Search

Objective: We aimed to determine the demographic data, mortality, and morbidity of early- and late-neonatal sepsis cases, the etiologic agents in these cases, and the antibiotic susceptibility of these agents. Methods: This study was conducted retrospectively in a tertiary neonatal intensive care unit (NICU). The demographic, clinical, and laboratory data of newborns diagnosed with culture-proven sepsis within 24 months were evaluated. Results: Two hundred and eleven culture data points belonging to 197 infants were evaluated. Forty percent of the infants had a history of premature birth. The most common clinical findings were respiratory distress and feeding intolerance. Coagulase-negative staphylococci (CoNS) were detected most frequently as early- and late-sepsis agents. The most common Gram-negative bacteria detected as late-sepsis agents were Klebsiella spp. and Escherichia coli (E. coli). The overall mortality rate was 10%. Conclusions: Neonatal sepsis continues to have high mortality rates in tertiary NICUs. CoNS was the most common agent, highlighting the importance of developing and maintaining personnel training and handwashing practices. It will be important to consider the resistance rates of Klebsiella spp., the most common Gram-negative agent in late-onset sepsis (LOS) cases, to commonly used antibiotics in empirical treatments.

Staphylococcus capitis has been recognized as a relevant opportunistic pathogen, particularly its persistence in neonatal ICUs around the world. Therefore, the aim of this study was to describe the epidemiological profile of clinical isolates of S. capitis and to characterize the factors involved in the persistence and pathogenesis of these strains isolated from blood cultures collected in a hospital in the interior of the state of São Paulo, Brazil. A total of 141 S. capitis strains were submitted to detection of the mecA gene and SCCmec typing by multiplex PCR. Genes involved in biofilm production and genes encoding enterotoxins and hemolysins were detected by conventional PCR. Biofilm formation was evaluated by the polystyrene plate adherence test and phenotypic resistance was investigated by the disk diffusion method. Finally, pulsed-field gel electrophoresis (PFGE) was used to analyze the clonal relationship between isolates. The mecA gene was detected in 99 (70.2%) isolates, with this percentage reaching 100% in the neonatal ICU. SCCmec type III was the most prevalent type, detected in 31 (31.3%) isolates and co-occurrence of SCCmec was also observed. In vitro biofilm formation was detected in 46 (32.6%) isolates but was not correlated with the presence of the ica operon genes. Furthermore, biofilm production in ICU isolates was favored by hyperosmotic conditions, which are common in ICUs because of the frequent parenteral nutrition. Analysis of the clonal relationship between the isolates investigated in the present study confirms a homogeneous profile of S. capitis and the persistence of clones that are prevalent in the neonatal ICU and disseminated across the hospital. This study highlights the adaptation of isolates to specific hospital environments and their high clonality.

Ripened sheep sausages are widely consumed in Italy, particularly in Sardinia. Despite their driving role in flavor and color development, coagulase-negative staphylococci in these products have been rarely investigated. A total of 70 CoNS cultures isolated from Sardinian sheep sausages were characterized by rep-PCR and M13-RAPD typing and identified by 16S rDNA sequencing. S. xylosus and S. equorum accounted for more than 70% of the total isolates, whilst S. pasteuri (8.5%), S. succinus (2.8%), and S. haemolyticus (2.8%) were less represented. The genes encoding the synthesis of putrescine, tyramine, cadaverine, and histamine were evaluated by PCR. None of the strains hosted genes for decarboxylases, except one S. pasteuri strain that was potentially a tyramine-producer. Antibiotic resistance was evaluated, along with nitrate reductase, lipolytic, and proteolytic activity, in a pool of selected cultures. Resistance to the primary antibiotics was rather widespread. S. xylosus, S. equorum, and S. pasteuri strains were all resistant to amoxicillin and kanamycin. S. equorum strains were sensitive to all tested antibiotics. S. xylosus strains were all resistant to penicillin B. Conversely, all S. pasteuri strains were resistant to both ampicillin and penicillin B, and four out of five strains exhibited tetracycline resistance. The high variability in the production of sheep sausages makes the search for adjunct cultures of crucial relevance. According to this perspective, the characterization of the autochthonous CSN population represents the first step to approach a starter selection.

This study aimed to genotypic and phenotypic analyses of the enterotoxigenic potential of Staphylococcus spp. isolated from raw milk and raw milk cheeses. The presence of genes encoding staphylococcal enterotoxins (SEs), including the classical enterotoxins (sea-see), non-classical enterotoxins (seg-seu), exfoliative toxins (eta-etd) and toxic shock syndrome toxin-1 (tst-1) were investigated. Isolates positive for classical enterotoxin genes were then tested by SET-RPLA methods for toxin expression. Out of 75 Staphylococcus spp. (19 Staphylococcus aureus and 56 CoNS) isolates from raw milk (49/65.3%) and raw milk cheese samples (26/34.7%), the presence of enterotoxin genes was confirmed in 73 (97.3%) of them. Only one isolate from cheese sample (1.3%) was able to produce enterotoxin (SED). The presence of up to eight different genes encoding enterotoxins was determined simultaneously in the staphylococcal genome. The most common toxin gene combination was sek, eta present in fourteen isolates (18.7%). The tst-1 gene was present in each of the analyzed isolates from cheese samples (26/34.7%). Non-classical enterotoxins were much more frequently identified in the genome of staphylococcal isolates than classical SEs. The current research also showed that genes tagged in S. aureus were also identified in CoNS, and the total number of different genes detected in CoNS was seven times higher than in S. aureus. The obtained results indicate that, in many cases, the presence of a gene in Staphylococcus spp. is not synonymous with the ability of enterotoxins production. The differences in the number of isolates with genes encoding SEs and enterotoxin production may be mainly due to the limit of detection of the toxin production method used. This indicates the need to use high specificity and sensitivity methods for detecting enterotoxin in future studies.

The objective of this review is to investigate the distribution and the characteristics of coagulase-negative Staphylococci (CoNS) implicated in ovine mastitis, and especially in subclinical cases, in order to provide a global perspective of the current research data and analyze specific critical aspects of the issue. PRISMA guidelines were implemented in the search of the last 20 years of the related literature in two databases. In total, 139 studies were included in this review. Relevant data were tracked down, assembled, and compared. Regarding the geographical distribution, most studies originated from Europe (68), followed by South America (33). Lacaune was the most examined breed, while S. epidermidis was the predominantly identified species, representing approximately 39% of the obtained isolates. Antibiotic resistance in the relevant bacteria was documented mostly for Penicillin (32.8%) and Amoxicillin (32.1%), while biofilm- and toxin-associated genes were encountered in variable rates because significant inequalities were observed between different articles. Significantly higher rates of antimicrobial resistance were detected in Asia and South America compared to Europe. Finally, the diagnostic procedures carried out in the respective studies were evaluated. Conventional culture and biochemical tests were mostly performed for simple strain identification; therefore, further molecular investigation of isolates should be pursued in future studies, as this will provide important data regarding specific aspects of the implication of CoNS in ovine mastitis.

The ecology and diversity of resistome in coagulase-negative staphylococci (CoNS) from healthy pigs and pig farmers are rarely available as most studies focused on the livestock-associated methicillin-resistant S. aureus. This study aims to characterize the antimicrobial resistance (AMR) mechanisms, intra-host species diversity (more than one species in a host), and intra-species AMR diversity (same species with more than one AMR profile) in CoNS recovered from the nasal cavities of healthy pigs and pig farmers. One-hundred-and-one CoNS strains previously recovered from 40 pigs and 10 pig farmers from four Spanish pig farms were tested to determine their AMR profiles. Non-repetitive strains were selected (n = 75) and their AMR genes, SCCmec types, and genetic lineages were analyzed by PCR/sequencing. Of the non-repetitive strains, 92% showed a multidrug resistance (MDR) phenotype, and 52% were mecA-positive, which were associated with SCCmec types V (46.2%), IVb (20.5%), and IVc (5.1%). A total of 28% of the pigs and pig farmers had intra-host species diversity, while 26% had intra-species AMR diversity. High repertoires of AMR genes were detected, including unusual ones such as tetO, ermT, erm43, and cfr. Most important was the detection of cfr (in S. saprophyticus and S. epidermidis-ST16) in pigs and pig farmers; whereas MDR-S. borealis strains were identified in pig farmers. Pig-to-pig transmission of CoNS with similar AMR genes and SCCmec types was detected in 42.5% of pigs. The high level of multidrug, within-host, and intra-species resistome diversity in the nasal CoNS highlights their ability to be AMR gene reservoirs in healthy pigs and pig farmers. The detection of MDR-S. borealis and linezolid-resistant strains underscore the need for comprehensive and continuous surveillance of MDR-CoNS at the pig farm level.

