Patients characteristics
The characteristics of 28 preterm infants included in this study were as follows: gestational age (weeks):mean ± SD:29.2 ± 3.3,median:29,range:25–36;birth weight (gram):mean ± SD:1,288 ± 606.2,median:1,075,range:592-2,800;age at onset of infection (days) :mean ± SD :19.8 ± 21.5 ,median :11, range :3-104 ; Lactate (µmol/L) :mean ± SD :38.2 ± 43.5 ,median :23.3 ,range :3.0-43.5 ;CRP (mg/L) :mean ± SD :3.28 ± 1.62,median:5.15,range :0-178 ;Leukocytes (/mm3):mean ± SD :26,775 ± 48,733.7 ,median :15,400 ,range :5,000-271,000 ; Platelets (/mm3) :mean ± SD :162,642.8 ± 98,180.5,median :54,500,range :60,000-286,000 (Table 1) ;maternal age at birth (years) :mean ± SD :29.7 ± 6.2 ,median :29.5,range :19–45.
Table 1
Characteristics of the 28 children with coagulase-negative Staphylococcus spp. included in the study
Patient No. | Gestational age*(WA) | Birth weight (g) | Age at onset of infection (days) | Gastrointestinal disorders | Lactates Mmol/L | *** CRP mg/L | Leukocytes /mm3 | Platelets /mm3 |
1 | 26 | 710 | 48 | yes | 7.2 | 0 | 9900 | 70000 |
2 | 25 + 4d** | 950 | 47 | yes | 7.3 | 5 | 9800 | 214000 |
3 | 36 | 2800 | 8 | yes | 5.5 | 53 | 9900 | 230000 |
4 | 26 + 6d | 700 | 8 | yes | 7.0 | 5,7 | 34200 | 173000 |
5 | 28 + 3d | 1200 | 30 | yes | 6.9 | 40 | 19900 | 65000 |
6 | 29 | 1370 | 9 | yes | 6.8 | 23 | 18400 | 200000 |
7 | 27 | 975 | 11 | yes | 7.5 | 62 | 25100 | 85000 |
8 | 28 + 5d | 960 | 13 | yes | 7.7 | 17 | 13400 | 103000 |
9 | 30 | 1060 | 7 | No | 3.5 | 0 | 12000 | 213000 |
10 | 31 + 2d | 1720 | 10 | No | 4.5 | 178 | 20500 | 70000 |
11 | 30 + 5d | 1730 | 10 | yes | 5.8 | 84 | 19000 | 60000 |
12 | 27 + 2d | 986 | 19 | No | 4.6 | 3,4 | 11800 | 316000 |
13 | 25 + 5d | 700 | 28 | yes | 6.5 | 0 | 11400 | 194000 |
14 | 35 | 2371 | 14 | yes | 6.8 | 99 | 10500 | 136000 |
15 | 26 | 592 | 15 | yes | 7.9 | 103 | 16100 | 72000 |
16 | 27 | 990 | 8 | yes | 2.9 | 89 | 271000 | 12000 |
17 | 29 + 4d | 1330 | 3 | yes | 3.4 | 58 | 37000 | 123000 |
18 | 29 + 2d | 755 | 6 | yes | 4.2 | 0 | 27600 | 98000 |
19 | 32 | 1090 | 53 | yes | 4.3 | 23,7 | 9500 | 298000 |
20 | 35 | 2400 | 6 | yes | 5.0 | 41,5 | 20400 | 73000 |
21 | 36 | 2730 | 104 | yes | 5.9 | 15 | 9100 | 199000 |
22 | 29 + 4d | 949 | 5 | yes | 5.3 | 31 | 10700 | 232000 |
23 | 34 | 1340 | 24 | yes | 3.5 | 45 | 14700 | 185000 |
24 | 30 | 1250 | 13 | yes | 3.7 | 10 | 18200 | 447000 |
25 | 29 | 1320 | 5 | No | 4.1 | 0 | 5000 | 120000 |
26 | 28 + 4d | 1192 | 11 | yes | 3.0 | 0 | 12000 | 286000 |
