Detection of multidrug-resistant bacteria in the nasal cavities and evaluation of sinus disorders in patients undergoing Le Fort I osteotomy.

INTRODUCTION: Orthognathic surgery can lead to sinus alterations, including sinusitis, attributed to the exposure of maxillary sinuses during Le Fort I osteotomy. Furthermore, being a hospital-based procedure, there is potential risk of complications arising from bacteria prevalent in such environments. This study evaluated maxillary sinusitis occurrence and the presence of multidrug-resistant bacteria in the nasal cavity before and after orthognathic surgery. METHODS: Ten patients with dentofacial deformities underwent Le Fort I osteotomy. Clinical evaluations using SNOT-22 questionnaire were performed, and nasal cavity samples were collected pre-surgery and 3-6 months post-surgery to quantify total mesophilic bacteria and detect Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae. Cone Beam Computed Tomography (CBCT) was performed pre- and post-operatively, and the results were evaluated using the Lund-Mackay system. This study was registered and approved by the Research Ethics Committee of PUCRS (No. 4.683.066). RESULTS: The evaluation of SNOT-22 revealed that five patients showed an improvement in symptoms, while two remained in the same range of interpretation. One patient developed post-operative maxillary sinusitis, which was not detected at the time of evaluation by SNOT-22 or CBCT. CBCT showed a worsening sinus condition in three patients, two of whom had a significant increase in total bacteria count in their nasal cavities. The Brodsky scale was used to assess hypertrophy in palatine tonsils, where 60% of the subjects had grade 1 tonsils, 20% had grade 2 and 20% had grade 3. None of the patients had grade 4 tonsils, which would indicate more than 75% obstruction. Two patients harboured S. aureus and K. pneumoniae in their nasal cavities. Notably, K. pneumoniae, which was multidrug-resistant, was present in the nasal cavity of patients even before surgery, but this did not result in maxillary sinusitis, likely due to the patients' young and healthy condition. CONCLUSION: There was an improvement in signs and symptoms of maxillary sinusitis and quality of life in most patients after orthognathic surgery. However, some patients may still harbour multidrug-resistant bacteria, even if they are asymptomatic. Therefore, a thorough pre-operative assessment is essential to avoid difficult-to-treat post-operative complications.

Detection and modeling of Staphylococcus aureus and fecal bacteria in Hawaiian coastal waters and sands.

Microbial pollution of recreational waters leads to millions of skin, respiratory, and gastrointestinal illnesses globally. Fecal indicator bacteria (FIB) are monitored to assess recreational waters but may not reflect the presence of Staphylococcus aureus, a global leader in bacterial fatalities. Since many community-acquired S. aureus skin infections are associated with high recreational water usage, this study measured and modeled S. aureus, methicillin-resistant S. aureus (MRSA), and FIB (Enterococcus spp., Clostridium perfringens) concentrations in seawater and sand at six beaches in Hilo, Hawai'i, USA, over 37 sample dates from July 2016 to February 2019 using culturing techniques. Generalized linear models predicted bacterial concentrations with physicochemical and environmental data. Beach visitors were also surveyed on their preferred activities. S. aureus and FIB concentrations were roughly 6-78 times higher at beaches with freshwater discharge than at those without. Seawater concentrations of Enterococcus spp. were positively associated with MRSA but not S. aureus. Elevated S. aureus was associated with lower tidal heights, higher freshwater discharge, onsite sewage disposal system density, and turbidity. Regular monitoring of beaches with freshwater input, utilizing real-time water quality measurements with robust modeling techniques, and raising awareness among recreational water users may mitigate exposure to S. aureus, MRSA, and FIB. PRACTITIONER POINTS: Staphylococcus aureus and fecal bacteria concentrations were higher in seawater and sand at beaches with freshwater discharge. In seawater, Enterococcus spp. positively correlated with MRSA, but not S. aureus. Freshwater discharge, OSDS density, water turbidity, and tides significantly predicted bacterial concentrations in seawater and sand. Predictive bacterial models based upon physicochemical and environmental data developed in this study are readily available for user-friendly application.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology.

