Complex class 1 integrons harboring CTX-M-2-encoding genes in clinical Enterobacteriaceae from a hospital in Brazil

Introduction: CTX-M enzymes are the most prevalent extended-spectrum beta-lactamases (ESBLs) in Brazil and around the world. The spread of CTX-M lies in their ability to be mobilized by insertion sequences and integrons. This study aimed to identify the mobile genetic structures associated with blaCTX-M genes from clinical Enterobacteriaceae strains. Methodology: Twenty-eight clinical non-clonal Enterobacteriaceae were screened by PCR for the presence of blaCTX-M genes and class 1 integrase (int1), and for the association of blaCTX-M with class 1 integrons. Plasmid incompatibility groups were assessed by PBRT. Wild-type plasmids were transformed into electrocompetent E. coli, and the S1-PFGE technique was used to verify the presence of high-molecularweight plasmids in both wild-type strains and E. coli transformants. Results: Sequencing showed that strains carried blaCTX-M-2 (n = 25) and blaCTX-M-59 (n = 3) genes inserted into the 3’-end of complex class 1 integrons. Thirteen strains also carried blaTEM and blaSHV genes. CTX-M-2/59-containing complex class 1 integrons were also present in E. coli transformants. The most frequent Inc groups were IncA/C (n = 10) and IncF (n = 8). Heavy plasmids were observed in both wild-type strains and E. coli transformants. Conclusions: The presence of the same blaCTX-M-2-group-containing genetic structure in seven Enterobacteriaceae species isolated at seven hospital wards shows the great mobility potential of complex class 1 integrons. Also, this is the first report of TEM-15, SHV-45, and SHV-55 in Latin America. The genetic environment of blaCTX-M-2 accounts for their maintenance and spread among Gram-negative bacteria.


Introduction
According to the last Latin American data reported by the SENTRY Antimicrobial Surveillance Program, extended-spectrum beta-lactamase (ESBL) production rates in E. coli and K. pneumoniae from Brazil are 12.8% and 49.9%, respectively [1].Among ESBL families, TEM, SHV, and CTX-M are the most reported around the world, and in Latin America, SHV and CTX-M variants are the most frequent [2][3][4].In Brazil, there is no report of TEM enzymes that express an ESBL phenotype, while some SHV ESBLs are frequently reported (SHV-5, SHV-12) [5][6].CTX-M enzymes are spread worldwide, and in Latin America, they are considered endemic and play an important role in the resistance landscape in Brazil, where several reports are continuously being published [5][6][7][8][9][10][11][12][13][14].There are five main clusters of CTX-M betalactamases: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25, and except for the last one, all of them have already been described in strains from Brazil.The most frequent encoding genes reported in our region are bla CTX-M-15 [6,12] from the CTX-M-1 cluster, bla CTX-M-2 [6][7][8][9][10][11][12][13][14] and bla CTX-M-59 [5,13] from the CTX-M-2 cluster, bla CTX-M-8 [8] from the CTX-M-8 cluster, and bla CTX-M-9 [10,[12][13] from the CTX-M-9 cluster.The reason for the spread of CTX-M lies in their ability to be mobilized by genetic structures, especially insertion sequences and integrons.CTX-M-2 group of enzymes, particularly, are encoded by genes frequently located in complex class 1 integrons, which are the result of the fusion of the insertion sequence ISCR1 to the 3'CS of class 1 integrons, a combinatorial event that allows the structure to become mobile at high frequency rates, and capable of moving great portions of DNA [15].Additionally, these structures are frequently mobilized to conjugative plasmids that facilitate their spread.Some resistance-associated plasmid incompatibility (Inc) groups have been considered epidemic due to their presence in bacteria from different sources and countries, and the most common Inc groups carrying ESBL-encoding genes are IncF, IncA/C, IncL/M, IncI1, and IncH12 [16].
The aim of this study was to characterize and map the mobile genetic structures associated with bla CTX-M genes from clinical Enterobacteriaceae strains, and to determine if the genes were located in plasmids or in the bacterial chromosome.

