Coagulase-negative Staphylococci Species Research Articles

P-1751. Why do we call in the Blues (and Purples)? Critical Result Reporting of Gram Stains for Gram Positive Cocci in Blood Cultures

Abstract Background Gram positive cocci (GPC) in blood cultures (BLCX) can represent pathogens or contaminants. Many laboratories notify care teams of a positive BLCX with the gram stain as a critical results report (CRR). However, PCR results about 90 minutes later and can provide useful information to distinguish a contaminant from a pathogen. The present study describes healthcare utilization surrounding CRR of gram stain result without the PCR result. Methods Retrospective observational study of patients with positive BLCX. Patients were excluded if their BLCX had yeast, polymicrobial growth, a gram-negative organism, or were < 18 years of age. Clinical and healthcare utilization information was collected using the electronic medical record. We defined the following as “true pathogens” (TP): MRSA, MSSA, Enterococcus, Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus pneumoniae. We also defined TP as a coagulase-negative staphylococcus or other Strep species with 2/2 positive BLCX with intraarticular or endovascular hardware present and with 4 SIRS criteria. A “likely contaminant” (LC) was defined as coagulase negative staphylococcus or Strep species (not included in the initial TP definition) with 1-2 BLCX positive, with or without intraarticular or endovascular hardware present, and < 4 SIRS criteria. Results Of 94 patients with BLCX positive for GPC, 54 were categorized as TP, and 40 as LC. 36 (67%) of patients with a TP on BLCX received vancomycin (VAN) before CRR and 19 (35%) received VAN after CRR. 12 (30%) patients with a LC on BLCX received VAN before CRR and 11 (28%) received VAN after CRR. For those not on VAN before CRR and were placed on VAN after the call, 10 (24%) had TP and (28%) had LC identified. Repeat BLCXs were performed in 93% of patients with TP and 83% of patients with LC. Infectious Disease (ID) consults were placed in 63% of TP and 73% for LC. Echocardiograms were performed in 57% for TP and 60% for LC. Conclusion Workup and treatment for GPC identified in BLCX were similar for TP and LC. VAN is being over-utilized for LC which could lead to patient harm. Unnecessary resources are being expended in the workup of LC. Using PCR results instead of the gram stain as the CRR may provide more complete information to providers to inform decision making. Disclosures All Authors: No reported disclosures

Tracing Staphylococcus capitis and Staphylococcus epidermidis strains causing septicemia in extremely preterm infants to the skin, mouth, and gut microbiota.

Septicemia is a major cause of morbidity in preterm infants. Coagulase-negative staphylococci (CoNS) can colonize skin, oral cavity, and intestines and are a common cause of septicemia in this group. The relation between CoNS colonization pattern at the species and strain level and septicemia has scarcely been studied. We mapped colonization of the skin, oral cavity, and intestines by CoNS species in extremely preterm infants and speciated and strain-typed the skin, mucosal, and blood isolates. Two-thirds of the CoNS septicemia blood strains, including a majority of S. capitis strains belonging to the NRCS-A clone, were tracked to the commensal microbiota. We demonstrated that CoNS species differ in their colonization patterns, whereby S. capitis was primarily a skin colonizer. However, its colonization of the oral cavity was enhanced among infants developing septicemia. Our study provides a starting point for further explorations of the relationship between CoNS colonization and septicemia in preterm infants.

Deciphering pathogenicity and virulence of the first Staphylococcus debuckii isolate from diabetic foot osteomyelitis.

This study identifies Staphylococcus debuckii as a new coagulase-negative staphylococcal species isolated from diabetic foot osteomyelitis (DFOM) and provides an in-depth analysis of its pathogenic and virulence profile, as well as demonstrating its potential to cause infection. The S. debuckii NSD001 strain was examined for its planktonic growth, biofilm production, and phagocytosis rates in murine macrophages compared to S. aureus NSA739. Additionally, persistence and replication within human osteoblasts were investigated, while the zebrafish embryo model was employed to assess virulence. Genomic sequencing and bioinformatic analysis were also conducted to identify genes associated with virulent potential. S. debuckii NSD001 exhibited robust planktonic growth and significant biofilm production, highlighting its capacity to initiate and maintain an infection, and demonstrated similar rates of phagocytosis as S. aureus NSA739 in murine macrophages, suggesting a mechanism for evading initial host defenses. The strain persisted and replicated within human osteoblasts, indicative of a strategy for intracellular survival and facilitation of chronic osteomyelitis. The zebrafish embryo model revealed a slower, yet fatal, virulence profile for S. debuckii NSD001 compared to the rapid lethality induced by S. aureus NSA739. Genomic sequencing and bioinformatic analysis uncovered various genes corroborating its virulence. S. debuckii NSD001 poses a significant concern in DFOM due to its ability to form biofilms and survive within host cells, presenting challenges for current treatment strategies. This underscores the need for updated clinical protocols and increased awareness among healthcare professionals to effectively manage infections caused by this emerging pathogen.

