Dual pathogenicity island transfer by piggybacking lateral transduction.

Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry.

Type III-A CRISPR immunity promotes mutagenesis of staphylococci.

Horizontal gene transfer and mutation are the two major drivers of microbial evolution that enable bacteria to adapt to fluctuating environmental stressors1. Clustered, regularly interspaced, short palindromic repeats (CRISPR) systems use RNA-guided nucleases to direct sequence-specific destruction of the genomes of mobile genetic elements that mediate horizontal gene transfer, such as conjugative plasmids2 and bacteriophages3, thus limiting the extent to which bacteria can evolve by this mechanism. A subset of CRISPR systems also exhibit non-specific degradation of DNA4,5; however, whether and how this feature affects the host has not yet been examined. Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. These mutations require the induction of the SOS response to DNA damage and display a distinct pattern. Our results demonstrate that by differentially affecting both mechanisms that generate genetic diversity, type III-A CRISPR systems can modulate the evolution of the bacterial host.

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Integrated genomic and functional analyses of human skin-associated Staphylococcus reveal extensive inter- and intra-species diversity.

Human skin is stably colonized by a distinct microbiota that functions together with epidermal cells to maintain a protective physical barrier. Staphylococcus, a prominent genus of the skin microbiota, participates in colonization resistance, tissue repair, and host immune regulation in strain-specific manners. To unlock the potential of engineering skin microbial communities, we aim to characterize the diversity of this genus within the context of the skin environment. We reanalyzed an extant 16S rRNA amplicon dataset obtained from distinct body sites of healthy volunteers, providing a detailed biogeographic depiction of staphylococcal species that colonize our skin. S. epidermidis, S. capitis, and S. hominis were the most abundant staphylococcal species present in all volunteers and were detected at all body sites. Pan-genome analysis of isolates from these three species revealed that the genus-core was dominated by central metabolism genes. Species-restricted-core genes encoded known host colonization functions. The majority (~68%) of genes were detected only in a fraction of isolate genomes, underscoring the immense strain-specific gene diversity. Conspecific genomes grouped into phylogenetic clades, exhibiting body site preference. Each clade was enriched for distinct gene sets that are potentially involved in site tropism. Finally, we conducted gene expression studies of select isolates showing variable growth phenotypes in skin-like medium. In vitro expression revealed extensive intra- and inter-species gene expression variation, substantially expanding the functional diversification within each species. Our study provides an important resource for future ecological and translational studies to examine the role of shared and strain-specific staphylococcal genes within the skin environment.

Differential survival of Staphylococcal species in macrophages.

Staphylococcus aureus is considered an extracellular pathogen, yet the bacterium is able to survive within and escape from host cells. An agr/sae mutant of strain USA300 is unable to escape from macrophages but can replicate and survive within. We questioned whether such "non-toxic" S. aureus resembles the less pathogenic coagulase-negative Staphylococcal (CoNS) species like S. epidermidis, S. carnosus, S. lugdunensis, S. capitis, S. warneri, or S. pettenkoferi. We show that the CoNS are more efficiently killed in macrophage-like THP-1 cells or in human primary macrophages. Mutations in katA, copL, the regulatory system graRS, or sigB did not impact bacterial survival in THP-1 cells. Deletion of the superoxide dismutases impaired S. aureus survival in primary macrophages but not in THP-1 cells. However, expression of the S. aureus-specific sodM in S. epidermidis was not sufficient to protect this species from being killed. Thus, at least in those cells, better bacterial survival of S. aureus could not be linked to higher protection from ROS. However, "non-toxic" S. aureus was found to be insensitive to pH, whereas most CoNS were protected when phagosomal acidification was inhibited. Thus, species differences are at least partially linked to differences in sensitivity to acidification.

Sticking together: independent evolution of biofilm formation in different species of staphylococci has occurred multiple times via different pathways.

