Acinetobacter baumannii is an important nosocomial pathogen, commonly causing infections in immunocompromised patients. It is increasingly reported as a multidrug-resistant organism, which is alarming because of its capability to resist all available classes of antibiotics including carbapenems. The aim of this study was to examine the genetic and epidemiological diversity of A. baumannii isolates from paediatric cancer patients in Egypt, by sequencing the intrinsic blaOXA–51-like gene, genotyping by pulsed-field gel electrophoresis and multi-locus sequence typing in addition to identifying the carbapenem-resistance mechanism. Results showed a large diversity within the isolates, with eight different blaOXA-51-like genes, seven novel sequence types and only 28% similarity by pulsed-field gel electrophoresis. All three acquired class-D carbapenemases (OXA-23, OXA-40 and OXA-58) were also identified among these strains correlating with resistance to carbapenems. In addition, we report the first identification of ISAba2 upstream of blaOXA-51-like contributing to high-level carbapenem resistance. This indicates the presence of several clones of A. baumannii in the hospitals and illustrates the large genetic and epidemiological diversity found in Egyptian strains.
Acinetobacter baumannii has emerged as an important nosocomial pathogen in the past decade, which in recent years has developed into a multidrug-resistant problematic pathogen . Acinetobacter baumannii is an opportunistic pathogen, frequently isolated from immunocompromised patients with prolonged hospitalization . As a consequence of immunoablative treatment, patients with cancer are at risk of developing A. baumannii infections, including sepsis, respiratory, wound and tissue infections, in addition to urinary tract infections [2, 3].
A major concern in A. baumannii is its worldwide clonal expansion and its ability to survive and disseminate in hospitals, with numerous outbreaks reported from different regions of the world . Acinetobacter baumannii is notably resistant to extreme environmental conditions, such as dryness, and can survive on surfaces for a long time, hence facilitating its spread [1, 4].
Resistance to carbapenems, the β-lactam drugs of last resort in treating A baumannii infections, has been attributed to the expression of carbapenem-hydrolysing oxacillinase genes, blaOXA23, blaOXA-40 and blaOXA58, which are usually plasmid encoded [5, 6]. The ubiquitous, chromosomally encoded blaOXA-51-like gene only confers resistance when an Insertion Sequence (IS) is present upstream of the gene .
Due to the prevalence of A. baumannii across the world, suitable typing methods to investigate the epidemiological distribution of the organism have been developed such as ribotyping, amplified fragment length polymorphisms, pulsed-field gel electrophoresis (PFGE) and, more recently, Multi-Locus Sequence Typing (MLST) . Additionally, amplification and sequencing of the ubiquitous blaOXA-51-like gene has also been used to determine clonal groups from diverse worldwide sources [7, 8].
Limited data were available concerning the epidemiological distribution of A. baumannii in the Middle East but, in the past few years, reports of strains in the United Arab Emirates, Iraq, Kuwait and Egypt harbouring diverse resistance mechanisms have emerged [9-12]. The aim of this study was to investigate the epidemiological and molecular diversity of A. baumannii strains isolated from two cancer centres in Cairo, Egypt.
Materials and Methods
Thirty-four non-duplicate A. baumannii were obtained from two centres; The Children's Cancer Hospital (CCH) and The National Cancer Institute (NCI), both located in Cairo, Egypt, from 2010 to 2011. Initial identification and susceptibility testing was done using VITEK and Phoenix automated machines. Genotypic identification was carried out by restriction analysis of 16s-23s rRNA spacer sequences using AluI and NdeII .
Detection of blaOXA-51-like genes
The intrinsic blaOXA-51-like genes were amplified for A. baumannii isolates using primers: OXA69A and B . Products were purified using a QIAquick PCR Purification Kit (Qiagen, Crawley, UK) and sequenced in both directions on a 3730 DNA Analyzer (Applied Biosystems, Warrington, UK). For isolates yielding a larger product size, a PCR was performed to screen for the associated upstream environment using primers FxOxa-F and FxOxa-R .
Detection of class D oxacillinases and genetic environment
Isolates were screened for the presence of acquired OXA carbapenemases by Multiplex PCR, as previously described . Isolates positive for the individual OXA groups were subsequently amplified and sequenced using primers for the full sequence of the genes. Associated genetic environment was also amplified and sequenced. Primers used are listed in Table 1.
