The spread of blaOXA-48 and blaOXA-244 carbapenemase genes among Klebsiella pneumoniae, Proteus mirabilis and Enterobacter spp. isolated in Moscow, Russia

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a great problem of healthcare worldwide. Study of the spread for blaOXA-48-like genes coding epidemically significant carbapenemases among hospital pathogens is important for the regional and global epidemiology of antimicrobial resistance. Antibacterial resistant isolates of Klebsiella pneumoniae (n = 95) from 54 patients, P.mirabilis (n = 32) from 20 patients, Enterobacter aerogenes (n = 6) from four patients, and Enterobacter cloacae (n = 4) from four patients were collected from January, 2013 to October, 2014 in neurosurgical intensive care unit (ICU) of the Burdenko Neurosurgery Institute, Moscow. Characteristics of the isolates were done using susceptibility tests, PCR detection of the resistance genes, genotyping, conjugation, DNA sequencing, and bioinformatic analysis. Major strains under study were multi drug resistant (MDR), resistant to three or more functional classes of drugs simultaneously—98.9 % K. pneumoniae, 100 % P.mirabilis, one E.aerogenes isolate, and one E.cloacae isolate. Molecular-genetic mechanism of MDR in K.pneumoniae and P.mirabilis isolates were based on carrying of epidemic extended-spectrum beta-lactamase blaCTX-M-15 gene (87.2 and 90.6 % accordingly), carbapenemase blaOXA-48-like gene (55.3 and 23.3 % accordingly), and class 1 (54.8 and 31.3 % accordingly) and class 2 (90.6 % P. mirabilis) integrons. The blaOXA-48-like-positive K. pneumoniae were collected during whole two-year surveillance period, while P. mirabilis and Enterobacter spp. carrying blaOXA-48-like genes were detected only after four and 18 months after the research start, respectively. The blaOXA-48-like gene acquisition was shown for P. mirabilis isolates collected from five patients and for E. cloacae isolate collected from one patient during their stay in the ICU, presumably from blaOXA-48-like-positive K. pneumoniae. The source of the blaOXA-244 gene acquired by E. aerogenes isolates and the time of this event were not recognized. The expanding of CPE in the surveyed ICU was associated with the spread of blaOXA-48 and blaOXA-244 carbapenemase genes documented not only among K.pneumoniae, well-known bacterial host for such genes, but among P.mirabilis, E.aerogenes, and E. cloacae.


Background
One of the main problems of healthcare worldwide is hospital-acquired infections (HAI) caused by multi drug resistant (MDR) pathogens [1,2] including carbapenemase-producing Enterobacteriaceae (CPE) [3]. The spread of CPE over the last decades is a great danger because carbapenems are the last treatment options for infections caused by MDR bacteria. Recently carbapenemases belonged to three Ambler-classes of beta-lactamases [4] were reported from CPE isolated in Russia: the KPC-type (class A), the NDM-type (class B), and the OXA-48-type (class D) [5]. The OXA-48 carbapenamase is enzyme that was first identified in Klebsiella pneumoniae clinical strains isolated in Turkey, 2003 [6]. Currently OXA-48-producers are described among many species of enterobacteria: K. pneumoniae, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii and Proteus mirabilis [7][8][9]. The endemic areas of OXA-48-producers are Turkey, the North Africa and India. Nosocomial outbreaks caused by OXA-48-producers are registered in many European countries including France, Germany, Switzerland, Spain, Holland, and Great Britain. Sporadic cases of such infections are observed in North and South America, China, Australia, and in the Middle East countries [10]. Wide spread of bla OXA-48 -like genes among enterobacteria is explained by their localization on the 62 kb conjugative plasmid belonged to IncL/M incompatibility group. Such plasmids are characterized by a high rate of transfer and a wide range of hosts. Their high conjugative potential is due to inactivation of tir gene coding the repressor of conjugative plasmid transfer, by the inserting of the composite transposon Tn1991/Tn1991.2 carrying the bla OXA-48-like gene [11]. In several studies is shown that bla OXA-48 -carrying plasmids provide the ability of both clonal and horizontal transfer [7]. There are several variants of bla OXA-48-like genes coding enzymes differ from each other on some amino acid substitutions [12]. For instance, the bla OXA-244 gene discovered in Spain in 2013 differs from the bla OXA-48 gene in a single nucleotide substitution A640G that result in a single amino acid substitution Arg214Gly [13]. It should be noted that carbapenemase activity of all OXA-48-like enzymes are low, so they cannot provide the high levels of carbapenem resistance without additional contribution of other factors such as porin mutations resulting in decrease of cell wall permeability [14].
In this paper we present phenotypic and genotypic characterization of antibacterial resistant K. pneumoniae, Proteus mirabilis, Enterobacter aerogenes, and E. cloacae isolates carrying bla OXA-48 and bla OXA-244 carbapenemase genes, and discuss K. pneumoniae as possibly source for bla OXA-48-like genes spreading in the ICU.

