Patients and bacteria isolates
We collected patients’ clinical information and isolated a total of 93 isolates of nonrepetitive S. maltophilia. Among them, we isolated 30 isolates of S. maltophilia from the ICU, and the rest were from a range of hospital departments (Figure 1), including 13 isolates from neurosurgery, 9 isolates from emergency internal medicine, 8 isolates from cadre health care, 6 isolates from cardiovascular surgery, 5 isolates from hematology, 4 isolates from liver surgery, 3 isolates from oncology, 3 isolates from nephrology, 2 isolates from emergency medicine, 2 isolates from neurology, 2 isolates from cholangio-pancreatic surgery, 1 from thoracic surgery, 1 from gastrointestinal surgery, 1 from respiratory medicine, 1 from digestive medicine, 1 from general surgery, and 1 from urology.
Among the patients with S. maltophilia infection, 62 males (66.7%) and 31 females (33.3%) were included. A total of 73 patients were age 60 years or older (78.5%). Before bacterial isolation, 35 patients (37.6%) were subjected to invasive examinations or treatments. Among all the S. maltophilia isolates, 81 isolates (87.1%) were isolated from sputum, 7 isolates (7.5%) from drains, 2 isolates from pleural effusion, 1 strain from ascites, 1 strain from urine, and 1 strain from blood.
Of the 93 patients, 7 (7.5%) had no basic diseases but did have fractures and malnutrition, and 86 (92.5%) had one to five underlying diseases. Of these, 25 had malignant tumors, 24 had hypertension, 12 had coronary heart disease, 10 had renal insufficiency, 5 had leukemia, 14 had head trauma, 20 had chronic bronchitis or pneumonia, and 12 had liver injury. All of the patients had a history of antibiotic use (one to six types), with an average of three antibiotics per person before isolation of S. maltophilia, of which 69 had used three or more antibiotics. The antibiotics primarily included cephalosporins (65/93), carbapenem (53/93), β-lactamase inhibitors (51/93), quinolones (35/93), glycopeptide (22/93), aminoglycosides (13/93), and tetracyclines (9/93) (Table 1).
Table 1. The clinical characteristics of the adult and pediatric patients
|
Adults(n = 93)
|
Demographics
|
|
Age (year, average, range)
|
66.3 (16–99)
|
Gender: male
|
62 (66.7%)
|
|
|
Baseline diseases, n (%)
|
|
Hypertension
|
24 (25.8)
|
Heart disease
|
12 (12.9)
|
Malignancy
|
25 (26.9)
|
Pulmonary disease
|
20 (21.5)
|
Liver disease
|
12 (12.9)
|
Leukemia
|
5 (5.4)
|
Head trauma
|
14 (15.1)
|
|
|
Strain isolation n (%)
|
|
ICU
|
30 (32.3)
|
Sputum
|
81 (87.1)
|
|
|
Invasive operation n (%)
|
35 (37.6)
|
|
|
Previous antibiotics usage n (%)
|
|
The number of antibiotics ≥3
|
69 (74.2)
|
Cephalosporins
|
65 (69.9)
|
Carbapenems
|
53 (57.0)
|
β-lactamase Inhibitors
|
51 (54.8)
|
Quinolones
|
35 (37.6)
|
Glycopeptides
|
22 (23.7)
|
Aminoglycosides
|
13 (14.0)
|
MLST analysis
The distribution of the clonal typing of S. maltophilia was relatively scattered. According to the different alleles, we assigned the isolates to 61 sequence types. Among them, 45 types of the 60 isolates were different from those published on the PubMLST database (recorded as STnew1-STnew45). The other 33 isolates consisted of existing types in the database, of which a relatively larger number was ST23 (n = 8). Some were isolates of ST5 (n = 3), ST15 (n = 3), ST24 (n = 3), ST3 (n = 2), ST84 (n = 2), ST89 (n = 2), and ST99 (n = 2), and some isolates were assigned to ST4, ST8, ST13, ST36, ST77, ST98, ST102, and ST112. The eight S. maltophilia isolates of ST23 were distributed in five different departments, and we classified the 30 isolates isolated from the ICU into 24 STs. We did not collected S. maltophilia isolates of the exact same sequence types in the other departments, which indicated the lack of obvious clonal transmission of S. maltophilia infections in this study [1]. The detailed results are shown in Figure 2.
PFGE typing results
According to the fragment diagnostic criteria of PFGE, PFGE types can be classified into a group or cluster if there are no more than three bands[8, 9]. In this study, the 93 SMA strains were scattered and could be divided into 73 clusters in this study. Among them, we divided 13 strains into the same cluster (from eight departments, not from the same department). We divided another five and two strains into the same cluster, but the others were all quite different. These results suggested that these isolates were not part of an outbreak in the department, and the detailed results are shown in Supplementary Figure 1.
Virulence gene detection
The results of the virulence gene detection showed that the positive rates of the four virulence genes were 79.6% (74/93) for Stmpr1, 91.4% (85/93) for Stmpr2, 94.6% (88/93) for Smf-1, and 52.7% (49/93) for Smlt3773. There were 31 isolates of S. maltophilia that carried all four of the genes.
Analysis of drug resistance
The resistance rates of S. maltophilia to levofloxacin and TMP/SMX were 4.3% and 9.7%, respectively. All of S. maltophilia isolates were susceptible to minocycline. Among these isolates, one isolate, numbered ji82, was resistant to both TMP/SMX and levofloxacin.
