A First Insight into Escherichia coli ST131 High-Risk Clone Among Extended-Spectrum Beta-Lactamase-Producing Urine Isolates in Istanbul with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry and Real-Time PCR

Abstract

Objectives:

We aim to investigate, as a first insight, the presence and rates of high-risk Escherichia coli ST131 clone in Istanbul and evaluate antimicrobial resistance and CTX-M-15 production of ST131 and non-ST131 isolates. The use of MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry) to detect E. coli ST131 clone is also evaluated.

Materials and Methods:

A total of 203 extended-spectrum beta-lactamase (ESBL)-producing urinary isolates from a training hospital in Istanbul were investigated. Detection of E. coli ST131 was done by MALDI-TOF MS and real-time PCR melting curve analysis. The presence of CTX-M and CTX-M-15 beta-lactamases was investigated by PCR and sequence analysis.

Results:

Of the 203 isolates, 81 (39.9%) and 75 (36.9%) isolates were identified as ST131 clone by PCR and MALDI-TOF MS, respectively. Resistance to ciprofloxacin was significantly higher among ST131 isolates. A total of 169 (83.5%) isolates produced CTX-M beta-lactamase, of which 72 (43%) were CTX-M-15. The production of CTX-M and CTX-M-15 were significantly higher among ST131 isolates.

Conclusions:

We have demonstrated, for the first time, high rates of ST131 clone among ESBL-producing E. coli isolates in Istanbul, a region with high rates of resistance to third-generation cephalosporins and fluoroquinolones. Further investigation of this high-risk clone and its contribution to high antimicrobial resistance in Turkey is essential. MALDI-TOF MS is a useful tool for detection of high-risk clones and associated resistance patterns, simultaneous to bacterial identification.

Introduction

The increasing rates of antimicrobial resistance worldwide is a serious public health problem leading to difficult-to-treat infections, increasing rates of disease transmission, longer hospital stays, and social and economical costs.¹ Particular clones, yielding multidrug resistance, spread “succesfully” and are powerful transmitters of vertical and horizontal resistance via integrons, transposons, and plasmids. The detection of these “high-risk clones” in a particular population, region, or hospital by molecular epidemiological methods is crucial for surveillance studies.²

Multilocus sequence typing (MLST) based on the sequence variations in multiple housekeeping genes yields sequence types (STs) and clonal complexes (CCs), which can be tracked internationally. Since 2000s, MLST studies have introduced Escherichia coli ST131 as a quintessential example of an international multiresistant high-risk clone and a pandemic clone asscociated with extended-spectrum beta-lactamase (ESBL), particularly CTX-M-15, production and quinolone resistance.^3–5

Although MLST is the gold standard molecular typing method to obtain population-based epidemological data, it is time consuming, expensive, and labor-intensive. Thus, cost-effective and simpler methods have been sought in clinical laboratories to track surveillance targets, one of which is E. coli ST131. Allele-specific PCR methods have been used and proved to be accurate to detect E. coli ST131.^6,7 A limited number of recent studies defined particular protein peaks generated by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry) to represent E. coli ST131 clone.^8,9

Turkey is a country with high rates of antimicrobial resistance among E. coli isolates.^10–12 In the Central Asian and Eastern European Surveillance of Antimicrobial Resistance 2016 annual report, 39% nonsucceptibility to third-generation cephalosporins and 48% nonsusceptibility to fluoroquinolones are documented for E. coli isolates from Turkey, which are higher than most of the European countries.¹³ Higher frequencies of ESBL-production in ciprofloxacin-resistant E. coli isolates has been reported from the country while CTX-M-1 group is reported in up to 86.8% of the isolates and emergence and spread of CTX-M-15-producing isolates in a very short period was demonstrated.^14–16 However, research on the presence and spread of ST131 is scarce.^17,18

In this study, we aim to investigate the presence and rates of high-risk E. coli ST131 clone among ESBL-producing urinary isolates in a training hospital in Istanbul, as a first insight, and evaluate antimicrobial resistance and CTX-M-15 production of ST131 and non-ST131 isolates. The use of MALDI-TOF MS to detect E. coli ST131 clone is also evaluated.

