blaTEM Genes in Extended-Spectrum β-Lactamase-Positive Strains of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis in Poland

1 Department of Microbiological Diagnostics and Infectious Immunology, Medical University of Bialystok, 15a Waszyngtona Street, 15-269 Bialystok, Poland 2Department of Anaesthesiology and Intensive Therapy, Medical University of Bialystok, 37 Szpitalna Street, 15-274 Bialystok, Poland 3Department of Microbiological Diagnostics and Infectious Immunology, University Hospital of Bialystok, 15a Waszyngtona Street, 15-269 Bialystok, Poland 4Hospital Emergency Department with Intensive Care Subdivision, University Hospital of Bialystok, 24a M. Skłodowskiej-Curie Street,15-276 Bialystok, Poland


Introduction
Bacteria belonging to the Enterobacteriaceae family have been reported worldwide as etiologic factors of many nosocomial infections [1].Infections caused by Enterobacteriaceae rods are difficult to manage because of the reduction of therapeutic possibilities, resulting from constantly increasing resistance of these organisms to antibiotics [2].Production of ESBLs is one of the most prevalent resistance mechanisms in Gram-negative bacilli.Initially, ESBLs were predominantly described in K. pneumoniae and E. coli strains, but recently the enzymes were found in other genera of the Enterobacteriaceae family [3].ESBL-producing bacteria exhibit effective hydrolyzation of -lactam antibiotics, including broad-spectrum -lactam drugs and monobactams, except cefamycins and -lactam inhibitors [4].The resistance usually depends on expression of  genes belonging to the inter alia  TEM ,  SHV , and  CTX-M genes family.The  TEM , 2 International Journal of Antibiotics  SHV , and  CTX-M genes are responsible for production of, respectively, TEM -lactamases, SHV -lactamases, and CTX-M -lactamases, large families of enzymes with evolutionary affinity.Since the first TEM-1 -lactamase was discovered, one hundred eighty-five new -lactamases of the TEM family have been reported worldwide, whereas ninety-three variants are responsible for production of ESBLs.Among one hundred seventy-two enzyme types of the SHV family, fortyfive have been reported as extended-spectrum -lactamases.The CTX-M family comprises more than sixty enzymes (http: //www.eucast.org/clinicalbreakpoints,[5,6]).
It is known that bla genes encoding antibiotic resistance may be placed on transferable elements such as plasmids or transposons.This localization of bla genes can facilitate a horizontal spreading of antibiotic resistance among bacterial strains [7].Due to the noticeable geographical differentiation of bla genes among ESBL-producers here we examined the prevalence of  TEM ,  SHV , and  CTX-M genes among ESBL-producing strains of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis, with the specification of their variants.The study was focused on the searching for genes  TEM ,  SHV , and  CTX-M based on reports in the literature describing them as the most commonly occurring among Enterobacteriaceae family [8,9].Moreover, due to the alarming reports in the literature about the emergence of Enterobacteriaceae ESBL-positive strains exhibiting multiresistance phenotype, the next aim of our study was to evaluate the sensitivity of tested strains to antibiotics, other groups than -lactam antibiotics, and to indicate the antibiotic with the highest activity.

Material and Methods
Tests were carried out on thirty-six ESBL-positive isolates including twelve strains of K. pneumoniae, twelve strains of P. mirabilis, and twelve strains of E. coli.All strains for the study were chosen on the basis of the screening test for the detection of ESBL-type enzymes.The isolates were collected from clinical specimens of patients hospitalized at the University Hospital in Bialystok (Poland) from the period of the first quarter of year 2013.The isolates were recovered from various clinical specimens mostly urine, tracheal secretions, throat swabs, rectal swabs, and wound swabs.The majority of collected strains originated from the intensive care unit, the hematology clinic, and the urology clinic.The identification of the strains was performed by the VITEK 2 GN cards and the automated identification system (bioMérieux, France) according to the procedure and following the manufacturer's instructions.Control strains used in this study included K. pneumoniae ATCC 700603, E. coli ATCC 35218, and E. coli ATCC 25922.