Objective: Recombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. Methods: Recombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. Results: The M-RAP method had sensitivity rates of 1, 10, and 1 copies/µL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively ( P < 0.05). Conclusion: An M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Clinical characteristics and risk factors for right-sided infective endocarditis in Korea: a 12-year retrospective cohort study.

Right-sided infective endocarditis (RSIE) is less common than left-sided infective endocarditis (LSIE) and exhibits distinct epidemiological, clinical, and microbiological characteristics. Previous studies have focused primarily on RSIE in patients with intravenous drug use. We investigated the characteristics and risk factors for RSIE in an area where intravenous drug use is uncommon. A retrospective cohort study was conducted at a tertiary hospital in South Korea. Patients diagnosed with infective endocarditis between November 2005 and August 2017 were categorized into LSIE and RSIE groups. Of the 406 patients, 365 (89.9%) had LSIE and 41 (10.1%) had RSIE. The mortality rates were 31.7% in the RSIE group and 31.5% in the LSIE group (P = 0.860). Patients with RSIE had a higher prevalence of infection with Staphylococcus aureus (29.3% vs. 13.7%, P = 0.016), coagulase-negative staphylococci (17.1% vs. 6.0%, P = 0.022), and gram-negative bacilli other than HACEK (12.2% vs. 2.2%, P = 0.003). Younger age (adjusted odds ratio [aOR] 0.97, 95% confidence interval [CI] 0.95-0.99, P = 0.006), implanted cardiac devices (aOR 37.75, 95% CI 11.63-141.64, P ≤ 0.001), and central venous catheterization  (aOR 4.25, 95%  CI 1.14-15.55, P = 0.029) were independent risk factors for RSIE. Treatment strategies that consider the epidemiologic and microbiologic characteristics of RSIE are warranted.

Molecular characterization of Staphylococcus aureus isolated from hospital-acquired infections in Ilam, Iran.

OBJECTIVE: This research study was undertaken to investigate antimicrobial resistance patterns and the prevalence of hospital-acquired infections (HAIs). The study focuses on common microorganisms responsible for HAIs and explores emerging challenges posed by antimicrobial drug-resistant isolates. METHODS: A comprehensive analysis of 123 patients with HAIs, hospitalized in surgical department and intensive care unit (ICU) at Imam Khomeini Hospital, Ilam, Iran, was conducted over a six-month period. Pathogenic bacterial isolates, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA), were isolated and subjected to antibiotic susceptibility testing. RESULTS: The study findings revealed a significant prevalence of multidrug-resistant (MDR) isolates, of which 73.3% were MRSA. Notably, 6.7% of S. aureus isolates exhibited resistance to vancomycin, indicating the emergence of VRSA. Respiratory infections were identified as the most prevalent HAI, constituting 34.67% of cases, often arising from extended ICU stays and invasive surgical procedures. Furthermore, patients aged 60 and above, particularly those associated with MDR, exhibited higher vulnerability to HAI. CONCLUSIONS: This research sheds light on the intricate interplay between drug resistance and HAI, highlighting the imperative role of rational antibiotic use and infection control in addressing this critical healthcare challenge.

Evaluation of the feasibility for replacing sheep blood with human blood in culture media used in microbiological diagnostics.

The present study aimed to suggest the replacement of animal blood with human blood in culture media, involving alternative methods and ethical considerations, such as animal welfare, in addition to potential laboratory cost reduction. Characteristics of growth and hemolysis development were compared in different culture media, using both sheep blood and human blood. Blood types from the ABO blood group system were tested, and commercially acquired sheep blood agar was used for comparison. Bacteria of the genus Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli were tested. It was observed that growth in media with type A and O positive blood showed closer similarities to those performed in agar with sheep blood. Depending on the bacterial species, the results were either more positive or not, with faster-growing and less demanding bacteria showing better results than, for example, S. pneumoniae, which demonstrated difficulty in the growth process and hemolysis generation in human blood agar. The research suggests that in some situations, sheep blood could be replaced, especially when the goal is growth and isolation, but may not be as suitable when the objective is to analyze hemolysis or when the studied species is demanding.

