Shelf life of pork from five different quality classes

A total of 117 loins were selected on the cutting line at 24 h post-mortem to study the long term shelf life (35 days, 4 °C) of vacuum packaged pork from five different quality classes (PSE: pale, soft, exudative; PFN: pale, firm, non-exudative; RSE: red, soft, exudative; RFN: red, firm, non-exudative; and DFD: dark, firm, dry). The microbial load at 0 d was not significantly different (P > 0.05) among the pork quality classes, indicating that the initial microflora was influenced by the dressing conditions at the plant, not by the meat quality class. But after 35 d of storage, total aerobic mesophilic and presumptive lactic acid bacteria counts were higher (P < 0.05) in DFD pork due to its higher ultimate pH. RSE was the second quality class most susceptible to spoilage, whereas PFN, RFN and PSE pork had similar microbial loads. Further research is needed to elucidate the causes of the shorter shelf life in RSE pork

Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections.

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMIs) that can evolve into difficult-to-treat chronic mastitis. To date, no vaccine formulation has shown high protective efficacy against S. aureus IMI, partly because this bacterium can efficiently evade the immune system. For instance, S. aureus small colony variants (SCVs) have intracellular abilities and can persist without producing invasive infections. As a first step towards the development of a live vaccine, this study describes the elaboration of a novel attenuated mutant of S. aureus taking advantage of the SCV phenotype. A genetically stable SCV was created through the deletion of the hemB gene, impairing its ability to adapt and revert to the invasive phenotype. Further attenuation was obtained through inactivation of gene vraG (SACOL0720) which we previously showed to be important for full virulence during bovine IMIs. After infection of bovine mammary epithelial cells (MAC-T), the double mutant (ΔvraGΔhemB) was less internalized and caused less cell destruction than that seen with ΔhemB and ΔvraG, respectively. In a murine IMI model, the ΔvraGΔhemB mutant was strongly attenuated, with a reduction of viable counts of up to 5-log10 CFU/g of mammary gland when compared to the parental strain. A complete clearance of ΔvraGΔhemB from glands was observed whereas mortality rapidly (48h) occurred with the wild-type strain. Immunization of mice using subcutaneous injections of live ΔvraGΔhemB raised a strong immune response as judged by the high total IgG titers measured against bacterial cell extracts and by the high IgG2a/IgG1 ratio observed against the IsdH protein. Also, ΔvraGΔhemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of mastitis associated spa types. This double mutant could serve as a live-attenuated component in vaccines to improve cell-mediated immune responses against S. aureus IMIs

In vitro antibiotic susceptibility and biofilm production of Staphylococcus aureus isolates recovered from bovine intramammary infections that persisted or not following extended therapies with cephapirin, pirlimycin or ceftiofur

International audienceAbstractStaphylococcus aureus intramammary infections (IMIs) have low cure rates using standard antibiotic treatment and increasing the duration of treatment usually improves therapeutic success. Chronic IMIs are thought to be caused by bacteria presenting a specific virulence phenotype that includes the capacity to produce greater amounts of biofilm. In this study, antibiotic susceptibility and biofilm production by S. aureus isolates recovered from IMIs that were cured or not following an extended therapy with cephapirin, pirlimycin or ceftiofur for 5, 8 and 8 days, respectively, were compared. An isolate was confirmed as from a persistent case (not cured) if the same S. aureus strain was isolated before and after treatment as revealed by the same VNTR profile (variable number of tandem repeats detected by multiplex PCR). The antibiotic minimal inhibitory concentrations (MICs) were determined for these isolates as well as the capacity of the isolates to produce biofilm. Isolates from persistent cases after extended therapy with cephapirin or ceftiofur had higher MICs for these drugs compared to isolates from non-persistent cases (p < 0.05) even though the antibiotic susceptibility breakpoints were not exceeded. Isolates of the ceftiofur study significantly increased their biofilm production in presence of a sub-MIC of ceftiofur (p < 0.05), whereas isolates from the pirlimycin group produced significantly less biofilm in presence of a sub-MIC of pirlimycin (p < 0.001). Relative antibiotic susceptibility of the isolates as well as biofilm production may play a role in the failure of extended therapies. On the other hand, some antibiotics may counteract biofilm formation and improve cure rates

Antibiofilm and antibacterial effects of specific chitosan molecules on Staphylococcus aureus isolates associated with bovine mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine intramammary infections (IMIs) and mastitis. Mastitis is the primary cause for the use of antibiotics in dairy farms but therapeutic failure is often observed. One of the reasons for the lack of effectiveness of antibiotic therapy despite the observed susceptibility of bacterial isolates in vitro are bacterial biofilms. In this study, we used chitosan of well-defined molecular weight (0.4-0.6, 1.3, 2.6 and 4.0 kDa) and investigated their antibiofilm and antibacterial activities in in vitro and in vivo models related to S. aureus IMIs. A chitosan of at least 6 units of glucosamine was necessary for maximum antibacterial activity. The 2.6 and 4.0 kDa forms were able to prevent biofilm production by the biofilm hyperproducer strain S. aureus 2117 and a bovine MRSA (methicillin-resistant S. aureus). The intramammary administration of the 2.6 kDa chitosan showed no adverse effects in mice or in cows, as opposed to the slight inflammatory effect observed in mammary glands with the 4.0 kDa derivative. The 2.6 kDa chitosan killed bacteria embedded in pre-established biofilms in a dose-dependent manner with a >3 log10 reduction in CFU at 4 mg/ml. Also, the 2.6 kDa chitosan could prevent the persistence of the internalized MRSA into the mammary epithelial cell line MAC-T. An in vitro checkerboard assay showed that the 2.6 kDa chitosan produced a synergy with the macrolide class of antibiotics (e.g., tilmicosin) and reduced the MIC of both molecules by 2-8 times. Finally, the intramammary administration of the 2.6 kDa chitosan alone (P<0.01) or in combination with tilmicosin (P<0.0001) reduced the colonization of mammary glands in a murine IMI model. Our results suggest that the use of chitosan alone or in combination with a low dose of a macrolide could help reduce antibiotic use in dairy farms

