Investigation of some changes and clonal relationship in enterococci isolates due to relocation of a hospital.

Objectives: To investigate the isolation rates, antimicrobial resistance rates, minimum inhibitory concentration values of antimicrobial agents, and clonal relationships of Enterococcus faecalis and Enterococcus faeciumdue to the relocation of a hospital to a newly constructed building. METHODS: The comparative, prospective study was conducted at adult general intensive care units of the Mus State Hospital, Mus, Turkey, in two phases; before the relocation from January 25 to December 1, 2014, and after the relocation from February 10 to May 24, 2015. Rectal swab samples were collected 72 hours post-hospitalisation. Identification of Enterococcus faecalis and Enterococcus faeciumisolates was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial resistance with minimum inhibitory concentration values was detected with Vitek 2 system. The clonal relatedness among the strains was investigated by pulsed-field gel electrophoresis. Data was analysed using SPSS 23. RESULTS: Of the 69 patients, 37(53.62%) were related to pre-relocation phase; 20(54.1%) females and 17(45.9%) males with mean age 62.81±21.71 years. There were 32(46.37%) patients in the post-relocation phase; 13(40.6%) females and 19(59.4%) males with mean age 62.69±21.35 years (p>0.05). Of the 84 enterococci strains isolated, 51(60.7%) were Enterococcus faecium; 28(55%) before relocation and 23(45%) after relocation (p=0.77). The remaining 33(39.3%) isolates were Enterococcus faecalis; 16(48.5%) before relocation and 17(51.5%) after relocation (p=0.73). Multiple strains were located in 7(18.9%) patients before relocation and in 7(21.9%) after relocation. In 1(3.1%) patient after relocation, 2(8.7%) Enterococcus faecium isolates with different resistance and pulsed-field gel electrophoresis patterns were detected. There were no significant differences between the isolation and antibiotic resistance rates before and after relocation (p>0.05), and a clonal relation between the isolates was not detected (p>0.05). Decreased minimum inhibitory concentration values were noted for some antibiotics. CONCLUSIONS: Clonal relationship between the isolates and change in the rates of isolation and antimicrobial resistance of Enterococcus faecalis and Enterococcus faecium was not detected due to relocation. Minimum inhibitory concentration values could be used to reveal relocation-related changes in isolates obtained from patients hospitalised in intensive care units.

Comparative analysis of proteomic adaptations in Enterococcus faecalis and Enterococcus faecium after long term bile acid exposure.

BACKGROUND: All gastrointestinal pathogens, including Enterococcus faecalis and Enterococcus faecium, undergo adaptation processes during colonization and infection. In this study, we investigated by data-independent acquisition mass spectrometry (DIA-MS) two crucial adaptations of these two Enterococcus species at the proteome level. Firstly, we examined the adjustments to cope with bile acid concentrations at 0.05% that the pathogens encounter during a potential gallbladder infection. Therefore, we chose the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA), as these are the most prominent bile acids. Secondly, we investigated the adaptations from an aerobic to a microaerophilic environment, as encountered after oral-fecal infection, in the absence and presence of deoxycholic acid (DCA). RESULTS: Our findings showed similarities, but also species-specific variations in the response to the different bile acids. Both Enterococcus species showed an IC50 in the range of 0.01- 0.023% for DCA and CDCA in growth experiments and both species were resistant towards 0.05% CA. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis (424 down-expressed proteins with DCA, 376 down-expressed proteins with CDCA) and in E. faecium (362 down-expressed proteins with DCA, 391 down-expressed proteins with CDCA). Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a "general bile acid response". Among these, various subunits of a V-type ATPase, different ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions instead of aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously. CONCLUSIONS: Overall, these findings provide a detailed insight into the proteomic stress response of two Enterococcus species and help to understand the resistance potential and the stress-coping mechanisms of these important gastrointestinal bacteria.

Enterococcus faecalis provides protection during scavenging in carrion crow ( Corvus corone).

