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Aims To investigate enterococci carrying linezolid and vancomycin resistance genes from fecal samples recovered from wild boars Methods and results Florfenicol- and vancomycin-resistant enterococci, isolated on selective agar plates, were screened by PCR for the presence of linezolid and vancomycin resistance genes. Five isolates carried optrA or poxtA linezolid resistance genes; one strain was resistant to vancomycin for the presence of vanA gene. All isolates were tested for their antibiotic susceptibility and subjected to Whole Genome Sequencing (WGS) analysis. In Enterococcus faecalis (E. faecalis) V1344 and V1676, the optrA was located on the new pV1344-optrA and pV1676-optrA plasmids, respectively, whereas in Enterococcus faecium (E. faecium) V1339 this gene was on a 22 354-bp chromosomal genetic context identical to the one detected in a human E. faecium isolate. In both E. faecium V1682 and E. durans V1343, poxtA was on the p1818-c plasmid previously found in a human E. faecium isolate. In E. faecium V1328, the vanA gene was on the Tn 1546 transposon in turn located on a new pV1328-vanA plasmid. Only E. faecium V1682 successfully transferred the poxtA gene to an enterococcal recipient in filter mating assays. Conclusions The occurrence of genetic elements carrying linezolid and vancomycin resistance genes in enterococci from wild boars is a matter of concern, moreover, the sharing of plasmids and transposons between isolates from wild animals, human, and environment indicates an exchange of genetic material between these settings. [ABSTRACT FROM AUTHOR]

Aims Enterocins K1 and EJ97 have specific antimicrobial activity against Enterococcus faecium and Enterococcus faecalis , respectively. The aim of this study was to investigate the utility of these enterocins for in vivo treatment of systemic enterococcal infections. Methods and results The antimicrobial effect in blood was analysed and compared against the effect in saline. Colony forming unit counts revealed that the enterocins killed all the bacteria within 1 hour. Additionally, the bactericidal effect against E. faecalis was more rapid in blood, indicating a possible synergy between EntEJ97 and blood. Importantly, no enterocin resistant mutants emerged in these experiments. Injecting the enterocins intraperitoneally in an in vivo mouse model and using fluorescence and minimum inhibitory concentration determination to estimate concentrations of the peptides in plasma, indicate that the enterocins exist in circulation in therapeutic concentrations. Alanine aminotransferase detection, and haemolysis analysis indicates that there is no detectable liver damage or haemolytic effect after injection. Conclusions The study revealed that EntK1 and EntEJ97 are able to kill all bacteria ex vivo in the presence of blood. In vivo experiments determine that the enterocins exist in circulation in therapeutic concentrations without causing liver damage or haemolysis. Future experiments should test these peptides for treatment of infection in a relevant in vivo model. [ABSTRACT FROM AUTHOR]

Aim The poultry industry represents an important economic sector in Tunisia. This study aims to determine the antimicrobial resistance phenotypes and genotypes and virulence factors of enterococci collected from chicken caecum in Tunisia. Methods and results Forty-nine composite chicken caecum samples were recovered in 49 different Tunisian farms (December 2019–March 2020). Each composite sample corresponds to six individual caecum from each farm. Composite samples were plated on Slanetz–Bartley agar both supplemented (SB-Van) and not supplemented (SB) with vancomycin and isolates were identified by matrix-assisted laser desorption/ionization time-of-flight. Antibiotic resistance and virulence genes were tested by Polymerase Chain Reaction (PCR) and sequencing and multilocus-sequence-typing of selected enterococci was performed. One hundred sixty seven enterococci of six different species were recovered. Acquired linezolid resistance was detected in 6 enterococci of 4/49 samples (8.1%): (A) four optr A-carrying Enterococcus faecalis isolates assigned to ST792, ST478, and ST968 lineages; (B) two poxt A-carrying Enterococcus faecium assigned to ST2315 and new ST2330. Plasmid typing highlighted the presence of the rep 10, rep 14, rep 7, rep 8, and pLG1 in these strains. One vancomycin-resistant E. faecium isolate (typed as ST1091) with van A gene (included in Tn 1546) was detected in SB-Van plates. The gelE, agg, esp , and hyl virulence genes were found in linezolid- and vancomycin-resistant enterococci. High resistance rates were identified in the enterococci recovered in SB plates: tetracycline [74.8%, tet (M) and tet (L) genes], erythromycin [65.9%, erm (B)], and gentamicin [37.1%, aac(6′)-Ie–aph(2″)-Ia ]. Conclusion The detection of emerging mechanisms of resistance related to linezolid and vancomycin in the fecal enterococci of poultry farms has public health implications, and further surveillance should be carried out to control their dissemination by the food chain. [ABSTRACT FROM AUTHOR]

Aim The purpose of this study is to assess the in vitro antimicrobial and anti-biofilm effects of the anti-protozoal agent tafenoquine (TAF) on Enterococcus and elucidate its underlying mode of action. Methods and results The present work investigated the susceptibility of TAF on 3 type strains and 11 clinical isolates of enterococci. The results indicated that TAF exhibited powerful antimicrobial activity against both of Enterococcus faecalis and Enterococcus faecium with minimum inhibitory and bactericidal concentrations ranging from 8 to 16 µg ml−1. Meanwhile, biofilm inhibition and eradication assays showed that TAF exhibited potent anti-biofilm activity against E. faecalis ATCC 29212 and E. faecium ATCC 19434. Ultra-microscopic observations revealed significant changes in bacterial morphology and structure caused by TAF, particularly for the disruption of plasma membrane. Mechanistic investigations also revealed that TAF altered both membrane permeability and potential while also impacting adenosine triphosphate production as well as reactive oxygen species generation. In addition, no detectable cytotoxicity of TAF on human cells was observed at concentrations near the minimal inhibitory concentration. Conclusions In summary, this study confirmed that TAF could effectively inhibit Enterococcus as well as its biofilm formation. [ABSTRACT FROM AUTHOR]

Aims This study aimed to evaluate the probiotic properties of Enterococcus strains isolated from Turkish traditional cheeses. Methods and results Fifty-two Enterococcus spp. were taxonomically determined as follows: Enterococcus faecium (26), Enterococcus faecalis (18), Enterococcus durans (6), and Enterococcus italicus (2). The ability of isolates/strains to survive the harsh conditions (acidity and in-vitro gastric solution) of the gastrointestinal tract was established. They also showed auto-aggregation, hydrophobicity, and co-aggregation ability. Hydrophobicities of the strains were found between 0.8%–21%, 0.7%–56%, and 2%–63% for xylene, chloroform, and ethyl acetate, respectively. Autoaggregation values of the Enterococcus strains were 4%–20%, 7%–30%, and 36%–98% after 2, 4, and 24-h incubation, respectively. In this study, the Enterococcus strains tested showed co-aggregation ability with the Escherichia coli ATCC 25922, Salmonella Typhimurium ATCC 14028, and Staphylococcus aureus ATCC 25923. The results of PCR amplification revealed that only five strains possess virulence factor genes (gelE,asa1,cyl A,esp). We determined antibiotic resistance, biofilm forming abilities, and hemolytic activity for safety evaluation of strains. Conclusions In this large and comprehensive study, we found that only few of Enterococcus strains have promising probiotic potential, among which E. faecalis ES1 and E. faecium EM1 showed the best probiotic properties (are the most promising probiotic candidates). [ABSTRACT FROM AUTHOR]

Aims: The objective of this study was to evaluate the inhibitory activity of compounds secreted by bacteria isolated from a hydrogen‐producing bioreactor to understand how these microorganisms interact in this community. Methods and Results: In vitro inhibitory assays were performed using samples secreted by bacteria subject to different treatments to determine if their inhibitory effect was due to organic acids, non‐proteinaceous compounds or bacteriocin‐like inhibitory substances (BLIS). Bacterial isolated were suppressed 43%, 30% and 27% by neutralized, precipitated and non‐neutralized cell‐free supernatants, respectively. Non‐hydrogen producers (non‐H2P) lactic acid bacteria (LAB) (Lactobacillus plantarum LB1, Lactobacillus pentosus LB7, Pediococcus acidilactici LB4) and hydrogen producers (H2P) LAB (Enterococcus faecium F) were inhibited by the production of organic acids, non‐proteinaceous compounds and BLIS. Meanwhile, the obligate anaerobe H2P (Clostridium beijerinckii B) inhibited by the production of non‐proteinaceous compounds and BLIS. The presence of BLIS was confirmed when proteolytic enzymes affected the inhibitory activity of secreted proteins in values ranging from 20% to 42%. The BLIS produced by L. plantarum LB1, P. acidilactici LB4, L. pentosus LB7 and E. faecium F showed molecular masses of ~11, 25, 20 and 11 kDa, respectively. Conclusions: It was demonstrated antagonistic interactions between Lactobacillus–Enterococcus and Pediococcus–Enterococcus species, generated by the secretion of organic acids, non‐proteinaceous compounds and BLIS. Significance and Impact of the Study: We report the interactions between LAB isolated from hydrogen‐producing bioreactors. These interactions might impact the dynamics of the microbial population during hydrogen generation. Our work lays a foundation for strategies that allow controlling bacteria that can affect hydrogen production. [ABSTRACT FROM AUTHOR]

