Temporal Dynamics and Impact of Manure Storage on Antibiotic Resistance Patterns and Population Structure of Escherichia coli Isolates from a Commercial Swine Farm

Many confined-livestock farms store their wastes for several months prior to use as a fertilizer. Storing manure for extended periods could significantly bias the composition of enteric bacterial populations subsequently released into the environment. Here, we compared populations of Escherichia coli isolated from fresh feces and from the manure-holding tank (stored manure) of a commercial swine farm, each sampled monthly for 6 months. The 4,668 confirmed E. coli isolates were evaluated for resistance to amikacin, ampicillin, cephalothin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and trimethoprim plus sulfamethoxazole. A subset of 1,687 isolates was fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) with the BOXA1R primer to evaluate the diversity and the population structure of the collection. The population in the stored manure was generally more diverse than that in the fresh feces. Half of the genotypes detected in the stored manure were never detected in the fresh fecal material, and only 16% were detected only in the fresh feces. But the majority of the isolates (84%) were assigned to the 34% of genotypes shared between the two environments. The structure of the E. coli population showed important monthly variations both in the extent and distribution of the diversity of the observed genotypes. The frequency of detection of resistance to specific antibiotics was not significantly different between the two collections and varied importantly between monthly samples. Resistance to multiple antibiotics was much more temporally dynamic in the fresh feces than in the stored manure. There was no relationship between the distribution of rep-PCR fingerprints and the distribution of antibiotic resistance profiles, suggesting that specific antibiotic resistance determinants were dynamically distributed within the population.     

Drug resistance in Vibrio cholerae strains isolated from clinical specimens

Cholera is a serious epidemic and endemic disease caused by the Gram-negative bacterium Vibrio cholerae. SXT is an integrative conjugation element (ICE) that was isolated from a V. cholerae; it encodes resistance to the antibiotics chloramphenicol, streptomycin and sulfamethoxazole/trimethoprim. One hundred seven V. cholerae O1 strains were collected from cholera patients in Iran from 2005 to 2007 in order to study the presence of SXT constin and antibiotic resistance.The study examined 107 Vibrio cholerae strains isolated from cholera prevalent in some Iranian provinces. Bacterial isolation and identification were carried out according to standard bacteriological methods. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) to four antibiotics (chloramphenicol, streptomycin, sulfamethoxazole, and trimethoprim) were determined by broth microdilution method. PCR was employed to evaluate the presence of established antibiotic resistance genes and SXT constin using specific primer sets.The resistance of the clinical isolates to sulfamethoxazole, trimethoprime, chloramphenicol, and streptomycin was 97%, 99%, 99%, and 90%, respectively. The data obtained by PCR assay showed that the genes sulII, dfrA1, floR, strB, and sxt element were present in 95.3%, 95.3%, 81.3%, 95.3%, and 95.3% of the V. cholerae isolates.The Vibrio strains showed the typical multidrug-resistance phenotype of an SXT constin. They were resistant to sulfamethoxazole, trimethoprime, chloramphenicol, and streptomycin. The detected antibiotic resistance genes included dfrA for trimethoprim and floR, strB, sulII and int, respectively, for chloramphenicol, streptomycin, sulfamethoxazole, as well as the SXT element.     

Genotypic-phenotypic discrepancies between antibiotic resistance characteristics of Escherichia coli isolates from calves in management settings with high and low antibiotic use

We hypothesized that bacterial populations growing in the absence of antibiotics will accumulate more resistance gene mutations than bacterial populations growing in the presence of antibiotics. If this is so, the prevalence of dysfunctional resistance genes (resistance pseudogenes) could provide a measure of the level of antibiotic exposure present in a given environment. As a proof-of-concept test, we assayed field strains of Escherichia coli for their resistance genotypes using a resistance gene microarray and further characterized isolates that had resistance phenotype-genotype discrepancies. We found a small but significant association between the prevalence of isolates with resistance pseudogenes and the lower antibiotic use environment of a beef cow-calf operation versus a higher antibiotic use dairy calf ranch (Fisher's exact test, P = 0.044). Other significant findings include a very strong association between the dairy calf ranch isolates and phenotypes unexplained by well-known resistance genes (Fisher's exact test, P < 0.0001). Two novel resistance genes were discovered in E. coli isolates from the dairy calf ranch, one associated with resistance to aminoglycosides and one associated with resistance to trimethoprim. In addition, isolates resistant to expanded-spectrum cephalosporins but negative for bla(CMY-2) had mutations in the promoter regions of the chromosomal E. coli ampC gene consistent with reported overexpression of native AmpC beta-lactamase. Similar mutations in hospital E. coli isolates have been reported worldwide. Prevalence or rates of E. coli ampC promoter mutations may be used as a marker for high expanded-spectrum cephalosporin use environments.     

