ISSN 0891-4168, Molecular Genetics, Microbiology and Virology, 2019, Vol. 34, No. 1, pp. 16–24. © Allerton Press, Inc., 2019. Russian Text © The Author(s), 2019, published in Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya, 2019, No. 1, pp. 17–24. EXPERIMENTAL WORKS Class 1 and 2 Integrons in Hospital Strains of Gram-Negative Bacteria Isolated in Moscow and in Regions of the Russian Federation E. S. Kuzinaa, *, E. I. Astashkina, A. I. Leva, E. N. Ageevaa, N. N. Kartseva, E. A. Svetocha, and N. K. Fursovaa a State Research Center for Applied Microbiology and Biotechnology, Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being, Obolensk, Moscow oblast, 142279 Russia *e-mail: e.leonova@mail.ru Received April 10, 2018; revised April 23, 2018; accepted November 15, 2018 Abstract—Natural systems of cloning and expression of mobile gene cassettes caught by site-specific recombination, class 1 and 2 integrons, play an important role in mobilization and spread of genetic determinants of antibiotic resistance in gram-negative bacterial human pathogens, especially in a hospital environment. The gene cassettes localized in variable parts of integrons determine resistance to antibacterial drugs (AD) of different functional classes. The aim of the work is the detection and characteristic of class 1 and 2 integrons in gram-negative bacteria isolated in multidisciplinary hospitals of Moscow and other regions of the Russian Federation in 2003–2015. Clinical strains of gram-negative bacteria (n = 1248) mainly had multidrug resistance phenotype (94%). An amount of 10% of strains were resistant to AD of three functional groups; 19%, four; 42%, five; 17%, six; and 7%, seven. A high level of resistance of the studied strains to beta-lactams is associated with the presence of beta-lactamase genes of blaTEM (35% strains), blaSHV (25%), blaCTX-M (38%), blaOXA (31%), blaVIM (3%), and blaNDM (2%) types; to AD of other functional groups, with the presence of class 1 integrons (59%) and class 2 integrons (8%). Most class 1 integrons (54%) and class 2 integrons (88%) contained in its variable part 22 variants of gene cassette arrays in class 1 integrons and 4 variants in class 2 integrons. During the study, 31 types of gene cassettes were identified (including the most widespread, aadB, aacA4, aacC1, aadA1, aadA2, aadA5, blaVIM-2, dfrA1, dfrA7, dfrA12, orfC, orfE, orfY, and sat1) associated with the resistance of strains to aminoglycosides, chloramphenicol, sulfonamides, and beta-lactams, as well as orf cassettes encoding the proteins with unknown functions. New gene cassette arrays were identified: dfrA12s-orfF-aadA2 (In1249) and dfrA1-IS911-sat1-aadA1 (not numbered). Keywords: gram-negative bacteria, multidrug resistance, mobile genetic elements, class 1 integrons, class 2 integrons, gene cassettes DOI: 10.3103/S0891416819010051 accumulating these genes as gene cassettes [9] and providing their expression from a strong integron promoter [10]. Integrons are widespread in the genomes of bacteria isolated in different ecological niches: in a hospital environment, in environmental objects, and in human and animal organisms [11–14]. To date, based on differences in the primary structure of the integrase gene, five integron classes have been described; among them, class 1 and 2 integrons are the most common [15]. A number of 31 078 class 1 integrons and 27 624 class 2 integrons were present in the GenBank NCBI database on February 14, 2018 (https://www.ncbi.nlm.nih.gov/). More than 130 gene cassettes and more than 80 gene cassette arrays in class 1 integrons and 6 types of gene cassette arrays in class 2 integrons have been described [16]. In the Russian Federation, class 1 and 2 integrons are also