The gut microbiota significantly influences host physiology and provides essential ecosystem services. While diet can affect the composition of the gut microbiota, the gut microbiota can also help the host adapt to specific dietary habits. The carrion crow ( Corvus corone), an urban facultative scavenger bird, hosts an abundance of pathogens due to its scavenging behavior. Despite this, carrion crows infrequently exhibit illness, a phenomenon related to their unique physiological adaptability. At present, however, the role of the gut microbiota remains incompletely understood. In this study, we performed a comparative analysis using 16S rRNA amplicon sequencing technology to assess colonic content in carrion crows and 16 other bird species with different diets in Beijing, China. Our findings revealed that the dominant gut microbiota in carrion crows was primarily composed of Proteobacteria (75.51%) and Firmicutes (22.37%). Significant differences were observed in the relative abundance of Enterococcus faecalis among groups, highlighting its potential as a biomarker of facultative scavenging behavior in carrion crows. Subsequently, E. faecalis isolated from carrion crows was transplanted into model mice to explore the protective effects of this bacterial community against Salmonella enterica infection. Results showed that E. faecalis down-regulated the expression of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin 6 (IL-6), prevented S. enterica colonization, and regulated the composition of gut microbiota in mice, thereby modulating the host's immune regulatory capacity. Therefore, E. faecalis exerts immunoregulatory and anti-pathogenic functions in carrion crows engaged in scavenging behavior, offering a representative case of how the gut microbiota contributes to the protection of hosts with specialized diets.

Fully Characterized Effective Bacteriophages Specific against Antibiotic-Resistant Enterococcus faecalis, the Causative Agent of Dental Abscess.

Background and Objectives: Enterococcus faecalis (E. faecalis) is a primary pathogen responsible for dental abscesses, which cause inflammation and pain when trapped between the crown and soft tissues of an erupted tooth. Therefore, this study aims to use specific phages as an alternative method instead of classical treatments based on antibiotics to destroy multidrug-resistant E. faecalis bacteria for treating dental issues. Materials and Methods: In the current study, twenty-five bacterial isolates were obtained from infected dental specimens; only five had the ability to grow on bile esculin agar, and among these five, only two were described to be extensive multidrug-resistant isolates. Results: Two bacterial isolates, Enterococcus faecalis A.R.A.01 [ON797462.1] and Enterococcus faecalis A.R.A.02, were identified biochemically and through 16S rDNA, which were used as hosts for isolating specific phages. Two isolated phages were characterized through TEM imaging, which indicated that E. faecalis_phage-01 had a long and flexible tail, belonging to the family Siphoviridae, while E. faecalis_phage-02 had a contractile tail, belonging to the family Myoviridae. Genetically, two phages were identified through the PCR amplification and sequencing of the RNA ligase of Enterococcus phage vB_EfaS_HEf13, through which our phages shared 97.2% similarity with Enterococcus phage vB-EfaS-HEf13 based on BLAST analysis. Furthermore, through in silico analysis and annotations of the two phages' genomes, it was determined that a total of 69 open reading frames (ORFs) were found to be involved in various functions related to integration excision, replication recombination, repair, stability, and defense. In phage optimization, the two isolated phages exhibited a high specific host range with Enterococcus faecalis among six different bacterial hosts, where E. faecalis_phage-01 had a latent period of 30 min with 115.76 PFU/mL, while E. faecalis_phage-02 had a latent period of 25 min with 80.6 PFU/mL. They were also characterized with stability at wide ranges of pH (4-11) and temperature (10-60 °C), with a low cytotoxic effect on the oral epithelial cell line at different concentrations (1000-31.25 PFU/mL). Conclusions: The findings highlight the promise of phage therapy in dental medicine, offering a novel approach to combating antibiotic resistance and enhancing patient outcomes. Further research and clinical trials will be essential to fully understand the therapeutic potential and safety profile of these bacteriophages in human populations.

Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated yvcJ-glmR-yvcL operon enhances fitness during cephalosporin exposure.

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.

Bactericidal Effect of Triple Antibiotic Paste against Enterococcus faecalis in Dentinal Tubules: An Ex Vivo Study.

