Prevalence and Risk Factors of ESBL-producing Enterobacteriaceae in The Community

Article Info History Received: 1 Jan 2021 Accepted: 19 Mar 2021 Available: 30 Apr 2021 Abstract Background: World Health Organization (WHO) data collection of Escherichia coli resistant to cephalosporin generation III already confirmed in 86 countries. Incredibly high carrier prevalence rates were also widely developed in Thailand, Egypt, and China. The Faculty of Medicine's research team at Jenderal Soedirman University, Purwokerto, discovered at the beginning of 2018 that Extended Spectrum β-Lactamase (ESBL) E. coli carriers throughout the 2015 class students were 26.8 percent. Objective: This research investigated the prevalence and associated factors of ESBLproducing Enterobacteriaceae (EPE) asymptomatic carriers in the community. Methods: The participant fill a questionnaire, and samples were taken from rectal swabs using Amies transport medium (Labware Charuzu), and then models were analyzed using HiCrome TM ESBL Agar Base (Himedia, India). Analysis of its Prevalence and Resistance Predictors used IBM SPSS Statistics Version 22.0 for Windows (Armonk, NY: IBM Corp). Results: The prevalence of EPE asymptomatic carriers in the community in Purwokerto was 66.7%. In the bivariate analysis, subjects who took antacids in the last eight weeks, history of hospitalization in the previous 12 months, the habit of consuming milk, yogurt, cheese, meat, seafood, and raw vegetables did not show any significant difference. Frequent chicken and freshwater fish consumption tended to be a risk factor for ESBL-producing Enterobacteriaceae with PR 1.462, 95% CI (1.1151.918); PR 1.666, 95% CI (0.936-2.966); however, in the multivariate logistic regression analysis, this was not significant. Conclusion: The prevalence of EPE asymptomatic carriers in the community in Purwokerto is 66.7%. None of the studied variables was correlated with ESBLproducing Enterobacteriaceae. However, ESBL remains an emerging antimicrobial resistance.


INTRODUCTION
Extended-spectrum β-Lactamase (ESBL) is an enzyme made by some bacteria. That enzyme can hydrolyze penicillin, first, second, third-generation cephalosporins, aztreonam (except cephamycin and carbapenem). 1 The bacteria that produce the most ESBL are bacteria from the Enterobacteriaceae family, especially Escherichia coli and Klebsiella pneumonia. 2 Based on substrate specificity the β-lactamase family split into four functional groups: penicillinase, ESBL, carbapenemase, and Amp-C enzyme. 3,4 The betalactamase (bla) gene is usually found on chromosomes, although it would also found in plasmids. 5 The ESBL coding gene residing in the plasmid is easily transferred to other germs, thereby accelerating resistance. 6 Community-acquired infections due to ESBL-producing Enterobacteriaceae (EPE) have increased. The flora fecal in the community is a vast potential reservoir for ESBL. 7 Based on a survey conducted by the CDC in 2013, each year, there are 26,000 infections caused by EPE, and around 1,700 of them die. * Corresponding author: E-mail: nia.krisniawati@unsoed.ac.id (Nia Krisniawati) Asymptomatic carriers of ESBL-producing bacteria in communities reported in several countries and continents with large differences in carrier rates between geographic areas and population characteristics studied.
Incredibly high carrier prevalence rates were also widely developed in Thailand (66%), Egypt (63%), and China (50%). 8 Low carrier levels shown from Sweden (3%). 9 In Indonesia as its own, a community carrier study by  10 The current study on the prevalence and risk factors of EPE carriers in Indonesia's healthy communities is rare. Each result of this study provides evidence for both the prevention and control of EPE in the population. Based on this context, specific, transparent, observable, and sustainable efforts need to be made in a single health approach across sectors, including the public health, livestock, fisheries, and agriculture sectors, to resolve this issue. These efforts involve critical surveillance data and strict implementation of field regulations to break the chain of EPE deployments. Therefore, prevalence data and related risk factors must establish firm policies to control antimicrobial resistance, especially EPE.

MATERIALS AND METHODS Population and sampling methods
The population in this study is the healthy people in Purwokerto. This research has received approval from the Faculty of Medicine's ethics committee, Universitas Jenderal Soedirman (Ref: 182/KEPK/VIII/2020). The sampling method is consecutive sampling. The minimum sample size for this analysis was 75. We conducted this research in 2020 for eight months at Purwokerto.

Questionnaire
Participants must fill out a questionnaire containing informed consent, name, age, sex, medical records (prior intake of antibiotics or antacids, initial hospitalization), consumption (milk, yogurt, cheese, meat, freshwater fish, seafood, chicken, etc.), and use of public toilets.

Isolation and identification
Samples isolated in HiCrome ™ ESBL Agar Base (Himedia, India) + HiCrome™ ESBL agar supplement and incubated for 24 hours at 37º C. Chromogenic mixture is used to distinguish the pigment ESBLproducing organisms. HiCrome™ ESBL Selective Supplement (FD278) allows suppressing some contaminating microorganisms. ESBL E.coli grow as either pink or purple colonies. ESBL producing members of the KESC group have bluish-green colonies; Proteus, Morganella, and Providencia. (Table. 1).

