Increased frequency of integrons and β-lactamase-coding genes among extraintestinal Escherichia coli isolated with a 7-year interval

We analyzed the level of antimicrobial resistance, and the presence of integrons and β-lactamase-coding genes in 69 clinically relevant Escherichia coli strains originating from extraintestinal infections isolated in 1999–2001 and 2008–2010. Comparison of the two groups showed significant differences in drug resistance frequency, and the presence of integron and β-lactamase-coding genes. The frequency of resistance to all antimicrobials beside imipenem, streptomycin, piperacillin/tazobactam, and sulfamethoxazole increased significantly, especially towards aminoglycosides, β-lactams and fluoroquinolones. Similarly, we noticed an increase in the number of strains with integrons from 31.6 to 80.7 %. The presence of integrase genes was associated with elevated frequency of resistance to each antimicrobial tested besides imipenem, piperacillin/tazobactam and ceftazidime. The presence of integrons was also associated with multidrug resistance phenotype. The genetic content of integrons comprised genes determining resistance toward aminoglycosides, sulfonamides and trimethoprim. Moreover, we noticed a significant increase in the frequency of blaCTX-M β-lactamases, with appearance of blaCTX-M-15 variant and newer plasmid-encoded β-lactamases like CMY-15 and DHA. The emergence of strains resistant to several classes of antimicrobials and carrying integrons, ESBL and AmpC β-lactamase-coding genes may predict the spread of isolates with limited treatment options.


Introduction
Escherichia coli is a facultative Gram-negative species commonly present as a commensal organism in intestinal tract of mammals, but also recognized as one of major pathogens of human and animals. Pathogenic E. coli strains capable of causing disease outside the intestinal tract are classified as extraintestinal pathogenic strains (ExPEC) (Russo and Johnson 2000;Kaper et al. 2004). ExPEC are divided into isolates causing urinary tract and bloodstream infections (uropathogenic strains, UPEC), and neonatal septicemia/meningitis (MNEC, meningitis associated E. coli) strains (Welch 2006). During the past years, the rates of antimicrobial resistance among ExPEC strains have increased substantially, leading to higher morbidity and mortality and substantially increasing treatment costs (Daikos et al. 2007). Cephalosporins, fluoroquinolones, and co-trimoxazole are often used to treat ExPEC infections and the surveillance studies in years 2000-2010 indicated that 20-40 % strains become resistant to that group of antimicrobials (Pitout 2012).
Antibiotic resistance may develop through mutations in chromosomal DNA or acquisition of plasmids or transposons carrying resistance determinants. Integrons play an important role in the spread of antimicrobial resistance of clinical Enterobacteriaceae strains, since they capture, integrate and express gene cassettes encoding proteins associated with antimicrobial resistance. The integron covers DNA fragment that consists of an integrase gene of the tyrosine recombinase family, primary recombination site called the attI, and a promoter P C that directs transcription of the captured genes (Hall and Collis 1995;Mazel 2006). Integrons are often associated with mobile DNA elements like transposons and plasmids, which enable lateral spread of resistance determinants. Five classes of integrons are recognized on the basis of integrase gene sequence (Cambray et al. 2010). All classes are associated with resistance determinants, three of them are responsible for multidrug resistance (MDR), with class 1 being most ubiquitous among clinical strains (Leverstein- van Hall et al. 2003;Mokracka et al. 2011). Over 130 different gene cassettes have been identified within integrons, providing resistance to most classes of antimicrobials, including b-lactams, aminoglycosides, amphenicols, macrolides, trimethoprim, quinolones and antiseptics (Partridge et al. 2009;Cambray et al. 2010). Enterobacteriaceae strains harbour numerous integron-embedded antimicrobial resistance determinants, including b-lactamase-coding genes (Weldhagen 2004;Eckert et al. 2006).
The aim of this research was to analyze the resistance patterns and characterize distribution and genetic content of integrons and b-lactamase encoding genes within the E. coli strains isolated from extraintestinal infections in the course of last decade.
The strains were stored in -80°C in BHI/glycerol (50/50). All phenotypic assays and determination of antimicrobial resistance were done immediately after collection. The interpretation of zone diameters was done according to the CLSI (2009) breakpoints.

