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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.

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.

In recent years, the number of foodborne infections with non-O1 and non-O139 Vibrio cholerae (NOVC) has increased worldwide. These have ranged from sporadic infection cases to localized outbreaks. The majority of case reports describe self-limiting gastroenteritis. However, severe gastroenteritis and even cholera-like symptoms have also been described. All reported diarrheal cases can be traced back to the consumption of contaminated seafood. As climate change alters the habitats and distribution patterns of aquatic bacteria, there is a possibility that the number of infections and outbreaks caused by Vibrio spp. will further increase, especially in countries where raw or undercooked seafood is consumed or clean drinking water is lacking. Against this background, this review article focuses on a possible infection pathway and how NOVC can survive in the human host after oral ingestion, colonize intestinal epithelial cells, express virulence factors causing diarrhea, and is excreted by the human host to return to the environment.

Campylobacter infection is the most commonly notified bacterial enteritis in Germany. We performed a large combined case-control and source attribution study (Nov 2011-Feb 2014) to identify risk factors for sporadic intestinal Campylobacter infections and to determine the relative importance of various animal sources for human infections in Germany. We conducted multivariable logistic regression analysis to identify risk factors. Source attribution analysis was performed using the asymmetric island model based on MLST data of human and animal/food isolates. As animal sources we considered chicken, pig, pet dog or cat, cattle, and poultry other than chicken. Consumption of chicken meat and eating out were the most important risk factors for Campylobacter infections. Additional risk factors were preparation of poultry meat in the household; preparation of uncooked food and raw meat at the same time; contact with poultry animals; and the use of gastric acid inhibitors. The mean probability of human C. jejuni isolates to originate from chickens was highest (74%), whereas pigs were a negligible source for C. jejuni infections. Human C. coli isolates were likely to originate from chickens (56%) or from pigs (32%). Efforts need to be intensified along the food chain to reduce Campylobacter load, especially on chicken meat.

Background Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in both human and veterinary medicine, presenting significant challenges in treatment because of biofilm production and its intrinsic resistance. This problem is exacerbated by the increase in acquired antimicrobial resistance. Bacteriophage (phage) therapy has emerged as a promising alternative for treating infection classically treated with antibiotics, offering a targeted approach to combat this infection. This study aimed to evaluate the therapeutic potential of 7 phages, focusing on their suitability for treating canine infections, as well as their purification and safety analysis for therapeutic use. Results Two self-isolated phages and five provided phages were analysed. All tested phages reduced bacterial load in vitro; however, their efficacy varied across different concentrations. The host range analysis revealed a spectrum between 9.8 and 68.6% of canine clinical P. aeruginosa isolates. In our in vitro tests 3 out of 7 phages were able to significantly reduce the biofilm biomass, achieving reductions up to 93.38%. The sequence analysis did not discover known virulence factors and genes connected to antimicrobial resistance mechanisms. The self-isolated phages were classified as lysogenic, whereas the other phages had a lytic infection cycle. Through the purification of the phages, high-titre phage preparations (> 10 11 PFU/ml) were generated with high stability for at least 1.5 years. The tested endotoxin units are below the regulatory limits. Conclusion Investigating phages as alternative treatment option seems promising with lytic phages covering a broad host range and a genomic potential for biofilm degradation. These findings support the development of phage cocktails as a targeted alternative for treating canine P. aeruginosa infections, particularly in cases of antibiotic resistance, and highlight the importance of selecting well-characterized lytic phages for therapeutic efficacy and safety.

Background Campylobacter ( C. ) jejuni is the leading cause of human campylobacteriosis worldwide. We performed a molecular epidemiological study to investigate the genetic relationship among C. jejuni strains isolated from human diarrhoeal patients, broiler products and dairy cattle in Lithuania. Methods The C. jejuni isolates from human clinical cases, dairy cattle and broiler products were genotyped using multilocus sequence typing (MLST). Allele numbers for each housekeeping gene, sequence type (ST), and clonal complex (CC) were assigned by submitting the DNA sequences to the C. jejuni MLST database ( http://pubmlst.org/campylobacter ). Based on the obtained sequence data of the housekeeping genes a phylogenetic analysis of the strains was performed and a minimum spanning tree (MST) was calculated. Results Among the 262 C. jejuni strains (consisting of 43 strains isolated from dairy cattle, 102 strains isolated from broiler products and 117 clinical human C. jejuni strains), 82 different MLST sequence types and 22 clonal complexes were identified. Clonal complexes CC21 and CC353 predominated among the C. jejuni strains. On ST-level, five sequence types (ST-5, ST-21, ST-50, ST-464 and ST-6410) were dominating and these five STs accounted for 35.9% ( n  = 94) of our isolates. In addition, 51 (19.5%) C. jejuni strains representing 27 (32.9%) STs were reported for the first time in the PubMLST database ( http://pubmlst.org/campylobacter ). The highest Czekanowski index or proportional similarity index (PSI) was calculated for C. jejuni strains isolated from human campylobacteriosis cases and broiler products (PSI = 0.32) suggesting a strong link between broiler strains and human cases. The PSI of dairy cattle and human samples was lower (PSI = 0.11), suggesting a weaker link between bovine strains and human cases. The calculated Simpson’s index of all C. jejuni isolates showed a high genetic diversity (D = 0.96). Conclusion Our results suggest that broiler products are the most important source of human campylobacteriosis in Lithuania. The study provides information on MLST type distribution and genetic relatedness of C. jejuni strains from humans, broiler products and dairy cattle in Lithuania for the first time, enabling a better understanding of the transmission pathways of C. jejuni in this country.