widespread in the genomes of multiresistant strains of gram-negative bacteria [17–21]. INTRODUCTION At present, hospital infections are a large problem for health care all over the world [1–3]. In Russia, the portion of isolates of gram-negative bacteria among bacterial causative agents of nosocomial infections (NI) was 76.5% in 2013–2014 [4–6]. In the last two decades, a trend towards an increase in the amount of multidrug-resistant (MDR), extremely resistant (ER), and panresistant (PR) bacteria among the causative agents of NI has been detected [7]. Mobile genetic elements (MGEs) (plasmids, bacteriophages, transposons, IS elements) play a large role in mobilization and spread of genetic determinants of antibiotic resistance in bacteria [8]. Integrons (natural systems of cloning and expression of mobile gene cassettes caught by site-specific recombination) are spread by means of MGE and play a special role, since they are an “antibiotic resistance gene depot” 16 CLASS 1 AND 2 INTEGRONS IN HOSPITAL STRAINS OF GRAM-NEGATIVE BACTERIA The aim of this work is the detection and characterization of class 1 and 2 integrons in the genomes of gram-negative bacteria isolated in Moscow and other regions of the Russian Federation in 2003–2015. MATERIALS AND METHODS 17 formed as instructed by “The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Vone Diameters. Version 7.1, 2017-03-10” (http://www.eucast.org/). Susceptible E. coli ATCC 25922 strain and highly resistant E. coli ATCC 35218 strain were used as internal standards. Ethical Requirements Our laboratory had no direct contact with patients from hospitals. The studied bacterial strains were obtained in collaboration with the microbiological laboratories of OOO National Agency for Clinical Pharmacology and Pharmacy, Moscow; Children’s Scientific and Clinical Center for Infectious Diseases, Federal Medical and Biological Agency, St. Petersburg; Burdenko National Medical Research Center for Neurosurgery, Ministry of Health, Moscow; and Infectious Clinical Hospital No. 1, Moscow Healthcare Department. In their names, the studied strains in their contain no personal data about patients, such as surname, first name, ethnicity, age, religion, or gender. In accordance with the legislation of Russian Federation, each patient signed an informed consent for medical procedures and diagnostic tests upon admission to the hospital. Detection of Antibiotic-Resistance Genes The genes encoding five types of beta-lactamases (blaCTX-M [22], blaTEM [23], blaSHV [19], blaOXA [24], blaVIM [25], and blaNDM [26]), as well as class 1 [27] and class 2 [28] integrases and cassette arrays of class 1 and 2 integrons [29], were determined by the PCR method with specific primers. The composition of the reaction mixture and amplification modes corresponded to those previously described for the designated primers. Thermolysates were used as matrices for amplification [19]. PCR was carried out in GradientPalmCycler (Corbertt Research, Mortlake, Australia) and Tertsik (DNA Technology, Protvino, Russia) with subsequent electrophoretic detection of amplification products in 1.5% agarose gel. DNA Sequencing Bacterial Strains and Cultivation Antibiotic resistant clinical strains of gram-negative bacteria were isolated in Moscow and other regions of Russia (n = 1248) in 2003–2015, including strains from the Enterobacteriaceae family (n = 694) and the group of nonfermenting gram-negative bacteria (n = 552). Bacteria were cultivated at a temperature of 37°C in a Mueller–Hinton broth and agar nutrient media (Himedia, Mumbai, India). The species identification of bacteria was conducted on Vitek-2 Compact (Biomerieux, Lyon, France) and MALDI-TOF Biotyper (Bruker, Karlsruhe, Germany) devices. Bacterial isolates were stored in 40% glycerol at a temperature of –70°C. Determination of Sensitivity to Antibacterial Drugs Minimum inhibitory concentrations (MICs) of the antibacterial drugs: ampicillin (AMP), amoxicillin/clavulanic acid (AMC), amoxicillin–sulbactam (AMS), cefuroxime (CXM), cefoxitin (CEX), cefotaxime (CTX), ceftriaxone (CRO), ceftazidime (CAZ), cefoperazone– sulbactam (CFP), cefepime (FEP), imipenem (IPM), meropenem (MEM), doxycycline (DOC), tigecycline (TGC), ciprofloxacin (CIP), chloramphenicol (CHL), gentamicin (GEN), tobramycin (TOB), amikacin (AMK), trimethoprim (TMP), cotrimoxazole (CTZ), nitrofurantoin (NIT), and colistin (CST) were determined on a Vitek-2 Compact device (Biomerieux, Lyon, France). Interpretation of the results was per- Sequencing reaction was performed by means of an ABI PRISM BigDyeTM Terminator v. 3.1 kit (Thermo Fisher Scientific, Waltham, United States) on an automatic ABI PRISM 3100-Avant DNA sequencer (SYNTOL, Moscow, Russia). Bioinformatics Analysis Computer analysis of DNA sequences was conducted by means of the Vector NTI9 (Invitrogen, Waltham, United States) and CHROMAS (Technelysium, Pty Ltd., http://technelysium.com.au) programs and the BLAST web resource (http://blast. ncbi.nlm.nih.gov/Blast.cgi). The analysis of integron structure was conducted by means of the INTEGRAL web resource (http://integrall.bio.ua.pt/?). Counting the number of references to integrones in GenBank database was conducted on March 10, 2018, by searching for the gene cassette names in the “Nucleotide” section of the NCBI web resource (https://www.ncbi.nlm.nih.gov/). Deposition of DNA Sequences in the GenBank Database Ninety-five nucleotide sequences of integron cassette arrays of 22 class 1 integrons and 20 nucleotide sequences of integron cassette arrays of four class 2 integrons have been deposited in the GenBank database (Table 1). MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 34 No. 1 2019 18 KUZINA et al. Table 1. Gene cassette arrays of class 1 and 2 integrons identified in clinical strains of gram-negative bacteria in the GenBank database Integron (INTEGRAL database) Integron class Gene cassette array 1 1 1 1 1 aacA4 aacA4-cmlA1j aacA4-orfD aacA7-smr2-orfD aacC1-orfX-orfY-aadA1 In46 In838 ND In673 In561 1 aadA1 In2 1 1 1 1 1 1 1 1 1 aadA2 aadA7 aadA6-orfD aadB aadB-aadA1y aadB-catB3 blaOXA30-aadA1 blaPSE1 dfrA12-orfF-aadA2 In127 In142 In51 In7 In822 In299 In322 In167 In27 1 1 dfrA12s-orfF-aadA2 dfrA17-aadA5 In1249 In54 1 1 dfrA1-aadA1 dfrA1-orfC In369 In363 1 1 1 1 dfrA5-ereA2 dfrA7 estX orfD2-aacA4'-17orfE14-catB8 dfrA12-sat2-aadA1 dfrA1-IS911-sat1-aadA1 dfrA1-sat2 dfrA1-sat2-aadA1 In398 In22 ND In609 2 2 2 2 In-2-4 ND In2-3 In-2-4 Number of placed nucleotide sequence of gene cassette array in GenBank database Number of references to integron in GenBank database on March 10, 2018 HQ832472, HQ832473, JN003857 HM043570, HM043571, HM569733 GQ924771 HQ832478, HQ832479 KM009103, KM009104, KM009105, KR610434 GQ924774, GQ924775, GQ924776, GQ924777, KP789949, KP902674, KU860564 GU001948 — HQ832477, KP713392, KU870999 GQ924772, GQ924773 HQ914241, KU901703, KY885012 HQ914240 GQ924769, JN003856 HQ832476 GQ924762, GQ924763, GQ924764, GQ924765, GQ924766, GQ924767, GU001949, HM043572, HM043573, HM043574, HM569734, KJ363320, KM009101, KM236804, KP789948, KP796139, KP902672, KP965723, KR610433 KT316808 GQ896493, GQ896494, GQ896495, GQ896496, GQ896497, GQ896498, GQ896499, GQ896500, GQ896501, GU055937, KF952266, KJ579283, KM009102, KM085438, KP713389, KP713390, KP713391, KP789947, KP789950, KP902673, KR610432, KT175892, KT175893, KT175894, KT175895, KT305944, KT305945, KT305943, KT316804, KT316805, KU860565 GQ924770, KR610435, KT305946 