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High prevalence of t895 and t9364 spa types of methicillin-resistant Staphylococcus aureus in a tertiary-care hospital in Mexico: different lineages of clonal complex 5
C. Negrete-González1,
E. Turrubiartes-Martínez1,2,
O. G. Galicia-Cruz3,
D. E. Noyola4,
G. Martínez-Aguilar5,
L. F. Pérez-González6,
R. González-Amaro7 &
P. Niño-Moreno ORCID: orcid.org/0000-0001-7335-71561,8
BMC Microbiology volume 20, Article number: 213 (2020) Cite this article
Staphylococcus aureus is a leading cause of broad-spectrum infections both in the community and within healthcare settings. Methicillin-resistant Staphylococcus aureus (MRSA) has become a global public health issue. The aim of this study was to examine the clinical and molecular characteristics of Staphylococcus aureus isolates and to define the population structure and distribution of major MRSA clones isolated in a tertiary-care hospital in Mexico.
From April 2017 to April 2018, 191 Staphylococcus aureus isolates were collected. The frequency of MRSA was 26.7%; these strains exhibited resistance to clindamycin (84.3%), erythromycin (86.2%), levofloxacin (80.3%), and ciprofloxacin (86.3%). The majority of MRSA strains harbored the SCCmec type II (76.4%) and t895 (56.8%) and t9364 (11.7%) were the most common spa types in both hospital-associated MRSA and community-associated MRSA isolates. ST5-MRSA-II-t895 (New York /Japan clone) and ST1011-MRSA-II-t9364 (New York /Japan-Mexican Variant clone) were the most frequently identified clones. Furthermore, different lineages of Clonal Complexes 5 (85.4%) and 8 (8.3%) were predominantly identified in this study.
Our study provides valuable information about the epidemiology of MRSA in a city of the central region of Mexico, and this is the first report on the association between t895 and t9364 spa types and ST5 and ST1011 lineages, respectively. These findings support the importance of permanent surveillance of MRSA aimed to detect the evolutionary changes of the endemic clones and the emergence of new strains.
Staphylococcus aureus (S. aureus) is a commensal and a pathogen in humans; approximately 30–50% of the population are transient nasal carriers and 10–20% of individuals are persistently colonized with this organism [1, 2]. Furthermore, colonization of the skin or mucosa with S. aureus may increase the risk of invasive infections [3]. In addition, S. aureus has been recognized as an extremely versatile pathogen in humans, causing three major syndromes: superficial lesions, such as impetigo and skin wound infections; deep and systemic infections, such as osteomyelitis, endocarditis, pneumonia, and bacteremia; and toxemic infections, such as toxic shock syndrome, scalded skin syndrome, and food poisoning [4].
In 1961, one year after the introduction of methicillin into medical practice, the first methicillin-resistant Staphylococcus aureus (MRSA) strain was identified; methicillin resistance is mediated by the Staphylococcal Cassette Chromosome mec (SCCmec) genetic element [5]. This element includes the mec and ccr gene complexes, which are flanked by three junkyard regions. SCCmec is inserted into a unique site of the bacterial chromosome by the action of Ccr proteins (encoded by the ccr gene complex), which induce the specific recombination between the attB sequence at the 3′ end and the attS homologous sequence of SCCmec [6]. Variations in the genetic content and structural organization of these elements result in 13 different types and subtypes of SCCmec [7,8,9].
An increasing number of MRSA strains were identified initially in hospital centers (HA-MRSA) and, several years later, cases of community-associated MRSA infections (CA-MRSA) were reported. In this regard, the epidemiology of MRSA infections has changed significantly with the global emergence and expansion of CA-MRSA strains [10].
The most frequently reported MRSA isolates belong to major Clonal Complexes (CC) CC1, CC5, CC8, CC22, CC30, CC45, and CC80 [11,12,13]. The most representative HA-MRSA clones are ST5-I/EMRSA-3/Cordobes-Chilean and ST5-II/USA100/New York/Japan clones (CC5), ST36-II/USA200 clone (CC30), ST45-II/USA600 clone (CC45), and ST239 III/ Brazilian/Hungarian clone (CC8), while the most representative CA-MRSA are ST1-IV/USA400 (CC1), ST5-IV/Pediatric clone (CC5), ST8-IV/USA300 and USA300-LA variant (CC8), EMRSA-15 clone (CC22), ST30-IV/Southwest Pacific clone (CC30), and ST80-IV/European clone (CC80) [12, 14]. The distribution of these clones varies in different countries and regions of the world; in Mexico, ST5-II/New York Japan and USA300 clones have been described [15, 16].
According to the World Health Organization (WHO), global surveillance of MRSA is essential for the identification of international transmission routes and the subsequent development of effective prevention and control strategies of this pathogen [17]. For this purpose, molecular typing methods are a valuable tool for the successful characterization of S. aureus isolates. In this regard, Next Generation Sequencing (NGS) has been used to identify S. aureus CCs and is considered the best laboratory technique for identification of DNA diversity in any organism. However, this methodology remains technically demanding and requires robust software to analyze the results [12]. Traditional typing methods include Multiple Locus Sequence Typing (MLST), Pulsed-Field Gel Electrophoresis (PGFE), and spa-typing. MLST is a great tool for evolutionary investigations and strain identification and is based on the allelic profile of the seven housekeeping genes. PFGE is based on the digestion of DNA with restriction endonucleases and the detection of the banding patterns. Although these two methods show a high discriminatory power, they are laborious and require intra-laboratory standardization protocols [12]. On the other hand, spa-typing is based on the detection of sequence variation in repeats at the X region of the staphylococcal protein A spa gene. This typing technique exhibits high discriminatory power, has a standardized nomenclature, is cost-effective, and shows an excellent reproducibility. Spa-typing can be used for the investigation of hospital outbreaks and to analyze the evolution of S. aureus [18]. However, this methodology has some limitations, mainly in regions where a particular clone or a small number of clones are endemic [11, 19].
The aim of this study was to estimate the prevalence of MRSA and to analyze the molecular characteristics, and antibiotic resistance profiles of CA- and HA-MRSA genotypes in San Luis Potosi, a large city (approximately 1.1 million inhabitants) in the center of Mexico.
S. aureus strains were obtained from one hundred ninety-one patients from the emergency department (n = 62), surgery (n = 47), intensive care unit (n = 31), internal medicine (n = 35), gynecology (n = 6), burn unit (n = 2), and outpatient service (n = 8); patients in whom samples were obtained in the outpatient service were subsequently admitted to the hospital. The clinical specimens were obtained from infections in skin and soft tissues (n = 79), respiratory tract (n = 53), blood (n = 36), bone and joints (n = 20), and cerebrospinal fluid (n = 3). Seventy-seven percent (147 out of 191) of strains were considered as HA and 23 % (44 out of 191) were classified as CA.
One hundred fourteen patients were male and seventy-seven were female. Forty isolates were identified in children, and 151 in adults. The median age was 44 years. The mean length of hospital stay was 18.4 ± 19.5 days (range 1–105 days). Table 1 shows comorbidities, surgical procedures, and history of hospital admission in the last two years before infection of participants in the study. The majority of patients (84.4%) were discharged due to clinical improvement, 2% of patients were transferred to another hospital, 1.6% of patients requested voluntary discharge, and 11.5% of patients infected with S. aureus died.
Table 1 Clinical and demographic characteristics of the patients with S. aureus infection included in the study
Ten (45.5%) of the 22 patients who died had an MRSA infection compared to 41 (24.3%) of the 169 patients who survived (P = 0.034). Patients who died were also older (mean 46.3 years) than those who survived (mean 35.2 years; P = 0.034). In contrast, there were no significant differences in the prevalence of underlying conditions (such as diabetes, malignancy, or renal disease) between patients with a fatal outcome and those who did not die (Additional file 1: Table S1).
Identification of MRSA strains
The mecA gene was detected in 51 out of 191 isolates (26.7%), and 45 of them showed resistance to oxacillin and were positive on cefoxitin-based screening. The study was carried out between epidemiological week (as defined by WHO) 14, 2017 and epidemiological week 17, 2018. The weekly number of S. aureus infections varied between 1 and 8 cases. As shown in Fig. 1, the largest number of cases was observed at week 37 (eight, two of them MRSA), whereas in weeks 25, 35, 38, 50, 9 and 12, six cases were identified. Moreover, the highest weekly number of MRSA cases was 4, in weeks 38 and 41, followed by weeks 20, 50, and 9 with 3 cases. Two MRSA cases were identified in weeks 18, 19, 23, 37, 39, 45, 46, 48, 5, and 13, whereas a single case was detected in weeks 14, 15, 17, 21, 25, 26, 29, 34, 35, 42, 51, 6, 8, 11, 12, and 14. No MRSA isolates were observed during weeks 16, 22, 24, 27, 28, 30–33, 36, 40, 43, 44, 47, 49, 52, 1–4, 7, 10, 15–17. Three different periods of MRSA detection were identified during the study. The first period occurred between weeks 14 and 29 (2017), the second between weeks 34 and 51(2017), and the third between weeks 5 and 14 (2018) Fig. 1.
Number of cases of S. aureus infection in each epidemiological week. Black bars correspond to MRSA isolates and grey bars to MSSA strains
Antimicrobial susceptibility
The antibiotic resistance pattern differed significantly between MRSA and MSSA isolates (P < 0.001 in most cases). Thus, most MRSA strains showed resistance to clindamycin (84.3%), erythromycin (86.2%), levofloxacin (80.3%), and ciprofloxacin (86.3%), with low resistance to gentamicin (13.7%) and rifampin (9.8%). In contrast, MSSA strains showed minimal resistance to clindamycin (7.1%), erythromycin (9.3%), ciprofloxacin (3.5%), levofloxacin (1.4%), and gentamicin (1.4%). None of MRSA or MSSA strains were resistant to vancomycin, linezolid, tigecycline, trimethoprim/sulfamethoxazole, and tetracycline (Table 2). Additional file 2: Table S2, shows the minimum inhibitory concentration for each antibiotic.
Table 2 Antibiotic resistant pattern of MSSA and MRSA isolates
SCCmec typing
Thirty-nine MRSA strains were classified as SCCmec type II (four CA-MRSA, and thirty-five HA-MRSA) and the SCCmec subtype IIb was identified in four strains (HA-MRSA). Two isolates harbored SCCmec type IVc/E and SCCmec type IVa was identified in one isolate (one CA-MRSA and two HA-MRSA). In five isolates it was not possible to identify the SCCmec types.
Spa-typing
MRSA isolates were classified in 11 different spa types, including t895 (n = 29, 56.8%), t9364 (n = 6, 11.7%), t008 (n = 2, 3.9%), t003 (n = 3, 5.8%), t4229, t002, t012, t040, t304, t111, and t509 (n = 1). The spa type t895 was the most common spa type among HA-MRSA and CA-MRSA isolates. In one strain, we identified a spa type not previously reported (spa type unknown). In three isolates, the PCR employed by us did not amplify the spa gene.
