Carbapenem- and Colistin-Resistant Enterobacter cloacae from Delta, Colorado, in 2015

Abstract

Resistance to carbapenems in Enterobacteriaceae is a clinical problem of growing significance. Difficulty in treating multidrug-resistant Gram-negative organisms with conventional antibiotics has led to a renewed and increasing use of polymyxin compounds, such as colistin. Here, we report the isolation of carbapenem- and colistin-resistant Enterobacter cloacae from a polymicrobial lower extremity wound in an ambulatory patient. Whole-genome sequencing demonstrated the presence of chromosomal bla_IMI-1 and bla_AmpC, as well as numerous efflux pump genes.

TEXT

Carbapenem resistance among Enterobacteriaceae is an increasing problem of great clinical concern (1). Imipenem-hydrolyzing β-lactamase (IMI) enzymes are Ambler class A serine β-lactamases with carbapenemase activity that are closely related to NMC-A-type β-lactamases (2–5). The chromosomal carbapenemase gene bla_IMI-1 was first reported in 1984 from two Enterobacter cloacae isolates in California and subsequently characterized in 1996 (5). IMI-1 confers resistance to penicillins, first- and second-generation cephalosporins, carbapenems (in particular, imipenem), and aztreonam. IMI-1 poorly hydrolyzes aminothiazole-containing cephalosporins (e.g., ceftriaxone and cefepime) and is inhibited by clavulanic acid (5, 6).

IMI-producing clinical isolates are rare but have been reported in the last decade in France, Finland, Singapore, and China (7–10). Recently, IMI-1-producing Enterobacter asburiae and E. cloacae isolates with concomitant colistin resistance were described in Ireland and China (11, 12). Historically, polymyxin resistance in Enterobacteriaceae has either been intrinsic or acquired via chromosomal mutation (13). This paradigm was recently challenged by the report of a plasmid-borne colistin resistance gene, mcr-1, in Escherichia coli from China (14).

In February 2015 in Delta, CO, a carbapenemase-producing E. cloacae strain was isolated from an outpatient wound culture from a 72-year-old woman with a history of cutaneous scleroderma, medically complicated obesity, and venous insufficiency. Antimicrobial susceptibility testing by agar dilution revealed broad β-lactam resistance, including resistance to penicillins, cephalosporins (with the exception of cefepime), and carbapenems (imipenem, meropenem, and ertapenem), but susceptibility to other classes of antibiotics (Table 1). She responded to outpatient trimethoprim-sulfamethoxazole and wound care management, with negative follow-up repeat cultures.

TABLE 1.

Antimicrobial susceptibility of the E. cloacae isolate from a wound swab, Delta, CO

Antibiotic(s)	MIC (μg/ml)a	Susceptibilityb
Amoxicillin-clavulanate	>16/8	R
Ampicillin	>16	R
Ampicillin-sulbactam	>16/8	R
Piperacillin-tazobactam	>64/4	R
Ticarcillin-clavulanate	>64/2	R
Cefazolin	>16	R
Cefoxitin	>16	R
Cefuroxime	>16	R
Cefotetan	>32	R
Cefotaxime	>32	R
Ceftazidime	>16	R
Ceftriaxone	>32	R
Cefepime	≤8	S
Imipenem	>8	R
Meropenem	>8	R
Ertapenem	>4	R
Aztreonam	>16	R
Ciprofloxacin	≤1	S
Levofloxacin	≤2	S
Tetracycline	≤4	S
Gentamicin	≤4	S
Amikacin	≤16	S
Tobramycin	≤4	S
Trimethoprim-sulfamethoxazole	≤2/38	S
Fosfomycin	>128	Rc
Colistin	>4	R

^aBreakpoint testing only.

^bR, resistant; S, susceptible.

^cSusceptible interpretation based on EUCAST 5.0 guidelines for Enterobacteriaceae.