KC862254, KC862255, KC862256, KF971879 GQ924768 KP789951 KP965724 HM485586 5960 5960 39 3 45 6652 4993 151 39 14597 14597 42 6543 0 196 214 1220 4 77 16 918 3298 482 KJ579284 HM592262 KP796141, KP796142 HM043575, HM043576, HM043577, KJ633801, KM009106, KM009107, KM085439, KM085440, KP271998, KP713393, KP796140, KP965725, KT175896, KT316806, KT316807, KX274124 13 2 1302 1093 ND, integron number not determined. MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 34 No. 1 2019 19 (a) 100 80 60 40 20 0 AMP AMC AMS CXM CEX CTX CRO CAZ CPS FEP IPM MEM DOC TGC CIP CHL GEN TOB AMK TMP CTZ NIT CST Portion of resistant strains, % CLASS 1 AND 2 INTEGRONS IN HOSPITAL STRAINS OF GRAM-NEGATIVE BACTERIA Portion of resistant strains, % Antibacterial drugs 25 (b) 20 15 10 5 0 1 2 3 4 5 6 7 Number of AD functional classes, pieces NGNB EB Fig. 1. Antibiotic resistance phenotypes of gram-negative bacterium strains. (a) Sensitivity to antibacterial drugs: AMP, ampicillin; AMC, amoxicillin/clavulanic acid; AMS, amoxicillin–sulbactam; CXM, cefuroxime; CEX, cefoxitin; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; CFP, cefoperazone–sulbactam; FEP, cefepime; IPM, imipenem; MEM, meropenem; DOC, doxycycline; TGC, tigecycline; CIP, ciprofloxacin; CHL, chloramphenicol; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; TMP, trimethoprim; CTZ, cotrimoxazole; NIT, nitrofurantoin; CST, colistin; (b) portion of strains simultaneously resistant to several functional classes: AD, antibacterial drugs; EB, enterobacteria; NGNB, nonfermenting gram-negative bacteria. RESULTS AND DISCUSSION Collection of Studied Strains and Their Sensitivity to Antibacterial Drugs Clinical strains of gram-negative bacteria (n = 1248), including Pseudomonas aeruginosa (n = 320), Klebsiella pneumoniae (n = 271), Acinetobacter baumannii (n = 232), Escherichia coli (n = 191), Enterobacter spp. (n = 132), Proteus spp. (n = 67), Citrobacter freundii (n = 13), Serratia spp. (n = 8), Morganella morganii (n = 7), Salmonella enterica (n = 2), Achromobacter xylosoxidans (n = 2), Providencia spp. (n = 2), and Shigella flexneri (n = 1), were isolated from the respiratory system (n = 493), urinary system (n = 379), surgical wounds (n = 159), digestive tract (n = 77), blood (n = 60), nervous system (n = 28), and skin and mucous membranes (n = 15) from patients of multidisciplinary hospitals in Moscow and other regions of the Russian Federation in 2003–2015, as well as from hospital environment. The analysis of sensitivity to antimicrobial drugs demonstrated the prevalence of strains resistant to beta-lactams, including to penicillins (99% strains), cephalosporins (95%), and carbapenems (20%); as well as to aminoglycosides (87%), chloramphenicol (74%), and sulfonamides (72%) (Fig. 1a). Multidrug resistance (MDR) phenotype (resistance to antimicrobial drugs of 3 and more functional classes according to the classification of A.P. Magiorakos et al. [7]) was detected in 94% strains of the collection. Among MDR strains, 10% were resistant to 3 functional groups of drugs; 19%, to 4; 42%, to 5; 17%, to 6; and 7%, to 7 (Fig. 1b). Genetic Determinants of Resistance to Beta-Lactams A high level of resistance of the studied strains to beta-lactams (penicillins, cephalosporins, and car- MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 34 No. 1 2019 KUZINA et al. bapenems) is associated with the presence of beta-lactamase genes of blaTEM (35% strains), blaSHV (25%), blaCTX-M (38%), blaOXA (31%), blaVIM (3%), and blaNDM (2%) types (Fig. 2). It should be emphasized that different beta-lactamase genes are typical for different bacterial species. Thus, blaSHV-type genes were detected only in the K. pneumoniae (92% of strains), blaVIM-type