Aims: Our objective was to determine how injectable antimicrobials affected populations of Salmonella enterica, Escherichia coli and Enterococcus spp. in feedlot cattle. Methods and Results: Two arrival date blocks of high‐risk crossbred beef cattle (n = 249; mean BW = 244 kg) were randomly assigned one of four antimicrobial treatments administered on day 0: sterile saline control (CON), tulathromycin (TUL), ceftiofur (CEF) or florfenicol (FLR). Faecal samples were collected on days 0, 28, 56, 112, 182 and study end (day 252 for block 1 and day 242 for block 2). Hide swabs and subiliac lymph nodes were collected the day before and the day of harvest. Samples were cultured for antimicrobial‐resistant Salmonella, Escherichia coli and Enterococcus spp. The effect of treatment varied by day across all targeted bacterial populations (p ≤ 0.01) except total E. coli. Total E. coli counts were greatest on days 112, 182 and study end (p ≤ 0.01). Tulathromycin resulted in greater counts and prevalence of Salmonella from faeces than CON at study end (p ≤ 0.01). Tulathromycin and CEF yielded greater Salmonella hide prevalence and greater counts of 128ERYRE. coli at study end than CON (p ≤ 0.01). No faecal Salmonella resistant to tetracyclines or third‐generation cephalosporins were detected. Ceftiofur was associated with greater counts of 8ERYREnterococcus spp. at study end (p ≤ 0.03). By the day before harvest, antimicrobial use did not increase prevalence or counts for all other bacterial populations compared with CON (p ≥ 0.13). Conclusions: Antimicrobial resistance (AMR) in feedlot cattle is not caused solely by using a metaphylactic antimicrobial on arrival, but more likely a multitude of environmental and management factors. [ABSTRACT FROM AUTHOR]

Aims: L‐tryptophan is an essential aromatic amino acid for the growth and development of animals. Studies about enteric L‐tryptophan‐producing bacteria are scarce. In this report, we characterized the probiotic potential of Enterococcus casseliflavus ECB140, focusing on its L‐tryptophan production abilities. Methods and results: ECB140 strain was isolated from the silkworm gut and can survive under strong alkaline environmental conditions. Bacterial colonization traits (motility and biofilm) were examined and showed that only ECB140 produced flagellum and strong biofilms compared with other Enterococcus strains. Comparative genome sequence analyses showed that only ECB140 possessed a complete route for L‐tryptophan synthesis among all 15 strains. High‐performance liquid chromatography and qRT‐PCR confirmed the capability of ECB140 to produce L‐tryptophan. Besides, the genome also contains the biosynthesis pathways of several other essential amino acids, such as phenylalanine, threonine, valine, leucine, isoleucine and lysine. These results indicate that ECB140 has the ability to survive passage through the gut and could act as a candidate probiotic. Conclusions: The study describes a novel, natural silkworm gut symbiont capable of producing L‐tryptophan. Enterococcus casseliflavus ECB140 physical and genomic attributes offer possibilities for its colonization and provide L‐tryptophan for lepidopteran insects. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the safety of irrigation water sources based on phenotypic antimicrobial resistance (AMR) in Enterococcus spp., a potential environmental reservoir for AMR determinants. Methods and Results: Eleven sites representing fresh and brackish water rivers, ponds and reclaimed water, were sampled over 2 years. Samples (n = 333) yielded 198 unique isolates of Ent. faecalis and Ent. faecium which were tested for antimicrobial susceptibility by microbroth dilution. Species distribution was influenced by water type and season. Enterococcus faecalis was more likely found in freshwater rivers and in summer, and Ent. faecium in reclaimed water and in spring. Only 11% of isolates were pansusceptible, while 48.5% and 26.3% were single (SDR) and multidrug resistant (MDR), respectively. MDR was more likely detected in Ent. faecium than Ent. faecalis. Winter isolates were more likely than summer isolates to exhibit MDR than SDR. Conclusions: Enterococcus faecalis and Ent. faecium in surface and reclaimed water exhibited diverse phenotypic AMR and a low‐level resistance to clinically important antimicrobials such as ampicillin, vancomycin and linezolid. Significance and Impact of the Study: Single and multidrug resistance in E. faecalis and E. faecium varied by season but not water type. Antimicrobial resistance prevalence can assist decisions on the safety of irrigation water sources for fresh produce crops. [ABSTRACT FROM AUTHOR]

Aims: To study the local intestinal lymphocyte immunity in mice with trichinellosis affected by probiotic bacteria. Methods and Results: Enterococcus faecium CCM8558, Enterococcus durans ED26E/7, Limosilactobacillus fermentum CCM7421 and Lactiplantibacillus plantarum 17 L/1 were administered daily (109 CFU ml−1) and mice were infected with Trichinella spiralis (400 larvae) on the 7th day of treatment. T. spiralis infection significantly inhibited lymphocyte subpopulations from 5 to 25 days postinfection (dpi). L. fermentum CCM7421 and L. plantarum 17 L/1 restored the CD4+ T cell numbers in the epithelium and lamina propria at the control level from 11 dpi. All strains stimulated the CD8+ T cells numbers in infected mice, which were restored in the lamina propria on 11 dpi and in the epithelium only on 32 dpi. B cells (CD19+) inhibition after T. spiralis infection was not affected by treatment till 25 dpi. Conclusions: The strain‐specific immunomodulatory effect of tested bacteria was confirmed. L. fermentum CCM7421 and L. plantarum 17 L/1 showed the greatest immunomodulatory potential on CD4+ and CD8+ T lymphocytes in trichinellosis. E. faecium CCM8558 and E. durans ED26E/7 activated only CD8+ T cells in the lamina propria. Significance and Impact of the Study: Positive modulation of the gut lymphocyte immunity in T. spiralis infection with bacterial strains showed their beneficial effect with the host′s antiparasitic defence. Significance and Impact of Study: Positive modulation of the gut lymphocyte immunity in T. spiralis infection with bacterial strains showed their beneficial effect with the host′s antiparasitic defence. [ABSTRACT FROM AUTHOR]

Aims: Childcare facilities act as microenvironments that facilitate and promote the selection, spread and transmission of antibiotic‐resistant micro‐organisms in the community. We focused on the study of antimicrobial resistance and genetic predispositions for β‐lactamase production in bacterial isolates from nursery teachers' clothing. Methods and Results: Antimicrobial resistance of bacterial strains belonging to Enterobacteriaceae, Enterococcus, Staphylococcus spp., Pseudomonas spp. and Bacillus spp. isolated from 80 samples of nursery teachers' clothing was determined. The selected ESβL genes were found in 30 (44.1%) of 68 strains examined. The CTX‐M type ESβL determinants were detected in 15.4%, 71.5% and 42.5% of the Enterobacteriaceae, Pseudomonas and Bacillus isolates, respectively. The OXA‐type coding genes were detected only in strains of the genera Pseudomonas (57.1%) and Bacillus (48.6%). Thus, most B. cereus strains were sensitive to the recommended antibiotics used to treat infections caused by these bacteria. Methicillin resistance was phenotypically confirmed in 27 (14.6%) of 185 staphylococcal isolates. Four isolates (2.2%) were identified as MRSA. Vancomycin resistance was not observed in any of the staphylococcal and enterococci strains. Conclusions: This study has shown that potential pathogens have been isolated from the clothing of nursery teachers, posing a risk of transmission to children. These clothes should be maintained and properly laundered to avoid cross‐contamination and the spread of multidrug‐resistant (MDR) bacteria in childcare centres. Significance and impact of the study: This study provides insight into the route of transmission of MDR micro‐organisms through the clothing of nursery teachers, to which greater importance should be given in the future. Proper procedures for the cleaning and use of clothing in daycare centres should be clarified and standardized. [ABSTRACT FROM AUTHOR]