BACKGROUND: Staphylococci cause a wide range of infections, including implant-associated infections which are difficult to treat due to the presence of biofilms. Whilst some proteins involved in biofilm formation are known, the differences in biofilm production between staphylococcal species remains understudied. Currently biofilm formation by Staphylococcus aureus is better understood than other members of the genus as more research has focused on this species. RESULTS: We assembled a panel of 385 non-aureus Staphylococcus isolates of 19 species from a combination of clinical sources and reference strains. We used a high-throughput crystal violet assay to assess the biofilm forming ability of all strains and assign distinct biofilm formation categories. We compared the prevalence of Pfam domains between the categories and used machine learning to identify amino acid 20-mers linked to biofilm formation. This identified some domains within proteins already linked to biofilm formation and important domains not previously linked to biofilm formation in staphylococci. RT-qPCR confirmed the expression of selected genes predicted to encode important domains within biofilms in Staphylococcus epidermidis. The prevalence and distribution of biofilm associated domains showed a link to phylogeny, suggesting different Staphylococcus species have independently evolved different mechanisms of biofilm production. CONCLUSIONS: This work has identified different routes to biofilm formation in diverse species of Staphylococcus and suggests independent evolution of biofilm has occurred multiple times across the genus. Understanding the mechanisms of biofilm formation in any given species is likely to require detailed study of relevant strains and the ability to generalise across the genus may be limited.

Genomic exploration of the fermented meat isolate Staphylococcus shinii IMDO-S216 with a focus on competitiveness-enhancing secondary metabolites.

BACKGROUND: Staphylococcus shinii appears as an umbrella species encompassing several strains of Staphylococcus pseudoxylosus and Staphylococcus xylosus. Given its phylogenetic closeness to S. xylosus, S. shinii can be found in similar ecological niches, including the microbiota of fermented meats where the species may contribute to colour and flavour development. In addition to these conventional functionalities, a biopreservation potential based on the production of antagonistic compounds may be available. Such potential, however, remains largely unexplored in contrast to the large body of research that is available on the biopreservative properties of lactic acid bacteria. The present study outlines the exploration of the genetic basis of competitiveness and antimicrobial activity of a fermented meat isolate, S. shinii IMDO-S216. To this end, its genome was sequenced, de novo assembled, and annotated. RESULTS: The genome contained a single circular chromosome and eight plasmid replicons. Focus of the genomic exploration was on secondary metabolite biosynthetic gene clusters coding for ribosomally synthesized and posttranslationally modified peptides. One complete cluster was coding for a bacteriocin, namely lactococcin 972; the genes coding for the pre-bacteriocin, the ATP-binding cassette transporter, and the immunity protein were also identified. Five other complete clusters were identified, possibly functioning as competitiveness factors. These clusters were found to be involved in various responses such as membrane fluidity, iron intake from the medium, a quorum sensing system, and decreased sensitivity to antimicrobial peptides and competing microorganisms. The presence of these clusters was equally studied among a selection of multiple Staphylococcus species to assess their prevalence in closely-related organisms. CONCLUSIONS: Such factors possibly translate in an improved adaptation and competitiveness of S. shinii IMDO-S216 which are, in turn, likely to improve its fitness in a fermented meat matrix.

Characterization of the resistome and predominant genetic lineages of Gram-positive bacteria causing keratitis.