Statistical analysis
This research uses an analytic observational study with a cross-sectional approach, which emphasizes measuring or observing data one time based on the dependent variable and the independent variable. Univariate analysis of the independent and dependent variables to obtain a descriptive picture of the variables studied in presentation distribution. Bivariate analysis to see the relationship between independent and dependent variables uses the Chi-Square or Fisher Exact test for categorical variables and the Mann-Whitney U test for numerical data. Multivariate analysis conducted with multiple logistic regression. Statistical analysis of Prevalence and determinants of resistance uses IBM SPSS Statistics Version 22.0 for Windows (Armonk, NY: IBM Corp). The analysis results described as Prevalence Risk (PR), p-value, and 95% Confidence Interval (CI). All p-values were 2-tailed, and p <0.05 would be considered significant.

RESULTS
The prevalence of asymptomatic carriers of Enterobacteriaceae ESBL from a total of seventy-five respondents who participated in this study was 50 (66.7%). The most common isolate was Escherichia coli 50 (66.7%) were two strains of organisms (Escherichia coli and Klebsiella pneumoniae) in one respondent were more often found with a percentage of 29 (38.7%) compared to one strain (Escherichia coli) 21 (28%). Most of the respondents had a senior high school education level, namely 32 (42.7%). (Table 2). Participants in this study, 43 (52,3%) were women, and 32 (42,7%) were men, with the mean age of the respondents was 32.35 ± 10.93 years. Women had a 67.4% higher frequency of ESBL colonization (29/43) than men 65.6% (21/32), but there was no statistically significant difference.  Subjects who took antacids in the last eight weeks and hospital admission in the previous 12 months had a higher incidence rate of ESBL carriers than other respondents (Table 3). However, the bivariate analysis showed no significant differences between these variables. In the bivariate analysis, the habits of consuming milk, yogurt, cheese, meat, seafood, and raw vegetables did not show any significant differences. In contrast, for subjects who took antibiotics over the last eight weeks, the overall incidence was 100 percent (11/11), significantly higher than several other respondents who were also asymptomatic carriers of Enterobacteriaceae ESBL 60.9 percent (39/64

DISCUSSION
Research into the asymptomatic carrier of ESBL E. coli is essential because it has significant clinical consequences for public health. 11 A healthy carrier can develop severe urinary tract, intra-abdominal, or bloodstream infections in the future. 12,13 Mortality rates following infection with ESBL E. coli are considerably higher. 14,15 The last review on carbapenems might be the only efficient treatment. 16 Often, moderate urinary tract infections occur to remain due to enhanced morbidity. 17 The cumulative combined Prevalence of ESBL E. coli carriers in the community (2003-2018) was 16.5%. 18 The combined Prevalence shows an increasing trend from 2.6% in 2003-2005 to 21.1% in 2015-2018. The highest carrier rates were observed in Southeast Asia throughout the period, while Europe was the lowest. 19 In the research we conducted, especially in Purwokerto, there was also an increase in ESBL E. coli carriers trends to 66.7% compared to previous studies conducted by a research team from the Faculty of Medicine, General Soedirman Purwokerto University in 2015 class students as much as 26.8%. In some other research, the fecal carrier rate of Enterobacteriaceae ESBL is about 62.7% among hospitalized patients and 33.8% among asymptomatic healthy people. A more frequently reported Enterobacteriaceae of ESBL seemed to be Escherichia coli (70.3% and 78.4%, in both patients and healthy subjects), followed by Klebsiella pneumoniae (26.8% and 17.0 %). 20 In our study, most isolates found were Escherichia coli 50 isolates (66.7%), most of which were found together with Klebsiella pneumoniae or two strains in one respondent 29 (38.7%).
Hijazi et al., in their study, found that thirty-one of the 125 participants (24.8%) were carriers of the Enterobacteriaceae ESBL. In the bivariate test, regular consumption (3 times per week or more) of milk, yogurt, cheese, chicken, or meat showed a significant effect. In multivariate logistic regression analysis, beef and chicken's routine consumption is also significantly related to increased Enterobacteriaceae ESBL carriers; the association of dairy products (milk, yogurt, cheese) was not significant. 7 In our study, frequent consumption of chicken and freshwater fish to be a risk factor for the carriers of ESBL-producing Enterobacteriaceae (Table. 3); however, in the logistic regression multivariate analysis, it was not significant. In the research developed by Otter et al., the risk factor for Enterobacteriaceae ESBL carriers among 1633 patients was receiving two or more antibiotic courses six months before the examination. 21 In our study, participants taking antibiotics in the last eight weeks had a 100% higher prevalence rate (11/11) than some other respondents who also were asymptomatic carriers of Enterobacteriaceae ESBL 60.9% (39/64) with PR 1.000, 95% CI (0.442-2.263). The above means that this variable is not a risk factor for the Enterobacteriaceae ESBL carriers or is protective, but this is not significant in multivariate logistic regression analysis. Shitrit et al. in their study found that of the 525 patients tested, 56 seemed to be positive for ESBL carriers, 4 different risk factors are associated: residency in a nursing facility, history of 1year previous hospitalization, history of antibiotic treatment, and last ESBL carriers. More than 50% of patients screened have at least one risk factor. 22

CONCLUSION
The prevalence of EPE asymptomatic carriers in the community in Purwokerto was 66.7%. These data are required to establish firm policies to control antimicrobial resistance. Consumption of chicken and freshwater fish is not a risk factor for ESBL-producing Enterobacteriaceae. However, ESBL remains an emerging antimicrobial resistance.