During the study period, 140 S. pneumoniae isolates were collected from inpatients which were cultured from hypopharyngeal aspirates (n=104), bronchoalveolar lavage (n=33), nasopharyngeal swabs (n=1) or ear discharge (n=2). Of the 693 pneumococcal strains isolated from outpatients, 140 were randomly selected using random number table method.
Serotype distribution and Vaccine coverage
The serotype distribution and vaccine coverage between the inpatient and outpatient S. pneumoniae isolates are shown in Table 1. The serotype was included in the table when its constituent ratio was greater than 5%. The serotype distribution was more concentrated among the inpatient isolates, in which only 15 serotypes were identified. The most common serotypes were 19F (32.9%), 19A (20.7%), 23F (10.7%), 6A (10.0%), 14 (8.6%) and 15B (6.4%), which accounted for 89.3%. Meanwhile, 29 serotypes were identified among the outpatient isolates. The most frequent serotypes were 19F (13.6%), 23F (12.9%), 6A (10.0%), 6B (10.0%), 19A (7.9%) and 34 (5.0%), which accounted for 59.3%. In addition, the serotype coverage rates of PCVs among the inpatient isolates were high.
Table 1
Serotype distribution and vaccines coverage rates of inpatient and outpatient S. pneumoniae isolates [n (%)]
Serotype /
coverage rate
|
Inpatient isolates
(n = 140)
|
Outpatient isolates
(n = 140)
|
19F
|
46 (32.9%)
|
19 (13.6%)
|
23F
|
15 (10.7%)
|
18 (12.9%)
|
14
|
12 (8.6%)
|
5 (3.6%)
|
6B
|
4 (2.9%)
|
14 (10.0%)
|
9V
|
1 (0.7%)
|
0
|
18C
|
0
|
1 (0.7%)
|
7F
|
1 (0.7%)
|
1 (0.7%)
|
19A
|
29 (20.7%)
|
11 (7.9%)
|
6A
|
14 (10.0%)
|
14 (10.0%)
|
3
|
1 (0.7%)
|
3 (2.1%)
|
15B
|
9 (6.4%)
|
5 (3.6%)
|
34
|
0
|
7 (5.0%)
|
Others
|
8 (5.7%)a
|
42 (30.0%)b
|
PCV7
|
78 (55.7%)
|
57 (40.7%)
|
PCV10
|
79 (56.4%)
|
58 (41.4%)
|
PCV13
|
123 (87.9%)
|
86 (61.4%)
|
Notes: a: the other types include serotypes 15C (2), 6C (2), 42 (1), 22F (1), and 15F (1); b: the other types include serotypes 6C (5), 23A (5), 15C (4), 42 (4), 15A (4), 11A (4), 29 (3), 22F (2), 13 (2), 20 (1), 23B (1), 8 (1), 24 (1), 31 (1), 19B (1), 11B (1), 10A (1), and 7C (1). |
Antimicrobial Susceptibility Testing
The antimicrobial susceptibilities of the inpatient and outpatient isolates are shown in Table 2. The non-susceptibility rate of penicillin in inpatients was high, and the penicillin MIC50 and MIC90 values were also higher. Among the inpatient isolates, the non-susceptibility rate of amoxicillin-clavulanic acid, imipenem, cefuroxime, cefaclor and trimethoprim-sulfamethoxazole were 7.1%, 65.7%, 92.8%, 93.6% and 85%, respectively. The corresponding date were 0.7%, 38.6%, 50%, 53.5% and 65.7%, respectively. All of the isolates showed high non-susceptibility to erythromycin and tetracycline. The resistant rate of chloramphenicol was low both in inpatient isolates and outpatient isolates. All pneumococcal isolates were susceptible to vancomycin, linezolid and levofloxacin.
