Characterization of resistance mechanisms of Enterobacter cloacae Complex co-resistant to carbapenem and colistin

Background The emergence of carbapenem-resistant and colistin-resistant ECC pose a huge challenge to infection control. The purpose of this study was to clarify the mechanism of the carbapenems and colistin co-resistance in Enterobacter cloacae Complex (ECC) strains. Results This study showed that the mechanisms of carbapenem resistance in this study are: 1. Generating carbapenemase (7 of 19); 2. The production of AmpC or ESBLs combined with decreased expression of out membrane protein (12 of 19). hsp60 sequence analysis suggested 10 of 19 the strains belong to colistin hetero-resistant clusters and the mechanism of colistin resistance is increasing expression of acrA in the efflux pump AcrAB-TolC alone (18 of 19) or accompanied by a decrease of affinity between colistin and outer membrane caused by the modification of lipid A (14 of 19). Moreover, an ECC strain co-harboring plasmid-mediated mcr-4.3 and blaNDM-1 has been found. Conclusions This study suggested that there is no overlap between the resistance mechanism of co-resistant ECC strains to carbapenem and colistin. However, the emergence of strain co-harboring plasmid-mediated resistance genes indicated that ECC is a potential carrier for the horizontal spread of carbapenems and colistin resistance. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-021-02250-x.


Background
Enterobacter cloacae complex (ECC) belongs to the Enterobacter genus of the Enterobacteriaceae family, which exists widely in nature and is also part of the common bacterial ora of the human gastrointestinal tract. In the past few decades, ECC was one of the most common pathogens in hospitals, often causing various infections, such as pneumonia, urinary tract infections, and sepsis (1). Enterobacter cloacae and Enterobacter hormaechei are the most common in clinical infected patients, especially in patients with immunocompromise and who admitted to the intensive care unit (ICU) (2). Due to the widespread use of antibiotics and the ability of ECC to be good at up-regulating or obtaining drug resistance determinants, multi-drug resistance (MDR) ECC strains have emerged and spread around the world. ECC infection accounts for 65% -75% of Enterobacter infections, which was called "ESKAPE" pathogen with ve other common pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) (3,4).
ECC is intrinsically resistant to penicillins and rst-and second-generation cephalosporins due to lowlevel expression of chromosomal ampC genes which encoding an inducible AmpC-type cephalosporinase (5). When ECC is exposed to β-lactam drugs for a long period of time, it can further lead to the highly induced phenotypes of AmpC cephalosporin, thus producing resistance to third-generation cephalosporins (6). The most common mechanism is that ampD mutation leads to structural overexpression of AmpC (7). At the same time, several studies have shown that mutations in the ampR gene, although relatively rare, can also cause overexpression of AmpC (8,9). In addition, the acquisition of a variety of plasmids mediated extended-spectrum β-lactamase (ESBL) genes conferred ECC resistance to most β-lactam drugs, making the treatment more di cult. Because ECC strains that overexpress AmpC enzymes and produce ESBLs were usually susceptible to carbapenem, carbapenem were considered to be the preferred antibacterial for the treatment of infections caused by high levels of cephalosporin-resistant ECC. The emergence of carbapenem-resistant ECC has aroused great clinical attention. Literature has shown that the resistance of Enterobacter cloacae to carbapenem can be mediated by AmpC expression and membrane permeability changes, but it may be more common to obtain carbapenemase gene transferred by plasmids (10). The plasmid-mediated mechanism signi cantly increases the spread of carbapenem resistance, while further limiting the choice of effective antibacterial drugs.
However, a number of recent studies have found that ECC strains carry carbapenemase genes while being resistant to colistin (11)(12)(13). Colistin is an old antibiotic which has been refocused in recent years.
Because of the good activity against Gram-negative bacteria, it is considered to be one of the last-line antimicrobials for treatment of MDR Gram-negative bacteria (14). According to previous reports, ECC may acquire colistin resistance by plasmid-mediated gene mcr, small protein gene ecr or two-component systems phoPQ, pmrAB (13,15,16). The emergence of carbapenem-resistant and colistin-resistant ECC will undoubtedly pose a huge challenge to infection control. And the mechanism of carbapenem and colistin resistance mediated by movable elements also increases the risk of widespread transmission.
The purpose of this study is to clarify the mechanism of the carbapenems and colistin co-resistance in ECC strains. We collected co-resistant ECC clinical isolates from a regional medical center, and performed this study through methods such as phenotype testing, gene identi cation, relative expression detection, and mass spectrometry analysis in order to provide further understanding for the resistance development of ECC strains.