Because of the detection of carbapenem resistance, additional characterization of the Enterobacter isolate was undertaken. Carbapenemase activity was confirmed by modified Hodge and Carba NP tests. PCR for bla_KPC and bla_NDM, performed as previously described, was negative (15). Purified nucleic acid from the isolate was assayed with the Check-MDR CT103XL microarray (Check-Points BV, Wageningen, The Netherlands); none of the 27 assay targets were detected. Additional PCR targeting genes encoding GES-, IMP-, OXA-48 group-, SME-, VIM-, NMC-, IMP-, SPM-, GIM-, and SIM-type enzymes revealed an 1,800-bp product amplified using the NMC-type primer set (6, 16–19). Amplified DNA was sequenced bidirectionally and aligned using BLAST to a reference IMI-1 carbapenemase (GenBank accession no. JX090311.1) with almost complete identity. Due to carbapenem resistance, colistin and fosfomycin susceptibility testing was performed by agar dilution (Table 1); the colistin MIC was >4 μg/ml (resistant), and the fosfomycin MIC was >128 μg/ml (resistant per EUCAST version 5.0 guidelines) (20).

Given the concomitant carbapenem and colistin resistance, whole-genome sequencing (WGS) was performed to better characterize the resistance determinants. WGS was performed on TruSeq version 3 paired-end and Nextera mate-pair libraries (target mate-pair insert size, 8 kbp) using a MiSeq platform (Illumina, Inc., San Diego, CA) with a 2 × 300-cycle kit, resulting in an average genomic coverage of 83×. Sequencing reads were processed for library adapter removal and filtering using Trimmomatic 0.32 (21), assembled with SPAdes 3.1.1 (22), processed with HMMER 3.1b2 (23), and resistance genes identified using ResFams 1.2 (24) and ResFinder 2.1 (25).

WGS revealed only chromosomal DNA and no plasmids. Multiple efflux pumps, a fosfomycin resistance determinant (fosA), a carbapenemase (bla_IMI-1), and a β-lactamase (bla_AmpC) were identified, with no nearby mobile elements (Table 2). The gene encoding IMI-1 had 100% sequence identity to a reference bla_IMI-1 gene (GenBank accession no. U50278) (5), while the bla_AmpC gene had 96% sequence identity (the highest level found) to an AmpC β-lactamase (bla_AZECL-27) from a Chinese urinary isolate of E. cloacae (GenBank accession no. KJ949108; UniProt: A0A0A0Q8L1) (26). fosA was 98% identical to a reference E. cloacae glutathione transferase (GenBank accession no. CP012167) (27). No sequences with significant identity to the newly described mobile polymyxin resistance gene mcr-1 were identified, nor were other chromosomal determinants of colistin resistance.

TABLE 2.

Antimicrobial resistance genes identified by whole-genome sequencing in an E. cloacae isolate from a wound swab, Delta, CO

Resistance mechanism	Function	NCBI accession no.	Identity (%)a
blaIMI-1	Class A carbapenem-hydrolyzing β-lactamase	U50278.1	100
blaAmpC	Class A β-lactamase	KJ949108	96
ampR	Transcriptional regulator of blaAmpC	CP009850	87
ampD	Enzymatic repressor of blaAmpC induction	CP009850	93
fosA	Fosfomycin resistance	CP012167	98
cat	Chloramphenicol acetyltransferase	CP009850.1	86
emrB	Multidrug efflux	CP012162.1	96
macB	Macrolide transporter	CP007546.1	93
mexE	Multidrug efflux	CP012162.1	95
mexX	Multidrug efflux	CP012162.1	95
acrA	Multidrug efflux	DQ679966.1	95
acrB	Multidrug efflux	DQ679966.1	94
tolC	Multidrug efflux	CP003737.1	95
robA	Multidrug efflux	CP007546.1	94
msbA	Multidrug efflux	CP011591.1	92
ompL	Porin	EGK63765	99*
oprD	Porin	WP_028014341	99*
ompC	Porin	WP_028013256	99*
phoP	Transcriptional regulatory protein	CP003737	95
phoQ	Sensor histidine kinase	CP003737	91

^aIdentity is nucleotide identity, except for those with an asterisk, which represents predicted amino acid identity.