Bacterial keratitis is a vision-threatening infection mainly caused by Gram-positive bacteria (GPB). Antimicrobial therapy is commonly empirical using broad-spectrum agents with efficacy increasingly compromised by the emergence of antimicrobial resistance. We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of GPB causing keratitis and to characterize their antimicrobial resistance patterns. A total of 161 isolates, including Staphylococcus aureus (n = 86), coagulase-negative staphylococci (CoNS; n = 34), Streptococcus spp. (n = 34), and Enterococcus faecalis (n = 7), were included. The population of S. aureus isolates consisted mainly of clonal complex 5 (CC5) (30.2%). Similarly, the population of Staphylococcus epidermidis was homogenous with most of them belonging to CC2 (78.3%). Conversely, the genetic population of Streptococcus pneumoniae was highly diverse. Resistance to first-line antibiotics was common among staphylococci, especially among CC5 S. aureus. Methicillin-resistant S. aureus was commonly resistant to fluoroquinolones and azithromycin (78.6%) and tobramycin (57%). One-third of the CoNS were resistant to fluoroquinolones and 53% to azithromycin. Macrolide resistance was commonly caused by erm genes in S. aureus, mphC and msrA in CoNS, and mefA and msr(D) in streptococci. Aminoglycoside resistance in staphylococci was mainly associated with genes commonly found in mobile genetic elements and that encode for nucleotidyltransferases like ant(4')-Ib and ant(9)-Ia. Fluroquinolone-resistant staphylococci carried from 1 to 4 quinolone resistance-determining region mutations, mainly in the gyrA and parC genes. We found that GPB causing keratitis are associated with strains commonly resistant to first-line topical therapies, especially staphylococcal isolates that are frequently multidrug-resistant and associated with major hospital-adapted epidemic lineages.

Comparative genome wise analysis of codon usage of Staphylococcus Genus.

The genus Staphylococcus encompasses a diverse array of bacteria with significant implications for human health, including disreputable pathogens such as Staphylococcus aureus and Staphylococcus epidermidis. Understanding the genetic composition and codon usage patterns of Staphylococcus species is crucial for unraveling their evolutionary dynamics, adaptive strategies, and pathogenic potential. In this study, we conducted a comprehensive analysis of codon usage patterns across 48 species within the Staphylococcus genus. Our findings uncovered variations in genomic G-C content across Staphylococcus species, impacting codon usage preferences, with a notable preference for A/T-rich codons observed in pathogenic strains. This preference for A/T-rich codons suggests an energy-saving strategy in pathogenic organisms. Analysis of dinucleotide pair expression patterns unveiled insights into genomic dynamics, with overrepresented codon pairs reflecting trends in dinucleotide expression across genomes. Additionally, a significant correlation between CAI and genomic G-C content underscored the intricate relationship between codon usage patterns and gene expression strategies. Amino acid usage analysis highlighted preferences for energetically cheaper amino acids, suggesting adaptive strategies promoting energy efficiency. This comprehensive analysis sheds light on the evolutionary dynamics and adaptive mechanisms employed by Staphylococcus species, providing valuable insights into their pathogenic potential and clinical implications. Understanding these genomic features is crucial for devising strategies to combat staphylococcal infections and improve public health outcomes.

Methicillin resistance in Staphylococcus pseudintermedius encoded within novel staphylococcal cassette chromosome mec (SCCmec) variants.

BACKGROUND: Staphylococcus pseudintermedius is a common opportunistic pathogen of companion dogs and an occasional human pathogen. Treatment is hampered by antimicrobial resistance including methicillin resistance encoded by mecA within the mobile genetic element SCCmec. OBJECTIVES: SCCmec elements are diverse, especially in non-Staphyloccocus aureus staphylococci, and novel variants are likely to be present in S. pseudintermedius. The aim was to characterize the SCCmec elements found in four canine clinical isolates of S. pseudintermedius. MATERIAL AND METHODS: Isolates were whole-genome sequenced and SCCmec elements were assembled, annotated and compared to known SCCmec types. RESULTS AND DISCUSSION: Two novel SSCmec are present in these isolates. SCCmec7017-61515 is characterized by a novel combination of a Class A mec gene complex and a type 5 ccr previously only described in composite SCCmec elements. The other three isolates share a novel composite SCCmec with features of SCCmec types IV and VI. CONCLUSIONS: S. pseudintermedius is a reservoir of novel SSCmec elements that has implications for understanding antimicrobial resistant in veterinary and human medicine.

Chromosomal and plasmid localization of ileS2 in high-level mupirocin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus isolated from canine and feline origins.

