Near Infrared Spectroscopy for Bacterial Detection in the Dairy Industry

[EN] This article discusses the use of near-infrared (NIR) spectroscopy combined with multivariate classification methods for detecting bacterial contamination in milk in the dairy industry. In the first experiment, the study found that NIR was accurate and reliable in detecting the presence of biofilms in milk. Our results showed that the technology was effective in distinguishing between contaminated and uncontaminated samples with an area under the receiver operating characteristic (ROC) curve (AUC) greater than 99%. It was also effective in classifying the samples belonging to different strains. In a second experiment, we used the same methodology to assess their effectiveness in detecting bacterial contamination proportions in milk. Our results showed that the technology was effective in classifying milk samples contaminated with four different bacteria and uncontaminated controls with an AUC greater than 97%. Moreover, results were still good when data from all bacteria were analyzed together, even at low bacterial concentrations, obtaining an average precision of 70%. These results demonstrate the potential of this technology to be used as a rapid and accurate method for identifying bacterial contamination in the dairy sector.SIPrincipado de Asturia

Evaluación de bacteriófagos como desinfectantes

La creciente necesidad de desarrollar nuevos productos antimicrobianos que permitan la eliminación de microorganismos tanto en el ámbito alimentario como en el hospitalario, ha impulsado la investigación de los bacteriófagos. Su eficacia como agentes antimicrobianos ya ha sido demostrada en el laboratorio, pero es necesario evaluar algunos aspectos importantes antes de su aplicación a nivel industrial. Aunque se trata de productos naturales, es preciso estudiar y optimizar su aplicación. En este trabajo nos hemos centrado en antimicrobianos fágicos frente a la bacteria patógena Staphylococcus aureus, en la interacción de los mismos con desinfectantes utilizados en la industria. En concreto, se ha evaluado la sensibilidad del bacteriófago phiIPLARODI a distintos desinfectantes industriales, resultando este muy resistente a todos excepto al peróxido de hidrógeno. También se ha estudiado las posibles sinergias y antagonismos entre el fago y los desinfectantes. Se observó una ligera sinergia no muy significativa en el caso del triclosán y un claro antagonismo en el caso de la clorhexidina. Por último, se comprobó si el efecto antagónico de la clorhexidina se debía a un aumento de la frecuencia de mutantes insensibles al bacteriófago con resistencia cruzada al desinfectante.Peer reviewe

Estabilidad de las proteínas líticas de bacteriófagos LysH5 y CHAP-SH3

La creciente necesidad de desarrollar nuevos productos antimicrobianos que permitan la eliminación de microorganismos tanto en el ámbito alimentario como en el hospitalario, ha impulsado la investigación de los bacteriófagos y sus proteínas líticas. Su eficacia como agentes antimicrobianos ya ha sido demostrada en el laboratorio, pero es necesario evaluar algunos aspectos importantes antes de su aplicación a nivel industrial. Aunque se trata de productos naturales, es preciso optimizar su aplicación a los alimentos, y adaptarlos a las condiciones de procesamiento de los mismos. En este trabajo nos hemos centrado en antimicrobianos fágicos frente a la bacteria patógena Staphylococcus aureus. Esta bacteria es la causa de infecciones graves y además, de toxiinfecciones alimentarias. En concreto, se ha optimizado un protocolo de concentración para el bacteriófago phiIPLA-RODI, mediante el cual se pueden obtener suspensiones fágicas de hasta 6,3 x 1010 ufp/mL. También se ha determinado la influencia de distintos parámetros sobre la estabilidad de las proteínas fágicas LysH5 y CHAP-SH3b. En cuanto a la temperatura, se ha visto que las proteínas mantienen su actividad a 25ºC y 37ºC mientras que la pierden a 50ºC. Por último, se comprobó que ambas proteínas también mantienen su actividad cuando se enfrentan a sobrenadantes de S. aureus.Peer reviewe

Phage-based strategy for staphylococcal biofilm removal

Trabajo presentado en el Break Biofilms Workshop, celebrado en Viena (Austria), del 16 al 18 de enero de 202

Deletion of the amidase domain of endolysin LysRODI enhances antistaphylococcal activity in milk and during fresh cheese production

