Comparison of clinical characteristics and outcomes of bloodstream infections due to multidrug-resistant Acinetobacter baumannii and other Gram-negative bacteria in ICU patients

Background: Multidrug-resistant (MDR) bloodstream infection (BSI) by Gram-negative bacteria (GNB) is an important cause of mortality in the intensive care unit (ICU). The purpose of this study was to compare the clinical characteristics of some GNB BSIs and to analyze their drug resistance, with an emphasis on the analysis of prognostic risk factors related to MDR-Acinetobacter baumannii (A. baumannii) BSI. Methods: A retrospective study was conducted in the ICU of lianyungang hospital in China. Patients with BSIs due to MDR-A. baumannii, MDR-Klebsiella pneumoniae (K. pneumoniae), MDR-Pseudomonas aeruginosa (P. aeruginosa) and MDR-Escherichia coli (E. coli) were included. Results: The overall drug resistance rate to imipenem of A. baumannii and K. pneumoniae was significantly higher than that of P. aeruginosa and E. coli (95.8% and 75.5% vs 44.6% and 9.2% respectively). The mortality rates were 71.9%, 63.3%, 41.5% and 38.1%, respectively. The multivariate analysis of MDR-A. baumannii BSI, APACHE II score, hormone use, development of septic shock were associated with the 30-day mortality, while high albumin level with survival. Conclusion: The treatment of MDR-A. baumannii and MDR-K. pneumoniae infection resulted difficult due to their high drug resistance rate. However, the understanding of the clinical characteristics of different BSIs might be helpful to predict, to some extent, the pathogenic bacteria involved so as to proceed with an early sensitive antibiotic treatment. The high mortality rate due to BSI MDR-A. baumannii might be correlated with APACHE II score, nutritional status, and hormone therapy, while septic shock was a warning sign of poor prognosis.


Clinical data collection
Pre-designed questionnaires were used to collect patient data from the Electronics Medical Records management system or clinical charts. The following information was reviewed: demographics; microbial drug resistance; comorbidities; cause of ICU admission; source of infection; microorganism colonization or infection prior to BSIs; Length of ICU stay within 30 days before infection; length of hospitalization before infection; albumin level before infection; previous chemotherapy or radiotherapy; antibiotics received before infection; transfusion before infection; previous steroid treatment; neutropenia before infection; parenteral nutrition before infection; acute physiological and chronic health assessment (APACHE II) score; all-cause 28-day mortality. As regard MDR-A. baumannii BSI, the immune response after infection by leukocyte and neutrophil changes was also evaluated, and the function of the organ or system after infection. Infections were not treated with colistin because it was not available in our hospital.

Definitions
The infection caused by MDR-A. baumannii or other GNB was defined as clinical signs of the systemic inflammatory response syndrome and positive culture. Infection onset was defined as the date of collection of the first positive blood culture for the observed pathogen. Primary bacteraemia was recorded if no source was identified. Before the infection onset, isolation of any microorganisms in other sites such as urine, lung, abdomen and skin were recorded as microorganism colonization or infection. Neutropenia was defined as an absolute neutrophil count < 1500/μl. Weak leukocyte response was defined as leucocyte lower than the pre-infection level by multiple tests within 2 days after the infection onset. Neutrophil deficiency was defined as an absolute neutrophil count < 500/μl. Prior antibiotic therapy was defined as the use of antibiotics for > 48 h within the 30 days before the infection onset. Steroid treatment was defined as an administration > 20 mg/day of prednisone (or its equivalent) for 3 or more days. Sensitive antibiotic therapy was defined as the therapy with at least one agent which has in vitro activity against the infecting pathogen. Length of ICU and hospitalization stay were expressed by the number of days from the date of admission to the date of the infection onset. Septic shock was defined according to the international consensus definition [14].
MDR was defined as the resistance to at least one agent in three or more categories of antibiotics [15].

Bacterial isolation and identification
Isolated bacteria were identified by the Vitek 2 system (bioMérieux, Marcy l'Etoile, France). According to the Clinical and Laboratory Standards Institute, the drug susceptibility test was performed by disk diffusion method or dilution method. Extended spectrum beta-lactamases (ESBLs) production was tested by disk diffusion method.
Colistin resistance test was not routinely carried out in our hospital, but the strains included in our study were all sensitive to colistin when the susceptibility test was performed.

Statistical analysis
Statistical analysis was performed using SPSS version 23.0. Continuous variables were presented as mean ± standard deviation (by Student's t-test) or as the median (the upper quartile, the lower quartile) (by Kruskal Wallis rank-sum test) when the distribution was not normal. Categorical variables were described by frequency (percentage), using the Chi-square test or Fisher exact test. Cox risk proportion model was used, variables with statistical significance in the univariate analysis were included in the multivariate model, and stepwise forward regression was used to screen variables; the entry criterion was 0.05, and the exclusion criterion was 0.10. Time to mortality was analysed using the Kaplan-Meier survival curve and the log-rank test by Graphpad Prism 6.0. P<0.05 was considered statistically significant, and all the tests were 2-tailed.

