mecA Research Articles

P-2166. Sensitivity and Specificity of Nanopore Sequencing for Detection of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus epidermidis in Positive Blood Culture Samples

Abstract Background Nanopore sequencing enables rapid DNA/RNA analysis without amplification. Implementing this for point-of-care diagnostics reduces time delays in traditional AST methods, potentially enhancing patient outcomes. We aim to evaluate the performance of Oxford Nanopore Technologies sequencing from positive blood culture for bacterial identification and antimicrobial susceptibility prediction in S. aureus and S. epidermidis by detecting the mecA gene. Methods We conducted a cross-sectional descriptive study on individuals with positive blood cultures identifying S. aureus and S. epidermidis over a 12-month period from April 2023 to March 2024. Bacterial DNA was extracted from the blood culture specimens, and then sequenced using the PromethION Flow Cell to identify the organisms and mecA genes, comparing the results to routine blood cultures and standard AST. Results 108 blood culture samples were initially collected. After applying the exclusion criteria, 80 samples qualified for DNA extraction and sequencing. 45 samples were identified as S. aureus (6 MRSA and 39 MSSA) and 35 as S. epidermidis (27 MRSE and 8 MSSE). Notably, 67.5% of samples contained less than 0.5 ng/μl of DNA. Sensitivity and specificity of mecA gene detection were 35.5% (95% CI: 18.6%–52.3%) and 83.7% (95% CI: 73.3%–94.0%), respectively, with an accuracy of 65.0% (95% CI: 54.5%–75.5%). Upon excluding samples with DNA less than 0.5 ng/μl accuracy improved to 73.1% (95% CI: 56.0%–90.1%). The specimen that showed a false-positive result will proceed to phenotypic confirmation using methicillin-infused mannitol-salt agar. After phenotypic confirmation, the accuracy improved to of 88.5% (95% CI: 76.2%–100.0%). In this study, we also found 5 samples in which the mecA gene was shown to be genotypically oxacillin-sensitive after phenotypic confirmation. The median time for detecting the mecA gene was 198 minutes (IQR: 37.5–458.7) after the onset of sequencing. Conclusion Our study on nanopore sequencing for MRSA and MRSE detection from blood cultures found promising but limited sensitivity and specificity in detecting mecA gene. Challenges in DNA extraction from Gram-positive bacteria affected overall accuracy. Further improvements are needed for enhanced diagnostic reliability. Disclosures All Authors: No reported disclosures

P-2097. Genomic basis of Oxacillin- and Cefoxitin-Susceptible PBP2a-Positive Staphylococcus aureus

Abstract Background Methicillin-resistance Staphylococcus aureus (MRSA) is resistant to all ß-lactam antibiotics except for the fifth-generation cephalosporin drug, ceftaroline. The mecA gene, which encodes an alternative penicillin-binding protein, PBP2a, is responsible for the high-level resistance to methicillin. However, mecA positive, oxacillin- and cefoxitin susceptible MRSA has been reported, with limited understanding of the genomic basis behind this contradictory phenomenon. Here, we studied the molecular base of a “stealth” MRSA from clinical specimen. Methods A MRSA isolate was routinely isolated from a deep wound specimen, which was positive for PBP2a by lateral flow assay but negative for cefoxitin screen test and susceptible to oxacillin (MIC of 0.5 ug/ml) by VITEK 2 AST-GP75 card. It was also susceptible to cefoxitin using disk diffusion method (diameter of 26 mm). The “stealth” MRSA isolate was sequenced on illumina NovaSeq 6000 platform using 150 bp paired end sequencing. Bacteria genome was assembled and annotated using pipelines on BV-BRC (https://www.bv-brc.org/). Results Genomic sequencing revealed a point mutation (G to A) in the mecA gene resulting in a Gly599Ser substitution located in the transpeptidase domain of PBP2a, which is close to the active site of Ser403 on the 3D structure of the protein. Additionally, an insertion in the methicillin resistance regulatory sensor-transducer mecR1 gene was identified, resulting in a frameshift mutation, and predicted to cause premature termination. However, the sequence of frame shift mutation in mecR1 has been described and was not associated with reversed cefoxitin susceptibility. Therefore, the point mutation in mecA gene is most likely responsible for loss of function of PBP2a, and lead to susceptible cefoxitin and oxacillin results despite a positive PBP2a reaction. Conclusion Whole genome sequencing is valuable to understand the genomic basis of unusual phenotypic testing results. The reversion of “stealth” MRSA strain to a high-level oxacillin- and cefoxitin-resistant strain has been reported, which highlights the importance of considering tests for mecA or PBP2a in routine laboratory practice to avoid the risk of missing such strains. Disclosures All Authors: No reported disclosures

