Abstract
Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous opportunistic organism that is hard to treat. This study aimed to investigate the association of blaVIM, blaIMP, and blaNDM prevalence with Cyclic di-GMP (c-di-GMP) in P. aeruginosa. To this end, 27 clinical isolates of P. aeruginosa were obtained from different hospitals in Baghdad, Iraq. The phenotypic detection of carbapenem and biofilm assays was performed by the M63 minimal medium, supplemented with glucose, magnesium sulfate. The polymerase chain reaction was utilized to detect carbapenem genes. The results showed that the isolates were highly resistant to Imipenem (37%) and Meropenem (63%). Imipenem (37%) and Meropenem (63%) demonstrated a moderate sensitivity against P. aeruginosa. The P. aeruginosa No.5 showed high resistance to carbapenem by blaVIM+, blaIMP+, and blaNDM+, followed by a robust biofilm confirmed with c-di-GMP levels and the twitching motility ability. Upon these findings, the use of antibiotics should be restricted to severe bacterial infections to avoid the rapid emergence of new resistant isolates, which leads to the hard treatment of infection with P. aeruginosa. It is highly recommended that these findings be notified for infectious control. Future studies can investigate the link between transferable resistant genes and c-di-GMP values.
Keywords: Cyclic di-GMP, blaVIM, blaIMP, blaNDM, Pseudomonas aeruginosa
1. Introduction
Pseudomonas aeruginosa (P. aeruginosa) is an important principled nosocomial pathogen. This organism is highly resistant to antimicrobials and causes difficulty in treating infections ( 1 ). Due to the small number of efficient antimicrobials as the intrinsic resistance, resistance is acquired through mutations and plasmids ( 2 ). The most important factor of resistance would be transferable resistance agents, such as Metallo-ß-lactamases (MβL) ( 3 ). It is well documented that carbapenem is pivotal to the treatment of MβL-resistant bacteria ( 4 ).
As a case of resistance genes, blaVIM, blaIMP, and blaNDM genes could be a plasmid or chromosomal region of class 1 integron used to encode blaVIM gene beside aminoglycoside-modifying genes. The global dissemination of MβL genes is considered a clinically important issue caused by integron. The emergence of isolates resistant to multiple antimicrobial agents is due to the ability of P. aeruginosa to merge variable resistance genes, which leads to strictly lowering the perspective treatment options of infections ( 5 , 6 ).
The P. aeruginosa in a biofilm could tolerate 1,000-fold antibiotic concentrations when compared to the same bacteria in an individual, free-living, planktonic state ( 7 ). As a result, within these concentrations, it may not clear biofilm infections, allowing the bacterial population to persist, recover, and spread ( 8 ). The biofilm formation, polysaccharide, and pellicle in P. aeruginosa are regulated by Cyclic di-GMP (c-di-GMP) ( 9 ). The c-di-GMP is a second messenger that promotes biofilm formation and contribution to pathogenic infection, as well as multidrug resistance in several Gram-negative pathogens. Therefore, the elevation of c-di-GMP in P. aeruginosa leads to the initial bacterial infection. On the other hand, low concentrations of c-di-GMP decrease the development of biofilm and infection ( 10 ). This study, therefore, attempted to investigate the association of blaVIM, blaIMP, and blaNDM with the level of c-di-GMP in clinical isolates of P. aeruginosa that form strong biofilm.
2. Materials and Methods
2.1. Collection and Identification
Twenty-seven clinical isolates of P. aeruginosa were collected and diagnosed in this study. Bacteria were isolated from different infected patients at three major teaching hospitals in Baghdad (Al-Kindey, Al-Kadhymiaand, and Ibn-Albalady). Five isolates were obtained from individuals suffering from urinary tract infections, seven isolates from sputum patients suffering from respiratory tract infections, five isolates from burn infections, five isolates from wound infections, and five isolates from bacteremia. The criteria described previously were applied for bacterial identification ( 11 ).
2.2. Antibiotic Susceptibility Tests
An antimicrobial susceptibility test was conducted on all isolates using the disc diffusion method by the CLSI ( 12 ). The study used two types of carbapenem discs: Imipenem and Meropenem. After 18 h, the inhibition zone’s diameter was measured, followed by its comparison with P. aeruginosa ATCC 27853 as the control strains.
