Small regulatory RNA RSaX28 promotes virulence by reinforcing the stability of RNAIII in community-associated ST398 clonotype Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a notorious pathogen that cause metastatic or complicated infections. Hypervirulent ST398 clonotype strains, remarkably increased in recent years, dominated Community-associated S. aureus (CA-SA) infections in the past decade in China. Small RNAs like RNAIII have been demonstrated to play important roles in regulating the virulence of S. aureus, however, the regulatory roles played by many of these sRNAs in the ST398 clonotype strains are still unclear. Through transcriptome screening and combined with knockout phenotype analysis, we have identified a highly transcribed sRNA, RSaX28, in the ST398 clonotype strains. Sequence analysis revealed that RSaX28 is highly conserved in the most epidemic clonotypes of S. aureus, but its high transcription level is particularly prominent in the ST398 clonotype strains. Characterization of RSaX28 through RACE and Northern blot revealed its length to be 533nt. RSaX28 is capable of promoting the hemolytic ability, reducing biofilm formation capacity, and enhancing virulence of S. aureus in the in vivo murine infection model. Through IntaRNA prediction and EMSA validation, we found that RSaX28 can specifically interact with RNAIII, promoting its stability and positively regulating the translation of downstream alpha-toxin while inhibiting the translation of Sbi, thereby regulating the virulence and biofilm formation capacity of the ST398 clonotype strains. RSaX28 is an important virulence regulatory factor in the ST398 clonotype S. aureus and represents a potential important target for future treatment and immune intervention against S. aureus infections.

Occurrence, antimicrobial susceptibility, and resistance genes of Staphylococcus aureus in milk and milk products in the Arsi highlands of Ethiopia.

BACKGROUND: In Ethiopia, milk production and handling practices often lack proper hygiene measures, leading to the potential contamination of milk and milk products with Staphylococcus aureus (S. aureus), including methicillin-resistant strains, posing significant public health concerns. This study aimed to investigate the occurrence, antimicrobial susceptibility profiles and presence of resistance genes in S. aureus strains isolated from milk and milk products. METHODS: A cross-sectional study was conducted in the Arsi highlands, Oromia, Ethiopia from March 2022 to February 2023. A total of 503 milk and milk product samples were collected, comprising 259 raw milk, 219 cottage cheese, and 25 traditional yogurt samples. S. aureus isolation and coagulase-positive staphylococci enumeration were performed using Baird-Parker agar supplemented with tellurite and egg yolk. S. aureus was further characterized based on colony morphology, Gram stain, mannitol fermentation, catalase test, and coagulase test. Phenotypic antimicrobial resistance was assessed using the Kirby-Bauer disc diffusion method, while the polymerase chain reaction (PCR) was employed for confirming the presence of S. aureus and detecting antimicrobial resistance genes. RESULTS: S. aureus was detected in 24.9% of the milk and milk products, with the highest occurrence in raw milk (40.9%), followed by yogurt (20%), and cottage cheese (6.4%). The geometric mean for coagulase-positive staphylococci counts in raw milk, yogurt, and cottage cheese was 4.6, 3.8, and 3.2 log10 CFU/mL, respectively. Antimicrobial resistance analysis revealed high levels of resistance to ampicillin (89.7%) and penicillin G (87.2%), with 71.8% of the isolates demonstrating multidrug resistance. Of the 16 S. aureus isolates analyzed using PCR, all were found to carry the nuc gene, with the mecA and blaZ genes detected in 50% of these isolates each. CONCLUSION: This study revealed the widespread distribution of S. aureus in milk and milk products in the Arsi highlands of Ethiopia. The isolates displayed high resistance to ampicillin and penicillin, with a concerning level of multidrug resistance. The detection of the mecA and blaZ genes in selected isolates is of particular concern, highlighting a potential public health hazard and posing a challenge to effective antimicrobial treatment. These findings highlight the urgent need to enhance hygiene standards in milk and milk product handling and promote the rational use of antimicrobial drugs. Provision of adequate training for all individuals involved in the dairy sector can help minimize contamination. These measures are crucial in addressing the threats posed by S. aureus, including methicillin-resistant strains, and ensuring the safety of milk and its products for consumers.

