Application of Molecular Diagnostics in Primary Detection of ESBL Directly from Clinical Specimens

Abstract

The infections caused by extended-spectrum β-lactamase (ESBL)-producing organisms are associated with increased mortality. The real-time polymerase chain reaction (PCR) method, which enables detection of ESBLs directly from patients' clinical material, was developed. This study focused on bla_CTX-M and bla_SHV determination in endotracheal aspirates. Each sample was identified with standard microbiological procedures and simultaneously analyzed for the presence of nucleic acids, which encode CTX-M and SHV ESBL enzymes using real-time PCR. A total of 341 samples were investigated. In the set, 27 ESBL-positive samples were identified by phenotypic methods, while 60 positive samples were identified by the PCR method. Of the 60 PCR-positive samples, 58 were positive for the bla_CTX-M. In two samples, the ESBL bla_SHV-ESBL gene was detected. One phenotypically positive sample was PCR negative. The real-time PCR assay does not require a cultivation step and therefore enables detection of ESBL in 6 hours. The rapid method is necessary for early and adequate antimicrobial treatment.

Introduction

Bacterial resistance to antibiotics is an old phenomenon, but today it represents one of the most important public health problems worldwide. In many cases, bacteria are resistant to multiple drug classes; therefore, our treatment choices are limited. Nosocomial infections caused by extended-spectrum β-lactamase (ESBL)-producing organisms pose a serious challenge. Delayed adequate treatment of infections caused by this type of resistant bacteria is associated with increased mortality of hospitalized patients.²² Treatment failure rate for patients infected with ESBL-producing enterobacteria is almost twice as high as that of the patients infected with non-ESBL-producing organisms.¹²

The typical character of ESBL is their ability to hydrolyze oxyimino-cephalosporins and aztreonam, while being inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam, or tazobactam.²³ ESBLs are often plasmid mediated and most are mutants of classic TEM or SHV β-lactamase enzymes, with one or more amino acid substitutions around the active site. Most of the SHV variants possessing the ESBL phenotype are characterized by substitution of a serine for glycine at position 238 and alanine for aspartic acid at position 179.⁴ A number of variants related to SHV-5 have substitution of lysine for glutamate at position 240.¹⁶ In the recent years, CTX-M enzymes became the most dominant group of β-lactamases. The majority of ESBLs have been found among Enterobacteriaceae representing the most important mechanisms of resistance to β-lactam antibiotics. To date, over 186 SHV- and 219 TEM-ESBL types and ∼157 CTX-M variants (clustered in five groups, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) have been identified.¹⁷

There is evidence that ESBL-producing bacteria represent a serious medical problem also in the Czech Republic. Data from the European Antimicrobial Resistance Surveillance Network (EARS-net)¹¹ database show an alarming number of Klebsiella pneumoniae isolates resistant to the third-generation cephalosporins (51.2%), especially due to the production of ESBLs. According to many authors, CTX-M β-lactamases seem to be the most prevalent ESBLs in this area. Enterobacteria with production of CTX-M enzymes were isolated from hospitalized patients, healthy community subjects, as well as from the animals.^9,10 SHV enzymes have also been detected and are the second most widespread ESBLs in this area. There have been only rare reports of TEM types of ESBLs from the Czech Republic.¹⁴

The phenotypic methods are currently the gold standard in determination of susceptibility or resistance of clinical isolates. The most widely used methods are disk diffusion and microdilution methods according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or Clinical and Laboratory Standards Institute (CLSI) criteria, E-test, or Double-Disc Synergy Test (DDST).¹³ Unfortunately, investigation of ESBLs by these microbiological procedures requires several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. The total time from primary incubation to test results could last for 3–4 days. The large majority of currently commercially available molecular methods require successful cultivation as a first step that extends the time of determination of potential ESBL-producing bacteria by 24 hours, for example, Check-MDR ESBL (Check-Points, PD Wageningen, The Netherlands) and the Multiplex ESBL Kit (Bioron Diagnostics, Ludwigshafen am Rhein, Germany). There are only a few diagnostic tools, for example, hyplexESBL ID (AmplexDiagnostics, Gars-Bahnhof, Germany) that are able to detect ESBLs from potentially infected human patient specimens.

