Novel Class 1 Integron in Clinical Isolates of Burkholderia cepacia Complex

INTRODUCTION

Burkholderia cepacia complex (BCC) is an opportunistic, nosocomial pathogen that causes significant disruption in immunocompromised individuals. They comprise nine genomovars and collectively form BCC, representing a group of 24 different species.^[1] These bacteria are highly host-specific and are present in soil, water, and plants. Metabolically diverse, they are linked to a varying clinical course in patients with cystic fibrosis (CF). This can include septicaemia, which is associated with a high mortality rate, necrotising pneumonia, and severe lung infections, collectively termed ‘cepacia syndrome’.^[2,3] It is one of the prime causes of sepsis in patients with non-CF in India, as it is observed to have a higher incidence (77%) in environmental samples.^[4] Originally identified by W. H. Burkholder as a plant pathogen that caused onion rot in the 1950s, it was later identified to cause human infection. The pathogen was initially known by the name Pseudomonas cepacia until 1992, when Yabuuchi and colleagues investigated and replaced it with the new genus Burkholderia.^[5] The transmission of B. cepacia complex in both healthcare and non-healthcare settings occurs through direct and indirect contact with infectious secretions and droplets.^[6] Due to various resistance mechanisms, BCC infections are resistant to major antibiotics, limiting treatment options.^[6] They are intrinsically resistant to polymyxins and aminoglycosides, which is mediated by efflux pump activity.^[7] Although first-line treatments for BCC infections include trimethoprim-sulfamethoxazole (TMP-SMX) and ceftazidime, considerable rates of resistance have been observed to these agents as well.^[8-10] The emergence of widespread antibiotic resistance has increased the rates of morbidity and mortality associated with serious infections caused by these organisms.

One such root cause of acquired resistance is the horizontal gene transfer (HGT) of the antibiotic resistance genes (ARGs).^[6] Mobile genetic elements (MGEs) are pivotal in carrying ARGs from one bacterium to another. They play a crucial role in the persistence and dissemination of antibiotic resistance in most bacteria.^[6,11] Integrons, among them, are DNA elements that capture open reading frames inserted in external gene cassettes and transform them into functional genes by ensuring proper expression.^[6,11] They are most widely recognised for their ability to spread ARGs across pathogens. Due to their potential to rapidly transmit resistance phenotypes, it is crucial to investigate whether integron-mediated features may impact human health in the future, such as enhanced virulence, pathogenicity, or resistance to emerging antimicrobial treatments in pathogens like Burkholderia. Against this background, our study aimed to screen clinical Burkholderia cepacia isolates from non-CF samples for integrons and to investigate the presence of ARGs within the gene cassettes.

MATERIAL AND METHODS

RESULTS

Antimicrobial susceptibility testing

Fourteen isolates tested showed the highest resistance to amoxicillin-clavulanic Acid (85%), followed by ampicillin (71%), chloramphenicol (57%), ceftazidime and co-trimoxazole (28%), piperacillin (21%), norfloxacin (7.1%), and minocycline (7%) [Figure 1]. No resistance was found against meropenem and clindamycin. Among the 14 isolates tested in this study, B6981, B591, B917, B2164, B5348, B13158/59, and B5505 exhibited resistance to four antibiotics out of the 10 tested, which was the highest count, and were considered MDR isolates. These 14 MDR isolates were further used to screen integrons in addition to the 41 MDR isolates from the previous study (n=55).

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Figure 1:

Graph depicting antibiotic resistance pattern of the 14 isolates against different antibiotics. MI: Minocycline, NX: Norfloxacin, PI: Piperacillin, COT: Co-trimoxazole, CAZ: Ceftazidime, C: Chloramphenicol, AMP: Ampicillin, AMC: Amoxicillin and clavulanic acid.

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Sequencing

The isolate B6981 was positive for a class 1 integron that carried an integrase gene and two ARGs: dihydrofolate reductase (dfrA5) and erythromycin esterase (ereA2). coding for trimethoprim and erythromycin, respectively [Figure 2]. Isolate B591 contained the integrase gene within its gene cassette. These three sequences were submitted to NCBI GenBank with the accession numbers PQ052888, PQ052889, and PQ166740. The remaining isolates yielded non-specific amplification, indicating the presence of OMP genes and a hypothetical protein. Hence, we considered them integron-negative.

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Figure 2:

Class 1 integron map of Burkholderia cepacia isolates (a) B6981 consisting of an integrase gene, dfrA5 gene encoding trimethoprim resistance, and ereA2 gene encoding resistance to erythromycin; (b) B591 consisting of an empty integron with just the integrase gene and without any gene cassette. att I stands for attachment site I.

