Agr Type Research Articles

Virulence and antimicrobial resistance genes profiles of spa type t605 methicillin-susceptible Staphylococcus aureus isolated from subclinical bovine mastitis.

Staphylococcus aureus is one of the most common pathogens associated with mastitis in dairy herds worldwide. This study evaluated the profile of virulence and antimicrobial resistance genes of spa type t605 methicillin-susceptible Staphylococcus aureus isolated from subclinical bovine mastitis in São Paulo, Brazil. A total of 57 S. aureus strains were screened by conventional PCR (Polymerase Chain Reaction) for 49 virulence genes. The most prevalent virulence genes detected were icaD (94.7%), fib (93%), fnbA (82.5%), clfA (80.7%), bap (78.9%), clfB (73.7%), icaA (66.7%), see (64.9%), and sed (61.4%). The blaZ (94.7%), aac6'aph2' (15.8%), and ant4 (12.3%) genes were the most common antimicrobial resistance genes; however, mecA and mecC genes were not found. All methicillin-susceptible S. aureus (MSSA) strains were characterized through spa and agr typing. The spa type t605 was found in all isolates. By agr typing, the most prevalent were type II (56.1%). Antimicrobial resistance was determined by the disk diffusion method, and 93% showed resistance to at least one antibiotic. Penicillin resistance was the most prevalent (87.7%), followed by tetracycline (12.3%), oxacillin (10.5%), and gentamicin (10.5%) resistance. Our study confirmed the spa type t605 as endemic, carrying a wide variety of virulence factors and high-level penicillin resistance. The profile seems to be associated with the colonization of MSSA and its persistence in subclinical mastitis.

Dissemination of epidemic ST239/ST241-t037-agrI-SCCmecIII methicillin-resistant Staphylococcus aureus in a Tunisian trauma burnintensive care unit.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing health care-infectionsin the world, especially in burns. The aim of this study was to assess the extent of dissemination of MRSA isolated from burn patients in Burn Intensive Care Unit in Tunisia and to evaluate the frequency of virulence and antibiotics resistance genes. Among the 72 S. aureus isolates analyzed in thestudy, 54% were MRSA. The majority of MRSA (94.8%) were multidrug resistant and they had a high resistance rates to kanamycin (94.8%), tobramycin (90%), tetracycline (94.8%) and ciprofloxacin and rifampicin (87%, each). The gene aac(6')-Ie-aph(2″)-Ia conferring resistance to kanamycine and tobtamycin were detected in all isolates and the aph(3')-Ia gene conferring resistance to gentamicin were detected in 2.8% of resistant isolates. Tetracycline resistance genes tet(M), tet(K) and tet(L) were detected in 100%, 10.8% and 2.8% of the isolates, respectively. The SCCmec type III and the agr type I were the most predominant (69.2% and 90%, respectively). The 27 SCCmecIII-agrI isolates were clustered into two PFGE types A and B. The two representative isolates of PFGE clusters A and B belonged to ST239-t037 and ST241-t037 respectively. As conclusion, our results showed a high prevalenceof MRSA in trauma burn intensive care unit belonging to two multidrug resistant clones ST239/ST241-agrI-t037-SCCmecIII MRSA. We also demonstrated that MRSA was disseminated betweenburn patients.

High Prevalence of Clonal Complex 398 Methicillin-Susceptible and -Resistant Staphylococcus aureus in Pig Farms: Clonal Lineages, Multiple Drug Resistance, and Occurrence of the Staphylococcal Cassette Chromosome mec IX

