Value of molecular typing in the assessment of bacterial translocation during coagulase-negative staphylococcal bacteremia in preterm infants

doi:10.21203/rs.2.19684/v1

Background

One hundred fifty-seven preterm infants enrolled in the study were hospitalized between 2012-2014 at Amiens-Picardie University Hospital.Only 28 (17.8%) of these children who had experienced at least one episode of secondary Coagulase-negative Staphylococcal bacteremia with concomitant positive stool cultures were included in this study.The purpose of this study was to assess the rate of intestinal bacterial translocation associated with these infections.

Methods

Blood cultures and stool cultures were performed in the context of this study. All isolates of Staphylococcus spp were examined by MALDI-TOF MS. Antibiotic susceptibility and genotyping were also performed.

Results

Sixteen resistance patterns were identified from blood and stool based on antibiotic susceptibility testing. Ten of the Coagulase-negative Staphylococcus strains isolated from blood samples exhibited R pattern e (35.7%) and eleven of the Coagulase-negative Staphylococcus strains isolated from stool samples exhibited R pattern e (39.2%). Blood culture results were concordant with stool culture results in 53.5% of cases and discordant in 46.5% of cases.Fifteen isolates exhibited three ERIC-2 (A, B, C) and three RAPD-PCR (D, E, F) patterns. ERIC-2 patterns comprised A ( S. epidermidis isolates); B ( S. haemolyticus isolates) and C ( unidentified Coagulase-negative Staphylococcus isolates). RAPD patterns consisted of D ( unidentified Coagulase-negative Staphylococcus isolates), E ( S. haemolyticus isolates), and F ( S. epidermidis isolates).

Conclusion

Bacterial translocation from the intestinal tract was likely source of Coagulase-negative Staphylococcal bacteremia in hospitalized preterm infants.

General Microbiology

Bacterial translocation

Coagulase-negative Staphylococcus

Blood culture

Stool culture

Preterm infants

Molecular typing

Bacterial translocation (BT) is defined as the passage of live bacteria, their products, or both, across the lamina propia to local mesenteric lymph nodes (MLN) and from there to extranodal sites [1–3]. The intestinal tract has multiple functions in the body apart from its primary function, the absorption of nutrients. It represents a barrier protecting the body from living microorganisms and antigens of the intestinal lumen.

Many physiological and pathological conditions, such as preterm infant, fasting, mesenteric ischemia-reperfusion, shock states, can alter intestinal functioning. Alteration of the intestinal barrier often results in a systemic inflammatory process and,more rarely,potentially fatal multiple organ dysfunction syndrome (MODS). Gastrointestinal tract (GIT) epithelial and immune cells play an essential role in initiation and resolution of the inflammatory process. Any adverse alteration of the intestinal barrier leads to disruption of this process, resulting in increased permeability of the intestinal barrier, promoting the passage of live bacteria, bacterial DNA or bacterial degradation products from the intestines to extraintestinal sites [4]. The route of BT has been examined in humans [1]. A study of human trauma patients [5] evaluated blood samples from portal vein (PV) catheters for evidence of translocating bacteria or endotoxins.The incidence of BT in humans undergoing emergency laparotomy has been reported to be 14 to 21% [6] and 15.4% [3].Active research has also been devoted to methods able to document the occurrence of BT.Numerous molecules have been evaluated as potential biomarkers for BT, including bacterial DNA, soluble CD14, lipopolysaccharide (LPS)/endotoxin, LPS binding protein (LBP), calprotectin [7].LPS and d-lactate have been identified as products of gut BT and systemic markers for increased gut permeability and BT [8]. BT has been proposed as the mechanism of bacteremia in clinical situations such as neutropenia in cancer patients [9] and hemorrhagic shock [10]. Translocation limited to MLN has also been described in patients with Crohn’s disease, colorectal cancer [11], premature children and newborns [12].The incidence of BT and its relationship to sepsis and MODS in preterm newborns remain unclear. Coagulase-negative Staphylococcal (CoNS) bacteremia is common in neonates and is often associated with the presence of a catheter. The mechanism responsible for intestinal BT remains poorly elucidated. It would therefore be interesting to study the rate of BT responsible for CoNS bacteremia in a population of preterm infants.

