Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches.

Antibiotic-resistant bacteria represent an emerging global health problem and are frequently detected in riverine environments. Analyzing the occurrence of corresponding antibioticresistant genes in rivers is of public interest as it contributes towards understanding the origin and dissemination of these emerging microbial contaminants via surface water. This is critical for devising strategies to mitigate the spread of resistances in the environment. Concentrations of bla CTX-M antibiotic resistance genes were quantified weekly over a 12-month period in Lahn River surface water at two sampling sites using quantitative real-time PCR. Gene abundances were statistically assessed with regard to previously determined concentrations of fecal indicator organisms Escherichia coli, intestinal enterococci and somatic coliphages, as well as influential environmental factors. Similar seasonal patterns and strong positive correlations between fecal indicators and bla CTX-M genes indicated identical sources. Accordingly, linear regression analyses showed that bla CTX-M concentrations could largely be explained by fecal pollution. E. coli provided the best estimates (75% explained variance) at the upstream site, where proportions of bla CTX-M genes in relation to fecal indicator organisms were highest. At this site, rainfall proved to be more influential, hinting at surface runoff as an emission source. The level of agricultural impact increased from downstream to upstream, linking increasing bla CTX-M concentrations after rainfall events to the degree of agricultural land use. Exposure assessment revealed that even participants in non-swimming recreational activities were at risk of incidentally ingesting bla CTX-M genes and thus potentially antibiotic resistant bacteria. Considering that bla CTX-M genes are ubiquitous in Lahn River and participants in bathing and non-bathing water sports are at risk of exposure, results highlight the importance of microbial water quality monitoring with an emphasis on antibiotic resistance not only in designated bathing waters. Moreover, E. coli might serve as a suitable estimate for the presence of respective antibiotic resistant strains.

Global ocean warming results in an increase of infectious diseases including an elevated emergence of Vibrio spp. in Northern Europe. The European Centre for Disease Prevention and Control reported annual periods of high to very high risks of infection with Vibrio spp. during summer months along the North Sea and Baltic Sea coasts. Based on those facts, the risk of Vibrio infections associated with recreational bathing in European coastal waters increases. To obtain an overview of the seasonal and spatial distribution of potentially human pathogenic Vibrio spp. at German coasts, this study monitored V. cholerae, V. parahaemolyticus, and V. vulnificus at seven recreational bathing areas from 2017 to 2018, including the heat wave event in summer 2018. The study shows that all three Vibrio species occurred in water and sediment samples at all sampling sites. Temperature was shown to be the main driving factor of Vibrio abundance, whereas Vibrio community composition was mainly modulated by salinity. A species-specific rapid increase was observed at water temperatures above 10°C, reaching the highest detection numbers during the heat wave event with abundances of 4.5 log10 CFU+1/100 ml of seawater and 6.5 log10 CFU+1/100 g of sediment. Due to salinity, the dominant Vibrio species found in North Sea samples was V. parahaemolyticus, whereas V. vulnificus was predominantly detected in Baltic Sea samples. Most detections of V. cholerae were associated with estuarine samples from both seas. Vibrio spp. concentrations in sediments were up to three log higher compared to water samples, indicating that sediments are an important habitat for Vibrio spp. to persist in the environment. Antibiotic resistances were found against beta-lactam antibiotics (ampicillin 31%, cefazolin 36%, and oxacillin and penicillin 100%) and trimethoprim-sulfamethoxazole (45%). Moreover, isolates harboring pathogenicity-associated genes such as trh for V. parahaemolyticus as well as vcg, cap/wcv, and the 16S rRNA-type B variant for V. vulnificus were detected. All sampled V. cholerae isolates were identified as non-toxigenic non-O1/non-O139 serotypes. To sum up, increasing water temperatures at German North Sea and Baltic Sea coasts provoke elevated Vibrio numbers and encourage human recreational water activities, resulting in increased exposure rates. Owing to a moderate Baltic Sea salinity, the risk of V. vulnificus infections is of particular concern.

Vibrio parahaemolyticus is a major cause of foodborne diseases and a significant threat to human health worldwide. Most of the infections caused by V. parahaemolyticus are usually associated with the consumption of raw or undercooked seafood. This study was conducted to determine the prevalence, quantitative load, and antimicrobial resistance of V. parahaemolyticus in retail seafood in Hanoi, Vietnam. A total of 120 seafood samples consisting of marine fish (n = 30), oysters (n = 30), shrimp (n = 30), and squid (n = 30) were purchased from different traditional markets in Hanoi between May and October 2020. Isolation of V. parahaemolyticus was based on ISO/TS 21872-1:2017, and the most-probable-number (MPN) method was used for quantification. The disk diffusion method was applied for antimicrobial susceptibility testing. Overall, V. parahaemolyticus was detected in 58.33% of the samples. V. parahaemolyticus was most commonly isolated in shrimp samples, with a prevalence of 86.67%, followed by fish (53.33%), squid (53.33%), and oysters (40%). One V. parahaemolyticus isolate from an oyster carrying the trh gene was detected. Of the positive samples, 27.14% contained V. parahaemolyticus counts of less than 2 log MPN/g, whereas 44.29% ranged from 2 to 4 log MPN/g and 28.57% contained more than 4 log MPN/g. Regarding antimicrobial resistance, 85.71% of V. parahaemolyticus isolates were resistant to at least one antibiotic tested. The highest rate of resistance was observed against ampicillin (81.43%), followed by cefotaxime (11.43%), ceftazidime (11.43%), trimethoprim-sulfamethoxazole (8.57%), and tetracycline (2.86%). The results demonstrate the high prevalence and quantitative load and the antimicrobial resistance of V. parahaemolyticus isolated from seafood sold in the study area. HIGHLIGHTS