Methodology
Twenty-eight non-clonal ESBL-producing Enterobacteriaceae strains, previously isolated [17] in a hospital set and representing, according to the institution's infection control committee, hospital infections (n = 9), community-acquired infections (n = 4), and colonization strains (n=15), were selected based on their distinct PFGE profiles and the detection of bla CTX-M genes by polymerase chain reaction (PCR) [18].These non-clonal strains were selected in order to demonstrate the mobility and spread potential of CTX-M-encoding genes even when there is no clonal dissemination.Thirteen of the 28 strains also carried other ESBL genes from TEM and SHV families (Table 1).The methodology described below was performed for all isolates.Minimum inhibitory concentrations (MICs) were determined by Etest strips (AB Biodisk, Solna, Sweden) for cefoxitin, cefotetan, ceftazidime, cefotaxime, aztreonam, cefepime, amoxicillin-clavulanate, piperacillin-tazobactam, meropenem, and ertapenem, according to the manufacturer's instructions.Results were interpreted based on Clinical and Laboratory Standards Institute (CLSI) 2014 guidelines [19].To search for CTX-Mencoding genes from clusters CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25, PCR for each cluster were separately carried out with a set of primers designed for this study (Table 2).Positive controls were included in each reaction (Table 2), and all PCR products were sequenced in order to validate the results.The PCR mixture was submitted to a denaturation step of 5 minutes at 94°C, followed by 30 cycles of amplification (45 seconds of denaturation at 94°C, 1 minute of annealing at 60°C, 1 minute of elongation at 72°C), and 10 minutes at 72°C for final elongation.A primary screening of class 1 integrases was carried out by PCR using previously described primers for intI1 gene amplification [20].The association of bla CTX-M with integrons was assessed using reverse primers for intI1 genes with reverse primers for bla CTX-M genes.Also, the 3'-ends of complex class 1 integron structures were amplified using a forward primer for sul1 gene and a reverse primer for qacEΔ1 gene [21], under the same conditions described above, except for a seven-minute elongation in each cycle.PCR products were purified and directly sequenced, including the previously amplified bla TEM and bla SHV genes [17].For the incompatibility group determination, plasmids were extracted by alkaline lysis (Wizard Plus SV Minipreps, Promega, Fitchburg, USA) followed by alcohol precipitation, and PCR-based replicon typing (PBRT) was carried out according to the methodology described by Carattoli et al. [22], which identifies replicons that represent 18 main Enterobacteriaceae incompatibility groups.Transformation experiments using One Shot TOP 10 Electrocomp E. coli (Invitrogen, Waltham, USA) as recipients were carried out, and transformed cells were selected in Luria agar enriched with 4 µg/mL of cefotaxime.PCR experiments that resulted positive for wild-type strains were also carried out for cefotaxime-resistant transformants.Plasmid sizes were determined by S1-PFGE technique: wild-type strains and transformants were grown overnight in 30 mL Luria broth containing cefotaxime 4 µg/mL.Cells were pelleted and resuspended in 400 µL TE buffer (Tris-HCl 10 mM, pH 7.5; EDTA 1 mM, pH 8.0), and then added to a microtube containing low-melting PFGE agarose (Bio-Rad, Hercules, USA), in a proportion of 1:4.Plug molds were filled with the cell-agarose mix and left at 4°C for solidification.Plugs were then put into a 5 mL lysis solution (EDTA 50 mM, pH 8.0; Tris-HCl 50 mM, pH 7.5; SDS 1%, Sarkosyl 1%, proteinase K 20 mg/mL), and incubated at 55°C for 2 hours.Then, plugs were subjected to two ultrapure water washes at 55°C for 15 minutes, followed by 4 TE buffer washes in the same conditions.Plugs were kept in TE buffer at 4°C.For the linearization of plasmids, plugs were digested with 10 U of S1 nuclease at 37°C for 45 minutes [23].After that, samples were subjected to pulsed field gel electrophoresis, for 20 hours at 6 V/cm, pulses ranging from 1 second to 40 seconds.PFGE gels were stained with ethidium bromide and visualized at UV light (Epi Chemi II Darkroom, UVP Bioimaging Systems, Upland, USA).

Results
Table 1 summarizes most of the present study's results.The highest MIC values expressed by both wild-type strains and transformants were for cefotaxime, cefepime, aztreonam, and piperacillintazobatam.PCR results showed that all strains carried CTX-M-2-group encoding genes, which were identified by sequencing as bla CTX-M-2 (n = 25) and bla CTX-M-59 (n = 3).All strains possessed class 1 integrases, and positive sul1-qacEΔ1 amplicons (4.5 kb) showed the association of bla CTX-M-2-group genes with complex class 1 integrons, which was confirmed by sequencing analysis.The genetic structure found was the insertion of bla CTX-M-2 or bla CTX-M-59 into the 3'-end of a complex class 1 integron, flanked upstream by sul1 and ISCR1 and downstream by a sequence having high homology to the Kluyvera spp.chromosome and the orf3::qacEΔ1 fusion gene (Figure 1).Regarding the incompatibility groups, the most common was IncA/C, which was present in ten (36%) strains, followed by IncF-like groups, found in eight (29%) strains.Nine isolates (32%) were not typeable by PBRT due to the limitations presented by this technique, which detects the 18 most common replicons that represent the main incompatibility groups in Enterobacteriaceae species, therefore not detecting less frequent or non-characterized groups [22].Transformation assays resulted in nine E. coli transformants that received plasmids extracted from E. coli, K. pneumoniae, M. morganii, and P. mirabilis.PCR and sequencing confirmed the presence of the CTX-M-2-group-containing complex class 1 integrons structures in all transformants.TEM and SHVencoding genes were not co-transferred with bla CTX-M- 2-group .S1-PFGE results (Figures 2 and 3, Table 1) showed that all but two wild-type strains possessed high-molecular-weight plasmids, ranging from 48.5 kb to 436.5 kb.This technique also showed that, except for strain 205/05, which transferred four plasmids to its respective transformant EC205, only one plasmid was transferred to each E. coli transformant, which harbored plasmids sizing from 48.5 kb to 179 kb that carried the bla CTX-M-2-group -containing complex class 1 integrons.

