AGNIESZKA CHMIELARCZYK, MONIKA POBIEGA*, JADWIGA WÓJKOWSKA-MACH, DOROTA ROMANISZYN, PIOTR B. HECZKO and MAŁGORZATA BULANDA

Chair of Microbiology, Kraków, Poland

• Corresponding author: M. Pobiega, Chair of Microbiology, Krakow, Poland; e-mail: monika.pobiega@gmail.com

Submitted 21 January 2014, revised 27 February 2015, accepted 17 March 2015
https://doi.org/10.5604/01.3001.0009.2117

Abstract

Bloodstream infections (BSIs) are associated with a significantly increased risk of fatality. No report has been found about the molecular epidemiology of Enterobacteriaceae causing BSI in neonates in Poland. The aim of this work was to determine the antibiotic resistance profiles, virulence gene prevalence, the epidemiological and genetic relationships among the isolates from Enterobacteriaceae causing BSI in neonates with birth weight < 1501 g. Antimicrobial susceptibility testing was performed. PCR was performed to identify the presence of common beta-lactamase genes, virulence genes. PFGE and MLST were performed. The surveillance group contained 1,695 newborns. The incidence rate for BSIs was 5.9%, the fatality rate 15%. The most common species were Escherichia coli (n = 24) and Klebsiella pneumoniae (n = 16). CTX-M-15 was found in 6 E. coli, 8 K. pneumoniae, 1 Enterobacter cloacae strains. Among E. coli fimH (83.3%), ibeA (37.5%), neuC (20.8%) were the most frequent. PFGE demonstrated unique pulsotypes among E. coli. E. coli ST131 clone was found in 7 E. coli strains. PFGE of 16 K. pneumoniae strains showed 8 pulsotypes. Five isolates from one NICU belonged to one clone. MLST typing revealed 7 dif­ferent ST with ST336 as the most prevalent. This study provides information about resistance, virulence and typing of Enterobacteriaceae strains causing BSI among neonates. E. coli and Klebsiella spp. isolated in this study have completely different epidemiology from each other.

Key words: Enterobacteriaceae, Escherichia coli ST131, Klebsiella pneumoniae, bloodstream infections, MLST, very-low birth weight neonates

Introduction

Bloodstream infections (BSIs) are the most com­mon nosocomial infections among neonates, associated with a significantly increased risk of fatality (Stoll et al., 2002). Newborns, especially those with very low birth weight (VLBW) are primarily at risk of develop­ing late-onset BSIs (LO-BSIs), caused by organisms acquired perinatally or postnatally, usually as a con­sequence of nosocomial transmission. The main risk factors for LO-BSI include prematurity, prolonged stay in a neonatal intensive care unit (NICU) and administration of invasive procedures (Ozkan et al., 2014). Consequently, the improved survival rates of small premature infants experiencing long stays in modern NICUs, which are better equipped for life-saving inten­sive care, has been a major factor in the increase inLO-BSI caused by Gram-negative bacteria (Cordero et al., 2004; Wojkowska-Mach et al., 2013). Enterobac­teriaceae are second only to coagulase-negative staphy­lococci (CoNS) in causing LO-BSIs (Karlowicz et al., 2000; Lutsar et al., 2014). Prolonged use of broad-spec­trum antibiotics in NICUs is associated with the occur­rence of multi-drug resistant (MDR) bacteria. Selec­tion of more broadly resistant Gram-negative enteric bacteria is linked with outbreaks of bacterial disease in NICUs (Cordero et al., 2004).

Nosocomial isolates such as Escherichia coli and Klebsiella sp. often have extended spectrum β-lactamase (ESBL) phenotypes, and E. coli strains carrying these enzymes have disseminated into the community. One of the best examples of this trend is the global spreadof the clonal E. coli sequence type (ST) 131 (ST131), which expresses CTX-M enzymes. (Birgy et al., 2013)In contrast, among Klebsiella strains, relatively few of the serotypes (particularly K1 and K2) appear to be linked with invasive strains. Also, the hypermucovis­cous phenotype, strongly associated with the presence of rpmA and magA genes, is a virulence marker in clinical strains (Jung et al., 2013). In Klebsiella pneumo­niae, CTX-M-15 is mainly associated with quinolone-resistant strains, belonging to ST11 clone (Damjanova et al., 2008; Oteo et al., 2009).