OBJECTIVES: To characterize the mobile genetic elements and genetic localization of ileS2 in high-level mupirocin-resistant (Hi-MupR) methicillin-resistant Staphylococcus pseudintermedius (MRSP) and MRSA isolates recovered from canine and feline clinical samples. METHODS: The identification of bacterial species and presence of mecA and ileS2 genes in MRSP and MRSA isolates were performed using MALDI-TOF MS and PCR, respectively. Antimicrobial resistance (AMR) phenotypes were determined by broth microdilution assays. The genome characteristics, ileS2-containing elements and staphylococcal cassette chromosome mec (SCCmec) were illustrated using complete circular genomes obtained from hybrid assembly of Illumina short-reads and Oxford Nanopore Technologies long-reads. These were analysed through phylogenetic and bioinformatics approaches. RESULTS: A total of 18 MRSP clinical isolates and four MRSA clinical isolates exhibited the Hi-MupR phenotype and carried multiple AMR genes, including mecA and ileS2 genes. MRSP ST182-SCCmec V (n = 6) and ST282-ΨSCCmec57395-t10 (n = 4) contained the ileS2 transposable unit associated with IS257 on the chromosome. Three MRSA ST398-SCCmec V-t034/t4652 isolates carried ∼42 kb pSK41-like ileS2 plasmids, whereas similar ileS2 plasmids lacking tra genes were found in MRSP ST282-ΨSCCmec57395-t72/t21 isolates. Furthermore, a new group of ileS2 plasmids, carried by MRSP ST45-ΨSCCmec57395, ST433-ΨSCCmecKW21-t05 and ST2165-SCCmec IV-t06, and by one MRSA ST398-SCCmec V-t034 strain, shared the plasmid backbone with the cfr/fexA-carrying plasmid pM084526_1 in MRSA ST398. CONCLUSIONS: This study provides the first evidence of ileS2 integration into the S. pseudintermedius chromosome, which is a rare occurrence in staphylococcal species, and plasmids played a pivotal role in dissemination of ileS2 in both staphylococcal species.

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.

Identification and characterization of Staphylococcus argenteus from Indonesia.

BACKGROUND: In 2015, Staphylococcus argenteus was reported for the first time as a novel species of the Staphylococcus aureus complex. While S. argenteus has been found in many countries, its presence in Indonesia has not been reported yet. Our aim is to confirm S. argenteus presence in Indonesia, describe its characteristics and analyze its genomic diversity. METHODS: The S. aureus isolates used in this study were collected from patients with skin and soft tissue infections in Indonesia, between July 2009 to February 2010. Randomly selected isolates were recultured from -80 C° stocks and analyzed using matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF). Isolates identified as S. argenteus, S. roterodami, or S. schweitzeri and S. aureus with a low score in the MALDI-TOF analysis were analyzed by a real-time PCR targeting the nucA gene able to identify true S. argenteus. Isolates identified as S. argenteus were further characterized by whole genome sequencing. Vitek®2 (bioMérieux) was used for antimicrobial susceptibility testing. RESULTS: Fifteen isolates were identified as S. argenteus, with the majority belonging to ST2250. Two pairs of isolates proved to be identical by core genome multilocus sequence typing analysis. Most isolates were susceptible to all antibiotics tested, except for seven isolates (46.7 %) that were resistant to benzylpenicillin, and one isolate was resistant to tetracycline (6.7 %). The presence of resistance genes blaZ and tet(45) correlated with these findings. Notably, the sey enterotoxin gene was prevalent in 80 % of the isolates. Other virulence factor genes were less prevalent. Plasmid replicon types in S. argenteus were also known to S. aureus. CONCLUSION: Our study reveals the occurrence of S. argenteus in Indonesia. The diversity within Indonesian S. argenteus matches the global diversity of S. argenteus. Identical isolates between patients indicate potential transmission events. A lower prevalence of a broad panel of virulence factors suggests that S. argenteus is less virulent than S. aureus.

Characterization of the major autolysin (AtlC) of Staphylococcus carnosus.

BACKGROUND: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated. RESULTS: AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC. CONCLUSIONS: In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.

Genetic recombination-mediated evolutionary interactions between phages of potential industrial importance and prophages of their hosts within or across the domains of Escherichia, Listeria, Salmonella, Campylobacter, and Staphylococcus.