Staphylococcus aureus bacteremia in alcoholics.

BACKGROUND: Alcoholism associates with increased Staphylococcus aureus bacteremia incidence and mortality. The objective was to compare disease progression, treatment and prognosis of Staphylococcus aureus bacteremia in alcoholics versus non-alcoholics. METHODS: The study design was a multicenter retrospective analysis of methicillin-sensitive Staphylococcus aureus bacteremia with 90-day follow-up. Patients were stratified as alcoholics or non-alcoholics based on electronic health record data. Altogether 617 Staphylococcus aureus bacteremia patients were included of which 83 (13%) were alcoholics. RESULTS: Alcoholics, versus non-alcoholics, were younger, typically male and more commonly had community-acquired Staphylococcus aureus bacteremia. No differences in McCabe´s classification of underlying conditions was observed. Higher illness severity at blood culture sampling, including severe sepsis (25% vs. 7%) and intensive care unit admission (39% vs. 17%), was seen in alcoholics versus non-alcoholics. Clinical management, including infectious disease specialist (IDS) consultations and radiology, were provided equally. Alcoholics, versus non-alcoholics, had more pneumonia (49% vs. 35%) and fewer cases of endocarditis (7% vs. 16%). Mortality in alcoholics versus non-alcoholics was significantly higher at 14, 28 and 90 days (14% vs. 7%, 24% vs. 11% and 31% vs. 17%), respectively. Considering all prognostic parameters, male sex (OR 0.19, p = 0.021) and formal IDS consultation (OR 0.19, p = 0.029) were independent predictors of reduced mortality, whereas ultimately or rapidly fatal comorbidity in McCabe´s classification (OR 12.34, p < 0.001) was an independent predictor of mortality in alcoholics. CONCLUSIONS: Alcoholism deteriorates Staphylococcus aureus bacteremia prognosis, and our results suggests that this is predominantly through illness severity at bacteremia onset. Three quarters of Staphylococcus aureus bacteremia patients we studied had identified deep infection foci, and of them alcoholics had significantly less endocarditis but nearly half of them had pneumonia.

Nasal carriage of virulent and multidrug resistant Staphylococcus aureus: a possible comorbidity of COVID-19.

BACKGROUND: Staphylococcus aureus (S. aureus) associated with COVID-19 has not been well documented. This cross-sectional study evaluated the association between nasal S. aureus carriage and COVID-19. METHODS AND RESULTS: Nasopharyngeal samples were collected from 391 participants presenting for COVID-19 test in Lagos, Nigeria, and S. aureus was isolated from the samples. Antimicrobial susceptibility test was done by disc diffusion method. All S. aureus isolates were screened for the presence of mecA, panton-valentine leucocidin (PVL) and toxic shock syndrome toxin (TSST) virulence genes by polymerase chain reaction. Staphylococcal protein A (spa) typing was conducted for all the isolates. Participants with COVID-19 had double the prevalence of S. aureus (42.86%) compared to those who tested negative (20.54%). A significant association was seen between S. aureus nasal carriage and COVID-19 (p = 0.004). Antimicrobial sensitivity results showed resistance to oxacillin (100%), cefoxitin (53%), and vancomycin (98.7%). However, only 41% of the isolates harbored the mecA gene, with SCCmecV being the most common SCCmec type. There was no association between the carriage of virulence genes and COVID-19. A total of 23 Spa types were detected, with t13249 and t095 being the two most common spa types. CONCLUSION: This study examined the association between nasal S. aureus carriage and SARS-COV-2 infection. Further research is required to fully explore the implications of S. aureus co-infection with COVID-19.

Antibiotic Resistance Profile of Staphylococcus aureus in Cancer Patients at Laquintinie Hospital in Douala, Littoral Region, Cameroon.