Clonal analysis by ERIC-PCR
The ERIC-PCR method utilizes primers complementary to enterobacterial repetitive intergenic consensus sequences of genomic DNA. The PCR reaction with primers ERIC 1 and ERIC 2 were done according to Versalovic et al. (1991). Computer analysis of electrophoretic patterns was carried out using GelCompar II version 3.5 software (Applied Maths). Similarity between fingerprints was calculated with the Dice coefficient. Cluster analysis was performed using the unweighted pair-group method with average linkages (UPGMA).
To check the gene cassette content, CS-PCR and Hep-PCR products were purified and sequenced. In case of two amplicons and amplicons longer than 1.7 kbp, the products were cloned by using pGEM Ò -T Easy Vector (Promega). Sequence data were analyzed with DNA Baser (HeracleSoftware) and aligned with available GenBank data using BLASTn. A gene cassette was identified if the percentage of similarity with GenBank data was higher than 95 %.
All PCR reactions were performed in a C1000 Thermal Cycler (BioRad). The PCR products were separated in 1.5 % agarose gel (Novazym). Molecular weight of PCR products was determined by Bio-Capt v. 99.04 software (Vilber Lourmat). All experiments were done in triplicate.

Statistical analysis
Association between the frequency of antibiotic resistance and integron presence was calculated using Pearson's v 2 test and Fisher's exact test. Association between integron presence and resistance ranges was determined with the Mann-Whitney U test (Statistica 10, StatSoft). P \ 0.05 was considered to indicate statistical significance.

Clonal analysis by ERIC-PCR
The fingerprints of E. coli isolates consisted of 1-17 bands ranging in size from 110 to 5,000 bp (Supplementary Fig. S1). Three pair of isolates (EC1 and EC3, EC45 and EC53, EC49 and EC62) had ERIC-PCR profiles with 100 % similarity, yet analysis of integrons and resistance patterns indicated differences between them. Strains EC1 and EC3 had different resistance patterns, EC45 and EC53 differed in bla gene content, whereas EC49 and EC62 had also different integron gene cassette arrays (Table 1).
The frequency of resistance to each of the antimicrobials beside imipenem, streptomycin, piperacillin/ tazobactam, and sulfamethoxazole was significantly higher in the second group of isolates (P \ 0.05).
We found AmpC cephalosporinase genes in five strains: four of them had bla CMY and one bla DHA . Sequencing of bla CMY and comparing the sequences versus available GenBank data identified them as bla CMY-15 . The bla CMY and bla DHA genes were present in isolates that had bla CTX-M and integrons.

