Staphylococcus Research Articles

Development and validation of a multiplex bead-based immunoassay for the simultaneous detection of fifteen pathogenic biological agents

In this study, a multiplex bead-based capture sandwich immunoassay (MIA) for the simultaneous detection of fifteen plant and bacterial Gram-positive and Gram-negative pathogenic biological agents (PBAs) was developed. This study included the selection of “sandwich” pairs of monoclonal antibodies, the development of an analytical protocol, and an evaluation of the specificity and sensitivity of detection. The working range, linearity, precision, and limits of detection of the developed assay were evaluated. The LLOQ of the multiplex assay was around 104 – 105 CFU/ml for bacteria and 0,4 ng/ml – 6,6 ng/ml for antigens. The assay can be used for the simultaneous detection of 15 PBAs, including Francisella tularensis, Brucella abortus, Bacillus anthracis, Legionella pneumophila, Staphylococcus aureus, Staphylococcus haemolyticus, Pseudomonas aeruginosa, and Salmonella typhimurium, as well as cholera toxin beta-subunit, viscumin, ricin, staphylococcal enterotoxins A and B, and botulinum toxins A and B.

Nasal carriage of Staphylococcus aureus in healthy dairy cows in Algeria: antibiotic resistance, enterotoxin genes and biofilm formation

BackgroundStaphylococcus aureus can colonize and infect a variety of animal species. In dairy herds, it is one of the leading causes of mastitis cases. The objective of this study was to characterize the S. aureus isolates recovered from nasal swabs of 249 healthy cows and 21 breeders of 21 dairy farms located in two provinces of Algeria (Tizi Ouzou and Bouira).MethodsThe detection of enterotoxin genes was investigated by multiplex PCRs. Resistance of recovered isolates to 8 antimicrobial agents was determined by disc-diffusion method. The slime production and biofilm formation of S. aureus isolates were assessed using congo-red agar (CRA) and microtiter-plate assay. Molecular characterization of selected isolates was carried out by spa-typing and Multi-Locus-Sequence-Typing (MLST).ResultsS. aureus was detected in 30/249 (12%) and 6/13 (28.6%) of nasal swabs in cows and breeders, respectively, and a total of 72 isolates were recovered from positive samples (59 isolates from cows and 13 from breeders). Twenty-six of these isolates (36.1%) harbored genes encoding for staphylococcal enterotoxins, including 17/59 (28.8%) isolates from cows and 9/13 (69.2%) from breeders. Moreover, 49.1% and 92.3% of isolates from cows and breeders, respectively, showed penicillin resistance. All isolates were considered as methicillin-susceptible (MSSA). Forty-five (76.3%) of the isolates from cows were slime producers and 52 (88.1%) of them had the ability to form biofilm in microtiter plates. Evidence of a possible zoonotic transmission was observed in two farms, since S. aureus isolates recovered in these farms from cows and breeders belonged to the same clonal lineage (CC15-ST15-t084 or CC30-ST34-t2228).ConclusionsAlthough healthy cows in this study did not harbor methicillin-resistant S. aureus isolates, the nares of healthy cows could be a reservoir of enterotoxigenic and biofilm producing isolates which could have implications in human and animal health.

Assessment of Microbial Contamination and Metabolite Exposure in Cosmetic Products Used in Women's Beauty Salons.