Antibiotic Susceptibility Testing and Determination of ESBL.
Susceptibility of clinical isolates to -lactams (amoxicillin, ampicillin, aztreonam, cefepime, ceftriaxone, ertapenem, and meropenem) and to ciprofloxacin, amikacin, gentamicin, levofloxacin, tobramycin, and trimethoprim/sulfamethoxazole was tested by using AST-N093 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). The thirty-six Enterobacteriaceae strains including twelve strains of K. pneumoniae, twelve strains of P. mirabilis, and twelve strains of E. coli from hospitalized patients were found to be ESBLproducers.The presence of ESBL phenotype was confirmed both by the double-disk-synergy test (DDST) [10] and VITEK 2 automated system.

Plasmid DNA Preparation.
The Enterobacteriaceae strains were cultured overnight on Trypticase Soy Broth, (Emapol, Poland) at 37 ∘ C. Plasmid DNA was extracted from Enterobacteriaceae strains by the alkaline method with the Plasmid Mini Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol.

PCR Amplification of bla Genes.
Prepared plasmid DNA was used as templates for  TEM ,  SHV , and  CTX-M gene amplification.Molecular detection of the bla genes was carried out with the Cyclone 96 (PEQLAB Biotechnology, GmbH, Germany) thermal cycler.The primers used for the polymerase chain reaction (PCR) assays were forward primer blaTEM-F (5  -GCTCACCCAGAAACGCTGGT-3  ), reverse primer blaTEM-R (5  -CCATCTGGCCCCAGT-GCTGC-3  ), forward primer blaSHV-F (5  -CCCGCAGCC-GCTTGAGCAAA-3  ), reverse primer blaSHV-R (5  -CAT-GCTCGCCGGCGTATCCC-3  ), forward primer blaCTX-M-F (5  -SCSATGTGCAGYACCAGTAA-3  ), and reverse primer blaCTX-M-R (5  -ACCAGAAYVAGCGGBGC-3  ).The oligonucleotide primers blaSHV and blaTEM were designed on the basis of the nucleotide sequence of  TEM and  SHV genes reported in the National Center for Biotechnology Information (NCBI) Gen Bank database, while blaCTX-M primers were synthesized according to a previously published protocol [11].The PCR mixture, in a final volume of 25 L, contained 10 pmol/L of each primer (1 L), 12.5 L of 2x PCR RED Master Mix (DNA-Gdansk, Poland), 3 L of template DNA, and 7.5 L of ultra pure H 2 O. PCR conditions for the  TEM ,  SHV , and  CTX-M genes were selected based on the properties of the primers and were, respectively, 5 min at 95 ∘ C, thirty-five cycles (1 min at 94 ∘ C, 1 min at 58 ∘ C, and 1 min at 72 ∘ C), and finally 10 min at 72 ∘ C; 5 min at 95 ∘ C, thirty-five cycles (1 min at 94 ∘ C, 1 min at 58.5 ∘ C, and 1 min at 72 ∘ C), and finally 10 min at 72 ∘ C; and 3 min at 94 ∘ C, thirty-five cycles (30 s at 94 ∘ C, 30 s at 55 ∘ C, and 45 s at 72 ∘ C), and finally 10 min at 72 ∘ C.

Detection of PCR Products and Sequence
Analysis.PCR amplicons were separated electrophoretically in Mini-Sub Cell GT (BIO-RAD, USA) at 5 V/cm for 90 min in 1.5% agarose gel (Basica LE GQT Agarose, PRONA Marine Research Institute, Spain) containing 0.5 g/mL of ethidium bromide (MP Biomedicals, Poland) in borate buffer (TBE, Tris-Borate-EDTA) and photographed using the ChemiDoc XRS imaging system (BIO-RAD) and Quantity One 1-D Analysis Software (Bio-Rad, USA).Then, the positions of amplification products were estimated with the position of the molecular weight marker.PCR products with a length of 686 bp ( TEM ), 733 bp ( SHV ), and 585 bp ( CTX-M ) were purified from the agarose gel using Gel-Out Kit (A&A Biotechnology) and then sequenced using 3130xls Genetic Analyser (Applied Biosystems, USA).Nucleotide sequences of  TEM ,  SHV , and  CTX-M genes were analyzed and compared with sequences available in the NCBI database using Basic Local Alignment Search Tool (BLAST) algorithms (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM= blastn&PAGE TYPE=BlastSearch&LINK LOC=blasthome).

