Prevalence and Molecular Characterization of Carbapenem and Colistin-Resistance Genes in Clinical Strains of Pseudomonas aeruginosa Isolated from Diabetic Patients

Revival of the Isolates

A total of 100 clinical isolates (wound: 64; sputum: 36) recovered from diabetes patients' wound and sputum samples and stored as glycerol stocks in −80°C were revived. The initial recovery of the isolates from the patients' sample was done by taking clearance from the Central Ethics Committee of the Nitte University (NU/CEC/2019/0229) and was conducted as per the ethical guidelines. Pus/wound swab and sputum samples from diabetic patients with respiratory infection were collected. The samples from diabetic patients older than 18 years and irrespective of sex with a history of diabetes (of more than 5 years) and with a blood glucose concentration more than 150 mg/dL were included in the study. The isolates were enriched in asparagine broth and streaked onto cetrimide agar to get a single colony. Further, the single colony was picked and subcultured on nutrient agar medium.

Antibiotic Susceptibility Testing

Pseudomonas aeruginosa strains were checked for their susceptibility to carbapenem using the Kirby-Bauer disc diffusion method following the Clinical and Laboratory Standards Institute guidelines (CLSI2020). The control strain used for antibiotic susceptibility testing was P. aeruginosa ATCC 27853. A 100 pL of 0.6 OD culture grown in Mueller-Hinton broth (MHB) was swabbed over a sterile Mueller-Hinton agar (MHA) plate. A 10-pg imipenem disc (HiMedia Laboratories Pvt. Ltd., India) was placed on the swabbed MHA plate and incubated at 37°C for 16 to 18 hours. The zone of inhibition (mm) around the disc was compared with the interpretive chart of CLSI, and the strains were designated as sensitive, intermediate, or resistant. Susceptibility to colistin was performed by microbroth dilution method as described in the CLSI guidelines. In brief, a serial twofold dilution of colistin (HiMedia Laboratories Pvt. Ltd.) was carried out in cation-adjusted Mueller-Hinton II broth (CA-MHBII) (500 pL) ranging from 0.125 to 256 μg/mL. A 100 μL of 10⁵ CFU/mL culture was inoculated to the medium with different dilutions of the antibiotic and incubated for 24 hours at 37°C. The isolates were classified as per the CLSI colistin breakpoints, as susceptible (2 μg/mL) or resistant (4 μg/mL) based on the turbidity.

Modified Hodge Test

Modified Hodge assay was performed as described by Amjad et al (2011).¹² A 100 μL of 10⁶ culture of Escherichia coli ATCC 25922 was inoculated to 5 mL of MHB and incubated at 37°C for 16 to 24 hours. After the incubation, 0.5 mL of the 10⁶ culture was added to another 4.5 mL of MHB to create a 1:10 dilution. A 100 μL of diluted culture was used to prepare a lawn on an MHA plate. Meropenem disk (10 pg) was placed at the center of the plate, and P. aeruginosa was streaked from the edge of the disc to the edge of the plate in a straight line. Four isolates were tested on a single plate. The plate was incubated for 16 to 24 hours at 37°C.

Molecular Detection of Antibiotic-Resistant Genes

The method of Ausubel et al (1992)¹³ with slight modifications was employed for the extraction of genomic DNA from the strains of P. aeruginosa and the reference strain PAO1. Precipitation of DNA was carried out by using isopropanol and 100 μL of 1× Tris-EDTA (pH 8.0) buffer was used to resuspend it. The extracted DNA was checked for its concentration and purity using the spectrophotometer at 260 and 280 nm (BioSpectrometer, Eppendorf, Germany). Specific primers for antibiotic-resistant genes were used to amplify the isolated DNA (Table 1). Polymerase chain reaction (PCR) was carried out in a 25-μL reaction mixture with 40 ng of DNA, 40 pmol of primer, 200 μM of deoxynucleotide triphosphates, 1.0 U of Taq DNA polymerase (HiMedia Laboratories Pvt. Ltd.), and 3 μL of 10X buffer. The PCR programming was with an initial denaturation of 5 minutes at 94°C, followed by 35 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 55°C, and extension for 1 minute at 72°C (Bio-Rad T100, United States). A 1.5% agarose gel was used for electrophoresis and documented using a gel documentation system.

Antibiotic Susceptibility Test

Antibiotic susceptibility testing of 64-wound and 36-sputum isolates against imipenem revealed greater resistance in sputum isolates (75%) followed by wound isolates (43.7%). More number of wound isolates (51%) was found to be sensitive to imipenem. Studies in the past have also observed resistance of P. aeruginosa strains to imipenem. Premanadham et al (2016)¹⁸ reported a resistance of 25.8%, Kaur et al. (2016)⁶ with 17.8%, Ahmed (2016)¹⁹ with 25%, and Lakum et al (2016)²⁰ with 21.6% resistance to imipenem in P. aeruginosa isolates. In comparison to previous studies, our study found a substantial increase in resistance in P. aeruginosa against imipenem. This could be attributed to the overuse and misuse of imipenem in treating infections with P. aeruginosa or ineffective infection prevention and control which has led to an increase in resistance to this drug. The transfer of resistance genes could be vertical, where the daughter cells inherit the genes, or horizontal, when the genetic material is shared between different species.²¹ The presence of more number of sputum isolates being resistant to imipenem could be attributed to the source of the sample from which it was isolated. For instance, carbapenem resistance is higher in isolates from respiratory infection than other sites of infection.²² Eighty-six percent of the sputum and 82% wound isolates were resistant to colistin. The number of isolates resistant to colistin was more as compared with imipenem. The resistance observed against colistin in our study was significantly greater than the studies of the past. In studies by Tahmasebi et al (2020)²³ and Abd El-Baky et al (2020),²⁴ P. aeruginosa isolates have shown resistance of 3.5 and 21.3%, respectively.