Rationally Designed Pyrimidine Compounds: Promising Novel Antibiotics for the Treatment of <i>Staphylococcus aureus</i>-Associated Bovine Mastitis

Staphylococcus aureus is one of the major pathogens causing bovine mastitis, and antibiotic treatment is most often inefficient due to its virulence and antibiotic-resistance attributes. The development of new antibiotics for veterinary use should account for the One Health concept, in which humans, animals, and environmental wellbeing are all interconnected. S. aureus can infect cattle and humans alike and antibiotic resistance can impact both if the same classes of antibiotics are used. New effective antibiotic classes against S. aureus are thus needed in dairy farms. We previously described PC1 as a novel antibiotic, which binds the S. aureus guanine riboswitch and interrupts transcription of essential GMP synthesis genes. However, chemical instability of PC1 hindered its development, evaluation, and commercialization. Novel PC1 analogs with improved stability have now been rationally designed and synthesized, and their in vitro and in vivo activities have been evaluated. One of these novel compounds, PC206, remains stable in solution and demonstrates specific narrow-spectrum activity against S. aureus. It is active against biofilm-embedded S. aureus, its cytotoxicity profile is adequate, and in vivo tests in mice and cows show that it is effective and well tolerated. PC206 and structural analogs represent a promising new antibiotic class to treat S. aureus-induced bovine mastitis

MOESM1 of In vitro antibiotic susceptibility and biofilm production of Staphylococcus aureus isolates recovered from bovine intramammary infections that persisted or not following extended therapies with cephapirin, pirlimycin or ceftiofur

Additional file 1. Examples of VNTR profiles for bacteriology-defined persistent cases from the cephapirin therapy study. S1 represents the VNTR profile of the S. aureus isolate before treatment with cephapirin while S2 represents the VNTR profile of the S. aureus isolate recovered after treatment. The asterisk (*) shows identical VNTR profile, i.e., the VNTR-validated persistent cases that were investigated in this study. The arrows indicate some of the differences between two profiles, and as such, the isolate recovered after treatment was considered to be the result of a new infection; the case was considered neither cured or persistent and was therefore not evaluated in this work

Effect of the 2.6 kDa chitosan on LDH release from MAC-T bovine mammary epithelial cells.

<p>Cells were exposed to the 2.6 kDa chitosan at different concentrations (0.5 mg/ml, 2 mg/ml and 8 mg/ml). Triton x-100 and culture medium were used as cytotoxic positive and negative (Ctrl) controls, respectively. The amount of formazan formed was normalized to the amount formed by the non-treated cells (Ctrl). Bars represent the means and vertical lines the standard deviation (SD). Significant differences in comparison to the negative control (Ctrl) are shown. Statistical analysis was performed using the Kruskal-Wallis test (non-parametric one way ANOVA) with Dunn’s multiple comparison test: *, <i>P</i><0.05; ***, <i>P</i><0.0001.</p

Efficacy of the 2.6 kDa chitosan used alone or in combination with tilmicosin on <i>S</i>. <i>aureus</i> in a mouse mastitis model.

<p>(A) Chitosan was administered twice at (0 h and 4 h post-inoculation) using 0.2, 2 or 10 mg/gland for the challenge against <i>S</i>. <i>aureus</i> strain Newbould. (B) A challenge with <i>S</i>. <i>aureus</i> strain 2117 was treated with the 2.6 kDa chitosan or tilmicosin (TIL) or a combination of both. Chitosan was administered twice at (0h and 4 h post-inoculation) whereas tilmicosin was administered 4 h post-inoculation. The middle bars indicate median values for each group of glands (n = 5–8) whereas the boxes specify quartiles Q1-Q3. The detection limit was 200 CFU (dotted line), below that, the glands were considered uninfected. Statistical analysis was performed using the Kruskal-Wallis test: *, <i>P</i><0.05; ***, <i>P</i><0.001.</p

Relative innocuity of different forms of chitosan in cows.

<p>Each quarter of cow’s udder has received 500 mg of chitosan (2.6 kDa or 4.0 kDa) or saline as the negative control. Milk samples and somatic cell counts (SCC) were determined 12 and 3 hours before the instillation of chitosan. After the intramammary instillation, milk samples were aseptically collected from cows at several points in time to evaluate inflammation by determining the SCC (A) and milk yields (B). Symbols represent the means and vertical lines the standard deviation. Data were analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). For the first experiment, time was used as a repeated effect and treatment (cow) was used as the subject. Orthogonal contrasts were performed to compare the effect of each treatment to control. No difference was observed between saline and the 2.6 kDa chitosan (SCC and quarter milk yield). Significant differences were observed between saline and 4.0 kDa for SCC: *, <i>P</i><0.05; **, <i>P</i>< 0.01; ***, <i>P</i><0.0001.</p

Antibiofilm and antibacterial effects of specific chitosan molecules on <i>Staphylococcus aureus</i> isolates associated with bovine mastitis - Fig 4

<p><b>Time-kill experiments showing the viability of <i>S</i>. <i>aureus</i> untreated (Ctrl) or in the presence of increasing concentrations of the 2.6 kDa chitosan, (A) for strain MRSA 1158c, and (B) for the biofilm hyperproducer strain 2117.</b> The CFU detection limit is 10 CFU/ml. Data are presented as means with standard deviations from three independent experiments.</p