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. 2015 Jun 12;59(7):4305–4307. doi: 10.1128/AAC.00565-15

First Report of an OXA-48-Producing Multidrug-Resistant Proteus mirabilis Strain from Gaza, Palestine

Liang Chen a, Nahed Al Laham a,b,✉, Kalyan D Chavda a, Jose R Mediavilla a, Michael R Jacobs c, Robert A Bonomo d,e, Barry N Kreiswirth a,✉
PMCID: PMC4468693  PMID: 25896692

Abstract

We report the first multidrug-resistant Proteus mirabilis strain producing the carbapenemase OXA-48 (Pm-OXA-48) isolated at Al-Shifa hospital in Gaza, Palestine. Draft genome sequencing of Pm-OXA-48 identified 16 antimicrobial resistance genes, encoding resistance to β-lactams, aminoglycosides, fluoroquinolones, phenicols, streptothricin, tetracycline, and trimethoprim-sulfamethoxazole. Complete sequencing of the blaOXA-48-harboring plasmid revealed that it is a 72 kb long IncL/M plasmid, harboring carbapenemase gene blaOXA-48, extended spectrum β-lactamase gene blaCTX-M-14, and aminoglycoside resistance genes strA, strB, and aph(3′)-VIb.

TEXT

Carbapenemase-producing Enterobacteriaceae (CRE) have spread worldwide and caused a significant public health alert (1, 2). However, our understanding of the dissemination of CRE in isolated regions, including the Palestinian territories, is limited. Here we report an OXA-48 type carbapenemase-producing Proteus mirabilis strain from Gaza, Palestine. OXA-48 was first identified in a Klebsiella pneumoniae strain isolated in Turkey in 2001 (3). Recently, OXA-48 has been reported in a number of countries in Europe, North Africa, and the Middle East, and it is regarded as endemic in certain areas (1, 4). blaOXA-48 has also been found in numerous other Enterobacteriaceae species, including Citrobacter freundii, Enterobacter spp., Escherichia coli, Klebsiella oxytoca, Providencia rettgeri, and Serratia marcescens (4). Transmission of blaOXA-48 and other carbapenemase genes is of particular concern within bacterial species exhibiting intrinsic antibiotic resistance, such as P. mirabilis (intrinsically resistant to tetracyclines and polymyxins), as it can lead to the emergence of extensively drug-resistant or pan-drug-resistant strains, further complicating clinical treatment. In this study, we used next-generation sequencing to characterize the genomes of the OXA-48-producing P. mirabilis strain (Pm-OXA-48) and the blaOXA-48-harboring plasmid. To the best of our knowledge, this is the first genomic characterization of an OXA-48-type carbapenemase in P. mirabilis.

The P. mirabilis strain Pm-OXA-48 was isolated from a urine sample obtained from a female outpatient at Al-Shifa hospital, the largest medical complex and central hospital in Gaza, in April 2012. Previous travel history was not recorded. Susceptibility testing was performed by CLSI broth microdilution (5, 6). The strain exhibited resistance or intermediate resistance to multiple antimicrobial agents, including cefotaxime (MIC, >32 μg/ml), cefepime (MIC, >16 μg/ml), ticarcillin-clavulanate (MICs, >128 and >2 μg/ml, respectively), imipenem (MIC, >8 μg/ml), doripenem (MIC, 2 μg/ml), ciprofloxacin (MIC, >2 μg/ml), levofloxacin (MIC, >8 μg/ml), gentamicin (MIC, >8 μg/ml), tobramycin (MIC, >8 μg/ml), amikacin (MIC, >32 μg/ml), minocycline (MIC, >16 μg/ml), doxycycline (MIC, >16 μg/ml), trimethoprim-sulfamethoxazole (MIC, >4 and >76 μg/ml, respectively), colistin (MIC, >4 μg/ml), polymyxin B (MIC, >4 μg/ml), and tigecycline (MIC, >4 μg/ml).

