Evaluation of Three Carbapenemase-Phenotypic Detection Methods and Emergence of Diverse VIM and GES Variants among Pseudomonas aeruginosa Isolates in Tunisia

Background: Since 2012, few reports on the molecular epidemiology of Pseudomonas aeruginosa were reported in Tunisia. Objectives: This study aimed to evaluate carbapenem-resistance determinants and molecular epidemiology and to compare the carbapenemase-phenotypic detection methods of multidrug-resistant P. aeruginosa isolates. Methods: During a period of four years (2014 to 2017), all imipenem-ceftazidime-resistant P. aeruginosa isolates were retrospectively selected at the microbial laboratory of Charles Nicolle hospital of Tunis. These isolates were examined by the modified Hodge test, modified carbapenem inactivation method (mCIM), and another mCIM, called CIMTris, and their performance was evaluated using PCR analysis as the gold standard. Results: A total of 35 isolates were recovered among patients hospitalized in different units. All strains were colistin-susceptible.All carbapenem-resistant isolates showed a high-level resistance to carbapenems. CIMTris and mCIM showed 96.15% and 46.15% sensitivity and 44.44% and 100% specificity, respectively, for detecting carbapenemase production.Conclusions: CIMTris is a promising approach for detecting carbapenemase activity in P. aeruginosa and merits further testing. Moreover, this study described the first detection of GES-5- and GES-9-producing P. aeruginosa in Tunisia as well as the co-occurrence of the blaGES-5 and blaVIM-11 carbapenemase genes in one isolate. These findings are of great concern because the rapid dissemination of MDR strains represents a major therapeutic and epidemiological threat.


Introduction
Pseudomonas aeruginosa is an important pathogen that causes various opportunistic and acute nosocomial-acquired infections, especially in immunocompromised patients. Its high intrinsic resistance and ability to develop multidrug resistance produce serious therapeutic problems. Carbapenems have been considered first-line agents to treat severe cases of P. aeruginosa infections [1]. Resistance to carbapenems can stem from the production of carbapenemases or other mechanisms such as mutation in the oprD gene, the overproduction of cephalosporinases, the over-expression of effluxpumps, or combinations of these mechanisms [2].
It should be noted that detecting carbapenemases is more difficult in P. aeruginosa compared to Enterobacteriaceae [10]. Currently, several phenotypic methods are available, but most of them are unsuitable for clinical laboratories to perform on a routine basis [10,11]. Thus, standardized carbapenemase detection methods using routine phenotypic screening tests are still controversial.
Since 2012, few reports on the molecular epidemiology of P. aeruginosa were reported in Tunisia [6]. The aim of our study was, therefore, to evaluate changes in carbapenemresistance determinants and molecular epidemiology and to compare carbapenemasephenotypic detection methods of multidrug-resistant P. aeruginosa isolates recovered at Charles Nicolle Hospital of Tunis, Tunisia, during a 4-year period.

Bacterial Isolates
A total of 80 imipenem-ceftazidime-resistant P. aeruginosa (ICRPA) isolates were retrospectively selected from the frozen stocks kept in brain heart infusion with 20% glycerol at −80 • C at the microbial laboratory of Charles Nicolle hospital of Tunis during a period of four years (2014 to 2017).
From frozen stock, each isolate was subcultured twice on tryptic soy agar (Biorad, Marnesta la Coquette, France), incubating each subculture in ambient air at 35 • C ± 2 • C for 18 to 24 h to ensure purity and viability. Thus, only 35 non-duplicated clinical ICRPA isolates were successfully subcultured and included in this study.
The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [M100-S25]. Colistin MICs were determined by the broth microdilution method using a commercialized kit (UMIC, Biocentric, Bandol-France).
Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains in antimicrobial susceptibility testing and MICs.

Phenotypic Detection of Carbapenemase Production
The phenotypic detection of carbapenemases was performed by a modified Hodge test, modified carbapenem inactivation method (mCIM), and another mCIM, called CIMTris, which uses 0.5 M Tris-HCl buffer rather than water for extraction, as previously reported [10]. All carbapenemase phenotypic methods were assessed twice by different raters.
The performance of the carbapenemase phenotypic tests was evaluated using PCR analysis for bla VIM and bla GES as the gold standard (Table 1) [12]. Phenotypic method sensitivities and specificities were calculated according to Ilstrup [12,13].