High prevalence of livestock-associated methicillin-susceptible and -resistant Staphylococcus aureus (LA-MSSA and LA-MRSA, respectively) in livestock farms, particularly pig farms, is an increasingly serious threat to food safety and public health. In this study, 173 S. aureus (84 MRSA and 89 MSSA) isolates from healthy pigs, farm environments, and farm workers in Korea were examined to determine the (1) genetic diversity of S. aureus isolates (sequence type [ST], spa, and agr types), (2) staphylococcal cassette chromosome mec (SCCmec) types of MRSA isolates, and (3) multidrug resistance (MDR) phenotypes of MRSA and MSSA isolates. Clonal complex 398 (CC398) genotypes of MRSA and MSSA isolates, particularly CC398-spa type t571-agr I lineages, displaying MDR phenotypes were highly prevalent in pig farms. High prevalence of CC398-t571 MRSA and MSSA was more frequently associated with weaning piglets and growing pigs. Moreover, the same clonal lineages of S. aureus isolates colonized both pigs and farm workers, suggesting the transmission of antimicrobial-resistant CC398 MRSA and MSSA between pigs and humans in the pig farms. Furthermore, two dominant SCCmec types, SCCmec V and SCCmec IX, were identified in CC398 MRSA isolates colonizing healthy pigs. To the best of our knowledge, this is the first report of a CC398 LA-MRSA isolate carrying SCCmec IX in Korea. Collectively, these results suggest widespread distribution of the CC398 lineage among MRSA and MSSA isolates in pigs, farm environments, and farm workers in Korea.

Molecular Characterization of Community- and Hospital- Acquired Methicillin-Resistant Staphylococcus aureus Isolates during COVID-19 Pandemic.

Methicillin-resistant Staphylococcus aureus (MRSA) is a drug-resistant superbug that causes various types of community- and hospital-acquired infectious diseases. The current study was aimed to see the genetic characteristics and gene expression of MRSA isolates of nosocomial origin. A total of 221 MRSA isolates were identified from 2965 clinical samples. To identify the bacterial isolates, the clinical samples were inoculated on blood agar media plates first and incubated at 37 °C for 18-24 h. For further identification, the Gram staining and various biochemical tests were performed once the colonies appeared on the inoculated agar plates. The phenotypic identification of antibiotic susceptibility patterns was carried out using Kirby-Bauer disk diffusion method by following the Clinical and Laboratory Standards Institute (CLSI) 2019 guidelines. The biofilm-producing potentials of MRSA were checked quantitatively using a spectrophotometric assay. All strains were characterized genotypically by SCCmec and agr typing using the specific gene primers. Furthermore, a total of twelve adhesion genes were amplified in all MRSA isolates. MRSA was a frequently isolated pathogen (44% community acquired (CA)-MRSA and 56% hospital acquired (HA)-MRSA), respectively. Most of the MRSA isolates were weak biofilm producers (78%), followed by moderate (25%) and strong (7%) biofilm producers, respectively. Prominent adhesion genes were clfB (100%), icaAD (91%), fib (91%), sdrC (91%) followed by eno (89%), fnbA (77%), sdrE (67%), icaBC (65%), clfA (65%), fnbB (57%), sdrD (57%), and cna (48%), respectively. The results of the current study will help to understand and manage the spectrum of biofilm-producing MRSA-associated hospital-acquired infections and to provide potential molecular candidates for the identification of biofilm-producing MRSA.

Resistance and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Heterogeneous Vancomycin-Intermediate Staphylococcus aureus.

To investigate the molecular epidemiology of Staphylococcus aureus (S. aureus) isolated from patients in the Yellow River Delta region and to construct antibiotic resistance profiles in different genetic backgrounds. Antibiotics susceptibility testing, staphylococcal protein A (spa) typing and accessory gene regulator (agr) typing were performed for all the 204 strains. Isolates with the positive mecA gene and heterogeneous vancomycin-intermediate S. aureus (hVISA) were subjected to multilocus sequence typing (MLST) and Staphylococcal chromosomal cassettes mec (SCCmec) typing. Thirty-nine MRSA strains were identified by mecA gene. Twenty-two hVISA isolates including 9 MRSA and 13 methicillin-sensitive S. aureus (MSSA) strains were confirmed, the rest isolates (n = 182) were vancomycin-susceptible S. aureus (VSSA). 94.9% of MRSA and 63.6% of hVISA belonged to agrI. Fifty-seven distinct spa types including 5 novel types were mainly t309 (30.9%), t078 (11.8%) and t437 (11.8%). Fourteen sequence types (STs) containing 3 new STs were classified into 3 clone complexes (CCs) and 7 singletons among MRSA and hVISA isolates. Most MRSA isolates (87.2%) belonged to type Ⅳ SCCmec. The predominant genotype among MRSA population was ST59-t437-agrI-IVa (53.8%), followed by ST72-t2431-agrI-IVF (15.4%). ST72 and CC5 (ST5/965/7197) were the most common hVISA clones. Both CC59 (ST59/7437) and ST72 clones were resistant to erythromycin and clindamycin among MRSA population. Strains of MSSA with phenotypic hVISA (MS-hVISA) exhibited a striking genetic diversity accompanied by the diversification of drug resistance patterns.