Mouse milk bacterial count in coagulase negative staphylococcus species induced mastitis

Bovine mastitis in an economically important disease of dairy cattle and caused by multi etiological factors. In the present study, milk viable bacterial count in mouse mastitis induced by Staphylococcus epidermidis, S. chromogenes, S. haemolyticus and S. aureusisolated from apparently normal bovine milk was studied. The 2 x 104CFUorganisms in 50 µl per teat were inoculated through intramammary route in 4th and 5th pairs of abdominal mammary gland in mice. The mouse milk ranging from 50 to 200 µl per mice wascollectedat 6, 12, 24, 48, 72 and 96 hrs after intramammary inoculation. The milk was diluted with sterile PBS and subjected to bacterial countby pour plate method. The viable bacterial count in mice milk showed significant (P<0.05) increase in bacterial colonies at 12, 24, 48 and 72hrs after S. aureus infection in mice. The three coagulase negative staphylococci(CNS) species showed initial increase in bacterial counts at 12 and 24 hrs but declined from 48 to 96 hrs after IMI in mice. Thus, CNS species can increase the mice viable bacterial count moderately but ten to fifteen fold increase was observed in S. aureus infected mice mammary gland.This indicated the subclinical nature of CNS intramammaryinfection in mice and also the host ability to overcome and eliminate the CNS infection. Mouse is a suitable model to study coagulase negative staphylococcus species induced bovine subclinical mastitis.

Novel sequence types and low levels of antimicrobial resistance associated with clinical mastitis in sheep flocks across Scotland.

This research paper aimed to demonstrate that mammary secretions provided by sheep farmers across Scotland from cases of clinical mastitis are free from environmental contamination, as well as to provide information on the major bacterial causes of disease and levels of antimicrobial resistance. Mastitis represents one of most significant diseases of small ruminant production worldwide. Staphylococcus aureus, Mannheimia haemolytica, Streptococcus uberis and coagulase-negative Staphylococcal species are common pathogens isolated from cases of sheep mastitis. Sampling kits supplied to 23 farms provided 33 samples for bacteriology, antimicrobial susceptibility testing and genetic analysis. Of the bacterial isolates identified, 60% were S. aureus, 23% M. haemolytica and 7% coagulase-negative staphylococci. Low levels of antimicrobial resistance were identified in the S. aureus isolates which provided novel multi-locus sequence types. In conclusion, this proof-of-concept survey demonstrated that mammary secretions free from environmental contamination may be provided by sheep farmers. It also provided data on the prevalence of antimicrobial resistance associated with clinical mastitis in sheep and will inform on the scale required for larger surveys aiming to improve current strategies for mastitis control in sheep flocks across the UK.

Microbial Trends and their Drug Resistance Responsible for Bloodstream Infections in a Superspeciality Transplant Hospital

Background & Objective: Bloodstream infections (BSIs) are critical healthcare-associated infections that lead to high morbidity and mortality, requiring rapid diagnosis and effective antimicrobial treatment. The increasing prevalence of multidrug-resistant organisms (MDROs) exacerbates this issue, particularly in developing countries. The purpose of this study is to assess the bacteriological profile and antimicrobial susceptibility trends of BSIs to establish an antibiogram for effective empirical treatment. Materials and Methods: This study was conducted retrospectively on 3,300 blood culture samples from a multispecialty hospital over 15 months. Cultures were performed using Bactec FX and identification and antibiotic susceptibility determined by Vitek2 and Kirby-Bauer methods following CLSI guidelines. Results: Overall, the positivity rate was (14.3%) with 473 isolates: (400 bacterial and 73 fungal). Gram-negative bacteria were predominant, led by Klebsiella pneumoniae (113 isolates) and Escherichia coli (100 isolates). Among the Gram-negative bacteria, antimicrobial susceptibility was found to be low for Cephalosporins (21%sensitivity) and Fluoroquinolones (19.3% sensitivity), with moderate susceptibility to Carbapenems (51.3%). Sensitivity was high for Colistin (98.9%), Amikacin (91.05%), Tigecycline (100%), Fosfomycin (100%) and Ceftazidime-avibactum Aztreonam (97.7%). Among Gram-positive bacteria, Coagulase-negative Staphylococcal Species (CONS) and Staphylococcus aureus were the most common. The overall sensitivity of Gram-positive bacteria to antibiotics tested was high compared to the Gram-negative bacteria. Sensitivity to antibiotics such as Linezolid was found to be 94.5% and Vancomycin was found to be 93.5%. Conclusion: The high incidence of MDROs especially among the Gram-negative bacteria highlights the need for continuous monitoring and antibiotic stewardship programs. Empirical therapy must consider local resistance patterns, and a multidisciplinary approach is essential to mitigate antimicrobial resistance.