Enumeration and antibiotic resistance patterns of fecal indicator organisms isolated from migratory Canada geese (Branta canadensis)

Thermotolerant fecal indicator organisms carried by migratory waterfowl may serve as reservoirs of antibiotic resistance. To determine the extent to which such antibiotic resistance markers were present in migratory Canada geese (Branta canadensis) on the Maryland Eastern Shore, we isolated Enterococcus spp. and Escherichia coli from fresh feces and examined the antibiotic resistance profiles of these bacteria. Samples were obtained in October 2002, January 2003, and March 2003. Thermotolerant E. coli counts ranged from 0 to 1.0x10(7) colony forming units (CFU)/0.1g (g-1) wet weight of feces, whereas Enterococcus spp. counts ranged from 1.0x10(2)-1.0x10(7) CFU g-1 wet weight of feces. Primary isolates of each indicator organism were tested against a panel of 10 antibiotics. Greater than 95% of E. coli isolates were resistant to penicillin G, ampicillin, cephalothin, and sulfathiazole; no E. coli were resistant to ciprofloxacin. Enterococcal isolates showed highest resistance to cephalothin, streptomycin, and sulfathiazole; no enterococci were resistant to chloramphenicol. The tetracyclines, streptomycin, and gentamycin provided the greatest discrimination among E. coli isolates; chlortetracycline, cephalothin, and gentamycin resistance patterns provided the greatest discrimination between enterococcal strains. Multiple antibiotic resistance (MAR) profiles were calculated: fall (E. coli=0.499; enterococci=0.234), winter (E. coli=0.487; enterococci=0.389), and spring (E. coli=0.489; enterococci=0.348). E. faecalis and E. faecium, which are recognized human nosocomial pathogens, were cultured from winter (44 and 56%, respectively) and spring (13 and 31%, respectively) fecal samples.     

Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant

Over 18 months, enterobacteria were isolated from the raw (189 isolates) and treated (156 isolates) wastewater of a municipal treatment plant. The isolates were identified as members of the genera Escherichia (76%), Shigella (7%), Klebsiella (12%) and Acinetobacter (4%). Antimicrobial susceptibility phenotypes were determined using the agar diffusion method for the antibiotics amoxicillin, gentamicin, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline and cephalothin, the disinfectants hydrogen peroxide, sodium hypochlorite, quaternary ammonium/formaldehyde and iodine, and the heavy metals nickel, cadmium, chromium, mercury and zinc. Class 1 integrons were detected by PCR amplification using the primers CS5 and CS3. Compared with the raw influent, the treated wastewater presented higher relative proportions of Escherichia spp. isolates resistant to ciprofloxacin and cephalothin (P<0.0001 and P<0.05, respectively). Except for mercury, which showed a positive correlation with tetracycline and sulfamethoxazole/trimethoprim, no significant positive correlations were observed between antibiotic, disinfectant and heavy metal resistance. The variable regions of class 1 integrons, detected in c. 10% of the Escherichia spp. isolates, contained predominantly the gene cassettes aadA1/dhfrI.     

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

AIMS: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. METHODS AND RESULTS: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek GNI+) and enterococci (API rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at >/=85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API(R) profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. CONCLUSIONS: Gull faeces could be a major contributor of E. coli (10(5)-10(9) CFU g(-1)) and enterococci (10(4)-10(8) CFU g(-)1) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. SIGNIFICANCE AND IMPACT OF THE STUDY: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.     

Occurrence and Abundance of Antibiotics and Resistance Genes in Rivers,Canal and near Drug Formulation Facilities – A Study in Pakistan

Antibiotic resistance (AR) is a global phenomenon that has severe epidemiological ramifications world-wide. It has been suggested that antibiotics that have been discharged into the natural aquatic environments after usage or manufacture can promote the occurrence of antibiotic resistance genes (ARG). These environmental ARGs could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. The aim of this study was to investigate the anthropogenic load of antibiotics in Northern Pakistan and study the occurrence of ARGs in selected samples from this region. 19 sampling sites were selected; including six rivers, one dam, one canal, one sewage drain and four drug formulation facilities. Our results show that five of the rivers have antibiotic levels comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics could be detected in the downstream river in close vicinity of the 10 million city Lahore, 1100, 1700 and 2700 ng L⁻¹ for oxytetracycline, trimethoprim, and sulfamethoxazole respectively. Highest detected levels were at one of the drug formulation facilities, with the measured levels of 1100, 4100, 6200, 7300, 8000, 27000, 28000 and 49000 ng L⁻¹ of erythromycin, lincomycin, ciprofloxacin, ofloxacin, levofloxacin, oxytetracycline, trimethoprim and sulfamethoxazole respectively. ARGs were also detected at the sites and the highest levels of ARGs detected, sulI and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. Highest levels of both antibiotics and ARGs were seen at a drug formulation facility, within an industrial estate with a low number of local residents and no hospitals in the vicinity, which indicates that the levels of ARGs at this site were associated with the environmental levels of antibiotics.     

