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J. Clinical Pediatrics and Mother Health Copy rights@ André Léké.et al
Auctores Publishing – Volume 1(1)-008 www.auctoresonline.org Page 1 of 11
Coagulase-Negative Staphylococcal Bacteremia by Bacterial
Translocation of Gastrointestinal Origin in Preterm Infants: Role
of Molecular Analysis
André Léké1,6*, Géraldine Amar2, Bertin Elion Dzon3, Gilles Morin4, Guy Kongolo5 , Maurice Biendo6
1Soins intensifs de Néonatologie et Médecine Néonatale, CHU Amiens Picardie, site Sud 80054, Amiens Cedex 1 (France)
2Réanimation Néonatale, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, (France)
3Service de Nutrition paraentérale et de Chirurgie vasculaire, CHU Lille, (France)
4Génétique Clinique Pédiatrique, CHU, Amiens Picardie, site SUD 80054, Amiens, Cedex 1 (France)
5Réanimation et Surveillance Continue Pédiatrique, CHU Amiens Picardie, site SUD 80054, Amiens Cedex 1 (France)
6Laboratoire Péritox UMR 101 CURS-UPJV, F-80054, Amiens Cedex 1 (France)
*Corresponding Author: André Léké, MD, PhD, Soins Intensifs de Néonatologie et Médecine Néonatale, CHU Amiens Picardie Site Sud, 80054
Amiens Cedex 1, France.
Received Date: June 05, 2021; Accepted Date: July 05, 2021; Published Date: July 09, 2021
Citation: André Léké., Géraldine Amar., Bertin Elion Dzon., Gilles Morin., Guy Kongolo., et al. (2021) Coagulase-Negative Staphylococcal
Bacteremia by Bacterial Translocation of Gastrointestinal Origin in Preterm Infants: Role of Molecular Analysis. J. Clinical Pediatrics and Mother
kanamycin (R < 14 mm); G: gentamicin (S ≥ 22 mm, R < 22 mm); T: tobramycin (S ≥ 22 mm, R < 22 mm); N: netilmicin (S ≥ 22 mm, R < 22 mm);
E: erythromycin (S ≥ 21 mm, R < 18 mm); L: lincomycin (S ≥ 22 mm, R < 19 mm); PT: pristinamycin (S ≥ 21 mm, R < 18 mm); RIF: rifampin (S ≥
26 mm, R< 23 mm); OFX: ofloxacin (S ≥ 20 mm, R < 20 mm); Van: vancomycin (S ≤ 2 mg/L); FA: fusidic acid (S ≥ 24 mm, R < 24mm); SXT:
trimethoprim-sulfamethoxazole (S ≥ 17 mm); FOS: fosfomycin (S ≥ 6mm, R < 6 mm); DOX: doxycycline (S ≥ 22 mm; R < 19 mm). *MIC: minimum
inhibitory concentrations of VAN were determined using E-test strips (BioMérieux, Marcy l’Etoile, France)
Table 3: Susceptibility of coagulase-negative Staphylococcus strains isolated from blood samples
The distribution of resistance patterns of these isolates showed sixteen antimicrobial resistance patterns (R patterns) a-p, and 10 of these strains exhibited
R pattern e [(35.7%) (isolates 5-11,13-15)] (Table 4).
R patterns Antimicrobial resistance Isolate numbers
a PR FOXR KR GR TR NR ER OFXR 1
b PR FOXR KR TR ER LR RIFR OFXR FAR 2
c PR FOXR KR GR TR NR ER OFXR 3
d PR FOXR KR GR TR NR ER LR RIFR OFXR 4, 24
e PR FOXR KR GR TR NR ER RIFR OFXR 5, 6, 7, 8, 9, 10, 11, 13, 14, 15,
f PR FOXR KR GR TR NR ER LR RIFR OFXR FOSR 12
g PR FOXR KR GR TR NR RIFR FOSR 16, 26
h PR FOXR KR GR TR NR ER RIFR OFXR FAR DOXR 17
i PR FOXR KR GR TR NR ER RIFR OFXR DOXR 18
j PR FOXR KR GR TR NR RIFR DOXR 19
k PR FOXR KR GR TR NR ER RIFR OFXR FOSR DOXR 20
l PR FOXR KR GR TR NR ER OFXR 21
m PR FOXR KR GR TR NR OFXR FAR FOSR 22
n PR FOXR KR ER FAR 23
o PR FOXR KR OFXR 25
p PR FOXR KR GR TR NR RIFR 27, 28
Sixteen antimicrobial resistance patterns were observed, 10 of which exhibited R pattern e [(35.7%) (Isolates 5-11, 13- 15)].
