Molecular epidemiology and antimicrobial resistance features of Acinetobacter baumannii clinical isolates from Pakistan

Background Acinetobacter baumannii is a Gram-negative opportunistic pathogen with a notorious reputation of being resistant to antimicrobial agents. The capability of A. baumannii to persist and disseminate between healthcare settings has raised a major concern worldwide. Methods Our study investigated the antibiotic resistance features and molecular epidemiology of 52 clinical isolates of A. baumannii collected in Pakistan between 2013 and 2015. Antimicrobial susceptibility patterns were determined by the agar disc diffusion method. Comparative sequence analyses of the ampC and blaOXA-51-like alleles were used to assign the isolates into clusters. The whole genomes of 25 representative isolates were sequenced using the MiSeq Desktop Sequencer. Free online applications were used to determine the phylogeny of genomic sequences, retrieve the multilocus sequence types (ST), and detect acquired antimicrobial resistance genes. Results Overall, the isolates were grouped into 7 clusters and 3 sporadic isolates. The largest cluster, Ab-Pak-cluster-1 (blaOXA-66 and ISAba1-ampC-19) included 24 isolates, belonged to ST2 and International clone (IC) II, and was distributed between two geographical far-off cities, Lahore and Peshawar. Ab-Pak-clusters-2 (blaOXA-66, ISAba1-ampC-2), and -3 (blaOXA-66, ISAba1-ampC-20) and the individual isolate Ab-Pak-Lah-01 (ISAba1-blaOXA-66, ISAba1-ampC-2) were also assigned to ST2 and IC II. On the other hand, Ab-Pak-clusters-4 (blaOXA-69, ampC-1), -5 (blaOXA-69, ISAba1-ampC-78), and -6A (blaOXA-371, ISAba1-ampC-3) belonged to ST1, while Ab-Pak-cluster-6B (blaOXA-371, ISAba1-ampC-8) belonged to ST1106, with both ST1 and ST1106 being members of IC I. Five isolates belonged to Ab-Pak-cluster-7 (blaOXA-65, ampC-43). This cluster corresponded to ST158, showed a well-delineated position on the genomic phylogenetic tree, and was equipped with several antimicrobial resistance genes including blaOXA-23 and blaGES-11. Conclusions Our study detected the occurrence of 7 clusters of A. baumannii in Pakistan. Altogether, 6/7 of the clusters and 45/52 (86.5%) of the isolates belonged to IC I (n = 9) or II (n = 36), making Pakistan no exception to the global domination of these two clones. The onset of ST158 in Pakistan marked a geographical dispersal of this clone beyond the Middle East and brought up the need for a detailed characterization.


Introduction
Acinetobacter baumannii is an important opportunistic pathogen that has increasingly been reported worldwide. The World Health Organization (WHO) has recently classified carbapenem resistant A. baumannii as a top priority organism for research and development of new antibiotics against antibiotic-resistant bacteria [1]. The

Open Access
Annals of Clinical Microbiology and Antimicrobials occurrence of A. baumannii in Pakistan was first reported in 2004, where it counted for 4.4% of the Gram-negative bacilli in a collection of 812 isolates obtained from a variety of clinical samples in Rawalpindi in 2002 [2]. Importantly, 72% of the reported A. baumannii isolates were found to be extended-spectrum beta-lactamase (ESBL) producers. Later, eight clusters of carbapenem-resistant isolates of A. baumannii were detected in two intensive care units in Karachi, Pakistan, between November 2007 and August 2008 [3]. Concurrently, A. baumannii was the most frequent bacterial pathogen among a collection of 50 carbapenem-resistant clinical isolates obtained in Rawalpindi in 2009 [4]. According to Kaleem et al., twenty-seven (84%) of the carbapenem-resistant A. baumannii isolates were metallo-beta-lactamase positive by the E-test strip method.
Recently, A. baumannii was isolated in 18% of patients with ventilator-associated pneumonia, making it the 4th most frequent pathogen, at the Khyber Teaching Hospital in Peshawar in 2013 [5]. Notably, A. baumannii was declared as the most resistant pathogen identified in this study. A. baumannii also ranked the second most commonly detected pathogen (24.7%) among a collection of carbapenemase producers obtained from one hospital in Lahore between 2015 and 2016 [6]. Furthermore, A. baumannii was the main isolated bacterium (39.8%) among a total of 113 isolates recovered from 80 ventilator-supported patients at the Foundation Hospital, Rawalpindi, in 2016 [7].
Nevertheless, only few of these studies have plotted the epidemiology of A. baumannii in Pakistan onto a global map. Most of the 2007-2008 hospital-acquired carbapenem-resistant A. baumannii belonged to either International clone (IC) I or II [3]. Similarly, 1/17 and 7/17 clinical isolates of A. baumannii, obtained in 2008, were linked to ICs I and II, respectively [8]. Two isolates collected in Norway in 2009 with a history of import from Pakistan were assigned to sequence types 2 (ST2) and ST15, using the multilocus sequence typing (MLST) scheme of Pasteur Institute [9,10]. ST2 is a key member of IC II while ST15 has often been reported in South America [11,12]. Our search in the A. baumannii MLST databases yielded records of 4 isolates from Pakistan (last accessed in 11 September 2019). The 4 isolates were entered in 2019, which might explain why we could not find published literature on them. Two of these isolates belonged to ST1310 (8,1,5,1,6,2,3) or ST1327 (13,1,5,26,7,1,29), for which there was no linkage to known ICs. The other two isolates belonged to ST1106 (2, 1, 2, 1, 5, 1, 1), a member of clonal complex CC1 corresponding to IC I [11].
The aim of this study was to investigate the molecular epidemiology and antibiotic resistance features of 52 A.
baumannii clinical isolates collected in Pakistan between 2013 and 2015. The clonality among our isolates was crossed over the global population of A. baumannii.