An important route of microbial transmission is the shared use of these products in beauty salons. We aimed to investigate level of contamination with microorganisms and their metabolites in shared-use cosmetics in several women's beauty salons. Bacterial and fungal strains from 320 opened/used cosmetic samples were identified according to the Iranian standards for microbial quality of cosmetic products in Golestan Province (North of Iran) during Jul-Sep 2021. In order to assess production of toxins and protease by the predominant bacterial and fungal isolates, multiplex-polymerase chain reaction and the Lowry protein assay were performed, respectively. Microbial contamination was detected in 180 samples (56.5%), and the highest and lowest rates of microbial contamination were related to skin products (63.88%) and eye beauty products (20%), respectively. The highest level of S. aureus contamination (> 4,000 colony-forming units/g) was found in toner and face wash samples, and the highest level of C. albicans contamination was seen in lipstick samples (>20,000 colony-forming units/g). Only one (2%) S. aureus isolate produced staphylococcal enterotoxin B, while 3 out of 12 (25%) C. albicans isolates were able to produce protease. The shared-used health and beauty products, face products, in the study area are heavily contaminated. Therefore, it is essential to store used cosmetics in dry and cool places, establish strict inspection regulations for cosmetic products before and after entering the market, and increase awareness of beauty salon workers regarding the appropriate use, sanitary control, and maintenance of health and beauty products.

Staphylococcal enterotoxin B exposed to pregnant rats inhibits the hedgehog signaling pathway in thymic T lymphocytes of the offspring

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 μg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.

Janus plasmonic-aggregation induced emission nanobeads as high-performance colorimetric–fluorescent probe of immunochromatographic assay for the ultrasensitive detection of staphylococcal enterotoxin B in milk

Herein, a colorimetric–fluorescent hybrid bifunctional nanobead with Janus structure (J-cf-HBN) was synthesized via one-pot microemulsification. Oleylamine-coated AuNPs and aggregation-induced emission luminogens (AIEgens) were suggested as building blocks to obtain high-performance colorimetric–fluorescent signals. The as-prepared J-cf-HBNs were used as a signal amplification probe to construct an immunochromatographic assay (J-cf-HBNs-ICA) platform for the ultrasensitive detection of staphylococcal enterotoxin B (SEB) in milk samples. Owing to the rational spatial distribution of AuNPs and AIEgens, the J-cf-HBNs present a highly retained photoluminescence and enhanced colorimetric signals. Combined with a pair of highly affinitive anti-SEB antibodies, the J-cf-HBN-ICA platform enabled the fast naked-eye visualization and fluorescent quantitative detection of SEB in various milk matrices. Given the advantages of the dual-mode high-performance J-cf-HBNs, the proposed strip achieved a high sensitivity for SEB qualitative determination with a visual limit of detection (LOD) of 1.56 ng mL−1 and exhibited ultrasensitivity for SEB quantitative detection with a LOD of 0.09 ng mL−1, which is 139-fold lower than that of ELISA using same antibodies. In conclusion, this work provides new insights into the construction of multimode immunochromatographic methods for food safety detection in the field.

Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry.

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25+CD69+CD40L+TNF-α- T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25+CD69+TNF-α- T cells were identified. Within CD8α+ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs.

In vitro selection of DNA aptamers against staphylococcal enterotoxin A

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Double-Enhanced Photothermal Lateral Flow Biosensor Based on Dual Gold Nanoparticle Conjugates.

Bacterial toxins emerge as the primary triggers of foodborne illnesses, posing a significant threat to human health. To ensure food safety, it is imperative to implement point-of-care testing methods. Lateral flow biosensors (LFBs) based on gold nanoparticles (GNPs) have been commonly used for rapid detection, but their applicationis limited by low sensitivity. Based on the localized surface plasmon resonance and photothermal effect of dual gold nanoparticle conjugates (DGNPs), we developed a smartphone-integrated photothermal LFB (PLFB) with double-enhanced colorimetric and photothermal sensitivity. Through numerical simulations, we verified that DGNPs have significantly enhanced photothermal performance compared to single 15 nm GNPs (SGNPs), and applied DGNPs in PLFB for the detection of staphylococcus enterotoxin A (SEA). The colorimetric and photothermal limits of detection of DGNPs-based PLFB for SEA were 0.091 and 0.0038 ng mL-1, respectively. Compared with the colorimetric detection of the SGNPs-based LFB, the colorimetric detection sensitivity of the DGNPs-based PLFB was increased by 10.7 times, and the photothermal detection sensitivity was further improved by 255.3 times. Moreover, the PLFB exhibits robust reproducibility and exceptional specificity and is applicable for detecting SEA in milk samples. This smartphone-integrated PLFB based on DGNPs allows users to detect toxins simply, conveniently, and quickly and has huge application potential in the field of food safety.