Dendrogram of MRSA strains
A dendrogram was constructed to analyze the relation among S. aureus strains based on their spa type (Fig. 2). The spa type t895 (cluster 1) was identified in twenty-nine isolates in patients from the surgery ward (n = 15), emergency department (n = 4), internal medicine (n = 3), intensive care unit (n = 1), and outpatient service (n = 1). In children, t895 was identified in the pediatric ward (n = 4) and neonatal intensive care unit (n = 1). Twenty-seven isolates harbored the SCCmec type II, one isolate harbored SCCmec type IIb, and in one strain the SCCmec type was not identified. Twenty-seven isolates of this cluster showed resistance to beta-lactams, fluoroquinolones (levofloxacin and ciprofloxacin), clindamycin, and erythromycin. The B-796 strain was rifampin-resistant, and the B-766 strain was gentamicin-resistant. The origin of MRSA strains in this cluster was predominantly HA-MRSA (n = 27), and only two cases were CA-MRSA (C-706 and C-708); the latter isolates were identified in the surgery ward, in weeks 13 and 14. In this cluster seven patients died.
Dendrogram of MRSA strains. Dendrogram constructed using the unweighted pair group method with arithmetic average (UPGMA) based on pairwise similarity values of spa types from 48 characterized MRSA strains. The scale corresponds to the percent of similarity. Blue branch corresponds to cluster 1, red branch to cluster 2, green branch to cluster 3, and orange branch to cluster 4
The cluster 2 (t9364) included six isolates, three from the surgery ward and one from the intensive care unit, the burn unit and the internal medicine ward. Five isolates in this cluster harbored the SCCmec type II, and one the SCCmec type IIb; strains from both SCC types showed resistance to fluoroquinolones, clindamycin, and erythromycin. Four of these strains (A-747, A-786, B-713, and B-773) were resistant to rifampin, and another (B-748) to gentamicin. The percent of similarity between spa types t895 and t9364 was 99.5%. The A-792 (t002) and B-706 (t509) isolates showed more than 98% of similarity with t985 and t9364, and A-701 (t111) showed 97.6% of similarity to spa types t895, t9364 and t003.
The cluster 3 (t003) included three isolates, two of them were detected in children (A-736 and C-703), and one (B-770) from a patient in the surgery ward. In this group, two isolates harbored SCCmec type IVc/E and were resistant to beta-lactams. In the other isolate we identified SCCmec type II.
The spa type t040 had a 92.7% similarity with the spa types t895, t9364, t003 and t002, and had 91.5% similarity with an unknown spa type that was identified in B-700. This strain was only resistant to beta-lactams.
The cluster 4 had 90.8% similarity with the previously mentioned spa types. This cluster included four isolates with t008, t4229 and t304 spa types. Two patients from the surgery and burn wards were infected with HA-MRSA-t008, A-713 strain was isolated in the eighteen week and harbored the SCCmec type II, and B-705 was isolated in the thirty-nine week and harbored the SCCmec type IVa.
Multilocus sequence typing
Spa types t895 and t9364, the major spa types identified in this study, have not previously been associated to sequence types (ST). In order to analyze this, we selected six isolates, and these were identified as ST1011 (n = 4) and ST5 (n = 2). The association analysis of spa types of clusters 3 to 4 with ST was performed in the Spa server.
In the present study, we have assessed the epidemiological characteristics of S. aureus isolates during one year of intra-hospital surveillance and we analyzed the molecular characteristics of MRSA strains. The most frequent S. aureus infections were those affecting the skin and soft tissues (n = 48, 25.1%) and bacteremia (n = 31, 17%). In contrast, the most frequent type of infection caused by MRSA isolates was surgical site infection (n = 14, 27%).
The mortality associated with staphylococcal infections in our study (11.5%) was lower than that previously reported (approximately 15 years ago) in Mexico (50%) [20]. MRSA infections were detected more frequently in fatal cases than in patients who survived. Study participants who died were also older than those who survived. Of note, while the presence of underlying diseases, history of surgical procedures, and health-care exposure have previously been reported to be associated with fatal infections [21, 22], we did not find significant differences for these conditions between patients who died and those who survived.
The recent epidemiology of S. aureus has focused on the increase and spread of MRSA strains in the healthcare setting and the community. In Denmark and Scandinavian countries the prevalence of MRSA is less than 1%. In contrast, in the east and southeast of Europe, the prevalence of MRSA is greater than 30% [22]. Peru has the highest reported prevalence in Latin America (80%) [17]. In Mexico, there are a limited number of studies about MRSA and the available information shows an increase in the prevalence of MRSA ranging from 7 to 53% between 1989 and 2017 [15, 23,24,25]. In our study, the prevalence of MRSA identified by molecular methods was higher than the prevalence identified by the oxacillin resistance phenotype, which has been the most used method in our country [17, 25, 26]. The 26.7% prevalence registered in our study is higher than that reported, between January and June 2018, in 47 hospitals in 20 states of Mexico (21.4%) [26], a study that did not include information from the state of San Luis Potosi. In a previous study, performed between 2005 and 2006 in six Mexican hospitals, the prevalence of MRSA ranged from 1 to 43%. The highest prevalence was recorded at the Hospital Central Dr. Ignacio Morones Prieto (HCIMP) [27]. In 11 years, the prevalence of MRSA decreased to 26.7% in this hospital. This fact can be explained by the infection control actions implemented. A study that highlights the importance of intra-hospital surveillance of MRSA was carried out between January 1997 and May 2003 at the Pediatric Hospital of the Centro Medico Nacional-Siglo XXI (Mexico City). At this hospital, the annual frequency of methicillin resistance ranged from 17 to 23% between 1997 and 2001, and dramatically decreased in 2002 (4%) and 2003 (0%), due to the intervention of the infection control committee at the end of 2001 [28].
Until February 2020 the Spa server has recorded 19,255 different spa types [29]. According to a literature review, in the last decade, the spa types t032/t008/t002 are the most prevalent in Europe, t037/t002 in Asia, t008/t002/t242 in America, t037/t084/t064 in Africa, and t020 in Australia [5]. Interestingly, in our study the prevalence of the spa types commonly described in America was lower than expected, and we mainly detected the spa types t895 and t9364.
Compared to other spa types, t895 has a low frequency (0.01%); however, in the last two years its detection has increased. Between 2017 and 2019, eight strains were reported in USA and another in Germany, according to the Spa Server [29]. Although data is scarce, previous reports have associated the t895 spa type with CC5 [30]. In this regard, our data suggest an apparent association between t895 spa type and the ST5 lineage of CC5. In 97% of the MRSA-t895 isolates, the SCCmec type II cassette was identified; these molecular characteristics correspond to the New York / Japan/ USA100 clone [15]. However, a limitation of this study is that the sequence types (MLST) of several of the t895 strains were not determined, which precluded a proper statistical analysis. Of note, SCCmec type I, type II, and type IV have been reported previously in MRSA-t895 strains [31].
The identification of t895 as the predominant spa type in our study is of relevance, since this may have clinical and epidemiological implications. Of interest, characterization of 21 MRSA strains isolated in Estado de Mexico (Mexico) in 2013 also showed t895 to be the predominant spa type, accounting for 76.2% of isolates [31]. In a study conducted in the United States, t895 spa type was predictive for the weak-biofilm producing phenotype, compared to t008 spa type, which was identified as a predictor of the strong-biofilm producing phenotype [30].
The spa type t9364 was registered in 2011 and corresponded to a strain detected in Mexico, in a region outside of the state of San Luis Potosi [29]. In this regard, our data describe, for the first time, the association between the t9364 spa type and the ST1011 sequence type. Sequence type ST1011 was registered in the MLST database in 2006; the first report of this ST included four clinical MRSA isolates which differed from the sequence type ST5 by the replacement of a nucleotide in the arcC gene. Three of these MRSA ST1011 isolates were identified at HCIMP and one at General Hospital of Durango [27]. Between 2008 and 2017, 14 isolates have been reported with the sequence type ST1011 and the SCCmec type II [15, 16]; all of these isolates have been identified in Mexico. In 2017, ST1011-II was classified as the New York / Japan clone because of its similarity to ST5-II [15], and in a subsequent phylogenetic analysis of CC5, it was observed that the clones identified in Mexico were grouped in a subclade that was subdivided into two subclades: ST5-II and ST1011-II. This suggests that ST1011-II is not a New York/ Japan clone, but it may be a variant of it that originated in the late 1990s, the period when the CC30 was replaced in Mexico [16]. In all, available data suggests that ST1011-II-t9364 may be a Mexican variant of the New York / Japan clone which has increased in prevalence in the last 11 years; however, more studies are required to determine the differences with ST5-II-t895 [16].
Other spa types identified with lower frequency in this study corresponded to t111, t509, t003, t012, t040, t4229, and t304. The spa type t003 has been related to ST225 and ST270 sequence types, which are part of CC5 and includes the Rhine Hesse, EMRSA-3 and New York/Japan clones [32]. In addition, the spa types t012 and t040 have been identified in strains belonging to CC30 and CC45, respectively. Furthermore, the spa types t4229 and t304 have been associated with ST8, ST247, ST250, and ST254 sequence types, which belong to CC8 and include the USA300, ORSA IV and Archaic/Iberian clones [33].
Diverse lineages of CC5 were predominant in our study. These strains are characterized by bearing the SCCmec I, II, and IV type cassettes with subtypes IVa, IVc/E, and IVg. In this regard, different studies have shown that most strains of this CC are multi-resistant, mainly to fluoroquinolones, aminoglycosides, macrolides, lincosamides, and streptogramins (as we detected in the isolates of our study), except for those that carry the IVc/E cassette that only show resistance to beta-lactams [15]. Moreover, to determine the relationship between MRSA strains, we classified them into clusters and analyzed their clinical and molecular characteristics. In this analysis, clusters 1 and 2 were distributed in all areas of the hospital within the three periods described previously; in this regard, it is possible that these three periods could be due to different introductions of clones into the hospital or be a consequence of intra-hospital transmission [34]. Although these two possibilities are plausible, the last one could have resulted from transfer of patients between different hospital wards during their stay. Moreover, all strains grouped in these two clusters were multi-resistant, and the highest number of deaths was recorded in cluster 1. Furthermore, most strains in cluster 3 were only resistant to beta-lactams and the methicillin resistance phenotype was not identified. Finally, three out of four isolates in cluster 4 were identified in weeks 38 and 39, the epidemiological weeks with the highest number of S. aureus infections. The use of efficient and accurate epidemiological typing methods is a requisite for monitoring the spread of epidemic clones within and between hospitals. In this case, spa-typing was a good tool for differentiate into CC5 lineages, because t895 and t9364 are not widespread spa types [19]. It is worth mentioning that if t895 and t9364 clones become endemic and spread to multiple regions of Mexico, the discriminating power of spa-typing to analyze nosocomial transmission would decrease. To overcome this limitation, recent studies suggest the use of a combination of different typing techniques to increase the ability to discriminate isolates [35].
