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。     

Antibiotic resistant bacteria/genes dissemination in lacustrine sediments highly increased following cultural eutrophication of Lake Geneva (Switzerland)

This study investigates faecal indicator bacteria (FIB), multiple antibiotic resistant (MAR), and antibiotic resistance genes (ARGs), of sediment profiles from different parts of Lake Geneva (Switzerland) over the last decades. MARs consist to expose culturable Escherichia coli (EC) and Enterococcus (ENT) to mixed five antibiotics including Ampicillin, Tetracycline, Amoxicillin, Chloramphenicol and Erythromycin. Culture-independent is performed to assess the distribution of ARGs responsible for, β-lactams (blaTEM; Amoxicillin/Ampicillin), Streptomycin/Spectinomycin (aadA), Tetracycline (tet) Chloramphenicol (cmlA) and Vancomycin (van). Bacterial cultures reveal that in the sediments deposited following eutrophication of Lake Geneva in the 1970s, the percentage of MARs to five antibiotics varied from 0.12% to 4.6% and 0.016% to 11.6% of total culturable EC and ENT, respectively. In these organic-rich bacteria-contaminated sediments, the blaTEM resistant of FIB varied from 22% to 48% and 16% to 37% for EC and ENT respectively, whereas the positive PCR assays responsible for tested ARGs were observed for EC, ENT, and total DNA from all samples. The aadA resistance gene was amplified for all the sediment samples, including those not influenced by WWTP effluent water. Our results demonstrate that bacteria MARs and ARGs highly increased in the sediments contaminated with WWTP effluent following the cultural eutrophication of Lake Geneva. Hence, the human-induced changing limnological conditions highly enhanced the sediment microbial activity, and therein the spreading of antibiotic resistant bacteria and genes in this aquatic environment used to supply drinking water in a highly populated area. Furthermore, the presence of the antibiotic resistance gene aadA in all the studied samples points out a regional dissemination of this emerging contaminant in freshwater sediments since at least the late nineteenth century.     

A comparison of antibiotics,antibiotic resistance genes,and bacterial community in broiler and layer manure following composting

Animal manure is an important source of antibiotics and antibiotic resistance genes (ARGs) in the environment. However, the difference of antibiotic residues and ARG profiles in layer and broiler manure as well as their compost remains unexplored. In this study, we investigated the profiles of twelve antibiotics, seventeen ARGs, and class 1 integrase gene (intI1) in layer and broiler manure, and the corresponding compost at large-scale. Compared with layer manure, broiler manure exhibited approximately six times more residual tetracyclines, especially chlortetracycline. The relative abundances of qnrS and ermA genes in broiler manure were significantly higher than those in layer manure. The concentration of tetracyclines not only had a significantly positive correlation with tetracycline resistance genes (tetA and tetC) but was also positively correlated with quinolone resistance (qepA, qnrB, and qnrS) and macrolide resistance (ermA and ermT). Most ARGs in manure were reduced after composting. However, the relative abundance of sulfonamide resistance gene sul1 increased up to 2.41% after composting, which was significantly higher than that of broiler (0.41%) and layer (0.62%) manure. The associated bacterial community was characterized by high-throughput 16S rRNA gene sequencing. The relative abundances of thermophilic bacteria had significant positive correlations with the abundance of sul1 in compost. The composting has a significant impact on the ARG-associated gut microbes in poultry manure. Variation partitioning analysis indicated that the change of bacterial community compositions and antibiotics contributed partially to the shift in ARG profiles. The results indicate that at industry-scale production broiler manure had more antibiotics and ARGs than layer manure did, and composting decreased most ARG abundances in poultry manure except for sulfonamide resistance genes.

     

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety

Foodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.     

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.     