BSI characteristics by different GNB
Patient characteristics are summarized in Table 1. Twenty-seven (35.5%) cases of MDR-E.
coli BSI were considered as community-acquired BSIs (which occurred before or within 48 hours after admission), all the rest were hospital-acquired BSIs.
The average age of patients with MDR-P. aeruginosa and MDR-E. coli BSIs was approximately 5 years younger than that of the other two groups (P=0.002). In patients with MDR-A. baumannii and MDR-K. pneumoniae BSIs, the prevalence rate of underlying diseases was high, especially chronic lung disease (P=0.003) and chronic kidney disease (P<0.001). In addition to MDR-E. coli BSI group, approximately half or more than half patients in other groups were admitted to ICU due to severe pneumonia and respiratory failure (both P<0.001). The proportion of patients with MDR-E. coli BSI admitted to ICU due to urinary tract infection, abdominal infection, shock, renal failure, and post-operation complications were higher than the proportion of other BSIs (shock P=0.009, other P<0.001). Admission to ICU due to burns was more commonly characterized by MDR-P.
pneumoniae, the resistance to tigecycline was the lowest (31.3%). The resistance rate of MDR-P. aeruginosa to piperacillin tazobactam, amikacin, ceftazidime and levofloxacin ranged from 23 to 31%. Overall, MDR-E. coli had the lowest drug resistance rate, but the resistance rate for levofloxacin was relatively high (71.9%).

Discussion
This study demonstrated that BSI caused by different GNB had significantly different clinical characteristics by comparing the risk factors such as causes of ICU admission, underlying diseases, sources of infection, treatment and invasive procedures.

MDR-K. pneumoniae BSIs
A meta study [16] found that the main risk factors for carbapenem-resistant K.
pneumoniae (CR K. pneumoniae) infection included: long hospital stay or ICU stay, previous use of carbapenems and steroids, CVC implantation, mechanical ventilation and tracheostomy, with the first three as the major risk factors. Our study found that all the above risk factors included a higher proportion of K. pneumoniae infections (mainly CR K. pneumoniae), but a less proportion of A. baumannii infection, compared with P. aeruginosa and E. coli infection. Other studies [8,17,18] have confirmed some of the above risk factors, and found that CR K. pneumoniae colonization is a strong risk factor for later infection. In our study, K. pneumoniae was found in 64.6% of patient specimens before the infection onset, and most of them were colonized bacteria. K. pneumoniae can asymptomatically colonize the skin, mouth, respiratory tract and intestines, and intestinal colonization is closely related to subsequent infections [19][20][21]. Thus, we suspected that K. pneumoniae in this study might mainly come from the direct or indirect gut microbiota.
One study found that the intestinal decontamination can decrease the incidence rate of ICU-acquired GNB BSI by 45%, while the selective oropharyngeal decontamination only decreased by 33% [22]. However, in a randomized trial of 8665 patients [23], oropharyngeal decontamination or SDD did not reduce mortality in ICU-acquired MDR-GNB BSI compared with the standard care. The all-cause mortality in our study was 63.3%, thus higher than 44.5% [8] (mortality due to CR K. pneumoniae BSI in ICU patients). Another study [24] found that mortality rates varied with severity, since in high-risk patients the mortality rate of CR K. pneumoniae BSI is 77%, while it is 45% in middle-risk patients.
Therefore, we hypothesized that the mortality was closely related to the general condition and treatment strategy.

MDR-E. coli BSIs
Although E. coli is present in the normal intestinal flora, it can enter and survive in a sterile parenteral environment, which often leads to urinary tract infections and BSI [25].
Indeed, E. coli is the most important pathogenic bacteria in urinary and abdominal infections [26] and BSI. One study reported that 76% of E. coli BSIs come from home, and the most common source of home-acquired E. coli BSIs was the urinary tract, while the gastrointestinal tract was the common source for nosocomial E. coli BSIs [27]. Our observations were similar with the above observations. Patients in the E. coli group were admitted to the ICU mainly for septic shock or renal failure secondary to urinary or abdominal infection, and BSI often occurred within 48 hours after the transfer from the general ward or community to the ICU, thus, the previous duration of hospital stay and ICU stay were short. In this study, most MDR-E. coli were ESBLs-producing strains and Ajao et al. [28] found that colonization and exposure to multiple antibiotics are independent risk factors for infection with E. coli-producing ESBLs. However, these factors were not significantly present in our study due to short hospital stay and unclear out-of-hospital medication for some patients. Overall, the E. coli group was significantly different from the other groups due to its special source of infection, and the mortality rate was low thanks to the ease of keeping its infections under control and easy access to sensitive antibiotics.