BACKGROUND: The in-depth understanding of the role of lateral genetic transfer (LGT) in phage-prophage interactions is essential to rationalizing phage applications for human and animal therapy, as well as for food and environmental safety. This in silico study aimed to detect LGT between phages of potential industrial importance and their hosts. METHODS: A large array of genetic recombination detection algorithms, implemented in SplitsTree and RDP4, was applied to detect LGT between various Escherichia, Listeria, Salmonella, Campylobacter, Staphylococcus, Pseudomonas, and Vibrio phages and their hosts. PHASTER and RAST were employed respectively to identify prophages across the host genome and to annotate LGT-affected genes with unknown functions. PhageAI was used to gain deeper insights into the life cycle history of recombined phages. RESULTS: The split decomposition inferences (bootstrap values: 91.3-100; fit: 91.433-100), coupled with the Phi (0.0-2.836E-12) and RDP4 (P being well below 0.05) statistics, provided strong evidence for LGT between certain Escherichia, Listeria, Salmonella, and Campylobacter virulent phages and prophages of their hosts. The LGT events entailed mainly the phage genes encoding for hypothetical proteins, while some of these genetic loci appeared to have been affected even by intergeneric recombination in specific E. coli and S. enterica virulent phages when interacting with their host prophages. Moreover, it is shown that certain L. monocytogenes virulent phages could serve at least as the donors of the gene loci, involved in encoding for the basal promoter specificity factor, for L. monocytogenes. In contrast, the large genetic clusters were determined to have been simultaneously exchanged by many S. aureus prophages and some Staphylococcus temperate phages proposed earlier as potential therapeutic candidates (in their native or modified state). The above genetic clusters were found to encompass multiple genes encoding for various proteins, such as e.g., phage tail proteins, the capsid and scaffold proteins, holins, and transcriptional terminator proteins. CONCLUSIONS: It is suggested that phage-prophage interactions, mediated by LGT (including intergeneric recombination), can have a far-reaching impact on the co-evolutionary trajectories of industrial phages and their hosts especially when excessively present across microbially rich environments.

Atopic dermatitis pediatric patients show high rates of nasal and intestinal colonization by methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

BACKGROUND: Atopic dermatitis (AD) patients have high rates of colonization by Staphylococcus aureus, which has been associated with worsening of the disease. This study characterized Staphylococcus spp isolates recovered from nares and feces of pediatric patients with AD in relation to antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, presence of pvl genes and clonality. Besides, gut bacterial community profiles were compared with those of children without AD. RESULTS: All 55 AD patients evaluated had colonization by Staphylococcus spp. Fifty-three (96.4%) patients had colonization in both clinical sites, whereas one patient each was not colonize in the nares or gut. Staphylococcus aureus was identified in the nostrils and feces of 45 (81.8%) and 39 (70.9%) patients, respectively. Methicillin-resistant Staphylococcus spp. isolates were found in 70.9% of the patients, and 24 (43.6%) had methicillin-resistant S. aureus (MRSA). S. aureus (55.6%) and S. epidermidis (26.5%) were the major species found. The prevalent lineages of S. aureus were USA800/SCCmecIV (47.6%) and USA1100/SCCmecIV (21.4%), and 61.9% of the evaluated patients had the same genotype in both sites. Additionally, gut bacterial profile of AD patients exhibits greater dissimilarity from the control group than it does among varying severities of AD. CONCLUSIONS: High rates of nasal and intestinal colonization by S. aureus and methicillin-resistant staphylococci isolates were found in AD patients. Besides, gut bacterial profiles of AD patients were distinctly different from those of the control group, emphasizing the importance of monitoring S. aureus colonization and gut microbiome composition in AD patients.

Coagulase-negative staphylococci from bovine milk: Antibiogram profiles and virulent gene detection.