Occurrence and abundance of tetracycline,sulfonamide resistance genes,and class 1 integron in five wastewater treatment plants

To understand the transport and fate of antibiotic resistance genes in wastewater treatment plants, 12 resistance genes (ten tetracycline resistance genes, two sulfonamides genes) and class 1 integron gene (intI1) were studied in five wastewater treatment plants with different treatment processes and different sewage sources. Among these resistance genes, sulfonamides genes (sul1 and sul2) were of the most prevalent genes with detection frequency of 100 %. The effluent water contained fewer types of resistance genes than the influent in most selected plants. The abundance of five quantified resistance genes (tetG, tetW, tetX, sul1, and intI1) decreased in effluent of plants treating domestic or industrial wastewater with anaerobic/aerobic or membrane bioreactor (MBR) technologies, but tetG, tetX, sul1, and intI1 increased along the treatment units of plants treating vitamin C production wastewater by anaerobic/aerobic technology. In plant treating cephalosporins production wastewater by UASB/aerobic process, the quantities of tetG, tetX, and sul1 first decreased in anaerobic effluent water but then increased in aerobic effluent water.     

The profile of antibiotics resistance and integrons of extended-spectrum beta-lactamase producing thermotolerant coliforms isolated from the Yangtze River basin in Chongqing

The spreading of extended-spectrum β-lactamases (ESBL)-producing thermotolerant coliforms (TC) in the water environment is a threat to human health but little is known about ESBL-producing TCs in the Yangtze River. We received 319 ESBL-producing stains obtained from the Chongqing basin and we investigated antibiotic susceptibility, bla gene types and the presence of integrons and gene cassettes. 16.8% of TC isolates were ESBL-producing bacteria and bla_TEM+CTx-M was the predominant ESBL type. 65.2% of isolates contained class 1 integrons, but only 3 carried intI 2. Gene cassettes were amplified and sequenced. aadA, drfA, cmlA, sat1, aar3 and two ORF cassettes were found. In conclusion, Yangtze River is heavily polluted by ESBL-producing TC bacteria and the combined bla gene type could enhance antibiotic resistance. Class 1 integrons were widespread in ESBL-producing isolates and play an important role in multi-drug resistance. Characterization of gene cassettes could reveal the dissemination of antibiotic resistance genes.     

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.     

Prevalence and antimicrobial resistance of Shiga toxin-producing Escherichia coli in milk and dairy products in Egypt

Abstract

Food contaminated with Shiga toxin-producing Escherichia coli (STEC) represents a hazardous public health problem worldwide. Therefore, the present study was performed to elucidate the virulent and antimicrobial resistance characteristics of STEC isolated from milk and dairy products marketed in Egypt. A total of 125 samples (raw market milk, bulk tank milk, Kareish cheese, white soft cheese, and small scale-produced ice cream, 25 each) were collected for determination the prevalence and antimicrobial resistance profiling of STEC. Thirty-six STEC isolates were recovered from milk and dairy products. Serological analysis illustrated that three isolates were E. coli O157:H7 and 33 isolates belonged to different serotypes. Molecular examination indicated that all isolates harboured stx1 and/or stx2 genes, 14 isolates expressed eaeA gene and 3 isolates possessed rfbE gene. Antimicrobial resistance profiling of the isolates was both phenotypically and genetically examined. Interestingly, 31 out of 36 (86.11%) isolates were multidrug-resistant and harboured the extended-spectrum β-lactamase encoding genes, namely, bla_CTX-M-15, bla_SHV-12 and bla_CTX-M-14. Moreover, 12 isolates (33.33%) harboured plasmid-mediated quinolone resistant gene, qnrS. The overall conclusion of the current investigation indicated insufficient hygienic measures adopted during milking, handling, and processing leading to development of pathogenic and multidrug-resistant STEC.     

Contamination of antibiotic resistance genes (ARGs) in a typical marine aquaculture farm: source tracking of ARGs in reared aquatic organisms

Abstract

Although the prevalence and concentrations of antibiotic resistance genes (ARGs) in aquaculture is receiving increasing scientific interest, there is little understanding of the direct sources and dissemination pathways of ARGs in marine aquaculture-reared organisms. This study investigated the dynamics of ARGs and the bacterial community throughout the rearing period in a typical marine aquaculture farm in South China. The results demonstrated that sul1 and qnrD were predominant in the sediment, and qnrD and qnrA were predominant in the intestinal tracts of shrimps. Network analysis showed that the chemical oxygen demand, total organic carbon, dissolved organic carbon, suspended solids, and total phosphorus were positively correlated with the predominant ARGs. The results of the network and source tracking analyses indicate that environmental factors and the bacterial community may drive the dissemination of ARGs dissemination in the environment and in shrimp reared by marine aquaculture, and sediment is the most direct and important medium in this dissemination. These results aid in improving our understanding of the sources, level, and dissemination of ARGs in marine aquaculture.     