27 | 26 | 995 | 33 | yes | 3.3 | 84 | 36900 | 75000 |
28 | 25 + 4d | 900 | 9 | No | 4.0 | 0 | 35700 | 205000 |
Mean | 29.2 | 1288 | 19.8 | Y: 82.2% N: 17.8% | 38.2 | 3.28 | 26775 | 162642.8 |
SD | 3.38 | 606.2 | 21.5 | | 43.5 | 1.62 | 48733.7 | 98180.5 |
Median | 29 | 1075 | 11 | | 23.3 | 5.15 | 15400 | 54500 |
*WA: weeks of amenorrhea; **d: days; ***CRP: C -reactive protein. The mean term of birth of the 28 preterm infants was 29 WA and 4 days with a mean weight birth of 1288 g. The CoNS infectious episode occurred at a mean age of 19.8 days. Mean CRP was 38.2 mg/L at the time of diagnosis. |
Blood culture and stool culture results
S. haemolyticus and S. epidermidis were isolated from 39.3% and 17.8% of blood cultures from these patients, respectively. Unidentified Coagulase-negative Staphylococcus spp.(uCoNS) was detected in 35.7% of blood cultures.S. haemolyticus and S.epidermidis were isolated from 42.8% and 17.8% of stool cultures from these patients, respectively .uCoNS was detected in 39.4% of stool cultures) ( Table 2).
Table 2
Distribution of coagulase-negative Staphylococcus trains isolated from blood culture and stool culture
Coagulase-negative Staphylococcus species | Blood culture n(%) | Stool culture n(%) | Total n(%) |
*S. epidermidis | 5(17.8) | 5(17.8) | 10(17.8) |
S. haemolyticus | 11(39.3) | 12(42.8) | 23(41.0) |
S. warneri | 1(3.6) | 0 | 1(1.8) |
S. capitis | 1(3.6) | 0 | 1(1.8) |
**uCoNS | 10(35.7) | 11(39.4) | 21(37.6) |
Total | 28(100.0) | 28(100.0) | 56(100.0) |
S. haemolyticus was the most widely represented species in these 28 cases of CoNS bacteremia (39.3% of blood cultures and 42.8% of stool cultures) followed by uCoNS (35.7% of blood cultures and 39.4% of stool cultures). |
*S. Staphylococcus; **uCoNS: Unidentified Coagulase-negative Staphylococcus |
Antibiotics susceptibility of CoNS strains
The 28 CoNS strains isolated from both blood cultures and stool cultures were resistant to methicillin (cefoxitin-resistant strains) and kanamycin (Tables 3 and 5).One hundred percent of the 28 CoNS strains isolated from blood samples were resistant to penicillin, cefoxitin and kanamycin,and 96.4% of isolates were resistant to gentamicin,tobramycin and netilmicin.The resistance of these strains to other antibiotics tested are shown in Table 3.The distribution of resistance patterns of these isolates showed sixteen antimicrobial resistance patterns ( R patterns) a to p,and10 of these strains exhibited R pattern e [(35.7%) ( isolates 5–11,13–15)] (Table 4).
