Genomic epidemiology reveals multiple mechanisms of linezolid resistance in clinical enterococci in China.

BACKGROUND: Infections caused by linezolid-resistant enterococci (LRE) are clinically difficult to treat and threaten patient health. However, there is a lack of studies on long time-span LRE strains in China. For this reason, our study comprehensively revealed the resistance mechanisms of LRE strains collected in a Chinese tertiary care hospital from 2011 to 2022. METHODS: Enterococcal strains were screened and verified after retrospective analysis of microbial data. Subsequently, 65 LRE strains (61 Enterococcus faecalis and 4 Enterococcus faecium, MIC ≥ 8 µg/ml), 1 linezolid-intermediate Enterococcus faecium (MIC = 4 µg/ml) and 1 linezolid-susceptible Enterococcus faecium (MIC = 1.5 µg/ml) were submitted for whole-genome sequencing (WGS) analysis and bioinformatics analysis. RESULTS: The optrA gene was found to be the most common linezolid resistance mechanism in our study. We identified the wild-type OptrA and various OptrA variants in 98.5% of LRE strains (61 Enterococcus faecalis and 3 Enterococcus faecium). We also found one linezolid-resistant Enterococcus faecium strain carried both optrA and cfr(D) gene, while one linezolid-resistant Enterococcus faecium only harbored the poxtA gene. Most optrA genes (55/64) were located on plasmids, with impB-fexA-optrA, impB-fexA-optrA-erm(A), fexA-optrA-erm(A), and fexA-optrA segments. A minority of optrA genes (9/64) were found on chromosomes with the Tn6674-like platform. Besides, other possible linezolid resistance-associated mechanisms (mutations in the rplC and rplD genes) were also found in 26 enterococcal strains. CONCLUSIONS: Our study suggested that multiple mechanisms of linezolid resistance exist among clinical LRE strains in China.

Detection and modeling of Staphylococcus aureus and fecal bacteria in Hawaiian coastal waters and sands.

Microbial pollution of recreational waters leads to millions of skin, respiratory, and gastrointestinal illnesses globally. Fecal indicator bacteria (FIB) are monitored to assess recreational waters but may not reflect the presence of Staphylococcus aureus, a global leader in bacterial fatalities. Since many community-acquired S. aureus skin infections are associated with high recreational water usage, this study measured and modeled S. aureus, methicillin-resistant S. aureus (MRSA), and FIB (Enterococcus spp., Clostridium perfringens) concentrations in seawater and sand at six beaches in Hilo, Hawai'i, USA, over 37 sample dates from July 2016 to February 2019 using culturing techniques. Generalized linear models predicted bacterial concentrations with physicochemical and environmental data. Beach visitors were also surveyed on their preferred activities. S. aureus and FIB concentrations were roughly 6-78 times higher at beaches with freshwater discharge than at those without. Seawater concentrations of Enterococcus spp. were positively associated with MRSA but not S. aureus. Elevated S. aureus was associated with lower tidal heights, higher freshwater discharge, onsite sewage disposal system density, and turbidity. Regular monitoring of beaches with freshwater input, utilizing real-time water quality measurements with robust modeling techniques, and raising awareness among recreational water users may mitigate exposure to S. aureus, MRSA, and FIB. PRACTITIONER POINTS: Staphylococcus aureus and fecal bacteria concentrations were higher in seawater and sand at beaches with freshwater discharge. In seawater, Enterococcus spp. positively correlated with MRSA, but not S. aureus. Freshwater discharge, OSDS density, water turbidity, and tides significantly predicted bacterial concentrations in seawater and sand. Predictive bacterial models based upon physicochemical and environmental data developed in this study are readily available for user-friendly application.

Spontaneous resolution or antibiotic effectiveness? Reflection on a case of pediatric urinary tract infections caused by Enterococcus raffinosus.

Enterococcus raffinosus, named by Collins et al. in 1989, is a cocci-shaped bacterium that typically appears in pairs or short chains. As a Gram-positive and non-motile bacterium, it grows at 10°C-45°C, exhibiting negative peroxidase activity [1]. It is a normal flora in the oropharynx and gastrointestinal tract of domestic cats [2] and can also be isolated from human rectal swabs [3], it belongs to the same genus Enterococcus as Enterococcus faecalis and Enterococcus faecium. Enterococcus faecalis and Enterococcus faecium constitute 90% of clinically isolated strains. However, the incidence of other enterococci, excluding E. faecalis and E. faecium, is on the rise [4]. In this case report, a patient with pediatric urinary tract infections caused by E. raffinosus was presented, and a summary of relevant literature was provided.

