Emergence of mobilized colistin resistance-1 in multidrug-resistant Klebsiella pneumoniae and Escherichia coli isolates from the Henan province in China: a multicentre study

Background The increased clinical use of polymyxin led to the emergence of polymyxin-resistant strains, especially those carrying plasmid-borne mobilized colistin resistance (mcr) gene variants. In this study, we aimed to evaluate the prevalence and characteristics of polymyxin-resistant Klebsiella pneumoniae and Escherichia coli isolates from the Henan province, China. A total of 16 polymyxin-resistant isolates among 2301 E. coli and K. pneumoniae isolates collected in 6 local hospitals in the Henan province were studied. The isolates were identied using matrix-assisted laser desorption/ionization time-of-ight mass spectrometry, and the minimum inhibitory concentrations (MICs) were determined using the microbroth dilution technique. Polymyxin-resistant isolates were further analysed for mcr-1 and carbapenemase-mediated resistance using the modied carbapenem inactivation method, the ethylenediaminetetraacetic acid-modied carbapenem inactivation method, and polymerase chain reaction. Pulsed-eld gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed to disclose the phylogenetic relationships of the polymyxin-resistant isolates. The clinical characteristics of patients infected with the polymyxin-resistant isolates were also retrospectively analysed.


Conclusion
The polymyxin resistance rate of K. pneumoniae was slightly higher than that of E. coli in the Henan province; however, mcr-1 was only detected in one K. pneumoniae isolate. Thus, close monitoring is needed to prevent and control the spread of PRCRKP.

Background
Antibiotic resistance has become a global public health priority in recent years. Colistin, also known as polymyxin, is one of the few therapeutic options available for the treatment of infectious diseases caused by multidrug-resistant gram-negative bacteria [1]. In China, injectable polymyxin was approved for the treatment of bacterial infections in January 2017. However, because of the increased usage of polymyxin in clinical settings, polymyxin-resistant strains, especially those carrying plasmid-borne mobilized colistin resistance (mcr) gene variants have emerged in China and various countries worldwide [2]. Moreover, the intraspecies transmission of resistant isolates has already been reported [1,3].
Since its discovery in southern China in late 2015 [4], mcr-1 has spread to over 40 countries and regions, implying that it plays a prevalent role in the transferability of polymyxin resistance. Of note, mcr-1-positive strains have also emerged in the Henan province, including in pigderived Escherichia coli isolates [5]. In fact, clinical E. coli isolates co-producing bla NDM and mcr-1 were previously reported by our laboratory [6], and a novel conjugative mcr-8.2-bearing plasmid was identi ed in an almost pan-resistant hypermucoviscous Klebsiella pneumoniae ST11 isolate [7]. However, overall, the reports of mcr in human-derived E. coli and K. pneumoniae isolates are mainly centred outside of Henan.
To better understand the epidemiological trends and characteristics of polymyxin-resistant clinical strains, here, we looked for polymyxin resistance among isolates collected at 6 hospitals in Henan from 2018 to 2019. A total of 16 polymyxin-resistant strains were collected, and their molecular resistance characteristics were analysed. To the best of our knowledge, this is the rst multi-centre study screening for polymyxin-resistant isolates among E. coli and K. pneumoniae in the Henan province, China.

Sample collection
Non-duplicated E. coli and K. pneumoniae strains were obtained from routine microbiological cultures of clinical samples including blood, urine, sputum, bronchoalveolar lavage uid (BAL), bile, hydrothorax, ascites, and various other specimens. A total of 2301 strains were isolated from 6 hospitals in Henan. Identi cation at the species level was performed by matrix-assisted laser desorption/ionization time-ofight mass spectrometry (MALDI-TOF MS; Bruker Daltonik GmbH, Bremen, Germany).
Susceptibility testing and determination of the minimum inhibitory concentrations (MICs) Susceptibility to polymyxin was screened using Etest strips (Antu, Zhengzhou, China); only isolates with MICs higher than 2 µg/mL were subjected to further susceptibility testing for validation using the microbroth dilution method based on the clinical breakpoints de ned by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2018) [11]. (CLSI) guidelines [12].

