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Molecular characteristics of global β-lactamase-producing Enterobacter cloacae by genomic analysis

Objective To analyze the characteristics of global β-lactamase-producing Enterobacter cloacae including the distribution of β-lactamase, sequence types (STs) as well as plasmid replicons. Methods All the genomes of the E. cloacae were downloaded from GenBank. The distribution of β-lactamase encoding genes were investigated by genome annotation after the genome quality was checked. The STs of these strains were analyzed by multi-locus sequence typing (MLST). The distribution of plasmid replicons was further explored by submitting these genomes to the genome epidemiology center. The isolation information of these strains was extracted by Per program from GenBank. Results A total of 272 out of 276 strains were found to carry β-lactamase encoding genes. Among them, 23 varieties of β-lactamase were identified, blaCMH (n = 130, 47.8%) and blaACT (n = 126, 46.3%) were the most predominant ones, 9 genotypes of carbapenem-hydrolyzing β-lactamase (CHβLs) were identified with blaVIM (n = 29, 10.7%) and blaKPC (n = 24, 8.9%) being the most dominant ones. In addition, 115 distinct STs for the 272 ß-lactamase-carrying E. cloacae and 48 different STs for 106 CHβLs-producing E. cloacae were detected. ST873 (n = 27, 9.9%) was the most common ST. Furthermore, 25 different plasmid replicons were identified, IncHI2 (n = 65, 23.9%), IncHI2A (n = 64, 23.5%) and IncFII (n = 62, 22.8%) were the most common ones. Notably, the distribution of plasmid replicons IncHI2 and IncHI2A among CHβLs-producing strains were significantly higher than theat among non-CHβLs-producing strains (p < 0.05). Conclusion Almost all the E. cloacae contained β-lactamase encoding gene. Among the global E. cloacae, blaCMH and blaACT were main blaAmpC genes. BlaTEM and blaCTX-M were the predominant ESBLs. BlaKPC, blaVIM and blaNDM were the major CHβLs. Additionally, diversely distinct STs and different replicons were identified. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-022-02667-y.


Introduction
Enterobacter (E. cloacae) belongs to facultative anaerobic Gram-negative bacilli, grouping into the E. cloacae complex group, the family Enterobacterale [1]. Generally, such bacteria colonize soil and water as well as the animal and human gut, representing one of the most leading species described in clinical infections, particularly in vulnerable patients [2]. It has been reported that E. cloacae is frequently associated with a multidrug resistance (MDR) phenotype, due to the inducible overproducing AmpC β-lactamases and acquisition of numerous genetic mobile elements containing resistance [3]. More worrisome, the production of carbapenem-hydrolyzing β-lactamase (CHβLs) rendering ineffective almost all β-lactams families have been continually acquired, resulting in the production of super-resistant bacteria carbapenem-resistant Enterobacter cloacae (CREL) [4].
β-lactamase is a predominant resistance determinant for β-lactam antibiotics in E. cloacae. To date, there are two classification schemes for β-lactamases, the more groupings in clinical laboratory generally correlate with broadly based molecular classification, where β-lactamases are divided into class A, B, C and D enzymes based on the amino acid sequence [5]. Currently, the most problematic enzymes are plasmid-mediated AmpC β-lactamases (pAmpCs) with bla ACT-like ampC genes being highly prevalent [6], extended-spectrum β-lactamases (ESBLs) with bla SHV and bla CTX-M being widely distributed [7], and CHβLs, all of which are challenging antibiotic effectiveness.
Globally, bla CHβLs such as bla KPC (class A), bla NDM/VIM/ IMP (class B) and bla OXA-48 (class D) are of grave clinical concern and proliferating [8]. It was reported that bla NDM-1 and bla NDM-5 were the main bla CHβLs , ST93, ST171 and ST145 was the predominant sequence types (STs) for CREL in a tertiary Hospital in Northeast China during 2010-2019 [9]. Whereas in Japan, bla IMP-1 was the dominant bla CHβLs conferring carbapenem resistance [10], and bla VIM was the main bla CHβLs in France between 2015-2018 [11]. However, the whole distribution of β-lactamase among global E. cloacae is unclear, and information on the clones of E. cloacae spreading internationally remains unknown. As we know that plasmids play an important role in horizontal gene transfer of antimicrobial resistance genes (ARG), and the identification of replicon types is helpful to analyze plasmid characteristics. Further, the association between plasmid replicons and different resistant determinants is essential to understand the role of plasmids in transmission of ARG [12]. For instance, IncN plasmids have been reported to be the predominant replicon types for bla IMP-4 -carrying strains [13], however, the prevalence of plasmid replicons among these bacteria were unknown. Notably, the association between IncIγ plasmid encoding bla CMY-2 ß-lactamase and the international ST19 was observed in multidrugresistant Salmonella Typhimurium [14]. Whether or not this phenomenon could be observed in E. cloacae needs to be confirmed. With the extensive use and development of antibacterial drugs, β-lactamases have evolved rapidly. Meanwhile, due to the rapid development of whole-genome sequencing (WGS) technology, the number of sequenced bacterial genomes has grown enormously, new β-lactamase variants continue to be described. As a common opportunistic pathogen [15], the information on the distribution of β-lactamase among E. cloacae was limited.
In this study, we first explored the distribution of β-lactamase including pAmpCs, bla ESBLs and bla CHβLs among E. cloacae isolates based on a global database. For β-lactamase positive strains, the sequence types (STs) and the distribution of plasmid replicons were further investigated. Furthermore, the prevalent characteristics of β-lactamase-producing E. cloacae were analyzed.