Cancer and chemotherapy predispose the patients to various bacterial infections. This study is aimed at isolating and establishing the distribution of antibiotic-resistant Staphylococcus aureus from fecal samples in subjects with cancer admitted to the Oncology Department at Laquintinie Hospital in Douala, in the Littoral Region of Cameroon. A cross-sectional study was conducted from October 2021 to March 2023. Cancer and noncancer patients were suffering from Staphylococcus aureus infection. The isolation of Staphylococcus aureus was based on culture on the specific medium. The Kirby-Bauer disk diffusion method was used for drug susceptibility testing. Of the 507 patients studied, 307 (60.55%) were cancer patients, compared to 200 (39.45%) noncancer patients. S. aureus was isolated in 81 (15.97%) participants, among which 62 (76.55%) were cancer patients and 19 (23.45%) were noncancer patients. In the study population, 31.92% of participants had breast cancer, followed by cervical cancer (13.68%) and leukemia (7.17%). Staphylococcus aureus isolates showed high resistance rates in cancer patients compared to noncancer patients to amoxicillin-clavulanic acid (AMC, 77.42% versus 31.58%), cefoxitin (FOX, 80.65% versus 63.16%), ciprofloxacin (CIP, 75.81% versus 26.32%), ofloxacin (OFX, 69.35% versus 31.58%), fusidic acid (FUS, 70.97% versus 53.63%), and tetracycline (TET, 85.48% versus 78.95%). Staphylococcus aureus showed a significant increase in multidrug-resistant (MDR) and methicillin-resistant (MRSA) phenotypes in cancer patients compared to noncancer patients (p < 0.05). The prevalence of MRSA was 76.54%, higher than that of methicillin-sensitive Staphylococcus aureus (MSSA) (23.46%). The frequency of MRSA was significantly higher (p < 0.001) in cancer patients (80.65%) than in noncancer patients (19.35%). This study showed that there is an association between antibiotic resistance and cancer status. Research and interventions must be focused on the cancer population to combat the appearance of MDR bacteria due to the loss of effectiveness of antibiotics.

Assessment of handwashing impact on detection of SARS-CoV-2, Staphylococcus aureus, Escherichia coli on hands in rural and urban settings of Côte d'Ivoire during COVID-19 pandemic.

BACKGROUND: Handwashing is the first line of hygiene measures and one of the oldest methods of preventing the spread of infectious diseases. Despite its efficacy in the health system, handwashing is often inadequately practiced by populations. This study aimed to assess the presence of SARS-CoV-2, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) on hands as indicators of lack of hand hygiene during COVID 19 pandemic. METHODS: A cross-sectional study was conducted in rural Taabo and urban Abidjan (Côte d'Ivoire) from January to September 2021. A total of 384 participants from 384 households were included in the study. The total households were distributed proportionally within various municipalities in the two study areas according to the number of households in each municipality, based on data of the National Institute of Statistics from the 2014 general population census. Hand swabbing of the 384 participants within households (320 in Abidjan and 64 in Taabo) was performed for the enumeration of E. coli and S aureus, using laboratory standard method and for the detection of SARS-CoV-2 by RT-qPCR. A binary logistic regression model was built with the outcome variable presence of Staphylococcus spp. on hands of respondents that was categorized into binary variables, Staphylococcus spp. (1 = presence, 0 = absence) for the Risk Ratio estimation. Place of living, sex, handwashing, education and age group were used to adjust the model to observe the effects of these explanatory variables. RESULTS: No presence of SARS-CoV-2 virus was detected on the hands of respondents in both sites. However, in urban Abidjan, only Staphylococcus spp. (Coagulase Negative Staphylococci) was found on the hands of 233 (72.8%, 95%CI: 67.7-77.4) respondents with the average load of 0.56 CFU/ Cm2 (95% CI, 0.52-0.60). Meanwhile, in rural Taabo, Staphylococcus spp. (Coagulase Negative Staphylococci) and E. coli were found on the hands of 40 (62.5%, 95%CI: 50.3-73.3) and 7 (10.9%, 95%CI: 5.4-20.9) respondents with the respective average load of 0.49 CFU/ Cm2 (95% CI, 0.39-0.59) and 0.08 CFU/ Cm2 (95% CI, 0.03-0.18). Participants living in rural Taabo were less likely to have Staphylococcus spp. on their hands (RR = 0.811; 95%IC: 0.661-0.995) compared to those living in urban Abidjan. CONCLUSIONS: No SARS-CoV-2 was detected on the hands of participants in both sites, suggesting that our study did not show direct transmission through hands. No E. coli was found in urban Abidjan while E. coli was found on the hands of participants in rural Taabo indicating poor hand washing and disinfection practices in rural Taabo. Living in urban Abidjan is statistically associated to having Staphylococcus spp. on hands. Further studies are necessary especially to understand to what extent the presence of Staphylococcus spp. on hands indicates a higher infection or fecal colonization rates in the case of E. coli.

