Abstract
Objectives:
Pseudomonas aeruginosa occupies a central position in nosocomial infections and remains a significant cause of morbidity and mortality. The aim of this study was to characterize carbapenem resistance mechanisms in P. aeruginosa isolates from clinical specimens collected at the University Hospital of Oran, western Algeria.
Materials and Methods:
The identification of 214 nonduplicated P. aeruginosa isolates (collected from January to December 2016) was confirmed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirteen antibiotics were tested using the disc diffusion method. Carbapenemase-encoding genes were detected with the GeneXpert system and multiplex polymerase chain reaction (PCR). Clonal relatedness was determined using multilocus sequence typing (MLST) and the seven housekeeping genes were further used for phylogenetic analysis of imipenem-resistant P. aeruginosa using concatenated gene fragments. The flanking regions of the blaVIM-4 gene were analyzed by whole-genome sequencing.
Results:
Eleven isolates (5.39%) were resistant to carbapenems. PCR amplification and sequencing showed that six of these isolates (2.94%) harbored the blaVIM-4 gene that was carried on a novel class 1 integron. MLST analysis assigned the tested isolates to seven different sequence types (STs), of which two were new (ST3349 and ST3350) and five were previously described (ST244, ST499, ST709, ST809, and ST1239).
Conclusion:
In this study, we reported P. aeruginosa isolates producing VIM-4 in an Algerian hospital. The blaVIM-4 is harbored in class 1 integron with a new arrangement of genes cassettes.
Introduction
Pseudomonas aeruginosa is a gram-negative bacterium widely diffused in the environment. It is an opportunistic human pathogen that can cause a wide range of acute and chronic infections, particularly in immunocompromised patients. It is one of the leading nosocomial pathogens. 1 Indeed, P. aeruginosa is the bacterium more frequently associated with ventilator-associated pneumonia and burn wound infections, and is a major cause of nosocomial bacteremia, with very high mortality rate (>30%). 2
P. aeruginosa is not only naturally resistant to many antibiotics but can also develop resistance to a wide range of antimicrobial agents. 3 The development of this resistance could be resulted either from the mutation of natural genes or the acquisition of foreign resistance genes via the horizontal gene transfer mediated by mobile genetic elements such as plasmids, transposons, and integrons. 4 Moreover, genomic islands are frequently mosaic, possessing features from multiple different mobile genetic elements, and contain several antibiotic resistance genes. 5 Since the 1980s, carbapenems have been recommended as last-resort drugs for the treatment of infections caused by this pathogen. 6 However, their intensive use has favored the spread of carbapenem-resistant P. aeruginosa (CRPA). Carbapenem resistance in P. aeruginosa has been most often associated with efflux pump protein overexpression and outer membrane porin OprD loss, and less frequently with carbapenemase production.7,8 Different classes of carbapenemases are produced by CRPA, mostly metallo-β-lactamases (MBL), including VIM, imipenem (IMP), AIM, SPM, GIM, SIM, DIM, and NDM, as well as KPC- and OXA-type enzymes. 6
Nosocomial outbreaks caused by MBL-producing P. aeruginosa have been reported in several countries; however, in Algeria, only few data are available. The aim of this study was to describe the prevalence and characterize CRPA isolates recovered from clinical specimens in the University Hospital of Oran, western Algeria.
Materials and Methods
Sample collection and strain identification
This study was carried out at the University Hospital of Oran, Algeria from January to December 2016. This hospital has a capacity of 1,085 beds that are distributed in 32 services with ∼60,000 hospitalizations per year. Among the 2,111 consecutive clinical specimens sent to the microbiology laboratory, 214 nonduplicated P. aeruginosa isolates were identified using first biochemical tests and then matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Bruker Daltonics, Bremen, Germany).
Antibiotic susceptibility testing
Antimicrobial susceptibility testing was performed using the disk diffusion method on Müller-Hinton agar and the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (version 9.2) on the EUCAST website. The following antibiotics were tested: ticarcillin (TIC, 75 μg), ticarcillin-clavulanic acid (TCC, 85 μg), piperacillin (PIP, 30 μg), piperacillin-tazobactam (TZP, 36 μg), imipenem (10 μg), meropenem (MEM, 10 μg), ceftazidime (CAZ, 10 μg), cefepime (FEP, 30 μg), aztreonam (ATM, 30 μg), gentamicin (GEN, 10 μg), tobramycin (TOB, 10 μg), amikacin (AMK, 30 μg), and ciprofloxacin (CIP, 5 μg).