Aims: Worldwide, studies regarding antimicrobial resistance in rabbits are scarce. In addition, it seems that rearing conditions have important impact on emergence and spread of antimicrobial‐resistant bacteria. Thus, the authors sought to (1) assess the role of rabbits residing across diverse ecosystems as potential reservoirs of antimicrobial‐resistant enterococci and (2) investigate the genetic background of detected resistances. Methods and Results: Faecal samples from 60 healthy farmed rabbits (one farm), 35 laboratory rabbits and 31 wild rabbits were analysed. Overall, 97 enterococci isolates were accumulated, as follows: 44 E. faecium, 37 E. faecalis, 7 E. gallinarum, 5 E. durans and 4 E. avium. E. faecalis isolates were statistically associated with farm rabbits and wild rabbits (p < 0.05). High rates of resistance were observed for tetracycline (60.8%; tetM [n = 48; 81.3%], tetO [n = 7; 11.8%] and tetL [n = 1; 1.7%]), erythromycin (43.3%; msr(A) [n = 14; 33.3%] and ermB [n = 13; 31%]), ampicillin (29.9%), streptomycin (26.8%; ant(6)‐Ia [n = 3, 11.5%]) and vancomycin (21.6%; vanA [one E. faecium + one E. faecalis; 9.5%]). Low frequencies of resistance were observed for teicoplanin (9.2%), linezolid (8.2%), ciprofloxacin (7.2%) and gentamicin (1%; aac(6′)‐Ie‐aph(2″)‐Ia). Resistance to ampicillin and vancomycin was associated with laboratory rabbits (p < 0.05). Int‐Tn (Tn916/1545) was detected in 27 (27.8%) isolates, of which 10 isolates co‐harboured tetM and ermB genes, while 16 comprised tetM. Conclusion: Findings indicate that clinically relevant enterococci species isolated from rabbits are frequently resistant to antimicrobials and harbour a range of genes associated with the Tn916/1545 family. Significance and Impact of the study: This study highlights the high rates of antimicrobial‐resistant enterococci from rabbits and the occurrence of both vancomycin‐ and linezolid‐resistant isolates, potentially representing a very serious threat to human and animal health. [ABSTRACT FROM AUTHOR]

Aims: The study aimed to investigate the prevalence and conservation of endocarditis and biofilm‐associated pili (ebp) genes in Enterococcus faecalis originated from animals and the potential of developing Ebp into serological diagnostic and vaccine targets. Methods and Results: In this work, we investigated the prevalence and conservation of ebp genes in 116 strains of E. faecalis originated from animals by using PCR and sequencing methods. The results demonstrated the presence of ebp genes (ebpA, ebpB and ebpC) in all 116 strains of E. faecalis, and their amino acid homology ranges from 96.6% to 100.0%. Moreover, the phylogenetic analysis of ebp genes in all 164 E. faecalis strains (including 48 reference strains) revealed that ebp genes show no significant correlation with species origins and regions of E. faecalis, indicating that ebp genes are conserved features in E. faecalis, even though it evolved under environmental pressures from various regions and origins. Given that EbpA1 as a part of the adhesion protein EbpA has immunogenicity, we further determined whether amino acid mutations have effects on the function and 3D structure of EbpA1. The results showed that two of the 26 mutations, at amino acids positions 178 and 387, had deleterious effects on the biological function of EbpA1 protein, while all mutations had no effect on the 3D structure or binding pockets of EbpA1 protein. Conclusions: This study suggests that ebp genes are prevalent and conserved in E. faecalis originated from diverse animal origins and regions. EbpA1 could be a potential target for serological diagnosis and vaccine development to prevent E. faecalis infection. Significance and Impact of Study: The current study provides data to support further research on Ebp as a serological diagnostic and vaccine target against E. faecalis infection. [ABSTRACT FROM AUTHOR]

Aims: Enterococci are implicated in hospital‐acquired infections and show high tenacity on inanimate objects in the hospital environment. This study investigated the prevalence of Enterococcus spp. in selected wards in public hospitals at four levels of healthcare from a district in KwaZulu‐Natal, South Africa. Methods and results: Swabs were collected from frequently touched areas in the paediatric wards and intensive care units (ICUs). Presumptive Enterococcus spp. were isolated and confirmed to genus and species levels, followed by Kirby‐Bauer disk diffusion against 14 antibiotics. The results showed that enterococci were recovered from all 11 surfaces tested with the highest contamination rate observed on occupied beds and mops used to clean floors. A total number of 295 Enterococcus was identified. Polymerase chain reaction identified Enterococcus faecalis 83.1% (245/295) and Enterococcus faecium 12.9% (38/295), while whole genome sequencing identified Enterococcus gallinarum 2.0% (6/295) and Enterococcus casseliflavus 2.0% (6/295). Significant prevalence was observed in paediatric wards 64.1% (189/295) compared with the ICUs 35.9% (106/295), p < 0.05, in central, regional and district hospitals. Collectively, 82.0% (242/295) of enterococcal isolates were multidrug resistant, and 80 different antibiograms were observed. The most prominent antibiogram for E. faecium was CIP‐RIF‐NIT‐TET‐ERY and for E. faecalis CIP‐TET‐ERY. Conclusion: E. faecalis was the most frequent enterococcal species isolated in all the hospitals investigated and correlates with studies conducted elsewhere. A substantially greater number of isolates were recovered from the paediatric wards compared with ICUs, and thus improved standards should be developed for infection control practices. It is suggested that the elevated use of antibiotics contributed to the increased nonsusceptible isolates observed from ICUs. This study highlighted the high recovery rate of enterococci in the hospital environment even in a nonoutbreak setting. Significance and impact of the study: Enterocci had a high prevalence rate on the surfaces within the hospitals studied. This study gives an insight into the possible roles all healthcare staff may play in infection control intervention, including proper handling of hospital cleaning equipment and lack of knowledge about the potential for bacteria dissemination. [ABSTRACT FROM AUTHOR]

Aims: The objective of this study was to isolate multifunctional bacteriocin‐producing strains; to characterize the expressed bacteriocin for the control of Listeria monocytogenes and vancomycin‐resistant Enterococcus; to evaluate the safety of studied strains; and to explore their antifungal activity. Methods and Results: Two Pediococcus strains were isolated from silage samples obtained from an organic farm in Belogradchik, Bulgaria. The strains were identified by 16S rRNA sequencing analysis and characterized as bacteriocins producers. Strong antimicrobial activity was detected against more than 74 different strains of Listeria monocytogenes, 27 different vancomycin‐resistant Enterococcus strains. In addition, studied strains were able to inhibit the growth of strains of Alternaria alternate, Aspergillus flavus, Aspergillus niger, Cladosporium sphaerospermum, Penicillium chrysogenum and Penicillium expansum. Some aspects of the antimicrobial mode of action were evaluated, including killing curves and aggregation properties. Both strains generated positive PCR results for the presence of pediocin PA‐1, but not for other bacteriocins evaluated in this screening process. Metabolomic analysis of the cell‐free supernatants from both strains was performed in order to explain the observed antifungal activity against different moulds. According to PCA and PLS‐DA score plot, P. acidilactici ST3522BG and P. pentosaceus ST3633BG were clearly clustered from control (MRS). Increases in the production of benzoic acid, 2‐hydroxyisocaproic acid, β‐phenyl‐lactic acid, α‐hydroxybutyric acid and 1,3‐butanediol were recorded, these metabolites were previously described as antifungal. Conclusions: Pediococcus acidilactici ST3522BG and P. pentosaceus ST3633BG were evaluated as producing bacteriocin strains with high specificity against Listeria and vancomycin‐resistant Enterococcus species. In addition, both investigated Pediococcus strains were evaluated as producer of effective antifungal metabolites with potential for the inhibition of mycotoxin‐producing moulds. Significance and Impact of the Study: To the best of our knowledge, this report is a pioneer in the evaluation of Pediococcus strains isolated from silage with highly specific bacteriocinogenic antimicrobial activity against Listeria spp. and vancomycin‐resistant Enterococcus spp., and antifungal activity against mycotoxin‐producing moulds. [ABSTRACT FROM AUTHOR]

Introduction: Nanoparticles (NPs) have been widely studied as an alternative to antibiotic use due to their antimicrobial properties at lower concentrations. Enterococcus faecalis is a facultative Gram‐positive microorganism inhabiting the gastrointestinal tract of humans and animals. It can also be present in other environments such as the oral cavity, water, sewage, soil and food. Aims: We evaluated whether E. faecalis could develop resistance to silver NPs (AgNPs) after exposure to sublethal concentrations of the NPs. Methods and Results: Proteomic analyses revealed that different pathways were activated during the acquired resistance under sublethal concentrations, and selected genes were validated by qPCR. Conclusions: The results of this study showed that E. faecalis is capable of generating resistance to AgNPs. Significance and Impact of the Study: To avoid the generation of resistance against AgNPs, future use of these NPs should be combined with other NPs prepared with different metals to prevent the dissemination of resistant strains. [ABSTRACT FROM AUTHOR]

