Bovine mastitis is a polymicrobial disease requiring a polydiagnostic approach

peer-reviewedBovine mastitis, an inflammation of the udder, is associated with increases in milk somatic cell count usually resulting from bacterial infection. We analysed 50 mastitic milk samples via cultivation, 16S rRNA sequencing and a combination of the two (culturomics) to define the complete microbial content of the milk. Most samples contained over 10,000 cfu mL-1 total bacterial counts including isolates that were haemolysin positive (n = 36). Among colonies isolated from blood agar plates, Streptococcus uberis was dominant (11/50) followed by Streptococcus dysgalactiae (6/50), Pseudomonas (6/50), Enterococcus faecalis (6/50), Escherichia coli (6/50), Staphylococcus argenteus (4/50), Bacillus (4/50) and Staphylococcus aureus (2/50). 16S rRNA profiling revealed that amplicons were dominated by Rhodococcus, Staphylococcus, Streptococcus and Pseudomonas. A higher inter-sample diversity was noted in the 16S rRNA readouts, which was not always reflected in the plating results. The combination of the two methods highlights the polymicrobial complexity of bovine mastitis

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments

Alternative treatments for bovine and human mastitis

Mastitis, an inflammation of the mammary gland normally caused by infection, is a painful condition with welfare implications recorded both in cows and lactating women. The main treatment for mastitis is the administration of antibiotics for both species. However, there is a compelling need for novel alternative therapies, considering the antibiotic resistance crisis and concomitant problems in the treatment of human and animal infections. Bacteriocins, are a heterogeneous group of small, peptide-based bacterium antimicrobials that have either broad or narrow range inhibition spectra and as such are a viable alternative to antibiotics in some cases. This thesis presents the current strategies to treat mastitis in cows and women. Chapter 1 provides an overview of alternative microbial treatments for bovine mastitis, with focus on probiotics, bacteriocins, phages, and phage endolysins. Chapter 2 examines the microbiology and treatment of human mastitis with Staphylococcus aureus, Staphylococcus epidermidis, and Corynebacterium sp. being identified as the major etiological agents in acute, subacute, and granulomatous mastitis, respectively. Moving forward, Chapter 3 sought to identify the major pathogenic species detected in fifty bovine mastitic milk samples by using cultivation and high-throughput sequencing (HTS). A combination of the two approaches illuminated the polymicrobial complexity of the disease and implied potential for multimicrobial origins of the symptoms. Chapter 4 investigated the milk microbiomes in healthy, subclinical and clinical lactating mothers and evaluated the immune status from these three groups with 37.8% of the asymptomatic women being subclinical. In Chapter 5 insights were provided into using alternative treatments, i.e. nisin A and vancomycin, on S. aureus for biofilm inhibition and eradication. None of the applied treatments were able to eradicate preformed biofilms while the combination treatment significantly inhibited biofilm formation compared with single treatments. Finally, in Chapter 6, 80 strains with antimicrobial activity were isolated from 37 asymptomatic human milk samples. Genome sequencing and in silico analysis of those isolates led to identification of sixteen novel bacteriocins representing all known bacteriocin subclasses. This study suggests that the milk microbiome is a rich source of strains with antimicrobial potential. Overall, the results of this work expand the large body of research that exists in the field of mastitis by exploring the microbiota composition in both mastitic cows and humans and by investigating the pathogens involved and relating it to local immune responses. Furthermore, the efficacy of a bacteriocin as a co-therapy against a mastitis pathogen along with the discovery of novel antimicrobial peptides within the human milk microbiome, suggests that bacteriocins could provide novel therapies for disease treatment and their deployment in the medical sector in the future is a likely prospect

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk.

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments

Monitoring Humoral Response Following BNT162b2 mRNA Vaccination against SARS-CoV-2 in Hematopoietic Stem-Cell Transplantation Patients: A Single-Center Prospective Study along with a Brief Review of Current Literature

Data on antibody response (AR) after vaccination against SARS-CoV2 in hematopoietic stem-cell transplantation setting (HSCT) were initially scarce, mainly due to the exclusion of such patients from approval studies. Shortly after the worldwide application of vaccination against SARS-CoV-2 in vulnerable populations such as patients with hematologic malignancies, limited single-center trials, including HSCT patients, were published. However, there was a great heterogeneity between them regarding the type of underlying malignancy, co-current treatment, type of vaccine, method of AR measurement, and time point of AR measurement. Herein, we present the results of a prospective study on AR after vaccination for SARS-CoV-2 using the BNT162b2 vaccine in a cohort of 54 HSCT recipients—mostly autologous from a single Unit—along with a broad review of the current literature. In our cohort, the AR positivity rate at 1 month was 80.8% and remained positive in 85.7% of patients at 3 months after vaccination. There were only nine non-responders, who were more heavily pretreated and more frequently hypogammaglobulinemic compared to responders. High antibody titers (AT), [AT ≥ 1000 U/mL], were detected in 38.5% and 30.6% of the patients at m1 and m3, respectively. A significant decline in AT between m1 and m3 was demonstrated—p 1 and AT3 were 480.5 and 293 U/mL, respectively. A novel finding of our study was the negative impact of IgA hypogammaglobulinemia on response to vaccination. Other negative significant factors were treatment with anti-CD20 antibody at vaccination and vaccination within 18 months from HSCT. Our data indicate that HSCT recipients elicit a positive response to the BNT162b2 vaccine against SARS-CoV-2 when vaccinated at 6 months post-transplant, and vaccination should be offered to this patient population even within the post-pandemic COVID-19 era