Table 4: Antimicrobial resistance patterns (R patterns) of the 28 strains isolated from blood samples
One hundred percent of the 28 CoNS strains isolated from stool samples were resistant to penicillin, cefoxitin, and kanamycin, and 96.4% of isolates
were resistant to gentamicin, tobramycin and netilmicin (Table 5).
Patient
No.
Antibiotics [Inhibition diameter (mm)]
Staphylococcus
Isolates P FOX K G T N E L PT RIF OFX VAN FA SXT FOS DOX
Resistance
phenotype
1 *S. epidermidis R R R R R R R S S S R S S S S S a
2 S. epidermidis R R R R R R R R S R R S R S S S b
3 S. haemolyticus R R R R R R R S S S R S S S S S c
J. Clinical Pediatrics and Mother Health Copy rights@ André Léké.et al
Auctores Publishing – Volume 1(1)-008 www.auctoresonline.org Page 6 of 11
4 S. epidermidis R R R R R R R R S R R S S S S S d
5 S. haemolyticus R R R R R R R S S R R S S S S S e
6 S. haemolyticus R R R R R R R S S R R S S S S S e
7 S. haemolyticus R R R R R R R S S R R S S S S S e
8 S. haemolyticus R R R R R R R S S R R S S S S S e
9 S. haemolyticus R R R R R R R S S R R S S S S S e
10 S. haemolyticus R R R R R R R S S R R S S S S S e
11 S. haemolyticus R R R R R R R S S R R S S S S S e
12 **CoNS R R R R R R R R S R R S S S R S f
13 CoNS R R R R R R R S S R R S S S S S e
14 CoNS R R R R R R R S S R R S S S S S e
15 S. haemolyticus R R R R R R R S S R R S S S S S e
16 CoNS R R R R R R R S S R R S S S R S e
17 CoNS R R R R R R R S S S R S S S S R g
18 S. haemolyticus R R R R R R R S S R R S S S S R h
19 CoNS R R R R R R R S S S R S S S S R g
20 S. haemolyticus R R R R R R R S S R R S S S S R h
21 CoNS R R R R R R R S S R S S S S S S l
22 CoNS R R R R R R S S S S R S R S R S j
23 CoNS R R R R R R S S S S S S S S S R k
24 S. epidermidis R R R R R R S S S R S S S S S S l
25 CoNS R R R S S S R S S S R S S S S S m
26 S. haemolyticus R R R R R R R S S R R S S S R S n
27 S. epidermidis R R R R R R R R S R S S S R S S o
28 CoNS R R R R R R S S S R R S S S S S p
One hundred percent of the 28 CoNS strains isolated from stool samples were resistant to penicillin cefoxitin, and kanamycin, and 96.4% of isolates
were resistant to gentamicin, tobramycin, and netilmicin.
Table 5. Susceptibility of coagulase-negative Staphylococcus strains isolated from stool samples
The resistance of these isolates to other antibiotics tested are shown in Table 5. All these isolates were classified into sixteen R patterns a-p, and 11 of
these strains exhibited R pattern e [(39.2%), (isolates 5-11,13-16)] (Table 6).
R patterns Antimicrobial resistance Isolate numbers
a PR FOXR KR GR TR NR ER OFXR 1
b PR FOXR KR GR TR NR ER LR RIFR OFXR FAR 2
c PR FOXR KR GR TR NR ER OFXR 3
d PR FOXR KR GR TR NR ER LR RIFR OFXR 4
e PR FOXR KR GR TR NR ER RIFR OFXR 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16
f PR FOXR KR GR TR NR ER LR RIFR OFXR FOSR 12
g PR FOXR KR GR TR NR ER OFXR DOXR 17, 19
h PR FOXR KR GR TR NR ER RIFR OFXR DOXR 18, 20
i PR FOXR KR GR TR NR ER RIFR 21
j PR FOXR KR GR TR NR OFXR FAR FOSR 22
J. Clinical Pediatrics and Mother Health Copy rights@ André Léké.et al
Auctores Publishing – Volume 1(1)-008 www.auctoresonline.org Page 7 of 11
k PR FOXR KR GR TR NR DOXR 23
l PR FOXR KR GR TR NR RIFR 24
m PR FOXR ER OFXR 25
n PR FOXR KR GR TR NR ER RIFR OFXR FOSR 26
o PR FOXR KR GR TR NR ER LR RIFR SXTR 27
p PR FOXR KR GR TR NR RIFR OFX R 28
All isolates were classified into 16 antimicrobial resistance patterns (designated R patterns (a to p), 11 of which exhibited R pattern e (39.2%).