Diagnostic value of BHI-V4 for heterogeneous and vancomycin-intermediate Staphylococcus aureus isolates: a systematic review and meta-analysis.

BACKGROUND: Brain-heart infusion agar supplemented with 4 µg/mL of vancomycin (BHI-V4) was commonly used for the detection of heterogeneous (hVISA) and vancomycin-intermediate Staphylococcus aureus (VISA). However, its diagnostic value remains unclear. This study aims to compare the diagnostic accuracy of BHI-V4 with population analysis profiling with area under the curve (PAP-AUC) in hVISA/VISA. METHODS: The protocol of this study was registered in INPLASY (INPLASY2023120069). The PubMed and Cochrane Library databases were searched from inception to October 2023. Review Manager 5.4 was used for data visualization in the quality assessment, and STATA17.0 (MP) was used for statistical analysis. RESULTS: In total, eight publications including 2153 strains were incorporated into the meta-analysis. Significant heterogeneity was evident although a threshold effect was not detected across the eight studies. The summary receiver operating characteristic (SROC) was 0.77 (95% confidence interval [CI], 0.74-0.81). The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic score and diagnostic odds ratio were 0.59 (95% CI: 0.46-0.71), 0.96 (95%CI: 0.83-0.99), 14.0 (95% CI, 3.4-57.1), 0.43 (95%CI, 0.32-0.57), 3.48(95%CI, 2.12-4.85) and 32.62 (95%CI, 8.31-128.36), respectively. CONCLUSION: Our study showed that BHI-V4 had moderate diagnostic accuracy for diagnosing hVISA/VISA. However, more high-quality studies are needed to assess the clinical utility of BHI-V4.

Periorbital cellulitis as the initial presentation of acute lymphoblastic leukaemia.

A girl in early childhood with no significant medical history developed left eye periorbital oedema and erythema. She was treated with intravenous antibiotics for suspected severe periorbital cellulitis. Despite treatment, the patient's cellulitis progressed into necrotising fasciitis, and she was transferred for ophthalmology review and imaging. A CT scan and eye swab culture-confirmed Staphylococcus aureus periorbital cellulitis. Incidentally, pathology revealed significant pancytopenia suspicious of leukaemia. The patient underwent bone marrow biopsy and was diagnosed with B-cell acute lymphoblastic leukaemia (ALL). A multidisciplinary specialist assessment revealed no ocular evidence of leukaemia and no intraocular concerns. In medical literature, it is consistently found that cases of ALL initially manifesting as proptosis or eyelid oedema are invariably due to neoplastic infiltration. This case represents unique documentation where periorbital cellulitis is the initial presentation of B-cell ALL, underscoring the necessity to consider periorbital cellulitis as a possible differential diagnosis in ophthalmic manifestations of ALL.

Toxigenic Staphylococcus aureus in some animal-originated food products marketed in Turkey: presence and Public Health concerns.