Whole genome sequencing of Pm-OXA-48 was performed using a MiSeq Desktop Sequencer (Illumina, San Diego, CA), and de novo assembly was performed using A5-miseq software (7). The Pm-OXA-48 assembly resulted in 91 contigs, with an N50 contig size of 114,052 nucleotides, and a total length of 4,137,208 bp. The average G+C content was 39.1%. Contigs were annotated using the Prokaryotic Genomes Automatic Annotation Pipeline available at NCBI (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/), providing a total of 3,843 genes, 3,662 coding DNA sequence genes, 20 rRNAs (5S, 16S, and 23S), and 80 tRNAs.

Analyses of acquired antimicrobial resistance genes were performed using ResFinder 2.1 (8), while antibacterial biocide resistance and metal resistance were determined using the Antibacterial Biocide and Metal Resistance Genes Database (BacMet) (9). This approach identified 16 antimicrobial resistance genes encoding resistance to β-lactams (blaOXA-48, blaCTX-M-14, and blaTEM-1), aminoglycosides [aph(3′)-Ia, aadA1, aac(3)-IIa, aph(3′)-VIb, strA, and strB], fluoroquinolones (qnrD), streptothricin (sat-1), phenicols (catA1 and cat), tetracycline [tet(J)], sulfonamide-trimethoprim (sul2 and dfrA1), and 27 genes related to antiseptic and toxic compound resistance, including arsR (arsenic resistance), cutEF (copper resistance), merA (mercury resistance), and emrD (benzalkonium resistance).

The quinolone resistance-determining region (QRDR) genes gyrA, gyrB, parC, and parE were further examined, and we observed mutations encoding amino acid substitutions at Ser83-Ile and Ser84-Arg within the QRDRs of GyrA and ParC, respectively. These genetic changes within Pm-OXA-48 correlated with the ciprofloxacin and levofloxacin resistance observed during susceptibility testing. In silico detection and typing of plasmids were performed using PlasmidFinder (10); three plasmid groups were identified in Pm-OXA-48, belonging to incompatibility groups L/M, Q1, and Col3M.

Unlike the spread of KPC-type carbapenemases, which are mainly associated with the clonal spread of K. pneumoniae sequence type 258 (ST258) strains (2), dissemination of OXA-48 is mainly associated with the horizontal transfer of a single IncL/M plasmid, pOXA-48a (4), although blaOXA-48 has also been reported in plasmids of other incompatibility groups (11). In order to further characterize the novel blaOXA-48-harboring plasmid from P. mirabilis strain Pm-OXA-48, we transferred it via conjugation to a recipient E. coli J53Az-resistant strain (12). The E. coli J53 transconjugant of Pm-OXA-48 displayed reduced susceptibility to cefotaxime (MIC, >32 μg/ml), cefepime (MIC, 16 μg/ml), ticarcillin-clavulanate (MIC, >128 and >2 μg/ml, respectively), imipenem (MIC, 2 μg/ml), doripenem (MIC, 1 μg/ml), tobramycin (MIC, 8 μg/ml), and amikacin (MIC, >32 μg/ml) but maintained susceptibility to minocycline (MIC, ≤2 μg/ml), doxycycline (MIC, ≤2 μg/ml), gentamicin (MIC, 4 μg/ml), trimethoprim-sulfamethoxazole (MIC, ≤1 and ≤19 μg/ml, respectively), colistin (MIC, 0.5 μg/ml), polymyxin B (MIC, 1 μg/ml), and tigecycline (MIC, ≤0.25 μg/ml) (5, 6).

Plasmid DNA from the E. coli J53 transconjugant harboring a single plasmid (subsequently named pOXA48-PM) was extracted using a Qiagen Plasmid Midi kit (Qiagen, Valencia, CA) and sequenced using the Illumina MiSeq system as described above (13). Sequencing reads were assembled de novo, gaps were closed, and annotations were performed as described previously (13). pOXA48-PM is 72,127 bp in length with an average G+C content of 50.7%, significantly higher than that of the genome (39.1%), harbors 91 predicted open reading frames (ORFs), and belongs to the IncL/M incompatibility group (Fig. 1). A BLAST search against all completely sequenced blaOXA-48-harboring plasmids in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) indicated that pOXA48-PM is closely related to other blaOXA-48-harboring IncL/M plasmids, including pOXA-48 (14), pKPoxa-48N1 (15), and pJEG011 (16) (Fig. 1). It displayed the highest query coverage (97%) and overall nucleotide identity (99%) to pJEG011, a blaOXA-48-harboring IncL/M plasmid from a K. pneumoniae ST101 strain (Kp002) isolated in Australia (16).

