Molecular and phenotypic evaluation of carbapenem resistance in Pseudomonas aeruginosa strains isolated from hospital infections

Authors

Abstract

Aim This study was planned to investigate the resistance pattern of P. aeruginosa isolated from nosocomial specimens and to find the most appropriate method to detect metallo-beta-lactamase resistance by comparing the MBL E-test and Modified Hodge test with PCR.
Materials and Methods In this study, 29 P.aeruginosa strains isolated from various clinics between June 2010 to June 2012 at Mersin University Research and Application Hospital, which were considered to be the causative agents of nosocomial infection according to CDC criteria and were considered to be resistant to carbapenem group antibiotics, were analyzed. Antibiotic susceptibility tests, metallo-beta-lactamase E-test, modified Hodge test, and polymerase chain reaction (PCR) tests were performed. In statistical analyses, the relationships between categorical data were analyzed using chi-square or Likelihood Ratio test statistics.
Results As a result of PCR experiments, the VIM-1 gene region responsible for metallo β-lactamase production was isolated in 11(37.9%). strains IMP-1, IMP-2 and VIM-2 gene regions were negative.
Discussion The results obtained in our study are consistent with other studies in Turkey and around the world. It is concluded that there is a need for well- standardized phenotypic tests with well-defined evaluation criteria and genotypic confirmation of these tests to reveal MBL production.

Keywords

Introduction

Pseudomonas aeruginosa is a prevalent gram-negative nosocomial infection. Particularly in individuals with immunodeficiency, it is a significant contributor to mortality due to its pronounced biofilm formation ability [1].
Given the restricted treatment alternatives and elevated morbidity and mortality rates linked to carbapenem-resistant P. aeruginosa (CRPA) infections, the World Health Organization has identified P. aeruginosa in its priority list of critical pathogens that urgently require new antibiotics, thereby underscoring the imperative for planning studies aimed at developing novel antimicrobial agents [2]. The primary acquired resistance mechanism contributing to carbapenem resistance is metallo-beta-lactamases (MBL) [3]. Verona integron-encoded metallo-β-lactamase (bla-VIM) and imipenemase (bla-IMP) are the most prevalent metallo-β-lactamase genes and are worldwide distributed. The proliferation of MBL-producing bacteria is anticipated to precipitate a range of worldwide issues in the future [4]. Metallo-beta-lactamases (MBL) are categorized in Class B of the Ambler classification and Group 3 of the Bush classification. These metalloenzymes are categorized into subgroups based on their structure or function. MBL activity relies on zinc and can be suppressed by EDTA or mercaptopropionic acid [4]. Metallo-beta-lactamases (MBLs) exhibit a high rate of hydrolysis for broad-spectrum beta- lactam antibiotics. Metalloenzymes exhibit effective hydrolysis of imipenem and meropenem, but show limited activity against aztreonam. Carbapenemases are enzymes that hydrolyze carbapenems via the zinc ion present in their active sites [5]. Routine tests for the identification of infrequently observed carbapenemases are conducted using various methods, such as carbapenem inactivation method-based (CIM-based) phenotypic tests or immunochromatographic tests to ascertain the presence of carbapenemase enzymes. Furthermore, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (available at: https://www.eucast.org/ast_of_ bacteria) and the Clinical and Laboratory Standards Institute (CLSI) (available at: https://clsi.org/standards/products/ microbiology/documents/m100/) recommend tests that assess carbapenem hydrolysis activity. It is established that molecular techniques, particularly real-time PCR analyses, serve as the reference standard for identifying carbapenemase genes [7]. This study aimed to identify the optimal method for detecting metallo-beta-lactamase resistance by examining the resistance patterns of P. aeruginosa isolated from nosocomial samples. It compared disk diffusion tests, E-tests, MBL E-tests, and Modified Hodge tests with PCR.