Despite many reports of ESBL-producing isolates among Polish hospitals, no report has been foundabout the molecular epidemiology of Enterobacteriaceae causing BSI in neonates. Here, we collected clinical iso­lates from Enterobacteriaceae family bacteria originat­ing from BSIs of neonates hospitalized in six Polish NICUs to determine their antibiotic resistance pro­files, virulence gene prevalence, and the epidemiologi­cal and genetic relationships among the isolates. Here we focused on detailed molecular studies, data about epidemiology of infections in new­borns are included in other manuscripts (Wojkowska-Mach et al., 2014).

Experimental
Materials and Methods

Study population. Prior to the study, a confirm­atory ethics vote for the data collected in the Polish Neonatology Surveillance Network (PNSN) for thescientific purpose was approved by the BioethicsCommittee of Jagiellonian University Medical College (No KBET/221/B/2011).

Continuous prospective target surveillance of infec­tions was conducted from 1/1/2009 through 12/31/2011 at Polish NICUs (only teaching hospitals) which par­ticipated in PNSN. These tertiary NICUs provided care for 20% of all VLBW infants born in Poland annually. The surveillance included infants hospitalized in these NICUs whose birth weights were < 1500 grams (VLBW) at birth until they achieved a weight of 1800 grams or died. The study covered 1,695 newborns. The general fatality case rate was 16.3%. All VLBW infants of sus­pected or documented infected were subject to registra­tion regardless of the time of occurrence according to criteria of Gastmeier et al. (2004) when they had clini­cal signs of a bloodstream infection (BSI) (Gastmeier et al., 2004).

• at least 2 of the following: temperature > 38°C or < 36.5°C or temperature instability, tachycardia or bradycardia, apnea, prolonged capillary refill, metabolic acidosis, hypoglycemia and other signs of bloodstream infections such as lethargy;
• and patients who had 1 of the following crite­ria: C-reactive protein > 2.0 mg/dl, immature/total neutrophil ratio (I/T ratio) > 0.2, leukocytes < 5000/μl and platelets < 10000/μl.

LO-BSI was defined when diagnosed > 72 hours after delivery. Clinical sepsis represented an infant wheresigns of infection existed but on blood culture a causa­tive organism was not identified.

Central venous catheter (CVC)-associated BSI (CVC-BSI) and peripheral intravenous catheter (PVC)-associated BSI (PVC-BSI) were defined as infections associated with the use of a central or peripheral venous catheter within the preceding 48 hours prior to the onset of the infection (Gastmeier et al., 2004).

Bacterial strains. The present study examined 55 Enterobacteriaceae family isolates originating from BSIs: 4 from early onset infections (EOI), 51 from LOI. The majority originated from NICUs VI and II (25 and 12, respectively). The remainder were from V (8), I (5), III (3) and IV (2).

Culture and species identification. All blood specimens of at least 1 ml were injected into an aerobic blood culture bottle (Bactec Plus 26 Aerobic; Becton Dickinson, Poland), and cultured on MacConkey agar, Columbia agar (at 37°C, each for 24 h) and Sabouraud agar (at 37°C, 48 hours) (all from BIOCORP, Poland). Collection and identification of the bacterial strains was performed in the local microbiology laboratories of each hospital. Isolates were identified by the automated identification system (VITEK 2; bioMérieux, Warsaw, Poland). Bacterial strains were stored at −70°C and sent to Department of Microbiology, Krakow.

Antimicrobial susceptibility tests. Isolates were tested using disk diffusion antimicrobial susceptibil­ity methods on Mueller-Hinton agar plates accord­ing to the current European Committee on Antimi­crobial Susceptibility Testing (EUCAST) guidelines using clinical breakpoint tables v. 3.1 (http://www.eucast. org v.3.1, accessed: 11.02.2013). All discs were obtained from Oxoid, Basingstoke. ESBL activity was detected using a modified double-disk synergy test (Drieux et al., 2008). ESBL-positive strains resistantto at least two other groups of antibiotics were consid­ered to be MDR.

DNA isolation. DNA templates were extracted using a Genomic Mini kit (A&A Biotechnology, Poland)according to manufacturer’s instructions.