2.3. Molecular Examination
The polymerase chain reaction (PCR) was used to detect three common carbapenems by a bacterial genomic DNA extraction kit (Bioneer, Daejeon, Korea). The oligonucleotide primer sequences used to detect the three genes were as follows: blaVIM (390 bp, amplified size), by VIM-F 5ʹ- GAT GGT GTT TGG TCG CAT A-3ʹ and VIM-R 5ʹ- CGA ATG CGC AGC ACCAG-3ʹ, blaIMP (740bp, amplified size) by IMP-F 5ʹ- TGA GCA AGT TAT CTG TAT TC -3ʹ and IMP-F 5ʹ- TTA GTT GCT TGG TTT TGA TG -3ʹ, as well as blaNDM (621 bp, amplified size) by NDM-F5ʹ- GGT TTG GCG ATC TGG TTT TC-3ʹ and NDM-R 5ʹ- CGG AAT GGC TCA TCA CGA TC-3ʹ, according to Wu, Lin ( 13 ), as well as Christensen, Simpson ( 14 ). Both positive and negative controls were used in the PCR.
2.4. Biofilm Quantification Assay
Biofilm formation assay was quantified as previously described by Musafer, Kuchma ( 15 ). The M63 minimal medium supplemented with glucose, Magnesium sulfate, and CAA was used to perform all biofilm assays.
2.5. Measurement of C-di-GMP
The c-di-GMP measurement was performed, according to the method previously described by Musafer ( 16 ). Within 18 h, the P. aeruginosa culture growth medium (1L of LB) was centrifuged at 250 rpm. Afterward, formaldehyde was added to prevent the degradation of c-di-GMP, and then, it was centrifuged for 10 min at 8,000 rpm at 4˚C. The nucleotide extraction was performed according to the method described by Amikam, Steinberger ( 17 ).
2.6. Statistical Analysis
Error bars±standard deviations of averages were measured for three independent experiments. Statistical analysis was performed using correlation coefficient (r) and LSD0.05 using the Microsoft EXCEL application (2016).
3. Results and Discussion
The sensitivity test for Imipenem and Meropenem antibiotics showed that the inhibition zone value of the resistant isolates was greater than the breakpoint, as defined by CLSI ( 12 ) while it would be susceptible if it was less than the breakpoint. The results in table 1 clearly showed that 27 isolates of P. aeruginosa were tested for carbapenem susceptibility, including Imipenem and Meropenem discs, which had a different range of resistance. Table 1 showed that No.6, No.19, and No.21 of P. aeruginosa isolates were resistant to Imipenem antibiotic whereas P. aeruginosa No.5, No.7, No.12, No.13, and No.19 were resistant to Meropenem antibiotic. The remarkable point is that P. aeruginosa No.19 showed resistance to both Imipenem and Meropenem. As Riera, Cabot ( 18 ) indicated in their study, the resistance of clinical isolates may contribute to the impermeability resistance, modification enzyme coded by plasmid or chromosome, the mutation in efflux pump that uptakes antibiotic, new carbapenem resistance mechanism, and target side changing for the antibiotic action. Resistance to Imipenem and Meropenem acquired by MβL genes are located on mobile genetic elements, such as plasmids or transposons, thereby enabling widespread dissemination ( 19 ).
Table 1.