Bacterial isolates
Two collections of A. baumannii clinical isolates were included in this study. The isolates, each recovered from one patient, were collected on a consecutive basis regardless of their antimicrobial susceptibility features ( Table 1). The first collection (n = 16) was obtained between February and September 2013 at the Combined Military Hospital in Lahore (CMH Lahore), while the second one (n = 36) was obtained between February and November 2015 at the Combined Military Hospital in Peshawar (CMH Peshawar). The geographic distance between the two hospitals is about 520 kilometres (Additional file 1: Figure S1). The isolates were cultured from pus (n = 17), respiratory secretions (n = 27), intravenous catheter tip (n = 5), Foley catheter tip (n = 2), and bile (n = 1). The clinical samples were processed according to standard in-house culture protocols. When needed, isolates were revived from frozen glycerol stocks onto Brain Heart Infusion agar plates or slants (Oxoid, Basingstoke, United Kingdom). Species identification was first determined using API 20 NE (BioMerieux, France) and later confirmed by detecting occurrence of the intrinsic bla OXA-51-like gene in all the isolates [13]. The identification was further verified by partial rpoB (zone 1, 352 bp) gene sequence analysis in 25 isolates selected for wholegenome sequencing [14].

Antimicrobial susceptibility testing
Susceptibility of the isolates to ampicillin/sulbactam, piperacillin/tazobactam, cefepime, cefotaxime, ceftazidime, ceftriaxone, meropenem, imipenem, gentamicin, amikacin, tobramycin, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, and minocycline was investigated by the agar disc diffusion method, using discs from Oxoid (Basingstoke, United Kingdom). The broth microdilution method was used for doxycycline (Pfizer Global pharmaceuticals), polymyxin B (Glaxosmith Kline pharmaceuticals), and colistin (Forest pharmaceuticals). The tests were performed and susceptibility patterns were interpreted following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [15].

Strain typing
The isolates were typed using two single-locus molecular schemes based on the allelic identity of the A. baumannii-intrinsic ampC and bla OXA-51-like genes [16,17]. PCR amplifications of ampC and bla OXA-51-like were performed using in-house designed primers (Additional Karah et al. Ann Clin Microbiol Antimicrob (2020) 19:2 file 2: Table S1) and followed by Sanger sequencing of the amplicons, as previously described [16,17]. This approach was able to detect the occurrence of insertion sequence (IS) elements, such as ISAba1, in the bordering regions of ampC and/or bla OXA-51-like .

Whole-genome sequence analyses
Twenty-five isolates were selected for whole-genome sequencing based on their antimicrobial resistance patterns and strain typing results. Sequencing was performed using the MiSeq Desktop Sequencer and MiSeq Reagent Kit v3 (Illumina, San Diego, CA, USA). DNA preparation, library construction, and genome sequencing were done according to the manufacturers' instructions. Sequence data were assembled and analysed using the CLC genomics workbench (v7.0.4; CLC bio, Aarhus, Denmark). The web server Reference sequence Alignment-based Phylogeny builder (REALPHY) was used to  construct a phylogenetic tree based on multiple alignments of the genomic sequence data [18]. Sequence reads were mapped to the genomes of three well-known reference strains: AYE (IC I, GenBank: NC_010410.1), ACICU (IC II, GenBank: NC_010611.1), and ATCC 17978 (sporadic ST437, GenBank: NZ_CP018664.1). An In-silico search was performed to detect the existence of 24 A. baumannii plasmid-borne replicase genes, according to the A. baumannii PCR-based replicon typing (AB-PBRT) scheme and a number of other studies on A. baumannii plasmids [19][20][21][22]. The MLST web-based search engine, hosted by the Center for Genomic Epidemiology in Denmark (http://www.genom icepi demio logy. org/), was used to assign the isolates into STs according to the Institute Pasteur's MLST scheme (http://www. paste ur.fr/mlst) [23]. The occurrence of acquired antimicrobial resistance genes was detected using the Res-Finder service, also hosted by the Center for Genomic Epidemiology in Denmark [24]. The occurrence of resistance genes was verified, and genetic surroundings were annotated based on the yields of nucleotide similarities obtained using the Basic Local Alignment Search Tool (http://blast .ncbi.nlm.nih.gov/Blast .cgi) against the "Nucleotide collection (nr/nt)" and/or "Whole-genome shotgun contigs (wgs)" databases [25]. The presence of neighbouring IS elements was detected using the ISfinder online application [26].