Objectives of our study were to assess the rate of intestinal BT and its role in causing sepsis in clinical conditions, and to evaluate the correlation between CoNS isolated from blood cultures and stool cultures using molecular typing.

Study design and study population

During the study period, a total of 157 neonates (less than 28 days of life) born prematurely (<37 weeks of gestation-WG) were immediately hospitalized in the neonatal intensive care units (nICUs) and in the pediatric intensive care units (pICUs) of Amiens-Picardie University Hospital (APUH), Amiens (France) between 2012 and 2014. Twenty-eight (17.8%) of these infants had experienced at least one episode of CoNS bacteremia with a concomitant positive stool culture. Only these 28 children were included in the study. However, 129 (82.2%) children including full term infants (≥ 37 WG),preterm infants with positive stool culture without CoNS bacteremia, preterm infants with CoNS bacteremia without stool cuilture positive, and preterm infants corresponding to duplicate cases were all excluded in this study.

Antibiotics used in patient treatment

Sixteen (57.1%) of the 28 preterm infants included in this study had received the following antibiotics during neonatal life: amoxicillin (9 cases ),vancomycin ( 6 cases ),amoxicillin + gentamicin ( 5 cases),fluoconazole (4 cases),piperacillin- tazobactam+ gentamicin (2 cases),vancomycin + amikacin ( 2 case),cefotaxime (2 cases),cilastine-imipenem + gentamicin ( 1 case),teicoplanin + amoxicillin (1 case),meropenem (1 case),ceftazidime (1 case),trimethoprim-sulfamethoxazole (1 case),metronidazole (1 case),and josamycin (1 case).These antibiotics were used individually or in combination in 15 suspected cases of maternal-fetal infections ,and piperacillin-tazobactam+gentamicin, vancomycin and metronidazole were used in one case of septic shock.Sixteen ( 57.1%) of the 28 mothers who delivered preterm had received the following antibiotics prior to delivery :amoxicillin ( 9 cases),ceftriaxone (2 cases), amoxycillin-clavulanic acid, piperacillin-tazobactam (1case),cefotaxime (1 case),ceftriaxone, cefixime and erythromycin (1 case),clindamycin and trimethoprim-sulfamethoxazole (1 case).These antibiotics were used individually or in combination to treat urinary tract infections,vaginal infections and premature rupture of membranes.

Data collection

All data were obtained from electronic medical records. Patient demographic characteristics, underlying conditions, and clinical and laboratory findings were collected.

Diagnostic criteria for bacteremia

One positive blood culture in the presence of suggestive clinical signs: bradycardia, oxygen desaturation, increased respiratory requirements, total leukocyte count > 18,000/mm³, C-reactive protein (CRP) > 10 mg/L, serum lactate> 1.65 mmol/L, temperature > 38.3°C or < 36°C.

Blood and stool samples

Blood was obtained for routine hematological, biochemical and bacteriological tests, including polymorphonuclear leukocyte and platelet counts and CRP. All blood cultures were performed for diagnostic purposes, and stool cultures were performed in the context of this study. Less than 0.5 mL of blood was inoculated into a Bactec Peds Plus F bottle and incubated in Bactec^TM Becton Dickinson instrument (BD Diagnostic Systems, Spark, MD, USA). Subcultures of initial blood culture broth were seeded on sheep blood (5%) Columbia agar and mannitol salt agar (MSA) (Oxoid, France) were incubated aerobically at 37°C for 24 hours.Anal or rectal samples using sterile swabs were seeded on sheep blood (5%) Columbia agar and MSA and were incubated aerobically at 37°C for 48 hours.

Identification

Staphylococcus spp. were identified after examining all colonies. All isolates negative for mannitol and for bound coagulase (Pastorex Staph Plus-Bio-Rad, France) and positive for catalase and Gram staining were classified as CoNS.

MALDI-TOF-MS

All Staphylococcus strains isolated by routine tests were examined by Matrix-Assisted-Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) (Brucker Daltonics, Bremen, Germany) according to a previously described procedure [13,14].

In the presence of a negative blood culture, the corresponding stool cultures and blood cultures were discarded. Conversely, when a Staphylococcus sp. was isolated from the patient’s blood culture, all morphologically distinct colonies detected by gross examination of stool cultures were identified by MALDI-TOF-MS.

Antibiotic susceptibility testing

Isolates were tested by the disk diffusion method on Mueller-Hinton (MH) agar according to the zone size criteria as recommended by the Antibiogram Committee of the French Microbiology Society (AC-FMS) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) – 2015 [15]. The antibiotics used were kanamycin (K), gentamicin (G), tobramycin (T), erythromycin (E), lincomycin (L), pristinamycin (PT), rifampin (RIF), ofloxacin (OFX), vancomycin (VAN), fusidic acid (FA), trimethoprim-sulfamethoxazole (SXT), and fosfomycin (FOS). Susceptibilities to benzylpenicillin (P) (disk loaded with 6 µg) and cefoxitin (FOX) (disk loaded with 30 µg) were determined by the disk diffusion method on salted MH at 37°C for 24 hours.

DNA isolation and molecular typing

Total nucleic acid extraction was performed using the bioMérieux NucliSENS easy MAG platform (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions.ERIC-PCR was performed as previously described [16]. Extracted DNA (100 ng from each isolate) was amplified in a final volume of 50 µL of the ERI-C2 primer (5’-AAG-TAA-GTG-ACT-GGG-GTG-AGC-G-3’) and 5 µL of CoralLoad PCR buffer. PCR cycling consisted of 94°C for 7 min followed by 45 cycles of 94°C for 1 min, 45°C for 1 min and 72°C for 2 min, and 72°C for 7 min. RAPD-PCR was performed as previously described [17]. The primer used was, 5’-GGT-TGG-GTG-AGA-ATT-GCA-CG-3’. Amplification reactions were carried out with a final volume of 50 µL containing 25 µL of Top Taq Master MIX (QIAGEN, Les Ulis, France), 1 µL of RAPD1 primer, 2 µL of DNA as template, 5 µL of CoralLoad PCR buffer. The cycling conditions were 95°C for 3 min, followed by 35 cycles of 94°C for 1 min, 40°C for 1 min, and 72°C for 2 min, and 72°C for 10 min. After amplifications, PCR products were resolved by electrophoresis on 1.2% agarose gels at 90 V for 6 h, followed by ethidium bromide staining and were visualized under UV light. A photograph was also taken.

Risk Factors (RFs) for gastrointestinal BT in preterm infants with CoNS bacteremia

The following clinical, laboratory and treatment parameters listed in Table 7 were analyzed as potential RFs for BT in this study. These RFs were categorized in two groups according to molecular typing: group1 included patients in whom BT was indicated; group 2, comprised patients in whom BT was not indicated.

Statistical analysis

We calculated the relative risk (RR) of BT among preterm infants with and without documented BT. Data are expressed as means ± standard deviation (SD) for quantitative variables and frequencies for qualitative variables. We calculated the RR of BT with associated 95% confidence intervals (CIs) using Fisher’s Exact test. Comparisons between documented BT and undocumented BT groups were performed with the Wilcoxon-Mann-Whitney test for quantitative data and Fisher’s Exact test for qualitative data. All tests of significance were two-sided and a p value of < 0.05 was considered to indicate statistical significance.

