Epidemiological Characteristics of Staphylococcus Aureus in Raw Goat Milk in Shaanxi Province, China.
ABSTRACT: Goat milk has been frequently implicated in staphylococcal food poisoning. The potential risk of raw goat milk contaminated by Staphylococcus aureus (S. aureus) in Shaanxi province of China is still not well documented. This study investigated the prevalence, antibiotic resistance, as well as virulence-related genes of S. aureus from raw goat milk samples in Shaanxi, China. A total of 68 S. aureus isolates were cultured from 289 raw goat milk. Most of the isolates were resistant to penicillin and oxacillin, although 41.18%, 33.82%, and 29.41% of the isolates expressed resistance to piperacillin, trimethoprim-sulfamethoxazole, and ciprofloxacin, respectively. Our data demonstrated that 91.18% of the isolates produced biofilm, of which 54.41% isolates belonged to high-biofilm producers. In addition, genotypic analysis of biofilm related genes (fnbA, clfB, fnbB, cna) revealed that 91.18% of the isolates harbored at least one of the genes, in which the most prevalent genes were fnbA (66. 17%), clfB (48.53%), and fnbB (26.47%). 94.8% of the isolates contained at least one toxin-related gene, of which seb (76.47%), tsst (36.76%), and sea (23.53%) genes were the more frequently detected. Further analysis revealed a positive association between fnbA, clfB, fnbB, seb, tsst, and sea genes and certain antibiotic resistance. The results indicated that raw goat milk samples contaminated by S. aureus can be a potential risk to public health.
Project description:Staphylococcus aureus encodes many proteins that act as virulence factors, leading to a variety of diseases, including mastitis in cows. Among these virulence factors, SpA, ClfA, ClfB, FnbA, and FnbB are important for the ability of S. aureus to adhere to and invade host cells as well as to evade host immune responses. The interaction between these S. aureus surface proteins and human immunoglobulin G and fibrinogen that are coupled to latex particles is utilized to induce latex agglutination reactions, which are used widely in diagnostic kits for confirmation of presumptive S. aureus isolates. In this study, the Staphaurex latex agglutination test was performed on a collection of confirmed bovine mastitis S. aureus isolates. Notably, 54% (43/79 isolates) of these isolates exhibited latex agglutination-negative phenotypes (Staphaurex-negative result). To gain insights into the reasons for the high frequency of Staphaurex-negative bovine mastitis S. aureus isolates, the spa, clfA, clfB, fnbA, and fnbB genes were examined. Specific genetic changes in spa, clfA, and fnbA, as well as a loss of fnbB, which may impair SpA, ClfA, FnbA, and FnbB functions in latex agglutination reactions, were detected in Staphaurex-negative S. aureus isolates. The genetic changes included a premature stop codon in the spa gene, leading to a truncated SpA protein that is unable to participate in S. aureus cell-mediated agglutination of latex particles. In addition, clfA and fnbA genetic polymorphisms were detected that were linked to ClfA and FnbA amino acid changes that may significantly reduce fibrinogen-binding activity. The genetic variations in these S. aureus isolates might also have implications for their bovine mastitis virulence capacity.
Project description:Staphylococcus aureus is the predominant bacterium responsible for various diseases in animals and humans. Preventive strategies could be better implemented by understanding the prevalence, genetic patterns, and the presence of enterotoxin and biofilm-producing genes along with the antibiotic susceptibility of this organism. This study was conducted in Rajasthan, the northwestern state of India, holding the largest population of cattle that makes it the second largest milk producer in India and no such prior information is available on these aspects.A total of 368 individual quarter bovine raw milk samples were collected from 13 districts of Rajasthan, and screened for the presence of S. aureus. Microbiological and molecular approaches were followed for bacterial identification. Genetic diversity was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of coagulase gene (coa), whereas enterotoxin and biofilm-producing genes were studied by PCR analysis. Antibiotic strips were employed to study the antibiotic resistance among strains.In all, 73 S. aureus strains were obtained from 368 bovine raw milk samples out of that only 30 showed the presence of coa. Nine types of coa patterns ranging from 730 to 1130 bp were observed among these isolates. PCR-RFLP of coa distinguished the isolates into 15 genotypic patterns, of which patterns I, IV, V, and VI were predominant. Of the isolates, 30% were positive for sec, 10% for sea, and 3.3% for seb; these genes are responsible for enterotoxin production, whereas all isolates were found positive for icaAD and eno. The prevalence rates of other biofilm-producing genes fnbA, clfB, ebpS, sasG, fnbB, sasC, cna, bap, fib and, bbp were 97, 93, 90, 80, 80, 77, 53, 27, 10, and 6.6%, respectively. Twenty-seven (90%) strains were multidrug resistant, of which 15 were methicillin resistant. Maximum sensitivity was reported for kanamycin and it could be considered as a drug of choice for controlling S. aureus mediated cattle infections in the studied regions.Overall, these strains could cause several diseases to humans, insisting the need for developing a stricter hygiene program for improving milking practices and animal health.
Project description:Nonsynonymous single nucleotide polymorphisms (SNPs) in fibronectin binding protein A (fnbA) of Staphylococcus aureus are associated with cardiac device infections. However, the role of fnbA SNPs in S. aureus arthroplasty infection is unknown.Bloodstream S. aureus isolates from a derivation cohort of patients at a single U.S. medical center with S. aureus bacteremia (SAB) and prosthetic hip or knee arthroplasties that were infected (PJI, n = 27) or uninfected (PJU, n = 43) underwent sequencing of fnbA and fnbB. A validation cohort of S. aureus bloodstream PJI (n = 12) and PJU (n = 58) isolates from Germany also underwent fnbA and fnbB sequencing.Overall, none of the individual fnbA or fnbB SNPs were significantly associated with the PJI or PJU clinical groups within the derivation cohort. Similarly, none of the individual fnbA or fnbB SNPs were associated with PJI or PJU when the analysis was restricted to patients with either early SAB (i.e., bacteremia occurring <1 year after placement or manipulation of prostheses) or late SAB (i.e., bacteremia >1 year after placement or manipulation of prostheses).In contrast to cardiac device infections, there is no association between nonsynonymous SNPs in fnbA or fnbB of bloodstream S. aureus isolates and arthroplasty infection. These results suggest that initial steps leading to S. aureus infection of cardiovascular and orthopedic prostheses may arise by distinct processes.
Project description:<i>Staphylococcus aureus</i> (<i>S. aureus</i>) infections are notoriously complicated by the ability of the organism to grow in biofilms and are difficult to eradicate with antimicrobial therapy. The purpose of the current study was to clarify the influence of sub-inhibitory concentrations (sub-MICs) of daptomycin and tigecycline antibiotics on biofilm adhesion factors and exoproteins expressions by <i>S. aureus</i> clinical isolates. Six clinical isolates representing positive biofilm <i>S. aureus</i> clones (3 methicillin-sensitive <i>S. aureus</i> (MSSA) and 3 methicillin-resistant <i>S. aureus</i> (MRSA)) were grown with sub-MICs (0.5 MIC) of two antibiotics (daptomycin and tigecycline) for 12 h of incubation. RNA extracted from culture pellets was used via relative quantitative real-time-PCR (qRT-PCR) to determine expression of specific adhesion (<i>fnbA</i>, <i>fnbB</i>, <i>clfA</i>, <i>clfB</i>, <i>fib</i>, <i>ebps</i>, <i>cna</i>, <i>eno</i>) and biofilm (<i>icaADBC</i>) genes. To examine the effect of sub-MIC of these antibiotics on the expression of extracellular proteins, samples from the culture supernatants of six isolates were collected after 12 h of treatment with or without tigecycline in order to profile protein production via 2D gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D gel-SDS-PAGE). Sub-MIC treatment of all clinical MRSA and MSSA strains with daptomycin or tigecycline dramatically induced or suppressed <i>fnbA</i>, <i>fnbB</i>, <i>clfA</i>, <i>clfB</i>, <i>fib</i>, <i>ebps</i>, <i>cna</i>, <i>eno</i>, and <i>icaADBC</i> gene expression. Furthermore, sub-MIC use of tigecycline significantly reduced the total number of separated protein spots across all the isolates, as well as decreasing production of certain individual proteins. Collectively, this study showed very different responses in terms of both gene expression and protein secretion across the various isolates. In addition, our results suggest that sub-MIC usage of daptomycin and tigecycline could signal virulence induction by <i>S. aureus</i> via the regulation of biofilm adhesion factor genes and exoproteins. If translating findings to the clinical treatment of <i>S. aureus</i>, the therapeutic regimen should be adapted depending on antibiotic, the virulence factor and strain type.
