Characterization of a carbapenem- and colistin-resistant Enterobacter cloacae carrying Tn6901 in bla NDM-1 genomic context.

We report a clinical strain of Enterobacter cloacae, PIMB10EC27, isolated in Vietnam in 2010 that was resistant to 21 of 26 tested antibiotics, including carbapenems (MICs >64 µg/mL) and colistin (MIC >128 µg/mL). The complete genome of strain PIMB10EC27 was sequenced by PacBio RSII and the Illumina Miseq system. Whole-genome analysis revealed that PIMB10EC27 contains a chromosome of the ST513 group (PIMBEC27, length 5,272,177 bp) and two plasmids, pEC27-1 of the IncX3 group (length 62,470 bp) and pEC27-2 of the IncHI1 group (length 84,602 bp). It also revealed that strain PIMB10EC27 carries 15 genes that confer resistance to at least 10 antibiotic groups. Particularly, the insertion of ISKpn19 and Tn6901 into the genomic context of bla NDM-1 was first identified and described. In another context, amino acid mutations G273D in PmrB and F515S in PmrC were first identified on the chromosome of PIMB10EC27, which may confer resistance to colistin in this strain.

Carbapenems are one of the broad-spectrum groups of β-lactam antibiotics and have been considered the best choice for treatment of infections caused by multidrug-resistant bacteria,1 however, the recent increase in the rate of carbapenem resistance has been a cause for concern.2 Encoded by the blaNDM-1 gene, New Delhi metallo-β-lactamase 1 (NDM-1), one of the most active and transmissible carbapenemases among the carbapenem-hydrolyzing β-lactamases, was first characterized by Yong et al in 20093 and has rapidly spread globally. To date, at least 17 NDM alleles have been characterized4 and the Tn125 composite transposon bracketed by two copies of ISAba125 appears to be the main vehicle for dissemination of the blaNDM-1 gene.5 The lack of new generations of antibiotics has positioned colistin, a decades-old antibiotic, as one of the treatments of last resort against multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacteria.6,7 Unfortunately, colistin resistance has been reported and is increasing.8–11

Enterobacter cloacae belongs to the Enterobacteriaceae family. These Gram-negative bacteria have been a frequent cause of nosocomial multidrug-resistant bacterial infections in the last decade.12,13 Carbapenem-resistant E. cloacae has been commonly reported in Vietnam and many other countries in the world.14–16 However, colistin resistance in E. cloacae has not been reported widely, particularly carbapenemase-producing colistin-resistant E. cloacae has very recently been reported from some countries including India,10 the United States,17 and China.18–20

In this study, the whole genome of a clinical carbapenem- and colistin-resistant E. cloacae strain, PIMB10EC27, was sequenced and analyzed for its genetic characteristics that may associate with its resistance phenotypes.

PIMB10EC27 was recovered from the urine of a 72-year-old male patient admitted to Binh Dan Hospital in Vietnam in December 2010 with diagnoses of invasive prostate cancer and urosepsis, he had undergone prostatectomy at the same hospital 15 days earlier. The patient was discharged at the request of the family in a state of acute urinary retention and respiratory failure 4 days after hospitalization. During hospitalization, the patient was treated with meropenem, cefoperazone, sulbactam, clavulanic acid, and amoxicillin, but not with colistin. The strain was isolated as a part of the routine hospital laboratory procedures and identified as E. cloacae using a Vitek II kit (BioMérieux, USA).

The in vitro antibiotic susceptibility of PIMB10EC27 was tested by the Kirby-Bauer (KB) method, and the minimum inhibitory concentration (MIC) using the agar dilution method was also determined according to recommendations of the Clinical and Laboratory Standards Institute (CLSI 2018)19 except for colistin, which was performed with broth microdilution method and interpreted by the guidelines of European Union Committee for Antimicrobial Susceptibility Testing (EUCAST 2019).20 The results showed that PIMB10EC27 was resistant to 21 of 26 tested antibiotics, particularly imipenem (MIC >64 µg/mL), ertapenem (MIC =128 µg/mL), meropenem (MIC =128 µg/mL), and colistin (MIC >128 µg/mL); susceptible to amikacin, levofloxacin nitrofurantoin; and showed intermediate resistance to ciprofloxacin, oxfloxacin (Table 1).

