Distribution of Pseudomonas-Derived Cephalosporinase and Metallo-β-Lactamases in Carbapenem-Resistant Pseudomonas aeruginosa Isolates from Korea

Abstract

The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-$\beta$-lactamases (MBLs) and AmpC $\beta$lactamases are responsible for high-level resistance to carbapenem and cephalosporin. We studied the diversity and frequency of $\beta$-lactamases and characterized chromosomal AmpC $\beta$lactamase from carbapenem-resistant P. aeruginosa isolates. Sixty-one carbapenem-resistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. Minimum inhibitory concentrations (MICs) of four antimicrobial agents were determined using the agar-dilution method. Polymerase chain reaction and sequencing were used to identify the various $\beta$-lactamase genes, class 1 integrons, and chromosomally encoded and plasmid-mediated ampC genes. In addition, the epidemiological relationship was investigated by multilocus sequence typing. Among 61 carbapenem-resistant P. aeruginosa isolates, 25 isolates (41.0%) were MBL producers. Additionally, 30 isolates producing PDC (Pseudomonas-derived cephalosporinase)-2 were highly resistant to ceftazidime (MIC₅₀ = $256{\mu}g/ml$) and cefepime (MIC₅₀ = $256{\mu}g/ml$). Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin and carbapenem resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (ceftazidime, p < 0.001; cefepime, p < 0.001; imipenem, p = 0.003; meropenem, p < 0.001). The coexistence of MBLs and AmpC $\beta$-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of $\beta$-lactamases.

Introduction

Pseudomonas aeruginosa is a major opportunistic pathogen responsible for hospital-acquired infections and is notorious for its capacity to develop resistance to multiple classes of β-lactams. Recently, carbapenems have been shown to be the most important and effective therapeutic options against serious infections caused by these pathogens, but resistance to these agents is increasingly reported worldwide [1, 2, 32].

Carbapenem resistance in P. aeruginosa is mainly due to a combination of different factors: low outer membrane permeability, overexpression of the efflux pump MexABOprM, hyperproduction of derepressed AmpC chromosomal β-lactamase, and the presence of transferable resistance determinants, in particular, carbapenem-hydrolyzing enzymes [6, 9, 19, 20]. Carbapenem-hydrolyzing enzymes are divided into two types based on molecular classification: serine enzymes, which are derivatives of class A or D enzymes, and metallo enzymes, which belong to class B [25].

Serine carbapenemases of the Klebsiella pneumoniae carbapenemase (KPC), Guiana extended-spectrum (GES), and oxacilinase (OXA) families have been occasionally reported in this species in certain parts of the world, whereas metallo-β-lactamases (MBLs), particularly Verona imipenemase (VIM), and IMP (active against imipenem) types, are the most widespread and have been reported globally [4, 12, 26, 29].

In addition to carbapenem-hydrolyzing enzymes, another important mechanism of resistance to β-lactams in P. aeruginosa is the production of chromosomal AmpC β-lactamases, which can be induced or derepressed to confer high-level penicillin and cephalosporin resistance [27]. Inducible AmpC can be upregulated by subinhibitory concentrations of certain β-lactams. Furthermore, mutations can occur in the regulatory components of AmpC, leading to stable hyperproduction of AmpC with concomitant high-level resistance to many classes of β-lactams [21, 24]. Several chromosomally mediated Pseudomonas-derived cephalosporinases (PDCs) with extended-spectrum cephalosporinase activities have been reported among P. aeruginosa [23]. There are several reports on the prevalence of MBL genes and molecular epidemiology in carbapenemresistant P. aeruginosa isolates from Korea, but the contribution of other mechanisms to carbapenem resistance such as class A and D β-lactamase and PDC genes is unknown.

The aim of this study was to determine the diversity and frequency of β-lactamases and characterize chromosomal AmpC β-lactamase in carbapenem-resistant P. aeruginosa isolates obtained from a tertiary hospital in Korea during a 4-year period. In addition, we investigated the epidemiological relationship and potential correlations between genetic characteristics and resistance to carbapenems.

