Characterization of Carbapenemase-Producing Enterobacteriaceae from Patients in Amman,Jordan

Bacterial isolates and identification

From May 2013 to April 2014, 2,759 nonduplicate Enterobacteriaceae were isolated from clinical specimens (blood culture, urine, fluid, wound, and sputum) from patients in five of the royal medical services hospitals in Amman, Jordan, ranging in size from 200 to 600 beds. Isolates were referred to the central hospital for antimicrobial susceptibility determinations using Vitek II (bioMérieux, Marcy l'Etoile, France). Susceptibilities were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) guidelines, 2014. ¹⁴ Additional susceptibilities for tigecycline and colistin were determined at Public Health England using agar dilution methodology and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) v4 breakpoints (www.eucast.org/ast_of_bacteria/previous_versions_of_documents date last accessed: September 20, 2017). Species identification was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS, Bruker Microflex LT; Bruker Daltonik GmbH, Bremen, Germany).

Screening for carbapenemase-producing isolates

Isolates that showed decreased susceptibility to one of the carbapenems (imipenem MIC ≥2 μg/ml, meropenem MIC ≥2 μg/ml, or ertapenem MIC ≥1 μg/ml) were considered as possible carbapenemase producers according to the CLSI guidelines (2014), and selected for further analysis.

Real-time PCR for carbapenemase genes (bla_NDM, bla_VIM, bla_KPC, and bla_OXA-48-like)

Carbapenemase genes were detected via real-time PCR ¹⁵ using lysates of freshly cultured bacteria. Assays were done using a Rotor-Gene Q instrument (QIAGEN, Crawley, UK) as described previously. ¹⁵

Variable number tandem repeat and pulsed-field gel electrophoresis typing

Variable number tandem repeat (VNTR) and pulsed-field gel electrophoresis (PFGE) typing methods were performed to determine the clonal relationships of the isolates. VNTR analysis of nine loci (A, E, H, J, K, D, N1, N2, and N4) was performed for K. pneumoniae isolates as described previously.^16,17 PFGE of XbaI-digested genomic DNA was performed as described previously ¹⁷ for Enterobacter spp.. VNTR profiles and PFGE gel images were analyzed and compared using BioNumerics (Applied Maths, Sint-Martens-Latem, Belgium) software, version 6.1. PFGE banding patterns were interpreted using a similarity cutoff of ≤86% to identify nonrelated isolates.

Plasmid transfers

Plasmids encoding carbapenemase enzymes were electroporated into Escherichia coli Alpha-Select recipients (Bioline, London, UK). Colonies selected on Luria-Bertani (LB) agar containing 100 mg/L ampicillin were screened for the presence of carbapenemase genes by RT-PCR. ¹⁵ Conjugation experiments were attempted from LB broth cultures at 37°C with a donor to recipient (E. coli J-53, azide resistant) ratio of 1:4, incubated on LB agar, followed by selection on agar containing azide and ampicillin (100 mg/L).

Plasmid DNA purification and plasmid restriction fragment length polymorphism

Plasmid DNA was purified from transformants using a phenol/chloroform extraction. ¹⁸ Plasmid DNA was digested with restriction enzyme PstI overnight and subjected to electrophoresis on a 0.85% agarose gel.

PCR-based replicon typing

PCR-based replicon typing (PBRT) was performed as previously described^19–21 on transformant DNA lysates (above). For PBRT-negative samples, an additional test for the FIB(pQIL) replicon (GenBank Accession No. GU595196) used FIB(pQIL)_F 5′- TCAGAAAAGGATCGGAATGG-3′ and FIB(pQIL)_R 5′- GCTCAGGAAGGCTCTCAATG-3′ (J. Findlay, unpublished).

Allele sequencing

Complete carbapenemase genes from representatives with each combination of carbapenemase gene family type, plasmid replicon, and plasmid restriction fragment length polymorphism (pRFLP) type were PCR amplified and sequenced. Alleles were identified via BLAST against the National Center for Biotechnology Information nucleotide database.

Bacterial isolates, susceptibility testing, and carbapenemase identification

From 2,759 isolates we identified 28 CPE (1%), comprising 23 (82.1%) K. pneumoniae, four (14.2%) Enterobacter cloacae complex, and one E. coli isolate. All were resistant to imipenem, meropenem, ertapenem, fosfomycin, ampicillin, amoxicillin/clavulanic acid, ticarcillin, piperacillin/tazobactam, cephalothin (≥16 mg/L), cefoxitin, cefotaxime, and ceftazidime. Most (60%) were resistant to ciprofloxacin, ofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole. At the time of testing (2015), additional antimicrobial susceptibilities to tigecycline and colistin were observed in 94% of the isolates. The 23 K. pneumoniae isolates harbored genes encoding OXA-48-like (n = 7), NDM-1 (n = 14), and both OXA-48 and NDM-1 (n = 2) genes. The four E. cloacae complex isolates encoded either NDM-1 (n = 3) or VIM-4 (n = 1) genes, and the E. coli isolate encoded an NDM-1 gene (Table 1).