Whole-genome phylogenetic tree
Among the 25/52 isolates selected for whole genome sequence analysis, Ab-Pak-clusters-1, -2, -3, -4, -5, -6A, -6B, -7 were represented by 6 (showing 3 different antimicrobial resistance patterns)/24, 3/7, 2/4, 2/3, 2/2, 2/2, 2/2, and 3/5 isolates, respectively. The three individual isolates were also included. The genome assembly features were presented in Additional file 5: Table S3. The whole-genome phylogenetic tree showed a branching pattern indorsing the assembly of Ab-Pak-cludters-2, -3, -4, -5 and -7 (Fig. 1). The distinction between Ab-Pak-cludters-6A and -6B was also confirmed. The six isolates of Ab-Pak-cluster-1 were distributed on two detached branches and a few sub-branches. Such a high discriminatory power of whole-genome phylogenetic analyses, compared to loci-focused strain typing approaches, has been noted in previous studies [36,37]. Nevertheless, the internal splits in Ab-Pak-cluster-1 were most likely related to a long-standing presence of this cluster. In addition, the tree showed a noticeably well-demarcated positioning of Ab-Pak-cluster-7 far from ICs I and II. Sequencing the whole genome of more isolates, especially from Ab-Pak-cluster-1, would have increased our capacity to confirm or exclude transmission events between patients [38]. However, such a limitation is generally tolerable when resources are limited.
Other replicase genes, such as repAci4 and the replicase genes of p2ABSDF and p4AYE, were present in 2/25, 5/25, and 4/25 of the isolates, respectively. Two isolates, Ab-Pak-Pesh-20 and Ab-Pak-Pesh-27, from Ab-Pak-cluster-4 carried a replicase gene with novel nucleotide identity, designated repAci24, showing only 58% nucleotide identity to the replicase gene of p3ABSDF (GenBank: CU468233.1). The three Ab-Pakcluster-7 isolates carried another novel replicase gene, designated repAci25, which will be further discussed below. The occurrence of partial sequences of some replicase genes was noted in the genomes of few isolates (Additional file 6: Table S4). Our in silico analysis was not able to confirm or exclude if these incomplete sequences represented or belonged to actual plasmids.

Antimicrobial resistance genes
A variety of antimicrobial resistance genes were detected in the genomes of the 25 isolates selected for whole genome sequencing (Additional file 7: Table S5). Class D ß-lactamase gene bla OXA-23 was present in 24/25 isolates. bla OXA-23 was located either in Tn2006 (12 isolates) or Tn2008 (12 isolates). Some isolates, such as Ab-Pak-Lah-04 and Ab-Pak-Lah-08 from Ab-Pak-cluster-1, carried three copies of bla OXA-23 . Of note, only one copy was intact while the other two were truncated at their 5′ extremities (data not published). A similar scenario was detected in strain AbPK1 (GenBank: CP024576). Interestingly, AbPK1 was collected in Pakistan in 2012. It also belonged to ST2 and had the ampC-19 and bla OXA-66 alleles [47]. Although it was isolated from infected animals, AbPK1 had a strong genotypic linkage to Ab-Pak-cluster-1.
The individual strain Ab-Pak-Pesh-22 was resistant to meropenem but susceptible to imipenem. Interestingly, Ab-Pak-Pesh-22 did not carry bla OXA-23 or any other commonly known mechanism for carbapenem resistance. On the other hand, Ab-Pak-Lah-01 was equipped with two mechanisms conferring carbapenem resistance, an acquired bla OXA-23 gene and an intrinsic bla OXA-66 gene proceeded by ISAba1 [48]. None of our isolates carried the class D ß-lactamase bla OXA-24-like or bla OXA-58-like genes.

Conclusion
Our study demonstrated that uncomplicated sequence typing schemes, based on comparative analysis of one or two intrinsic loci, such as ampC and bla OXA-51-like , could be a practical approach for rapid grouping of bacterial clinical isolates, such as A. baumannii. However, further validation of the results is commonly needed. The occurrence of a 100% rate of multidrug-resistant strains is alarming and worth a rapid action plan, including regular follow ups and urgent management procedures. The study enabled us to detect seven clusters of A. baumannii prevailing in two clinical settings in Pakistan. Two of these clusters, Ab-Pak-cluster-1 and -6, lasted for more than 2 years and were able to spread between Lahore and Peshawar. The predominance of IC II in Pakistan was in line with the intensive circulation of this clone worldwide (11). The frequent occurrence of isolates belonging to IC I underlined that this clone is still a key trouble-maker in several parts of the world, including Pakistan. Significantly, Pakistan and the Middle East could be a reservoir for under-detected clones of carbapenem-resistant A. baumannii, including the bla OXA-23 -and bla GES-11 -positive ST158. The clinical significance and virulence features of ST158, represented by Ab-Pak-cluster-7 in Pakistan, is worth further investigations.