The presence of carbapenemase was investigated phenotypically by the modified Carbapenem Inactivation Method (mCIM) as previously described. 9
Molecular characterization of carbapenems resistance mechanisms
In P. aeruginosa isolates showing decreased susceptibility to carbapenems, the presence of blaVIM, blaKPC, blaOXA-48, blaIMP, and blaNDM-1 was assessed with the GeneXpert system (Cepheid®) according to the manufacturer's instructions. 10 In addition, blaGES, blaSIM, blaIMP, and blaGIM were detected by multiplex polymerase chain reaction (PCR) followed by sequencing. 11
PCR amplification of oprD among carbapenem-resistant isolates was performed using specific primers. 12 Sequences were compared with that of reference P. aeruginosa PAO1.
Multilocus sequence typing
P. aeruginosa strains were genotyped by multilocus sequence typing (MLST) using seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE), as previously described. 13 Sequence alignment and analysis were done at the P. aeruginosa MLST website.
Genetic environment of blaVIM
In VIM-producing P. aeruginosa isolates, the presence of integrons was detected by quantitative PCR (qPCR), as previously described. 14 Two strains (Nos. 332 and 306) were characterized also by Whole-Genome Sequencing (WGS) with the Ion Proton™ technology (Thermo Fisher Scientific), according to the manufacturer's instructions. Reads were assembled using the Mimicking Intelligent Read Assembly (MIRA) software. Contigs were analyzed with the Geneious software (Biomatters).
The nucleotide sequence data reported in this study have been submitted to the GenBank database under the accession number MN395487.
Results
Antibiotic resistance in P. aeruginosa isolates
Among the 214 P. aeruginosa isolates identified, 11 showed resistance to imipenem and were further characterized (Table 1). These isolates were isolated from different clinical specimens and from different wards (Table 1).
Phenotypic and Genotypic Features of the 11 Imipenem-Resistant Pseudomonas aeruginosa Isolates
AMK, amikacin; CAZ, ceftazidime; FEP, cefepime; GEN, gentamicin; IMP, imipenem; MEM, meropenem; NA, not available; PIP, piperacillin; ST, sequence type; TCC, ticarcillin-clavulanic acid; TOB, tobramycin; TZP, piperacillin-tazobactam.
Among the eleven imipenem-resistant P. aeruginosa isolates, eight (72.72%) isolates showed resistance to ticarcillin, seven (63.63%) isolates were resistant to gentamicin, tobramycin, and ticarcillin-clavulanic acid, six (54.54%) isolates were resistant to piperacillin-tazobactam, ceftazidime, cefepime, meropenem, and piperacillin, and five (45.45%) isolates were found resistant to amikacin (Table 1). All strains were susceptible to aztreonam and ciprofloxacin. The presence of carbapenemase was observed in 6 of the 11 isolates tested by the mCIM.
Characterization of carbapenemase-encoding genes and blaVIM-4 genetic environment
Molecular characterization and sequencing revealed the presence of a unique type of MBL corresponding to blaVIM-4 in six isolates. In the other five isolates, no carbapenemase genes were detected. To investigate for mutations of the oprD gene, the entire gene was amplified and sequenced for these five strains. As a result, these strains were found to have frameshift mutations (1-bp nucleotide insertion) of the oprD gene compared with the reference oprD gene of PAO1. The qPCR showed the presence of integrons in the six isolates producing VIM-4. WGS performed on two of the VIM-producing isolates showed that they harbored the same complex class 1 integron containing four gene cassettes (Fig. 1). In this integron (In1777), the blaVIM-4 gene cassette is in first position, followed by aacA7 [a gene encoding aminoglycoside N(6′)-acetyltransferase type 1 that causes resistance to tobramycin and amikacin], cmlA5 (a gene encoding an efflux transporter that leads to chloramphenicol resistance), and a cassette with two fused genes (blaOXA-10 that encodes an oxacillinase leading to narrow-spectrum β-lactam resistance, and aadA1 that encodes aminoglycoside nucleotidyltransferase causing resistance to streptomycin and spectinomycin). The IS26 gene is located upstream of the In1777 integron, and the dfrA23 gene (resistance to trimethoprim) is located downstream of the ISCR1 gene.