Polymerase chain reaction (PCR) for extended-spectrum β-lactamase genes and virulence factor screening. PCR was performed to identify the presence of blaCTX-M, blaSHV and blaTEM genes (Chmielarczyk et al., 2013; Monstein et al., 2007), products were sequenced by commercial company (Genomed, Warsaw, Poland).

E. coli was checked for the presence of selected viru­lence genes as described elsewhere (Chmielarczyk et al., 2013; Johnson and Stell, 2000; Watt et al., 2003). The isolates were classified to 1 of the 4 main E. coli phylogenetic groups (A, B1, B2,and D) (Chmielarczyk et al., 2013; Clermont et al., 2000).

K. pneumoniae was tested for the presence of rmpA (regulator of mucoid phenotype A), magA (mucovis­cosity-associated gene A), wabG (involved in the bio­synthesis of the outer core lipopolysaccharide), uge (encoding uridine diphosphate galacturonate 4-epime­rase, which is responsible for capsule biosynthesis),kfu (iron uptake system), and allS (encoding the acti­vator of the allantoin regulon) (Brisse et al., 2009). PCR was performed with primer pairs specific forthe K. pneumoniae capsule gene cluster (cpsK1, cpsK2) (Fang et al., 2007; Turton et al., 2008). NCTC 5054 and NCTC 5055 were used as positive controls for cpsK1 and cpsK2, respectively.

Pulsed-field gel electrophoresis (PFGE). All iso­lates were analyzed using the standardized PFGE pro­tocol developed at the Centers for Disease Control and Prevention by the PulseNet program. XbaI (Thermo Scientific) was used for DNA digestion. The digested products were separated on a CHEF III PFGE system (BioRad, Warsaw, Poland) in 0.5 × Tris-borate-EDTA buffer at 14°C at 6 V for 20 h with a ramped pulse time of 2.2–54.2 s for E. coli and 14°C at 6 V for 22 h with a ramped pulse time of 2–35 s for K. pneumoniae, Kleb­siella oxytoca, Enterobacter cloacae and Serratia marcescens. GelCompar (Applied Maths, Kortrijk, Belgium) was used for cluster analysis with the unweighted pair group method with an arithmetic mean and the Dice coefficient similarity require to be > 90% for the pattern to be considered as belonging to the same type.

Multilocus sequence typing (MLST). MLST for E. coli was performed in accordance with (Wirth et al., 2006) (http://mlst.ucc.ie/mlst/dbs/Ecoli), for K. pneu­moniae was performed in accordance with (Diancourt et al., 2005) (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html).

Results

The surveillance group contained 1,695 newborns with birth weight below 1500 g. Data on gestational age, mode of delivery, birth weight, multiple birth were pre­viously published (Wojkowska-Mach et al., 2014). In the study group, 100 cases of Enterobacteriaceae BSI were registered. Among all the etiological factors for BSI, bacteria belonging to Enterobacteriaceae family were responsible for 15.2% of such infections. The inci­dence rate for Enterobacteriaceae BSIs was 5.9%, while the fatality rate was 15%. The fatality rate for E. coli was 26.0% and for Klebsiella sp. 10.0% (p = 0.09). Of the 55 Enterobacteriaceae isolates included in our study, there were 24 E. coli, 16 K. pneumoniae, 4 K. oxytoca, 5 E. cloacae, 1 Enterobacter sakazakii, 4 S. marcescens and 1 Morganella morganii (Table I).

The CVC utilization rate was 0.45 and the PVC utili­zation rate was 0.16 (calculated by dividing the number of days of CVC/PVC by the total number of patient days). Among the infections caused by E. coli, 20.8% of LO-BSIs were associated with CVC use, while 8.3% were associated with PVC use. Among the LO infec­tions caused by K. pneumoniae, 43.75% were associated with CVC and 18.75% with PVC.

Table I
Participation of particular isolates in species and NICU.