Acquisition of E. cloacae genomes and strain information
A total of 296 E. cloacae genomes were downloaded in batches from NCBI using Aspera software on 16 th , Dec 2021 [16]. The genomic quality of these 296 strains was further filtered by Checkm and Quast software [17,18]. The high-quality genome was defined as "completeness > 90% and containment < 5%". Meanwhile, the quantity of contigs is required to be " ≤ 500, and N50 ≥ 40,000". Twenty genomes that did not meet the above conditions were filtered out. The investigated strains were collected from different years shown in Figure S1A, the collected dates of 58 strains were "blank" meaning that the information was missing. These strains were submitted by 32 countries, mainly from USA (n = 58), France (n = 30), United Kingdom (n = 27), China (n = 24), Japan (n = 18), Singapore (n = 13) and Nigeria (n = 12), other countries were also involved ( Figure S1B). The countries of 26 strains remained unknown. Notably, 158 out of 272 strains were hosted by Homo sapiens (n = 158, 58.1%), mainly from gastrointestinal tract (n = 57, 21.0%).

Investigation of β-lactamase among global E. cloacae
To avoid differences in genome gene prediction by different annotation methods. All the 276 genomes were annotated by Prokka software [19], which is a fast prokaryotic genome annotation software. All the strains containing β-lactamase encoding genes were further analyzed.

Analysis on the sequence type of β-lactamase carrying E. cloacae
The self-made Perl program was used to extract the nucleotide coding sequence of genes from each genome sequence file (GBK format) [20]. The allele sequences and allelic profiles of 7 conserved genes of E. cloacae were downloaded from website https:// pubml st. org/. The sequence of the genome was set as "query", the seven conserved gene sequence files were set as "subject "(database). Blastn alignment analysis was then implemented between query and subject. The thresholds set were as follows: E-value = 1e-5, identity = 100%, matching length = subject gene length.

