Bloodstream infections caused by ST2 Acinetobacter baumannii over 6 years in China: risk factors, antibiotic regimens and virulence

Acinetobacter baumannii (Ab) is an important pathogen of medical-related infections, A.baumannii sequence type 2 (ST2) has spread all over the world. To the best of our knowledge, this is the rst study to analyze the clinical and microbiological characteristics of patients with bloodstream infection (BSI) due to ST2 A.baumannii. the and of with due to


Background
In recent years, bloodstream infections due to multidrug-resistant gram-negative bacteria such as Acinetobacter baumannii have been increasingly observed among hospitalized patients [1], resulting in very limited or even no remaining options for the treatment of blood infection (BSI) caused by multidrugresistant A. baumannii [2]. It prompted the World Health Organization (WHO) to recognize MDR-AB as the critical, number one priority among a published list of 12 antibiotic-resistant bacteria [3].
Providing appropriate treatment for infections caused by MDR-AB is a challenge. The lack of new antimicrobial agents in the research and development of the pharmaceutical industry has increased the incidence of MDR-AB [4]. MDR-AB usually require therapy with colistin, an older and relatively toxic polymyxin antimicrobial [5]. Meanwhile, tigecycline attains poor serum levels and has a limited record of treating serious infections [6]. While colistin and tigecycline are considered rst-line therapeutics for MDR-AB, there is still no optimal treatment plan for MDR-AB thus far [7].
The population of MDR-AB is characteristic of clonal dissemination, as revealed by multi-locus sequence typing (MLST). Clonal complex (CC) 2 is by far the most widely disseminated A. baumannii population in more than 30 countries, including China [8], and ST2 is dominant in CC2 [9]. ST2 has been reported to be associated with antimicrobial resistance [10][11]. Furthermore, the mortality of BSI caused by ST2 was more than 60% [12].
It is urgent to identify the risk factors for the death of patients with ST2 A. baumannii-induced bloodstream infection and to nd the appropriate treatment options. Therefore, the aim of the present study was to analyse clinical features, antimicrobial treatments and outcomes of patients with ST2 A. baumannii-induced BSI. Furthermore, we evaluated the virulence of ST2 A. baumannii and analyzed its impact on the prognosis of these patients.

Study location and patient
This study was conducted at the First A liated Hospital of Wenzhou Medical University, a 4100-bed teaching hospital (Wenzhou, China), over a 6-year period (1 January 2013 to 31 December 2018). Inclusion criteria of A. baumannii-induced bloodstream infection include [13]: (1) age ≥ 18 years; (2) blood culture positive for MDR-AB; (3) clinical signs consistent with infection; (4) the infection occurred ≥ 48 h after hospital admission. Only the rst episode was included if the patient had multiple episodes of A. baumannii-induced BSI. Study design and data collection A retrospective study design was employed, with the main outcome measure being 30-day in-hospital mortality. Clinical information and laboratory parameters were collected from medical charts that used prede ned de nitions of variables. The collected data included demographic characteristics, underlying diseases, length of stay in hospital, treatments and procedures performed, laboratory results, antimicrobial therapies, and all-cause 30-day mortality.
The results of tigecycline were interpreted according to the recommendation of the Food and Drug Administration, with MICs of ≤2, 4, and ≥8 μg/ml interpreted as susceptible, intermediate, and resistant, respectively. MDR-AB were de ned as non-susceptible to three or more different antimicrobial categories.
All these strains were stored at -80 °C for further research.

Multi-locus sequence typing MLST
In the present study, seven housekeeping genes (cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB) were ampli ed and sequenced to characterize the genotypes of all isolates according to the provided protocols (www.pasteur.fr/mlst). The alleles and STs were assigned according to the online database of the Institut Pasteur's MLST web site of A. baumannii.

Bio lm formation
The bio lm formation ability of the A. baumannii isolates on 96-well polystyrene microtiter plates was assessed using the crystal violet staining, as formerly described [14]. For evaluating bio lm formation, Muller-Hinton Broth and A. baumannii ATCC19606 were used as negative and positive controls, respectively. The ratio of the OD 570 of the experimental isolate to the OD value of the negative control OD C was de ned as the bio lm forming ability of the isolate. All the experiments were conducted in triplicate. The results were classi ed into the four following categories [15]: a) OD 570 ≤ OD C = nonbio lm producer; b) OD C < OD 570 ≤ 2OD C = weak bio lm producer; c) 2OD C < OD 570 ≤ 4OD C = medium bio lm producer; d) 4OD C < OD 570 = strong bio lm producer.