In our study, all S. aureus strains were susceptible to tetracycline, doxycycline and minocycline [36], and trimethoprim-sulfamethoxazole. This observation is of relevance, since these are alternatives for ambulatory treatment of MRSA infections, such as skin and soft tissue infections [37]. In contrast, tetracycline resistance was reported in 6% of MSSA strains and 17% of MRSA strains collected globally between 1997 and 2016 [38]. Resistance to this antibiotic in S. aureus is encoded by the tetK and tetM genes [39], mainly detected in SCCmec III, IV, and V MRSA strains [9, 40, 41]. The majority of MRSA strains in our study had SCCmec type II, and this might explain, in part, the absence of tetracycline resistance.
Our data indicate that the most prevalent clones in all areas of our hospital were ST5-MRSA-II-t895 (New York /Japan clone) and ST1011-MRSA-II-t9364 (New York/Japan-Mexican Variant clone), which belong to CC5. In the HCIMP, the dominance of two CC5 lineages is evident; however, MRSA isolates with molecular characteristics consistent with Irish (weeks 18, 38 and 39), USA300 (week 39) and Pediatric (week 13) clones, that are considered epidemic MRSA clones, were identified. We consider that this study further supports continuous molecular monitoring of S. aureus infections as a valuable tool for epidemiological surveillance of MRSA since it allows the evaluation of evolutionary changes of endemic clones and the introduction of emerging clones that can cause hospital outbreaks. In addition, subsequent studies that assess the correlation between the phenotype and the MRSA genotype are required, as well as characterization of additional features of these clusters, including virulence factors and resistance genes.
This cross-sectional study was conducted at HCIMP in San Luis Potosi, Mexico, after approval by the Research Committee [COFEPRIS 14 CI 24028083] and the Research Ethics Committee of the HCIMP [CONBIOETICA-24-CEI-001-20,160,427]. The registration number was 29–17. Informed consent was obtained from all participants or legal guardians.
The city of San Luis Potosi is located in central Mexico and is the capital of the state of San Luis Potosi. HCIMP provides medical services to mid- and low-income populations from all over the state; it has 250 beds and 32 beds in the intensive care unit (ICU).
From April 2017 to April 2018, a total of 191 non-repeated S. aureus isolates were obtained from different patients in all hospital wards. These isolates were identified by using the Vitek 2C (bioMérieux) system and confirmed by PCR amplification of the nuc gene.
Demographic and clinical data, including sex, age, date of hospitalization, type of infection, date of isolation, underlying disease and outcome of infection were collected from medical records. Patients were classified in groups according to their age, as follows: infants (0 to 1-year-old), children (2 to 10 years old), adolescents (11 to 17 years old), young adults (18 to 35 years old), adults (36 to 60 years old), and seniors (more than 60 years old). An infection was considered as CA when symptoms presented < 48 h of a patient's hospital admission, in the absence of previous healthcare exposure, whereas an infection was considered as HA when occurred 48 h after patient admission or when it was associated with the following risk factors: hospitalization in an acute care unit for at least 48 h in the last year, chemotherapy administration, hemodialysis, wound care, enteric nutrition or specialized nursing care 30 days before the infection [15, 42, 43].
Antimicrobial susceptibility testing was performed using Vitek 2C (bioMérieux) and results were interpreted using the Clinical and Laboratory Standards Institute guidelines. Antibiotics tested included benzylpenicillin, clindamycin, erythromycin, levofloxacin, ciprofloxacin, moxifloxacin, rifampin, gentamicin, vancomycin, tetracycline, linezolid, oxacillin, and cefoxitin test [36].
Three colonies of an overnight culture were suspended in 100 μL of DNase free water and incubated at 94 °C for 5 min and − 70 °C for additional 5 min. Then, tubes were centrifuged at 13,000 rpm for 5 min and the supernatant was used as DNA template.
nuc and mecA identification
All S. aureus strains were screened by targeting the nuc and mecA genes by multiplex PCR (Table 3) [44, 45]. PCR reactions were performed in a 25 μL volume containing 1x of Buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 4 mM of MgCl2, 10 pmol of each primer, 200 μM of each dNTP's, 1 U of Taq DNA polymerase and bacterial genomic DNA. The PCR conditions were maintained at 95 °C for 5 min for initial denaturation followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Then, 20 μL aliquots of each sample were subjected to electrophoresis on 2% agarose gel.
Table 3 PCR primers used in this study
Identification of SCCmec types was performed by multiplex PCR using the genomic DNA from each MRSA isolate, according to a previously described method and primers (Table 3) [46, 47]. DNA amplification was carried out with a 2 min denaturation step at 94 °C, followed by 30 cycles of 60 s at 94 °C for denaturation, 60 s at 55 °C for annealing, and 60 s at 72 °C for extension, and then 5 min at 72 °C for final extension. Then, 20 μL aliquots of each sample were subjected to electrophoresis on 2% agarose gel.
The X region of the spa gene of each MRSA isolate was amplified by PCR with the primers 1095F and 1517R, as described previously (Table 3) [48]. The amplified products were sequenced, and the results were analyzed using the Ridom Staph Type software version 1.4 (Ridom, GmbH, Wurzburg, Germany [http://spa.ridom.de/index.shtml]) to determine the repeat profile and the spa type of each isolate [29, 49].
Dendrogram was constructed based on spa types data using a temporary BioNumerics evaluation license from Applied Maths (version 7.6, bioMérieux).
MLST was performed on six MRSA strains of the spa types t9364 (n = 4) and t895 (n = 2). Seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) of S. aureus were used for MLST typing (Table 3). PCRs were carried out in 50 μl reaction volumes containing 10 ng of chromosomal DNA, 10 pmol of each primer, 1 U of Taq DNA polymerase, 5 μl of 10x PCR buffer, and 200 μM each of dNTPs. PCR was performed with an initial denaturation at 95 °C for 5 min, followed by 37 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, followed by a final extension step of 72 °C for 5 min [50]. After amplification, the PCR products were purified and sequenced by dideoxynucleotides method (3500 Genetic Analyzer, Applied Biosystems). The consensus sequences were assembled, and the allelic profile was matched using the MLST database (https://pubmlst.org/saureus/).
Comparisons between groups was carried using Fisher's exact test or the chi-squared test (for categorical variables) and Student's t test of Mann-Whitney U test (for continuous variables) using Statistical Package for Social Sciences software for Mac OS, version 25.0 (SPSS, IBM, Inc., Chicago, IL, USA). P value < 0.05 were considered statistically significant.
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
S. aureus :
Methicillin-resistant Staphylococcus aureus
HA-MRSA:
Healthcare-associated MRSA
CA-MRSA:
Community-associated MRSA
MSSA:
Methicillin-sensible Staphylococcus aureus
Clonal complex
Sequence type
MLST:
Multiple Locus Sequence Typing
PFGE:
Pulsed-Field Gel Electrophoresis
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We thank to María Anita de Lira Torres, Andrés Flores Santos, and Laura Cerda Ramos, the staff of the Microbiology Laboratory of the HCIMP, for their support in the collection of strains.
We thank to Adriana Rodríguez Martínez and Miriam Briano Macias for their valuable technical support in this project.
This work was supported by the Grant 142334 from CONACyT-Salud, Mexico to PNM. CNG was a recipient of a scholarship 443025 from CONACyT, Mexico. The funding agency had no role in the study design, sample collection, data collection and analysis, decision to publish, or preparation of the manuscript.
Sección de Genómica Médica, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
C. Negrete-González, E. Turrubiartes-Martínez & P. Niño-Moreno
Laboratorio de Hematología, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
E. Turrubiartes-Martínez
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
O. G. Galicia-Cruz
Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
D. E. Noyola
Unidad de Investigación Biomédica, Instituto Mexicano del Seguro Social, Durango, Mexico
G. Martínez-Aguilar
Hospital Central "Dr. Ignacio Morones Prieto", San Luis Potosí, Mexico
L. F. Pérez-González
Sección de Medicina Molecular y Traslacional, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
R. González-Amaro
Laboratorio de Genética, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
P. Niño-Moreno
C. Negrete-González
CNG conceived the study, acquired clinical data and samples, performed the experiments, interpreted results, and drafted the manuscript. ETM and PNM co-designed and supervised the study and interpreted the results of experiments. DEN and OGC analyzed and interpreted data. DEN and RGA critically revised and edited the manuscript. GMA and LPG analized and interpreted the patient data. All authors have read and approved the manuscript.
Correspondence to P. Niño-Moreno.
This study was approved by the Research Committee [COFEPRIS 14 CI 24 028 083] and the Research Ethics Committee [CONBIOETICA-24-CEI-001-20160427] of the HCIMP. The registration number was 29–17.
Written informed consent was obtained from all participants or legal guardians/parents for those under the age of 16 years.
Additional file 1: Table S1.
Demographic and clinical characteristics of patients with Staphylococcus aureus infections who died or survived. Table S1 shows the demographic and clinical characteristics of patients with Staphylococcus aureus infections who died or survived.
Minimum Inhibitory Concentration (μg/mL) data for the MRSA strains. Table S2 shows the MIC for each antibiotic for the MRSA strains.
Negrete-González, C., Turrubiartes-Martínez, E., Galicia-Cruz, O.G. et al. High prevalence of t895 and t9364 spa types of methicillin-resistant Staphylococcus aureus in a tertiary-care hospital in Mexico: different lineages of clonal complex 5. BMC Microbiol 20, 213 (2020). https://doi.org/10.1186/s12866-020-01881-w
SCCmec type II
Clonal complex 5-ST1011
Spa type t895
Spa type t9364
New York/Japan-Mexican variant clone


