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Background: Mature lysostaphin (~28-kDa Lss) from Staphylococcus simulans proves effective in killing methicillin-resistant Staphylococcus aureus (MRSA) which is endemic in hospitals worldwide. Lss is Zn2+-dependent endopeptidase, but its bacteriolytic activity could be affected by exogenously added Zn2+. Objective: To gain greater insights into structural and functional impacts of Zn2+and Ni2+on Lss-induced bioactivity. Methods: Lss purified via immobilized metal ion-affinity chromatography was assessed for bioactivity using turbidity reduction assays. Conformational change of metal ion-treated Lss was examined by circular dichroism and intrinsic fluorescence spectroscopy. Co-sedimentation assay was performed to study interactions between Zn2+-treated Lss and S. aureus peptidoglycans. Metal ionbinding prediction and intermolecular docking were used to locate an extraneous Zn2+-binding site. Results: A drastic decrease in Lss bioactivity against S. aureus and MRSA was revealed only when treated with Zn2+, but not Ni2+, albeit no negative effect of diethyldithiocarbamate—Zn2+-chelator on Lss-induced bioactivity. No severe conformational change was observed for Lss incubated with exogenous Zn2+ or Ni2+. Lss pre-treated with Zn2+ efficiently bound to S. aureus cell-wall peptidoglycans, suggesting non-interfering effect of exogenous metal ions on cell-wall targeting (CWT) activity. In silico analysis revealed that exogenous Zn2+, but not Ni2+, preferably interacted with a potential extraneous Zn2+-binding site (His253, Glu318 and His323) placed near the Zn2+-coordinating Lssactive site within the catalytic (CAT) domain. Conclusion: Our present data signify the adverse influence of exogenous Zn2+ ions on Lss-induced staphylolytic activity through the exclusive presence within the CAT domain of an extraneous inhibitory Zn2+-binding site, without affecting the CWT activity.

Objectives To evaluate the in vitro activity of dalbavancin compared with vancomycin, daptomycin and other agents against a large collection of coagulase-negative staphylococci (CoNS) isolates. Methods A total of 5088 CoNS causing clinically significant infection were consecutively collected from 122 medical centres in the USA and Europe over 6 years (2014–2019). Isolates were tested for susceptibility by the reference broth microdilution method. Species identification was confirmed by MALDI-TOF. Most isolates were from bloodstream infections (BSIs) (53.5%) or skin/skin structure infections (28.5%). Results Staphylococcus epidermidis was the most common species overall (54.6%) and for BSI (61.3%). The second most common species were Staphylococcus lugdunensis overall (12.3%) and Staphylococcus hominis for BSI (14.7%). Dalbavancin (MIC50/90, 0.03/0.06 mg/L) inhibited >99.9% of CoNS isolates at ≤0.25 mg/L (susceptible breakpoint for Staphylococcus aureus per CLSI). All species were inhibited at ≤0.25 mg/L dalbavancin, except some S. epidermidis (>99.9%) and Staphylococcus warneri (98.9%) isolates. Staphylococcus capitis and Staphylococcus simulans exhibited the lowest dalbavancin MIC50/90 values (0.015/0.03 mg/L) and Staphylococcus haemolyticus and Staphylococcus saprophyticus the highest (MIC50/90, 0.06/0.12 mg/L); 47.8% of S. epidermidis and 34.7% of S. haemolyticus exhibited decreased susceptibility to vancomycin (MIC ≥ 2 mg/L) and 23.2% of S. capitis and 28.4% of S. warneri showed decreased susceptibility to daptomycin (MIC ≥ 1 mg/L). Conclusion Antimicrobial susceptibility varied widely among CoNS species. Dalbavancin inhibited >99.9% and 99.1% of isolates at the US-FDA and EUCAST breakpoints, respectively. Clinical studies of dalbavancin for treatment of CoNS infections should be considered based on these in vitro data.