(1) Background: Coagulase-negative staphylococci (CoNS) are an important group of organisms that can cause bloodstream infection (BSI) and infective endocarditis (IE). The prevalence of IE in patients with BSI due to different CoNS species, however, has received limited attention; (2) Methods: A retrospective study of adults with monomicrobial CoNS BSI who had undergone echocardiography and a risk factor analysis was done to determine the most common CoNS species that cause definite IE; (3) Results: 247 patients with CoNS BSI were included in the investigation; 49 (19.8%) had definite IE, 124 (50.2%) possible IE, and 74 (30.0%) BSI only. The latter two entities were grouped in one category for further analysis. The most common species in CoNS BSI was Staphylococcus epidermidis (79.4%) and most patients (83.2%) had possible IE/BSI only. 59.1% of patients with BSI due to S. lugdunensis had definite IE. The majority of CoNS were healthcare-associated/nosocomial bacteremia. Multivariable analysis demonstrated that valve disease (p = 0.002) and a foreign cardiovascular material (p < 0.001) were risk factors associated with definite IE. Patients with S. lugdunensis BSI had an 8-fold higher risk of definite IE than did those with S. epidermidis BSI and nearly a 13-fold higher risk than did patients with BSI due to other species of CoNS (p = 0.002); (4) Conclusions: The prevalence of definite IE in patients with BSI due to different CoNS species was significant. CoNS bacteremia, particularly with S. lugdunensis, confers a significant risk of IE, particularly in patients with a valve disease or intravascular foreign body material and should not be immediately dismissed as a contaminant.

While modern molecular methods have decisively accelerated and improved microbiological diagnostics, phenotypic variants still pose a challenge for their detection, identification and characterization. This particularly applies if they are unstable and hard to detect, which is the case for the small-colony-variant (SCV) phenotype formed by staphylococci. On solid agar media, staphylococcal SCVs are characterized by tiny colonies with deviant colony morphology. Their reduced growth rate and fundamental metabolic changes are the result of their adaptation to an intracellular lifestyle, regularly leading to specific auxotrophies, such as for menadione, hemin or thymidine. These alterations make SCVs difficult to recognize and render physiological, biochemical and other growth-based methods such as antimicrobial susceptibility testing unreliable or unusable. Therefore, diagnostic procedures require prolonged incubation times and, if possible, confirmation by molecular methods. A special approach is needed for auxotrophy testing. However, standardized protocols for SCV diagnostics are missing. If available, SCVs and their putative parental isolates should be genotyped to determine clonality. Since their detection has significant implications for the treatment of the infection, which is usually chronic and relapsing, SCV findings should be specifically reported, commented on, and managed in close collaboration with the microbiological laboratory and the involved clinicians.

Background: The abuse of antibiotics during the SARS-CoV-2 pandemic might have disrupted efforts to curb the further development and spread of the antimicrobial resistance of Staphylococcus aureus infection and Staphylococcus spp. coagulase-negative (CoNS) agents of nosocomial bloodstream infections (NBSIs). The purpose of our work was to study the resistance patterns of Staphylococcus aureus and CoNS through the analysis of blood cultures in hospitalized SARS-CoV-2-positive and SARS-CoV-2-negative patients (pts.). Materials and methods: During the period January 2018–June 2021, a retrospective case–control study was performed on blood cultures positive for Staphylococcus spp. detected in 177 adult pts. (≥18 years old) hospitalized for >48 hours at Sant’Elia Hospital, Caltanissetta. Results: Staphylococcus aureus was isolated in 33.9% of blood culture samples, and among CoNS, the most frequent strains were Staphylococcus capitis (18.6%) and Staphylococcus hominis (18.1%). Patients aged ≥ 65 years, with a greater number of males, comprised the SARS-CoV-2-negative pts. (71.8% vs. 52.2%, p = 0.0154). Among the SARS-CoV-2-positive patients, the significant resistance of Staphylococcus aureus was only observed for erythromycin (57.1%). The oxacillin resistance of Staphylococcus capitis was higher in SARS-CoV-2-positive than in negative pts. (90% and 78.3%, respectively). Comparing the two groups, we found an increase in resistance in SARS-CoV-2-negative patients for the following antibiotics: gentamicin for Staphylococcus aureus (p = 0.007), clindamycin and erythromycin (p = 0.012) for Staphylococcus hominis and oxacillin and rifampicin for Staphylococcus haemoliticus (p = 0.012). Conclusions: Our study confirms the relevance of oxacillin-resistant Staphylococcus aureus in being responsible for bloodstream infection and draws attention to highly oxacillin-resistant CoNS such as Staphylococcus capitis. The presence of resistant strains of CoNS in hospitals can be worrying, as it limits treatment options and worsens outcomes. The Infection Control Committee (ICC) recommends new treatment strategies to decrease colonization and infections. As part of the implementation of a bloodstream infection prevention program, the authors encourage the introduction of a report on the antimicrobial resistance of hospital bacteremia due to CoNS.

According to clinical guidelines, the management of catheter-related bloodstream infections (CRBSI) due to coagulase-negative staphylococci (CoNS) includes catheter removal and antibiotic treatment for 5 to 7 days. However, in low-risk episodes, it remains uncertain whether antibiotic therapy is necessary. This randomized clinical trial aims to determine whether the non-administration of antibiotic therapy is as safe and effective as the recommended strategy in low-risk episodes of CRBSI caused by CoNS. With this purpose, a randomized, open-label, multicenter, non-inferiority clinical trial was conducted in 14 Spanish hospitals from 1 July 2019 to 31 January 2022. Patients with low-risk CRBSI caused by CoNS were randomized 1:1 after catheter withdrawal to receive/not receive parenteral antibiotics with activity against the isolated strain. The primary endpoint was the presence of any complication related to bacteremia or to antibiotic therapy within 90 days of follow-up. The secondary endpoints were persistent bacteremia, septic embolism, time until microbiological cure, and time until the disappearance of a fever. EudraCT: 2017-003612-39 INF-BACT-2017. A total of 741 patients were assessed for eligibility. Of these, 27 were included in the study; 15 (55.6%) were randomized to the intervention arm (non-antibiotic administration) and 12 (44.4%) to the control arm (antibiotic therapy as per standard practice). The primary endpoint occurred in one of the 15 patients in the intervention group (septic thrombophlebitis) and in no patients in the control group. The median time until microbiological cure was 3 days (IQR 1–3) in the intervention arm and 1.25 days (IQR 0.5–2.62) in the control arm, while the median time until fever resolution was zero days in both arms. The study was stopped due to the insufficient number of recruited patients. These results seem to indicate that low-risk CRBSI caused by CoNS can be managed without antibiotic therapy after catheter removal; efficacy and safety are not affected.

House flies (Musca domestica) are very diffuse insects attracted by biological materials. They are abundantly present in farm environments and can frequently come in contact with animals, feed, manure, waste, surfaces, and fomites; consequently, these insects could be contaminated, carry, and disperse several microorganisms. The aim of this work was to evaluate the presence of antimicrobial-resistant staphylococci in house flies collected in poultry and swine farms. Thirty-five traps were placed in twenty-two farms; from each trap, 3 different kinds of samples were tested: attractant material present in the traps, the body surface of house flies and the body content of house flies. Staphylococci were detected in 72.72% of farms, 65.71% of traps and 43.81% of samples. Only coagulase-negative staphylococci (CoNS) were isolated, and 49 isolates were subjected to an antimicrobial susceptibility test. Most of the isolates were resistant to amikacin (65.31%), ampicillin (46.94%), rifampicin (44.90%), tetracycline (40.82%) and cefoxitin (40.82%). Minimum Inhibitory concentration assay allowed to confirm 11/49 (22.45%) staphylococci as methicillin-resistant; 4 of them (36.36%) carried the mecA gene. Furthermore, 53.06% of the isolates were classified as multidrug-resistant (MDR). Higher levels of resistance and multidrug resistance were detected in CoNS isolated from flies collected in poultry farms than in swine farms. Therefore, house flies could carry MDR and methicillin-resistant staphylococci, representing a possible source of infection for animals and humans.

Infections are one of the most significant complications of neonates, especially those born preterm, with sepsis as one of the principal causes of mortality. Coagulase-negative staphylococci (CoNS), a group of staphylococcal species that naturally inhabit healthy human skin and mucosa, are the most common cause of late-onset sepsis, especially in preterms. One of the risk factors for the development of CoNS infections is the presence of implanted biomedical devices, which are frequently used for medications and/or nutrient delivery, as they serve as a scaffold for biofilm formation. The major concerns related to CoNS infections have to do with the increasing resistance to multiple antibiotics observed among this bacterial group and biofilm cells’ increased tolerance to antibiotics. As such, the treatment of CoNS biofilm-associated infections with antibiotics is increasingly challenging and considering that antibiotics remain the primary form of treatment, this issue will likely persist in upcoming years. For that reason, the development of innovative and efficient therapeutic measures is of utmost importance. This narrative review assesses the current challenges and emerging diagnostic tools and therapies for the treatment of CoNS biofilm-associated infections, with a special focus on late-onset sepsis.