Materials and Methods

Bacterial isolates

A total of 203 ESBL-producing E. coli isolates obtained from urine samples sent to Şişli Hamidiye Etfal Training and Research Hospital Clinical Microbiology Laboratory between January 2015 and February 2016 were included in the study. The recurring isolates obtained from the same patients were excluded. For routine identification procedures, MALDI-TOF MS Microflex LT (Bruker Daltonics, Germany) platform and MALDI Biotyper 3.1 software (Bruker Daltonics, Germany) was used. Antimicrobial susceptibility testing was performed by BD Phoenix™ automated AST system (Becton Dickinson, USA) or Kirby-Bauer disk diffusion method, following Clinical and Laboratory Standards Institute recommendations¹⁹ and double-disk synergy method was used for ESBL definition.²⁰ The isolates were stored at −80°C in trypticase soy broth until molecular tests were performed. MLST-defined clinical isolates of one ST131 and one non-ST131 E. coli were used as control isolates.

Real-time PCR for E. coli ST131 detection

Real-time PCR was done on RotorGene Q instrument (Qiagen, Hilden, Germany) as described by Tchesnokova et al. to detect E. coli ST131 clone with a modification that melting curve analysis was performed instead of probe detection.⁷ In brief, we amplified the 83 bp region specific for ST131 and performed high-resolution melting analysis with Epitect HRM PCR Kit (Qiagen). The melting temperature of the product was determined as 83.3°C. The modified method was tested for one positive and one negative MLST defined control and 20 positive and 20 negative clinical isolates defined by the original protocol by Tchesnokova et al.⁷ After obtaining concordant results, all isolates were tested with the modified method.

MALDI-TOF MS for E. coli ST131 detection

Detection of E. coli ST131 by MALDI-TOF MS was done as recently described by Lafolie et al.⁹ As a modification, isolates from chromagar instead of Mueller–Hinton agar were used. Analysis by MALDI-TOF MS was done on a Microflex LT platform (Bruker Daltonik GmbH, Germany) after formic acid extraction. Each sample was analyzed in triplicate. Mass spectra were obtained by the flexControl 3.4 software platform operating in linear positive-ion mode at a laser frequency of 60 Hz over a mass range of 2,000 to 20,000 Da. Analysis of the mass spectra was performed using the spectrum view of flexAnalysis 3.4 software with baseline subtraction. Peaks that differentiate ST131 and non-ST131 isolates were identified by visual comparison of the specific spectra. The presence of ST131 discriminatory peaks at about m/z 9,713 and 10,474 were sought and the mass spectra of each isolate were noted.

Detection of CTX-M and CTX-M-15 beta-lactamases

The isolates were tested for the presence of CTX-M gene as described by Kiratisin et al. and PCR for CTX-M Group 1 was done using group 1 primers from the multiplex PCR described by Woodford et al. in a singleplex PCR to identify potential CTX-M-15 carriers.^21,22 When positive DNA bands for CTX-M group 1 gene were obtained, the 909 bp amplicon of the CTX-M PCR were extracted from the PCR gel using QIAquick Gel Extraction kits (Qiagen) for sequencing. Forward and reverse sequencing was performed by using the Bigdye Terminator V3.1 cycle sequencing kit with an automated DNA sequencing on ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Finally, the produced chromatogram files comprising sequencing results were transferred to UGENE software (hhtp://ugene.unipro.ru), and necessary corrections were made. The genetic sequence was compared with the reference CTX-M-15 gene in GeneBank (GeneBank accession number AY013478) by using Basic Local Alignment Tool (http://blast.ncbi.nlm.nih.gov/Blast).

