Coagulase-negative staphylococci in ewe udder surface and raw milk samples

Coagulase-negative staphylococci (CNS) are among the major causes of subclinical mastitis in dairy ewe flocks. This has a financially significant impact on the ewe dairy sector and consumer health. The determination of the bacterial count, particularly CNS, is critical in terms of the quantity and quality of ovine milk. Thus, the purpose of this study was to quantify the CNS count in the udder surface and raw milk samples of the ewe, in addition to identifying CNS strains (n=8) collected from udder surface and individual raw milk samples by 16S rRNA gene sequencing. A total of 164 samples of udder surface and raw milk originated from four sheep farms were tested. The obtained values were compared between the different farms. Furthermore, values during 2018 and 2019 in the case of Farm I were compared. Significantly higher (p<0.05) average count was observed in udder surface samples taken from Farm I (2.8±1.0 lg CFU/cm2) than that of Farm III samples (2.3±0.6 lg CFU/cm2). In the case of individual raw milk, the higher value was observed in samples derived from Farm III (3.5±0.9 lg CFU/mL), while the obtained value was significantly lower (p<0.05) in samples originated from Farm IV (1.8±0.4 lg CFU/mL). In the bulk tank milk samples, the highest mean value was 5.3±0.4 CFU/mL, and there was no significant (p>0.05) variation between farms. Coagulase-negative staphylococci counts were decreased to a certain extent in both sample types tested during 2019 except for individual raw milk derived from the Tsigai breed. The correlation between the mean CNS counts of udder surface and individual raw milk was very weak (r=0.048). Staphylococcus simulans, Staphylococcus auricularis, and Staphylococcus equorum were identified by molecular sequencing and Staphylococcus simulans were the most frequently identified CNS species. A higher CNS count of bulk tank milk than individual raw milk indicates possible contamination during milking and storage. Therefore, further studies are required to investigate the other sources of bulk tank milk contamination to improve the hygienic quality of milk.

Staphylococcus simulans bloodstream infection following CIED extraction

A 78-year-old man with an implantable cardioverter-defibrillator (ICD) presented with chills and malaise. His history was significant for heart failure with reduced ejection fraction and complete heart block. He had undergone permanent pacemaker placement that was later upgraded to an ICD 5 years before his presentation. Physical examination revealed an open wound with surrounding erythema overlying the device site. Blood cultures obtained on admission were negative. Transesophageal echocardiogram did not show valve or lead vegetations. He underwent a prolonged extraction procedure. Postoperatively, he developed septic shock and cultures from the device, and repeat peripheral blood cultures grew Staphylococcus simulans and Staphylococcus epidermidis. He was treated with intravenous vancomycin but had refractory hypotension, leading to multiorgan failure. He later expired after being transitioned to comfort care. The patient may have acquired S. simulans by feeding cows on a nearby farm, and the prolonged extraction procedure may have precipitated the bacteraemia.

Toxic shock syndrome with a cytokine storm caused by Staphylococcus simulans: a case report