Aims: The streptococcal disease has been associated with serious mortality and significant global economic loss in the tilapia farming industry. The overall goal of this work was to test herbal hydrogels based on encapsulated Enterococcus faecium ABRIINW.N7 for potential probiotic anti‐microbial activity against Streptococcus iniae in red hybrid tilapia. Methods and Results: Abnormal behaviour, clinical signs, postinjection survival and histopathology (kidney, liver, eye and brain) were measured. Cumulative mortality of CON+, free cells, ALG and treatments (F1‐F7) was 30, 24, 22, 19, 17, 17, 16, 14, 14 and 12 out of 30 fish and the survival rates for E. faecium ABRIINW.N7 microencapsulated in an alginate‐BS blend with 0·5, 1, 1·5, 2, 2·5 and 3% fenugreek were 43, 43, 47, 53, 53 and 60%, respectively. After the incorporation of fenugreek with the alginate‐BS blend, there was an 8–21% increase in probiotic cell viability. Furthermore, the survival rate for the alginate‐BS blend with 2·5 and 3% fenugreek (F6 and F7) was significantly (P ≤ 0·05) higher than other blends. The highest encapsulation efficiency, viability in gastrointestinal conditions and during storage time and excellent antipathogenicity against S. iniae were observed in alginate‐BS +3% fenugreek formulation (F7). Conclusions: It is recommended that probiotic strains like E. faecium ABRIINW.N7 in combination with local herbal gums, such as BS and fenugreek plus alginate, can be used as a suitable scaffold and an ideal matrix for the encapsulation of probiotics. Significance and Impact of the Study: This study proposes models connecting process parameters, matrix structure and functionality. [ABSTRACT FROM AUTHOR]

Aims: N‐chlorotaurine (NCT) is a body‐own mild oxidizing antiseptic that can be applied topically as a well‐tolerated anti‐infective at many body sites. The objective of this study was to demonstrate its activity against representative nosocomial multidrug‐resistant bacteria. Methods and Results: The bactericidal activity of NCT was tested in quantitative killing assays against a panel of multiresistant Gram‐positive and Gram‐negative clinical isolates. N‐chlorotaurine (1%, 55 mmol l−1) reduced the number of CFU of strains of methicillin‐resistant Staphylococcus aureus, linezolid‐resistant Staphylococcus epidermidis, vancomycin‐resistant, and linezolid‐ and vancomycin‐resistant Enterococcus faecium, 3MRGN and 4MRGN Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae by at least 2 log10 steps after 15 min and completely or nearly to the detection limit after 30 min at pH 7·1 and 37°C. Conclusion: The activity of NCT against these clinical isolates is similar to that against non‐resistant ATCC strains and therefore not influenced by antibiotic resistance. This can be explained by the oxidizing and chlorinating mechanism of action of NCT, which leads to an attack of multiple targets in the microorganisms. Significance and Impact of the Study: The bactericidal spectrum of NCT is not restricted by resistance against antibiotics. Therefore, it can be used against resistant strains, too. [ABSTRACT FROM AUTHOR]

Aim: The objective of this study was to characterize lactic acid bacteria (LAB) from rabbits to be used as potential autochthonous probiotic. Methods and Results: Fifteen faecal samples were collected from wild and laboratory rabbits. One hundred and eight isolates were collected and tested for their inhibitory power against eight pathogenic bacteria. Among them, 43 Enterococcus isolates were able to inhibit at least one pathogen. Enterocine genes entA, entB and entP were detected in 14, 17 and 22 isolates, respectively. These isolates were tested for their antibiotic susceptibility and genes encoding virulence factors. Relevant phenotypes of antibiotic resistance were observed especially for ampicillin, vancomycin and linezolid. The following virulence genes were detected (number of positive isolates): hyl (5), esp (8), gelE (30), agg (2), ace (21), efa (6), CylLL/s (5), cob (26), cpd (32) and ccf (33). Five isolates were considered as safe and showed tolerance to both acid and bile salt. Conclusion: Bacteriocinogenic enterococci isolates from rabbits may show relevant resistance phenotypes and virulence factors. In addition, one Enterococcus durans isolate presents promising autochthonous probiotic candidate. Significance and Impact of the study: This study reveals interesting properties for E. durans isolate and supports their utilization as autochthonous probiotic in rabbit husbandry. [ABSTRACT FROM AUTHOR]

Aims: The objective of this study was to isolate a bacteriocin‐producing strain and to characterize the expressed bacteriocin for the control of Listeria monocytogenes with aim of biopreservation application. Methods and Results: Soil samples from a Korean organic farm were subjected to microbiological analysis for isolation of potential bacteriocinogenic LAB, based on a three‐level approach, using L. monocytogenes ATCC 15313 as an indicator test micro‐organism. From a total of 17 isolates with inhibitory potential, seven were confirmed to be bacteriocin producers. The selected isolates were differentiated based on their morphology, catalase reaction, sugar fermentation profile obtained by API50CHL and by RAPD‐PCR generating two unique profiles. One of the isolates, ST110LD, a specific strong producer of anti‐Listeria bacteriocins (12 800 AU ml−1) was identified as Leuconostoc citreum. The proteinaceous nature of the inhibitory compound produced by Leuc. citreum ST110LD was confirmed through treatment with pepsin and α‐chymotrypsin. Bacteriocin activity was observed to be not affected by the presence of milk, NaCl, SDS, Tween 80 or glycerol. Bacteriocin ST110LD effectively inhibited the growth of exponentially growing L. monocytogenes ATCC 15313 during a 10‐h incubation period in BHI at 37°C. In addition, this bacteriocin showed specific inhibition of only Listeria spp., but did not inhibit the growth of beneficial cultures included in the microbial test panel for assessment of the spectrum of activity. Conclusions: Leuconostoc citreum ST110LD was evaluated as safe bacterium strain, producing bacteriocin with high specificity against listerial and enterococcal species. Specificity of producer strain and expressed bacteriocin can be explored in biopreservation of different fermented food products or applied in biotherapy of antibiotic resistant listerial or enterococcal infections. Significance and Impact of the Study: To the best of our knowledge, this is the first report of bacteriocin produced by Leuc. citreum strain with highly specific antimicrobial activity against Listeria sp. and Enterococcus sp. [ABSTRACT FROM AUTHOR]

Aims: Cellulase‐producing Enterococcus species isolated from the rumen of Tibetan yak (Bos grunniens) were characterized, and their combined effects on the silage quality of various forages were studied. Methods and Results: The two isolated strains with high cellulolytic potential were identified as Enterococcus faecalis (EF85) and E. faecium (EF83) by 16S rRNA sequencing. Both EF85 and EF83 could grow well at 15–55°C, pH 3·0–6·0 and in 3·0–6·5% NaCl. The cellulase secreted by EF85 and EF83 showed good stability at temperatures from 20 to 45°C and pH from 4·5 to 7·0. A commercial inoculant (CLP), a commercial cellulase (CE) and the two cellulolytic strains (EF85 + EF83) were added to whole‐crop corn, sweet sorghum and Napier grass ensiling for 120 days respectively. In Napier grass silage, all inoculants significantly increased lactic acid content and ratio of lactic to acetic acid and decreased pH, butyric acid and ammonia nitrogen contents. The acid detergent fibre and cellulose contents in EF85 + EF83 treatment were significantly lower than those in the other treatments. In whole‐crop corn and sweet sorghum silages, all additives had no significant effect on the fermentation quality, while CE and EF85 + EF83 markedly enhanced cellulose degradation and increased free sugar content. Conclusion: The combined inoculation of the cellulolytic strain EF85 and EF83 to various forages reduced the fibre content of the resulting silages. Significance and Impact of the Study: Few studies involved inoculation of silage with Enterococcus species in different forage types. The isolated cellulolytic strains of E. faecalis EF85 and E. faecium EF83 could be a great alternative for commercial inoculants and enzymes in silage production. [ABSTRACT FROM AUTHOR]

Aims: To better understand the persistence dynamics of enteropathogenic bacteria in freshwater wetland habitats, we constructed lab‐scale mesocosms planted with two different wetland plant species using a subsurface flow wetland design. Mesocosms were treated with either a high‐quality or a poor‐quality water source to examine the effects of water quality exposure and plant species on Escherichia coli, Salmonella spp. and Enterococcus spp. in the rhizoplane, rhizosphere and water of wetland habitats. Methods and Results: Quantities of study micro‐organisms were detected using real‐time PCR in wetland mesocosms. A combination of molecular and culture‐based methods was also used to enumerate these organisms from surface water and plant material at high, medium and poor water quality sites in the field. We found that all three enteropathogenic micro‐organisms were influenced by microhabitat type and plant species. Organisms differed with respect to their predominant microhabitat and the extent of persistence associated with wetland plant species in the mesocosm study. Of the monitored pathogens, only E. coli was influenced by both water quality treatment and plant species. Salmonella spp. quantities in the rhizoplane consistently increased in all treatments over the course of the mesocosm experiment. Conclusions: Plant species selection appears to be an overlooked aspect of constructed wetland design with respect to the removal of enteropathogenic micro‐organisms. Escherichia coli and Enterococcus concentrations in wetland outflow were significantly different between the two plant species tested, with Enterococcus concentrations being significantly higher in mesocosms planted with Phalaris arundinaceae and E. coli concentrations being higher in mesocosms planted with Veronica anagallis‐aquatica. Furthermore, there is evidence that the rhizoplane is a significant reservoir for Salmonella spp. within wetland habitats. Significance and Impact of the Study: This is the first time that Salmonella spp. has been shown to proliferate under natural conditions within the rhizoplane. This will contribute to our understanding of wetland removal mechanisms for enteropathogenic bacteria. This study identifies the rhizoplane as a potentially important reservoir for human pathogenic micro‐organisms and warrants additional study to establish whether this finding is applicable in non‐wetland habitats. [ABSTRACT FROM AUTHOR]

