Antibiotic Resistance of Escherichia coli Isolated from Conventional, No Antibiotics, and Humane Family Owned Retail Broiler Chicken Meat.

The use of antibiotics for therapeutic and especially non-therapeutic purposes in livestock farms promotes the development of antibiotic resistance in previously susceptible bacteria through selective pressure. In this work, we examined E. coli isolates using the standard Kirby-Bauer disk diffusion susceptibility protocol and the CLSI standards. Companies selling retail chicken products in Los Angeles, California were grouped into three production groupings-Conventional, No Antibiotics, and Humane Family Owned. Humane Family Owned is not a federally regulated category in the United States, but shows the reader that the chicken is incubated, hatched, raised, slaughtered, and packaged by one party, ensuring that the use of antibiotics in the entire production of the chicken is known and understood. We then examined the antibiotic resistance of the E. coli isolates (n = 325) by exposing them to seven common antibiotics, and resistance was seen to two of the antibiotics, ampicillin and erythromycin. As has been shown previously, it was found that for both ampicillin and erythromycin, there was no significant difference (p > 0.05) between Conventional and USDA (United States Department of Agriculture)-certified No Antibiotics chicken. Unique to this work, we additionally found that Humane Family Owned chicken had fewer (p ≤ 0.05) antibiotic-resistant E. coli isolates than both of the previous. Although not considered directly clinically relevant, we chose to test erythromycin because of its ecological significance to the environmental antibiotic resistome, which is not generally done. To our knowledge, Humane Family Owned consumer chicken has not previously been studied for its antibiotic resistance. This work contributes to a better understanding of a potential strategy of chicken production for the overall benefit of human health, giving evidentiary support to the One Health approach implemented by the World Health Organization.

Multi-tiered approach utilizing microbial source tracking and human associated-IMS/ATP for surveillance of human fecal contamination in Baja California, Mexico.

As both the need for reuse of reclaimed wastewater and the burden placed on existing wastewater treatment plants increase, so does the need for methods that can reliably, rapidly and economically identify human-associated contamination. A survey of surface water quality was conducted in Baja California, Mexico where inadequate infrastructure or its inefficient operation leads to poor water quality. The HF183 and Bacteroides thetaiotaomicron (B. theta) human-associated gene markers were detected in 84% and 82% of samples collected during dry weather, illustrating evidence of widespread human fecal contamination. In addition, an inversely-coupled (Inv-IMS/ATP) viability-based assay for detection of B. theta was developed and applied for rapid detection and screening of human-associated fecal contamination. The Inv-IMS/ATP assay was able to effectively differentiate between surface waters impacted with human fecal contamination, and B. theta levels measured by Inv-IMS/ATP were highly correlated with HF183 and B. theta human marker measurements (r = 0.76; r = 0.82) in complex surface water samples. In areas with widespread human fecal contamination and limited access to more expensive methods, a multi-pronged approach utilizing a combination of methods including the Inv-IMS/ATP assay for rapid evaluation and screening of surface water quality alongside human-associated genetic markers may improve risk assessment and surveillance capabilities.

Effect of freshwater sediment characteristics on the persistence of fecal indicator bacteria and genetic markers within a Southern California watershed.

In this study, the aging of culturable FIB and DNA representing genetic markers for Enterococcus spp. (ENT1A), general Bacteroides (GB3), and human-associated Bacteroides (HF183) in freshwater sediments was evaluated. Freshwater sediment was collected from four different sites within the upper and lower reach of the Topanga Creek Watershed and two additional comparator sites within the Santa Monica Bay, for a total of six sites. Untreated (ambient) and oven-dried (reduced microbiota) sediment was inoculated with 5% sewage and artificial freshwater. Microcosms were held for a 21-day period and sampled on day 0, 1, 3, 5, 7, 12, and 21. There were substantial differences in decay among the sediments tested, and decay rates were related to sediment characteristics. In the ambient sediments, smaller particle size and higher levels of organic matter and nutrients (nitrogen and phosphorus) were associated with increased persistence of the GB3 marker and culturable Escherichia coli (cEC) and enterococci (cENT). The HF183 marker exhibited decay rates of -0.50 to -0.96 day-1, which was 2-5 times faster in certain ambient sediments than decay of culturable FIB and the ENT1A and GB3 markers. The ENT1A and GB3 markers decayed at rates of between -0.07 and -0.28 and -0.10 to -0.44 day-1, and cEC and cENT decayed at rates of between -0.22 and -0.81 and -0.03 and -0.40 day-1, respectively. In the oven-dried sediments, increased persistence of all indicators and potential for limited growth of culturable FIB and the GB3 and ENT1A markers was observed. A simplified two-box model using the HF183 marker and cENT decay rates generated from the microcosm experiments was applied to two reaches within the Topanga Canyon watershed in order to provide context for the variability in decay rates observed. The model predicted lower ambient concentrations of enterococci in sediment in the upper (90 MPN g-1) versus lower Topanga watershed (530 MPN g-1) and low ambient levels of the HF183 marker (below the LLOQ) in sediments in both lower and upper watersheds. It is important to consider the variability in the persistence of genetic markers and FIB when evaluating indicators of fecal contamination in sediments, even within one watershed.

Disparate Antibiotic Resistance Gene Quantities Revealed across 4 Major Cities in California: A Survey in Drinking Water, Air, and Soil at 24 Public Parks.

Widespread prevalence of multidrug and pandrug-resistant bacteria has prompted substantial concern over the global dissemination of antibiotic resistance genes (ARGs). Environmental compartments can behave as genetic reservoirs and hotspots, wherein resistance genes can accumulate and be laterally transferred to clinically relevant pathogens. In this work, we explore the ARG copy quantities in three environmental media distributed across four cities in California and demonstrate that there exist city-to-city disparities in soil and drinking water ARGs. Statistically significant differences in ARGs were identified in soil, where differences in blaSHV gene copies were the most striking; the highest copy numbers were observed in Bakersfield (6.0 × 10-2 copies/16S-rRNA gene copies and 2.6 × 106 copies/g of soil), followed by San Diego (1.8 × 10-3 copies/16S-rRNA gene copies and 3.0 × 104 copies/g of soil), Fresno (1.8 × 10-5 copies/16S-rRNA gene copies and 8.5 × 102 copies/g of soil), and Los Angeles (5.8 × 10-6 copies/16S-rRNA gene copies and 5.6 × 102 copies/g of soil). In addition, ARG copy numbers in the air, water, and soil of each city are contextualized in relation to globally reported quantities and illustrate that individual genes are not necessarily predictors for the environmental resistome as a whole.

Long-term monitoring of molecular markers can distinguish different seasonal patterns of fecal indicating bacteria sources.

Elevated levels of fecal indicator bacteria (FIB) have been observed at Topanga Beach, CA, USA. To identify the FIB sources, a microbial source tracking study using a dog-, a gull- and two human-associated molecular markers was conducted at 10 sites over 21 months. Historical data suggest that episodic discharge from the lagoon at the mouth of Topanga Creek is the main source of bacteria to the beach. A decline in creek FIB/markers downstream from upper watershed development and a sharp increase in FIB/markers at the lagoon sites suggest sources are local to the lagoon. At the lagoon and beach, human markers are detected sporadically, dog marker peaks in abundance mid-winter, and gull marker is chronically elevated. Varied seasonal patterns of FIB and source markers were identified showing the importance of applying a suite of markers over long-term spatial and temporal sampling to identify a complex combination of sources of contamination.

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters.

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment.