Te prevalence of MRSA in our study was 20% which is similar to Chmiel et al. who reported the prevalence of MRSA infection at 25% in the USA, as compared to 11% in Canada and Europe [39]. MRSA infections have been increasingly reported among populations with CF worldwide [40]. According to the annual reports of CFF Patient Registry on 2020, the highest prevalence of MRSA occurs in individuals between the ages of 10 and 30, whereas MSSA peaks among those younger than 15. Te pattern was different in our study, in which MSSA was predominant in 11 to 15 age group and MRSA peaked in younger patients, mostly in 2 to 5-year-old patients (Figure 2(c)). Te high frequency of MRSA in younger children in our study may probably be caused by a circulating MRSA clone in the community. Another possible explanation may be the fact that in countries where care protocols have improved, MRSA colonization has shifted toward older ages which refects the critical role of health care policies and appropriate treatment in improving the CF population health. Te results of spa typing showed a great diversity across S. aureus isolates infecting individuals with CF (Figure 2(c) and supplementary Table 1). In this study, spa t037 was the most frequent lineage, which is diferent from results reported from the USA and Argentina, where spa t002 was more prevalent in pediatric CF patients [41,42]. To the best of our knowledge, there is no information on the pattern of common spa types of S. aureus isolates from cystic fbrosis patients in Iran, and this is the frst report. Based on the analysis, we conclude that the S. aureus clinical isolates surveyed here are not from clonal lineages that transmit between CF patients but instead are from multiple lineages. Te majority of patients that were coinfected with two or more phenotypically diferent S. aureus isolates, harbored the same spa type except for patient numbers 11 and 53 that were colonized with two diferent spa types (supplementary Table 1). Patient number 13 had two visits with an interval of 8 month in both of which S. aureus t632 was isolated that may imply the stability of initial strain (supplementary Table 1).
Te second-most prevalent isolate was P. aeruginosa 41.7% (43/103). With more rapid detection and development of antipseudomonal antibiotics among the pediatric population, new P. aeruginosa acquisitions can be more efectively eradicated [43]. Annual report of the CFF Patient Registry on 2020 demonstrated the percentage of individuals with a positive culture for P. aeruginosa has continued to decline over time, with the largest decrease observed among individuals younger than 18 years (44.5 percent had a positive culture in 2000 compared with 18.1 percent in 2020) [44,45]. In our study the prevalence varied signifcantly by age, from 4.8% in patients under 2 years of age to 30.4% in 16 to 20-year-old patients (p > 0.00). In a study, conducted at Mofd Children's Hospital in Iran from 2004 to 2010, the main infecting pathogen was P. aeruginosa (38.8%) [32] which is not identical to our results. A recent study from Iran reported P. aeruginosa as the most common bacterial isolate from CF patients with a prevalence of 55.5% [34]. Te most important underlying reason for this discrepancy may be the time of sampling and the diferent population age composition.
As shown in Figure 3(d), the overall resistance rate is low among P. aeruginosa isolates. Isolates within the frst cluster which show a high level of resistance to amikacin are mostly isolated from adolescent patients. Higher amikacin resistance in this group in comparison to others may have been caused by a higher treatment frequency with this antibiotic for chronic infections with P. aeruginosa. A relative high level of resistance is apparent in the third and fourth cluster, where there are more adults that are likely to have been received more antibiotics throughout their life span. In our study, ceftazidime and imipenem were the most efective antibiotics against P. aeruginosa isolates, and the least effective antibiotics were cefepime and levofoxacin (Figure 3(a) and supplementary Table 3). A study in Iran showed the most efective antibiotics against P. aeruginosa isolated from CF patients included rifampin, vancomycin, imipenem, ciprofoxacin, ofoxacin, and ceftazidime and the less efective antibiotics were penicillin, ampicillin, cephalothin, and cefxime, respectively [32]. Te latest report from Iran on P. aeruginosa isolates from CF patients showed the highest resistance rate was observed for gentamicin, followed by amikacin, imipenem, and ceftazidime, and the lowest resistance rates were observed in piperacillintazobactam [34].
A multi-center study conducted in the United Kingdom, Belgium, and Germany on the antimicrobial susceptibility of P. aeruginosa isolates showed high resistance levels of 54% for penicillins (ticarcillin, piperacillin, and piperacillintazobactam), 59% for ceftazidime, 46% for amikacin, 27% for ciprofoxacin, 20% for carbapenems, and 16% for tobramycin. Resistance levels were notably much higher than that which have been reported before [46][47][48]. High levels of resistance were mainly attributed to the epidemic clone the Liverpool Epidemic Strain (LES) which was prevalent in the UK, and four MDR sequence type 958 (ST958) isolates that were found to be spread over the three countries [49]. Difering antibiotic treatment strategies, demographics, genetic characteristics of the population, laboratory methods, and hygiene practice may justify the diference observed between studies.
By rep-PCR typing, P. aeruginosa isolates were divided into 41 types with a 95% similarity cut-of level (supplementary Figure 1) [28]. Analysis of rep-PCR results indicate that the majority of our patients are infected with diferent lineages (Figure 3(d) and supplementary Table 3). Te great diversity may be associated with microevolutionary events in the airway environment of CF patients [50]. Te surveillance of the diferent morphotypes of P. aeruginosa isolated from individual CF patients to recognize when new morphotypes that may be more resistant to antimicrobial agents emerge is of great concern. As for patients' number 6, 7, 10, and 14, who had multiple referrals, diferent morphotypes with a higher resistance rate were isolated (supplementary Table 3).
S. aureus and P. aeruginosa are the two most commonly recognized bacterial pathogens associated with chronic lung infections in patients with CF [51]. A number of studies have shown that coinfection is associated with diminished lung function and more rapid pulmonary decline [2,17,52]. In our study, 22.3% (23/103) of the cases were coinfected with S. aureus and P. aeruginosa (Figure 1). Te global rates of S. aureus/P. aeruginosa coinfection around the world range from 8.6% to 60% with an average of 28.3% which is highest for patients in their mid-twenties [41].
Almost all P. aeruginosa and S. aureus (more than 98%) were bioflm producers (Figures 2(a), 2(c), 3(a), 3(c) and 3(d)) which exemplify the important role of bioflm mode of growth as a key factor facilitating persistence of infection in the CF respiratory tract. A report from Iran showed that 76% and 67% of P. aeruginosa and S. aureus isolates from CF patients were bioflm producers, respectively [26]. As it is evident in Figure 3(c), contrary to expectations, nonmucoid showed a greater ability to form more strong bioflm structures than mucoid isolates. Tis may be due to the inadequacy of the microtiter plate method for measuring the ability of P. aeruginosa from CF patients to form bioflms. As previously mentioned, P. aeruginosa strains in a CF lung are more likely to form aggregate structures known as microcolonies by connecting to each other and to the mucin rather than attaching to a surface [53].
In spite of signifcant advances in treatment procedures focusing on CFTR potentiator drugs, respiratory infections remain an important cause of lung disease [39]. Tus, monitoring of such infections and the changing trend of infectious agents that may play a role in pulmonary exacerbation is vitally important in CF patients. Te limitations of our study are the small number of patients from a single CF center and the unavailability of clinical histories and previous culture results. Lack of corroboration of culture methods with molecular approaches is another limitation which could help to have a better scheme of the bacterial profle during exacerbation. Furthermore, anaerobic bacteria, viruses, and fungi which may play a crucial role in proceeding exacerbation of respiratory parameters were not assessed in our study.