Table 1

Antibiotic susceptibility of the E. cloacae PIMB10EC27

Antibiotic	Kirby-Bauer method	MIC (µg/mL)
Gentamycin	R
Tobramycin	R
Amikacin	S	2
Kanamycin	R
Piperacillin-tazobactam	R
Ticarcillin-clavulanic acid	R
Imipenem	R	>64
Ertapenem	R	128
Meropenem	R	128
Cefuroxime	R
Cefotaxime	R	>256
Ceftriaxone	R	>256
Ceftazidime	R	>256
Ciprofloxacin	I
Levofloxacin	S
Ofloxacin	I
Trimethoprim-sulfamethoxazole	R
Sulfonamide	R
Aztreonam	R
Nitrofurantoin	S
Piperacillin	R
Chloramphenicol	R	>256
Tetracycline	R	>256
Doxycycline	R
Cefoxitin		>256
Colistin		>128

Abbreviations: R, Resistant; S, Sensitive; I, Intermediate.

A Nextera XT DNA Library Prep Kit (Illumina Inc., USA) and SMRTbell Template Prep Kit (Pacific Biosciences, USA) were used to prepare libraries for the whole genome sequencing of PIMB10EC27 using simultaneously the MiSeq System (Illumina Inc.) with MiSeq Reagent Kit v.2 (2×150 cycles), and the PacBio RSII (Pacific Biosciences) together with Sequel Sequencing Kit 2.0 (8 rxn).

In total, 3.8Gb of filtered subreads were obtained from PacBio RSII sequencing platform. Using PacBio’s pre-patching program, we obtained 353Mb of 35,932 reads with the sizes of 500–27,847 bp. The pre-assemble reads were further packaged with HGAP program, which yielded a 5,272,177 bp chromosome and two plasmids of 62,318 bp and 84,480 bp. MiSeq data was used for the error correction of PacBio consensus sequences. By integrating these two platforms, we were able to obtain the complete genome which consisted of a chromosome (5,272,177 bp) belonging to the ST513 group and two plasmids, pEC27-1 of the IncX3 group (62,470 bp) and pEC27-2 of the IncHI1 group (84,602 bp) (Figure 1). The complete genome sequence was annotated with NCBI Prokaryotic Genome Annotation Pipeline servers and analyzed by bioinformatics programs or software, including ResFinder 2.1,21 MLST,22 Isaga,23 Galaxy,24 and Plasmid Finder25 for antibiotic resistance genes identification, chromosome classification, sequences of insertion sequence (IS) determination, integrons finding, and plasmids classification, respectively. In total, 15 coding genes conferring resistance to 10 antibiotic groups were found on PIMB10EC27 (Table 2). Among these genes, blaNDM-1, blaSHV-12, blaCMH, aac, and tet(D) were associated with resistance phenotypes of PIMB10EC27.On the other hand qnrS1 equivalent to the intermediate fluoroquinolone-resistance phenotype of PIMB10EC27 has been proven to reduce the antibiotic susceptibility of bacteria to quinolones or fluoroquinolone.26–28 In addition, 160 putative transposase open reading frames (tORFs) were identified in which the number of tORFs located on the chromosome, pEC27-1 and pEC27-2 were 113, 10 and 37, respectively. Antibiotic resistance genes closely located to these transposase sequences may have contributed to the accumulation and increase in the antibiotic resistance of PIMB10EC27. A 1722-bp class 1 integron with gene cassette dfrA14 flanked by IS6 sequences was also found on plasmid pEC27-2.

