Prevalence and risk factors of fecal carriage of extended-spectrum [beta]-lactamase (ESBL)-producing enterobacteriaceae in hospitalized and ambulatory children/ Cocuklarda genisletilmis spektrumlu beta laktamaz ureten enterobacteriaceae fekal tasiyiciligi prevalansi ve risk faktorleri.Introduction
Extended-spectrum [beta]-lactamases (ESBLs) are a heterogeneous group of plasmid mediated bacterial enzymes that can hydrolyze oxymino-[beta]-lactams and are responsible for bacterial resistance against extended-spectrum [beta]-lactam antibiotics. Production of ESBL was first described in K. pneumoniae isolates in 1983. Especially with the excessive worldwide use of cephalosporins, the incidence of multidrug-resistant ESBL-producing Enterobacteriaceae is markedly elevated (1,2). These organisms are major nosocomial pathogens, which cause urinary tract infections, bacteremia or intra-abdominal infections. Because of the cross-resistance that they show to other groups of antibiotics, few antimicrobial agents are available as therapeutic options for these infections (3). At the same time, they are important community-acquired urinary pathogens, and their spread in the community is an unique public health problem (2). Use of Carbapenems, which are still effective against ESBL-producing microorganisms, has been associated with a low risk of mortality in cases of serious infections caused by these pathogens (4-6).
Colonization with ESBL-producing strains is considered a prerequisite for infection (1). The aim of this study was to prospectively investigate the prevalence and possible risk factors for fecal carriage of ESBL-producing E. coli and Klebsiella spp. Hospitalized and ambulatory patients at a tertiary care center in Eskisehir, Turkey, were studied during a non-outbreak period between May and October 2007.
Materials and Methods
This study was performed between May 2007 and October 2007 in a tertiary care hospital in Eskisehir, Turkey. This university hospital serves the city (population approximately 700 000), and surrounding cities. The pediatric and neonatal intensive care units (ICUs) located in this hospital are the only tertiary ICUs for these cities. Nearly 150 new patients were hospitalized in pediatric services (including ICUs) and 2 000 children were admitted to our outpatient clinic and emergency unit every month. We prospectively evaluated our inpatient clinics for fecal carriage of ESBL-producing microorganisms every week. Children seen in the outpatient clinics or emergency department with no history of hospital admission in the preceding month were enrolled in the outpatient group. Patients were excluded if an ESBL-producing microorganism had been previously isolated from sterile sites. This study was approved by the local ethical committee and informed consent was obtained from at least one of the parents of each patient.
For the inpatient group, the following data were recorded: age, gender, primary diagnosis, chronic underlying conditions, presence of co-morbidity, total hospital stay, intensive (pediatric or neonatal) care stay, history of receiving broad-spectrum antibiotics, history of cephalosporin usage (before hospitalization), total parenteral nutrition, indwelling catheter, surgical intervention, urinary catheterization and presence of neutropenia. For outpatients, age, gender, chronic underlying disease (diabetes mellitus, renal or cardiac disease) and history of 3rd or 2nd generation cephalosporin usage in the previous 3 months were recorded. The prevalence of carriage was calculated as the percentage of carriers among participants of each group. To investigate the risk factors associated with fecal carriage within groups, carriers were compared with non-carriers in terms of exposure to the different variables studied.
Fecal carriage was studied either by performing a rectal swab or by immediately sending stool samples to the microbiology unit. Patients with an ESBL-producing E. coli isolate on a rectal swab were considered carriers. The stool samples were inoculated onto EMB agar plates without antibiotics for evaluation of aerobic gram-negative flora and with 2 [micro]g/ml cefotaxime for selection of ESBL-producing strains. Inoculated plates were incubated at 35[degrees]C for 24 to 48 h under aerobic conditions, and colonies consistent with E. coli or Klebsiella were subjected to identification with the MicroScan WalkAway-96 SI System (Dade Behring, USA). E. coli and Klebsiella isolates that showed positive results with the screening test were tested for ESBL production using the combination disk test, based on CLSI methodology. All ESBL-producing strains were also evaluated for imipenem susceptibility by the agar dilution method, according to CLSI (7,8). The range of antimicrobial resistance concentrations tested by agar dilution was 0.03 to 32 [micro]g/ml. Antibiotics were supplied as powders of known potency by Merck Sharp & Dohme(r). E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as the reference strains for quality control.
Statistical analyses were performed with SPSS 13.0 for Windows. For independent samples, categorical variables were compared using the Chi square test or Fisher's exact test. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated from 2-by-2 contingency tables. A p-value of <0.05 was considered significant.
Four hundred and sixty-four samples were collected between May and October 2007. Two hundred and seventy (58%) were from hospitalized patients, and 194 (42%) were from ambulatory patients.