PoconoGeek needed a new banner ad design and created a contest on 99designs.
A winner was selected from 46 designs submitted by 13 freelance designers.
Padlock Software LLC is a one man software shop selling a license management solution for Java developers. The company is small but serious, as technical proficiency and overall professionalism are important.
The target audience for these banner ads are Java developers who make purchasing decisions for their product line. They are technically proficient and spend a lot of time in front of a computer. Therefore, the ads should not be be flashy, gaudy, or contain loud/bright/florescent colors. It should not hurt your eyes, and should leave plenty of "whitespace".
These guys will quickly tune out ads that look offensive in their design, especially ads that look less than professional. This ad should resemble something that Oracle would put out.
I need two PNG ad banners with dimensions of 728x90 and 220x250 pixels. Both ads should be identical in content and overall design (obviously the layout will have to differ a bit).
1) "Your code on your terms"
2) "Java(tm) powered license management"
Capitalization is left to you to decide.
The product name is officially "Padlock License Manager for Java", and either that or "Padlock License Manager" should be clearly readable on the ads along with the Padlock logo (see attachments).
Color pallet is up to you guys!


San Francisco, CA: As Nazis and white supremacists continue to plague our communities with marches and protests, let us be clear that we condemn their actions, beliefs, and rhetoric. What happened in Charlottesville last weekend was despicable and deplorable. It was an act of domestic terrorism.