Aims: Our aim was to analyse the survival of Enterococcus cecorum (EC) at various temperatures, relative air humidities and on different substrates commonly existing in broiler houses. Methods and Results: A pathogenic EC isolate (EC14) was used to inoculate sterile litter, polyvinyl chloride (PVC) and dust samples. Incubation at 37, 25 or 15°C with either 32% relative humidity (RH) or 78% RH followed. At defined time points (0–4272 h post‐inoculation), samples were examined in triplicate for the total viable count. Selected combinations were repeated for a non‐pathogenic and two additional pathogenic EC strains. For EC14, the measured survival time ranged from 48 to 4272 h (178 days) depending on the substrate–humidity–temperature combination. The longevity was the highest on litter, followed by dust and then PVC. Lower temperatures facilitated its survival, lower relative air humidity favoured the survival only in combination with 25 or 15°C. All three pathogenic strains showed longer survival times (up to 432 h, 18 days) compared to the non‐pathogenic EC strain (168 h, 7 days) under the same conditions. Conclusions: Enterococcus cecorum demonstrates a high persistence in the environment especially at 15°C and 32% RH. Significance and Impact of the Study: Hygiene management plans should consider the durability of EC and the risk of a carry‐over to control consecutive EC outbreaks. [ABSTRACT FROM AUTHOR]

Aims: Attenuated Total Reflection Fourier Transform Infrared (ATR‐FT‐IR) Spectroscopy and chemometric modelling, including soft independent modelling by class analogy (SIMCA), partial least squares discriminant analysis (PLS‐DA) and support vector machine (SVM), were applied to attempt to discriminate 60 clinical isolates of Enterococcus faecium and Enterococcus faecalis and hence evaluate the performance of the spectroscopic approach in identifying enterococci infections. Methods and Results: The bacterial samples were identified by polymerize chain reaction (PCR) amplification and their ATR‐FT‐IR spectra acquired. Spectra were processed to the second derivative using the Savitzky–Golay algorithm and normalized using extended multiplicative signal correction employing the UnscramblerX (CAMO, Norway) software package. Multivariate classification models and their performance were evaluated using Cohen's Kappa coefficient. Principal component analysis (PCA) score plots showed separate clusters of spectra related to membership to E. faecium and E. faecalis, with this explained by bands assigned to PO2 (1230 cm−1), P‐O‐C (1114 cm−1), monosubstituted alkene (997, 987 cm−1) and C‐O (1070, 1055, 1036 cm−1) corresponding to teichoic acids, polysaccharides and peptidoglycan from the cell wall in PCA and PLS‐DA loading plots. The best classification model for E. faecium and E. faecalis is SVM, indicating via highest Kappa score. The classification coefficient between SIMCA, PLS‐DA, SVM and PCR as reference method were 0·59, 0·9 and 1, respectively, shown as the Kappa scores. Conclusions: The main spectral differences observed between the two clinically relevant enterococci species were associated with changes in the teichoic acid content of cell walls. With regard to the binary classification method, SVM was found to be the best performing classification model, providing the highest correlation with the PCR results. Significance and Impact of the Study: The study shows that ATR‐FT‐IR spectroscopy in combination with chemometric modelling can be applied for the phenotypic identification and discrimination of clinically relevant and similar enterococcal species. [ABSTRACT FROM AUTHOR]

Aims: This study was conducted to early detect the negative culture bacterial pathogens causing subclinical mastitis for the fast diagnosis of the disease and the reduction of some milk‐transmitted pathogenic bacteria to human consumers. Methods and Results: A total of 171 positive California mastitis test (CMT) milk samples collected from asymptomatic dairy cows in Sharkia Governorate, Egypt were examined by conventional bacteriological methods. The obtained results revealed that Streptococcus species (77·2%), followed by Staphylococcus species (48·6%) and Escherichia coli (25·7%) were the most predominant bacterial pathogens isolated from positive culture milk samples, whereas Enterobacter and Pseudomonas species were the lowest ones (1·2%, for each). Herein, 13 (7.6%) negative culture milk samples were subjected to propidium monoazide (PMA) conventional PCR assay, followed by DNA sequencing of purified PCR amplicons. Sequence analysis identified seven different types of negative culture bacterial pathogens comprising as following; 4 Enterococcus hirae, 2 Bacillus cereus, 2 Staphylococcus aureus, 1 Bacillus mycoides, 1 Bacillus subtilis, 1 Enterococcus faecium and 1 Escherichia coli. Conclusions: All the detected negative culture bacterial pathogens by PMA‐PCR assay, followed by DNA sequencing were incriminated in causing subclinical mastitis disease and had serious implications on human public health through consumption of milk contaminated with those recovered bacterial pathogens. Significance and Impact of the Study: The used methods could be useful in the routine detection of negative culture bacterial pathogens present in milk and consequently, it will help in the rapid diagnosis of subclinical mastitis disease and the reduction of many milk‐transmitted diseases to human. [ABSTRACT FROM AUTHOR]

Aim: The aim of the study was to characterize phenotypically and genotypically Enterococcus faecium strains collected from bovine mastitis milk and to evaluate one of them for its virulence in a murine mastitis model. Methods and Results: A total of five E. faecium isolates were collected from cows with subclinical mastitis. EF‐7A showed resistance to antibiotics tested, it presented alpha haemolysin and did not present gelatinase activity. It yielded cyA, efafm and gelE1 genes and it could be characterized as a moderate biofilm producer. It was able to internalize in MAC‐T cells and 1×108 colony forming unit ml‐1 was able to establish an intramammary infection in mice. The strain could be recovered from liver, kidney and blood samples. RAPD profiles showed different bands with respect to the inoculated strain. Histopathology analyses showed different grades of polymorphonuclear neutrophils infiltration in mammary glands. Conclusion: This is the first report that studied E. faecium strain in a lactating mouse model of mastitis and showed that the experimental inoculation was able to stimulate an inflammatory response resulting in mastitis. Results contribute to a better understanding of intramammary infections caused by E. faecium. Significance and Impact of the Study: This investigation shows that mice represent a valuable model for the study of the mastitis pathogenesis caused by E. faecium considering the high costs of using cows for mastitis research. [ABSTRACT FROM AUTHOR]

Aim: Infiltration experiments aimed to show the effectivity of coral sand to remove micro‐organisms in septic tank wastewater treatment system, in South Tarawa, Kiribati. Methods and Results: Laboratory experiments evaluated effective microbial removal properties of a packed bed of coral beach sand conditioned with domestic effluent. Bacterial and viral indicators Escherichia coli J6‐2, Enterococci faecalis and f‐RNA (MS2) bacteriophage, along with viral pathogens adenovirus, echovirus, norovirus and rotavirus, were dosed (at 106–107 CFU, PFU or genome copies per ml concentration) with effluent to unsaturated coral sand‐packed columns. For the conditions simulated, all organisms showed removal efficiencies of >4‐Log Removal Values (LRVs) (i.e. >99·99% effective reduction in number). Results revealed that the conditioned coral sand had a higher affinity for attenuating viruses than the bacteria tested. MS2 phage, adenovirus, echovirus, norovirus and rotavirus were absent in leachate from the base of the sand columns. E. coli J6‐2 and E. faecalis were recovered at 100–101 CFU (colony forming units) per ml in the column effluent, following a dosage of 106 CFU per ml. Destructive sampling of the columns after experiments revealed that a high proportion of viral pathogens were retained in the top 30 mm of the sand. Conclusions: On the basis of the relative spatial distribution of trace organisms in the sand, we speculate that adsorption processes likely dominated attenuation of the viral pathogens, more so than physical straining effects. Further study is required to fully elucidate the removal mechanisms. Significance and Impact of the Study: To the best of our knowledge this is the first study of the effective microbial removal capacity of coral sand under unsaturated conditions. The work represents an initial exploratory step of developing some standardized design practice of on‐site wastewater treatment systems in Kiribati, to offer enhanced protection of groundwater resources and reduce diarrhoeal disease. [ABSTRACT FROM AUTHOR]

