One Health-One Biofilm.

Bacterial biofilms are structured clusters of bacterial cells enclosed in a self-produced polymer matrix that are attached to a biotic or abiotic surface. This structure protects bacteria from hostile environmental conditions. There are also accumulating reports about bacterial aggregates associated but not directly adherent to surfaces. Interestingly, these bacterial aggregates exhibit many of the same phenotypes as surface-attached biofilms. Surface-attached biofilms as well as non-attached aggregates are ubiquitous and found in a wide variety of natural and clinical settings. This strongly suggests that biofilm/aggregate formation is important at some steps in the bacterial lifecycle. Biofilm/aggregate formation might therefore be important for some bacterial species for persistence within their host or their environment, while for other bacterial species it might be more important for persistence in the environment between infection of different individuals or even between infection of different hosts (humans or animals). This is strikingly similar to the One Health concept which recognizes that the health and well-being of humans, animals and the environment are intricately linked. We would like to propose that within this One Health concept, the One Biofilm concept also exists, where biofilm/aggregate formation in humans, animals and the environment are also intricately linked. Biofilm/aggregates could represent the unifying factor underneath the One Health concept. The One Biofilm concept would support that biofilm/aggregate formation might be important for persistence during infection but might as well be even more important for persistence in the environment and for transmission between different individuals/different hosts.

Untargeted and targeted metabolomics reveal that adenosine nucleotides released in Actinobacillus pleuropneumoniae supernatant inhibit porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses in the swine industry and causes major economic losses. To date, there has not been an effective antiviral treatment for the disease. We have shown in previous studies that culture supernatant of Actinobacillus pleuropneumoniae (App), the causative agent of porcine pleuropneumonia, possesses antiviral activity in vitro against PRRSV, and we have clearly established that the antiviral activity was mediated by small molecular weight (i.e., <1 kDa), heat resistant metabolites present in the App supernatant ultrafiltrates. However, the identity of those metabolites remains unknown. The objective of the current study was to identify the active metabolites using untargeted and targeted mass spectrometry-based metabolomics and test their respective antiviral activity against PRRSV in the Jude Porcine Lung Epithelial Cell Line (SJPL). The results presented reveal very significant antiviral activity of App supernatant ultrafiltrates against PRRSV in SJPL cells. Consequently, we identified and quantified several adenosine nucleotide metabolites present in App supernatant ultrafiltrates using mass spectrometry-based metabolomics, and the concentrations detected were very high. SJPL cells infected with PRRSV and treated with 2'-adenosine monophosphate (2-AMP), 3'-adenosine monophosphate (3-AMP) or 5'-adenosine monophosphate (5-AMP) significantly reduced PRRSV infection. Interestingly, many antiviral drugs or prodrugs are adenosine analogs, and the mechanism of action was previously elucidated. Currently marketed nucleoside analog drugs could potentially be used to treat PRRSV infection.

Secondary Staphylococcus aureus intramammary colonization is reduced by non-aureus staphylococci exoproducts.

Non-aureus staphylococci (NAS) and Staphylococcus aureus are pathogens that cause bovine mastitis, a costly disease for dairy farmers, however; many NAS are considered part of the normal udder microbiota. It has been suggested that through a mechanism that remains to be elucidated, NAS intramammary colonization can prevent subsequent infection with other bacterial pathogens. This study shows that in a murine mastitis model, secondary Staph. aureus intramammary colonization is reduced by exoproducts from Staph. chromogenes and Staph. simulans, both NAS, while Streptococcus spp. exoproducts have much less ability to affect the course of the infection caused by S. aureus.

Counteracting Bacterial Motility: A Promising Strategy to Narrow Listeria monocytogenes Biofilm in Food Processing Industry.