WinLep compresses JPEG images to LEP. This compression format called Lepton allows the compression of JPEG images saving on average a 22% of size without losing quality in the process of compression and when it is decompressed the image is exactly the original one.
Lepton is a lossless open source algorithm developed by Dropbox capable of compressing images at 5 MB/s and decompressing them at 15 MB/s. WinLep takes advantage of these qualities to offer a fast and lossless quality compressor/decompressor.
WinLep compresses JPEG and decompresses LEP, moreover it can display JPEG and LEP compressed files.


Per earlier post (see discussion and chart), corporate spreads in the US are grinding lower - with new post-recession lows for both IG and HY spreads. The Merrill HY Index spread is now below 400bp and the investment grade equivalent is below 130bp. For those who track fixed income ETFs, the following chart comparing treasuries with corporate bonds (LQD vs. IEF and HYG vs. IEI) illustrates the extend of spread compression.
In fact US corporate credit is outperforming other forms of credit assets such as commercial real estate (see post). A good way to see that outperformance vs. global risk assets is in the components of the Credit Suisse Risk Appetite Index. For the first time since Bernanke began hinting about taper, US Credit Risk Appetite is at the level of "euphoria".
But some argue that these credit spread levels are justified given where the US stock market is currently valued. The scatter plot below shows the S&P500 index vs the HY index spread over the past 10 years. The last time spreads were at these levels (2007), equities were priced much lower (S&P500 was around 1550). Of course corporate revenue has grown substantially since then making this comparison less relevant. Nevertheless some are suggesting that it's the stock market which is overvalued relative to credit.
Whatever the case, as monetary conditions in the US begin to tighten and interest rates rise, credit spread compression has to slow or reverse. The current trend is simply not sustainable.


text:
Still Alice Essay
"Alzheimer's disease (AD) is a slowly progressive disease of the brain that is characterized by impairment of memory and eventually by disturbances in reasoning, planning, language, and perception." (Howard Crystal) In Health 1000 we were asked to read the book Still Alice. I have never dealt with or have done any study on Alzheimer's disease before reading this book. After finishing this book it has really opened my eyes to how bad of a disease and how it cripples the mind. I never imagined the effect of this disease on a patient and the patient family. This book is about a upper middle aged lady named Alice who is diagnosed with Alzheimer's disease, and how she and her family learn how to ...view middle of the document...
Toward the end of the book he decides to take a job in New York. He doesn't do this to be selfish but he knew that's what she would want for him. John holds on to hope the whole book until he reads how the Amylix wasn't able to stabilize the Alzheimer patients. John starts to cry after finding out, because this is basically the final straw. It was his last ditch effort and in knowing it failed it symbolized that he failed and that Alice was doomed. This forced him to admit to himself that the women loved and cared so much for was no longer there and will never be there again.
Alice ended up telling her colleagues about her diagnosis much later than when she told John. She only told them when she realized how much her teaching has slipped up and knowing it was only going to get worse with time. Alice would forget a whole lecture even though she just spent hours reviewing and studying her notes before class. One time she even went into her classroom and sat down for a few minutes just to get up and walk out of her classroom without teaching one thing. Alice ends up sending a formal letter to all her colleagues letting them know that she has Alzheimer and is going to be stepping down. Most of them stop by her office and offer their condolences after receiving her letter. After finding out her colleagues pretty much avoided Alice but if they did run across her they were polite, but quick to excuse themselves from her presence. This was hurtful for Alice because she once considered everybody in her department almost like a second family.
I feel the one most effected my Alice diagnosis of the children was Lydia. Lydia and Alice always have had a rough relationship for the most part. Alice believes that Lydia should be going to college and getting an education instead of running around L.A. taking acting classes. Every time they talked or visited each other it always ended up in some heated argument. Of the three children Lydia and Alice relationship improved after finding out about everything. Lydia even came home for a summer to help out with her mother. During that summer Lydia and Alice becomes closer and they are able to hold conversations that didn't end up in a fight. Alice evens picks up reading plays and scripts. So she finds some common ground with her daughter that leads to them become closer and helping in settling somewhat the lifelong dispute of acting and college. Alice even begins to want to see Lydia act in a play but is unable to attend one for a while due to that fact that she can's travel alone and John was really busy. One...
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913 words - 4 pages Personal Writing It was a year I had been with Alice. We did everything together. I cared for that girl like no one could imagine. I would of done anything for her. There wasn't a time, I would not think about her when we weren't together. Everyone knew us for being the happiest couple in our town, Little Wood. I thought we would be together until we grew old…I was wrong. I finished work last Thursday earlier
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For handwork, using a quality embroidery hoop makes a world of difference. These have nice rounded edges and an easy to turn brass screw. These feel good and sturdy in your hand. I always wrap my inner hoop in strips of muslin or twill tape to help keep my work taut in the hoop.