genes were detected only in the P. aeruginosa (17%), and blaOXA-type genes (blaOXA-40-, blaOXA-23-, and blaOXA-51-type) were detected only in the A. baumannii (88%). Class 1 and 2 Integrons During the study, 842 integrons were detected, including 737 class 1 integrons (59% strains) and 105 class 2 integrons (8% strains). In addition, integrons were detected in 43% of strains of enterobacteria and in 25% of strains of nonfermenting gram-negative bacteria. The largest number of class 1 integrons was detected in E. coli, P. aeruginosa, and K. pneumomiae, and the largest number of class 2 integrons was found in P. mirabilis (Table 2). Most class 1 integrons (54%) and class 2 integrons (88%) contained in their variable part arrays of the gene cassettes associated with strain resistance to antibacterial drugs of different functional groups (aminoglycosides, chloramphenicol, sulfonamides, and beta-lactams), as well as orf cassettes encoding proteins with unknown functions. Gene Cassette Arrays of Class 1 and 2 Integrons During the study, 22 variants of gene cassette arrays in class 1 integrons and 4 variants in class 2 integrons were detected (Fig. 3). Class I integrons had arrays consisting of one gene cassette (aacA4, aadA1, aadA2, aadB, blaPSE1, dfrA7, estX), two gene cassettes (aacA4-cmlA1j, aacA4-orfD, aadA6-orfD, aadB-aadA1y, aadB-catB3, bla-OXA30-aadA1, dfrA17-aadA5, dfrA1aadA1, dfrA1-orfC, dfrA5-ereA2), three gene cassettes (aacA7-smr2-orfD, dfrA12-orfF-aadA2, dfrA12s-orfFaadA2), and four gene cassettes (aacC1-orfX-orfYaadA1; orfD2-aacA4'-17-orfE14-catB8). Class 2 integrons contained arrays out of two gene cassettes (dfrA1-sat2) and three gene cassettes (dfrA12-sat2aadA1, dfrA1-IS911-sat1-aadA1, dfrA1-sat2-aadA1). Estimation of the prevalence of the gene cassette arrays identified in this study based on a representation of annotated integron sequences in bacterial genomes in the GenBank database demonstrated that class 1 integrons (n = 62597) are much more common than class 2 integrons (n = 2410). Among the class 1 integrons that we identified, the integrons carrying one gene cassette (n = 33120) and two gene cassettes (n = 28537) were most represented in GenBank database on March 10, 2018, while the integrons with three cassettes (n = 413) and four cassettes (n = 527) were Portion of strains, % 20 EB 40 NGNB 30 20 10 0 blaTEM blaSHV* blaCTX-M blaOXA** blaOXA-48 blaVIM*** blaNDM Beta-lactamase genes Fig. 2. Representation of beta-lactamase genes in gramnegative bacterium strains. *, blaSHV-type genes detected only in K. pneumoniae; **, blaVIM-type genes detected only in P. aeruginosa; ***, blaOXA (blaOXA-40-, blaOXA-23-, and blaOXA-51-type) genes detected only in A. baumannii; EB, enterobacteria; NGNB, nonfermenting gram-negative bacteria. less represented. The arrays with two gene cassettes (n = 1302) and with three gene cassettes (n = 1108) were described in class 2 integrons (Table 1). Identification of New Integron Gene Arrays A new class 1 integron, which was assigned the number In1249 in the INTEGRAL database, was identified in E. coli I-7433 clinical strain isolated from a patient’s urine in a Moscow hospital in 2014. Sequencing of variable part of this integron detected the presence of three gene cassettes (dfrA12s-orfF-aadA2), and the dfrA12s gene cassette (GenBank KT316808) is a new Table 2. Representation of class 1 and 2 integrons in gramnegative bacteria Number Species of bacteria of strains, pcs Number of strains carrying integrons (portion, %) class 1 class 2 E. coli 191 131 (69) 22 (12) K. pneumoniae 267 140 (52) 10 (4) 64 27 (42) 48 (75) Other enterobacteria 172 129 (75) 22 (13) A. baumannii 228 113 (50) 1 (0.4) P. aeruginosa 320 196 (61) 1 (0.3) 6 