P-2112. Development and Validation of a Cheap, Portable Assay to Rapidly Profile Bacterial Infections of the Bloodstream

Abstract Background Antimicrobial resistant (AMR) bacterial infections pose a significant and growing health threat worldwide, and their burden is most severe in lower income areas where diagnostic infrastructure is lacking. The development of cost-effective and accessible diagnostic tools for the rapid identification of resistant infections in low-resource settings is crucial. Specific High-sensitivity Enzymatic Reporter Unlocking, or SHERLOCK, is a promising CRISPR-based system that can aid in the diagnosis of infections and support clinical decision making. Here, we present an assay termed BADLOCK (Bacteria and AMR Detection by SHERLOCK) that uses this technology to rapidly identify bacterial species and AMR genes on blood cultures. BADLOCK workflow demonstrating simplicity of approach and rapid turnaround. Methods We combine isothermal amplification of targets with CRISPR/Cas13-based detection into a single step, allowing for a streamlined workflow and a total time-to-identification of less than two hours from positive cultures using lateral flow strips (Fig. 1). The assay primarily targets topA for species identification, a highly conserved gene within species with sufficient variability between them to enable species-level typing. A panel of clinically important AMR genes, including the ESBL blaCTX-M-15, three major carbapenemases, and the GPC resistance genes mecA and vanA, is also included. Pilot study assessing assay performance. A) each column represents a different isolate while each row represents a different CRISPR gene target, which are enumerated at the bottom. Each species name refers to the topA gene sequence unique to that species. The top box shows the expected gene content based on prior sequencing, while the bottom box shows the results of each assay. The blue shading shows relative fluorescence intensity of the generated signal. B) Basic test characteristics for each target gene. Results In addition to the AMR resistance genes, we have validated a panel of 9 gram-negative rods, 6 staphylococci, 2 enterococci, and 2 streptococcal species with further targets being developed, and have performed a laboratory pilot study on a subset of these (Fig. 2). We have additionally collected over 600 sequential positive blood cultures and are in the process of performing a large clinical validation study on patient samples, thereby elucidating the precise test characteristics of our assay. Conclusion We have developed an assay capable of rapidly detecting bacterial species causing bloodstream infections and their major epidemic AMR genes on low-cost paper strips. Our work has the potential to improve the diagnostic landscape for resistant infections in low-and-middle-income countries and serve as an adjunctive diagnostic to traditional approaches in highly resourced setting, which would represent a major step forward in infectious disease diagnostics. Disclosures All Authors: No reported disclosures

From Farm to Community: Dispersal of Potentially Pathogenic Staphylococcus and Mammaliicoccus Species and Antimicrobial Resistance Across Shared Environments.

Staphylococcaceae are a diverse bacterial family with important implications for human and animal health. This study highlights the One Health relevance of their environmental dispersal, particularly, by identifying closely related or genetically identical strains circulating between farm and community environments. Environmental Staphylococcaceae strains were isolated from animal farms and interconnected areas within a university setting, both influenced by anthropogenic activities. Genetic diversity analysis via GTG5-PCR revealed that specific strains circulated across these environments, likely facilitated by human interaction, supporting the potential for cross-environmental bacterial transfer. The strains were further tested for antimicrobial resistance against a panel of antibiotics, biofilm formation, and the presence of resistance-associated genes. We identified a wide array of Staphylococcaceae species (n = 16), with over half being pathogenic to humans and animals, including S. aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus. Notably, 55.5% of the strains exhibited multidrug resistance, and 15.9% harbored the methicillin resistance gene mecA, underscoring the challenges these infections might pose to treatment. Biofilm-forming capabilities further enhance the environmental persistence of these strains. These findings underscore the critical need for surveillance and targeted intervention strategies in a One Health framework to mitigate antimicrobial resistance across shared environments.