January was a great month for p2p lending as far as publicity goes. There were several TV segments, news articles and posts on major blogs. In case you missed some of these, here is a breakdown of recent news.
I have been watching CNBC for almost 20 years and it was great to see Renaud Laplanche, CEO of Lending Club, interviewed on their morning show. Renaud did a great job despite the fact the interviewers knew so very little about Lending Club or p2p lending.
Interview with Michael Garrity, CEO of CommunityLend in Canada.
Interview with a Lending Club borrower and an investor who were on either side of the loan transaction.
This same article was picked up by Entrepreneur.com, and Fox Small Business Center ran a similar article.
Daily Kos – So you really want to hurt the financial industry?
The interviewers' relative cluelessness as to what Lending Club is or actually does in my opinion speaks volumes about what is necessary if Lending Club is to end up reaching a break-even origination volume.
I feel that fairly soon they need to step up their "name recognition" game a fairly high degree, and shift the capital they were once pouring into bonus payouts into more traditional marketing (billboards, subway / bus ads, television commercials, etc.). For example, simply a well-placed ad in a Manhattan subway line could possibly yield hundreds if not thousands of investors and borrowers for close to the same they were spending on bonus incentive offers, while at the same time massively increase name recognition. National advertising is probably not within their budget capacities at this time, but only once Lending Club becomes a "household name," so to speak, can it begin to truly compete with banks on a meaningful level (and of course get to the 20 million monthly origination volume, or 25 million, or wherever the break-even level is).
20, 25 million a month? I'd say it's closer to a BILLION a year in loan originations before one can start talking about being a competitor on a meaningful level. Even that figure would work out to just $3 million a month in origination fees.
And at that point I guarantee you that every XYZ bank will find a way to throw their hat in the p2p ring & take a chunk of the market.
The $20-25 million/month figure I'm talking about the "break-even" figure I've heard tossed around before, whereby origination fees plus loan service fees finally equal operations cost and they can begin to talk about profitability. As things stand now they are operating at a fairly large net loss, and there is no guarantee that they ever will make a profit.
I agree that when (if) the p2p market explodes everyone will want a piece of the action, but if Lending Club can secure a reputation now as "the" p2p lending outfit, it will be a great aid to their maintaining a competitive moat in the future.
@David, I have heard the $20-25 million bandied around as well as a break even point. I will be surprised if LC doesn't surpass $20 million a month before the end of the year. And Laplanche has said in these interviews the he expects LC will turn cash flow positive in 2012.
I have also heard that LC will be moving away from investor bonuses this year. I don't know about a national advertising campaign, but I am sure they know they need to do more. Zopa in the UK just launched a national TV campaign and this week did record volumes in originations. I agree more needs to be done to get their name out there and all these PR appearances help.
@Dan, I think you have your calculations wrong. At $1 billion a month in loan originations that will put revenue from originations at $30 million a month (assuming an average 3% origination fee). Then with investor service fees running at 1% of the entire active loan portfolio, you would probably have another $5 million a month in service fees. So, that would be a business grossing $400 million a year roughly.
Sure there will be more competition by then. But LC will have the advantage of a longer track record of loan history. And with the SEC registration hurdle not every XYZ bank is going to want to jump into the ring.
Attention to detail Peter. I said a billion a year.
I stand corrected. My mistake.


CARRIS' Museum is pleased to inform that from the 17th to the 21st may the Second Part of the Museum is hosting a Contemporary Art Fair - JustLx.
For this reason the Second Part is restricted to those who buy tickets.
The First and Third Part of the Museum are opened in regular function and can be visit for free.


