Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood. - Related Documents




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583401.0000Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood. The genus Staphylococcus includes pathogenic and non-pathogenic facultative anaerobes. Due to the plethora of virulence factors encoded in its genome, the species Staphylococcus aureus is known to be the most pathogenic. S. aureus strains harboring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, however, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbor mecA, the genetic driver for staphylococcal methicillin-resistance. In this chapter, the detailed practical procedure for operating a real-time pentaplex PCR assay in blood cultures is described. The pentaplex real-time PCR assay simultaneously detects markers for the presence of bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl), and methicillin resistance (mecA).201728600770
583310.9998Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing. Up to now, blood culturing systems are the method of choice to diagnose bacteremia. However, definitive pathogen identification from positive blood cultures is a time-consuming procedure, requiring subculture and biochemical analysis. We developed a microarray for the identification of Staphylococcus aureus comprising PCR generated gene-segments, which can reduce the blood culture post-processing time to a single day. Moreover, it allows concomitant identification of virulence factors and antibiotic resistance determinants directly from positive blood cultures without previous amplification by PCR. The assay unambiguously identifies most of the important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA-aphD, blaZ and ermA. To obtain positive signals, 20 ng of purified genomic S. aureus DNA or 2 microg of total DNA extracted from blood culture was required. The microarray specifically distinguished S. aureus from gram-negative bacteria as well as from closely related coagulase negative staphylococci (CoNS). The microarray-based identification of S. aureus can be accomplished on the same day blood cultures become positive in the Bactec. The results of our study demonstrate the feasibility of microarray-based systems for the direct identification and characterization of bacteria from cultured clinical specimens.200717141897
583220.9998New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.200415528678
579730.9997PCR-reverse blot hybridization assay for screening and identification of pathogens in sepsis. Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections.201323447637
578340.9997Molecular Investigation and Virulence Determination of Methicillin and Vancomycin Resistant Clinical Staphylococcus Aureus Isolates. Staphylococcus aureus is an opportunistic pathogen that provides conditions for host invasion due to various virulence factors and plays a role in causing various infections. The pathogenicity of these bacteria may vary depending on the host's susceptibility. This study investigates the sensitivity of S. aureus strains isolated from clinical samples to methicillin and vancomycin, and it evaluates the presence of resistance, virulence and toxin-producing genes, and their expression level in the methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-intermediate S. aureus (VISA) isolates. A cross-sectional study was conducted, encompassing 502 S. aureus isolates obtained from diverse infections over the course of a year. The methicillin and vancomycin sensitivities of the isolates were ascertained by disk diffusion and microdilution broth methods, respectively. The presence of genes associated with resistance, adhesion, and toxin production was subsequently investigated through the implementation of multiplex polymerase chain reaction (PCR) methodology. The expression levels of virulence and resistance genes were detected in resistant and sensitive isolates using real-time quantitative PCR (qPCR). Among the 502 S. aureus isolates, 168 (33.6%) were identified as MRSA. Furthermore, a total of six isolates (1.2%) were identified as VRSA, and two isolates (0.4%) were identified as VISA. The distribution of virulence and resistance-related genes varied among the isolates. The results of the gene expression study demonstrated that the expression levels of the majority of the studied genes were significantly higher in resistant isolates (MRSA and VRSA) compared to sensitive isolates. It is imperative to acknowledge that VRSA and MRSA are regarded as grave hazards to human health. The present study underscores the necessity for enhanced sanitary measures to more effectively control this hospital pathogen, particularly in light of the presence and expression of genes encoding virulence factors in S. aureus isolates.202540980455