Antimicrobial activity of adipose-derived mesenchymal stromal cell secretome against methicillin-resistant Staphylococcus aureus

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is still a growing concern in the field of antimicrobial resistance due to its resistance to conventional antibiotics and its association with high mortality rates. Mesenchymal stromal cells (MSCs) have been shown as a promising and attractive alternative treatment for bacterial infections, due to their antibacterial properties and potential to bypass traditional resistance mechanisms. This study aims to shed light on the antibacterial potential of adipose-derived mesenchymal stromal cell (AD-MSC) secretome against clinical isolates of Staphylococcus spp., including MRSA strains.MethodsUsing the Kirby-Bauer disk diffusion method, broth microdilution assays, and colony-forming unit (CFU) counting, the antibacterial activity of AD-MSC secretome was assessed. These tests were first conducted on Staphylococcus (S.) aureus ATCC 25923, then on 73 clinical isolates including MRSA strains. Further molecular analysis was performed to identify resistant genes in MRSA isolates.ResultsThe AD-MSC secretome demonstrated significant antibacterial activity against S. aureus ATCC with a 32 mm inhibition zone. 96% of the collected staphylococcal clinical isolates showed susceptibility to the secretome with 87.5% inhibition observed in MRSA isolates, along with 100% in MSSA, MSSE, and MRSE strains. Molecular analysis revealed that MRSA strains resistant to the secretome harbored mecA, ermA, and ermB genes. Additionally, the mecA-negative MRSA strains remained susceptible to the secretome, suggesting alternative resistance mechanisms.ConclusionThese findings emphasize the ability of AD-MSCs secretome as a promising alternative for treating antibiotic-resistant infections, with potential applications in combating MRSA. However, further research is required to explore its clinical applications as a complementary or standalone therapy for resistant infections.

Genetic resistance factors and antimicrobial resistance phenotypes in methicillin- resistant Staphylococcus aureus isolates of animals and humans

Objective: To investigate the prevalence, genetic resistance factors, and antimicrobial resistance phenotypes of methicillin-resistant Staphylococcus (S.) aureus (MRSA) from clinically healthy animals (horses, dogs, and cats) and their human handlers, providing a baseline for broader genes sequencing based on One Health studies to the emergence of resistance as well as guides regarding specific therapies, hospital antibiotic usage/stewardship, and target-specific infection control measures. Methods: This cross-sectional study involved both human and animals. S. aureus isolates derived were characterised for their antimicrobial phenotypes through antimicrobial susceptibility testing with PCR detection of mecA, mecC, and DNA sequencing of mepR, mepA, mepB, and sapep genes for correlation with their antimicrobial resistance phenotypes. Results: Seventy S. aureus isolates from 149 human handlers, 240 horses, and 206 companion animals, including dogs and cats were studied. The prevalence of resistance was highest for penicillins (100.0%) and amoxicillin (94.3%), followed by erythromycin (87.7%), trimethoprim (78.6%), azithromycin (77.1%), imipenem (61.4%), and tetracycline (40.0%). Lower resistance prevalences were observed for ciprofloxacin (27.1%), chloramphenicol (20.0%), trimethoprim-sulfamethoxazole (12.9%), and gentamicin (2.9%). Twenty-six isolates had their DNA sequenced for mepR, mepA, mepB, and sapep genes for correlation with their antimicrobial phenotypes. Transcriptional profiling revealed that both animal and human MRSA isolates exhibited a gene cluster mepRAB (multidrug export protein gene), encoding a MarR-likc transcriptional regulator (mepR), a M20/ M25/M40 metallo-hydrolase protein gene (sapep) encoding resistance to biocides and carbapenems, and a hypothetical protein gene of unknown function (mepB). Conclusions: This study demonstrated extensive multidrug resistance in MRSA, revealing similarities in the resistance patterns and multiple antibiotic resistance indices among the isolates. These findings suggest the potential presence of non-mec resistance mechanisms in MRSA, in addition to the mec gene mechanism.

Antimicrobial Resistance, Virulence Gene Profiling, and Spa Typing of Staphylococcus aureus Isolated from Retail Chicken Meat in Alabama, USA

Antibiotic-resistant Staphylococcus aureus (S. aureus) in retail meat poses a public health threat requiring continuous surveillance. This study investigated the frequency of isolation, toxin genes, and antibiotic resistance profile of S. aureus recovered from retail poultry meat samples and presented results beneficial to public health interventions. Of 200 samples collected, 16% (32/200) tested positive for S. aureus, and these were recovered from thigh 37.5% (12/32), wing 34.4% (11/32), gizzard (15.6% (5/32), and liver 12.5% (4/32) samples. Findings of spa typing analysis revealed that 68.8% (22/32), 18.8% (6/32), 9.4% (3/32), and 3.0% (1/32) of the isolates belonged to the spa types t267, t160, t548, and t008, respectively. For antibiotic susceptibility testing, 12.5% (4/32) of the isolates were resistant to only penicillin, but one isolate (1/32; 3%) showed resistance to the antibiotics penicillin, erythromycin, ampicillin, and oxacillin. PCR analysis revealed that 9.4% (3/32) of the isolates carried the mecA gene associated with methicillin-resistant Staphylococcus aureus (MRSA) isolates. One MRSA isolate was identified as a t008 spa type, and harbored a 26,974 bp-sized plasmid, which was the source of its resistance to penicillin, ampicillin, erythromycin, and oxacillin. The staphylococcal enterotoxin (SE) genes seg, sei, sek, seb, selm, and seln were also identified among the isolates, and mostly the antimicrobial and enterotoxin genes were carried on plasmids of the isolates. This study raises awareness on the continuous circulation of pathogenic microbes like S. aureus in retail poultry meat.