Aim: It is well‐known that enterococci are abundant in the environment; however, the role of surface water as a reservoir of antimicrobial‐resistant enterococci remains largely undefined. In this study, surface water samples were collected over a 2‐year period from the Upper Oconee watershed, Athens, GA to examine enterococci and their antimicrobial resistance. Methods and Results: Approximately 97% (445/458) of the samples were positive for enterococci and a total of 637 enterococci were isolated. The predominant species were Enterococcus casseliflavus (33·6%) followed by Enterococcus faecalis (26·5%) and Enterococcus hirae (13·2%). Regardless of species, the highest levels of resistance were to lincomycin (88·5%) and tetracycline (13%); isolates also exhibited resistance to newer antimicrobials, daptomycin (8·9%) and tigecycline (6·4%). Multidrug resistance (resistance ≥3 antimicrobial classes) was observed to as many as five classes of antimicrobials. Resistant enterococci appeared to be randomly dispersed over the seasons rather than clustered by species or antimicrobial resistance. Conclusions: This study demonstrated that surface waters contain a large population of diverse species of antimicrobial‐resistant enterococci, including resistance to new antimicrobials. Significance and Impact of the Study: These results may indicate the potential of human intestinal illness and/or colonization of the human gut with resistant enterococci as enterococci correlate with increased disease risk to humans during recreational exposure to water. [ABSTRACT FROM AUTHOR]

Aims: The intestinal microbiota of newborn calves is largely unexplored even if it is of great significance for their future health. Therefore, the aim of the study was to gain a better insight into the development dynamics of certain bacterial groups during the first week of life. Methods and Results: Faecal samples of healthy Simmental calves (dual‐purpose breed; n = 80), bottle fed and raised in a dairy farm were taken immediately after birth and at 6/12/24/48/72/168 h (h) after birth. Samples were analysed using cultural, biochemical and molecular–biological methods. The aerobe, anaerobe, Enterobacteriaceae and Enterococcus counts of healthy calves increased significantly between 6 and 24 h postpartum (P <0·05). Apart from the anaerobes, bacterial counts decreased after reaching a plateau at 24–48 h. Enterococcus faecalis was detected in significantly higher counts compared to E. faecium (P <0·05). Lactobacilli developed more slowly and increased until day 7 after birth to a mean value of 6·8 × 107 CFU per g. MALDI‐TOF analysis of 2338 lactobacilli isolates resulted in 36 different species. Conclusions: Lactobacillus reuteri became the most common Lactobacillus sp. during the first week of life. Significance and Impact of the Study: This fact seems to be very important for the calf's intestinal health because L. reuteri is known to show in vitro bactericidal effects against bacterial pathogens and anti‐infective activities against rotaviruses and Cryptosporidium parvum. [ABSTRACT FROM AUTHOR]

Aims: To elucidate the antibiotic resistance and virulence genes of nisin‐resistant Enterococcus faecalis isolated from raw buffalo milk and to study the effect of nisin‐sensitive and ‐resistant E. faecalis on the innate immunity of rats. Methods and Results: Slanetz–Bartley agar plates containing nisin were used to isolate nisin‐resistant E. faecalis. The virulence factors were ascertained using quantitative real‐time polymerase chain reaction. Cell viability, phagocytosis, intracellular survival and enzyme assays were performed to investigate the interaction of E. faecalis with rat macrophages. Nisin‐resistant E. faecalis was less prone to phagocytosis and survived longer inside the macrophages, due to reduced production of reactive oxygen species and nitric oxide. The viability and activation of macrophages was also reduced in the presence of resistant E. faecalis, as observed by enhanced lactate dehydrogenase production and reduced β‐galactosidase. Conclusions: Nisin‐resistant E. faecalis and its virulence factors were reported in raw buffalo milk. This study shows that nisin‐resistant variants exhibited cross resistance to antibiotics and suppressed the innate immune responses of rats by directly affecting macrophage activity. Significance and Impact of the Study: This study elucidated the contamination of raw buffalo milk by nisin‐resistant E. faecalis, which may pose food safety risk. [ABSTRACT FROM AUTHOR]

Aims: Investigate the interactions of organic acids (OAs), acetic, butyric, citric, formic, lactic and propionic acid against 50 Gram‐positive vancomycin‐resistant Enterococcus faecium (VRE) strains to determine whether pH, undissociated or dissociated acid forms correlate with bacterial inhibition. Methods and Results: Concentrations of undissociated and dissociated OAs at the molar minimum inhibitory concentrations (MICMs) of the VRE were calculated using the Henderson–Hasselbalch equation. The pH at the MICMs of all VRE strains against acetic, butyric, formic and propionic acids was similar, 4·66 ± 0·07, but there was a 1·1 pH unit difference for all six OAs. Inhibition of VRE by all six OAs did not appear to be solely dependent on pH or on the undissociated OA species. The inhibition of VRE by all six dissociated acids was within Δ = 3·1 mmol l−1. Conclusions: Vancomycin‐resistant Enterococcus faecium inhibition correlated with the dissociated OA species. A small decrease in the concentration of the dissociated OAs from optimum may result in allowing VRE strains to escape disinfection. Significance and Impact of the Study: When an OA is used to disinfect VRE strains, the concentration of the dissociated OA should be carefully controlled. A concentration of at least 20 mmol l−1 dissociated OA should be maintained when disinfecting VRE. [ABSTRACT FROM AUTHOR]

Aims: Isolation and characterization of Enterococcus phages and application of phage cocktail to control E. faecalis in milk., Methods and Results: For phage isolations, double layer agar method was used. Host range of the phages were determined by the spot test. Twelve phages with varying host ranges were isolated. Phages PEF1, PEF7b, and PEF9 with different host ranges and lytic activities were selected for phage cocktails. Compared to two-phages cocktails tested, the cocktail containing all the three phages displayed stronger antibacterial and biofilm removal activities. The cocktail treatment reduced viable E. faecalis in biofilm by 6 log within 6 h at both 30°C and 4°C. In milk, the cocktail gradually reduced the viable count of E. faecalis and the count reached below the lower limit of detection at 48 h at 4°C., Conclusion: The strong bactericidal and biofilm removal activities of the phage cocktail suggest the potential of this cocktail as a natural biocontrol agent for combating E. faecalis in milk., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Abstract: Aim: The study aimed to isolate and characterize Enterococcus species from apparently healthy waste attendants, cattle and cattle waste in Tanzania. Emphasis was given to antimicrobial resistance and in particular occurrence of vancomycin (VA)‐resistant enterococci. Methods and Results: Faecal samples were collected from healthy cattle, cattle waste attendants and cattle house wastes, and isolation of Enterococcus species was performed using Slanetz Bartley agar. Isolates were characterized with regard to species, antimicrobial susceptibility and presence of VA resistance genes. Enterococcus faecalis was the most prevalent species from all sources of isolation (43·5%), followed by Enterococcus faecium (38·4%). Isolates of E. faecium showed a higher number of phenotypic antimicrobial resistance than isolates of E. faecalis. Fifty‐eight isolates, which showed resistance or intermediate resistance to VA by disc diffusion test, were analysed for VA‐resistant Enterococcus (VRE) by PCR. The vanA gene was detected in 14 isolates of E. faecium and 12 isolates of E. faecalis, while vanB was detected in three isolates. No isolates were found to carry vanC1‐gene. Conclusion: VRE was detected in both human and cattle samples, despite no known use of antimicrobial agents that can select for VRE in livestock in Tanzania. Enterococcus faecalis was the most commonly isolated species from cattle and humans. Significance and Impact of the Study: The study provides information on the prevalence of VRE in human and nonhuman samples in Tanzania calling for further studies on the origin of VRE in such isolates, since no selection mechanism in Tanzania are known. [ABSTRACT FROM AUTHOR]

Aim Investigate dynamics of culturable mesophilic bacteria and selected food-contaminating bacteria from three herbs and their production environment. Methods and Results Marjoram, basil and thyme were investigated during one growing season by sampling plants, organic fertilizers, soil, irrigation water and marketed products. Mesophilic bacteria and selected food-contaminating bacteria ( Escherichia coli, Enterococcus spp., Bacillus cereus group) were cultured and identified by MALDI biotyping. Culturable mesophilic bacteria on marjoram and basil plants decreased over time by two orders of magnitude starting at above 106 colony forming units per gram ( CFU per g), while they remained constant on thyme (~104 CFU per g). Compared to the last field sample, mesophilic bacteria were increased on all market-ready products by one order of magnitude. Marjoram and basil were dominated by B. cereus group, Enterobacter spp. and Pseudomonas spp. , thyme by Bacillus spp. and Pseudomonas spp. All selected food-contaminating bacteria were detected in soil and reservoir-sourced irrigation water, whereas in municipal water, only B. cereus group and rarely Enterococcus spp. were found. Escherichia coli was detected only on young marjoram and basil plants (5 × 102 and 5 × 101 CFU per g, respectively), whereas Enterococcus spp. and B. cereus group were consistently detected on these two herbs. Thyme plants only contained B. cereus group consistently (above 103 CFU per g). Marketed marjoram and thyme contained Enterococcus spp. (5 × 102 and 104 CFU per g) and B. cereus group (~5 × 102 CFU per g), while no selected food-contaminating bacteria were found on marketed basil. Conclusions Overall, culturable mesophilic bacteria were dominated by Pseudomonas spp. and Bacillus spp., with increased numbers on market-ready products. Selected food-contaminating bacteria were readily detectable, however, only the B. cereus group was found throughout in all systems. Significance and Impact of the Study Insight into composition and development of mesophilic bacterial communities and selected food-contaminating bacteria of fresh herbs contributes to estimating consumer exposure. [ABSTRACT FROM AUTHOR]