Table 2
Susceptibility and MICs to 13 antibiotics of inpatient and outpatient S. pneumoniae isolates
Antimicrobiala
|
Inpatient isolates
(n = 140)
|
Outpatient isolates
(n = 140)
|
Penicillin
|
|
|
Parenteral non-meningitis
|
|
R%
|
2.1
|
0.7
|
I%
|
5
|
0
|
Oral non-meningitis
|
|
|
R%
|
40.0
|
20.7
|
I%
|
54.3
|
38.6
|
MIC range (mg/L)
|
0.016-12
|
0.004-6
|
MIC50 (mg/L)
|
1
|
0.5
|
MIC90 (mg/L)
|
2
|
1.5
|
Amoxicillin-clavulanic acid
|
|
R%
|
0.7
|
0
|
I%
|
6.4
|
0.7
|
MIC range (mg/L)
|
0.016-12
|
0.016-3
|
MIC50 (mg/L)
|
1
|
0.38
|
MIC90 (mg/L)
|
2
|
1.5
|
Cefuroxime
|
|
|
R%
|
87.1
|
43.6
|
I%
|
5.7
|
6.4
|
MIC range (mg/L)
|
0.023-48
|
0.016-12
|
MIC50 (mg/L)
|
3
|
0.5
|
MIC90 (mg/L)
|
8
|
4
|
Cefaclor
|
|
|
R%
|
90.7
|
51.4
|
I%
|
2.9
|
2.1
|
MIC range (mg/L)
|
0.19->256
|
0.125->256
|
MIC50 (mg/L)
|
24
|
3
|
MIC90 (mg/L)
|
64
|
96
|
Ceftriaxone
|
|
|
R%
|
1.4
|
0.7
|
I%
|
17.9
|
10.7
|
MIC range (mg/L)
|
0.012-4
|
0.006-3
|
MIC50 (mg/L)
|
0.75
|
0.38
|
MIC90 (mg/L)
|
1.5
|
1.5
|
Imipenem
|
|
|
R%
|
0.7
|
0
|
I%
|
65
|
38.6
|
MIC range (mg/L)
|
0.008-2
|
0.004–0.38
|
MIC50 (mg/L)
|
0.19
|
0.094
|
MIC90 (mg/L)
|
0.25
|
0.25
|
Vancomycin
|
|
|
MIC range (mg/L)
|
0.19–0.5
|
0.25-1
|
MIC50 (mg/L)
|
0.5
|
0.5
|
MIC90 (mg/L)
|
0.5
|
0.5
|
Linezolid
|
|
|
MIC range (mg/L)
|
0.38-2
|
0.25-2
|
MIC50 (mg/L)
|
0.75
|
1
|
MIC90 (mg/L)
|
1
|
1.5
|
Levofloxacin
|
|
|
MIC range (mg/L)
|
0.38-2
|
0.38-2
|
MIC50 (mg/L)
|
0.75
|
0.75
|
MIC90 (mg/L)
|
1
|
1
|
Erythromycin
|
|
|
R%
|
100
|
96.4
|
MIC range (mg/L)
|
4->256
|
0.094-256
|
MIC50 (mg/L)
|
> 256
|
> 256
|
MIC90 (mg/L)
|
> 256
|
> 256
|
Tetracycline
|
|
|
R%
|
92.9
|
93.6
|
I%
|
4.2
|
1.4
|
Chloramphenicol
|
|
|
R%
|
4.3
|
10.7
|
Trimethoprim-sulfamethoxazole
|
|
R%
|
85
|
65.7
|
I%
|
2.1
|
11.4
|
Notes: |
a: The breakpoint of penicillin was based on the parenteral non-meningitis (intermediate [4mg/L], resistant [≥ 8mg/L]), and oral non-meningitis (intermediate [0.12-1mg/L], resistant [≥ 2mg/L]). The breakpoint of ceftriaxone was based on only parenteral non-meningitis(intermediate [2mg/L], resistant [≥ 4mg/L]). |
The multi-drug resistant pattern of the pneumococcal isolates is shown in the Table 3. Approximately 92.1% (129/140) of all inpatient isolates and 85.0% (119/140) of all outpatient isolates were MDRSP. The most prevalent antibiotic resistant pattern of macrolides/β-lactams/tetracyclines/sulfonamides was observed both in inpatient and outpatient isolates.