The purpose of this work was to find out if the application of real-time polymerase chain reaction (PCR) is possible for primary detection of ESBL genetic determinants directly from clinical material. This study focused on the most prevalent ESBLs in the Czech Republic (CTX-M, SHV).

Materials and Methods

Clinical samples and bacterial strains

The retrospective study was performed at the Department of Anesthesiology and Intensive Care Medicine, University Hospital Olomouc, Czech Republic. A total of 341 endotracheal aspirates from 155 patients suffering from respiratory tract infections or respiratory failure were collected during a 6-month period from 1st March to 31st August 2010. Only patients with mechanical ventilation were included into the study. Healthcare staff collected duplicate samples from patients-one for microbiological investigation and one for genetic detection. The number of obtained samples from single patients could differ because of the length of the hospitalization stay and clinical diagnosis (from 1 to 10 aspirates).

Bacterial strains were isolated from the clinical samples using standard microbiological procedures. For the cultivation, common culture media (meat peptone agar, blood agar, MacConkey agar, etc.) were used to obtain a pure culture of microorganisms. Bacterial isolates were identified according to the characteristic appearance and on the basis of biochemical properties. Further identification of bacteria was made by automated systems like the Phoenix automated system (BD Diagnostics, San Jose, CA) and MALDI-TOF Biotyper (Brucker Daltonics, Bremen, Germany).

Phenotypic detection of ESBL

The bacterial isolates were determined as ESBL producers using the DDST, which was modified by including a disc with cefepime and another disc with ceftazidime and ceftazidime/clavulanic acid. Jarlier's DDST was modified by including a disc with cefepime and by simultaneous use of a disc with ceftazidime and clavulanic acid.^15,18

Control strains

For optimization of the real-time PCR, well-known characterized strains were used as positive controls: K. pneumoniae NCTC 13368 (bla_SHV-18), Escherichia coli NCTC 13461 (bla_CTX-M group 1), E. coli NCTC 13462 (bla_CTX-M group 2), E. coli NCTC 13463 (bla_CTX-M group 8), Enterobacter cloacae NCTC 13464 (bla_CTX-M group 9), and K. pneumoniae NCTC 13465 (bla_CTX-M group 25) All strains were acquired from the National Collection of Type Cultures (Salisbury, United Kingdom).

In addition to those control strains, some ESBL-positive clinical isolates from the collection of the Department of Microbiology, Faculty of Medicine and Dentistry, Palacký University, Olomouc, were sequenced for ESBL genotype determination and used as another positive control: E. coli (bla_CTX-M-1), E. coli (bla_CTX-M-15, bla_TEM-1), E. coli (bla_CTX-M-15), E. coli (bla_CTX-M-27, bla_TEM-1), E. coli (bla_CTX-M-9), E. coli (bla_CTX-M-14), E. coli (bla_SHV-12), K. pneumoniae (bla_SHV-2), Enterobacter aerogenes (bla_SHV-1), and K. pneumoniae (bla_SHV-1).

DNA extraction and real-time PCR assay

All endotracheal aspirates (n=341) were isolated by the GeneProof PathogenFree DNA Isolation Kit (GeneProof a.s., Brno, Czech Republic). One primer pair was designed to amplify whole bla_SHV-1 gene (non-ESBL) or other related variants with size 591 bp. Three probes discriminate the main single-nucleotide polymorphisms leading to change of amino acids (position 179, 238, and 240), which determine the ESBL genotype. Four primer pairs were designed to amplify all five clusters of bla_CTX-M genes (amplification product=336 bp). The CTX-M-25 cluster is amplified from the primer CTX-M-1 for. and from the primer CTX-M-8 rev. Five FAM-labeled probes determine five different clusters in a single multiplex reaction. Locked nucleic acid (LNA) modification (+) was incorporated into the probe to increase melting temperature (T_m) and specificity. All primers and probes used in this study are listed in Table 1. Each PCR mix (30 μl) contained the Maxima™ Probe qPCR Master Mix (2×) (Thermo Scientific, Waltham, MA), primers, probes, internal control, and 10 μl of the isolated DNA. Optimal reaction conditions had been determined empirically using the SLAN real-time PCR system (Shanghai Hongshi Medical Technology Co., Shanghai, China). Cycling conditions were a 2-minute uracil-DNA glycosylase (UDG) decontamination period at 37°C, 10-minute incubation period at 95°C, and followed by 45 cycles of PCR, each cycle consisting of 5 seconds at 95°C and 40 seconds at 64°C with a single fluorescence reading for three channels being taken at the end of the extension stage 20 seconds at 72°C.