Staphylococcus aureus chronic intramammary infections in dairy cows: Pathogen-specific characteristics

Abstract Staphylococcus aureus is one of the main causes of mastitis in dairy cows worldwide. The most common form of the disease is characterized by chronic subclinical infection with sporadic clinical episodes that can persist throughout the life of the animal. Once infection evolves to chronicity, the bacteriological cure rate following classic antibiotic therapy is low and persistently infected cows become the main reservoir of the organism in a dairy herd. The establishment and persistence of S. aureus intramammary infection (IMI) has been associated with certain characteristics of the pathogen that allow it to evade the immune response or induce a deficient immune response that is unable to eliminate the pathogen. The main pathogen characteristics that have been suggested to be linked to S. aureus persistence in the bovine mammary gland (MG), include the capacity to invade cells and/or survive intracellularly and to form biofilms, the capsular polysaccharides (CP) production, the ability to form small colony variants (SCVs), the accessory gene regulator ( agr ) type of the strain and the capacity to acquire antibiotic resistance. This review summarizes current knowledge about the most relevant pathogen-specific characteristics that are associated with the establishment and chronicity of S. aureus IMI. A better understanding of these pathogen characteristics will allow for the development of intervention strategies aimed at improving different aspects of the control of this disease, including diagnostic methods, therapeutics and modulation of the host immune response.

Determination of Virulence Factors and Resistance Profile of Methicillin-Resistant Staphylococcus aureus Strains among Different Types of spa, agr, and SCCmec.

In order to restrict the spread of methicillin-resistant S. aureus (MRSA) in hospitals, it is necessary to characterize isolates rapidly and precisely. The objective of this study was to determine virulence factors and resistance profiles of MRSA strains among spa, agr, and SCCmec types. In total, 55 MRSA isolates were collected from clinical specimens. The MRSA isolates were characterized by antimicrobial susceptibility testing, virulence genes, agr typing, spa typing, and SCCmec typing. According to our findings, all MRSA strains were resistant to cefoxitin; 88% and 86.7% of which were resistant to erythromycin and clindamycin, respectively. Type II agr was predominant with 54.54% frequency. Among 27 different spa types, type t030 was most frequently (25.45%). Most MRSA isolates (63.3%) were SCCmec type III. The pvl and tst genes were found in 25.3% and 32.7% of MRSA isolates, respectively. Among the MRSA strains, ermA, ermB, and ermC were present in 50%, 33.3%, and 57.3% of cases, respectively. In addition, 43 of the 55 MRSA strains (78%) harbored aminoglycoside resistance genes. The results of our study revealed that the MRSA rate in our region is dramatically high. Better infection control guidelines in hospitals, as well as ongoing epidemiological surveillance studies, could be strongly suggested for effective prevention of the spread of MRSA to inpatients.

Characterization of the virulence, agr typing and antimicrobial resistance profile of Staphylococcus aureus strains isolated from food handlers in Brazil.

Staphylococcus aureus is one of the main pathogens associated with foodborne outbreaks in Brazil and food handlers can carry toxigenic and resistant S. aureus strains. The aims of this study were to verify the frequency of virulence genes, to identify the agr groups and to determine the antimicrobial resistance profile of S. aureus strains isolated from food handlers of pilot kitchens located in São Paulo, Brazil. A total of 74 strains of the Staphylococcus genus were detected and 50% were identified as of the species S. aureus. The enterotoxin genes detection, tst and luk-PV detection, agr typing, mecA detection, ccr complex detection and SCCmec typing were performed using PCR. The antimicrobial resistance testing was performed by the disk diffusion method. The enterotoxin genes were identified in 36 S. aureus, including sea (83.8%). The tst gene was detected in 18.92% of the strains and the luk-PV was detected in only one isolate. Agr typing classified 58.3% of the strains as type I. Seven (18.92%) strains were classified as MRSA and the ccr2 complex was detected in six of these isolates. The SCCmec typing characterized strains as type II, III, IV and V. Moreover, there were also a greater number of resistant strains to penicillin (83.78%) and clarithromycin (67.57%). In conclusion, the study revealed a significant prevalence of S. aureus, and the presence of different virulence genes and a worrying resistance profile in S. aureus strains isolated from food handlers in this country.

