A New OXA-66-harboring Acinetobacter Baumannii Sub-Clonal Complex 195 is Spreading Widely in Guangzhou, China, A Multicenter Study

Objectives The clonal spread of Acinetobacter baumannii (A. baumannii) is an emerging problem. We analyzed the molecular epidemiology of A. baumannii isolated from 5 teaching hospitals in Guangzhou, China. Methods 138 A. baumannii isolates were collected. Multilocus sequence typing (MLST) was used to assess the genetic relationships among the isolates. The bla OXA-51-like gene was amplied and then sequenced. Results Most of the isolates (55.8%, 77/138) were obtained from intensive care units (ICUs). The respiratory system was the most common site where A. baumannii was found (72.5%, 100/138). A. baumannii remained susceptible to polymyxin and tigecycline, but the susceptibility to other antimicrobial agents was below 30%. We used MLST to group the A. baumannii isolates into 8 existing sequence types (STs) and 17 new STs. With the predicted founder ST195 (accounted for 35.5% of all isolates, 49/138), Clonal complex (CC) 195 was the most prevalent and widely spread STs in the hospital environment. All ST195 isolates harbored OXA-66 according to the bla OXA-51-like gene sequencing. But each hospital had its unique epidemiological feature. Conclusions OXA-66 gene harboring CC195 was the most epidemic STs in Guangzhou, China. Health care facilities should develop their own management strategy.


Introduction
The clonal spread of multiple drug-resistant A. baumannii is an emerging problem worldwide [1][2][3][4]. A. baumannii is remarkable in its ability to acquire antibiotic resistance. It is also recognized as one of the most di cult health care-associated infections to control and treat [1,5,6]. Epidemiological studies and monitoring of the spread of A. baumannii may improve infection prevention and control. Previously, studies [7,8]revealed CC92 represented the most epidemic sequence types (STs) in China. ST92 is the founder of CC92 and the predominant ST, while other STs belonging to CC92 vary by area. ST75 may be the most common epidemic ST in eastern China [9], whereas ST138 may be the most common ST in western China [10]. However, our study showed ST195 may be the major ST in Guangzhou and we did not detect any ST92 [11]. Another study also showed that ST195 and ST365 were the predominant STs from two hospitals in Guangzhou, China [12]. It seems ST195 but not ST92 may be more common in Guangzhou, China. However, these studies are limited by either single-center study or small sample size. Additionally, some studies have shown that sequence-based typing (SBT) of bla OXA−51−like gene variants could be used to assess the epidemiological characterization of A. baumannii [13][14][15], but more data are needed. Therefore, a multicenter study is needed to better evaluate the issue. Here, we investigated 138 A. baumannii isolates from 5 teaching hospitals to determine the epidemic STs of the isolates in Guangzhou, the largest city of southern China. Road, Guangzhou, China). Among the 138 A. baumannii isolates, 52 isolates had been reported in our previous study [5]. All A. baumannii isolates were obtained from clinical samples, such as bronchoalveolar lavage uid, blood, sputum, cerebrospinal uid, and urine. Duplicate isolates from the same patient were excluded.
Multilocus sequence typing and bla OXA−51−like gene sequencing According to Bartual et al. [17], MLST was performed as follows. 7 housekeeping genes, gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were ampli ed and then sequenced. The resulting sequences were assigned to STs using the Pubmlst database [18]. The eBURST algorithm (version 3) [19]was used to assess the genetic relationships by assigning STs to clonal complexes (CCs). CCs are de ned as including any STs sharing alleles at > = 6 of 7 loci. The CC comprises a predicted founding ST as a common ancestor with other closely related STs descending from the predicted founding ST.
SBT-bla OXA−51−like genes were carried out as follows. The bla OXA−51−like genes were ampli ed and sequencing [20]. The sequences were analyzed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the genetic diversity of the bla OXA−51−like genes [5,15]. The primer sequences used in this study are listed in Supplementary table 1.

Results
Geographical distribution of the ve teaching hospitals in Guangzhou City 5 teaching hospitals are from the central region of Guangzhou ( Figure 1B). GFPH and FAH are located in the Yuexiu district; TAH is located in the Tianhe district; GXH and GRCH are located in the Haizhu district. Those three districts have more than 70% of the medical resources of Guangzhou [21] ( Figure 1A).

