Next Article in Journal
Fallopia japonica Root Extract Ameliorates Ovalbumin-Induced Airway Inflammation in a CARAS Mouse Model by Modulating the IL-33/TSLP/NF-κB Signaling Pathway
Next Article in Special Issue
Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain
Previous Article in Journal
A Brief Review on the Potential of Psychedelics for Treating Alzheimer’s Disease and Related Depression
Previous Article in Special Issue
Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 15
10.3390/ijms241512515
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt

by
Sandra Sánchez-Urtaza
1,
Alain Ocampo-Sosa
2,3,*,
Ainhoa Molins-Bengoetxea
1,
Jorge Rodríguez-Grande
2,
Mohammed A. El-Kholy
4,
Marta Hernandez
5,
David Abad
5,
Sherine M. Shawky
6,
Itziar Alkorta
7 and
Lucia Gallego
1,*
1
Department of Immunology, Microbiology and Parasitology, Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Spain
2
Microbiology Service, Health Research Institute (IDIVAL), University Hospital Marqués de Valdecilla, 39008 Santander, Spain
3
CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, 28029 Madrid, Spain
4
Division of Clinical and Biological Sciences, Department of Microbiology and Biotechnology, College of Pharmacy, Arab Academy for Science, Technology & Maritime Transport (AASTMT), Alexandria P.O. Box 1029, Egypt
5
Laboratory of Molecular Biology and Microbiology, One Health, Agrarian Technological Institute of Castile and Leon (ITACyL), 47009 Valladolid, Spain
6
Medical Research Institute, Alexandria University, Alexandria 5422031, Egypt
7
Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, 48940 Leioa, Spain
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(15), 12515; https://doi.org/10.3390/ijms241512515
Submission received: 3 July 2023 / Revised: 30 July 2023 / Accepted: 5 August 2023 / Published: 7 August 2023
(This article belongs to the Special Issue Antimicrobial Resistance, Molecular Epidemiology and Emerging Therapeutic Strategies in Carbapenem-Resistant Gram-Negative Bacteria)
Browse Figures
Review Reports Versions Notes

Abstract

:
The increasing rates of antimicrobial resistance among carbapenem-resistant Acinetobacter baumannii in the Middle East and North Africa are one of the major concerns for healthcare settings. We characterised the first A. baumannii isolate harbouring five β-lactamases identified in Egypt. The isolate Ale25 was obtained from an ICU patient of a hospital from Alexandria. The isolate was phenotypically and genotypically screened for carbapenemase genes. The isolate was resistant to carbapenems, aminoglycosides, fluoroquinolones and cefiderocol. Whole-Genome Sequencing identified five β-lactamase genes, blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57, together with other antibiotic resistance genes, conferring resistance to sulfonamides, macrolides, tetracyclines, rifamycin and chloramphenicol. Virulome analysis showed the presence of genes involved in adhesion and biofilm production, type II and VI secretion systems, exotoxins, etc. Multi-Locus Sequence Typing analysis identified the isolate as Sequence Types 113Pas and 2246Oxf, belonging to International Clone 7. Sequencing experiments revealed the presence of four plasmids of 2.7, 22.3, 70.4 and 240.8 Kb. All the β-lactamase genes were located in the chromosome, except the blaPER-7, gene which was found within the plasmid of 240.8 Kb. This study highlights the threat of the emergence and dissemination of these types of isolates.

1. Introduction

Carbapenem-resistant Acinetobacter baumannii (CRAB) constitutes one of the major challenges for healthcare settings due to its high rates of antimicrobial resistance, heading the list of critical pathogens published by The World Health Organization. The main mechanisms of carbapenem resistance in A. baumannii are OXA-type carbapenemases [1]. However, the number of A. baumanni isolates harbouring class B metallo-β-lactamases, such as New Delhi Metallo-Beta-lactamases (NDM), have dramatically increased [1]. In the Middle East and North Africa, A. baumannii clinical outbreaks are usually caused by isolates endemically producing carbapenemases NDM and OXA-23 [2]. Furthermore, in A. baumannii clinical isolates, class A β-lactamases, such as PER-type β-lactamases, can also be responsible for resistance to carbapenems and the last-resort antibiotic cefiderocol [3]. Furthermore, as demonstrated by other authors, a plasmid containing blaPER-1 gene was transferred to a susceptible A. baumannii CIP70.10 resulted in a ≥16-fold increase in the MIC of cefiderocol [3,4]. Despite the alarming situation, there is little information or studies about A. baumannii in Egypt [2]. The aim of the present study was the molecular characterisation of the first A. baumannii isolate harbouring five β-lactamases in Egypt, obtained from a 63-year-old renal dialysis female patient who entered the ICU of a hospital from Alexandria.

