Participant population and baseline characteristics
Six hundred and fifty-one isolates (406 E. coli and 245 Klebsiella spp) were isolated from 568 children and subjected to AST (Figure 1). Among the 568 children, 343 (60.4%) were discharged from Kisii Teaching and Referral Hospital (KTRH) and 103 (39.6%) were discharged from Homabay County Referral Hospital (HCRH), 348 (60%) were less than two years of age and 348 (61.3%) were female (Table 1). Prior to discharge, the median duration of hospitalization was 3 days [IQR 2,6 days] with 203 (35.7%) children being hospitalized for ≥ 5 days, 12 (2.1%) were HIV infected while 70 (12.3%) were HIV exposed. There were more HIV-exposed children in HCRH 51 (22.7%) compared to KTRH 19, (5.5%). Common diagnoses at discharge were pneumonia 164 (28.9%), malaria 155 (27.3%), and diarrhoea 108 (19%) (Table 1). The majority of the children 502 (88.4%) had an antibiotic prescribed during their hospitalization with penicillins 359 (63.2%) being the most prescribed antibiotic followed by gentamicin 316 (55.6%) and ceftriaxone 187 (32.9%). Fluoroquinolones were rarely administered during hospitalization with ciprofloxacin being prescribed to only 2 (0.9%) children in HCRH and none among children from KTRH. Children at KTRH 328 (95.6%) compared to HCRH 174, (88.4%) were more likely to be prescribed an antibiotic. Nearly half of the children, 266 (46.8%) had ciprofloxacin non-susceptible isolates (46.3% E. coli and 37.6% Klebsiella spp). Of the 83 children who had both E. coli and Klebsiella isolated, 14 (2.46%) had ciprofloxacin non-susceptibility in both isolates.
Table 1: Participant Baseline Characteristics
|
Kisii
|
Homa Bay
|
Total
|
n
|
N:343
|
N:225
|
N:568
|
Sociodemographic
|
Age(months)
|
1 to 5
|
53
|
15.5%
|
20
|
8.9%
|
73
|
12.9%
|
6 to 11
|
73
|
21.3%
|
44
|
19.6%
|
117
|
20.6%
|
12 to 23
|
87
|
25.4%
|
71
|
31.6%
|
158
|
27.8%
|
24 to 59
|
130
|
37.9%
|
90
|
40%
|
220
|
38.7%
|
Sex
|
Male
|
205
|
59.8%
|
133
|
59.1%
|
338
|
59.5%
|
Female
|
138
|
40.2%
|
92
|
40.9%
|
230
|
40.5%
|
Duration of Hospitalization(days)
|
0 to 2
|
100
|
29.2%
|
66
|
29.3%
|
166
|
29.2%
|
2 to 4
|
121
|
35.3%
|
73
|
32.4%
|
194
|
34.2%
|
>5
|
117
|
34.1%
|
86
|
38.2%
|
203
|
35.7%
|
Unknown a
|
5
|
1.5%
|
0
|
0
|
5
|
0.9%
|
median (25%, 75%)
|
3
|
(2, 6)
|
4
|
(2, 6)
|
3
|
(2, 6)
|
HIV Status
|
HIV unexposed
|
309
|
90.1%
|
164
|
72.9%
|
473
|
83.3%
|
HIV exposed, uninfected
|
19
|
5.5%
|
51
|
22.7%
|
70
|
12.3%
|
HIV infected
|
6
|
1.7%
|
6
|
2.7%
|
12
|
2.1%
|
HIV-uninfected/exposure status unknown
|
9
|
2.6%
|
4
|
1.8%
|
13
|
2.3%
|
Diagnosis at Discharge b, c
|
Diarrhea
|
65
|
19%
|
43
|
19.1%
|
108
|
19%
|
Lower respiratory tract infection
|
117
|
34.1%
|
47
|
20.9%
|
164
|
28.9%
|
Malaria
|
68
|
19.8%
|
87
|
38.7%
|
155
|
27.3%
|
Malnutrition
|
24
|
7%
|
18
|
8%
|
42
|
7.4%
|
Pneumonia
|
117
|
34.1%
|
47
|
20.9%
|
164
|
28.9%
|
Upper respiratory tract infection
|
36
|
10.5%
|
10
|
4.4%
|
46
|
8.1%
|
Any antibiotic used during admission (enrollment visit)
|
Any antibiotics used
|
328
|
95.6%
|
174
|
77.3%
|
502
|
88.4%
|
Azithromycin
|
2
|
0.6%
|
2
|
0.9%
|
4
|
0.7%
|
Ceftriaxone
|
102
|
29.7%
|
85
|
37.8%
|
187
|
32.9%
|
Penicillin
|
268
|
78.1%
|
91
|
40.4%
|
359
|
63.2%
|
Gentamicin
|
241
|
70.3%
|
75
|
33.3%
|
316
|
55.6%
|
Ciprofloxacin
|
0
|
0
|
2
|
0.9%
|
2
|
0.4%
|
a Missing either admission or discharge dates (n=5)
|
b Diagnosis are not mutually exclusive
c No documented diagnosis at discharge (n=23)
|
Correlates for carriage of fluoroquinolone non-susceptible E. coli or Klebsiella at Hospital discharge.
