Analysis of blaCHDL Genes and Insertion Sequences Related to Carbapenem Resistance in Acinetobacter baumannii Clinical Strains Isolated in Warsaw, Poland

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D β-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance.


Introduction
In February 2017, the World Health Organization (WHO) published a list of the antibiotic-resistant "priority pathogens" that pose the greatest threat to human health [1]. Carbapenem-resistant Acinetobacter baumannii was classified as the most pressing threat in "the critical group". This non-fermentative, Gram-negative coccobacillus is one of the main causes of severe nosocomial infections, such as ventilator-associated pneumonia, bloodstream infections, bacteremia, urinary tract infections and wound infections, especially in burn patients and post-surgical procedures [2,3]. The increased incidence of carbapenem resistance among clinical isolates, which has risen dramatically over the last twenty years, is of major concern. Resistance to carbapenems in A. baumannii is mediated mainly by the production of various carbapenem-hydrolyzing β-lactamases belonging to Ambler classes A (KPC and GES groups), B (IMP, VIM, SIM and NDM groups) and D (OXA-51-like, OXA-23-like, OXA-24-like, OXA-58-like, OXA-143-like and OXA-235-like) [4]. Among the clinical isolates of A. baumannii, carbapenem-hydrolyzing class D β-lactamases (CHDLs), also called serine oxacillinases, are the most prevalent. Although they exhibit a weak hydrolysis of carbapenems [3], CHDLs may confer high levels of carbapenem resistance when an insertion sequence (IS) is located upstream of the gene, providing a strong promoter that leads to the overexpression of bla CHDL genes [5]. Genes coding for OXA-51-like enzymes, unlike other CHDL enzymes, occur naturally in all A. baumannii strains [6].
In this study, the contribution of different carbapenemases to carbapenem resistance among clinical A. baumannii strains isolated in one tertiary hospital in Warsaw, Poland, was determined, and 61 imipenem-nonsusceptible isolates were characterized using phenotypic and molecular methods, including genotyping of the isolates.

Distribution of bla CHDL Genes and Insertion Sequences
Among all 61 of the collected non-duplicate, imipenem-nonsusceptible clinical isolates of the Acinetobacter calcoaceticus-A. baumannii complex, the identification of the A. baumannii species was confirmed using gyrB multiplex PCR. PCR analysis of the bla CHDL genes occurrence among these isolates revealed the presence of intrinsic bla OXA-51-like genes and following acquired bla CHDL genes: bla OXA-23-like in 14 of the 61 (23%) isolates, bla OXA-24-like in 39 of the 61 (64%) isolates and bla OXA-58-like in 6 of the 61 (10%) isolates. Furthermore, 59 of the 61 isolates harbored no more than two bla CHDL genes (bla OXA-51-like and one of the acquired bla OXA-23/OXA-24/OXA-58 genes). The bla OXA-143-like genes were not detected in any of the isolates. Two isolates (3%) carried only bla OXA-51-like genes, and in these two cases, ISAba1 was found upstream of the genes encoding the OXA-51-like enzymes. Each of the bla OXA-23-like and bla OXA-58-like genes had an upstream ISAba1 and ISAba3 insertion respectively. Whole genome sequencing was performed on 15 selected A. baumannii isolates, including the representative isolates containing each acquired OXA-possessing group, with differing β-lactam sensitivity profiles and CarbAcineto NP assay results, including the time after which positive carbapenemase activity was detected (Table S1). The whole genome datasets of the 15 strains generated and analyzed during the current study are available at the NCBI BioProject repository (SubmissionID: SUB9082120, BioProject ID: PRJNA701882). Whole genome sequencing (WGS) analysis identified the bla OXA-66 gene encoding intrinsic OXA-66 belonging to the OXA-51-like family, as well as the bla ADC-30 gene encoding ADC-30 from the AmpC cephalosporinase family, in all of the isolates. Among the acquired CHDLs and IS elements, the genome analysis revealed the presence of bla OXA-72 (OXA-24-like family) in six isolates (no. 76, 81, 159, 165, 176 and 195), ISAba1 upstream of the bla OXA-23 gene (OXA-23-like family) in five isolates (no. 96, 113, 118, 129 and 185) and ISAba3 upstream and downstream of the bla OXA-58 (OXA-58-like family) in two isolates (no. 43 and 52; Figure 1). Additionally, in the case of five isolates (no. 86, 87, 96, 129 and 185), the presence of a bla TEM-1 gene encoding a β-lactamase with a narrow spectrum of activity was demonstrated.  Table 1 shows the susceptibility pattern of 61 A. baumannii isolates for 13 antibacterial agents. The minimal inhibitory concentration (MIC) distribution for the carbapenems, imipenem and meropenem, is shown in Table 2. All 61 A. baumannii isolates were determined to be colistin-sensitive, and resistant to ciprofloxacin, piperacillin and piperacillin  Table 1 shows the susceptibility pattern of 61 A. baumannii isolates for 13 antibacterial agents. The minimal inhibitory concentration (MIC) distribution for the carbapenems, imipenem and meropenem, is shown in Table 2. All 61 A. baumannii isolates were determined to be colistin-sensitive, and resistant to ciprofloxacin, piperacillin and piperacillin with tazobactam, regardless of the bla CHDL gene carriage. The majority of isolates were resistant to trimethoprim-sulfamethoxazole, with the exception of 5 out of the 39 bla OXA-24-likecarrying isolates. Of the isolates carrying the acquired bla CHDL genes, all isolates containing the ISAba1-bla OXA-23-like and bla OXA-24-like genes were resistant to both of the carbapenems tested. The highest imipenem and meropenem MIC values were obtained for bla OXA-24-like carrying isolates (64 mg/L and 128 mg/L, respectively).