Results and Discussion
The analysis of selected strains was concerned with the searching for  SHV ,  CTX-M , and  TEM genes in plasmid materials of tested strains, which were confirmed by phenotypic tests as producers of extended-spectrum -lactamases.Regarding the literature reports describing a substantial number of genes encoding -lactamases inter alia ESBLs, we focused our research on the families of bla genes which are presented in literature as the most common among Enterobacteriaceae strains [12].Moreover, limited number of reports regarding the prevalence of bla genes among Enterobacteriaceae strains in Poland meant that this topic has been the subject of research.
Performed analysis with use of PCR reaction and specific primers for the  SHV ,  CTX-M , and  TEM genes revealed variation in occurrence of bla genes among tested strains (Table 1).Studies have shown that the genes responsible for the production of CTX-M -lactamases were more prevalent among tested strains in comparison to the genes encoding SHV-type or TEM-type -lactamases.The presence of  CTX-M genes was observed among all tested Enterobacteriaceae strains, whereas, the presence of the  SHV genes was limited to all tested strains of K. pneumoniae.Moreover, PCR results based on primers specific for the blaTEM-type -lactamases revealed the presence of  TEM genes among twenty-five of thirty-six tested strains, including two strains of E. coli, twelve strains of K. pneumoniae, and eleven strains of P. mirabilis.
In the next stage of the study, the PCR products of the expected size, which were observed in electrophoresis, were sequenced.The sequencing process led to the determination of the nucleotide sequences of the obtained products of PCR reactions and the identification of different types of genes whose presence was detected.Sequences of  CTX-M genes detected by electrophoretic analysis were identified as  CTX-M-15 in all tested strains of E. coli and among 91.7% of K. pneumoniae strains.In 8.3% of K. pneumonia strains the presence of  CTX-M-3 genes responsible for production of extended-spectrum -lactamase CTX-M-3 was noticed.Moreover, DNA sequencing revealed the prevalence of  CTX-M-91 genes encoding CTX-M-91 extendedspectrum -lactamase that were detected in 66.7% of P. mirabilis.Among the remaining 33.3% of P. mirabilis the presence of  CTX-M-89 encoding CTX-M-89 extended-spectrum -lactamase was observed.Additionally, DNA sequencing revealed that 16.7% of E. coli, 100% of K. pneumoniae, and 91.7% of P. mirabilis strains harbored  TEM-1 genes encoding TEM-1 enzyme with activity of broad-spectrum -lactamase.Furthermore, K. pneumoniae strains were found to carry the following  SHV genes:  SHV-18 (41.7%),  SHV-7 (8.3%),  SHV-2 (58.3%), and  SHV-5 (25%), encoding different extended-spectrum -lactamses.The results provide information about the diversity of bla genes presence among K. pneumoniae, P. mirabilis, and E. coli strains, in the North-Eastern Polish, that were harboring mainly  CTX-M-15 genes.As reported, world literature, both  CTX-M-15 and  CTX-M-3 genes, detected among tested strains, belong to CTX-M-1 group, which is often described as Enterobacteriaceae.Global reports describe, in addition to the CTX-M-15 and CTX-M-3 detected in our study, CTX-M-9 and CTX-M-14 as the dominant variants among the CTX-M family and the most widespread enzymes among non-TEM and non-SHV plasmid mediated ESBLs [13].These results are consistent with reports describing CTX-M-family enzymes as the group that, during the last few years, has become predominant [14].Furthermore, in the present study  CTX-M-91 and  CTX-M-89 among P. mirabilis were identified.These genes exhibit genetic similarity with  CTX-M-25 encode enzymes belonging to the sub-CTX-M-25 with extended-spectrum -lactamase activity [15,16].Genes  CTX-M-91 and  CTX-M-89 are uncommon but their occurrence in P. mirabilis and Enterobacter cloacae was reported [17,18].Moreover, our results are in agreement with the literature that  TEM-1 genes and TEM-1 -lactamase are a prevalent plasmid-mediated -lactamase in Gram-negative bacteria [19].As reported in the literature, the occurrence of  TEM genes in Enterobacteriaceae can be as high as 61% [20].Strains of Enterobacteriaceae showing the presence of  TEM genes responsible for the production of particular ESBL enzymes such as TEM-47, TEM-4, TEM-29, TEM-85, TEM-93, and TEM-94 have also been described [21].Moreover, the presence of the  SHV genes was observed only in tested strains of K. pneumoniae, which confirms the position of K. pneumoniae bacteria species as organisms commonly harboring genes encoding enzymes of the SHV family.The results of our study showed a lack of  SHV genes in strains of E. coli and P. mirabilis, which does not exclude the possibility of the occurrence of these genes among species of these bacteria, which is supported by the published literature describing the strains of E. coli showing the presence of genes  SHV-5 and  SHV-12 [22].The SHVfamily enzyme variants of  SHV-18 ,  SHV-7 ,  SHV-2 , and  SHV-5 detected in our study are also revealed among ESBLproducing Enterobacteriaceae strains in Europe [23].The presence of  SHV ,  CTX-M , and  TEM genes among tested rods of the Enterobacteriaceae family is presented in Table 1.
It should be noted that tested strains of K. pneumoniae have the genes responsible for the production of two ESBLs.They had the following genotypes:  CTX-M-15 and  SHV-18 ;  CTX-M-15 and  SHV-7 ;  CTX-M-3 and  SHV-2 ;  CTX-M-15 and  SHV-5 ;  CTX-M-15 and  SHV-2 .The phenomenon of multiple ESBL-production is becoming more common.It has been reported that, among strains of ESBL-positive Enterobacteriaceae, 44% harbored bla genes for multiple ESBLs [24].
Table Overview over all strains, identified bla genes, and resistance phenotypes.