Listeria monocytogenes (L. monocytogenes) is often associated with processed food as it can form biofilms that represent a source of contamination at all stages of the manufacturing chain. The control and prevention of biofilms in food-processing plants are of utmost importance. This study explores the efficacy of prospect molecules for counteracting bacterial mechanisms leading to biofilm formation. The compounds included the phytomolecule tomatidine, zinc chloride (ZnCl2), ethylenediaminetetraacetic acid (EDTA), and a more complexed mixture of bacterial compounds from coagulase-negative staphylococci (CNS exoproducts). Significant inhibition of L. monocytogenes biofilm formation was evidenced using a microfluidic system and confocal microscopic analyses (p < 0.001). Active molecules were effective at an early stage of biofilm development (≥50% of inhibition) but failed to disperse mature biofilms of L. monocytogenes. According to our findings, prevention of surface attachment was associated with a disruption of bacterial motility. Indeed, agar cell motility assays demonstrated the effectiveness of these molecules. Overall, results highlighted the critical role of motility in biofilm formation and allow to consider flagellum-mediated motility as a promising molecular target in control strategies against L. monocytogenes in food processing environments.

Actinobacillus pleuropneumoniae Interaction With Swine Endothelial Cells.

Actinobacillus pleuropneumonia is a swine (host) specific respiratory pathogen and the etiological agent of swine pleuropneumonia which affects pigs of all ages, many being asymptomatic carriers. This pathogen has high morbidity and mortality rates which generates large economic losses for the pig industry. Actinobacillus pleuropneumoniae is a widely studied bacterium, however its pathogenesis is not yet fully understood. The prevalence of the 18 serotypes of A. pleuropneumoniae varies by geographic region, in North American area, more specifically in Mexico, serotypes 1, 3, 5b, and 7 show higher prevalence. Actinobacillus pleuropneumoniae is described as a strict extracellular pathogen with tropism for lower respiratory tract. However, this study depicts the ability of these serotypes to adhere to non-phagocytic cells, using an endothelial cell model, as well as their ability to internalize them, proposing it could be considered as an intracellular pathogen.

A Siderophore Analog of Fimsbactin from Acinetobacter Hinders Growth of the Phytopathogen Pseudomonas syringae and Induces Systemic Priming of Immunity in Arabidopsis thaliana.

Siderophores produced in soil by plant growth-promoting rhizobacteria (PGPRs) play several roles, including nutrient mobilizers and can be useful as plants defense elicitors. We investigated the role of a synthetic mixed ligand bis-catechol-mono-hydroxamate siderophore (SID) that mimics the chemical structure of a natural siderophore, fimsbactin, produced by Acinetobacter spp. in the resistance against the phytopathogen Pseudomonas syringaepv tomato DC3000 (Pst DC3000), in Arabidopsis thaliana. We first tested the antibacterial activity of SID against Pst DC3000 in vitro. After confirming that SID had antibacterial activity against Pst DC3000, we tested whether the observed in vitro activity could translate into resistance of Arabidopsis to Pst DC3000, using bacterial loads as endpoints in a plant infection model. Furthermore, using quantitative polymerase chain reaction, we explored the molecular actors involved in the resistance of Arabidopsis induced by SID. Finally, to assure that SID would not interfere with PGPRs, we tested in vitro the influence of SID on the growth of a reference PGPR, Bacillus subtilis. We report here that SID is an antibacterial agent as well as an inducer of systemic priming of resistance in A. thaliana against Pst DC3000, and that SID can, at the same time, promote growth of a PGPR.

Prevalence and characteristics of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) isolated from chicken meat in the province of Quebec, Canada.

This study was conducted to estimate the prevalence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) in retail chicken meat and broiler chickens from the Province of Quebec, Canada, and to characterize LA-MRSA isolates. A total of 309 chicken drumsticks and thighs were randomly selected in 2013 from 43 retail stores in the Monteregie. In addition, nasal swabs and caeca samples were collected in 2013-2014 from 200 broiler chickens of 38 different flocks. LA-MRSA was not detected in broiler chickens. Fifteen LA-MRSA isolates were recovered from four (1.3%) of the 309 chicken meat samples. Multi-Locus Sequence Typing (MLST) and SCCmec typing revealed two profiles (ST398-MRSA-V and ST8-MRSA-IVa), which were distinct using pulse-field gel electrophoresis (PFGE) and microarray (antimicrobial resistance and virulence genes) analyses. In addition to beta-lactam resistance, tetracycline and spectinomycin resistance was detected in all isolates from the 3 positive samples of the ST398 profile. Southern blot hybridization revealed that the resistance genes aad(D) and lnu(A), encoding resistances to aminoglycosides and lincosamides respectively, were located on plasmid. All isolates were able to produce biofilms, but biofilm production was not correlated with hld gene expression. Our results show the presence of two separate lineages of MRSA in retail chicken meat in Quebec, one of which is likely of human origin.