Milk contamination with Staphylococcus aureus can lead to food poisoning in consumers. One strategy to minimize this risk is the use of phage-derived lysins, which are innocuous for humans and do not readily select for resistant variants. However, it remains necessary to find new candidate lysins and define the conditions for their utilization. This study compares the potential of LysRODI and its derivative LysRODIΔAmi (lacking the amidase domain), which displays high activity and storage stability, to successfully decrease staphylococcal contamination in milk under different conditions. Our results show that the engineered protein is more efficacious than the parent endolysin in practically all cases. For instance, while LysRODI only decreased the number of cells by 1–2 log units in different types of commercial milk and contamination levels, the chimeric lysin eliminated them below detection. Also, LysRODIΔAmi was more active against four strains with varying degrees of susceptibility. Regarding incubation temperature, both proteins were faster at 32 °C and 37 °C. Significantly, the engineered lysin eliminated detectable contamination in just 15 min. Finally, LysRODIΔAmi proved very successful at reducing staphylococcal contamination below detection during lab-scale fresh cheese production by enzymatic coagulation. Our data show that LysRODIΔAmi is a promising candidate for biocontrol in milk.This work was funded by grants PID2019-105311 RB-I00 (MICIU/AEI/FEDER, EU, Spain501100011033) to P.G. and A.R, and AYUD/2021/52120 (Program of Science, Technology and Innovation, 2021–2023 and FEDER EU, Principado de Asturias, Spain). S.A. has an FPI fellowship (Ministry of Science and Innovation, Spain)

Actividad estafilocócica de las endolisinas LysRODI y LysRODI¿Ami en leche y queso fresco

Trabajo presentado en la VII Reunión Red Española de Bacteriófagos y Elementos Transductores - RED FAGOMA, celebrada en Trujillo, Cáceres (España), del 26 al 28 de octubre de 202

Staphylococcus aureus biofilms formed in milk display resistance to the phage lytic protein LysRODI¿Ami

Trabajo presentado en la ASM Conference on Biofilms, celebrada en Charlotte, NC (Estados Unidos), del 13 al 17 de noviembre de 202

Activity of endolysin lysRODI and its derived lysin LysRODI¿Ami against Staphylococcus lactis in milk and fresh cheese

Resumen del trabajo presentado en el Viruses of Microbes (VoM 2022), celebrado en Guimaraes (Portugal), del 18 al 22 de julio de 202

Impact of the calcium concentration on the efficacy of phage phiIPLA-RODI, LysRODI¿Ami and nisin for the elimination of Staphylococcus aureus during lab-scale cheese production

Staphylococcus aureus is a Gram-positive human opportunistic pathogen that may also cause food poisoning because of the ability of some strains to produce heat stable enterotoxins that can persist in food even after the pathogen is successfully eliminated. In this context, biopreservation may be a forward-looking strategy to help eliminate staphylococcal contamination in dairy products by using natural compounds. However, these antimicrobials exhibit individual limitations that may be overcome by combining them. This work investigates the combination of a virulent bacteriophage, phiIPLA-RODI, a phage-derived engineered lytic protein, LysRODIΔAmi, and the bacteriocin nisin for the elimination of S. aureus during lab-scale cheese production at two CaCl2 concentrations (0.2 % and 0.02 %), and subsequent storage at two different temperatures (4 °C and 12 °C). In most of the assayed conditions, our results demonstrate that the combined action of the antimicrobials led to a greater reduction of the pathogen population than the compounds individually, albeit this effect was additive and not synergistic. However, our results did show synergy between the three antimicrobials for reducing the bacterial load after 14 days of storage at 12 °C, temperature at which there is growth of the S. aureus population. Additionally, we tested the impact of the calcium concentration on the activity of the combination treatment and observed that higher CaCl2 levels led to a notable increase in endolysin activity that allowed the utilization of approximately 10-times less protein to attain the same efficacy. Overall, our data show that the combination of LysRODIΔAmi with nisin and/or phage phiIPLA-RODI, and an increase in the calcium concentration are successful strategies to decrease the amount of protein required for the control of S. aureus contamination in the dairy sector with a low potential for resistance selection, thereby reducing costs.This work was funded by grants PID2019-105311RB-I00 (MICIU/AEI/FEDER, UE, Spain) to P.G. and A.R., and AYUD/2021/52120 (Program of Science, Technology and Innovation 2021–2023, FICYT and FEDER EU, Principado de Asturias, Spain). S.A. has an FPI fellowship (Ministry of Science and Innovation, Spain)