Antimicrobial resistance pattern of Uro-Pathogens emphasizing non-lactose fermenting gram negative bacilli.

Objectives: To identify various species of non-lactose fermenting gram-negative bacilli involved in urinary tract infections, and to determine their antimicrobial resistance pattern. METHODS: The retrospective, descriptive, cross-sectional study was conducted from January 1 to April 1, 2022, at the Dow University of Health Sciences, Karachi, and comprised data from the institutional diagnostic laboratory that was related to urine samples regardless of age and gender from January 1, 2020, to December 31, 2021. Data was analysed using SPSS version 25. RESULTS: Of the 103,887 urine samples, 41,280(39.7%) were positive, 51,146(49.2%) showed no bacterial growth, 11,000(10.6%) had non-significant bacterial growth and 461(0.4%) had mixed bacterial growth. Of the positive samples, 18359(44.5%) were positive in 2020, and 22,921(55.5%) in 2021. Gram-negative lactose fermenting bacteria included escherichia coli 23,123(22.3%) and klebsiella pneumoniae 2,993(2.9%), gram-negative non-lactose fermenting bacteria included pseudomonas aeruginosa 1,110(1.07%), and gram-positive bacteria included enterococcus 8,008(7.7%). Pseudomonas aeruginosa was most resistant against tobramycin 880(79.3%) and least resistant against piperacillin-tazobactam 146(13%). CONCLUSIONS: Piperacillin-tazobactam was highly sensitive drug against non-lactose fermenting uro-pathogens.

In Vitro Evaluation of Phytobiotic Mixture Antibacterial Potential against Enterococcus spp. Strains Isolated from Broiler Chicken.

Enterococcus spp. are normal intestinal tract microflorae found in poultry. However, the last decades have shown that several species, e.g., Enterococcus cecorum, have become emerging pathogens in broilers and may cause numerous losses in flocks. In this study, two combinations (H1 and H2) of menthol, 1,8-cineol, linalool, methyl salicylate, γ-terpinene, p-cymene, trans-anethole, terpinen-4-ol and thymol were used in an in vitro model, analyzing its effectiveness against the strains E. cecorum, E. faecalis, E. faecium, E. hirae and E. gallinarum isolated from broiler chickens from industrial farms. To identify the isolated strains classical microbiological methods and VITEK 2 GP cards were used. Moreover for E. cecorum a PCR test was used.. Antibiotic sensitivity (MIC) tests were performed for all the strains. For the composition H1, the effective dilution for E. cecorum and E. hirae strains was 1:512, and for E. faecalis, E. faecium and E. gallinarum, 1:1024. The second mixture (H2) showed very similar results with an effectiveness at 1:512 for E. cecorum and E. hirae and 1:1024 for E. faecalis, E. faecium and E. gallinarum. The presented results suggest that the proposed composition is effective against selected strains of Enterococcus in an in vitro model, and its effect is comparable to classical antibiotics used to treat this pathogen in poultry. This may suggest that this product may also be effective in vivo and provide effective support in the management of enterococcosis in broiler chickens.

Spatio-Temporal Variation in the Exceedance of Enterococci in Lake Burley Griffin: An Analysis of 16 Years' Recreational Water Quality Monitoring Data.

Recreational waterbodies with high levels of faecal indicator bacteria (FIB) pose health risks and are an ongoing challenge for urban-lake managers. Lake Burley Griffin (LBG) in the Australian Capital city of Canberra is a popular site for water-based recreation, but analyses of seasonal and long-term patterns in enterococci that exceed alert levels (>200 CFU per 100 mL, leading to site closures) are lacking. This study analysed enterococci concentrations from seven recreational sites from 2001-2021 to examine spatial and temporal patterns in exceedances during the swimming season (October-April), when exposure is highest. The enterococci concentrations varied significantly across sites and in the summer months. The frequency of the exceedances was higher in the 2009-2015 period than in the 2001-2005 and 2015-2021 periods. The odds of alert-level concentrations were greater in November, December, and February compared to October. The odds of exceedance were higher at the Weston Park East site (swimming beach) and lower at the Ferry Terminal and Weston Park West site compared to the East Basin site. This preliminary examination highlights the need for site-specific assessments of environmental and management-related factors that may impact the public health risks of using the lake, such as inflows, turbidity, and climatic conditions. The insights from this study confirm the need for targeted monitoring efforts during high-risk months and at specific sites. The study also advocates for implementing measures to minimise faecal pollution at its sources.

Both biofilm cytotoxicity and monocytes' adhesion may be used as estimators of enterococcal virulence.