2. Results

The isolate was identified by MALDI-TOF, gyrB multiplex PCR and was confirmed by whole genome sequencing as A. baumannii, and it also tested positive for blaOXA-51-like gene.
The susceptibility testing results are shown in Table 1.
Molecular typing assigned the isolate to STs 113 by Pasteur Scheme and ST 2246 by Oxford Scheme. These results in combination with the blaOXA-51-like variant revealed that the isolate belonged to IC7.
Three recognised carbapenemase genes were detected by Whole Genome Sequencing, blaOXA-64, blaNDM-1 and blaOXA-23, as well as the Extended Spectrum Beta-Lactamase coding gene blaPER-7 and the cephalosporinase gene blaADC-57. The isolate also harboured several aminoglycoside resistance genes conferring resistance to streptomycin, gentamycin, tobramycin, amikacin, kanamycin and spectinomycin, as well as genes conferring resistance to sulfonamides, macrolides, tetracyclines, rifamycin and chloramphenicol, respectively (Table 2).
Analysis of the genetic surroundings of the β-lactamase genes located the blaNDM-1 within the truncated isoform of transposon Tn125 (ΔTn125) (Figure 1a). The blaPER-7 gene was found within a complex structure connecting the ISCR1 element and a class 1 integron with part of IS26 upstream of the integron. The gene-cassette variable regions contained the antibiotic resistance genes arr-2 and cmlA5 genes (Figure 1b). Upstream of blaPER-7, we found the ISCR1 element. A gst gene and an abc transporter gene were detected in the ISCR1 linked genes variable region downstream of blaPER-7. Interestingly, another ISCR1 element, IS5, and part of IS10A were found downstream of the 3′-CS. The blaOXA-23 gene was located within transposon Tn2006 (Figure 1c). No insertion sequences were found in close proximity to blaOXA-64 or blaADC-57 genes (Figure 1d,e).
Virulome analysis revealed the presence of a wide variety of genes involved in adherence and biofilm production (type IV pili, biofilm-controlling response regulator, AdeFGH efflux pump, quorum sensing, Csu fimbriae, biofilm-associated protein, outer-membrane protein, Poly-N-acetyl-D-glucosamine), immune modulation (capsule, lipopolysaccharide and penicillin-binding Protein G), effector delivery systems (type II and type VI secretion systems), exotoxins (phospholipases C and D) and iron uptake (acinetobactin) (Table 3).
Two structures of around 26 and 2.4 Kb (Figure 2A) and a plasmid of approximately 240 Kb (Figure 2B) were observed by conventional plasmid extraction and S1-Pulsed Field Gel Electrophoresis experiments, respectively. Further sequencing experiments revealed three conjugative plasmids of 22.3, 70.4 and 240.8 Kb as well as a non-conjugative plasmid of 2.7 Kb. PCR-based and whole-genome-sequence-based Acinetobacter Replicon typing experiments identified repAci6 (homology group GR6) in the 70.4 Kb plasmid and a rep gene encoding a Rep_3 family protein R3-T60 in the 240.8 Kb plasmid. We were not able to identify the rep genes of the 2.7 and 22.3 Kb plasmids using these methods.
Plasmid sequencing experiments located all the β-lactamase genes in the chromosome (Supplementary Materials Tables S1–S5), except the blaPER-7 gene, which was found within the 240.8 Kb plasmid pAbAle25.1 together with aminoglycoside (armA, strA, strB), sulfonamide (sul1, sul2), macrolide (msrE, mphE), tetracycline (tet(B)), chloramphenicol (cmlA5) and rifamycin (arr-2) resistance genes (Figure 3). The aminoglycoside resistance gene aph (3′)-VI was found within the 70.4 Kb plasmid. Hybridisation experiments also confirmed the chromosomal location of the blaNDM-1 gene (Figure 2B).

3. Discussion

Consistent with the recent studies reporting a carbapenem resistance rate of 98% among Egyptian A. baumannii isolates [5], our isolate was resistant to imipenem and meropenem. To our knowledge, no cefiderocol-resistant A. baumannii isolates have been previously reported in Egypt, turning the spotlight on the emergence and dissemination of resistance to cefiderocol in Egypt.
The isolate belonged to IC7, which is especially interesting considering that isolates belonging to IC7 are frequently reported in South America [6,7] but not in Egypt, where IC2 is the most prevalent international clone [8].
The presence of ISAba125 upstream of the blaNDM-1 may enhance its expression [9]. In our isolate, blaNDM-1 gene was located in the chromosome, which is the most frequent localization of these genes, as previously reported by other authors [1,10]. By BLASTn, we observed that the genetic environment of the blaNDM-1 gene was 100% similar to an A. baumannii isolated in France in 2011 (Acc. No. JX000237.3), an A. baumannii isolated in Lebanon in 2015 (Acc. No. CP082952.1) and an A. baumannii isolated in India in 2018 (Acc. No. CP038644) assigned to ST85 Pasteur Scheme, ST1089 Oxford Scheme with blaOXA-94, and which harboured a single copy of blaNDM-1. These three isolates belonged to IC9, suggesting that this genetic context is circulating and being transmitted among different ICs.
The structure containing the ISCR1 element and a class 1 integron, in which the blaPER-7 gene was located, has been previously described, and it is closely related to multidrug-resistant bacteria [11]. The presence of this betalactamase gene in a high-molecular-weight conjugative plasmid of 240.8 Kb has not been previously reported, although the blaPER-1 variant has been described by other authors within a similar structure [12]. Our concern is that it is the first time one obtains the sequence of this structure coding for multiple resistance and virulence genes. Despite the fact that the carbapenemase activity of PER-type and ADC-like enzymes is doubtful, recent studies have shown that PER-7 may exhibit carbapenemase activity [9] and that ADC-57 may hydrolyse ertapenem [13], which, in our isolate, may be contributing to carbapenem resistance.
The blaOXA-23 gene was found within the transposon Tn2006, which is the most frequently reported transposon harbouring this gene and the only one that has been experimentally proven to transpose [14,15]. The presence of ISAba1 upstream of the blaOXA-23 gene might lead to the overexpression and mobilisation of this carbapenemase gene, enhancing carbapenem resistance [16].
The results we obtained by different plasmid extraction methods showed that in order to obtain the complete profile, a combination of all of them is needed, as this improves the number of structures that can be detected. Each technique is not able to provide a complete landscape of the plasmidome that was finally revealed by sequencing experiments. Plasmid extraction kits and conventional lysis are useful for low-molecular-weight plasmids isolation and for a first screening, but it becomes necessary to complete these results with additional experiments, such as S1-PFGE or sequencing techniques. The combination of using short and long-read technologies to perform hybrid assemblies has been demonstrated to be a powerful tool for plasmids resolution [17].
To the best of our knowledge, only a single isolate harbouring four different classes of β-lactamase genes has been previously reported in Bangladesh [18], so this would be the first report of an A. baumannii clinical isolate harbouring four different classes of β-lactamase genes in Egypt, and it would also be the first cefiderocol-resistant isolate reported in Egypt. This study highlights the ability of A. baumannii to acquire and accumulate multiple antibiotic resistance genes, and it also turns the spotlight on the emergence and threat of the dissemination of IC7 isolates, not frequently reported in Egypt, and the importance of controlling the dissemination of these types of isolates.