MDR-P. aeruginosa BSIs
Unlike other bacteria, P. aeruginosa not only has high virulence, but also has inherent resistance to some antibiotics and is prone to drug resistance. Our study found that P.

MDR-A. baumannii BSIs
It is well known that ICU is a very common place to contract an infection, especially from A. baumannii, which mainly includes the following two reasons. As regard patient's factors: long hospital stay [5], experience of invasive procedures (CVC [35], surgery [1,36], mechanical ventilation [37]), exposure to antibiotics [1,36] such as carbapenems, weak immunity (previous hormone therapy [36], tumor patients with neutropenia [38]). As regard the contamination of wards [39] A. baumannii is a bacterium commonly present in ICU that is difficult to eliminate and it often spreads among the ward crowd, resulting in a high risk of A. baumannii colonization for patients [40,41]. Some of these aspects were also found in our study. Compared with other groups, A. baumannii group had the longest hospitalization or ICU stay, the highest total proportion of invasive operations, and exposure to more than 3 classes of antibiotics (carbapenem and piperacillin tazobactam were the main antibiotics used). Our hypothesis is that it is difficult to reduce the occurrence of A. baumannii infection in ICU, except for the decontamination and improvement of the awareness of the cleaning operations of the medical workers.

Drug resistance
Carbapenems were often selected for empirical treatment in our study, which is an important factor causing the onset of carbapenem-resistant A. baumannii, K. pneumoniae and P. aeruginosa. Carbapenems and tigacycline were rarely used in E. coli group when admitted to hospital, thus, the resistance rate was significantly lower in this group.
However, with drug-resistant genes resulting in drug-resistant bacteria, they can widely spread in hospitals and communities, consequently resulting in an increased incidence of carbapenem-resistant E. coli and CR K. pneumoniae infections in China [42]. In China, ST11 has been considered as the dominant CR K. pneumoniae strain, but new ST11 CR A. baumannii is emerging [43], which is hypervirulent, multidrug resistant, and transmissible, potentially resulting a real superbacteria that could pose a serious threat to public health.
The polycolisin-resistant E. coli has been found in China [42], which undoubtedly adds to the current sad situation.

Prognostic analysis of MDR-A. baumannii BSIs
Overall, previous studies found that the prognosis of A. baumannii BSI in ICU patients is different depending on the underlying disease, illness severity, drug resistance, therapies used, post-infection appearance and infection source. The following characteristics often lead to poor prognosis: improper initial antibiotic treatment [9,38], comorbidities (tumours [38,44,45], liver cirrhosis [46], chronic obstructive pulmonary disease and chronic renal failure [37]), high drug resistance, neutropenia [38], previous high-dose hormone therapy [47], previous surgery [9], infection originated from the respiratory tract [35,46], high SAPS score [8], Pitt score [35], APACHE II score [37,38,46]. However, the development of septic shock [47] and the severity of the disease are the factors most closely related to prognosis, and our study also found these two key points. A study [9] focused on ICU patients found that patients developing septic shock after MDR-A.
baumannii BSI have a 23.8% higher mortality rate than patients without septic shock (82.5% vs 58.7%). An early study [10] grouped by APACHE II score found that the mortality of 15 < APACHE II scores ≤ 25 group was 14.3%, and the mortality of 25 < APACHE II scores ≤ 35 group was 3 times of that of 15 < APACHE II scores ≤ 25 group. In addition, Yang et al. found that in case of A. baumannii BSI, the death rate of the carbapenem MICs ≥ 8 mg/l group was twice that of the MICs ≤ 48 mg/l group [48]. In our study, hypoalbuminemia and a previous hormone therapy were independently associated with death, suggesting that nutritional and immune statuses are very important. Because the access to colistin was limited in our hospital, common drug treatments mainly included tigecycline in combination with cefoperazone/sulbactam, and monotherapy with amikacin or sulfamethoxazole trimethoprim, but none of the monotherapies has been reported as associated with survival. In addition, in vitro treatments with sensitive antibiotics that improved the prognosis were not found.

Limitations and strengths
Some limitations are present in this study. Firstly, this was an observational research with its inherent defects. Secondly, it is a single-centre study with relatively few cases included, thus, differences between groups could not reflect a common situation worldwide. Thirdly, in the analysis of the prognosis, the adjustment for the severity of the disease and antibiotic regimen was not performed. However, this study was based on ICU

Conflict of interest:
The authors declare that they have no conflict of interest.

Source of funding: This work was supported by the Lianyungang Health Commission Plan
Funding Project of Jiangsu province (QN1804).

Acknowledgements:
We thank to the the nurses in our department, also thank to the staff of all microbiology laboratories in our hospital.