Bioethical requirements
The materials used in the work do not contain personal data of patients, because of clinical isolates were marked without name, date of birth, address, number of the disease history, personal documents and other personal materials. At the same time, in accordance with the Requirements of the Russian Federation Bioethical Committee, each patient signed a contract with hospital at admission to the clinic. The contract contained consent to treatment and laboratory examination, including a detailed investigation using instrumental methods.

Conjugation experiment
Conjugation was performed using previously described method [15]. Donor and recipient overnight cultures were grown under aeration conditions at 120 rpm/min during 18 h at 37 °C on LB broth containing 100 mg/L cefotaxime (Sigma, USA) for donor strain, and 200 mg/L rifampicin (Sigma, USA) for recipient strain. Five milliliters of fresh LB broth without antibiotic were inoculated by 0.05 ml overnight cultures and incubated with aeration to OD 600 = 0.6. Then 1 ml each donor and recipient cultures were combined into one tube and incubated at 37 °C for 3 h without shaking. After that the conjugating mixture was diluted with tenfold steps and 0.1 ml was plated in triplicate on LB agar containing 100 mg/L cefotaxime and 200 mg/L rifampicin. Bacteria were incubated at 37 °C for 48 h. To determine the efficiency of conjugation the conjugation mixture was inoculated in parallel on cefotaxime + rifampicin and rifampicin. Frequency of plasmid transfer was calculated as the number of transconjugant cells per number of recipient cells.

DNA sequencing
Cycle sequencing reactions were performed using the ABI PRISM BigDye Terminator v.3.1 kit. Purified products were analysed on an ABI PRISM 3100-Avant automated DNA Sequencer in the SINTOL Center for collective use (Moscow, Russia).

GenBank accession numbers
The
The most K. pneumoniae and P. mirabilis isolates in this study were resistant to the majority of used antibacterial agents: AMC, SAM, CXM, CTX, CRO, CIP, CHL, GEN, TOB, TMP, SXT and NIT. Additionally, the K. pneumoniae isolates were resistant to FOX, CAZ, CFS, FEP, ETP, MEM and TET, and the P. mirabilis isolates were resistant to IPM, TET, TGC and CST (Fig. 1). It is noteworthy the high proportion of the pathogens were resistant to TGC (32.4 % K. pneumoniae and 50.0 % P. mirabilis isolates) and to CST (87.5 % P. mirabilis isolates), that are the drugs using for the treatment of severe hospital infections. So, only one antibiotic, CST remains good activity against K. pneumoniae (89.5 % isolates), and only three drugs, CFS (100 % isolates), ETP (100 % isolates) and CAZ (87.1 % isolates) were effective against P. mirabilis (Fig. 1). The antibacterials used in this study belong to the eight functional classes: beta-lactams (penicillins, cephalosporins, and carbapenems), tetracyclines, fluoroquinolones, phenicoles, aminoglycosides, polymyxins, sulfonamides, and nitrofurans. According to Magiorakos et al. [25] many strains under study were categorized as multi drug resistant (MDR) pathogens because of 98.9 % K. pneumoniae and 100 % P. mirabilis isolates were resistant to three or more functional classes of drugs simultaneously (Fig. 2). It should be noted that a large part of both K. pneumoniae (35.9 %) and P. mirabilis (54.8 %) strains were resistant to seven functional antibacterial classes. One E. aerogenes isolate was estimated as an extremely drug resistant because of resistance to all used antibiotics except TGC; three isolates were resistant to cefalosporins, carbapenems and NIT; two isolates were resistant to NIT only. Among E. cloacae isolates one isolate was MDR with sensitivity to IPM and AMK only; other isolates were resistant only to penicillins and cephalosporins.