Table 6: Antimicrobial resistance patterns (R patterns) of the 28 strains isolated from stool samples
Clinical/laboratory/
treatment
parameters
Documented
translocation
(n=15)
Undocumented
translocation
(n=13)
Wilcoxon-
Mann -Whitney
test (p-value)
Fisher’s exact test
(p-value)
Gestational age (weeks)
mean±SD
median (range)
28.6±3.35
28 (25-36)
29.9±3.40
29 (25-36)
P=0.27
Birth weight (g)
mean±SD
median (range)
1254.9±644.3
986 (592-2800)
1326.2±582.8
1192 (755-2730)
P=0.0098
Delivery mode
Vaginal
Cesarean
46.6% (7/15)
53.4% (8/15)
53.8% (7/13)
46.2% (6/13)
P=1
OR: 0.7878 95%CI
[0.1323; 4.2017]
Gastrointestinal
Disorders
80.0% (12/15)
84.6% (11/13) P=1
OR: 0.7355 95%CI
[0.0521; 7.7488]
Age at onset of
Infection (days)
mean±SD
median (range)
18.4±13.65
13(7-48)
1.5±28.61
9 (3-104)
P=0.015
CRP (mg/L)
mean±SD
median (range)
44.8±52.15
23(0-178)
30.5±31.35
23.7 (0-89)
P=0.59
Leukocytes (/mm3)
mean±SD
median (range)
15120±7799.0
12000 (1900-
34200)
39061.5±70588
18200 (5000-
271000)
P=0.042
Platelets (/mm3)
mean±SD
median (range)
142733±8385
136000(25000-
316000)
181000±11705
185000 (12000-
447000)
P=0.33
Lactate (mmol/L)
mean±SD
median (range)
6.36±1.30
6.8 (3.5-7.9)
4.04±0.90
4.0 (2.9-5.9)
P=0.0002
Lipid emulsion 12 (80%) 4 (30.7%) P=0.02
OR: 8.1821 95%CI
[1.2555; 73.4536]
Antenatal corticosteroid 14 (93.4%)
11 (84.6%)
P=0.58
OR: 2.4627 95% CI [0.1141; 160.529]
Proton pump inhibitor
13 (86.6%)
10 (76.9%)
P=1
OR: 0.6605 95% CI
[0.01; 14.4325]
Patent ductus arteriosus
7 (46.6%)
10 (76.9%)
P=0.03
OR: 0.0961 95% CI
[0.0018; 0.9895]
Hemodynamic disorders
10 (66.6%)
2 (15.3%)
P=0.009
OR: 9.9418 95% CI
[1.3872; 127.034]
History of jaundice
14 (93.3%) 7 (53.8%)
P=0.02
OR: 10.9341 5% CI
J. Clinical Pediatrics and Mother Health Copy rights@ André Léké.et al
Auctores Publishing – Volume 1(1)-008 www.auctoresonline.org Page 8 of 11
[1.0273; 587.9893]
Antenatal antibiotic
therapy
9 (60%) 7 (53.8%)
P=1
OR: 1.2742 95% CI
[0.2241; 7.3857]
Neonatal antibiotic
therapy
12 (80%) 4 (30.7%)
P=0.02
OR: 8.1821 95% CI
[1.2555; 73.4236]
APUH: Amiens Picardie University Hospital; SD: Standard Deviation; CRP: C-reactive protein; OR: Odds Ratio, CI: confidence interval;
RR: relative risk
Table 7: Rates and relative risk factors for digestive bacterial translocation in preterm infants hospitalized in APUH, classified according
to two groups: documented translocation and undocumented translocation
Comparison of blood culture and stool culture results
according to phenotype resistance pattern
In this series of 28 Staphylococcus isolates, blood culture results were
concordant with stool culture results in 53.5% (15/28) of cases and
discordant in 46.5% (13/28) of cases. Ten of the fifteen concordant strains
exhibited R pattern e and corresponded to eight S. haemolyticus and two
uCoNS isolates; five strains exhibited R patterns a, b, d, e, and f, and
corresponded to three S. epidermidis, one S. haemolyticus and one uCoNS
isolates, respectively. The following resistance patterns were detected on
blood cultures from the 12 discordant cases: g (S. capitis isolate), h
(uCoNS isolate), j (S. epidermidis isolate), k (S. haemolyticus isolate), l
(uCoNS isolate), m (uCoNS isolate), n (uCoNS isolate), d (S. haemolyticus
isolate), o (uCoNS isolate), g (uCoNS isolate), p (S. warneri and uCoNS
isolates). Similarly, the following R patterns were detected on stool
cultures from the 12 discordant cases: e (uCoNS isolate), g (uCoNS
isolate), h (S. haemolyticus isolate), i (uCoNS isolate), j (uCoNS isolate),
k (uCoNS isolate), l (S. epidermidis isolate), m (uCoNS isolate), n (S.