Characterization of mcr-1and carbapenemase mediated resistance
The modi ed carbapenem inactivation method (mCIM) and ethylenediaminetetraacetic acid-modi ed carbapenem inactivation method (eCIM), which are recommended by the CLSI, were used for the phenotypic detection of carbapenemase production. The presence of carbapenem resistance genes (bla VIM , bla IMP , bla KPC , bla NDM , and bla oxa-48-like ) and of the polymyxin resistance gene mcr-1 in polymyxinresistant isolates were screened by polymerase chain reaction using the methods described previously [13,14].

Pulsed-eld gel electrophoresis (PFGE)
Molecular epidemiology of all polymyxin-resistant strains was determined by PFGE after total chromosomal DNA digestion with XbaI in accordance with a previous report [15]. The PFGE patterns were analysed using the BIONUMERICS software (Applied Maths NV, Sint-Martens-Latem, Belgium) using the Dice similarity coe cient. Isolates were considered as the same strain (PFGE type) if they possessed a genetic similarity of ≥ 95%.

Results
Overall prevalence of polymyxin-resistant strains Over the course of the study, 16 out of the 2301 E. coli and K. pneumoniae isolates (0.7%) were found to be polymyxin-resistant: 5 E. coli and 11 K. pneumoniae isolates, collected from 6 different hospitals. The prevalence of polymyxin resistance in E. coli and K. pneumoniae was 0.3% and 1.4%, respectively (Table 1).

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing showed that all of the isolates (100%) were resistant to AMP, KZ, and CTX; 93.3% were resistant to LEV; 86.6% were resistant to CAZ, FEP, and ATM; 80% were resistant to SAM and TZP; 66% were resistant to GN and AK; 62.5% were resistant to IPM and MEM; and 60% were resistant to SXT. Only one isolate was resistant to CZA (6.3%), and all of them were susceptible to TGC (100%) ( Figure 1).
The MICs of polymyxin in the context of these 16 strains ranged from 4-64 μg/mL: the full range in the context K. pneumoniae isolates (median: 64 μg/mL) and 4 μg/mL in the context of E. coli isolates ( Table 2).

Detection of antimicrobial resistance genes
Among the 16 polymyxin-resistant isolates, 5 carried the mcr-1 gene, including 1 K. pneumoniae and 4 E. coli isolates. In addition, 9 K. pneumoniae and 1 E. coli isolates were carbapenemase-positive. The mCIM and eCIM results showed that the 9 K. pneumoniae isolates were serine carbapenemase-positive, and the E. coli isolate was metallo-carbapenemase-positive. Furthermore, the PCR results showed that the 9 K. pneumoniae isolates were bla KPC-2 -positive, but none of them carried mcr-1. On the other hand, the E. coli isolate was both bla NDM-5 -and mcr-1-positive. Of note, no other carbapenemase genes, such as bla IMP , bla VIM , and bla OXA48-like , were detected (Table 2).

Epidemiological characterization
MLST analysis showed that the nine K. pneumoniae carbapenem-resistant isolates all belonged to sequence type (ST) 11. On the other hand, among the ve E. coli isolates, two belonged to ST132 and the other three belonged to ST2, ST983, and ST632 (Table 2).
Of note, dendrogram analysis of PFGE at 95% similarity revealed that homology among the 5 E. coli and 11 K. pneumoniae isolates was low and sporadic, suggesting a very low likelihood of clonal spread (Figure 2).