Phenotypic Detection of Carbapenemases
Positive carbapenemase activity results using the CarbAcineto NP Test were obtained for the majority of ISAba1-bla OXA-23-like -(n = 12/14) and bla OXA-24-like -carrying (n = 34/39) isolates. For the other bla OXA-23-like -and bla OXA-24-like -carrying isolates, as well as all of the isolates containing ISAba3-bla OXA-58-like (n = 6) and ISAba1-bla OXA-51-like genes (n = 2), uninterpretable (the optical reading indicated only a slight change in color for the phenol red solution in the test tube) results were observed. In these cases, compared to the internal control (red color tube), after the required incubation time (maximum 2 h), only the development of a red-orange color was observed in the test tube, but not the expected yellow or orange color. The test was performed in duplicate.
Moreover, none of the 61 studied isolates showed MBL-type enzymes based on the results obtained from the double disc diffusion method (DDST) using ethylenediaminetetraacetic acid (EDTA) assay, imipenem and ceftazidime discs.

Molecular Typing and Genome Analysis of the Isolates
To understand how closely related all 61 A. baumannii isolates are, they were genotyped using pulsed field gel electrophoresis (PFGE) ( Figure 2). Twenty-one pulsotypes (PTs) were determined. The isolates were considered to be a cluster when their similarity was at least 80%. Isolates harboring the bla OXA-24-like , ISAba1-bla OXA-23-like and ISAba3-bla OXA-58-like genes were clustered into 12 PTs, 5 PTs and 3 PTs, respectively. Two isolates carrying only the ISAba3-bla OXA-51-like gene were clustered into two different PTs. Among isolates harboring the bla OXA-24-like gene, the majority were isolated in 2012 and 2013 (31/39, 80%) and belonged to 11 different clusters. Most isolates obtained in 2010 and 2011with the bla OXA-24-like gene formed a separate cluster (PT A) and for five of these isolates a similarity of over 98% was observed. One isolate from 2012 was also mapped to the PT A pulsotype.

