Article pubs.acs.org/est
Antibiotic Body Burden of Chinese School Children: A Multisite Biomonitoring-based Study Hexing Wang,†,∇ Bin Wang,†,∇ Qi Zhao,† Yanping Zhao,‡ Chaowei Fu,† Xin Feng,§ Na Wang,† Meifang Su,∥ Chuanxi Tang,⊥ Feng Jiang,† Ying Zhou,*,† Yue Chen,# and Qingwu Jiang† †
Key Laboratory of Public Health Safety of Ministry of Education, School of Public Health, Fudan University, Shanghai 200032, China ‡ Minhang District Center for Disease Control and Prevention, Minhang District, Shanghai 201101, China § Haimen City Center for Disease Control and Prevention, Haimen City, Jiangsu Province 226100, China ∥ Yuhuan County Center for Disease Control and Prevention, Yuhuan County, Zhejiang Province 317600, China ⊥ Changning District Center for Disease Control and Prevention, Changning District, Shanghai 200051, China # School of Epidemiology, Public Health and Preventive Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H8M5, Canada S Supporting Information *
ABSTRACT: To explore the antibiotic body burden of Chinese school children, total urinary concentrations (free and conjugated) of 18 representative antibiotics (5 macrolides, 2 β-lactams, 3 tetracyclines, 4 quinolones, and 4 sulfonamides) were measured by ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry among 1064 school students recruited from 3 economically and geographically distinct areas in east China in 2013. All 18 antibiotics were detected in urine samples with the detection frequencies ranging from 0.4 to 19.6%. The antibiotics were detected in 58.3% of urine samples overall, and this detection frequency reached at 74.4% in one study site. Of them, 47.8% of the urine samples had a sum of mass concentration of all antibiotics between 0.1 (minimum) and 20.0 ng/mL, and 8 antibiotics had their concentrations of above 1000 ng/mL in some urine samples. Three veterinary antibiotics, 4 human antibiotics, and 11 human/veterinary antibiotics were found overall in 6.3, 19.9, and 49.4% of urine samples, respectively. The detection frequencies and concentration levels of antibiotics in urine samples differed by study areas. Concerning mixed exposures, a total of 137 combinations of antibiotics and 20 combinations of antibiotic categories were found overall. Two or more antibiotics or categories were concurrently detected in more than 20% of urine samples. On the basis of a usage analysis, contaminated food or environment might be relevant exposure sources for tetracyclines, quinolones, and sulfonamides.
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INTRODUCTION
the microbial ecosystem, which has led to environmental reservoirs of resistant genes and bacteria, which have brought us to the dawn of a postantibiotic era.4−6 These reservoirs now exist in the bodies of humans and food animals; in sewage plants, rivers, and farms; and in households and hospitals, and
As one group of pharmaceuticals and personal care products (PPCPs), antibiotics have been emerging contaminants in the environment with much concern for their possible threats to aquatic environment and human health.1 Since the discovery of penicillin in 1928, a large number of antibiotics has been produced and used in human and veterinary medicine worldwide.2 Antibiotics consumed in human and animal medicine can directly or indirectly enter into humans, animals, food, and the environment.3 This creates selection pressure on © XXXX American Chemical Society
Received: December 7, 2014 Revised: April 1, 2015 Accepted: April 1, 2015
A
DOI: 10.1021/es5059428 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Table 1. Descriptive Analysis of Selected Antibiotics in All Subjects (n = 1064) percentile antibiotics macrolidesb azithromycin clarithromycin erythromycin roxithromycin tylosin β-lactamsb ampicillin cefaclor tetracyclinesb oxytetracycline chlortetracycline tetracycline quinolonesb ofloxacin ciprofloxacin enrofloxacin norfloxacin sulfonamidesb sulfamethazine trimethoprimd sulfamethoxazole sulfadiazine all antibioticse
usage human human human/veterinary human veterinary human/veterinary human human/veterinary veterinary human/veterinary human/veterinary human/veterinary veterinary human/veterinary human/veterinary human/veterinary human/veterinary human/veterinary
n (%)a 205 174 18 11 4 11 72 47 29 40 24 13 7 281 142 113 45 37 321 209 107 49 5 620
(19.3) (16.4) (1.7) (1.0) (0.4) (1.0) (6.8) (4.4) (2.7) (3.8) (2.3) (1.2) (0.7) (26.4) (13.3) (10.6) (4.2) (3.5) (30.2) (19.6) (10.1) (4.6) (0.5) (58.3)
50th
75th
− − − − − − − − − − − − − − − − − − − − − − − 0.9 (1.5)
− − − − − − − − − − − − − 0.3 (0.6) − − − − 1.1 (1.9) − − − − 4.4 (9.1)
90th 4.9 3.8 − − − − − − − − − − − 1.4 0.6 0.3 − − 3.8 3.2 0.2 − − 22.8
(12.0)c (8.5)
(3.5) (1.1) (0.7)