Table 1.

Oligonucleotides Used in This Study

Oligonucleotide	Sequence (5′-3′) ^{a b}	Reference
SHV-1	F-GAAAGATCCACTATCGCCAGCAGG	This study
SHV-1	R-GTTGCCAGTGCTCGATCAGCGC	This study
SHV-238c	FAM-CT+CGC+CAG+CT+CC-BHQ1	This study
SHV-238t	FAM-TT+CGC+CAG+CT+CC-BHQ1	This study
SHV-179	Cy5-TG+TCG+CG+GGCG-BHQ2	This study
CTX-M-1	F-ATGTGCAGCACCAGTAAAGTGATGGC	This study
CTX-M-2	F-ATGTGCAGTACCAGTAAGGTGATGGC	This study
CTX-M-8	F-ATGTGCAGCACCAGTAAGGTGATGGC	This study
CTX-M-9	F-ATGTGCAGTACCAGTAAAGTTATGGC	This study
CTX-M-1	R-ATCACGCGGATCGCCCGGAAT	This study
CTX-M-2	R-ATCACGCGGGTCGCCTGGAAT	This study
CTX-M-8	R-ATCGCGCGGGTCGCCGGGGAT	This study
CTX-M-9	R-GTCTCTCGGGTCGCCGGGAAT	This study
CTX-M-1	FAM-CCCGACAGCTGGGAGACGAAACGT-BHQ1	Modified³
CTX-M-2	FAM-C+GA+CAA+TA+CTG+CC-BHQ1	This study
CTX-M-8	FAM-C+GA+TAA+TA+CGG+CC-BHQ1	This study
CTX-M-9	FAM-C+GA+CAATA+CCG+CC-BHQ1	This study
CTX-M-25	FAM-C+GA+TAA+TA+CT+GC+CA-BHQ1	This study
ESBL-IS	HEX-CAGCGGGTA/ZEN/CTCCTACCTGATT-3IABkFQ	This study

+, LNA modification.

ZEN, ZEN internal quencher (IDT).

ESBL, extended-spectrum β-lactamase; LNA, locked nucleic acid.

The Quality Control for Molecular Diagnostics (QCMD) panel was used for evaluation of the PCR method. The aim of External Quality Assessment programs (EQA), which QCMD (www.qcmd.org) provide, is to help monitor and improve quality within the clinical laboratory by assessing a laboratory's ability to use molecular diagnostic technologies within the routine clinical setting. In our study, the ESBL and Carbapenemase EQA Pilot Study (QCMD) 2013 was used. Ten blind samples contained different types of ESBLs and carbapenemases: K. pneumoniae TEM-1 (non-ESBL), SHV-11 (non-ESBL), SHV-12 (ESBL), KPC-2; K. pneumoniae SHV-1 (non-ESBL); E. coli TEM-1 (non-ESBL), CTX-M-2 (group 2), E. aerogenes TEM-24 (ESBL), SHV-1 (non-ESBL); E. cloacae CTX-M-9 (group 9), VIM-31 (VIM-2-like); K. pneumoniae TEM-1 (non-ESBL), TEM-52 (ESBL), SHV-1 (non-ESBL), K. pneumoniae TEM-1 (non-ESBL), SHV-12 (ESBL), CTX-M-15 (group 1), NDM-1; negative sample; Serratia marcescens TEM-1 (non-ESBL), VIM-4 (VIM-1 like), Citrobacter koseri (negative for ESBLs and carbapenemases).