Biofilm formation ability
The average biofilm formation ability of S. maltophilia was OD492 = 0.54 ± 0.49 (0.044–2.34). The OD values of S. maltophilia isolated from the male and female patients were OD492 of 0.52 ± 0.51 and OD492 of 0.57 ± 0.47, respectively, and we did not find any significant difference between the two groups. We also did not find a significant difference in the biofilm formation ability between people age 60 or older and those younger than 60 years old, as shown in Figure 3. In addition, we analyzed the drug resistance and biofilm formation ability of the isolates and did not find an obvious correlation between the drug-resistant phenotype and the biofilm formation ability, as shown in
Table 2. The positive rates of the three biofilm genes rmlA, spgM, and rpfF were 82.8% (77/93), 92.5% (86/93), and 64.5% (60/93), respectively. The point mutations of the spgM gene in the isolates with strong biofilm formation abilities were relatively consistent and significantly different from those with weak biofilm formation abilities. The detailed sequencing results of some of the isolates are shown in Figure 4 (the base pairs of the two isolates with different biofilm formation abilities were selected as the representatives). The other two biofilm genes, however, did not have obvious point mutations in the isolates with different biofilm formation abilities.
Table 2. Drug-resistant rates and the relationship between the drug resistance and biofilm formation
Antibiotics
|
Resistant rate
|
Pearson's correlation
|
Levofloxacin
|
4.3%
|
0.02
|
TMP/SMX
|
9.7%
|
0.04
|
Minocycline
|
0%
|
NA
|
The carriage of the virulence genes
The carriage of the four virulence genes Stmpr1, Stmpr2, smf-1, and Smlt3773locus were 79.6%, 91.4%, 94.6%, and 52.7%, respectively.
Analysis of the risk factors in ICU patients infected with S. maltophilia
The results of a univariate analysis demonstrated that the changes in lymphocytes, albumin, and the use of antibiotics were infection risk factors in the ICU patients (Table 3). After the multivariate analysis, we found the type of antibiotic use and lymphocyte count to be independent risk factors of infection with S. maltophilia (Table 4). These findings may be used as a new reference index for clinical sensitivity and control of S. maltophilia.
Table 3. Univariate analysis of risk factors of S. maltophilia infections in the ICU
Items
|
Patients (n = 30)
|
Control (n = 60)
|
P value
|
OR(95%CI)
|
Male (sex)
|
23 (76.7%)
|
38 (63.3%)
|
0.263
|
0.565 (0.208-1.534)
|
Age (years)
|
64.8 ± 19.1
|
65.5 ± 16.9
|
0.873
|
|
Leukocyte
|
11.5 ± 5.4
|
10.9 ± 4.1
|
0.777
|
|
Neutrophil
|
9.4 ± 4.9
|
9.4 ± 3.9
|
0.767
|
|
Lymphocyte
|
1.3 ± 0.9
|
0.9 ± 0.4
|
0.012
|
|
Monocyte
|
0.7 ± 0.5
|
0.5 ± 0.4
|
0.536
|
|
Albumin
|
30.6 ± 4.2
|
28.3 ± 5.7
|
0.033
|
|
Globulin
|
29.0 ± 6.4
|
28.2 ± 7.0
|
0.286
|
|
Prealbumin
|
129.0 ± 52.3
|
124.8 ± 49.9
|
1.000
|
|
Surgeries
|
14 (46.7%)
|
27 (45.0%)
|
0.496
|
0.724 (0.286-1.835)
|
Organ transplantation
|
5 (16.7%)
|
9 (15.0%)
|
0.987
|
0.990 (0.296-3.310)
|
Malignant tumor
|
8 (26.7%)
|
15 (25.0%)
|
0.894
|
0.933 (0.337-2.585)
|
Hypertension
|
7 (23.3%)
|
15 (25.0%)
|
0.923
|
0.949 (0.328-2.748)
|
Diabetes
|
3 (10.0%)
|
9 (15.0%)
|
0.397
|
0.547 (0.135-2.213)
|
Pulmonary infection
|
9 (30.0%)
|
16 (26.7%)
|
0.990
|
1.007 (0.374-2.712)
|
Cardiopathy
|
4 (13.3%)
|
9 (15.0%)
|
0.841
|
0.875 (0.238-3.213)
|
Liver injury
|
4 (13.3%)
|
7 (11.7%)
|
0.972
|
1.024 (0.272-3.856)
|
Trachea intubation
|
12 (40.0%)
|
21 (35.0%)
|
0.941
|
1.036 (0.406-2.640)
|
Chemotherapy
|
2 (6.7%)
|
3 (5.0%)
|
0.843
|
1.205 (0.189-7.681)
|
Immunosuppressor
|
9 (30.0%)
|
17 (28.3%)
|
0.867
|
0.920 (0.343-2.464)
|
Number of antibiotics
|
3.6 ± 1.2
|
3.0 ± 1.1
|
0.029
|
|
Carbapenems
|
21 (70.0%)
|
40 (66.7%)
|
0.731
|
1.187 (0.445-3.167)
|
Cephalosporins
|
20 (66.7%)
|
45 (75.0%)
|
0.604
|
0.771 (0.289-2.059)
|
Quinolones
|
16 (53.3%)
|
30 (50.0%)
|
1.000
|
1.000 (0.396-2.523)
|
Table 4. Multivariate logistic regression analysis associated with S. maltophilia infections in the ICU
Risk factors
|
B value
|
Wals
|
P value
|
OR value
|
95% CI
|
lower limit
|
upper limit
|
Lymphocyte
|
1.077
|
4.208
|
0.04
|
2.937
|
1.049
|
8.222
|
Albumin
|
0.099
|
3.05
|
0.081
|
1.104
|
0.988
|
1.234
|
Antibiotics
|
0.596
|
5.956
|
0.015
|
1.814
|
1.124
|
2.927
|