Phenotypic and Genotypic Characterization of Staphylococcus aureus Isolated from Nasal Samples of Healthy Dairy Goats in Algeria.

The present study aimed to determine the phenotypic and genotypic characteristics of S. aureus isolates from the nasal swabs of goats. A total of 232 nasal samples (one per animal) were collected from goats on 13 farms located in two regions of Algeria and were analyzed for the presence of S. aureus. The detection of virulence factors was carried out using PCR. The antibiotic susceptibility of the recovered isolates was assessed using the disc diffusion method. The biofilm formation ability was assessed by the Congo red agar method and a microtiter plate assay, and the molecular characterization of isolates was carried out by spa-typing, and for selected isolates also by multilocus sequence typing (MLST). Overall, 36 out of 232 nasal swabs (15.5%) contained S. aureus, and 62 isolates were recovered. Regarding the virulence factors, at least one staphylococcal enterotoxin gene was detected in 30 (48.4%) isolates. The gene tst encoding the toxic shock syndrome toxin was detected in fifteen isolates (24.2%), but none of the isolates harbored the gene of Panton-Valentine leukocidin (lukF/S-PV). Nine different spa-types were identified, including the detection of a new one (t21230). The recovered isolates were assigned to three clonal complexes, with CC5 (51.8%) being the most common lineage. Two isolates were methicillin-resistant (MRSA) and belonged to ST5 (CC5) and to spa-types t450 and t688. Moreover, 27 (43.5%) of the S. aureus isolates were found to be slime producers in Congo red agar, and all of the recovered isolates could produce biofilms in the microtiter plate assay. Our study showed that the nares of healthy goats could be a reservoir of toxigenic and antibiotic-resistant strains of S. aureus isolates, including MRSA, which could have implications for public health.

Phage K exposure generates phage-neutralizing activity in a rabbit model-humoral receptiveness may impact efficacy of phage therapy.

Phage therapy has not been established in the clinical routine, in part due to uncertainties concerning efficacy and immunogenicity. Here, three rabbits were immunized against staphylococcal phage K to assess viral potency in the presence of immunized serum. Three rabbits received weekly intramuscular injections of ~1010±1 pfu/mL phage K. Phage K-specific IgG formation was measured by an enzyme-linked immunosorbent assay (ELISA); phage inactivation was assessed by calculating K-rates. Using transmission electron microscopy (TEM) and immunogold labeling, antibody binding to phage K was visualized. This was numerically assessed by objective imaging analysis comparing the relative distances of each gold particle to the nearest phage head and tail structure. Immunization led to a strong IgG response, plateauing 7 days after the last phage injection. There was no significant correlation between K-rate and antibody titer over time. TEM showed IgG binding to the head structure of phage K. Image analysis showed a significant reduction in relative distances between antibodies and phage head structures when comparing samples from day 0 and day 28 (P < 0.0001). These results suggest that while individual serum analysis for antibodies against therapeutic phage bears consideration prior to and with prolonged therapy, during phage application, the formation of specific antibodies against phage may only partially explain decreased phage potency in the presence of immunized serum. Instead, other factors may contribute to an individual's "humoral receptiveness" to phage therapy. Future investigations should be directed toward the identification of the humoral factors that have the most significant predictive value on phage potency in vivo.

Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

OCCURRENCE OF VIRULENCE GENES OF Staphylococcus aureus AND Escherichia coli IN VENDED FOODS IN SELECTED MOTOR-PARKS IN OBIO/AKPOR, RIVERS STATE

The study aimed at evaluating the microbiological safety of vended foods in selected motor parks in Obio/Akpor Local Government Area, Rivers State, Nigeria. Forty-eight samples obtained from four motor parks (Choba, Rumuokoro, Eliozu and Oil-mill Park) were analyzed using standard microbiological methods for total heterotrophic bacteria count, virulent and antibiotic resistant profiles of Staphylococcus aureus and Escherichia coli. Total heterotrophic bacteria count ranged from 4.60 to 8.39 log10CFU/g. A total of 10 (20.83%) samples were positive for S. aureus while 14 (29.17%) were positive for E. coli. Total staphylococcal and Escherichia coli counts ranged from 4.89 to 7.01 and 2.70 to 5.31 log10cfu/g, respectively. Both bacteria were 100% resistant to augmentin. The confirmed E. coli showed varying resistant to cefixime (50%), ceftazidime (42.86%), cefuroxim (14.29%) and ofloxacin (7.14%); while S. aureus also showed varying resistant to erythromycin (10.00%), ceftriaxone (90.00%), cloxacillin (100.00%) and gentamycin (0.00%) The confirmed E. coli produced the expected bands for the E. coli attaching and effusing (eaeA), aggregative adherence fimbriae (aggR) and antimicrobial sensitivity testing (astA) virulence genes while S. aureus did not produce the expected bands with staphylococcal enterotoxin A (sea) and staphylococcal enterotoxin B (seb) genes. The detection of virulence gene bearing E. coli and multiple antibiotic resistant E. coli and S. aureus portends danger for commuters who patronizes these foods, hence the need for urgent public health interventions.

Staphylococcus aureus Isolated From Traditional Artisanal Raw Milk Cheese from Southern Brazil: Diversity, Virulence, and Antimicrobial Resistance Profile

Staphylococcus aureus is one of the primary pathogenic agents found in cheeses produced with raw milk. Some strains of S. aureus are enterotoxigenic, possessing the ability to produce toxins responsible for staphylococcal food poisoning when present in contaminated foods. This study aimed to genotypically characterize, assess the antimicrobial resistance profile, and examine the enterotoxigenic potential of strains of S. aureus isolated from artisanal colonial cheese. Additionally, a bacterial diversity assessment in the cheeses was conducted by sequencing the 16S rRNA gene. The metataxomic profile revealed the presence of 68 distinct species in the cheese samples. Fifty-seven isolates of S. aureus were identified, with highlighted resistance to penicillin in 33% of the isolates, followed by clindamycin (28%), erythromycin (26%), and tetracycline (23%). The evaluated strains also exhibited inducible resistance to clindamycin, with nine isolates considered multidrug-resistant (MDR). The agr type I was the most prevalent (62%) among the isolates, followed by agr type II (24%). Additionally, ten spa types were identified. Although no enterotoxins and their associated genes were detected in the samples and isolates, respectively, the Panton-Valentine leukocidin gene (lukS-lukF) was found in 39% of the isolates. The presence of MDR pathogens in the artisanal raw milk cheese production chain underscores the need for quality management to prevent the contamination and dissemination of S. aureus strains.

Multiplex Detection of Seven Staphylococcal Enterotoxins Using Liquid Chromatography–Mass Spectrometry Combined with a Novel Capture Molecule

