INTRODUCTION
Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) have become increasingly prevalent nosocomial pathogens [1-3]. During recent years, community-onset infections due to ESBL-EC have emerged worldwide [4,5]. ESBL-EC are also frequently resistant to non-β-lactam antibiotics, such as fluoroquinolones (FQs), trimethoprim-sulfamethoxazole (TMP-SMX), and aminoglycosides [2,4]. FQs, cephalosporins, and TMP-SMX are recommended as empirical therapies for community-onset infection due to E. coli [6-8]. Most patients with community-onset infections caused by these organisms have urinary tract infections (UTIs) or intra-abdominal infections [5,9]. FQs and TMP-SMX may be regarded as the treatment of choice or as alternative antimicrobial therapy for UTIs or intra-abdominal infections due to ESBL-EC, if the bacteria are susceptible in vitro to these agents [4].
The clinical relevance of multidrug resistance (MDR) among ESBL-EC (i.e., those resistant to multiple other antibiotics or antibiotic classes in addition to the oxymino β-lactams) is of great concern because therapeutic options are severely limited [10]. As the frequency of infection caused by MDR-ESBL-EC is increasing, so the use of carbapenems is rising, which may contribute to the spread of carbapenem resistance. Moreover, isolation of carbapenem resistant ESBL-producing Enterobacteriaceae has been increasingly reported [11]. In addition, empirical antibiotic treatment of community-onset infections may be inappropriate if MDR-ESBL-EC are highly prevalent in the community.
Even though MDR-ESBL-EC poses significant therapeutic challenges, there is still little clinical information about community-onset MDR-ESBL-EC infections. Although several studies have reported on hospital-acquired MDR-ESBL-EC infections, data regarding community-onset MDR-ESBL-EC infections are limited [10,12,13].
This study was conducted to determine risk factors and the molecular epidemiology of MDR among ESBL-EC causing community-onset infections. We wanted to identify risk factors associated with the acquisition of community-onset MDR-ESBL-EC infections. In addition, we wanted to identify risk factors associated with acquisition of a MDR-ESBL-EC among patients with community-onset ESBL-EC infections. For this purpose, we employed the case-control-control study design, which enables more accurate identification of risk factors for multidrug-resistant pathogens than the standard case-control study design [14]. Using data collected in a 9-month period at two university hospitals, we conducted parallel analyses in which patients who presented with an infection caused by MDR-ESBL-EC were compared to patients who presented with an infection caused by non-MDR-ESBL-EC and patients with non-ESBL-EC infections.
METHODS
Study design and patients
We performed a post hoc analysis of individual patients included in a previous study [15]. In the current study, a case-control-control design was used to evaluate risk factors associated with MDR-ESBL-EC in community-onset infections. Study subjects were prospectively identified as previously described [15] through a computerized database maintained by the clinical microbiology laboratory. The study was conducted between September 2010 and May 2011 at Samsung Medical Center in Seoul (a 1,950-bed tertiary care university hospital) and at Samsung Changwon Hospital (a 700-bed community-based university-affiliated hospital in Changwon, Korea). Patients with community-onset infection due to E. coli were included in the study. The inclusion and exclusion criteria were the same as reported previously [15]. A case patient was defined as a person whose clinical sample yielded MDR-ESBL-EC. A control group I (CG I) patient was defined as a person whose clinical sample yielded ESBL-EC that did not meet the criteria for MDR. A control group II (CG II) patient was defined as a patient with a non-ESBL-EC infection. For each patient who presented with an infection caused by ESBL-EC, one CG II was randomly chosen from a group of outpatients from whom non-ESBL-EC had been isolated from a clinical sample that had been sent to the same laboratory for culturing during the week following presentation of the case patient. The CG II patients were matched according to the culture specimen and acquisition unit. The patient data collected included age, sex, underlying disease, severity of underlying diseases as classified by McCabe and Jackson criteria [16], presence of severe sepsis or septic shock at presentation, severity of illness at presentation according to the Pitt bacteremia score [2], and receipt of any antimicrobial therapy prior to onset of the infection. The presence of any of the following comorbid conditions was also documented: neutropenia, recent surgical procedure within the prior 3 months, corticosteroid use within the prior 1 month, immunosuppressive therapy within the prior 1 month, or the presence of an indwelling urinary catheter or percutaneous tube. Because the study was observational, the attending physician decided on indications for cultures, other tests, and treatments based on each patient’s individual clinical situation. The study was approved by the Institutional Review Board of Samsung Medical Center, Seoul, Korea. Written informed consent was not required because of the observational nature of the study.