Evaluating the impact of genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) on hospital infection prevention and control decisions.

Genomic epidemiology enhances the ability to detect and refute methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in healthcare settings, but its routine introduction requires further evidence of benefits for patients and resource utilization. We performed a 12 month prospective study at Cambridge University Hospitals NHS Foundation Trust in the UK to capture its impact on hospital infection prevention and control (IPC) decisions. MRSA-positive samples were identified via the hospital microbiology laboratory between November 2018 and November 2019. We included samples from in-patients, clinic out-patients, people reviewed in the Emergency Department and healthcare workers screened by Occupational Health. We sequenced the first MRSA isolate from 823 consecutive individuals, defined their pairwise genetic relatedness, and sought epidemiological links in the hospital and community. Genomic analysis of 823 MRSA isolates identified 72 genetic clusters of two or more isolates containing 339/823 (41 %) of the cases. Epidemiological links were identified between two or more cases for 190 (23 %) individuals in 34/72 clusters. Weekly genomic epidemiology updates were shared with the IPC team, culminating in 49 face-to-face meetings and 21 written communications. Seventeen clusters were identified that were consistent with hospital MRSA transmission, discussion of which led to additional IPC actions in 14 of these. Two outbreaks were also identified where transmission had occurred in the community prior to hospital presentation; these were escalated to relevant IPC teams. We identified 38 instances where two or more in-patients shared a ward location on overlapping dates but carried unrelated MRSA isolates (pseudo-outbreaks); research data led to de-escalation of investigations in six of these. Our findings provide further support for the routine use of genomic epidemiology to enhance and target IPC resources.

Relapsing bronchopneumonia due to community-associated methicillin-resistant Staphylococcus aureus: a case report.

BACKGROUND: The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has increased the incidence of community-onset MRSA infection. Respiratory tract infections caused by MRSA has been noted for their severity; however, repeated relapses that require extended antibiotic therapy are rare. CASE PRESENTATION: We report a case of relapsing bronchopneumonia caused by CA-MRSA in a 56-year-old man. The patient responded to antibiotics, but repeatedly relapsed after stopping treatment. MRSA was consistently isolated from airway specimens during each relapse. Extended oral antibiotic treatment with trimethoprim/sulfamethoxazole (TMP/SMX) for 6 months achieved infection control. Whole-genome sequencing of the isolated strain revealed that the causative agent was sequence type (ST)1/staphylococcal cassette chromosome mec (SCCmec) type IVa, a clone that is rapidly increasing in Japan. DISCUSSION AND CONCLUSIONS: This patient had an unusual course of MRSA bronchopneumonia with repeated relapses. Although the choice of antibiotics for long-term use in MRSA respiratory tract infections has not been well established, TMP/SMX was effective and well tolerated for long-term therapy in this case. The clinical course of infections related to the rapid emerging clone, ST1/SCCmec type IVa warrants further attention.

Allosteric Probe-Based Colorimetric Assay for Direct Identification and Sensitive Analysis of Methicillin Resistance of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistance of Staphylococcus aureus (SA) holds significant clinical importance. However, the methicillin-resistance detection strategies commonly require complicated cell lysis and gene extraction. Herein, we devised a novel colorimetric approach for the sensitive and accurate identification of methicillin-resistance of SA by combining allosteric probe-based target recognition with self-primer elongation-based target recycling. The PBP2a aptamer in the allosteric probe successfully identified the target MRSA, leading to the initiation of self-primer elongation based-cascade signal amplification. The peroxidase-like hemin/G-quadruplex undergo an isothermal autonomous process that effectively catalyzes the oxidation of ABTS2- and produces a distinct blue color, enabling the visual identification of MRSA at low concentrations. The method offers a shorter duration for bacteria cultivation compared to traditional susceptibility testing methods, as well as simplified manual procedures for gene analysis. The overall amplification time for this test is 60 min, and it has a detection limit of 3 CFU/ml. In addition, the approach has exceptional selectivity and reproducibility, demonstrating commendable performance when tested with real samples. Due to its advantages, this colorimetric assay exhibits considerable potential for integration into a sensor kit, thereby offering a viable and convenient alternative for the prompt and on-site detection of MRSA in patients with skin and soft tissue infections.

