Local characteristics of molecular epidemiolgy of Acinetobacter baumannii in Jilin province (northeast China)

A. Baumannii is an opportunistic nosocomial pathogen which has severe antibiotic resistance. However, the epidemiology is less clearly understood in Jilin province and China. Thus, 89 A. baumannii isolates from a single hospital in Jilin province between 2013–2017 were performed by MLST. In order to better understanding of the epidemiology of Jilin isolates, Chinese strains originated from other domestic regions and worldwide isolates in MLST database were analyzed by silico phylogenetic tools together. A total of 22 STs in Jilin were identified, and 10 STs were found to be novel. The top three predominant sequence types are ST195 (n = 34, 38.2%), ST208 (n = 14, 15.7%) and ST540 (n = 13, 14.6%). ST369 is predicted to be group founder and ST195, ST540 are subgroup founders of the majority STs in Jilin Province. Some newly discovered singletons showed close relationship with strains from other countries, which suggest that nation-cross transmission is one of important origin of Jilin strains. The majority of Jilin STs showed clonality and close relationship with the majorities from other regions of China. But occupation of individual STs in Jilin were different from that of other domestic regions. The aggregation trend and genetic relationship proved that predominant Jilin STs continue to mutate during transmission. Drug resistance facilitated transmission of Jilin A.baumannii isolates because more than 94% of isolates are resistant to at least one carbapenem and the STs with strong resistance to carbapenems usually has more isolates. In conclusion, high diversity and different occupation of STs, and occupation of novel STs proved that epidemiology of A. baumannii in Jilin has special regional characteristics, and drug resistance facilitated transmission of domestic strains and foreign strains.


Introduction
The gram-negative bacterium A. baumannii is an opportunistic nosocomial pathogen inducing a vast array of infections, of which the most common infections are ventilator-associated pneumonia and bloodstream infections with high mortality rate [1]. Multidrug resistance (MDR) limits therapeutic options and impacts on clinical care. Although carbapenems were considered the mainstay against MDR A. baumannii infections, carbapenem resistant A. baumannii has been prioritized as the top one of WHO priority pathogens list that are urgent to research and develop new antibiotics [2,3]. However, infection prevention and control are most effective when applied at the earliest possible stage. Molecular epidemiology is an indispensable tool to early monitor the connection of pathogen clusters with infections, which is also helpful to identify the origin of causative organisms and investigate healthcare-associated infections [4].
MLST is a proven tool for the molecular typing of A. baumannii, MLST helps not only in tracking the sequence types (STs) and outbreaks, but also in understanding the microbial evolution [5]. The relevant cluster analysis based on STs also presents several advantages, such as the ease of interpretation and the creation of hierarchical grouping of the isolates. Moreover, it provides a global overview of the relatedness of the isolates and how the defined clusters are connected to each other [6].
In China, carbapenems resistant A. baumannii is also on the rise, which has become a serious threat to public health [7]. Some papers on MLST showed that clonal complex 92 (CC92) is the predominant group with the widest distribution in Chinese mainland [8][9][10]. However, to our knowledge, the strains in these studies were mainly isolated from other regions of China.
The epidemiology of A. baumannii is less clearly understood in Jilin province as only six Jilin strains with same ST were registered in MLST database before our study. Therefore, STs of 89 A. baumannii isolates from a single hospital in Jilin between 2013-2017 were analyzed by MLST and other silico phylogenetic tools. For the sake of analyzing regional characteristics, previously registered Chinese isolates and large amount of global collection of A. baumannii from MLST database (https:// pubml st. org/ organ isms/ acine tobac ter-bauma nnii) were also covered in this study.

Results
Among obtained STs, the number of alleles in each locus varied from 5 to 14 and that of SNPs ranged from 5 to 63. The sequence diversity of each locus ranged from 2.98 to 6.40% with average value of 3.83%. Furthermore, nucleotide diversity (π) in this study was in the range of 0.00413 to 0.03514 (Table 1).

Antimicrobial susceptibility
The result of antimicrobial susceptibility is shown in Table 2. Totally, the resistant portions of Jilin isolates to ertapenem, imipenem, meropenem, tigecycline, and colistin B are 91.01% (n = 81), 87.64% (n = 78), 89.89% (n = 80), 7.86% (n = 7), and 13.48% (n = 12), respectively. Twelve isolates were found to be sensitive to at least one of tested carbapenems. The overwhelming majority in our Jilin isolates (77/89) were resistant to all tested carbapenems, and 75 of them were at least sensitive to one or both of tigecycline and colistin B. But one isolate was resistant to all tested antibiotics including tigecycline and colistin B.