BACKGROUND: Coagulase-negative Staphylococcus species are an emerging cause of intramammary infection, posing a significant economic and public health threat. The aim of this study was to assess the occurrence of coagulase-negative Staphylococcus species in bovine milk and dairy farms in Northwestern Ethiopia and to provide information about their antibiotic susceptibility and virulence gene profiles. METHODS: The cross-sectional study was conducted from February to August 2022. Coagulase-negative Staphylococcus species were isolated from 290 milk samples. Species isolation and identification were performed by plate culturing and biochemical tests and the antimicrobial susceptibility pattern of each isolate was determined by the Kirby-Bauer disc diffusion test. The single-plex PCR was used to detect the presence of virulent genes. The STATA software version 16 was used for data analysis. The prevalence, proportion of antimicrobial resistance and the number of virulent genes detected from coagulase-negative Staphylococcus species were analyzed using descriptive statistics. RESULTS: Coagulase-negative Staphylococcus species were isolated in 28.6%, (95% CI: 23.5-34.2) of the samples. Of these, the S. epidermidis, S. sciuri, S. warneri, S. haemolyticus, S. simulans, S. chromogens, S. cohnii, and S. captis species were isolated at the rates of 11, 5.2, 3.4, 3.1, 3.1, 1, 1, and 0.7% respectively. All the isolates showed a high percentage (100%) of resistance to Amoxicillin, Ampicillin, and Cefotetan and 37.5% of resistance to Oxacillin. The majority (54.2%) of coagulase-negative isolates also showed multidrug resistance. Coagulase-negative Staphylococcus species carried the icaD, pvl, mecA, hlb, sec, and hla virulent genes at the rates of 26.5%, 22.1%, 21.7%, 9.6%, 9.6% and 8.4% respectively. CONCLUSION: The present study revealed that the majority of the isolates (54.2%) were found multidrug-resistant and carriage of one or more virulent and enterotoxin genes responsible for intramammary and food poisoning infections. Thus, urgent disease control and prevention measures are warranted to reduce the deleterious impact of coagulase-negative species. To the best of our knowledge, this is the first study in Ethiopia to detect coagulase-negative Staphylococcus species with their associated virulent and food poisoning genes from bovine milk.

Integrating multi-wet laboratory diagnostics to study staphylococci in animals in Uganda.

BACKGROUND: Several diagnostic environments in Uganda lack real-time, robust and high-throughput technologies for comprehensive typing of microbes, which is a setback to infectious disease surveillance. This study combined various wet laboratory diagnostics to understand the epidemiology of pathogenic staphylococci isolated from animals in Uganda and the implications for global health security priorities. METHODS: A retrospective study was conducted employing records and pathogenic staphylococci (from animals) archived at the Central Diagnostic Laboratory (CDL), Makerere University, Uganda, between January 2012 and December 2019. The bacteria were speciated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tested for virulence factors [beta lactamases, lecithinase, deoxyribonuclease (DNase), haemolysins] and resistance to ten antimicrobials of clinical and veterinary relevance. Tetracycline and methicillin resistance genes were also tested. RESULTS: The prevalent diseases were mastitis in cattle and skin infections in dogs. Of the 111 staphylococci tested by MALDI-TOF MS, 79 (71.2%) were Staphylococcus aureus, 27 (24.3%) were Staphylococcus pseudintermedius and 5 (4.5%) were Staphylococcus schleiferi. All these strains expressed haemolysins. The prevalence of strains with lecithinase, penicillinase, cephalosporinase and DNase was 35.9% (14/39), 89.7% (35/39), 0.0% (0/39) and 87.2% (34/39), respectively. Staphylococci were primarily resistant to early penicillins (over 80%), tetracycline (57.7%), and chloramphenicol (46.2%). Minimal resistance was noted with cloxacillin (0.0%), ciprofloxacin (9.6%), and cefoxitin (3.8%). The prevalence of multidrug resistance (MDR) was 78.8% for general staphylococci, 82.2% for S. aureus, 73.1% for S. pseudintermedius, and 60.0% for S. schleiferi. Multidrug resistant staphylococci were significantly more prevalent in the cattle isolates than in the dog isolates (P < 0.05). The prevalence of methicillin-resistant staphylococci (MRS) tested by resistance to cefoxitin and mecA carriage was 3.8%. These four strains were all isolated from dog skin infections. The tetK gene was the most predominant (35.4%), followed by tetM (25.0%). CONCLUSION: In resource-constrained settings, the approach of integrated diagnostics promises sustainable disease surveillance and the addressing of current capacity gaps. The emergence of MRS (zoonotic bacteria) in companion animals creates a likelihood of reduced treatment options for related human infections, a threat to global health.