Serum resistance assay
Serum sensitivity assays were performed on clinical isolates [16]. 1 mL bacterial cultures were incubated until the bacterial suspension reached an OD 600 of 0.5. Then washed and suspended in 1 mL of phosphate-buffered saline (PBS). Next, 100 μL of the bacterial suspension was mixed with 300 μL of normal human serum (NHS) and the mixture was incubated at 37°C for 3 h. Finally, calculate the colonyforming units (CFUs) of bacteria by single plate-serial dilution spotting (SP-SDS) [17]. The serum bactericidal effect was expressed as the ratio of the CFUs in the serum-bacteria suspension to the CFUs in a bacterial suspension without NHS. All experiments were performed in triplicate, and results were expressed as percent survival.

Galleria mellonella larva infection assay
The virulence of A. baumannii was estimated by infecting G. mellonella larvae, as described previously [8].
Brie y, a suspension of A. baumannii was prepared in PBS at approximately 1×10 8 CFU/mL, and 10 μL of this suspension (approximately 1×10 6 CFU) was injected through the last proleg of each larva. The control larvae were injected with 10 μL of PBS without bacteria. The survival rate of the G. mellonella was recorded for 144 hours. The larvae were considered dead when they were unresponsive to touch. 10 larvae were used for each experiment in triplicate Statistical analysis All statistical analyses were performed using SPSS 22.0 software (IBM, Armonk, NY, USA). The categorical variables were listed as percentages and the continuous data were expressed as mean± standard deviation (mean ± SD) or median (25th-75th percentile) appropriately. The Chi-squared test or the Fisher's exact test was used for categorical variables, and the odds ratio (OR) was calculated with con dence intervals (CIs) of 95%. The continuous data were analyzed using the Student's t test or Mann-Whitney U test. P-value < 0.05 was considered statistically signi cant. All tests were two-tailed. To determine the risk factors for A. baumannii-induced BSI, univariate logistic regression analyses were performed. All variables with a P-value <0.05 were included in the multivariate model.

Results
Clinical factors associated with 30-day mortality among patients with ST2 A. baumannii-induced BSI A total of 108 patients with A. baumannii-induced BSI were enrolled during the 6-year study period. 75 patients died within 30 days of A. baumannii BSI, with a mortality rate of 69.4%. The demographics between the non-survivor group and survivor group were similar, as shown in Table 1. Brie y, the majority of patients in both groups were elderly and male patients. The most common comorbidity was pneumonia. MDR-AB was also isolated from sputum in 86 patients (79.6%) during the hospitalization. Empirical antimicrobial therapy was performed on all patients.  (Table 1). Furthermore, deep vein intubation and isolation of other microbials were not signi cantly associated with survival (P = 0.247, fungi P = 0.890, and bacteria P = 0.642, respectively), while change antimicrobial within 48 h after isolating from blood, use of antibacterial combination, and more inpatient days were signi cantly associated with survival (P < 0.001, P = 0.037, and P = 0.007, respectively) ( Table 1).

Pathogenicity of A. baumannii
Thirty A. baumannii isolated from the survivors and 30 A. baumannii isolated from the non-survivor were randomly selected for bio lm formation experiment and serum resistance assay. In the group of the survivors, the percent of weak bio lm producer, medium bio lm producer and strong bio lm producer were 13.3%, 66.7%, and 20%, respectively. Also, in the group of the non-survivors, the percent of weak bio lm producer, medium bio lm producer and strong bio lm producer were 6.7%, 83.3%. and 10%, respectively. However, the bio lm formation ability of the two groups was not statistically different (3.36 ± 1.30 vs 3.13 ± 0.77; P = 0.417) (Fig. 1). On the other hand, after a 3 h incubation with normal human serum, the survival rate of A. baumannii isolated from non-survivors was higher than A. baumannii isolated from survivors, but the survival rates were not statistically different among the two groups, either (17.5% [6.9% − 38.0%] vs 10.5% [6.2% − 27.0%]; P = 0.209 ) (Fig. 2). Furthermore, 10 A. baumannii isolated from the survivors and 10 A. baumannii isolated from the non-survivor were randomly selected for G. mellonella larva infection assay. G. mellonella survival data were shown in Fig. 3. The survival rates of the larvae infected between two groups were not statistically different in every observation between day 1 and day 6 (P = 0.522). The pathogenicity of ST2 was signi cantly higher than ATCC 19606 (P < 0.001).