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

Fifty-eight clinical Salmonella typhi strains isolated from patients suspected of suffering from typhoid fever were obtained at the Korle-Bu Teaching Hospital and the Noguchi Memorial Institute for Medical Research, both located in Ghana, Africa. Each isolate was examined for susceptibility to ampicillin, chloramphenicol, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole by the disk diffusion assay. Five of the isolates were resistant to all five antibiotics while 10 isolates were resistant to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, which are considered 'first line' antibiotics in the treatment of typhoid fever. Thirty-four isolates were resistant to at least one of the antibiotics tested and 62% of these isolates possessed conjugable plasmids belonging to incompatibility group IncHI. Ninety percent of the conjugable plasmids conferred a multiple drug-resistant phenotype on the strains harboring them. Additionally, 14 strains contained plasmids that were transformable and six of them encoded multiple drug resistance. Our findings indicate that multiple drug resistance to the 'first line' antibiotics in S. typhi may be more prevalent in Africa than previously thought.     

Occurrence of antibiotic resistance in Escherichia coli from surface waters and fecal pollution sources near Hamilton, Ontario

Antibiotic resistance was examined in 462 Escherichia coli isolates from surface waters and fecal pollution sources around Hamilton, Ontario. Escherichia coli were resistant to the highest concentrations of each of the 14 antibiotics studied, although the prevalence of high resistance was mostly low. Two of 12 E. coli isolates from sewage in a CSO tank had multiple resistance to ampicillin, ciprofloxacin, gentamicin, and tetracycline above their clinical breakpoints. Antibiotic resistance was less prevalent in E. coli from bird feces than from municipal wastewater sources. A discriminant function calculated from antibiotic resistance data provided an average rate of correct classification of 68% for discriminating E. coli from bird and wastewater fecal pollution sources. The preliminary microbial source tracking results suggest that, at times, bird feces might be a more prominent contributor of E. coli to Bayfront Park beach waters than municipal wastewater sources.     

Loss of virulence genes in Escherichia coli populations during manure storage on a commercial swine farm

Confined livestock production farms typically store their wastes prior to land application. Here, we employed three complementary approaches to evaluate changes in the population structure and stability of virulence genes in Escherichia coli during manure storage on a commercial farm that housed healthy swine. Isolates were genotyped by repetitive extragenic palindromic PCR using the BOXA1R primer and evaluated for the presence of selected virulence genes by PCR. Isolates obtained from the manure holding tank (n = 392) carried estB, fedA, stx(2e), astA, paa, aida-I, and sepA at lower frequencies than isolates obtained from fresh feces (n = 412). Fresh fecal material from the barn was added into diffusion chambers and immersed in the manure holding tank for 7 weeks. The fecal E. coli population was initially dominated by a single genotype, all isolates of which carried fedA and aida-I. After 7 weeks, a genotype that did not carry any virulence genes dominated the surviving population. In a second experiment, 48 fecal isolates of E. coli that varied in their genotypes and virulence gene complement were incubated in diffusion chambers in the manure holding tank for 3 weeks. Over 95% of the inoculum population carried at least one virulence gene, whereas after 3 weeks 90% of the recovered isolates carried no virulence genes. Taken together, these results indicate that during commercial manure storage, there was a significant reduction in the carriage of these virulence genes by E. coli. We propose that loss of virulence genes from enteric pathogens in the farm and in natural environments may, if generalized, contribute to the attenuation of a public health risk from contamination with agricultural wastes.     

Resistance patterns of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains isolated from animals, food and asymptomatic human carriers in Switzerland

AIMS: The objective of the present study was to determine the resistance patterns of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains isolated from different sources in Switzerland during the period 1997-99 as an epidemiological marker. METHODS AND RESULTS: The disk diffusion method was used to test 82 non-O157 STEC strains for susceptibility to 13 antibiotics. Ten strains were resistant to one and 20 strains to two and more antibiotics. The most frequent resistance types were streptomycin (14 strains), cephalothin (14 strains), sulfamethoxazole (14 strains) and tetracycline (14 strains). Three O100:H- STEC strains isolated from healthy slaughter pigs were resistant to eight antibiotics: streptomycin, sulfamethoxazole, trimethoprim, tetracycline, ampicillin, chloramphenicol, neomycin and gentamicin. CONCLUSION: Periodic surveillance of the antibiotic susceptibilities would be an important measure in detecting emergence and spread of resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic susceptibility testing can be a useful tool for typing strains and should be used in combination with other phenotypic and genotypic methods.     