Food poisoning caused by Staphylococcal enterotoxins (SEs) is prevalent globally, making efficient detection of these toxins very important. Traditionally, liquid chromatography–mass spectrometry required immunosorbent enrichment by magnetic bead-coupled antibodies obtained by animal-specific immunization. However, this method is time-consuming and costly. In this study, two recombinant protein capture molecules were designed based on the principle of toxins binding to Major Histocompatibility Complex (MHCII) and T cell receptor (TCR) molecules. The two capture molecules are called MHCII and MHCII-D10. The design of the MHCII and TCR-D10 was achieved through searching for the binding site protein sequence of Staphylococcal enterotoxins in the relevant literature, and MHCII-D10 was to link MHCII sequence with TCR-D10 sequence using linker (G4S)3 linking peptide. These capture molecules were shown to effectively bind to seven types of toxins and to capture SEs in various matrices. The digestion time, ratio, and temperature were further optimized, reducing the overall digestion time to just 2 h. The specificity, linearity, sensitivity, precision (RSD%), and recovery of the two methods were verified by liquid chromatography–mass spectrometry. When the MHCII and MHCII-D10 captured the toxins, the limit of quantification (LOD) in the 1 × PBS, plasma, and milk matrices ranged from 1.5625 to 100 fmol/µL, with the recovery rate ranging from 18.4% to 96%. The design of these capture molecules eliminates the need for animal-specific immunization, simplifying the pre-detection process and avoiding ethical concerns. This development holds significant promise for clinical diagnosis and reference.

Microbiological Risks of Traditional Raw Cow's Milk Cheese (Koryciński Cheeses).

Traditional and regional foods have been increasing in popularity among consumers in Poland for many years. The observed trend of searching for natural and authentic taste encourages many producers to craft products from raw milk, including Koryciński cheeses. The aim of this study was to assess the microbiological hazards resulting from the presence of pathogenic bacteria in Koryciński cheeses available in retail trade. The tests were carried out using accredited methods, including the detection of the presence of Salmonella spp., the enumeration of Listeria monocytogenes, the enumeration of coagulase-positive staphylococci, and the detection of staphylococcal enterotoxins in food when the number of coagulase-positive staphylococci in the sample exceeded the limit of 105 cfu/g. The research material consisted of 45 Koryciński cheeses. The tests conducted revealed that Salmonella spp. was not detected in any of the examined cheeses. However, coagulase-positive staphylococci were present in 68.9% of the samples. In as many as 15 tested cheeses, the level of S. aureus contamination was above 105 cfu/g; therefore, these samples were tested for the presence of staphylococcal enterotoxins. The presence of staphylococcal enterotoxins was found in one Koryciński cheese. In four cheeses, the number of L. monocytogenes exceeded the level of 102 cfu/g, the limit specified in Regulation 2073/2005 on microbiological criteria for foodstuffs. The obtained research results confirm the validity of monitoring the microbiological quality of Koryciński cheeses and the need to increase awareness of ensuring proper hygienic conditions of production, including the increased risk associated with unpasteurized milk products.

Prevalence, characterization, and implications of methicillin-resistant Staphylococcus aureus (MRSA) in ready-to-eat foods from Delta, Nigeria: a concern for consumer safety

Abstract Ready-to-eat (RTE) foods are susceptible to contamination with methicillin-resistant Staphylococcus aureus (MRSA), presenting significant health risks to consumers. This study aimed to isolate, identify, and characterize MRSA from RTE foods in Delta, Nigeria, and assess their implications for consumer safety. Four hundred RTE food samples were collected from food outlets, and MRSA presence was determined using oxacillin resistance screening agar supplemented with polymyxin B and oxacillin. Polymerase chain reaction confirmed and characterized MRSA isolates for virulence potentials and antimicrobial resistance genes. Out of the 400 samples, 57(14.25%) tested positive for MRSA. The prevalence of virulence genes varied, with Panton-Valentine Leukocidin (pvl) detected in 40.51% of isolates, along with the detection of several staphylococcal enterotoxin genes. Antimicrobial resistance genes, including tetracycline (tetM, 43.04%), erythromycin (ermC, 32.91%), and methicillin (mecA, 100%; mecC, 29.11%) were detected. Staphylococcal cassette chromosome mec (SCCmec) typing revealed diverse profiles, with type V being predominant (32.9%). MRSA isolates exhibited resistance to multiple antibiotics, with 83.54% of them classified as multidrug-resistant. Extracellular virulence factors were common, with strong biofilm producers observed in 69.62% of isolates. These findings underscore the complexity of MRSA contamination in RTE foods, highlighting the need for enhanced surveillance and control measures to safeguard public health.