4. Materials and Methods

A 63-year-old renal dialysis female patient with acute-on-chronic kidney disease was admitted to the hospital and entered the ICU on 13 November 2020. On 29 November 2020, a swab from around the abdominal pigtail catheter was obtained by the Microbiology Service, Alexandria University Medical Research Institute. Being a renal patient, she was treated with tigecycline injection as well as topical polymyxin. The patient was discharged home after 40 days at the ICU.
Species identification was assessed by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF/Vitek-MS with SARAMIS MS-IVD v2, Biomérieux, Marcy-l’Étoile, France), and the detection of blaOXA-51-like gene was conducted by PCR [19,20,21] and gyrB multiplex PCR [22] and confirmed by whole genome sequencing. Minimal inhibitory concentrations of ticarcillin, ticarcillin/clavulanic acid, piperacillin, piperacillin/tazobactam, imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, aztreonam, tigecycline, cefiderocol, minocycline, colistin and trimethoprim/sulfamethoxazole were determined by broth microdilution method following CLSI guidelines. MICs were interpreted using the resistance breakpoints for Acinetobacter spp. from EUCAST (Version 13.1, June 2023, http://www.eucast.org/clinical_breakpoints/, accessed on 14 July 2023). Antimicrobial activity of cefiderocol was also determined by disk diffusion method using 30 µg cefiderocol discs (ThermoFisher Scientific, Waltham, MA, USA). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control strains.
In order to know the genetic basis of antimicrobial resistance, carbapenemase-encoding genes were first analysed by three multiplex PCRs, including primers for blaOXA-23-like, −40-like, −51-like, −58-like, −143-like, −235-like, blaVIM, blaKPC, blaNDM, blaOXA-48, blaIMI, blaGES, blaGIM, blaIMP and ISAba-1/blaOXA-51-like [21,23]. Then, the Ale25 genome was fully sequenced using both the Illumina Miseq system with the v3 chemistry for 2 × 300 paired-end libraries (Illumina Inc., San Diego, CA, USA) [24] and the MinION Mk1C sequencing device with the Rapid Barcoding Kit (SQK-RBK004) with an R9 flow cell (FLO-MIN106) (Oxford Nanopore Technologies, Cambridge, UK).
Total DNA was purified with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and sequenced on a Illumina MiSeq device using reagents kit v3 for 2 × 300 paired-end libraries (Illumina Inc., San Diego, CA, USA) [24]. Raw reads from the sequencing platform were directly analysed using the in-house bioinformatics pipeline TORMES® [25]. A. baumannii ATCC 17978 was used as a reference strain. Quality control and filtering of the reads were assessed using Trimmomatic [26], Prinseq [27] and Kraken [28]. Genome assembly was performed with SPAdes [29] and Quast [30] and genome annotation with Prokka software tool version 1.14.6 [31]. Multi-Locus Sequence Typing (MLST) was performed following Pasteur and Oxford typing schemes using Ridom SeqSphere+ software version 8.5.1 (Ridom© GmbH, Münster, Germany). The blaOXA-51-like variant combined with the Sequence Type (ST) were used to assign the isolate to an International Clone (IC), as these variants have been previously described as being related to ICs [32]. The search for antibiotic resistance genes was conducted using Beta-Lactamase DataBase (BLDB) [33], NCBI BLAST [34] and ABRicate (Seemann T, Abricate, Github https://github.com/tseemann/abricate, accessed on 14 July 2023) against ResFinder [35], CARD [36] and ARG-ANNOT [37] databases. Genetic environments of the β-lactamase genes were edited and visualised by the use of SnapGene Viewer 6.0.5. Virulence factors were screened using Virulence Factors Database (VFDB) search tool [38] and Ridom SeqSphere+ software (Ridom© GmbH).
Basecalling and barcoding of Oxford Nanopore Technology reads was performed using guppy v.6.5.7+ca6d6af and minimap2 version 2.24-r112. Basecalling was conducted using super-quality mode. Quality checks and filtering of reads shorter than 250 pb or with an average quality under Q10 was carried out with NanoPack v.1.4.1 [39]. Read contamination was also checked by using Kraken v.2.1.2 8 [40]. The reads were assembled by using Flye v2.9.2-b1786 [41] and polished using medaka v.1.7.2 (https://github.com/nanoporetech/medaka, accessed on 14 July 2023). Assembly completion was inspected visually using Bandage [42], and quality was checked using Quast v.5.2.0 [30]. Then, assemblies were annotated using Bakta v.1.7.0 [43]. Abricate v1.0.1 (https://github.com/tseemann/abricate, accessed on 14 July 2023) was used to find antimicrobial resistance genes by using the following databases: NCBI-AMRFinderPlus [44], CARD [45], ResFinder [35] and ARG-ANNOT [37]. Finally, plasmids were detected and classified by using MOBsuite:3.0.3 [46] and Copla.py v.1.0 [47]. Plasmids were plotted by using Proksee.ca [48]. Genome sequences of Ale25 were generated by combining data from both the Illumina and MinION datasets using Unicycler v0.5.0 (https://github.com/rrwick/Unicycler, accessed on 14 July 2023). Finally, both A. baumannii PCR-based replicon typing (AB-PBRT) and Acinetobacter Plasmid Typing database were used against the draft genome to determine the presence of replicase genes, as previously described [49,50]. This assembled genome was submitted to NCBI under the accession number JANBZS000000000.
To investigate the presence of plasmids, we first performed plasmid extractions using GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s indications, and S1-Pulsed-Field Gel Electrophoresis (PFGE) experiments. The Bacterial DNA embedded in agarose plugs was digested using 14 units of S1-nuclease (Takara Bio, Kusatsu, Japan) per plug, and then it was run on a CHEF-DR III system (Bio-Rad, Munich, Germany) for 18 h at 6 V/cm and 14 °C. CHEF DNA Size Standard Lambda Ladder (Bio-Rad) was used as a molecular weight marker. Southern-blot hybridisation of the resulting PFGE was performed to locate blaNDM-1 gene with specific digoxigenin-labelled DNA probes (Roche, Mannheim, Germany). Signal detection was performed using DIG Nucleic Acid Detection Kit (Roche).