578250.9997The Efficacy of Bacteriocins Against Biofilm-Producing Bacteria Causing Bovine Clinical Mastitis in Dairy Farms: A New Strategy. Using an alternative bio-product is one of the most promising ways to control bovine mastitis and avoid new intra-mammary infections. The aims of this study were to ascertain the prevalence of biofilm-forming bacteria responsible for causing clinical mastitis in dairy herds and to assess the effectiveness of bacteriocins, produced by Bacillus subtilis, in controlling the growth of these bacteria in the milk of animals. A total of 150 milk samples were collected from cows and buffalos suffering from mastitis and the etiological agents were isolated and identified by the VITEK-2-COMPACT-SYSTEM®. Additionally, the capability of the bacterial isolates to produce biofilms was determined. RT-PCR was used to detect enterotoxin-producing genes (sed and seb), resistance genes (mecA and blaZ), and biofilm-associated genes (icaA and fnbA) in the isolated bacteria. The susceptibility patterns of the bacterial isolates to bacteriocins were assessed using an agar well-diffusion assay. S. aureus was significantly more capable of producing biofilms than coagulase-negative Staphylococcus isolates. S. ubris was the strongest biofilm producer among the Streptococcus species. The sensitivity profiles of the Staphylococcus spp. (S. aureus and coagulase-negative Staphylococcus) and their biofilm producers to bacteriocins were significantly higher (100% and 90%, respectively) at the same concentration. Bacteriocins had a lethal effect on Staphylococci, Streptococci, and biofilm development at a dose of 250 µg/mL. In dairy farms, bacteriocins are a viable alternative treatment for the prevention and control of bovine clinical mastitis.202337256384
577960.9997Development of a One-Step Multiplex qPCR Assay for Detection of Methicillin and Vancomycin Drug Resistance Genes in Antibiotic-Resistant Bacteria. The most common antibiotic-resistant bacteria in Korea are methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Pathogen identification in clinical laboratories can be divided into traditional phenotype- and genotype-based methods, both of which are complementary to each other. The genotype-based method using multiplex real-time polymerase chain reaction (PCR) is a rapid and accurate technique that analyzes material at the genetic level by targeting genes simultaneously. Accordingly, we aimed to develop a rapid method for studying the genetic characteristics of antibiotic-resistant bacteria and to provide an experimental guide for the efficient antibiotic resistance gene analysis of mecA detection for MRSA and vanA or vanB detection for VRE using a one-step multiplex qPCR assay at an early stage of infection. As a result, the sensitivity and specificity of the mecA gene for clinical S. aureus isolates, including MRSA and methicillin-susceptible S. aureus, were 97.44% (95% CI, 86.82-99.87%) and 96.15% (95% CI, 87.02-99.32%), respectively. The receiver operating characteristic area under the curve for the diagnosis of MRSA was 0.9798 (*** p < 0.0001). Therefore, the molecular diagnostic method using this newly developed one-step multiplex qPCR assay can provide accurate and rapid results for the treatment of patients with MRSA and VRE infections.202439452724
550470.9997Whole Genome Sequencing of Staphylococci Isolated From Bovine Milk Samples. Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non-aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus. However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus. The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus, which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infections.202134987483
597480.9997Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus. As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.201021083822
596890.9997A PCR assay for rapid detection of vancomycin-resistant enterococci. Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.200212007446
5778100.9996A Simple and Rapid Low-Cost Procedure for Detection of Vancomycin-Resistance Genes in Enterococci Reveals an Outbreak of Vancomycin-Variable Enterococcus faecium. The detection of resistance to vancomycin in enterococci cultured from patients is important for the treatment of individual patients and for the prevention of hospital transmission. Phenotypic antimicrobial resistance tests may fail to detect potential vancomycin-resistant enterococci. We have developed and tested a PCR based procedure for routine screening for vancomycin-resistance genes in clinical samples with enterococci. Primary cultures from diagnostic samples reported with growth of Enterococcus faecium or E. facalis were tested for vanA and vanB genes by real-time PCR without