分离自母婴肠道的假小链双歧杆菌的耐药性分析

【背景】由于滥用抗生素导致细菌耐药性日益严重。对于双歧杆菌,人们往往注重其益生功能的挖掘而忽视了对其耐药性的研究,存在一定的安全隐患。【目的】检测母婴肠道中假小链双歧杆菌的耐药性,探究婴儿肠道中假小链双歧杆菌耐药性的来源。【方法】利用微量肉汤稀释法测定48株分离自母婴肠道的假小链双歧杆菌对14种抗生素的耐药性,比较分离自不同家庭母婴肠道中假小链双歧杆菌的耐药性。【结果】48株母婴肠道分离株对四环素、氯霉素、新霉素、环丙沙星100%耐药,对其余10种抗生素耐药率依次为：卡那霉素98%、利福平80%、克林霉素78%、甲氧苄啶63%、红霉素59%、庆大霉素43%、链霉素16%、万古霉素14%、氨苄西林6%、利奈唑胺2%。母婴肠道分离株的耐药性无显著差异,分离自同一家庭母婴肠道的菌株具有相似的耐药表型。【结论】分离自母婴肠道的假小链双歧杆菌对多种抗生素具有耐药性,婴儿肠道中假小链双歧杆菌的耐药性可能是由母亲肠道垂直传递而来。     

Antimicrobial susceptibilities of Aeromonas spp. isolated from environmental sources

Aeromonas spp. are ubiquitous aquatic bacteria that cause serious infections in both poikilothermic and endothermic animals, including humans. Clinical isolates have shown an increasing incidence of antibiotic and antimicrobial drug resistance since the widespread use of antibiotics began. A total of 282 Aeromonas pure cultures were isolated from both urban and rural playa lakes in the vicinity of Lubbock, Texas, and several rivers in West Texas and New Mexico. Of these, at least 104 were subsequently confirmed to be independent isolates. The 104 isolates were identified by Biolog and belonged to 11 different species. The MICs of six metals, one metalloid, five antibiotics, and two antimicrobial drugs were determined. All aeromonads were sensitive to chromate, cobalt, copper, nickel, zinc, cefuroxime, kanamycin, nalidixic acid, ofloxacin, tetracycline, and sulfamethoxazole. Low incidences of trimethoprim resistance, mercury resistance, and arsenite resistance were found. Dual resistances were found in 5 of the 104 Aeromonas isolates. Greater numbers of resistant isolates were obtained from samples taken in March versus July 2002 and from sediment versus water. Plasmids were isolated from selected strains of the arsenite- and mercury-resistant organisms and were transformed into Escherichia coli XL1-Blue MRF'. Acquisition of the resistance phenotypes by the new host showed that these resistance genes were carried on the plasmids. Mercury resistance was found to be encoded on a conjugative plasmid. Despite the low incidence of resistant isolates, the six playa lakes and three rivers that were sampled in this study can be considered a reservoir for antimicrobial resistance genes.     

Novel method for rapid assessment of antibiotic resistance in Escherichia coli isolates from environmental waters by use of a modified chromogenic agar

We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% +/- 4% overall), sulfamethoxazole, tetracycline (21% +/- 3% overall), and ciprofloxacin (6% +/- 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.     

1939株革兰阴性杆菌的耐药监测分析

目的 了解广州华侨医院2007年至2009年分离的1 939株革兰阴性杆菌的耐药状况,为临床合理使用抗生素提供依据.方法 利用梅里埃VITEK-2 Compact微生物分析仪鉴定细菌,K-B法进行药敏试验,采用WHONET 5.4 软件统计分析药敏结果.结果 2007年至2009年分离出的革兰阴性杆菌1 939株,居前...     

