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Frontiers in Molecular Biosciences 2018Lysostaphin from and its family enzymes rapidly acquire prominence as the next generation agents in treatment of infections. The specificity of lysostaphin is promoted...
Lysostaphin from and its family enzymes rapidly acquire prominence as the next generation agents in treatment of infections. The specificity of lysostaphin is promoted by its C-terminal cell wall targeting domain selectivity toward pentaglycine bridges in cell wall. Scission of these cross-links is carried out by its N-terminal catalytic domain, a zinc-dependent endopeptidase. Understanding the determinants affecting the efficiency of catalysis and strength and specificity of interactions lies at the heart of all lysostaphin family enzyme applications. To this end, we have used NMR, SAXS and molecular dynamics simulations to characterize lysostaphin structure and dynamics, to address the inter-domain interaction, the enzyme-substrate interaction as well as the catalytic properties of pentaglycine cleavage in solution. Our NMR structure confirms the recent crystal structure, yet, together with the molecular dynamics simulations, emphasizes the dynamic nature of the loops embracing the catalytic site. We found no evidence for inter-domain interaction, but, interestingly, the SAXS data delineate two preferred conformation subpopulations. Catalytic H329 and H360 were observed to bind a second zinc ion, which reduces lysostaphin pentaglycine cleaving activity. Binding of pentaglycine or its lysine derivatives to the targeting domain was found to be of very low affinity. The pentaglycine interaction site was located to the N-terminal groove of the domain. Notably, the targeting domain binds the peptidoglycan stem peptide Ala-d-γ-Glu-Lys-d-Ala-d-Ala with a much higher, micromolar affinity. Binding site mapping reveals two interaction sites of different affinities on the surface of the domain for this peptide.
PubMed: 30018958
DOI: 10.3389/fmolb.2018.00060 -
Journal of Dairy Science Jun 2018The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of...
The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.
Topics: Animals; Cattle; Cell Count; Electrophoresis, Gel, Pulsed-Field; Female; Mammary Glands, Animal; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus
PubMed: 29525303
DOI: 10.3168/jds.2017-13910 -
Frontiers in Microbiology 2019Biogenic amines (BAs) in sausages represent a health risk for consumers, and thus investigating the BAs accumulation mechanism is important to control the BAs. In this...
Biogenic amines (BAs) in sausages represent a health risk for consumers, and thus investigating the BAs accumulation mechanism is important to control the BAs. In this study, the BAs profiles of 16 typical Chinese sausage samples were evaluated, and 8 kinds of common BAs were detected from different samples. As a whole, the BAs contents of the majority of Chinese sausage samples were within the safe dosage range, except that the total BAs and histamine concentrations of sample HBBD were above the toxic dosage levels. Furthermore, the bacterial and fungal communities of the Chinese sausage samples were investigated by high-throughput sequencing analysis, and , , , , , and were identified as the predominant genera. Accordingly, 13 representative strains were selected from the dominant genera, and their BAs formation and degradation properties were evaluated. Finally, the results of fermented meats model experiment indicated that the isolates including Sp, Se, Sc1, Sc2, and Ss could significantly reduce BAs, possessing the potential as the starter cultures to control the BAs in fermented meat products. The present study not only helped to explain the BAs accumulation mechanism in Chinese sausage, but also developed the candidates for potential BAs control in fermented meat products.
PubMed: 31130922
DOI: 10.3389/fmicb.2019.00872 -
Foods (Basel, Switzerland) May 2022and screened from Guizhou specialty food were used to prepare fermented pork loin ham. The sensory qualities and flavor profiles of fermented pork loin hams from 0 to...
and screened from Guizhou specialty food were used to prepare fermented pork loin ham. The sensory qualities and flavor profiles of fermented pork loin hams from 0 to 42 days were investigated in order to reveal the dynamics of fermented pork loin ham. The results show that total free amino acids (TFAA) content reached the highest value on the 35th day, and the umami amino acids, including aspartic acid (ASP), glutamic acid (GLU), glycine (GLY), and alanine (ALA), were the main amino acids in all periods. Notably, the RV coefficient (0.875) indicates that free amino acids (FAA) are highly correlated with the sensory score of the E-tongue. In terms of the volatile compounds identified, the esters content gradually increased between 7 and 42 days, and ethyl octanoate was the most abundant compound during all periods. These esters imparted a characteristic aroma component to the fermented pork loin ham. The most important finding was that the increase in the content of esters represented by octanoic acid-ethyl ester might be related to the increase in the content of FAA with the increase in fermentation time. Both the E-nose and E-tongue showed good discrimination ability for fermented tenderloin ham with different fermentation times, which was crucial in cases with large clusters. In addition, the multiple factor analysis (MFA) indicated that the E-nose aroma value might be the key factor in distinguishing fermented pork loin ham with different fermentation times.