Results

Phenotypic detection

During the period mentioned above, a total number of 341 endotracheal aspirates were collected from 155 patients. From the obtained bacterial isolates, 125 belonged to the Enterobacteriaceae family. The most frequent isolated bacterial species were E. coli and K. pneumoniae (Table 2). These two species appeared in 53 patients. ESBL producers were determined in 27 patients, with predominant representation of K. pneumoniae strains. There was not determined any clinical sample containing more than one enterobacteria with production of these types of β-lactamases. In addition to enterobacteria, other bacterial strains causing respiratory tract infections in hospitalized patients were also detected: Pseudomonas aeruginosa (n=35), Burkholderia cepacia complex (n=28), Staphylococcus aureus (n=14), Streptococcus pneumoniae (n=9), Stenotrophomonas maltophilia (n=7), Acinetobacter sp. (n=5), and Haemophilus sp. (n=4).

Table 2.

Distribution of Bacterial Species of Enterobacteriaceae Family, Including Phenotypic Detection of ESBL

Bacterial species	Total number of isolates	Number of phenotypically ESBL-positive isolates
Citrobacter koseri	2	0
Citrobacter freundii	1	0
Citrobacter braakii	1	0
Enterobacter cloacae	3	0
Enterobacter spp.	16	7
Escherichia coli	28	3
Klebsiella oxytoca	7	0
Klebsiella pneumoniae	47	17
Morganella morganii	5	0
Proteus mirabilis	5	0
Proteus vulgaris	1	0
Providencia rettgeri	1	0
Serratia marcescens	7	0
Serratia ficaria	1	0

Genetic detection

Real-time PCR method for the detection of ESBL producers identified 60 positive samples (endotracheal aspirates), which contained genes encoding SHV and CTX-M β-lactamases (Table 3). Each sample was tested at least three times during PCR optimization. Fifty-eight samples were bla_CTX-M positive and 2 samples were positive to the bla_SHV-ESBL gene. Out of all PCR-positive samples, in 27, enterobacteria with production of ESBL were detected. In the rest of the endotracheal aspirates, either no ESBL-positive enterobacteria were detected (n=23) or no bacterial culture was identified (n=9). Only one phenotypically ESBL-positive sample was negative using real-time PCR.

Table 3.

Comparison of ESBL-Positive Isolates Determined by Cultivation Methods and Real-Time PCR Method

		Cultivation results
		Positive	Negative	Total
PCR results	Positive	26	34	60
	Negative	1	280	281
	Total	27	314	341

PCR, polymerase chain reaction.

The real-time PCR method also determined presence of bla_SHV (non-ESBL) genes. These genes were detected in 87 aspirates. Among them, K. pneumoniae strains were cultured from 37 aspirates. Fifty samples were positive to bla_SHV (non-ESBL) genes, but no K. pneumoniae was detected using phenotyping tests. On the other hand, if the K. pneumoniae species were indicated in the sample (47 endotracheal aspirates), only 37 bla_SHV (non-ESBL) genes were detected. QCMD panel was tested by SHV and CTX-M mastermixes. Two SHV-ESBL and three CTX-M ESBL were detected. That means all SHV- or CTX-M-positive (ESBL) samples were detected by our PCR method.

Standard culture was considered as the gold standard. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined. PPV of the real-time PCR method is 43% and NPV=99.6%. Specificity was determined as 96% and sensitivity as 89%.

Discussion

The alarming spread of ESBL-producing enterobacteria poses a serious public health threat. Increasingly, it has captured the scientists' attention to search a rapid and adequate method for their detection. ESBL-positive enterobacteria are emerging worldwide making the accurate identification crucial, especially for correct antimicrobial therapy. The routine laboratory detection of ESBLs in microbiology practice is based on the use of various phenotypic tests such as disk diffusion and microdilution methods, E-test or DDST,¹⁵ or using some chromogenic screening plates, for example, Brilliance ESBL agar (Oxoid, Hampshire, United Kingdom) or chromID ESBL from bioMérieux (Marcy l'Etoile, France). However, standard laboratory methods are too slow and may lack the required sensitivity.²¹ Real-time PCR can provide results in 6 hours and it has significantly higher sensitivity.