Discussion
In this study, all MDR-AB isolated from BSI belong to ST2. Historically, the widespread spread of MDR-AB across the globe is mainly due to the spread of two main clones, which were called global clones 1 and 2 [18], although new lineages are now common in some parts of the world [8,19]. The analysis of all publicly available genome sequences shows that ST2, ST1, ST79 and ST25 account for more than 71% of all genomes sequenced so far, of which ST2 is by far the most dominant type [20]. However, the MDR-AB population structure isolated from bloodstream infection in this study was very single, all the isolates were ST2. This meant the dissemination of ST2 in the hospital. Of notice, A. baumannii usually comes from the medical environment (patient, medical staff or medical device). Several studies have shown that A. baumannii can spread between hospitals [2,21], which means the prevalence of ST2 is more serious in this region, may not just the hospitals involved in this study.
The clinical factors associated with 30-day mortality among patients with ST2 A. baumannii-induced BSI were analyzed. The use of mechanical ventilation, ICU stay and thrombocytopenia may cause higher mortality. Previous studies demonstrated that the mortality risk factors associated with poor prognosis include older age, severity of the underlying disease, septic shock, a high Pitt bacteremia score, previous surgery, mechanical ventilation, and inappropriate antimicrobial treatment [12,[22][23]. The ICU was the highest risk unit of this nosocomial outbreak of MDR-AB, which could subsequently disseminate within the hospital [24]. Our data also shows that ST2 BSI is a special ICU acquired infection, which may be caused by the impaired immune function of ICU patients. Thrombocytopenia indicated that the patient has a serious infection in these situation, which also makes the patient's prognosis worse. Change antimicrobial within 48 h after isolating from blood, use of antibacterial combination, and more inpatient days were signi cantly associated with survival. Early appropriate antibacterial therapy is essential to reduce mortality caused by severe MDR-AB bacteremia [25]. However, for bloodstream infections caused by MDR-AB, the risk of inappropriate early antibacterial treatment will greatly increase [26]. In our study, all patients had taken empirical antimicrobial use, but almost all inappropriate. Due to the long time required for blood culture, clinically, doctors usually use or change antibacterial drugs according to the symptoms and other auxiliary examinations of patients. Although all strains are resistant to carbapenems, carbapenems are still important therapeutic options for treating multidrug resistant A. baumannii infections in China, where colistin was not available until recently and tigecycline was not available until 2012. The detailed antibiotic therapy after isolating from blood were also analyzed. Although the use of cefoperazone/sulbactam and tigecycline does not improve the prognosis of patients, the combination of cefoperazone/sulbactam and tigecycline is still the most effective option. It is reported that the combination of cefoperazone/sulbactam and tigecycline has synergistic effect in vitro, but there is no support from clinical data [27]. Our data support that this combination can improve the prognosis of patients.
The pathogenicity of ST2 isolates were evaluated, ST2 isolates from the survival group and the death group had no statistical difference in the bio lm formation, serum resistance and virulence models. A. baumannii has always been considered as a hypovirulent opportunistic pathogen [28]. Nevertheless, we found that the virulence and serum resistance of ST2 are signi cantly higher than that of the model strain. Because ST2 has multidrug resistance and high pathogenicity, it is di cult to evaluate the respective contributions of virulence and drug resistance in the infection process.
This study has several limitations. Firstly, it was conducted in a single center. The ndings need to be validated by a larger multicenter study. Secondly, although there was a trend for lower mortality with the combination of cefoperazone/sulbactam and tigecycline, sample size was small, the results must be interpreted carefully.
Conclusion ST2 A. baumannii has spread widely in hospitals, BSI caused by ST2 A. baumannii strains represents a challenge for physicians, considering the high mortality associated with the infections. A priori prediction of resistance is di cult as cultivation of A. baumannii from bloodstream infection takes a long time. Empiric coverage for ST2 strains will likely be required even prior to determination of antimicrobial susceptibilities in critically ill patients. The combination of cefoperazone/sulbactam and tigecycline may be a meaningful treatment option.