Molecular epidemiology of bacterial isolates

Of 23 K. pneumoniae isolated, 17 were available for further characterization, and of these 14 encoded NDM alone, two encoded OXA-48 alone, and one isolate encoded both NDM-1 and OXA-48. VNTR typing for the 17 K. pneumoniae isolates indicated seven different VNTR profiles as follows: 5,3,5,14,14,2,4,2,1 (corresponding to sequence type (ST) 147) (n = 7 isolates; hospital 1), 5,4,1,1,-,2,4,4,1 (corresponding to ST101) (n = 3; hospital 4), 3,5,2,13,2,1,3,4,1 (corresponding to ST11) (n = 2; hospitals 2 and 5), 2,4,2,8,0,2,2,3,1 (corresponding to ST48) (n = 2; hospitals 1 and 3), 3,2,4,9,2,2,2,4,1 (corresponding to ST327) (n = 1; hospital 1), 6,3,4,0,1,1,4,1,1 (corresponding to ST14) (n = 1; hospital 1), and 3,3,3,0,1,1,4,1,1 (corresponding to ST15) (n = 1; hospital 1). Of the NDM-positive K. pneumoniae isolates, seven were obtained from a single hospital and had VNTR profiles that corresponded to ST147; two isolates with VNTR profiles corresponding to ST11 were identified from separate hospitals. Single isolates of types corresponding to STs 14, 15, and 327 were all obtained from hospital 1, while an isolate with a type corresponding to ST48 was obtained from hospitals 1 and 3 (Table 2). Three K. pneumoniae with OXA-48 were obtained from a single hospital (hospital 4) and had a VNTR type that corresponded to ST101, one of the isolates encoded both OXA-48 and NDM-1. Each of the four E. cloacae complex isolates (Supplementary Fig. S1; Supplementary Data available online at www.libertpub.com/mdr) was distinct by PFGE. Three E. cloacae encoded NDM-1 (from hospitals 3 and 4) and one encoded VIM-4 (from hospital 4) (Table 2). A single isolate of E. coli, which encoded NDM, was obtained from hospital 1 (Table 2).

Molecular epidemiology of carbapenemase-encoding plasmids

From our previous work, ⁹ NDM plasmids were detected in patients of three different nationalities, hospitalized in a single hospital. Conjugation assays did not identify a self-transferable carbapenemase-encoding plasmid, but transformation did reveal NDM was encoded on IncFIB(pQIL) or IncFII(K)/FIB(pQIL) plasmid replicons in 13 K. pneumoniae and within each replicon type there was a high degree of pRFLP relatedness. For example, seven ST147 isolates (from a single hospital) encoded NDM on IncFIB(pQIL) plasmids that were mostly indistinguishable as pRFLP type B (Fig. 1). Six K. pneumoniae isolates encoded NDM on IncFII(K)/FIB(pQIL) plasmids that were indistinguishable as pRFLP type C. The corresponding isolates were diverse by MLST, representing at least four STs. The pRFLP type C profile (Table 2) from the IncFII(K)/FIB(pQIL) NDM replicon found in the E. coli (isolate no. 1, Fig. 1) was indistinguishable from other pRFLP type C plasmids. One NDM plasmid with a novel pRFLP profile and an unknown replicon type occurred in an ST11 isolate (isolate no. 14, Fig. 1). OXA-48 was encoded on IncL/M plasmids that differed by two pRFLP bands in two isolates from the same hospital, which included an ST101 isolate. Another ST101 isolate from the same hospital encoded OXA-48 and NDM-1, of which NDM was shown to be encoded on an IncFII(K)/FIB(pQIL) replicon. OXA-48 was not encoded on the incFII(K)/FIB(pQIL) replicon and no transformant with OXA-48 was isolated from this sample.

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FIG. 1.

pRFLP of the 20 NDM and OXA-48-like carrying CPE isolates. pRFLP type (B) and (C)—NDM plasmids of Klebsiella pneumoniae and Escherichia coli (n = 1); pRFLP type (A)—OXA-48-like plasmids of K. pneumoniae; pRFLP type (D) and (E)—NDM plasmid of K. pneumoniae (n = 14) and Enterobacter cloacae; M, Markers. CPE, carbapenemase-producing Enterobacteriaceae; pRFLP, plasmid restriction fragment length polymorphism.

The carbapenemase-encoding plasmids from three NDM-1- and one VIM-4-positive E. cloacae complex isolates were nontypable via PBRT. The NDM-encoding plasmids from three isolates were typable by pRFLP as either type D or E. The two isolates with pRFLP type E patterns occurred in different hospitals (Fig. 1, Table 2).