NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas

ABSTRACT

Among Gram-negative bacteria, carbapenem-resistant infections pose a serious and life-threatening challenge. Here, the CRACKLE network reports a sentinel detection and characterization of a carbapenem-resistant Klebsiella pneumoniae ST147 isolate harboring bla_NDM-5 and bla_OXA-181 from a young man who underwent abdominal surgery in India. bla_NDM-5 was located on an IncFII plasmid of ≈90 kb, whereas bla_OXA-181 was chromosomally encoded. Resistome and genome analysis demonstrated multiple copies of the transposable element IS26 and a “hot-spot region” in the IncFII plasmid.

TEXT

Carbapenem-resistant Klebsiella pneumoniae (CR Kp) infections are among the most problematic clinical challenges worldwide, since few antimicrobials retain activity against them, and they are associated with high morbidity and mortality (1, 2). The New Delhi metallo-beta-lactamase (NDM-1) was first identified in 2008 (3). bla_NDM has emerged as a major global health challenge not only because of its ability to confer resistance to nearly all beta-lactam antibiotics but also because of its rapid spread worldwide. At present, 16 bla_NDM-5 variants are reported in >40 countries, and it is endemic in several areas (Southeast Asia, the Balkans, and the Middle East) (4). The NDM-5 variant differs from NDM-1 at amino acid positions Val88Leu and Met154Leu, and this metallo-beta-lactamase was first identified in a multidrug-resistant Escherichia coli ST648 isolate from a patient in the United Kingdom who had a recent history of hospitalization in India (5). NDM-5 has since been detected in Algeria Spain, Japan, Australia, China, and Egypt (6–10).

The OXA-48 beta-lactamase was initially identified in a K. pneumoniae isolate from a patient residing in Istanbul, Turkey, in 2001 (11). Soon after, the gene encoding this carbapenemase (the “phantom menace”) rapidly disseminated through Turkish hospitals, with outbreaks in the main cities of the country reaching endemic levels (12, 13). Since then, 11 variants of bla_OXA-48-like have been designated and have spread to the Middle East, North Africa, and Europe. In addition, several nosocomial outbreaks were reported in Mediterranean countries, including France, Spain, Lebanon, Israel, Tunisia, Morocco, and Malta, and have reached an endemic situation (14). OXA-181, a variant of OXA-48 that differs by four amino acid substitutions (Thr104Ala, Asn110Asp, Glu175Gln, and Ser179Ala), was initially identified in Enterobacter cloacae and K. pneumoniae isolates that were recovered in 2007 in India (15). Since then, OXA-181-producing Enterobacteriaceae were reported in several other countries on the Indian subcontinent, including Bangladesh, Sri Lanka, and Nepal (16, 17), followed by Canada, France, the Netherlands, New Zealand (in a K. pneumoniae isolate from a patient from Nepal), Norway (in a patient from Romania), Oman, Romania, Singapore, South Africa, and the United Kingdom (in a patient from India) (18).

Herein, we report a K. pneumoniae isolate harboring bla_NDM-5 and bla_OXA-181 and a paired isolate from the same patient exhibiting highly plastic genomic regions associated with the IS26 insertion element involved in the loss of bla_NDM-5 and several other resistance determinants. These isolates were detected as part of a surveillance network dedicated to characterizing the K. pneumoniae carbapenemase (KPC) epidemic in the United States and elsewhere.

A young man underwent sleeve gastrectomy for management of obesity in India in 2014. The surgery was complicated by a postoperative anastomotic leak, and the patient was transferred to a U.S. hospital a month after surgery. An abdominal washout was required, and the patient was placed on bowel rest and started on total parenteral nutrition. However, he developed an enterocutaneous fistula and was transferred to one of the hospitals participating in CRACKLE (The Consortium on Resistance against Carbapenems in Klebsiella pneumoniae), a prospective, multicenter, observational study in the Great Lakes Region of the United States (2), for a Roux-en-Y gastric bypass procedure and resection of the fistula. This surgery was performed 4 months after his initial operation. Cultures sent from abdominal fluid revealed a carbapenem-resistant K. pneumoniae strain (CR Kp-1) (Table 1) and Candida albicans. The patient was initially treated with ciprofloxacin, metronidazole, and micafungin. After CR Kp-1 was confirmed, ciprofloxacin was switched to tigecycline. He remained febrile, and a repeat abdominal computed tomography scan revealed persistent fluid collections, which were drained percutaneously. Cultures from this procedure again revealed a carbapenem-resistant K. pneumoniae strain (CR Kp-2) (Table 1). He was treated for a total of 16 days with tigecycline. His symptoms resolved, and he was discharged in stable condition and off of antibiotics after a 28-day admission.