Role of sodium salicylate in Staphylococcus aureus quorum sensing, virulence, biofilm formation and antimicrobial susceptibility.

The widespread threat of antibiotic resistance requires new treatment options. Disrupting bacterial communication, quorum sensing (QS), has the potential to reduce pathogenesis by decreasing bacterial virulence. The aim of this study was to investigate the influence of sodium salicylate (NaSa) on Staphylococcus aureus QS, virulence production and biofilm formation. In S. aureus ATCC 25923 (agr III), with or without serum, NaSa (10 mM) downregulated the agr QS system and decreased the secretion levels of alpha-hemolysin, staphopain A and delta-hemolysin. Inhibition of agr expression caused a downregulation of delta-hemolysin, decreasing biofilm dispersal and increasing biofilm formation on polystyrene and titanium under static conditions. In contrast, NaSa did not increase biofilm biomass under flow but caused one log10 reduction in biofilm viability on polystyrene pegs, resulting in biofilms being twice as susceptible to rifampicin. A concentration-dependent effect of NaSa was further observed, where high concentrations (10 mM) decreased agr expression, while low concentrations (≤0.1 mM) increased agr expression. In S. aureus 8325-4 (agr I), a high concentration of NaSa (10 mM) decreased hla expression, and a low concentration of NaSa (≤1 mM) increased rnaIII and hla expression. The activity of NaSa on biofilm formation was dependent on agr type and material surface. Eight clinical strains isolated from prosthetic joint infection (PJI) or wound infection belonging to each of the four agr types were evaluated. The four PJI S. aureus strains did not change their biofilm phenotype with NaSa on the clinically relevant titanium surface. Half of the wound strains (agr III and IV) did not change the biofilm phenotype in the 3D collagen wound model. In addition, compared to the control, ATCC 25923 biofilms formed with 10 mM NaSa in the collagen model were more susceptible to silver. It is concluded that NaSa can inhibit QS in S. aureus, decreasing the levels of toxin production with certain modulation of biofilm formation. The effect on biofilm formation was dependent on the strain and material surface. It is suggested that the observed NaSa inhibition of bacterial communication is a potential alternative or adjuvant to traditional antibiotics.

Clonal distribution and antimicrobial resistance of methicillin-susceptible and -resistant Staphylococcus aureus strains isolated from broiler farms, slaughterhouses, and retail chicken meat

Colonization of food-producing animals by antimicrobial-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has become a serious public health problem worldwide. In the current study, clonal diversities of livestock-associated S. aureus isolates collected from broiler farms, slaughterhouses, and retail chicken meat were examined. Two-hundred S. aureus isolates (43 MRSA and 157 methicillin-susceptible S. aureus [MSSA] isolates) were analyzed to determine 1) the genotypes of the isolates (multilocus sequence, agr, and spa types), 2) the methicillin resistance phenotype and staphylococcal cassette chromosome mec (SCCmec) types, 3) the antimicrobial resistance profiles, and 4) the mutational changes in gyrA, gyrB, parC, and parE in fluoroquinolone-resistant isolates. Fifteen different sequence types (STs) of MSSA strains displaying a relatively high degree of genetic diversity were detected in broiler farms, slaughterhouses, and retail chicken meat. In contrast to MSSA, 2 dominant genetic lineages of MRSA (ST692-SCCmecV with t2249 spa type, and ST188-SCCmecIVa with spa type t189) were found in healthy broilers. The high prevalence of ST692 and ST188 in healthy broilers is associated with high levels of multiple antimicrobial-resistance phenotypes, particularly fluoroquinolone resistance. All fluoroquinolone-resistant isolates carried double point mutations in gyrA (S84L) and parC (S80F), regardless of STs or methicillin resistance. Notably, only the ST188 lineage carried an additional third mutation in gyrB (D494N), correlating with enhanced ciprofloxacin minimum inhibitory concentration values versus the strains with double mutations. These results provide important insights into the genetic diversity of antimicrobial-resistant S. aureus strains associated with the chicken meat production chain, including healthy broilers, in Korea.