The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus.

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.

High prevalence of ST5-SCCmec II-t311 clone of methicillin-resistant Staphylococcus aureus isolated from bloodstream infections in East China.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is a challenging global health threat, resulting in significant morbidity and mortality worldwide. This study aims to determine the molecular characteristics and antimicrobial susceptibility of 263 MRSA isolates in Zhejiang Province, east China. METHODS: From 2014 to 2019, a total of 263 MRSA isolates from bloodstream infections (BSIs) were collected from 6 hospitals in 4 cities in Zhejiang province, east China. Antimicrobial susceptibility tests were conducted according to the guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI). To characterize and analyze these isolates, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing and virulence genes gene profiles were performed. RESULTS: The most predominant clone was ST5-SCCmec II-t311, which accounted for 41.8% (110/263), followed by ST59 (44/263, 16.7%). Compared with non-ST5-II-t311 isolates, ST5-II-t311 isolates were more resistant to erythromycin, tetracycline, levofloxacin, moxifloxacin, and ciprofloxacin, but more susceptible to clindamycin. Moreover, the rates of multidrug resistance were higher in ST5-II-t311 isolates compared to the non-ST5-II-t311 isolates. In comparison to the non-ST5-II-t311 isolates, ST5-II-t311 isolates showed no significant difference in virulence genes detected. CONCLUSIONS: MRSA ST5-II-t311 clone has become the most predominant clone in Zhejiang Province, east China and has higher rates of multidrug resistance than other isolates, that should be kept in mind when treating BSI. Moreover, MRSA ST59 clone shows an upward trend and has begun to spread into hospitals. Our findings highlight the importance of epidemiological studies of S. aureus carriage in the eastern region.

Insights from a decade of surveillance: Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Norway from 2008 to 2017.

AIM: Norway has a low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and reporting of all MRSA cases has been mandatory, including infections and carriage, since 1995 and 2005 accordingly. This provides a unique window to study the spread of MRSA in Norway over time. The aim of this study was to analyze the nationwide trends in the molecular epidemiology of MRSA in Norway over a period of 10 years. METHODS: Clinical and epidemiological data as well as bacterial genotype (spa-type and PVL) were analyzed for all reported MRSA cases in Norway in the period 2008-2017. RESULTS: During the study period, there were 15,200 MRSA cases reported in Norway, from 14,386 patients. The notification rate per 100,000 population increased by 15% annually, rising from 14.2 in 2007 to 48.6 in 2017. This increase was primarily driven by MRSA carriage and community-associated MRSA cases. The incidence of invasive infections remained stable and low, at less than 0.5. The incidence of healthcare-associated MRSA showed an increasing trend, while the number of outbreak-related cases, particularly those associated with nursing homes, decreased. Overall, there were significantly more MRSA infections in males than females. Interestingly, there was a significantly higher prevalence of MRSA infections in female young adolescents compared to males. spa-typing revealed a very heterogeneous MRSA population (D = 0.97), predominantly impacted by international travel and migration patterns, and less by domestic spread in the community. CONCLUSIONS: This study highlights that Norway, while still classified as a low-prevalence country, has experienced a significant increase in the incidence of MRSA between 2008 and 2017, which can predominantly be attributed to CA-MRSA and MRSA carriage.

Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Santa Catarina (SC), Brazil.

The study investigated the characteristics of Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Santa Catarina. Findings revealed prevalent SCCmecII and IV, multiresistance, Leucocidin ED genes, and one ST105 isolate. The results indicated that the in-state MRSA isolates showed the same characteristics as the out-of-state isolates among the investigated features.

Distribution of Staphylococcus aureus carriage among healthcare workers in a Japanese convalescent and rehabilitation hospital.