1 (17) 0 (0) 1248 737 (59) 105 (8) P. mirabilis Other NGNB Total MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 34 No. 1 2019 CLASS 1 AND 2 INTEGRONS IN HOSPITAL STRAINS OF GRAM-NEGATIVE BACTERIA Intl1 attl aacA4 In46 Intl1 attl aadA1 ND In2 In127 aadA2 Intl1 attl Intl1 attl aadA7 aacA4 cmlA1 attC In673 Intl1 attl Intl1 attl Intl1 attl aacA4 attC orfD aadB Intl1 attl In299 Intl1 attl Intl1 attl Intl1 attl dfrA7 attl estX∆ In54 In369 In363 In398 Intl1 aadA2 dfrA12s orfF aadA2 Intl1 attl Intl1 attl Intl1 attl Intl2 attl Intl2 attl Intl2 attl Intl2 attl attC attC attC orfD attC attC attC aacA4 orfE attC aacC1 attC catB8 attC attC orfC In561 attC aadA1 drfA7 attl orfF attl blaOXA-1 In322 Intl1 catB3 attC dfrA12 Intl1 attC orfD attC aadA1 aadB smr attl In1249 In609 aadB aacA7 Intl1 In27 aacA6 attC orfD In822 In7 ND attl In51 In142 In22 Intl1 In838 21 attC orfY attC aadA1 dfrA1 sat1 dfrA1 sat1 aadA1 dfrA12 sat1 aadA1 attC In2-3 aadA5 attC attC In2-4 Intl1 attl drfA1 attC aadA1 attC attC In2-4 Intl1 drfA1 attl attC orfC dfrA1attC sat1attC aadA1 ND Intl1 drfA5 attl attC ereA2 IS911 Fig. 3. Variants of gene cassette arrays in class 1 and 2 integrons in gram-negative bacterium strains. attI, primary integron recombination site; attC, gene cassette recombination site. allele of the gene encoding dehydrofolate reductase, which provides resistance to trimethoprim. The analysis of the gene primary structure demonstrated the presence of significant nucleotide substitution T305C, which led to Val102-Ala amino acid substitution in the composition of the encoded enzyme. A new class 2 integron, in which the structure of the dfrA1 gene cassette is damaged by the insertion of IS911 sequence (1256 bp), was identified in the S. flexneri Y-5 clinical strain isolated during a dysentery outbreak in Yakutsk in 2010. This gene cassette structure (dfrA1-IS911-sat1-aadA1) was not previously described, and we deposited it in the GenBank database under number HM592262. The uniqueness of the structure and the presence in all S. flexneri isolates isolated with a dysentery outbreak allowed the class 2 integron to be used as a molecular genetic marker for epidemiological analysis and to concluded about the clonality of this outbreak. Gene Cassettes of Antibiotic Resistance Gene cassettes of 31 types were identified during the study. The analysis of representation of these cassette types in GenBank database demonstrated that the gene cassettes aadB, aacA4, aacC1, aadA1, aadA2, aadA5, blaVIM-2, dfrA1, dfrA7, dfrA12, orfC, orfE, orfY, and sat1 were more common in bacteria on March 10, 2018, while aacA1, aadA6, aadA7, blaPSE1, dfrB4, ereA2, smr2, and dfrA12s were less common (Table 3). The following gene cassettes were the most common in a study by Italian authors in 2009: aadA1 (259 references), aacA4 (204 references), dfrA1 (162 references), aadA2 (150 references), and aadB (89 references) [16]. Over the past 9 years, representation of these gene cassettes in GenBank database increased by 25, 40, 27, 34, and 164 times, respectively. CONCLUSIONS Class 1 and 2 integrons are an important molecular genetic mechanism of the formation of MDR phenotype in gram-negative bacteria isolated from patients and from the hospital environment of multidisciplinary hospitals in Moscow and other regions of the Russian Federation in 2003–2015. During the study, generally accepted role of integrons as a kind of “depot” of genetic determinants of antibiotic resistance and “reserve” for creating new gene cassette combinations was confirmed. The described new gene cassette modifications (dfrA12s and dfrA1-IS911) can be useful molecular genetic markers to track the prevalence of cassettes and evolution of gene cassette arrays, as