Table 3
Multi-drug resistant pattern of inpatient and outpatient S. pneumoniae isolates [n (%)]
Class of antibiotic
|
Resistance pattern*
|
Inpatient isolates
(n = 140)
|
Outpatients isolates
(n = 140)
|
5
|
Macrolides/β-lactams/tetracyclines/chloramphenicols/sulfonamides
|
3 (2.1%)
|
7 (5.0)
|
4
|
Macrolides/tetracyclines/chloramphenicols/sulfonamides
|
1 (0.7%)
|
5 (3.6%)
|
Macrolides/β-lactams/tetracyclines/sulfonamides
|
109 (77.9%)
|
52 (37.1%)
|
Macrolides/β-lactams/tetracyclines/chloramphenicols
|
1 (0.7%)
|
0
|
Macrolides/β-lactams/sulfonamides/chloramphenicols
|
1 (0.7%)
|
0
|
3
|
Macrolides/β-lactams/tetracyclines
|
7 (5.0%)
|
9 (6.4%)
|
Macrolides/β-lactams/sulfonamides
|
3 (2.1%)
|
3 (2.1%)
|
Macrolides/tetracyclines/sulfonamides
|
4 (2.9%)
|
40 (28.6%)
|
Macrolides/tetracyclines/chloramphenicols
|
0
|
3 (2.1%)
|
Total
|
——
|
129 (92.1%)
|
119 (85.0%)
|
* The breakpoint of penicillin was based on the parenteral non-meningitis. |
MLST
The MLSTs of the inpatient and outpatient isolates were shown in table 4. Among the inpatient isolates, forty-five STs were detected, the predominant STs were ST271 (24.3%, 34/140), ST320 (19.3%, 27/140), ST81 (7.1%, 10/140), ST876 (7.1%, 10/140) and ST3397 (5.7%, 8/140), which were mainly associated with serotypes 19F, 19A, 23F, 14 and 15B, respectively. Eighty-one STs were detected among the outpatient isolates, the predominant STs were ST81 (8.6%, 12/140), ST271 (7.9%, 11/140), and ST320 (6.4%, 9/140), which were associated with serotypes 23F, 19F, and 19A, respectively.
The population snapshot of the S. pneumoniae isolates as determined by eBURST analysis is shown in fig. 1. The eBURST analysis results found five CCs and twenty-four singletons among the inpatient isolates, fifteen CCs and forty-two singletons among the outpatient isolates. CC271 was the most common CC bonth in the inpatient isolates and outpatient isolates. Among the inpatient isolates, thirteen STs were newly assigned (9765, 9781 - 9786, 9790, 9791, and 10136) via MLST analysis; three of the new STs (9765, 9781, and 9782) were novel combinations of known alleles, whereas the remainder had new local alleles (i.e., aroE 296, gdh 420, gdh 405, gdh 421, xpt 553, ddl 619, and ddl 620). Among the outpatient isolates, twenty-seven were newly assigned (10080 - 10106) via MLST analysis. Sixteen of the new STs (10080 - 10095) were novel combinations of known alleles, whereas the remainder contained new local alleles (gdh 431 - 433, gki 439, spi 423 - 425, xpt 594 - 595, and ddl 638 - 639).
The size of the dot was proportional to the number of strains included in the ST. The line represent that there was a single site mutation between the two clones. The black ST numbers represent the isolates from inpatients, the green ST numbers represent the isolates from outpatients, and the purple ST numbers represent the isolates from the two patients group.
Table 4
MLST of inpatient and outpatient S. pneumoniae isolates [n (%)]
MLST
|
Inpatient isolates
(n = 140)
|
Outpatient isolates
(n = 140)
|
271
|
34 (24.3%)
|
11 (7.9%)
|
320
|
27 (19.3%)
|
9 (6.4%)
|
81
|
10 (7.1%)
|
12 (8.6%)
|
876
|
10 (7.1%)
|
3 (2.1%)
|
3397
|
8 (5.7%)
|
6 (4.3%)
|
others
|
51 (36.4%)
|
99 (70.7%)
|