Our study aimed to identify markers of enterococci's virulence potential by evaluating the properties of strains of different sites of isolation. Enterococcal strains were isolated as commensals from faeces and as invasive strains from the urine and blood of patients from the University Clinical Centre, Gdansk, Poland. Changes in monocytes' susceptibility to the cytotoxic activity of isolates of different origins and their adherence to biofilm were evaluated using a flow cytometer. The bacterial protein profile was estimated by matrix assisted laser desorption ionization-time of flight mass spectrometer. The cytotoxicity of biofilm and monocytes' adherence to it were the most accurate factors in predicting the prevalence of the strain in the specific niche. Additionally, a bacterial protein with mass-to-charge ratio (m/z) 5000 was found to be responsible for the increased bacterial cytotoxicity, while monocytes' decreased adherence to biofilm was linked with the presence of proteins either with m/z 3330 or 2435. The results illustrate that monocytes' reaction when exposed to the bacterial biofilm can be used as an estimator of pathogens' virulence potential. The observed differences in monocytes' response are explainable by the bacterial proteins' profile. Additionally, the results indicate that the features of both bacteria and monocytes impact the outcome of the infection.

Dissemination, virulence characteristic, antibiotic resistance determinants of emerging linezolid and vancomycin-resistant Enterococcus spp. in fish and crustacean.

Enterococci are emerging nosocomial pathogens. Their widespread distribution causes them to be food contaminants. Furthermore, Enterococci can colonize various ecological niches and diffuse into the food chain via contaminated animals and foods because of their remarkable tolerance to unfavorable environmental circumstances. Due to their potential dissemination to humans, antimicrobial-resistant Enterococci in fish are a worldwide health issue. This study characterized AMR, ARGs, VAGs, gelatinase activity, and biofilm formation in Enterococcus spp. recovered from fish and seafood and evaluated potential correlations. 54 Enterococcus spp. strains(32.73 %)were isolated from 165 samples (75 Oreochromis niloticus, 30 Argyrosomus regius, and 60 Shrimp), comprising 30 Enterococcus faecalis (55.6 %) and 24 Enterococcus faecium (44.4 %) with total 32.73 % (54/165), The maximum prevalence rate of Enterococcus spp. was observed in Nile tilapia (34/54; 63 %), followed by shrimp (14/54; 25.9 %) and Argyrosomus regius (6/54; 11.1 %). The maximum prevalence rate of E. faecalis was observed in Nile tilapia (22/30; 73.3 %), followed by shrimp (8/30; 26.7 %) with significant differences. The prevalence rate of E. faecium was observed in Nile tilapia (12/24; 50 %), followed by shrimp (6/24,25 %). E. faecium is only isolated from Argyrosomus regius (6/24,25 %). Isolates exhibited high resistance against both tetracycline (90.7 %) and erythromycin(88.9 %), followed by gentamycin (77.8 %), ciprofloxacin (74.1 %), levofloxacin (72.2 %), penicillin (44.4 %), vancomycin (37 %), and linezolid (20.4 %). 50 strains (92.6 %) exhibited resistance to more than two antibiotics, 5 strains (10 %) were XDR, and the remaining 45 strains (90 %) were classified as MDR. 92.6 % of the isolates had MARindices >0.2, indicating they originated in settings with a high risk of contamination. Additionally, ten ARGs were identified, with tet(M) 92.6 %, followed by erm(B) (88.9 %), aac(6')-Ie-aph(2″)-Ia(77.8 %), tet(K) (75.9 %), gyrA (74.1 %), blaZ (48.1 %), vanA (37 %), vanB (31.5 %), optrA (20.4 %), and catA(3.7 %). Biofilm formation and gelatinase activity were observed in 85.2 %, and 61.1 % of the isolates, respectively. A total of 11 VAGs were detected, with gelE as the most prevalent (83.3 %) followed by agg(79.6 %), pil (74.1 %), both sprE and asa1 (72.2 %), hyl (70.4 %), eps(68.5 %), EF3314 (57.4 %), ace (50 %), and cylA (35.2 %) with no detection of cylB. In conclusion, the emergence of linezolid-resistant -vancomycin-resistant enterococci recovered from Egyptian fish and shrimp, suggests that fish and seafood might participate a fundamental part in the emergence of antimicrobial resistance among humans.

Transplanting human infant gut microbiome species into Galleria mellonella.