NICU	Escherichia coli (n=24)			Klebsiella pneumoniae (n=16)			Enterobacter sp. (n=6)	Klebsiella oxytoca (n=4)	Serratia marcescens (n=4)	Morganella morganii (n=1)
		ST	Pulsotype		ST	Pulsotype
I (n=5)	1 1	ST141 ST73	unique unique	1	ST153	unique	2
II (n=12)	4 2   1 1	ST131 ST69 ST69 ST75	unique identical (the same patient) unique unique				1	3
III (n=3)				1	ST336	unique			1	1
IV (n=2)									2
V (n=8)	2 1 1 1	ST131 ST141 ST69 ST543	unique unique unique unique	2	ST17	identical (the same patient)		1
VI (n=25)	3 1 1 1 1 1 1	ST998 ST131 ST141 ST69 ST95 ST569 non typeable	unique unique unique unique unique unique unique	5 2 2 1 1 1	ST336 ST11 ST6 ST6 ST153 ST870	clone A clone B clone C unique unique unique	3		1

The highest level of resistance among E. coli isolates was observed against AMP (91.7%), AMC (54.2%), SXT (41.7%) and ATM (33.3%). The highest level of resistance among K. pneumoniae isolates was observed against AMP (100%), ATM (81.3%), AMC (56.3%), all the cephalosporins investigated herein (56.3%), and CIP (43.8%) (Table II). The ESBL phenotype was found among 16 isolates (29%), with nine K. pneumoniae, six E. coli and one E. cloacae.

Molecular characterization showed that all ESBL-positive isolates carried the blaCTX-M gene: 15 of them had CTX-M-15 and one had CTX-M-3. Eleven iso­lates (68.8%) harbored the TEM-1 gene together with the CTX-M gene. Five isolates harbored the SHV-11 gene and one harbored SHV-1 together with CTX-M. Four isolates harbored CTX-M-15 gene together with TEM-1 gene and SHV-11 gene. One isolates harbored CTX-M-15 gene together with TEM-1 gene and SHV-1 gene. Ten of the ESBL-positive strains (5 E. coli and 5 K. pneumoniae) were regarded as MDR types.

The most frequently detected genes among the E. coli isolates were iron-related genes including fhuA (91.7%), fecA (75%), iutA (58.3%), fyuA (54.2%), iroN (50%) and iucC (50%), iha (8.3%). The most frequently detected adhesion gene was fimH (found in 83.3% of the isolates). The ibeA gene was found in 37.5% of isolates, while the neuC gene was found in 20.8% of isolates.

Sixteen (66.6%) isolates clustered in the ECOR group B2, 6 (25.0%) in D, 1 (4.2%) in A, and 1 (4.2%) in group B1.

None of the K. pneumoniae strains tested here were the capsular K1 or K2 type. Additionally, the magA gene was not identified in the isolates. In contrast, the gene uge was detected in all of the K. pneumoniae strains, while wabG was present in 11 of them (68.8%). Only one isolate harbored allS (no 75), while another harbored kfu and rpm genes (no 220).

PFGE typing demonstrated that almost all of the 24 E. coli isolates had unique pulsotypes. Two strains with identical pulsotypes were isolated from the same patient.

Table II
Non-susceptible strains among different species of Enterobacteriaceae family

	Escherichia coli n=24(%)	Klebsiella pneumoniae n=16 (%)	Enterobacter sp. n=6 (%)	Klebsiella oxytoca n=4 (%)	Serratia marcescens n=4 (%)	Morganella morganii n=1 (%)
Penicillins
Ampicillin	22 (91.7)	16 (100)	nd	4 (100)	nd	nd
Amoxicillin-clavulanate	13 (54.2)	9 (56.3)	nd	2 (50)	nd	nd
Piperacillin-tazobactam	0 (0)	2 (12.5)	1 (16.7)	0 (0)	0 (0)	0 (0)
Cephalosporins
Cefepime	6 (25)	9 (56.3)	0 (0)	0 (0)	0 (0)	0 (0)
Cefotaxime	5 (20.8)	9 (56.3)	2 (33.3)	0 (0)	1 (25)	1 (100)
Ceftazidime	6 (25)	9 (56.3)	1 (16.7)	0 (0)	1 (25)	0 (0)
Cefuroxime	6 (25)	9 (56.3)	nd	0 (0)	nd	nd
Carbapenems
Doripenem	1 (4.2)	3 (18.8)	0 (0)	0 (0)	0 (0)	0 (0)
Ertapenem	2 (8.3)	3 (18.8)	2 (33.3)	0 (0)	0 (0)	1 (100)
Imipenem	1 (4.2)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Meropenem	1 (4.2)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Monobactams
Aztreonam	8 (33.3)	13 (81.3)	2 (33.3)	1 (25)	1 (25)	1 (100)
Fluoroquinolones
Ciprofloxacin	5 (20.8)	7 (43.8)	0 (0)	0 (0)	0 (0)	0 (0)
Aminoglycosides
Amikacin	2 (8.3)	2 (12.5)	0 (0)	0 (0)	0 (0)	0 (0)
Gentamicin	3 (12.5)	3 (18.8)	0 (0)	0 (0)	0 (0)	0 (0)
Tobramycin	6 (25)	8 (50)	0 (0)	0 (0)	0 (0)	1 (100)
Tetracyclines
Tigecycline	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Miscellaneous
Chloramphenicol	1 (4.2)	3 (18.8)	0 (0)	0 (0)	0 (0)	0 (0)
Trimethoprim-sulfamethoxazole	10 (41.7)	6 (37.5)	0 (0)	0 (0)	0 (0)	0 (0)