well as in epidemiological analysis. The conducted analysis of gene cassettes on the basis of representation in GenBank database and of those identified during the study indicates the prevalence of class 1 and 2 integrons in the genomes of clinical strains of bacteria isolated in different regions of the world. MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 34 No. 1 2019 22 KUZINA et al. Table 3. Gene cassettes of antibiotic resistance of class 1 and 2 integrons identified in clinical strains of gram-negative bacteria Gene cassette Synonyms Encoded enzyme Resistance Representation in GenBank database on March 10, 2018 aacA1 aac(6')-Ia Aminoglycoside (6') acetyltransferase AG 116 aacA4 aac(6')-Ib Aminoglycoside (6') acetyltransferase AG 8151 aacA7 aac(6')-II Aminoglycoside (6') acetyltransferase AG 666 aacC1 aac(3)-Ia Aminoglycoside (3) acetyltransferase AG 1836 aadA1 ant(3'')-Ia Aminoglycoside (3) adenylyltransferase STR, SPE 6699 aadA2 – Aminoglycoside (3) adenylyltransferase STR, SPE 5092 aadA5 – Aminoglycoside (3) adenylyltransferase STR, SPE 1438 Aminoglycoside (3) adenylyltransferase STR, SPE 261 Aminoglycoside (3) adenylyltransferase STR, SPE 149 Aminoglycoside (2'') adenylyltransferase STR, SPE 14597 aadA6 aadA11 – aadA7 aadB ant(2'')-Ia blaOXA-1 blaOXA-30 Class D OXA-type beta-lactamase BL 391 blaPSE1 – Class B PSE-type metal beta-lactamase BL 25 blaVIM-2 – Class B VIM-type metal beta-lactamase BL 7589 catB3 – Chloramphenicol acetyltransferase СМ 685 catB8 – Chloramphenicol acetyltransferase СМ 700 cmlA1 – Chloramphenicol exporter СМ 939 dfrA1 dhfrIb, dfr1, dhfr1 Type A dihydrofolate reductase THR 4295 dfrA5 dhfrV, dfrV Type A dihydrofolate reductase THR 447 dfrA7 dhfrVII, dfrVII, dfrA17 Type A dihydrofolate reductase THR 2398 dfrA12 dhfrXII, dfr12 Type A dihydrofolate reductase THR 2666 dfrB4 dhfr2, dfr2b Type B dihydrofolate reductase THR 58 Erythromycin esterase ERI 36 – ereA2 orfC gcuC, orfX Hypothetical protein UK 6550 orfD gcuD Hypothetical protein UK 952 orfE gcuE Hypothetical protein UK 1556 orfF gcuF Hypothetical protein UK 960 orfY gcuQ, orfX`, orfXB, orf10 Hypothetical protein UK 3955 sat1 sat2 Streptomycin acetylase STT 4632 Esterase Insecticides 3298 Small multiple resistance protein BL, AG, CM, THR, QNL Type A dihydrofolate reductase THR – estX smr2 dfrA12s smr, orfO – 191 0 AG, aminoglycosides; BL, beta-lactams; CM, chloramphenicol; ERI, erythromycin; UK, unknown; SPE, spectinomycin; STR, streptomycin; STT, streptothricin; THR, trimethoprim; QNL, fluoroquinolones MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 34 No. 1 2019 CLASS 1 AND 2 INTEGRONS IN HOSPITAL STRAINS OF GRAM-NEGATIVE BACTERIA ACKNOWLEDGMENTS We are grateful to A.N. Kruglov, Cand. of Biology, senior scientist (OOO National Agency for Clinical Pharmacology and Pharmacy, Moscow); S.V. Sidorenko, Dr. of Biology, professor (Children’s Scientific and Clinical Center for Infectious Diseases, Federal Medical and Biological Agency, St. Petersburg); O.N. Ershova, Dr. of Medicine, associate professor (Burdenko National Medical Research Center for Neurosurgery, Ministry of Health, Moscow); and V.E. Malikov, Cand. of Medicine (Infectious Clinical Hospital No. 1, Moscow Healthcare Department). 6. 7. FUNDING 8. This work was performed as a part of the Federal Theme of Research of the Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being “Monitoring and Study of the Properties of Causative Agents of Food and Hospital Infections, Development of Diagnostic Tools” (2016–2020). 9. COMPLIANCE WITH ETHICAL STANDARDS Conflict of interests. The authors declare that they have no conflict of interest. Statement on the welfare of animals. 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