haemolyticus isolate), o (S. epidermidis isolate), and p (uCoNS isolate).
The S. epidermidis (R pattern i) and S. haemolyticus (R pattern h) strains
were isolated from blood culture and stool culture of patient 18,
respectively.
Molecular typing results
Phenotyping results suggested BT from the GIT to the circulatory system
in 15 preterm infants. When the same Staphylococcus spp. were isolated
from both stool and peripheral blood, and exhibited the same resistance
pattern, they were further genotyped by ERIC-PCR and RAPD-PCR to
confirm BT. Fifteen isolates were selected to obtain a diverse sample of
patients (blood and stool samples), and R patterns. These 15 selected
Staphylococcus strains were compared by ERIC-PCR and RAPD-PCR.
Three different ERIC-2 patterns (A, B, C) (Figure 1) and three different
RAPD patterns (D, E, F) (Figure 2) were identified in the 15 selected
isolates. ERIC-2 patterns comprised A [S. epidermidis (isolates 1, 2 and
4)]; B [S. haemolyticus (isolates 3,5-11, and 15)], and C [uCoNS (isolates
12-14)].
Figure 1: Representative ERIC- PCR types of 15 CoNS spp.isolated from blood cultures. Isolate numbers are Indicated below
(MW) are expressed in base pairs (bp). The 15 isolates were differentiated into three distinct ERIC-PCR types called A, B, and C.
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Figure 2: Representative RAPD-PCR types of 15 CoNS spp. isolated from stool cultures. Isolate numbers are indicated below [CoNS (Isolates 12-14);
S. haemolyticus (isolates 3, 5-11, 15); S. epidermidis (1, 2, 4)]. Pattern types are indicated below (D, E, F). Molecular weights (MW) are expressed in
base pairs (bp). The 15 CoNS spp. isolated were differentiated into three distinct RAPD-PCR types, called D, E and F.
RAPD patterns consisted of D [uCoNS (isolates 12-14)], E [S.
haemolyticus (isolates 3,5-11, and 15)], and F [S. epidermidis (isolates 1,
2, and 4)]. The three S. epidermidis R patterns a, b, and d exhibited the
AF genotype; The three other uCoNS strains with resistance patterns e and
f exhibited the CD genotype. Finally, nine S. haemolyticus phenotype e
strains exhibited the BE genotype. This major epidemic BE profile
included 60% of S. haemolyticus strains (9/15) isolated in both blood
culture and stool culture, and was identified in both units participating in
this study. The remaining strains (three S. epidermidis and three uCoNS)
exhibiting AF and CD genotypes, respectively, were considered to be
sporadic cases.
Combined analysis of ERIC-2 and RAPD results identified three different
genomic groups (gg): I to III. The strains isolated from blood culture and
stool culture in each group were more similar to each other than to the
other strains.
Bacterial Translocation results
Translocation from the GIT to the circulatory system was documented in
53.5% (15/28) of preterm infants. The same Staphylococcus spp. was not
found in blood and stool in 46.5% (13/28) of preterm infants, strongly
suggesting the absence of BT in these preterm infants. In patient 18, blood
culture was positive for S. epidermidis, and stool culture was positive for
S. haemolyticus, although culture of a nasopharyngeal sample taken prior
to the onset of bacteremia isolated S. epidermidis, suggesting that the
respiratory tract was the probable source of bacteremia in this child (this
source was not included in this study).
Risk factors for the occurrence of BT in preterm infants
with CoNS bacteremia
Comparison of documented (group1) and undocumented (group2)
gastrointestinal BT is shown in Table 7. Two tests, Wilcoxon-Mann-
Whitney test and Fisher’s Exact test, identified the presence of BT RFs,
such as: birth weight (p=0.0098); age at onset of infection (p=0.01);
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