Sixty-six (24%) of 270 hospitalized patients (including 70 newborns, 13 of whom were ESBL positive) harbored ESBL-producing bacteria, including 25 (37%) who harbored Escherichia coli, 39 (60%) who harbored Klebsiella pneumoniae and 2 (3%) who harbored Klebsiella oxytoca. Thirty-four (51%) patients in this group were male, and 32 (49%) were female; no significant difference was found between genders (99 male and 105 female p>0.05). Duration of hospital stay was between 3 and 351 days (median 40 days). Intensive care unit stay length varied between 1 and 190 days (median 110 days). Mechanical ventilation duration in ICU patients was between 1 and 290 days (median 79 days). Fifty-three patients (80%) had at least one previously described co-morbidity, while 13 (20%) had none. Nineteen (28%) had taken total parenteral nutrition, 27 (40%) had urinary catheterization, 24 (36%) had indwelling catheterization, 32 (48%) had an ICU stay, 26 (39%) had surgical intervention, 12 (18%) had neutropenia, and 11 (16%) had prior cephalosporin use (10 with ceftriaxone and one with cefepim) before hospitalization. For the hospitalized patients, univariate analysis showed that ICU stay (p<0.001), urinary catheterization (p=0.008), indwelling catheterization (p=0.007), surgical interventions (p<0.001) and prior 3rd-generation cephalosporin usage (p=0.004) were associated with ESBL positivity (Table 1). In this group, imipenem MIC values for E. coli ranged from 0.06-0.5 [micro]g/ml: the MIC50 was 0.125 [micro]g/ml, and the MIC90 was also 0.125 [micro]g/ml. For Klebsiella spp. against imipenem, MIC values ranged from 0.125-4 [micro]g/ml: the MIC50 was 0.5[micro]g/ml, and MIC90 was 2 [micro]g/ml.
Fourteen isolates from 194 ambulatory patients (7.2%) were identified as ESBL producers, including 10 (72%) E. coli, 3 (21%) K. pneumoniae and 1 (7%) Klebsiella oxytoca. Ten patients had a history of 2nd and 3rd-generation cephalosporin use (5 cefuroxime axetil, 1 cefaclor, 4 ceftriaxone). However, there was no statistically significant difference in gender, age or presence of underlying disease (renal disease, hepatic failure, malignancy, cardiac disease etc.) (p>0.05). Among ambulatory patients positive for ESBL producers, the most remarkable result is the prior 2nd and 3rd-generation cephalosporin usage as an independent risk factor (p=0.003) for fecal carriage (Table 1). In the outpatient group, E. coli MIC values for imipenem varied between 0.06 and 0.125 [micro]g/ml, with 3 K. pneumoniae and 3 K. oxytoca isolates at the highest level of 0.125 [micro]g/ml.
The rate of fecal carriage of ESBL-producing isolates was 24.4% in hospitalized children, and the rate of occurrence of ESBL-producing isolates among ambulatory children was 7.2%. According to another study including adults and children from our country, fecal carriage rates are 47% in hospitalized patients and 15% in outpatients (9). Valverde et al. (1) reported dramatically increased levels of ESBL-producing isolates in recent years and showed that fecal carriage rates of ESBL-producing Enterobacteriaceae in Spain increased from 0.6% in 1991 to 7.0% in 2003. According to previous reports similar to our study, E. coli accounts for the vast majority of isolates. Recently, in Spain, Rodiriguez-Bano et al. (10) showed that 7.4% of patients admitted to the emergency department have fecal carriage of ESBL-producing E. coli. The prevalence of fecal carriage varies between different geographical areas; in 2003, it was 1.4% in the UK, 2.4% in Lebanon, 7% in India and 15.4% in Saudi Arabia (11-14). A higher prevalence of 4.2% was found in hospitalized neonates upon admission to a neonatal ICU in the United States (15). Ben-Ami et al. (16) found that 10.8% of the patients studied on hospital admission in Israel were fecal carriers. A significant increase in the prevalence of fecal carriage was also observed in healthy children in Bolivia and Peru, from 0.1% in 2001 to 1.7% in 2005 (17). Rates from different geographical regions vary according to different antibiotic policies and whether the study was performed during nosocomial outbreak situations. Our study result of a 7.4% rate of fecal carriage of ESBL-producing isolates among ambulatory children is similar with the recent study from Spain, but higher than other previous reports (except from Saudi Arabia) (10,14).
