Detection of antibiotic resistance and tetracycline resistance genes in Enterobacteriaceae isolated from the Pearl rivers in South China

This study investigated antibiotic resistance profiles and tetracycline resistance genes in Enterobacteriaceae family isolates from the Pearl rivers. The Enterobacteriaceae isolates were tested for susceptibility to seven antibiotics ampicillin, chloramphenicol, ciprofloxacin, levofloxacin, sulphamethoxazole/trimethoprim, tetracycline and trimethoprim. In Liuxi reservoir, with an exception to ampicillin resistant strains (11%) no other antibiotic resistance bacterial strains were detected. However, multiple drug resistance in bacterial isolates from the other sites of Pearl rivers was observed which is possibly due to sewage discharge and input from other anthropogenic sources along the rivers. Four tetracycline resistance genes tet A, tet B, tet C and tet D were detected in the isolates from the rivers. The genes tet A and tet B were widely detected with the detection frequencies of 43% and 40% respectively. Ciprofloxacin and levofloxacin resistant enteric bacteria were also isolated from the pig and duck manures which suggest a wider distribution of human specific drugs in the environment. This investigation provided a baseline data on antibiotic resistance profiles and tetracycline resistance genes in the Pearl rivers delta.     

Human health implications of clinically relevant bacteria in wastewater habitats

The objective of this review is to reflect on the multiple roles of bacteria in wastewater habitats with particular emphasis on their harmful potential for human health. Indigenous bacteria promote a series of biochemical and metabolic transformations indispensable to achieve wastewater treatment. Some of these bacteria may be pathogenic or harbour antibiotic resistance or virulence genes harmful for human health. Several chemical contaminants (heavy metals, disinfectants and antibiotics) may select these bacteria or their genes. Worldwide studies show that treated wastewater contain antibiotic resistant bacteria or genes encoding virulence or antimicrobial resistance, evidencing that treatment processes may fail to remove efficiently these bio-pollutants. The contamination of the surrounding environment, such as rivers or lakes receiving such effluents, is also documented in several studies. The current state of the art suggests that only some of antibiotic resistance and virulence potential in wastewater is known. Moreover, wastewater habitats may favour the evolution and dissemination of new resistance and virulence genes and the emergence of new pathogens. For these reasons, additional research is needed in order to obtain a more detailed assessment of the long-term effects of wastewater discharges. In particular, it is important to measure the human and environmental health risks associated with wastewater reuse.     

Relationship between antibiotic residues and occurrence of resistant bacteria in Nile tilapia (Oreochromisniloticus) cultured in cage-farm

The aim of this study was to investigate the relationship between antibiotic residues found in the muscle of cage-farm-raised Nile tilapia (Oreochromisniloticus), the occurrence of resistant bacteria, and the sanitary practices adopted by farmers in Ilha Solteira reservoir, Brazil. Nine fish (three small fish, 40–200 g; three medium-sized fish, 200–500 g; and three large fish, 500–800 g) were collected from four cage farms every three months from April 2013 to January 2014. Ten antibiotic residues were determined using liquid chromatography-mass spectrometry, and bacteria were isolated and tested for antibiotic resistance to determine the multiple antibiotic resistance (MAR) index. Only three antibiotics (oxytetracycline, tetracycline, and florfenicol) were detected in the muscle of Nile tilapia, and their residues were the highest in small fish; however, the MAR index was higher in large fish. In addition, a direct positive relationship between the MAR index and the concentration of antibiotic residues in Nile tilapia was found. Overall, the adoption of prophylactic management practices improved the sanitary status of cage farms, reducing bacterial infections and hampering the development of antibiotic-resistant bacteria.     