Intramammary infection (IMI) from the environment and infected quarters can cause co-infection. The objective of this study was to determine the ability of coagulase-negative staphylococci (CNS) to survive in the same environment as Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli as major pathogens. In total, 15 and 242 CNS strains were used in Experiment I and Experiment II, respectively. Both experiments were separated into three conditions: culture with CNS 24 h before (PRIOR), after (AFTER), and at the same time (EQUAL). The lack of a clear zone, regardless of size, was determined to be the key to the survival of both. The CNS species’ percentages of survival against major pathogens were tested using Fisher’s exact test. Differences in the percentages of survival were evident among the CNS species in all conditions. For the PRIOR condition, all CNS mostly survived when living with major strains; however, S. chromogenes could degrade S. agalactiae. Although most CNS strains were degraded in the AFTER and EQUAL conditions, some strains of S. hominis and S. simulans could resist S. aureus and S. agalactiae. In conclusion, some specific strains of CNS are able to survive in an environment with major pathogens. Research into the survival strains may indicate that the concept of novel bacteria with bacteriolytic capabilities might be possible as a novel mastitis treatment.

Clinical outcomes of persistent staphylococcal bacteremia vary depending on the causative organism. This secondary data analysis study compared the clinical characteristics of persistent Staphylococcus aureus (S. aureus)- and coagulase-negative staphylococci (CoNS)-caused bacteremia, focusing on the methicillin-resistant status. This study used data collected from patients who underwent blood cultures between January 2012 and December 2021 at Tohoku University Hospital, Japan. Patients with persistent staphylococcal bacteremia were divided into groups based on the pathogen and methicillin-resistant status, and their characteristics were analyzed. The primary outcomes were early (30-day), late (30–90 days), and 90-day mortality rates. The early, late, and 90-day mortality rates were similar between the persistent CoNS and S. aureus bacteremia groups. Patients with persistent methicillin-resistant S. aureus (MRSA) bacteremia tended to have higher early, late, and 90-day mortality rates than those with persistent methicillin-susceptible S. aureus bacteremia (not statistically significant). No differences were observed between the methicillin-resistant and-susceptible CoNS groups. In patients with persistent CoNS bacteremia, mortality tended to increase, especially in debilitated or immunocompromised patients with distant metastases, underscoring the importance of infection source control. Mortality tended to be high in patients with persistent MRSA bacteremia, especially when persistent bacteremia clearance was not confirmed, illustrating the need for careful therapeutic management.

Objective: We aimed to determine the demographic data, mortality, and morbidity of early- and late-neonatal sepsis cases, the etiologic agents in these cases, and the antibiotic susceptibility of these agents., Methods: This study was conducted retrospectively in a tertiary neonatal intensive care unit (NICU). The demographic, clinical, and laboratory data of newborns diagnosed with culture-proven sepsis within 24 months were evaluated., Results: Two hundred and eleven culture data points belonging to 197 infants were evaluated. Forty percent of the infants had a history of premature birth. The most common clinical findings were respiratory distress and feeding intolerance. Coagulase-negative staphylococci (CoNS) were detected most frequently as early- and late-sepsis agents. The most common Gram-negative bacteria detected as late-sepsis agents were Klebsiella spp. and Escherichia coli ( E. coli ). The overall mortality rate was 10%., Conclusions: Neonatal sepsis continues to have high mortality rates in tertiary NICUs. CoNS was the most common agent, highlighting the importance of developing and maintaining personnel training and handwashing practices. It will be important to consider the resistance rates of Klebsiella spp., the most common Gram-negative agent in late-onset sepsis (LOS) cases, to commonly used antibiotics in empirical treatments.

Ventricina del Vastese is a traditional dry fermented sausage from Central Italy not yet characterized for the occurrence, identity and safety of coagulase-negative staphylococci (CNS), a bacterial group technologically important for this kind of product. Therefore, in this study, 98 CNS isolates from four manufacturers were differentiated using repetitive element palindromic PCR (Rep-PCR) and identified using 16S rRNA gene sequencing. These were examined for genes encoding biogenic amine (BA) production, resistance to aminoglycosides, β-lactams, tetracyclines and staphylococcal enterotoxins (SEs). Staphylococcus succinus (55%) predominated, followed by S. xylosus (30%), S. epidermidis (7.4%), S. equorum (3.1%), S. saprophyticus (3.1%) and S. warneri (1%). One S. succinus subsp. casei isolate was slightly β-hemolytic. SEs and the histidine decarboxylase gene hdcA were not detected, whereas the tyrosine decarboxylase gene tdcA was detected in four S. xylosus isolates. The blaZ beta-lactamase gene in an S. equorum isolate, tetracycline resistance genes tetK in six S. succinus isolates and tetA in one S. succinus isolate also bearing tetK were found. The product examined is characterized by a peculiar CNS species ratio and a low occurrence and diversity of AR transferable genes than found in other studies, as a probable consequence of production only with meat from animals raised in small farms with extensive rearing systems in which antibiotic usage is infrequent.

The presence of biofilms can negatively affect several different areas, such as the food industry, environment, and biomedical sectors. Conditions under which bacteria grow and develop, such as temperature, nutrients, and pH, among others, can largely influence biofilm production. Staphylococcus species survive in the natural environment due to their tolerance to a wide range of temperatures, dryness, dehydration, and low water activity. Therefore, we aimed to evaluate the influence of external environmental factors on the formation of biofilm of staphylococci isolated from hospital wastewater and surface waters. We investigated the biofilm formation of methicillin-resistant and -susceptible S. aureus (MRSA and MSSA) and coagulase-negative staphylococci (CoNS) under various temperatures, pH values, salt concentrations, glucose concentrations, and under anaerobic and aerobic conditions. CoNS had the ability to produce more biofilm biomass than MSSA and MRSA. All environmental factors studied influenced the biofilm formation of staphylococci isolates after 24 h of incubation. Higher biofilm formation was achieved at 4% of NaCl and 0.5% of glucose for MSSA and CoNS, and 1% of NaCl and 1.5% of glucose for MRSA isolates. Biofilm formation of isolates was greater at 25 °C and 37 °C than at 10 °C and 4 °C. pH values between 6 and 8 led to more robust biofilm formation than pH levels of 9 and 5. Although staphylococci are facultative anaerobes, biofilm formation was higher in the presence of oxygen. The results demonstrated that multiple environmental factors affect staphylococci biofilm formation. Different conditions affect differently the biofilm formation of MRSA, MSSA, and CoNS strains.

Background: For years, coagulase-negative staphylococci (CoNS) were not considered a cause of bloodstream infections (BSIs) and were often regarded as contamination. However, the association of CoNS with nosocomial infections is increasingly recognized. The identification of more than 40 different CoNS species has been driven by the introduction of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Yet, treatment guidelines consider CoNS as a whole group, despite increasing antibiotic resistance (ABR) in CoNS. This retrospective study provides an in-depth data analysis of CoNS isolates found in human blood culture isolates between 2013 and 2019 in the entire region of the Northern Netherlands. Methods: In total, 10,796 patients were included that were hospitalized in one of the 15 hospitals in the region, leading to 14,992 CoNS isolates for (ABR) data analysis. CoNS accounted for 27.6% of all available 71,632 blood culture isolates. EUCAST Expert rules were applied to correct for errors in antibiotic test results. Results: A total of 27 different CoNS species were found. Major differences were observed in occurrence and ABR profiles. The top five species covered 97.1% of all included isolates: S. epidermidis, S. hominis, S. capitis, S. haemolyticus, and S. warneri. Regarding ABR, methicillin resistance was most frequently detected in S. haemolyticus (72%), S. cohnii (65%), and S. epidermidis (62%). S. epidermidis and S. haemolyticus showed 50–80% resistance to teicoplanin and macrolides while resistance to these agents remained lower than 10% in most other CoNS species. Conclusion: These differences are often neglected in national guideline development, prompting a focus on ‘ABR-safe’ agents such as glycopeptides. In conclusion, this multi-year, full-region approach to extensively assess the trends in both the occurrence and phenotypic resistance of CoNS species could be used for evaluating treatment policies and understanding more about these important but still too often neglected pathogens.