Project description:Staphylococcus aureus is one of the most important food-borne pathogens globally. It produces various toxins and invasive enzymes and can be found in numerous food products. Milk is an important source of staphylococcal food poisoning. After pasteurization, this microorganism or its enterotoxins might still remain in pasteurized milk. Therefore, this study was to investigate the contamination of S. aureus in 258 pasteurized milk from 39 cities of China. The prevalence and levels of S. aureus in these samples as well as antibiotic susceptibility profiles, virulence genes, biofilm formation, and biofilm related genes, spa typing and MLST were used to determine the characterization among the isolates. It was found 3.9% of samples were detected S. aureus in 8 of 39 cities in China. The contaminated level were not very excessive which showed the MPN values of the most positive samples (9/10) were less than 1 MPN/g. All pasteurized milk-related S. aureus isolates have ability to produce biofilm and harbored icaA, icaD, eno, clfA, clfB, fnbA, fnbB, fib genes, other biofilm related genes icaC were showed in 91.7% of isolates and cna gene were showed in 50%, except bap gene which were free in all isolates. The antibiotic susceptibility test showed that all isolates were resistant or intermediate-resistant to different concentrations of the antibiotics. Furthermore, 75.0% of the isolates were resistant to three or more antibiotic classes, which indicated multidrug resistance. The isolates had virulence potential, which showed 66.7% (8/12) of the isolates carried one or more virulence-associated genes. Molecular typing by MLST and spa typing enabled classification of these isolates into a total of 11 sequence types (STs) and spa types, which indicated high genetic diversity. Most of these types were related to various clinical S. aureus infections. Thus, the findings of this study reflect the potential risk of S. aureus infection in China. Our study also provides comprehensive analysis of the prevalence of S. aureus in pasteurized milk and helps ensure more accurate treatment of human infection with effective antibiotics.
Project description:Staphylococcus aureus isolates collected from sites of intramammary infection during a 10-month period and from extramammary sites (dairy cow teat skin, teat canals, and skin lesions; milking liners; and hands and nostrils of milking personnel) at two separately managed Finnish dairy herd establishments were analyzed to study the sources and reservoirs of bovine S. aureus intramammary infection. Selected isolates were subjected to pulsed-field gel electrophoresis (PFGE) typing and PCR analysis for genes encoding hemolysins (hla to hlg), leukocidins (lukED and lukM), superantigens (sea, sec, sed, seg to seo, seu, and tst), adhesins (fnbA and fnbB), and penicillin and methicillin resistance (blaZ and mecA). S. aureus was found throughout the herds in 94% of the cows. Nine PFGE types were found, with the herds each having their own predominant type and sharing one type. The degree of diversity of PFGE types in herd II, which integrated foreign heifers, was higher than that in herd I. For both herds, the majority of the PFGE-typed isolates both from milk and from extramammary sites represented the predominant PFGE types. In isolates from herd I, the most prevalent genes were hla-hlg, lukED, and fnbA; in those from herd II, they were hla, hld, hlg, lukED, and fnbA. The other genes were pulsotype linked within the herds. The predominant PFGE types carried both fnbA and fnbB; only fnbA was detected in the other PFGE types. No connection between specific virulence genes and the origins of isolates was found. The results suggest that for the two herds, most S. aureus isolates from extramammary sites were indistinguishable from the isolates infecting the mammary gland and that those sites can thus act as origins and reservoirs of intramammary infections. However, contamination in the opposite direction cannot be excluded.
