Chromosomal coharboring of blaIMP-60 and mcr-9 in Enterobacter asburiae isolated from a Japanese woman with empyema: a case report.

BACKGROUND: Polymyxin E (colistin) is a last-resort antibiotic to treat infections caused by carbapenemase-producing Enterobacteriaceae (CPE). However, reports of CPEs resistant to colistin have been increasing, and the mcr genes are emerging as resistance mechanisms. Among them, plasmid-mediate mcr-9 is known to be associated with colistin resistance, whereas reports on chromosomal mcr-9 and its association with colistin resistance in humans are few. CASE
PRESENTATION: We identified Enterobacter asburiae harboring mcr-9 and blaIMP-60 in the pleural fluid of a patient with empyema. The long-read sequencing technique revealed that these genes were located on its chromosome. Despite the lack of exposure to colistin, the organism showed microcolonies in the inhibition circle in the E-test and disk diffusion test. Antibiotic susceptibility testing by broth microdilution confirmed its resistance to colistin.
CONCLUSION: Our case report showed that mcr-9 can be present not only on plasmids but also on the chromosome in E. asburiae, and that the presence of mcr-9 on its chromosome may influence its susceptibility to colistin.

Background

Carbapenemase-producing Enterobacteriaceae (CPE) are a threat for healthcare providers because they can be resistant to many antimicrobial agents including carbapenems, and there are few reliable antimicrobial treatment options [1]. Reports of colistin-resistant CPE have improved our understanding of the mechanisms of resistance, including the acquisition of mobile colistin-resistance (mcr) genes (mcr-1 to mcr-10) [2-4]. The mcr genes, especially mcr-9, are frequently found in Enterobacter cloacae complex strains, and several of these strains co-harbor metallo-beta-lactamase genes, such as bla and bla [5]. Plasmid-borne mcr genes conferring resistance to colistin pose a serious threat to international public health [3]; in particular, IncHI2 plasmids bearing mcr-9 play a central role in the global spread of resistant strains [6]. While many reports of plasmid-borne mcr are available, there are only a few case reports on the detailed clinical course of infection and antibiotic susceptibility testing of Enterobacteriaceae carrying mcr and metallo-beta-lactamase genes on their chromosomes. Here, we report the case of a patient with empyema who carried E. asburiae harboring bla and mcr-9 on its chromosome.

Case presentation

The patient was a 68-year-old previously healthy woman who had suffered from a severe sore throat for a week and was admitted after being diagnosed with septic shock, acute epiglottitis, descending mediastinitis, and bilateral pleural empyema due to Streptococcus constellatus and Bacteroides thetaiotaomicron. Although the patient received an intensive care with treatment by percutaneous drainage and intravenous piperacillin/tazobactam, she experienced a relapse of empyema due to E. asburiae resistant to meropenem and colistin (Table 1). Multiple resistant microcolonies were present within the zone of clearing by the E-test and disc diffusion method (Additional file 1 and 2: Fig. S1, S2). We suspected the strain was heteroresitant to colistin at first, but antibiotic susceptibility testing using broth microdilution (BMD) confirmed colistin resistance (Table 2).

Table 1

MICs of different antibiotics (except colistin) for E. asburiae strain performed using the Microscan WalkAway system

Antibiotics	MIC (μg/ml)
Ampicillin	≥ 32
Ampicillin/sulbactam	≥ 32
Piperacillin/tazobactam	≥ 256
Cefazolin	≥ 32
Cefpodoxime proxetil	≥ 16
Cefmetazole	≥ 64
Ceftriaxone	≥ 16
Ceftazidime	≥ 32
Cefepime	≥ 32
Latamoxef	≥ 16
Cefpodoxime/clavulanate	≥ 4
Aztreonam	≥ 16
Meropenem	8
Amikacin	≤ 8
Gentamicin	≤ 1
Tigecycline	2
Nalidixic acid	≥ 64
Levofloxacin	2
Fosfomycin	≥ 64
Trimethoprim/sulfamethoxazole	≤ 2

MIC minimum inhibitory concentration

Table 2

Colistin susceptibility of E. asburiae

Methods for colistin susceptibility	Colistin MIC (μg/ml)
	Enterobacter asburiae
Disc diffusion method (mm)	No inhibition zone*
E-test	> 256*
Broth microdilution (BMD)	> 1024

MIC minimum inhibitory concentration

*Multiple micro-colonies were detected within the inhibition circle (Additional file 1 and 2: Fig. S1, S2)