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241512515/s1.

Author Contributions

Conceptualization, S.S.-U. and L.G.; methodology, S.S.-U., A.O.-S., J.R.-G., A.M.-B., M.H. and D.A.; formal analysis, S.S.-U., A.O.-S., J.R.-G. and L.G.; investigation, S.S.-U., A.O.-S., J.R.-G. and L.G.; resources, S.M.S. and M.A.E.-K.; data curation, S.S.-U., J.R.-G., M.H. and D.A.; writing—original draft preparation, S.S.-U. and L.G.; writing—review and editing, S.S.-U., L.G. and A.O.-S.; supervision, L.G.; project administration, I.A.; funding acquisition, I.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the MINISTRY OF SCIENCE AND INNOVATION (MCIN/AEI/10.13039/501100011033), grant number PID2020-116495RB-I00; the DEPARTMENT OF EDUCATION OF THE BASQUE GOVERNMENT (Research Groups of the Basque University System 2021), grant number Group IT1578-22, GIC21/18; and the ARAB ACADEMY FOR SCIENCE, TECHNOLOGY AND MARITIME TRANSPORT, grant number 2072.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of the Basque Country (M30_2022_235, 17 June 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The assembled genome is available in NCBI under the accession number JANBZS000000000.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Xanthopoulou, K.; Urrutikoetxea-Gutiérrez, M.; Vidal-Garcia, M.; Diaz de Tuesta del Arco, J.-L.; Sánchez-Urtaza, S.; Wille, J.; Seifert, H.; Higgins, P.G.; Gallego, L. First Report of New Delhi Metallo-Β-Lactamase-6 (NDM-6) in a Clinical Acinetobacter baumannii Isolate from Northern Spain. Front. Microbiol. 2020, 11, 589253. [Google Scholar] [CrossRef]
  2. Hassan, R.M.; Salem, S.T.; Hassan, S.I.M.; Hegab, A.S.; Elkholy, Y.S. Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates from Egyptian Patients. PLoS ONE 2021, 16, e0251508. [Google Scholar] [CrossRef]
  3. Poirel, L.; Sadek, M.; Nordmann, P. Contribution of PER-Type and NDM-Type Β-Lactamases to Cefiderocol Resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2021, 65, e0087721. [Google Scholar] [CrossRef] [PubMed]
  4. Naeimi Mazraeh, F.; Hasani, A.; Sadeghi, J.; Samadi Kafil, H.; Soroush Barhaghi, M.H.; Yeganeh Sefidan, F.; Rishi Sharabiani, H.; Hematyar, Y.; Ahangarzadeh Rezaee, M. High Frequency of blaPER-1 Gene in Clinical Strains of Acinetobacter baumannii and its Association with Quorum Sensing and Virulence Factors. Gene Rep. 2021, 24, 101232. [Google Scholar] [CrossRef]
  5. Said, H.S.; Benmahmod, A.B.; Ibrahim, R.H. Co-Production of AmpC and Extended Spectrum Beta-Lactamases in Cephalosporin-Resistant Acinetobacter baumannii in Egypt. World J. Microbiol. Biotechnol. 2018, 34, 189. [Google Scholar] [CrossRef] [PubMed]
  6. Cerezales, M.; Xanthopoulou, K.; Wille, J.; Bustamante, Z.; Seifert, H.; Gallego, L.; Higgins, P.G. Acinetobacter baumannii Analysis by Core Genome Multi-Locus Sequence Typing in Two Hospitals in Bolivia: Endemicity of International Clone 7 Isolates (CC25). Int. J. Antimicrob. Agents 2019, 53, 844–849. [Google Scholar] [CrossRef]
  7. Kurihara, M.N.L.; de Sales, R.O.; da Silva, K.E.; Maciel, W.G.; Simionatto, S. Multidrug-Resistant Acinetobacter baumannii Outbreaks: A Global Problem in Healthcare Settings. Rev. Soc. Bras. Med. Trop. 2020, 53, e20200248. [Google Scholar] [CrossRef]
  8. Al-Hassan, L.; Zafer, M.M.; El-Mahallawy, H. Multiple Sequence Types Responsible for Healthcare-Associated Acinetobacter baumannii Dissemination in a Single Centre in Egypt. BMC Infect. Dis. 2019, 19, 829. [Google Scholar] [CrossRef] [Green Version]
  9. Wang, Y.; Wu, C.; Zhang, Q.; Qi, J.; Liu, H.; Wang, Y.; He, T.; Ma, L.; Lai, J.; Shen, Z.; et al. Identification of New Delhi Metallo-Β-Lactamase 1 in Acinetobacter lwoffii of Food Animal Origin. PLoS ONE 2012, 7, e37152. [Google Scholar] [CrossRef] [Green Version]