A Contemporary Picture of Coagulase-Negative Staphylococcal Endocarditis: A Nationwide GAMES Cohort Study.

Coagulase-negative staphylococci (CoNS) are an increasingly common cause of infective endocarditis (IE) and lack recent data from large studies. Our aim was to describe the epidemiology, clinical characteristics, and outcomes of staphylococcal IE in a contemporary nationwide cohort study, while comparing coagulase-negative staphylococcal IE (CoNSIE) to IE from Staphylococcus aureus (SAIE), and among IE caused by Staphylococcus epidermidis (SE), S.lugdunensis (SL), and other CoNS. We completed a post hoc analysis of a prospectively collected cohort of 4,567 consecutive definitive IE episodes from 44 Spanish centers between 2008 and 2022 (GAMES ["Grupos de Apoyo al Manejo de la Endocarditis Infecciosa en España"] cohort). A total of 842 cases of CoNSIE were compared with 1,109 cases of SAIE. Additionally, 647 episodes caused by SE were compared with 54 caused by SL and 109 caused by 9 other known CoNS species. Multivariate analyses were performed to investigate prognostic factors for in-hospital and 1-year mortality. Staphylococci accounted for 1,951 (42.7%) episodes of IE. The predominant CoNS etiology was SE (76.8%), followed by SL (6.4%), S.capitis (3.5%), S.haemolyticus (3.2%), S.hominis (3.2%), S.warneri (1.5%), and 5 other species. CoNSIE showed a distinct clinical profile from SAIE (older age, higher rates of prior heart disease, aortic and prosthetic valve involvement, nosocomial acquisition, methicillin resistance, intracardiac complications, and cardiac surgery), while in-hospital mortality was higher in SAIE (32.8% vs 37.1%; P = 0.049), with no significant differences in 1-year mortality. S.lugdunensis displayed a shorter course of infection and higher rates of leaflet perforation/rupture than S.epidermidis and other CoNS, but cardiac surgery rates (60.4% vs 61.1% vs 56.0%; P = 0.850), as well as in-hospital (33.1% vs 37.0% vs 27.5%; P = 0.540) and 1-year mortality rates were high and similar in all groups. Septic shock, heart failure, and cardiac surgery (protective) were consistently identified as independent prognostic factors, whereas none of the staphylococcal species were independently associated with mortality. Last, each 5-year study period was independently associated with a reduction in staphylococcal in-hospital and 1-year mortality. CoNSIE was a relatively common (18.4%) and life-threatening entity with particularities by species yet generally high rates of surgery and mortality, although its prognosis improved over time.

Antibiotic resistance profile of <i>Coagulase-negative staphylococci</i> species isolated from subclinical mastitis in Colombian dairy herds

Boyacá is Colombia's fifth-largest milk-producing department. Previous studies have shown the importance of Coagulase-negative Staphylococci (CNS) species as causes of subclinical mastitis in dairy herds. This work aimed to determine the antibiotic resistance profile in CNS obtained from subclinical mastitis, and the presence of the mecA gene. CNS DNA was taken from a previous study of the research group, and specific primers were used for the detection of the mecA gene in isolates that showed antibacterial resistance to at least one of the following antibiotics: ampicillin, tetracycline, streptomycin, oxacillin, penicillin, and enrofloxacin. The results show different patterns of antibiotic resistance in the CNS isolates despite being obtained from the same herd. In addition, S. chromogenes and S. epidermidis were the most prevalent pathogens, and with the highest presence of the mecA gene (33.4 and 37.5% respectively). The results are like those found in other countries and worldwide studies, however, the presence of the mecA gene was not associated in some species such as S. capitis, S. sciuri, and S. haemolyticus with phenotypic resistance to oxacillin or penicillin, therefore, more research is required on other genes possibly associated with this.

Characterization of Chlorhexidine Resistance Among Clinical Isolates of Coagulase-Negative Staphylococcus Species in a Tertiary Care Center, Chennai.