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Why I Hate The Dating Site/App Called…..
Why I Love The Dating Site/App Called…..
#onlinedating | Harry Styles and Olivia Wilde Spark Dating Rumors After Holding Hands At a Wedding | #bumble | #tinder | #pof
January 4, 2021 Author: Dating Scams 101
After Harry Styles and Olivia Wilde were spotted holding hands at a small, intimate wedding ceremony over the weekend, rumors started to fly that there might be something more than a professional relationship brewing between the pair.
The singer brought Wilde as his guest to his manager Jeffrey Azoff's 16-person wedding in Montecito, California, where they were photographed strolling hand-in-hand. For the event, Harry wore a black suit with a slightly ruffled white button-up and Western-style black bow tie from Gucci's Spring 2021 collection, while Wilde opted for a much more colorful look that also could have been a subtle tribute to the pop star. The actress wore a rainbow-hued floral and lace maxi dress from Gucci's Resort 2021 collection, a brand that Styles has regularly served as both the face and muse for in the past. Both stars also wore matching face masks by The Vampire's Wife.
The pair were spotted again on Monday at Styles's home returning from the trip with their luggage in tow. According to a source who spoke to the Daily Mail, "Olivia and Harry have become close and the pair have been spending a lot of time together in recent weeks. She has been seen visiting his home on several occasions."
The two got to know each other on the set of Don't Worry Darling, Wilde's next directorial project which is also shooting in Montecito. (She cast Styles to replace Shia LaBeouf.) Wilde previously told Vogue that she "did a little victory dance" when Styles was officially cast in the role, saying, "To me, he's very modern and I hope that this brand of confidence as a male that Harry has—truly devoid of any traces of toxic masculinity—is indicative of his generation and therefore the future of the world…It's pretty powerful and kind of extraordinary to see someone in his position redefining what it can mean to be a man with confidence."
In November, it was reported that Wilde and her fiancé Jason Sudeikis had called off their engagement after over eight years together, but remain friendly exes. According to a source who spoke to People, "The split happened at the beginning of the year. It's been amicable and they've transitioned into a great co-parenting routine. The children are the priority and the heart of the family's relationship." The former couple shares a 6-year-old son Otis and four-year-old daughter Daisy.
Representatives for both Styles and Wilde were not immediately available for comment.
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Summary Prospectus
Semi-Annual Report
IRA Application
ROTH IRA Application
Regular Account Application
Account Options Application
Change of Registration Guidelines Application
Cost Basis Application
Designation of IRA Application
IRA Distribution Application
IRA Transfer Rollover Application
Letter of Instruction Application
Non-Qualified Asset Transfer Application
SEP IRA Allocation Application
Transaction Request Application
Transfer on Death Application
Entity Account Application
Oct. 31, 2022 HCMAX Tax Summary
Oct. 31, 2021 Estimated Year End Tax Information as of October 31, 2021
Principal Risks of Investing in the Fund
An investment in the Fund is subject to investment risks, including the possible loss of some or all of the principal amount invested. There can be no assurance that the Fund will be successful in meeting its investment objective. Generally, the Fund will be subject to the following additional risks: Cybersecurity Risk, Investment Advisor Risk, Market Risk, Risks from Writing Options, Sector Focus Risk, COVID-19 Risk, Small-Cap and Mid-Cap Companies Risk, and Value Risk. More information about these risks and other risks can be found in the Fund's prospectus.
An investor should consider the investment objectives, risks, and charges and expenses of the Fund carefully before investing. The prospectus contains this and other information about the Fund. A copy of the prospectus is available at the link below or by calling Shareholder Services at 1-855-400-5944. The prospectus should be read carefully before investing.
Hillman Capital Management, Inc. (the "Adviser") has contractually agreed to limit the Total Annual Fund Operating Expenses of the Fund (exclusive of (i) any front-end or contingent deferred loads; (ii) brokerage fees and commissions; (iii) acquired fund fees and expenses; (iv) fees and expenses associated with investments in other collective investment vehicles or derivative instruments (including for example option and swap fees and expenses); (v) borrowing costs (such as interest and dividend expense on securities sold short); (vi) taxes; and (vii) extraordinary expenses, such as litigation expenses (which may include indemnification of Fund officers and Trustees and contractual indemnification of Fund service providers (other than the Adviser)) to an annual rate of not more than: 0.95% of the Fund's average daily net assets for the Fund's No Load Class. This agreement is in effect through at least January 31, 2023, and will automatically continue upon annual approval by the board for successive twelve-month periods unless (i) it is terminated earlier by the Board of Trustees, or (ii) the Adviser provides at least 30 days written notice of its non-continuance prior to the end of the then effective term. Except due to the Adviser's notice of non-renewal, this Agreement may only be amended or terminated with the approval of the Board of Trustees of the Trust.
The Fund may not be available for sale in all states. The information presented on this page is not an offer to sell or a solicitation of an offer to purchase the Fund.
ALPS Distributors, Inc. is the distributor of the Hillman Capital Management Funds, 1290 Broadway, Suite 1000, Denver, CO 80203.
ALPS Distributors, Inc. is not affiliated with Hillman Capital Management, Inc.
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ABOUT RIO 2016
Six countries join Rio 2016 shooting qualification race
Following the first IPC Shooting World Cup Paralympic qualifier of the season, 34 National Paralympic Committees have quota places for the next Paralympic Games. 31 Jul 2015
Samira Eram won gold in the women's P2 in Croatia, upsetting top-ranked compatriot Javanmardidodmani.
ⒸIPC
IPC Shooting has confirmed that 34 National Paralympic Committees have so far secured coveted quota places for shooting at the Rio 2016 Paralympic Games, including four that have qualified directly for the first time.
The news follows the first Paralympic qualifier of 2015 – the IPC Shooting World Cup in Osijek, Croatia, in early July, with two more to come in Australia and the USA in September and November respectively.
In Osijek, shooters from Spain and Italy won places for their countries, with Kuwait, Bulgaria and Uzbekistan all heading to a Paralympic shooting range for the first time. Ireland obtained their inaugural direct qualification, having competed as a bipartite entry at London 2012.
Athletes were first able to obtain quota spots at the 2014 IPC Shooting World Championships in Suhl, Germany, where 28 countries successfully began their road to Rio 2016.
Sarah Bond, IPC Shooting Sport Manager, said: "The athletes and teams seem to be delighted with the new qualification system, which is working to ensure that more countries have the chance to compete at the highest level.
"We have seen the quality of competition increase consistently in line with an increased investment by countries to develop shooting and their athletes. We are pleased that this is now all coming together to create what I am sure will be one of the most competitive Paralympic Games in history at Rio 2016."
Also in Osijek, 11 countries added to their quota numbers with South Korea currently leading the way with 11 places. China have seven whilst Great Britain have achieved six spots.
Australia also have six, having already equalled their number of quota places obtained for London 2012, with a home World Cup in Sydney, Australia, from 15-19 September still to come.
The last IPC Shooting World Cup of the season in Fort Benning, USA, will take place between 3-7 November, offering athletes the final opportunity to qualify.
Once the qualification period ends, countries will begin the process of nominating which athletes will travel to Rio de Janeiro.
The full list of countries that have achieved quota places can be found on IPC Shooting's website.
Slovakia NPC hosts series of youth sports camps
New Zealand team announced for IPC Shooting World Cup in Sydney
Doroshenko, Bevis squeeze out golds at World Cup in Croatia
Eram surprises in Iranian showdown at World Cup
Top shooters on their road to Rio 2016


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Last updated 05 Jul 2014
The Healing Power of Macrobiotic Tea
3-year Organic Hira Bancha 3-年有机番茶
There is an old saying that those who drink the tea made from tea leaves harvested on the eighty-eighth day after the first day of spring will stay in a perfect state of health and live longer. The tea leaves gathered on this day are the best in their quality, fairly mature and containing a rich mix of aroma and flavor.
Tea leaves are harvested several times a year. The first harvest tea is considered the best, called Ichi-ban-cha (No. 1 tea), then followed by the second harvest or Ni-ban-cha (No. 2 tea), and then the third harvest or San-ban-cha (No. 3 tea). Tea harvested in the later part of the season is called Bancha or Aki-Bancha, which means Autumn Bancha in Japanese. These Bancha are usually used to produce dried tea leaves by means of roasting, producing products such as Hira Bancha or Houjicha.
Hira Bancha is made using second harvest tea leaves mixed together with the twigs of the tea tree and then roasted. The caffeine content is very low in bancha tea leaves and the tea has a warming effect on the body.
The benefits of Hira Bancha
Bancha tea is very rich in calcium content. Daily consumption will improve calcium absorption in the body.
Relieves fatigue.
Increases the alkalinity of the body, thus preventing acidosis.
Discharges excess fat from the body, thus preserving the health of the liver and heart.
Alleviates skin atopic conditions.
You can prevent pollen contamination by rinsing the eyes with bancha tea.
Leftover bancha tea can be used to cleanse the face.
Discharges mucus from the body.
Spent bancha tea leaves can be dried and used as a deodorizer.
Organic Kukicha
Kukicha is made from stems and branches that are separated in the refining process of Sencha. The biggest difference from commercialized Kukicha is that Unsui's Kukicha is roasted. Unsui's Kukicha has over 30 years of history and, until now, has been sold exclusively in the Macrobiotic field. Because stems and branches of tea leaves are roasted, its roasting aroma and mild flavor are the main characteristics. During hot summer, it is best enjoyed as cold tea. Unsui's Kukicha has been the most popular kind of tea because it has less caffeine, so that it does not have any stimulating effect. Unsui's Kukicha can be enjoyed by people with sickness, children, and all other tea drinkers.
Organic Sencha (green tea)
It is the most representative kind of Japanese tea that occupies about 80% of tea production in Japan. Harvested tea leaves are steamed, rubbed thoroughly, and then formed into long thin shapes. Sencha tea leaves have a vivid deep green color, a refreshing aroma, and a light bitterness in its sweet flavor, and thus it pairs well with sweet desserts as well as when you would like to refresh your palette. It contains the most protein and dietary fiber amongst all teas.
Unsui's organic genmaicha consists of a mixture of toasted brown rice and green tea leaves. This special blend balances the yin energy of the green tea, and is suitable for those with a sensitive digestive system.
Organic Green Tea Matcha Powder
Rejuvenate, Focus, Nourish. Deeply steeped in Japanese tradition, Unsui's premium grade matcha powder is one of the world's most potent natural sources of antioxidants, as well as immune boosting and detoxifying flavanols and catechins (EGCG). We select only the finest Japanese matcha powdered green teas made using ceremonial- and traditional- certified organic whole tea leaves to form our special blend, resulting in an organic, regal, full-bodied, rich and balanced flavor experience.
100% benefit from the tea leaves. Matcha is made by grinding the entire leaf into powder, preserving all the nutrients and benefits that are taken away by tea bags, ensuring that the benefits of the tea leaves can be consumed as you drink. This ensures that matcha drinkers consume a higher concentration of antioxidants, vitamins, minerals and fiber than any other type of tea.
What makes matcha special
Provides sustained energy.
Contains 10 times the amount of antioxidants and amino acids of steeped green tea.
Full-bodied with a rich flavor and texture.
Helps burn calories, regulate blood sugar and detoxification.
Healthy alternative to coffee.
How to prepare green tea matcha
Matcha Tea is unique in that it is a fine green powder. Traditional Matcha is prepared by placing a portion of the tea in a bowl or cup, and then adding hot water. The tea is then dissolved and aerated using a traditional bamboo whisk (known as cha-sen in Japanese) until it is creamy and frothy. You can also use a hand mixer to whisk the matcha powder.
Posted by June Ka Lim, macrobiotic chef and counselor
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If you cling to someone or something, you hold onto them tightly.
If someone clings to a position or a possession they have, they do everything they can to keep it even though this may be very difficult.
Clothes that cling to you stay pressed against your body when you move.
Something that is clinging to something else is stuck on it or just attached to it.
If someone clings to a person they are fond of, they do not allow that person to be free or independent.
If you cling to an idea or way of behaving, you continue to believe in its value or importance, even though it may no longer be valid or useful.
Uso común. cling es una de las 10000 palabras más usadas del Diccionario Collins.
Another man was rescued as he clung to the riverbank.