  10. Fernández-Cuenca, F.; Pérez-Palacios, P.; Galán-Sánchez, F.; López-Cerero, L.; López-Hernández, I.; López Rojas, R.; Arca-Suárez, J.; Díaz-de Alba, P.; Rodríguez Iglesias, M.; Pascual, A. First Identification of blaNDM-1 Carbapenemase in blaOXA-94-Producing Acinetobacter baumannii ST85 in Spain. Enfermedades Infecc. Microbiol. Clín. 2020, 38, 11–15. [Google Scholar] [CrossRef]
  11. Cheng, C.; Sun, J.; Zheng, F.; Lu, W.; Yang, Q.; Rui, Y. New Structures Simultaneously Harboring Class 1 Integron and ISCR1-Linked Resistance Genes in Multidrug-Resistant Gram-Negative Bacteria. BMC Microbiol. 2016, 16, 71. [Google Scholar] [CrossRef] [Green Version]
  12. Leungtongkam, U.; Thummeepak, R.; Tasanapak, K.; Sitthisak, S. Acquisition and Transfer of Antibiotic Resistance Genes in Association with Conjugative Plasmid or Class 1 Integrons of Acinetobacter baumannii. PLoS ONE 2018, 13, e0208468. [Google Scholar] [CrossRef] [Green Version]
  13. Ingti, B.; Upadhyay, S.; Hazarika, M.; Khyriem, A.B.; Paul, D.; Bhattacharya, P.; Joshi, S.R.; Bora, D.; Dhar, D.; Bhattacharjee, A. Distribution of Carbapenem Resistant Acinetobacter baumannii with blaADC-30 and Induction of ADC-30 in Response to Beta-Lactam Antibiotics. Res. Microbiol. 2020, 171, 128–133. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, Z.; Li, H.; Zhang, J.; Wang, H. Co-Occurrence of bla(OXA-23) in the Chromosome and Plasmid: Increased Fitness in Carbapenem-Resistant Acinetobacter baumannii. Antibiotics 2021, 10, 1196. [Google Scholar] [CrossRef]
  15. Nigro, S.J.; Hall, R.M. Structure and Context of Acinetobacter Transposons Carrying the OXA23 Carbapenemase Gene. J. Antimicrob. Chemother. 2016, 71, 1135–1147. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Hamidian, M.; Nigro, S.J. Emergence, Molecular Mechanisms and Global Spread of Carbapenem-Resistant Acinetobacter baumannii. Microb. Genom. 2019, 5, e000306. [Google Scholar] [CrossRef]
  17. Khezri, A.; Avershina, E.; Ahmad, R. Hybrid Assembly Provides Improved Resolution of Plasmids, Antimicrobial Resistance Genes, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Clinical Isolates. Microorganisms 2021, 9, 2560. [Google Scholar] [CrossRef] [PubMed]
  18. Jahan, M.I.; Rahaman, M.M.; Hossain, M.A.; Sultana, M. Draft Genome Sequence of a Carbapenem-Resistant Clinical Acinetobacter baumannii Revealing Co-Existence of Four Classes of Β-Lactamases. J. Glob. Antimicrob. Resist. 2021, 27, 329–331. [Google Scholar] [CrossRef] [PubMed]
  19. Woodford, N.; Ellington, M.J.; Coelho, J.M.; Turton, J.F.; Ward, M.E.; Brown, S.; Amyes, S.G.B.; Livermore, D.M. Multiplex PCR for Genes Encoding Prevalent OXA Carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 2006, 27, 351–353. [Google Scholar] [CrossRef]
  20. Higgins, P.G.; Lehmann, M.; Seifert, H. Inclusion of OXA-143 Primers in a Multiplex Polymerase Chain Reaction (PCR) for Genes Encoding Prevalent OXA Carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 2010, 35, 305. [Google Scholar] [CrossRef]
  21. Higgins, P.G.; Pérez-Llarena, F.J.; Zander, E.; Fernández, A.; Bou, G.; Seifert, H. OXA-235, a Novel Class D Β-Lactamase Involved in Resistance to Carbapenems in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2013, 57, 2121–2126. [Google Scholar] [CrossRef] [Green Version]
  22. Higgins, P.G.; Lehmann, M.; Wisplinghoff, H.; Seifert, H. gyrB Multiplex PCR to Differentiate between Acinetobacter calcoaceticus and Acinetobacter Genomic Species 3. J. Clin. Microbiol. 2010, 48, 4592–4594. [Google Scholar] [CrossRef] [Green Version]
  23. Cerezales, M.; Biniossek, L.; Gerson, S.; Xanthopoulou, K.; Wille, J.; Wohlfarth, E.; Kaase, M.; Seifert, H.; Higgins, P.G. Novel Multiplex PCRs for Detection of the most Prevalent Carbapenemase Genes in Gram-Negative Bacteria within Germany. J. Med. Microbiol. 2021, 70, 001310. [Google Scholar] [CrossRef]