Tuning Ion Current Rectifying Nanopipettes for Sensitive Detection of Methicillin-Resistant Staphylococcus aureus.

Infectious diseases pose a growing challenge in healthcare, with the increasing rate of antimicrobial resistance limiting therapeutic options available for treatment. Rapid detection of infections at the earliest opportunity can significantly improve patient outcomes. In this report, ion current rectifying quartz nanopipettes with ca. 109 nm orifices were utilized for the label-free detection of DNA indicative of methicillin-resistant Staphylococcus aureus (MRSA). By immobilizing probe DNA complementary to the mecA gene on the internal walls of the nanopipette, the detection was achieved by monitoring changes in ion current rectification (ICR) following probe-target hybridization. We demonstrate enhanced sensitivity by controlling the surface probe density, resulting in a tunable, sensitive sensor technology with a detection limit as low as 0.35 pM. Finite element simulations are used to support our experimental findings, revealing that to maximize target-induced changes to ICR, the probe surface density must be minimized. This sensitive and label-free methodology was integrated with the polymerase chain amplification reaction to achieve selective identification of this pathogen from laboratory-grown cultures, highlighting that ion current rectifying nanopipette sensors offer the potential to be a cost-effective and rapid tools for infectious disease detection.

Whole-genome analysis of a ST45-SCCmec IVa (2B)-t116 methicillin-resistant Staphylococcus aureus strain isolated from the sputum of a 5-year-old child with pneumonia.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 is a major global MRSA lineage with huge strain diversity and a high clinical impact. In Hainan and Guangzhou of China, the ST45-MRSA was mainly associated with t116. The MRSA strain SA2107 was isolated from the sputum of a 5-year-old child with pneumonia. The whole genome of SA2107 was sequence using Illumina (Novaseq 6000) and PacBio (Sequel IIe) sequencers, and the sequences were assembled using hybrid assembly. The carriage of antibiotic resistance genes, virulence genes, and mobile genetic elements were identified using bioinformatics tools. The comparative genomic analyses of MRSA strain SA2107 with other MRSA strains worldwide were performed. The genome size of ST45-SCCmec IVa (2B)-t116 MRSA strain SA2107 was ~2.9 Mb. Mobile genetic elements analysis of SA2107 revealed two plasmids (30,064-bp pSA2107-1 and 8,033-bp pSA2107-2), three prophages, two integrative and conjugative elements (ICEs), and two insertion sequences (ISs, IS431 and IS1272). The SCCmec IVa (2B) carried by SA2107 contained the class B mec gene complex (IS431-mecA-ΔmecR1-IS1272) and type 2 ccr gene complex (ccrA2 and ccrB2). Besides mecA, another beta-lactam resistance gene blaZ was found to located on six copies of bla complex (blaZ, blaR1, and blaI) on the chromosome of SA2107. Three kinds of virulence factors were detected on the chromosome of SA2107, including genes encoding toxins, exoenzyme, and immune-modulating protein. Notably, the three prophages harbored by the chromosome of SA2107 all carried virulence genes. Thus far, only three complete genomes available for ST45-SCCmec IVa (2B)-t116 strain from United States, Germany, and Australia, respectively. The strain SA2107 was the first complete genome data (CP104559) from China for ST45-SCCmec IVa (2B)-t116 MRSA.

