Antimicrobial sensitivity pattern of children with cystic fibrosis in Bangladesh: a lesson from a specialized Sishu (Children) Hospital

Background: Infection control in cystic fibrosis (CF) patients plays a crucial role in improving the survival of patients with CF. Antimicrobial sensitivity patterns in these patient groups in our country are currently lacking. Therefore, the purpose of the study was to evaluate the microbiological cultures and antimicrobial susceptibility pattern of pediatric CF patients. Method: A total of 50 respiratory samples were prospectively collected from the period between February 2021 and October 2021. Sputum and oropharyngeal swabs were processed for culture and microbiological testing. Sample collection and evaluation were performed according to the Good Laboratory Practice guidelines (GLP). Informed written consent was ensured before participation. Statistical analysis was performed with SPSS v 26. Result: The median age of the children was 30 months (6–120) months, with a male predominance (66% vs 34%). Single and two organisms were isolated in 72% ( n = 36) and 12% ( n = 6) of cases, respectively. During the study period, 36% of the patients harbored Pseudomonas aeruginosa , 18% harbored Klebsiella pneumoniae , and both Staphylococcus aureus and Escherichia coli were detected in 16% of cases. Levofloxacin was found to be the most active antibiotic agent with 100% susceptibility. In contrast, nearly all isolates were resistant to amoxicillin, erythromycin and rifampicin. Conclusion: Levofloxacin is the most effective agent to treat CF patients. Active surveillance of the resistance pattern should always continue to be promoted.


Background
Repeated bacterial lung infection leads to the progressive development of respiratory incompetency in patients with cystic fibrosis (CF), resulting in expanded mortality and morbidity [1,2]. In cystic fibrosis, mutation of the CFTR gene impairs innate defenses, such as the mucociliary clearance system and bactericidal activity of the airways, resulting in accelerated pulmonary colonization and chronic infection with harmful bacteria [3]. Near one-third of patients with CF experience lung problems since their early childhood and develop radiological evidence of bronchiectasis [4]. Hemophilus influenzae and Staphylococcus aureus are the dominant causative agents in the early stage of the disease, but with the progression of age and disease, Pseudomonas aeruginosa becomes the most prevalent and notorious for CF

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Egyptian Pediatric Association Gazette lungs [5,6]. In addition, other opportunistic microbes, including Stenotrophomonas maltophilia, Achromobacter spp., and Burkholderia cepacia, are also responsible for exacerbations of pulmonary symptoms [7,8]. Moreover, as the patient ages, frequent use of antibiotics facilitates antimicrobial resistance along with alteration of microbiome diversity in the affected lung milieu. Thus, classical microbes are being replaced by newly emerged pathogens, and benign colonizers are becoming the culprits of disease advancement [7,9,10]. Therefore, considering such dynamic microbiology, it is often difficult to control CF airway infection [11]. However, during an exacerbation, to regain optimum pulmonary efficacy, there is no alternative of suitable antibiotics, and it should be under the guidance of an in vitro antibiotic susceptibility test (AST) [12].
On the other hand, it is currently not a disease of the West; rather, it is fairly reported from different corners of the world, with an incidence ranging from 1:2000 to 1:35,000 in new births. Despite limited comprehensive documentation of CF cases in Bangladesh, approximately 100 cases have been reported in the last decade [11,[13][14][15][16]. To establish standard care of these infectionprone children, knowing the microbiological profile and antibacterial resistance pattern is therefore essential. Therefore, studying the microbiological pattern and susceptibility to antibiotics among the collected samples of children with CF was the objective of the study.

Materials and methods
This observational study was carried out over a period between February 2021 and October 2021 among the patients admitted to the inpatient department of Dhaka Shishu Hospital, a tertiary level hospital in Bangladesh designated to care for the children. Approval from the institutional review board (IRB) was sought before the commencement of the study. Written informed consent was obtained from the parents of the affected child. Patients were included in this investigation on conditions in which they had CF, authenticated by their measured sweat chloride level ≥ 90 mmol/L (performed on SAN-ASOL (SM-01) sweat analyzer, Sanasol Ltd. Hungary) along with characteristic clinical features [17]. Children with suggestive clinical features of asthma, aspiration pneumonia, pulmonary tuberculosis, foreign body aspiration, primary immunodeficiency, congenital heart disease, pulmonary abscess, congenital diaphragmatic hernia, and congenital lobar emphysema were excluded from this study.
Parents were interviewed, and a structured questionnaire was employed to fill out for all cases. Complete blood counts (CBCs), random blood sugar (RBS), sputum and oropharyngeal swab cultures with antibiograms, Mantoux tests (MTs), and chest X-rays (CXRs) were performed in all patients. Other supporting investigations, such as stool for fat droplets, serum albumin level, liver function test (LFT), and echocardiography, were also performed. Reviewing of the previous medical records was also performed.

Specimen collection and culture
Sputum samples were collected into sterile containers provided by the laboratory, screwed tightly, and labeled. Parents were directed to keep their child refrain from food for 1 h before collection. Subjects who were unable to spontaneously expectorate oropharyngeal swabs were collected with the assistance of parents or by asking the child to open the mouth, gently rubbing a sterile swab stick over the pharyngeal wall and replacing that in a sterile plastic container.
Within 1 h of collection, samples were transported to the microbiology laboratory of the same institution. Samples were inoculated on Mac-Conkey's agar, chocolate agar, and blood agar and incubated at 37 °C for 24 h. After completion of 24 h, colonies were identified, subcultured, and subjected to biochemical reactions for identification of microorganisms as per standard technique [18].