Staphylococcus spp. remain the leading biofilm-forming agents causing orthopedic implant-associated infections (OIAI). This is a descriptive study of phenotypic and genomic features identified in clinical isolates of S. aureus and coagulase-negative Staphylococcus (CoNS) recovered from OIAIs patients that progressed to treatment failure. Ten isolates were identified by matrix-time-of-flight laser-assisted desorption mass spectrometry (MALDI-TOF-MS) and tested for antibiotic susceptibility and biofilm formation. Genotypic characteristics, including, MLST (Multi Locus Sequence Typing), SCCmec typing, virulence and resistance genes were assessed by whole-genome sequencing (WGS). All S. aureus harbored mecA, blaZ, and multiple resistance genes for aminoglycosides and quinolones. All MRSA were strong biofilm producers harboring the complete icaADBC and icaR operon. Seven CoNS isolates comprising five species (S. epidermidis, S. haemolyticus, S. sciuri, S. capitis and S. lugdunensis) were analyzed, with mecA gene detected in five isolates. S. haemolitycus (isolate 95), and S. lugdunensis were unable to form biofilm and did not harbor the complete icaADBCR operon. High variability of adhesion genes was detected, with atl, ebp, icaADBC operon, and IS256 being the most common. In conclusion, MRSA and CoNS isolates carrying genes for biofilm production, and resistance to β-lactam and aminoglycosides are associated with treatment failure in OIAIs.

The aim of this study was to characterize methicillin-resistant coagulase-negative staphylococci (MRCoNS) isolates from the healthy staff of a university veterinary hospital in order to assess their importance as a reservoir of antimicrobial resistance and to determine their population structure and evolution. The study duration was over two years (2020–2021), 94 individuals were analyzed in duplicate, and 78 strains were obtained. The overall prevalence of methicillin-resistant strains detected throughout the study was 61.7%, with point prevalence values of 53.2% in 2020 and 31.5% in 2021. A total of 19.1% of the individuals analyzed were carriers throughout the study. The most frequently identified MRCoNs were Staphylococcus epidermidis (92.3%) and S. warneri (3.8%). A total of 75.6% of the isolates obtained showed the development of multi-resistance, preferentially against erythromycin, gentamicin, and tetracycline, and to a lesser extent against fusidic acid, norfloxacin, and clindamycin; these antimicrobials are frequently used in the veterinary field. Although most of the S. epidermidis isolates obtained showed wide genetic variability and low dispersion, which are characteristic of community-associated isolates, a small number of strains spread between individuals in close physical proximity and were maintained over time, forming stable clones. These clones generally maintained the same type of staphylococcal cassette chromosome mec (SCCmec) and had a similar antimicrobial resistance pattern.

Several different species of animals host staphylococci as normal microbiota. These animals can be a source of staphylococci zoonotic infections. People with routine or occupational exposure to infected/colonized animals are at risk of a potential transmission. Therefore, we aimed to investigate the presence of S. aureus and other staphylococci in camels used for recreational purposes as well as their antimicrobial resistance, virulence factors and genetic lineages. A total of 172 samples were collected from 86 healthy camels (nose and mouth) from different farms located in the Canary Islands, Spain. Antimicrobial susceptibility testing was performed against 14 antimicrobial agents. The presence of virulence genes was studied by PCR. Multilocus sequence typing, spa typing and agr typing were performed in all S. aureus isolates. From the 86 camels tested, 42 staphylococci were isolated, of which there were 11 S. aureus, 13 S. lentus, 12 S. sciuri, 3 S. xylosus, S. epidermidis, S. hominis and S. chromogenes. Staphylococci isolates were resistant to penicillin, ciprofloxacin, clindamycin and fusidic acid. All S. aureus isolates harbored the hla, hlb and hld virulence genes. S. aureus isolates were ascribed to three sequence types (STs) and three spa types. All S. aureus isolates belonged to agr type III. Camels from Gran Canaria used in recreational purposes have a moderate prevalence of S. aureus and other coagulase-negative staphylococci. Nevertheless, S. aureus isolates are susceptible to almost all antibiotics tested.

In this paper, we describe a new quantitative method to evaluate and quantify in vitro growth inhibition of mastitis-related bacteria. Colony-forming units of Staphylococcus (S.) aureus (n = 10), Escherichia (E.) coli (n = 10), and Streptococcus (S.) uberis (n = 10) were quantified after their growth on top of layers of trypticase soy agar (TSA) containing six different concentrations (varying from 102 to 107 CFU/mL) of bovine non-aureus staphylococci (NAS), i.e., S. chromogenes (n = 3) and S. simulans (n = 3) isolates. Growth inhibition of the mastitis-related major bacterial pathogens, including E. coli, was confirmed by all NAS, an effect that varied highly among NAS isolates and was not evident from the semiquantitative method with which the new method was compared. By subsequent application of the new method on a larger set of 14 bovine NAS isolates, we observed that S. simulans and NAS originating from teat apices (especially S. epidermidis) required lower concentrations to inhibit both methicillin-sensitive (MSSA) (n = 5) and methicillin-resistant S. aureus (MRSA) isolates (n = 5) originating from milk. Therefore, the new assay is a promising tool to precisely quantify the intra- and inter-species differences in growth inhibition between NAS.

Natural aquatic environments represent one of the most important vehicles of bacterial dissemination. Therefore, we aimed to isolate staphylococci from surface waters and to investigate the presence of antimicrobial resistance genes and virulence factors as well as the genetic lineages of all Staphylococcus aureus isolates. Staphylococci were recovered from water samples collected from 78 surface waters, including rivers, streams, irrigation ditches, dams, lakes, and fountains. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Multilocus sequence typing and spa-typing were performed in all S. aureus isolates. From the 78 water samples, 33 S. aureus, one S. pseudintermedius, and 51 coagulase-negative staphylococci (CoNS) were identified. Among the S. aureus isolates, four MRSA were identified, and all harbored the mecC gene. Fourteen S. aureus were susceptible to all antimicrobials tested and the remaining showed resistance to penicillin, erythromycin and/or tetracycline encoded by the blaZ, ermT, msr(A/B), tetL, and vgaA genes. Regarding the clonal lineages, one mecC-MRSA isolate belonged to spa-type t843 and sequence type (ST) 130 and the other three to t742 and ST425. The remaining S. aureus were ascribed 14 spa-types and 17 sequence types. Eleven species of CoNS were isolated: S. sciuri, S. lentus, S. xylosus, S. epidermidis, S. cohnii spp. urealyticus, S. vitulinus, S. caprae, S. carnosus spp. Carnosus, S. equorum, S. simulans, and S. succinus. Thirteen CoNS isolates had a multidrug resistance profile and carried the following genes: mecA, msr(A/B), mph(C), aph(3′)-IIIa, aac(6′)-Ie–aph(2′’)-Ia, dfrA, fusB, catpC221, and tetK. A high diversity of staphylococci was isolated from surface waters including mecCMRSA strains and isolates presenting multidrug-resistance profiles. Studies on the prevalence of antibiotic-resistant staphylococci in surface waters are still very scarce but extremely important to estimate the contribution of the aquatic environment in the spread of these bacteria.

Background: Empiric antibiotic therapy for suspected vertebral osteomyelitis (VO) should be initiated immediately in severely ill patients, and might be necessary for culture-negative VO. The current study aimed to identify differences between community-acquired (CA) and healthcare-associated (HA) VO in terms of clinical presentation, causative pathogens, and antibiotic susceptibility. Methods: Cases of adult patients with VO treated at a German university orthopaedic trauma center between 2000 and 2020 were retrospectively reviewed. Patient history was used to distinguish between CA and HA VO. Susceptibility of antibiotic regimens was assessed based on antibiograms of the isolated pathogens. Results: A total of 155 patients (with a male to female ratio of 1.3; and a mean age of 66.1 ± 12.4 years) with VO were identified. In 74 (47.7%) patients, infections were deemed healthcare-associated. The most frequently identified pathogens were Staphylococcus aureus (HAVO: 51.2%; CAVO: 46.8%), and Coagulase-negative Staphylococci (CoNS, HAVO: 31.7%; CAVO: 21.3%). Antibiograms of 45 patients (HAVO: n = 22; CAVO: n = 23) were evaluated. Significantly more methicillin-resistant isolates, mainly CoNS, were found in the HAVO cohort (27.3%). The highest rate of resistance was found for cefazolin (HAVO: 45.5%; CAVO: 26.1%). Significantly higher rates of resistances were seen in the HAVO cohort for mono-therapies with meropenem (36.4%), piperacillin–tazobactam (31.8%), ceftriaxone (27.3%), and co-amoxiclav (31.8%). The broadest antimicrobial coverage was achieved with either a combination of piperacillin–tazobactam + vancomycin (CAVO: 100.0%; HAVO: 90.9%) or meropenem + vancomycin (CAVO: 100.0%; HAVO: 95.5%). Conclusion: Healthcare association is common in VO. The susceptibility pattern of underlying pathogens differs from CAVO. When choosing an empiric antibiotic, combination therapy must be considered.