Viable but non-culturable (VBNC) state is referred to as a dormant state of non-sporulating bacteria enhancing the survival in adverse environments. To our knowledge, only few studies have been conducted on whole genomic expression of Vibrio parahaemolyticus VBNC state. Since a degradation of nucleic acids in V. vulnificus non-culturable state has been detected, we hypothesize that gene regulation of VBNC cells is highly reduced, downregulation of gene expression is dominant and only metabolic functions crucial for survival are kept on a sustained basis. Hence, we performed the whole transcriptomic profiles of V. parahaemolyticus in three phases (exponential, early stationary phase and VBNC state). Compared with exponential and early stationary phase, in V. parahaemolyticus VBNC cells we found 509 induced genes and 309 repressed by more than 4-fold among 4820 investigated genes. Upregulation was dominant in most of non-metabolism functional categories, while five metabolism-related functional categories revealed downregulation in VBNC state. To our knowledge, this is the first study of comprehensive transcriptomic analyses of three phases of V. parahaemolyticus RIMD2210633. Although the mechanism of VBNC state is not yet clear, massive regulation of gene expression occurs in VBNC state compared with expression in other two phases, indicating VBNC cells are active. Our study shows whole genome expression data and explains beside biochemic impacts, why the viable but non-culturable state: usually so-called dormant state is actually a wrong term.

Human campylobacteriosis results from foodborne infections with Campylobacter bacteria such as Campylobacter jejuni and Campylobacter coli, and represents a leading cause of bacterial gastroenteritis worldwide. After consumption of contaminated poultry meat, constituting the major source of pathogenic transfer to humans, infected patients develop abdominal pain and diarrhea. Post-infectious disorders following acute enteritis may occur and affect the nervous system, the joints or the intestines. Immunocompromising comorbidities in infected patients favor bacteremia, leading to vascular inflammation and septicemia. Prevention of human infection is achieved by hygiene measures focusing on the reduction of pathogenic food contamination. Molecular targets for the treatment and prevention of campylobacteriosis include bacterial pathogenicity and virulence factors involved in motility, adhesion, invasion, oxygen detoxification, acid resistance and biofilm formation. This repertoire of intervention measures has recently been completed by drugs dampening the pro-inflammatory immune responses induced by the Campylobacter endotoxin lipo-oligosaccharide. Novel pharmaceutical strategies will combine anti-pathogenic and anti-inflammatory effects to reduce the risk of both anti-microbial resistance and post-infectious sequelae of acute enteritis. Novel strategies and actual trends in the combat of Campylobacter infections are presented in this review, alongside molecular targets applied for prevention and treatment strategies.

Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria. In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log10 unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log10 units. The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.

Background Arcobacter cryaerophilus is considered an emerging foodborne pathogen and is associated primarily with infectious gastrointestinal disease in humans. However, the underlying pathogenic mechanisms remain poorly understood. Therefore, the aim of the present study was to investigate the pathogenic potential of twelve A. cryaerophilus strains using various in vitro assays in two human colonic cell lines, HT-29/B6 and T84. Results All strains tested were able to adhere to and invade into both cell lines, with strain-dependent differences in their adhesion and invasion rates. In addition, two strains showed cytotoxic effects on both cell lines. The ability to disrupt the epithelial barrier function of T84 cell monolayers was shown for two strains by measurement of transepithelial electrical resistance. As structural factors correlate with the barrier dysfunction, immunofluorescence staining of the tight junction domain was performed, and revealed an altered distribution of claudin-5 in infected cells. Conclusions The results highlight the strain-dependent pathogenic mechanisms of A. cryaerophilus that may contribute to key symptoms such as diarrhoea. These findings also highlight the importance of further research into the pathogen A. cryaerophilus .