OBJECTIVE: Study of the human infant gut microbiome requires the use of surrogate mammalian species such as mice. We sought to investigate the usefulness of the greater wax moth larva, Galleria mellonella, as an alternative. RESULTS: We have analysed the native gut microbiome of Galleria and developed methods for clearing the native microbiome and introducing species from human infant faecal samples. We find that some species, e.g. enterococci, are more successful at recolonisation, but that others, e.g. Bifidobacterium, are less so. The work paves the way for using Galleria rather than mice in this and similar work.

CLI: A new protocol for the isolation of Lactic Acid Bacteria from complex plant samples.

Lactic Acid Bacteria (LAB) are predominantly probiotic microorganisms and the most are Generally Recognized As Safe (GRAS). LAB inhabit in the human gut ecosystem and are largely found in fermented foods and silage. In the last decades, LAB have also has been found in plant microbiota as a new class of microbes with probiotic activity to plants. For this reason, today the scientific interest in the study and isolation of LAB for agronomic application has increased. However, isolation protocols from complex samples such as plant tissues are scarce and inefficient. In this study, we developed a new protocol (CLI, Complex samples LAB Isolation) which yields purified LAB from plants. The sensitivity of CLI protocol was sufficient to isolate representative microorganisms of LAB genera (i.e. Leuconostoc, Lactococcus and Enterococcus). CLI protocol consists on five steps: i) sample preparation and pre-incubation in 1% sterile peptone at 30 °C for 24-48 h; ii) Sample homogenization in vortex by 10 min; iii) sample serial dilution in quarter-strength Ringer solution, iv) incubation in MRS agar plates with 0.2% of sorbic acid, with 1% of CaCO3, O2 < 15%, at pH 5.8 and 37 °C for 48 h.; v) Selection of single colonies with LAB morphology and CaCO3-solubilization halo. Our scientific contribution is that CLI protocol could be used for several complex samples and represents a useful method for further studies involving native LAB.

An analysis of culture-based methods used for the detection and isolation of Salmonella spp., Escherichia coli, and Enterococcus spp. from surface water: A systematic review.

Identification of methods for the standardized assessment of bacterial pathogens and antimicrobial resistance (AMR) in environmental water can improve the quality of monitoring and data collected, support global surveillance efforts, and enhance the understanding of environmental water sources. We conducted a systematic review to assemble and synthesize available literature that identified methods for assessment of prevalence and abundance of bacterial fecal indicators and pathogens in water for the purposes of monitoring bacterial pathogens and AMR. After screening for quality, 175 unique publications were identified from 15 databases, and data were extracted for analysis. This review identifies the most common and robust methods, and media used to isolate target organisms from surface water sources, summarizes methodological trends, and recognizes knowledge gaps. The information presented in this review will be useful when establishing standardized methods for monitoring bacterial pathogens and AMR in water in the United States and globally.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

Challenges for heat stress: Intestinal culturable bacteria of Lohmann Brown chickens.

This study aimed to assess how heat stress, specifically within the range of 35-38 °C, affects the populations of culturable intestinal lactobacilli, enterococci, and Escherichia coli, as well as the expression of Heat Shock Proteins (HSP70), in Lohmann Brown chickens. It also explored the influence of the chickens' blood transferrin and ceruloplasmin genotypes on these responses. Thirty chickens underwent eight hours of heat stress, maintained at an average temperature of 37 °C and a relative humidity of 75-80%, with continuous access to food and water. Behavioral monitoring was conducted throughout to prevent excessive heat-related mortality. The Lohmann Brown chickens from the Yerevan "Arax" poultry farm were initially classified based on their blood transferrin and ceruloplasmin genotypes to investigate potential correlations between intestinal bacterial composition and variations in these polymorphisms. A significant correlation was found between heat stress and the abundance of culturable enterococci within the intestinal microbiota, regardless of chicken TfAB, TfBC, CpAB, CpCC and TfAB, TfBC, CpAB, CpCD genotypes. Heat stress led to nearly double the HSP70 levels in chicken blood, along with a reduction in the culturable enterococci population by at least 10,000-fold in the intestinal microbiota. These findings are significant for targeted management strategies to mitigate heat stress in chicken populations.

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.

Identification and biological characteristics of Enterococcus casseliflavus TN-47 isolated from Monochamus alternatus.