(8.5) (6.3) (0.1)
(51.5)
95th 39.9 31.5 − − − − 1.2 − − − − − − 3.2 1.1 0.7 − − 6.7 4.9 0.5 − − 87.5
(99.9) (81.5)
(1.8)
(6.4) (2.3) (2.2)
(12.9) (10.0) (1.3)
(208.7)
99th 3092.8 2401.7 1.2 0.6 − 0.7 61.4 4.9 35.3 4.6 2.0 1.1 − 25.2 4.9 5.5 2.1 7.1 21.2 13.3 3.9 3.9 − 3585.7
(5759.1) (4684.9) (3.2) (1.5) (1.2) (118.9) (5.3) (69.9) (10.5) (6.0) (1.4) (51.6) (9.1) (7.5) (5.8) (10.1) (44.9) (24.4) (8.3) (11.5) (8927.7)
max 19608.6 7056.8 1240.7 19605.2 6.8 4.1 42689.5 42689.5 25758.2 2682.6 2626.7 361.2 55.8 499.8 499.8 42.3 10.6 45.2 3021.1 23.6 1268.8 1750.0 66.2 42689.8
(113458.9) (18025.0) (25063.0) (111721.1) (19) (8.8) (78005.5) (78005.5) (69254.6) (9478.8) (9281.5) (184.3) (197.3) (323.3) (323.3) (60.0) (16.6) (79.1) (6510.2) (66.7) (2734.0) (3771.0) (201.2) (113460.3)
a
Number of positive detection (detection frequency, %). bMass sum of antibiotics in corresponding antibiotic categories. cVolume-based concentration value, ng/mL (creatinine-corrected concentration value, μg/g creatinine). dBecause it is usually mixed use with sulfonamides in practical application, trimethoprim was analyzed together with sulfonamides. eMass sum of all antibiotics. −, < limit of detection (LOD).
environment such that the antibiotic profiles among them are correlated with each other,4 and the biomonitoring of antibiotics in humans can also reflect the comprehensive utilization status of antibiotics in various sectors to some extent. As the human body metabolizes antibiotics, a considerable proportion is excreted in urine by free or conjugated species; although, antibiotics entering the human body can be metabolized to multiple metabolites.16,17 Thus, urinary antibiotics are potential exposure biomarkers of antibiotics, which provides a convenient and reliable way to assess antibiotic body burdens in human populations.18 China is one country that produces and uses a very large amount of antibiotics; it is also one country that seriously misuses or overuses antibiotics.19,20 Financial incentives, lack of knowledge about antibiotic safety, and poor management of antibiotic use in animal husbandry and aquaculture may be direct causes of the misuse or overuse of antibiotics.21−25 Consequently, many antibiotics have been frequently found in the environment and in food.1,26 Children have a high metabolic rate and a high mobility, which make it easy for them to come into contact with adverse compounds and pathogens from the environment or food.27 Children are also more susceptible to respiratory and some other infections.28 The majority of families in China have only one or two children due to family planning policies,29 and there is often an overconcern of children’s health. Parents’ expectancy of prescribing more drugs further adds the risk of inappropriate use of antibiotics in children;30 therefore, it is likely that children have a heavier body burden of antibiotics compared with adults in China. Moreover, children are in rapid growth and development, which makes them more susceptible than
the resistant genes and bacteria can transfer among them by multiple pathways.5 For humans, antibiotics entering the body through medical use and/or contaminated food or environment can reach the respiratory tract by blood circulation and can reach the urinary and intestinal tracts by the excretion in urine and feces to facilitate the emergence or immigration of resistant genes and bacteria in these sites, which poses a potential risk of the infection by resistant bacteria.7,8 Moreover, the antibiotic exposure also poses other potential health hazards on humans, such as drug side effects, childhood-onset asthma, childhood obesity, inflammatory bowel disease, or colorectal carcinoma.9−12 One mechanism is proposed to disturb the gut microbiota and has been recently found to have a profound influence on body physiology.13 However, the exposure of human to antibiotics is not well explored across the globe. Prescription examination and questionnaire surveys are common means of assessment for the exposure of antibiotics by clinical use and selfmedication.14,15 Examination of prescriptions related to antibiotics is useful in clinical setting, but it is not suitable in veterinary medicine because the record system is unavailable. A questionnaire survey is sometimes difficult to implement and may be subject to great variation. Compared to two exposure assessment methods, the biomonitoring of antibiotics can reflect an overall exposure dose of antibiotics not only from clinical use and self-medication, but also from food and the environment, and measure the internal exposure dose, which can be used to examine the association between exposure and effects more accurately than prescription and questionnaire. Because antibiotics consumed in human and veterinary medicine can circle among human, animal, food, and the B