  24. Hernández, M.; Iglesias, M.R.; Rodríguez-Lázaro, D.; Gallardo, A.; Quijada, N.; Miguela-Villoldo, P.; Campos, M.J.; Píriz, S.; López-Orozco, G.; de Frutos, C.; et al. Co-Occurrence of Colistin-Resistance Genes mcr-1 and mcr-3 among Multidrug-Resistant Escherichia coli Isolated from Cattle, Spain, September 2015. Eurosurveillance 2017, 22, 30586. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Quijada, N.M.; Rodríguez-Lázaro, D.; Eiros, J.M.; Hernández, M. TORMES: An Automated Pipeline for Whole Bacterial Genome Analysis. Bioinformatics 2019, 35, 4207–4212. [Google Scholar] [CrossRef]
  26. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A Flexible Trimmer for Illumina Sequence Data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [Green Version]
  27. Schmieder, R.; Edwards, R. Quality Control and Preprocessing of Metagenomic Datasets. Bioinformatics 2011, 27, 863–864. [Google Scholar] [CrossRef] [Green Version]
  28. Wood, D.E.; Salzberg, S.L. Kraken: Ultrafast Metagenomic Sequence Classification using Exact Alignments. Genome Biol. 2014, 15, R46. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A New Genome Assembly Algorithm and its Applications to Single-Cell Sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Gurevich, A.; Saveliev, V.; Vyahhi, N.; Tesler, G. QUAST: Quality Assessment Tool for Genome Assemblies. Bioinformatics 2013, 29, 1072–1075. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  31. Seemann, T. Prokka: Rapid Prokaryotic Genome Annotation. Bioinformatics 2014, 30, 2068–2069. [Google Scholar] [CrossRef] [Green Version]
  32. Zander, E.; Nemec, A.; Seifert, H.; Higgins, P.G. Association between Β-Lactamase-Encoding bla(OXA-51) Variants and DiversiLab Rep-PCR-Based Typing of Acinetobacter baumannii Isolates. J. Clin. Microbiol. 2012, 50, 1900–1904. [Google Scholar] [CrossRef] [Green Version]
  33. Naas, T.; Oueslati, S.; Bonnin, R.A.; Dabos, M.L.; Zavala, A.; Dortet, L.; Retailleau, P.; Iorga, B.I. Beta-Lactamase Database (BLDB)—Structure and Function. J. Enzym. Inhib. Med. Chem. 2017, 32, 917–919. [Google Scholar] [CrossRef] [Green Version]
  34. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic Local Alignment Search Tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  35. Zankari, E.; Hasman, H.; Cosentino, S.; Vestergaard, M.; Rasmussen, S.; Lund, O.; Aarestrup, F.M.; Larsen, M.V. Identification of Acquired Antimicrobial Resistance Genes. J. Antimicrob. Chemother. 2012, 67, 2640–2644. [Google Scholar] [CrossRef] [PubMed]
  36. Alcock, B.P.; Huynh, W.; Chalil, R.; Smith, K.W.; Raphenya, A.R.; Wlodarski, M.A.; Edalatmand, A.; Petkau, A.; Syed, S.A.; Tsang, K.K.; et al. CARD 2023: Expanded Curation, Support for Machine Learning, and Resistome Prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 2023, 51, D690–D699. [Google Scholar] [CrossRef] [PubMed]
  37. Gupta, S.K.; Padmanabhan, B.R.; Diene, S.M.; Lopez-Rojas, R.; Kempf, M.; Landraud, L.; Rolain, J. ARG-ANNOT, a New Bioinformatic Tool to Discover Antibiotic Resistance Genes in Bacterial Genomes. Antimicrob. Agents Chemother. 2014, 58, 212–220. [Google Scholar] [CrossRef] [Green Version]
  38. Liu, B.; Zheng, D.; Jin, Q.; Chen, L.; Yang, J. VFDB 2019: A Comparative Pathogenomic Platform with an Interactive Web Interface. Nucleic Acids Res. 2019, 47, D687–D692. [Google Scholar] [CrossRef] [PubMed]
  39. De Coster, W.; Rademakers, R. NanoPack2: Population-Scale Evaluation of Long-Read Sequencing Data. Bioinformatics 2023, 39, btad311. [Google Scholar] [CrossRef]
  40. Wood, D.E.; Lu, J.; Langmead, B. Improved Metagenomic Analysis with Kraken 2. Genome Biol. 2019, 20, 257. [Google Scholar] [CrossRef] [Green Version]
  41. Kolmogorov, M.; Yuan, J.; Lin, Y.; Pevzner, P.A. Assembly of Long, Error-Prone Reads using Repeat Graphs. Nat. Biotechnol. 2019, 37, 540–546. [Google Scholar] [CrossRef]
  42. Wick, R.R.; Schultz, M.B.; Zobel, J.; Holt, K.E. Bandage: Interactive Visualization of De Novo Genome Assemblies. Bioinformatics 2015, 31, 3350–3352. [Google Scholar] [CrossRef] [Green Version]