Materials and Methods

This study investigated 29 P. aeruginosa strains, identified as causative agents of nosocomial infections according to CDC criteria and recognized for their resistance to carbapenem antibiotics. These strains were isolated from various clinics at Mersin University Research and Application Hospital over 2 years, between June 2010 to June 2012. P. aeruginosa isolates were identified using classical microbiological techniques and the automated commercial system VITEK 2 (bioMérieux, France). The isolates were maintained at -70°C in broth medium with 10% glycerol until the study commenced.
Antibiotic Susceptibility Tests
The antibiotic susceptibilities of the strains were determined using disk diffusion and E-test methods. The susceptibilities of the isolated strains to imipenem were assessed using the Kirby- Bauer disk diffusion method, following EUCAST guidelines. Standard strains E. coli ATCC 25922 and P. aeruginosa 27853 were utilized for quality control purposes [9]. Phenotypic identification of metallo-beta-lactamase Numerous tests have been developed to date for the detection of metallo- beta-lactamases in routine microbiology laboratories, and they have been developed by utilizing the inhibition of these enzymes in the presence of metal chelators such as EDTA or 2-mercaptopropionic acid.
MBL E-Test
The E-test (AB Biodisk, Solna, Sweden) employed in this study is a commercially available assay designed for the phenotypic identification of the metallo-beta-lactamase enzyme. A test is deemed positive for the metallo-beta-lactamase enzyme if the MIC value of imipenem+EDTA diminishes by at least eight- fold relative to the MIC value of imipenem. Another criteria for a positive test is the identification of a pattern known as the “ghost zone” in the central region of the E-test strip, which lacks imipenem or EDTA [6]. The quality control of the test was conducted using metallo-beta-lactamase-positive and metallo- beta-lactamase-negative bacteria employed in previous assays.
Modified Hodge Test
The “Modified Hodge Test” (MHT) differs from previous tests as it does not rely on the inhibition of the metallo-beta- lactamase enzyme by EDTA. The MHT operates on the basis that a bacterial isolate sensitive to imipenem can proliferate in the presence of imipenem when coexisting with a metallo- beta-lactamase generating bacterium [10]. E. coli ATCC 35218 was utilized as the imipenem-susceptible bacterial strain in this assay (available at: https://www.ecdc.europa.eu/sites/ default/files/documents/surveillance-antimicrobial-resistance- Europe-2018.pdf ). A uniform inoculation of the E. coli strain’s bacterial suspension was performed on MHA with a sterile swab. After the medium surface had dried, a meropenem disk (10μg) was positioned at the center of the medium. Bacterial colonies were obtained using a sterile swab from the fresh culture of the P. aeruginosa isolate for testing on blood agar, followed by a thick bacterial streak extending from one end of the meropenem disk to the border of the medium. Thus, each plate was examined for a total of two isolates, one serving as a positive control. After inoculation, the plates were incubated at 35°C for 18 hours. The reduced inhibitory zone related to the E. coli strain in the area of P. aeruginosa proliferation, along with the presence of E. coli growth in this region, was regarded as a favorable test outcome.
Molecular Analysis of Resistance Genes by Polymerase Chain Reaction (PCR)
The investigation assessed whether P. aeruginosa strains deemed imipenem-resistant by the disk diffusion technique had the IMP-1, IMP-2, VIM-1, and VIM-2 resistance genes through PCR analysis. The target areas for gene investigation and the corresponding primer sequences were derived from the research conducted by Shibata et al (2003) in Table 1 [12].
Following the amplification of gene areas associated with metallo-β-lactamase synthesis using PCR, the resultant products were subjected to electrophoresis, visualized under a UV transilluminator at a wavelength of 312 nm, and documented using gel imaging equipment.
Statistical Analysis
Statistical analyses assessed correlations between categorical variables with the chi-square or Likelihood Ratio test statistic. The sensitivity, specificity, positive predictive value, and negative predictive value of the E-Test, MHT, and MBLE-Test (IMPEDTA) were determined based on PCR positivity. The Mann- Whitney U test was employed to assess differences in age, duration, and treatment duration between the PCR-negative and PCR-positive groups. Statistical analyses were conducted using SPSS version 11.5.1 and MedCalc version 11.5.1 software tools. In statistical analysis, a p-value of less than 0.05 indicates significant results.
Ethical Approval
This study has been approved by the Mersin University Clinical Research Ethics Committee (Date: 2009-11-25, No:2009-26).

