During the first semester of 2012, an unexpected increase in the frequency of isolation of ESBL-E. coli was observed in the NICU of the University Hospital “Azienda Ospedaliero-Universitaria Policlinico P. Giaccone”, Palermo, Italy. The NICU annually admits 250 infants approximately of all gestational ages (mean 36.7, range 26–42). Because it is associated to the regional reference centre for genetic diseases, the NICU has a high prevalence of neonates with malformations (20% approximately) as well as of outborn admissions (35% approximately). Moreover, a further 20% proportion of patients has complex conditions requiring subspecialty medical or surgical care. The NICU includes one intensive care room consisting of 8 beds and one intermediate care room including 8 further beds. The average nurse to patient ratio is 1:3 to 1:4 in the two sections, respectively.
Since June 2009, a surveillance protocol is routinely in place which includes rectal swabs obtained on a weekly basis (every Tuesday) from all infants staying in the NICU to monitor the prevalence of colonization with multidrug resistant (MDR) Gram negatives.
All children admitted to the NICU and colonized by ESBL-E. coli between January and June 2012, were included in the study. Cases were defined as infants colonized by E. coli simultaneously resistant to third generation cephalosporins and fluoroquinolones. Microbiology database was queried as far back as January 2010 to identify additional ESBL-E. coli isolates.
The study protocol was approved by the Ethics Committee of the Azienda Ospedaliero-Universitaria Policlinico “P. Giaccone”, Palermo, Italy, and informed consent was sought in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from the parents of each patient.
A case–control study was performed to look for possible risk factors for the colonization by the ESBL-E.coli strain. Controls were the infants who were being admitted between the admission of the index case and the discharge of the last case and had negative surveillance cultures for ESBL-E.coli. For cases, risk factors were assessed for the interval between admission and the first positive culture for ESBL-E. coli. For controls, risk factors were assessed for the entire NICU stay. Data on demographics, way of delivery, gestational age, birth weight, singleton or twin pregnancy, antibiotics administration, surgical procedures, exposure to devices and length of stay were collected. Univariate analysis was carried out by the EpiInfo software (ver.7; Centers for Disease Control and Prevention, Atlanta, GA, US). Tests of significance were performed with χ2 or Student’s t test where appropriate. The assessment of certain risk factors such as birth weight, length of stay, device exposures, and length of antibiotic treatment, analysis of variance (ANOVA) was used. Stepwise logistic regression analysis of variables found significant in the univariate analysis (P <0.10) was performed using StatPlus 2009 software. All significance tests were two-tailed, and P <0.05 was considered significant.
Intestinal colonization by antibiotic-resistant Gram negatives was assessed by culturing broth-enriched rectal swabs onto MacConkey agar. Four antibiotic disks, containing gentamicin (10 μg), amoxicillin-clavulanic acid (20–10 μg), meropenem (10 μg) and ceftazidime (30 μg) were placed on each plate before incubation. These antimicrobial drugs were selected in order to include the two more frequently used antibacterial drugs in UTIN, gentamicin and amoxicillin-clavulanic acid, and two further antibiotics, such as ceftazidime and meropenem, that could allow for timely detecting Gram negative organisms resistant to cephalosporins and/or carbapenems. Selective media were not employed, so that Gram negative colonization could be detected and ceftazidime-susceptible and –non susceptible organisms could be isolated. Colonies growing into antibiotic inhibition halos were subcultured, biochemically identified using API20E strips (Biomérieux, Marcy l'Étoile, France) and submitted to antibiotic susceptibility testing . Antibiotic susceptibility testing and ESBL detection were first performed by disk diffusion and double disk synergy test and then confirmed by Etest (Biomérieux, Marcy l'Étoile, France) methods according with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines . An isolate was defined as resistant to third generation cephalosporins, when the inhibition zone diameter of ceftriaxone (30 μg) and cefotaxime (5 μg) was < 20 mm and 17 mm, respectively . This isolate was defined as simultaneously resistant to fluoroquinolones when the inhibition zone diameter of ciprofloxacin (5 μg) was < 19 mm . E. coli ATCC 25922 was used as quality control strain. An alert email was sent to NICU in the event of growth of colonies or no inhibition zone around the ceftazidime and/or meropenem disk.
E. coli phylogenetic group was determined by a triplex polymerase chain reaction (PCR) assay as described previously .
Multiplex PCRs were used to amplify genes encoding ESBLs . blaCTX-M genes were amplified with specific primers and sequenced. In addition, the aac(6’)-Ib-cr aminoglycoside-quinolone resistance gene was characterized by PCR amplification and sequencing. Conjugation experiments were performed using the E. coli strain K12J5 Rifr as a recipient. Plasmids were classified according to their incompatibility group by using a PCR-based replicon typing scheme .
Clonality of the ESBL-E. coli isolates was determined by rep-PCR using the DiversiLab Escherichia kit for DNA fingerprinting (BioMerieux, Marcy-l'Etoile, France) . E. coli isolates were further characterized by multilocus sequence typing (MLST) according to the University College Cork (Cork, Ireland) scheme for E. coli (http://mlst.ucc.ie/mlst/dbs/Ecoli).