Human Herpes Virus-6 Encephalitis Following Autologous Blood and Marrow Transplant.

Human herpesvirus-6 (HHV-6) is a highly prevalent virus that establishes lifelong latency in human hosts. Symptomatic HHV-6 reactivation rarely occurs in immunocompetent individuals and is best described in immunosuppressed patients such as recipients of bone marrow transplants (BMT). In that setting, HHV-6 reactivation has been associated with fever, rash, pneumonitis, encephalitis, and delayed engraftment. While these complications are well documented in allogeneic transplant, the clinical impact of such reactivation is not well known in autologous BMT. We described a case of HHV-6-associated encephalitis in a previously heavily treated patient with multiple myeloma (MM) following a second autologous BMT, and discuss the need for clinicians to be aware of the potential clinical impact of HHV-6 following autologous BMT in the era of immunomodulatory agents.

Regulation of waaH by PhoB during Pi Starvation Promotes Biofilm Formation by Escherichia coli O157:H7.

In open environments such as water, enterohemorrhagic Escherichia coli O157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. This activates the phosphate-specific transport (Pst) system that contains a high-affinity Pi transporter. In the Δpst mutant, PhoB is constitutively activated and regulates the expression of genes in the Pho regulon. Here, we show that Pi starvation and deletion of the pst system enhance E. coli O157:H7 biofilm formation. Among differentially expressed genes of EDL933 grown under Pi starvation conditions and in the Δpst mutant, we have found that a member of the PhoB regulon, waaH, predicted to encode a glycosyltransferase, was highly expressed. Interestingly, WaaH contributed to biofilm formation of E. coli O157:H7 during both Pi starvation and in the Δpst mutant. In the Δpst mutant, the presence of waaH was associated with lipopolysaccharide (LPS) R3 core type modifications, whereas in E. coli O157:H7, waaH overexpression had no effect on LPS structure during Pi starvation. Therefore, waaH participates in E. coli O157:H7 biofilm formation during Pi starvation, but its biochemical role remains to be clarified. This study highlights the importance of the Pi starvation stress response to biofilm formation, which may contribute to the persistence of E. coli O157:H7 in the environment.IMPORTANCE Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen that causes bloody diarrhea that can result in renal failure. Outside of mammalian hosts, E. coli O157:H7 survives for extended periods of time in nutrient-poor environments, likely as part of biofilms. In E. coli K-12, the levels of free extracellular Pi affect biofilm formation; however, it was unknown whether Pi influences biofilm formation by E. coli O157:H7. Our results show that upon Pi starvation, PhoB activates waaH expression, which favors biofilm formation by E. coli O157:H7. These findings suggest that WaaH is a target for controlling biofilm formation. Altogether, our work demonstrates how adaptation to Pi starvation allows E. coli O157:H7 to occupy different ecological niches.

The Mastitis Pathogens Culture Collection.

The Mastitis Pathogen Culture Collection contains more than 16,000 mastitis-causing bacterial isolates from milk samples taken from cow quarters and bulk tanks in a national cohort of 91 dairy farms across Canada over a period of 2 years. These isolates are linked to demographic and production data that were recorded at the mammary gland, cow, and farm levels.

Incorporation of Actinobacillus pleuropneumoniae in Preformed Biofilms by Escherichia coli Isolated From Drinking Water of Swine Farms.

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, represents one of the most important health problems in the swine industry worldwide and it is included in the porcine respiratory disease complex. One of the bacterial survival strategies is biofilm formation, which are bacterial communities embedded in an extracellular matrix that could be attached to a living or an inert surface. Until recently, A. pleuropneumoniae was considered to be an obligate pathogen. However, recent studies have shown that A. pleuropneumoniae is present in farm drinking water. In this study, the drinking water microbial communities of Aguascalientes (Mexico) swine farms were analyzed, where the most frequent isolated bacterium was Escherichia coli. Biofilm formation was tested in vitro; producing E. coli biofilms under optimal growth conditions; subsequently, A. pleuropneumoniae serotype 1 (strains 4074 and 719) was incorporated to these biofilms. Interaction between both bacteria was evidenced, producing an increase in biofilm formation. Extracellular matrix composition of two-species biofilms was also characterized using fluorescent markers and enzyme treatments. In conclusion, results confirm that A. pleuropneumoniae is capable of integrates into biofilms formed by environmental bacteria, indicative of a possible survival strategy in the environment and a mechanism for disease dispersion.