Figure 1

Structure of plasmid pEC27-1 and pEC27-2. pEC27-1 is an IncX3 plasmid that possesses highly syntenic plasmid backbone compare to IncHI1 plasmid pEC27-2. The outer circle shows ORFs on forward and reverse strands. Resistance genes, transposase genes and resolvase genes are depicted by red, black, blue arrows, respectively. The two inner circles show the GC content (purple circle) and GC skew (blue indicates positive values, red indicates negative values) information.

Table 2

Distribution of coding genes conferring to antibiotic resistance in PIMB10EC27

Gene	Description	Resistance mechanism	Antibiotic group/agent
Chromosome
blaCMH	CMH family class C β-lactamase	Antibiotic inactivation	β-lactams
fosA	FosA/FosA2 family fosfomycin resistance glutathione transferase	Antibiotic inactivation	Fosfomycin
pEC27-1
blaNDM-1	New Delhi metallo-β-lactamase NDM-1	Antibiotic inactivation	β-lactams
blaSHV-12	Extended-spectrum beta-lactamase SHV-12	Antibiotic inactivation	β-lactams
bleMBL	Bleomycin-resistance protein	Sequestering effect of the bleomycin-binding protein	Bleomycin
pEC27-2
aac(3)-IId	Aminoglycoside acetyltransferase	Antibiotic inactivation	Aminoglycosides
blaLAP-2	Class A β-lactamase LAP-2	Antibiotic inactivation	β-lactams
blaTEM-1B	Class A broad-spectrum β-lactamase TEM-1	Antibiotic inactivation	β-lactams
catA2	Chloramphenicol acetyltransferase	Antibiotic inactivation	Phenicols
dfrA14	Dihydrofolate reductase	Altered affinity of reductase	Trimethoprim
qnrS1	Quinolone resistance pentapeptide repeat protein	Antibiotic target protection	Quinolones, fluoroquinolones
strA	Aminoglycoside phosphotransferase	Antibiotic inactivation	Aminoglycosides
strB	Aminoglycoside phosphotransferase	Antibiotic inactivation	Aminoglycosides
sul2	Sulfonamide resistant dihydropteroate synthase	Antibiotic target replacement	Sulphonamides
tet(D)	Tetracycline efflux pump	Antibiotic efflux	Tetracyclines

We further analyzed the genes in PIMB10EC27 that confer resistance to carbapenem and colistin. As a result, a blaNDM-1 gene was found on pEC27-1 of PIMB10EC27 along with a blaSHV-12 and bleMBL. A comparison of the nucleotide sequence of pEC27-1 and those of other IncX3 plasmids carrying blaNDM-1 and blaSHV-12, including pKPN5047 (KC311431), pNDM-HF727 (KF976405), pNDM-HN380 (JX104760), and RJA274 plasmid NDM-1 (KF877335) was carried out in order to identify if any unique structure characteristics contributed to the multidrug-resistance of PIMB10EC27. Comparative results of the genomic context surrounding the resistance genes revealed that all of the plasmids had a conserved sequence carrying ΔISAba125, blaNDM-1, bleMBL, trpF, dsbC, cutA1, groES, groEL, and ISCR21 (Figure 2). This conserved sequence is also a Tn125 composite transposon, in which the blaNDM-1 gene lies downstream of a truncated ISAba125 element that provides the −35 region for the promoter of blaNDM-1.29 A 93-bp sequence separates the right-hand inverted repeat of ISAba125 from the start codon of blaNDM-1, and the deletion of an IS5 was identified in the plasmid pEC27-1. An ISCR21-like element was also found downstream of blaNDM-1 and it is suggested that this element may be responsible for initial gene capture.5 The IS26-bounded region of pEC27-1 containing ygbI, ygbJ, and blaSHV-12 was found to be very distinct from the other plasmids. This region carried three copies of IS26, and the local ygbJ gene was split into two parts, whereas other plasmids carried only two copies of IS26 and the full ygbJ gene. The isolation and reverse with a copy of IS26 in the plasmid pEC27-1 of the 424-bp region of the ygbJ gene might propose there is an IS26-mediated re-organization. Interestingly, an insertion of ISKpn19 and Tn6901 into the mpr gene (encoding zinc metalloproteinase) lying further upstream of blaNDM-1 was also observed (Figure 2). Belonging to ISKra4 family, ISKpn19 is 2851 bp in length and has been found to lie downstream of blaOXA-181 in an IncX3-type plasmid.30 Tn6901, first described in plasmid Rts1 of Proteus vulgaris,31 is 6.9 kb in length and harbors 6 genes, including a transposase gene (tnpA), a resolvase gene (tnpR), an alcohol dehydrogenase gene (adh), a glyoxalase/bleomycin resistance gene (glo), an S-formylglutathione hydrolase gene (fgh), and a regulatory protein gene (frmR). We report here the first case, to our knowledge, of a 9.8-kb insertion sequence harboring ISKpn19 and Tn6901 in the genomic context of blaNDM-1. This insertion made the genomic context of blaNDM-1 in PIMB10EC27 more complex with 6 resistance genes, including adh, glo, fgh, blaNDM-1, bleMBL, and blaSHV-12, which may also affect the dissemination and expression of blaNDM-1. Further research is required to clarify these assumptions.