PubMed: 35627071
DOI: 10.3390/foods11101501 -
BMC Veterinary Research Nov 2013Coagulase-negative staphylococci (CNS) cause usually subclinical or mild clinical bovine mastitis, which often remains persistent. Symptoms are usually mild, mostly only...
BACKGROUND
Coagulase-negative staphylococci (CNS) cause usually subclinical or mild clinical bovine mastitis, which often remains persistent. Symptoms are usually mild, mostly only comprising slight changes in the appearance of milk and possibly slight swelling. However, clinical mastitis with severe signs has also been reported. The reasons for the differences in clinical expression are largely unknown. Macrophages play an important role in the innate immunity of the udder. This study examined phagocytosis and killing by mouse macrophage cells of three CNS species: Staphylococcus chromogenes (15 isolates), Staphylococcus agnetis (6 isolates) and Staphylococcus simulans (15 isolates). Staphylococcus aureus (7 isolates) was also included as a control.
RESULTS
All the studied CNS species were phagocytosed by macrophages, but S. simulans resisted phagocytosis more effectively than the other CNS species. Only S. chromogenes was substantially killed by macrophages. Significant variations between isolates were seen in both phagocytosis and killing by macrophages and were more common in the killing assays. Significant differences between single CNS species and S. aureus were observed in both assays.
CONCLUSION
This study demonstrated that differences in the phagocytosis and killing of mastitis-causing staphylococci by macrophages exist at both the species and isolate level.
Topics: Animals; Cattle; Feeder Cells; Female; Macrophages; Mastitis, Bovine; Mice; Mice, Inbred BALB C; Phagocytosis; Staphylococcal Infections; Staphylococcus
PubMed: 24207012
DOI: 10.1186/1746-6148-9-227 -
Veterinary World Jun 2017This study was devoted to elucidate the tetracycline resistance of coagulase-negative staphylococci (CNS) derived from normal and subclinical mastitic (SCM) buffaloes'...
AIM
This study was devoted to elucidate the tetracycline resistance of coagulase-negative staphylococci (CNS) derived from normal and subclinical mastitic (SCM) buffaloes' milk in Egypt.
MATERIALS AND METHODS
A total of 81 milk samples from 46 normal buffalo milk samples and 35 SCM buffalo milk samples at private dairy farms of Egypt were used in this study. CNS were identified using phenotypic and molecular methods (polymerase chain reaction [PCR]). CNS isolates were tested for tetracycline resistance using routine methods and multiplex PCR targeting tetracycline () resistance genes followed by sequencing of positive PCR products and phylogenetic analysis.
RESULTS
Isolation and identification of 28 (34.5%) CNS from normal and SCM buffaloes' milk, namely, (39.2%), (25.0%), (10.7%), (10.7%), and 3.5% to each of , , , and . Using nested PCR, all the 28 CNS isolates revealed positive for 16srRNA gene specific for genus staphylococci and negative for thermonuclease () gene specific for species. The presence of tetracycline resistance-encoding genes (K, L, M, and O) was detected by multiplex PCR. All isolates were negative for L, M, and O genes while 14 (50%) CNS isolates were positive for K gene, namely, (100%), (100%), (66.6%), (45.4%), and (42.8%). Nucleotide sequencing of K gene followed by phylogenetic analysis showed the high homology between our CNS isolates genes of tetracycline resistance with isolates including Egyptian ones. This proves the transfer of the tetracycline resistance encoding genes between coagulase-negative and coagulase positive spp.
CONCLUSION
CNS isolates have distinguishingly high resistance to tetracycline. Abundant tetracycline usage for mastitis treatment leads to the spread of genetic resistance mechanisms inside CNS strains and among all spp. Consequently, tetracycline is not effective anymore.
PubMed: 28717325
DOI: 10.14202/vetworld.2017.702-710 -
Journal of Global Antimicrobial... Mar 2021To evaluate the in vitro activity of dalbavancin compared with vancomycin, daptomycin and other agents against a large collection of coagulase-negative staphylococci...