The MIC of imipenem and meropenem were determined using an agar double dilution technique according to the British Society of Antimicrobial Chemotherapy (BSAC) guidelines . Pseudomonas aeruginosa NCTC 10662, Escherichia coli NCTC 10418 and Staphylococcus aureus NCTC 6571 were used as control strains.
Pulsed-field gel electrophoresis
All isolates were typed by PFGE according to the procedure previously described by Seifert et al. . Briefly, plugs were incubated in 30 U ApaI at 37° overnight, and subsequently run on 1% pulsed-field-certified agarose gel (Bio-Rad, Hertfordshire, UK) in 0.5 × TBE buffer with an initial pulse of 5 s and a final pulse of 20 s for 20 h. The gels were stained with Gel-Red solution and visualized using the Diversity Database (Bio-Rad) software image-capturing system.
Multi-locus sequence typing
The PCR for the seven housekeeping genes: gltA, gyrB, gdhB, rpoD, recA, gpi and cpn60 was performed according to the scheme developed by Bartual et al. . Products were purified and sequenced as described above. MLST was performed for ten isolates, representatives of the blaOXA-51-like gene variants identified. If isolates from different hospitals harboured similar blaOXA-51-like genes, an isolate from each hospital was selected randomly for comparison. Isolates chosen for MLST were: 8357, 9925-SAM, 1780, 634, 21174, 22055, 161, P38-YSF, P67-AZ and 14611.
Diversity of blaOXA-51-like genes
All isolates were confirmed as A. baumannii, and sequencing of the intrinsic blaOXA-51-like revealed the presence of eight different genes: blaOXA-64, blaOXA-65, blaOXA-66, blaOXA-69, blaOXA-71, blaOXA-78, blaOXA-94 and blaOXA-89 (Table 2). blaOXA-65 was the most prevalent, found in 14 isolates, obtained from both hospitals. blaOXA-64 is now commonly found in the Middle East (A. Al Hasan, and S.G.B. Amyes, unpublished results; ), it was found in seven isolates obtained from both hospitals. There were representatives from the three worldwide clones (formally known as the European clones). blaOXA-66 was found in four isolates, three of which were from CCH. blaOXA-69 was identified in two isolates at the intensive care unit (ICU) of CCH and were part of an A. baumanniii outbreak in early 2011. blaOXA-71 was found in two isolates from different hospitals. blaOXA-78 and blaOXA-89 were both found in strains from CCH, whereas blaOXA-94 was from two isolates from NCI, recovered from the same floor, 1 day apart.
Table 2. Isolates harbouring blaOXA-51-like genes, with isolation details. carbapenem-hydrolysing class D β-lactamase (CHDL) genes, minimum inhibitory concentration (MIC) and sequence type. Isolates in bold were in the A. baumannii outbreak in early 2011
Insertion sequences associated with blaOXA-51-like
Sequencing upstream of the blaOXA-51-like gene, blaOXA-89 in isolate 22055 revealed the presence of ISAba2, with the -35 (ttatat) and -10 (ttgtaggat) promoters 29 bp apart, and located 102 bp and 82 bp upstream of blaOXA-89, respectively. No other insertion sequences were identified upstream of the blaOXA-51-like genes.
The PFGE analysis revealed a large diversity within the strains. Some isolates with similar blaOXA-51-like genes had very distinct PFGE patterns, suggesting no epidemiological similarity between the strains. As seen in Figure 1, only six isolates harbouring blaOXA-65 show > 80% similarity in their PFGE pattern. Additionally, blaOXA-64 isolates all shared less than 80% similarity. Even isolates with blaOXA-94, which were collected from patients on the same floor of the same hospital 1 day apart, had distinct PFGE patterns. On the other hand, the blaOXA-71 containing isolates, although from different hospitals, had similar PFGE patterns. The similarity for all the isolates was calculated by Dice coefficient to be 28.7%.
Seven housekeeping genes were amplified and sequenced as described above for ten isolates. Ten distinct sequence types (STs) were identified, seven of which are novel and assigned ST408–ST414. The remaining three STs were identified as ST331, ST108 and ST208. Typing by MLST further illustrated the large diversity found within the strains, as isolates with similar blaOXA-51-like genes had different STs. This is clear for isolates 9925-SAM and NCI-P67, both were from the NCI and possessed blaOXA-64, but they belonged to different STs: 409 and 411, respectively. When compared with another blaOXA-64-positive isolate, 8357, which was from a patient at CCH, another ST was identified, ST408.