 

Materials and Methods

Bacterial Isolation and Identification

A total of 61 consecutive and non-duplicated carbapenemresistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. The isolates were identified with the Vitek 2 automated ID system (BioMérieux, Hazelwood, MO, USA), and carbapenemresistant P. aeruginosa isolates were selected based on resistance to imipenem and meropenem.

Antimicrobial Susceptibility Testing

In the antimicrobial susceptibility tests, the minimum inhibitory concentration (MIC) was determined by using the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [3]. Four antimicrobial agents were tested, including imipenem, meropenem, ceftazidime, and cefepime (Sigma-Aldrich, St. Louis, MO, USA). The interpretation of susceptibility was performed according to the CLSI breakpoints. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains.

Multilocus Sequence Typing

Multilocus sequence typing (MLST) was performed according to the methods described on the P. aeruginosa MLST database website (http://pubmlst.org/paeruginosa/). PCR and sequencing were performed for seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE). The nucleotide sequences of these genes were compared with the sequences submitted to the MLST database to determine the allelic numbers and sequence types (STs).

Identification and Analysis of β-Lactamase Genes and Integrons

PCR assays were performed to amplify the sequence of MBLs, including the blaIMP, blaVIM, blaGIM, blaSPM, blaSIM, blaNDM, blaAIM, blaDIM, and blaFIM genes, as described previously (Table 1) [17, 18]. PCR detection of class A and D β-lactamase genes (blaTEM, blaSHV, blaGES, blaVEB, blaKPC, blaPSE, blaPER, blaCTX-M-1,2,9 group, blaOXA-I,II,III group, blaOXA-23, blaOXA-24, blaOXA-48, blaOXA-51, and blaOXA-58) and class 1, 2, and 3 integrons was also performed as previously described [7, 11, 20, 31]. Sequence analyses were confirmed with the BLAST program at the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/). The structure of variable regions of integrons was determined by PCR mapping and sequencing.

Table 1.Oligonucleotides used as primers for amplication and sequencing in this study.

Genotypic Detection and Sequencing of Chromosomally Encoded and Plasmid-Mediated ampC Gene

The genotypes of all 61 carbapenem-resistant P. aeruginosa isolates were analyzed for the presence of chromosomal PDC genes and for different families of plasmid-mediated ampC genes by multiplex PCR as described previously [15, 23]. Amplified PCR products were purified and sequenced; the results of DNA sequencing were compared with known β-lactamase gene sequences using the BLAST program.

Statistical Analysis

The data were analyzed using SPSS ver. 21.0 (SPSS, Chicago, IL, USA) with one-way analysis of variance. The differences were considered statistically significant at p < 0.05.

 

Results

MLST Analysis of Carbapenem-Resistant P. aeruginosa

Among the 61 carbapenem-resistant P. aeruginosa isolates, the sites of isolation were sputum (29 isolates, 47.5%), urine (22 isolates, 36.1%), blood (4 isolates, 6.6%), wounds (3 isolates, 4.9%), bile (2 isolates, 3.3%), and pus (1 isolate, 1.6%) (Table 2). A total of 61 carbapenem-resistant P. aeruginosa isolates were identified as 17 different STs by MLST experiments. ST235 (30 isolates, 49.2%) was the most frequently detected clone. According to frequency, five other detected STs were ST245 (5 isolates, 8.2%), ST654 (5 isolates, 8.2%), ST357 (4 isolates, 6.6%), ST111 (3 isolates, 4.9%), and ST257 (3 isolates, 4.9%). The remaining 11 STs (ST179, ST195, ST244, ST267, ST274, ST589, ST645, ST708, ST1062, ST1455, and ST1663) were each represented by one isolate (1.6%).

Table 2.Abbreviations: ST, sequence type.