OBJECTIVES
To evaluate the in vitro activity of dalbavancin compared with vancomycin, daptomycin and other agents against a large collection of coagulase-negative staphylococci (CoNS) isolates.
METHODS
A total of 5088 CoNS causing clinically significant infection were consecutively collected from 122 medical centres in the USA and Europe over 6 years (2014-2019). Isolates were tested for susceptibility by the reference broth microdilution method. Species identification was confirmed by MALDI-TOF. Most isolates were from bloodstream infections (BSIs) (53.5%) or skin/skin structure infections (28.5%).
RESULTS
Staphylococcus epidermidis was the most common species overall (54.6%) and for BSI (61.3%). The second most common species were Staphylococcus lugdunensis overall (12.3%) and Staphylococcus hominis for BSI (14.7%). Dalbavancin (MIC, 0.03/0.06 mg/L) inhibited >99.9% of CoNS isolates at ≤0.25 mg/L (susceptible breakpoint for Staphylococcus aureus per CLSI). All species were inhibited at ≤0.25 mg/L dalbavancin, except some S. epidermidis (>99.9%) and Staphylococcus warneri (98.9%) isolates. Staphylococcus capitis and Staphylococcus simulans exhibited the lowest dalbavancin MIC values (0.015/0.03 mg/L) and Staphylococcus haemolyticus and Staphylococcus saprophyticus the highest (MIC, 0.06/0.12 mg/L); 47.8% of S. epidermidis and 34.7% of S. haemolyticus exhibited decreased susceptibility to vancomycin (MIC ≥ 2 mg/L) and 23.2% of S. capitis and 28.4% of S. warneri showed decreased susceptibility to daptomycin (MIC ≥ 1 mg/L).
CONCLUSION
Antimicrobial susceptibility varied widely among CoNS species. Dalbavancin inhibited >99.9% and 99.1% of isolates at the US-FDA and EUCAST breakpoints, respectively. Clinical studies of dalbavancin for treatment of CoNS infections should be considered based on these in vitro data.
Topics: Anti-Bacterial Agents; Coagulase; Europe; Microbial Sensitivity Tests; Staphylococcus; Teicoplanin
PubMed: 33285311
DOI: 10.1016/j.jgar.2020.11.020 -
Research in Veterinary Science May 2023Single-layer centrifugation (SLC) with a low-density colloid is an efficient method for removing contaminating microorganisms from boar semen while recovering most...
Single-layer centrifugation (SLC) with a low-density colloid is an efficient method for removing contaminating microorganisms from boar semen while recovering most spermatozoa from the original sample. This study tested the performance of this technique, using 50-ml tubes, by spiking commercial semen doses prepared without antibiotics with selected bacterial species followed by storage at 17 °C. The doses were spiked up to 10/ml CFU (colony forming units) of the bacteria Burkholderia ambifaria, Pseudomonas aeruginosa, and Staphylococcus simulans. The semen was processed by SLC (15 ml of sample and 15 ml of colloid) with the colloid Porcicoll at 20% (P20) and 30% (P30), with a spiked control (CTL) and an unspiked control (CTL0), analyzing microbiology and sperm quality on days 0, 3 and 7. SLC completely removed B. ambifaria and S. simulans, considerably reducing P. aeruginosa and overall contamination (especially P30, ∼10 CFU/ml of total contamination on day 7, median). Sperm viability was lower in P20 and P30 samples at day 0, with higher cytoplasmic ROS. Still, results were similar in all groups on day 3 and reversed on day 7, indicating a protective effect of SLC (possibly directly by removal of damaged sperm and indirectly because of lower bacterial contamination). Sperm chromatin was affected by the treatment (lower DNA fragmentation and chromatin decondensation) and storage (higher overall condensation on day 7 as per chromomycin A3 and monobromobimane staining). In conclusion, SLC with low-density colloids can remove most bacteria in a controlled contamination design while potentially improving sperm quality and long-term storage at practical temperatures.
Topics: Male; Animals; Swine; Semen; Spermatozoa; Semen Analysis; Semen Preservation; Centrifugation; Colloids; Chromatin; Burkholderia; Sperm Motility
PubMed: 37031470
DOI: 10.1016/j.rvsc.2023.03.024 -
Journal of Dairy Science Dec 2019Coagulase-negative staphylococci (CNS) are one of the most common bovine mastitis pathogens found worldwide. In this study, we investigated the prevalence and...
