The emergence of a novel sequence type of MDR Acinetobacter baumannii from the intensive care unit of an Egyptian tertiary care hospital

Background and aim of work Acinetobacter baumannii is known for nosocomial outbreaks worldwide. In this study, we aimed to investigate the antibiotic susceptibility patterns and the clonal relationship of A. baumannii isolates from the intensive care unit (ICU) of an Egyptian hospital. Methods In the present study, 50 clinical isolates of multidrug resistant (MDR)-A. baumannii were obtained from patients admitted into the ICU from June to December 2015. All isolates were analyzed for antimicrobial susceptibilities. Multiplex PCR was performed to detect genes encoding oxacillinase genes (bla OXA-51-like, bla OXA-23-like, bla OXA-24-like, and bla OXA-58-like). Multilocus sequence typing (MLST) based on the seven-gene scheme (gltA, gyrB, gdhB, recA, cpn60, gpi, rpoD) was used to examine these isolates. Results All A. baumannii clinical isolates showed the same resistance pattern, characterized by resistance to most common antibiotics including imipenem (MIC ≥ 8μ/mL), with the only exception being colistin. Most isolates were positive for bla OXA-51-like and bla OXA-23-like (100 and 96%, respectively); however, bla OXA-24-like and bla OXA-58-like were not detected. MLST analysis identified different sequence types (ST195, ST208, ST231, ST441, ST499, and ST723) and a new sequence type (ST13929) with other sporadic strains. Conclusions MDR A. baumannii strains harboring bla OXA-23-like genes were widely circulating in this ICU. MLST was a powerful tool for identifying and epidemiologically typing our strains. Strict infection control measures must be implemented to contain the worldwide spread of MDR A. baumannii in ICUs.


Methods
The study was carried out in EL Sheikh Zayed hospital which provides tertiary care from specialists and consultants after referral (in orthopedic, trauma, neuro/spine surgeries) from primary care and secondary care hospitals in Egypt. A lab-based surveillance was performed over a period of 6 months (June-December 2015) after the isolation of five MDR A. baumannii strains in a period of 1 week showing the same phenotypic characteristics.

Bacterial strains
All clinical samples of the patients admitted during the above-mentioned period were processed at the microbiology unit. All samples were cultured on blood agar and MacConkey agar (Oxoid Co. England). All culture plates were incubated aerobically at 35 °C for 24-48 h. Identification of isolated organisms was performed by conventional biochemical reactions. During the experimental period, 50 A. baumannii non-duplicate strains were isolated.
Multidrug resistance was defined in this analysis as resistance to three or more representatives of the following classes of antibiotics: fluoroquinolones, extendedspectrum cephalosporins, aminoglycosides, and carbapenems [8].
Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213 were used as reference strains for susceptibility testing per Clinical and Laboratory Standards Institute (CLSI, 2015) guidelines and interpretations [7].

Multilocus sequence typing
MLST analysis was performed per the protocol of the Pasteur Institute. Fragments of seven internal housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) were amplified and sequenced as previously described [10]. Briefly, PCR amplifications were performed with a MasterCycler Nexus (Eppendorf, Hamburg, Germany) with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min, and a 4-min final extension at 72 °C. The amplicons were verified by agarose gel electrophoresis and were subsequently purified for bidirectional Sanger sequencing reactions. Multiple allele sequences were assigned for each locus with an arbitrary allele number to obtain characterization of sequence types (STs) for each A. baumannii isolate. Each sequence was compared with sequences deposited in the Institute of Pasteur MLST schema (http://pubmlst.org/perl/bigsdb/ bigsdb.pl?db=pubmlst_abaumannii_pasteur_seqdef ).