Discussion
CTX-M enzymes are spread worldwide, and the CTX-M-2 group is frequently reported in Brazil and other Latin American countries [2][3][4].In the present study, bla CTX-M-2-group genes were found disseminated through 28 non-clonal Enterobacteriaceae strains from seven different species, isolated from patients at  seven distinct wards of a healthcare institution.Also, bla CTX-M-2-group genes were found to be associated with TEM and SHV-encoding genes, and this is the first report of TEM-15, SHV-45, and SHV-55 in Latin America.The high mobility rate of bla CTX-M-2 genes among Gram-negative bacteria, as well as the exposure of strains to a severe antimicrobial selective pressure in the hospital, are the probable reasons for the spread of these genes.Besides bla CTX-M-2 , another CTX-M-2-group variant, bla CTX-M-59 , was also detected herein, in association with bla SHV-12 , bla SHV-27 , and bla SHV-110 .CTX-M-59 was first described in a K. pneumoniae outbreak in Brazil [5], and its spread in our country has been recently associated with IncA/C plasmids [13], which were detected in two of three CTX-M-59-producing strains in this study, and had also been transferred to CTX-M-59-producing E. coli transformants EC248 and EC276 (Table 1).The successful mobilization of complex class 1 integrons is mainly due to the presence of ISCR1, which can move big portions of DNA by its rolling circle mechanism [15,[24][25].This structure has already been reported in Brazil and other Latin American countries, and it is seriously implicated in the spread of CTX-M-2 enzymes in this region [26][27].Brazilian strains carrying complex class 1 integrons that contain bla CTX- M-2 genes inserted in their second variable region date from 2000, as described by Clímaco et al. [10], so these structures have been present among Brazilian isolates for at least 15 years.The CTX-M-2-producing strains selected for this study were isolated in 2004 and 2005, from patients hospitalized in seven different wards of an institution [17], and the presence of the same bla CTX-M-2-group -containing genetic structure in 28 non-clonal Enterobacteriaceae isolates shows its spread potential.In addition, the CTX-M-2-containing complex class 1 integrons were not exclusively located in plasmids, so the integration properties of the structure into the bacterial chromosome also account for its maintenance among the strains within the environment.Also, the dissemination of these integrons was not due to a particular plasmid, as different plasmid sizes and PBRT patterns were found among wild-type strains and E. coli transformants.Therefore, it is possible that the spread of CTX-M-2 and CTX-M-59 through the hospital isolates had occurred mostly due to the mobilization of class 1 integrons to high-molecular-weight plasmids, which were frequent among the studied strains and may have been spread by conjugation events (Figures 2 and 3).Transformation experiments were carried out using cefotaxime (4 µg/mL) selection, so the probable location of bla CTX-M-2 and bla CTX-M-59 in those strains that did not yield any E. coli transformant (n = 19) would be the bacteria chromosome, which therefore could result in clonal dissemination of these genes.Regarding EC205, which received four highmolecular-weight plasmids from its parental strain FSP205/05, the high number of plasmids harbored by FSP205/05 (five plasmids) likely favored this transference.
As shown in Table 1, CTX-M-2-group-containing strains were present in nosocomial and communityacquired infections, as well as in colonizing bacteria.Therefore, the genetic environment of CTX-M-2cluster encoding genes accounts for their maintenance and spread among Gram-negative bacteria present in healthcare environments.

Conclusions
CTX-M-2 cluster of enzymes are encoded by bla genes, which are commonly part of complex class 1 integrons, conferring these enzymes' success in both vertical and horizontal dissemination.The present work shows that these structures can be spread among several Enterobacteria species through highmolecular-weight plasmids; they can be located in the bacterial chromosome and have clonal spread potential, thus posing a challenge to the control of resistant infections in the hospital environment.Finally, the finding of the same genetic structure in bacteria isolated from several wards of an institution alerts for the need for reinforcement in hospital infection control measures to avoid the spread of these easily transferable resistance genes.

14 a
-25 F CTX-M-25 R ATGAGAAAAAGCGTAAGGCGGG CCGTCGGTGACWATTCTG bla CTX-M-25 and other bla CTX-M .865 FSP164/Positive control strains belong to the Public Health School Laboratory Culture Collection from the Public Health School, University of São Paulo.Dropa et al. -CTX-

Figure 3 .
Figure 3. PFGE agarose gel containing five of the nine CTX-M-2-group-producing E. coli transformants after total DNA extraction followed by S1 digestion for plasmid linearization.

Table 1 .
Summary of strain characteristics and most relevant results

Table 1 (
continued).Summary of strain characteristics and most relevant results

Table 2 .
Primers designed for this study