Earlobes, nasal cavities, and fingers of 145 healthcare workers in convalescent and rehabilitation hospital (60 nurses and 85 rehabilitation healthcare workers) were sampled. Of the 3 sites sampled, Staphylococcus aureus was detected in one or more sites in 25 nurses and 27 rehabilitation workers. S. aureus was detected in all 3 sites in 2 (8.0%) nurses and 2 (7.4%) rehabilitation workers, and the S. aureus isolates in each case showed related PFGE pattern. S. aureus was detected in both the fingers and nasal cavities of 5 (18.5%) of the rehabilitation healthcare workers; in all 5 cases, the PFGE patterns of the S. aureus isolates from each site belonged to same cluster based on PFGE. Of the 2 cases in which methicillinresistant S. aureus (MRSA) was recovered from earlobes, fingers, and nasal cavities, in both cases, MRSA isolates from the 3 sites were the same clone according to PFGE analysis and SCCmec type IV. As S. aureus was detected in pierced earlobes of nurses, hand hygiene must be practiced after touching pierced earlobes and before patient contact. The same S. aureus clone in the nasal cavity and earlobes indicates that the route of transmission is through the fingers.

Antimicrobial resistance and virulence profiling of Staphylococcus pseudintermedius isolated from cats, Bangladesh.

Staphylococcus pseudintermedius is a significant bacterial pathogen that frequently colonizes different body sites and mucous membranes of pets. The objectives of the cross-sectional study were to estimate the prevalence, antimicrobial resistance pattern, and detection of diverse resistance as well as virulence genes of S. pseudintermedius in cats. A standard bacteriological method, species-specific gene and different antimicrobial resistance as well as virulence genes were confirmed by PCR assay. A total of 233 swab samples were collected from different body sites of 102 cats, among them 146 swabs from 73 healthy cats, and 87 from 29 diseased cats. Overall, prevalence of S. pseudintermedius in cats was 12.01%, while dermatitis and otitis affected cats were 26.08% and 33.33%, respectively. The highest antimicrobial resistance was observed against penicillin (96.42%) followed by streptomycin (85.71%) and erythromycin (78.57%). Moreover, 89.28% of S. pseudintermedius isolates exhibit multi-drug resistance (MDR) (≥ 3 classes' antimicrobial resistant). In addition, 17.86% isolates harbored the mecA gene; thus, were classified as methicillin-resistant S. pseudintermedius (MRSP). Furthermore, the erythromycin resistance genes ermA and ermB were harbored by 25% and 10.71% of isolates, while 42.86% and 17.86% of isolates carried tetK and tetL (tetracycline resistance) genes, respectively. In virulence profiling, 32.14% (sea) and 10.71% (seb) of isolates were found positive for enterotoxin genes, whereas, the toxic shock syndrome toxin-1 (tst-1) gene and the Panton-Valentine leukocidin gene (pvl) were detected in 25% and 14.29% of isolates, respectively. To our knowledge, this is the first report of cats in Bangladesh for MDR S. pseudintermedius, MRSP, and their virulence profiling.

Emergence and clonal expansion of a qacA-harbouring sequence type 45 lineage of methicillin-resistant Staphylococcus aureus.

The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.

Antimicrobial Resistance and the Prevalence of the Panton-Valentine Leukocidin Gene among Clinical Isolates of Staphylococcus aureus in Lithuania.

This study aimed to determine resistance to antimicrobials of Staphylococcus aureus strains isolated from clinical specimens in Lithuanian hospitals and to identify the genes conferring resistance and virulence. The study was carried out from June 2019 to September 2021. S. aureus strains were isolated from skin, soft tissues, blood, lower respiratory tract, urine and other specimens. Antibiotic susceptibility testing was performed using the disc diffusion method according to EUCAST guidelines. All isolates were analyzed for detection of the ermA, ermC, mecA, mecC, tetK, tetM, and lukF-PV genes by multiplex real-time PCR. The 16S rRNA coding sequence was applied as an internal PCR control. Altogether, 745 S. aureus strains were analyzed. Antimicrobial susceptibility testing revealed that all isolates were susceptible to rifampin and vancomycin. Of the 745 strains, 94.8% were susceptible to tetracycline, 94.5% to clindamycin, and 88.3% to erythromycin. The lowest susceptibility rate was found for penicillin (25.8%). Six percent of the tested strains were methicillin-resistant S. aureus (MRSA). The majority of methicillin-resistant strains were isolated from skin and soft tissues (73.3%), with a smaller portion isolated from blood (17.8%) and respiratory tract (8.9%). The ermC gene was detected in 41.1% of erythromycin-resistant S. aureus strains, whereas ermA was detected in 32.2% of erythromycin-resistant S. aureus strains. 69.2% of tetracycline-resistant S. aureus strains had tetK gene, and 28.2% had tetM gene. 7.3% of S. aureus isolates harbored lukF-PV gene. The frequency of the pvl gene detection was significantly higher in MRSA isolates than in methicillin-susceptible S. aureus isolates (p < 0.0001).