Results

Isolates
The investigation comprised 29 P. aeruginosa isolates obtained from various clinical specimens: tissue 14(48.3%), urine 4(13.8%), deep tracheal aspirate 4(13.8%), pleural fluid 3(10.3%), sputum 1(3.4%), blood 1(3.4%), and abscess 2(9.8%). The MBL E-test (IMP-EDTA) was utilized as one of the methods for phenotypic determination of metallo-beta-lactamase in the isolates examined in the study. A total of 29 isolates were subjected to the E-test. Of the 29 isolates subjected to the E-test, 6(20.7%) exhibited imipenem: imipenem+EDTA MIC ratios of ≥8. The samples were assessed as MBL positive and were found to phenotypically produce metallo-beta-lactamase. Of the 29 isolates, 23(79.3%) were assessed as MBL negative and were found not to phenotypically produce metallo-beta- lactamase. Figure 1 shows an isolate identified as phenotypically producing metallo-beta-lactamase via the E-test.
Modified Hodge Test
The MHT is another assay employed for the phenotypic identification of metallo-beta-lactamase in P. aeruginosa isolates. Among the 29 isolates analyzed in the research, as shown in Figure 2 “6(20.7%)” tested positive using the modified Hodge test, whereas “23(79.3%)” tested negative. All six isolates that tested positive by MHT were also confirmed positive by the EDTA-containing E-test.
Genotypic Demonstration of Metallo-Beta-Lactamase Enzyme PCR tests resulted in the detection of the VIM-1 gene region associated with metallo-β-lactamase synthesis in 11(37.9%) strains. The gene regions IMP-1, IMP-2, and VIM-2 were determined to be absent. The bands generated from the PCR tests are seen in Figure 3.
Comparisons of PCR with Other Tests
Positive predictive value (PPV) an be defined as the number of true positives divided by the sum of true positives and false positives. PPV is related to the sensitivity and specificity of the test. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MBL for the detection of carbapenemase production were 54%, 100%, 100%, and 78.3%, respectively. (Table 2).
The relationship between the MHT and PCR was found to be statistically significant, P=0.001. The sensitivity of MHT was determined to be 54% and the specificity was 100%. The PPV was calculated as 100% and the NPV as 78.3% as shown in Table 3. 100% concordance was found between the results of the two tests performed.