Microbiological Quality and Safety of Brazilian Mozzarella Cheese During Production Stages.

This study assessed the microbiological quality and safety of mozzarella during various production stages in northern Tocantins, Brazil, by identifying critical biological points in the industrial environment within a tropical climatic region. Batches of mozzarella were evaluated, from raw milk to primary packaging, with a shelf life of 120 d at 4°C. Indicator microorganisms were quantified, and through microbiological and biomolecular approaches, Salmonella spp. and Listeria monocytogenes were identified. In addition, the toxigenic potential of coagulase-positive staphylococci (CPS) was characterized. Results indicated that the raw milk used for mozzarella production had low microbiological quality; pasteurization of raw milk effectively eliminated all identified pathogens and reduced microbiological counts (p > 0.05). An increase in bacterial counts (>2 log colony-forming unit [CFU]/g) and recontamination with Salmonella spp. and CPS, which potentially produce staphylococcal enterotoxin B, were observed during milk coagulation and curd draining. Stretching of the fermented curd reduced the enterobacteria, total coliforms, and Escherichia coli median values by 2.56, 2.64, and 2.3 log CFU/mL, respectively. Similarly, brining the pieces by immersion reduced the quantity of enterobacteria and total coliforms by 2.3 and 1.6 log CFU/mL, respectively. Of interest, in the freshly finished product, Salmonella spp. was present but L. monocytogenes was absent; however, after the shelf-life period, L. monocytogenes was present but Salmonella spp. was absent. Considering the environmental conditions that can promote the multiplication and preservation of pathogens and spoilage of dairy products in tropical climates, it is necessary to review operational hygiene procedures, particularly in milk coagulation vats and fermentation tables. This will ensure the production of high-quality mozzarella cheese with a reduced consumption risk.

Polarization of HIV-1- and CMV-Specific IL-17-Producing T Cells among People with HIV under Antiretroviral Therapy with Cannabis and/or Cocaine Usage.

This study evaluated the influence of cannabis and/or cocaine use in human immunodeficiency virus (HIV)- and cytomegalovirus (CMV)-specific T-cell responses of people with HIV (PWH). There was a higher percentage of IL-17-producing HIV-Gag-specific CD8+ T-cells in all drug users than that in PWH non-drug users. Stratifying the drug-user groups, increased percentages of IL-17-producing HIV-Gag-specific CD4+ and CD8+ T-cells were found in PWH cannabis plus cocaine users compared to PWH non-drug users. In response to CMV, there were higher percentage of IL-17-producing CMV-specific CD8+ T-cell in PWH cocaine users than that in PWH non-drug users. Considering all drug users together, there was a higher percentage of SEB-stimulated IL-17-producing CD4+ T-cells than that in PWH non-drug users, whereas cannabis users had higher percentages of IL-17-producing CD4+ T-cells compared to non-drug users. Cryopreserved peripheral blood mononuclear cells from 37 PWH undergoing antiretroviral therapy (ART) using cannabis (10), cocaine (7), or cannabis plus cocaine (10) and non-drug users (10) were stimulated with HIV-1 Gag or CMV-pp65 peptide pools, or staphylococcal enterotoxin B (SEB) and evaluated for IFN-γ- and/or IL-17A-producing CD4+ and CD8+ T-cells using flow cytometry. Cannabis plus cocaine use increased HIV-specific IL-17 producing T-cells and cocaine use increased IL-17 CMV-specific CD8+ T-cell responses which could favor the inflammatory conditions associated with IL-17 overproduction.