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DISCUSSION

Burkholderia is a potent nosocomial pathogen causing sepsis in non-CF patients with poor clinical outcomes. Over the past few decades, researchers worldwide have conducted epidemiological, taxonomic, and molecular biology studies on BCC strains known to cause persistent infections in CF patients.^[13] The intrinsic resistance of BCC isolates to many antibiotics poses a serious global challenge for clinicians treating these infections. The rise of widespread antibiotic resistance has led to higher rates of morbidity and mortality associated with these illnesses. In the current study, the clinical isolates were resistant to most of the antibiotics tested, including ampicillin (71%), chloramphenicol (57%), ceftazidime and co-trimoxazole (28%), piperacillin (21%), norfloxacin (7.1%), and minocycline (7%). Thus, the resistance pattern observed here demands the need for combinational therapy. However, the antibiotic amoxicillin-clavulanic acid used in this study showed maximum resistance (85%), which raises concerns regarding the use of antibiotics in combination therapy. This observation disagrees with the study by Tamma et al.^[6] wherein the treatment with another combination, ceftazidime-avibactam, could completely eradicate bacteraemia in a 2-month-old infant. Also, Branstetter et al.^[14] revealed that a 17-year-old patient who suffered from CGD and presented with cepacia syndrome could completely recover on the administration of tobramycin, enteral minocycline, ceftazidime, IV sulfamethoxazole-trimethoprim, and corticosteroids. Therefore, understanding the antibiotic susceptibility profiles of organisms is crucial for targeted therapy and effective antimicrobial stewardship.

To delve deeper into the root cause of the above resistance mechanism, our study focused on screening for the presence of integrons within these MDR isolates. Out of the 55 MDR isolates evaluated, integrons were found only within 14 isolates by PCR. However, Sanger sequencing revealed the presence of class 1 integrons only in one isolate, while an integrase gene in another. This depicts decreased prevalence of the integrons among the MDR BCC. Furthermore, the resistance observed in the remaining bacterial isolates could be attributed to alternative mechanisms, including efflux pumps, alterations in metabolic pathways, and genetic mutations. In the present study, among the 14 integron-positive isolates, B6981 and B591 contained a class 1 integron and integrase, respectively. However, none of the isolates carried a class 3 integron by PCR, which highlights the increased prevalence of class 1 integrons. This finding aligns with the study by Zarei-Yazdeli et al.^[15], which reported that 82.6% of the Pseudomonas aeruginosa isolates carried intI1 genes, while none of the isolates carried the intI3 gene. Zhang et al.^[16] In China, a study revealed the existence of class 1 and class 2 integrons in 252 (81%) and 7 (2.3%) strains, respectively, with the absence of Class 3 integrons.

Currently, in the investigation of the genetic bases of MDR Burkholderia, a significant aspect under consideration is the integron and its associated gene cassettes. These elements play a crucial role in horizontal gene acquisition and gene expression, serving as a gene reservoir, and have been linked to the development of antibiotic resistance among clinical bacterial isolates.^[11] Also, there have been numerous studies that have utilised Sanger sequencing as a standard technique for the determination of unknown gene function within the gene cassettes.^[17,18] Hence, we examined the presence of the gene cassettes by subjecting the integron-positive isolates to Sanger sequencing.

The gene cassettes investigated in this study belonged to the family of dihydrofolate reductase (dfrA5), which encodes resistance to trimethoprim, and erythromycin esterase (ereA2), which encodes for erythromycin resistance. The nucleotide sequence of the integron revealed 99% and 100% similarity with the sequences from the NCBI GenBank (CP156941.1, CP162903.1). The gene cassette encoding trimethoprim resistance discovered in our study is one of the most common genes integrated within class 1 integrons in the clinical isolates.^[19,20] This prevalence may also reflect the selection pressure exerted when these antibiotics were more extensively used in the past.^[21-29] However, the presence of the gene encoding for erythromycin resistance belongs to the macrolide family, to which species of BCC are innately resistant.^[24-26] Hence, these gene cassettes, even though not commonly found in class 1 integrons, were detected in the current study. The presence of the integrase gene in another isolate, B591, indicates that it can readily accommodate resistance genes through the natural acquisition and dissemination mechanism of the integrons.

The limitation of the present study is that the genes identified within the gene cassette can only support the resistance patterns of trimethoprim as observed in antimicrobial susceptibility profiling, but cannot support the entirety of their resistance pattern. Nevertheless, this study has evaluated the presence of class 1 integrons in MDR BCC and the potential of integrons in AMR dissemination.

Compliance with ethical standards: The Burkholderia isolates used in the study were obtained from the institutional repository. However, all relevant ethical standards were considered before conducting the experiments.

CONCLUSION

In conclusion, BCC, an opportunistic pathogen, poses a significant threat to immunocompromised individuals due to its propensity to cause severe infections. The inherent resistance of BCC to a broad spectrum of antibiotics remains a formidable challenge, necessitating urgent attention and intervention. Integron-mediated resistance is likely a key contributor to the antimicrobial resistance observed in these isolates, underscoring the need to further explore the genetic mechanisms at play. Moreover, HGT mechanisms, which facilitate the dissemination of resistance genes, must be rigorously monitored. This continuous surveillance is crucial for developing and implementing robust AMR management strategies, which are essential to mitigate the growing threat BCC poses in clinical settings.