OBJECTIVE: The aim of this study was to investigate the bactericidal effect of various concentrations of triple antibiotic paste (TAP) against Enterococcus faecalis (E. faecalis) in dentinal tubules using a bacterial culture assay and confocal laser scanning microscope (CLSM). METHODS: Ninety human teeth were contaminated with E. faecalis (ATCC 29212) and randomly allocated into 5 groups; the negative control (without TAP), 1 mg/ml, 5 mg/ml, 7.5 mg/ml and 10 mg/ml TAP (n=18). After a 3-week TAP treatment, samples were collected from the root canal space, root dentin at 100-µm and 200-µm depth. The collected samples were subjected to a bacterial culture assay (n=10). Eight roots from each group underwent CLSM analysis to determine the live/dead bacterial cells. RESULTS: The bacterial culture assay results indicated that the negative control samples were all culturable. The number of culture-positive samples decreased after TAP treatment at 1, 5, 7.5 and 10 mg/ml, with 2, 2, 1 and 0 culturable samples, respectively. However, there was no significant difference among the TAP treatments. Surprisingly, the CLSM analysis demonstrated live bacteria in the dentinal tubules in all samples. The negative control had 52.36%+-3.24 live bacteria. TAP treatment at 10 mg/ml had the lowest percentage of live bacterial cells (40.58%+-5.40), followed by 7.5 mg/ml (44.14%+-6.03), 5 mg/ml (46.31%+-5.32) and 1 mg/ml (52.55%+-8.82). The percentage of live cells in the 10 mg/ml, 7.5 mg/ml and 5 mg/ml TAP groups were significantly lower than the 1 mg/ml TAP and negative control groups. CONCLUSION: TAP treatment significantly decreased the percentage of viable E. faecalis cells in the dentinal tubules and its bactericidal effect was dose-dependent.

Comparison of Antibacterial Efficacy of Triple Antibiotic-Loaded Hydrogel Versus Modified Triple Antibiotic-Loaded Hydrogel as Intracanal Medicament Against Enterococcus faecalis: An In vitro Study.

OBJECTIVE: Triple antibiotic paste (TAP) is known to have an essential role in the success of endodontic treatment by eliminating pathogens from the root canal system. Unfortunately, it causes discolouration and cytotoxicity at high concentrations. The objective of this research was to assess and compare the antimicrobial effectiveness of various concentrations (1 mg, 5 mg, 10 mg) of TAP, TAP hydrogel (TAPH), M-TAP, and M-TAP hydrogel (MTAPH) against Enterococcus faecalis. METHODS: The agar well diffusion method was used to assess the antibiotic sensitivity of the following intracanal medicaments: TAP (ciprofloxacin, metronidazole, and minocycline) mixed in a ratio of 1: 1: 1; TAPH, M-TAP (ciprofloxacin, metronidazole, and amoxicillin), M-TAPH and plain hydrogel. Each tested medicament was individually evaluated for its antimicrobial activity against Enterococcus faecalis. Structural and topographical characterisation were analysed using a Scanning Electron Microscope (SEM) and interpreted using ImageJ software. A microdilution broth test was performed to examine the minimum inhibitory concentration and minimum bactericidal concentration (MBC) of M-TAP and TAP. RESULTS: Except for the plain hydrogel, M-TAP and hydrogel and TAP and hydrogel showed significantly varied inhibitory zones at different concentrations. M-TAPH showed the highest mean zone of inhibition of 21.6, 33.33 and 38.0 mm at a concentration of 1, 5, and 10 mg/mL when compared to TAPH, which showed a mean zone of inhibition of 3.3 mm,12.3 mm, 21.3 mm at the respective concentrations. The MIC study shows that more than 75% of Enterococcus faecalis growth was inhibited by M-TAP at a concentration of 5 µg/mL, whereas TAP showed inhibition at a concentration of 35 µg/mL. MBC results indicate that almost 99.9% of the bacterial population was killed at a concentration of 100 µg/mL (10-1) for TAP and 10 µg/mL (10-2) for M-TAP. CONCLUSION: The antibacterial efficacy of M-TAP was significantly higher than TAP. Application of M-TAP at lower doses is advised to overcome the disadvantages seen with TAP.

Mapping the widespread distribution and transmission dynamics of linezolid resistance in humans, animals, and the environment.