  43. Schwengers, O.; Jelonek, L.; Dieckmann, M.A.; Beyvers, S.; Blom, J.; Goesmann, A. 2. Bakta: Rapid and Standardized Annotation of Bacterial Genomes Via Alignment-Free Sequence Identification. Microb. Genom. 2021, 7, 000685. [Google Scholar] [CrossRef] [PubMed]
  44. Feldgarden, M.; Brover, V.; Haft, D.H.; Prasad, A.B.; Slotta, D.J.; Tolstoy, I.; Tyson, G.H.; Zhao, S.; Hsu, C.; McDermott, P.F.; et al. Validating the AMRFinder Tool and Resistance Gene Database by using Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of Isolates. Antimicrob. Agents Chemother. 2019, 63, e00483-e19. [Google Scholar] [CrossRef] [Green Version]
  45. Jia, B.; Raphenya, A.R.; Alcock, B.; Waglechner, N.; Guo, P.; Tsang, K.K.; Lago, B.A.; Dave, B.M.; Pereira, S.; Sharma, A.N.; et al. CARD 2017: Expansion and Model-Centric Curation of the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 2016, 45, D566–D573. [Google Scholar] [CrossRef] [PubMed]
  46. Robertson, J.; Nash, J.H. MOB-Suite: Software Tools for Clustering, Reconstruction and Typing of Plasmids from Draft Assemblies. Microb. Genom. 2018, 4, e000206. [Google Scholar] [CrossRef]
  47. Redondo-Salvo, S.; Bartomeus-Peñalver, R.; Vielva, L.; Tagg, K.A.; Webb, H.E.; Fernández-López, R.; de la Cruz, F. COPLA, a Taxonomic Classifier of Plasmids. BMC Bioinform. 2021, 22, 390. [Google Scholar] [CrossRef] [PubMed]
  48. Grant, J.R.; Enns, E.; Marinier, E.; Mandal, A.; Herman, E.K.; Chen, C.; Graham, M.; Van Domselaar, G.; Stothard, P. Proksee: In-Depth Characterization and Visualization of Bacterial Genomes. Nucleic Acids Res. 2023, 51, W484–W492. [Google Scholar] [CrossRef] [PubMed]
  49. Bertini, A.; Poirel, L.; Mugnier, P.D.; Villa, L.; Nordmann, P.; Carattoli, A. Characterization and PCR-Based Replicon Typing of Resistance Plasmids in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2010, 54, 4168–4177. [Google Scholar] [CrossRef] [Green Version]
  50. Lam Margaret, M.C.; Jonathan, K.; Holt Kathryn, E.; Hall Ruth, M.; Mehrad, H. Detection and Typing of Plasmids in Acinetobacter baumannii using rep Genes Encoding Replication Initiation Proteins. Microbiol. Spectr. 2022, 11, 2478. [Google Scholar]
Ijms 24 12515 g001
Figure 1. Genetic environments of the β-lactamase genes harboured by the isolate Ale25: (a) blaNDM-1; (b) blaPER-7; (c) blaOXA-23; (d) blaOXA-64; (e) blaADC-57. The numbers above the arrows represent the base pairs of the sequences. The three dots in (d,e) represent that the sequences are larger than the fragment showed.
Figure 1. Genetic environments of the β-lactamase genes harboured by the isolate Ale25: (a) blaNDM-1; (b) blaPER-7; (c) blaOXA-23; (d) blaOXA-64; (e) blaADC-57. The numbers above the arrows represent the base pairs of the sequences. The three dots in (d,e) represent that the sequences are larger than the fragment showed.
Ijms 24 12515 g001
Ijms 24 12515 g002
Figure 2. (A) Conventional plasmid extraction of the isolate Ale25. Lanes 1 & 2: control strains E. coli 678 (NCTC 50193) and 679 (NCTC 50192); lane 3: Ale25 profile showing plasmids of ~26 Kb and 2.4 Kb (green arrows); (B) S1 nuclease—Pulsed Field Gel Electrophoresis of the isolate Ale25. Lane 1: molecular weight marker; Lane 2: chromosome and a plasmid of ~240 Kb (green arrow); Lane 3: southern-blot hybridisation with a blaNDM-1 DNA probe showing the chromosomal location of the gene (green arrow). Molecular weights are expressed in Kb.
Figure 2. (A) Conventional plasmid extraction of the isolate Ale25. Lanes 1 & 2: control strains E. coli 678 (NCTC 50193) and 679 (NCTC 50192); lane 3: Ale25 profile showing plasmids of ~26 Kb and 2.4 Kb (green arrows); (B) S1 nuclease—Pulsed Field Gel Electrophoresis of the isolate Ale25. Lane 1: molecular weight marker; Lane 2: chromosome and a plasmid of ~240 Kb (green arrow); Lane 3: southern-blot hybridisation with a blaNDM-1 DNA probe showing the chromosomal location of the gene (green arrow). Molecular weights are expressed in Kb.
Ijms 24 12515 g002
Ijms 24 12515 g003
Figure 3. Plasmid pAbAle25.1 of 240.8 Kb harbouring, among other virulence genes, blaPER-7 gene.