High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Antibiotic Multiresistance Analysis of Mesophilic and Psychrotrophic Pseudomonas spp. Isolated from Goat and Lamb Slaughterhouse Surfaces throughout the Meat Production Process

The aim of this study was to investigate the phenotypic and genotypic antibiotic resistance profiles of pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which were representative of areas that are possible sources of meat contamination. Mesophilic (85 isolates) and psychrotrophic (37 isolates) pseudomonads identified at the species level generally were resistant to sulfamethoxazole, erythromycin, amoxicillin, ampicillin, chloramphenicol, trimethoprim, rifampin, and ceftazidime (especially mesophiles), as well as colistin and tetracycline (especially psychrotrophes). However, they generally were sensitive to ciprofloxacin, gentamicin, imipenem, and kanamycin regardless of species identity. Worryingly, in the present study, we found multidrug resistance (MDR) to up to 13 antibiotics, which was related to intrinsic and acquired resistance mechanisms. Furthermore, a link between various antimicrobial resistance genes was shown for beta-lactams and tetracycline, trimethoprim, and sulfonamides. The distribution and resistome-based analysis of MDR pseudomonads in different slaughterhouse zones indicated that the main sources of the identical or related pseudomonad strains were the animals (feet and wool) and the slaughterhouse environment, being disseminated from the beginning, or entrance environment, to the environment of the finished meat products. Those facts must be taken into consideration to avoid cross-contamination with the subsequent flow of mobile resistance determinants throughout all slaughterhouse zones and then to humans and the environment by the application of adequate practices of hygiene and disinfection measures, including those for animal wool and feet and also the entrance environment.     

Temporal Variability of Antibiotics Fluxes in Wastewater and Contribution from Hospitals

Significant quantities of antibiotics are used in all parts of the globe to treat diseases with bacterial origins. After ingestion, antibiotics are excreted by the patient and transmitted in due course to the aquatic environment. This study examined temporal fluctuations (monthly time scale) in antibiotic sources (ambulatory sales and data from a hospital dispensary) for Lausanne, Switzerland. Source variability (i.e., antibiotic consumption, monthly data for 2006–2010) were examined in detail for nine antibiotics – azithromycin, ciprofloxacin, clarithromycin, clindamycin, metronidazole, norfloxacin, ofloxacin, sulfamethoxazole and trimethoprim, from which two main conclusions were reached. First, some substances – azithromycin, clarithromycin, ciprofloxacin – displayed high seasonality in their consumption, with the winter peak being up to three times higher than the summer minimum. This seasonality in consumption resulted in seasonality in Predicted Environmental Concentrations (PECs). In addition, the seasonality in PECs was also influenced by that in the base wastewater flow. Second, the contribution of hospitals to the total load of antibiotics reaching the Lausanne Wastewater Treatment Plant (WTP) fluctuated markedly on a monthly time scale, but with no seasonal pattern detected. That is, there was no connection between fluctuations in ambulatory and hospital consumption for the substances investigated.     

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment. To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators - including inorganic ions, antibiotics, and antibiotic resistance genes - were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 microg/L. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes - tet(M), tet(O), tet(Q), and tet(W) - were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

Carriage of antibiotic-resistant enteric bacteria varies among sites in Galapagos reptiles

Increased overlap between humans and wildlife populations has increased the risk for novel disease emergence. Detecting contacts with a high risk for transmission of pathogens requires the identification of dependable measures of microbial exchange. We evaluated antibiotic resistance as a molecular marker for the intensity of human-wildlife microbial connectivity in the Galápagos Islands. We isolated Escherichia coli and Salmonella enterica from the feces of land iguanas (Conolophus sp.), marine iguanas (Amblyrhynchus cristatus), giant tortoises (Geochelone nigra), and seawater, and tested these bacteria with the use of the disk diffusion method for resistance to 10 antibiotics. Antibiotic-resistant bacteria were found in reptile feces from two tourism sites (Isla Plaza Sur and La Galapaguera on Isla San Cristóbal) and from seawater close to a public use beach near Puerto Baquerizo Moreno on Isla San Cristóbal. No resistance was detected at two protected beaches on more isolated islands (El Miedo on Isla Santa Fe and Cape Douglas on Isla Fernandina) and at a coastal tourism site (La Lobería on Isla San Cristóbal). Eighteen E. coli isolates from three locations, all sites relatively proximate to a port town, were resistant to ampicillin, doxycycline, tetracycline, and trimethoprin/sulfamethoxazole. In contrast, only five S. enterica isolates showed a mild decrease in susceptibility to doxycycline and tetracycline from these same sites (i.e., an intermediate resistance phenotype), but no clinical resistance was detected in this bacterial species. These findings suggest that reptiles living in closer proximity to humans potentially have higher exposure to bacteria of human origin; however, it is not clear from this study to what extent this potential exposure translates to ongoing exchange of bacterial strains or genetic traits. Resistance patterns and bacterial exchange in this system warrant further investigation to understand better how human associations influence disease risk in endemic Galápagos wildlife.