Virulence and host specificity of staphylococci from Staphylococcus intermedius group of pigeon origin with an emphasis on Staphylococcus intermedius.

The Staphylococcus intermedius group (SIG) includes coagulase-positive staphylococci commonly found in animals. The taxonomic classification within the SIG has evolved with molecular techniques distinguishing five species. Despite their similarities, these species exhibit varied host affinities, with unclear implications for virulence and host interaction. This study aimed to investigate the presence of coagulase-positive staphylococci in pigeons and to detect genes encoding for selected virulence factors in isolated strains. Another goal was to determine the adhesion capabilities of randomly selected pigeon S. intermedius, S. delphini, and canine S. pseudintermedius strains to canine and pigeon corneocytes and their adhesion and invasion abilities to canine keratinocytes in vitro. In total, 121 coagulase-positive strains were isolated from domestic and feral pigeons. The most prevalent species were S. delphini B and S. intermedius in domestic and feral pigeons, respectively. We proved that pigeon strains carried genes encoding for exfoliative toxin SIET and leukotoxin Luk-I. Moreover, we found that S. intermedius showed higher adherence to pigeon than to canine corneocytes, aligning with its presumed natural host. No difference in adherence abilities of S. pseudintermedius to canine and pigeon corneocytes was observed. In this study, we also observed that S. pseudintermedius could successfully invade the canine keratinocytes, in contrary to S. delphini and S. intermedius. Moreover, only S. intermedius was not able to invade canine keratinocytes at all. These findings highlight the complex interplay between SIG bacteria, and their hosts, underscoring the need for further research to understand the mechanisms of host adaptation and pathogenicity within this group.

Unravelling the complex mechanisms of multidrug resistance in bovine mastitis pathogens: Insights into antimicrobial resistance genes, biofilm dynamics, and efflux systems.

Mastitis remains a paramount economic threat to dairy livestock, with antibiotic resistance severely compromising treatment efficacy. This study provides an in-depth investigation into the multidrug resistance (MDR) mechanisms in bacterial isolates from bovine mastitis, emphasizing the roles of antimicrobial resistance genes (ARGs), biofilm formation, and active efflux systems. A total of 162 Staphylococci, eight Escherichia coli, and seven Klebsiella spp. isolates were obtained from 215 milk samples of clinical and subclinical mastitis cases. Antibiotic susceptibility testing identified Twenty Staphylococci (12.35 %), six E. coli (75 %) and seven Klebsiella (100 %) identified as MDR displaying significant resistance to ß-lactams and tetracyclines The Multiple Antibiotic Resistance (MAR) index of these isolates ranged from 0.375 to 1.0, highlighting extensive resistance. Notably, 29 of the 33 MDR isolates produced biofilms on Congo red agar, while all exhibited biofilm formation in the Microtitre Plate assay. Critical ARGs (blaZ, blaTEM, blaCTX-M, tetM, tetA, tetB, tetC, strA/B, aadA) and efflux pump genes (acrB, acrE, acrF, emrB, norB) regulating active efflux were identified. This pioneering study elucidates the synergistic contribution of ARGs, biofilm production, and efflux pump activity to MDR in bovine mastitis pathogens. To our knowledge, this comprehensive study is the first of its kind, offering novel insights into the complex resistance mechanisms. The findings underscore the imperative need for advanced antibiotic stewardship and strategic interventions in dairy farming to curb the rise of antibiotic-resistant infections, thereby protecting both animal and public health.