Genetic relatedness
ST195, ST208, ST218, ST368, ST369, and ST540, which are predominant STs in Jilin could be classified into CC92 group. However, other main STs in Jilin including ST1779, ST1926, and ST2370, could not be classified into CC92 group because of lacking sufficient alleles of the loci in housekeeping genes. It must be pointed out that although ST1779 (n = 6) is not a novel ST, it has never been founded in MLST database before. The goeBURST analysis within the scope of Jilin isolates showed that 78 our isolates in nine STs and six previous isolates in ST75 could be classified into a big group with ST369 as group founder and ST195, ST540 as subgroup founders ( Fig. 2A). And among 10 novel STs, 8 newly identified STs are singletons without any association with other Jilin isolates ( Fig. 2A). The details of locus variation in goeBURST algorithm were displayed in Table 3.
In the nationwide analysis, ST369 was also a group founder with ST136, ST195, ST208, ST350 and ST540 as subgroup founders (Fig. 2B). The locations and distribution of Jilin STs in nationwide unrooted tree were like that in Jilin tree, but there was no nationwide subgroup founders ST136, ST350 and their downstream branches in Jilin tree. Subgroup founder ST208 had several variant STs, which located in three downstream branches. But in Jilin, as a predominant ST (14/89), ST208 did not occupy any variant and branches. This phenomenon suggested that ST208 in Jilin possibly was not a local isolate. It came from other domestic regions and induced an outbreak in our sample-taking hospital. Same to ST469, which was identified as a singleton in Jilin, but in nationwide, ST469 was variant of ST350, which was not detected in Jilin isolates (Fig. 2).
In worldwide analysis (Fig. 3), the majority of Jilin STs are classified in Group 1 along with predominant STs from Australia, France, Germany, Iraq, Italy, Japan,
If the analysis was performed worldwide, all STs were divided into six groups as shown in Fig. 5. All Chinese A. baumannii predominant STs belonged to Group F (Fig. 6).

Discussion
In this study, all isolates came from single hospital, the overall diversity (STs/isolate) was about 0.247 (22/89) was not high, but the proportion of newly discoved ST and their origin were over our expectation. Ten novel STs were identified in 89 isolates, it could be said that the results of this study contributed newer STs to the MLST database. And many of newly discovered STs just with 1 isolate were regarded as singleton lacking relationship with previous discovered Chinese STs, but had closer relationship with foreign STs, which suggested that they STs are obtained in this study, * refers to novel ST that is found in this study). And Group B (Chinese Major ST): genetic relationship of the majority A. baumannii isolates of China using goeBURST. Yellow node refers to group founder, red node refers to subgroup founder, light blue refers to common node are maybe the variant of foreign strains. The new immigrant with strong resistance, such as ST2933, should be paid more attention to continuously monitor and control its epidemiology because it will get more opportunities to spread and are more likely to become dominant strains in the future.
But isolates with domestic origin, such as ST208, which could be considered as a model to evaluate nosocomial infection of A. baumannii in this hospital. As a nation level major ST, ST208 had many variants and evolution branches (Fig. 2B), but in Jilin it had high occupation (14/89) without any variants and evolution branches ( Fig. 2A), which suggested that it spread to this hospital and caused a nosocomial outbreak within short period. So, the samples being taken from single hospital was still helpful to epidemiological research, prevent and control A. baumannii infection. And the causative pathogen could be identified quickly, and infection prevention and control measures could be applied earlier if daily routine molecular typing is implemented [4].
The majority (81.1%) of Jilin STs belonged to CC92 group, which is the largest and most widely distributed A. baumannii clone in China [8]. According to the published articles about A. baumannii research in China [8,[11][12][13][14][15][16][17], epidemic STs in Jilin appeared earlier than non-epidemic STs in Jilin. But in Jilin isolates, some major Chinese STs in CC92 group were not founded, such as ST191 (Beijing and Hunan) [18,19] and ST1145 (Guangdong) [20]. And the epidemic ST92, which was predicted to be the group founder of CC92 group, was never found in Jilin province too.
Multidrug resistance strains (MDR) of A. baumannii have increased because of the acquirement of mobile genetic elements such as transposons, plasmids, and integrons. Especially, carbapenems resistant A. baumannii (CR-Ab) is a worldwide problem because these strains are often resistant to all other commonly used antibiotics [21]. In the hospital, the antibiotic susceptibility of all Jilin isolates was routinely tested by K-B method. The tested antibiotics include amikacin, gentamicin, imipenem, meropenem, cefazolin, ceftazidime, cefotaxime, cefepime, aztreonam, ampicillin, piperacillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, piperacillin/ tazobactam, polymyxin, compound sulfonamide, chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Almost all isolates are resistant to cephalosporins,   . 3 The evolutionary history inferred using the UPGMA method. The analysis involved 22 STs of this study from Jilin province which are shaded in red, another 10 predominant STs of China and selected 120 predominant STs from other countries. Jilin STs with * were newly discovered STs. Excepting ST2370, 2396 and 2398, that were distributed in the first group together with the predominant STs in China, the other newly discovered Jilin STs were distributed in other groups, which showed that these STs had closer relationship with foreign isolates penicillins, gentamicin, and ciprofloxacin. As carbapenem is used as last line antibiotic treatment to eliminate infections with multidrug-resistant Gram-negative bacteria [21], we tested the carbapenems resistance in the lab again. At same time, resistance against tigecycline and colistin B were also tested again because they usually are members of final choices in China. The STs with earlier appearance and the stronger resistance, had more isolates, such as ST195 and ST540. Four isolates sensitive to all tested carbapenems distributed in ST373, ST2397,  ST2399, and ST2402. STs, including ST373, ST2397, ST2399 and ST2402, which just occupied one isolates that sensitive to all tested carbapenems. Although the single isolates of ST373 appeared early in 2013, it was not found in following years because the sensitivity to carbapenems made it lost the chance of spreading. However, for single isolates of ST2397, ST2399 and ST2402, as singletons they appeared in 2017 and did not have links with local isolates. They were sporadic nation-cross spreading isolates because the result of evolutionary history by UPGMA method (Fig. 3) also showed they have close relationship with STs of other countries. The sensitivity to carbapenem also made them lost continuous dissemination. So, the reason for the small number of sensitive STs was gradually eliminated by carbapenems, while the reason for newly discovered STs was that new variant or new immigrant have not yet had time to reproduce. Beside Jilin province, the patients also came from other domestic regions and neighboring countries. All strains were identified by Matrix Assisted Laser Desorption Ionization (MALDI)-Time of Light (TOF)-Mass Spectrometer (MS) and 16SrRNA encoding sequence analysis. However, there are still some strains that cannot be identified as A. baumannii or A. colcaoaceticus, which makes it impossible to include such strains in our study.
Although the results of this study showed the unique molecular epidemiological characteristics of A. baumannii in Jilin Province, a single origin of isolates and a low number of isolates are also the shortcomings of this study. It will be more meaningful to obtain more isolates from more hospitals for similar study in the future.