the isolation of specific bacteria. Up to ten samples were pooled and tested in each real-time PCR reaction, with subsequent individual testing of cultures from positive pools. In a one-month test period in 2017 vanA gene was detected in one out of 340 urine samples with vancomycin-susceptible enterococci reported from diagnostic culture. A second test period in 2018 included 357 urine samples, and vanA gene was detected in samples from eight patients. Subsequently, all urine samples reported with growth of E. faecium during a period of one year were tested. Fifty-eight individuals were identified with enterococci, carrying the vanA gene not previously detected. Routine molecular testing of primary culture material from patient samples may improve the detection of hospitalized patients carrying E. faecium with resistance genes to vancomycin.202236140520
2403110.9996Characterization of coagulase-negative staphylococci and macrococci isolated from cheese in Germany. Cheese, especially ripened varieties, harbor a very complex and heterogeneous microbiota. In addition to the desired microorganisms (starter cultures) added during cheese production, potentially harmful bacteria may also enter the production chain. Regarding the latter, the focus of this study was on coagulase-negative staphylococci (CNS) and Macrococcuscaseolyticus. Both are known to harbor a variety of genes coding for antibiotic resistance, including mecA, mecB, mecC, and mecD. Coagulase-negative staphylococci or macrococci carrying such genes or other virulence factors should not be present in cheese. Cheese samples (101 in total) were collected from retail sources. Coagulase-negative staphylococci and M. caseolyticus were isolated utilizing selective agars, and species were identified by phenotypical tests and partial sequencing of the sodA gene. The results allowed identification of 53 CNS strains and 19 M. caseolyticus strains. Among the CNS, 11 isolates of Staphylococcus saprophyticus and one Staphylococcus epidermidis isolate were obtained. Both species are potential human pathogens and may thus adversely affect the safety of these food products. Screening for antimicrobial resistance was performed by application of disc diffusion tests, a gradient strip-test, and 14 different PCR tests. Evidence for methicillin resistance (by either positive disc diffusion assay for cefoxitin or by mec PCR) was found in CNS isolates and M. caseolyticus (9 isolates each). Regarding other virulence factors, no genetic determinants for coagulase or the most common staphylococcal enterotoxins sea, seb, sec, sed, and see were detected in any of the CNS or M. caseolyticus isolates by PCR testing. In conclusion, the presence of facultatively pathogenic CNS and carriers of genes for antibiotic resistance in both groups of microorganisms, especially mec genes, and the respective food safety issues need further evaluation and surveillance.202235965117
5776120.9996Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae. BACKGROUND: Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macrolide resistance genes. METHODOLOGY: One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay. RESULTS: A multiplex PCR comprising the antibiotic resistance genes, ermB and and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics. CONCLUSION: The characterization of pneumococcal isolates for penicillin and erythromycin resistance genes could be useful to predict the susceptibility of these isolates to other antibiotics, especially beta-lactams drugs. We have developed an assay with a shorter turnaround time to determine the species and resistance profile of Streptococcus pneumoniae with respect to penicillin and macrolides using the Real Time PCR format with fluorescent labeled Taqman probes, hence facilitating earlier and more definitive antimicrobial therapy which may lead to better patient management.200819738350
5539130.9996Staphylococcus aureus from Subclinical Cases of Mastitis in Dairy Cattle in Poland, What Are They Hiding? Antibiotic Resistance and Virulence Profile. Bovine mastitis is a common disease worldwide, and staphylococci are one of the most important etiological factors of this disease. Staphylococcus aureus show adaptability to new conditions, by which monitoring their virulence and antibiotic resistance mechanisms is extremely important, as it can lead to the development of new therapies and prevention programs. In this study, we analyzed Staphylococcus aureus (n = 28) obtained from dairy cattle with subclinical mastitis in Poland. The sensitivity of the isolated strains to antibiotics were confirmed by the disc diffusion method. Additionally, minimum inhibitory concentration values were determined for vancomycin, cefoxitin and oxacillin. Genotyping was performed by two