In our study, pediatric or neonatal ICU stay, urinary catheterization, indwelling catheter, surgical interventions (including tracheotomy) and prior 3rd-generation cephalosporin use were associated with ESBL positivity for hospitalized patients. Demir et al. (18) reported that the strongest independent predictors of ESBL-producing K. pneumoniae colonization were mechanical ventilation and hospitalization for longer than 14 days. Celebi et al. (19) defined pediatric risk factors and clinical outcomes associated with Bacteremia from ESBL-producing Klebsiella spp. in Bursa, Turkey, between 2003 and 2007. The overall incidence was 4.7 per 1000 admissions; 57% of the isolates were ESBL producers, and malignancy was the common underlying condition according to their study. They described aspects of the previous therapy with broad-spectrum antibiotics, prolonged hospitalization, the presence of a central venous catheter and total parenteral nutrition as risk factors for colonization with ESBL-producing Klebsiella spp.
Physicians should be aware that ESBL-producing organisms are not only circulating in hospital environments but in the community as well. This situation can supply the causative agents for both community acquired and nosocomial infections. Our study suggests that 7.2% of outpatients have fecal carriage of ESBL-producing bacteria. Although there were limited patients for statistical analysis (14 patients), the imipenem MIC values for these isolates were considerably lower than those from hospitalized patients. The increase in the proportion of carriers in the community raises the risk that other individuals will become carriers as a consequence of human-to-human transmission of resistant bacteria or through the environment, enriching the resistance gene pool and thus facilitating the acquisition of resistant genes by susceptible bacteria.
One way to decrease ESBL prevalence in an institute is a strict antibiotic policy that reduces the usage of cephalosporins. A history of 3rd generation cephalosporin use is one of the risk factors for fecal carriage of ESBL-producing microorganisms in our hospitalized patients. Interestingly, for non-hospitalized patients, 2nd or 3rd generation cephalosporin use incurs a 2.4-fold increased risk for fecal carriage of ESBL-producing microorganisms. Rodriguez-Bano et al. (10) investigated the risk factors associated with fecal carriage in healthy individuals in non-outbreak situations, and they suggested that home food may be a source of these isolates in patients with community acquired UTI caused by ESBL-producing E. coli. Furthermore, Ben-Ami et al. (16) found that poor functional status, current antibiotic use, chronic renal insufficiency, liver disease and use of histamine 2-receptor antagonists were the independent risk factors for fecal carriage; their study included a wide range of ESBL-producing Enterobacteriaceae, and one-third of their patients were from a long-term care facility. Colodner et al. (20) reported that 2nd and/or 3rd generation cephalosporins are risk factors for urinary tract infections due to ESBL-producing microorganisms. A history of 3rd generation cephalosporin usage was the only previously known independent risk factor for bloodstream infections with ESBL-producing E. coli and K. pneumoniae (21).
Our findings for outpatients show that strict antimicrobial agent usage policy, especially restricting extended-spectrum cephalosporins, could help to control the high carriage rates for these organisms. These findings warrant further studies about the consequences of colonization with ESBL-producing bacteria, both in the community and the hospital setting.
This study was presented as a poster presentation 48th Annual ICAAC/IDSA 46th Annual Meeting, October 25-28, 2008, Washington DC, United States.
Conflict of Interest
No conflict of interest was declared by the authors.
Gelis Tarihi: 05.10.2010
Kabul Tarihi: 02.03.2011
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Abdurrahman Kiremitci , Ener Cagri Dinleyici , Zeynel Abidin Yargic , Gul Durmaz , Neslihan Tekin , Askin Derya Aybey , Mehmet Arif Aksit 
 Department of Medical Microbiology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey
Department of Pediatrics, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey
Department of Pediatrics, Division of Neonatology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey
Dr. Ener Cagri Dinleyici Department of Pediatrics, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey,
Phone: +90 222 229 00 64
Table 1. Univariate analysis of factors influencing fecal carriage of ESBL producing Enterobacteriaceae in hospitalized and ambu-latory patients Hospitalized patients ESBL (+) ESBL (-) OR Lower n=66 n=204 Total parenteral nutrition 19 43 1.51 0.77 Urinary catheterization 27 49 2.19 1.17 Indwelling catheterization 24 41 2.27 1.18 Intensive care unit stay 32 17 10.35 4.92 Surgical intervention 26 37 2.93 1.53 Neutropenia 12 20 2.04 0.88 Prior cephalosporin usage 11 11 3.51 1.33 Ambulatory patients ESBL (+) ESBL (-) OR Lower n=14 n=180 Presence of chronic illness 8 76 1.82 0.55 Prior 2nd and 3rd generation cephalosporins 10 54 5.83 1.59 Upper p Total parenteral nutrition 2.87 p=0.196 Urinary catheterization 4.10 p=0.008 Indwelling catheterization 4.35 p=0.007 Intensive care unit stay 22.0 p<0.001 Surgical intervention 5.64 p<0.001 Neutropenia 4.73 p=0.067 Prior cephalosporin usage 9.27 p=0.004 Upper p Presence of chronic illness 6.23 p=0.278 Prior 2nd and 3rd generation cephalosporins 23.23 p=0.003