Resistance genotypes
Detection of the bla CTX-M , bla TEM , bla SHV , bla OXA-48like , bla NDM , and bla KPC beta-lactamase genes as well as class 1 and class 2 integrons in the genomes of the strains has been done for study of the resistance molecular genetic mechanisms (Fig. 3). The bla CTX-M genes were detected in 87.2 % K. pneumoniae and 90.6 % P. mirabilis isolates. Among 38 bla CTX-M genes major (n = 36) were Mobile genetic elements, class 1 integrons were detected in 54.8 % K. pneumoniae and in 31.3 % P. mirabilis isolates (Fig. 3). Variable regions of class 1 integrons identified in K. pneumoniae carried three types of the gene cassette array: (dfrA1-orfC), (dfrA17-aadA5), and (dfrA12-orfF-aadA2). The class 1 integrons detected in P. mirabilis carried only one type of the gene cassette array (dfrA17-aadA5). Class 2 integrons obtained in P. mirabilis (90.6 %) isolates carried a gene cassette array (dfrA12-sat2-aadA1). However, one class 1 integron and one class 2 integron revealed in E. cloacae and E. cloacae isolates respectively did not have any gene cassette arrays, that might be considered as a reserve for future accumulating of the resistance genes. The integron identification numbers have been obtained in accordance with the nomenclature described by Moura et al. [26] using a specialized database INTEGRAL. The (dfrA1-orfC) gene cassette array was attributed to the In263 integron, the (dfrA17-aadA5) array to the In392-395 integron, the (dfrA12-orfF-aadA2) array to the In27 integron, and the (dfrA12-sat2-aadA1) array to the In2-4 integron. According to the report of Partridge et al. from the University of Sydney, Australia, the vast majority of gene cassettes submitted into the GenBank database to date are extremely rare (presented less than 10 times), while a small group of gene cassettes are extremely frequent, they were named "successful" cassettes [27]. All gene cassettes identified in clinical isolates collected from the ICU in our study belong to this group of "successful" cassettes. Thus, on the date Feb 6, 2015, the aadA1 cassette has been presented in the GenBank database 910 times, the aadA2 cassette 601 times, the aadA5 cassette 248 times, the dfrA1 cassette 348 times, the dfrA12 cassette 245 times, the orfF cassette 218 times, the dfrA17 cassette 209 times, the orfC cassette 135 times, and the sat2 cassette 79 times. These data suggest the high prevalence of the gene cassettes detected in our study among bacterial pathogens.  Since the permeability of bacterial cell wall is important for multi drug resistance [23], we carried out the detection of porin gene ompK36 and obtained this gene in 92.4 % K. pneumoniae isolates.

Diversity of RAPD-genotypes
Great genetic heterogeneity of the pathogen populations was shown by RAPD-PCR genotyping for K. pneumoniae (n = 72) and P. mirabilis (n = 32). It was revealed 36 K. pneumoniae genotypes and 8 P. mirabilis genotypes. Four K. pneumoniae genotypes were prevalent in different periods of the study. The K23 genotype (n = 14) was major in 2013, while the K26 genotype (n = 5) was major in the first half of 2014, and the K30 (n = 6) and the K31 (n = 5) genotypes were prevalent in the second half of 2014. Interestingly, K. pneumoniae isolates belonged to K23, K26 and K31 genotypes carried the bla OXA-48-like genes, whereas isolates belonged to K30 genotype did not have these genes. Among all P. mirabilis isolates collected in our study, the P1 RAPD-PCR-genotype (n = 23) was prevalent. It is noteworthy that all P. mirabilis isolates carried bla OXA-48-like genes belonged to this prevalent genotype (Table 1). So, great variety of K. pneumoniae and P. mirabilis genotypes indicates no single source for dissemination of these pathogens. Perhaps presence of bla OXA-48-like genes in bacterial genome provides some populational advantage for certain genotypes. Two RAPD-genotypes (E1 and E2) were identified for E. aerogenes isolates, but all three bla OXA-244 -positive isolates were attributed to one genotype E1. Only one RAPDgenotype (E3) was determined for all E. cloacae isolates, including bla OXA-48 -positive one.