Definitions
The community-onset infections were further classified as healthcare-associated (HCA) or community-associated (CA). Episodes were considered to be HCA if any of the following criteria were present: a history of a 48 hours or longer hospital admission in the previous 90 days, hemodialysis, intravenous medication, home wound care in the previous 30 days, or residence in a nursing home or long-term care facility [9,17]. Community-onset infections that did not meet these criteria were considered to be CA. MDR-ESBL-EC was defined as an ESBL-EC that demonstrated in vitro resistance to all of the following three antibiotics or antibiotic classes: TMP-SMX, FQs (i.e., ciprofloxacin and levofloxacin), and gentamicin. Non-MDR-ESBL-EC was defined as ESBL-EC that did not meet the criteria for MDR. Community-onset infection was defined as an infection diagnosed within the first 48 hours of hospitalization. The sites of infection were determined by the physicians on the basis of clinical evaluation and the isolation of EC from the presumed portal of entry [9,18]. Primary bacteremia was defined according to Centers for Disease Control and Prevention definitions [18]. Patients with immunosuppression included those who were on immunosuppressive therapy (chemotherapeutic agents, immunosuppressive agents, or radiation therapy). Neutropenia was defined as an absolute neutrophil count < 500 neutrophils/mm3. Sepsis was defined as the presence of a systemic inflammatory response syndrome (any two of the following: tachypnea > 20 breaths per minute, white blood cell count < 4,000 or > 12,000 cells/µL, heart rate > 90 beats per minute, and fever > 38.0°C or hypothermia < 36.0°C) in addition to a documented or presumed infection [19]. Severe sepsis was defined as sepsis with one or more clinical signs of organ dysfunction and septic shock defined as sepsis with hypotension despite adequate fluid resuscitation along with the presence of perfusion abnormalities [19]. Prior antibiotic therapy was defined as the receipt of any systemic antibiotics for more than 48 hours in the preceding 30 days.
Microbiologic analysis
Identification of microorganisms, antimicrobial susceptibility testing, and ESBL confirmatory testing were performed as previously described [15]. Quality control was performed using the strains E. coli 25922 and Pseudomonas aeruginosa 27853.
Detection of ESBL genes and sequencing of the polymerase chain reaction product
For the characterization of ESBL types, polymerase chain reactions (PCRs) and sequencing of PCR products were performed using available stored isolates, as described previously [15]. ESBL-related genes, such as TEM, SHV, CTX-M, and OXA, were amplified by PCR from clinical isolates as described in previous studies [20,21]. The types of ESBL genes were identified by comparing the sequences to those in the database of G. Jacoby and K. Bush (http//lahey.org/Studies/).
Genetic typing by pulsed-field gel electrophoresis
Available MDR-ESBL-EC isolates were evaluated for genetic relatedness by pulsed-field gel electrophoresis (PFGE). For PFGE, agarose-embedded bacterial genomic DNA was digested with 20 U Xba l. The restriction fragments were separated by electrophoresis in 0.5 × Tris/borate/ethylenediaminetetraacetic acid (TBE) buffer. The subsequent PFGE analyses were performed on a CHEF-MAPPER XA apparatus (Bio-Rad Laboratories, Hercules, CA, USA) as described previously [22]. The isolates were considered genetically related if the Dice coefficient correlation was 80% or greater, which corresponds to the possibly related criterion of Tenover et al. [23].
Statistical analysis
The Student t test or the Mann-Whitney test was used to compare continuous variables, and the chi-square or Fisher exact test was used to compare categorical variables. We used a backward stepwise logistic regression analysis to control for the effects of confounding variables. All risk factors with a p < 0.1 at the bivariate level were included in the multivariate logistic model predicting MDR-ESBL-EC infection. All variables for which the p value was less than 0.05 in the multivariate analysis were retained in the final model. Interactions between variables were not introduced into the models. Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated. All p values were two-tailed, and p < 0.05 was considered statistically significant. IBM SPSS version 19 (IBM Co., Armonk, NY, USA) was used for these analyses.