The carbapenem sensitivity test results P. aeruginosa isolates
| Clinical Isolates | Clinical Source | Imipenem | Meropenem | ||||
|---|---|---|---|---|---|---|---|
| S | I | R | S | I | R | ||
| 23 | 20-22 | 19 | 23 | 20-22 | 19 | ||
| PAO1 Wild type | Wild type | - | - | - | - | - | - |
| P. aeruginosa#1 | Sputum | - | 21 | - | 25 | - | - |
| P. aeruginosa#2 | - | 22 | - | 30 | - | - | |
| P. aeruginosa#3 | - | 22 | - | 26 | - | - | |
| P. aeruginosa#4 | - | 22 | - | 30 | - | - | |
| P. aeruginosa#5 | - | 20 | - | - | - | No I.Z | |
| P. aeruginosa#6 | Urine | - | - | 19 | - | 20 | - |
| P. aeruginosa#7 | 24 | - | - | - | - | 15 | |
| P. aeruginosa#8 | 25 | - | - | 25 | - | - | |
| P. aeruginosa#9 | 23 | - | - | 25 | - | - | |
| P. aeruginosa#10 | - | 21 | - | 23 | - | - | |
| P. aeruginosa#11 | - | 22 | - | 30 | - | - | |
| P. aeruginosa#12 | 23 | - | - | - | - | 18 | |
| P. aeruginosa#13 | 26 | - | - | - | - | 18 | |
| P. aeruginosa#14 | 25 | - | 30 | - | - | ||
| P. aeruginosa#15 | 21 | - | 32 | - | - | ||
| P. aeruginosa#16 | 25 | - | - | 34 | - | - | |
| P. aeruginosa#17 | 22 | - | 20 | - | |||
| P. aeruginosa#18 | 34 | - | - | 24 | - | - | |
| P. aeruginosa#19 | Blood | - | - | 14 | - | - | No I.Z |
| P. aeruginosa#20 | - | 22 | - | 30 | - | - | |
| P. aeruginosa#21 | Burn | - | - | 18 | - | 21 | -- |
| P. aeruginosa#22 | - | 22 | - | 30 | - | - | |
| P. aeruginosa#23 | Wound | - | 20 | - | - | 21 | - |
| P. aeruginosa#24 | - | 20 | - | - | 22 | - | |
| P. aeruginosa#25 | - | 20 | - | 25 | - | - | |
| P. aeruginosa#26 | 23 | - | - | 30 | - | - | |
| P. aeruginosa#27 | 24 | - | - | 30 | - | - | |
| 37% | 51% | 11% | 62.9% | 18.5% | 18.5% | ||
R, resistant; I, intermediate; S, sensitive; I.Z, Inhibition zone
As shown in table 2, there are two main findings. First, all carbapenem-resistant isolates showed a positive prevalence of blaVIM. Second, P. aeruginosa No.5, No.7, No.12, No.13, and No.19 were blaVIM, blaIMP, and blaNDM positive, resistant to Meropenem, and sensitive to Imipenem. On the other hand, P. aeruginosa No.21 and No.1 were negative for blaIMP and blaNDM. The findings have been compared to the P. aeruginosa (wild type) standard strain for P. aeruginosa. These results were in agreement with previous studies mentioning that resistance to Meropenem was related to Metallo-carbapenems encoded by carbapenem genes, and resistance to Imipenem antibiotic alone was related to porins gene ( 20 ).
Table 2.
Carbapenem resistance pattern and the existence of related genes
| Isolates | Carbapenem pattern | bla VIM | bla IMP | bla NDM | |
|---|---|---|---|---|---|
| PAO1 | IPM-S | MEM-S | - | - | - |
| P. aeruginosa#5 | IPM-I | MEM-R | + | + | + |
| P. aeruginosa#7 | IPM-S | MEM-R | + | + | + |
| P. aeruginosa#12 | IPM-S | MEM-R | + | + | + |
| P. aeruginosa#13 | IPM-S | MEM-R | + | + | + |
| P. aeruginosa#19 | IPM-R | MEM-R | + | + | + |
| P. aeruginosa#21 | IPM-R | MEM-I | + | - | - |
| P. aeruginosa#1 | IPM-I | MEM-S | + | - | - |
| Percentage | 57% | 86% | 100% | 71% | 71% |
PAO1: Pseudomonas aeruginosa, IPM-S: Imipenem-Sensitive, IPM-I: Imipenem-Intermediate, IPM-R: Imipenem-Resistance, MEM-S: Meropenem-Sensitive, MEM-I: Meropenem-Intermediate, MEM-R: Meropenem-Resistance, P. aeruginosa: Pseudomonas aeruginosa
The present study found that the biofilm producer in P. aeruginosa No.5, No.7, No.12, No.13, and No.19 had all carbapenem genes blaVIM+, blaIMP+, and blaNDM+ positive, compared to P. aeruginosa No.6, No.11, No.26, and No.18 with negative carbapenem genes. The correlation coefficient (r) was estimated at 0.96, meaning there is a strong positive correlation between the biofilm formation values and the presence of blaVIM+, blaIMP+, and blaNDM+ genes, as shown in table 3. These findings were in line with the findings of a study by Drenkard and Ausubel ( 21 ), pinpointing that the antibiotic-resistant variants of phenotypic P. aeruginosa could promote biofilm formation in the lungs of cystic fibrosis patients and in in vitro. The horizontal gene transmission within bacteria turns into a biofilm producer and multidrug resistance mechanisms against beta-lactam, aminoglycosides, and fluoroquinolones antibiotics ( 22 ).