Impact of treated wastewater irrigation on antibiotic resistance in the soil microbiome

The reuse of treated wastewater (TWW) for irrigation is a practical solution for overcoming water scarcity, especially in arid and semiarid regions of the world. However, there are several potential environmental and health-related risks associated with this practice. One such risk stems from the fact that TWW irrigation may increase antibiotic resistance (AR) levels in soil bacteria, potentially contributing to the global propagation of clinical AR. Wastewater treatment plant (WWTP) effluents have been recognized as significant environmental AR reservoirs due to selective pressure generated by antibiotics and other compounds that are frequently detected in effluents. This review summarizes a myriad of recent studies that have assessed the impact of anthropogenic practices on AR in environmental bacterial communities, with specific emphasis on elucidating the potential effects of TWW irrigation on AR in the soil microbiome. Based on the current state of the art, we conclude that contradictory to freshwater environments where WWTP effluent influx tends to expand antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes levels, TWW irrigation does not seem to impact AR levels in the soil microbiome. Although this conclusion is a cause for cautious optimism regarding the future implementation of TWW irrigation, we conclude that further studies aimed at assessing the scope of horizontal gene transfer between effluent-associated ARB and soil bacteria need to be further conducted before ruling out the possible contribution of TWW irrigation to antibiotic-resistant reservoirs in irrigated soils.     

Practical survey on antibiotic-resistant bacterial communities in livestock manure and manure-amended soil

Through livestock manure fertilization, antibiotics, antibiotic-resistant bacteria and genes are transferred to agricultural soils, resulting in a high prevalence of antibiotic-resistant bacteria in the soil. It is not clear, however, whether a correlation exists between resistant bacterial populations in manure and manure-amended soil. In this work, we demonstrate that the prevalence of cephalexin-, amoxicillin-, kanamycin- and gentamicin-resistant bacteria as well as bacteria simultaneously resistant to all four antibiotics was much higher in manure-amended soils than in manure-free soil. 454-pyrosequencing indicated that the ARB and multiple antibiotic-resistant bacteria (MARB) in swine or chicken manure and manure-amended soil were mainly distributed among Sphingobacterium, Myroides, Enterococcus, Comamonas and unclassified Flavobacteriaceae. The genus Sphingobacterium was highly prevalent among ARB from swine manure and manure-amended soil, and was also the most dominant genus among MARB from chicken manure and manure-amended soil. Other dominant genera among ARB or MARB populations in manure samples, including Myroides, Enterococcus and Comamonas, could not be detected or were detected at very low relative abundance in manure-amended soil. The present study suggests the possibility of transfer of ARBs from livestock manures to soils and persistence of ARB in these environments.     

Environmental pollution by antibiotics and by antibiotic resistance determinants

Antibiotics are among the most successful drugs used for human therapy. However, since they can challenge microbial populations, they must be considered as important pollutants as well. Besides being used for human therapy, antibiotics are extensively used for animal farming and for agricultural purposes. Residues from human environments and from farms may contain antibiotics and antibiotic resistance genes that can contaminate natural environments. The clearest consequence of antibiotic release in natural environments is the selection of resistant bacteria. The same resistance genes found at clinical settings are currently disseminated among pristine ecosystems without any record of antibiotic contamination. Nevertheless, the effect of antibiotics on the biosphere is wider than this and can impact the structure and activity of environmental microbiota. Along the article, we review the impact that pollution by antibiotics or by antibiotic resistance genes may have for both human health and for the evolution of environmental microbial populations.     

Tetracycline resistance in semi-arid agricultural soils under long-term swine effluent application

