Dynamics of integron structures across a wastewater network - Implications to resistance gene transfer.

Class 1 and other integrons are common in wastewater networks, often being associated with antibiotic resistance genes (ARGs). However, the importance of different integron structures in ARG transfer within wastewater systems has only been implied, especially between community and hospital sources, among wastewater treatment plant compartments, and in receiving waters. This uncertainty is partly because current clinical class 1 integron qPCR assays (i.e., that target human-impacted structures, i.e., clintI1) poorly delineate clintI1 from non-impacted class 1 integron structures. They also say nothing about their ARG content. To fill these technical gaps, new real-time qPCR assays were developed for "impacted" class 1 structures (called aint1; i.e., anthropogenic class 1 integrons) and empty aint1 structures (i.e., carry no ARGs; called eaint1). The new assays and other integron assays then were used to examine integron dynamics across a wastewater network. 16S metagenomic sequencing also was performed to characterise associated microbiomes. aint1 abundances per bacterial cell were about 10 times greater in hospital wastewaters compared with other compartments, suggesting aint1 enrichment with ARGs in hospital sources. Conversely, the relative abundance of eaint1 structures were over double in recycled activated sludge compared with other compartments, except receiving waters (RAS; ∼30% of RAS class 1 structures did not carry ARGs). Microbiome analysis showed that human-associated bacterial taxa with mobile integrons also differed in RAS and river sediments. Further, class 1 integrons in RAS bacteria appear to have released ARGs, whereas hospital bacteria have accumulated ARGs. Results show that quantifying integron dynamics can help explain where ARG transfer occurs in wastewater networks, and should be considered in future studies on antibiotic resistance in the environment.

Spatial ecology of a wastewater network defines the antibiotic resistance genes in downstream receiving waters.

Wastewater treatment plants (WWTPs) are an effective barrier in the protection of human and environment health around the world, although WWTPs also are suggested to be selectors and-or reservoirs of antibiotic resistance genes (ARGs) before entering the environment. The dogma about WWTPs as "ARG selectors" presumes that biotreatment compartments (e.g., activated sludge; AS) are single densely populated ecosystems with elevated horizontal gene transfer. However, recent work has suggested WWTP biotreatment compartments may be different than previously believed relative to antibiotic resistance (AR) fate, and other process factors, such as bacterial separation and specific waste sources, may be key to ARGs released to the environment. Here we combined 16S rRNA metagenomic sequencing and high-throughput qPCR to characterise microbial communities and ARGs across a wastewater network in Spain that includes both community (i.e., non-clinical urban) and hospital sources. Contrary to expectations, ARGs found in downstream receiving waters were not dominated by AS biosolids (RAS), but more resembled raw wastewater sources. In fact, ARGs and microbial communities in liquid-phase WWTP effluents and RAS were significantly different (Bray-Curtis dissimilarity index = 0.66 ±â€¯0.11), with a consequential fraction of influent ARGs and organisms passing directly through the WWTP with limited association with RAS. Instead, ARGs and organisms in the RAS may be more defined by biosolids separation and biophysical traits, such as flocculation, rather than ARG carriage. This explains why RAS has significantly lower ARG richness (47 ±â€¯4 ARGs) than liquid-phase effluents (104 ±â€¯5 ARGs), and downstream water column (135 ±â€¯4 ARGs) and river sediments (120 ±â€¯5 ARGs) (Tukey's test, p < 0.001). These data suggest RAS and liquid-phase WWTP effluents may reflect two parallel ecosystems with potentially limited ARG exchange. As such, ARG mitigation in WWTPs should more focus on removing bacterial hosts from the liquid phase, AR source reduction, and possibly disinfection to reduce ARG releases to the environment.

Assessment of food safety using a new real-time PCR assay for detection and quantification of virulence factors of enterococci in food samples.

AIMS: Development of Taqman MGB real-time PCR (q-PCR) assays for the quantitative detection of virulence factor genes in pure culture and food samples with regard to food safety assessment. METHODS AND RESULTS: New Taqman primers and probes were designed for the ace, esp and gelE genes based on the determinants of virulence profiles of enterococcal strains from GenBank. The high specificity and accuracy of the Taqman probe assay was confirmed. The limit of detection for the different virulence genes was 102  CFU ml-1 or CFU g-1 for pure culture and meat samples, and 103  CFU g-1 for cheese samples. CONCLUSION: This method provides the specific and rapid detection and quantification of ace, esp and gelE genes compared to conventional PCR assays, thus allowing the rapid and direct safety assessment of Enterococcus genus in food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents efficient methods that can be used directly on food products for the rapid quantification and tracing of virulence genes, regarding food safety assessment. Moreover, this is the first study to quantify these virulence factors using a specific Taqman q-PCR assay in food samples.

Development of a real-time PCR assay for direct detection and quantification of Bacillus sporothermodurans in ultra-high temperature milk.

In our study, a new and highly sensitive real-time PCR Taqman assay was developed for the direct and specific detection of Bacillus sporothermodurans in UHT milk. The target region was selected based on the 16S rRNA gene profiles of 11 B. sporothermodurans from GenBank. A standard curve was created using a reference strain of B. sporothermodurans, DSM 10599. A low limit of detection for B. sporothermodurans in UHT milk (10 cfu/mL) was obtained. Furthermore, a total of 110 UHT milk samples from several supermarkets were directly assessed to detect and quantify B. sporothermodurans using the real-time PCR Taqman developed. The B. sporothermodurans counts obtained were highly correlated with the microbial plate counts in the UHT milk samples. This is the first time that B. sporothermodurans has been quantified directly from UHT milk. This technique could be applicable as a routine tool for preventing the growth of these bacteria by allowing for the rapid screening of raw milk samples in dairy plants. As expected, the probability of bacterial growth in UHT milk packages increased with the B. sporothermodurans counts in the raw milk.