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)]
Patient (number)	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)]
Patient (number)	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	P^R FOX^R K^R G^R T^R N^R E^R OFX^R	1
b	P^R FOX^R K^R T^R E^R L^R RIF^R OFX^R FA^R	2
c	P^R FOX^R K^R G^R T^R N^R E^R OFX^R	3
d	P^R FOX^R K^R G^R T^R N^R E^R L^R RIF^R OFX^R	4, 24
e	P^R FOX^R K^R G^R T^R N^R E^R RIF^ROFX^R	5, 6, 7, 8, 9, 10, 11, 13, 14, 15,
f	P^R FOX^R K^R G^R T^R N^R E^R L^R RIF^R OFX^R FOS^R	12
g	P^R FOX^R K^R G^R T^R N^R RIF^R FOS^R	16, 26
h	P^R FOX^R K^R G^R T^R N^R E^R RIF^R OFX^R FA^R DOX^R	17
i	P^R FOX^R K^R G^R T^R N^R E^R RIF^R OFX^R DOX^R	18
j	P^R FOX^R K^R G^R T^R N^R RIF^R DOX^R	19
k	P^R FOX^R K^R G^R T^R N^R E^R RIF^R OFX^R FOS^R DOX^R	20
l	P^R FOX^R K^R G^R T^R N^R E^R OFX^R	21
m	P^R FOX^R K^R G^R T^R N^R OFX^R FA^R FOS^R	22
n	P^R FOX^R K^R E^R FA^R	23
o	P^R FOX^R K^R OFX^R	25
p	P^R FOX^R K^R G^R T^R N^R RIF^R	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	P^R FOX^R K^R G^R T^R N^R E^R OFX^R	1
b	P^R FOX^R K^R G^R T^R N^R E^R L^R RIF^R OFX^R FA^R	2
c	P^R FOX^R K^R G^R T^R N^R E^R OFX^R	3
d	P^R FOX^R K^R G^R T^R N^R E^R L^R RIF^R OFX^R	4
e	P^R FOX^R K^R G^R T^R N^R E^R RIF^R OFX^R	5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16
f	P^R FOX^R K^R G^R T^R N^R E^R L^R RIF^R OFX^R FOS^R	12
g	P^R FOX^R K^R G^R T^R N^R E^R OFX^R DOX^R	17, 19
h	P^R FOX^R K^R G^R T^R N^R E^R RIF^R OFX^R DOX^R	18, 20
i	P^R FOX^R K^R G^R T^R N^R E^R RIF^R	21
j	P^R FOX^R K^R G^R T^R N^R OFX^R FA^R FOS^R	22
k	P^R FOX^R K^R G^R T^R N^R DOX^R	23
l	P^R FOX^R K^R G^R T^R N^R RIF^R	24
m	P^R FOX^R E^R OFX^R	25
n	P^R FOX^R K^R G^R T^R N^R E^R RIF^R OFX^R FOS^R	26
o	P^R FOX^R K^R G^R T^R N^R E^R L^R RIF^R SXT^R	27
p	P^R FOX^R K^R G^R T^R N^R RIF^R 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.

Clinical/biological/

Therapeutic

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

Lactates/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]

Arterial canal

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]

Jaundice history

14 (93.3%)

7 (53.8%)

P=0.02

OR : 10.9341 5% CI

[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-Protein reactive ; OR : Odds Ratio, CI : confidence intervals ; RR : relative risk

Table 7. Rates and relative risk factors for digestive bacterial translocation in preterm infants hospitalized in APUH Center belonging to two groups : documented translocation and undocumented translocation

In this study, S.haemolyticus and S.epidermidis were the CoNS species most isolated, with rates of 41.8% and 17.8% respectively. These pathogens are a major cause of nosocomial bacteremia and catheter infections in nICUs [18]. CoNS are the most common cause of late-reported sepsis [19]. Sepsis accounts for 45% of severe infections in neonatology units [20].

