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). The MSSA carriage rate was 13.3% (99/742).
Spa Types, resistance patterns and antibiotypes
In this study, all the isolates were susceptible to rifampicin, vancomycin and linezolid, while all the MRSA were susceptible to vancomycin, linezolid and clindamycin (Tables S1 & S2). Compared to MSSA isolates (68.8%, 99/144), MRSA isolates were more resistant to non-beta-lactam antimicrobials i.e. trimethoprim/sulfamethoxazole (co-trimoxazole) 73.3% (33/45) vs. 27.3% (27/99) [p<0.0001], erythromycin 75.6% (34/45) vs. 24.2% (24/99) [p<0.0001], chloramphenicol 60% (27/45) vs. 19.2% (19/99) [p<0.0001], gentamicin 55.6% (25/45) vs. 25.3% (25/99) [p=0.0004] and ciprofloxacin 35.6% (16/45) vs. 2% (2/99) [p<0.0001]. 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). The proportion of PVL+ MSSA and PVL+ MRSA was 9/99 (9.1%) and 19/45 (13.2%), respectively. All PVL+ 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 (Table 1). A general description of the number of clusters observed on PFGE analysis and diversity of the collection is shown in Figure S1.
Table 1: Frequency of spa types among S. aureus isolates 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
|
9
|
20
|
15
|
10.4
|
= 0.0118
|
t645
|
06 (55.4)
|
05 (45.5)
|
11
|
11.1
|
0
|
0
|
11
|
7.6
|
= 0.0205
|
t4353
|
06 (60)
|
04 (40)
|
9
|
9.1
|
1
|
2.2
|
10
|
7
|
= 0.1324
|
t002
|
06 (85.7)
|
01 (14.3)
|
6
|
6.1
|
1
|
2.2
|
7
|
5
|
= 0.3158
|
t037
|
06 (85.7)
|
01 (14.3)
|
0
|
0
|
7
|
15.6
|
7
|
5
|
= 0.0001
|
t078
|
-
|
-
|
2
|
2
|
0
|
0
|
2
|
1.4
|
|
t355
|
-
|
-
|
1
|
1
|
1
|
2.2
|
2
|
1.4
|
|
t3092
|
-
|
-
|
1
|
1
|
1
|
2.2
|
2
|
1.4
|
|
t12939
|
-
|
-
|
0
|
0
|
1
|
2.2
|
1
|
0.7
|
|
t3662
|
-
|
-
|
1
|
1
|
0
|
0
|
1
|
0.7
|
|
t318
|
-
|
-
|
1
|
1
|
0
|
0
|
1
|
0.7
|
|
t1456
|
-
|
-
|
1
|
1
|
0
|
0
|
1
|
0.7
|
|
t10394
|
-
|
-
|
1
|
1
|
0
|
0
|
1
|
0.7
|
|
t1476
|
-
|
-
|
1
|
1
|
0
|
0
|
1
|
0.7
|
|
t2168
|
-
|
-
|
1
|
1
|
0
|
0
|
1
|
0.7
|
|
t213
|
-
|
-
|
1
|
1
|
0
|
0
|
1
|
0.7
|
|
Unknown
|
-
|
-
|
4
|
-
|
0
|
0
|
4
|
-
|
|
NT
|
-
|
-
|
17
|
-
|
4
|
-
|
21
|
-
|
|
ND
|
-
|
-
|
35
|
-
|
20
|
-
|
55
|
-
|
|
Total
|
-
|
-
|
99
|
-
|
45
|
-
|
144
|
-
|
|
The predominant spa types are depicted in bold font. RF denotes Relative Frequency (%) i.e., the number of times that the event (i.e. spa lineage) occurred, divided by the total in that category. NT, not type-able; ND, not determined.
Ninety nine (68.8%, 99/144) isolates were MSSA as they were cefoxtin susceptible and mecA negative (Tables S1 & S2). The percentage and proportion of MSSA isolates resistant to penicillin, tetracycline, cotrimoxazole, erythromycin, gentamicin and chloramphenicol are 78.8% (78/99), 79.8% (79/99), 27.3% (27/99), 24.2% (24/99), 25.3% (25/99) and 19.2% (19/99), respectively. Three (3%, 3/99) MSSA isolates (K277-1, K251 and 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). 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). There were other spa types that exclusively occurred in MSSA e.g. t078, t3662, t318, t1456, t10394, t1476, t2168 and t213 (Table 1) but their frequencies were too low (i.e. ≤2 occurrences) to allow meaningful analysis.
Table 2 summarizes the antibiotypes and their relationship with spa and SCCmec types. Overall, a total of 38 and 28 antibiotypes were detected among MSSA and MRSA isolates, respectively. 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 and MRSA and their relationship with spa types
|
Antibiotype
|
Resistance profile
|
No. 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, gentamicin; 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 from Mulago Hospital in Kampala, but slightly different from rural Western Uganda (Figure S2). The number of isolates from the other sites (i.e. Mulago Hospital and rural Western Uganda) were as follows: 105 (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 and 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 and t037 (IMHDSS); t645, t4353, t064, t084, t355, t3772 and t4609 (Mulago Hospital); and t318, t064, t645, t186, t11656, t127, t786 and t2771 (rural Western Uganda) (Figure S2). Spa type t037 exclusively occurred in MRSA and it was detected only at IMHDSS and Mulago Hospital. On the other hand, spa types t645 and 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 and t355 were neither associated with MRSA or MSSA. Interestingly, spa type t064 that was significantly associated with MRSA at IMHDSS was not associated with MRSA at Mulago Hospital (Table S3).