N-Acetyl-glucosamine influences the biofilm formation of Escherichia coli.

BACKGROUND: The intestinal mucous layer is a physical barrier that limits the contact between bacteria and host epithelial cells. There is growing evidence that microbiota-produced metabolites can also be specifically sensed by gut pathogens as signals to induce or repress virulence genes. Many E. coli, including adherent and invasive (AIEC) strains, can form biofilm. This property can promote their intestinal colonization and resistance to immune mechanisms. We sought to evaluate the impact of mucus-derived sugars on biofilm formation of E. coli. RESULTS: We showed that the mucin sugar N-acetyl-glucosamine (NAG) can reduce biofilm formation of AIEC strain LF82. We demonstrated that the inactivation of the regulatory protein NagC, by addition of NAG or by mutation of nagC gene, reduced the biofilm formation of LF82 in static condition. Interestingly, real-time monitoring of biofilm formation of LF82 using microfluidic system showed that the mutation of nagC impairs the early process of biofilm development of LF82. Thus, NAG sensor NagC is involved in the early steps of biofilm formation of AIEC strain LF82 under both static and dynamic conditions. Its implication is partly due to the activation of type 1 fimbriae. NAG can also influence biofilm formation of other intestinal E. coli strains. CONCLUSIONS: This study highlights how catabolism can be involved in biofilm formation of E. coli. Mucus-derived sugars can influence virulence properties of pathogenic E. coli and this study will help us better understand the mechanisms used to prevent colonization of the intestinal mucosa by pathogens.

Invited review: Incidence, risk factors, and effects of clinical mastitis recurrence in dairy cows.

Clinical mastitis (CM) is one of the most frequent and costly diseases in dairy cows. A frustrating aspect of CM is its recurrent nature. This review was conducted to synthesize knowledge on risk of repeated cases of CM, effects of recurrent CM cases, and risk factors for CM recurrence. A systematic review methodology was used to identify articles for this narrative review. Searches were performed to identify relevant scientific literature published after 1989 in English or French from 2 databases (PubMed and CAB Abstracts) and 1 search platform (Web of Science). Fifty-seven manuscripts were selected for qualitative synthesis according to the inclusion criteria. Among the 57 manuscripts selected in this review, a description of CM recurrence, its risk factors, and effects were investigated and reported in 33, 37, and 19 selected manuscripts, respectively. Meta-analysis and meta-regression analyses were used to compute risk ratio comparing risk of CM in cows that already had 1 CM event in the current lactation with risk of CM in healthy cows. For these analyses, 9 manuscripts that reported the total number of lactations followed and the number of lactations with ≤1 and ≤2 CM cases were used. When summarizing results from studies requiring ≥5 d between CM events to consider a CM event as a new case, we observed no significant change in CM susceptibility following a first CM case (risk ratio: 0.99; 95% confidence interval: 0.86-1.14). However, for studies using a more liberal CM recurrence definition (i.e., only 24 h between CM events to consider new CM cases), we observed a 1.54 times greater CM risk (95% confidence interval: 1.20-1.97) for cows that already had 1 CM event in the current lactation compared with healthy cows. The most important risk factors for CM recurrence were parity (i.e., higher risk in older cows), a higher milk production, pathogen species involved in the preceding case, and whether a bacteriological cure was observed following the preceding case. The most important effects of recurrent CM were the milk yield reduction following a recurrent CM case, which was reported to be similar to that of the first CM case, and the increased risk of culling and mortality, which were reported to surpass those of first CM cases.

Actinobacillus pleuropneumoniae culture supernatant antiviral effect against porcine reproductive and respiratory syndrome virus occurs prior to the viral genome replication and transcription through actin depolymerization.