Background Coagulase-negativeStaphylococci(CoNS) are potential pathogens and are often associated with healthcare-associated infections (HAIs). Chlorhexidine (CHX) is the most widely used antiseptic to reduce colonization and infection by allStaphylococci, including CoNS. Resistance to CHXamong CoNS has been observed over the past few years, consequent to its widespread use. Phenotypic tolerance or reduced susceptibility to CHX is conferred by plasmid-mediatedqacgroup of genes, mainlyqacA/Bandsmr, which cause activation of efflux pumps over the bacterial cell wall. This study aims to characterize the phenotypic and genotypic resistance exhibited by CoNS species against CHX. Methods After ethical approval, 148 consecutive, non-repetitive isolates of clinically significant CoNS species of hospitalized patients, isolated from blood samples and exudative specimens, were included in the study. Speciation was performed by conventional biochemical identification and automated methods. Antimicrobial susceptibility testing was performed by disc diffusion technique and for vancomycin by minimum inhibitory concentration (MIC) determination, as per Clinical Laboratory Standards Institute (CLSI) M-100 2023 guidelines. Methicillin resistance was detected using a cefoxitin disc. MIC for CHX was performed by agar dilution method; reduced susceptibility was considered when MIC to CHX ≥4 µg/mL. The simplex polymerase chain reaction (PCR) was carried out with suitable controls to detectqacA/Bandsmr. Statistical analysis was conducted to determine the association ofqacA/Bandsmrgenes with MIC of CHXin the study isolates. Results Fifteen different species of CoNS were obtained from clinical samples. A high percentage of resistance was observed against various classes of antibiotics. Methicillin resistance was observed in 69.6% (103/148) of isolates. Of 148 CoNS, 52.7% (78/148) of isolates exhibited reduced susceptibility to CHX with an MIC ≥4 µg/mL. These isolates exhibited a higher percentage of methicillin resistance (75.6%, 59/78). By PCR, 34.5% (51/148) of isolates carried either or both genes. GeneqacA/Bwas solely detected in 27.02% (40/148) of isolates, of which 14 were CHX-tolerant and the remaining 26 were CHX-susceptible. Genesmrwas solely detected in 4.1% (6/148) of isolates comprising three isolates each in CHX-tolerant and susceptible categories.There were 3.4% (5/148) of isolates that harbored bothgenes, of which only one isolate was CHX-susceptible, while the other four were CHX-tolerant. A proportion of isolates that were phenotypically tolerant to CHX did not carry either or both genes. A significant statistical association was found between reduced susceptibility to CHX and the presence of antiseptic resistance genesin the study isolates (p-value=0.033942). Conclusion To our knowledge, this is the first study from South India to investigate CHX resistance among CoNS using phenotypic and genotypic methods. The rise of antiseptic resistance among CoNS is an emerging threat to current infection control practices. The presence ofqacA/Bandsmrgenes, especially in CHX susceptible isolates, is concerning since these resistance genes are located on transferable plasmids, and the isolates can develop resistance eventually upon exposure to CHX.

Genomic analysis of multi-drug resistant coagulase-negative staphylococci from healthy humans and animals revealed unusual mechanisms of resistance and CRISPR-Cas system.

Coagulase-negative staphylococci (CoNS) are evolving as major reservoirs and vectors of unusual and critical antimicrobialresistance (AMR) mechanisms. In this study, the genomic characterization of 26 multidrug-resistant (MDR)-CoNS (S. borealis, S. saprophyticus, S. sciuri, S. hominis, S. epidermidis, S. pasteuri, S. hyicus, S. simulans, S. haemolyticus, and S. arlettae) previously obtained from the nasal cavity of healthy nestling storks, humans who had no contact with animals, pigs, and pig farmers, as well as dogs and dog owners from Spain was performed. High-quality draft genomes obtained by Illumina sequencing technology were used to determine their resistome, virulome, mobile genetic elements, and CRISPR-Cas types. The relatedness of three CoNS species with publicly available genomes was assessed by core-genome single nucleotidepolymorphisms (SNPs). AMR genes to all classes of antibiotics in staphylococci were detected including unusual ones (mecC, ermT, and cfr), of which their corresponding genetic organizations were analyzed. About 96.1% of the MDR-CoNS strains harbored diverse adherence or immune evasion genes. Remarkably, one enterotoxin-C and -L-carrying S. epidermidis-ST595 strain from a nestling stork was detected. Moreover, various plasmid bound-biocide resistance genes (qacACGJ) were identified in 34.6% of the MDR-CoNS. Two genes that encode for cadmium and zinc resistance (cadD, czrC) were found, of which czrC predominated (42.3%). Complete CRISPR-Cas system was detected in 19.2% of the CoNS strains, of which cas-1, -2, and -9 predominated, especially in 75% of the S. borealis strains. The phylogenetic analysis identified clusters of related S. epidermidis lineages with those of other countries (SNP < 100). Also, highly related S. borealis isolates (SNP < 10) from pigs was confirmed for the first time in Spain. These findings showed that various ecological niches harbor CoNS that presented MDR phenotypes mediated by multiple AMR genes carried by mobile genetic elements with relatively low frequency of intact CRISPR-Cas systems. Furthermore, the transmission of some CoNS species in humans and animals is strongly suggested.

Phenotypic Variation in Staphylococcus aureus during Colonisation Involves Antibiotic-Tolerant Cell Types.