Antimicrobial susceptibility test
Susceptibility to antimicrobial agents of all isolates was determined by the Kirby Bauer modified disc diffusion technique using Mueller-Hinton agar plates, and zones of inhibition were interpreted according to Clinical and Laboratory Standards Institute (CLSI), 2017 guidelines. Antibiotic discs such as amoxicillin, ceftazidime, cefepime, vancomycin, piperacillin, imipenem, meropenem, linezolid, cotrimoxazole, gentamicin, amikacin, erythromycin, ciprofloxacin, levofloxacin, rifampicin clindamycin, tobramycin, timentin, and aztreonam were used, and proper concentrations of the drugs were maintained [19].

Statistical analyses
Statistical analyses were conducted using SPSS software: v-26. Continuous variables are expressed as the mean and standard deviation. Categorical variables are expressed as counts (percentages). Nonparametric test, e.g., Fischer exact test whenever necessary. Statistical significance was determined at p ≤ 0.05.

Result
A total of 50 children with cystic fibrosis were included in the study. The median age of the participants was 30 months, with a range of 6 to 120 months. Most of the children were aged between > 1 and 5 years (n = 30, 60%), and the majority were male (n = 33, 66%). Among all, 57.14% of children (n = 28) had a family history of cystic fibrosis, and the parents of 41.67% of children (n = 15) had consanguinity of marriage (Table 1).
Death was not significantly associated with any particular microorganism isolated from respiratory samples among children with cystic fibrosis (p > 0.05 for all) ( Table 4).

Discussion
CF patients are afflicted by a vicious cycle of infection and inflammation of the airway due to decreased airway surface liquid volume and impaired mucociliary clearance [20]. A small group of bacterial species colonizes the airway in CF, causing chronic infection [9]. Understanding the prevalence and antimicrobial susceptibility pattern of these colonizing bacteria in the context of the country is important for the appropriate management of infection in CF patients. We aimed to study this pattern in the case of children with CF in Bangladesh.
Our study revealed that Pseudomonas aeruginosa is the predominant isolate from respiratory pathogens, and nearly three-quarters of children with CF were infected with a single microorganism. Among all the antimicrobial agents tested, levofloxacin was the most sensitive, and amoxicillin, erythromycin, and rifampicin were the most resistant. There was no association of outcome with species of bacteria isolated from the respiratory specimens.
We isolated P. aeruginosa and S. aureus in 36% and 16% of specimens, respectively. Our findings corroborate those of Kabir et al. [15], who conducted a largescale clinico-epidemiologic study of CF children in Bangladesh and found a high prevalence of P. aeruginosa (27%). Nobandegani et al. [21] found a predominance of S. aureus isolates in 54% of specimens and found P. aeruginosa in 30% of specimens. Likewise, Valenza et al. [22] found a high prevalence of S. aureus (63.3%) and P. aerugisona (50%) in their participants. However, the latter two studies included both children and adults, and Santos et al.
[23] previously found that P. aeruginosa and S. aureus were the most frequently found organisms in these two groups of patients. The use of antimicrobials might also affect the distribution of causative microbes in children with CF. Hauser et al. [24] noted that during the early half of the twentieth century in children with CF, when potent antistaphylococcal drugs were not available, S. aureus was the predominant isolate from respiratory pathogens and was subsequently replaced by P. aeruginosa with the development of potent antistaphylococcals.
Another noticeable observation in this study was the presence of K. pneumoniae, a gram-negative bacterium, in the respiratory specimens of our participants, which is usually associated with healthcare-acquired pneumonia and is not a typical pathogen of CF [25]. However, the genetic flexibility of K. pneumoniae enables it to evade host immunity and colonize respiratory epithelia, which might have been the reason behind such a high prevalence in our study participants.
Antimicrobial sensitivity pattern analysis in the present study showed that some of the first-generation antimicrobials, such as amoxicillin, erythromycin, and rifampicin, were nearly completely resistant against different microorganisms isolated from children with CF. Alarmingly, the emergence of carbapenem resistance was evident from our analysis, and none of the imipenem and meropenem isolates were 100% sensitive against any organisms. A northern European study [26] among cystic fibrosis patients found the development of high resistance rates of P. aeruginosa against different antimicrobials, including carbapenems. Another study in Germany noted carbapenem resistance in both mucoid and nonmucoid P. aeruginosa [22]. Frequent use of carbapenems as a broad-spectrum antibiotic in the hospital setting might lead to the development of high resistance rates to these drugs in children with CF. This is of concern, as the options of broad-spectrum antimicrobials are becoming narrower daily.
We noted high methicillin resistance among the staphylococcal isolates in our study, which conforms to the findings of previous studies [21]. Even vancomycin was found to be highly resistant (87.5%) against  S. aureus. Only fluoroquinolones such as levofloxacin, ciprofloxacin, and fourth-generation cephalosporine (cefepime) were found to be effective against these strains. All beta-lactam antibiotics were resistant to E. coli. Only levofloxacin and linezolid were effective against it. Levofloxacin was the only drug found to be 100% sensitive against all species of bacteria found in respiratory specimens of children with CF. This, although good news for clinicians, necessitates judicial and cautious use of antibiotics in cystic fibrosis patients.
The present study did not find any association between bacterial species and the outcome of cystic fibrosis patients. However, studies [24] concerning microbial species and clinical outcome in cystic fibrosis are limited by the fact that any association, if found, does not imply causation, it is impossible to find a comparison group without microbial infection, and there is lack of antimicrobial specification because of polymicrobial infection of the patients.
Excessive use of antibiotics along with lack of patient compliance leads to rapid development of resistant strains, some of which are resistant to multiple drugs. Therefore, antibiotic guidelines should be updated regularly in light of continuously updated research findings for the context of a country. Here, we found that levofloxacin is the most sensitive drug that should be preserved for the most resistant cases. Additionally, specific antibiotics should be chosen for a specific set of Percentage was calculated among available data * p < 0.05