Statistical analysis

SPSS 15.0 programme is used for statistical analysis. The descriptive statistics of the ST were given as numbers and percentages. Assessment of concordance was done by Kappa analysis. To examine the relationship between ST131/nonST131 isolates and antimicrobial resistances and CTX-M/CTX-M-15 beta-lactamase production, cross-tables were constructed. Since all the variables are binary, Fisher's exact test for 2 × 2 tables was used (significance level 0.05).

Results

Patients and isolates

Of 203 ESBL-producing E. coli, 62 (30.5%) were from male patients while 141 (69.5%) were from female. Of the patients, 38, 40, 14, 28, and 83 patients were aged between 0–5, 6–15, 16–29, 30–59, and >60, respectively. The numbers of patients from outpatient and inpatient clinics were 168 (82.8%) and 35 (17.2%), respectively.

Detection E. coli ST131 clone

Of the 203 isolates, 81 (39.9%) and 75 (36.9%) isolates were identified as ST131 clone by PCR and MALDI-TOF MS, respectively. When compared to PCR, MALDI-TOF MS presented 92.6% sensitivity and 100 specificity while positive and negative predictive values were 100% and 95.3%, respectively.

Antimicrobial susceptibility

Of the 203 ESBL-producing isolates, all were susceptible to carbapenems (ertapenem, imipenem, and meropenem), and amikacin while variable levels of resistance to other antibacterials were noted, that is, 4.5% to nitrofurantoin, 31.5% to gentamicin, 62.1% to ciprofloxacin, and 65.5% to trimethoprim/sulfamethoxazole. Statistical analyses yielded enough evidence to claim that ST131 clone, that is, defined by PCR, and ciprofloxacin and gentamicin were not independent. Resistance to ciprofloxacin was significantly higher among ST131 isolates than non-ST131 isolates, while resistance to gentamicin was higher in non-ST131 isolates (Table 1).

Table 1.

Antibotic Susceptibilities, CTX-M and CTX-M-15 Beta-Lactamase Production of ST131 and Non-ST131 Escherichia coli Isolates

	Antibiotic									Gene
	Ciprofloxacin			Gentamicin			SXT			CTX-M ^a			CTX-M-15
	R	S	Total	R	S	Total	R	S	Total	N	P	Total	N	P	Total
ST131 PCR
ST131
Count	59	22	81	19	62	81	52	29	81	4	77	81	30	51	81
%	72.8	27.2	100	23.5	76.5	100	64.2	35.8	100	4.9	95.1	100	37.0	63.0	100
Non-ST131
Count	67	55	122	45	77	122	81	41	122	30	92	122	101	21	122
%	54.9	45.1	100	36.9	63.1	100	66.4	33.6	100	24.6	75.4	100	82.8	17.2	100
Total
Count	126	77	203	64	139	203	133	70	203	34	169	203	131	72	203
%	62.1	37.9	100	31.5	68.5	100	65.5	34.5	100	16.7	83.3	100	64.5	35.5	100
p-Value	0.012			0.044			0.765			0.000			0.000

The CTX-M column involves all CTX-M beta-lactamases including CTX-M 1 group (and CTX-M 15).

N, negative; P, positive.

Detection of CTX-M and CTX-M-15 beta-lactamases

A total of 169 isolates were found to be positive for CTX-M beta-lactamase gene (83.5% of all isolates). Of these 169 isolates, 72 (43%) were positive for CTX-M-1 PCR, all of which were CTX-M-15 by sequence analysis. The presence of CTX-M and CTX-M-15 genes was significantly higher among ST131 isolates when compared to non-ST131 isolates (Table 1).