Staphylococcus aureus is a bacterial species that is commonly found colonising healthy individuals but that presents a paradoxical nature: simultaneously, it can migrate within the body and cause a range of diseases. Many of these become chronic by resisting immune responses, antimicrobial treatment, and medical intervention. In part, this ability to persist can be attributed to the adoption of multiple cell types within a single cellular population. These dynamics in the S. aureus cell population could be the result of its interplay with host cells or other co-colonising bacteria-often coagulase-negative Staphylococcal (CoNS) species. Further understanding of the unique traits of S. aureus alternative cell types, the drivers for their selection or formation during disease, as well as their presence even during non-pathological colonisation could advance the development of diagnostic tools and drugs tailored to target specific cells that are eventually responsible for chronic infections.

Nasopharyngeal carriage, antimicrobial susceptibility patterns, and associated factors of Gram-positive bacteria among children attending the outpatient department at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia.

Gram-positive bacteria residing in the nasopharynx can lead to severe illnesses in children, such as otitis media, pneumonia, and meningitis. Despite the potential threat, there is a lack of comprehensive data regarding the carriage rates of these bacteria among children in outpatient departments in the study area. This study aimed to assess the nasopharyngeal carriage, antimicrobial resistance patterns, and associated factors of Gram-positive bacteria among children attending the outpatient department at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. A hospital-based cross-sectional study was conducted from May 1, 2023, to August 30, 2023. A total of 424 nasopharyngeal swab samples were collected using sterile nasopharyngeal swabs, inoculated on Blood Agar and Mannitol Salt Agar plates, and identified through colony morphology, Gram stain, and biochemical tests. Antimicrobial susceptibility of the identified bacterial isolates was determined employing both the Kirby-Bauer and modified Kirby-Bauer methods. D-tests were conducted using clindamycin and erythromycin discs to detect inducible clindamycin resistance, while cefoxitin disc tests were utilized to ascertain methicillin resistance. Data entry was executed using Epi-Data version 4.6, and subsequent analysis was performed utilizing SPSS version 25. Bivariable and multivariable logistic regression analyses were employed to identify associated factors. An adjusted odds ratio at a 95% confidence interval with a P-value of < 0.05 was considered statistically significant. The overall nasopharyngeal carriage rate of Gram-positive bacteria was 296/424 (69.8%, 95% CI: 65.3-74.0). Staphylococcus aureus was the most prevalent 122/424 (28.8%), followed by Streptococcus pneumoniae 92/424 (21.7%). Methicillin resistance was observed in 19/122 (15.6%) of S. aureus and 3/60 (5%) of coagulase-negative staphylococcus (CoNS) species. Inducible clindamycin resistance was 10/122 (8.2%) in S. aureus and 4/53 (7.5%) in coagulase-negative staphylococcus species. Multidrug resistance was found in 146/296 (49.3%, 95% CI: 43.6-55.0) of the isolates. Associated factors with a bacterial carriage were large family size (AOR = 3.061, 95% CI: 1.595-5.874, P = 0.001), having siblings under five years old (AOR = 1.991, 95% CI: 1.196-3.313, P = 0.008), indoor cooking (AOR = 2.195, 95% CI: 1.275-3.778, P = 0.005), an illiterate mother (AOR = 3.639, 95% CI: 1.691-7.829, P = 0.001), and hospital visits (AOR = 2.690, 95% CI: 1.405-5.151, P = 0.003). The study found a high nasopharyngeal carriage of Gram-positive bacteria in outpatient children, including notable levels of methicillin-resistant S. aureus and multi-drug-resistant isolates. Clindamycin, rifampin, and erythromycin were the most effective antimicrobials for the tested isolates. Factors contributing to bacterial carriage include visits to healthcare facilities, larger family sizes, having younger siblings, maternal illiteracy, and indoor cooking. This emphasizes the need for methicillin-resistant S. aureus surveillance in pediatric outpatient settings and community health education, especially for children's guardians. Additionally, improving household ventilation by separating kitchens from sleeping areas and regular screening of younger siblings in healthcare environments were recommended to reduce bacterial transmission within family members. The study also called for studies with advanced procedures like minimum inhibitory concentration testing and molecular characterization to better comprehend the resistance patterns and genes in circulating bacteria.

Isolation of Aerobic Bacterial Species Associated with Palpable Udder Defects in Non-Dairy Ewes.