A detailed next-generation sequencing study was carried out to examine the mechanism of drug resistance to colistin in the E. asburiae strain. The long-read sequencing technique revealed that in this case, E. asburiae co-harbored mcr-9 and bla on its chromosome (Fig. 1). By applying error correction twice using Pilon on short reads, an improvement in the overall sequence and length was achieved. Specifically, we performed the following analyses: (a) long-read sequencing reads were demultiplexed by Porechop v0.2.4 (https://github.com/rrwick/Porechop), and the reads were adaptor-trimmed and quality-filtered by Nanofilt (Q score, 9; minimum length, 1000 bp), (b) chimeric reads were removed using yacrd v0.6.0 (https://github.com/natir/yacrd), (c) the reads of errors were corrected by short-read sequencing reads with LoRDEC v0.8 software [7] with default parameters, (d) de novo assembly was performed by Flye v2.6 [8] (with default parameters using corrected long-read sequencing reads), (e) assembled contigs of errors were corrected by short-read sequencing reads with Pilon v1.23 [9] twice with default parameters, (f) genome and plasmid sequences were annotated using DFAST (https://dfast.nig.ac.jp), and (g) antimicrobial resistance genes were detected using ResFinder v4.1 with default parameters on the CGE server (http://www.genomicepidemiology.org). Subsequent long-read sequence analysis of E. asburiae using MinION revealed a circular chromosome (accession no. AP024281), which contained bla and mcr-9. There were also resistance genes on the chromosome, such as aac(6')-llc, blaACT-6, and sul1_2. E. asburiae, in this case, had only one plasmid (AP024282), but there were no resistance genes on it. A linear comparison of the mcr-9-surrounding regions of the chromosome of E. asburiae (AP024281) and a plasmid of Salmonella Infantis pRH-R27 was performed by BLAST and visualized using Easyfig v2.2.2 (https://mjsull.github.io/Easyfig/) (LN555650) (Fig. 1). The core structure of the reported mcr-9 cassette, rcnR-rcnA-pcoE-IS903-mcr-9-wbuC [6], was also observed in the chromosome of E. asburiae.

Fig. 1

mcr-9-surrounding regions on the chromosome of E. asburiae and in a plasmid of Salmonella Infantis. Colored arrows represent open reading frames, with brown, green, and red arrows representing heavy metal resistance genes, mobile genetic elements, and mcr-9, respectively. The remaining genes are shown in gray

The patient underwent repeated percutaneous drainage and intravaenous antimicrobial therapy with piperacillin/tazobactam, gentamicin and levofloxacin for two months, then we switched oral therapy with trimethoprim/sulfamethoxazole and levofloxacin until resolution of the empyema in imaging studies. After being discharged on day 168, she is now living on her own after the rehabilitation therapy and has had no recurrences.

Discussion and conclusions

The novelty of E. asburiae in this case is the demonstration of colistin resistance with mcr-9 on the chromosome. Most mcr-9 genes are located on plasmids; a previous report stated that only 7.8% (6/78) of identified mcr-9 genes are located on chromosomes of Enterobacteriaceae strains from human, animal, food, and the environment [6]. The mcr gene is believed to be mobile in various genetic elements and can be integrated into chromosomes, making colistin resistance prevalent in a large number of Enterobacteriaceae [1]. To our knowledge, this is the first report of colistin resistance in strains expressing chromosomal mcr-9 and bla. Reports of mcr-9 on human chromosomes are rare; we summarize three cases in Additional file 3: Table S1 [10-12]. Whether there are differences in characteristics, such as susceptibility to colistin, between Enterobacteriaceae carrying mcr-9 on plasmids and on their chromosomes is unclear.

Surprisingly, the patient in this case was infected with an E. asburiae strain resistant to colistin even though she had not been exposed to colistin. In our opinion, mcr-9 and colistin resistance have not been adequately investigated. In the current case, BMD showed colistin resistance, and multiple micro-colonies were also present within the zone of inhibition circle in both E test and disk diffusion method. Napier et al. reported colistin susceptibility of E. cloacae determined using the E test; they showed that small colonies were found in the zone of inhibition [13], as in this case, which they described as “heteroresistant”. Although colistin susceptibility testing for CPE is difficult, BMD is the most reliable test [14, 15] and we judged the strain was resistant to colistin. Strains that are heteroresistant to colistin are considered to become resistant to the drug after treatment with colistin and regain sensitivity in the absence of colistin exposure. Furthermore, Kieffer et al. reported that the expression of cloned mcr-9 in Enterobacteriaceae does not significantly affect colistin susceptibility and that exposure of bacteria to colistin, in addition to the introduction of mcr-9, may induce mcr-9 expression and lead to colistin resistance [16]. Three previous reports showed that Enterobacteriaceae isolated from humans with mcr-9 on the chromosome were colistin-sensitive (Additional file 3: Table S1) [10-12], whereas E. asburiae in the current case was resistant to colistin despite the lack of exposure to the drug. Further studies are needed to determine whether the presence of mcr-9 on the chromosome influences clinical colistin susceptibility. Moreover, It is possible that mcr-9 and bla60 gene were transferred from the B. thetaiotaomicron detected from the patient, although it was susceptible to carbapenem and ampicillin/sulbactam. We could not conduct a further investigation because we first initiated treatment with piperacillin/tazobactam and after the empyema recurred, microbiology testing results could not detect B. thetaiotaomicron. This is a limitation of this study.

In conclusion, we identified a colistin-resistant strain of E. asburiae that simultaneously possesses mcr-9 and bla genes on its chromosome. The presence of mcr-9 on the chromosome may be associated with colistin resistance.

Additional file 1: Supplementary Figure 1. Image of Enterobacter asburiae isolate colR/S plated on Mueller-Hinton agar with a colistin E test strip (left) and the disc diffusion method (right). Resistant colonies were present within the zone of clearing.

Additional file 2: Supplementary Figure 2. Magnified image of E test's micro-colonies. Red arrows indicate resistant colonies.

Additional file 3: Supplementary Table 1. Case Reports of Enterobacteriaceae carrying chromosomally located mcr-9 isolated from humans.