MIC and carbapenem-hydrolysing class D β-lactamase (CHDL) genes
The majority of isolates (n = 25), representing 73%, were resistant to imipenem and/or meropenem (MIC ≥8 mg/L). This resistance could be correlated with the presence of the acquired class-D oxacillinases: blaOXA-23, blaOXA-58 and blaOXA-40 (Table 2).
Genes encoding all three transferable OXA types associated with resistance were identified in these strains: blaOXA-23 in 18 isolates, blaOXA-58 in five isolates and blaOXA-40 in one isolate. All isolates, except one, possessing blaOXA-23 were resistant to imipenem and meropenem (MIC ≥8 mg/L). ISAba1 was detected upstream of blaOXA-23 in the resistant isolates, hence providing a promoter for the expression of the gene (Figure 2). However, this IS element was not found upstream in the blaOXA-23-containing isolate that was carbapenem sensitive. The analysis of the A. baumannii outbreak in the ICU at CCH in early 2011 revealed that although the strains harboured distinct blaOXA-51-like types and were epidemiologically different, they all possessed blaOXA-23 as the resistance mechanism.
blaOXA-58-positive isolates were also found in both hospitals and all were resistant to meropenem and imipenem, with the exception of isolate 14298, which was intermediate to meropenem (MIC 4 mg/L). The genetic environment of the blaOXA-58 showed that the gene was flanked by two copies of ISAba3 (Figure 2). Two isolates harboured an interrupted sequence of ISAba3 upstream of the blaOXA-58 gene (L. Al-Hassan, H. El Mehallawy and S. G. B. Amyes, unpublished results).
A single isolate, 14611 from CCH, was positive for blaOXA-40 and it was also resistant to carbapenems. No insertion element was detected upstream of the blaOXA-40 gene.
Eight of the 11 isolates that did not harbour acquired carbapenemase genes were sensitive to carbapenems (MIC <8 mg/L). One isolate, 22055, lacking these genes was resistant to carbapenems and harboured the chromosomal OXA-89 β-lactamase. ISAba2 was found upstream of the blaOXA-89 gene (Figure 2).
Acinetobacter baumannii is a problematic, multidrug-resistant pathogen identified in healthcare environments worldwide . The remarkable ability of A. baumannii to capture and express resistance genes has allowed it to become one of the major threats in hospitals, as it becomes resistant to all available antibiotics, including carbapenems . Resistance mechanisms such as modification of target site, efflux pumps and enzymatic inactivation have all been reported in A. baumannii . Of major concern is the presence of several classes of β-lactamases within the A. baumannii genome. The localization of these resistance genes on plasmids facilitates their movement from one bacterium to another . Class D oxacillinase genes: blaOXA-23, blaOXA-40 and blaOXA-58 have been repeatedly reported in A. baumannii outbreaks from different parts of the world [1, 19].
The construction of a linkage map based on the intrinsic OXA-51-like β-lactamases was reported by Evans et al. . The sequence relationship was determined for 37 distinct members of the OXA-51-like β-lactamase family. This study identified three large groups around OXA-66, OXA-69 and OXA-98 in addition to other unrelated branched enzymes . In the current study a large diversity was found in the sequences of blaOXA-51-like with eight different gene variants identified. This is particularly interesting given the short duration of isolate collection (1 year) as well as the isolates deriving from only two hospitals. In fact seven different blaOXA-51-like genes were identified in CCH alone. When looking at the distribution of blaOXA-51-like genes in the linkage map, it is clear that they have different origins as the genes identified are not clustered in closely related groups. Fourteen isolates, accounting for 41%, harboured blaOXA-65, which according to the linkage map forms a ‘central hub’ from which all other groups radiate and is thought to be ancestral to all blaOXA-51-like genes . This subsequently indicates the presence of the potential ancestral blaOXA-51-like gene in A. baumannii in Egypt, which is in the current collection of strains and is the major gene identified. Additionally, this may explain that the large diversity found is an outcome of the evolution of the ancestral blaOXA-65 gene in some cases, rather than the of ‘foreign carriage’ of clones into the country.