FIG 1.

FIG 1

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Plasmid structures of blaOXA-48-harboring IncL/M plasmids pOXA-48a (JN626286), pJEG011 (KC354801), pOXA48-PM (KP025948, this study), and pKPoxa-48N1 (KC757416). Light-blue shading indicates shared regions of homology, while green shading indicates inversely displayed regions of homology. Open reading frames (ORFs) are indicated by arrows and are colored based on predicted gene function; brown arrows indicate genes involved with plasmid stability and partition, and white arrows indicate other scaffolding genes. Genes associated with the tra locus are indicated by green arrows, and replication-associated genes are indicated by dark-blue arrows. Antimicrobial resistance genes are indicated by red arrows, and accessory genes are indicated by yellow arrows.

The main difference between plasmids pOXA48-PM and pJEG011 is that their respective blaOXA-48 genes are carried on different Tn1999 elements. While blaOXA-48 was initially identified on composite transposon Tn1999.1 flanked by two copies of insertion sequence IS1999 (14), three additional Tn1999 variants have since been reported (3, 17–20). Tn1999.2, identified in various enterobacterial isolates from Turkey, differs from Tn1999.1 by the insertion of IS1R into the upstream region of blaOXA-48 (Fig. 1) (20); Tn1999.3 differs from Tn1999.2 by an additional IS1R integration into the downstream region of blaOXA-48 (19), while Tn1999.4 is a mosaic transposon encoding OXA-48 and CTX-M-15 (17). blaOXA-48 in pOXA48-PM is carried by Tn1999.2, as is the case in pKPoxa-48N1, whereas blaOXA-48 is harbored by Tn1999.1 in pJEG011. In addition, the Tn1999.2 element in pOXA48-PM is in the opposite orientation relative to pJEG011 (Fig. 1) (confirmed by PCR and Sanger sequencing [data not shown], demonstrating the plasticity of blaOXA-48-harboring plasmids. Similarly to pJEG011, pOXA48-PM also belongs to the IncL/M plasmid incompatibility group and harbors multiple antimicrobial resistance genes, including β-lactam resistance genes blaOXA-48 and blaCTX-M-14 and aminoglycoside resistance genes strA, strB, and aph(3′)-VIb. Among these, blaCTX-M-14 is harbored on an ISEcp1 transposition unit in pOXA48-PM. The cooccurrence of blaOXA-48 and blaCTX-M-14 within the same conjugative plasmid also highlights the propensity of a highly transmissible plasmid capable of hydrolyzing all available β-lactams.

In summary, this study describes the first report of a carbapenemase-producing Enterobacteriaceae strain in Gaza, Palestine, a highly populated region with poor public services and limited medical resources. To the best of our knowledge, it is also the first description of blaOXA-48 in Proteus spp. The pan-resistant nature of P. mirabilis strain Pm-OXA-48 underlies the significance of detection and mitigation of OXA-48 and other carbapenemases in this resource-poor region of the world before either the strains or the plasmids have the opportunity to spread further.

Nucleotide sequence accession numbers.

The draft genome sequence of P. mirabilis strain Pm-OXA-48 has been deposited in the GenBank whole-genome shotgun (WGS) database under the accession no. JSCB00000000. The complete nucleotide sequence of plasmid pOXA48-PM has been deposited under the GenBank accession no. KP025948.

ACKNOWLEDGMENTS

This study was supported by a grant (to B.N.K.) from the National Institutes of Health (grant R01AI090155). This work was also supported by grants R01AI072219 and R01AI063517 (to R.A.B.) from the National Institutes of Health and by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, the Veterans Affairs Merit Review Program, and the Geriatric Research Education and Clinical Center VISN 10 (to R.A.B.).

N.A.L. thanks the Fulbright Scholar Program for a grant sponsored by the Department of State, Bureau of Educational and Cultural Affairs, United States, which supported his visit to the Public Health Research Institute, Rutgers University, United States.

B.N.K. holds two patents that focus on using DNA sequencing to identify bacterial pathogens.

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