Table 3
Susceptibility of coagulase-negative Staphylococcus strains isolated from blood samples
Patient (number) | 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 | S | 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 |
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 | S. capitis | R | R | R | R | R | R | S | S | S | R | S | S | S | S | R | S | g |
17 | CoNS | R | R | R | R | R | R | R | S | S | R | R | S | R | S | S | R | h |
18 | S. epidermidis | R | R | R | R | R | R | R | S | S | R | R | S | S | S | S | R | i |
19 | S. epidermidis | R | R | R | R | R | R | S | S | S | R | S | S | S | S | S | R | j |
20 | S. haemolyticus | R | R | R | R | R | R | R | S | S | R | R | S | S | S | R | R | k |
21 | CoNS | R | R | R | R | R | R | R | S | S | S | R | S | R | S | S | S | l |
22 | CoNS | R | R | R | R | R | R | S | S | S | S | R | S | R | S | R | S | m |
23 | CoNS | R | R | R | S | S | S | R | S | S | S | S | S | R | S | S | S | n |
24 | S. haemolyticus | R | R | R | R | R | R | R | R | S | R | R | S | S | S | S | S | d |
25 | CoNS | R | R | R | S | S | S | S | S | S | S | R | S | S | S | S | S | o |
26 | CoNS | R | R | R | R | R | R | S | S | S | R | S | S | S | S | R | S | g |
27 | S. warneri | R | R | R | R | R | R | S | S | S | R | S | S | S | S | S | S | p |
28 | CoNS | R | R | R | R | R | R | S | S | S | R | S | S | S | S | S | S | p |
One hundred percent of the 28 CoNS strains isolated from blood samples were resistant to penicillin,cefoxitin and kanamycin,and 96.4% of isolates were resistant to gentamicin,tobramycin,and netilmicin. |
R: resistant; S: susceptible; CoNS: Coagulase-negative Staphylococcus; P: Benzyl-penicillin (R < 26 mm); FOX: Cefoxitin (R < 24 mm); K: 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 < 24 mm); SXT: trimethoprim-sulfamethoxazole(S ≥ 17 mm); FOS: fosfomycin (S ≥ 6 mm, 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 4
Susceptibility of coagulase-negative Staphylococcus strains isolated from stool samples
Patient (number) | 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 |
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 | S | 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 and cefoxitin, and 96.4% of isolates were resistant to kanamycin,gentamicin,tobramycin and netilmicin. |
Abbreviations: see Table 3 |
Table 5
Antimicrobial resistance patterns (R patterns) of the 28 strains isolated from blood samples
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 RIFROFXR | 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)]. |
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).The resistance of these isolates to other antibiotics tested are shown in Table 5.All these isolates were classified into sixteen R patterns a to p,and 11 of these strains exhibited R pattern e [(39.2%),(isolates 5–11,13–16)] (Table 6).
Table 6
Antimicrobial resistance patterns (R patterns) of the 28 strains isolated from stool samples
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 |
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%). |
The most common resistance patterns of the strains isolated combined heterogeneous resistance to methicillin, kanamycin, gentamicin, tobramycin and netilmicin,and resistance to erythromycin, rifampin and ofloxacin.
Comparison of blood culture and stool culture results according to 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).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 patterns (A, B, C) (Fig. 1) and three different RAPD patterns (D, E, F) (Fig. 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)].The 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. The remaining strains (three S. epidermidis and three uCoNS) exhibiting AF and CD genotypes,respectively, were considered to be sporadic cases. The BE genotype was identified in both units participating in this study.
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. were not found in blood and stool in 46.5% (13/28) of preterm infants, strongly suggesting the absence of BT in these preterm infants, and that the intestinal tract would not constitute the only or direct source of bacteremia. 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); leucocytes/mm3 (p = 0.042); lactate/mmol/L (p = 0.0002) (Wilcoxon-Mann-Whitney); intravenous perfusion lipid emulsion (OR:8.1821; 95% CI [1.2555; 73.4536]), p = 0.02; treated patent ductus arteriosus (OR: 0.0961; 95% CI [0.0018; 0.9895]),p = 0.03;hemodynamic disorders (OR: 9.9418; 95% CI [1.3972; 127.034]), p = 0.009;history of jaundice (OR: 10.9341; 95% CI [1.0273; 587.9893], p = 0.02; neonatal antibiotic therapy (OR: 8.1821;95% CI [1.2555; 73.4236]), p = 0.02 (Fisher’s Exact test) were direct independent RFs for the occurrence of gastrointestinal BT. None of the other RFs tested were significant.