The objectives of these studies were to identify associations between udder half defects (hard or lump) and bacteria isolated from milk or mammary tissue swabs, to compare with samples from normal udder halves at different physiological time points and to compare bacterial species isolated via milk and swabs of mammary tissue from within the same udder halves. A total of 1054 samples were aseptically collected from each udder half of 199 non-dairy breed (Romney) ewes from three different studies (Study A, n = 77; Study B, n = 74; and Study C, n = 48). Conventional bacterial culture and MALDI-ToF mass spectrometry were used for bacterial identification. Of the 225 samples from which bacteria were isolated, Mannheimia haemolytica and Streptococcus uberis were the dominantly identified species from defective udder halves, whereas coagulase-negative staphylococcus (CNS) species, mostly Staphylococcus simulans and Staphylococcus chromogenes, were more frequently isolated from normal udder halves. The ongoing presence of bacterial species over time was variable, although less frequently identified species showed less stability over time. A very high agreement (91.5%) of bacterial species identified was observed between the mammary tissue swab and udder half milk samples during post-weaning. In summary, palpable udder half defects were associated with bacterial positivity, and the ongoing presence of the bacteria over time was dependent on the species involved. Hence, culling ewes with palpable udder half defects that had more stable bacterial species could contribute to reducing the recurrence of palpable defects or mastitis.

Prevalence, genetic characteristics, and antimicrobial resistance of staphylococcal isolates from oral cavity and skin surface of healthy individuals in northern Japan

BackgroundOral cavity is an ecological niche for colonization of staphylococci, which are a major bacterial species causing community-acquired infections in humans. In this study, prevalence, and characteristics of staphylococci in oral cavity and skin of healthy individuals were investigated in northern Japan. MethodsSaliva from oral cavity and swab from skin surface of hand were collected and cultured on selective media. Species of the isolates were identified genetically, and ST was determined for S. aureus and S. argenteus. Genes associated with antimicrobial resistance were detected by PCR. ResultsAmong 166 participants, a total of 75 S. aureus isolates were obtained from 61 individuals (37 %), and recovered more frequently in oral cavity (n = 48) than skin (n = 27). Among 23 STs identified in S. aureus isolates, ST8 (CC8), ST15 (CC15), and ST188 (CC1) were the most common (10 isolates each), with STs of CC1 being dominant (17 isolates). Methicillin-resistant S. aureus (MRSA) was isolated in the skin of two individuals and belonged to ST1 and ST6. Resistance to erythromycin and gentamicin associated with erm(A) and aac(6’)-Ie-aph(2”)-Ia, respectively, was more commonly found in ST5 and ST8 isolates. One S. argenteus isolate (ST2250, mecA-negative) was recovered from oral cavity of a participant (0.6 %). A total of 186 isolates of coagulase-negative staphylococci (CoNS) were recovered from 102 participants and identified into 14 species, with S. warneri being the most common (n = 52), followed by S. capitis (n = 42), S. saprophyticus (n = 20) and S. haemolyticus (n = 19). mecA was detected in S. saprophyticus, S. haemolyticus, and S. caprae, while arginine-catabolic mobile element (ACME) in only S. capitis and S. epidermidis. ConclusionS. aureus was more prevalent in oral cavity than skin surface, belonging to three major STs, with CC1 being a dominant lineage. The prevalence of antimicrobial resistance was distinct depending on CoNS species.

Oral Commensals in Healthy Individuals: A Clinicocytological Study.

Background Each human being has a specific group of microorganisms that are necessary for both sustaining health and causing illness. Normally, these microorganisms maintain bio-communalism, do not harm the host, and lead to a state known as symbiosis or eubiosis. The commensal nature of these bacteria is always maintained in symbiosis and attains pathogenic potential when there is an imbalance between host immunity and microorganisms. Our study focuses on the identification and differentiation of the various commensals present in the oral cavity of healthy individuals over a given period of time. Aims and objectives This study aims to: (i) identify various commensal bacterial species present in the oral cavity; (ii) differentiate each commensal bacterial species present in the oral cavity of healthy individuals using cytological and culturing methods; (iii) identify the presence of different types of commensal bacterial species in the same individuals with the specific time intervals; (iv) compare and correlate the presence or absence of bacterial species present as a commensal in both male and female; (v) identify and characterize the commensal bacterial species present in the oral cavity of healthy individuals; (vi) investigate the consistency of commensal bacterial species presence over time and between genders. Methodology We included sixty healthy individuals between the ages of 20 and 24 from both genders, took buccal smears once every two days for ten days, stained them with Gram stain, and grew them in blood agar and Mac Conkey agar. Results The most common commensals include Gram-positive cocci, and among them, Coagulase-negative staphylococcus species (85%) are predominant, followed by Staphylococcus aureus (13.33%), and Streptococcus species (1.67%). The presence of colonies remains the same in all three samples obtained from the same healthy individuals. Conclusion Loss of balance between commensals and pathogens can lead to dysbiosis, which results in disease.