BACKGROUND: The rise of linezolid resistance has been widely observed both in clinical and non-clinical settings. However, there were still data gaps regarding the comprehensive prevalence and interconnections of linezolid resistance genes across various niches. RESULTS: We screened for potential linezolid resistance gene reservoirs in the intestines of both humans and animals, in meat samples, as well as in water sources. A total of 796 bacteria strains out of 1538 non-duplicated samples were identified to be positive for at least one linezolid resistance gene, optrA, poxtA, cfr, and cfr(D). The prevalence of optrA reached 100% (95% CI 96.3-100%) in the intestines of pigs, followed by fish, ducks, and chicken at 77.5% (95% CI 67.2-85.3%), 62.0% (95% CI 52.2-70.9%), and 61.0% (95% CI 51.2-70.0%), respectively. The meat and water samples presented prevalences of 80.0% (95% CI 70.6-87.0%) and 38.0% (95% CI 25.9-51.9%), respectively. The unreported prevalence of the cfr(D) gene was also relatively higher at 13.0% (95% CI 7.8-21.0%) and 19.0% (95% CI 10.9-25.6%) for the feces samples of ducks and pigs, respectively. Enterococci were the predominant hosts for all genes, while several non-enterococcal species were also identified. Phylogenetic analysis revealed a significant genetic distance among linezolid resistance gene reservoirs, with polyclonal structures observed in strains within the same niche. Similar genetic arrays harboring assorted insertion sequences or transposons were shared by reservoirs displaying heterogeneous backgrounds, though large diversity in the genetic environment of linezolid resistance genes was also observed. CONCLUSIONS: The linezolid resistance genes were widespread among various niches. The horizontal transfer played a crucial role in driving the circulation of linezolid resistance reservoirs at the human-animal-environment interfaces. Video Abstract.

Effect of sodium hypochlorite gel on bacteria associated with periodontal disease.

OBJECTIVES: An adjunct in non-surgical periodontal therapy might be sodium hypochlorite (NaOCl)-based agents. The purpose of the present in vitro study was to get deeper knowledge on the influence of different parameters as time after mixing, pH, and chemical composition of an amino acid 0.475% NaOCl (AA-NaOCl) gel consisting of two components on its anti-biofilm activity. MATERIALS AND METHODS: Six-species biofilms were cultured for 5 days, before AA-NaOCl gel was applied. In the different series, the influence of the time after mixing of the two components before application, of the concentration of NaOCl in the gel mixture, of the pH of the gel mixture, and of an exchange of the amino acid component by hyaluronic acid (HA), was analyzed. RESULTS: Mixing time point experiments showed that the AA-NaOCl gel is capable of statistically significantly reducing colony-forming unit (cfu) counts up to 30 min after mixing, but only up to 20 min after mixing the reduction was more than 2 log10 cfu. The pH experiments indicate that a reduced pH results in a reduced activity of the NaOCl formulation. NaOCl concentrations in the formulation in the range from 0.475 to 0.2% provide adequate activity on biofilms. A HA/NaOCl gel was equally active against the biofilm as the AA-NaOCl gel. CONCLUSION: Mixing of the components should be made in a timeframe of 20 min before applications. An optimization of the composition of the NaOCl formulation might be possible and should be a topic in further in vitro studies. CLINICAL RELEVANCE: The AA-NaOCl gel formulation can be mixed up to 20 min before application. Further, the study indicates that the composition of the NaOCl gel formulation can be optimized.

Data-driven prediction of colonization outcomes for complex microbial communities.

Microbial interactions can lead to different colonization outcomes of exogenous species, be they pathogenic or beneficial in nature. Predicting the colonization of exogenous species in complex communities remains a fundamental challenge in microbial ecology, mainly due to our limited knowledge of the diverse mechanisms governing microbial dynamics. Here, we propose a data-driven approach independent of any dynamics model to predict colonization outcomes of exogenous species from the baseline compositions of microbial communities. We systematically validate this approach using synthetic data, finding that machine learning models can predict not only the binary colonization outcome but also the post-invasion steady-state abundance of the invading species. Then we conduct colonization experiments for commensal gut bacteria species Enterococcus faecium and Akkermansia muciniphila in hundreds of human stool-derived in vitro microbial communities, confirming that the data-driven approaches can predict the colonization outcomes in experiments. Furthermore, we find that while most resident species are predicted to have a weak negative impact on the colonization of exogenous species, strongly interacting species could significantly alter the colonization outcomes, e.g., Enterococcus faecalis inhibits the invasion of E. faecium invasion. The presented results suggest that the data-driven approaches are powerful tools to inform the ecology and management of microbial communities.

Leaf Extracts of Moringa oleifera Cultivated in Baghdad: Characterization and Antimicrobial Potential against Endodontic Pathogens.