Deep learning model for personalized prediction of positive MRSA culture using time-series electronic health records.

Methicillin-resistant Staphylococcus aureus (MRSA) poses significant morbidity and mortality in hospitals. Rapid, accurate risk stratification of MRSA is crucial for optimizing antibiotic therapy. Our study introduced a deep learning model, PyTorch_EHR, which leverages electronic health record (EHR) time-series data, including wide-variety patient specific data, to predict MRSA culture positivity within two weeks. 8,164 MRSA and 22,393 non-MRSA patient events from Memorial Hermann Hospital System, Houston, Texas are used for model development. PyTorch_EHR outperforms logistic regression (LR) and light gradient boost machine (LGBM) models in accuracy (AUROCPyTorch_EHR = 0.911, AUROCLR = 0.857, AUROCLGBM = 0.892). External validation with 393,713 patient events from the Medical Information Mart for Intensive Care (MIMIC)-IV dataset in Boston confirms its superior accuracy (AUROCPyTorch_EHR = 0.859, AUROCLR = 0.816, AUROCLGBM = 0.838). Our model effectively stratifies patients into high-, medium-, and low-risk categories, potentially optimizing antimicrobial therapy and reducing unnecessary MRSA-specific antimicrobials. This highlights the advantage of deep learning models in predicting MRSA positive cultures, surpassing traditional machine learning models and supporting clinicians' judgments.

A look at staphylococci from the one health perspective.

Staphylococcus aureus and other staphylococcal species are resident and transient multihost colonizers as well as conditional pathogens. Especially S. aureus represents an excellent model bacterium for the "One Health" concept because of its dynamics at the human-animal interface and versatility with respect to host adaptation. The development of antimicrobial resistance plays another integral part. This overview will focus on studies at the human-animal interface with respect to livestock farming and to companion animals, as well as on staphylococci in wildlife. In this context transmissions of staphylococci and of antimicrobial resistance genes between animals and humans are of particular significance.

Methicillin-resistant Staphylococcus spp. investigation in hospitalized horses and contacting personnel in a teaching veterinary hospital.

Staphylococci are well-known opportunistic pathogens associated with suppurative diseases in humans and animals. Antimicrobial resistance is an emergent threat to humans and animals worldwide. This study investigated the prevalence of methicillin-resistant Staphylococcus spp. (MRS) in hospitalized horses and contacting personnel (veterinarians and staff), and assessed possible interspecies transmission in a teaching veterinary hospital. Nasal swabs from horses (n = 131) and humans (n = 35) were collected. The microorganisms were identified by traditional biochemical tests and genotypic methods, i.e., PCR, internal transcript spacer PCR (ITS-PCR), and gene sequencing. Staphylococcal species were isolated in 18% (23/131) of the horses, of which 8% (11/131) were S. hyicus, 4 % (5/131) were S. aureus, 4% (5/131) were S. pseudintermedius, and 2% (2/131) were S. schleiferi subsp. coagulans. The mecA gene was detected in an S. pseudintermedius isolate. Staphylococcus spp. was isolated in 40% (14/35) of the human samples, all of which were S. aureus. In four samples of S. aureus, the clonal profile ST398 was identified; among them, a clonal similarity of 98.1% was observed between a horse and a contacting human. This finding supports the need for biosecurity measures to avoid the spread of multidrug-resistant staphylococci in humans and horses.