A total of 394 strains of staphylococci found in humans and pets in Novosibirsk, Siberian Russia, were characterized in terms of antibiotic resistance and corresponding genes. Two coagulase-positive and 17 coagulase-negative species were identified. The majority of isolates, with the exception of S. haemolyticus and hospital S. epidermidis isolates, were sensitive to most of the tested antibiotics, and isolates from pets displayed the lowest level of resistance. Nevertheless, methicillin-resistant (MRS) and/or multidrug-resistant (MDR) isolates were found in all prevailed species, including coagulase-negative. A set of genes corresponding to the detected resistance was identified: mecA (beta-lactam resistance), aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa, ant(4′)-Ia (aminoglycoside-modifying enzymes), ermA/ermC, and msrA (macrolide resistance). Complete genome analysis for ten MDR S. epidermidis and five MDR S. haemolyticus isolates revealed additional antibiotic resistance genes mphC, qacA/qacB, norA, dfrC/dfrG, lnuA, BseSR, and fosB. NorA, dfrC, and fosB were present in all S. epidermidis genomes, whereas mphC and msrA were identified in all S. haemolyticus ones. All investigated MDR S. epidermidis and four of five S. haemolyticus strains were moderate or strong biofilm producers, whereas multiple genes responsible for this function and for virulence and pathogenicity were identified mostly in S. epidermidis, but were less frequently represented in S. haemolyticus.

Staphylococcal food poisoning (SFP), caused by the contamination of staphylococcal enterotoxins, is a common foodborne disease worldwide. The aims of this study were: (1) to investigate classical staphylococcal enterotoxin genes, sea, seb, sec, sed, and see, among Staphylococcus aureus and coagulase-negative staphylococci (CNS) associated with bovine mastitis; (2) to determine the effect of temperature on the expression of classical staphylococcal enterotoxin genes in staphylococci in milk. The detection of classical staphylococcal enterotoxin genes was performed using S. aureus (n = 51) and CNS (n = 47). The expression of classical enterotoxin genes, including sea, seb, sec, and see, was determined during the growth of staphylococci in milk subjected to ultra-high-temperature processing at two different temperatures: 8 °C and room temperature. Classical staphylococcal enterotoxin genes were expressed more frequently in S. aureus (35.30%) than in CNS (12.77%). The sec gene was most frequently detected in S. aureus (29.41%) and CNS (6.38%). Moreover, the expression of sea and sec was significantly higher at room temperature than at 8 °C after 16 h of incubation (p < 0.05). These results emphasize the importance of maintaining the storage temperature of milk below 8 °C to reduce the risk of SFP.

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) have become the main causative agents of medical device-related infections due to their biofilm-forming capability, which protects them from the host’s immune system and from the action of antimicrobials. This study evaluated the ability of RNA III inhibiting peptide (RIP) to inhibit biofilm formation in 10 strains isolated from clinical materials, including one S. aureus strain, two S. epidermidis, two S. haemolyticus, two S. lugdunensis, and one isolate each of the following species: S. warneri, S. hominis, and S. saprophyticus. The isolates were selected from a total of 200 strains evaluated regarding phenotypic biofilm production and the presence and expression of the ica operon. The isolates were cultured in trypticase soy broth with 2% glucose in 96-well polystyrene plates containing catheter segments in the presence and absence of RIP. The catheter segments were observed by scanning electron microscopy. The results showed inhibition of biofilm formation in the presence of RIP in all CoNS isolates; however, RIP did not interfere with biofilm formation by S. aureus. RIP is a promising tool that might be used in the future for the prevention of biofilm-related infections caused by CoNS.

Macrolide-resistant methicillin-resistant Staphylococcus aureus (MAC-MRSA) is one of the most clinically relevant pathogens due to its significant ability of resistance acquisition to different antimicrobial agents. This study aimed to evaluate antimicrobial susceptibility and the use of different combinations of azithromycin with other antibiotics for combating MAC resistance. Seventy-two Staphylococci (38.5%) (n = 187), showed resistance to MACs; of these, 53 isolates (73.6%, n = 72) were S. aureus and 19 (26.4%, n = 72) were coagulase-negative staphylococci (CoNS). Out of the 53 S. aureus and 19 CoNS isolates, 38 (71.7%, n = 53) and 9 (47.4%, n = 19) were MRSA and methicillin-resistant CoNS, respectively. The constitutive MACs, lincosamides and streptogramin-B (cMLS) comprised the predominant phenotype among S. aureus isolates (54.7%) and CoNS isolates (78.9%). The PCR analysis showed that the ermC gene was the most prevalent (79.2%), followed by msrA (48.6%), and ermA (31.9%). Azithromycin combinations with either linezolid, ceftriaxone, gentamicin, or cefotaxime provided synergy in 42.1%, 44.7%, 31.6% and 7.9% of the 38 MAC-MRSA isolates, respectively. Statistical analysis showed significant association between certain MAC resistance genotypes and the synergistic effect of certain azithromycin combinations (p value < 0.05). In conclusion, azithromycin combinations with either linezolid, or ceftriaxone showed synergism in most of the MAC-resistant MRSA clinical isolates.

Ripened sheep sausages are widely consumed in Italy, particularly in Sardinia. Despite their driving role in flavor and color development, coagulase-negative staphylococci in these products have been rarely investigated. A total of 70 CoNS cultures isolated from Sardinian sheep sausages were characterized by rep-PCR and M13-RAPD typing and identified by 16S rDNA sequencing. S. xylosus and S. equorum accounted for more than 70% of the total isolates, whilst S. pasteuri (8.5%), S. succinus (2.8%), and S. haemolyticus (2.8%) were less represented. The genes encoding the synthesis of putrescine, tyramine, cadaverine, and histamine were evaluated by PCR. None of the strains hosted genes for decarboxylases, except one S. pasteuri strain that was potentially a tyramine-producer. Antibiotic resistance was evaluated, along with nitrate reductase, lipolytic, and proteolytic activity, in a pool of selected cultures. Resistance to the primary antibiotics was rather widespread. S. xylosus , S. equorum , and S. pasteuri strains were all resistant to amoxicillin and kanamycin. S. equorum strains were sensitive to all tested antibiotics. S. xylosus strains were all resistant to penicillin B. Conversely, all S. pasteuri strains were resistant to both ampicillin and penicillin B, and four out of five strains exhibited tetracycline resistance. The high variability in the production of sheep sausages makes the search for adjunct cultures of crucial relevance. According to this perspective, the characterization of the autochthonous CSN population represents the first step to approach a starter selection.

Staphylococcus capitis has been recognized as a relevant opportunistic pathogen, particularly its persistence in neonatal ICUs around the world. Therefore, the aim of this study was to describe the epidemiological profile of clinical isolates of S. capitis and to characterize the factors involved in the persistence and pathogenesis of these strains isolated from blood cultures collected in a hospital in the interior of the state of São Paulo, Brazil. A total of 141 S. capitis strains were submitted to detection of the mecA gene and S CCmec typing by multiplex PCR. Genes involved in biofilm production and genes encoding enterotoxins and hemolysins were detected by conventional PCR. Biofilm formation was evaluated by the polystyrene plate adherence test and phenotypic resistance was investigated by the disk diffusion method. Finally, pulsed-field gel electrophoresis (PFGE) was used to analyze the clonal relationship between isolates. The mecA gene was detected in 99 (70.2%) isolates, with this percentage reaching 100% in the neonatal ICU. SCC mec type III was the most prevalent type, detected in 31 (31.3%) isolates and co-occurrence of SCC mec was also observed. In vitro biofilm formation was detected in 46 (32.6%) isolates but was not correlated with the presence of the ica operon genes. Furthermore, biofilm production in ICU isolates was favored by hyperosmotic conditions, which are common in ICUs because of the frequent parenteral nutrition. Analysis of the clonal relationship between the isolates investigated in the present study confirms a homogeneous profile of S. capitis and the persistence of clones that are prevalent in the neonatal ICU and disseminated across the hospital. This study highlights the adaptation of isolates to specific hospital environments and their high clonality.

Coagulase-negative staphylococci (CoNS) are frequently occurring ocular opportunistic pathogens that are not easily identifiable to the species level. The goal of this study was to speciate CoNS and document antibiotic susceptibilities from cases of endophthalmitis (n = 50), keratitis (n = 50), and conjunctivitis/blepharitis (n = 50) for empiric therapy. All 150 isolates of CoNS were speciated using (1) API Staph (biochemical system), (2) Biolog GEN III Microplates (phenotypic substrate system), and (3) DNA sequencing of the sodA gene. Disk diffusion antibiotic susceptibilities for topical and intravitreal treatment were determined based on serum standards. CoNS identification to the species level by all three methods indicated that S. epidermidis was the predominant species of CoNS isolated from cases of endophthalmitis (84–90%), keratitis (80–86%), and conjunctivitis/blepharitis (62–68%). Identifications indicated different distributions of CoNS species among endophthalmitis (6), keratitis (10), and conjunctivitis/blepharitis (13). Antibiotic susceptibility profiles support empiric treatment of endophthalmitis with vancomycin, and keratitis treatment with cefazolin or vancomycin. There was no clear antibiotic choice for conjunctivitis/blepharitis. S. epidermidis was the most frequently found CoNS ocular pathogen, and infection by other CoNS appears to be less specific and random. Antibiotic resistance does not appear to be a serious problem associated with CoNS.

