Persistence of Staphylococcus spp. in milk from cows undergoing homeopathy to control subclinical mastitis.

BACKGROUND: Mastitis is one of the major diseases in dairy cattle, as it causes great economic losses to producers due to the reduction of milk production and changes in the quality of the product. The disease is mainly caused by bacteria of the genus Staphylococcus spp., these microorganisms can express various virulence factors, such as biofilms for example. In herds with organic management, producers and technicians use unconventional ways to treat and control the disease, such as homeopathy. However, it is not known if this type of treatment is able to control pathogenic bacteria such as those of the genus Staphylococcus, of relevance to animal and human health. Thus, the objective of this study was to investigate the production of biofilm in vitro and its genes by Staphylococcus spp. isolated in the milk of cows treated with homeopathy, as well as the persistence of microorganisms in animals. METHODS: Ninety-nine isolates of Staphylococcus spp. from cows treated and not treated with homeopathy were identified by internal transcribed space-polymerase chain reaction and investigated for the presence of the icaABCD, bap, aap, atlE, and bhp genes and in vitro biofilm production using the adhesion method on polystyrene plates. The enzyme restriction profile was determined by Pulsed-Field Gel Electrophoresis. Clusters of S. aureus and S. epidermidis with three or more isolates had an isolate selected for Multilocus Sequence Typing. RESULTS: The frequency of S. aureus isolations was similar in treated and untreated cows, while 71.4% of the coagulase-negative identified were isolated in cows treated with homeopathy. The distribution of the operon ica genes was similar in animals with and without treatment, except for the icaD gene, more frequent in treated cows. Production of biofilm was associated with presence of one or more genes from the icaADBC operon. S. aureus revealed a greater diversity and greater dissemination in cows treated and not treated with homeopathy. Sequence Types ST1, ST5, and ST126 were identified in S. aureus. CONCLUSIONS: The presence of biofilm-associated genes and the in vitro production of biofilms, combined with the persistence of clonal profiles of Staphylococcus spp. demonstrate other forms of control for bovine mastitis should be researched for organic production herds.

Staphylococcus epidermidis Pathogenesis: Interplay of icaADBC Operon and MSCRAMMs in Biofilm Formation of Isolates from Pediatric Bacteremia in Peshawar, Pakistan.

Background and Objective: Staphylococcus epidermidis is an opportunistic pathogen from pediatric bacteremia that is commonly isolated. Biofilm is the major virulence factor of S. epidermidis; however, the role of biofilm determinants in biofilm formation is highly contradictory and diverse. The current study aimed to investigate the role of polysaccharide-dependent and polysaccharide-independent pathogenic determinants in biofilm formation under physiological stress conditions. Materials and Methods: The isolates (n = 75) were identified and screened for the icaADBC operon, IS256, and an array of MSCRAMMs (Microbial Surface Component Recognizing Adhesive Matrix Molecules) through PCR analysis. The activity of the icaADBC operon was detected by Congo red assay, and the biofilm formation was analyzed through microtiter plate assay. Results: S. epidermidis isolates produced biofilm (n = 65; 86.6%) frequently. The icaA was the major representative module of the actively expressing icaADBC operon (n = 21; 80.7% sensitivity). The MSCRAMMs, including fbe (n = 59; 90.7%; p = 0.007), and embp (n = 57; 87.6%; p = 0.026), were highly prevalent and associated with biofilm positive S. epidermidis. The prevalence of icaADBC operon in biofilm positive and negative S. epidermidis was not significant (n = 41; 63%; p = 0.429). No significant association was found between IS256 and actively complete icaADBC operon (n = 10; 47.6%; p = 0.294). In the presence of 5% human plasma and glucose stress, S. epidermidis produced a strong biofilm (n = 55; 84.6%). Conclusion: The polysaccharide-dependent biofilm formation is significantly replaced (n = 21; 28%; p = 0.149) by a polysaccharide-independent mechanism (n = 59; 90.7%; p = 0.007), in which the MSCRAMMs might actively play their role. The fibrinogen-binding protein and extracellular matrix-binding protein might be potential anti-biofilm drug targets, markers of rapid diagnosis, and potential vaccine candidates of S. epidermidis involved in pediatric bacteremia.