TABLE 1.

MICs for CR Kp-1 and CR Kp-2, evaluated by Etest

Antibiotic	MIC (mg/liter) for:
	CR Kp-1	CR Kp-2
Tigecycline	0.5	0.5
Amikacin	>256	2
Gentamicin	>256	2
Ampicillin-sulbactam	>256	>256
Piperacillin/tazobactam	256	>256
Ceftazidime/avibactam	>256	0.5
Ceftazidime	>256	48
Cefepime	>256	96
Ceftriaxone	>32	>32
Cefotaxime	>32	>32
Ertapenem	>32	>32
Imipenem	>32	4
Meropenem	>32	24
Doripenem	>32	8
Polymyxin Ba	≤0.5	1

^aPolymyxin B susceptibility was evaluated by broth microdilution.

The Check MDR CT103 XL assay (Check-Points, Wageningen, Netherlands) was used to detect beta-lactamase genes, including extended-spectrum beta-lactamases (bla_SHV and bla_TEM ESBLs), plasmid-mediated AmpCs, and carbapenemases, including bla_KPC, bla_NDM, bla_VIM, bla_IMP, bla_OXA-48-like, bla_GES, bla_GIM, bla_SPM, bla_OXA-23-like, bla_{OXA-24/40-like}, bla_OXA-48-like. CR Kp-1 contained bla_NDM, bla_OXA-48-like, bla_{CTX-M-15-like}, bla_SHV-WT, and bla_TEM-WT. CR Kp-2 contained bla_OXA-48-like, bla_{CTX-M-15-like}, and bla_SHV-WT. PCR amplification and sequencing performed to identify bla_NDM and bla_OXA-48-like variants confirmed that CR Kp-1 carried bla_NDM-5 and bla_OXA-181, whereas CR Kp-2 carried only bla_OXA-181.

Conjugation experiments were performed using K. pneumoniae clinical strains as donors and the azide-resistant E. coli J53 as a recipient; however, transconjugants were not obtained. Therefore, plasmid DNA was extracted and electroporated into E. coli DH10B (19). Transformants were selected on ampicillin-containing lysogeny broth agar, and the presence of bla_NDM-5 and bla_OXA-181 was confirmed by PCR amplification. Plasmid characterization on CR Kp-1 and CR Kp-2 isolates, as well as on transformants, was performed by PCR-based replicon typing using the PCR-based replicon typing kit (Diatheva, Fano, PU, Italy) following the manufacturer's instructions. Only an IncFII amplicon was obtained for both strains and transformants. S1 nuclease pulsed-field gel electrophoresis (PFGE) followed by Southern hybridization with bla_NDM-5, bla_OXA-181, and IncFII replicon probes for clinical strains and transformants (see Fig. S1 in the supplemental material) and I-CeuI PFGE followed by Southern hybridization with bla_OXA-181 and 16S probes for clinical strains (see Fig. S2 in the supplemental material) suggested that bla_NDM-5 was located on an IncFII plasmid of ≈90 kb, while bla_OXA-181 was encoded chromosomally (20, 21).

Draft whole-genome sequences were obtained from Illumina paired-end reads. Use of ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/) with assemblies as input confirmed that CR Kp-2 possesses a resistome smaller than that of CR Kp-1 (Table 2) (22). Likewise, BLAST revealed that for CR Kp-1, bla_NDM-5 was localized on an IncFII plasmid of ≈90 kb (99% similarity with pCC1409, a bla_NDM-5-harboring IncFII plasmid from a K. pneumoniae ST147 isolated in Poland; KT725789.1) (Fig. 1A). Interestingly, this same plasmid was present in CR Kp-2; however, it was slightly smaller due to the loss of an ≈25-kb region, including not only bla_NDM-5 but also several other resistance determinants (e.g., bla_TEM-1, mphA, ermB, dfrA12, aadA2, and rmtB) (Fig. 1A). ISFinder (https://www-is.biotoul.fr) showed the presence of multiple transposable elements, including IS26, distributed along the above-mentioned region (Fig. 1B) (23). Of note, the finding of rmtB is manifested by the difference in susceptibility to aminoglycosides (256 versus 2 mg/liter) (Table 1). In addition, the presence of numerous resistance determinants may indicate that this is a “hot-spot region.” Given the replicative transposition mechanism of IS26 and its previously shown critical role in the mobilization and reorganization of antibiotic resistance genes in Gram-negative bacteria, we hypothesize that the excision of this region might have been aided by IS26 (24, 25).