To our knowledge, this is the first report in the United States of an IMI carbapenemase-producing E. cloacae strain that was also resistant to colistin. Recent surveillance for colistin resistance in the United Kingdom and Ireland revealed prevalences of 7% and 14% in blood and lower respiratory tract Enterobacter isolates, respectively (28). Similarly, 15% of the Enterobacter isolates from Canadian hospitals in 2008 were colistin resistant (29). The selection of carbapenem and colistin resistance in Enterobacter species undergoing antibiotic therapy has been reported (30), possibly due to the existence of environmental heteroresistant Enterobacter populations (31, 32). The mechanisms of colistin resistance in Enterobacter species are not well understood. Colistin resistance in Gram-negative bacilli is associated with mutations in the two-component regulatory proteins PhoPQ and PmrAB that result in alterations in lipopolysaccharide biosynthesis or overexpression of EptA-like phosphoethanolamine transferase proteins (13, 14, 33, 34). The phoPQ sequences from this isolate were similar to those of reference Enterobacter sequences, being without the loss-of-function mutations associated with colistin resistance (13). As with other recent reports, our patient was not documented to have ever received a carbapenem or polymyxin.

IMI-1 carbapenemases and AmpC β-lactamases are typically poor hydrolyzers of third- and fourth-generation cephalosporins, although resistance to third-generation cephalosporins via stable AmpC derepression is well known in Enterobacter species (35). Sequence analysis revealed no mutations in the IMI-1 regulatory (imiR) or enzymatic (imiA) genes. The chromosomal AmpC was relatively novel by sequence alignment (96% nucleotide identity to GenBank accession no. KJ949108 and 98% amino acid identity to Enterobacter AmpC GenBank accession no. KSX59524) and did not possess mutations in active-site residues (36). Derepression of ampC can result from mutations in ampR or ampD; dysfunction of AmpD has also been reported to result in the overexpression of nmcA-type carbapenemases (37). Analysis of the ampR nucleotide and predicted protein sequences did not reveal any mutations previously determined to result in increased ampC expression. Compared with a wild-type ampD reference sequence (GenBank accession no. U40785, ampD from E. cloacae 029), the ampD of this isolate contained several predicted amino acid substitutions, including Pro175Arg (previously described in temperature-sensitive or hyperinducible ampD variants) and substitutions Ala71Arg and Arg138His (38–41). Further investigation is required to determine the significance of these mutations. No mutations were identified in the detected porins (OprD, OmpL, and OmpC). Several efflux pumps were present by genomic analysis; expression-level analysis would be required to fully characterize their significance.

A majority of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing isolates of Enterobacteriaceae are susceptible to fosfomycin (42), with susceptibility in 47 to 72% E. cloacae isolates (EUCAST criteria) (43, 44). This isolate was resistant to fosfomycin, consistent with the chromosomally encoded glutathione transferase (fosA). FosA family proteins are responsible for a majority of enzymatic fosfomycin resistance among Enterobacteriaceae, but the overall prevalence of fosA in Enterobacter species is not well defined. With increased clinical interest in the use of intravenous fosfomycin to treat multidrug- and carbapenem-resistant Enterobacteriaceae, the surveillance of fosfomycin susceptibility in carbapenem-resistant Enterobacter strains will be important (45).

Our report highlights the isolation of carbapenem- and colistin-resistant E. cloacae from a patient being treated in a rural health care setting with minimal or no exposure to those drugs. Health care providers should be aware of the potential for isolation of carbapenem- and/or colistin-resistant organisms in health care settings not typically associated with their emergence. We also highlight the value of phenotypic testing for carbapenemase production to detect unusual carbapenemases unlikely to be targeted by commonly used nucleic acid amplification tests.

Nucleotide sequence accession number.

The sequencing reads and assembly were deposited in DDBJ/ENA/GenBank under BioProject accession no. PRJNA310238.