Characterization of Pseudomonas aeruginosa isolated from chronically infected children with cystic fibrosis in India

Cystic fibrosis (CF) is the recessive genetic disorder of Caucasian race. CF in Asians and Indians was thought to be almost nonexistent in the past. However, recent reports have suggested CF to be more common in Indian population than previously thought. For proper management, it is important to diagnose the infection in the patients with CF [1–3].

The mutations in the cystic fibrosis transmembrane regulator gene in CF patients lead to recurrent and chronic respiratory tract infections, which serves as a major cause of morbidity and mortality [4]. If appropriate antibiotics are used, the quality of life improves in CF patients. Thus, the knowledge of the etiological agents and their antimicrobial susceptibility can help in planning the appropriate antibiotic therapy to be instituted in CF patients.

Infection due to Pseudomonas aeruginosa (P. aeruginosa) has been implicated as a major cause of morbidity and mortality in patients with CF [5–8]. Once P. aeruginosa colonizes the respiratory passages, it is rarely possible to eradicate it. Prolonged and repeated administration of drugs lead to the development of multidrug resistance in P. aeruginosa strains. To detect emergence of the resistant strains, it is important to monitor the antibiotic susceptibility pattern of P. aeruginosa isolates. Increased resistance to gentamicin, quinolone and cephalosporins has been reported in chronically infected patients [9, 10].

With severity of pulmonary infection, the organism undergoes phenotypic modification, which helps in its persistence under adverse environment of the respiratory tract. The organism diversifies into different colony morphotypes [11, 12] and develops antibiotic resistance for its survival. This may be due to an adaptation of the organism to the host defense mechanisms and antibiotic selective pressure [13]. Under recurrent and chronic endobronchial P. aeruginosa infections, the initial colonization is with nonmucoid forms of an organism but with progressive lung tissue damage, it gets converted to the mucoid form. The mucoidity helps an organism to grow as biofilm, which serves as a protective niche and helps to evade the host immune response and action of antibiotics. The organism also becomes auxotrophic for amino acid due to high amino acid concentration in the respiratory passages. The most commonly required amino acid reported in literature is methionine while other amino acids-leucine, arginine and ornithine have been required by a minority of P. aeruginosa strains [14].

During the course of infection, the organism may undergo genotypic changes. The chronically infected patients have been found to have long-term colonization by one predominant genotype but variation in genotypes within a single patient has also been observed [13, 15].

Since no data is available on the characterization of P. aeruginosa from CF patients in India, the present study was undertaken to study phenotypic and genotypic variations of P. aeruginosa isolated from CF patients attending the Pediatric Chest Clinic at All India Institute Of Medical Sciences (AIIMS), New Delhi, INDIA.

Cystic fibrosis (CF) is the most common inherited fatal disease in Caucasian population. This generalized disorder of salt and water transport of exocrine glands is caused by mutations in cystic fibrosis transmembrane regulator gene in the airway epithelial cells and submucosal glands. These patients are very prone to bronchopulmonary infection due to lack of the innate defensive mechanisms. Pulmonary infection is the most common cause of morbidity and mortality in CF patients [16].

CF was thought to be extremely rare in India. However, recent reports from Indian subcontinent suggested CF to be more common in Indian population than what was previously thought [1]. In our study, we have followed all 56 patients attending the Pediatric Chest Clinic at All India Institute Of Medical Sciences, New Delhi, INDIA between August 1998-August 2001. The median age of diagnosis of patients was 4.5 years, which was much higher than what was earlier reported in the literature where the median age was found as 6 and 12 months among Caucasian American and Indian American children respectively [17]. This may be due to delayed diagnosis of CF patients in India.

P. aeruginosa has been the predominant bacterium associated with pulmonary infection in patients with CF [18–22]. In our study, P. aeruginosa was also found as an implicated organism in 82% culture positive patients and its rate of infection was significantly higher as compared to the other organisms in CF patients. The colonization with P. aeruginosa has been linked with pulmonary deterioration of CF patient resulting in an increased risk of hospitalization [21]. In the study, all the patients (n = 9) who required hospitalization were chronically infected with P. aeruginosa.

Besides morbidity, P. aeruginosa has been associated with increased mortality [18–22]. In the study all the 4 patients who died were chronically infected with P. aeruginosa. Thus significant higher rates of hospitalization and death were observed among patients chronically infected with P. aeruginosa.

Although prolonged and frequent administration of antibiotics has been useful in the treatment of pulmonary exacerbations, there is risk of development of multiple drug resistance in P. aeruginosa strains. In the study, 41% isolates were multidrug resistant and were recovered from chronically infected patients, indicating the possible role of antibiotic pressure. Similar findings have been reported elsewhere [23–25].

All three patients infected with ESBL positive isolates were found sensitive to meropenam and imipenam. The similar finding has been reported earlier where carbapenams have been found the most effective and reliable against ESBL positive isolates [26].