The use of medicinal plant preparations to clean and disinfect root canal infection is gaining popularity. The aim of this study was to evaluate the bioactive composition of leaf extracts of Moringa oleifera plants cultivated in Iraq (specifically Baghdad) and their antimicrobial activity against selected root canal pathogens for potential application in endodontic treatment. Materials and Methods. Moringa leaf extracts were prepared either through cold maceration or warm digestion techniques to perform an ethanolic or aqueous extraction, respectively. Phytochemical detection was performed before thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to measure flavonoids and phenolic compounds within both extracts. Then, their antimicrobial activities were investigated against Streptococcus mutans, Enterococcus faecalis, and Candida albicans through minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and agar well diffusion assay in comparison to NaOCl and Ca(OH)2. Results. Phytochemical screening showed several active ingredients but with higher expression of flavonoids and phenolic compounds. Also, different types of these compounds were detected through TLC and quantified by HPLC. MIC values for ethanolic extract against Streptococcus mutans, Enterococcus faecalis, and Candida albicans were 60, 65, and 55, respectively, while for aqueous extract, MIC values were 70, 80, and 50, respectively. Aqueous extract showed a higher inhibition zone than ethanolic extract for both Streptococcus mutans and Enterococcus faecalis with a statistically significant difference (p ≤ 0.001) for all tested materials except with NaOCl and Ca(OH)2 in Streptococcus mutans and Enterococcus faecalis, respectively. The ethanolic extract showed a higher inhibition zone against Candida albicans, with a statistically significant difference (p ≤ 0.001) for all tested materials. Conclusion. Ethanolic and aqueous extracts of Moringa oleifera leaves cultivated in Baghdad contain considerable quantities of phytochemicals, especially flavonoid and phenolic compounds, and demonstrated antimicrobial activities against selected endodontic pathogens. Therefore, Moringa leaf extracts could be suggested as an alternative antimicrobial material in endodontic treatment.

Characteristics of intestinal bacteriophages and their relationship with Bacteria and serum metabolites during quail sexual maturity transition.

BACKGROUND: Bacteriophages are prokaryotic viruses that rank among the most abundant microbes in the gut but remain among the least understood, especially in quails. In this study, we surveyed the gut bacteriophage communities in 22 quails at different ages (days 20 and 70) using shotgun metagenomic sequencing. We then systematically evaluated the relationships with gut bacteria and host serum metabolites. RESULTS: We discovered that Myoviridae and Siphoviridae were the dominant bacteriophage families in quails. Through a random forest and LEfSe analysis, we identified 23 differential bacteriophages with overlapping presence. Of these, 21 bacteriophages (e.g., Enterococcus phage IME-EFm5 and Enterococcus phage IME-EFm1) showed higher abundances in the day 20 group, while two bacteriophages (Bacillus phage Silence and Bacillus virus WPh) were enriched in the day 70 group. These key bacteriophages can serve as biomarkers for quail sexual maturity. Additionally, the differential bacteriophages significantly correlated with specific bacterial species and shifts in the functional capacities of the gut microbiome. For example, Enterococcus phages (e.g., Enterococcus phage EFP01, Enterococcus phage IME-EFm5, and Enterococcus phage IME-EFm1) were significantly (P < 0.001, FDR) and positively correlated with Enterococcus faecalis. However, the relationships between the host serum metabolites and either bacteriophages or bacterial species varied. None of the bacteriophages significantly (P > 0.05, FDR) correlated with nicotinamide riboside and triacetate lactone. In contrast, some differential bacterial species (e.g., Christensenella massiliensis and Bacteroides neonati) significantly (P < 0.05, FDR) correlated with nicotinamide riboside and triacetate lactone. Furthermore, characteristic successional alterations in gut bacteriophages, bacteria, and host serum metabolites across different ages highlighted a sexual maturity transition coexpression network. CONCLUSION: This study improves our understanding of the gut bacteriophage characteristics in quails and offers profound insights into the interactions among gut bacteriophages, bacteria, and host serum metabolites during the quail's sexual maturity transition.

Genome-wide analysis of acid tolerance genes of Enterococcus faecalis with RNA-seq and Tn-seq.

