PME and Other ESBL-Positive Multiresistant Pseudomonas aeruginosa Isolated from Hospitalized Patients in the Region of Kurdistan,Iraq

Abstract

Nosocomial infections occur worldwide and also in the Kurdistan region. Frequently patients colonized with multiresistant Pseudomonas aeruginosa isolates are encountered in many hospitals. As information is lacking with respect to the mechanisms of resistance responsible for the multiresistant character of the P. aeruginosa isolates and their genetic relationship, isolates were prospectively collected and characterized with respect to their mechanism of resistance. During 2012 and 2013, 81 P. aeruginosa isolates were collected from three teaching hospitals in the city of Erbil, Iraq. Susceptibility testing was performed using the VITEK-2 system. Isolates were screened for the presence of extended-spectrum β-lactamases (ESBLs) and for the presence of metallo β-lactamases (MBLs). The presence of serine carbapenemases was detected by PCR. The genetic relationship of the isolates was demonstrated by amplified fragment length polymorphism (AFLP). Susceptibility results revealed high rates of resistance against all classes of antibiotics except polymyxins. Genetic characterization demonstrated the presence of ESBL-genes, that is, bla_VEB (30%) and bla_PER (17%), also ESBL bla_PME was detected in four isolates. AFLP typing revealed clonal spread of bla_VEB, bla_PER, and three clusters of bla_OXA-10-positive isolates. Only one isolate was MBL (bla_VIM) positive. Of a selected number of isolates (n = 11), whole-genome sequencing analysis revealed that these isolates belonged to “high-risk” MLSTs ST244, ST235, ST308, and ST654. This study reveals the presence and clonal spread of widely resistant high-risk clones of P. aeruginosa in Iraqi Kurdistan. As far as we are aware, this is the first report of multiple, polyclonal, PME producing P. aeruginosa outside the Arabian Peninsula.

Introduction

Pseudomonas aeruginosa is a common cause of opportunistic infections, particularly in patients with burn wounds, cystic fibrosis, and patients on mechanical ventilation. P. aeruginosa infections occur frequently, are difficult to treat, and are associated with substantial mortality and morbidity.¹ Antimicrobial treatment is hampered as P. aeruginosa is intrinsically resistant to multiple classes of antibiotics due to low permeability of the outer membrane combined with efflux pumps and presence of chromosomal β-lactamases.^2–7 Antimicrobial resistance can even increase further by acquisition of genetic elements, for example, plasmids carrying SHV-, TEM-, and CTX-M-type class A extended-spectrum β-lactamases (ESBLs) or by PER-, VEB-, GES-, BEL-, and PME-type ESBLs.^2,8–10 These intrinsic or acquired mechanisms of β-lactam resistance result in ceftazidime and piperacillin resistance. Also, resistance toward last-resort antibiotics such as carbapenems can occur and is mostly due to OprD porin loss or presence of carbapenemase enzymes. Carbapenemases, in particular class B VIM- and IMP-type metallo-β-lactamases (MBLs), are increasingly reported worldwide.¹¹

To get insight into the mechanisms of resistance involved in the multiresistant organisms, we prospectively collected 81 P. aeruginosa isolates from different hospitals and wards in the region of Kurdistan. In the present study, these isolates were characterized with respect to the mechanisms of resistance and genotyped by amplified fragment length polymorphism (AFLP) analysis.

Materials and Methods

Bacterial isolate collection, identification, and susceptibility testing

P. aeruginosa isolates were collected from February 2012 until January 2013 in teaching hospitals Rozhawa, Rizgari, and Hawler, all located in the city of Erbil, Iraq. A total of 81 consecutively obtained P. aeruginosa isolates cultured from a variety of clinical samples (burn wound, urine, skin and soft tissue, blood, aspiration, sputum, and ear swab) were, regardless of the susceptibility results, included in the present study. Identification of all isolates was confirmed by using the MALDI Biotyper system (Bruker, Leiderdorp, Netherlands). Antimicrobial susceptibility was determined using the VITEK^® 2 system (bioMérieux VITEK Systems, Inc., Hazelwood, MO) by means of the AST-N199 panel. EUCAST breakpoints were applied for categorization of the minimal inhibitory concentration (MIC) results. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were included as quality control isolates. The susceptibility for colistin was determined by the broth microdilution method and performed in accordance with the EUCAST guidelines.