A strain of Staphylococcus simulans D14 (S. simulans D14) showed the highest nitrate reductase activity (NRA) of 4.52 mM NO2−/mg dry weight by the spectrophotometric method, which was screened from traditional Chinese sausage. When the UV mutagenesis time was 25 s, the positive mutation rate was the highest at 26.60%. The NRA of the obtained positive mutant UV-11 was 9.21 mM NO2−/mg dry weight, and the activity was found to be 1.04-fold higher than that of the original strain S. simulans D14. A Plackett–Burman design (PBD) was employed to screen the significant variables pH, KNO3 (%) and incubation time (h), and response surface methodology (RSM) was used to optimize the significant variables using a Box–Behnken design (BBD). The results showed that the NRA of S. simulans UV-11 was 15.22 mM NO2−/mg dry weight under optimum conditions of 37 °C, pH 6.5, incubation time 15 h, KNO3 0.045%, NaCl 5%, NaNO2 0.015%, peptone 1%, and D–mannitol 1%, which increased by 65.2% compared with the unoptimized medium. Natural curing agents (containing 107 CFU/g S. simulans UV-11 under optimal conditions and 1.40% celery powder, T2) were added to the cured meat model. T2 produced significantly lighter and redder signals than the control group (C) and the addition of 150 ppm NaNO2 group (T1). The thiobarbituric acid reactive substance (TBARS) of T2 was 2.30 mg malonaldehyde/kg product and residual nitrite of T2 was 7.1 ppm after 14 days of storage,which were lower than those groups of C and T1. Taking into account the results of cured meat models, S. simulans UV-11 could be selected as a potential starter culture for the processing of natural meat products.

Chromium(vi) contaminated sites have been targeted for studying highly chromate-resistant bacteria. From a total of 23 Cr(vi)-resistant bacteria isolated on Luria-Bertani agar medium supplemented with K2CrO4 (1,500 μg/mL), only one strain UT8 (Staphylococcus simulans) was able to tolerate high concentration of chromate, i.e., up to 200 mg/mL in agar medium from tannery effluent. In acetate minimal medium (AMM), it showed maximum tolerance of up to 2,500 μg/mL. Cr(vi) removal potential was 94.6% after 24 h (K2CrO4 1,500 μg/mL). Parametric conditions were optimized in AMM to attain maximum chromate removal. Exopolysaccharides extracted from bacterial cells exposed to chromate exhibited major absorption shifts from 2,500 to 500 cm−1 revealed by Fourier transform infrared spectroscopy. Energy-disperse X-ray spectroscopy further confirmed the adsorption of oxyanions to the bacterial cells. Surface topography of the Cr(vi) treated cells showed transformation into concave shape by scanning electron microscopy. The presence of resistance genes, i.e., chromate reductase (chrR) and class I integrase (intI1), further confirmed tolerance toward chromate. Microarray data analysis of transcriptional gene expression suggested upregulation of cys gene cluster under chromate exposure. Concisely, the present investigations revealed the potential of S. simulans to be an effective candidate for chromate reclamation of wastewater.

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. Plasmid pACK1 of S. simulans was extracted using the 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 27-kDa r-lysostaphin. Protein content was estimated 91 mg/L by Bradford assay. The recombinant lysostaphin represented 90% of its maximum activity at 40 °C and displayed good thermostability by keeping about 80% of its maximum activity at 45 °C. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40 °C and showed good stability at 40 °C for 16 h incubation.

The aim of this study was to identify and evaluate the bacteria load and the level of Staphylococcus Species and Staphylococcus aureus contamination in raw cow´s milk distributed in Khartoum state. One hundred and eighty samples were taken from Khartoum, Omdurman and Khartoum North, 60 samples of raw milk from healthy apparently cows in farms, 60 samples of raw milk vended by donkeys, 60 samples of raw milk vended by cars. Total Viable Bacterial count was done using standard plate count. The samples were detected for the presence of Staphylococcus Species using conventional methods, cultured in Baird-Parker and Mannitol agar, Gram Stain, biochemical tests were done. The TVBC showed that the highest bacterial load was detected in the raw milk vended by Donkey (6.90±.03 log10 cfu/ml) vended by cars (6.78±0.12 log10 cfu/ml) then the lowest bacterial load detected in the milk collected from farm (6.63±0.07 log10 cfu/ml). Out of 180 samples of raw milk studied, 130 showed contamination by Staphylococcus species corresponding to 72.2% of the samples being contaminated and out of 180 samples of raw cow´s milk 80 was contaminated with staphylococcus aureus corresponding to 44.4% of the samples.The isolated Staphylococcus species was confirmed further by using the Polymerase Chain Reaction (PCR) targeting the partial sequence of 16s rRNA gene. Sequencing identified Staphylococcus aureus, staphylococcus epidermidis, Staphylococcus hominies and Staphylococcus simulans. The results showed a high level of contamination by Staphylococcus Spp. and staphylococcus aureus in raw cow´s milk that distributed in Khartoum state.