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Microsoft working on a smartwatch to counter Samsung's Galaxy Gear
The US patent's office has released a news that technological giant Microsoft has filed for a patent for Smart Watch. According to the reports Microsoft had filed the Patent application in 2012 itself but US patents and trademark office released the news now. Microsoft's Smart watch would be a counter attack for Samsung's Galaxy Gear last year and towards facing future competition from much anticipated Apple's iWatch which is also under the works.
Microsoft's smartwatch project is believed to be headed by former Nokia CEO, Stephen Elop who runs Microsoft's devices division.
Images released from US patent office says-wristwatch has a features of fitness tracker which notes heart rates using optical sensors, inbuilt GPS to measure movement with motion sensors, just like the smartphones which has these kind of features. It also says it has features of music player, phone and messaging services.
This is not the first time that Microsoft tried its hands in smartwatch, in 2004, Microsoft released a device by collaborating with watchmakers Fossil, Citizen, Suunto and technology used in the device was termed as SPOT short for smart personal objects technology. These watches used to display news, stock closing and sports scores using FM radio receivers, But Microsoft discontinued the project in 2008.
Images also appear to show that it has detachable wrist straps and also face of the device can be docked into external charger and another device.
There is a big market for smartwatches as more and more people are getting tech savy and miniturisation is the way to the future as per many tech analysts. This was proved to be true with the startup Pebble whose not so great smart watch notched up sales of 400,000 units as per the makers, last year. Samsung released a wrist watch in the name of its Galaxy Gear last year but it has not made any inroads as of yet. Rumours are in air that Apple will launch its iWatch by year end or in 2015. And search giant Google is to follow suit with the release of Moto 36- smartwatch in USA this summer.
Microsoft refused to comment anything regarding the smartwatch's release date or even it's availability in market.
Reach me via Twitter @knithaurs
Labels: Hardware, hot, New Product, Tech News


We're so frickin' grateful for his pecks!
In the spoof, Corden rocks silky pajamas, and looks at a "Thank U, Jeff" book, imitating Grande in the music video.
"First saw him in The Fly, where his face was grotesque. Then he won independence from an alien mess," Corden sings. "In Thor: Ragnarok, his performance was classic. But who could forget the hot doctor that he played in Jurassic?"
Goldblum gets in on the fun, taking over Kris Jenner's Amy Poehler impression from the music video and holding up a video camera in the audience as Corden dances with men dressed as the 66-year-old actor's most iconic characters.
It seems she'll have to pry the book from Corden's hands as he addresses it at the end of the video. "That journal with all the pictures of me, have you had that for a while? You compiled that?" Goldblum jokingly asks.
"No… I'll get rid of it," Corden says bashfully.
"I thought it was very sweet," Goldblum replies.
"I'll keep it," Corden says hurriedly before rushing out of the dressing room.
Grande's "Thank U, Next" music video was released last week and has already broken records on YouTube. Lots of stars, including Mark Ruffalo, Jennifer Garner, Reese Witherspoon, Tina Fey and more have reacted to it.


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La CB 125 F est un modèle de moto, de type roadster, du constructeur japonais Honda. Annoncée en , et révélée le , elle remplace la , un modèle produit depuis 2008. Le prix de lancement était inférieur à celui de la  considérant que ce nouveau modèle est produit en Chine (sous le nom de « Shine »), alors que la  fut produite en Inde. À cause des nouvelles normes, le freinage de la  a évolué en 2017 avec le freinage couplé CBS.

Évolution par rapport à la CBF 125
Look typé roadster, inspiré des CB 500 F et CB 650 F, optique de phare mise en valeur.
Selle d'un seul tenant abaissée à 775 mm au lieu de 792.
Nouvelles jantes à six bâtons dédoublés en 18 pouces (17 sur la CBF).
Pneus en 80 mm à l'avant et 90 mm à l'arrière (au lieu de 90 et 100).
L'empattement, l'angle de chasse et la traînée ont été légèrement augmentés.
La fourche a vu son diamètre augmenté (31 mm au lieu de 30), ainsi que son débattement (120 mm au lieu de 115). Les amortisseurs sont désormais réglables sur cinq positions au lieu de trois.
Le moteur a perdu un peu en puissance maxi mais elle arrive un peu plus tôt.
Rapports de boite plus courts, lui donnant une nervosité pour la ville et le péri-urbain.
Un balancier d'équilibrage se charge de réduire les vibrations.

Mise à niveau Euro 5
En octobre 2020, la CB 125 F évolue pour intégrer les nouvelles normes européennes d'émissions applicables en 2021 (Euro 5). Cette intégration inclut également un léger restylage esthétique dans trois coloris (rouge, blanc et noir).

Les principales modifications esthétiques concernent :
 restylage de la tête de fourche ;
 projecteurs avant à lampe électroluminescente ;
 modification des écopes contre le réservoir.

Une commande d'appel de phare apparaît, et l'autonomie est augmentée via les modifications apportées au moteur malgré un réservoir passant de .

Le moteur reste un monocylindre à quatre temps à deux soupapes refroidi par air. Il est désormais équipé d'un alterno-démmareur, lequel permet entre autres améliorations de réduire sa masse de . Les performances indiquées par Honda sont :
 puissance maximale : 8 kW à  (en augmentation de 0,2 kW) ;
 couple maximal :  à  (en augmentation de ) ;
 consommation : 1,54 L/100 km (1,96 L/100 km sur la version précédente).

125 F
Motocyclette des années 2010
Motocyclette des années 2020

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Poten-Cee Gummies may look beary cute, but don't be fooled by their candy-like appearance.
Each Poten-Cee gummy bear is actually loaded with more than the Vitamin C content of some other similar brands. Poten-Cee Gummies have 60mg Vitamin C which meets the 45mg requirement of children 10 to 12 years, based on RENI (Recommended Energy and Nutrient Intake 2002 edition).
Vitamin C is essential for normal growth and development, including that of energetic children.
It is also one of many antioxidants — nutrients that block damage caused by free radicals.
Poten-Cee Gummies have rosehip extract and beta-carotene.


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