Figure 3. Plasmid pAbAle25.1 of 240.8 Kb harbouring, among other virulence genes, blaPER-7 gene.
Ijms 24 12515 g003
Table 1. Minimum Inhibitory Concentrations (MICs) and interpretations of the A. baumannii isolate Ale25. “R” and “S” corresponds to resistant and susceptible, respectively. “IE” indicates that there is insufficient evidence that the organism or group is a good target for therapy with the agent.
Table 1. Minimum Inhibitory Concentrations (MICs) and interpretations of the A. baumannii isolate Ale25. “R” and “S” corresponds to resistant and susceptible, respectively. “IE” indicates that there is insufficient evidence that the organism or group is a good target for therapy with the agent.
AntibioticMIC (mg/L)Interpretation
Ticarcillin/clavulanic acid>64IE
Piperacillin>64IE
Piperacillin/tazobactam>64IE
Imipenem>64R
Meropenem>64R
Amikacin>64R
Gentamicin>64R
Tobramycin>64R
Ciprofloxacin32R
Trimethoprim/sulfamethoxazole>32R
Tigecycline0.125IE
Minocycline0.5IE
Colistin0.125S
Cefiderocol16R
Table 2. Resistome of the A. baumannii isolate Ale25. Genes are represented in italics.
Table 2. Resistome of the A. baumannii isolate Ale25. Genes are represented in italics.
Β-Lactamase GenesTetracyclinesSulfonamidesAminoglycosidesMacrolidesRifamycinChloramphenicol
blaOXA-641tet(B)sul1armAmph(E)arr-2cmlA5
blaOXA-231 sul2strAmsr(E)
blaNDM-1 1 strB
blaPER-7 aph (3′)-VI
blaADC-57 ant (3″)-IIa
1 Recognised carbapenemase genes.
Table 3. Virulome of the A. baumannii isolate Ale25. Genes are represented in italics. BCRR—Biofilm-controlling response regulator; BAP—Biofilm-associated protein; OMP—Outer-membrane proteins; PNAG—Poly-N-acetyl-D-glucosamine; PBPG—Penicillin-Binding Protein G; LPS—Lipopolysaccharide; T2SS—Type II secretion system; T6SS—Type VI secretion system.
Table 3. Virulome of the A. baumannii isolate Ale25. Genes are represented in italics. BCRR—Biofilm-controlling response regulator; BAP—Biofilm-associated protein; OMP—Outer-membrane proteins; PNAG—Poly-N-acetyl-D-glucosamine; PBPG—Penicillin-Binding Protein G; LPS—Lipopolysaccharide; T2SS—Type II secretion system; T6SS—Type VI secretion system.
Adherence and Biofilm ProductionImmune
Modulation		Effector Delivery SystemsExotoxinsIron Uptake
Type IV PiliBCRREfflux PumpQuorum SensingCsu FimbriaeBAPOMPPNAGCapsulePBPGLPST2SST6SSPhospholipases C and DAcinetobactin
pilBbfmRadeFabaRcsuAbapompApgaA,tviBpbpGlpxAgspCtssAplc1,barA
pilCbfmSadeGabaIcsuB pgaBgalE lpxBgspDtssBplc2barB
gsp0/pilD adeH csuA/B pgaCgalU lpxCgspE1tssCplcDbauA
pilF csuC pgaDpgi lpxDgspE2hcp/tssD bauB
pilG csuD lpxLgspFtssE basA
pilH csuE lpxMgspGtssF basB
pilI lpsBgspHtssG basC
pilJ gspIclpV/tssH basD
basG
pilM gspJvgrG/tssI basJ
pilN gspKtssK entE
pilO gspLtssL
pilP gspNtssM
pilQ gspMtagX
pilR tse2
pilS
pilT
pilU
pilV
pilW
pilX
PilY1
fimT
fimU
fimV
tsaP
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Sánchez-Urtaza, S.; Ocampo-Sosa, A.; Molins-Bengoetxea, A.; Rodríguez-Grande, J.; El-Kholy, M.A.; Hernandez, M.; Abad, D.; Shawky, S.M.; Alkorta, I.; Gallego, L. Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt. Int. J. Mol. Sci. 2023, 24, 12515. https://doi.org/10.3390/ijms241512515

AMA Style

Sánchez-Urtaza S, Ocampo-Sosa A, Molins-Bengoetxea A, Rodríguez-Grande J, El-Kholy MA, Hernandez M, Abad D, Shawky SM, Alkorta I, Gallego L. Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt. International Journal of Molecular Sciences. 2023; 24(15):12515. https://doi.org/10.3390/ijms241512515

Chicago/Turabian Style

Sánchez-Urtaza, Sandra, Alain Ocampo-Sosa, Ainhoa Molins-Bengoetxea, Jorge Rodríguez-Grande, Mohammed A. El-Kholy, Marta Hernandez, David Abad, Sherine M. Shawky, Itziar Alkorta, and Lucia Gallego. 2023. "Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt" International Journal of Molecular Sciences 24, no. 15: 12515. https://doi.org/10.3390/ijms241512515

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop