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Gene emrC Associated with Resistance to Quaternary Ammonium Compounds Is Common among Listeria monocytogenes from Meat Products and Meat Processing Plants in Poland
(1) Background: L. monocytogenes is a food pathogen of great importance, characterized by a high mortality rate. Quaternary ammonium compounds (QACs), such as benzalkonium chloride (BC), are often used as disinfectants in food processing facilities. The effectiveness of disinfection procedures is crucial to food safety. (2) Methods: A collection of 153 isolates of L. monocytogenes from meat processing industry was analyzed for their sensitivity to BC using the agar diffusion method. Genes of interest were detected with PCR. (3) Results: Genes emrC, bcrABC, and qacH were found in 64 (41.8%), 6 (3.9%), and 1 isolate (0.7%), respectively, and 79 isolates (51.6%) were classified as having reduced sensitivity to BC. A strong correlation between carrying QACs resistance-related genes and phenotype was found (p-value < 0.0001). Among 51 isolates originating from bacon (collected over 13 months), 48 had the emrC gene, which could explain their persistent presence in a processing facility. Isolates with the ilsA gene (from LIPI-3) were significantly (p-value 0.006) less likely to carry QACs resistance-related genes. (4) Conclusions: Reduced sensitivity to QACs is common among L. monocytogenes from the meat processing industry. Persistent presence of these bacteria in a processing facility is presumably caused by emrC-induced QACs resistance.
Phage-Based Control of Listeria innocua in the Food Industry: A Strategy for Preventing Listeria monocytogenes Persistence in Biofilms
Listeria innocua, though considered non-pathogenic, frequently coexists with Listeria monocytogenes in industrial environments, aiding its survival in biofilms. These biofilms pose a significant challenge in food processing facilities, as they protect bacteria from disinfectants and facilitate their spread. The aim of this review was to identify bacteriophages as a promising method for eliminating Listeria biofilms from the food industry. Lytic bacteriophages show great potential in combating Listeria biofilms. Commercially available products, such as PhageGuard Listex™ (P100) (Micreos Food Safety, Wageningen, The Netherlands), effectively reduce both L. monocytogenes and L. innocua in food products and on production surfaces. Additionally, phage-derived enzymes, such as endolysins, can degrade biofilms, eliminating bacteria without compromising food quality. The following article highlights that although bacteriophages present a promising biocontrol method, further research is necessary to assess their long-term effectiveness, particularly regarding bacterial resistance. To maximize efficacy, a combination of strategies such as phage cocktails and disinfectants is recommended to enhance biofilm eradication and minimize food contamination risks.
The Genetic Determinants of Listeria monocytogenes Resistance to Bacteriocins Produced by Lactic Acid Bacteria
Background: Listeria monocytogenes is a Gram-positive bacterium responsible for listeriosis, a serious foodborne disease that can lead to serious health complications. Pregnant women, newborns, the elderly, and patients with weakened immune systems are particularly susceptible to infection. Due to the ability of L. monocytogenes to survive in extreme environmental conditions, such as low temperatures, high salinity, and acidity, this bacterium poses a serious threat to food production plants and is particularly difficult to eliminate from these plants. One of the promising solutions to reduce the presence of this bacterium in food products is bacteriocins as natural control agents. These are substances with antibacterial activity produced by other bacteria, mainly lactic acid bacteria (LAB), which can effectively inhibit the development of pathogens such as L. monocytogenes. The use of bacteriocins in the food industry is beneficial due to their natural origin, specificity of action, and consumer safety. However, the problem of resistance to these substances exists. Results: This review focuses on the mechanisms of bacteriocin resistance, such as modifications of bacteriocin docking receptors, changes in the structure of the cell wall and membrane, and the occurrence of cross-resistance to different bacteriocins. Genetic factors determining these mechanisms and strategies to cope with the problem of resistance are also presented. Conclusions: Research on this issue is crucial for developing effective preventive methods that will enable the safe and long-term use of bacteriocins in food production.
Characteristics of Intestinal Barrier State and Immunoglobulin-Bound Fraction of Stool Microbiota in Advanced Melanoma Patients Undergoing Anti-PD-1 Therapy
The gut microbiota is recognized as one of the extrinsic factors that modulate the clinical outcomes of immune checkpoint inhibitors (ICIs) in cancer patients. However, the role of the intestinal barrier, which mutually interacts with the gut microbiota, in shaping anti-cancer immune responses has not been extensively studied so far. Therefore, the primary goal of our study was to investigate the relationship between intestinal barrier functionality and clinical outcomes of anti-PD-1 therapy in patients with advanced melanoma. Fecal samples were collected from 64 patients before and during anti-PD-1 therapy. The levels of zonulin, calprotectin, and secretory immunoglobulin A (SIgA), which reflect intestinal permeability, inflammation, and immunity, respectively, were measured in fecal samples (n = 115) using ELISA. Moreover, the composition of the immunoglobulin (Ig)-bound (n = 108) and total stool microbiota (n = 117) was determined by the V3-V4 region of 16S rRNA gene sequencing. ELISA indicated a higher baseline concentration of fecal SIgA in patients with favorable clinical outcomes than those with unfavorable ones. Moreover, high baseline concentrations of intestinal barrier state biomarkers correlated with survival outcomes. In the cases of fecal zonulin and fecal SIgA, there was a positive correlation, while in the case of fecal calprotectin, a negative correlation. Furthermore, there were differences in the microbial profiles of the Ig-bound stool microbiota between patients with favorable and unfavorable clinical outcomes and their changes during treatment. Collectively, intestinal barrier functionality was associated with clinical outcomes of anti-PD-1 therapy in advanced melanoma patients. Future studies are warranted to elucidate whether intestinal barrier modification could improve ICI efficacy and estimate the clinical value and utility of biomarkers reflecting its state.
Genoserotyping of Listeria monocytogenes strains originating from meat products and meat processing environments
Background. Listeria monocytogenes is a foodborne human pathogen and a causative factor of listeriosis, which is an illness with a high mortality rate. Serotyping is a method for differentiating L. monocytogenes isolates based on unique combinations of somatic (O) and flagellar (H) antigens on the surface of their cells. Standard serotyping involves agglutination methods, which require using antisera. However, there are also genoserotyping methods which allow to categorise L. monocytogenes isolates into particular groups of serotypes (referred to as serogroups) based on genetic analyses. Differentiating L. monocytogenes isolates is an important issue in terms of food safety, surveillance and traceability of contamination sources. In this work, we present results of the genoserotyping of 153 L. monocytogenes isolates originating from meat products and meat processing environments at Polish processing plants. Two protocols were used for genoserotyping analyses: the first one allows to differentiate between four most common serotypes (1/2a, 1/2b, 1/2c and 4b) and the second one allows to distinguish hipervirulent serovar 4h from other serotypes. Results and conclusion. Results achieved using both methods were consistent and all isolates were categorised into corresponding serogroups within the two methodologies. Most of the isolates (73.9 %) were characterised as members of the IIa serogroup (representing the 1/2a, 3a serovars). The IVb (4b, 4d, 4e) serogroup was the second most common (and comprised 18.3 % of isolates), followed by IIb (1/2b, 3b, 7) and IIc (1/2c, 3c), however, the last two groups were equally numerous (and each of them comprised 3.9 % of all isolates). None of the collected isolates belonged to the serogroup representing the 4a, 4c, 4ab and 4h serotypes.
Circulating Microbial Cell-Free DNA in Health and Disease
Human blood contains low biomass of circulating microbial cell-free DNA (cfmDNA) that predominantly originates from bacteria. Numerous studies have detected circulating cfmDNA in patients with infectious and non-infectious diseases, and in healthy individuals. Remarkable differences were found in the microbial composition of healthy subjects and patients compared to cohorts with various diseases or even patients with diversified prognoses, implying that these alterations may be associated with disease development. Although the function of circulating cfmDNA needs to be elucidated (whether it acts as a bystander of dysbiosis or a key player in disease development), several studies have demonstrated its potential as a non-invasive biomarker that may improve diagnosis and treatment efficacy. The origin of circulating cfmDNA is still the subject of much deliberation, but studies have identified members of various microbiome niches, including the gut, oral cavity, airways, and skin. Further studies investigating the origin and function of circulating cfmDNA are needed. Moreover, low-biomass microbiome studies are prone to contamination, therefore stringent negative experimental control reactions and decontamination frameworks are advised in order to detect genuine circulating cfmDNA.
Lactiplantibacillus plantarum, Duodenal Hydroxyphenyllactic Acid and Iron: Insights from a Rat Model of a High-Fat Iron-Deficient Diet
A Clinical Outcome of the anti-PD-1 Therapy of Melanoma in Polish Patients Is Mediated by Population-Specific Gut Microbiome Composition
Gut microbiota is considered a key player modulating the efficacy of immune checkpoint inhibitor therapy. The study investigated the association between response to the anti-PD-1 therapy and the baseline gut microbiome in the Polish cohort of melanoma patients, alongside selected agents modifying the microbiome. Sixty-four melanoma patients enrolled for the anti-PD-1 therapy and 10 healthy subjects were recruited. Response to the treatment was assessed according to the response evaluation criteria in solid tumors, and patients were classified as responders or non-responders. The association between selected extrinsic factors and response was investigated using questionnaire-based analysis, and metataxonomics of the microbiota. The Bacteroidota to Firmicutes ratio was higher, and the richness was decreased in the responders. The abundance of Prevotella copri and Bacteroides uniformis was related to the response, whereas non-responder gut microbiota was enriched with Faecalibacterium prausnitzii and Desulfovibrio intestinalis, and some unclassified Firmicutes. Dietary patterns, including plant, dairy, and fat consumption, but also gastrointestinal tract functioning were significantly associated with the therapeutic effects of the therapy. The specific gut microbiota alongside diet were found associated with response to the therapy in the Polish population of melanoma patients.
Characteristics of Intestinal Barrier State and Immunoglobulin-Bound Fraction of Stool Microbiota in Advanced Melanoma Patients Undergoing Anti-PD-1 Therapy
The gut microbiota is recognized as one of the extrinsic factors that modulate the clinical outcomes of immune checkpoint inhibitors (ICIs), such as inhibitors targeting programmed cell death protein 1 (PD-1), in cancer patients. However, the link between intestinal barrier, which mutually interacts with the gut microbiota, and therapeutic effects has not been extensively studied so far. Therefore, the primary goal of this study was to investigate the relationship between intestinal barrier functionality and clinical outcomes of anti-PD-1 therapy in patients with advanced melanoma. Fecal samples were collected from 64 patients before and during anti-PD-1 therapy. The levels of zonulin, calprotectin, and secretory immunoglobulin A (SIgA), which reflect intestinal permeability, inflammation, and immunity, respectively, were measured in fecal samples (n = 115) using an Enzyme-Linked Immunosorbent Assay (ELISA). Moreover, the composition of the immunoglobulin (Ig)-bound (n = 108) and total stool microbiota (n = 117) was determined by the V3–V4 region of 16S rRNA gene sequencing. ELISA indicated a higher baseline concentration of fecal SIgA in patients with favorable clinical outcomes than those with unfavorable ones. Moreover, high baseline concentrations of intestinal barrier state biomarkers correlated with survival outcomes. In the cases of fecal zonulin and fecal SIgA, there was a positive correlation, while in the case of fecal calprotectin, there was a negative correlation. Furthermore, there were differences in the microbial profiles of the Ig-bound stool microbiota between patients with favorable and unfavorable clinical outcomes and their changes during treatment. Collectively, these findings indicate an association between intestinal barrier functionality and clinical outcomes of anti-PD-1 therapy in advanced melanoma patients.
