Bacteria belonging to the Enterobacteriaceae family that produce extended-spectrum β-lactamase (ESBL) enzymes are important pathogens of infections. Increasing numbers of ESBL-producing bacterial strains exhibiting multidrug resistance have been observed. The aim of the study was to evaluate the prevalence of , , and genes among ESBL-producing Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis strains and to examine susceptibility to antibiotics of tested strains. In our study, thirty-six of the tested strains exhibited genes , , , and . Moreover, twelve ESBL-positive strains harbored genes , , , and , and the presence of a gene in twenty-five ESBL-positive strains was revealed. Among K. pneumoniae the multiple ESBL genotype composed of , and genes encoding particular ESBL variants was observed. Analysis of bacterial susceptibility to antibiotics revealed that, among β-lactam antibiotics, the most effective against E. coli strains was meropenem (100%), whereas K. pneumoniae were completely susceptible to ertapenem and meropenem (100%), and P. mirabilis strains were susceptible to ertapenem (91.7%). Moreover, among non-β-lactam antibiotics, gentamicin showed the highest activity to E. coli (91.7%) and ciprofloxacin the highest to K. pneumoniae (83.3%). P. mirabilis revealed the highest susceptibility to amikacin (66.7%).

1. 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 β-lactam antibiotics, including broad-spectrum β-lactam drugs and monobactams, except cefamycins and β-lactam inhibitors [4]. The resistance usually depends on expression of genes belonging to the inter alia , , and genes family. The , , and genes are responsible for production of, respectively, TEM β-lactamases, SHV β-lactamases, and CTX-M β-lactamases, large families of enzymes with evolutionary affinity. Since the first TEM-1 β-lactamase was discovered, one hundred eighty-five new β-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, forty-five have been reported as extended-spectrum β-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 , , and 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 , , and 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 β-lactam antibiotics, and to indicate the antibiotic with the highest activity.

2. 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.

2.1. Antibiotic Susceptibility Testing and Determination of ESBL

Susceptibility of clinical isolates to β-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 ESBL-producers. The presence of ESBL phenotype was confirmed both by the double-disk-synergy test (DDST) [10] and VITEK 2 automated system.

2.2. 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.

2.3. PCR Amplification of bla Genes

Prepared plasmid DNA was used as templates for , , and 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′-CCATCTGGCC CCAGTGCTGC-3′), forward primer blaSHV-F (5′-CCCGCAGCCGCTTGAGCAAA-3′), reverse primer blaSHV-R (5′-CATGCTCGCCGGCGTATCCC-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 and 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 μL, contained 10 pmol/μL of each primer (1 μL), 12.5 μL of 2x PCR RED Master Mix (DNA-Gdansk, Poland), 3 μL of template DNA, and 7.5 μL of ultra pure H2O. PCR conditions for the , , and 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.

2.4. 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 μg/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 (), 733 bp (), and 585 bp () 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 , , and 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).

3. Results and Discussion

The analysis of selected strains was concerned with the searching for , , and genes in plasmid materials of tested strains, which were confirmed by phenotypic tests as producers of extended-spectrum β-lactamases. Regarding the literature reports describing a substantial number of genes encoding β-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 , , and 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 β-lactamases were more prevalent among tested strains in comparison to the genes encoding SHV-type or TEM-type β-lactamases. The presence of genes was observed among all tested Enterobacteriaceae strains, whereas, the presence of the genes was limited to all tested strains of K. pneumoniae. Moreover, PCR results based on primers specific for the blaTEM-type β-lactamases revealed the presence of 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 genes detected by electrophoretic analysis were identified as 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 genes responsible for production of extended-spectrum β-lactamase CTX-M-3 was noticed. Moreover, DNA sequencing revealed the prevalence of genes encoding CTX-M-91 extended-spectrum β-lactamase that were detected in 66.7% of P. mirabilis. Among the remaining 33.3% of P. mirabilis the presence of encoding CTX-M-89 extended-spectrum β-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 genes encoding TEM-1 enzyme with activity of broad-spectrum β-lactamase. Furthermore, K. pneumoniae strains were found to carry the following genes: (41.7%), (8.3%), (58.3%), and (25%), encoding different extended-spectrum β-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 genes. As reported, world literature, both and 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 and among P. mirabilis were identified. These genes exhibit genetic similarity with encode enzymes belonging to the sub-CTX-M-25 with extended-spectrum β-lactamase activity [15, 16]. Genes and 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 genes and TEM-1 β-lactamase are a prevalent plasmid-mediated β-lactamase in Gram-negative bacteria [19]. As reported in the literature, the occurrence of genes in Enterobacteriaceae can be as high as 61% [20]. Strains of Enterobacteriaceae showing the presence of 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 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 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 and [22]. The SHV-family enzyme variants of , , , and detected in our study are also revealed among ESBL-producing Enterobacteriaceae strains in Europe [23]. The presence of , , and 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: and ; and ; and ; and ; and . 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].