Modified Hodge Test

All the isolates that showed resistance to imipenem by disc diffusion method were further confirmed by Hodge test. The increased growth of the indicator strain toward the carbapenem disk and the clover leaf-like indentation at the spot where the isolate and the indicator strain intersected were signs of a positive result. Of the 55 isolates (wound = 28 and sputum = 27) subjected to the test, all the isolates were positive indicating the elaboration of the carbapenemase enzyme conferring them with resistance to the antibiotic (Supplementary Fig. S1). In the present investigation, all the 55 isolates were positive for the test, whereas studies by Jayalakshmi and Pandurangan (2016)²⁵ and Datta et al (2017)²⁶ observed a positive result in nearly 50% of the isolates. The increase in the number of isolates producing carbapenemase enzyme as observed in the present study might be correlated with the rise in the horizontal transfer of plasmid between the isolates carrying genes encoding the enzyme. Moreover, horizontal gene transfer has been shown to play an important role in the spread of multidrug resistance in gram-negative bacteria.²⁷

Molecular Detection of Antibiotic-Resistant Genes

All the isolates resistant to imipenem and colistin were screened for the presence of carbapenem- (bla_VIM, bla_NDM, bla_OxA-48) and colistin- (mcr-1, mcr-5) resistant genes. The representative gel images and prevalence of resistance genes are given in Table 2 and Fig. 1. All the resistant isolates of wound and sputum were found to possess bla_OXA-48 gene. None of the sputum and wound isolates were found to harbor bla_NDM. Of the 28 resistant wound isolates, bla_OXA-48 was observed in 20 isolates, and 8 isolates were shown to be with both bla_OXA-48 and bla_VIM. None of the wound isolates was found to be with only bla_VIM. The same trend was seen in sputum isolates also. Of the 27 sputum isolates, bla_OXA-48 was found in 20 of the strains, and bla_OXA-48 and bla_VIM was seen in 5 isolates. In contrast to our findings, Datta et al (2017)²⁶ observed the presence of 74 and 34% of isolates with bla_VIM and bla_OXA-48, respectively. The higher prevalence of OXA-48 and VIM as seen in the current investigation is following a study by Suwantarat and Carroll (2016)²⁸ who has shown these genes as the predominant carbapenemases occurring in Southeast Asia.

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Fig. 1

Prevalence of carbapenem- and colistin-resistant genes in Pseudomonas aeruginosa (A) wound and (B) sputum isolates.

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Table 2 Representative agarose gel electrophoresis image of PCR amplification of antibiotic-resistant genes

Gene	M L1 L2 L3 L4 L5 L6 L7	Product size (bp)
blaVIM		390
blaOXA		599
mcr-5		650
mcr-1		375

Abbreviations: L1-L6, bacterial isolates; L7, negative control; M, 100 bp marker; PCR, polymerase chain reaction.

Of the 53 resistant wound isolates, mcr genes (mcr-1 and mcr-5) were found in 30 isolates. In 18 isolates, only mcr-5 gene was found and in 4 isolates, only mcr-1 gene was observed. In eight isolates, both mcr-1 and mcr-5 were found. Of the 27 resistant sputum isolates, mcr-5 was noticed in 10 strains and 4 isolates were with mcr-1. Both mcr-1 and mcr-5 were detected in six isolates. The number of isolates with mcr-5 gene exceeded that of mcr-1. The mcr genes widely reported in Enterobacteriaceae are very rare in P. aerugi-nosa.²⁹ Although, P. aeruginosa strains have been recognized to be with carbapenemase genes, resistance to polymyxins and the detection of mcr genes is not frequent. As the study has indicated the presence of mcr genes in a large number of isolates, their detection becomes very important to improve the treatment of infected cases with MDR and XDR P. aeruginosa strains. The lower prevalence of mcr-1 gene in the isolates could be possibly a mechanism to reduce changes in the membrane permeability that occurs due to phosphoe-thanolamine transferase encoded by mcr-1 gene that alters the structure of lipopolysaccharide making it susceptible to hydrophobic antibiotics.³⁰ In both wound and sputum isolates, there was a coexistence of colistin and carbapenemase genes in 16 and 31% of the strains, respectively. Similar results have been observed in a study by Chen et al (2022) ³¹ where the isolates coharbored bla_NDM-1 and mcr-1 genes.