Spread of bla OXA-48-like genes in ICU
The case of the bla OXA-48 gene acquisition by K. pneumoniae within ICU was demonstrated for the Patient K. The first isolate belonged to K29 genotype (May 21, 2013) had no the bla OXA-48 gene; the next two isolates of K29 genotype (Oct 31, 2013 and Dec 12, 2013) have already acquired the bla OXA-48 gene (Table 1; Fig. 4). The first case of the bla OXA-48 gene emergence in a new bacterial host, P. mirabilis, was registered in the Patient H (Apr 08, 2013). It was happened four months after the start of the study, while such genes in K. pneumoniae isolates were detected during the entire two year period. Two months later, Jun 13, 2013, the bla OXA-244 gene was identified in P. mirabilis isolate collected from the Patient C. Unfortunately, a retrospective epidemiological analysis has not allowed concluding relatedness of occurrence the bla OXA-244 gene in Russia and in Spain where this gene was first described in 2013 [13]. The first case of the bla OXA-244 gene emergence in a bacterial host E. aerogenes was registered in the Patient F (Jun 17, 2014), i.e. 18 months after the start of the study. The first case of the bla OXA-48 gene identification in E. cloacae was obtained in the Patient O (Aug 30, 2014), i.e. 20 months after the start of the study. So, two bla OXA-48-like alleles have disseminated in the ICU simultaneously.
Comparative analysis of two or more P. mirabilis isolates collected from the same patient has been done for four patients: E, H, I, and J. All of these patients had no the bla OXA-48-like gene in their first isolates. The second isolates (Patients H and J) and the second and the third isolates (Patients E and I) carried the bla OXA-48-like genes. So, the bla OXA-48-like genes acquisition by P. mirabilis occurred in the ICU, presumably from bla OXA-48-like -positive K. pneumoniae which is half of all K. pneumoniae isolates under study. Additional proof of this hypothesis is the concurrent isolation of bla OXA-244 -positive K. pneumoniae and P. mirabilis from urine of the Patient C (Fig. 4), as well as isolation of bla OXA-48 -positive K. pneumoniae and E. cloacae from endotracheal aspirate and urine correspondently of the Patient N. Unfortunately, such clear hypothesis cannot to explain the fact of the bla OXA-48 -positive K. pneumoniae isolation from endotracheal aspirate and feces of the Patient F and-on the same period-the bla OXA-244 -positive E. aerogenes isolates from liquor of this patient (Table 1).

Plasmid localization of bla OXA-48-like genes
It is known that the bla OXA-48-like genes localized on the IncL/M conjugative plasmids [11,22]. PCR-detection of the specific IncL/M plasmid markers-repA and traUshowed the presence of the plasmid in all bla OXA-48-likepositive P. mirabilis (n = 7) and Enterobacter spp. (n = 4) isolates, and the absence of such plasmid in the bla OXA-48-like -negative isolates. The same correlation between bla OXA-48-like gene and the markers of the IncL/M plasmid was revealed for K. pneumoniae strains. So the bla OXA-48like genes in bacterial isolates under study were located on IncL/M conjugative plasmids.
The acquisition of the bla OXA-48-like gene consisting plasmid IncL/M has been demonstrated by the experimental conjugation at intra-and inter-species transfer. Two clinical K. pneumoniae strains, B-500 and B-757 K, resistant to cefotaxime were used as donors. The K. pneumoniae M-9 Rif strain was used as a recipient at intraspecies conjugation, and the E. coli HB101 Rif was used as a recipient at interspecies conjugation. Selection of transconjugants was done by selective markers cefotaxime and rifampicin. The efficiency of conjugation was 2.0-8.5 × 10 −4 at intra-and 2.0-3.0 × 10 −3at inter-species conjugation. The IncL/M transfer from the donor to the recipient was detected by the plasmid markers repA and traU appearance in the transconjugants. The bla CTX-M , bla TEM , bla SHV , and class 1 integrase Table 1 The features  genes were detected in the transconjugant cells ( Table 2). The distribution of genetic markers in the transconjugants shows that both donor K. pneumoniae strains carried two conjugative plasmids: the IncL/M plasmid (bla + OXA-244 repA + traU + ) and the unidentified plasmid (bla + CTX-M bla + TEM bla + SHV ) ( Table 2).

Conclusions
The molecular genetic mechanisms for multi-drug resistance of K. pneumoniae, P. mirabilis, E. aerogenes and E. cloacae isolates collected from a Moscow neurosurgical ICU in 2013-2014 were shown associated with bla CTX-M , bla TEM , bla SHV beta-lactamase genes, class 1 and class 2 integrons, as well as with bla OXA-48-like carbapenemase genes. Two alleles genes, the bla OXA-48 and the bla OXA-244 , have been identified in the isolates under study. Moreover, P. mirabilis, E. aerogenes and E. cloacae are first determined as bacterial hosts for the of bla OXA-48-like genes in Russia. The bla OXA-48-like genes were found to be integrated into the well-known conjugative plasmids of IncL/M incompatibility group that probably were transferred from K. pneumoniae to P. mirabilis and to E. cloacae during the period of the patient stay in the ICU. The source of the bla OXA-244 gene acquired by E. aerogenes isolates and the time of this event were not recognized. So, this study highlights ongoing expanding of CPE pathogens in Russia. Obtained knowledge is important for an adequate evaluation of the epidemiological situation, for predicting the development of the situation in the future, and for the correct choice of the optimal strategy for the antibacterial therapy.