Risk factors for the acquisition of extended-spectrum beta-lactamase-producing Enterobacteriaceae in hospitalized children

1 Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay 2 Departamento de Pediatría, Centro Hospitalario Pereira Rossell, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay 3 Departamento de Farmacología y Terapéutica. Hospital de Clínicas “Dr. Manuel Quintela”, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay 4 Laboratorio Central del Hospital Pereira Rossell, Ministerio de Salud Pública, Montevideo, Uruguay


Background
Infections by extended-spectrum Beta-lactamases (ESBL)-producing Enterobacteriaceae (EPE) are an increasing problem in pediatrics and are usually associated with higher hospital costs, failure of the empirical antibiotic treatment, and higher mortality rates [1][2][3][4].
Data concerning prevalence and risk factors (RF) for EPE infections in the general pediatric population are scarce, mostly associated with bacteremia, and only a few include molecular identification of ESBLs [2,8].

The study
We performed a case-control study (1:2 ratio) in the Hospital Pediátrico Centro Hospitalario Pereira Rossell (HP-CHPR), Montevideo, Uruguay, enrolling patients admitted into the pediatrics wards (PW) and the intensive care unit (ICU) between 1 May 2009 and 30 April 2010.
Patients with clinical isolates of EPE and non-EPE were defined as cases and controls, respectively.Both groups were paired according to the origin of the sample to ensure similar epidemiological conditions.Due to the proportion of EPE infections within the ICU, we extended the collection period to gather the required number of controls.
Only one isolate per patient was taken into consideration for this study.
Identification and antibiotic susceptibility testing were performed using the Vitek2 Compact system (bioMérieux, Marcy l'Etoile, France) and interpreted following CLSI recommendations [9].
Patient data was obtained from clinical records (age; gender; ward; total length of stay; length of stay since acquisition of infection; presence of nosocomial infection [10]; hospitalization in PW or ICU in the previous six months; use of third-generation cephalosporins (TGC) during the last month; presence of underlying chronic illnesses).We also registered the empirical antibiotic therapy (EAT), adjusted therapy according to the susceptibility report, and noted the success of the empirical and/or adjusted therapy (i.e., apyrexia or clinical improvement after 72 hours of antibiotic therapy, and/or negative bacterial cultures).
Numeric variables were compared using the T-test;  2 or Fisher's exact test were used for variables divided into categories.
A multivariate analysis was performed using logistic regression including variables with a P value less than 0.2 on the univariate analysis.A two-tailed P value less than 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 17.0 software (IBM SPSS Inc., Chicago, IL, USA).
This study was approved by the Direction Board of the HP-CHPR.
A total of 104 Enterobacteriaceae strains were isolated from patients admitted into the PW and the ICU.Fifteen strains were identified as EPE, 6 out of 82 (7%) in the PW and 9 out of 22 (40%) in the ICU.
Distribution of strains from 15 cases and 30 controls is shown in Table 1.
EPE acquisition was associated with underlying chronic illnesses ( The mean of hospitalization for detection of Enterobacteriaceae was 11 days for controls versus 23 days for cases (P = 0.007).
The number of different antibiotic therapies used was significantly higher for cases than for controls (P < 0.0001).As shown in and one with the correct EAT.In contrast, three patients with urinary tract infections (two K. pneumoniae and one E. coli) responded favorably to the EAT regardless of being infected by antibioticresistant microorganisms.
The EAT was more successful for the control group (P = 0.009); globally, antibiotic resistance was higher in EPE than in non-EPE (Table 2).
The following ESBL genes were detected by PCR: four bla CTX-M-2 (two in E. coli and two in K. pneumoniae); four bla CTX-M-9 (two in E. cloacae and two in K. pneumoniae); three bla SHV-5 (two in S. marcescens and one in E. coli); two bla CTX-M-8 (two K. pneumoniae); one bla CTX-M-15 ( in K. pneumoniae); and one bla SHV-2 (K.pneumoniae).
Seven of the eight amikacin-resistant isolates harbored the aac(6´)-Ib gene.
Plasmid-mediated quinolone resistance (PMQR) genes were detected in four strains, one K. pneumoniae strain with qnrB along with bla CTX-M-8 , and three bla CTX-M-9 -producing strains carrying qnrA (two E. cloacae and one K. pneumoniae).
Different from previous reports, we observed an increase in the relative frequency of CTX-M enzymes, other than CTX-M-2, accompanied by PMQR genes [5][6][7].
EPE acquisition has been linked to hospitalization, presence of severe illnesses, presence of prosthetics, and prior exposure to antibiotics, especially TGC [2,11,12].
Multivariate analysis showed that previous hospitalization in the ICU was the only independent RF for EPE acquisition.However, to improve the detection of patients with EPE infection, the four predisposing factors detected by univariate analysis should be kept in mind.The presence of three or more of these predisposing factors (for children older than 30 days), has a sensitivity to detect patients with EPE ~84.6% and a specificity ~96.4%.RF for EPE infections in newborns involves different aspects, mostly related to the perinatal period [13].
The acquisition of EPE in this study was associated with an inadequate EAT and to the usage of Concomitantly, EPE were significantly more resistant to ciprofloxacin and aminoglycosides, leading to failure of the EAT, regardless of the administration of TGC.Although quinolone usage is restricted in the pediatric population [14], five out of fifteen EPE showed co-resistance to ciprofloxacin.

Conclusion
Our work provides clinical and epidemiological information that may facilitate the characterization of EPE acquisition in the HP-CHPR.The risk factors detected in this work can be used to optimize both diagnosis and treatment of patients with probable EPE infections, regardless of hospital ward or type of infection.

Table 1 .
General characteristics of the study patients and strains a: non significance

Table 2 .
Antimicrobial resistance and antimicrobials used for treatment in case and control groups treatment and antibiotic susceptibility profile