TABLE 2.

Resistome of CR Kp-1 and CR Kp-2

Strain	Genome size (bp)	No. of contigs	Resistance determinants
CR Kp-1	5,608,317	87	oqxA, oqxB, blaSHV-11, fosA, rmtF, aacA4, aac(6′)Ib-cr, qnrB12, dfrA14, blaOXA-181, ARR-2, blaCTX-M-15, mph(A), sul1, erm(B), dfrA12, aadA2, rmtB, blaTEM-1, blaNDM-5
CR Kp-2	5,587,222	79	oqxA, oqxB, blaSHV-11, fosA, rmtF, aacA4, aac(6′)Ib-cr, qnrB12, dfrA14, blaOXA-181, ARR-2, blaCTX-M-15

FIG 1.

(A) Coverage of Illumina reads from K. pneumoniae isolates CR Kp-1 and CR Kp-2 mapped using Bowtie2 (33) across the reference IncFII plasmid pCC1409 (KT725789.1). (B) Organization of the 25-kb bla_NDM-5-containing region of the IncFII plasmid present only in CR Kp-1. This entire region was excised out of the IncFII plasmid present in CR Kp-2.

ISEcp1 was identified upstream of bla_OXA-181, as previously described (18); I-CeuI PFGE and probe hybridization suggested that this carbapenemase was chromosomally located in both clinical strains. Interestingly, transformation experiments revealed that bla_OXA-181 was also carried by a plasmid on the CR Kp-2 E. coli transformant, which, based on size and IncFII probe hybridization, is the same plasmid that lost the above-mentioned ≈25-kb bla_NDM-5-harboring region. This might indicate a transposition event aided by the flanking ISEcp1 accompanying this gene, highlighting the plasticity of this plasmid to incorporate or lose genes (Fig. S1 and S2 in the supplemental material).

In the United States, bla_NDM has been circulating since 2010 (26), whereas bla_OXA-48 was imported in 2012 by patients who were initially hospitalized in Saudi Arabia and India (27). The rapid and widespread dissemination of bla_OXA-181 throughout Southeast Asia, the recent finding of bla_OXA-181 (associated with the ISEcp1 mobile genetic element) in the chromosome of a wastewater Shewanella xiamenensis isolate (28), and the recognition that most cases outside of this area of the world are from patients that had a recent travel history to Southeast Asia and the Asian Pacific region suggest that this is the likely place of origin for bla_OXA-181 and an important reservoir of this carbapenemase gene. Multilocus sequence typing (MLST) (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) revealed that CR Kp-1 and CR Kp-2 both belong to ST147. A K. pneumoniae isolate belonging to the same ST type (ST147) and carrying an IncFII plasmid with bla_OXA-181 and bla_NDM-5 was previously reported in South Korea from a patient transferred from a tertiary care hospital in Abu Dhabi, United Arab Emirates (29). However, the first report of a K. pneumoniae isolate harboring bla_OXA-181 and bla_NDM-5 occurred in Singapore in 2013 and was isolated from the urine of a patient that was transferred from Bangladesh (16). Another isolate harboring both carbapenemase genes was described in Egypt; however, it was an E. coli that carried each gene on a different plasmid (30). In the United States, a K. pneumoniae isolate carrying bla_NDM-1 and bla_OXA-232 was reported in another patient transferred from India (31). However, bla_NDM-5 and bla_OXA-181 have been reported only once before in the same patient but in different E. coli isolates (32).

In summary we describe the occurrence of two clinically important carbapenemases (the “evil twins”), bla_OXA-181 (chromosomally encoded) and bla_NDM-5 (plasmid encoded), in a carbapenem-resistant K. pneumoniae clinical isolate in the United States. Our results also underscore the ability of ceftazidime/avibactam to indicate the presence of metallo-beta-lactamases when carbapenem-resistant isolates show resistance to this combination. These findings underscore the importance of molecular surveillance programs such as CRACKLE that characterize resistant strains and highlight the emergence of novel genotypes in the United States.

Accession number(s).

The nucleotide sequence of the draft genomes for CR Kp-1 (1699_2676) and CR Kp-2 (1699_2677) were deposited in GenBank under accession numbers NZ_MPYK00000000.1 and NZ_MPYL00000000.1, respectively.