Enterococcus faecalis, a formidable nosocomial and community-acquired opportunistic pathogen, can persist a wide range of extreme environments, including low pH and nutrient deficiency. Clarifying the survival mechanism of E. faecalis in low-pH conditions is the key to combating the infectious diseases caused by E. faecalis. In this study, we combined transcriptome profiling (RNA-seq) and transposon insertion sequencing (TIS) to comprehensively understand the genes that confer these features on E. faecalis. The metadata showed that genes whose products are involved in cation transportation and amino acid biosynthesis were predominantly differentially expressed under acid conditions. The products of genes such as opp1C and copY reduced the hydrion concentration in the cell, whereas those of gldA2, gnd2, ubiD, and ubiD2 mainly participated in amino metabolism, increasing matters to neutralize excess acid. These, together with the folE and hexB genes, which are involved in mismatch repair, form a network of E. faecalis genes necessary for its survival under acid conditions.

IMPORTANCE: As a serious nosocomial pathogen, Enterococcus faecalis was considered responsible for large numbers of infections. Its ability to survive under stress conditions, such as acid condition and nutrient deficiency was indispensable for its growth and infection. Therefore, understanding how E. faecalis survives acid stress is necessary for the prevention and treatment of related diseases. RNA-seq and TIS provide us a way to analyze the changes in gene expression under such conditions.

Possibility of transfer and activation of 'silent' tetracycline resistance genes among Enterococcus faecalis under high-pressure processing.

In this study, the tetracycline resistance of Enterococcus faecalis strains isolated from food was determined and molecular analyses of the resistance background were performed by determining the frequency of selected tetracycline resistance genes. In addition, the effect of high-pressure stress (400 and 500 MPa) on the expression of selected genes encoding tetracycline resistance was determined, as well as changes in the frequency of transfer of these genes in isolates showing sensitivity to tetracyclines. In our study, we observed an increase in the expression of genes encoding tetracyclines, especially the tet(L) gene, mainly under 400 MPa pressure. The study confirmed the possibility of transferring genes encoding tetracyclines such as tet(M), tet(L), tet(K), tet(W) and tet(O) by horizontal gene transfer in both control strains and exposed to high-pressure. Exposure of the strains to 400 MPa pressure had a greater effect on the possibility of gene transfer and expression than the application of a higher-pressure. To our knowledge, this study for the first time determined the effect of high-pressure stress on the expression of selected genes encoding tetracycline resistance, as well as the possibility and changes in the frequency of transfer of these genes in Enterococcus faecalis isolates showing sensitivity to tetracyclines and possessing silent genes. Due to the observed possibility of increased expression of some of the genes encoding tetracycline resistance and the possibility of their spread by horizontal gene transfer to other microorganisms in the food environment, under the influence of high-pressure processing in strains phenotypically susceptible to this antibiotic, it becomes necessary to monitor this ability in isolates derived from foods.

Biodiversity and antibiotic resistance profile provide new evidence for a different origin of enterococci in bovine raw milk and feces.

Enterococci are widely distributed in dairy sector. They are commensals of the gastrointestinal tract of animals, thus, via fecal contamination, could reach raw milk and dairy products. The aims of this study were: 1) to investigate the enterococcal diversity in cow feces and milk samples and 2) to evaluate the antibiotic resistance (AR) of dairy-related enterococci and their ability to transfer resistance genes. E. faecalis (59.9%), E. faecium (18.6%) and E. lactis (12.4%) were prevalent in milk, while E. faecium (84.2%) and E. hirae (15.0%) were dominant in bovine feces. RAPD-PCR highlighted a high number of Enterococcus biotypes (45 from milk and 37 from feces) and none of the milk strains exhibited genetic profiles similar to those of feces biotypes. A high percentage of enterococci isolated from milk (71%) were identified as multidrug resistant and resistance against streptomycin and tetracycline were widespread among milk strains while enterococci from feces were commonly resistant to linezolid and quinupristin/dalfopristin. Only E. faecalis strains were able to transfer horizontally the tetM gene to Lb. delbrueckii subsp. lactis. Our results indicated that Enterococcus biotypes from milk and bovine feces belong to different community and the ability of these microorganisms to transfer AR genes is strain-dependent.

Identification of ISVlu1-derived translocatable units containing optrA and/or fexA genes generated by homologous or illegitimate recombination in Lactococcus garvieae of porcine origin.