Conclusions
Overall, the antibiotics resistance of A. baumannii is very serious in Jilin Province, the proportion of bacteria resistant to at least one carbapenems antibiotic has exceeded 94% (84/89). Antibiotic resistance is a helpful factor for predominant STs spreading. The proportion of predominant STs in Jilin are different from some domestic regions, such as Guizhou, Beijing and Hunan, and suspicious nation cross new STs occupied high proportion in Jilin. Epidemiological research by MLST in single hospital is helpful to monitor and control nosocaomial infection of A. baumannii because newly discovered STs, genetic relatedness and geographical distribution reflected sufficient spreading clues of A. baumannii.

Ethics statement
All bacteria are routinely collected for diagnosis in the Affiliated Hospital of Changchun University of Chinese Medicine. No extra samples and personal information about patients were used in the study. Therefore, a written personal informed consent and ethics committee approval were not required, and the study was in line with Chinese laws.

Antimicrobial susceptibility tests
All collected A. baumannii isolates were subjected to antimicrobial susceptibility test against ertapenem, imipenem, meropenem, tigecycline, and colistin B according broth dilution method following M100 Performance standards for antimicrobial susceptibility testing thirtieth edition of the Clinical and Laboratory Standards Institute (CLSI) [22]. between STs and the antimicrobial susceptibility among the study population was carried out using Fisher's precision probability test. And all antibiotics were the products of Sigma.

Phylogenetic analysis
The relatedness analysis was performed by online goeBURST and eBURST under the criteria of single locus variant (SLV) as both goeBURST and eBURST allow unrooted tree-based relationship representation of the analyzed isolates ( http:// eburst. mlst. net/) [24,25]. The criteria ensures that even single locus variation of housekeeping genes will be displayed in unrooted tree. Factors such as the number of alleles, variable sites in each allele, frequency of single nucleotide polymorphism (SNPs) were also considered in the algorithm of two online methods. As the distance between original genotype and variant usually suggests the degree of variation, the distance from triple locus variants (TLVs) to original genotype is longer than that from SLVs and double locus variants (DLVs) to original genotype. Nucleotide sequence diversity (π) was calculated using DNAsp V5.1 software [26]. All Chinese STs in including Jilin isolates were compared to isolates from other countries by PHYLOViZ Online of MLST site (http:// pubml st. org/ bpseu domal lei), which is a flexible tool for relationship and regional characteristic analysis [27].
To further determine the molecular evolutionary relationship between Chinese STs and prevalent STs in other countries, 22 Jilin STs, another 10 Chinese epidemic STs (isolates ≥ 8), and 120 epidemic STs (isolates ≥ 2) from other countries were selected and analyzed by Unweighted Pair Group Method (UPGM) of PHYLOViZ program [28,29]. In other article, this analysis also was performed by Arithmetic average method in molecular evolutionary genetic analysis (MEGA) software [30,31]. The topology and grouping of all STs were displayed on constructed boot-strapped phylogenetic tree.