DISCUSSION
Our data showed that 27.8% of community-onset ESBL-EC isolates were MDR and significant risk factors for MDR among ESBL-EC isolates causing community-onset infections were prior exposure to FQs, receipt of immunosuppressive therapy, and HCA infection. Previous studies have suggested that the prevalence of community-onset ESBL-EC infections varies widely by geographic region, and most ESBL-producing isolates show co-resistance to multiple antibiotics [5,9,24,25]. However, data on risk factors for community-onset MDR-ESBL-EC infections are limited. Although two previous studies evaluated risk factors for infections by MDR-ESBL-EC, these studies included mainly nosocomial infections and the proportion of community-onset infections was less than 10% of the study population [10,13]. To the best of our knowledge, this is the first study to investigate risk factors and molecular epidemiology of MDR-ESBL-EC isolates among community-onset infections.
In the current study, we sought to identify risk factors for MDR-ESBL-EC among community-onset infections. The rate of MDR among ESBL-EC isolates was similar to rates found in previous studies [10,12]. We found that the receipt of immunosuppressive therapy, prior exposure to FQs, and HCA infection were associated with community-onset MDR-ESBL-EC infections. Patients with community-onset infection due to an MDR organism were more likely to be prescribed inactive empirical therapy. The MDR-ESBL-EC group received more inappropriate empirical therapy than the non-MDR-ESBL-EC group (90.0% vs. 66.7%, p = 0.015). Patients on immunosuppressive therapy, those with prior exposure to FQs, and those with HCA infections should be offered carbapenems as empirical therapy even for community-onset infections.
In our previous study, prior receipt of FQs was an independent risk factor for nosocomial MDR-ESBL-EC and Klebsiella pneumoniae infections [12]. In our previous studies, HCA infection and prior receipt of FQs were associated with community-onset ESBL-EC infections [15]. In other previous studies, FQ use has been associated with the acquisition of other MDR gram-negative infections, such as P. aeruginosa and Acinetobacter baumannii [26,27]. In addition, Villers et al. [27] showed that the institution of a policy restricting the use of intravenous FQs could decrease the rate of infection with A. baumannii. Interestingly, a recent study showed a correlation between FQ resistance and MDR in ESBL-EC infections [28]. Because this has been previously reported for MDR gram-negative infections [26,29,30], we can speculate that prior receipt of a FQ either results in selective pressure or facilitates the activation of intrinsic mechanisms that confer resistance to multiple antibiotic drug classes, such as the drug efflux mechanism.
In our study, immunosuppressive therapy was also associated with community-onset MDR-ESBL-EC infections. Immunosuppression presents an especially troublesome situation for critically ill patients as it has been identified as an independent risk factor for infectious morbidity and mortality [31]. Not surprisingly, a significantly greater proportion of immunosuppressed patients received antibiotic therapy during their treatment. Patients treated with immunosuppressive agents had more exposure to antibiotics than immunocompetent patients, which might be related to their acquiring MDR-ESBL-EC infections.
We observed CTX-M-15 was significantly associated with MDR-ESBL-EC (59.1% vs. 32.5%, p = 0.028). In recent studies, CTX-M-14 and CTX-M-15 were the predominant types of ESBLs, and CTX-M-15 was significantly associated with ST131 [32,33]. ST131 E. coli isolates had a trend toward greater nonsusceptibility to ciprofloxacin and cefepime [32,33]. Therefore, our data suggests that CTX-M-15 isolates show more MDR patterns than CTX-M-14.
We performed PFGE to examine the molecular epidemiology of community-onset MDR-ESBL-EC. Ten isolates among 20 MDR-ESBL-EC were not clonally related. The remaining 10 isolates were classified into four closely related groups. Taking the PFGE results into consideration, it is reasonable to assume that the MDR-ESBL-EC isolates identified in this study belong to diverse clones.