This study aimed to detect the presence of Staphylococcus aureus in some animal source food (ASF) including emulsified meat products (sausage and salami), dry fermented meat product (soudjouk), semi dry meat product (pastrami) and raw chicken meat. Sixty six  (38.8%) of 170 samples were found to be positive for S. aureus. It was determined that S. aureus was found in 17 (56.6%) salami, 27 (54%) raw chicken meat, 9 (30%) soudjouk, 9 (30%) pastrami, 4 (13.3%) sausage samples. Staphylococcal enterotoxins (SEs) were identified in 5 out of 66 (7.5 %) isolates food matrices including 3 (4.5%) SEA, 2 (3.03%) SEC. The sea and sec genes were detected in 3 (4.5%) of 66 isolates. The results of this study highlight the need to provide suitable control strategies concerning production, sales, and storage to prevent the spread of enterotoxigenic S. aureus isolates in ASF. The key contribution of this study is its revelation of the presence of S. aureus in animal products sold in Turkish local markets, highlighting the potential public health risks associated with animal foods.

Alarming multidrug resistance in Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis: Antibiotic resistance patterns and occurrence of selected resistance genes.

Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.

Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) for the diagnosis of bacterial periprosthetic joint infection.

BACKGROUND: Periprosthetic joint infection (PJI) is one of the most serious and debilitating complications that can occur after total joint arthroplasty. Therefore, early diagnosis and appropriate treatment are important for a good prognosis. Recently, molecular diagnostic methods have been widely used to detect the causative microorganisms of PJI sensitively and rapidly. The Multiplex Loop-Mediated Isothermal Amplification (LAMP) method eliminates the complex temperature cycling and delays caused by temperature transitions seen in polymerase chain reaction (PCR) methods, making it faster and easier to perform compared to PCR-based assays. Therefore, this study developed a multiplex LAMP assay for diagnosing bacterial PJI using LAMP technology and evaluated its analytical and clinical performance. METHODS: We developed a multiplex LAMP assay for the detection of five bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa, and Escherichia coli, frequently observed to be the causative agents of PJI. The method of analytical sensitivity and cross-reactivity were determined by spiking standard strains into the joint synovial fluid. The analytical sensitivity of the multiplex LAMP assay was compared with that of a quantitative real-time PCR (qPCR) assay. Clinical performance was evaluated using 20 joint synovial fluid samples collected from patients suspected of having bacterial PJI. RESULTS: The analytical sensitivity of the gram-positive bacterial multiplex LAMP assay and qPCR were 105/104 CFU/mL, 103/103 CFU/mL, and 105/104 CFU/mL against S. agalactiae, S. epidermidis, and S. aureus, respectively. For P. aeruginosa and E. coli, the analytical sensitivity of the multiplex LAMP and qPCR assays were 105/104 and 106/104 CFU/mL, respectively. The multiplex LAMP assay detects target bacteria without cross-reacting with other bacteria, and exhibited 100% sensitivity and specificity in clinical performance evaluation. CONCLUSIONS: This multiplex LAMP assay can rapidly detect five high-prevalence bacterial species causing bacterial PJI, with excellent sensitivity and specificity, in less than 1 h, and it may be useful for the early diagnosis of PJI.

Emergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of Staphylococcus aureus, Belgium, 2013 to 2023.