Charakterystyka frakcji mikrobioty jelitowej związanej z wydzielniczymi immunoglobulinami A (SlgA) u pacjentów z zaawansowanym czerniakiem poddanych immunoterapii anty-PD-1
Analiza wolnego pozakomórkowego DNA o pochodzeniu bakteryjnym u pacjentów z zaawansowanym czerniakiem poddanych immunoterapii anty-PD-1
Antibiotic Resistance Profiles and Genetic Determinants of Listeria innocua Isolated from Food Sources in Poland
Background: Antimicrobial resistance (AMR) is a growing public health concern affecting both medicine and food safety. While Listeria monocytogenes is the primary pathogen of concern, Listeria innocua—commonly found in food and food-processing environments—may serve as a reservoir for resistance genes and a useful indicator of species for surveillance. This study aimed to assess the phenotypic antibiotic susceptibility and detect resistance-associated genes in L. innocua isolates from meat products and processing environments in Poland. Methods: A total of 51 L. innocua isolates were analyzed, originating from raw and processed meat products as well as meat-processing environments. Antimicrobial susceptibility was determined using the disc diffusion method against 18 antibiotics representing multiple classes. Phenotypic resistance was interpreted following CLSI guidelines (CLSI, 2020). Isolates exhibiting resistance or intermediate resistance were further screened for resistance-associated genes using PCR. Results: All isolates were fully susceptible to ampicillin, benzylpenicillin, chloramphenicol, gentamicin, rifampin, trimethoprim-sulfamethoxazole, and vancomycin. High susceptibility was observed for ciprofloxacin, erythromycin, meropenem, trimethoprim, and nitrofurantoin, with only sporadic intermediate responses. Moderate resistance levels were noted for streptomycin (10%) and tetracycline (12%). The lowest susceptibility was recorded for clindamycin and linezolid, with most isolates exhibiting intermediate or resistant phenotypes. Universal resistance to cefotaxime and oxacillin was found. Eighteen distinct resistance patterns were identified. PCR confirmed the presence of several resistance-associated genes, including mecA, lnuA, lnuB, cfr, optrA, and poxtA, consistent with observed phenotypes. Conclusions: This study provides the first detailed characterization of AMR in L. innocua from Polish meat and processing environments. The findings highlight its heterogeneous resistance profiles and potential role as a reservoir of clinically relevant resistance genes. Incorporating L. innocua into surveillance programs may strengthen early detection of emerging resistance and enhance food safety monitoring.
Influence of supplementation with probiotic bacteria Lactiplantibacillus plantarum and Latilactobacillus curvatus on selected parameters of duodenum iron metabolism in rats on a high-fat, iron-deficient diet
A Clinical Outcome of the Anti-PD-1 Therapy of Melanoma in Polish Patients Is Mediated by Population-Specific Gut Microbiome Composition
The gut microbiota is considered a key player modulating the efficacy of immune checkpoint inhibitor therapy. The study investigated the association between the response to anti-PD-1 therapy and the baseline gut microbiome in a Polish cohort of melanoma patients, alongside selected agents modifying the microbiome. Sixty-four melanoma patients enrolled for the anti-PD-1 therapy, and ten healthy subjects were recruited. The response to the treatment was assessed according to the response evaluation criteria in solid tumors, and patients were classified as responders or non-responders. The association between selected extrinsic factors and response was investigated using questionnaire-based analysis and the metataxonomics of the microbiota. In the responders, the Bacteroidota to Firmicutes ratio was higher, and the richness was decreased. The abundance of Prevotella copri and Bacteroides uniformis was related to the response, whereas the non-responders’ gut microbiota was enriched with Faecalibacterium prausnitzii and Desulfovibrio intestinalis and some unclassified Firmicutes. Dietary patterns, including plant, dairy, and fat consumption as well as gastrointestinal tract functioning were significantly associated with the therapeutic effects of the therapy. The specific gut microbiota along with diet were found to be associated with the response to the therapy in the population of melanoma patients.