Evaluation of Inoculating Sterile Pericardial Fluid into Blood Culture Bottles

Background: There is limited data regarding the benefits of direct inoculation of sterile pericardial fluid into blood culture bottles. We discovered widespread adoption of this practice at our institution during pericardiocenteses and became concerned about over-capturing of skin flora contaminants. We aimed to understand how organisms detected in pericardial fluid inoculated into blood culture bottles were interpreted clinically. Methods: We investigated a cluster of four patients with coagulase-negative Staphylococcus (CoNS) isolated in pericardial fluid inoculated blood culture bottles (PF-BCxBs) over a 2-week period; three of these patients had recent cardiac surgery and were initially flagged as potential SSIs. We further expanded to a retrospective review and identified 28 patients with ≥1 organism isolated from PF-BCxBs from 7/2021 to 6/2023. Clinical, microbiological, and pharmacy data were abstracted. The primary outcome was the proportion of patients with a clinically diagnosed infection. Results: Investigation into the initial cluster revealed a pseudo-outbreak - three of four patients had no clinical evidence of infection (CoNS was deemed a contaminant); one was treated for a potential infection. All patients had concomitant negative routine fluid cultures. Discussions with the cardiology teams revealed areas for improvement in the process for inoculating fluid into blood culture bottles. From the two-year review, 18% (5/28) of patients were clinically diagnosed with an infection (two Staphylococcus aureus; two CoNS; one Candida rugosa). Of the patients without Staphylococcus aureus, all three had a concomitant negative routine fluid culture, were receiving antibiotics prior to pericardiocentesis, and had white blood cell counts (WBC) &gt;12 K/uL. The remaining 82% (23/28) of patients were deemed not to have an infection. Of these 23 patient without infection, organisms isolated were 16 CoNS (70%) and seven Cutibacterium species (30%). None of these patients had a fever, one (4%) was receiving pre-pericardiocentesis antibiotics, and three (9%) had WBC &gt;12 K/uL. 70% (16/23) of these patients were started on antibiotics after gram-stain results; all were eventually discontinued (mean antibiotic days = 2, range 1-5 days). 83% (19/23) of these patients had a concomitant negative routine fluid culture. Conclusion: The majority of patients with an organism isolated from PF-BCxBs had either CoNS or Cutibacterium species and were deemed not to have a clinical infection. Within the small cohort limitations, clinical utility of blood culture bottle inoculation seems highest for patients with pre-procedural concern for infection. IPC teams should be aware of the non-pathogenic skin flora frequency and potential implication on SSI surveillance.

Evaluating the Clinical Impact of Species-Level Identification in Coagulase-Negative Staphylococci Positive Blood Cultures

Background: Coagulase-negative staphylococci (CoNS) are often considered contaminants when isolated from blood cultures. While criteria exist to distinguish true bacteremia from contamination (Table 1), clinical judgement is often necessary. Clinical microbiology laboratories have traditionally identified CoNS to the species level only if present in multiple cultures. There have been concerns that blood cultures positive for rare or less familiar CoNS species might be misinterpreted as true bacteremia. Tufts Medical Center (TMC) clinical microbiology laboratory started reporting all CoNS to the species level in January 2023. We studied the impact of species-level identification of CoNS on clinically relevant outcomes following this change. Methods: The study evaluated inpatients at TMC aged ≥ 18 years with CoNS isolated from blood cultures between July 2022 and June 2023. The primary outcome was the difference in anti-staphylococcal antibiotic utilization between the pre- and post-intervention groups. Secondary outcomes included differences in true bacteremia diagnosis, length of hospital stay, and mortality between the two groups. We also compared the performance of Souvenir’s criteria with clinical judgement at distinguishing contamination from true bacteremia. A total of 100 patients were included in the pre- and post-intervention groups to detect an estimated effect size of 15% with a power of 81%. Results: Most patients were male, White, and English speaking (Table 2). No differences were found between the two groups in terms of infectious disease consultation frequency, blood culture collection department, or the presence of central venous catheters (Table 2). Staphylococcus epidermidis was the predominant CoNS in the post-intervention group. Blood cultures were repeated before and after starting antibiotics in 24% and 74% (pre-intervention) and 18% and 84% (post-intervention) of cases, respectively. Anti-staphylococcus antibiotic use was the same in both groups (82%). The median antibiotic therapy duration was 4.5 days pre- vs 3 days post-intervention (p =0.39). There were no differences in hospital length of stay or mortality between the two groups (Table 3). The clinical diagnosis of true bacteremia was established in 28% of cases in the pre- vs 25% in the post-intervention group (p= 0.63). Compared to clinical judgement, Souvenir’s true bacteremia criteria demonstrated a sensitivity of 80.3%, negative PV of 91.9%, and positive PV of 55.2%. Conclusions: Species-level identification of CoNS positive blood cultures did not impact antibiotic utilization, diagnosis of true bacteremia, length of hospital stay, or mortality. Further studies with larger cohorts and prospective designs are needed to validate these findings and assess the long-term implications in patients.

The Microbiology of Knee Prosthetic Joint Infection and its Influence on Persistent Infection.