Antibiotic resistance and genetic diversity of Escherichia coli isolates from traditional and integrated aquaculture in South China

This study investigated antibiotic resistance profiles including antibiotic resistance frequencies, resistance genes and resistance patterns in Escherichia coli strains isolated from traditional and integrated aquaculture systems in South China by using antibiotic susceptibility testing and real time polymerase chain reaction (PCR) technique. The E. coli isolates were found to be resistant to at least one antibiotic among 12 antibiotics. Higher resistance frequencies to ampicillin, sulfamethoxazole, trimethoprime, streptomycin and tetracycline were found compared to the rest antibiotics. Among the 10 tetracycline resistance genes detected in the resistant isolates, the most prevalent tetracycline resistance genes were tetA, tetW and tetB with the frequency of 69.7%, 63.5% and 21.9%, respectively. Three sulfonamide resistance genes were detected in these resistant isolates, with their detection frequencies in the following order: sul2 (55.3%) > sul3 (28.2%) > sul1 (6.2%). Four resistance genes mainly encoding extended-spectrum β-lactamases (ESBLs) were detected in these resistant isolates, with the detection frequencies of blaTEM (28.4%) > blaOXA (9.7%) > blaCTX (9.3%) > blaCARB (5.2%) > blaSHV (0.0%). It was found that the integrated aquaculture system exhibited generally higher prevalence of antibiotic resistance than the traditional aquaculture system. An integrated aquaculture system could facilitate development of bacterial resistance and spread of the antibiotic resistance genes, and consequently become an important reservoir of resistance genes.     

Dynamics of antibiotic resistance genes and microbial community in shortcut nitrification–denitrification process under antibiotic stresses

In this study, the performance of shortcut nitrification–denitrification (SCND) at different TC and SD stress conditions (0 μg/L, 1–97 days; 100 μg/L, 98–138 days; 500 μg/L, 139–175 days) was investigated. Higher level antibiotic stress (500 μg/L) led to the serious deterioration of nitrogen removal, and denitrification was more sensitive to antibiotic stress than nitrification. The dynamics of antibiotic resistance genes (ARGs) and microbial community were revealed by quantitative real-time PCR and 16S rDNA high-throughput sequencing, respectively. Tet-genes (tetA, tetQ, tetW), sul-genes (sulI, sulII), and mobile genetic element (intI1) in activated sludge increased by 1.2?~?2.5 logs with long-term exposure of antibiotic stress, and sulI, tetA, tetQ, and tetW were significantly positively correlated with intI1. Long-term antibiotics stress caused the decrease of most denitrifiers, and five genera were identified as the potential host of ARGs. The key impact factors of SCND drove the dynamics of ARGs and microbial community. Except for sulII gene, DO and FA were significantly positively correlated with ARGs, while FNA, NAR, and NO₂^?-N showed opposite effects to ARGs. Overall, maintaining relative lower DO, higher FNA, NAR, and NO₂^?-N conditions are not only benefit to the stable operation of SCND, but may also conducive to the control of ARG dissemination. This study provides theoretical basis on the control of ARGs in the SCND process.

     

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.     

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.     