Schematic structure of the class 1 integron harboring the blaVIM4 cassette in Pseudomonas aeruginosa No. 332 strain. blaVIM-4 gene cassette encoding MBL VIM-4, aacA7 gene cassette encoding aminoglycoside N(6′)-acetyltransferase type 1, cmlA5 gene cassette encoding an efflux transporter that leads to chloramphenicol resistance, blaOXA-10 gene cassette encoding an oxacillinase leading to narrow spectrum β-lactam resistance, aadA1 gene cassette encoding an aminoglycoside nucleotidyltransferase. The IS26 is located upstream, and the dfrA23 gene (resistance to trimethoprim) is located downstream of the ISCR1 gene. MBL, metallo-β-lactamases.
Genotyping
MLST analysis showed that the 11 isolates resistant to imipenem could be classified in 7 sequence types (ST) (Table 1), of which 5 were previously described (ST809 [n = 3], ST449 [n = 3], ST244 [n = 1], ST709 [n = 1], and ST1239 [n = 1]) and 2 were new (ST3349 and ST3350). Concerning P. aeruginosa ST809, analysis of patients' medical records suggested a probable nosocomial outbreak among these three patients being in the same hospital at the same time.
Discussion
The emergence of CRPA strains is a public health issue worldwide. In the present study, CRPA prevalence in Oran University Hospital was 5.14%. CRPA prevalence in the Gulf Cooperation Council countries varied from 3% to 21% between 2002 and 2015. 15 In African countries, CRPA prevalence was highest in Egypt (51% of 586 isolates), Libya (56% of 36 isolates), and Algeria (50% of 398 isolates), and lowest in Tunisia (19% of 3,119 isolates) and Morocco (28% of 155 isolates). 15 Previous studies in Algeria reported different CRPA prevalence rates: 39.33% between 2009 and 2012 in western Algeria, 16 and 18.75% in eastern Algeria between 2012 and 2013. 17
In our research, MBL production was detected in 6 isolates (2.8% of the 214 isolates analyzed, and 54.54% of the 11 imipenem-resistant isolates). The molecular characterization of these six isolates showed that they all produced VIM-4. The blaVIM-4 gene cassette was first identified in a P. aeruginosa isolate in Greece in April 2001. This gene is a variant of blaVIM-1, with one nucleotide difference resulting in a serine to arginine substitution at position 175 of the VIM-1 sequence. 18 In their review, Moghnieh et al. reported that MBL production (mostly VIM and IMP) is the most important mechanism of carbapenem resistance in P. aeruginosa throughout the Arab League countries. 15 In Algeria, few reports on MBL-producing P. aeruginosa have been published. Touati et al. described fourteen VIM-2-producing P. aeruginosa strains in eastern Algeria. 19 In western Algeria, Sefraoui et al. detected clinical P. aeruginosa isolates that produced VIM-2. 16 Meradji et al. 17 and Mellouk et al. 20 reported P. aeruginosa harboring the blaVIM-4 gene in eastern Algeria. Moreover, the transfer between an Algerian and a French hospital of four multidrug-resistant bacterial strains, including VIM-4-producing P. aeruginosa, by a single patient has been reported. 21
Until now, blaVIM genes have been always embedded in class 1 integrons. 22 More than 100 different class 1 integron structures associated with blaVIM have been described in Enterobacteriaceae and nonfermenting isolates. 23 In this study, we described a new cassette array arrangement in the integron containing the blaVIM-4 gene.
The MLST analysis revealed the presence of five already described ST clones (809, 499, 1239, 244, and 709) and also two new STs (ST3349 and ST3350). Thus, none of the STs found in this work is related either to CC111 or CC235. It is reported that CC111 and CC235 have been shown to dominate among MBL-producing P. aeruginosa isolates. 24 ST809 has been already reported in Malesia 25 and Spain, 26 whereas ST1239 was described only in south-west China in carbapenem-sensitive P. aeruginosa strains. 27 ST499 was reported in non-MBL-producing P. aeruginosa strains in Spain25,28 and China.27,29 The ST244 has been identified in several countries 30 and was associated with VIM-2, and extended-spectrum β-lactamases, such as PER-1 and VEB-1. In Algeria, ST244 was reported in Oran 16 and Annaba. 20
Conclusion
In conclusion, the CRPA prevalence in this study was lower than in previous studies. We reported in this study P. aeruginosa-producing VIM-4 isolates in an Algerian hospital. The blaVIM-4 is harbored in class 1 integron with a new arrangement of genes cassettes.
Footnotes
Disclosure Statement
No competing financial interests exist.
Funding Information
This work was supported by the Programme algéro-francais de Bourses doctorales (PROFAS) B+ 2018 (PROFAS B+ to Miss. ZAIDI F.Z.)