Results
Out of 358 patients admitted to ICU from June to December 2015, 56 (15.6%) patients were diagnosed with various types of hospital-acquired infections (HAI) as shown in Table 1. A total of 50 non-duplicate A. baumannii strains were isolated from different patient samples. Phenotypic antibiotic susceptibility testing for all A. baumannii isolates showed the same drug resistance pattern, characterized by resistance to all antibiotics used including imipenem, except for colistin. Genotypic analysis of bla OXA-51 -like, bla OXA-23 -like, bla OXA-24 -like, and bla OXA-58 -like genes by multiplex PCR (Fig. 1) showed that bla OXA-51 -like and bla OXA-23 -like were the most prevalent genes with 100 and 96% prevalence, respectively. However, bla OXA-24 -like and bla OXA-58 -like were not detected in the current study. Multilocus sequence typing (MLST) of the 50 clinical isolates of A. baumannii has yielded different sequence types; ST195, ST208, ST231, ST441, ST499, and ST723. Interestingly, a new sequence type ST13929 was identified among A. baumannii clinical isolates as shown in Table 2 and Fig. 2. A. baumannii ST13929 has been isolated from a young male (21 years old) patient who had no history of overseas travel. He was admitted to ICU at El Sheikh Zayed Specialized Hospital, Giza, Egypt on 5th of December 2014. The patient had multiple traumas due to motor car accident. After 7 days of ventilation the patient diagnosed to have ventilator associated pneumonia (VAP). Empirical antibiotic therapy of intravenous ceftriaxone/cefotaxime had been initiated. Endotracheal aspiration has been cultured on blood, chocolate and MacConkey agars. The recovered colonies had been identified as A. baumannii. Further genotypic identification was done by restriction analysis of 16S-23S rRNA spacer sequences using AluI and NdeII. The isolate exhibited XDR towards imipenem (MIC > 32 mg/L), meropenem (MIC > 32 mg/L), ceftazidime (>256 mg/L), cefepime (MICs > 256 mg/L), gentamicin (MICs > 256 mg/L), amikacin (MICs > 256 mg/L), and ciprofloxacin (MICs > 32 mg/L). Tigecycline susceptibility was observed at MIC of 1 mg/L. The antimicrobial therapy was changed to tigecycline on day 11. The patient had spent 107 days in the hospital. The patient was alive after the hospitalization period. There was an ongoing XDR-A. baumannii outbreak in the institution in the same period and multiple isolates had been investigated.

Discussion
MDR A. baumannii is a problematic, multidrug-resistant pathogen identified in healthcare settings worldwide, especially in ICUs [12]. A. baumannii has a notable ability to capture and express resistance genes. All resistance mechanisms including target modification, efflux pump expression, and enzymatic inactivation have been described in A. baumannii [13].
In the current study, five MDR A. baumannii strains were isolated over 1 week from the same ICU. All isolates showed the same phenotypic characteristics which prompted us to start a survey study of the antimicrobial susceptibility and clonal relationship of A. baumannii strains isolated from this ICU.
All our isolates were resistant to imipenem. The main role of the A. baumannii resistance to carbapenems is mediated by oxacillinases and, less frequently, by metalloβ-lactamases [4,5].
bla OXA-23 -like, bla OXA-24/40 -like, and bla OXA-58 -like genes have been repetitively reported in A. baumannii outbreaks from diverse parts of the world. The  localization of numerous β-lactamase genes on plasmids facilitates their horizontal mobilization from one bacterium to another [13,14].
All our isolates harbored the bla OXA-51 -like gene, which is ubiquitous in A. baumannii [15]. bla OXA-23 was the most universally identified gene, while bla OXA-24 -like and bla OXA-58 -like genes were not detected in any strain. bla OXA-23 is the most prevalent carbapenemaseencoding gene in the Mediterranean region. This might be explained by the higher carbapenemase activity of bla OXA-23 and/or acquisition of carbapenem resistance through horizontal gene transfer [16][17][18]. The bla OXA-23 gene was either encoded on the chromosome or on plasmids and was associated with four dissimilar genetic structures, with the most common being transposons Tn2006. bla OXA-23 has been reported in different regions of the Middle East, The United Arab Emirates, Algeria, Libya, Bahrain, and recently, Qatar [16,19].
Taking into consideration that ST208 is the ancestor strain of several STs including ST89, ST88, ST190, ST225, and ST75, it has been identified in different parts of the world such as Japan, China, Thailand, Korea, Italy, Australia, Portugal, and the Czech Republic [23].
In conclusion, MDR A. baumannii strains harboring the bla OXA 23 -like gene were widely circulating in our ICU. MLST provided us with a powerful tool for identifying and epidemiologically typing our strains. Studying the epidemiology of HAIs is urgent to prevent the clonal dissemination of antibiotic-resistant pathogens, not only in hospital settings, but in the community, as well. Strict infection control measures and antimicrobial stewardship programs are necessary to contain the worldwide spread of MDR A. baumannii. Proving the clonal relation between clinical isolates emphasizes the importance of surveillance programs and strict IC measures that would influence decision-making and health policy.