Methicillin-resistant Staphylococcus aureus contamination of high-touched surfaces in a university campus.

Methicillin-resistant Staphylococcus (MRSA) is a public and occupational health concern, both in community and healthcare settings. In recent years, community-acquired MRSA (CA-MRSA) has emerged as a major causative agent of infections in individuals with no health care exposure or any of the classical risk factors associated with infections. Environmental surfaces frequently touched by hands play a role in the transmission of CA-MRSA, where inanimate objects are considered potential reservoirs and the source of MRSA infections. The purpose of this study was to examine the prevalence of MRSA on environmental surfaces inside a university campus. A total of 1078 high-touch surface samples were collected from door handles, light switches, desks, keyboards and restroom surfaces. MRSA isolates were identified and confirmed by PCR, utilizing the Staph. aureus nuc and mecA genes. Antibiotic resistance profiles were determined using disc diffusion and minimum inhibitory concertation methods. In addition, the ability to form biofilms was investigated by the 96-well plate microdilution technique. PCR assays were performed to detect enterotoxin and antibiotic-resistant genes. The genetic diversity of MRSA was determined through multi-locus sequence typing (MLST), spa and agr typing methods. The overall contamination of Staph. aureus and MRSA was 14.6% (157/1078) and 2.8% (30/1078), respectively. The highest rate of MRSA contamination was detected in restroom sinks and door handles. All MRSA isolates were MDR, with the highest resistance observed was against trimethoprim-sulfamethoxazole. Most MRSA isolates (29/30, 97%) carried at least one gene encoding for staphylococcal enterotoxins (SE), with 10 different SE genotypes were observed. A total of 16 different spa types were detected among the 30 MRSA isolates. Multi-locus sequence typing revealed that 21 MRSA isolates belonged to eight known sequence types (ST), while nine isolates were novel strains. The most detected ST and spa types were ST22 and t223, respectively. Agr types I and III were represented in 28 out of the 30 isolates. The majority of the isolates carried SCCmec type IV, but only one isolate was positive for PVL. Our findings signify the potential of the high-touch surfaces in harbouring and transmitting MRSA to campus staff and students. Thus, the implementation of effective prevention measures outside the healthcare setting is needed to reduce the risk of acquiring CA-MRSA infections. MRSA infections impose a profound economic burden due to illness and productivity loss. The results of this study not only help us to better understand the environmental reservoirs of this pathogen, but also provide information about its transmission pathways and healthcare settings entry routs.

Staphylococcus aureus and Methicillin-Resistant Coagulase-Negative Staphylococci in Nostrils and Buccal Mucosa of Healthy Camels Used for Recreational Purposes.