blaOXA-69, blaOXA-66 and blaOXA-71 have been associated with Worldwide [European] Clones I, II and III, respectively, and all have been identified in the current study [6, 7]. blaOXA-66 and blaOXA-71 genes were identified in both hospitals, which may indicate local distribution in Egyptian hospitals. blaOXA-69, on the other hand, was found in two isolates in the ICU outbreak in early 2011 at CCH only. This illustrates the extent of spread of the major lineages of A. baumannii.
blaOXA-89 is a member of the blaOXA-98 cluster and contains the resultant protein showing three amino acid substitutions from OXA-98. In the current study, one isolate from CCH was found positive for blaOXA-89, and harboured ISAba2 upstream. The presence of an insertion sequence upstream of other blaOXA-51-like genes has been reported to enhance the expression and cause resistance to carbapenems [20, 21]. ISAba2 has only been reported upstream of blaOXA58 . With no other resistance mechanism identified, the presence of ISAba2 was responsible for high-level resistance to both imipenem and meropenem (MIC 128 mg/L and 256 mg/L, respectively). Furthermore, this shows the ability of IS to insert upstream of these genes and act as promoters.
blaOXA genes that are not part of previously identified clusters have also been identified in the current study: blaOXA–94 in two isolates from the NCI and blaOXA-64 in eight isolates from both hospitals. OXA-64 is closely related to OXA-71 and is now commonly found in the Middle East [7, 9] (A. Al-Hasan and S.G.B. Amyes, unpublished results). blaOXA-94, on the other hand, forms a branch of blaOXA-65 cluster with three amino acid substitutions in the resultant protein.
As expected from this large diversity of isolates, there is considerable variation in their PFGE profiles. Notably, isolates harbouring similar blaOXA-51-like genes have different PFGE profiles and no epidemiological linkage can be inferred. This could be a result of the localization of the patients in different wards and at different times in the hospital. Even for isolates recovered from the ICU at different times, there seems to be significant variability in profiles suggesting the presence of different clones within the same hospital. Turton et al. found a correlation between PFGE and sequence typing, in contrast to Evans et al. who later noted major differences between PFGE typing and sequence typing in their study [7, 23].
MLST further illustrated the diversity within the isolates as eight out of ten isolates typed were assigned to novel STs. Previous reports have shown that typing with blaOXA-51-like was more consistent with MLST than with PFGE . In the current study, isolates 8357, P67-AZ and 9925-SAM had similar blaOXA-51-like genes but, when they were typed with MLST, they showed three different novel STs, 408, 409 and 411, respectively. The PFGE patterns were also different for these isolates. This could indicate the presence of three distinct clones in the two hospitals, especially that they were isolated in different months and in different wards. MLST, in this case, correlated with the epidemiological data of PFGE. Hamouda et al.  found MLST to be more accurate than PFGE when studying isolates on a global scale.
Seventy-three percent of the isolates were resistant to carbapenems, and this is associated with all three CHDL genes found in this study. Different genetic structures are associated with the upstream environment of blaOXA-58 and blaOXA-23 and they have been identified in different regions of the world [22, 24]. In the current study, blaOXA-23 is associated with ISAba1 in the upstream environment and blaOXA-58 is flanked by ISAba3. The effective mobilization of these genes by insertion sequences upstream together with the localization on plasmid largely contribute the spread of these resistance genes .
In conclusion, the data presented show the large diversity of A. baumannii isolated from two centres in Cairo, Egypt. The genetic plasticity of A. baumannii is represented by the presence of several insertion sequences upstream of the resistance genes, thereby facilitating the expression and causing resistance to carbapenems. Several clones seem to be present in Egyptian hospitals requiring increased awareness of the healthcare personnel and stricter infection control policies to prevent the dissemination of these isolates.
Nucleotide Sequence Accession Number
The ISAba2-blaOXA-89 sequence of strain 22055 has been deposited under the accession number JX499236.
We are grateful to the hospital staff at The Children's Cancer Hospital, Egypt and The National Cancer Institute for providing us with the samples and allowing part of the work to be undertaken at their centres.
A part of this work was presented at the 22nd European Congress of Clinical Microbiology and Infectious Diseases, London, 2012.