DOI: 10.1021/es5059428 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Table 2. Descriptive Analysis of Antibiotic Categories by Usage in All Subjects (n = 1064) percentiles antibiotics
n (%)a
50th
75th
90th
95th
99th
max
human veterinary human/veterinary
212 (19.9) 67 (6.3) 526 (49.4)
− − −
− − 2.3 (5.3)
7.2 (15.6)b − 6.7 (12.6)
53.9 (133.4) 0.8 (2.0) 13 (23.1)
3092.8 (5759.1) 3.7 (9.1) 80.3 (175.8)
25758.2 (69254.6) 361.2 (184.3) 42689.8 (111722.6)
a Number of positive detection (detection frequency, %). bVolume-based concentration value, ng/mL (creatinine-corrected concentration value, μg/ g creatinine). Human antibiotics: azithromycin, clarithromycin, roxithromycin, and cefaclor. Veterinary antibiotics: tylosin, chlortetracycline, and enrofloxacin. Human/veterinary antibiotics: 11 other antibiotics except for human and veterinary antibiotics. −, < limit of detection (LOD).
adults to the exposure to antibiotics and pathogens.27 Therefore, much concern should be paid to the exposure of children to antibiotics. In this study, we aimed to explore the antibiotic body burden of Chinese school children based on the measurement of 18 representative urinary antibiotics from 5 common categories (Table 1) among more than 1000 children selected from 3 economically and geographically distinct areas in east China.
meat).34−36 However, due to a lack of suitable analytical methods, 7 of 25 antibiotics were not determined, including 5 β-lactams (penicillin G, penicillin V, amoxicillin, cefuroxime, and ceftriaxone) and 2 aminoglycosides (gentamicin and streptomycin). Finally, 18 antibiotics were measured in this study, including five macrolides, two β-lactams, three tetracyclines, four quinolones, and four sulfonamides (Table 1). Among them, four are used only as human antibiotics, three are only used as veterinary antibiotics, and the rest are used as both human and veterinary antibiotics (Table 1).31−33 Urine Collection and Analysis. First morning urine was used in this study. After collection, urine samples were immediately aliquoted, transported to the lab in an ice chest as soon as possible, and frozen at −80 °C in the dark until analysis. Urinary creatinine was measured by an enzymatic method on Architect C8000 biochemical analyzer (Abbott Laboratories, Abbott Park, IL). Total urinary concentrations (free and conjugated) of 18 antibiotics were determined by the isotope dilution two-dimensional ultraperformance liquid chromatography coupled to high-resolution quadrupole timeof-flight mass spectrometry (UPLC-Q/TOF MS), following the method previously established in our lab.18 Briefly, after an aliquot (1.0 mL) of urine was spiked with 12 isotopically internal standards and hydrolyzed by β-glucuronidase, the mixture was purified by solid-phase extraction and analyzed by two-dimensional UPLC-Q/TOF MS (detailed information is provided in Supporting Information). All analyses of urine samples were performed between March and June 2014 by the same analytical team in our lab. Ninety urine samples were analyzed in each batch, and one isotopedilution standard curve was freshly prepared for each batch. The correlation coefficients ranged from 0.991 to 0.998. The limit of detection (LOD), calculated as a signal-to-noise ratio of 3, ranged from 0.04 to 1.99 ng/mL. Four spiked urine samples and two solvent blanks were prepared and analyzed to monitor the background interferences, precision, and trueness of analytical procedure for each batch. Two spiked concentrations of 5 and 20 ng/mL were set for each antibiotic, and each spiked concentration was duplicated twice. The interbatch recoveries of 18 antibiotics in spiked urine samples varied between 76.1 and 122.6% with the interbatch relative standard deviations ranging from 8.2 to 19.7%. Only trimethoprim was found to have interference from background noise, and the final concentration values of trimethoprim were subtracted with this interference. To further confirm the positive detection of antibiotics in urine by UPLC-Q/TOF MS, five representative antibiotics from five categories (azithromycin from macrolides, cefaclor from β-lactams, oxytertacycline from tetracyclines, ofloxacin from quinolones, and sulfamethazine from sulfonamides) were reanalyzed using UPLC coupled to triple quandrupole mass spectrometry (UPLC-QqQ MS) in 10% of