MLST typing revealed 10 different sequence types; these included ST131 (7 isolates), ST69 (5), ST141 (3), ST998 (3), ST73 (1), ST75 (1), ST95 (1), ST543 (1) and ST569 (1). One isolate could not be typed. Four among the ST131 isolates were from NICU II (Fig. 1). PFGE typ­ing of 16 K. pneumoniae strains showed eight different pulsotypes. Five isolates from NICU VI belonged to one clone A. Those isolates were detected in 2009, 2010 and 2011. The mortality connected with those strains was 21.4%. Clone B consisted of two isolates similarly to clone C while the remaining five isolates had unique pul­sotypes. MLST typing revealed seven different sequence types: ST 336 (6 isolates), ST6 (3), ST 11 (2) ST17 (2), ST153 (1), ST321 (1), and ST 870 (1). The main clone identified by PFGE was compatible with ST 336 (Fig. 2). PFGE typing of the E. cloacae strains revealed unique pulsotypes. Among K. oxytoca, two strains with identi­cal pulsotypes were from the same patient, as was in the case for S. marcescens (data not shown).

Discussion

Enterobacteriaceae remain one of the most impor­tant, albeit not the most frequent, cause of BSIs. Sep­ticaemia or BSI in neonates is an important causeof neonatal mortality and morbidity in developing countries and K. pneumoniae and E. coli are prominent causative agents. In a study of 6215 infants admitted to the National Institute of Child Health and Human Development (NICHD) Neonatal Research Network (NRN) centers, 70% of first episode late-onset infec­tions were caused by Gram-positive organisms, with coagulase-negative staphylococci accounting for 48% of the infections; however, the death rates were highest for infants infected with E. coli. (Karlowicz et al., 2000) Studies conducted by Makhoul et al. (2005) showed that mortality rate was about 4 times higher for infections caused by E. coli and 6 times higher for K. pneumo­niae than for CoNS infections. Gram-negative bacilli (GNB) have been shown to be the non-dominant group of etiological risk factors for BSI, representing 20–30% of the infections in some studies (Graham et al., 2006; Mitt et al., 2014; Nagao, 2013). A study conducted byCordero et al. (2004) showed that 20% of BSI episodes were caused by GNB, of which 15% were Enterobacte­riaceae. Identification of these microorganisms in our study group was 15.2% and, importantly, infection with these etiological agents has been shown to be connected with high mortality rates in neonates (15%). Stoll et al. (2011) has described E. coli as the major causative patho-gen of BSIs in preterm infants and the second most common cause of BSIs in term infants. Vergnano et al. (2011) indicated that E. coli was the third most common microorganism isolated from neonates and reported a prevalence of 13% in the UK, whereas in Germany the prevalence was 4.8% (Geffers et al., 2008), which is about three times less than that observed in NICUs in the present study. Unfortunately, E. coli is frequently associated with severe infections and is the leading cause of sepsis-related mortality among VLBW infants. Indeed, E. coli accounted for 24.5% of EOI of neonates in the United States (Weston et al., 2011) and 14.6% in Polish VLBW infants (Chmielarczyk et al., 2014). In the present study, other Enterobacteriaceae family microorganisms such as Klebsiella spp. and Enterobacter spp. were markedly less common, similar to other stud­ies (Cordero et al., 2004; Geffers et al., 2008; Lombardi et al., 2014; Nagao, 2013; Vergnano et al., 2011).