Aims The thermal resistance of desiccation-adapted Salmonella Senftenberg 775/W was compared with those of indigenous enterococci and total aerobic bacteria in poultry litter. Methods and Results Aged broiler litter and composted turkey litter with 20, 30, 40 and 50% moisture contents were inoculated with desiccation-adapted Salm. Senftenberg 775/W, and then heat-treated at 75 and 85°C. Compared to total aerobic bacteria, there were better correlations between mean log reductions of desiccation-adapted Salm. Senftenberg 775/W and indigenous enterococci in broiler litter samples with 20, 30, 40 and 50% moisture contents at 75°C ( R2 > 0·91), and 20, 30 and 40% moisture contents at 85°C ( R2 > 0·87). The mean log reductions of Salm. Senftenberg 775/W were better correlated with those of indigenous enterococci in turkey litter samples with 20, 30, 40 and 50% moisture contents at 75°C ( R2 > 0·88), and 20 and 30% moisture contents at 85°C ( R2 = 0·83) than those of total aerobic bacteria, which had a better correlation in turkey litter sample with 40% ( R2 = 0·98) moisture content at 85°C. Conclusion Indigenous enterococci may be used to validate the thermal processing of poultry litter, as it predicts the survival behaviour of Salmonella under some treatment conditions. Significance and the Impact of the Study This study provides some scientific data for poultry litter processors when validating the effectiveness of thermal processing. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB). Methods and Results Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl- d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 ( pNZ: LR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS- PAGE analysis. Conclusions Lactococcus and Enterococcus transformed with pNZ: LR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications. Significance and Impact of the study The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aim The objective of this study was to identify and characterize lactic acid bacteria isolated from human milk, with an emphasis on their probiotic properties. Methods and Results The strains were tested for their ability to inhibit growth of Enterococcus faecalis, Salmonella enterica subsp. enterica serotype Enteritidis , Listeria monocytogenes, Staphylococcus aureus and Escherichia coli, as well as for susceptibility to antimicrobial agents and for acid pH and bile salt tolerance. Gram-positive and catalase-negative were selected and identified as Enterococcus (83·3%) after sequencing the 16S rDNA gene. All the isolates inhibited growth of Ent. faecalis and S. serotype Enteritidis, 97% inhibited growth of L. monocytogenes and Staph. aureus and 78·8% inhibited growth of E. coli. Most of the isolates were resistant to gentamicin (50%) and vancomycin (47%). Twelve isolates grew when subjected to pH 3·0 and 0·1% bile salts. At lower pH (2·5-2·0), Ent. faecalis F1 and Weissella confusa F8 were more efficient. Conclusion It was possible to isolate from human milk the lactic acid bacteria with potential for use as probiotics. Significance and Impact of the Study Lactic acid bacteria isolated of nursing mothers have probiotic properties. [ABSTRACT FROM AUTHOR]

Aims The goal was to determine the effect of starch source (corn, oats and wheat) and concentration on: (i) total amylolytic bacteria, Group D Gram-positive cocci ( GPC), lactobacilli and lactate-utilizing bacteria, and (ii) fermentation by equine microbiota. Methods and Results When faecal washed cell suspensions were incubated with any substrate amylolytics increased over time. However, at 24 h there were 10 and 1000-fold more amylolytics with corn than wheat or oats respectively. Predominant amylolytics isolated were Enterococcus faecalis (corn, wheat) and Streptococcus bovis (oats). GPC increased with any substrate, but decreased during stationary phase in oats only. Lactobacilli decreased during stationary phase with corn only. By 24 h, oats had more lactate-utilizers and lactobacilli and fewer GPC than corn and wheat. More gas was produced from oats or wheat than from corn. Conclusions These results indicate that the growth of bacteria and fermentative capacity associated with starch metabolism is starch source dependent. Significance and Impact of the Study This study demonstrates a relationship between starch source and microbial changes independent of host digestion. However, future research is needed to evaluate the effect of starch source on the hindgut microbial community in vivo. [ABSTRACT FROM AUTHOR]

Aims To investigate the safety and technological traits of previously isolated bacteriocinogenic enterococci strains for potential use as starter/adjunct cultures in foods. Methods and Results Fifty-five bacteriocinogenic enterococci strains previously isolated from different origins in Tunisia were screened for safety. Twenty-two strains did not harbour the genes coding for virulence traits, were susceptible to relevant antibiotics such as vancomycin, and tested negative for haemolysis, histamine production, gelatinase activity and DNase activity. These strains were further assessed for some technological properties, demonstrating low milk-acidifying ability, low proteolytic activity, high peptidolytic activity and diacetyl production in milk. Conclusions This study revealed that 22 bacteriocinogenic enteroccoci strains did not present virulence features and could be safely applied in food preservation. Some strains also showed good technological potential as adjunct/protective cultures in milk fermentation and cheese production. Significance and Impact of the Study This is one of very few studies that identified safe Enterococcus strains capable of producing a wide variety of enterocins against different spoilage and pathogenic micro-organisms that have good potential for application as adjunct/protective cultures in foods. [ABSTRACT FROM AUTHOR]

Aims The objective was to investigate whether in-feed supplementation of copper, at elevated level, co-selects for macrolide resistance in faecal enterococci. Methods and Results The study was conducted in cattle ( n = 80) with a 2 × 2 factorial design of copper (10 or 100 mg kg−1 of feed) and tylosin (0 or 10 mg kg−1 of feed). Thirty-seven isolates (4·6%; 37/800) of faecal enterococci were positive for the tcrB and all were Enterococcus faecium. The prevalence was higher among cattle fed diets with copper and tylosin (8·5%) compared to control (2·0%), copper (4·5%) and tylosin (3·5%) alone. All tcrB-positive isolates were positive for erm(B) and tet(M) genes. Median copper minimum inhibitory concentrations ( MICs) for tcrB-positive and tcrB-negative enterococci were 20 and 4 mmol l−1, respectively. Conclusions Feeding of elevated dietary copper and tylosin alone or in combination resulted in an increased prevalence of tcrB and erm(B)-mediated copper and tylosin-resistant faecal enterococci in feedlot cattle. Significance and Impact of the Study In-feed supplementation of elevated dietary copper has the potential to co-select for macrolide resistance. Further studies are warranted to investigate the factors involved in maintenance and dissemination of the resistance determinants and their co-selection mechanism in relation to feed-grade antimicrobials' usage in feedlot cattle. [ABSTRACT FROM AUTHOR]

Aims To screen and identify wine-isolated LAB strains for bacteriocin production, and to identify and characterize bacteriocins. Methods and Results One hundred and fifty-five LAB strains isolated from South African red wines undergoing spontaneous malolactic fermentation were screened for bacteriocin production. Eight isolates were identified to be bacteriocin producers and were identified as Enterococcus faecium. All eight isolates had the same phenotypic and genotypic profiles. The peptides were preliminarily identified as enterocin P using mass spectrometry and further confirmed by PCR-amplifying enterocin P gene. The enterocin activity was inhibited by α-Chymotrypsin, papain and proteinase K treatments. It was heat stable at 37, 60, 80 and 100°C and showed activity over a broad pH range of 2-10. The production of the enterocin followed that of primary metabolite kinetics and, it showed bactericidal effect to some wine spoilage LAB strains. Conclusions Our study identified the presence of the enterocin-producing Enterococcus in wine. The enterocin was heat stable; with broad pH range and bactericidal effects to sensitive strains. Significance and Impact of the Study This is one of very few studies that isolated Enterococcus species from wine. It is, however, the first to report presence of bacteriocin-producing Enterococcus in wine fermentation. [ABSTRACT FROM AUTHOR]

Aims To assess the antimicrobial properties of lactic acid bacteria from Tunisian raw bovine milk. Methods and Results A bacteriocin-producing Enterococcus faecium strain was isolated from raw cow milk with activity against Gram-positive and Gram-negative bacteria. Antimicrobial substances produced by this strain were sensitive to proteolytic enzymes and were thermostable and resistant to a broad range of pH (2-10). Mode of action of antimicrobial substances was determined as bactericidal. Maximum activity was reached at the end of the exponential growth phase when checked against Listeria ivanovii BUG 496 (2366·62 AU ml−1). However, maximum antimicrobial activity against Pseudomonas aeruginosa 28753 was recorded at the beginning of the exponential growth phase. Enterococcus faecium GGN7 was characterized as free from virulence factors and was susceptible to tested antibiotics. PCR analysis of the micro-organism's genome revealed the presence of genes coding for enterocins A and B. Mass spectrometry analysis of RP-HPLC active fractions showed molecular masses corresponding to enterocins A (4835·77 Da) and B (5471·56 Da), and a peptide with a molecular mass of 3215·5 Da active only against Gram-negative indicator strains. The latter was unique in the databases. Conclusions Enterococcus faecium GGN7 produces three bacteriocins with different inhibitory spectra. Based on its antimicrobial properties and safety, Ent. faecium GGN7 is potentially useful for food biopreservation. Significance and Impact of the Study The results suggest the bacteriocins from GGN7 strain could be useful for food biopreservation. [ABSTRACT FROM AUTHOR]