Annually, millions pounds of antibiotics are released unmetabolized into environment along with animal wastes. Accumulation of antibiotics in soils could potentially induce the persistence of antibiotic resistant bacteria. Antibiotics such as tetracyclines and tetracycline-resistant bacteria have been previously detected in fields fertilized with animal manure. However, little is known about the accumulation of tetracyclines and the development of tetracycline resistance in semi-arid soils. Here we demonstrate that continuous land application with swine effluent, containing trace amounts of chlortetracycline, does not necessarily induce tetracycline resistance in soil bacteria. Based on the testing of more than 3,000 bacteria isolated from the amended soils, we found no significant increase in the occurrence and level of chlortetracycline resistant bacteria in soils after 15 years of continuous swine effluent fertilization. To account for a possible transfer of tetracycline-resistant bacteria originated from the swine effluent to soils, we analyzed two commonly found tetracycline resistant genes, tet(O) and tet(M), in the swine effluent and fertilized soils. Both genes were present in the swine effluent, however, they were not detectable in soils applied with swine effluent. Our data demonstrate that agronomic application of manure from antibiotic treated swine effluent does not necessarily result in the development of antibiotic bacterial resistance in soils. Apparently, concentrations of chlortetracycline present in manure are not significant enough to induce the development of antibiotic bacterial resistance.     

Abundance and removal of antibiotic resistance genes (ARGs) in the rearing environments of intensive shrimp aquaculture in South China

Although research regarding antibiotic resistance genes (ARGs) in aquaculture environments has gained increasing scientific interest, further studies are required to understand the abundances and removal mechanisms of ARGs during the entire rearing period of shrimp aquaculture. Thus, in this study, abundances, distributions and removal rates of ARGs in different environmental compartments of intensive shrimp farms in South China were investigated during the entire rearing period. The results indicated that sul1 and cmlA were the predominant ARGs in the water and sediment samples. Additionally, the total abundance of ARGs was higher in shrimp pond water than in the source water and farm effluent. Moreover, sediment samples indicated significantly higher ARG abundances than water samples from the shrimp ponds (P?<?0.05). Environmental factors were found to significantly affect the distribution of ARGs in shrimp rearing environments. Furthermore, stable ponds aided the removal of ARGs from shrimp pond water. This study accounted for temporal variations in ARG abundances as well as removal of ARGs in different environmental compartments during the entire shrimp rearing period. However, additional research is required to optimize the water treatment process for removal of ARGs from the aquaculture.     

Distribution of antibiotic-resistant bacteria in chicken manure and manure-fertilized vegetables

Veterinary manure is an important pollution reservoir of antibiotics and antibiotic-resistant bacteria (ARB). However, little is known of the distribution of ARB in plant endophytic bacteria and the number/types of ARB in chicken manure. In this study, 454-pyrosequencing was used to investigate the distribution and composition of ARBs in chicken manure and fertilized vegetables. The prevalence of ARB in the samples of the chicken manure compost recovered from farms on which amoxicillin, kanamycin, gentamicin, and cephalexin were used was 20.91–65.9 % for ARBs and 8.24–20.63 % simultaneously resistant to two or more antibiotics (multiple antibiotic resistant bacteria (MARB)). Antibiotic-resistant endophytic bacteria were widely detected in celery, pakchoi, and cucumber with the highest rate of resistance to cephalexin. The pyrosequencing indicated that the chicken manure dominantly harbored Firmicutes, Bacteroidetes, Synergistetes, and Proteobacteria and that Bacteroidetes was significantly enhanced in farms utilizing antibiotics. In the total cultivable colonies, 62.58–89.43 % ARBs and 95.29 % MARB were clustered in Bacteroidetes with the dominant species (Myroides ordoratimimus and Spningobacterium spp., respectively) related to human clinical opportunistic pathogens.     