Statistical analysis
The differences on the distribution of major resistant determinants and plasmid replicons among bla CHβLscarrying strains and strains without bla CHβLs was analyzed by Chi-square test. Distribution difference on resistant determinants and plasmid replicons among all the β-lactamase-producing and among the bla CHβLscarrying strains were checked by McNemar test. The distribution rates were statistically different when p value was less than 0.05.
Notably, the prevalence of replicons IncHI2 and IncHI2A among bla CHβLs -carrying strains were significantly higher than that among the strains without bla CHβLs (p < 0.05), whereas no significant difference on the prevalence of plasmid replicons IncCOI, IncFII, IncFIB and IncR among these two groups were observed ( Table 2).
The distribution of bla SHV was consistent with plasmid replicon IncR, and prevalence of bla CTX-M was in accordance with the prevalence of IncFII, IncFIB and IncHI2A (p > 0.05). Additionally, the prevalence of oxacillin-hydrolyzing-bla OXA and IncFIB as well as IncCOI was accordant (Table 3). Moreover, the prevalence of bla KPC and bla VIM were consistent with the distribution of IncCOI, IncFII, IncFIB, IncHI2 and IncHI2A, and no differences  were observed on the distribution of bla IMI , bla NDM and those of IncCOI, IncFII and IncFIB (p > 0.05) ( Table 4).