Background Exotoxins secreted from Staphylococcus aureus or Streptococcus pyogenes act as superantigens that induce systemic release of inflammatory cytokines and are a common cause of toxic shock syndrome (TSS). However, little is known about TSS caused by coagulase-negative staphylococci (CoNS) and the underlying mechanisms. Here, we present a rare case of TSS caused by Staphylococcus simulans (S. simulans). Case presentation We report the case of a 75-year-old woman who developed pneumococcal pneumonia and bacteremia from S. simulans following an influenza infection. The patient met the clinical criteria for probable TSS, and her symptoms included fever of 39.5 °C, diffuse macular erythroderma, conjunctival congestion, vomiting, diarrhea, liver dysfunction, and disorientation. Therefore, the following treatment was initiated for bacterial pneumonia complicating influenza A with suspected TSS: meropenem (1 g every 8 h), vancomycin (1 g every 12 h), and clindamycin (600 mg every 8 h). Blood cultures taken on the day after admission were positive for CoNS, whereas sputum and pharyngeal cultures grew Streptococcus pneumoniae (Geckler group 4) and methicillin-sensitive S. aureus, respectively. However, exotoxins thought to cause TSS, such as TSS toxin-1 and various enterotoxins, were not detected. The patient’s therapy was switched to cefazolin (2 g every 8 h) and clindamycin (600 mg every 8 h) for 14 days based on microbiologic test results. She developed desquamation of the fingers on hospital day 8 and was diagnosed with TSS. Conventional exotoxins, such as TSST-1, and S. aureus enterotoxins were not detected in culture samples. The serum levels of inflammatory cytokines, such as neopterin and IL-6, were high. CD8+ T cells were activated in peripheral blood. Vβ2+ population activation, which is characteristic for TSST-1, was not observed in the Vβ usage of CD8+ T cells in T cell receptor Vβ repertoire distribution analysis. Conclusions We present a case of S. simulans-induced TSS. Taken together, we speculate that no specific exotoxins are involved in the induction of TSS in this patient. A likely mechanism is uncontrolled cytokine release (i.e., cytokine storm) induced by non-specific immune reactions against CoNS proliferation.

Cloning and Expression of Staphylococcus Simulans Lysostaphin Enzyme Gene in Bacillus Subtilis WB600

Background: Lysostaphin is a glycylglycine endopeptidase, secreted by Staphylococcus simulans, capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the Staphylococcus aureus cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in Bacillus subtilis WB600 host using pWB980 expression system.Results: Plasmid pACK1 of S. simulans was extracted using alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into Escherichia coli DH5α. The amplified gene fragment and uncloned pWB980 vector were digested using PstI and XbaІ enzymes and purified. The restricted gene fragment was ligated into the pWB980 expression vector by the standard protocols, then the recombinant plasmid was transformed into B. subtilis WB600 using electroporation method. The recombinant protein was evaluated by the SDS-PAGE method and confirmed by western immunoblot. Analysis of the target protein showed a band corresponding to an about 27-kDa r-lysostaphin. Protein content was estimated 91µg/ml by Bradford assay.Conclusions: The recombinant lysostaphin represented 90% of its maximum activity at 40℃ and displayed good thermostability by keeping about 80% of its maximum activity at 45℃. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40℃ and showed good stability at 40℃ for 16 h incubation.

Susceptibility to Bacteriocins in Biofilm-Forming, Variable Staphylococci Isolated from Local Slovak Ewes’ Milk Lump Cheeses

Seventeen staphylococci isolated from 54 Slovak local lump cheeses made from ewes’ milk were taxonomically allotted to five species and three clusters/groups involving the following species: Staphylococcus aureus (5 strains), Staphylococcus xylosus (3 strains), Staphylococcus equorum (one strain) Staphylococcus succinus (5 strains) and Staphylococcus simulans (3 strains). Five different species were determined. The aim of the study follows two lines: basic research in connection with staphylococci, and further possible application of the bacteriocins. Identified staphylococci were mostly susceptible to antibiotics (10 out of 14 antibiotics). Strains showed γ-hemolysis (meaning they did not form hemolysis) except for S. aureus SAOS1/1 strain, which formed β-hemolysis. S. aureus SAOS1/1 strain was also DNase positive as did S. aureus SAOS5/2 and SAOS51/3. The other staphylococci were DNase negative. S. aureus SAOS1/1 and SAOS51/3 showed biofilm formation on Congo red agar. However, using quantitative plate assay, 12 strains out of 17 showed low-grade biofilm formation (0.1 ≤ A570 < 1), while five strains did not form biofilm (A570 < 0.1). The growth of all strains, including those strains resistant to enterocins, was inhibited by nisin and gallidermin, with high inhibition activity resulting in the inhibition zone in size from 1600 up to 102,400 AU/mL (arbitrary unit per milliliter). This study contributes to microbiota colonization associated with raw ewe’s milk lump cheeses; it also indicates bacteriocin treatment benefit, which can be used in prevention and/or elimination of staphylococci.