Phenotypic β-lactamase screening

The screening for the presence and expression of ESBL and AmpC β-lactamase expression was determined by combined disk tests on Mueller–Hinton (MH) agar (MH II, Becton Dickinson, Franklin Lakes) and MH agar supplemented with 400 mg/L cloxacillin sodium salt (Sigma-Aldrich, St. Louis, MO), using disks containing 30 μg ceftazidime and 30 μg ceftazidime +10 μg clavulanic acid (NEO-SENSI TABS™, Rosco Diagnostica A/S, Taastrup, Denmark). Isolates with an increased zone of inhibition (ZOI) of ≥5 mm with the addition of clavulanic acid were considered ESBL positive. A ≥5 mm increase in ZOI of ceftazidime on cloxacillin-containing plates was interpreted as a derepressed AmpC phenotype.^12,13

Screening for MBLs was performed using doripenem/ethylenediaminetetraacetic acid (EDTA) and imipenem/EDTA-based combined disk tests as previously described.¹⁴

Whole-genome sequencing

Illumina MiSeq 251-bp paired-end sequencing was performed on 11 selected isolates to obtain information as to which PCRs had to be performed on all 81 isolates. The selection of isolates for whole-genome sequencing (WGS) was based on differences in susceptibility for ceftazidime/cefepime in combination with or without reduced susceptibility to imipenem or meropenem combined with the results of the phenotypic ESBL assay. Reads were assembled using a pipeline from Statens Serum Institute based on SPAdes (http://cab.spbu.ru/software/spades).¹⁵ Acquired resistance genes were identified against the ResFinder 2.1 database (https://cge.cbs.dtu.dk/serices/ResFinder).¹⁶ MLST types were identified using MLST (https://github.com/tseemann/mlst). WGS information was also used to obtain information on the organization of the OprD-gene.

PCR detection of β-lactamases

DNA was extracted using InstaGene™ Matrix (Bio-Rad Laboratories, Veenendaal, Netherlands) according to the manufacturer's protocol for DNA preparation from bacteria. PCR amplification was used to detect bla_VEB, bla_PER, bla_BEL, bla_GES, bla_PME, bla_TEM, bla_KPC, bla_OXA-10-like, bla_OXA-48-like, bla_OXA-50-like, bla_VIM, and bla_IMP.^9,17–25 Reactions were performed in 25 μL volumes containing 2.5 μL 10 × Taq buffer (with KCl, Thermo-Fisher Scientific, Landsmeer, Netherlands), 2.5 μL 25 mM MgCl₂ (Thermo-Fisher Scientific), 0.25 μL dNTP Mix (10 mM each, Thermo-Fisher Scientific), 0.25 μL of each primer (50 μM), 0.04 μL Taq DNA polymerase (recombinant, Thermo-Fisher Scientific), 2.5 μL template DNA, and PCR-grade water up to total reaction volume. Thermocycling conditions were 1 min at 95°C, followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, with a final extension step at 72°C for 7 min. PCR products were visualized on 2% agarose gel containing 1:20,000 SYBR^® Safe DNA gel stain (Thermo-Fisher Scientific).

AFLP typing

DNA was extracted using the MagNA Pure 96 DNA and Viral NA Small Volume Kit with the MagNA Pure 96 system (Roche Diagnostics, Almere, Netherlands). Restriction/ligation (R/L) reactions were performed in 20 μL volumes containing 2 μL 10 × T4 DNA ligase buffer (New England Biolabs, Ipswich, MA), 0.2 μL T4 DNA ligase (400 U/μL, New England Biolabs), 0.2 μL HpyCH4IV and MseI (both 10 U/μL, New England Biolabs), 1 μL of each oligonucleotide adapter (50 μM), 2 μL of DNA-extract, and 13.4 μL PCR-grade water. R/L reactions were incubated for 1 hr at room temperature and diluted 1:5 in AE-buffer (QIAGEN, Valencia, CA). PCRs were performed in 20 μL volumes containing 10 μL PCR Master (Roche Diagnostics), 2 μL of each primer (HpyCHIV4-G-FAM, MseI-GG, 10 μM each), 2 μL 1:5 diluted R/L reaction, and 4 μL PCR-grade water. Thermocycling conditions were 30 sec at 56°C, 4 min at 94°C, followed by 50 cycles of 30 sec at 94°C, 30 sec at 66°C (−0.5°C/cycle touchdown to 56°C), and 1 min at 72°C, with a final extension step at 72°C for 60 min. PCR products were diluted 1:10 in PCR-grade water. For fragment analysis, 2 μL 1:10 diluted PCR product was added to 18 μL Hi-Di™ Formamide (Applied Biosystems, Foster City, CA) and 0.1 μL Internal Lane Standard 600 (ILS600; Promega Benelux, Leiden, Netherlands), incubated for 1 min at 95°C, and followed by rapid cooling to 20°C. Fragment analysis was performed on the ABI 3130xl genetic analyzer (Applied Biosystems), using POP-7™ Polymer and a 36 cm 16-capillary array. Electropherograms were normalized and cluster analysis was performed using BioNumerics software, Version 7.5 (Applied Maths, St.-Martens-Latem, Belgium). Similarity coefficients were determined by Pearson correlation and unweighted pair group method with arithmetic mean (UPGMA) algorithms.

Results

Antimicrobial susceptibility and phenotypic β-lactamase screening

Susceptibility determinations revealed high rates of resistance against all tested antibiotics (Table 1). Seventy isolates (86%) could be defined as multidrug resistant (MDR) and 57 (70%) isolates as extensively drug resistant (XDR) according to the European Centre for Disease Prevention and Control criteria.²⁶ The colistin MICs were as follows: 72 isolates with MICs of 0.5 or 1 μg/mL, 6 isolates with MICs of 2 μg/mL, and 3 isolates with MICs of 4 μg/mL. According to the 2018 guidelines, three isolates would be regarded resistant. According to the 2014 guidelines, these three isolates would have been regarded as susceptible. At the moment, only two isolates were susceptible to all tested antibiotics.

Table 1.

Categorization of Susceptibility Results of 81 Pseudomonas aeruginosa Isolates

	Susceptible	Intermediate	Resistant
Antimicrobial agent	No. (%)	No. (%)	No. (%)
Piperacillin/tazobactam	11 (14)	0	70 (86)
Ceftazidime	19 (25)	0	62 (77)
Cefepime	18 (22)	0	63 (78)
Imipenem	19 (25)	6 (7)	56 (68)
Meropenem	15 (19)	21 (26)	45 (56)
Gentamicin	10 (12)	0 (0)	71 (88)
Tobramycin	24 (30)	0 (0)	57 (70)
Ciprofloxacin	26 (32)	0 (0)	55 (68)
Colistin	78 (96)	0 (0)	3 (4)

Susceptibility results were obtained by VITEK 2, and the susceptibility results of colistin were obtained by the broth microdilution technique and subsequently interpreted according to the 2018 EUCAST guidelines.

To obtain information with respect to the mechanisms causing resistance toward ceftazidime, we applied the combined disk test to reveal the presence of ESBLs and derepressed AmpCs. Thirty-eight of 81 isolates (47%) were found to be phenotypically ESBL positive, 12 isolates (15%) showed a derepressed AmpC phenotype, and three isolates (4%) were ESBL positive in combination with a derepressed AmpC phenotype. ESBL and AmpC activity could not be determined for six isolates, because ZOI was absent or too small for interpretation, even with the addition of 400 mg/L cloxacillin. Only one isolate was phenotypically MBL positive in the screening assay.

Molecular characterization

By performing specific PCRs, we inventoried which genes were actually present in these isolates. However, to prevent missing less prevalent genes by PCR, we first selected, on basis of the in the Materials and Methods mentioned criteria, different phenotypes for WGS analysis (n = 11). Numerous acquired resistance genes were identified (Table 2), including multiple β-lactamases and aminoglycoside modifying enzymes.

Table 2.