Discussion
β-lactamase is a primary resistance determinant, widely disseminating on mobile genetic elements across the opportunistic pathogens including E. cloacae [21]. Exploring the spread characteristics of β-lactamase among E. cloacae based on the global genome database of GenBank is quite important for illustrating the resistance characteristics of such strains and guiding rational drug use in clinic. Our analysis showed that the number of E. cloacae has been continuously increasing since the genome of first one was submitted in 2003. More than 32 countries all over the world submitted the genomes, indicating the representativeness of these strains. To note, the host of these β-carrying E. cloacae strains were predominantly Homo sapiens, with the gastrointestinal tract being the major isolation resource, suggesting that Homo sapiens were the dominant host and gastrointestinal tract was predilection seat. Importantly, 106 bla CHβLs -carrying E. cloacae strains isolated during 2010-2020 were scattered among global 27 countries and 5 continents, indicating a rapid emergence and wide distribution of such strain, which alerts us the urgency of implementation of prevention and control measures.
Our analysis showed that bla CMH was the most frequent β-lactamase gene. However, literature search with bla CMH as the key word showed that bla CMH-1 was first detected in E. cloacae as a novel bla AmpC gene at a Medical Center in Southern Taiwan [22]. Since then, bla CMH-2 and bla CMH-3 were sequentially identified in India and Europe [22,23]. Thereafter, no bla CMH was reported in PubMed database albeit genomic analysis showed the widest distribution of these enzyme. To our surprise, the most prevalent bla CMH-6 , bla CMH-4 , bla CMH-5 and bla CMH-3 were not identified among E. cloacae at all. Moreover, bla ORN-1 , Fig. 4 The sequence types of 272 β-lactamase-producing Enterobacter cloacae identified in the chromosome of Raoultella. ornithinolytica in 2004 [24,25], has never been reported in E. cloacae. Interestingly, bla CARB-2 as a carbenicillin-hydrolyzing enzyme, has been identified within multiple strains including Klebsiella. pneumonia [26], Achromobacter xylosoxidan [27], Escherichia. coli [28], Acinetobacter pittii [29] and E. cloacae [30] in a variety of countries, however, was quite rare in our study. Which may be related Fig. 5 The sequence types of 106 carbapenem-hydrolyzing β-lactamase-producing Enterobacter cloacae Fig. 6 The sequence types of dominant carbapenem-hydrolyzing β-lactamase-producing Enterobacter cloacae. 6A, the sequence types (STs) of bla VIM -carrying strains; 6B, The STs of bla KPC -carrying strains; 6C, STs of bla NDM -carrying strains; 6D, STs of bla IMI -carrying strains; 6E, STs of bla IMP -carrying strains; 6F, STs of bla OXA -carrying strains to its clinical importance. bla LAP-2 as a narrow-spectrum β-lactamase was also rare in our study, albeit it has been reported [31] [32]. Furthermore, bla SCO-1 was a novel plasmid-mediated class A β-lactamase with carbenicillinase characteristics in E. coli [33], has not been reported in E. cloacae until now. As we know that bla ACT was also a plasmid-encoded ampC gene [34]. Although the prevalence of bla ACT was secondary to bla CMH in our study, distribution of exact bla ACT -variants was not so high. Note worthily, the most common bla ACT-59 in our study has never been reported. Which may be due to the limitation of screening methods. It was reported that bla VEB-3 was encoded by the chromosome and located in an integron, and only 2 bla VEB-2 genes were detected in our study.
However, outbreak of infection caused by bla VEB-3 -carrying-E. cloacae has been reported in China [35], Additionally, bla KPC , bla VIM , bla NDM , bla IMI and bla IMP were the major bla CHβLs accounting for carbapenem resistance in global E. cloacae. Among them, bla KPC-2 and bla VIM-4 were the most predominant ones. Which is a light different from previous report showing bla  and bla IMP-8 was the main bla CHβLs within E. cloacae in China [36]. Noteworthily, 28 bla VIM-4 -carrying E. cloacae ST873 were only found in Homo sapiens in France, indicating that there was a clonal dissemination of such strain among Homo sapiens in France during 2010-2020, which was not reported previously, albeit nosocomial infections caused by E. cloacae ST873 in 2 hospitals in France has been reported [37]. As a novel bla CHβLs , bla FLC-1 belongs to Ambler class A β-lactamases, has been identified an E. cloacae Complex isolated from food products [38]. Interestingly, such enzyme displayed a distinctive substrate profile, hydrolyzing penicillin, narrow-and broad-spectrum cephalosporins, aztreonam, and carbapenems but not extended-spectrum cephalosporin. In addition, bla NMC-A , a class A bla CHβL , has been frequently detected in E. cloacae [39,40] [41,42], albeit we just found 2 bla NMC-A in this study. As bla CHβLs , bla GES-24 seems to have a broader host than bla GES-2 although we only found 2 bla GES-2 and 1 bla GES-24 in this study. To date, all reports on bla IMI-1 focus on E. cloacae, indicating that E. cloacae may be the best host for bla IMI .  Table 3 The differences on the distribution of plasmid replicons and resistant determinants among the β-lactamase producing Enterobacter cloacae The higher prevalence of bla TEM and bla SHV among bla CHβLs -carriers in our study was in accordance with a previous report to some degree, which showed that bla CTX-M , bla TEM and bla SHV were mostly detected concurrently with bla CHβLs [43]. Albeit no distribution difference of bla CTX-M was observed. Notably, the significantly higher distribution of bla CMH among non-CHβLs-producers may indicate that bla CMH may be the predominant gene conferring β-lactams among the strains without bla CHβLs.
Furthermore, the multiple STs identified in our study displayed a genetic diversity of β-lactamase producing E. cloacae. It seemed that clonal dissemination for such strain was rare except for bla VIM-4 -carrying ST873 ones, suggesting that the spread of CREL was mainly mediated by mobile elements such as plasmids.
Additionally, variously distinct plasmid replicons detected in our study indicate their dissemination potential for resistant determinants. Noteworthily, the obviously higher prevalence of IncHI2 and IncHI2A among bla CHβLs -carrying strains may suggest association between bla CHβLs and IncHI2. It was reported that IncHI2 widely detected in global CRE genomes, was termed as 'super-plasmids' resulting from the large size and prolific carriage of resistance determinants [44]. And the consistent distribution of such plasmid and bla KPC and bla VIM may indicate a good cost fitness between them.
There were several limitations in this study. First, the number of E. cloacae was relatively small which may result from the reason that E. cloacae was only little part of E. cloacae complex. Second, the resistance profiles of these strains were not available for us to compare the difference between the genotypes and phenotypes. Some of the strain information were missing, which was not beneficial for us to fully illustrate the characterization of E. cloacae. Third, some new enzymes are devoid of further phenotypic descriptions because they were directly obtained from whole-genome sequencing studies. Anyway, it is currently difficult to draw an accurate global picture of this bacteria, highlighting the need for more comprehensive genome sequence data and genomic analysis.
In summary, almost all the E. cloacae contained β-lactamase encoding gene. Among the global E. cloacae, bla CMH and bla ACT were main bla AmpC genes. Bla TEM and bla CTX-M were the predominant ESBLs. Bla KPC , bla VIM and bla NDM were the major CHβLs. Additionally, diversely distinct STs and different replicons were identified.