Discussion
An increase in the number of A. baumannii strains that are resistant to carbapenems is one of the major therapeutic problems worldwide [3,7,8]. Most of these carbapenemresistant strains exhibit multidrug-resistance (MDR) or extensively drug-resistance (XDR) profiles. According to the European Centre for Disease Prevention and Control (ECDC) data, more than 50% of invasive Acinetobacter spp. strains isolated in Poland in 2015 were defined as carbapenem resistant [9]. Among the 61 carbapenem-nonsusceptible isolates analyzed in our study, all were classified as MDR strains, as defined by Magiorakos et al. [10]. More worryingly, several isolates carrying the genes ISAba1-bla OXA-23-like (10 isolates), bla OXA-24-like (6 isolates) and ISAba1-bla OXA-51-like (1 isolate), showed an XDR resistance profile. An in-depth understanding of the mechanisms causing this widespread resistance is one of the main challenges in the fight to limit the occurrence and spread of carbapenem-resistant strains.
An analysis of the occurrence of bla CHDL genes among the studied isolates revealed the presence of the intrinsic bla OXA-51-like gene in all of the isolates. The majority of isolates also carried one of the following acquired bla CHDL genes: bla OXA-24-like , ISAba1-bla OXA-23-like or ISAba3-bla OXA-58-like . In our study, the ISAba1 sequence was also detected upstream of bla OXA-51-like genes in two isolates in which no acquired genes encoding the remaining CHDL enzymes were found. The presence of an insertion sequence located upstream of bla CHDL leads to an overexpression of this gene, and contributes to the resistance of A. baumannii strains to β-lactams, including carbapenems [5]. So far, the occurrence of the following IS elements ISAba1, ISAba2, ISAba3, ISAba4, ISAba9, ISAba10, IS18 and ISAba825, associated with carbapenem resistance in A. baumannii clinical strains, has been described [11][12][13][14]. ISAba1 is the most common insertion sequence identified in A. baumannii, as well as in other Acinetobacter species, worldwide [12,14]. In clinical A. baumannii strains, the ISAba1 element was found primarily upstream of the bla OXA-23-like genes and, less often, upstream of the bla OXA-51-like and bla OXA-58-like genes. The group of OXA-23-like enzymes is the most commonly acquired resistance CHDL carbapenemase worldwide [3,[15][16][17][18][19]. In our study, all of the isolates with the bla OXA-23-like genes possessed ISAba1 upstream. This is the first data concerning the frequency of ISAba1 upstream of bla OXA-23-like genes in A. baumannii isolates from Poland. WGS analysis confirmed the presence of ISAba1bla OXA-23 gene sequences. So far, in isolates studied worldwide, the presence of other insertion sequences such as ISAba4 and ISAba10 upstream of bla OXA-23-like genes has been described [12]. Interestingly, among the strains examined in this study, more were carrying the bla OXA-24-like gene than the ISAba1-bla OXA-23-like gene (64% versus 23%, respectively). A few studies on A. baumannii strains producing OXA-24 in Poland also indicated a high frequency of occurrence among strains isolated from hospitals. Chmielarczyk et al. reported that 79% of the isolates they studied possessed the bla OXA-24 gene, whereas in the study by Nowak et al., only 49% of the isolates studied possessed the bla OXA-24 gene [20,21]. Reports from Poland appear to be contrary to the data from other countries. OXA-24-like carbapenemases produced by A. baumannii have been found in many European (Bulgaria, France, Portugal, Spain and Sweden) and non-European (Israel, United States and Colombia) countries; however, in most reports, OXA-24 gave way to OXA-23, making the latter more frequently occurring [15,17,[22][23][24][25][26]. An insertion sequence has never been found upstream of bla OXA-24-like genes. The genome analysis revealed the presence of the bla OXA-72 gene (from bla OXA-24-like family) in six isolates analyzed in this study. This variant retains its carbapenemase activity. According to Liu et al., imipenem MIC values for isolates producing this enzyme is at least 64 mg/L, which is concordant with our results [17]. A third group of the acquired bla CHDL genes identified among the Polish isolates tested was ISAba3-bla OXA-58-like . The bla OXA-58 gene is currently being reported worldwide, and its increased expression is always associated with IS elements upstream of this gene. In A. baumannii strains isolated from different European countries, the following insertion elements have been found in the bla OXA-58-like gene promoters: ISAba1, ISAba2, ISAba3, ISAba3like , IS18 and ISAba825 [8,13,27,28]. To date, there are only sparse data on the occurrence of bla OXA-58-like genes in A. baumannii strains isolated in Poland, but none contained information about the presence of IS elements upstream of these genes [15,29]. This is the first time the ISAba3 sequence has been detected upstream of the bla OXA-58-like genes in A. baumannii isolates from Poland, as well as in all six of the OXA-58-producing isolates. Moreover, WGS analysis showed the presence of ISAba3 elements both upstream and downstream of the bla OXA-58 gene in the two isolates tested. It has been reported that often the bla OXA-58-like genes in A. baumannii strains are flanked on two sides with the same or different IS elements [4,7,12].
Overall, using PFGE for genotyping all 61 isolates collected over 2010-2014 did not show any long-term epidemiological incidents caused by any carbapenem resistant A. baumannii strains among patients in the tested hospital. Only a few isolates showed a correlation with a 95% similarity, indicating the transmission of strains between several patients (among two, three, and five patients). The largest number (n = 4) of identical (100% similarity in PFGE) isolates (OXA-24-producing) from different patients was received in 2010. The MLST results revealed that all whole genome sequenced isolates belonged to ST2 (by the Pasteur scheme) which is a highly predominant clone in Poland, as well as in other European countries [15,29,30]. In Poland, carbapenem-resistant strains of A. baumannii belonging to other clones, such as ST1, ST5 and ST193, have rarely been isolated [29]. Analysis of the other four out of seven house-keeping genes in the Oxford scheme typing in relation to the Pasteur scheme allows for the differentiation of strains, especially within ST2, that dominate in Europe. The Oxford scheme revealed diversity among studied isolates and distinguished five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). So far, genetic typing of Polish strains of A. baumannii has not been carried out using the Oxford scheme. Among the A. baumannii strains isolated in Europe, the following above-mentioned sequence types have been described: ST195 in Croatia, ST208 in Bulgaria and Greece, ST348 in Germany and ST425 in Greece [31][32][33][34][35]. Carbapenem-resistant A. baumannii ST195 and ST208 are widespread in the world, and are dominant mainly in the Far East, e.g., in China [36][37][38][39][40]. Furthermore, the ST208/ST1806 strains have been isolated in South Korea [41].
In our study, the majority of isolates carrying the bla OXA-24-like and ISAba1-bla OXA-23-like genes demonstrated the highest carbapenem MIC values, and were the only isolates that presented a positive CarbAcineto NP test result. According to Dortet et al., this test is capable of detecting the production of various CHDLs, such as OXA-23, OXA-24, OXA-58 and OXA-143 groups, by A. baumannii strains with a high level of resistance to carbapenems, i.e., MICs 8 ->32 mg/L [42]. Positive carbapenemase activity results in the CarbAcineto NP Test were obtained for the majority of the ISAba1-bla OXA-23-like (n = 12/14) and bla OXA-24-likecarrying (n = 34/39) isolates. In this study, the results of the CarbAcineto NP test for several bla OXA-23-like -and bla OXA-24-like -carrying isolates (2/14 and 5/39, respectively), as well as for all isolates with the ISAba3-bla OXA-58-like (n = 6) and ISAba1-bla OXA-51-like genes (n = 2), were uninterpretable. Even though the CarbAcineto NP test is able to detect A. baumannii CHDL-producing isolates, it is still possible that the results would be ambiguous. Literacka et al., in their study, demonstrated that for one OXA-24-like, three OXA-23-like and two OXA-58-like-producing A. baumannii isolates, the results of the CarbAcineto NP Test were uninterpretable [43]. In such cases, expression levels of the genes encoding CHDL enzymes may be low or inhibited, which would suggest that some other resistance mechanism affecting carbapenem activity dominates in those isolates, giving high carbapenem MIC values. Perhaps various resistance mechanisms interact simultaneously in these few isolates, such as efflux pumps, changes in outer membrane proteins and, to a lesser extent, carbapenemases.
The MICs of imipenem and meropenem were also determined with the microdilution method in a Mueller Hinton II (MH II) broth medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), according to CLSI guidelines (2012, 2018) [46,47]. Escherichia coli ATCC 25,922 was used as the reference strain for quality control.