Table 3.
Biofilm formation and carbapenem genes of Pseudomonas aeruginosa isolates
| Isolates No. | Biofilm values (A490 ± SDb) | Carbapenem gene patterns |
|---|---|---|
| PAO1 | 0.11±0.0169 | blaVIM-, blaIMP-, blaNDM- |
| P. aeruginosa#5 | 0.92±0.0124 | blaVIM+, blaIMP+, blaNDM+ |
| P. aeruginosa#7 | 0.92±0.0141 | blaVIM+, blaIMP+, blaNDM+ |
| P. aeruginosa#12 | 0.85±0.0249 | blaVIM+, blaIMP+, blaNDM+ |
| P. aeruginosa#13 | 0.85±0.0205 | blaVIM+, blaIMP+, blaNDM+ |
| P. aeruginosa#19 | 0.80±0.0169 | blaVIM+, blaIMP+, blaNDM+ |
| P. aeruginosa#21 | 0.53±0.0163 | blaVIM+, blaIMP-, blaNDM- |
| P. aeruginosa#1 | 0.54±0.0124 | blaVIM+, blaIMP-, blaNDM- |
| P. aeruginosa#6 | 0.35±0.0163 | blaVIM-, blaIMP-, blaNDM- |
| P. aeruginosa#11 | 0.36±0.0235 | blaVIM-, blaIMP-, blaNDM- |
| P. aeruginosa#26 | 0.37±0.0216 | blaVIM-, blaIMP-, blaNDM- |
| P. aeruginosa#18 | 0.24±0.0518 | blaVIM-, blaIMP-, blaNDM- |
P. aeruginosa: Pseudomonas aeruginosa
PAO1: Pseudomonas aeruginosa
Concentrations of the cellular c-di-GMP of P. aeruginosa were measured as follows: P. aeruginosa No.5, No.11, and No.26 were measured by high performance liquid chromatography (HPLC), as stated previously by Musafer ( 16 ). Figure 1 shows c-di-GMP levels (0.02 pmol/mg) in standard P. aeruginosa. The lowest c-di-GMP levels (0.007pmol/mg and 0.008 pmol/mg) in P. aeruginosa were found in No.5 and No.26, respectively, whereas the highest level was 0.03 pmol/mg in P. aeruginosa No. 5. The finding confirmed that the high biofilm (blaVIM+, blaIMP+, and blaNDM+) exists in P. aeruginosa No.5. In addition, the experiment on twitching motility confirmed the results of high biofilm since twitching is an important step in biofilm formation ( 15 ). Based on the findings, this study showed significant levels of c-di-GMP in isolates with a high biofilm producer and the three carbapenem genes in P. aeruginosa clinical isolates. These results are in agreement with the findings of Musafer ( 16 ) on c-di-GMP.
Figure 1.
Cellular c-di-GMP’s quantification and concentrations by HPLC from 30 mg of cells. A significant difference was considered when P<0.05
Imipenem (37%) and Meropenem (63%) demonstrated a moderate sensitivity to P. aeruginosa, as a clinically important antibiotic. The P. aeruginosa No.5 showed high resistance to carbapenem by blaVIM+, blaIMP+, and blaNDM+, followed by a robust biofilm that confirmed c-di-GMP levels and the twitching motility ability (Figure 2). Upon these findings, using antibiotics should be restricted to severe bacterial infections to avoid the rapid emergence of new resistant isolates, which leads to the hard treatment of infection with P. aeruginosa. It is highly recommended that these findings be notified by infectious control departments. Future studies can investigate the link between transferable resistance genes and c-di-GMP values.
Figure 2.
Explanation of the ability of Pseudomonas aeruginosa with a strong biofilm of twitching motility
Authors' Contribution
Study concept and design: H. K. M. and F. N. J.
Acquisition of data: H. K. M.
Analysis and interpretation of data: M. B. A. A.
Drafting of the manuscript: M. B. A. A.
Critical revision of the manuscript for important intellectual content: H. K. M. and F. N. J.
Statistical analysis: M. B. A. A.
Administrative, technical, and material support: H. K. M.
Ethics
The study protocol was approved by the medical ethics board of the Al-Mustansiriyah University, Ministry of Higher Education and Research, Baghdad, Iraq. Written informed consents were provided by all the subjects participated in the study
Conflict of Interest
The authors declare that they have no conflict of interest.
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