ANALYSIS OF HOSPITAL-ACQUIRED MRSA INFECTIONS IN PUS SAMPLES: A COMPREHENSIVE DISCUSSION

Background: Hospital-acquired infections (HAIs) represent a significant healthcare challenge globally, with Staphylococcus aureus (S. aureus) being a leading cause of skin and soft tissue infections. Methicillin-resistant Staphylococcus aureus (MRSA) poses an even greater threat due to its resistance to commonly used antibiotics. Understanding the prevalence and resistance patterns of MRSA is crucial to developing effective infection control strategies, especially in resource-limited settings like Peshawar, Pakistan. Objective: To determine the frequency of hospital-acquired infections caused by S. aureus in skin abscesses and evaluate the antibiotic resistance patterns of MRSA in patients from District Peshawar. Methods: A total of 525 pus samples were collected from patients with skin abscesses during hospital stays in Peshawar. Samples were processed in a microbiology laboratory using standard protocols, including Gram staining, colony morphology assessment, and biochemical tests to identify S. aureus. Methicillin resistance was confirmed using cefoxitin and oxacillin disc diffusion tests, and the presence of the mecA gene was validated through PCR. Antibiotic susceptibility testing was performed for 14 commonly prescribed antibiotics following Clinical and Laboratory Standards Institute (CLSI) guidelines. Statistical analysis was conducted using SPSS version 26. Results: Out of 525 pus samples, 100 isolates were confirmed as MRSA. The prevalence of MRSA was higher in males (54%) than females (46%). The highest infection rate was observed in the 21–40 years age group (35%), followed by 1–20 years (27%), 41–60 years (25%), and above 60 years (11%). Resistance was highest to cefoxitin (100%), oxacillin (94%), ciprofloxacin (81%), and amoxicillin (79%). However, isolates showed high susceptibility to vancomycin (93%), linezolid (95%), and teicoplanin (90%). Conclusion: The study highlights the alarming prevalence of MRSA in hospital-acquired skin abscesses in Peshawar, coupled with significant antibiotic resistance. Strengthened infection control measures, antibiotic stewardship programs, and ongoing surveillance are essential to curb the spread of resistant pathogens.

Isolation and Molecular Detection of Antimicrobial Resistance Genes in Escherichia coli and Staphylococcus Species from Joint Ill Cases of Calves in Puducherry

Background: Joint ill is a septicemic polyarthritis condition due to the localization of pathogenic bacteria within the joints of young calves. The emergence of antibiotic resistant pathogen in such cases may lead to further complication in treatment. Thus, the present study was aimed to isolate and identify the bacterial pathogens and its antimicrobial resistant genes from joint ill cases in calves. Methods: A total of 31 pus swabs were collected from joint ill cases from calves and subjected for bacterial isolation and identification by phenotypic and genotypic method followed by determination of its antimicrobial resistant genes (tet and mecA gene) and antibiogram. Result: Based on colony characters, microscopic observation, biochemical tests, 17/31 (54.83%) Escherichia coli and 14/31 (45.16%) Staphylococcus aureus were isolated and identified phenotypically. All the isolates were further confirmed by polymerase chain reaction using E. coli and S. aureus species specific primers. The antimicrobial resistant genes carrying E. coli and S. aureus isolates were also detected, in which 4/17 (23.5%) isolates were positive for tet gene and 4/14 (28.57%) were positive for the mecA gene. The antibiotic susceptibility test showed that the isolates were highly sensitive to Enrofloxacin (100%) and Gentamicin (77%), but were resistant to Amoxyclav (70%) and tetracycline (50%). On conclusion, E. coli and S. aureus are the most common bacterial pathogens identified from this study. The presence of antimicrobial resistant genes (tet and mecA) and antibiogram pattern of the bacterial isolates indicating the possible treatment failure and serious public health risks in future.

Loop-mediated isothermal amplification assay coupled with lateral flow dipstick for the rapid detection of methicillin-resistant Staphylococcus pseudintermedius from dogs.

Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.

Molecular Epidemiological Characteristics of Staphylococcus pseudintermedius, Staphylococcus coagulans, and Coagulase-Negative Staphylococci Cultured from Clinical Canine Skin and Ear Samples in Queensland.