Results
Results of antimicrobial susceptibility testing and e ux inhibitors assay Antimicrobial susceptibility testing was used to determine the minimum inhibitory concentrations (MICs) of Carbapenem and Colistin. As shown in table S2, all 19 ECC strains were resistant to ertapenem (MIC 50 = 4 μg/mL) and colistin (MIC 50 = > 64 μg/mL). Besides, 5 ECC strains were resistant to meropenem, 6 ECC strains were resistant to imipenem, and 4 of them were resistant to 3 carbapenems. The results obtained from the e ux inhibitors assay were shown in Figure 1. In the presence of e ux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP), the MICs of colistin to 19 ECC strains decreased signi cantly and returned to susceptible, while CCCP showed no effect on the MICs of ertapenem. Taken together, these results suggest that there is an association between colistin resistance of ECC strains and e ux pump.

Relative expression of genes encoding outer membrane protein and e ux pump protein
To assess the relationship between carbapenem resistance and the expression of outer membrane proteins OmpC and OmpF and the relationship between colistin resistance and expression of e ux pump proteins AcrA and AcrB, 19 ECC strains co-resistant to carbapenem and colistin, 1 carbapenem and colistin co-susceptible ECC strain CG37 and Enterobacter cloacae ATCC 700323 were used. As shown in Figure 2, compared with ATCC 700323, the expression level of ompC was reduced in 15 co-resistant ECC strains. Furthermore, the expression of ompF was de cient in all 19 co-resistant ECC strains. The e ux pump gene acrA was found to be signi cantly overexpressed in 18 co-resistant ECC strains. Conversely, the acrB gene was not found to be signi cantly overexpressed in co-resistant ECC strains (except CG1249).

Prevalence of mcr, ecr genes and structural modi cation of wild-type lipid A
To investigate whether the colistin resistance of the ECC strains in this study is related to the modi cation of lipid A, we detected the relevant resistance genes and analyzed the ion peak spectrum of lipid A using matrix-assisted laser desorption/ionization time of ight mass spectrometry (MALDI-TOF MS). From Figure 1 we can see that the ECC strain CG175 harboring mcr-4.3, which belongs to the plasmid-mediated mcr family gene. Moreover, a novel transmembrane protein gene ecr was found in other 11 ECC strains (Y541, CG737, CG741, CG701, CG864, CG884, CG934, CG1038, CG1050, CG1051, CG1506). The structures of lipid A in ECC strains in this study showed diversity ( Figure 3 and Figure 4). Among them, colistin-susceptible ATCC 700323 possessed wild-type lipid A with three ion peaks of m/z 1796, 1824, and 2062, respectively ( Figure 3A). Wild-type lipid A were also present in 19 co-resistant ECC strains, of which the ion peak of m/z 1824 is predominant. Interestingly, 14 of the 19 ECC strains contained modi ed lipid A related to colistin resistance. Taken together, the ion peak spectrum of 14 co-resistant ECC strains can be divided into 3 different modes ( Figure 3B, Figure 3C. Figure 3D), including 4 ion peaks of m/z 1919, 1947, 1955, and 2185. From the data in Figure 4, the groups that modify wild-type lipid A and cause resistance to colistin are phosphoethanolamine (pETN, m/z 123) and 4-amino-4-deoxy-Larabinose (-L-Ara4N, m/z 131).