methods: PCR melting profile and MLVF-PCR (multiple-locus variable-number tandem-repeat fingerprinting). Furthermore, the presence of antibiotic resistance and virulence genes were checked using PCR reactions. The analyzed strains showed the greatest resistance to penicillin (57%), oxytetracycline (25%) and tetracycline (18%). Among the analyzed staphylococci, the presence of 9 of 15 selected virulence-related genes was confirmed, of which the icaD, clfB and sea genes were confirmed in all staphylococci. Biofilm was observed in the great majority of the analyzed bacteria (at least 70%). In the case of genotyping among the analyzed staphylococci (combined analysis of results from two methods), 14 patterns were distinguished, of which type 2 was the dominant one (n = 10). This study provides new data that highlights the importance of the dominance of biofilm over antibiotic resistance among the analyzed strains.202236558738
5831140.9996Development of a nucleic acid lateral flow immunoassay (NALFIA) for reliable, simple and rapid detection of the methicillin resistance genes mecA and mecC. The gene mecA and its homologue mecC confer methicillin resistance in Staphylococcus aureus and other staphylococci. Methicillin-resistant staphylococci (MRS) are considered resistant to all β-lactam antibiotics. To avoid the use of β-lactam antibiotics for the control of MRS infections, there is an urgent need for a fast and reliable screening assay for mecA and mecC that can easily be integrated in routine laboratory diagnostics. The aim of this study was the development of such a rapid detection method for methicillin resistance based on nucleic acid lateral flow immunoassay (NALFIA) technology. In NALFIA, the target sequences are PCR-amplified, immobilized via antigen-antibody interaction and finally visualized as distinct black bars resulting from neutravidin-labeled carbon particles via biotin-neutravidin interaction. A screening of 60 defined strains (MRS and non-target bacteria) and 28 methicillin-resistant S. aureus (MRSA) isolates from clinical samples was performed with PCR-NALFIA in comparison to PCR with subsequent gel electrophoresis (PCR-GE) and real-time PCR. While all samples were correctly identified with all assays, PCR-NALFIA was superior with respect to limits of detection. Moreover, this assay allowed for differentiation between mecA and mecC by visualizing the two alleles at different positions on NALFIA test stripes. However, since this test system only targets the mecA and mecC genes, it does not allow to determine in which staphylococcal species the mec gene is included. Requiring only a fraction of the time needed for cultural methods (i.e. the gold standard), the PCR-NALFIA presented here is easy to handle and can be readily integrated into laboratory diagnostics.201727569992
2393150.9996Detection of a mecC-positive Staphylococcus saprophyticus from bovine mastitis in Argentina. INTRODUCTION: Bovine mastitis causes important economic losses in the dairy industry. Coagulase-negative staphylococci (CNS) are a group of bacteria commonly isolated from bovine mastitis and can display resistance to a wide range of antimicrobial agents. OBJECTIVES: The objective of this study was to determine staphylococcal resistance towards β-lactam, macrolide and lincosamide antimicrobials in quarters previously treated with third-generation cephalosporin and after lincosamide intramammary therapy. METHODS: Sick quarters of eighteen cows from Villaguay, Entre Ríos (Argentina) with clinical mastitis were studied. All staphylococcal isolates were tested by disk diffusion for their antimicrobial susceptibilities. Cefoxitin resistance was investigated by PCR and sequencing for both the mecA and mecC genes. RESULTS: Resistances to penicillin, oxacillin and cefoxitin were observed, whereas no resistance to macrolide and lincosamide was detected. A cefoxitin-resistant Staphylococcus saprophyticus was found to be mecA-negative but mecC-positive. CONCLUSIONS: This study reports for the first time the mecC gene from a CNS in bovine mastitis in South America. Because CNS may act as reservoirs of antimicrobial resistance genes, they can be seen as a potential public health threat with respect to antimicrobial resistance and the development of multiple resistance. Also, the emergence of methicillin-resistant phenotypes will limit therapeutic options.201728732791