Fate of antimicrobial resistance genes in response to application of poultry and swine manure in simulated manure-soil microcosms and manure-pond microcosms

This study aimed to determine the occurrence, abundance, and fate of nine important antimicrobial resistance genes (ARGs) (sul1, sul2, tetB, tetM, ermB, ermF, fexA, cfr, and Intl1) in the simulated soil and pond microcosms following poultry and swine manure application. Absolute quantitative PCR method was used to determine the gene copies. The results were modeled as a logarithmic regression (N?=?mlnt?+?b) to explore the fate of target genes. Genes sul1, Intl1, sul2, and tetM had the highest abundance following the application of the two manure types. The logarithmic regression model fitted the results well (R ² values up to 0.99). The reduction rate of all genes (except for the genes fexA and cfr) in manure-pond microcosms was faster than those in manure-soil microcosms. Importantly, sul1, intl1, sul2, and tetM had the lowest reduction rates in all the samples and the low reduction rates of tetM was the first time to be reported. These results indicated that ARG management should focus on using technologies for the ARG elimination before the manure applications rather than waiting for subsequent attenuation in soil or water, particularly the ARGs (such as sul1, intl1, sul2, and tetM investigated in this study) that had high abundance and low reduction rate in the soil and water after application of manure.     

Assessment of vancomycin resistance transfer among enterococci of clinical importance in milk matrix

Abstract

Dissemination of vancomycin resistance in enterococci has been associated with horizontal transfer of mobile genetic elements. Aim of the study was to evaluate if milk matrix is a suitable environment to support transferability of vancomycin resistance (vanA) gene from clinical vancomycin-resistant Enterococcus faecium to vancomycin-sensitive Enterococcus faecalis. Enterococci strains were firstly screened for the presence of cpd (inducible sex pheromone determinant) gene, vanA and tetL genes (vancomycin and tetracycline resistance markers, respectively) and the gelE (extracellular metalloendopeptidase) gene to define the mating pairs. Based on these selection markers, we investigated the transferability of eight plasmid-borne vanA harbored by E. faecium (vanA+, cpd-, tetL- and gelE-) into two E. faecalis (vanA-, cpd+, tetL?+?and gelE+) recipient strains in milk matrix. The strains were mated in a 1:1 ratio in 7% reconstituted milk and incubated at 37?°C. Transconjugants emerged from all 16 matings within 2?h of incubation and were evidenced by dual antibiotic resistance (vancomycin and tetracycline). The vancomycin-resistance of trasconjugants was maintained even after ten subsequent passages on nonselective medium. Transconjugants were positive for vanA, tetL and gelE genes. This study indicates milk matrix as suitable environment to support gene exchange between Enterococcus species.     

Enterotoxin production and antimicrobial susceptibility in Staphylococci isolated from traditional raw milk cheeses in Serbia

This study was undertaken to determine the prevalence of coagulase positive staphylococci (CPS) by examining a total of 71 raw milk cheeses. Additionally, enterotoxigenicity, antimicrobial susceptibility and the presence of mecA and mecC genes in the staphylococcal isolates were investigated. The isolation and enumeration procedure of CPS followed the International Organization for Standardization (ISO) standard. The presumptive staphylococci were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the VITEK MS system. VIDAS® Staph enterotoxin II assay was used for the detection of classical enterotoxins. Antimicrobial susceptibility testing (AST) was accomplished performing the disk diffusion method. All suspected methicillin resistant staphylococci were investigated for the presence of mecA and mecC genes by PCR assay. A high prevalence (87.32%) of CPS was detected in the cheeses at contamination levels up to 5.58 log CFU g^?1. Among 47 staphylococcal isolates screened for enterotoxin production, only one isolate, identified as S. hyicus, was confirmed as being enterotoxigenic. Resistance to penicillin (63.70%) was the most common resistance among the tested Staphylococcus aureus isolates. The dominant phenotypic resistance patterns in coagulase negative staphylococci (CNS) were resistance to ofloxacin and fusidic acid. All CNS isolates were susceptible to the clinically important antibiotics clindamycin, chloramphenicol, gentamicin, linezolid, rifampicin and trimethoprim-sulfamethoxazole. The mecA and mecC genes were not detected. To the best of our knowledge, this is the first study concerning evaluation of the presence of methicillin resistant staphylococci (MRS) in dairy products in Serbia.     