Coagulase-negative staphylococci (CNS) are one of the most common bovine mastitis pathogens found worldwide. In this study, we investigated the prevalence and distribution of CNS species in mastitis milk samples and further characterized the methicillin-resistant (MR) CNS. A total of 311 CNS were isolated from 3,692 quarter milk samples from 1,373 dairy cattle at 81 farms between 2013 and 2017. Further evaluation of the CNS isolates revealed 14 CNS species among the samples and 3 predominant species-namely, Staphylococcus chromogenes, Staphylococcus simulans, and Staphylococcus epidermidis. Resistance was higher in S. epidermidis than in other CNS species except for resistance against oxacillin in Staphylococcus sciuri. Resistance to β-lactams was the most common in all CNS species (8.4% in ampicillin, 21.2% in oxacillin, and 13.5% in penicillin). Conversely, only minimal resistance to cephalothin, ceftiofur, and pirlimycin/novobiocin was found. Twenty-one isolates from 4 species were mecA-carrying MRCNS strains, including 18 S. epidermidis and 1 each of S. sciuri, Staphylococcus equorum, and Staphylococcus hominis. The majority of the mecA-carrying MRCNS isolates were produced in the biofilm. Furthermore, multidrug-resistant sequence type 179 isolate produced the strongest biofilm. Seven genotypes were detected in the 18 MR S. epidermidis strains, the most predominant of which persisted on a farm for 2 yr. Our findings for the antimicrobial susceptibility profiles and genotypic characterization of the MRCNS isolates could provide valuable information for controlling the spread of resistance and the selection of appropriate antimicrobial therapies for mastitis in the future. Further, strategic antibiotic use for mastitis treatment and hygienic management practices aimed at the prevention of the growth of resistant bacteria are urgently needed on dairy farms.
Topics: Animals; Anti-Bacterial Agents; Cattle; Coagulase; Drug Resistance, Bacterial; Farms; Female; Genotype; Mastitis, Bovine; Milk; Oxacillin; Prevalence; Republic of Korea; Staphylococcal Infections; Staphylococcus; Staphylococcus epidermidis
PubMed: 31548061
DOI: 10.3168/jds.2019-17028 -
Microbial Drug Resistance (Larchmont,... Sep 2018This study was conducted to determine the occurrence of antimicrobial resistance and enterotoxin-encoding genes (EEGs) in Staphylococcus spp. recovered from equipment...
This study was conducted to determine the occurrence of antimicrobial resistance and enterotoxin-encoding genes (EEGs) in Staphylococcus spp. recovered from equipment used to prepare hospital meals, in a university hospital in Rio de Janeiro, Brazil. Sixty samples were collected from semi-industrial equipment (one blender and one mixer) in the hospital's kitchen. Resistance genes and SCCmec types were detected by PCR. From the 40 isolates of Staphylococcus spp. identified, 8 were Staphylococcus aureus. Thirty-two (80%) Staphylococcus spp. isolates were resistant to at least one antimicrobial agent. Resistance genetic determinants were detected: erm gene (Staphylococcus epidermidis [n = 2]; Staphylococcus hominis [n = 1]), mecA gene (S. epidermidis [n = 2]), and aa(6')-aph(2'') gene (Staphylococcus caprae [n = 1], S. epidermidis [n = 2], S. hominis [n = 1], Staphylococcus pausteri [n = 1], Staphylococcus simulans [n = 1], and Staphylococcus warneri [n = 1]). The presence of at least one EEG in 83% (n = 33) of the isolates was identified. Two strains of S. epidermidis were methicillin-resistant S. epidermidis (MRSE) and harboring SCCmec type IV. Staphylococcus spp. contaminated some hospital kitchen's equipment, indicating that hygiene procedures should be improved. Results also indicate that meals can be a vehicle to disseminate multiresistant Staphylococcus spp., including MRSE, and Staphylococcus with EEGs.
Topics: Adult; Anti-Bacterial Agents; Bacterial Proteins; Brazil; DNA, Bacterial; Enterotoxins; Female; Hospitals, University; Humans; Male; Methicillin Resistance; Microbial Sensitivity Tests; Middle Aged; Staphylococcal Infections; Staphylococcus
PubMed: 29653479
DOI: 10.1089/mdr.2016.0309