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MATERIALS AND METHODS Study Population. Three economically and geographically distinct areas were selected in the core of the Yangtze River delta in east China between April and November 2013: Shanghai City (high economic development level), Haimen City (medium economic development level) in Jiangsu Province, and Yuhuan County (low economic development level) in Zhejiang Province. In cooperation with the local Centers for Disease Control and Prevention, two study sites were established in Shanghai Cityone site in a suburb (Minhang District) and another downtown (Changning District)and one study site was established in each Haimen City and Yuhuan County. After a primary school was randomly selected in each of the four study sites, three or four classes were randomly selected from each of third, fourth, and fifth grades. There were a total of 1103 students in these selected classes. Data on sex, date of birth, and ethnicity of the participants were obtained from school archives. After the students without written informed consents from her/his parent/guardian and the students with liver or kidney diseases were excluded, 1078 students were included in this study. A small number of children who were more than 12 years old, minority, or lack of data on age, sex, or ethnicity, were excluded from the analysis. Finally, a total of 1064 Han students aged 8−11 years were included in this analysis. Of them, 149, 337, 350, and 228 students were aged 8, 9, 10, and 11 years, respectively; 511 were girls and 553 were boys; and 226 were from Changning District, 309 were from Minhang District, 264 were from Yuhuan County, and 265 were from Haimen City. The study was reviewed and approved by the Institutional Review Board of Fudan University. Selection of Antibiotics. Eight antibiotic categories are commonly used in human health care or animal husbandry in China: macrolides, β-lactams, tetracyclines, quinolones, sulfonamides, chloramphenicols, lincomycins, and aminoglycosides. In these categories, there are about 90 human prescription drugs,31 about 40 veterinary prescription drugs,32 about 12 used as feed additives,33 and a total of about 120 antibiotics that are available in human health care or animal husbandry. We first considered 25 common and representative antibiotics that are frequently used in human or veterinary medicine or frequently detected in the environment or food (surface water, milk, or C
DOI: 10.1021/es5059428 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology positive urine samples. The positive agreement rates were between 93.1 and 100%. Statistical Analysis. Antibiotics were divided into different categories by their antibacterial mechanisms or human/ veterinary usages, and new variables were generated by a mass concentration sum. The five new variables by their antibacterial mechanisms were macrolides, β-lactams, tetracyclines, quinolones, and sulfonamides (Table 1). Three new variables by their usages were human antibiotics, veterinary antibiotics, and human/veterinary antibiotics that are used as both human and veterinary antibiotics in practice (Table 2). All 18 antibiotics were summed as a new variable. Because it is usually mixed use with sulfonamides in practical application, trimethoprim was analyzed together with sulfonamides. A positive outcome of new variable was defined as a detection of any antibiotic(s) included in new variable in one urine sample. The dilution of urinary concentrations of antibiotics was corrected by urinary creatinine. The creatinine-corrected concentration (μg/g creatinine) is calculated by dividing urinary concentrations (μg/L) of antibiotics by urinary concentrations of creatinine (g/L). We provided the median, maximum, and selected percentiles of volume-based (ng/mL) and creatinine-corrected concentrations and the distributions of two kinds of concentrations were compared. The overall detection frequency distribution of all antibiotics was demonstrated by their volume-based concentration ranges. The detection frequencies of antibiotics or antibiotic categories were calculated for all subjects or by sex, age group (8−9 and 10−11 years of age), and study site. Pearson chi-square tests were used to test the detection frequencies of urinary antibiotics in relation to sex, age group, and study site. The profiles of the concentration distribution of urinary antibiotics were tested in relation to sex, age group, and study site by nonparametric tests. We used logistic regression models to estimate the associations of urinary antibiotics with sex, age, and study site after adjusting for some potential confounders. The detection frequencies of mixed exposures were examined in all subjects according to the number of mixed antibiotics or antibiotic categories. All statistical analyses were performed using the statistical software packages SPSS (version 17; SPSS, Inc., Chicago, IL). A p-value of less than 0.05 was considered statistically significant.