Engineering hybrid lantibiotics yields the highly stable and bacteriocidal peptide cerocin V.

Antimicrobial peptides (AMPs) show promise as alternatives to traditional antibiotics for treating drug-resistant infections. Their adaptability and diverse sequence possibilities allow for rational design by modulating physicochemical determinants to achieve desired biological properties, transforming them into peptides for potential new therapies. Nisin, one of the best-studied AMPs, is believed to have potential to be used as a therapeutic, particularly against antibiotic-resistant bacteria. However, its instability in physiological conditions limits its use in clinical applications and pharmaceutical development. Exploration of new natural variants of nisin has uncovered diverse properties using different domains. Shuffling peptide modules can fine-tune the chemical properties of these molecules, potentially enhancing stability while maintaining or improving antimicrobial activity. In this study, hybrid AMPs were created by combining domains from three unique nisin variants, i.e. nisin A, cesin and rombocin, leading to the identification of a promising variant, named cerocin A, which harbours only 25 amino acids compared to the typical 31-35 amino acid length of nisin. Cerocin A demonstrates potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), approaching that of nisin itself. Cerocin A's mode of action involves a dual mechanism through the combination of two domains, consisting of a small ring/domain (6 amino acids) from the C-terminal end of rombocin attached to the preceding peptide of cesin, changing it from a bacteriostatic to a bactericidal peptide. Further mutation studies identified a new variant, cerocin V, with significantly improved resistance against trypsin degradation, while maintaining high potency. Importantly, cerocin V showed no undesired toxic effects on human red blood cells and remained stable in human plasma. In conclusion, we demonstrate that peptide construction using domain engineering is an effective strategy for manipulating both biological and physicochemical aspects, leading to the creation of novel bioactive molecules with desired properties. These constructs are appealing candidates for further optimization and development as novel antibiotics.

Persistence and evolution of linezolid- and methicillin-resistant Staphylococcus epidermidis ST2 and ST5 clones in an Italian hospital.

OBJECTIVES: Staphylococcus epidermidis is a member of the human skin microbiome. However, in recent decades, multidrug-resistant and hospital-adapted S. epidermidis clones are increasingly involved in severe human infections associated with medical devices and in immunocompromised patients. In 2016, we reported that a linezolid- and methicillin-resistant S. epidermidis ST2 clone, bearing the G2576T mutation, was endemic in an Italian hospital since 2004. This study aimed to retrospectively analyse 34 linezolid- and methicillin-resistant S. epidermidis (LR-MRSE) strains collected from 2018 to 2021 from the same hospital. METHODS: LR-MRSE were typed by Pulsed-Field Gel Electrophoresis and multilocus sequence typing and screened for transferable linezolid resistance genes. Representative LR-MRSE were subjected to whole-genome sequencing (WGS) and their resistomes, including the presence of ribosomal mechanisms of linezolid resistance and of rpoB gene mutations conferring rifampin resistance, were investigated. RESULTS: ST2 lineage was still prevalent (19/34; 55.9%), but, over time, ST5 clone has been widespread too (15/34; 44.1%). Thirteen of the 34 isolates (38.2%) were positive for the cfr gene. Whole-genome sequencing analysis of relevant LR-MRSE displayed complex resistomes for the presence of several acquired antibiotic resistance genes, including the SCCmec type III (3A) and SCCmec type IV (2B) in ST2 and ST5 isolates, respectively. Bioinformatics and polymerase chain reaction (PCR) mapping also showed a plasmid-location of the cfr gene and the occurrence of previously undetected mutations in L3 (ST2 lineage) and L4 (ST3 lineage) ribosomal proteins and substitutions in the rpoB gene. CONCLUSION: The occurrence of LR-MRSE should be carefully monitored in order to prevent the spread of this difficult-to-treat pathogen and to preserve the efficacy of linezolid.

Historic methicillin-resistant Staphylococcus aureus: expanding current knowledge using molecular epidemiological characterization of a Swiss legacy collection.