Bacterial antibiotic resistance and biofilm formation are mechanisms usually involved in the pathogeny of implant-related infections. Worldwide, antibiotic susceptibility tests are usually carried out using nutrient-rich media. Clinical routine laboratories and even research centers use for example EUCAST or CLSI for guidelines. In this study, we investigated the effect of different nutrient media on the antibiotic susceptibility and icaADBC gene expression of bacteria in biofilm. As media, Müller-Hinton Bouillon (MHB), Tryptic Soy Broth (TSB) and human synovial fluid (SF) diluted 1:4 in phosphate buffered saline (PBS), each also supplemented with 1% glucose, were used. The influence of different nutrient media on the antibiotic susceptibility of coagulase-negative staphylococci (CoNS) was evaluated by counting of colony-forming units (CFU) and by checking the metabolic activity of the bacteria. We used reverse transcriptase and real-time qPCR to investigate the influence of nutrient media on the biofilm gene expression. We used two-way analysis of variance (ANOVA). p < 0.05 was considered to be statistically significant. Significant differences in growth and antibiotic susceptibility were detected in all strains tested among the different media used. The nutrient media showed influence on the cell viability of all bacteria after antibiotic treatment. IcaADBC gene expression was significantly influenced by glucose and all nutrient media. The results highlight the influence of glucose on the antibiotic susceptibility, growth and gene expression of all strains tested. For all strains, a significant difference in bacterial recovery, viability and gene expression were found when compared to biofilm grown in SF.

Coagulase-negative staphylococci (CoNS) may be considered contaminants when isolated from clinical specimens but may also be a cause of true infection. This study aimed to compare the clonality and SCCmec type of a collection of CoNS isolated from blood cultures of inpatients, nasal swabs of healthy individuals, and patients with chronic wounds, all from the same community, using SCCmec typing, pulsed-field gel electrophoresis (PFGE), and MLST. Staphylococcus epidermidis, exhibited high clonal diversity, but hospital and community clusters were observed. Nosocomial S. epidermidis clones belonged to sequence types ST2, ST6, and ST23. Some Staphylococcus haemolyticus clones were found to circulate in the hospital and community, while Staphylococcus saprophyticus exhibited very high clonal diversity. Staphylococcus lugdunensis, Staphylococcus warneri, and Staphylococcus capitis revealed several isolates belonging to the same clone in the hospital and community. The detection of different SCCmec types within the same cluster indicated high diversity. S. epidermidis was associated with SCCmec I and III, S. haemolyticus with I and II, S. capitis with type V, Staphylococcus hominis with mec complex type A and ccr1, and S. warneri and S. saprophyticus with SCCmec I. The generation of elements and new combinations of cassette genes were highly associated with CoNS isolates, suggesting that SCCmec may not be a good marker of clonality in these bacteria.

Coagulase-negative staphylococci (CoNS) are among the most frequently recovered bacteria in routine clinical care. Their incidence has steadily increased over the past decades in parallel to the advancement in medicine, especially in regard to the utilization of foreign body devices. Many new species have been described within the past years, while clinical information to most of those species is still sparse. In addition, interspecies differences that render some species more virulent than others have to be taken into account. The distinct populations in which CoNS infections play a prominent role are preterm neonates, patients with implanted medical devices, immunodeficient patients, and those with other relevant comorbidities. Due to the property of CoNS to colonize the human skin, contamination of blood cultures or other samples occurs frequently. Hence, the main diagnostic hurdle is to correctly identify the cases in which CoNS are causative agents rather than contaminants. However, neither phenotypic nor genetic tools have been able to provide a satisfying solution to this problem. Another dilemma of CoNS in clinical practice pertains to their extensive antimicrobial resistance profile, especially in healthcare settings. Therefore, true infections caused by CoNS most often necessitate the use of second-line antimicrobial drugs.

The aim of the study was to analyze the biofilm-production capacity of 87 coagulase-negative Staphylococcus strains (CoNS) isolated from broiler chickens and to determine the occurrence of biofilm-associated genes. The biofilm production capacity of staphylococci was assessed using the microtiter plate method (MTP), and the frequency of genes was determined by PCR. The ability to form a biofilm in vitro was shown in 79.3% of examined strains. Strong biofilm capacity was demonstrated in 26.4% of strains, moderate capacity in 25.3%, weak capacity in 27.6%, and a complete lack of biofilm production capacity in 20.7% of strains. The icaAB gene responsible for the production of extracellular polysaccharide adhesins was detected in 6.9% of strains. The other four genes, i.e., bap (encoding biofilm-associated protein), atlE (encoding cell surface protein exhibiting vitronectin-binding activity), fbe (encoding fibrinogen-binding protein), and eno (encoding laminin-binding protein) were detected in 5.7%, 19.5%, 8%, and 70.1% of strains, respectively. Demonstration of genes that play a role in bacterial biofilm formation may serve as a genetic basis to distinguish between symbiotic and potentially invasive coagulase-negative staphylococcal strains.

Background: Implantable medical devices, such as prosthetics, catheters, and several other devices, have revolutionized medicine, but they increase the infection risk. In previous decades, commercially available antibiotics lost their activity against coagulase-negative Staphylococci (CoNS) and several other microorganisms. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the two major omega-3 polyunsaturated fatty acids (ω-3 PUFAs) with antimicrobial properties. Materials and Methods: In this study, we tested the EPA and the DHA for its antibacterial and anti-biofilm activity in vitro against Staphylococcus epidermidis, Staphylococcus aureus, and different CoNS as reference strains and isolated from patients undergoing orthopedic treatment for implant infections. The tests were carried out with the strains in planktonic and biofilm form. Cytotoxicity assay was carried out with EPA and DHA using human gingival fibroblasts HGF-1. Results: The highest concentration of EPA and DHA promoted the complete killing of S. epidermidis 1457 and S. aureus ATCC 25923 in planktonic form. The fatty acids showed low activity against P. aeruginosa. EPA and DHA completely killed or significantly reduced the count of planktonic bacteria of the patient isolated strains. When incubated with media enriched with EPA and DHA, the biofilm formation was significantly reduced on S. epidermidis 1457 and not present on S. aureus ATCC 25923. The reduction or complete killing were also observed with the clinical isolates. The pre-formed biofilms showed reduction of the cell counting after treatment with EPA and DHA. Conclusion: In this study, the ω-3 PUFAs EPA and DHA showed antimicrobial and anti-biofilm activity in vitro against S. aureus, S. epidermidis, and P. aeruginosa, as well as against multi-drug resistant S. aureus and CoNS strains isolated from patients undergoing periprosthetic joint infections (PJI) treatment. Higher concentrations of the fatty acids showed killing activity on planktonic cells and inhibitory activity of biofilm formation. Although both substances showed antimicrobial activity, EPA showed better results in comparison with DHA. In addition, when applied on human gingival fibroblasts in vitro, EPA and DHA showed a possible protective effect on cells cultured in medium enriched with ethanol. Further studies are required to confirm the antimicrobial activity of EPA and DHA against multi-drug resistant strains and pan-drug resistant strains.

The anaerobic coagulase-negative staphylococcal species Staphylococcus saccharolyticus is a member of the normal skin microbiota. However, S. saccharolyticus is rarely found in clinical specimens and its pathogenic potential is unclear. The clinical data of prosthetic hip (n = 5) and shoulder (n = 2) joint implant-associated infections where S. saccharolyticus was detected in periprosthetic tissue specimens are described. The prosthetic hip joint infection cases presented as “aseptic” loosening and may represent chronic, insidious, low-grade prosthetic joint infections (PJIs), eventually resulting in loosening of prosthetic components. All cases were subjected to one-stage revision surgery and the long-term outcome was good. The shoulder joint infections had an acute onset. Polymicrobial growth, in all cases with Cutibacterium acnes, was found in 4/7 patients. All but one case were treated with long-term administration of beta-lactam antibiotics. Whole-genome sequencing (WGS) of the isolates was performed and potential virulence traits were identified. WGS could distinguish two phylogenetic clades (clades 1 and 2), which likely represent distinct subspecies of S. saccharolyticus. Little strain individuality was observed among strains from the same clade. Strains of clade 2 were exclusively associated with hip PJIs, whereas clade 1 strains originated from shoulder PJIs. It is possible that strains of the two clades colonize different skin habitats. In conclusion, S. saccharolyticus has the potential to cause PJIs that were previously regarded as aseptic loosening of prosthetic joint devices.