Determining the dispersion time in Staphylococcus epidermidis biofilm using physical and molecular approaches.

Despite being an innocuous commensal of human skin and mucous membranes, Staphylococcus epidermidis, infects surgical wounds and causes infections through biofilm formation. This study evaluates, in a time-dependent experiment, the self-dispersion of S. epidermidis CIP 444 biofilm when formed on borosilicate glass (hydrophilic) and polystyrene (hydrophobic) surfaces, using physical and molecular approaches. During a seven-day period of incubation, absorbance measurement revealed a drop in biofilm optical density on both studied surfaces on day 4 (0.043-0.035 nm/cm2, polystyrene), (0.06-0.053 nm/cm2, borosilicate glass). Absorbance results were correlated with crystal violet staining that showed a clear detachment from day 4. The blue color increases again on day 7, with an increase in biofilm optical density indicating the regeneration of the biofilm. Changes in gene expression in the S. epidermidis biofilm were assessed using a real-time reverse transcription-polymerase chain reaction. High expression of agr genes was detected on days 4 and 5, confirming our supposition of dispersion in this period, autolysin genes like atlE1 and aae were upregulated from day 3 until day 6 and the genes responsible for slime production and biofilm accumulation, were upregulated on days 4, 5, and 6 (ica ADBC) and on days 5, 6 and 7 (aap), indicating a dual process taking place. These findings suggest that S. epidermidis CIP 444 biofilms disperse at day 4 and reform at day 7. Over the course of the seven-day investigation, 2-ΔΔCt results showed that some genes in the biofilm were dramatically enhanced while others were significantly decreased as compared to planktonic ones.

Various mutations in icaR, the repressor of the icaADBC locus, occur in mucoid Staphylococcus aureus isolates recovered from the airways of people with cystic fibrosis.

Staphylococcus aureus is one of the major pathogens isolated from the airways of people with cystic fibrosis (pwCF). Recently, we described a mucoid S. aureus phenotype from respiratory specimens of pwCF, which constitutively overproduced biofilm that consisted of polysaccharide intercellular adhesin (PIA) due to a 5bp-deletion (5bp-del) in the intergenic region of the intercellular adhesin (ica) locus. Since we were not able to identify the 5bp-del in mucoid isolates of two pwCF with long-term S. aureus persistence and in a number of mucoid isolates of pwCF from a prospective multicenter study, these strains were (i) characterized phenotypically, (ii) investigated for biofilm formation, and (iii) molecular typed by spa-sequence typing. To screen for mutations responsible for mucoidy, the ica operon of all mucoid isolates was analyzed by Sanger sequencing. Whole genome sequencing was performed for selected isolates. For all mucoid isolates without the 5 bp-del, various mutations in icaR, which is the transcriptional repressor of the icaADBC operon. Mucoid and non-mucoid strains belonged to the same spa-type. Transformation of PIA-overproducing S. aureus with a vector expressing the intact icaR gene restored the non-mucoid phenotype. Altogether, we demonstrated a new mechanism for the emergence of mucoid S. aureus isolates of pwCF.

Differences in Biofilm Formation by Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains.

Staphylococcus aureus (S. aureus) is a common pathogen involved in community- and hospital-acquired infections. Its biofilm formation ability predisposes it to device-related infections. Methicillin-resistant S. aureus (MRSA) strains are associated with more serious infections and higher mortality rates and are more complex in terms of antibiotic resistance. It is still controversial whether MRSA are indeed more virulent than methicillin-susceptible S. aureus (MSSA) strains. A difference in biofilm formation by both types of bacteria has been suggested, but how only the presence of the SCCmec cassette or mecA influences this phenotype remains unclear. In this review, we have searched for literature studying the difference in biofilm formation by MRSA and MSSA. We highlighted the relevance of the icaADBC operon in the PIA-dependent biofilms generated by MSSA under osmotic stress conditions, and the role of extracellular DNA and surface proteins in the PIA-independent biofilms generated by MRSA. We described the prominent role of surface proteins with the LPXTG motif and hydrolases for the release of extracellular DNA in the MRSA biofilm formation. Finally, we explained the main regulatory systems in S. aureus involved in virulence and biofilm formation, such as the SarA and Agr systems. As most of the studies were in vitro using inert surfaces, it will be necessary in the future to focus on biofilm formation on extracellular matrix components and its relevance in the pathogenesis of infection by both types of strains using in vivo animal models.