Coagulase-negative staphylococci (CNS) are one of the most common bovine mastitis pathogens found worldwide. In this study, we investigated the prevalence and distribution of CNS species in mastitis milk samples and further characterized the methicillin-resistant (MR) CNS. A total of 311 CNS were isolated from 3,692 quarter milk samples from 1,373 dairy cattle at 81 farms between 2013 and 2017. Further evaluation of the CNS isolates revealed 14 CNS species among the samples and 3 predominant species-namely, Staphylococcus chromogenes, Staphylococcus simulans, and Staphylococcus epidermidis. Resistance was higher in S. epidermidis than in other CNS species except for resistance against oxacillin in Staphylococcus sciuri. Resistance to β-lactams was the most common in all CNS species (8.4% in ampicillin, 21.2% in oxacillin, and 13.5% in penicillin). Conversely, only minimal resistance to cephalothin, ceftiofur, and pirlimycin/novobiocin was found. Twenty-one isolates from 4 species were mecA-carrying MRCNS strains, including 18 S. epidermidis and 1 each of S. sciuri, Staphylococcus equorum, and Staphylococcus hominis. The majority of the mecA-carrying MRCNS isolates were produced in the biofilm. Furthermore, multidrug-resistant sequence type 179 isolate produced the strongest biofilm. Seven genotypes were detected in the 18 MR S. epidermidis strains, the most predominant of which persisted on a farm for 2 yr. Our findings for the antimicrobial susceptibility profiles and genotypic characterization of the MRCNS isolates could provide valuable information for controlling the spread of resistance and the selection of appropriate antimicrobial therapies for mastitis in the future. Further, strategic antibiotic use for mastitis treatment and hygienic management practices aimed at the prevention of the growth of resistant bacteria are urgently needed on dairy farms.

Chromium is a carcinogenic toxicant widely used in many industries. The concentration of chromium is increasing in various areas of the world causing threat to living beings. Bioremediation is an inexpensive and eco-friendly approach to detoxify such contaminants. Many bacteria can live both as free cells (planktonic) and as biofilms (sessile), and the pattern of chromium reduction is different in these live forms. In the present study, chromium reduction by planktonic cells and biofilms of bacteria was determined in a comparative manner. Chromium-resistant bacteria capable of biofilm formation were isolated from contaminated soil and wastewater. The bacteria were characterized morphologically and biochemically, and identified by 16S rRNA gene sequencing. Cr(VI) reduction by planktonic cells and biofilms was determined over different periods of incubation. The wastewater isolates showed significant resistance against Cr(VI). Minimum inhibitory concentration (MIC) of Cr (VI) was found to be 900 μg ml−1 for Staphylococcus simulans KW1 and Staphylococcus hominis KW2. Whereas, Bacillus cereus KW4 and Staphylococcus equorum KS1 showed MIC values of 800 μg ml−1 and 850 μg ml−1 respectively. Among all the isolates, maximum biofilm formation was shown by Staphylococcus equorum KS1 both qualitatively and quantitatively. Planktonic cells of these bacteria were more efficient in Cr(VI) reduction as compared to their biofilms in 24 h. Maximum Cr(VI) reduction in planktonic form was shown by Staphylococcus simulans KW1 (27.9 μg ml−1, 55%), whereas in biofilm mode of growth the maximum Cr(VI) reduction was shown by Staphylococcus equorum KS1 (4.6 μg ml−1, 9.2%). The results show that for faster Cr(VI) reduction in wastewater the planktonic form of these bacteria is more suited.