There has been an increase in the prevalence of gram-positive bacteremia in neonates in the last two decades. However, as a consequence of better care, there has been an increase in the survival of premature neonates. Coagulase-negative staphylococci (CoNS) is the most prevalent bacteria, responsible for up to 60% of late-onset sepsis (LOS). Daptomycin, a lipopeptide antimicrobial agent, is active against CoNS. This was an observational, retrospective case series study carried out in the Pediatric Hospital of King Saud Medical City, Riyadh, Saudi Arabia. The medical records of 21 neonates, aged 0–28 days, who were treated in Neonatal Intensive Care Unit (NICU) with intravenous daptomycin as monotherapy or combination therapy for at least 4 days for proven gram-positive infection between June 2019 to July 2020, were included. The median gestational and chronological age were 27 weeks and 5 days, respectively. The most frequent diagnosis in neonates was infective endocarditis (42.9%). Of the 21 patients who received daptomycin therapy, 13 (62%) recovered and 8 died. The clinical cure rate was higher in Staphylococcus hominis (100%) and in patients who received 6 mg/kg/dose twice daily (62.5%). The mean of aspartate aminotransferase significantly elevated after starting daptomycin (p = 0.048). However, no muscular or neurological toxicity of daptomycin was documented in any of the cases. Overall, daptomycin was well tolerated, even with long-term treatment.

Coagulase-negative staphylococci (CoNS) are increasingly associated with nosocomial infections, especially among the immunocompromised and those with invasive medical devices, posing a significant concern. We report on clinical multidrug-resistant CoNS from the uMgungundlovu District, KwaZulu-Natal Province, South Africa, as emerging pathogens. One hundred and thirty presumptive CoNS were obtained from blood cultures. Culture, biochemical tests, and the Staphaurex™ Latex Agglutination Test were used for the initial identification of CoNS isolates; confirmation and speciation were undertaken by the VITEK 2 system. Susceptibilities of isolates against a panel of 20 antibiotics were determined using the Kirby-Bauer disk diffusion method, and the multiple antibiotic resistance (MAR) indices of the isolates were determined. The polymerase chain reaction (PCR) was used to amplify the mecA gene to confirm methicillin resistance. Overall, 89/130 presumptive CoNS isolates were confirmed as CoNS by the VITEK 2 system. Of these, 68 (76.4%) isolates were putatively methicillin-resistant by the phenotypic cefoxitin screen test and 63 (92.6%) were mecA positive. Staphylococcus epidermidis (19.1%), S. hominis ssp. hominis (15.7%), and S. haemolyticus (16.9%) were the most common CoNS species. Isolates showed high percentage resistance against penicillin (100.0%), erythromycin (74.2%), and azithromycin (74.2%) while displaying high susceptibilities to linezolid (95.5%), gentamicin (95.5%), and tigecycline (94.4%). Multidrug resistance (MDR) was observed in 76.4% of isolates. MAR index calculation revealed 71.9% of isolates with MAR index >0.2 and 20.2% >0.5. Isolates with the highest MAR indices (0.7 and 0.8) were recovered from the neonatal intensive care unit. Fifty-one MDR antibiograms were observed. The high prevalence of methicillin resistance and multidrug resistance in several species of CoNS necessitates surveillance of this emerging pathogen, currently considered a contaminant of microbial cultures.

The skin microbiota of atopic dermatitis (AD) patients is characterized by increased Staphylococcus aureus colonization, which exacerbates disease symptoms and has been linked to reduced bacterial diversity. Skin bacterial communities in AD patients have mostly been described at family and genus levels, while species-level characterization has been limited. In this study, we investigated the role of the bacteria belonging to the Staphylococcus genus using targeted sequencing of the tuf gene with genus-specific primers. We compared staphylococcal communities on lesional and non-lesional skin of AD patients, as well as AD patients with healthy controls, and determined the absolute abundance of bacteria present at each site. We observed that the staphylococcal community, bacterial alpha diversity, and bacterial densities were similar on lesional and non-lesional skin, whereas AD severity was associated with significant changes in staphylococcal composition. Increased S. aureus, Staphylococcus capitis, and Staphylococcus lugdunensis abundances were correlated with increased severity. Conversely, Staphylococcus hominis abundance was negatively correlated with severity. Furthermore, S. hominis relative abundance was reduced on AD skin compared to healthy skin. In conclusion, various staphylococcal species appear to be important for skin health.

Coagulase-negative staphylococci (CoNS) have emerged as major pathogens in healthcare-associated facilities, being S. epidermidis, S. haemolyticus and, more recently, S. lugdunensis, the most clinically relevant species. Despite being less virulent than the well-studied pathogen S. aureus, the number of CoNS strains sequenced is constantly increasing and, with that, the number of virulence factors identified in those strains. In this regard, biofilm formation is considered the most important. Besides virulence factors, the presence of several antibiotic-resistance genes identified in CoNS is worrisome and makes treatment very challenging. In this review, we analyzed the different aspects involved in CoNS virulence and their impact on health and food.

Staphylococci, like Staphylococcus aureus and S. epidermidis, are common colonizers of the human microbiota. While being harmless in many cases, many virulence factors result in them being opportunistic pathogens and one of the major causes of hospital-acquired infections worldwide. One of these virulence factors is the ability to form biofilms—three-dimensional communities of microorganisms embedded in an extracellular polymeric matrix (EPS). The EPS is composed of polysaccharides, proteins and extracellular DNA, and is finely regulated in response to environmental conditions. This structured environment protects the embedded bacteria from the human immune system and decreases their susceptibility to antimicrobials, making infections caused by staphylococci particularly difficult to treat. With the rise of antibiotic-resistant staphylococci, together with difficulty in removing biofilms, there is a great need for new treatment strategies. The purpose of this review is to provide an overview of our current knowledge of the stages of biofilm development and what difficulties may arise when trying to eradicate staphylococcal biofilms. Furthermore, we look into promising targets and therapeutic methods, including bacteriocins and phage-derived antibiofilm approaches.

Although coagulase-positive staphylococci are considered to be the main factor responsible for food poisoning, an increasing role for the coagulase-negative staphylococci in the production of enterotoxins has been observed in recent years. This study was conducted to assess the occurrence of genes responsible for the production of staphylococcal enterotoxins (SE), enterotoxin-like toxins (SEI) and toxic shock syndrome toxin-1 (TSST-1) in coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food from bars and restaurants. One hundred and eighteen CoNS strains were tested using polymerase chain reaction (PCR) to five superantigenic toxin genes, including five different types of classical enterotoxins (sea, seb, sec, sed and see) and the toxic shock syndrome toxin-1 (tsst-1) as well as to supertoxin-like genes. PCR-positive isolates were then tested using immunoenzymatic methods (SET-RPLA, Vidas SET 2) for toxin expression. Out of 118 CoNS strains, the presence of staphylococcal enterotoxins was confirmed in 72% of them. The most frequently found enterotoxin-like genotype was ser, selu. Two of the tested strains had up to ten different enterotoxin genes in the genome at the same time. Although no production of enterotoxins was detected in the CoNS, which means that their possible role in the epidemiology of food-borne diseases is minimal, the data demonstrated that the toxigenic capacity of the CoNS should not be ignored, and that this group of microorganisms should be continuously monitored in food.

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signals in Gram-negative bacteria. Many genes encoding AHL-degrading enzymes have been cloned and characterized in various microorganisms. Coagulase-negative staphylococci (CNS) are present on the skin of animals and are considered low-virulent species. The AHL-lactonase gene homologue, ahlS, was present in the genomes of the CNS strains Staphylococcus carnosus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus sciuri. We cloned the candidate ahlS homologue from six CNS strains into the pBBR1MCS5 vector. AhlS from the CNS strains showed a higher degrading activity against AHLs with short acyl chains compared to those with long acyl chains. AhlS from S. sciuri was expressed and purified as a maltose-binding protein (MBP) fusion. Pseudomonas aeruginosa is an opportunistic pathogen that regulates several virulence factors such as elastase and pyocyanin by quorum-sensing systems. When MBP-AhlS was added to the culture of P. aeruginosa PAO1, pyocyanin production and elastase activity were substantially reduced compared to those in untreated PAO1. These results demonstrate that the AHL-degrading activity of AhlS from the CNS strains can inhibit quorum sensing in P. aeruginosa PAO1.