Antibiofilm Activity of Omega-3 Fatty Acids and Its Influence on the Expression of Biofilm Formation Genes on Staphylococcus aureus.

Background: Currently, 1-2% of all prosthetic joint surgeries are followed by an infection. These infections cause approximately 4% of deaths in the first year after surgery, while the 5-year mortality rate is up to 21%. Prosthetic joint infections are mainly caused by Staphylococcus aureus or Staphylococcus epidermis strains. Both species share the capability of biofilm formation and methicillin resistance. The formation of biofilm helps bacterial cells to withstand critical environmental conditions. Due to their tolerance against antibacterial substances, biofilms are a significant problem in modern medicine. Alternatives for the use of methicillin as a therapeutic are not yet widespread. The use of omega-3 fatty acids, such as docosahexaenoic acid, may help against prosthetic joint infections and lower mortality rates. The aim of this study is to evaluate if docosahexaenoic acid offers a safe anti-biofilm activity against Staphylococcus aureus and MRSA without enhancing icaADBC-dependent biofilm formation or additional stress responses, therefore enhancing antibiotic tolerance and resistance. Methods: In this study, we examined the gene expression of biofilm-associated genes and regulators. We performed RT-qPCR after RNA extraction of Staphylococcus aureus ATCC 29213 and one clinical MRSA strain. We compared gene expression of icaADBC, SarA, SigB, and agrAC under the influence of 1.25 mg /L and 0.625 mg/L of docosahexaenoic acid to their controls. Results: We found a higher expression of regulatory genes such as SarA, SigB, agrA, and agrC at 1.25 mg/L of docosahexaenoic acid in ATCC 29213 and a lower increase in gene expression levels in clinical MRSA isolates. icaADBC was not affected in both strains at both concentration levels by docosahexaenoic acid. Conclusions: Docosahexaenoic acid does not enhance icaADBC-dependent biofilm formation while still reducing bacterial CFU in biofilms. Docosahexaenoic acid can be considered an option as a therapeutic substance against biofilm formation and may be a good alternative in reducing the risk of MRSA formation.

The Toxin-Antitoxin MazEF Drives Staphylococcus aureus Biofilm Formation, Antibiotic Tolerance, and Chronic Infection.

Staphylococcus aureus is the major organism responsible for surgical implant infections. Antimicrobial treatment of these infections often fails, leading to expensive surgical intervention and increased risk of mortality to the patient. The challenge in treating these infections is associated with the high tolerance of S. aureus biofilm to antibiotics. MazEF, a toxin-antitoxin system, is thought to be an important regulator of this phenotype, but its physiological function in S. aureus is controversial. Here, we examined the role of MazEF in developing chronic infections by comparing growth and antibiotic tolerance phenotypes in three S. aureus strains to their corresponding strains with disruption of mazF expression. Strains lacking mazF production showed increased biofilm growth and decreased biofilm antibiotic tolerance. Deletion of icaADBC in the mazF::Tn background suppressed the growth phenotype observed with mazF-disrupted strains, suggesting the phenotype was ica dependent. We confirmed these phenotypes in our murine animal model. Loss of mazF resulted in increased bacterial burden and decreased survival rate of mice compared to its wild-type strain demonstrating that loss of the mazF gene caused an increase in S. aureus virulence. Although lack of mazF gene expression increased S. aureus virulence, it was more susceptible to antibiotics in vivo Combined, the ability of mazF to inhibit biofilm formation and promote biofilm antibiotic tolerance plays a critical role in transitioning from an acute to chronic infection that is difficult to eradicate with antibiotics alone.IMPORTANCE Surgical infections are one of the most common types of infections encountered in a hospital. Staphylococcus aureus is the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate, as the bacteria form biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria that are planktonic, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate that mazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allows S. aureus to transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host.

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL-1, including 15.7% with an MIC of 8 μg mL-1 and 2% with an MIC of 16 μg mL-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.