Name of strain
bla CTX-M genes bla TEM genes bla SHV genes Resistance phenotype to non--lactam antibiotics Active -lactam antibiotics  To summarize, DNA sequencing has allowed us to identify specific variants of the bla genes among ESBL-positive Enterobacteriaceae.The majority of the bla genes detected in our study was encoding enzyme variants that are commonly found in Europe.This may indicate the possibility of the dissemination of bla genes among Enterobacteriaceae, which may be associated with the occurrence of bla genes on the mobile genetic elements.
Epidemiological data indicate that, over the past few years, antimicrobial resistance has dramatically increased.Additionally, more and more studies present data about the constantly increasing resistance to both -lactams and other groups of antibiotics in the bacteria of the Enterobacteriaceae family [26,27].The constant increase of simultaneous resistance to various classes of antibiotics significantly reduces the possibility of therapeutic treatment of infections caused by ESBL-producers [28][29][30].That prompted us to analyze the level of resistance among ESBL-positive tested strains.
ESBL-positive strains showing simultaneous resistance to both -lactams and antibiotics of other groups are defined as multidrug-resistant strains [30].Frequent occurrence of multidrug-resistant strains as an etiological factor of infections is described in numerous reports [34,35].Characteristic location of genes responsible for resistance is considered to be the reason for the prevalence of this phenomenon.Resistant genes for -lactamases are often located in mobile genetic elements such as plasmids and integrons, whereby the horizontal transfer of these genes is possible not only in bacteria of the same species but also between bacteria of Enterobacteriaceae species and nonfermenting rods [36,37].In Enterobacteriaceae, resistant genes are located on plasmids, where genes responsible for resistance to different groups of antibiotics may be located in a close neighborhood and thus may be transmitted at the same time to other bacteria [38,39].
The majority of tested clinical isolates possessing particular bla genes were resistant to at least one antibiotic from three different classes of antibiotics, which classifies them as multidrug-resistant bacteria.The occurrence of ESBLpositive strains expressing multidrug resistance to antibiotics has remained the dominant problem in the therapy of