Resistance Genes for 11 Pseudomonas aeruginosa Isolates Detected by Whole-Genome Sequencing

	Antibiotic class
MLST (n)	β-lactamase	Aminoglycoside	Fluoroquinolone and aminoglycoside	Fosfomycin	Sulfonamide	Trimethoprim	Chloramphenicol	Tetracycline
ST244 (7)	bla _OXA-10	aac(3)-Id, aadA1, aph(3′)-Ib, aph(3′)-IIb		fosA	sul1	dfrB5	catB7	tetA
ST244 (1)	bla _OXA-10	aac(3)-Id, aadA1, aph(3′)-Ib, aph(3′)-IIb		fosA	sul1	dfrB5	catB7, cmlA1	tetA
ST235 (1)	bla_TEM-1B, bla_VEB-1	aac(3)-IIa, aadA6, aph(3′)-IIb	aac(6′)Ib-cr	fosA	sul1		catB7, cmlA1	tetA
ST308 (1)	bla _DHA-1 , bla _OXA-10	aadA1, aph(3′)-Ia, aph(3′)-IIb	aac(6′)Ib-cr	fosA	sul1		catB7, cmlA1	tetA
ST654 (1)	bla _PME-1	aadB, aph(3′)-IIb, aph(3′)-VIa, strA, strB		fosA	sul1		catB7	tetA, tetG

The obtained WGS data provided information on what kind of acquired mechanisms could be expected and guided which additional PCRs were needed for characterization of all collected isolates with respect to the β-lactamase genes. Genes for bla_OXA-10, bla_VEB, bla_PER, and bla_TEM were detected by PCR in 47 (58%), 24 (30%), 14 (17%), and 16 (20%) isolates, respectively (Fig. 1). The single phenotypically MBL-positive isolate proved to be due to a bla_VIM. Almost all isolates were positive for bla_OXA-50. All isolates were negative for bla_BEL, bla_GES, bla_KPC, and bla_OXA-48.

FIG. 1.

Dendrogram of AFLP fingerprints provided with genotypic results. (A–E) represent five different AFLP clusters. The image can be magnified online for improved readability. AFLP, amplified fragment length polymorphism.

As WGS results revealed that also a PME β-lactamase was present, all isolates were screened using primers designed by Tian et al.⁹ The screening for PME revealed four bla_PME-positive isolates in our collection of 81 isolates.

Besides information involved on the mechanisms of resistance to ceftazidime, we also deduced from the WGS data, in the subset of isolates (n = 11), the repression of the OprD-gene as alternative mechanism of imipenem resistance. The WGS data revealed that in seven P. aeruginosa ST244 isolates, a single-nucleotide insertion (c.T54_C55insT) in the structural gene was present, resulting in a frameshift and leading to a premature stop. In two isolates, several mutations were demonstrated and in the remaining isolate many mutations were found in combination with a deletion of 10 amino acids probably also resulting in a nonfunctional OprD protein (Table 3).

Table 3.

Nonsynonymous Mutations in OprD

	Nonsynonymous mutations ^a
MLST (n)	OprD
ST244 (7)	c.T54_C55insT, frameshift, nonsense from AA 19, stop codon at AA 54
ST244 (1)	WT
ST235 (1)	T103S; K115T; F170L; E185Q; P186G; V189T; R310E; A315G; G425A
ST308 (1)	T103S; K115T; F170L; E185Q; P186G; V189T; R310E; A315G; G425A
ST654 (1)	V127L; E185Q; P186G; V189T; E202Q; I210A; E230K; S240T; N262T; T276A; A281G; K296Q; Q301E; R310E; G312R; A315G; L347M; p.M372_G383delinsVDSSSSYAGL; S403A; Q424E

Compared with reference genome P. aeruginosa PAO1 (NC_002516).

Genotyping

As already indicated, in a limited number (n = 11) of isolates, WGS was performed. For these particular isolates, we were able to deduce the MLST type. These isolates belonged to MLSTs ST244 (n = 8), ST235, ST308, and ST654 (Table 2).

As performing WGS for all isolates was not feasible, the rest of the isolates were typed by means of AFLP. AFLP typing revealed five clusters of isolates (n > 3) with a correlation coefficient of >90% (Fig. 1), indicating dissemination of widely resistant clones throughout the three hospitals. All but one isolate in cluster A (n = 22) were positive for bla_VEB, 15 were positive for bla_OXA-10, and 12 were bla_TEM positive. Cluster B comprised 11 bla_PER-positive isolates. All isolates in clusters C (n = 4), D (n = 8), and E (n = 17) were positive for OXA-10, except for one isolate in cluster D. Cluster E almost completely comprised isolates with a phenotype of derepressed AmpC or indeterminate results in the AmpC/ESBL-assay. The four bla_PME-positive isolates were distributed over clusters B and D, and an isolated cluster, indicating a horizontal spread of the bla_PME gene. Identical clones were found in different patients and in different hospitals.