Gene Primer a Sequence (5 -3 ) c Product Size (bp) b Reference
a F, sense primer; R, antisense primer. b Nucleotide numbering begins at the initiation codons of genes. c D = A or G or T; Y = C or T.
The testing of mCIM in ICRPA isolates showed that 12 of 26 (46.15%) isolates harboring carbapenemase genes were positive on mCIM. All the 14 mCIM-negative isolates harbored acquired carbapenemase genes. Nine of the nine(100%) isolates not harboring acquired carbapenemase genes were negative on mCIM. Thus, mCIM showed a sensitivity of 46.15% and a specificity of 100% for detecting carbapenemase activities (Tables 5 and 6).

Discussion
In our study, most of the collected ICRPA strains were isolated from low respiratory samples in ICU patients, confirming the data of a previous study [17]. It has been reported that most of the nosocomial infections caused by carbapenemase-producing P. aeruginosa (CPPA)most frequently affect patients with pneumonia associated with mechanical ventilation, and this is the main cause of chronic respiratory infection in immunocompromised patients.
Our study revealed that all isolated ICRPA remained susceptible only to colistin, indicating the dissemination of multidrug-resistant (MDR) strains and an emerging problem in our hospital. The problem of bacterial resistance to commonly used antibiotics is worldwide [2,5,8,17]. The management of ICRPA infections represents a difficult therapeutic challenge due to the increasing resistance levels of these organisms to most classes of antimicrobial agents.
The acquisition of carbapenemase genes by P. aeruginosa is an important cause of MDR, and therefore, the rapid and correct detection of carbapenemases is crucial [2]. Molecular methods are the gold standard in the identification of carbapenemase-producing strains, but phenotypic methods have been developed due to the high cost of molecular methods and their inability to detect new carbapenemase genes [2]. However, some phenotypic assays are still not accepted for non-fermentative Gram-negative bacilli [10]. In this study, we evaluate the performance of three phenotypic methods in the detection of carbapenemaseproducing P.aeruginosa, including the modified Hodge test, mCIM, and CIMTris. The CIMTris differed from the mCIM by the Tris-HCl buffer that was used instead of water during the MEM inactivation step. The CIMTris showed markedly higher sensitivity than the mCIM and modified Hodge test (96.1% vs. 46.1 and 12.5%). The Tris-HCl buffer used in the CIMTris seemed to effectively extract carbapenemases of class A and B. However, the specificity of the CIMTris was lower than the other two methods.This could probably be explained by the degradation of meropenem by other carbapenemases that werenot detected in this study.Our results show that CIMTris is useful, simple, and accessible to clinical laboratories for detecting carbapenemase production in ICRPA, but PCR is needed to confirm the presence of carbapenemase-encoding genes, as previously reported [10].
Of the 35 ICRPA strains, 26 were harboring carbapenemase-encoding genes, and 16 strains were carrying class A beta-lactamases. The coexistence of bla GES-5 and bla VIM-11 was found in one strain. Thus, our results show a predominance of bla GES-5 , which is not in agreement with earlier studies carried out in Tunisia [6,8] and worldwide [2][3][4] that showeda dissemination of bla VIM-2 . Interestingly, we report here for the first time in North Africa the emergence of bla GES-5 and bla GES-9 harboring CPPA isolates thathad been reported in European [18,19], Asian [20], South African [21], and South American [22] studies.
The high levels of resistance among isolated P. aeruginosa, especially in ICUs where there are critically ill patients who underwent invasive procedures using multiple devices and broader spectrum antibiotics, emphasizes the need for measures to prevent the clinical dissemination of these isolates. A similar scenario was described by Koutsogiannou et al., who also reported the clonal dissemination of MDR P. aeruginosa in a university hospital [23].

Conclusions
Carbapenemase enzymes among P. aeruginosa isolates in Tunisia remain poorly investigated. This study reveals new information about carbapenemase enzymes among P. aeruginosa isolates in Tunisia by demonstrating the first detection of of GES-5 and GES-9 carbapenemases as well as the co-occurrence of bla GES-5 and bla VIM-11 in one isolate. Moreover, the CIMTris is a promising approach for detecting carbapenemase activity in P. aeruginosa and merits further testing. These findings are of great concern because the rapid dissemination of MDR strains represents a major therapeutic and epidemiological threat and requires the implementation of strict hygiene procedures and regular surveillance studies.  Informed Consent Statement: Patient consent was waived, only specimen from routine diagnostic were used.