The demand on rapid, ready-to-use, and flexible methods in molecular detection of ESBLs has resulted in some commercial assays such as Check-MDR ESBL (Check-Points), the Multiplex ESBL Kit (Bioron Diagnostics), and Verigene^® Gram-Negative Blood Culture Test (Nanosphere, Northbrook, IL). However, there are only a few diagnostic tools that are able to detect ESBLs from potentially infected human patient specimens.

The aim of our study was to develop a rapid real-time PCR system for primary detection of genes encoding SHV and CTX-M types of ESBLs directly from clinical material. We focused on the analysis of the most prevalent enzyme types in our region. This study confirmed higher sensitivity of PCR methods for detection of the ESBL genotype, which is not cultivation demanding compared to the standard microbiological tests.²⁷ Almost double the amount (n=60) of samples were ESBL-positive using the PCR method. PPV was established as 43%, but it must be emphasized that not all ESBL determining genes, which were detected in clinical specimens, have to be exprimed.

Thirty-four clinical samples were ESBL-positive by the real-time PCR method, but negative by phenotypic detection (either negative by DDST or no bacterial culture was observed). The absence of bacterial culture can be the result of ongoing antibiotic therapy; therefore, no viable bacterial cells are observed.²⁰ In 10 patients, the records from previous sample collection revealed the presence of ESBL-positive bacteria in clinical specimens from the respiratory tract as well as from other types of materials. That is why we suppose that only genes encoding a specific type of β-lactamase could be detected. In the same amount of samples (n=9), no enterobacteria, only P. aeruginosa and S. maltophilia isolates were identified. It was published that the bla_CTX-M gene can also be found in P. aeruginosa strains. CTX-M-1 producing P. aeruginosa isolate has been reported from The Netherlands,¹ CTX-M-2 from Bolivia and Brazil,^5,24,26 and CTX-M-43 from Bolivia.⁵ According to the study of Bahmani and Ramazanzadeh² out of the 123 P. aeruginosa isolates, 12 were SHV positive using the PCR method. This could probably explain the presence of bla_CTX-M and bla_SHV genes in aspirates that were not confirmed as ESBL positive by DDST.

One phenotypically positive sample was PCR false-negative. This probably occurred due to repeated freezing and refrigeration of the sample. This sample was slightly positive in former experiments during the real-time PCR method optimization. Most likely DNA degradation occurred. This has also been reported in the study of Podivinsky et al.²⁵ where significant loss of DNA occurred in samples with lower amount of DNA after storage at −20°C.

The PCR assay was designed to detect ESBL. However, the method of detection also allows discrimination of non-ESBL bla_SHV genes. The majority of K. pneumoniae isolates are characterized by the production of chromosomally encoded SHV-1 β-lactamase with narrow spectrum activity and responsible for ampicillin resistance.⁶ Using our PCR assay, bla_{SHV ESBL} genes encoding SHV types of β-lactamases with narrow spectrum activity were detected in 87 samples. However, only 47 K. pneumoniae strains were identified by cultivation methods. In the set of 87 bla_SHV (non-ESBL)-positive samples, no bacteria were detected in 15 samples by cultivation methods. This could also be explained by ongoing antibiotic treatment.²⁰ In some cases, K. pneumoniae was found in the patient's history, in other types of clinical samples. This could probably be the reason why bla_SHV (non-ESBL) genes were detected. We suppose that in the rest of the endotracheal aspirates these genes could be plasmid encoded and present in other types of bacteria. The second reason could be the inability of cultivation methods to establish K. pneumoniae strains.

In conclusion, a number of molecular biological methods based on PCR have been developed for detection of SHV and CTX-M derivates.^(3,7,8,>,19) Nevertheless, all assays demand successful cultivation of clinical material. This prolongs the period about 24 hours to get results. In our study, we offer a real-time PCR-based method for primary detection of SHV and CTX-M types of ESBLs directly from clinical material for rapid identification of resistance patterns and subsequent application of adequate antibiotic therapy.

Footnotes

Acknowledgments

Supported by the grant IGA LF_2014_021 and Knowledge Transfer Program, Czech Ministry of Industry and Trade, and project “Development of Diagnostic Kits for Rapid Detection of Selected Types of Bacterial Resistance.”

Disclosure Statement

No competing financial interests exist.

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