Demographic characteristics
The characteristics of children sampled were described previously [10, 20]. Briefly, about half of the children were less than 2 years old and 50% were female with a mean age of 30 months. Majority (82%) of the children lived in rural areas and their primary source of water was spring water (74%) and well water (26%); only 1% of the children had access to piped water. The median number of children per household was 2 while the median number of household members per bedroom (as a measure of crowding) was 4. Based on reports of mothers 90% of the children had been sick in the previous 2 weeks and the most common symptoms were fever, running nose and cough. Of the children who had been sick around 30% were reported to have been given antibiotics, mostly cotrimoxazole and ampicillin.
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). Furthermore, 45 (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 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%, 34/99), chloramphenicol (60%, 19/99), 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 multidrug resistant (MDR –resistance to three or more classes of antimicrobials) including the mupirocin resistant isolate, Tables S1 & S2. Again, compared to MSSA, slightly more MRSA isolates (13.2%, 19/45) carried 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. On PFGE analysis isolates of spa type t064 clustered together, 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 not as resistant to ciprofloxacin (2%, 2/99). 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 and MRSA, were susceptible to rifampicin, vancomycin and linezolid but only three (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
|
FOX-PEN-TET-SXT-ERY-CHL-GEN
|
7 (15.6)
|
- (4), t064 (3)
|
I (4), IV (3)
|
R2
|
FOX-PEN-TET-SXT-ERY-CHL-GEN-CIP
|
4 (9)
|
-
|
I (3), IV (1)
|
R3
|
FOX-PEN-TET-SXT-ERY-CHL-GEN
|
4 (9)
|
-
|
I (3), IV (1)
|
R4
|
FOX-PEN-TET-SXT-ERY
|
3 (6.7)
|
t002 (1), t064 (1)
|
I (1), II (1), IV (1)
|
R5
|
FOX-PEN-TET-SXT-ERY-CIP
|
3 (6.7)
|
t064 (1)
|
IV (3)
|
R6
|
FOX-PEN-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 aureus; MRSA, Methicillin resistant S. aureus.
When we compared the genotypes of S. aureus isolates at the IMHDSS to genotypes of previously characterized isolates in Uganda, we found that the spa types we 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. Thus, the predominant lineages in each of the three settings 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. Overall, 40 spa types account for clinical and colonizing MSSA/MRSA clones circulating in Uganda, of which t645, t064, t4353, t002, t318, t037, t355, t084, t3772, t127 and t186 are predominant, Figure 2 & Table S3. Spa type t037 was detected only at Mulago Hospital & IMHDS but not in Western Uganda; again, it exclusively occurred in MRSA. Spa types t645 & t4353 occurred in all the three sites and they were significantly associated with MSSA, Table S3 & Figure 2. When we analyzed isolates from the three sites together, 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, Table S3.