Single center study of ESBL-related strains of Enterobacteriaceae collected from clinical specimens of infants with the congenital heart disease using multiplex PCR amplification

G. V. Filonenko, O. S. Talalaiev, D. L. Kyryk © 2017 G. V. Filonenko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 616.12-007.1-053.2-022.7:579.842.17


Introduction
Acquired antibiotic resistance of the Enterobacteriaceae represents globally escalating problem in treatment of healthcare-as-sociated infections.The rapid evolution of bacterial resistance among Enterobacteriaceae to the beta-lactam class of antibiotics has reached epidemic proportions [1].The background component of this growing resistance Biomedicine ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2017. Vol. 33.N 2. P 92-101 doi: http://dx.doi.org/10.7124/bc.000947 Single center study of ESBL-related strains of Enterobacteriaceae from infants with congenital heart disease is the expression of enzymes known as extended-spectrum β-lactamases (ESBL) by a bacterial cell.These enzymes are generally defined as beta-lactamases and confer bacterial resistance to penicillins, first-, second-, third-ge ne ra tion cephalosporins and to aztreonam, but not the carbapenems and cephamycins [2].Widespread use of these groups of antibiotics has led to the expansion of β-lactamaseencoding genes in Enterobacteriaceae like TEM, CTX-M, and SHV which can be encoded on mobile genetic elements, including plasmids and transposons that also often encrypt adventive resistance genes including multiple classes of antibiotics [3].
Risk factors for ESBL infection development are patient's age, prolonged in-hospital stay and previous stay at an intensive care unit (ICU) stay, prior use of antibiotics, and indwelling devices such as urinary catheters, central venous catheters, tracheostomy, and endotracheal tubes [4].
ESBL-related hospital infections are associated with negative outcomes for patients, resulting in prolonged hospital stay, increased hospitalization / inpatient costs, and increased morbidity rates [2].The main driver of this increased morbidity is insufficient antibiotic therapy.So, early monitoring of groups of surgical patients who are at risk for infection by ESBL-producing bacteria is crucial for a choice of antibiotic treatment strategy and important point for infection spread control.
Modern data indicate a growing prevalence of ESBL's isolated from different patient groups globally [5][6][7][8][9][10].However, neither single-nor multicenter studies have been conducted on ESBL-related hospital infections in Ukraine.The resistance is determined by the expression of bla genes belonging to bla TEM , bla SHV , and bla CTX-M genes family.To date, CTX-M enzymes are characterized as the most clinically important groups of ESBLs, which is followed by SHV-and TEM-derived ESBLs.The bla TEM , bla SHV , and bla CTX-M genes are responsible for production of evolutionarily relative enzymes -TEM β-lactamases, SHV β-lactamases, and CTX-M β-lactamases, respectively.To date, there are about 100 derivatives of TEM-1 enzymes; 185 new β-lactamases of the TEM family have been reported worldwide, while only ninety-three variants are responsible for production of ESBLs.According to data reported recently, among one hundred seventy-two types of SHV family of enzymes, forty-five were represen ted as extended-spectrum beta-lactamases.The CTX-M family includes more than sixty enzymes (http: //www.eucast.org/clinicalbreakpoints).
Multiple molecular typing methods, including PCR-based assays, have been developed for detection and identification of the growing number of bla TEM , bla SHV and bla CTX-M genes.Though, there is still limited scientific data on epidemiology of ESBL spread in the Ukraine.To the best of our knowledge, this is the first study on the molecular epidemiology and antimicrobial susceptibilities of ESBL-producing Enterobacteriaceae among surgical patients in Ukraine.
The purpose of this study was to explore feasibility of simultaneous identification of bla TEM , bla SHV and bla CTX-M genes by multiplex PCR detection in a series of clinical isolates of Enterobacteriaceae with previously characterized ESBL phenotype, to im-plement routing monitoring of ESBLproducing Enterobacteriaceae strains at the Ukrainian Children's Cardiac Center (Kyiv, Ukraine), to determine predominant genotypes among ESBL-producing Enterobacteriaceae strains.

Materials and Methods
All study strains were selected based on the screening tests for the detection of ESBL-type enzymes.The isolates were collected from clinical specimens of patients hospitalized at the Ukrainian Children's Cardiac Center (Kyiv, Ukraine) during the period from January to December, 2015.The isolates were recovered from various clinical specimens, mostly tracheal discharges, throat swabs, wounds, urine and blood.The majority of the collected strains were obtained during cardiac surgery in 704 patients representing different regions of Ukraine.The average age in the studies was 128 ± 106.5 days (0 days to 1 year) (Table 1).
The ESBL phenotype of the bacterial strains was determined by the VITEK 2 GN cards and the automated identification system (bio Merieux, France) in accordance with internal SOPs and manufacturer's protocols.

Testing of antibiotic susceptibility and ESBL determination
Susceptibility of clinical isolates to β-lactams (ampicillin, cefazolin, cefuroxime, cefepime, ceftriaxone, imipenem, and meropenem) and to ciprofloxacin, amikacin, ofloxacin, levofloxacin, tobramycin, and trimethoprim / sulfamethoxazole was tested by using AST-N076 cards and the automated VITEK 2 system.Susceptibility was interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations (http://www.eucast.org/clinicalbreakpoints). Thirty Enterobacteriaceae strains including twenty-two strains of K. pneumoniae, four strains of E.coli, and four strains of other Enterobacteriaceae from hospitalized patients were found to be ESBL producers.The presen ce of ESBL phenotype was confirmed by VITEK 2 automated system.