Periprosthetic joint infection (PJI) following total knee arthroplasty (TKA) is a severe complication causing a significant burden. The study aims were to establish the epidemiology of microorganisms in TKA PJI, the rate of persistent infection requiring further surgery, and the risk factors for this. Microbiological specimens between June 2002 and March 2017 at five regional hospital sites were identified with revision TKA procedures in the National Joint Replacement Registry. The time between procedures, type of revision strategy, and any subsequent further revision operations were collected. At minimum 2-year follow-up, 174 revision TKA were identified, with a mean patient age of 69 ± 11 years. A broad range of pathogens were identified. Fifty cases (29%) had persistent infection requiring at least one further operative procedure, 13 cases required 3 or more. Coagulase-negative Staphylococcus species (CNS) was seen most with failed surgery, polymicrobial infections also posing a significant risk factor. The best chance of a successful PJI surgical strategy was < 12 months from primary TKA, with the greatest risk between 3 and 5 years (p < 0.05). Younger age significantly increased the risk of further surgery (p < 0.05). Management varied; 103 (59%) debridement, antibiotic therapy and implant retention, with further surgery in 29%; 45 (17%) single-stage revision, with further surgery in 13%; and 26 (15%) two-stage revision, with further surgery in 12%. This study presents the most common causative pathogens for PJI in TKA, and the high rate of persistent infection after initial revision surgery. Risk factors for persistent infection and further revision surgery were polymicrobial and CNS infections, patients who presented between 3 and 5 years following primary TKA, and younger age. This study therefore raises important risk factors and areas for future research to reduce the burden of multiple operations after PJI.

Association of coagulase-negative staphylococci with orthopedic infections detected by in-house multiplex real-time PCR.

Clinical significance of coagulase-negative staphylococci (CoNS) has been gradually acknowledged in both healthcare and clinical research, but approaches for their precise discrimination at the species level remain scarce. The current study aimed to evaluate the association of CoNS with orthopedic infections, where accurate and prompt identification of etiology is crucial for appropriate diagnosis and treatment decision-making. A 16S rRNA-based quantitative PCR (qPCR) assay was developed for the detection of Staphylococcus genus and two panels of 3-plex qPCR assays for further differentiation of six CoNS species with remarkable clinical significance, including S. epidermidis, S. haemolyticus, S. simulans, S. hominis, S. capitis, and S. caprae. All the assays exhibited excellent analytical performance. ΔCq (quantification cycle) between 16S rRNA and CoNS species-specific targets was established to determine the primary CoNS. These methods were applied to detect CoNS in wound samples from orthopedic patients with and without infection. Overall, CoNS were detected in 17.8% (21/118) of patients with clinically suspected infection and in 9.8% (12/123) of patients without any infection symptom (p < 0.05). Moreover, the association with infection was found to be bacterial quantity dependent. S. epidermidis was identified as the predominant species, followed by S. simulans, S. haemolyticus, and S. hominis. Male sex, open injury, trauma, and lower extremity were determined as risk factors for CoNS infections. CoNS-positive patients had significantly longer hospitalization duration (20 days (15, 33) versus 13 days (7, 22) for Staphylococcus-negative patients, p = 0.003), which could be a considerable burden for healthcare and individual patients. Considering the complex characteristics and devastating consequences of orthopedic infections, further expanding the detection scope for CoNS may be pursued to better understand the etiology of orthopedic infections and to improve therapeutic strategies.

Coagulase-negative staphylococci enhance the colour of fermented meat through a complex cross-talk between the arginase and nitric oxide synthase activities

The nitric oxide synthase (NOS) activity was investigated in 35 staphylococcal strains from five distinct coagulase-negative staphylococci (CNS) species. NOS pathway is related with the production of nitric oxide, potentially leading to the formation of nitrosomyoglobin imparting a pinkish-red colour to fermented meats. NOS activity was investigated employing both genotypic and phenotypic methods. Among the strains studied, 16 were found to contain the gene encoding NOS, while 25 strains possessed the arginase gene. Strains from Staphylococcus saprophyticus, S. equorum, and S. carnosus species exhibited phenotypic NOS-like activity. This was evidenced by the production of l-citrulline and a comparably lower release of l-ornithine in meat simulation media fermented under microaerobic conditions by those staphylococcal cultures. The authentic NOS activity was further examined using lab-scale meat models. The characteristic red colouration, typically associated with sodium nitrite-cured meat products, was observed. Strains L31, L49, and V10 (S. saprophyticus) demonstrated a pronounced effect on meat colouration, achieving shades comparable to nitrite-enhanced meat; however, a concurring up-accumulation of putrescine and spermine (likely originating from ornithine) was also outlined. These findings suggest the potential of meat-associated CNS to promote colour development in nitrite-free meats, offering a promising area for enhancing the visual appeal of such products.