5596160.9996Enterotoxigenicity and Antibiotic Resistance of Coagulase-Negative Staphylococci Isolated from Raw Buffalo and Cow Milk. Staphylococcal food poisoning is considered to be one of the most common foodborne illnesses worldwide. Because milk is rich in nutrients and its neutral pH, it leads to the growth of various bacteria. To date, the correlation between enterotoxigenic potential in Staphylococcus species and antimicrobial resistance (AMR), using bioinformatics analysis in buffalo and cow raw milk and the possible health risks from these bacteria, has not been examined in Egypt. A total of 42 Staphylococcus isolates representing 12 coagulase-positive staphylococci (Staphylococcus aureus and Staphylococcus intermedius) and 30 coagulase-negative staphylococci (Staphylococcus capitis, Staphylococcus xylosus, Staphylococcus carnosus, Staphylococcus saccharolyticus, and Staphylococcus auricularis) were isolated. An assay of the antimicrobial resistance phenotypes indicated low resistance against vancomycin (9.5%). The blaZ gene was associated with penicillin G and methicillin resistance and not with sulbactam + ampicillin. The presence of the gene ermB presented the correlation with erythromycin resistance and tetK with tetracycline resistance (correlation index: 0.57 and 0.49, respectively), despite the absence of the same behavior for ermC and tetM, respectively. Interestingly, the gene mecA was not correlated with resistance to methicillin or any other β-lactam. Correlation showed that slime-producing isolates had more resistance to antibiotics than those of nonslime producers. The multiple correlations between antibiotic resistance phenotypes and resistance genes indicate a complex nature of resistance in Staphylococcus species. The antimicrobial resistance could potentially spread to the community and thus, the resistance of Staphylococcus species to various antibiotics does not depend only on the use of a single antimicrobial, but also extends to other unrelated classes of antimicrobials.202031750778
5781170.9996Antibiotic susceptibility of human-associated Staphylococcus aureus and its relation to agr typing, virulence genes, and biofilm formation. BACKGROUND AND OBJECTIVE: Carriage of virulence factors confers some evolutionary benefit to bacteria, which favors the resistant strains. We aimed to analyze whether antibiotic susceptibility of Staphylococcus aureus strains is affected by agr typing, biofilm formation ability, and virulence profiles. METHODS: A total of 123 S. aureus clinical isolates were subjected to antimicrobial susceptibility testing by disk diffusion method, biofilm formation by microtiter plate method, as well as polymerase chain reaction screening to identify virulence genes and the accessory gene regulator (agr) types I-IV. A P value < 0.05 was considered significant. RESULTS: The most prevalent virulence gene was staphyloxanthin crtN, followed by hemolysin genes, capsular cap8H, toxic shock toxin tst, and enterotoxin sea, respectively. Resistant isolates were more commonly found in the agr-negative group than in the agr-positive group. Isolates of agr type III were more virulent than agr I isolates. Strong biofilm producers showed more antibiotic susceptibility and carried more virulence genes than non-strong biofilm producers. Associations were found between the presence of virulence genes and susceptibility to antibiotics. Carriage of the virulence genes and agr was higher in the inpatients; while, resistance and strong biofilms were more prevalent in the outpatients. CONCLUSION: These findings indicated the presence of several virulence factors, biofilm production capacity, agr types and resistance to antibiotics in clinical S. aureus isolates. Considering the importance of S. aureus for human medicine, an understanding of virulence and resistance relationships would help to reduce the impact of S. aureus infections.202134210263
5502180.9996Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk. The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.201930928272
5807190.9996ST8-t008-SCC (mec) IV methicillin-resistant Staphylococcus aureus in retail fresh cheese. This study reports the finding of 3 ST8-t008-SCC (mec) IVa (2B) methicillin-resistant Staphylococcus aureus (MRSA) strains in fresh cheese purchased within a single market in Costa Rica. In line with the finding of the resistance genes mecA, blaZ, mph(C), and msr(A) in their genomes, these bacteria showed phenotypic resistance to multiple β-lactams and erythromycin. In addition, they carry genes for acquired resistance to aminoglycosides (aph(3')-III) and fosfomycin (fosD), and genes for a myriad of virulence factors, including adhesins, hemolysins, and exotoxins. Our strains share multiple genomic features with MRSA from the USA300 lineage, which is a widely distributed and highly virulent strain implicated in community infections. As a result, consuming these or similar products could lead to multidrug infections in susceptible individuals. These results highlight safety deficiencies in cheese production practices and emphasize the risk of foodborne transmission of hard-to-treat ST8 MRSA strains.202439650008