Nonhemolytic Listeria monocytogenes - Prevalence Rate, Reasons Underlying Atypical Phenotype, and Methods for Accurate Hemolysis Assessment
Listeria monocytogenes is a foodborne pathogen that typically presents β-hemolytic activity. However, there are literature reports indicating that L. monocytogenes strains are sometimes nonhemolytic or their zones of hemolysis are perceivable only after removal of the colonies from the agar plate. Nonhemolytic L. monocytogenes are most commonly encountered in food products, but some have also been detected in clinical samples. Usually, atypical bacteria of this species belong to serotype 1/2a. Mutations of the prfA gene sequence are the most common reason for changed phenotype, and mutations of the hly gene are the second most common cause. There are also reports that the methodology used for detecting hemolysis may influence the results. Sheep or horse blood, although most commonly used in modern studies, may not allow for the production of clear hemolytic zones on blood agar, whereas other types of blood (guinea pig, rabbit, piglet, and human) are more suitable according to some studies. Furthermore, the standard blood agar plate technique is less sensitive than its modifications such as bilayer or top-layer (overlay) techniques. The microplate technique (employing erythrocyte suspensions) is probably the most informative when assessing listerial hemolysis and is the least susceptible to subjective interpretation.
Listeria monocytogenes Isolates from Meat Products and Processing Environment in Poland Are Sensitive to Commonly Used Antibiotics, with Rare Cases of Reduced Sensitivity to Ciprofloxacin
Antibiotic resistance is a global health problem, causing not only an increased mortality rate of bacterial infections but also economic losses due to, among other reasons, the need for longer hospital stays. Listeria monocytogenes is one of the foodborne pathogens with the ability to induce a serious illness called listeriosis, with approximately 20–30% fatal outcomes. The treatment regimen of listeriosis in humans includes the administration of antibiotics (in most cases, ampicillin or trimethoprim with sulfamethoxazole in case of allergies to β-lactams), so the resistance of this pathogen to antibiotics can potentially lead to increased mortality. The antibiotic sensitivity status of n = 153 L. monocytogenes isolates originating from meat food samples (raw and processed) and meat-processing environment (both contacting and non-contacting with food) collected between October 2020 and November 2021 in Poland was examined in this study. Susceptibility to antibiotics was determined using the disc diffusion method on Mueller–Hinton agar plates. All collected samples were susceptible to 9 antibiotics: ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), penicillin (10 IU), streptomycin (10 µg), sulfamethoxazole/trimethoprim (1.25/23.75 µg), tetracycline (30 µg) and vancomycin (30 µg). Some of the isolates (n = 10; 6.5%) showed reduced susceptibility to ciprofloxacin (5 µg), which was classified as an intermediate response. All these ten isolates were collected from surfaces contacting with food in food-processing facilities.
Influence of supplementation of probiotic bacteria Lactobacillus plantarum and Lactobacillus curvatus on selected parameters of liver iron metabolism in rats on high-fat iron-deficient diet
High Prevalence of Virulence-Associated Genes and Length Polymorphism in actA and inlB Genes Identified in Listeria monocytogenes Isolates from Meat Products and Meat-Processing Environments in Poland
Listeria monocytogenes is a human pathogen that has the ability to cause listeriosis, a disease with possible fatal outcomes. The typical route of infection is ingestion of the bacteria with contaminated food. In this study, 13 virulence-associated genes were examined with PCR in the genomes of 153 L. monocytogenes isolates collected from meat products and processing environments in Poland. All isolates possessed genes from LIPI-1—hly, actA, plcA, plcB and mpl—as well as four internalins: inlA, inlB, inlC, inlJ. Invasion-associated protein iap, as well as genes prfA and sigB, encoding regulatory proteins, were also detected in all isolates. Gene flaA, encoding flagellin, was detected in 113 (74%) isolates. This was the only gene that was not detected in all isolates, as its presence is serotype-dependent. Gene actA showed polymorphism with longer and shorter variants in PCR amplicons. Two isolates were characterized by truncated inlB genes, lacking 141 bp in their sequence, which was confirmed by gene sequencing. All isolates were positive in hemolysis assays, proving the synthesis of functional PrfA and Hly proteins. Four genotypes of L. monocytogenes based on actA polymorphism and two genotypes based on inlB polymorphism were distinguished within the isolates’ collection.