Discussion
ECC can adapt to the environment quickly and obtain resistance to various antibacterial drugs by inducing resistance determinants and gaining exogenous resistance genes. The emergence of MDR ECC poses a huge challenge for the effective clinical treatment.
Carbapenem is an atypical β-lactam antibiotic with the broadest resistance spectrum and has the best antibacterial effects at present. It also maintains good susceptibility to Enterobacteriaceae carrying ESBLs or overexpressing AmpC-type cephalosporinase, and is the choice for clinical control of MDR ECC infections (17,18). However, with the widespread use of carbapenem, the number of carbapenemresistant ECC clinical strains is increasing, which can lead to failure of clinical anti-infection treatment. As mentioned in the literature review, the main mechanisms that mediate the resistance of ECC strains to carbapenem are: 1. Generating carbapenemase ; 2. The production of AmpC or ESBLs combined with loss or decreased expression of out membrane protein (19,20). In addition, studies have found that the susceptibility of Escherichia coli and Enterobacter cloacae to carbapenem can be increased when the AcrAB-TolC e ux pump is inhibited (21,22).
Colistin is an "old" antibiotic, which has been re-applied to the clinic because of its good antibacterial activity against MDR gram-negative bacteria. In fact, it is the last line of drug for the treatment of serious infections caused by MDR gram-negative bacteria. As reported by Mirelis, the total resistance rate of Enterobacteriaceae to colistin was 0.67% but the resistance rate of Enterobacter cloacae 4.2% was much higher than that of Escherichia coli and Klebsiella pneumoniae (23). The resistance mechanism of ECC strains to colistin can be mediated by the mcr-1 gene carried by the plasmid (15,24,25), and may also be related to the mutations of two-component system encoded by pmrAB and phoPQ (13). Recently, Zong et al. found that a novel small transmembrane protein Ecr may mediate the heterogeneous resistance of ECC to colistin through the PhoPQ two-component system (16), but the distribution of ecr gene in ECC strains and its correlation with colistin resistance still need further study. In reviewing the literature, colistin resistance mediated by mcr series genes or mutations in pmrAB and phoPQ is related to structural modi cation of lipid A of ECC strains, such as phosphoethanolamine (pETN) or 4-amino-4deoxy-L-arabinose (-L-Ara4N). The modi cation of wild-type lipid A can reduce the potential of the outer membrane in Escherichia coli and reduce the a nity of colistin to the outer membrane (26,27). Furthermore, the overexpression of e ux pump AcrAB-TolC may also lead to the resistance of ECC strains to colistin (28). In recent years, carbapenems and colistin co-resistant ECCstrains have been found in multiple regions around the world (11)(12)(13)(29)(30)(31), which will further increase the di culty of treating MDR ECC infection. And some of the co-resistant ECC strains have the ability to spread drug resistance gene horizontally, which posing a huge threat to public health.
The current study found that 7 out of 19 carbapenems and colistin co-resistant ECC strains carried the carbapenemase gene, of which 3 strains carried bla KPC-2 and 4 strains carried bla NDM-1 . It can be found that the ECC strains carrying the carbapenemase gene are all resistant or intermediary to meropenem, imipenem, and ertapenem, but most of the ECC strains that do not carry the carbapenemase gene were only resistant to ertapenem in a low level, and were susceptible to meropenem and imipenem. This nding is in agreement with CLSI guidelines which suggested that resistance of E. cloacae to imipenem and meropenem is usually related to the carbapenemase gene (32). This study found that the predominant mechanism of carbapenem resistance in ECC strains is not generating carbapenemase. The results of this study showed that AmpC or ESBLs production combined with decreased expression of out membrane protein confer low-level resistance to ertapenem in ECC strains that do not produce carbapenemase. In addition no correlation was found between the e ux pump AcrAB-TolC and carbapenem resistance of ECC strains in this study.
This study found that 11 (57.9%) of the 19 ECC strains carry the ecr gene, which is thought to be related to colistin heterogeneity resistance by regulating lipid A modi cation. Further analysis of lipid A by MALDI-TOF mass spectrometry revealed that all ECC strains carrying the ecr gene undergo lipid A modi cation, suggesting that ecr may mediate colistin resistance in this study. Interestingly, of the 11 strains carrying the ecr gene, except for the wild-type lipid A of Y541, which was modi ed with phosphoethanolamine (pETN), and the remaining strains were all modi ed with 4-amino-4-deoxy-Larabinose (-L-Ara4N). We speculate that Y541 may be involved in other colistin resistance mechanisms, which need further study. The results of the e ux inhibitor assay suggest that colistin resistance may also be related to the e ux pump, and the expression of the e ux pump genes acrB showed that except for the strain CG741, the expression levels of acrB in other ECC strains did not increase signi cantly. However, the expression levels of acrA gene in 18 ECC strains increased in varying degrees. This result is different from telk et al.'S report that the regulation of sosRS can simultaneously upregulate expression of acrA and acrB to induce heterogeneous resistance in Enterobacter cloacae. This inconsistency may be due to acrA, acrB and tolC genes are not co-regulatory genes. Their expression is usually regulated by multiple levels of different regulatory factors (33)(34)(35).
Notably, CG175 carries the bla NDM-1 gene along with the mcr-4.3 gene. The genomic characteristics of this strain have been reported in the study of Chavda et al. (12). This is the rst Enterobacter cloacae isolate co-harboring mcr-4.3 and bla NDM-1 that reported in China. Although in the study by Chavda  To screen for suspected carbapenemase production in the 19 ECC strains, the mCIM was performed. As recommended by CLSI (32)

Polymerase chain reaction (PCR) detection for resistance genes
The genomic DNA of the 19 ECC strains were extracted using the Biospin Bacterial Genomic DNA Extraction kit (Bio ux, Tokyo, Japan were recorded for optimal ion signals in negative-ion mode using a Bruker auto ex MALDI-TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). Data were acquired and processed by exControl and exAnalysis 3.4 (Bruker Daltonics Inc.).

Funding
This work was supported by the Planned Science and Technology Project of Wenzhou (no. Y20170204).
The funding body provided funds for the purchase of consumption materials for the study but had no role in the design of the study and collection, analysis, and interpretation of data and writing of the manuscript.
Authors' contributions SXL and RCF contributed equally to this study. SXL and RCF wrote the manuscript under supervision of TLZ, JMC. SXL, RCF, YZ, LJC, NH, KHY and CZ performed the research. SXL and RCF performed the data analysis. The research plan for this project was conceived based on several rounds of discussions among all co-authors. All authors read and approved the nal version of the manuscript.  Results of MALDI-TOF mass spectrometry of lipid A. There are 4 different types of MS pro le as shown by (A)(B)(C)(D). The black labeled ion peak is the original peak or modi ed peak not related to colistin resistance, and the red labeled ion peak is the modi ed peak related to colistin resistance.

Figure 4
Lipid A structures with corresponding m/z values found in ECC isolates.

Supplementary Files
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