Numerous studies have demonstrated that most ESBL-producing Enterobacteriaceae are resistant to multiple antibiotic classes. In addition to the agents included in our definition of MDR, we also noted a high prevalence of resistance to piperacillin/tazobactam and tobramycin among the MDR-ESBL-EC isolates (36.7% and 93.3%, respectively).
The types of infection due to MDR-ESBL-EC were generally those expected for E. coli, with UTIs predominating, followed by intra-abdominal or pancreaticobiliary tract infections. FQs, TMP-SMX, and aminoglycosides are commonly used in community-onset infections, and if the infection is not severe, can be additional treatment options for UTIs.
We found a high prevalence of community-onset MDR-ESBL-EC isolates that also demonstrated resistance to additional antibiotics. Of great concern is the fact that one in four ESBL-EC isolates in this study was MDR. As the prevalence of MDR-ESBL-EC infections increases, inappropriate antibiotic treatment will also increase. A previous study demonstrated that increased mortality is not related to ESBL production but to the fact that the empirical therapies used were more frequently inappropriate among patients with ESBL-EC [5]. Reliance on carbapenems will continue to increase because other effective treatment options are increasingly limited. However, the increasing use of carbapenems has been paralleled by the rapid emergence of carbapenem resistance [11]. We suggest the empirical use of carbapenems only for patients with community-onset UTIs or intra-abdominal sepsis with any risk factor for MDR-ESBL-EC. However, other classes of antibiotics, especially amikacin and piperacillin/tazobactam, can be used for empirical therapy for patients with less severe infections caused by gram-negative bacilli, based on antimicrobial susceptibility results [34,35].
It has been shown previously that an ESBL-producing organism can be transferred to a hospital environment from the community [36]. Failure to consider the emergence of drug-resistant organisms in the community could undermine infection-control efforts in hospitals and render empirical antibiotic therapy inadequate. Interventions to limit the emergence of ESBL-producing isolates have traditionally focused on restricting certain antimicrobial agents, such as third-generation cephalosporins, associated with ESBL infections. Although such interventions are certainly an important component in efforts to control the emergence of ESBL-producing isolates, infection-control measures are also likely to be critical in interrupting the spread of such organisms.
There are several recognized limitations to our study. First, our study was observational, and thus, unknown risk factors might have been unequally distributed between the study groups. Harris et al. [37] suggested that using control patients infected with susceptible pathogens biases studies from the null hypothesis. Hence, we used a case-control-control study to assess the identifiable risk factors associated with community-onset MDR-ESBL-EC. Limitations related to the case-control-control study design may also include the fact that the patients in CG II may not truly represent the population. We matched case and control patients on the basis of hospital and time period but intentionally did not match for variables such as age, sex, or severity of underlying conditions, which might themselves have been risk factors [38]. Second, not all ESBL-EC isolates were available for further molecular analysis. Therefore, the ESBL characterization results may not be representative of all ESBL-EC isolates. However, there was no systematic process affecting which isolates were not available and more than 91% of isolates were available for molecular analysis. Third, plasmid analysis was not done. Thus, the possibility of dissemination of MDR-ESBL-EC isolates through the spread of such extrachromosomal genetic elements (even in the presence of unrelated PFGE patterns) cannot be excluded. Fourth, because previous exposure to FQs is likely to be associated with nosocomial acquisition, a well-established risk factor for resistance, our findings may have been affected by known or unknown confounders. Finally, our study was conducted in a large referral care medical center and a smaller community hospital. Thus, the results may not be generalizable to other types of institutions.
In conclusion, the emergence of MDR among community-onset ESBL-EC is particularly troublesome. We determined that 27.8% of ESBL-EC isolates, even in community-onset infections, were MDR and that the independent risk factors for infections caused by such isolates were prior exposure to FQs, receipt of immunosuppressive therapy, and HCA infection. Clinicians should be aware of the occurrence of community-onset MDR-ESBL-EC among predisposed patients and select initial appropriate empirical antimicrobial therapy to reduce morbidity and mortality in such patients. Given that a limited number of new antimicrobial agents are being developed to treat MDR-ESBL-EC, rigorous infection control measures are advocated to prevent the spread of this microorganism. Also, an assessment of the effectiveness of strategies implemented to minimize the exposure to FQs in patients at risk for developing community-onset ESBL-EC infections would be of interest.