The objective of this study was to determine the prevalence and diversity of coagulase-negative staphylococci (CoNS) species from wild birds in Spain, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content. During 2015–2016, tracheal samples of 242 wild birds were collected in different regions of Spain for staphylococci recovery. The species identification was performed using MALDI-TOF. The antimicrobial resistance phenotype and genotype was investigated by the disk diffusion method and by PCR, respectively. The presence of the virulence genes lukF/S-PV, tst, eta, etb, etd and scn was investigated by PCR. Moreover, CoNS carrying the mecA gene were subjected to SCCmec typing. Of the tested animals, 60% were CoNS-carriers, and 173 CoNS isolates were recovered from the 146 positive animals, which belonged to 11 species, with predominance of S. sciuri (n = 118) and S. lentus (n = 25). A total of 34% of CoNS isolates showed a multidrug resistance phenotype, and 42 mecA-positive methicillin-resistant CoNS (MRCoNS) were detected. The isolates showed resistance to the following antimicrobials (percentage of resistant isolates/antimicrobial resistance genes detected): penicillin (49/ blaZ, mecA), cefoxitin (24/ mecA), erythromycin and/or clindamycin (92/ erm(B), erm(C), erm(43), msr(A), mph(C), lnu(A), lsa(B), vga(A) and sal(A)), gentamicin and/or tobramycin (5/ aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia), streptomycin (12/str), tetracycline (17/ tet(K), tet(L), tet(M)), ciprofloxacin (4), chloramphenicol (1/ fexA), fusidic acid (86/ fusB, fusD) and trimethoprim–sulfamethoxazole (1/ dfrK). None of the isolates harbored the lukF/S-PV, eta, etb, etd and scn genes, but two S. sciuri isolates (1%) carried the tst gene. Wild birds are frequently colonized by CoNS species, especially S. sciuri. We identified scavenging on intensively produced livestock and feeding on landfills as risk factors for CoNS carriage. High proportions of MRCoNS and multidrug resistant CoNS were detected, which coupled with the presence of important virulence genes is of concern.

The capacity for biofilm formation is one of the crucial factors of staphylococcal virulence. The occurrence of biofilm-forming staphylococci in raw milk may result in disturbances in technological processes in dairy factories as well as the contamination of finished food products. Therefore, this study aimed to determine the prevalence and characteristics of staphylococcal biofilm formation in raw milk samples and to explore the genetic background associated with biofilm formation in those isolates. The material subjected to testing included 30 cow’s milk samples acquired from farms in the central part of Poland. A total of 54 staphylococcal strains were isolated from the samples, of which 42 were classified as coagulase-negative (CoNS) staphylococci belonging to the following species: S. haemolyticus, S. simulans, S. warneri, S. chromogenes, S. hominis, S. sciuri, S. capitis, S. xylosus and S. saprophyticus, while 12 were classified as S. aureus. The study examined the isolates’ capacity for biofilm formation and the staphylococcal capacity for slime production and determined the presence of genetic determinants responsible for biofilm formation, i.e., the icaA, icaD, bap and eno and, additionally, among coagulase-negative staphylococci, i.e., the aap, bhp, fbe, embP and atlE. Each tested isolate exhibited the capacity for biofilm formation, of which most of them (79.6%) were capable of forming a strong biofilm, while 5.6% formed a moderate biofilm, and 14.8% a weak biofilm. A capacity for slime production was demonstrated in 51.9% isolates. Most of the tested staphylococcal strains (90.7%) had at least one of the tested genes. Nearly half (47.6%) of the CoNS had the eno gene, while for S. aureus, the eno gene was demonstrated in 58.3% isolates. The frequency of the bap gene occurrence was 23.8% and 25% in CoNS strains and S. aureus, respectively. The fbe gene was demonstrated in only three CoNS isolates. The presence of the icaA was only demonstrated in CoNS strains (24.1%), while the icaD was found in both CoNS strains (21.4%) and S. aureus (100%). Among the CoNS, the presence of the embP (16.7%), aap (28.6%) and atlE (23.8%) was demonstrated as well. The obtained study results indicate that bacteria of the Staphylococcus spp. genus have a strong potential to form a biofilm, which may pose a hazard to consumer health.

Coagulase-negative staphylococcal species constitute an important part of the human skin microbiota. In particular, facultative anaerobic species such as Staphylococcus epidermidis and Staphylococcus capitis can be found on the skin of virtually every human being. Here, we applied a culture-independent amplicon sequencing approach to identify staphylococcal species on the skin of healthy human individuals. While S. epidermidis and S. capitis were found as primary residents of back skin, surprisingly, the third most abundant member was Staphylococcus saccharolyticus, a relatively unstudied species. A search of skin metagenomic datasets detected sequences identical to the genome of S. saccharolyticus in diverse skin sites, including the back, forehead, and elbow pit. Although described as a slow-growing anaerobic species, a re-evaluation of its growth behavior showed that S. saccharolyticus can grow under oxic conditions, and, in particular, in a CO2-rich atmosphere. We argue here that S. saccharolyticus was largely overlooked in previous culture-dependent and -independent studies, due to its requirement for fastidious growth conditions and the lack of reference genome sequences, respectively. Future studies are needed to unravel the microbiology and host-interacting properties of S. saccharolyticus and its role as a prevalent skin colonizer.

Coagulase-negative staphylococci (CNS) make up a diverse bacterial group, appearing in a myriad of ecosystems. To unravel the composition of staphylococcal communities in these microbial ecosystems, a reliable species-level identification is crucial. The present study aimed to design a primer set for high-throughput amplicon sequencing, amplifying a region of the tuf gene with enough discriminatory power to distinguish different CNS species. Based on 2566 tuf gene sequences present in the public European Nucleotide Archive database and saved as a custom tuf gene database in-house, three different primer sets were designed, which were able to amplify a specific region of the tuf gene for 36 strains of 18 different CNS species. In silico analysis revealed that species-level identification of closely related species was only reliable if a 100% identity cut-off was applied for matches between the amplicon sequence variants and the custom tuf gene database. From the three primer sets designed, one set (Tuf387/765) outperformed the two other primer sets for studying Staphylococcus-rich microbial communities using amplicon sequencing, as it resulted in no false positives and precise species-level identification. The method developed offers interesting potential for a rapid and robust analysis of complex staphylococcal communities in a variety of microbial ecosystems.

Coagulase-negative staphylococci (CoNS) are an important group of opportunistic pathogenic microorganisms that cause infections in hospital settings and are generally resistant to many antimicrobial agents. We report on phenotypic and genotypic virulence characteristics of a select group of clinical, mecA-positive (encoding penicillin-binding protein 2a) CoNS isolates. All CoNS were resistant to two or more antimicrobials with S. epidermidis strain 214EP, showing resistance to fifteen of the sixteen antimicrobial agents tested. Aminoglycoside-resistance genes were the ones most commonly detected. The presence of megaplasmids containing both horizontal gene transfer and antimicrobial resistance genetic determinants indicates that CoNS may disseminate antibiotic resistance to other bacteria. Staphylococcus sciuri species produced six virulence enzymes, including a DNase, gelatinase, lipase, phosphatase, and protease that are suspected to degrade tissues into nutrients for bacterial growth and contribute to the pathogenicity of CoNS. The PCR assay for the detection of biofilm-associated genes found the eno (encoding laminin-binding protein) gene in all isolates. Measurement of their biofilm-forming ability and Spearman’s rank correlation coefficient analyses revealed that the results of crystal violet (CV) and extracellular polymeric substances (EPS) assays were significantly correlated (ρ = 0.9153, P = 3.612e-12). The presence of virulence factors, biofilm-formation capability, extracellular enzymes, multidrug resistance, and gene transfer markers in mecA-positive CoNS clinical strains used in this study makes them powerful opportunistic pathogens. The study also warrants a careful evaluation of nosocomial infections caused by CoNS and may be useful in studying the mechanism of virulence and factors associated with their pathogenicity in vivo and developing effective strategies for mitigation.

Staphylococcus aureus demonstrates considerable membrane lipid plasticity in response to different growth environments, which is of potential relevance to response and resistance to various antimicrobial agents. This information is not available for various species of coagulase-negative staphylococci, which are common skin inhabitants, can be significant human pathogens, and are resistant to multiple antibiotics. We determined the total fatty acid compositions of Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and Staphylococcus aureus for comparison purposes. Different proportions of branched-chain and straight-chain fatty acids were observed amongst the different species. However, growth in cation-supplemented Mueller–Hinton broth significantly increased the proportion of branched-chain fatty acids, and membrane fluidities as measured by fluorescence anisotropy. Cation-supplemented Mueller–Hinton broth is used for routine determination of antimicrobial susceptibilities. Growth in serum led to significant increases in straight-chain unsaturated fatty acids in the total fatty acid profiles, and decreases in branched-chain fatty acids. This indicates preformed fatty acids can replace biosynthesized fatty acids in the glycerolipids of coagulase-negative staphylococci, and indicates that bacterial fatty acid biosynthesis system II may not be a good target for antimicrobial agents in these organisms. Even though the different species are expected to be exposed to skin antimicrobial fatty acids, they were susceptible to the major skin antimicrobial fatty acid sapienic acid (C16:1Δ6). Certain species were not susceptible to linoleic acid (C18:2Δ9,12), but no obvious relationship to fatty acid composition could be discerned.