Discussion
Polymyxin has been used against aggressive infections caused by multidrug-resistant bacteria; however, its use has been severely compromised by the emergence of plasmid-mediated polymyxin resistance in Enterobacteriaceae. Hence, in this study, we surveyed the polymyxin resistance rates in E. coli and K. pneumoniae isolates from hospitalized patients at six local hospitals in the Henan province, China.
Among the total 2301 E. coli and K. pneumoniae isolates, 16 (0.7%) strains were polymyxin-resistant, 5 of which carried the mcr-1 gene. Of note, of the 1499 E. coli isolates, 5 (0.3%) were polymyxin-resistant and 4 were mcr-1-positive; on the other hand, of the 802 K. pneumoniae isolates, 11 (1.4%) were polymyxin-resistant, 1 of which carried mcr-1. Previously, 0.88% (34/3854) of the E. coli isolates and 0.21% (5/2410) of the K. pneumoniae isolates carrying mcr-1 were reported in the China Antimicrobial Resistance Surveillance Trial [16]. Additionally, another study found that 1% (20/1495) of the E. coli isolates and 0.18% (1/571) of the K. pneumoniae isolates recovered from bloodstream infections in China were mcr-1-positive [17]. Our results are, therefore, in line with the previous ones, with rates not exceeding 1.5%. Although mcr-1 was more common in E. coli isolates than in K. pneumoniae isolates, the polymyxin resistance rate of K. pneumoniae was slightly higher than that of E. coli in our study, which is presumably due to antibiotic selection because the detection rate (32.8%) of carbapenem-resistant K. pneumoniae (PRCRKP) in Henan ranked rst among all Chinese provinces in 2019 (http://www.carss.cn/Report/Details?aId=770). Of note, compared to polymyxin-resistant E. coli, polymyxin-resistant K. pneumoniae were associated with 8-64 times higher MICs, suggesting chromosomal mutations in related genes, such as phoP/phoQ, pmrA/pmrB, and mgrB. Additionally, other intrinsic mechanisms might also play important roles in increasing polymyxin resistance in K. pneumoniae [17].
Two carbapenemase genes, bla KPC and bla NDM , are responsible for the phenotypic resistance of 90% of carbapenem-resistant Enterobacteriaceae strains in China [18]. The co-existence of mcr and carbapenemase genes, such as bla NDM-5 [19], bla NDM-4 [20], bla KPC [21], and bla OXA [22], has been sporadically reported in different countries. In the national monitoring data from China, one report showed that the mcr-1 gene was detected in 4.6% (13/282) of carbapenem-resistant E. coli isolates and coexisted with the New Delhi metallo-enzyme (NDM)-5 in one strain [23]. In another study, the mcr-1 prevalence among carbapenem-resistant E. coli and PRCRKP isolates was 3.7% (14/376) and 0% (0/1134), respectively, and 14 carbapenem-resistant E. coli isolates coproduced bla NDM4/5/9 with mcr-1 [24]. In this study, only one E. coli isolate coproduced mcr-1 and bla NDM-5 . An E. coli isolate belonging to ST167 that co-expressed bla NDM and mcr-1 was previously reported in Henan [6,25], but in our study, the aforementioned coproducing E. coli isolate belonged to ST2. The other E. coli strains in this study belonged to ST132, ST983, and ST632. Additionally, nine PRCRKP isolates belonged to ST11, but PFGE showed different types, suggesting these strains, all isolated from the same hospital, were unrelated. Altogether, our results demonstrate that polymyxin-resistant isolates are non-clonal and have different virulence and resistance potentials.
The patients carrying polymyxin-resistant isolates had varying severities of illness, and 33.3% of them had a history of polymyxin use.
Moreover, 66.7% of them were cured, and these positive outcomes could be explained by the nding that most polymyxin-resistant isolates remained susceptible to other antimicrobials, such as CZA, SXT, and TGC.
In conclusion, here, we show that polymyxin resistance rate of K. pneumoniae is slightly higher than that of E. coli, while the presence of mcr-1 is lower in polymyxin-resistant K. pneumoniaeversus E. coli in the Henan province, China. Further molecular investigations and studies are warranted to elucidate the polymyxin resistance mechanism of PRCRKP. In addition, continuous and close monitoring is required to prevent the dissemination of polymyxin-resistant K. pneumoniae and E. coli strains.

Consent for publication
No personally identi able information was collected in this study.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.    Table 3 Clinical characteristics of the patients carrying polymyxin-resistant isolates