PURPOSE: Recently, the strong antiviral activity of an Actinobacillus pleuropneumoniae (App) culture supernatant against porcine reproductive and respiratory syndrome virus (PRRSV) was discovered. Following this finding, the objective of the present study was to understand how the App culture supernatant inhibits PRRSV replication in its natural targeted host cells, i.e. porcine alveolar macrophages (PAMs). METHODOLOGY: Several assays were conducted with App culture supernatant-treated PRRSV-infected cell lines, such as PAM, St-Jude porcine lung and MARC-145 cells. RT-qPCR assays were used to determine the expression levels of type I and II IFN mRNAs, viral genomic (gRNA) and sub-genomic RNAs (sgRNAs). Proteomic, Western blot and immunofluorescence assays were conducted to determine the involvement of actin filaments in the App culture supernatant antiviral effect.Results/Key findings. Type I and II IFN mRNA expressions were not upregulated by the App culture supernatant. Time courses of gRNA and sgRNA expression levels demonstrated that the App culture supernatant inhibits PRRSV infection before the first viral transcription cycle. Western blot experiments confirmed an increase in the expression of cofilin (actin cytoskeleton dynamics regulator) and immunofluorescence also demonstrated a significant decrease of actin filaments in App culture supernatant-treated PRRSV-infected PAM cells. App culture supernatant antiviral activity was also demonstrated against other PRRSV strains of genotypes I and II. CONCLUSION: App culture supernatant antiviral effect against PRRSV takes place early during PRRSV infection. Results suggest that App culture supernatant antiviral effect may take place via the activation of cofilin, which induces actin depolymerization and subsequently, probably affects PRRSV endocytosis. Other experiments are needed to fully validate this latest hypothesis.

Actinobacillus pleuropneumoniae biofilms: Role in pathogenicity and potential impact for vaccination development.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that belongs to the family Pasteurellaceae. It is the causative agent of porcine pleuropneumonia, a highly contagious respiratory disease that is responsible for major economic losses in the global pork industry. The disease may present itself as a chronic or an acute infection characterized by severe pathology, including hemorrhage, fibrinous and necrotic lung lesions, and, in the worst cases, rapid death. A. pleuropneumoniae is transmitted via aerosol route, direct contact with infected pigs, and by the farm environment. Many virulence factors associated with this bacterium are well characterized. However, much less is known about the role of biofilm, a sessile mode of growth that may have a critical impact on A. pleuropneumoniae pathogenicity. Here we review the current knowledge on A. pleuropneumoniae biofilm, factors associated with biofilm formation and dispersion, and the impact of biofilm on the pathogenesis A. pleuropneumoniae. We also provide an overview of current vaccination strategies against A. pleuropneumoniae and consider the possible role of biofilms vaccines for controlling the disease.

Interactions of Intestinal Bacteria with Components of the Intestinal Mucus.

The human gut is colonized by a variety of large amounts of microbes that are collectively called intestinal microbiota. Most of these microbial residents will grow within the mucus layer that overlies the gut epithelium and will act as the first line of defense against both commensal and invading microbes. This mucus is essentially formed by mucins, a family of highly glycosylated protein that are secreted by specialize cells in the gut. In this Review, we examine how commensal members of the microbiota and pathogenic bacteria use mucus to their advantage to promote their growth, develop biofilms and colonize the intestine. We also discuss how mucus-derived components act as nutrient and chemical cues for adaptation and pathogenesis of bacteria and how bacteria can influence the composition of the mucus layer.

Detection of a mecC-positive Staphylococcus saprophyticus from bovine mastitis in Argentina.