Conclusion
Tis study provides a picture of the bacterial profle from the respiratory tract of CF patients.
S. aureus and P. aeruginosa were the most common isolated bacteria during pulmonary exacerbation episodes. Our study demonstrated a signifcant decline in the prevalence of P. aeruginosa infections in comparison to previous studies. We found S. aureus to be more prevalent in younger patients, whereas mucoid P. aeruginosa was the dominant species in adults.
Te polymicrobial nature of airway infections in CF patients makes it problematic to isolate fastidious and slowgrowing microorganisms such as H. infuenzae and B. cepacia complex, which may be hampered by more dominant species. It is suggested to use molecular techniques to detect these less frequent and fastidious organisms. Te current study highlights the importance of epidemiological surveillance of CF pulmonary exacerbations. Early detection and prompt eradication treatment at the very onset of infection may efectively prevent the establishment of chronic infection and reduce the risk of morbidity, mortality, recurrent pulmonary exacerbation, and hospitalization.

Data Availability
Te data used to support the fndings of this study are included within the article and the supplementary information fles.

Consent
All participants included in the study and/or parents (in case of children) were provided with informed consent before enrollment in the study. All sputum specimens were produced voluntarily.

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
Soroor Erfanimanesh carried out all laboratory experiment, collected data and drafted the manuscript. Mohammad Reza Modaresi and Mohammad Mehdi Feizabadi participated in the design of the study and advised in all parts of the study. Shahnaz Halimi, Reza Bigverdi and Vajiheh Sadat Nikbin analyzed data and performed the quality control of data. Fereshteh Jabalameli and Mohammad Emaneini supervised all parts of the study. Te authors read and approved the fnal manuscript.