Figure 1. Detection frequency distribution of mass concentration sum of 18 selected antibiotics (LOD: limit of detection).
distribution profiles of volume-based concentration were similar to that of creatinine-corrected concentration for five generated variables (Figure S1, Supporting Information). Figures 2 and 3 show the overall urinary detection frequencies of antibiotic categories by antimicrobial mechanisms in relation to age group, sex, and study site. The detection frequency of β-lactams was higher in the 8−9 year age group (8.4%), and a higher detection frequency was seen in boys for sulfonamides (33.1%; Figure 2). Significant regional differences were seen for all antibiotic categories (Figure 3). The detection frequencies of macrolides (24.9%), β-lactams (10.0%), sulfonamides (50.2%), and all antibiotics (74.4%) in Minhang were higher than those in other sites, while the detection frequencies of tetracyclines (6.1%) and quinolones (37.9%) were higher in Haimen (Figure 3). Similar differences were observed for the concentration sum of antibiotic categories in relation to sex, age, and study site (Table S1, Supporting Information). After we adjusted for study site, age, and/or sex in logistical regression models, some associations changed. In addition to β-lactams, a higher detection frequency was found for macrolides and all antibiotics in young children than old children (Table S2, Supporting Information). The significant associations of antibiotic categories with study site and sex remained (Table 3 and Table S2, Supporting Information). Figures 4 and 5 show the overall urinary detection frequencies of antibiotic categories by usage in relation to study site, sex, and age group. Human antibiotics were more detected in urine from young children (22.8%) than in urine from older children (17.5%), and no significant differences were seen in detection frequencies between young and old children for veterinary antibiotics and human/veterinary antibiotics and between girls and boys for three antibiotic categories (Figure 4). The detection frequencies were different among study sites for three antibiotic categories (Figure 5). Of four sites, human and human/veterinary antibiotics were more detected in Minhang (28.2 and 65.0%, respectively) than in the other three sites, and veterinary antibiotics were more detected in Haimen (11.0%).
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RESULTS The detection frequencies of 18 antibiotics ranged from 0.4% for roxithromycin to 19.6% for sulfamethazine in urine (Table 1). The overall detection frequency of all antibiotics was 58.3% and those of macrolides, β-lactams, tetracyclines, quinolones, and sulfonamides ranged from 3.8% to 30.2%. Azithromycin, ciprofloxacin, ofloxacin, sulfamethazine, or trimethoprim were detected in more than 10% of urine samples. Eight of 18 antibiotics had some extreme urinary concentrations of above 1000 ng/mL, with the highest one being more than 40000 ng/ mL for ampicillin. Of four human antibiotics, the azithromycin had the highest detection frequency of 16.4%, and of three veterinary antibiotics, the enrofloxacin had the highest detection frequency of 4.2%. The overall detection frequencies of human antibiotics, veterinary antibiotics, and human/ veterinary antibiotics ranged from 6.3 to 49.4% (Table 2). As shown in Figure 1, the overall detection frequencies of all antibiotics within the concentration ranges of 0.1 (minimum)− 1.0 ng/mL, 1.0−20 ng/mL, and 20.0−42689.8 ng/mL (maximum) were 10.3, 37.5, and 10.6%, respectively. The D
DOI: 10.1021/es5059428 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology
Figure 2. Detection frequency of antibiotic categories by antimicrobial mechanism in relation to (a) age group and (b) sex (*: 0.01 < p-value