Discussion

Pseudomonas aeruginosa is a microorganism prevalent in the environment. While it is not typically part of the human microbiota, it has the capacity to colonize the gastrointestinal tract, upper respiratory tract, or skin flora, particularly in hospitalized patients. This pathogen is associated with hospital-acquired opportunistic infections that exhibit high mortality rates, especially in intensive care units. Despite a significant rise in carbapenem resistance across European countries in prior years, recent data indicate a notable decline in these trends. Despite the overall decrease, country-specific analyses reveal that higher resistance percentages are reported in numerous southern and eastern European nations compared to their northern counterparts [12]. The World Health Organization reported a carbapenem resistance rate of 37% among Pseudomonas aeruginosa isolates in Turkey as part of the Central Asia and Eastern Europe Antimicrobial Resistance Surveillance (CAESAR 2017) (available at studyhttps://apps.who. int/iris/bitstream/handle/10665/344085/9789289052252- eng.pdf?sequence=2&isAllowed=y ) The primary mechanism underlying the development of resistance to carbapenems is the presence of carbapenemases. Carbapenemases are beta- lactamases that effectively hydrolyze at least one carbapenem, a subgroup of the beta-lactam class, including imipenem or meropenem. The most clinically significant group of enzymes is the Group B carbapenemases, that is, metallo-β-lactamases (MBLs), due to their resistance to almost all β-lactam antibiotics [14].
Among the several classifications of metallo-beta-lactamases, the IMP (imipenemases) and VIM (Verona integron-encoded MBL) enzymes are the most commonly identified [15].
The accelerated global dissemination of IMP- or VIM-type MBLs in recent years can be primarily attributed to the fact that the genes encoding these enzymes are frequently located on mobile genetic elements, such as integrons. These genes can disseminate horizontally between different strains due to their transposon- or plasmid-mediated mobility. It has been demonstrated that, owing to their mobility, these organisms are capable of conferring resistance not only to carbapenems but also to all beta-lactams, aminoglycosides, and fluoroquinolones. [16, 17]. This study examined the presence of metallo-beta- lactamase (MBL) production genes in Pseudomonas aeruginosa, focusing on the prevalence of blaIMP-1 and blaVIM-1 genes in isolates through genotypic and phenotypic methods. The production of MBL enzymes was detected at a rate of 20.7% using two distinct phenotypic methods. Ejikeugwu et al. (2018) reported MBL positivity rates of 34.9% using the phenotypic method and 27.9% via PCR [18]. In our study, this rate was determined to be 37.9% through PCR analysis. This study confirmed the phenotypic production of metallo-β-lactamase (MBL) enzymes in 15 (34.9%) isolates of P. aeruginosa. Multiplex PCR technique that demonstrated MBL production in 27.9% (12 isolates) of the P. aeruginosa isolates possessing the blaVIM-1 MBL genes [18]. VIM-type variants are prevalent in strains obtained from Europe and Southeast Asia. The evolutionary trees of VIM-type enzymes are organized according to amino acid similarity. Approximately 41 variants have been identified. The two clearly delineated primary clusters are VIM-1 and VIM-2. VIM-1 and VIM-2 have 90% amino acid similarity. VIM- 2 is the predominant variant of the VIM family and has been identified in several European nations [19].
Studies conducted in our country to date indicate that VIM-type MBLs are the most commonly reported types in P. aeruginosa isolates. Bahar et al.(2004) isolated VIM-5 from an imipenem- resistant Pseudomonas aeruginosa strain, whereas Malkoçoglu et al.(2017) isolated VIM-1, VIM-2. In our study, VIM-1 positivity was observed in 37.9% of cases. [20, 21]. Conversely, numerous studies assessing phenotypic methods for detecting MBL production indicate that both MHT and the E-test demonstrate comparable effectiveness [22].
Given that MHT is a straightforward and cost-effective test, it remains applicable in areas where carbapenemases predominate. It is important to recognize that the interpretation of MHT is inherently subjective and necessitates a considerable amount of time for accurate detection. Furthermore, additional research involving larger sample sizes is necessary to validate the efficacy of these tests. The E-test MBL strip is a quantitative method for the phenotypic detection of metallo-β-lactamases (MBLs), utilizing a combination of a β-lactam substrate and a β-lactam or MBL inhibitor. Numerous studies in the literature have evaluated the performance of the MBL E-test. Khosravi et al. (2012) and Behera et al. (2008) reported that the MBL E-test demonstrated a sensitivity of 91.3% and a specificity of 100% when compared to PCR for detecting MBL production [23, 24]. In another study, however, the sensitivity of the MBL E-Test was found to be 91.3%, and the specificity was found to be 100% [21].
In alignment with other published studies, our research determined the sensitivity of the MBL E-Test to be 54% and the specificity to be 100%. The positive predictive value (PPV) was determined to be 100%, while the negative predictive value (NPV) was calculated at 78.2%. This study contributes to the literature by demonstrating that the MBL E-test exhibits high sensitivity in detecting MBLs. The E-test demonstrates high sensitivity for the detection of metallo-beta-lactamases (MBLs) in Pseudomonas aeruginosa and is the sole method that facilitates the determination of minimum inhibitory concentration (MIC). However, due to cost constraints and availability issues, it may not be feasible for all laboratories to conduct the E-test.

Limitations

The limitation of our study includes its retrospective natüre and the limited number of patients.

Conclusion

This study provides epidemiological data that will inform strategies for understanding the antimicrobial resistance patterns in P. aeruginosa clinical isolates in Turkey and for controlling the dissemination of carbapenemases. PCR was found to be more effective than the MBL E-test and MHT. It indicates that dependable, straightforward, rapid, and economical phenotypic tests suitable for routine use in clinical diagnostic laboratories in developing countries remain effective for detecting carbapenemases. While no single phenotypic test has been established that fulfills all criteria of an ideal test, it would be advantageous to enhance research on novel phenotypic techniques applicable in clinical microbiology laboratories of varying scales.

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