Prevalence of β-Lactamases

Of the 61 isolates, 10 isolates (16.4%) harbored OXA-type and 2 (3.3%) harbored Pseudomonas-specific enzyme (PSE)-type enzyme (Table 3). Of the OXA β-lactamases,OXA-10was the most prevalent, followed by OXA-1 and OXA-2. Fourteen isolates (23.0%) harbored two different β-lactamases, and 12 isolates (19.7%) harbored three enzymes. Of these, MBL genes were identified in 25 isolates (41.0%) harboring blaOXA-1. Two MBL genes, blaIMP-6 and blaVIM-2, were identified in 22 (36.1%) and 3 isolates (4.9%), respectively. All 22 isolates carrying the blaIMP-6 gene belonged to ST235 and three isolates carrying the blaVIM-2 gene belonged to ST357.

Table 3.revalence of Ambler class A, B, and D β-lactamases in 61 carbapenem-resistant Pseudomonas aeruginosa isolates.

Structure of Class 1 Integrons

Class 1 integrons were detected in 36 (59.0%) of the 61 carbapenem-resistant P. aeruginosa isolates and no class 2 or 3 integrons were found. The gene cassettes found in this study were divided into six types (Type A, B, C, D, E, and F) according to the cassette composition (Table 4). Type A (4.0 kb), obtained in 18 isolates, carried the aadB-cmlA-blaOXA-10-aadA1 gene cassette. Eleven isolates of type B (5.5 kb) belonged to ST235 and carried blaIMP-6 -qac-aacA4-blaOXA-10- aadA2. Type C (1.8 kb), found in two isolates, carried aacA4-blaOXA-2-orfD, and type D (1.2 kb) had aadA6-orfD (2 isolates). Two isolates contained type E (1.0 kb) carrying the aadA4 gene cassette. Type F (2.5 kb) was detected in only one isolate and carried aadA4-blaOXA-10- aadA2.

Table 4.Schematic representation of gene cassette structures located in the class 1 integron isolated from 61 carbapenem-resistant Pseudomonas aeruginosa isolates.

Identification of AmpC Variants

On performing PCR for the presence of chromosomal ampC gene, all 61 isolates of P. aeruginosa harbored PDC gene while the plasmid-mediated ampC gene was not present. Sequencing analysis of the PCR product of the chromosomal ampC gene revealed that 61 isolates obtained six variants (PDC-1, PDC-2, PDC-3, PDC-5, PDC-7, and PDC-8) (Table 5).

Table 5.Abbreviations: N, number of isolates; CAZ, ceftazidime; CFP, cefepime; IPM, imipenem; MEM, meropenem.

The most frequent variant was PDC-2 (30 isolates, 49.2%) and PDC-3 was the second most frequently detected variant (10 isolates, 16.4%). Thirty isolates producing PDC-2 were highly resistant to ceftazidime (MIC50 = 256 µg/ml) and cefepime (MIC50 = 256 µg/ml). Meanwhile, the remaining 31 isolates showed full or intermediate susceptibility, with the ceftazidime MIC50 ranging from 8 to 16 µg/ml. Additionally, 25 isolates harboring MBL genes were highly resistant to ceftazidime (MIC range 64 to >256 µg/ml), cefepime (MIC range 64 to >256 µg/ml), imipenem (MIC range >256 µg/ml), and meropenem (MIC range >256 µg/ml) (Table 6). In contrast, 36 isolates that did not contain MBL genes showed relatively low resistance, with MICs ranging from 2 to 128 µg/ml for ceftazidime, 2 to >256 µg/ml for cefepime, 8 to >256 µg/ml for imipenem, and 8 to >256 µg/ml for meropenem.

Table 6.Abbreviations: N, number of isolates; CAZ, ceftazidime; CFP, cefepime; IPM, imipenem; MEM, meropenem

 

Discussion

Resistance to β-lactams (particularly carbapenem and cephalosporin) in P. aeruginosa has been increasingly reported worldwide, and this is also the case in Korea. According to previous Korean National Surveillance Antimicrobial Resistance (KONSAR) studies, from 2005 to 2011, resistance rates of P. aeruginosa to imipenem increased from 19% to 26%, and to ceftazidime increased from 19% to 23% [33]. This study analyzed various genes of P. aeruginosa isolates that are responsible for resistance to carbapenems. Class D OXA β-lactamases were more frequently detected than class A in P. aeruginosa (16.4% versus 3.3%). In particular,OXA-10was only observed in ST235 isolates (19 isolates), and was accompanied by blaIMP-6 in 12 isolates (63.2%). Similarly, a previous study found that 35 (60.3%) of 58 OXA-10-producing isolates harbored blaIMP-6 and/or blaVIM-2, and belonged to only ST235 [1]. In addition, 25 (41.0%) carbapenem-resistant P. aeruginosa isolates were MBL producers.