人工湿地中抗生素抗性大肠杆菌和抗性基因的去除与分布

抗生素的滥用导致抗生素抗性菌和抗性基因随生活污水和养殖废水的排放在环境中肆意散播,其去除及环境行为越来越受到关注。采用K-B纸片法测定了9套不同工艺构型模拟人工湿地中大肠杆菌对7种抗生素的抗性率,并应用多重PCR检测磺胺类sul1、2、3与四环素tetA、B、C、D抗性基因,探究人工湿地对抗性菌的去除效率及抗性菌、抗性基因的分布规律。结果显示,人工湿地能有效去除污水中70%左右的抗性大肠杆菌,有效降低了细菌抗性的传播风险;共计分离出535株大肠肝菌中有378株对一种以上抗生素有抗性性,以四环素、磺胺类和氨苄西林抗性率最高,达到25%以上,其他4种抗性率较低,不足20%;2种抗性基因的检出率都在70%以上;对不同采样点大肠杆菌的抗性性及抗性基因的比较发现,各部分大肠杆菌的抗性水平、多重抗性指数(MRI)以及抗性基因(sul、tet)检出率和组合数表现出:基质≥出水>进水,推测抗性菌被湿地基质截留,在基质生物膜上发生抗性基因的重组,并释放抗性菌,提高了出水中抗性水平和抗性基因检出率。     

The effect of residential and agricultural runoff on the microbiology of a Hawaiian ahupua'a.

The objective of this project was to study the relationship between environmental runoff and the incidence of antibiotic-resistant microorganisms (ARMO) in freshwater streams. Five water systems along the windward coast of the island of O'ahu were evaluated. Samples were collected from sites upstream of residential or agricultural areas, throughout these areas, and at sites of entrance into oceans or bays. It was hypothesized that the incidence of ARMO would increase as the stream received runoff from residential and agricultural areas. The percentage of ARMO did not increase as the streams passed through residential or agricultural areas. Surprisingly, pristine sites, well upstream from residential or agricultural areas, contained bacteria resistant to at least one antibiotic. Areas most affected by runoff did not show a significant increase in the incidence of antibiotic-resistant organisms, suggesting that the incidence of antibiotic resistance is not simply a function of contamination with agricultural or residential runoff. The correlation of antibiotic resistance with heavy metal resistance was evaluated, because others (Fasim et al., 1999; Lazar et al., 2002; Nies, 1999) have shown that antibiotic and heavy metal resistance are each carried on extrachromosomal plasmids. The vast majority of ARMO were also resistant to concentrations of heavy metals reported in the sediments of indicator streams (Waihee, system III), suggesting that an antibiotic-resistant bacterium has a high probability of having dual resistance to a heavy metal. A 3.2-kb plasmid (pSTAMP) was isolated from a bacterium with dual antibiotic and heavy metal resistance. Further analysis of the plasmid is currently in progress.     

Microcosm investigations of stormwater pond sediment toxicity to embryonic and larval amphibians: variation in sensitivity among species

Stormwater ponds have become common features of modern development and often represent significant amounts of open space in urbanized areas. Although stormwater ponds may provide habitat for wildlife, factors responsible for producing variation in wildlife use of ponds have received limited attention. To investigate the role of variation in species tolerances of pollutants in structuring pond-breeding amphibian assemblages, we exposed species tolerant (Bufo americanus) and not tolerant (Rana sylvatica) of urbanization to pond sediments in laboratory microcosms. Pond microcosms had elevated sediment metal levels and chloride water concentrations. Among R. sylvatica embryos, exposure to pond sediments resulted in 100% mortality. In contrast, B. americanus embryos and larvae experienced only sublethal effects (i.e., reduced size at metamorphosis) due to pond sediment exposure. Our results suggest variation in pollutant tolerance among early developmental stages of amphibians may act in concert with terrestrial habitat availability to structure amphibian assemblages associated with stormwater ponds.     

Antibiotic resistance,virulence factors and biofilm formation ability in Escherichia coli strains isolated from chicken meat and wildlife in the Czech Republic