Enhancement of antitumor response of staphylococcal enterotoxin C2 mutant 2M-118 by promoting cell-mediated antitumor immunity

BackgroundStaphylococcal enterotoxin C2 (SEC2) is used as an immunotherapeutic drug in China. However, SEC2 are limited due to its immunosuppressive and toxic effects. A SEC2 2M-118 (H118A/T20L/G22E) mutant generated by site-directed mutagenesis was studied to elucidate the underlying antitumor mechanism. MethodsThe effects of 2M-118 on mouse fibrosarcoma (Meth-A) cells and cytokine responses were tested in vitro using a transwell assay and ELISA, respectively. 2M-118 effect on immune function in tumor-bearing mice was tested. Cytokine levels and antitumor responses were measured using ELISA and flow cytometry, respectively. TUNEL staining and immunohistochemistry were employed to detect the tumor apoptosis and CD4+ and CD8+ tumor infiltrating lymphocytes (TILs) in tumor tissue. Results2M-118 demonstrated the growth inhibition on tumor cells, increase of cytokines production (IL-2, IFN-γ, and TNF-α) and splenocyte proliferation in vitro. 2M-118 effectively inhibited tumor development and increased lymphocytes and cytokines in a tumor-bearing mouse model. Additionally, 2M-118 regulated the tumormicroenvironment by reducing the number of myeloid-derived suppressor cells (MDSCs), increasing the number of TILs, and inducing tumorcell apoptosis. Conclusion2M-118 promotes immune function and enhances antitumor response. This indicates that 2M-118 could potentially be developed as a novel anti-tumor drug with-highefficiencyandlowtoxicity.

Physicochemical and Bacteriological Analyses of Eniong River Water and Sediment in Southern Nigeria

The diversity of bacteria in freshwater is influenced by some important physicochemical factors and autotrophic nutrients. Their diversity can be employed as a sensitive pollution indicator in the environment and change because bacteria respond quickly to changes in the ecosystem. Analysis of physicochemical and bacteriological quality of humic freshwater and sediment was carried out on samples from three (3) geo-referenced sections of Eniong River, AkwaIbom State designated ST3 (Upstream), ST2 (Midstream) and ST1 (Downstream) using standard analytical methods. The results obtained were compared with World Health Organization (WHO) and Federal Ministry of Environment (FMENV) standards. The study revealed that thesamples had a mean heterotrophic bacterial count of 5.95±0.41 log10 CFU/g and heavy pollution with faecal bacteria such as (4.31±2.51 log10CFU/g TCC and 5.19 ±0.33 log10CFU/g FCC), including mean counts of Staphylococcus spp (5.90±1.16log10CFU/g), Vibrio spp (.21±1.04 log10CFU/g), Salmonella-Shigella (3.15±0.33 log10CFU/g), and Actinomycetes (3.29±2.49 log10CFU/g) that considerably exceeded the recommended range of WHO/FMENV for portability. The most abundance bacteria in were Bacillus subtilis, S. aureus and Shigella spp (55.6%) followed by Pseudomonas aeruginosa, Bacillus subtilis, Salmonella, Nocardia sp, Proteus sp., Staphylococcu sspp, Burkholderia pseudomallei and Enterococcus faecalis with a prevalence rate of 44.4%. The results showed the mean DO (7.67±0.037 mg/ml)), EC (85.07±0.52 μS/cm) and temperature (27.480C±0.049) of Eniong river water were within acceptable standard values while TSS (12.5 mg/ml) and TDS (19.0±1.30 mg/ml) values were not. Also, Pb, Cd, Vn, Ni and Cu recorded higher values while the concentrations of Fe2+, K+, Na+, Ca2+ and Mg2+ were remarkably low in both water and sediment. The mean pH of the river and sediment were 6.48±0.018 and 6.42±0.019, respectively. Similarly, among the anionic surfactant, the mean values for nitrate, carbonate and sulphate were higher in sediment than in water, while carbonate was not detected in water. The findings imply that the quality of Eniong River water and sediment is heavily compromised. Therefore, the creation of awareness on the importance of water safety and hygiene, regular monitoring of drinking water quality and enforcement of compliance with the standards by regulatory agencies are recommended to eschew possible health hazards.