An understanding of the spatio-temporal distribution of several groups of mastitis pathogens can help to inform programs for the successful control and management of mastitis. However, in the absence of an active surveillance program such information is not readily available. In this retrospective study we analyzed passive surveillance data from a diagnostic laboratory with an aim to describe the spatio-temporal trend of major mastitis pathogens between 2008 and 2017 in Ontario dairy cattle. Data for all milk culture samples submitted to the Animal Health Laboratory (AHL) at the University of Guelph between 2008 and 2017 was accessed. Descriptive analyses were conducted to identify the major pathogens and Chi-square goodness-of-fit tests were used to compare between multiple proportions. Likewise, univariable logistic regression analysis was performed to determine if there was a change in the probability of isolating the major mastitis pathogens depending on geography or time. Seasonality was assessed by calculating the seasonal relative risk (RR). Of a total of 85,979 milk samples examined, more than half of the samples (61.07%) showed no growth and the proportion of samples that showed no growth almost halved during the study period. Of the samples (36.21%, n = 31,133) that showed any growth, the major bacterial pathogens were Staphylococcus aureus (15.60%), Non-aureus Staphylococci (NAS) (5.04%), Corynebacterium spp. (2.96%), and Escherichia coli (2.00%). Of the NAS, the major species reported were Staphylococcus chromogenes (69.02%), Staphylococcus simulans (14.45%), Staphylococcus epidermidis (12.99%), and Staphylococcus hyicus (2.13%). A temporal change in the prevalence of contagious pathogens like S. aureus and Corynebacterium spp. was observed with an increasing odds of 1.06 and 1.62, respectively. Likewise, except for Trueperella pyogenes, the prevalence of all the major environmental mastitis pathogens increased during the study period. The isolation of most of the pathogens peaked in summer, except for S. aureus, T. pyogenes, and Streptococcus dysgalactiae which peaked in spring months. Interestingly, a regional pattern of isolation of some bacterial pathogens within Ontario was also observed. This study showed a marked spatio-temporal change in the prevalence of major mastitis pathogens and suggests that a regional and seasonal approach to mastitis control could be of value.

Bulk tank milk from 58 dairy goat and sheep flocks located in southern Spain was examined to determine the prevalence and distribution of Staphylococci. A total of 45 isolates were obtained and characterized to determine the species, antimicrobial resistance profile, and genetic similitude by pulse-field gel electrophoresis (PFGE) using SmaI. Staphylococcus aureus isolates were confirmed by polymerase chain reaction (PCR) analysis of nuc, and resistance to methicillin was determined by PCR analysis of mecA. A total of 10 different staphylococcal species were identified, 22.2% and 77.8% of which were coagulase positive and negative, respectively. Twenty-two (48.89%) isolates were resistant to at least one antimicrobial agent. Higher antimicrobial resistance values were obtained against tetracycline (28.9%) and penicillin (22.2%). Two isolates (S. aureus and Staphylococcus lentus) were resistant to cefoxitin; however, none of the 45 isolates harbored mecA. Thirty pulsotypes were detected by PFGE. Interestingly, some isolates of S. aureus, S. lentus, Staphylococcus simulans, and Staphylococcus caprae showed high genetic similarity (>80%). These data suggest that genetically similar staphylococcal isolates circulate among goat and sheep dairy herds, and their different resistance patterns could be influenced by the management systems used.

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 degrees C. The production cost of 1000U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.

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.

Staphylococcus simulans is a coagulase-negative Staphylococcus species that is a commensal of domestic animals. We report the case of a 58 year old man with an aggressive form of S. simulans endocarditis involving native aortic and mitral valves, and the aortic root. Blood cultures were positive for S. simulans on presentation. The presence of S. simulans was confirmed by 16S PCR of the explanted aortic valve. Due to development of paravalvular leak causing left sided heart failure, and of a large thrombus in the left ventricle the patient required further surgery with replacement of the metallic valves with bio-prosthetic valves two months after the initial surgery. He died shortly after the re-do operation due to due to gram negative sepsis and cardiogenic shock. In addition to our case, we summarise the clinical presentation and outcome of the four additional cases of S. simulans endocarditis described in the literature to date. We also report virulence factors associated with other S. simulans isolates. The aim of this case report and literature review is to highlight the potentially increased pathogenicity of S. simulans.

Recent studies highlight the abundance of commensal coagulase-negative staphylococci (CoNS) on healthy skin. Evidence suggests that CoNS actively shape the skin immunological and microbial milieu to resist colonization or infection by opportunistic pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), in a variety of mechanisms collectively termed colonization resistance. One potential colonization resistance mechanism is the application of quorum sensing, also called the accessory gene regulator (agr) system, which is ubiquitous among staphylococci. Common and rare CoNS make autoinducing peptides (AIPs) that function as MRSA agr inhibitors, protecting the host from invasive infection. In a screen of CoNS spent media, we found that Staphylococcus simulans, a rare human skin colonizer and frequent livestock colonizer, released potent inhibitors of all classes of MRSA agr signaling. We identified three S. simulans agr classes and have shown intraspecies cross talk between noncognate S. simulans agr types for the first time. The S. simulans AIP-I structure was confirmed, and the novel AIP-II and AIP-III structures were solved via mass spectrometry. Synthetic S. simulans AIPs inhibited MRSA agr signaling with nanomolar potency. S. simulans in competition with MRSA reduced dermonecrotic and epicutaneous skin injury in murine models. The addition of synthetic AIP-I also effectively reduced MRSA dermonecrosis and epicutaneous skin injury in murine models. These results demonstrate potent anti-MRSA quorum sensing inhibition by a rare human skin commensal and suggest that cross talk between CoNS and MRSA may be important in maintaining healthy skin homeostasis and preventing MRSA skin damage during colonization or acute infection.

Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn2+-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn2+ ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni2+ affinity chromatography (IMAC). The purified monomeric bacteriocin exhibited concentration-dependent bioactivity against S. aureus and its methicillin-resistant strain through cell-wall hydrolysis rather than membrane perturbation. Following pre-incubation of the purified lysostaphin with exogenous Zn2+, a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn2+-chelator), the adverse effect of the exogenous Zn2+ on bioactivity was greatly decreased. Conversely, lysostaphin pre-treated with excess PNT retained relatively high bioactivity, indicating ineffective chelation of PNT to detach the catalytic Zn2+ from the active-site pocket. Structural analysis of the lysostaphin-catalytic domain together with amino acid sequence alignments of lysostaphin-like endopeptidases revealed a potential extraneous Zn2+-binding site found in close proximity to the Zn2+-coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn2+ ions on staphylolytic activity of the purified Zn2+-dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.

Lysostaphin is a peptidoglycan hydrolase, produced by Staphylococcus simulans, which has illustrated significant bactericidal activities against Staphylococcus aureus species. Currently, recombination is the common approach of lysostaphin production. However, the recombinant produced lysostaphin shows weak antibacterial activities. The reason can be the aggregation of produced lysostaphin which leads to the destruction of natural protein folding. The most common strategy providing the best situation for correct refolding of the recombinant protein is dialysis. In this study, based on the computer simulations different condition of dialysis was applied to achieve the most significant antibacterial activity. In this study, lysostaphin was expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS cells and purified by affinity chromatography. Various dialysis methods were employed to enable the protein to refold to its natural form. The results of final protein antibacterial activity evidenced the high efficiency of computer simulations estimation ability in predicting best dialysis buffer according to the interaction between protein and buffer additive compounds. Finally, it was confirmed that the buffer containing proline 0.15 M and glucose 0.2 M caused the best lysostaphin refolding. Employing computer simulation before initiating the dialysis process would be a novel efficient and economic pathway of protein folding recovery.

The most common pathogens causing intramammary infections (IMI) in dairy goats are staphylococci. Gene sequencing has been the reference method for identification of staphylococcal species, but MALDI-TOF mass spectrometry could represent a rapid and cost-effective alternative method. The objectives were to evaluate the typeability and accuracy of partial gene sequencing and MALDI-TOF for identifying staphylococci isolated from caprine milk samples, and to evaluate the relationship between staphylococcal species IMI, milk somatic cell score (SCS), and milk yield (MY). A composite (goat-level) milk sample was collected from all 940 lactating goats in a single herd. Dairy Herd Information Association test-day data for parity, days in milk, SCS, and MY were retrieved from Dairy Herd Information Association records. Milk samples were cultured on Columbia blood agar, and isolates from samples that yielded a single colony type of a presumptively identified Staphylococcus spp. were identified by PCR amplification and partial sequencing of rpoB, tuf, or 16S-rRNA, and MALDI-TOF. Mixed linear models were used to evaluate the relationship between staphylococcal IMI, SCS, and MY. The goat-level prevalence of staphylococcal IMI based on isolation of a single colony type was 24.4% (213/874). Seventeen goats had a contaminated sample. Among the remaining goats (n = 857), the most common species causing single colony-type IMI were Staphylococcus simulans (7.9%), Staphylococcus xylosus (3.5%), Staphylococcus caprae (3.6%), Staphylococcus chromogenes (2.9%), and Staphylococcus epidermidis (2.2%). The typeability of staphylococcal isolates with partial housekeeping gene sequence analysis (rpoB, complemented by tuf and 16S as needed) was 97.7%. The typeability and accuracy of MALDI-TOF were 84 and 100%, respectively. Overall, only Staphylococcus chromogenes IMI was associated with a higher SCS than goats with no growth. After adjusting for parity and stage of lactation, staphylococcal IMI status was not significantly associated with MY. For the staphylococci isolated from goats in this herd, MALDI-TOF proved an accurate method of speciation with a relatively high typeability. An association between staphylococcal IMI, SCS, and MY was not defined using goat-level data with the exception of S. chromogenes IMI, which was associated with a higher SCS than goats with no growth.