During chronic infection with the organism, there is progressive anatomical deterioration of the CF lung, which provides a highly spatial structured environment leading to diversification of the organism into six different colony morphological types-Type1-Type 6[12]. This diversification enhances the capacity of an organism to survive in lower respiratory tract of CF patients [13]. In the present study, 7/8(87.5%) chronically infected patients harbored different colony morphological types in the sputum samples.

Once P. aeruginosa has colonized the respiratory passages of CF patients, it leads to the induction of mucoidity, resulting in persistent infections. In the study, 6/8(75%) patients who were chronically infected with P. aeruginosa harbored mucoid P. aeruginosa in their respiratory samples. Our finding in agreement with the earlier reports where an increased association of mucoidity with chronicity of infection was observed [4, 27, 28]. Out of 4 patients who died, 3(75%) harbored mucoid colonies in their respiratory samples signifying the association of mucoidy with high mortality in CF patients. Our finding was in accordance with the earlier study where association of mucoidity with high risk of death was observed [26].

The antimicrobial susceptibility among mucoid and nonmucoid isolates was compared. The mucoid isolates showed increased resistance against gentamicin and lower resistance against beta lactam (7.4% to piperacillin and no resistance to ceftazidime). The results were analogy to the studies where high resistance to aminoglycosides [29, 30] and low resistance to beta lactam [31, 32] was observed among mucoid isolates.

With chronicity of infection, there is an increase in concentration of amino acids in bronchial secretions, which is due to active epithelial transport, plasma exudation and protease activity in lung tissue and surface fluid. This results in development of auxotrophy for certain amino acids required for growth. In the present study, auxotrophic P. aeruginosa was found in 2/27(7.4%) patients in contrary to an earlier report where auxotrophy of P. aeruginosa was found in 86% patients infected with the organism [33]. The reason for low percentage of auxotrophs among our patients may be due to lower number of chronic patients in this study.

Furthermore in the study, 3/5 auxotrophs had arginine requirement and the remaining 2 were auxotrophic for methionine. This was in agreement with the earlier reports where methionine and arginine were considered as the most commonly required amino acids by auxotrophs [14, 33, 34]. As these strains could be more resistant to antibiotics, it is important to study auxotrophy of isolates in CF patients with severe P. aeruginosa infection in order to plan out appropriate therapy for the management of CF patients.

Bacterial typing schemes are based on phenotypic or genotypic analysis of multiple isolates within a particular species. Such analysis helps in investigation of outbreaks and determines the recurrence or relapse of an infection. The epidemiology of P. aeruginosa infection in CF patients has been hampered since the conventional phenotyping methods-phage and pyocin typing have poor discriminatory power and poor reproducibility respectively. Genetic typing methods have shown to be more discriminatory than phenotypic methods for typing P. aeruginosa CF isolates [35]. Various genotypic typing methods- restriction fragment length polymorphism (RFLP) analysis of tox A I and pil A loci, pulse field gel electrophoresis (PFGE), random amplified DNA polymorphism (RAPD) PCR have been used to identify the number of strains colonizing the CF patient [13].

PCR-ribotyping is based on the uniform spacer region in rRNA operons of P. aeruginosa [36] resulting in low degree of heterogeneity of rRNA operons. In the study, majority of isolates showed the single PCR-ribotyping Pattern P1, probably due to the above reason. Hence for the study of dynamic of P. aeruginosa strain variation, ERIC-PCR may be a useful tool.

By ERIC-PCR, all 27 culture positive patients except one were found colonized with the unique genotype (E1-E33). This indicates that cross-colonization among unrelated CF patients was uncommon. The results were concordant to the earlier studies where cross-infection between unrelated patients was considered unusual [37, 38]. All 27 patients except 4 were colonized with the single persistent genotype. These 4 patients were chronically infected and were colonized with 2 or more genotypes. Our results were in agreement with the previous studies where majority of CF patients were colonized with a predominant genotype for longer period of time but high degree of genotypic variability within patients have been observed suggesting that the colonizing strain may occasionally be replaced [13, 39–41].

Thus we have found that CF is a problem in our country but delayed diagnosis and low index of suspicion have delayed the appropriate management of these patients. Although all the patients attending the Pediatric chest clinic were included in the study, the number of patients, especially the chronic ones, was limited in India, which further limited the number of chronic patients. Although the small number of patients in our study is not sufficient to answer all the epidemiological questions, these results indicate that a larger scale CF study should be conducted in India.

P. aeruginosa is the major colonizer of the respiratory passages of CF patients. The patients with chronic infection showed significantly higher rates of hospitalization and death. With chronicity of infection, the organism undergoes phenotypic and genotypic variations. Among phenotypic variations, the organism becomes multidrug resistant, diversifies its colony morphology, acquires mucoidity and becomes auxotrophic for various amino acids. The multiple genotypes have been found to colonize the respiratory passages of CF patients.

Thus in a particular clinical set up, high index of suspicion should be there for diagnosis of CF patients so as to prevent the delay in diagnosis and management of CF patients. The routine microbiological examination of respiratory samples from CF patients are advised to study the characteristics of the infecting agents of which P. aeruginosa is the most common.