Attachment of pathogenic bacteria to food contact surfaces and the subsequent biofilm formation represent a serious threat for the food industry, since these bacteria are more resistant to antimicrobials or possess more virulence factors. The main aim of this study was to investigate the correlation between antibiotic resistance against 13 antibiotics, distribution of 10 virulence factors and biofilm formation in 105 Escherichia coli strains according to their origin. The high prevalence of antibiotic resistance that we have found in wildlife isolates could be acquired by horizontal transfer of resistance genes from human or domestic or farm animals. Consequently, these commensal bacteria might serve as indicator of antimicrobial usage for human and veterinary purposes in the Czech Republic. Further, 46 out of 66 resistant isolates (70%) were able to form biofilm and we found out statistically significant correlation between prevalence of antibiotic resistance and biofilm formation ability. The highest prevalence of antibiotic resistance was observed in weak biofilm producers. Biofilm formation was not statistically associated with any virulence determinant. However, we confirmed the correlation between prevalence of virulence factors and host origin. Chicken isolates possessed more virulence factors (66%), than isolates from wildlife (37%). We can conclude that the potential spread of antibiotic resistance pattern via the food chain is of high concern for public health. Even more, alarming is that E. coli isolates remain pathogenic potential with ability to form biofilm and these bacteria may persist during food processing and consequently lead to greater risks of food contamination.     

Evaluating the vulnerability of surface waters to antibiotic contamination from varying wastewater treatment plant discharges

Effluents from three wastewater treatment plants with varying wastewater treatment technologies and design were analyzed for six antibiotics and caffeine on three sampling occasions. Sulfamethoxazole, trimethoprim, ciprofloxacin, tetracycline, and clindamycin were detected in the effluents at concentrations ranging from 0.090 to 6.0 microg/L. Caffeine was detected in all effluents at concentrations ranging from 0.19 to 9.9 microg/L. These findings indicate that several conventional wastewater management practices are not effective in the complete removal of antibiotics, and their discharges have a large potential to affect the aquatic environment. To evaluate the persistence of antibiotics coming from the wastewater discharges on the surrounding surface waters, samples were collected from the receiving streams at 10-, 20- and 100-m intervals. Ciprofloxacin, sulfamethoxazole, and clindamycin (0.043 to 0.076 microg/L) were found as far as 100 m from the discharge point, which indicates the persistence of these drugs in surface waters.     

Antibiotics pollution in Jiulong River estuary: source, distribution and bacterial resistance

To gain insight into the antibiotic pollution in the Jiulong River estuary and the pollutant sources, we analyzed the concentration of 22 widely-used antibiotics in water samples collected from the river and estuary, 17 and 18 sampling sites, respectively. Contamination with sulfonamides, quinolones and chloramphenicols was frequently detected and the distribution pattern of antibiotics suggested that most of the pollutants are from the Jiulong River, especially from the downstream watersheds. To reveal the ecological effects, we isolated 35 bacterial strains from the estuary and analyzed their antibiotic resistance to the eight most frequently detected antibiotics. The bacteria were subsequently classified into seven different genera by 16SrDNA sequencing. Up to 97.1% of the bacteria showed resistance and 70.6% of strains showed multi-resistance to these antibiotics, especially to sulfonamides. This study demonstrated a pattern of antibiotic contamination in the Jiulong River and its estuary and illustrated high bacterial antibiotic resistance which was significantly correlated with the average antibiotics concentrations and detected frequencies in the estuary.     

Trends in the occurrence of human and veterinary antibiotics in the sediments of the Yellow River, Hai River and Liao River in northern China

The occurrence of four classes of 17 commonly used antibiotics (including fluoroquinolones, tetracycline, sulfonamides, and macrolides) was investigated in the sediments of the Yellow River, Hai River and Liao River in northern China by using rapid resolution liquid chromatography-tandem mass spectrometry. Higher concentrations were detected for most antibiotics in the sediments of the Hai River than in the sediments of the other rivers. Norfloxacin, ofloxacin, ciprofloxacin and oxytetracycline in the three rivers were most frequently detected with concentrations up to 5770, 1290, 653 and 652 ng/g, respectively. High frequencies and concentrations of the detected antibiotics were often found in the downstream of large cities and areas influenced by feedlot and fish ponds. Good fitted linear regression equations between antibiotic concentration and sediment physicochemical properties (TOC, texture and pH) were also found, indicating that sediment properties are important factors influencing the distribution of antibiotics in the sediment of rivers.     