Detection of Metallo-β-Lactamases (MBL)
The MBL mechanism of resistance was detected using the double disc diffusion method (DDST) [48]. Disks of 10 µg of imipenem (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and 30 µg of ceftazidime (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) were placed at a distance of 20 mm (center to center) from a disk containing 0.01 mL of 0.5 mM EDTA solution. The presence of an extended inhibition zone of antibiotics towards the disc with EDTA was interpreted as a positive result for metallo-β-lactamase-producing strains screening.

CarbAcineto NP Test
The CarbAcineto NP test was adapted from the updated version of the Carba NP test, used for the detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas spp. The test was performed as described by Dortet et al. [42].

Molecular Detection of bla OXA-like Genes and ISs Upstream of bla CHDL Genes
Genomic DNA was extracted using the Genomic Mini isolation kit (A&A Biotechnology, Gdynia, Poland), according to manufacturer's instructions. Detection of the bla CHDL genes was performed using (1) duplex PCR for bla OXA-51-like and bla OXA-23-like , (2) duplex PCR for bla OXA-24-like and bla OXA-58-like and (3) singleplex for bla OXA-143-like , and (4) singleplex was used for detection of the insertion sequences (ISs) upstream of bla OXA-51-like , bla OXA-23-like , bla OXA-24-like and bla OXA-58-like . The primers used are listed in Table 4

Pulsed Field Gel Electrophoresis (PFGE)
All 61 A. baumannii isolates were typed by PFGE according to a previous protocol [50] with modifications. An overnight culture of bacteria was suspended in 150 µL of a cell suspension buffer and was mixed with 20 µL of 20 mg/mL Proteinase K (Promega GmbH, Walldorf, Germany) and 170 µL of 1.5% low melting agarose (SeaKem Gold Agarose (Lonza, Basel, Switzerland)) and distributed in a plug mold. The genomic DNA in agarose plugs was lysed in 2.5 mL of cell lysis solution supplemented with 20 µL of 20 mg/mL Proteinase K (Promega GmbH, Walldorf, Germany) and 7.5 µL of 10 mg/mL RNase (Sigma, St. Louis, MO, USA), and was washed and digested with 20 U ApaI restriction enzyme (ABO, Gdańsk, Poland). Electrophoresis was performed using the CHEF DR II system (Bio-Rad, Hercules, CA, USA). The migration conditions were as follows: switch angle 120, voltage 6 V/cm, temperature 14 • C and a two-blocks program with a total run time of 22 h (first-block 1-8 s for 15 h, and second-block 5-20 s for an additional 7 h). The genomic DNA of Salmonella serotype Braenderup strain (H9812) digested with XbaI (ABO, Gdańsk, Poland) and Lambda-DNA Ladder PFG Marker (New England BioLabs, Ipswich, MA, USA) were used as the DNA molecular-weight markers [51]. The PFGE patterns were analyzed using GelCompar II software (Applied Maths, Sint-Martens-Latem, Belgium) with the Dice coefficient and clustering by unweighted pair group method with arithmetic mean (UPGMA) with 1% tolerance. The isolates were clustered in the PFGE pulsotypes (PTs) in accordance with the recommendations of Tenover et al. [52].

Whole Genome Sequencing (WGS) and Multi-Locus Sequence Typing (MLST)
Several A. baumannii isolate genomes were whole genome sequenced. This was performed using Public Health England -Genomic Services and Development Unit (PHE-GSDU) on the HiSeq 2500 System (Illumina, CambridgeZ, UK) with paired end read lengths of 150 bp. A minimum of 150 Mb of Q30 quality data were obtained for each isolate. FastQ files were quality trimmed using Trimmomatic 0.32 [53]. SPAdes 3.1.1 was used to produce draft chromosomal assemblies, and contigs less than 500 bp were filtered out [54]. All of the above sequencing analyses were performed using PHE Galaxy [55]. Using the WGS data, the sequence types (STs) were determined using both the Pasteur and Oxford MLST databases [56]. In the case of the Pasteur MLST scheme, the cpn60, fusA, gltA, pyrG, recA, rplB and rpoB gene sequences were analyzed, while the Oxford MLST scheme analysis included the gltA, gyrB, gdhB, recA, cpn60, gpi and rpoD gene sequences.

Conclusions
From the A. baumannii clinical strains isolated in Poland, the dominating carbapenem resistance mechanism was CHDL enzymes, mainly those from the OXA-24-like family. The presence of insertion sequences flanking bla OXA-58-like (ISAba3) and bla OXA-23-like (ISAba1) genes, which enhance the gene expression, was demonstrated. All tested isolates belong to sequence type ST2 (of the Pasteur MLST scheme) from the global IC2 clone. The Oxford MLST scheme allowed for the differentiation of the sequence types of these isolates.