Summary of 138 A. baumannii isolates
Among the 138 A. baumannii isolates, 66 isolates were from GFPH, 32 isolates were from FAH 29 isolates were from GRCH, 6 isolates were from TAH and 5 isolates were from GXH (  (Figure 2A and Table 1). However, each hospital had its unique pattern. Compared to other hospitals, the GFPH had more patients from Department of Respiratory medicine while FAH showed more patients in Neurology department. In GRCH, most patients came from Emergency department (34.5%, 10/29 (Figure 2A).
The respiratory system (72.5%, 100/138) was the most common site where A. baumannii was found, followed by the blood (14.5%, 20/138 and wounds (6.5%, 9/138 . When comparing among hospitals, FAH had a higher portion of A. baumannii obtained from blood, while GFPH had more respiratory samples, and GRCH had more wound samples ( Figure 2B).
We analyzed 138 A. baumannii isolates from 5 hospitals. CC195, with its predicted founder ST195 (accounted for 35.5% of all isolates, 49/138), was the most epidemic STs ( Figure 4A and Table 1). We combined 138 A. baumannii isolates with 225 A. baumannii isolates from MLST database (Accessed 22 Mar 2017) submitted by other researches from China and ran the clonal relationship analysis ( Figure 4B, Table 1and Supplementary table 3). The results showed CC92, with its founder ST92, was the most epidemic STs in China. Additionally, we also found a new sub-CC195 is forming in Guangzhou, China, and derived from CC92. (Figure 4B).

Discussion
A. baumannii has emerged as one of the most troublesome pathogens in health care institutions globally. Due to antibiotic resistance and clonal spread, identifying potential reservoirs of the organism and modes of transmission to control the spread of A. baumannii are urgently needed. Currently, data from Guangzhou is very limited. To our knowledge, this study is the rst multicenter epidemiological study regarding A. baumannii spreading in Guangzhou, the largest city in southern China. It provided an insight view of looking into the development of customized strategies for infection control.
The increasing resistance of A. baumannii to many antimicrobial agents in China has been noticed by the CHINET surveillance system. Especially, resistance to imipenem and meropenem has rapidly rising trend from 32.9% in 2005 to 77.1% in 2018 and from 41.3% in 2005 to 78.1% in 2018, respectively [22,23]. According to our data, things are getting worse in Guangzhou, more than 80% of A. baumannii isolates showed resistance to imipenem and meropenem, and only tigecycline and polymyxin remained effective. However, tigecycline and polymyxin resistance A. baumannii has been reported and the resistance rate kept rising [24][25][26][27]. Therefore, monitoring and controlling the spread of A. baumannii is urgent.
Our study revealed CC195, with the predicted founder ST195, which was the most common CC identi ed within the 5 hospitals. It suggested the current epidemic strains in Guangzhou, China is CC195.
Previously, studies found CC92 was the most epidemic STs in multiple provinces in China [7,8]. Interestingly, ST92 is the predicted founder of CC92, and ST195 is the single locus variant of ST92 (Fig. 4B). However, we did not detect any ST92 in our previous report [11] or this study with larger sample size. ST92 was not detected in other studies from China [28] or South Africa [29] either.
Additionally, studies showed ST195, ST208, ST365, and ST191 (but not ST92) are the most common STs in hospitals across China [11,12,28]. One explanation for the absence of ST92 was ST195 evolved from ST92 and obtained more adaptation capacity to the hospital environment. With this evolution, ST195 may gradually replace ST92 and became the dominant clone (Fig. 4B). In our study, most of A. baumannii isolates including all ST195 carried OXA-66, which was similar to the previous report that 18 representative isolates from China carried the same OXA-66 allele [30]. This nding indicated OXA-66 gene harboring CC195 had strong adaptability to the environment and served as a new epidemic marker in Guangzhou City, China.
Our study revealed CC195 was spreading in the hospital. But each hospital had its unique epidemiological feature. Most patients of GRCH were from Emergency department while FAH had more patients in neurology department. This nding may reveal potential different reservoirs for CC195 within hospital settings. It suggests each hospital should develop its own management strategy.

Conclusion
OXA-66 gene harboring CC195 was the most epidemic STs in Guangzhou, China, and serves as a new epidemic marker. Health care facilities should develop their own management strategy.

Declarations
Ethics approval and consent to participate The Guangzhou First People's Hospital, School of Medicine, South China University of Technology Ethics Committee approved the study. All the patients were provided informed consent for inclusion in the study.

Consent for publication
All authors read and approved the nal manuscript for publication Availability of data and material All data generated or analysed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
YjL supervised the study, performed the susceptibility testing and MLST, and wrote the manuscript. XmH, WyY and CzP discussed the data and helped nalize the manuscript. ZxM, McH and CqF contributed to the susceptibility testing and sequence-based typing of the bla OXA-51-like genes. HlC and PhG provided advice regarding the susceptibility testing technology. ZwZ and SqW planned and supervised the experiments.