MSSA and MRSA carriage rates
The processed samples yielded 600 Gram positive and catalase positive isolates (one isolate per sample/child), of which 144 were confirmed to be S. aureus (one isolate per sample/child) (Figure 1 and Tables S1 & S2). Thus, the nasopharyngeal carriage rate of S. aureus in the children was 19.4% (144/742). Forty five (31.3%, 45/144) of the isolates were confirmed to be MRSA yielding a carriage rate of 6.1% (45/742) in the children.
Spa Types, resistance patterns and antibiotypes
All MRSA isolates were susceptible to vancomycin, linezolid and generally to clindamycin though two exhibited intermediate resistance to this drug (Tables S1 & S2). Compared to MSSA, MRSA isolates were more resistant to non-β-lactam antimicrobial agents i.e. tetracycline (91.1%, 41/45), SXT (73.3%, 33/45), erythromycin (75.6%, 34/45), chloramphenicol (60%, 27/45), gentamicin (55.6%, 25/45) and ciprofloxacin (35.6%, 16/45). One MRSA isolate (K2283) exhibited high-level mupirocin resistance (HLMupr) while 42 (93.3%, 42/45) were MDR including the mupirocin resistant isolate (Tables S1 & S2). Again, compared to MSSA, slightly more MRSA isolates (13.2%, 19/45) carried the PVL genes but the difference was not statistically significant (P=0.4562). All PVL-positive MRSA isolates were MDR and generally the presence of PVL genes was associated with the MDR phenotype (P=0.0332). Seven spa types (t064, t4353, t002, t037, t355, t3092 and t12939) were detected among MRSA isolates, of which t064 (20%, 9/45) and t037 (15.6%, 7/45) were predominant (Tables S1 & S2). Spa types t037 and t064 were significantly associated with MRSA and SCCmec types I & IV respectively, with t037 exclusively occurring in MRSA isolates, Table 1. A general description of the number of clusters observed on PFGE analysis, and the diversity of the collection, is shown in Figure S1.
Table 1: Distribution of spa types in S. aureus from children in IMHDSS, Eastern Uganda
Spa type
|
MDR (%)
|
MSSA
|
MRSA
|
Total
|
P-value
|
Yes
|
No
|
Frequency
|
RF
|
Frequency
|
RF
|
Frequency
|
RF
|
|
t064
|
10 (66.7)
|
05 (33.3)
|
06
|
6.1
|
09
|
20
|
15
|
10.4
|
P = 0.0118
|
t645
|
06 (55.4)
|
05 (45.5)
|
11
|
11.1
|
0
|
0
|
11
|
7.6
|
P = 0.0205
|
t4353
|
06 (60)
|
04 (40)
|
09
|
9.1
|
01
|
2.2
|
10
|
7
|
P = 0.1324
|
t002
|
06 (85.7)
|
01 (14.3)
|
06
|
6.1
|
01
|
2.2
|
07
|
5
|
P = 0.3158
|
t037
|
06 (85.7)
|
01 (14.3)
|
0
|
0
|
07
|
15.6
|
07
|
5
|
P = 0.0001
|
t078
|
-
|
-
|
02
|
2
|
0
|
0
|
02
|
1.4
|
|
t355
|
-
|
-
|
01
|
1
|
01
|
2.2
|
02
|
1.4
|
|
t3092
|
-
|
-
|
01
|
1
|
01
|
2.2
|
02
|
1.4
|
|
t12939
|
-
|
-
|
0
|
0
|
01
|
2.2
|
01
|
0.7
|
|
t3662
|
-
|
-
|
01
|
1
|
0
|
0
|
01
|
0.7
|
|
t318
|
-
|
-
|
01
|
1
|
0
|
0
|
01
|
0.7
|
|
t1456
|
-
|
-
|
01
|
1
|
0
|
0
|
01
|
0.7
|
|
t10394
|
-
|
-
|
01
|
1
|
0
|
0
|
01
|
0.7
|
|
t1476
|
-
|
-
|
01
|
1
|
0
|
0
|
01
|
0.7
|
|
t2168
|
-
|
-
|
01
|
1
|
0
|
0
|
01
|
0.7
|
|
t213
|
-
|
-
|
01
|
1
|
0
|
0
|
01
|
0.7
|
|
Unknown
|
-
|
-
|
04
|
-
|
0
|
0
|
04
|
-
|
|
NT
|
-
|
-
|
17
|
-
|
04
|
-
|
21
|
-
|
|
ND
|
-
|
-
|
35
|
-
|
20
|
-
|
55
|
-
|
|
Total
|
-
|
-
|
99
|
-
|
45
|
-
|
144
|
-
|
|
The predominant spa types are depicted in bold font. RF denotes Relative Frequency (%).
Ninety nine (68.8%, 99/144) isolates were MSSA as they were cefoxtin susceptible and mecA negative (Tables S1 & S2). Nevertheless, MSSA isolates were highly resistant to penicillin (78.8%, 78/99), tetracycline (79.8%, 79/99), SXT (27.3%, 27/99), erythromycin (24.2%, 24/99), gentamicin (25.3%, 25/99) and chloramphenicol (19.2%, 19/99) but compared to MRSA, they were less resistant to ciprofloxacin i.e. 2% (2/99) vs. 35.6% (16/45) for MSSA & MRSA isolates, respectively. Three (3%, 3/99) MSSA isolates (K277-1, K251 & K1064) exhibited high-level mupirocin resistance and were also clindamycin resistant (Tables S1 & S2). A total of 61 (61.6%, 61/99) MSSA were MDR including the three mupirocin resistant isolates (Tables S1 & S2). Generally, all isolates (MSSA & MRSA) were susceptible to rifampicin, vancomycin and linezolid but only three MSSA (R16, R180 & R716) were pan-susceptible to antibiotics. Fourteen spa types were detected among MSSA, of which t645 (11.1%, 11/99), t4353 (9.1%, 9/99), t064 (6.1%, 6/99) and t002 (6.1%, 6/99) were predominant (Tables S1 & S2). Spa type t645 exclusively occurred in MSSA (Table 1).