Simple SummaryAnimal-associated staphylococci have been isolated in human infections. Therefore, these strains may pose a zoonotic risk in addition to constituting a reservoir for antimicrobial resistance genes. In this study, we isolated Staphylococcus aureus and other species of staphylococci from camels used for recreational activities in the Canary Islands. Most S. aureus lacked the antimicrobial resistance genes, but some staphylococci species carried the mecA gene which confers resistance to methicillin. The carriage of this gene conferring resistance to methicillin in staphylococci isolated from camels may be a public health concern since there is a risk of bacterial transmission to humans during recreational activities. Furthermore, since the Canary Islands are the only camel exporter to the European Union, camels could constitute a source of zoonotic agents to the rest of the European countries.Several different species of animals host staphylococci as normal microbiota. These animals can be a source of staphylococci zoonotic infections. People with routine or occupational exposure to infected/colonized animals are at risk of a potential transmission. Therefore, we aimed to investigate the presence of S. aureus and other staphylococci in camels used for recreational purposes as well as their antimicrobial resistance, virulence factors and genetic lineages. A total of 172 samples were collected from 86 healthy camels (nose and mouth) from different farms located in the Canary Islands, Spain. Antimicrobial susceptibility testing was performed against 14 antimicrobial agents. The presence of virulence genes was studied by PCR. Multilocus sequence typing, spa typing and agr typing were performed in all S. aureus isolates. From the 86 camels tested, 42 staphylococci were isolated, of which there were 11 S. aureus, 13 S. lentus, 12 S. sciuri, 3 S. xylosus, S. epidermidis, S. hominis and S. chromogenes. Staphylococci isolates were resistant to penicillin, ciprofloxacin, clindamycin and fusidic acid. All S. aureus isolates harbored the hla, hlb and hld virulence genes. S. aureus isolates were ascribed to three sequence types (STs) and three spa types. All S. aureus isolates belonged to agr type III. Camels from Gran Canaria used in recreational purposes have a moderate prevalence of S. aureus and other coagulase-negative staphylococci. Nevertheless, S. aureus isolates are susceptible to almost all antibiotics tested.

Staphylococcus aureus Causing Skin and Soft Tissue Infections in Companion Animals: Antimicrobial Resistance Profiles and Clonal Lineages.

Staphylococcus aureus is a relevant agent of skin and soft tissue infections (SSTIs) in animals. Fifty-five S. aureus comprising all SSTI-related isolates in companion animals, collected between 1999 and 2018 (Lab 1) or 2017 and 2018 (Lab 2), were characterized regarding susceptibility to antibiotics and heavy metals and carriage of antimicrobial resistance determinants. Clonal lineages were established by PFGE, MLST and agr typing. Over half of the isolates (56.4%, 31/55) were methicillin-resistant S. aureus (MRSA), and 14.5% showed a multidrug resistance (MDR) phenotype. Resistance was most frequently observed for beta-lactams (81.8%, related to blaZ and/or mecA), fluoroquinolones (56.4%) and macrolides/lincosamides (14.5%, related to erm(A) or erm(C)). The distributions of heavy-metal MICs allowed the detection of non-wild-type populations associated with several resistance genes. The collection showed genetic diversity, with prevalence of clonal lineage ST22-agrI (45.5%, 25/55), comprising only MRSA isolates, and several less frequently detected clones, including ST5-agrII (14.6%, 8/55), ST398-agrI (9.1%, 5/55) and ST72-agrI (7.3%, 4/55). This work highlights the high frequency of SSTI-related MRSA strains that reflect the clonal lineages circulating both in companion animals and humans in Portugal, reinforcing the need for a One Health approach when studying staphylococci causing infections in companion animals.

Antibiotic Resistance and Molecular Profiling of the Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis from India.

Staphylococcus aureus is an opportunistic bacterium known to cause severe infections in humans and animals. It is one of the major bacteria causing subclinical and clinical mastitis, leading to significant economic losses in livestock industry. In this study, we have isolated and characterized 80 S. aureus clinical isolates from mastitis-infected animals. The analysis of antimicrobial susceptibility, molecular typing, biofilm production and genetic determinants was performed to understand molecular and phenotypic features of the prevalent pathogen. Our antibiotic susceptibility assays showed the majority (57.5%) of isolates to be multidrug-resistant (MDR), 38.75% resistant and 3.75% sensitive. We found 25% isolates to be methicillin-resistant S. aureus (MRSA) based on oxacillin susceptibility assays. In the MRSA group, maximum isolates (95%) were MDR compared to 45% in MSSA. Multilocus sequence typing (MLST) revealed 15 different STs; ST-97 was the most common ST, followed by ST-2459, ST-1, ST-9 and ST-72. The agr typing showed agr-I as the most common type, followed by type II and III. Most isolates developed biofilms, which ranged in intensity from strong to weak. The presence or absence of lukS, a virulence-related gene, was found to have a substantial relationship with the biofilm phenotype. However, no significant association was found between biofilm formation and antimicrobial resistance or other virulence genes. We also found four MRSA isolates that were mecA negative based on molecular assays. Our findings reveal the prevalence of multidrug-resistant S. aureus clinical isolates in India that are biofilm positive and have critical genetic factors for disease pathogenesis causing bovine mastitis. This study emphasizes the need for the comprehensive surveillance of S. aureus and other mastitis-causing pathogens to control the disease effectively.