Figure 2

Genetic context of blaNDM-1 on IncX3 plasmids pKPN5047, pNDM-HF727, pNDM-HN380, RJA274 plasmid NDM-1, and pEC27-1. Gray shading denotes shared regions of homology: light gray indicates a forward match and dark gray indicates a reverse match. Notably, ygbJ is split into two parts, and the IS26-ΔygbJ region is inverted in pEC27-1 relative to the other plasmids. The mpr gene in pEC27-1 is interrupted by ISKpn19 and Tn6901 insertions. Open reading frames are portrayed by arrows and are colored. Resistance genes are indicated in red arrows, including blaNDM-1, blaSHV-12, bleMBL, fgh, glo, and adh.

Genome analysis was also performed to identify the genes that confer resistance to colistin of PIMB10EC27. In this research, we did not find any mcr gene that recently characterized as a novel mobile gene responsible for colistin resistance among Gram negative bacteria.32 In addition, our transformation experiments revealed that none of the plasmids of PIMB10EC27 was associated with colistin resistance (data not shown). In another context, encoding genes involved in the attachment of L-Ara-4N and P-EtN to LPSs of PIMB10EC27, including phoP, phoQ, pmrB, pmrA, pmrC, mgrB, lpx, and arn were examined, and amino acid replacements G273D in PmrB and F515S in PmrC of PIMB10EC27 were recorded. The role of these mutations in colistin resistance remains to be investigated.

In summary, we successfully constructed the complete genome of multidrug-resistant E. cloacae strain PIMB10EC27 carrying the novel genomic context of blaNDM-1. To our knowledge, this is the first report in Vietnam of a carbapenemase-producing E. cloacae strain that was also resistant to colistin. PIMB10EC27 was isolated from a patient not being treated with colistin and not identified as a case of a plasmid-mediated colistin-resistance mechanism.

In the battle against multidrug-resistant bacteria, particularly carbapenem-resistant bacteria, colistin has been considered to remain effective. In such context, this report of a clinical isolate that is resistant to both colistin and carbapenem raises a high concern over future clinical management and infection control.

Nucleotide sequence accession numbers. The complete nucleotide sequences of PIMB10EC27 have been deposited in GenBank under accession numbers CP020089-CP020091.

(https://www.ncbi.nlm.nih.gov/nuccore/CP020089.1/, https://www.ncbi.nlm.nih.gov/nuccore/CP020090, https://www.ncbi.nlm.nih.gov/nuccore/CP020091).