Crossing the skin barrier provides a direct route of invasion for bacteria; therefore, insertion of a CVC and the length of its duration in situ are risk factors for the development of a BSI. Lombardi et al. (2014) showed that in the neonatal pathology ward CVC infections reached 36.3% among Gram-negative bacteria, and Klebsiella spp. were the most frequently isolated. This result is similar to our data. E. coli and Klebsiella spp. isolated from NICUs in this study have different epidemiology to each other; hence, they require different surveillance and infection con­trol. According to PFGE results, E. coli comprised dif­ferent clones, while among the K. pneumoniae isolates, some showed high genetic similarity. Similar findings have been reported previously by other authors. (Castro et al., 2010; Wojkowska-Mach et al., 2013) Of the six NICUs we investigated, the majority of the Enterobac­teriaceae isolates were from NICU VI, where Klebsiella dominated; this finding may be related to clonal spread of these bacteria. The Klebsiella clones detected in our study belong to four sequence types (ST): ST6, ST11, ST17 and ST336, of which ST11 and ST17 are known pathogens and colonizers widely distributed in Europe (Damjanova et al., 2008; Oteo et al., 2009).

The MLST technique showed that seven E. coli strains belonged to ST131, but according to the PFGE results, these strains had unique pulsotypes and came from three different centers; hence, they are assumed not to be clonally spread. It has been reported that strains belonging to the same ST do not always cluster in a single branch based on cluster analysis of PFGE patterns (Vimont et al., 2008). ST131 is often reported in adults and recently also in children and neonates, and is probably transmitted between mothers and neonates (Denkel et al., 2014). The spread of ESBL-producing ST131 E. coli in the community, especially during the neonatal period, is a cause for serious concern (Birgy et al., 2013). Recently, a CTX-M-15 ESBL-positive E. coli ST131 clone, belonging to the B2 phylogenetic group and characterised by a particular antimicrobial resistance and high virulence potential, became a major public health problem especially in developing coun­tries (Brisse et al., 2012; Clermont et al., 2009).

The microorganisms responsible for neonatal BSI have changed over time and vary from place to place. Prolonged use of broad-spectrum antibiotics in NICUs is associated with the current epidemic of MDR Gram-negative bacteria. Biedenbach et al. (2004) reported that the neonatal sepsis rate from Klebsiella spp. was higher in Latin America than in North America and was cor­related with the use of extended-spectrum antibiotics. Klebsiella spp. and E. coli had the highest level of resist­ance to ampicillin, amoxicillin and aztreonam, which may have been caused by frequent administration of these drugs. Studies about antibiotic consumption con­ducted on the Polish NICUs have shown that beta-lac­tams were administred in more than 50% of infections (Rozanska et al., 2012). In our study, higher levels of antibiotic resistance (and more ESBL-positive strains) were observed among Klebsiella strains, probably as a result of the epidemic clone detected in NICU VI.

The E. coli isolates studied here were more likely to posses the ibeA gene than those derived from urinary and respiratory tract infections (Chmielarczyk et al., 2013). Soto et al. found that strains causing EOI harbored a higher frequency of the ibeA gene (Soto et al., 2008). Among Klebsiella sp. only some of the isolates possessed virulence genes such as rpmA or kfu. None of the iso­lates possessed K1 or K2 serotypes. As the capsule isa major virulence factor for K. pneumoniae (Cortes et al., 2002), information about the prevalence of the capsular types in such infections is an essential component of infection prevention and control (Pan et al., 2013).

According to the results, contact isolation is required when a patient is colonized or infected by a microor­ganism which is transmitted by direct physical contact. The presented results have shown that reliable adher­ence to that principle by the personnel is necessary when Klebsiella spp. is highly level of endemic or in case of epidemic. However, data about E. coli showed that precautions should be different and regimen about hand hygiene is insufficient. In the case of E. coli sur­veillance on people who take care of a neonate includ­ing mother and people performing Kangaroo care, could be performed. Infection prevention should also require intensive education and preparing procedures about Kangaroo care, feeding and milk expression (including milk storage) (Guzman-Cottrill et al., 2013).

Acknowledgements
The authors wish to thank the staff in NICUs for their help and interest in the study.

Funding
This work was supported by a grant from the Ministry ofScience and Higher Education (DEC-2011/01/D/N27/00104). The sponsors provided the funding for the project only.

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