Diagnosis of bovine intramammary infection (IMI) has traditionally been based on bacterial culture, but currently IMI can also be detected with DNA based methods, such as multiplex real-time PCR. The aim of this study was to describe the elimination of bacteria in experimentally induced IMI on the quarter level, using conventional bacterial culture (BC) and multiplex real-time PCR. Two coagulase-negative staphylococcal species, Staphylococcus epidermidis and Staphylococcus simulans, were experimentally inoculated into 14 healthy quarters of 8 dairy cows during 4 consecutive study periods. Intramammary infections were followed with 20 milk samplings per each quarter. Milk somatic cell count was monitored to evaluate the inflammation process in the quarters. Four quarters cured spontaneously during the study period according to the culture. The PCR detected staphylococcal DNA from these quarters for several days after they were defined as cured in BC. Agreement between BC and PCR results varied from substantial to almost perfect agreement for the first 36 h postchallenge, decreasing to moderate levels toward the end of the sampling period. Based on this study, we recommend collecting possible follow-up samples to assess the bacteriological cure from IMI not until 2 to 3 wk after the onset of mastitis or after the quarter milk somatic cell count has normalized when PCR is used.

The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.

Objectives To investigate the presence and molecular characteristics of oxazolidinone resistance genes cfr and optrA in staphylococci from household animals in rural China. Methods Various samples were collected from household animals in 12 rural villages. Staphylococcal isolates showing florfenicol MICs ≥10 mg/L were identified and screened for the presence of cfr and/or optrA. PCR-positive isolates were characterized by antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blotting. WGS data were analysed to identify the core-genome phylogenetic profile of each isolate as well as the genetic environment of cfr and/or optrA. Results Nine optrA-positive (seven Staphylococcus sciuri and two Staphylococcus simulans) and 10 cfr-positive staphylococci were identified from eight and five villages, respectively. The gene optrA was chromosomally encoded in all nine isolates, whereas cfr was located on a plasmid in one S. sciuri and three Staphylococcus saprophyticus and in the chromosomal DNA of single Staphylococcus cohnii and Staphylococcus lentus isolates and two S. sciuri isolates. The remaining two cfr-carrying Staphylococcus haemolyticus isolates were indistinguishable by PFGE. Most optrA- or cfr-carrying staphylococci also harboured phenicol, tetracycline and/or macrolide-lincosamide-streptogramin B resistance genes. Genetic environment analysis showed that, for the first time, optrA was associated with transposon Tn6261, while cfr was adjacent to both a tnp (transposase) gene and a Tn558 transposon. Conclusions The current study reveals for the first time the wide distribution of oxazolidinone resistance genes optrA and cfr in household animals in rural areas of China and is the first identification of optrA in S. simulans isolates.

The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with subclinical mastitis caused by coagulase-negative staphylococci and from healthy cows. The blood and milk samples were obtained from 35 midlactation, multiparous (between parities 2 and 4) Holstein-Friesian cows. In the milk samples from 20 cows with subclinical mastitis, the following species of Staphylococcus were detected: Staphylococcus xylosus (8 samples), Staphylococcus chromogenes (6 samples), Staphylococcus haemolyticus (2 samples), Staphylococcus simulans (2 samples), and Staphylococcus sciuri (2 samples). The results of the present study indicate that the level of IL-6 in cows suffering from subclinical mastitis tended to be high in both serum and milk (432.09 and 254.32 pg/mL) compared with the level in healthy cows (164.47 and 13.02 pg/mL, respectively). Amyloid A value also was significantly higher in milk of unhealthy cows compared with cows without subclinical mastitis (790.2 and 360.5 ng/mL). No significant differences were found in levels of amyloid A in serum of both tested groups of cows (2,680.0 and 2,720.0 ng/mL). In contrast, concentration of IL-4 was significantly lower both in serum and in milk of cows with staphylococcal mastitis (86.1 and 123.17 pg/mL) compared with control animals (413.5 and 670.2 pg/mL). The level of IL-10 also was significantly higher in milk of healthy cows than in infected cows (39.78 and 22.5 pg/mL); however, differences in serum levels of this cytokine between tested groups were significantly less important (220.6 and 175.1 pg/mL).