The results of this study established that birth weight, age at onset of infection, leucocytes,serum lactates, intravenous lipid emulsion perfusion, treated patent ductus arteriosus, hemodynamic disoders, history jaundice, and neonatal antibiotic therapy were independent RFs for the occurrence of BT in preterm infants with CoNS bacteremia.MacFie J et al [6] investigated factors independently associated with BT in humans and showed that the following variables were associated with an increased prevalence of BT based on univariate analysis: intestinal obstruction, jaundice, inflammatory bowel diasease, malignancies, preoperative total parenteral nutrition (TPN) and emergency surgery. However,multivariate analysis showed that only emergency surgery and preoperative TPN were independently associated with translocation [7].These results differ from those of our study in that our study population consisted of only a sample of preterm infants due to our inclusion criteria, whereas the participants in MacFie’study with BT had a median age of 71 years

Our study also showed that BT in preterm infants with sepsis is responsible of secondary bacteremia and is driven by external factors such as length of stay in the nICUs or prolonged feeding by an enteral feeding tube. Such results have been observed by Jezioski E et al.[21].

According to the study conducted by Pappof P et al [2],prematurily appears to play a significant predisposing role by reducing mucosal barrier function.Other factors reported in the literature to influence BT are as follows:(i) bacterial overgrowth in small bowel, use of antibiotics, obstructive jaundice, intra-abdominal hypertension; (ii) damage to the gut barrier, systemic inflammatory response syndrome, or direct injury (abdominal surgery); (iii) systemic immunosuppression; (iv) immaturity of the intestinal barrier per se, and immaturity of host defense function [22, 23].Various publications have identified BT in a wide group of diseases, such as acute pancreatitis, cirrhosis, malignancy, heart failure, aortic aneurysm repair, cardiopulmonary bypass and bowel transplant [22–24].

In this study, the presence of BT from the gut to the circulatory system in 15 of 28 preterm infants with CoNS bacteremia represents a prevalence of 53.5% according to molecular typing results. Some studies have reported BT from the gut to MLN in 4 to 59% of patients with various clinical conditions [25].O’Boyle CJ et al observed this phenomenon in 15.4% of cases of patients undergoing laparotomy [3].Moharem HA et al [26] detected BT in 33% of liver transplant patients. Bellot P et al found an incidence of BTof 38% among cirrhotic patients [27].Other studies have shown that the presence of BT is associated with significant hemodynamic changes [26].In the absence of clinical infections,BT is due to the release of inflammatory mediators such as tumor necrosis factors (TNF)-α and IL-17 [28].

In this series of 28 CoNS strains, the predominant antibiotic resistance pattern comprised 10 strains, including 8 S. haemolyticus strains, mainly presenting a major epidemic profile including the BE genotype that spread rapidly after emergence in the two units participating in this study. The results of this study demonstrate the predominance of this major epidemic profile, corresponding to 28.6% (8/28) of the strains studied. Furthermore, a close correlation was observed between the various genotypes and the associated antibiotic resistance patterns of the CoNS strains isolated. The isolated CoNS strains demonstrated the emergence of multidrug resistance (resistance to β-lactams, aminoglycosides and fluoroquinolones). The selection pressure related to the increasing use of β-lactams and aminoglycosides accounts for the antibiotic resistance of these strains.

The results of this study demonstrate that CoNS species belonging to intestinal microbiota were the most likely source of CoNS bacteremia in hospitalized preterm infants. Published data have also described neutropenia in cancer patients |9] and hemorrhagic shock [10] as likely sources of bacteremia. In the present study,BT was essentially demonstrated by comparison of the ERIC-PCR and RAPD-PCR genomic patterns of the same Staphylococcus species, isolated concomitantly from the patient’s blood and stool. Pulsed-field gel electrophoresis (PFGE) typing has been shown to be a useful technique to establish relatedness between stool and blood isolates from children [29]. ERIC-PCR and RAPD-PCR are powerful genomic typing methods, which have not been previously used in any published studies on BT related to clinical events in preterm infants.In this study,53.5% of the preterm infants with bacteremia caused by BT of an intestinal CoNS undoubtedly underestimates the true prevalence of BT in the study population. It is difficult to demonstrate a correlation between BT and bacteremia, as these forms of bacteremia only appear to have clinical repercussions in the presence of massive BT, when the body’s capacity to clear the organism is exceeded, when the organism responsible is particularly virulent [29] or when the host’s immune defense mechanism is altered [9]. Under these conditions, micro-organisms from the GIT may be responsible for localized or systemic infections. Molecular biology techniques can now be used to noninvasively study BT in humans [30, 31]. ERIC-PCR and RAPD-PCR confirmed the presence of BT in 15 phenotypically selected patients included in the molecular typing study. CoNS isolated from blood and stool presented a high level of phenotypic and genotypic similarity in 53.5% of cases. In this population of patients with CoNS bacteremia, stools constituted a definite source of CoNS. In the 46.5% of discordant cases with or without bacteremia obtained from blood cultures and stool cultures, BT could not formally be excluded because of the existence of other non-intestinal sources that were not analyzed in this study.No significant difference was observed between clinically or microbiologically severe bacteremia in preterm infants with and without documented BT.