DNA isolation
Purified DNA from overnight bacterial cultures was isolated using the QIAGEN DNeasy Blood and Tissue Kit (Catalog# 69504) and NucleoSpin® Blood QuickPure kit (MACHEREY NAGEL, Germany).Qualitative analysis of DNA was performed by electrophoresis in 1 % agarose gel with 0.5 % TBE buffer (Tris-Borate-EDTA).

Multiplex PCR amplification
Multiplex PCR-based genotyping tests were conducted on thirty ESBL-positive isolates (Table 1) including twenty-two strains of K. pneumoniae, four strains of E.coli, and four strains of other Enterobacteriaceae (Enterobac ter cloacae, Serratia marcescens and Klebsiella oxytoca).
For genotyping tests, we used sequence primers-pair and technique of multiplex PCR, with some modifications, described previously [11].

Results and Discussion
In this study, we examined the prevalence of ESBL producing Enterobacteriaceae and carried out molecular characterization and antimicrobial susceptibility testing of clinical samples from patients during their admission to the cardiac hospital.
In our study, the main ESBL producers were found to be K. pneumoniae and E. coli and other species, which is consistent with previously published results [9,14,15], overall part of ESBL-producing pathogens among Ente robac teriaceae strains was substantially lower (10.9 %) than that reported in other studies in other geographical regions [14,15] and higher than reported in other hospital-associated infection studies [11,13].The majority of CTX-M-U1 CTX-M-U2 atgtgcagyaccagtaargtkatggc tgggtraartargtsaccagaaycagcgg 593 [11,13] Single center study of ESBL-related strains of Enterobacteriaceae from infants with congenital heart disease these infections were due to E. coli rather than Klebsiella pneumoniae, as described in the non-transplant literature in many cases and as was observed in our study.However, during screening of 1065 patients at the UCLA Medical Center who had undergone heart or lung transplantation between 1996 and 2010, the incidence of ESBL-related infections in these groups appeared to be 2.2 %, 5.5 %, and 10.7 % of patients, respectively [13].It can be explained by methodological differences in the measured prevalence levels, difference in age groups of patients and time-depended distribution of resistance.In this study, the respiratory tract was the major source of ESBL-producing isolates, followed by the blood and other sampling media.Similar findings have previously been repor ted for the ICUs at hospitals in Mexico, India and Qatar where the major source of ESBLproducing isolates were the respiratory tract and blood [9,16,17].However, in other geographical regions, urine and blood were reported as the major source of ESBL-producing bacteria [6,12].
Our epidemiological data show a dramatic increase in antimicrobial resistance.Addi tionally, more and more studies show continuous evolution of resistance to β-lactams and other groups of antibiotics in the bacteria of the Enterobacteriaceae family [6-16, 18, 19].
The constant increase of simultaneous resistance to various classes of antibiotics significantly reduces the possibility of therapeutic treatment of infections caused by ESBLproducers [6-10, 12, 14, 15, 18].That prompted us to analyze the level of resistance among ESBL-positive tested strains.
ESBL-positive strains showing simultaneous resistance to β-lactamases and antibiotics of other groups are defined as multidrug-resistant strains [1,2].Global spread of multidrugresistant strains as an etiological factor of infections is described in numerous reports .Characteristic location of genes responsible for resistance is the reason contributing to the prevalence of this phenomenon.Genes encoding β-lactamases are often located in mobile genetic elements such as plasmids, allowing horizontal transfer of these genes between bacteria of Enterobacteriaceae species and non-fermenting bacteria [3].
In Enterobacteriaceae, resistant genes responsible for resistance to different groups of antibiotics are often located on the same plasmids in a close neighborhood and, thus can be transmitted at the same time to other bacteria [21].
The ESBL-producing pathogens identified by phenotypic methods were also analyzed by using PCR methods.Of 30 ESBL isolates, 6.6 % harbored multiple bla genes simultaneously and the prevalence of bla TEM was as high as 70.0 %, followed by bla SHV at 46.6 %, and bla CTX-M at 46.6 %.The majority of the TEMpositive isolates were K. pneumoniae (81.8 %), E. coli (50.0 %), and other Enterobacteriaceae, (33.1 %); however, all CTX-M and SHV positive isolates were K. pneumoniae (50.0 % and 59.0 % respectively).Furthermore, all three bla genes (TEM, SHV, and CTX-M) were detected in only 9.0 % of K. pneumoniae isolates, while two genes (SHV/CTX-M) were present in 9.0 % of K. pneumoniae, with TEM/ CTX-M being present in 22.5 % of K. pneumoniae and TEM/SHV being detected in 45.5 % of K. pneumoniae, and 33.3 % in other Enterobacteriaceae isolates (Table 1, Fig. 1).

Conclusions
Compared with previous data from other geographical regions, our study shows relatively low prevalence (10.9 %) of ESBL-producing Enterobacteriaceae.Though lower than in other countries and regions, our results suggest that there is sufficient global infection burden to warrant public health interventions.Notably, majority of isolates were multi-drug resistant and belonged to TEM plasmid-type.The emergence of TEM producing Enterobacteriaceae isolates is of major concern and necessitates further control and research in this area.As meropenem shows good activity against these ESBL producers, it should be restricted for managing patients with suspected Gramnegative bacterial infections with ESBL production.Additionally, antimicrobial control and early detection by active surveillance in combination with effective infection monitoring programs and methods are key steps for reducing or controlling the spread of ESBLpositive hospital-acquired infections in Ukraine.