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.

With the ability of bacteria to produce ESBL enzymes, the phenomenon of broad spectrum resistance to β-lactam antibiotics is observed and confirmed by numerous reports [25]. That relationship was also shown in our analysis. The antibiotic susceptibilities of ESBL-producers are presented in Table 2. Among E. coli strains expressing ESBL activity, high levels of resistance to ampicillin (100%), amoxicillin/clavulanic acid (100%), ceftriaxone (100%), and aztreonam (16.7%) were observed. Tested strains of E. coli were susceptible to cefepime (75%), ertapenem (83.3%), and meropenem (100%). All K. pneumoniae ESBL-positive strains were resistant to ampicillin (intrinsic resistance), amoxicillin/clavulanic acid, ceftriaxone, and aztreonam. Furthermore, ESBL-positive K. pneumoniae strains were fully susceptible to meropenem (100%) and ertapenem (100%). Additionally, P. mirabilis ESBL-positive strains were completely resistant to ampicillin, amoxicillin/clavulanic acid, and ceftriaxone. High activity to P. mirabilis revealed aztreonam (91.7%), ertapenem (91.7%), and meropenem (100%) (Table 2).

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 β-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 [2830]. That prompted us to analyze the level of resistance among ESBL-positive tested strains.

ESBL-producers revealed high resistance to antibiotic groups other than β-lactams. The results of our study are in line with global reports [28, 3133]. Our analysis showed a significant degree of ESBL-positive E. coli resistance to such antibiotics as ciprofloxacin, levofloxacin, and tobramycin. The percentages of resistant strains were, respectively, 91.7%, 91.7%, and 75%. K. pneumoniae ESBL-positive strains appeared to be resistant to such antibiotics as levofloxacin (75%), amikacin (58.3%), gentamicin (100%), and tobramycin (75%). P. mirabilis strains revealed resistance towards ciprofloxacin (100%), levofloxacin (100%), gentamicin (100%), and tobramycin (100%). Moreover, all tested strains were fully resistant to trimethoprim/sulfamethoxazole (100%) (Table 2).

ESBL-positive strains showing simultaneous resistance to both β-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 β-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 ESBL-positive strains expressing multidrug resistance to antibiotics has remained the dominant problem in the therapy of infections caused by Gram-negative bacilli [40]. Bacterial resistant phenotypes and the presence of bla genes among tested strains are summarized in Table 1. The analysis revealed that among E. coli strains carrying the gene, the most prevalent phenotype of resistance included resistance towards ciprofloxacin, levofloxacin, tobramycin, and trimethoprim/sulfamethoxazole. Moreover, all strains of P. mirabilis carrying and genes appeared to be resistant to ciprofloxacin, gentamicin, levofloxacin, tobramycin and trimethoprim/sulfamethoxazole. K. pneumoniae strains with simultaneous presence of the bla genes of the SHV (, , , and ), TEM (), and CTX-M ( and ) families presented a resistance profile including resistance against amikacin, gentamicin, levofloxacin, tobramycin, and trimethoprim/sulfamethoxazole (Table 1).

Observations of a high level of antibiotic resistance among strains analyzed in the present study are disturbing but in agreement with previously published results that showed susceptibility of multiple ESBL K. pneumoniae strains to meropenem, ertapenem, imipenem, and ciprofloxacin [41].

Our data demonstrated the prevalence of particular bla genes responsible for production appropriate β-lactamases in tested ESBL-positive Enterobacteriaceae strains. The dominance of the genes among E. coli and K. pneumoniae and genes among P. mirabilis was revealed. Both genes and genes are responsible for production of extended-spectrum β-lactamases. Moreover, the prevalence of genes responsible for the production of broad-spectrum β-lactamases among K. pneumoniae and P. mirabilis has been shown, and bla genes of the SHV family (, , , and ) responsible for the production of ESBL enzymes have been detected in strains of K. pneumoniae. Among tested rods, only K. pneumoniae strains revealed the simultaneous presence of and genes in combination with and particular types of (, , , and ) genes. It is noteworthy that among K. pneumoniae the multiple-ESBL genotype composed of , , , , , and genes encoding particular ESBL variants was observed. Moreover, our study revealed a high level of resistance to antibiotics and the prevalence of multidrug-resistant bacteria among ESBL producers.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Paweł Sacha and Piotr Wieczorek contributed equally to this work.


The authors are grateful to Steven Snodgrass for editorial assistance.