Direct Quantitative Immunochemical Analysis of Autoinducer Peptide IV for Diagnosing and Stratifying Staphylococcus aureus Infections.

An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureusagr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I–III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I–III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus. The research strategy here reported can be extended to the rest of the AIP types of S. aureus, allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients.

Comparison of Automated Ribotyping, spa Typing, and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods.

Species-Wide Phylogenomics of the Staphylococcus aureus Agr Operon Revealed Convergent Evolution of Frameshift Mutations.

ABSTRACTStaphylococcus aureus is a prominent nosocomial pathogen that causes several life-threatening diseases, such as pneumonia and bacteremia. S. aureus modulates the expression of its arsenal of virulence factors through sensing and integrating responses to environmental signals. The agr (accessory gene regulator) quorum sensing (QS) system is a major regulator of virulence phenotypes in S. aureus. There are four agr specificity groups each with a different autoinducer peptide sequence encoded by the agrD gene. Although agr is critical for the expression of many toxins, paradoxically, S. aureus strains often have nonfunctional agr activity due to loss-of-function mutations in the four-gene agr operon. To understand patterns in agr variability across S. aureus, we undertook a species-wide genomic investigation. We developed a software tool (AgrVATE; https://github.com/VishnuRaghuram94/AgrVATE) for typing and detecting frameshift mutations in the agr operon. In an analysis of over 40,000 S. aureus genomes, we showed a close association between agr type and S. aureus clonal complex. We also found a strong linkage between agrBDC alleles (encoding the peptidase, autoinducing peptide itself, and peptide sensor, respectively) but not agrA (encoding the response regulator). More than 5% of the genomes were found to have frameshift mutations in the agr operon. While 52% of these frameshifts occurred only once in the entire species, we observed cases where the recurring mutations evolved convergently across different clonal lineages with no evidence of long-term phylogenetic transmission, suggesting that strains with agr frameshifts were evolutionarily short-lived. Overall, genomic analysis of agr operon suggests evolution through multiple processes with functional consequences that are not fully understood.IMPORTANCE Staphylococcus aureus is a globally pervasive pathogen that produces a plethora of toxic molecules that can harm host immune cells. Production of these toxins is mainly controlled by an active agr quorum-sensing system, which senses and responds to bacterial cell density. However, there are many reports of S. aureus strains with genetic changes leading to impaired agr activity that are often found during chronic bloodstream infections and may be associated with increased disease severity. We developed an open-source software called AgrVATE to type agr systems and identify mutations. We used AgrVATE for a species-wide genomic survey of S. aureus, finding that more than 5% of strains in the public database had nonfunctional agr systems. We also provided new insights into the evolution of these genetic mutations in the agr system. Overall, this study contributes to our understanding of a common but relatively understudied means of virulence regulation in S. aureus.

Çeşitli Klinik Örneklerden İzole Edilen Metisiline Dirençli ve Duyarlı Staphylococcus aureus Suşlarının Toksin Profillerinin Araştırılması

Aim: This study aimed to investigate the superantigenic (SAg) toxin, exfoliative toxin (ET), hemolysin (HLY), leukotoxin (LUK) genes and accessory gene regulator (agr) types in Staphylococcus aureus isolates from various clinical materials. Material and Methods: A total of 190 S. aureus isolates were investigated for the presence of toxin genes, mecA gene and agr types using by polymerase chain reaction (PCR). Results: mecA gene was detected in 87 (45.8%) isolates. Of the 190 S. aureus isolates examined, 83.7% (n=159) were found to be positive for SAg genes. The seg (41.1%) was determined to be the most common toxin gene, followed by sei (38.9%), selo (38.9%), selm (28.4%), sea (%25.8), and tst (18.4%) genes, respectively. Seventy one different SAg toxin profiles were identified. Type I νSaβ encoding seg, sei, selm, seln and selo was the most common mobile genetic element (MGE), which was detected in 37 isolates (19.5%). The hla, hlb, hld, hlg and hlg2 genes were detected in 92.6% (n=176), 1.6% (n=3), 98.9% (n=188), 1.1% (n=2) and 31.6% (n=60) of the isolates, respectively. The pvl gene was detected in 12.6% (n=11) of methicillin resistant S. aureus (MRSA) and 14.6% (n=15) of methicillin sensitive S. aureus (MSSA), respectively (p=0.701). While none of the isolates carried lukM gene, 67% (n=69) of MSSA and 69% (n=60) of MRSA isolates were found to be positive for lukED gene (p=0.519). Conclusion: High occurrence and diversity of toxin genes among S. aureus isolates could be explained by horizontal transmission of toxin genes through MGEs.