Discussion
Sixty-nine clinically relevant E. coli strains originating from extra-intestinal infections were comprised in the study. We analyzed the level of antimicrobial resistance, the presence of integrons and b-lactamases-coding genes. Comparison of two groups of strains: one isolated in 1999-2001 and another in 2008-2010 showed significant differences in drug resistance frequency, presence of integrons and b-lactamasecoding genes. The frequency of antimicrobial resistance to all antimicrobials beside imipenem, streptomycin, piperacillin/tazobactam, and sulfamethoxazole increased significantly, reaching high levels toward aminoglycosides, b-lactams and fluoroquinolones. It generally mirrors the trends in E. coli resistance, yet we must emphasize the fact that the frequency of resistance of strains isolated in 1999-2001 was already    (Gagliotti et al. 2011). That regarded resistance to third-generation cephalosporins and combined resistance, i.e. resistance to two, three or four antimicrobials classes (aminoglycosides, aminopenicilins, third-generation cephalosporins and fluoroquinolones). We noted an increase in resistance to aminoglycosides, beta-lactams and fluoroquinolones. The EARSS survey from 2008 has reported the resistance against third-generation cephalosporines to be the most dynamic in Europe, which predicted increase in the number of ESBL-producing strains (European Centre for Disease Prevention and Control 2009). The EARS-Net report from 2010 has supported the remarkable Europe-wide decline of antimicrobial susceptibility in E. coli: in several countries both multidrug resistance and resistance frequency were increasing. The proportion of E. coli isolates resistant to third-generation cephalosporins increased significantly during 2006-2010 in half of the reporting countries. Among these isolates, a high proportion (65-100 %) was identified as ESBL producers. These data indicate that ESBL production is highly prevalent in third-generation cephalosporinresistant E. coli in European hospitals (European Centre for Disease Prevention and Control 2011). In our research, the percentage of ESBL-positive strains increased significantly from 21.1 to 93.5 % between 1999-2001 and 2008-2010. Most of the ESBLproducing isolates (75.7 %) had a bla CTX-M gene: the frequency of strains with bla CTX-M grew significantly in group 2 in comparison with group 1. The analysis of the resistance frequency and the presence of bla CTX-M genes reflected another tendency: the strains producing CTX-M b-lactamases acquired resistance to other than b-lactams, classes of antimicrobials namely tetracycline (P = 0.002) and fluoroquinolones (P \ 0.001). Surveys conducted worldwide have shown a growing resistance frequency to antimicrobials like tetracycline, gentamicin, tobramycin and ciprofloxacin in CTX-M producing E. coli (Pitout and Laupland 2008).
We also noticed the increase in the number of strains with integrons from 31.6 to 80.7 %. The presence of integrase genes was associated with increased frequency of resistance to each antimicrobial tested besides imipenem, piperacillin/tazobactam and ceftazidime (P \ 0.05). The presence of integrons was also associated with multidrug resistance and the presence of ESBL phenotype and ESBL-encoding genes (P \ 0.001). The genetic content of integrons comprised genes determining resistance toward aminoglycosides, sulfonamides and trimethoprim but the resistance of intI-positive isolates was far broader. In the first group of E. coli we detected three arrays of genes within integrons: dfrA1-aadA, dfrA17-aadA5, and dfrA12-orfF-aadA2. Gene cassette arrays like that are commonly appearing in E. coli isolates (Machado et al. 2005;El-Najjar et al. 2010). In the genomes of isolates from 2008 to 2010 we observed greater versatility of gene cassettes, more gene cassettes within an integron, appearance of class 2 integrons, and presence of more than one integron in bacterial genome. We detected six different integron-embedded gene cassette arrays: dfr2d, aadA1, dfrA1-aadA1, dfrA17-aadA5, dfrA12-orfF-aadA2, and aacA4-aacC1-orfA-orfB-aadA1 as well as cassettes coding for hypothetical proteins. All intI1-positive strains produced ESBL. The most often identified gene determining b-lactamase production was bla CTX-M type, identified by sequencing as bla CTX-M-1 , bla CTX-M-3 , bla CTX-M-15 , and bla CTX-M-55 . In strains isolated from 1998 to 2001, only bla CTX-M-3 was present. The CTX-M b-lactamases are now predominant in Poland and were noted for the first time in the late 1990s and identified as CTX-M-3. (Gniadkowski et al. 1998b;Livermore et al. 2007). In the following years, bla CTX-M-15 appeared possibly by point mutation in bla CTX-M-3 . The CTX-M-15 lactamase is 100-fold more active against ceftazidime than CTX-M-3 (Cartelle et al. 2004).
The genetic environment of bla CTX-M genes in most of our isolates was homogenous and consisted of ISEcp1 upstream bla CTX-M-1 , bla CTX-M-3 , and bla CTX-M-15 . ISEcp1 is frequently associated with bla CTX-M genes and as it includes promoter sequences, it enhances otherwise poor bla CTX-M expression (Poirel et al. 2008). That element may also transpose downstream located fragments and thus facilitate the spread of bla CTX-M genes (Partridge 2011). In the isolates from 2008 to 2010 beside integrons and bla CTX-M , there were also plasmid-mediated AmpC b-lactamases: bla CMY-15 and bla DHA . AmpC b-lactamases at high levels, hydrolyse penicillins, most cephalosporins, cephamycins and monobactams (Pitout 2012). In a survey comprising 13 Polish hospitals, bla CMY were identified in Proteus mirabilis only, and CMY-15 was the type of enzyme most common among them (Empel et al. 2008).
In summary, we found significant increase in resistance frequency including resistance to first line antibiotics like cephalosporins and fluoroquinolones, integron presence, and ESBL phenotype frequency. We also noticed significant increase in the frequency of bla CTX-M b-lactamases with appearance of bla CTX-M-15 variant and newer plasmid-encoded b-lactamases like CMY and DHA. We observed the emergence of strains with resistance to several classes of antimicrobials simultaneously with integrons, ESBL and AmpC b-lactamases coding genes. That may predict the spread of strains resistant to main classes of antimicrobials with no options for treatment apart from monobactams.
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