Discussion

Multiresistant P. aeruginosa isolates have been reported worldwide and also in the region of Kurdistan.^27,28 To obtain information with respect to the mechanisms of resistance causing the multiresistant character of the isolates and their genetic relationship, isolates in several hospitals of the city of Erbil were collected. It appeared that 86% were multidrug resistant and 70% XDR. As most of the isolates were resistant to carbapenems and aminoglycosides, we focused first on the demonstration of the presence of carbapenemase genes. However, in only one isolate of 81, the presence of the VIM-gene could be demonstrated. As no additional carbapenemases were demonstrated, we concluded that the resistance to carbapenems was probably due to loss of OprD porins. Proof for this particular mechanism was obtained by demonstrating in a subset of isolates the presence of amino acid changes and/or deletions in the structural OprD gene, resulting in a nonfunctional OprD gene product.²⁹

Besides these intrinsic mechanisms of resistance, P. aeruginosa is also capable of acquiring ESBL genes such as TEM, SHV, CTX-M, OXA-10-like, PER, and VEB resulting in ceftazidime-reduced susceptibility. The presence of PER and VEB genes was demonstrated in a considerable number of isolates. In addition, in almost 50% of the isolates, also an OXA-10-like gene was demonstrated.³⁰ The presence of PER and VEB genes in the same geographical region has already been reported by Mirsalehian et al.³¹ and Shahcheraghi et al.³² The spread of these PER- and VEB-positive isolates was clonally related.³³ PER was thought to be concentrated in Turkey, but has been reported in the Far East and also in the Balkan region as well as Poland.³³

Most remarkable was the detection of four PME-positive isolates. PME-producing P. aeruginosa isolates have first been described in The United Arabian Peninsula by Tian et al.⁹ and later in Qatar.¹⁰ These positive PME specimens belonged to clusters A and D and in two other genetically distinct isolates, showing that the gene is capable of horizontal spreading. The isolates were retrieved mainly from the Rozhawa hospital and one isolate from the Hawler hospital, suggesting the presence of an epidemiological link between both hospitals. As no epidemiological data were collected, we were not able to prove this finding.

As one of the PME-positive isolates was sequenced (WGS), we were able to deduce the MLST type that belonged to ST654. ST654 has been described and identified as a high-risk clone. This clone has also been shown to be very successful in the spread of VIM-positive and also KPC-positive isolates.^34,35

Besides the presence of the PME gene in different AFLP clusters, the presence of the VEB gene was mainly concentrated in cluster A (n = 22). In this cluster A, also one PME isolate together with a PER gene was demonstrated. The majority of the VEB-positive isolates also contained an OXA-10 gene, sometimes in combination with a TEM β-lactamase. The majority of VEB-positive isolates were obtained from patients admitted to the Rozhawa hospital. Two isolates were obtained from Rzgari and one isolate from Hawler hospital.

Furthermore, 28 isolates contained only OXA-10 β-lactamase and belonged to three different AFLP clusters, that is, C, D, and E. Isolates belonging to clusters C and D were again collected at the three different hospitals. While the isolates of cluster E only were collected from the Rozhawa hospital.

In conclusion, during a period of 1.5 years, multiresistant P. aeruginosa isolates carrying different ESBL genes such as bla_OXA-10, bla_VEB, bla_PER, and bla_PME were demonstrated in patients admitted to different hospitals in the city of Erbil. Furthermore, it has been shown that these multiresistant organisms were clonally related, indicating that either a common source is involved in the spreading of these isolates or spreading is due to cross transmission between patients at the different wards.

As no epidemiological data are available, we were not able to link the related isolates in the different hospitals or in the different wards. However, the data at least demonstrate that identical clones were found within one hospital and between different hospitals. It would be interesting to study the effect of increased intensified infection control measurement on the number and spread of these multiresistant clones.³⁶

Footnotes

Acknowledgment

We are grateful to Marian ten Kate for her technical assistance in performing colistin MICs.

Disclosure Statement

No competing financial interests exist.

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