INTRODUCTION: Bovine mastitis causes important economic losses in the dairy industry. Coagulase-negative staphylococci (CNS) are a group of bacteria commonly isolated from bovine mastitis and can display resistance to a wide range of antimicrobial agents. OBJECTIVES: The objective of this study was to determine staphylococcal resistance towards ß-lactam, macrolide and lincosamide antimicrobials in quarters previously treated with third-generation cephalosporin and after lincosamide intramammary therapy. METHODS: Sick quarters of eighteen cows from Villaguay, Entre Ríos (Argentina) with clinical mastitis were studied. All staphylococcal isolates were tested by disk diffusion for their antimicrobial susceptibilities. Cefoxitin resistance was investigated by PCR and sequencing for both the mecA and mecC genes. RESULTS: Resistances to penicillin, oxacillin and cefoxitin were observed, whereas no resistance to macrolide and lincosamide was detected. A cefoxitin-resistant Staphylococcus saprophyticus was found to be mecA-negative but mecC-positive. CONCLUSIONS: This study reports for the first time the mecC gene from a CNS in bovine mastitis in South America. Because CNS may act as reservoirs of antimicrobial resistance genes, they can be seen as a potential public health threat with respect to antimicrobial resistance and the development of multiple resistance. Also, the emergence of methicillin-resistant phenotypes will limit therapeutic options.

Impact of Nutrient Restriction on the Structure of Listeria monocytogenes Biofilm Grown in a Microfluidic System.

Biofilm formation by the pathogen Listeria monocytogenes is a major concern in food industries. The aim of this work was to elucidate the effect of nutrient limitation on both biofilm architecture and on the viability of the bacteria in microfluidic growth conditions. Biofilm formation by two L. monocytogenes strains was performed in a rich medium (BHI) and in a 10-fold diluted BHI (BHI/10) at 30°C for 24 h by using both static conditions and the microfluidic system Bioflux. In dynamic conditions, biofilms grown in rich and poor medium showed significant differences as well in structure and in the resulting biovolume. In BHI/10, biofilm was organized in a knitted network where cells formed long chains, whereas in the rich medium, the observed structure was homogeneous cellular multilayers. Biofilm biovolume production in BHI/10 was significantly higher than in BHI in these dynamic conditions. Interestingly, biovolume of dead cells in biofilms formed under limited nutrient conditions (BHI/10) was significantly higher than in biofilms formed in the BHI medium. In the other hand, in static conditions, biofilm is organized in a multilayer cells and dispersed cells in a rich medium BHI and poor medium BHI/10 respectively. There was significantly more biomass in the rich medium compared to BHI/10 but no difference was noted in the dead/damaged subpopulation showing how L. monocytogenes biofilm could be affected by the growth conditions. This work demonstrated that nutrient concentration affects biofilm structure and the proportion of dead cells in biofilms under microfluidic condition. Our study also showed that limited nutrients play an important role in the structural stability of L. monocytogenes biofilm by enhancing cell death and liberating extracellular DNA.

Coagulase-negative staphylococci species affect biofilm formation of other coagulase-negative and coagulase-positive staphylococci.

Coagulase-negative staphylococci (CNS) are considered to be commensal bacteria in humans and animals, but are now also recognized as etiological agents in several infections, including bovine mastitis. Biofilm formation appears to be an important factor in CNS pathogenicity. Furthermore, some researchers have proposed that CNS colonization of the intramammary environment has a protective effect against other pathogens. The mechanisms behind the protective effect of CNS have yet to be characterized. The aim of this study was to evaluate the effect of CNS isolates with a weak-biofilm phenotype on the biofilm formation of other staphylococcal isolates. We selected 10 CNS with a weak-biofilm phenotype and 30 staphylococcal isolates with a strong-biofilm phenotype for this study. We measured biofilm production by individual isolates using a standard polystyrene microtiter plate assay and compared the findings with biofilm produced in mixed cultures. We confirmed the results using confocal microscopy and a microfluidic system with low shear force. Four of the CNS isolates with a weak-biofilm phenotype (Staphylococcus chromogenes C and E and Staphylococcus simulans F and H) significantly reduced biofilm formation in approximately 80% of the staphylococcal species tested, including coagulase-positive Staphylococcus aureus. The 4 Staph. chromogenes and Staph. simulans isolates were also able to disperse pre-established biofilms, but to a lesser extent. We also performed a deferred antagonism assay and recorded the number of colony-forming units in the mixed-biofilm assays on differential or selective agar plates. Overall, CNS with a weak-biofilm phenotype did not inhibit the growth of isolates with a strong-biofilm phenotype. These results suggest that some CNS isolates can negatively affect the ability of other staphylococcal isolates and species to form biofilms via a mechanism that does not involve growth inhibition.

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.