Aims: The aim was to develop an optimized detachment method for separating Bacteroidales from sediments to allow enumeration via PMA-qPCR. The effectiveness of four different detachment treatments in removing Bacteroides fragilis was compared as a function of time as well as in relation to Enterococcus faecalis and Escherichia coli as detected by cultivation and qPCR. Methods and Results: Cells were inoculated into four sediments from sea water (SW) and freshwater (FW) beaches. Sediment samples were taken on days 1 and 7 and subjected to four different treatments for separation of micro-organisms. On day 1, the detachment treatments performed equally well in removing intact Bact. fragilis cells. In contrast, 7 days later the detachment treatment with Tween 80 and handshaking (TH) resulted in up to eightfold higher 16S rRNA gene concentrations of intact and total Bact. fragilis cells compared to other detachment treatments. Total Ent. faecalis cells based on the 23S rRNA gene were also preferentially recovered by treatment TH. Cultivable Ent. faecalis or E. coli numbers detached from sediments were similar for all methods in most sediments tested. Conclusions: Handshaking and 1% Tween 80/NaOH (pH 7·0) eluant was the most efficient technique to recover intact as well as total Bact. fragilis cells in sediment samples with different salinities and after prolonged sediment cell contact time. Significance and Impact of the Study: The optimized detachment method enables the application of PMA-qPCR to sediment samples to detect the presence of Bacteroidales cells and their DNA in future microbial source tracking studies. [ABSTRACT FROM AUTHOR]

Aims To determine the extent to which discrepancies between qPCR and culture-based results in beach water quality monitoring can be attributed to: (i) within-method variability, (ii) between-method difference within each method class ( qPCR or culture) and (iii) between-class difference. Methods and Results We analysed 306 samples using two culture-based ( EPA1600 and Enterolert) and two q PCR ( Taqman and Scorpion) methods, each in duplicate. Both q PCR methods correlated with EPA1600, but regression analyses indicated approximately 0·8 log10 unit overestimation by q PCR compared to culture methods. Differences between methods within a class were less than half of this and were minimal for between-replicate within a method. Using the 104 Enterococcus per 100 ml management decision threshold, Taqman qPCR indicated the same decisions as EPA1600 for 87% of the samples, but indicated beach posting for unhealthful water when EPA1600 did not for 12% of the samples. After accounting for within-method and within-class variability, 8% of the samples exhibited true between-class discrepancy where both q PCR methods indicated beach posting while both culture methods did not. Conclusion Measurement target difference ( DNA vs growth) accounted for the majority of the qPCR- vs-culture discrepancy, but its influence on monitoring application is outweighed by frequent incorrect posting with culture methods due to incubation time delay. Significance and Impact of the Study This is the first study to quantify the frequency with which culture- vs- qPCR discrepancies can be attributed to target difference - vs - method variability. [ABSTRACT FROM AUTHOR]

Aims To evaluate phenotypic resistance to macrolides-lincosamides and streptogramin B ( MLSB) antibiotics and to determine their localization as well as transferability of erythromycin resistance genes in enterococcal isolates of naturally fermented food- Idli batter. Methods and Results Diverse MLSB phenotypes observed among the enterococcal spp. ( n = 32) were analysed through double disc and triple disc test. Standard minimum inhibitory concentration tests along with induction studies displayed synergistic or cross-resistance among MLSB antibiotics. Plasmid profiling and Southern hybridization revealed that erm( B) and msr( C) genes were localized either on chromosome or on high molecular weight plasmids and showed co-localization of these genes with lnu( B), tet( L) and tet( W) in one of the isolate. In vitro conjugation experiments demonstrated plasmid-mediated transfer of erm(B) gene from three Enterococcus durans strains to Enterococcus faecalis JH2-2. Conclusions The study illustrated diverse MLSB phenotypes, multiple resistance genes and transferable plasmids among enterococci isolated from naturally fermented foods. Significance and Impact of the Study From a public health point of view, the study identified that naturally fermented foods could represent a source of antibiotic resistance enterococci that can spread through foods. These results also suggest that vigilance may be exercised with the use of combination or novel MLSB antibiotics in treating enterococcal infections. [ABSTRACT FROM AUTHOR]

Aims In this study, faecal indicator bacteria ( FIB) namely Escherichia coli and Enterococcus spp. were seeded into slurries of possum faeces and placed on the roof and in the gutter of a roof-captured rainwater ( RCR) system. The persistence of FIB in these circumstances was determined under ambient climatic conditions. FIB persistence was also determined under in situ conditions in tank water using diffusion chambers. Methods and Results The numbers of surviving FIB at different time intervals were enumerated using culture-based methods. Both FIB were rapidly inactivated on the roof under sunlight conditions ( T90 = 2 h) compared with shade conditions ( T90 = 9-53 h). Significant differences were observed between sunlight and shade conditions on the roof for both T90 values of E. coli ( P < 0·001) and Enterococcus spp. ( P < 0·001). E. coli showed biphasic inactivation patterns under both clean and unclean gutter conditions. Enterococcus spp., however, showed rapid inactivation ( T90 = 2 h for the clean gutter and T90 = 6 h for the unclean gutter) compared with E. coli ( T90 = 22 h for the clean gutter and T90 = 20 h for the unclean gutter). Significant differences were also observed between the T90 values of E. coli and Enterococcus spp. for both clean ( P < 0·001) and unclean ( P < 0·001) gutters. Both E. coli and Enterococcus spp. showed nonlinear biphasic inactivation in tank water. Significant difference was observed between the T90 value of E. coli inactivation compared with Enterococcus spp. ( P < 0·001) in the tank water. Conclusions In this study, FIB were observed to survive longer ( T90 = 9-53 h) on the roof under shade conditions compared with sunlight conditions ( T90 = 2 h). If there is a rainfall event within two to three days after the deposition of faecal maters on the roof, it is highly likely that FIB would be transported to the tank water. When introduced into the tank, a relatively slow inactivation process may take place ( T90 = 38-72 h). Significance and Impact of the Study The presence of FIB in water indicates faecal pollution and potential presence of enteric pathogens. Therefore, the information on the resilience of FIB, as obtained in this study, can be used for indirect assessment of health risks associated with using roof-captured rainwater for potable and nonpotable purposes. [ABSTRACT FROM AUTHOR]

Aims The relationship between land use (undeveloped, cattle grazing, urban), faecal indicator bacteria ( FIB) levels and microbial source tracking ( MST) marker detection was investigated in lakes created following phosphate mining. Methods and Results Faecal coliforms and enterococci were cultured, and MST markers were detected by PCR [ Methanobrevibacter smithii, human polyomaviruses ( HPy Vs), ruminant, human ( HF183) and general Bacteroidales]. FIB levels varied significantly by sampling date and were correlated with antecedent rainfall. FIB levels varied with land use category only in the case of faecal coliform levels in sediments of urban lakes, which were significantly greater than those in undeveloped or cattle-impacted lakes. Ruminant Bacteroidales were detected consistently in cattle-impacted lakes (57%) and rarely in other lakes. HPy Vs was the only human source marker detected. Conclusions Rainfall was more strongly associated with FIB levels than land use category. The detection frequency of only the ruminant MST marker was associated with land use. Significance and Impact of Study Microbial source tracking ( MST) can fine-tune the assessment of human health risk from recreational use of inland waters, particularly when similar FIB levels but different surrounding land use and probable impacts exist. [ABSTRACT FROM AUTHOR]

Aims To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid-producing engineered MG1363 strain under stress condition was investigated. Methods and Results We cloned carotenoid biosynthesis genes from yellow-pigmented Enterococcus gilvus. The cloned genes consisted of crt N and crt M and its promoter region were inserted into the shuttle vector p RH100, and the resulting plasmid was named p RC. The cloned crt NM was expressed using p RC in noncarotenoid-producing L. lactis ssp. cremoris MG1363. The expression of crt NM led to the production of C30 carotenoid 4,4′-diaponeurosporene. After exposure to 32 mmol l−1 H2 O2, low pH (1.5, acidified with HCl), 20% bile acid and 12 mg ml−1 lysozyme, the survival rates of the MG1363 strain harbouring pRC were 18.7-, 6.8-, 8.8- and 4.4-fold higher, respectively, than those of MG1363 strain harbouring the empty vector p RH100. Conclusions The expression of carotenoid biosynthesis genes from Ent. gilvus improves the multistress tolerance of L. lactis. Significance and Impact of the study First report of the improvement of multistress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production. [ABSTRACT FROM AUTHOR]