Background/Objectives: Infections in dogs caused by methicillin-resistant staphylococci (MRS) present limited treatment options. This study's objective was to investigate the molecular epidemiology of Staphylococcus spp. cultured exclusively from clinical canine skin and ear samples in Queensland, Australia, using whole-genome sequencing (WGS). Methods: Forty-two Staphylococcus spp. isolated from clinical canine skin and ear samples, from an unknown number of dogs, were sourced from two veterinary diagnostic laboratories between January 2022 and May 2023. These isolates underwent matrix-assisted laser desorption ionisation- time of flight bacterial identification, minimum inhibitory concentration testing using SensititreTM plates and WGS. Phylogenetic trees and core genome multilocus sequence typing (cgMLST) minimum spanning trees (MSTs) were constructed. Results: The isolates included methicillin-resistant and -sensitive S. pseudintermedius (MRSP: 57.1%, 24/42; and MSSP: 19.1%, 8/42), methicillin-resistant and -sensitive S. coagulans (MRSC: 14.3%, 6/42; and MSSC: 2.4%, 1/42) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS: 7.1%, 3/42). Thirty-nine isolates were included after WGS, where all MRS harboured the mecA gene. Eighteen sequence types (STs) were identified, including three novel MRSP and six novel MSSP STs. MRSP ST496-V-VII (23%; 9/39) and MRSP ST749-IV-(IVg) (12.8%; 5/39) were commonly isolated. Phylogenetic analysis of single nucleotide polymorphisms showed that MRSP, MRSC and MSSC were similar to globally isolated staphylococci from canine skin and ear infections. Using cgMLST MSTs, MRSP isolates were not closely related to global strains. Conclusions: Our findings revealed a genotypically diverse geographical distribution and phylogenetic relatedness of staphylococci cultured from clinical canine skin and ear samples across Queensland. This highlights the importance of ongoing surveillance to aid in evidence-based treatment decisions and antimicrobial stewardship.

In Silico Exploration of Staphylococcal Cassette Chromosome mec (SCCmec) Evolution Based on Phylogenetic Relationship of ccrAB/C.

As the mobile cassette carrier of the methicillin resistance gene mecA that is transported across staphylococci species, the evolution and origin of Staphylococcal Cassette Chromosome mec (SCCmec)-and in particular, the composition of mecA and SCCmec-have been extensively discussed in the scientific literature; however, information regarding its dissemination across geographical limits and evolution over decades remains limited. In addition, whole-genome sequencing-based macro-analysis was unable to provide sufficiently detailed evolutionary information on SCCmec. Herein, the cassette chromosome recombinase genes ccrAB/C, as essential components of SCCmec, were employed to explore the evolution of SCCmec. This work established the basic taxonomy of 33 staphylococci species. The CUB of mecA, ccrAB/C of 12 SCCmec types and core genome of 33 staphylococci species were subsequently compared; the phylogenetic relationship of ccrAB/C was observed via SCCmec typing on a temporal and geographical scale; and the duplicate appearance of ccrAB/C was illustrated by comparing SCCmec compositions. The results highlighted a deviation in the CUB of mecA and ccrAB/C, which evidenced their exogenous characteristics to staphylococci, and provided theological support for the phylogenetic analysis of ccrAB/C as representative of SCCmec. Importantly, the phylogenetic relationship of ccrAB/C did not exhibit centralization over time; instead, similarly to mecA, ccrAB/C with similar identities had close clades across decades and geographical limits and different SCCmec types, which enabled us to discriminate SCCmec based on the sequence identity of ccrAB/C. In addition, the duplicate appearance of ccrAB/C and fixed composition of the ccrAB/C complex among different strains were indicative of more complicated transmission mechanisms than targeting direct repeats of SCCmec.

Emerging Resistance Mechanisms in Gram-Positive Bacteria Isolated From Septicemia Cases in ICUs: A Focus on Genotypic Insights.

Background Bloodstream infections (BSIs) are associated with high morbidity and mortality, especially in intensive care unit (ICU) settings. The most common Gram-positive pathogens, methicillin-resistantStaphylococcus aureus (MRSA) and coagulase-negative Staphylococci (CoNS), are likely to cause BSIs. The phenotypic and genotypic characteristics of multidrug-resistant (MDR)Staphylococcal species isolated from patients admitted to the ICUwith septicemia were evaluated for better treatment outcomes. Materials and methods A cross-sectional study was conducted at Sree Balaji Medical College and Hospital over two years (July 2022 to June 2024). Blood samplesin blood culture bottles received in the laboratory from ICU patients with suspected sepsis were included in this study. The BacT/ALERT® 3D system (bioMérieux, France) was used to assess the bacterial growth. TheVITEK®2 system (bioMérieux, France) and conventional methods were used to identify Gram-positive isolates. Antibiotic susceptibility was determined by the Kirby-Bauer disc diffusion method, and polymerase chain reaction (PCR) was used to detect genes like mecA, icaA, and icaD genes. Results Out of 274 blood samples, eight (2.9%) were contaminants, and 121 (44.2%) were culture-positive as true pathogens. Eighty-seven (71.9%) Gram-positive isolates were identified from the positive blood cultures, of whichCoNS was predominant (51, 58.6%), followed by Staphylococcus aureus(25, 28.7%). Methicillin resistance was observed in 10 (13.1%) Staphylococcusaureus and 14 (18.4%) CoNS isolates. PCR detected the mecA gene in 20 (83.3%) methicillin-resistant isolates and biofilm-related genes icaD in 64 (84.2%) and icaA in 58 (76.3%). Vancomycin and teicoplanin showed high effectiveness. Conclusions This study has emphasized the importance of molecular screening for the mecA, icaA, and icaD genes in framing antibiotic regimens. The findings emphasize the efficacy of vancomycin, teicoplanin, and linezolid in combating MDR Staphylococcus infections in ICUs of hospitals and demonstrate the importance of antibiotic stewardship.