Discussion

In the last decade, E. coli ST131 has been defined as a significant example of a high-risk clone and has been a major target for global surveillance as it is associated with both CTX-M beta-lactamase production and resistance to oral antibacterials including fluoroquinolones.^5,23–26 The prevalance of E. coli ST131 varies by geographic region and host population.⁵ A range of 12.5–30% is reported among E. coli isolates worlwide, accounting for about half of the ESBL-producing E. coli isolates and about 80% of fluoroquinolone-resistant isolates.²⁷ A study, conducted at the beginning of 2000s in Turkey, reported that 70% of ESBL-producing E. coli isolates from Turkey exhibited resistance to floroquinolones.²⁸ A meta-analysis including studies between 1996 and 2012 on E. coli urinary isolates from Turkey reported increasing trends in ESBL production and fluoroquinolone resistance rates in the last decade.¹⁰ In 2009, Ögedey et al. reported in another study from the country, an ESBL production rate of 34% and high floroquinolone resistance among E. coli isolates and demonstrated that 96% of the ESBLs were CTX-M enzymes, the majority being CTX-M-15.²⁹ Dissemination of CTX-M-15 beta-lactamase genes carried on Inc FI and FII plasmids, which we now know to be associated with ST131, among clinical isolates of E. coli was reported in 2008 in a university hospital in Istanbul, Turkey.^15,30 However, there is scarce research on the presence, rates, and spread of ST131 in Turkey.

The first report of ST131 from Turkey declared one isolate among 17 ESBL-producing E. coli isolates from İzmir.¹⁷ Since then, there have been two studies including E. coli isolates isolated in 2011 from a single hospital in Ankara by Can et al. reporting rates of 12% ST131 among E. coli isolates and 31% ST131 among ESBL-producing E. coli isolates obtained from urinary tract infections.^18,31 Our study is the first report of ST131 isolates from Istanbul, the city with the highest number of inhabitants in Turkey and which is a bridge between Asia and Europe. The isolates were from urine samples of patients admitted to one of the biggest training and research hospitals, and a high number of ESBL-producing isolates obtained in 1 year were included in the study. In this study, we report that ∼40% of ESBL-producing isolates belong to ST131 clone. Resistance to ciprofloxacin and the production of CTX-M and CTX-M-15 enzymes were significantly higher among ST131 isolates than non-ST131 isolates, suggesting that ST131 contributes significantly to the current extensiveness of drug resistance and associated resistance genes in E. coli isolates.

Although MLST is the gold standard molecular typing method for the surveillance of ST131 clone, cost-effective and simpler methods have been used including PCR methods and MALDI-TOF MS.^6–9 We have used the real-time PCR method described by Tchesnokava et al.⁷ with a modification that instead of probe detection, we used melting curve analysis with success, which led further decrease in time to detection of the ST131 clone, that is, a total of 2 hours. Likewise, we modified the MALDI-TOF MS detection described by Lafolie et al.⁹ as we used chromagar instead of Mueller–Hinton agar for the colonies to be tested. Further testing with Mueller–Hinton agar did not add more to the detection of ST131 by MALDI-TOF MS. We conclude that ST131 detection can be performed simultaneous to bacterial growth on culture.

The retrospective nature of our study and the involvement of solely the ESBL-producing urinary isolates can be considered as the limitations of our study. Even so, it gives the very first data about the presence of ST131 in Istanbul. Another limitation may be that the infections are not comprehensively categorized as “community” or “healthcare setting” acquired, however, ∼83% of the patients included in the study are outpatients and the results may mostly represent the circulation of this clone in the community. A prospective study including all E. coli isolates from urine and blood cultures, with the categorization of infections as community and healthcare associated, is ongoing.

In conclusion, we have demonstrated, for the first time, high rates of ST131 clone among ESBL-producing E. coli isolates in Istanbul, a region with high rates of resistance to third-generation cephalosporins and fluoroquinolones. Further investigation of this high-risk clone and its contribution to high antimicrobial resistance in Turkey is essential for developing strategies for the control of antimicrobial resistance. MALDI-TOF MS is a useful tool for detection of high-risk clones and associated resistance patterns, simultaneous to bacterial identification.

Footnotes

Acknowledgments

The authors thank Füsun Can for MLST-defined ST131 and non-ST131 control isolates. Caner Özdemir is acknowledged for statistical analysis. This research was performed with the ethical approval of Şişli Hamidiye Etfal Training and Research Hospital Ethics Committee (Decision No. 1136).

Disclosure Statement

No competing financial interests exist.

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