Overall, a total of 38 and 28 antibiotypes were detected among MSSA and MRSA isolates, respectively. Table 2 summarizes the antibiotypes and their relationship with spa types and SCCmec types. The most prevalent antibiotypes in MSSA and MRSA had the resistance patterns PEN-TET (17.2%) and FOX-PEN-TET-SXT-ERY-CHL-GEN (15.6%), respectively (Table 2).
Table 2: Antibiotypes among MSSA & MRSA and their relationship with Spa types
|
Antibiotype
|
Resistance profile
|
# isolates showing this pattern (%)
|
Major Spa types (frequency)
|
SCCmec type (frequency)
|
MSSA
|
S1
|
PEN-TET
|
17 (17.2)
|
t064 (4), t4353 (3), t645 (2), t355 (1)
|
Not applicable
|
S2
|
PEN-TET-ERY
|
11 (11.1)
|
t002 (3),
t645 (1), t078 (1), t4353 (1), t2168 (1)
|
S3
|
PEN-TET-GEN
|
9 (9.1)
|
t318 (1), t213 (1), t1476 (1)
|
S4
|
PEN
|
6 (6.1)
|
t002 (1), t645 (1), t4353 (1)
|
S5
|
TET
|
6 (6.1)
|
t4353 (1)
|
S6
|
PEN-TET-CHL-GEN
|
4 (4.4)
|
t645 (1)
|
S7
|
PEN-TET-SXT
|
4 (4.4)
|
t002 (1), t1456 (1)
|
S8
|
PEN-TET-SXT-CHL
|
3 (3)
|
-
|
S9
|
PEN-SXT-ERY-CLI-MUP
|
3 (3)
|
-
|
S10
|
PEN-TET-CHL
|
2 (2)
|
t645 (1), t4353 (1)
|
S11
|
TET-SXT-CHL
|
2 (2)
|
t3662 (1), t10394 (1)
|
S12
|
PEN-TET-ERY-CHL
|
2 (2)
|
t064 (1), t3092 (1)
|
S13
|
PEN-SXT
|
2 (2)
|
t4353 (1)
|
S14
|
PEN-TET-SXT-ERY
|
2 (2)
|
t645 (1)
|
S38
|
- (Pan-susceptible)
|
3 (3)
|
t064 (1)
|
MRSA
|
R1
|
PEN-FOX-TET-SXT-ERY-CHL-GEN
|
7 (15.6)
|
- (4), t064 (3)
|
I (4), IV (3)
|
R2
|
PEN-FOX-TET-SXT-ERY-CHL-GEN-CIP
|
4 (9)
|
-
|
I (3), IV (1)
|
R3
|
PEN-FOX-TET-SXT-ERY-CHL-GEN
|
4 (9)
|
-
|
I (3), IV (1)
|
R4
|
PEN-PEN-TET-SXT-ERY
|
3 (6.7)
|
t002 (1), t064 (1)
|
I (1), II (1), IV (1)
|
R5
|
PEN-FOX-TET-SXT-ERY-CIP
|
3 (6.7)
|
t064 (1)
|
IV (3)
|
R6
|
PEN-FOX-TET-SXT-ERY-GEN
|
2 (4.4)
|
t064 (1)
|
I (1), IV (1)
|
Shown are antibiotypes depicted by two or more isolates. Antibiotypes depicted by only one isolate are shown in Table S1. FOX, cefoxitin; PEN, penicillin; TET, tetracycline; SXT, trimethoprim/sulfamethoxazole or co-trimoxazole; ERY, erythromycin, CHL, chloramphenicol; GEN, gentamycin; CIP, ciprofloxacin; CLI, clindamycin; RIF, rifampicin; MUP, Mupirocin High level; VAN, vancomycin; LZD, linezolid; MSSA, Methicillin susceptible S. aureus; MRSA, Methicillin resistant S. aureus.
When the genotypes of S. aureus isolates were compared with previously characterized isolates in Uganda, it was observed that the spa types detected at IMHDSS were previously reported for S. aureus isolates from Mulago Hospital in Kampala, but they were slightly different from spa types for isolates from rural Western Uganda (Figure S2). The total number of isolates from the two other sites was105 (Mulago Hospital, 64 & 41 from Seni et al [9] & Kateete et al [8], respectively) and 73 (rural Western Uganda) [18]; of these, MRSA isolates were 113 (65 Mulago Hospital & 48 rural Western Uganda). Overall, 40 spa types accounted for MSSA/MRSA clones, of which t645, t064, t4353, t002, t318, t037, t355, t084, t3772, t127 and t186 were predominant (Figure 2 & Table S3). The frequent spa lineages in each of the sites were t064, t645, t4353, t002 & t037 (IMHDSS); t645, t4353, t064, t084, t355, t3772 & t4609 (Mulago Hospital); and t318, t064, t645, t186, t11656, t127, t786 & t2771 (rural Western Uganda) (Figure S2). Spa type t037 exclusively occurred in MRSA and it was detected only at IMHDSS & Mulago Hospital. On the other hand, spa types t645 & t4353 occurred in all the three sites and they were significantly associated with MSSA (Table S3 & Figure 2). When isolates from the three sites were analyzed, spa types t4353, t002 & t355 were neither associated with MRSA nor MSSA; interestingly, spa type t064 that was significantly associated with MRSA at IMHDSS was not associated with MRSA at Mulago Hospital (Table S3).