Characterization of Staphylococcus pseudintermedius Isolates from Skin Infections of Dogs

Background: Staphylococcus pseudintermedius is a part of the canine skin microflora and an opportunistic pathogen. It plays a central role in canine pyoderma, otitis and surgical wound infections. These conditions correlate with virulence genes distributed in the bacterial genome. These genes determine strain variability on typing, in turn aiding epidemiological surveillance. The aim of this study was to isolate, identify and characterize Staphylococcus pseudintermedius (SP) and Methicillin resistant Staphylococcus pseudintermedius (MRSP) from dogs with skin infections in Chennai, India. Methods: SP and MRSP positive isolates were identified by multiplex PCR for nuc and mecA genes respectively. Characterization of the isolates for virulence genes responsible for biofilm formation (icaA, icaD), cell wall adherence (SpsO, SpsK, SpsP, SpsQ, SpsF), toxins (ExpA, ExpB, SIET, Sel, Se-int, LukS, LukF) and gene regulation (Agr, SarA) was performed. Result: Out of 275 samples, 120 SP and 8 MRSP positive isolates were identified. Only one isolate could be typed as SCCmec Type V whereas other MRSP isolates were non typeable. Agr typing of MRSP isolates revealed type II in 7 isolates and type III in one isolate. Our study revealed that there was no significant difference in the detection of virulence genes between MSSP and MRSP.

Molecular Characterization, Virulence Determinants, and Antimicrobial Resistance Profile of Methicillin-Resistant Staphylococcus aureus in the North of Iran; a High Prevalence of ST239-SCCmec III/t037 Clone

Objectives: Emergence and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) have become a major universal health concern, limiting therapeutic options. Methods: A total number of 37 MRSA isolates, including 19 clinical isolates from hospitalized patients and 18 colonizing isolates from health care workers were identified from 3 hospitals, in Gorgan, North of Iran. Antimicrobial susceptibility test was performed using the disk diffusion method and E-test. The presence of virulence and antibiotic resistance determinants were evaluated by PCR. The genotypical characterization was further analyzed using multi-locus sequence, spa, staphylococcal cassette chromosome, mec (SCCmec), and agr typing. Results: The frequency of MRSA among S. aureus isolates was 38.14% (37/97). The most frequent S. aureus resistant isolates were found to be obstinate against penicillin (98%) and gentamicin (82.5%). Additionally, the lowest resistance rates were found against daptomycin (0%), vancomycin (2.7%), and quinupristin-dalfopristin (5.4%). All MRSA isolates were susceptible to daptomycin with minimum inhibitory concentration (MIC)50/MIC90 of 0.25/0.5 μg/mL. One isolate belonging to sequence type 239 (ST239)-SCCmecIII/t037 clone (MIC ≥16 μg/mL) was resistant to vancomycin. All but 1 isolate that shares ST22-SCCmec IV/t790 strain were positive for both tsst and pvl genes. The most predominant MRSA isolates (27%) were associated with ST239-SCCmec III/t037, and ST239-SCCmec III/t924 (16.2%) clones, subsequently. In our study, circulating MRSA strains were genetically diverse with a high prevalence of ST239-SCCmec III/t037 clone. Conclusion: These findings emphasize the need for future and continuous surveillance studies on MRSA to prevent the dissemination of existing multidrug resistance MRSA clones in an effective manner.