BackgroundAntimicrobial resistance to mupirocin and fusidic acid, which are used for treatment of skin infections caused by Staphylococcus aureus, is of concern.AimTo investigate resistance to fusidic acid and mupirocin in meticillin-susceptible S. aureus (MSSA) from community-acquired skin and soft tissue infections (SSTIs) in Belgium.MethodsWe collected 2013-2023 data on fusidic acid and mupirocin resistance in SSTI-associated MSSA from two large Belgian laboratories. Resistant MSSA isolates sent to the Belgian Staphylococci Reference Centre were spa-typed and analysed for the presence of the eta and etb virulence genes and the mupA resistance gene. In addition, we whole genome sequenced MSSA isolates collected between October 2021 and September 2023.ResultsMupirocin resistance increased between 2013 and 2023 from 0.5-1.5% to 1.7-5.6%. Between 2018 and 2023, 91.4% (64/70) of mupirocin-resistant isolates were co-resistant to fusidic acid. By September 2023, between 8.9% (15/168) and 10.1% (11/109) of children isolates from the two laboratories were co-resistant. Of the 33 sequenced isolates, 29 were sequence type 121, clonal and more distantly related to the European epidemic fusidic acid-resistant impetigo clone (EEFIC) observed in Belgium in 2020. These isolates carried the mupA and fusB genes conferring resistance to mupirocin and fusidic acid, respectively, and the eta and etb virulence genes.ConclusionWe highlight the spread of a mupirocin-resistant EEFIC in children, with a seasonal trend for the third quarter of the year. This is of concern because this variant is resistant to the two main topical antibiotics used to treat impetigo in Belgium.

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

SaLTy: a novel Staphylococcus aureus Lineage Typer.

Staphylococcus aureus asymptomatically colonises 30 % of humans but can also cause a range of diseases, which can be fatal. In 2017 S. aureus was associated with 20 000 deaths in the USA alone. Dividing S. aureus isolates into smaller sub-groups can reveal the emergence of distinct sub-populations with varying potential to cause infections. Despite multiple molecular typing methods categorising such sub-groups, they do not take full advantage of S. aureus genome sequences when describing the fundamental population structure of the species. In this study, we developed Staphylococcus aureus Lineage Typing (SaLTy), which rapidly divides the species into 61 phylogenetically congruent lineages. Alleles of three core genes were identified that uniquely define the 61 lineages and were used for SaLTy typing. SaLTy was validated on 5000 genomes and 99.12 % (4956/5000) of isolates were assigned the correct lineage. We compared SaLTy lineages to previously calculated clonal complexes (CCs) from BIGSdb (n=21 173). SALTy improves on CCs by grouping isolates congruently with phylogenetic structure. SaLTy lineages were further used to describe the carriage of Staphylococcal chromosomal cassette containing mecA (SCCmec) which is carried by methicillin-resistant S. aureus (MRSA). Most lineages had isolates lacking SCCmec and the four largest lineages varied in SCCmec over time. Classifying isolates into SaLTy lineages, which were further SCCmec typed, allowed SaLTy to describe high-level MRSA epidemiology. We provide SaLTy as a simple typing method that defines phylogenetic lineages (https://github.com/LanLab/SaLTy). SaLTy is highly accurate and can quickly analyse large amounts of S. aureus genome data. SaLTy will aid the characterisation of S. aureus populations and ongoing surveillance of sub-groups that threaten human health.

Exploring the virulence potential of Staphylococcus aureus CC121 and CC152 lineages related to paediatric community-acquired bacteraemia in Manhiça, Mozambique.

Staphylococcus aureus is a frequent agent of bacteraemia. This bacterium has a variety of virulence traits that allow the establishment and maintenance of infection. This study explored the virulence profile of S. aureus strains causing paediatric bacteraemia (SAB) in Manhiça district, Mozambique. We analysed 336 S. aureus strains isolated from blood cultures of children younger than 5 years admitted to the Manhiça District Hospital between 2001 and 2019, previously characterized for antibiotic susceptibility and clonality. The strains virulence potential was evaluated by PCR detection of the Panton-Valentine leucocidin (PVL) encoding genes, lukS-PV/lukF-PV, assessment of the capacity for biofilm formation and pathogenicity assays in Galleria mellonella. The overall carriage of PVL-encoding genes was over 40%, although reaching ~ 70 to 100% in the last years (2014 to 2019), potentially linked to the emergence of CC152 lineage. Strong biofilm production was a frequent trait of CC152 strains. Representative CC152 and CC121 strains showed higher virulence potential in the G. mellonella model when compared to reference strains, with variations within and between CCs. Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL+ and other lineages, as they display important virulence traits that may negatively impact the management of SAB paediatric patients in Manhiça district, Mozambique.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.