Children who received an antibiotic during hospitalization were 69% more likely to have a ciprofloxacin non-susceptible E. coli isolate (PR 1.69, [95%CI=1.09, 2.63], p=0.01) and over two times more likely to have a ciprofloxacin non-susceptible Klebsiella spp isolate (PR 2.61, [95%CI=1.05, 6.53], p=0.01). The presence of ESBL carriage was also associated with the presence of either a ciprofloxacin non-susceptible E. coli or Klebsiella spp isolate. Length of hospital stay was associated with ciprofloxacin non-susceptible E. coli and children with hospitalizations extending 4 or more days were nearly 40% more likely to have ciprofloxacin non-susceptible E. coli (PR 1.38 [95%CI 1.07, 1.78] p= 0.01). The use of either ciprofloxacin or ceftriaxone were equally associated with ciprofloxacin non-susceptible Klebsiella or E. coli. Children hospitalized for diarrhoea were 27% less likely to have a ciprofloxacin non-susceptible E. coli compared to those who did not present with diarrhea (PR 0.73 [95%CI 0.53, 1.0] p=0.03). Similar magnitudes of association for hospital length and diarrhea diagnosis were observed in Klebsiella isolates but were not statistically significant. (Table S1)
Distribution of ciprofloxacin non-susceptible isolates.
Among the 266 children included in this study, we isolated 188 and 92 ciprofloxacin-susceptible E. coli and Klebsiella spp, respectively, totaling 280 ciprofloxacin non-susceptible isolates. Of the 266 children, 14 had both a ciprofloxacin non-susceptible E. coli and Klebsiella spp isolated. Among the 92 Klebsiella spp, 86 were Klebsiella pneumoniae and 6 were Klebsiella oxytoca (Figure 1).
Table 2: Distribution of MIC (µg/mL) per organism
|
Frequency of isolates with indicated MIC value
|
CIP MIC50 (µg/mL)
|
CIP MIC90(µg/mL)
|
MIC values (µg/mL)
|
0.25
|
0.5
|
1
|
2
|
4
|
8
|
16
|
32
|
|
|
Bacterial species
|
|
|
|
|
|
|
|
|
E. coli
|
2
|
51
|
9
|
7
|
10
|
2
|
5
|
102
|
32
|
32
|
K. oxytoca
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
4
|
32
|
32
|
K. pneumoniae
|
1
|
10
|
23
|
23
|
2
|
1
|
2
|
24
|
2
|
32
|
No. of occurrences(%)
|
4
(1.4)
|
61 (21.8)
|
32 (11.4)
|
30
(10.7 )
|
12 (4.3)
|
4 (1.4)
|
7
(2.5)
|
130 ( 46.4)
|
|
|
CIP MICs values for the 280 ciprofloxacin non-susceptible isolates (188 E. coli and 92 Klebsiella [86 Klebsiella pneumoniae and 6 Klebsiella oxytoca) ranged between 0.25 – 32 µg/mL with CIP MIC50 (µg/mL) of 32 µg/mL. Of the 280 isolates, 214 (76.4%) were resistant while 61 (21.8 %) were intermediate. Among the resistant isolates, high-level CIP resistance (MIC ≥32µg/mL) was common in almost half of the isolates (46.4%) most commonly among E. coli 102 (54.3%) and slightly less in Klebsiella spp (28/92, 30.4%). The CIP MIC distribution and MIC50 among the isolates are shown in Table 2.
Distribution of PMQR determinants
Six different PMQR determinants: qnr (qnrB, and qnrS), enzyme modifying aac(6′)-Ib-cr, and efflux pumps (qepA, oqxA, and oqxB) were detected (Table 3). At least one of the PMQR genes was detected in nearly all (224/280, 80%) of the screened isolates. Most E. coli and Klebsiella spp (40%) isolates had at least one qnr determinant detected. Of the qnr genes, qnrB was the most commonly detected qnr gene (20/188, 11%) and 47/92(51%) in E. coli and Klebsiella spp, respectively. In E. coli, qnrS was the most detected qnr gene (27/188, 14%). qnrA was not detected in any of the E. coli or Klebsiella spp isolates.