Molecular investigation of Staphylococcus aureus isolated from inanimate surfaces in Jordanian hospitals.

Staphylococcus aureus is a ubiquitous nosocomial bacterium, which confers hospital-associated infections ranging from moderate to life-threatening disorders. The pathogenicity of the microorganism is attributed to various camouflage mechanisms harbored in its genome. Methicillin-resistant Staphylococcus aureus strains have become significant pathogens in nosocomial and community settings. In the current study, we aimed to determine the prevalence of S. aureus, and more specifically, MRSA at different departments in four major hospitals in Jordan. A total of 500 inanimate surfaces located in the intensive care unit ICU, kidney department, surgery department, internal department, sterilization ‎department, burn department, and operation department were swabbed.‎ All isolates were identified by using routine bacterial culture, Gram staining, and a panel of biochemical tests such as; catalase, coagulase, DNase, urease, oxidase, and hemolysin production were performed. In terms of PCR, three main genes were screened, the 16S rRNA gene targeting Staphylococcus spp as a housekeeping gene, the coA gene was used as a specific gene to detect S. aureus, and the mecA gene used to identify MRSA isolates. Results revealed the prevalence of Staphylococcus spp was 212 (42.4%), ‎S. aureus ‎prevalence by coA gene 198 (39.6%), and MRSA by mecA gene in 81 samples ‎‎ (16.2%)‎. There was a strong positive connection (P < 0.01) found between department site and bacterial contamination.‎ It was concluded that inanimate hospital environments contain a relatively high number of S. aureus and MRSA. Proper sterilization techniques, infection prevention, and control management strategies should be implemented.

Molecular Characterization of Cefoxitin‐Resistant Coagulase‐Negative Staphylococci From Frequently Touched Surfaces of Hospital and Urban‐Built Environments of Central India

Coagulase‐negative staphylococci (CoNS) are the major pathogen (hospital as well as environmental) and their emerging multidrug‐resistant (MDR) strains complicate the treatment process. In this study, we investigated the prevalence and antibiotic resistance of CoNS on frequently touched surfaces in hospital and urban built environments (UBEs) in Vidarbha, Maharashtra, India. A total of 200 isolates screened for Staphylococcus species and 55 methicillin‐resistant staphylococci isolates were identified, and among them, 19 were classified as cefoxitin‐resistant CoNS. These 19 cefoxitin‐resistant CoNS isolates were tested for the presence of the mecA gene by conventional PCR and only nine (47.36%) were found to be mecA-positive. mecA-positive strains were tested to check MIC for various antibiotics and three marker gene characteristics, namely, ß‐lactamase, cefoxitin screen, and inducible clindamycin resistance via the VITEK 2 system. These strains were 100% resistant to benzylpenicillin and oxacillin, and approximately 50% were resistant to vancomycin. Amplified mecA gene fragments were sequenced, and SNP analysis was performed alongside a standard sequence from Staphylococcus aureus (Acc no. NG_047938.1). In total, among the 466 nucleotides, 386 sequences were found to be invariable, and 80 polymorphic variables were identified (46 singleton variable sites and 34 parsimony information sites). The spread of antibiotic resistance is very common in both UBEs and hospital environments; thus, our study concluded that a surveillance program is recommended for the Vidarbha region for the assessment of co‐occurring CoNS and better infection control of the environment for future reduction in contact infection.

Assessment of biofilm formation, antibiotic resistance patterns, and the prevalence of adhesion-related genes in clinical Staphylococcus aureus isolates.