This study was performed on a limited sample of 28 preterm infants who had at least bacteremia with concomitant CoNS positive stool culture.This inclusion criterion is thery restrictive.This study only concerned BT from intestinal origin .Other sites, such as respiratory or skin,were not investigated in this study. Staphylococcus spp. are the predominant germ within the microbiota of very preterm baby and therefore it can be considered that all preterm infants are carriers of detectable CoNS or not in standard culture .This sample of patients can therefore be considered to be a random sample representative of the population of preterm newborns with CoNS bacteremia.

This study clearly demonstrates that BT from the intestinal tract was the most likely source of CoNS bacteremia in hospitalized preterm infants.BT appears to be an important early step in sepsis in debilitated preterm patients. Reinforcement of the intestinal barrier, regulation of the intestinal microbiota by breast milk and using prebiotics or probiotics constitute possible approaches to the prevention of intestinal BT.

Authors’ contributions

AL,GA,BED,GM,SG,GK,MB participated in meeting and follow-up discussions that culminated in the preparation of this manuscript.They contributed to the study conception,design,and drafted the manuscript.All authors participated in the acquisition,analysis and the interpretation of data and also in editing and final revisions. All authors read and approved the final manuscript.

Author details

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)

³Services de Nutrition parentérale et de Chirurgie vasculaire, CHU de Lille 59000 Lille (France)

⁴Génétique Clinique Pédiatrique, CHU, Amiens Picardie, Site Sud 80054 Amiens Cédex 1 (France)

⁵Réanimation et Surveillance Continue Pédiatrique, CHU Amiens Picardie Site Sud,80054 Amiens Cédex (France)

⁶Laboratoire Péritox INERIS UMR 101 CURS-UPJV F-80054, Amiens Cedex 1 (France)

Acknowledgments

The authors would like to thank the Clinical Research and Innovation Department for proofreading and correcting the English of the manuscript.

Funding

The authors have no financial relationships relevant to this article to disclose

Ethical approval and consent to participate

All procedures performed in studies involving human participants were conducted in accordance with the guidelines laid down in the Declaration of Helsinki and/or national research committee (Ethical Committee of the APUH,n° 139).Clinical and Laboratory data concerning the preterm infants were included in this study.Informed consent from legal guardians of the minors included in the study was not specifically requested.

Conflict of interest

The authors declare that they have no conflict of interest

Availability of data and materials

All data generated or analyzed during this study are included in this published article and the additional information files.

Consent for publication Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Value of molecular typing in the assessment of bacterial translocation during coagulase-negative staphylococcal bacteremia in preterm infants

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Study design and study population

Antibiotics used in patient treatment

Data collection

Diagnostic criteria for bacteremia

Blood and stool samples

Identification

MALDI-TOF-MS

Antibiotic susceptibility testing

DNA isolation and molecular typing

Risk Factors (RFs) for gastrointestinal BT in preterm infants with CoNS bacteremia

Statistical analysis

Results

Patients characteristics

Blood culture and stool culture results

Antibiotics susceptibility of CoNS strains

Comparison of blood culture and stool culture results according to resistance pattern

Molecular typing results

Bacterial Translocation results

Risk factors for the occurrence of BT in preterm infants with CoNS bacteremia

Discussion

Study Limitations

Conclusions

Declarations

References

Status:

Version 1