Table 3: Distribution of PMQR determinants per organism
|
E. coli
|
K. oxytoca
|
K. pneumoniae
|
Total
|
PMQR genes
|
N=188
|
N=6
|
N=86
|
N=280
|
qnrB
|
20 (11%)
|
2 (33%)
|
45 (52%)
|
67 (24%)
|
qnrS
|
27 (14%)
|
1 (17%)
|
18 (21%)
|
46 (16%)
|
aac(6′)-Ib-cr
|
89 (47%)
|
5 (83%)
|
73 (85%)
|
167 (59%)
|
qepA
|
16 (9%)
|
0 (0%)
|
0 (0%)
|
16 (6%)
|
OqxA
|
2 (1%)
|
4 (67%)
|
83 (97%)
|
89 (32%)
|
OqxB
|
0 (0%)
|
3 (50%)
|
59 (69%)
|
62 (22%)
|
OqxAB
|
0 (0%)
|
3 (50%)
|
58 (67%)
|
61 (22%)
|
The most predominant plasmid-mediated quinolone resistance gene was aac-(6’)-lb (167/280, 59%) identified in more than half of all fluoroquinolone non-susceptible isolates. Klebsiella spp had more aac(6’)-lb positive isolates with (78/89, 85%) Klebsiella compared to (89/188, 47%) of E. coli isolates. All isolates carrying the aac(6’)-lb gene were positive for the cr variant. qepA was only detected in (16/188, 9%) E. coli fluoroquinolone non-susceptible isolates (Figure 2). The oqxAB complex was the most dominant efflux pump detected (61/92, 66.3%) in Klebsiella, however, none were detected in E. coli. Only (2/188, 1%) E. coli isolates had the oqxA gene, however, both of these isolates lacked the oqxB gene therefore none of the E. coli isolates carried the oqxAB complex.
QepA sequence analysis
DNA sequencing of the qepA gene from 16 E. coli isolates revealed amino acid substitutions at codons 95 and 134. Double amino acid substitutions F95L and V134I were common in 9/16 (56.3%) E. coli isolates. Six qepA positive E. coli isolates carried had no amino acid substitution while one isolate had only V134I amino acid substitution (Table 4). Assigned accession numbers for the qepA gene from 8 representative isolates submitted to GenBank are as follows: ACC.No:OP918677, and ACC.No OQ031499-OQ031505 (sequences awaiting processing by NCBI). Additional details (See Additional file 4)
Table 4: MICs and amino acid changes in qepA E. coli isolates
Isolate ID
|
Accession No.
|
Site
|
Organism
|
CIP MIC
|
INTER
|
qepA Variants
|
Amino acid variation
|
F95
|
V134
|
E24
|
OQ031502
|
Kisii
|
E. coli
|
32
|
R
|
-
|
F95
|
V134I
|
E28
|
OQ031503
|
Kisii
|
E. coli
|
2
|
R
|
qepA1
|
F95
|
V134
|
E40
|
OQ031504
|
Kisii
|
E. coli
|
2
|
R
|
qepA1
|
F95
|
V134
|
E42
|
OQ031505
|
Kisii
|
E. coli
|
32
|
R
|
qepA1
|
F95
|
V134
|
E49
|
OQ031499
|
Homabay
|
E. coli
|
32
|
R
|
qepA4
|
F95L
|
V134I
|
E66
|
OP918677
|
Kisii
|
E. coli
|
32
|
R
|
qepA4
|
F95L
|
V134I
|
E79
|
OQ031500
|
Homabay
|
E. coli
|
32
|
R
|
qepA4
|
F95L
|
V134I
|
E80
|
OQ031501
|
Homabay
|
E. coli
|
32
|
R
|
qepA4
|
F95L
|
V134I
|
Distribution of co-carriage of PMQR determinants per organism
A total of 225/280 (80%) isolates had at least one PMQR gene including; all 92 Klebsiella spp and most E. coli 133/189 (70.37%). Interestingly, qnrB and qnrS co-occurred in two Klebsiella spp (0.71%) isolates. Co-carriage of qnrB with acc (6’) lb-cr was present in 12/188 (6.4%) E. coli and aac(6′)-Ib-cr with oqxAB detected in 47/92 (51%) Klebsiella spp were the most prevalent combination of PMQR gene combinations. E. coli isolates had three notable combinations of different PMQR determinants with qnr combination being predominant in the co-existence of genes. On the other hand, Klebsiella spp had as many as nine different combinations and similarly, qnr gene combinations were predominant in the different combinations of determinants. The most common co-carriage in both bacterial species was qnrB with acc (6’) lb-cr found in 56/280 (20%). Additionally, acc (6’) lb-cr co-existed with a majority of PMQR genes in both E. coli and Klebsiella spp isolates (Table 5).
Table 5: Co-carriage of PMQR determinants per organism
|
Organism
|
PMQR genes present
|
E. coli
|
Klebsiella spp
|
No. of occurrences
|
At least one PMQR
|
133/188
|
92/92
|
225/280
|
qnrB + qnrS
|
0
|
2
|
2
|
qnrB + qnrS + aac(6′)-Ib-cr
|
0
|
1
|
1
|
qnrB + qnrS + aac(6′)-Ib-cr + oqxAB
|
0
|
1
|
1
|
qnrB + aac(6′)-Ib-cr
|
12
|
44
|
56
|
qnrB + acc (6’) lb-cr + oqxAB
|
0
|
25
|
25
|
qnrB + oqxAB
|
0
|
13
|
13
|
aac(6′)-Ib-cr + oqxAB
|
0
|
47
|
47
|
qnrS + aac(6′)-Ib-cr
|
6
|
15
|
21
|
qnrS + aac(6′)-Ib-cr + oqxAB
|
0
|
10
|
10
|
qepA + qnrS
|
3
|
0
|
3
|
qepA + aac(6′)-Ib-cr
|
3
|
0
|
3
|