This study aimed to evaluate the biofilm formation abilities of clinical Staphylococcus aureus strains, assess their antibiotic susceptibility patterns, and identify the prevalence of adhesion-associated genes. In this study, a total of 60S. aureus strains were collected from urine, pus, wounds, blood, body fluid, and sputum in health centers affiliated with Abadan University of Medical Sciences, Iran. Strains were identified via microbiological methods and polymerase chain reaction (PCR) to target the nuc gene. Antibiotic susceptibility testing (AST) was conducted via the disc diffusion method. Methicillin-resistant S. aureus (MRSA) strains were identified by cefoxitin disc diffusion and PCR targeting the mecA gene. Biofilm formation was assessed via a microtiter plate assay, and the prevalence of adhesion-encoding genes was evaluated via PCR. The data were analyzed in Excel and SPSS via statistical methods, with P-values <0.05 considered significant. Using AST, daptomycin and linezolid were the most effective antibiotics (100% susceptibility rate). According to the results of the cefoxitin disc test, 48.3% (n=29/60) of the strains were MRSA. All the MRSA strains harbored the mecA gene. In total, 32% of the strains were biofilm producers. Moreover, 56.2%, 28.1%, and 15.6% of the strains produced weak, moderate, and strong biofilms, respectively. There were no significant differences between the MRSA and MSSA strains in terms of the association of biofilm formation with antibiotic resistance except for erythromycin (P-value=0.0087), gentamicin (P-value=0.0009), and penicillin (P-value=0.0009). The most prevalent biofilm-encoding genes were icaA (76.7%), followed by icaD (70%), clfA (65.0%), and fnbA (53.3%). This study identified MRSA strains with biofilm-forming abilities that possess adhesion-associated genes. The most prevalent biofilm-encoding gene was icaA. To prevent further spread of these strains, regional preventive measures are needed.

Methicillin-resistant Staphylococcus aureus isolated from mobile phones of nursing students in Cuenca, Ecuador

Staphylococcus aureus is an important pathogen in healthcare facilities, with its resistance to a number of antibiotics currently being a global concern. In this report the presence of S.aureus, resistance gene virulence and antibiotic susceptibility profiles were determined in the mobile phones of senior nursing students. S.aureus was isolated in 11.84% (9/76) of the samples. Furthermore, 44.44% of the mobile phones carried the mecA (MRSA) gene, while none carried the vanA gene. Virulence genes identified were 100% hla, 88.89% hlb, 22.22% tst and sec, and 11.11% sea. The antibiogram revealed that 33.33% of the strains were resistant to cefoxitin and 44.44% showed inducible resistance to clindamycin (ICRSA). The mobile phones of senior nursing students represent an important reservoir of drug-resistant and virulent strains of S.aureus, which could act as infectious foci for the transmission of this pathogen.

Antimicrobials profiling, biofilm formation, and clonal lineage of methicillin-resistant Staphylococcus aureus isolated from cockroaches.

Cockroaches are widely recognized as vectors for transmitting pathogenic microorganisms in hospital and community environments due to their movement between contaminated and human-occupied spaces. Staphylococcus aureus (S. aureus), particularly methicillin-resistant Staphylococcus aureus (MRSA), is a primary global health concern because of its capacity to cause a wide range of infections and its resistance to many antibiotics. Despite efforts to control nosocomial infections, the role of cockroaches in disseminating antibiotic-resistant bacteria has not been fully explored. This study aims to investigate the antibiotic resistance patterns, biofilm formation, and genetic characteristics of S. aureus isolated from cockroaches in hospital environments. Understanding the role of cockroaches as vectors of drug-resistant S. aureus can contribute to developing more effective infection control strategies in healthcare settings. This study examined 386 cockroaches, including 230 American and 156 German cockroaches. Antibiotic sensitivity, inducible resistance, and biofilm formation were evaluated. The presence of mecA, ermA, ermB, ermC, msrA, icaA, icaB, icaC, icaD, SCCmec, mupA, mupB, and iles-1 genes was determined. Randomly amplified polymorphic DNA typing was performed to determine genetic relatedness. Fifty S. aureus isolates were identified, with 48% confirmed as MRSA. No isolate exhibited constitutive resistance to clindamycin. However, 96% of the isolates displayed inducible clindamycin resistance (iMLSB phenotype) when tested using the D-test. The prevalence of icaA, icaB, icaC, and icaD genes were 34%, 8%, 0%, and 0%, respectively. So, 29.1%, 16.6%, 12.5%, and 8.3% of isolates had SCCmec gene cassettes of types I, II, III, and IV, respectively. The prevalence of ermA, ermB, ermC, and msrA genes was found to be 18%, 16%, 58%, and 4%, respectively. Seven different clusters were found in the RAPD-PCR, with cluster A (5 isolates) being the most common. These results show that cockroaches are important in transmitting resistance factors as mechanical vectors. Therefore, taking sanitary measures to control the insect population is unavoidable.