With the widespread use of antibiotics, the incidence of antibiotic resistance in microorganisms has increased. Monochamus alternatus is a trunk borer of pine trees. This study aimed to investigate the in vitro antimicrobial and biological characteristics of Enterococcus casseliflavus TN-47 (PP411196), isolated from the gastrointestinal tract of M. alternatus in Jilin Province, PR China. Among 13 isolates obtained from the insects, five were preliminarily screened for antimicrobial activity. E. casseliflavus TN-47, which exhibited the strongest antimicrobial activity, was identified. E. casseliflavus TN-47 possessed antimicrobial activity against Staphylococcus aureus USA300 and Salmonella enterica serovar Pullorum ATCC 19945. Furthermore, E. casseliflavus TN-47 was sensitive to tetracyclines, penicillins (ampicillin, carbenicillin, and piperacillin), quinolones and nitrofuran antibiotics, and resistant to certain beta-lactam antibiotics (oxacillin, cefradine and cephalexin), macrolide antibiotics, sulfonamides and aminoglycosides. E. casseliflavus TN-47 could tolerate low pH and pepsin-rich conditions in the stomach and grow in the presence of bile acids. E. casseliflavus TN-47 retained its strong auto-aggregating ability and hydrophobicity. This strain did not exhibit any haemolytic activity. These results indicate that E. casseliflavus TN-47 has potential as a probiotic. This study provides a theoretical foundation for the future applications of E. casseliflavus TN-47 and its secondary metabolites in animal nutrition and feed.

The dysregulation of biliary tract microflora is closely related to primary choledocholithiasis: a multicenter study.

Bile microecology changes play an important role in the occurrence and development of choledocholithiasis. At present, there is no clear report on the difference of bile microecology between asymptomatic patients with gallbladder polyps and choledocholithiasis. This study compared bile microecology between gallbladder polyp patients and patients with choledocholithiasis to identify risk factors for primary choledocholithiasis. This study was conducted in 3 hospitals in different regions of China. Bile samples from 26 patients with gallbladder polyps and 31 patients with choledocholithiasis were collected by laparoscopic cholecystectomy and endoscopic retrograde choledocholithiasis cholangiography (ERCP), respectively. The collected samples were used for 16S ribosomal RNA sequencing and liquid chromatography mass spectrometry analysis. The α-diversity of bile microecological colonies was similar between gallbladder polyp and choledocholithiasis, but the ß-diversity was different. Firmicutes, Proteobacteri, Bacteroidota and Actinobacteriota are the most common phyla in the gallbladder polyp group and choledocholithiasis group. However, compared with the gallbladder polyp patients, the abundance of Actinobacteriota has significantly lower in the choledocholithiasis group. At the genera level, the abundance of a variety of bacteria varies between the two groups, and Enterococcus was significantly elevated in choledocholithiasis group. In addition, bile biofilm formation-Pseudomonas aeruginosa was more metabolically active in the choledocholithiasis group, which was closely related to stone formation. The analysis of metabolites showed that a variety of metabolites decreased in the choledocholithiasis group, and the concentration of beta-muricholic acid decreased most significantly. For the first time, our study compared the bile of gallbladder polyp patients with patients with choledocholithiasis, and suggested that the change in the abundance of Actinobacteriota and Enterococcus were closely related to choledocholithiasis. The role of Pseudomonas aeruginosa biofilm in the formation of choledocholithiasis was discovered for the first time, and some prevention schemes for choledocholithiasis were discussed, which has important biological and medical significance.

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.

Multidrug-resistant Enterococcus faecium strains enter the Norwegian marine environment through treated sewage.

This study aimed to understand the antibiotic resistance prevalence among Enterococcus spp. from raw and treated sewage in Bergen city, Norway. In total, 517 Enterococcus spp. isolates were obtained from raw and treated sewage from five sewage treatment plants (STPs) over three sampling occasions, with Enterococcus faecium as the most prevalent (n = 492) species. E. faecium strains (n = 307) obtained from the influent samples, showed the highest resistance against quinupristin/dalfopristin (67.8%). We observed reduced susceptibility to erythromycin (30.6%) and tetracycline (6.2%) in these strains. E. faecium strains (n = 185) obtained from the effluent samples showed highest resistance against quinupristin/dalfopristin (68.1%) and reduced susceptibility to erythromycin (24.9%) and tetracycline (8.6%). We did not detect resistance against last-resort antibiotics, such as linezolid, vancomycin, and tigecycline in any of the strains. Multidrug-resistant (MDR) E. faecium strains were detected in both influent (2.3%) and effluent (2.2%) samples. Whole genome sequencing of the Enterococcus spp. strains (n = 25) showed the presence of several antibiotic resistance genes, conferring resistance against aminoglycosides, tetracyclines, and macrolides, as well as several virulence genes and plasmid replicons. Two sequenced MDR strains from the effluents belonged to the hospital-associated clonal complex 17 and carried multiple virulence genes. Our study demonstrates that clinically relevant MDR Enterococcus spp. strains are entering the marine environment through treated sewage.

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.