漂浮植物塘协同处理农村分散生活污水研究

通过实验考察了漂浮植物塘对经模拟化粪池简易处理的农村分散生活污水的深度净化效果，结果表明：水葫芦、大薸、聚草塘对上述污水均具有良好的净化效果，水葫芦塘对TP的去除效果最好，大薸塘对TN和NH⁺₄-N的去除效果最好。当水力停留时间为20 d时，3种漂浮植物塘的出水污染物含量可低于GB/T 18921-2002观赏性景观环境用水河道类标准。在南方农村宅沟园河内种植漂浮植物是构建农村实用生活污水收集处理系统、控制农村生活污水对河道污染的一种有效措施。     

Diversity of antibiotic resistance genes and encoding ribosomal protection proteins gene in livestock waste polluted environment

The rapid development and increase of antibiotic resistance are global phenomena resulting from the extensive use of antibiotics in human clinics and animal feeding operations. Antibiotics can promote the occurrence of antibiotic resistance genes (ARGs), which can be transferred horizontally to humans and animals through water and the food chain. In this study, the presence and abundance of ARGs in livestock waste was monitored by quantitative PCR. A diverse set of bacteria and tetracycline resistance genes encoding ribosomal protection proteins (RPPs) from three livestock farms and a river were analyzed through denaturing gradient gel electrophoresis (DGGE). The abundance of sul(I) was 10³ to 10⁵ orders of magnitude higher than that of sul(II). Among 11 tet-ARGs, the most abundant was tet(O). The results regarding bacterial diversity indicated that the presence of antibiotics might have an evident impact on bacterial diversity at every site, particularly at the investigated swine producer. The effect of livestock waste on the bacterial diversity of soil was stronger than that of water. Furthermore, a sequencing analysis showed that tet(M) exhibited two genotypes, while the other RPPs-encoding genes exhibited at least three genotypes. This study showed that various ARGs and RPPs-encoding genes are particularly widespread among livestock.     

不同生物炭对猪粪堆肥过程中抗生素及抗生素抗性基因的削减规律研究

为探讨不同类型生物炭对猪粪堆肥过程中抗生素和抗生素抗性基因(ARGs)的削减规律,分别将玉米秸秆生物炭、竹炭和猪粪生物炭添加到猪粪中进行好氧堆肥,解析不同类型生物炭添加对猪粪堆肥过程中抗生素浓度和ARGs丰度的影响.研究发现,添加竹炭堆肥对抗生素的平均去除率最高(97％),其中对四环素类抗生素的去除率高达98％;添加玉...     

The dissemination of antibiotic resistance in various environmental objects (Russia)

Environmental objects (surface and groundwater, soil, bottom sediments, wastewater) are reservoirs in which large-scale multidirectional exchange of determinants of antibiotic resistance between clinical strains and natural bacteria takes place. The review discusses the results of studies on antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) isolated from environmental objects (water, soil, sewage, permafrost) of the Russian Federation. Despite the relevance of the topic, the number of available publications examining the resistomes of Russian water bodies and soils is small. The most studied environmental objects are surface waters (rivers, lakes), permafrost deposits. Soil resistomes are less studied. Data on ARG and ARB in wastewater are the least covered in publications. In most of the studies, antibiotic resistance of isolated pure bacterial cultures was determined phenotypically. A significant number of publications are devoted to the resistance of natural isolates of Vibrio cholerae, since the lower reaches of the Volga and Don rivers are endemic to cholera. Molecular genetic methods were used in a small number of studies. Geographically, the south of the European part of Russia is the most studied. There are also publications on the distribution of ARG in water bodies of Siberia and the Russian Far East. There are practically no publications on such developed regions of Russia as the center and northwest of the European part of Russia. The territory of the country is very large, anthropogenic and natural factors in its various regions vary significantly; therefore, it seems interesting to combine all available data in one work.