Project description:: Staphylococcus aureus (S. aureus) is one of the most clinically important zoonotic pathogens, but an understanding of the prevalence, biofilm formulation ability, virulence, and antimicrobial resistance genes of S. aureus from veterinary hospitals is lacking. By characterizing S. aureus in different origins of veterinary hospitals in Guangzhou, China, in 2019, we identified with the presence of S. aureus in pets (17.1%), veterinarians (31.7%), airborne dust (19.1%), environmental surfaces (4.3%), and medical device surfaces (10.8%). Multilocus sequence typing (MLST) and Staphylococcus protein A (spa) typing analyses demonstrated methicillin-sensitive S. aureus (MSSA) ST398-t571, MSSA ST188-t189, and methicillin-resistant S. aureus (MRSA) ST59-t437 were the most prevalent lineage. S. aureus with similar pulsed-field gel electrophoresis (PFGE) types distributed widely in different kinds of samples. The crystal violet straining assays revealed 100% (3/3) of MRSA ST59 and 81.8% (9/11) of MSSA ST188 showed strong biofilm formulation ability, whereas other STs (ST1, ST5, ST7, ST15, ST88, ST398, ST3154 and ST5353) showed weak biofilm production ability. Polymerase chain reaction (PCR) confirmed the most prevalent leucocidin, staphylococcal enterotoxins, ica operon, and adhesion genes were lukD-lukE (49.0%), sec-sel (15.7%), icaA-icaB-icaC-icaR (100.0%), and fnbB-cidA-fib-ebps-eno (100.0%), respectively. Our study showed that the isolates with strong biofilm production ability had a higher prevalence in clfA, clfB, fnbA and sdrC genes compared to the isolates with weak biofilm production ability. Furthermore, 2 ST1-MRSA isolates with tst gene and 1 ST88-MSSA isolate with lukS/F-PV gene were detected. In conclusion, the clonal dissemination of S. aureus of different origins in veterinary hospitals may have occurred; the biofilm production capacity of S. aureus is strongly correlated with ST types; some adhesion genes such as clfA, clfB, fnbA, and sdrC may pose an influence on biofilm production ability and the emergence of lukS/F-PV and tst genes in S. aureus from veterinary hospitals should raise our vigilance.
Project description:We used genomic fingerprinting to investigate an outbreak of methicillin-resistant Staphylococcus aureus in the neonatal intensive care units (NICUs) of two hospitals. The hospitals are located in the same city and are part of the same medical care system. Fingerprinting was done by Southern blot hybridization with DNA probes for the genes encoding the S. aureus collagen adhesin (cna), fibronectin-binding proteins (fnbA and fnbB), and beta-toxin (hlb). Genomic DNA was digested with HaeIII (cna and fnbA-fnbB probes) or HindIII (hlb probe). Hybridization patterns could be distinguished on the basis of (i) the presence or absence of cna, (ii) the size of the restriction fragment containing the cna gene, (iii) restriction fragment length polymorphisms within fnbA and fnbB, (iv) the presence of a lysogenic phage within hlb, and (v) the sizes of the restriction fragments containing the phage-bacterial DNA junction fragments. Over a period of 4 months we examined a total of 46 isolates obtained from various wards within each hospital. Among these 46 isolates, we observed a total of 4 cna patterns, 11 fnbA-fnbB patterns, and 11 hlb patterns. Southern blots with HaeIII-digested genomic DNA and a combination of all three gene probes revealed a total of 16 clearly distinguishable patterns. A total of 22 of the 46 isolates were identical with respect to every genomic marker examined. A total of 21 of these 22 isolates were obtained from patients within an NICU. Nineteen of 21 isolates also exhibited identical antibiotic resistance profiles (antibiogram). Although 5 of the remaining 24 strains exhibited an antibiogram identical to those of the NICU isolates, all 24 strains could be distinguished from the NICU isolates by at least one genomic marker. These results suggest that the NICU isolates had a common origin and that genomic fingerprinting with the cna, fnbA, fnbB, and hlb gene probes can provide an important epidemiological tool for the identification of clinical isolates of S. aureus.
Project description:Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) isolates have previously been classified into major epidemic clonal types by pulsed-field gel electrophoresis in combination with multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec typing. We aimed to investigate whether genetic variability in potentially polymorphic domains of virulence-related factors could provide another level of differentiation in a diverse collection of epidemic MRSA clones. The target regions of strains representative of epidemic clones and genetically related methicillin-susceptible S. aureus isolates from the 1960s that were sequenced included the R domains of clfA and clfB; the D, W, and M regions of fnbA and fnbB; and three regions in the agr operon. Sequence variation ranged from very conserved regions, such as those for RNAIII and the agr interpromoter region, to the highly polymorphic R regions of the clf genes. The sequences of the clf R domains could be grouped into six major sequence types on the basis of the sequences in their 3' regions. Six sequence types were also observed for the fnb sequences at the amino acid level. From an evolutionary point of view, it was interesting that a small DNA stretch at the 3' clf R-domain sequence and the fnb sequences agreed with the results of MLST for this set of strains. In particular, clfB R-domain sequences, which had a high discriminatory capacity and with which the types distinguished were congruent with those obtained by other molecular typing methods, have potential for use for the typing of S. aureus. Clone- and strain-specific sequence motifs in the clf and fnb genes may represent useful additions to a typing methodology with a DNA array.