Compared with a previous study, the rates of MBL production showed a 2.5-fold increase from 16.2% in 2008– 2012 to 41.0% in 2011–2014 among carbapenem-resistant P. aeruginosa isolates [2]. Among the 22 ST235 IMP-6-producing isolates, 11 isolates (50.0%) shared an identical class 1 integron with a gene cassette array (blaIMP-6-qac-aacA4-blaOXA-1-aadA1) between the 5’ and 3’ conserved sequence. The blaVIM-2gene was identified in three isolates of ST357.

Mechanisms of drug resistance in AmpC β-lactamase can either be chromosomally or plasmid-mediated. The majority of AmpC β-lactamases are chromosomally mediated and are found in Serratia, Pseudomonas, Acinetobacter, Citrobacter, and Enterbacter spp. Chromosomally mediated resistance is due to mutation(s) in the bacterial DNA, and such genes are not easily transferable to other bacterial species [5, 8]. In the present study, six variants of the chromosomally mediated ampC enzyme PDC were identified in all 61 carbapenem-resistant P. aeruginosa isolates. The most frequent AmpC-type variant was PDC-2, containing the substitutions G27D, A97V, T105A, and V205L. Substitutions in this region have been previously linked to the broadening of the enzyme’s hydrolytic spectrum, facilitating the degradation of compounds such as ceftazidime [10, 22]. In our study, 30 isolates harboring PDC-2 exhibited high levels of resistance to ceftazidime (MIC50 = 256 µg/ml) and cefepime (MIC50 = 256 µg/ml). Additionally, of the 30 isolates, 20 (66.7%) belonged to ST235 and most of the ST235 isolates were recovered from urine (14 isolates, 70%). Another study from France reported 10 variants of a PDC (PDC 1-10) gene, in which several variants showed reduced susceptibility to ceftazidime, cefepime, and imipenem [23, 28].

Plasmid-mediated AmpC β-lactamases can spread laterally, making them transferable to other bacteria. Therefore, they are frequently seen in many bacterial species such as E. coli, K. pneumonia, Salmonella spp., Citrobacter freundii, Enterobacter aerogenes, and Proteus mirabilis [5, 16]. In this study, we were unable to detect plasmid-mediated ampC genes, which is consistent with a previous study by Wang et al. [30], who reported that no plasmid-mediated ampC genes were detected among 258 carbapenem-resistant P. aeruginosa isolates.

Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin (MIC range for ceftazidime and cefepime, 64 to >256 µg/ml) and carbapenem (MIC range for imipenem and meropenem, >256 µg/ml) resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (MIC range for ceftazidime, 2–128 µg/ml; cefepime, 2 to >256 µg/ml; imipenem and meropenem, 8 to >256 µg/ml). These results highlight the importance of MBL genes in cephalosporin and carbapenem resistance in P. aeruginosa (ceftazidime, cefepime, and meropenem, p < 0.001; imipenem, p = 0.003).

Similarly, Bae et al. [1] reported that production of IMP-6 and VIM-2 MBLs is the main mechanism for acquiring resistance to ceftazidime and carbapenems in P. aeruginosa isolates. In addition, Neyestanaki et al. [11] found that the production of MBLs and AmpC β-lactamases was the major emerging mechanism of resistance to carbapenem among P. aeruginosa isolates in Teharan, Iran. Similarly, combinations of various β-lactamases have recently been reported in studies from India, Brazil, Italy, and Argentina [13, 14].

In conclusion, the coexistence of MBLs and AmpC β-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of β-lactamases.