BACKGROUND: Few methicillin-resistant Staphylococcus aureus (MRSA) from the early years of its global emergence have been sequenced. Knowledge about evolutionary factors promoting the success of specific MRSA multi-locus sequence types (MLSTs) remains scarce. We aimed to characterize a legacy MRSA collection isolated from 1965 to 1987 and compare it against publicly available international and local genomes. METHODS: We accessed 451 historic (1965-1987) MRSA isolates stored in the Culture Collection of Switzerland, mostly collected from the Zurich region. We determined phenotypic antimicrobial resistance (AMR) and performed whole genome sequencing (WGS) using Illumina short-read sequencing on all isolates and long-read sequencing on a selection with Oxford Nanopore Technology. For context, we included 103 publicly available international assemblies from 1960 to 1992 and sequenced 1207 modern Swiss MRSA isolates from 2007 to 2022. We analyzed the core genome (cg)MLST and predicted SCCmec cassette types, AMR, and virulence genes. RESULTS: Among the 451 historic Swiss MRSA isolates, we found 17 sequence types (STs) of which 11 have been previously described. Two STs were novel combinations of known loci and six isolates carried previously unsubmitted MLST alleles, representing five new STs (ST7843, ST7844, ST7837, ST7839, and ST7842). Most isolates (83% 376/451) represented ST247-MRSA-I isolated in the 1960s, followed by ST7844 (6% 25/451), a novel single locus variant (SLV) of ST239. Analysis by cgMLST indicated that isolates belonging to ST7844-MRSA-III cluster within the diversity of ST239-MRSA-III. Early MRSA were predominantly from clonal complex (CC)8. From 1980 to the end of the twentieth century, we observed that CC22 and CC5 as well as CC8 were present, both locally and internationally. CONCLUSIONS: The combined analysis of 1761 historic and contemporary MRSA isolates across more than 50 years uncovered novel STs and allowed us a glimpse into the lineage flux between Swiss-German and international MRSA across time.

Cross-sectional analysis of risk factors associated with Mugil cephalus in retail fish markets concerning methicillin-resistant Staphylococcus aureus and Aeromonas hydrophila.

Introduction: Aeromonas hydrophila and methicillin-resistant Staphylococcus aureus (MRSA) are potent bacterial pathogens posing major hazards to human health via consuming fish harboring these pathogens or by cross-contamination beyond the contaminated environment. The aim of this study was to determine risk variables associated with the presence of certain pathogenic bacteria from Mugil cephalus fish in retail markets in Egypt. The virulence genes of A. hydrophila and S. aureus were also studied. Furthermore, the antibiotic sensitivity and multidrug resistance of the microorganisms were evaluated. Methods: In a cross-sectional investigation, 370 samples were collected from mullet skin and muscle samples, washing water, fish handlers, knives, and chopping boards. Furthermore, fish handlers' public health implications were assessed via their response to a descriptive questionnaire. Results: S. aureus and Aeromonas species dominated the investigated samples with percentages of 26.76% and 30.81%, respectively. Furthermore, A. hydrophila and MRSA were the predominant recovered bacterial pathogens among washing water and knives (53.85% and 46.66%, respectively). The virulence markers aerA and hlyA were found in 90.7% and 46.5% of A. hydrophila isolates, respectively. Moreover, the virulence genes nuc and mec were prevalent in 80% and 60% of S. aureus isolates, respectively. Antimicrobial susceptibility results revealed that all A. hydrophila isolates were resistant to amoxicillin and all MRSA isolates were resistant to amoxicillin and ampicillin. Remarkably, multiple drug resistance (MDR) patterns were detected in high proportions in A. hydrophila (88.37%) and MRSA (100%) isolates. The prevalence of Aeromonas spp. and S. aureus had a positive significant correlation with the frequency of handwashing and use of sanitizer in cleaning of instruments. MRSA showed the highest significant prevalence rate in the oldest age category. Conclusion: The pathogenic bacteria recovered in this study were virulent and had a significant correlation with risk factors associated with improper fish handling. Furthermore, a high frequency of MDR was detected in these pathogenic bacteria, posing a significant risk to food safety and public health.