Comparison of Spontaneous Antibiotic Resistance Frequency of Salmonella Typhimurium Growth in Glucose Amended Continuous Culture at Slow and Fast Dilution Rates

Abstract

The objective of the study was to determine the frequency of spontaneous acquisition of resistance to select antibiotics by Salmonella Typhimurium (ST) when grown in glucose amended continuous flow culture at slow (D = 0.025 h^{? 1}) or fast (D = 0.27 h^{? 1}) dilution rates. The bacterium was grown in LB minimal medium (pH 6.25) containing no antibiotics. Upon achieving steady state, samples were plated to tryptic soy agar (TSA) alone or supplemented (per ml) with 2 and 16 μg oxytetracycline, 4 and 16 μg tetracycline, 2 and 64 μg kanamycin, and 0.25 and 2 μg enrofloxacin. Regardless of growth rate, CFU of resistant ST from the TSA containing antibiotics was less than 2 × 10¹ except for 2 μg kanamycin and 0.25 μg enrofloxacin treatments (higher than 1 × 10⁹ and 4 × 10⁷ CFU of resistant ST for trials 1 and 2, respectively). Frequency of recovering resistant ST from the TSA containing the higher antibiotic concentrations was less than 1 in 10⁹ for all antibiotics, but was higher on the media containing 2 μg kanamycin and 0.25 μg enrofloxacin at both slow and fast growth rates. In general, minimal susceptibility differences were detected for isolates from slow and fast dilution rates.     

Environmental fate of tetracycline resistance genes originating from swine feedlots in river water

Tetracyclines are antibiotics commonly used in swine farms to treat disease and promote growth. However, there are growing concerns regarding the discharge of animal feces into the environment owing to the potential for development and dissemination of tetracycline resistance genes (TRGs). In this study, farming wastewater from one Chinese swine farm as well as river water from seven locations downstream of the farm was sampled. Polymerase chain reaction (PCR) showed that 12 TRGs, including six efflux pump genes (tet(B), tet(C), tet(D), tet(E), tet(G) and tet(L)), five ribosomal protection proteins (RPPs) genes (tet(O), tet(M), tet(Q), tet(W) and tet(S)), and one enzymatic modification gene (tet(X)), were present in all wastewater and river water samples. Quantitative real-time PCR (qPCR) showed that the abundance of tet(C), tet(X), tet(O), tet(M), tet(Q) and tet(W) decreased with downstream flow. Among the detected TRGs, tet(C) had the highest abundance, ranging from 459.5 copies/16S rRNA gene copies in wastewater to 33.8 copies/16S rRNA gene copies in river water samples collected from the last location. Furthermore, pig-specific Bacteroidales 16S rRNA genetic marker was quantified by qPCR to determine the level of fecal pollution in the river water. Bivariate correlation analysis confirmed that the total relative abundance of the six TRGs was significantly correlated with the level of swine feces in the aquatic environment (R² = 0.63, P < 0.05), suggesting that swine feces mainly contributed to the spread of TRGs in the river water.

Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Environmental Science and Health, Part A to view the free supplemental file.     

Resistance in bacteria and indirect beta-lactamase detection in E. coli isolated from Culex pipiens detected by matrix-assisted laser desorption ionization time of flight mass spectrometry

The aim of this study was detections of antibiotic resistance and resistance mechanism in bacteria isolated from mosquitos (Culex pipiens) living near humans. Therefore, antibiotic resistance in bacteria isolated from Culex pipiens was investigated by disk diffusion test and MIC E-test in this study. MALDI-TOF mass spectrometry was used for detection of resistant mechanism. In this study, hydrolytic breakdown products after a few hours of incubation of the bacteria isolated from Culex pipiens were detected. Results show that enzymatic destruction of ampicillin by beta-lactamases is able to be detected by MALDI-TOF mass spectrometry from wild strains of potential pathogens. The MALDI-TOF mass spectrometry is useful method for routine detection of beta-lactamases resistant mechanism, but overnight incubation of pure culture is necessary. The results are important for proper and fast intervention to limit the spread of beta-lactamase-producing wild bacteria and provide information for appropriate initial therapy of the infections caused by these microbes.