The optrA gene encodes an ABC-F protein which confers cross-resistance to oxazolidinones and phenicols. Insertion sequence ISVlu1, a novel ISL3-family member, was recently reported to be involved in the transmission of optrA in Vagococcus lutrae. However, the role of ISVlu1 in mobilizing resistance genes has not yet fully explored. In this study, two complete and three truncated copies of ISVlu1 were found on plasmid pBN62-optrA from Lactococcus garvieae. Analysis of the genetic context showed that both optrA and the phenicols resistance gene fexA were flanked by the complete or truncated ISVlu1 copies. Moreover, three different-sized ISVlu1-based translocatable units (TUs) carrying optrA and/or fexA, were detected from pBN62-optrA. Sequence analysis revealed that the TU-optrA was generated by homologous recombination while TU-fexA and TU-optrA+fexA were the products of illegitimate recombinations. Importantly, conjugation assays confirmed that pBN62-optrA was able to successfully transfer into the recipient Enterococcus faecalis JH2-2. To our knowledge, this is the first report about an optrA-carrying plasmid in L. garvieae which could horizontally transfer into other species. More importantly, the ISVlu1-flanked genetic structures containing optrA and/or fexA were also observed in bacteria of different species, which underlines that ISVlu1 is highly active and plays a vital role in the transfer of some important resistance genes, such as optrA and fexA.

Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

The efficacy of 2780 nm Er,Cr;YSGG and 940 nm Diode Laser in root canal disinfection: A randomized clinical trial.

OBJECTIVES: Effective disinfection of the root canals is the cornerstone of successful endodontic treatment. Diminishing the microbial load within the root canal system is crucial for healing in endodontically treated teeth. The aim of this study was to evaluate the effect of 2780 nm Er,Cr:YSGG and 940 nm diode lasers on the eradication of microorganisms from single-rooted teeth with asymptomatic apical periodontitis. MATERIALS AND METHODS: Thirty participants conforming to the inclusion criteria were randomly divided into 3 groups according to the disinfection protocol used; Conventional group: 2.5% Sodium Hypochlorite (NaOCl) and 17% EDTA solution NaOCl/EDTA, Dual laser group: 2780 nm Erbium, chromium: yttrium scandium-gallium-garnet (Er,Cr:YSGG) laser and 940 nm diode laser Er,CrYSGG/Diode, and Combined group: 17% EDTA and 940 nm diode laser EDTA/Diode. Bacterial samples were collected before and after intervention. The collected data were statistically analyzed using Friedman's test and Kruskal-Wallis test (P ≤ 0.05). RESULTS: The results of the study showed that both dual laser Er,CrYSGG/Diode and combined laser EDTA/Diode groups showed significantly less mean Log10 CFU/ml of aerobic and anaerobic bacterial counts than the conventional NaOCl/EDTA group. CONCLUSIONS: In this study we evaluated in vivo the bactericidal efficacy of three disinfection protocols for endodontic treatment of single-rooted teeth with apical periodontitis. The results indicated that both dual laser Er,CrYSGG/Diode and combined laser EDTA/Diode groups provide superior bactericidal effect compared to the conventional NaOCl/EDTA group. CLINICAL RELEVANCE: The integration of lasers into root canal disinfection protocols has demonstrated significant bacterial reduction which might promote healing and long-term success.

A preliminary report on critical antimicrobial resistance in Escherichia coli, Enterococcus faecalis, and Enterococcus faecium strains isolated from healthy dogs in Chile during 2021-2022.

Antimicrobial Resistance (AMR) represents one of the main current threats to global public health; where production animals, companion animals, humans, and the environment play a significant role in its dissemination. However, little attention has been given to companion animals as reservoirs and disseminators of relevant antimicrobial resistant bacteria, especially in South American countries such as Chile. For this reason, this research aimed to estimate the prevalence of AMR to different critical antibiotics at a screening level in commensal bacteria such as E. coli and Enterococcus spp., isolated from healthy pet dogs in the Metropolitan Region of Chile, studying their geographical distribution and evaluating associations of phenotypic resistance to different antibiotics. Thus, in E. coli we detected AMR to all critical drugs assessed, including 34.1% to amoxicillin, 20.1% to colistin, 15.7% to enrofloxacin, and 9.2% to cefotaxime. On the other hand, AMR prevalence in E. faecalis was 8.1% for ampicillin and 3.4% for vancomycin; while for E. faecium the AMR prevalence was 19.1% for ampicillin and 10.2% for vancomycin. Additionally, significant differences in prevalence of the different possible AMR were detected according to their geographical distribution, suggesting the existence of various risk factors and stressing the need to establish mitigation measures specific to the differences identified.

Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.