These results contrast with an earlier study from Cote d’Ivoire that reported more than half of the participants declaring sexual abstinence and those who were sexually active having a CB-839 nmr low frequency

of sexual intercourse (once a month or less) [30]. A major finding of the current study is that South Indian patients with higher viral loads were more likely to transmit HIV to their seronegative partners, and during the 12 months of follow-up, patients in seroconverting relationships continued to have significantly higher viral loads than patients in serodiscordant relationships. Although studies from other regions have documented that PVL is a marker for HIV transmission [10–12,14], the findings of the current study differ from an earlier study from Zambia in which PVL was only weakly predictive of male-to-female transmission within couples and rates of male-to-female and female-to-male transmission were similar [16]. In an earlier study at our centre, men with PVLs >100 000 were more likely to be in concordant relationships [31]. Although the

most important justification for expanding access to ART in resource-limited settings has been to prolong the life of HIV-infected patients, a secondary outcome could also be a reduction in the risk of HIV transmission because AZD6244 in vitro ART dramatically suppresses peripheral blood levels of HIV-1 RNA [32]. The incidence of HIV infection among the initially seronegative partners was 6.52 per 100 person-years. An earlier study from Western India documented a lower incidence rate of seroconversion (1.22 per 100 person-years) among serodiscordant couples, which was attributed to high rates of condom use, low rates of STIs and high CD4 T lymphocyte counts [21]. However, a study from Zambia documented a similar transmission rate between couples (7.7 per 100 person-years) [16]. The Rakai study reported an even higher incidence of 11.8 AZD9291 order per 100

person-years [9]. These varying incidences of HIV transmission in different settings are likely to reflect different numbers of partners, varying duration of relationships, availability of ART, coital frequency, availability of clinical care and the structures of various sexual networks [33]. Herpes simplex virus type-2 (HSV-2) co-infection has been identified as a key risk factor for the heterosexual transmission of HIV [13,34], and HSV-2 infection reactivation in HIV-infected individuals can lead to a rise in HIV viral load and increased rates of HIV seroconversion [8,35]. In the current study, a substantial number of patients presented with genital HSV-2 at enrolment and patients in relationships that seroconverted between 6 and 12 months of follow-up had a higher period prevalence of genital HSV-2. Acyclovir suppressive therapy can suppress genital and plasma HIV RNA levels [36], and could be used as prophylactic therapy in populations with high HSV-2 burdens to reduce the transmission of HIV.

For women enrolled in both the MoCHiV and the SHCS, precise information on ART prior to and during pregnancy as well as on clinical characteristics (e.g. CD4 cell count, viral load and the presence of opportunistic infections) and possible (behavioural) risk factors for premature birth (such as smoking and illicit drug use) before and during pregnancy has become available. All data were reported prospectively on structured worksheets and entered into the national database at the coordinating centre. Informed consent was obtained from each woman participating in the SHCS and for each child’s parents or legal guardians before enrolment into the MoCHiV, and local institutional ethics committee CDK inhibitor approval

was obtained for both the SHCS and the MoCHiV. Analyses were restricted to HIV-1-positive women with a history of at least one pregnancy that was completed to live birth, excluding multiple (twin) pregnancies, which are commonly of shorter duration. Figure 1 shows a flow chart for further data selection. We excluded pregnancies that were terminated through elective caesarean section before 37 weeks of gestation (61 pregnancies in 30 mothers). The primary outcome ‘premature birth’ was defined as delivery before completion of the 37th week of pregnancy. We investigated the effects of different ART regimens on prematurity in several ways. Analysis 1 included all available data, i.e. 1180 pregnancies in 1040 mothers, and

examined the association between prematurity and type click here of ART exposure (no therapy, mono or dual therapy, and cART) without consideration of potential confounding maternal risk factors for prematurity, as such information was commonly incomplete in the early years of the MoCHiV (i.e. in women

exclusively enrolled in the MoCHiV). In analysis 2 we compared rates of premature birth in 418 pregnancies in 366 mothers exclusively on cART, who initiated treatment before or during pregnancy. Analysis 3 was further restricted to 334 pregnancies in 294 women under follow-up in the SHCS during pregnancy. For these women, a detailed treatment history was available, which allowed us to investigate the relationship between the duration of cART, both prior to and during pregnancy, and prematurity or pregnancy duration. The aim Carnitine palmitoyltransferase II of analysis 4 was to control for a number of potential maternal confounders for prematurity and we therefore excluded 762 (of the initial 1180) pregnancies in 695 women who were not under follow-up in the SHCS during pregnancy. We further excluded 43 pregnancies in 41 mothers who did not receive ART during pregnancy and 10 pregnancies in 10 mothers without viral load measurement during pregnancy. The adjusted analysis was based on 365 pregnancies in 318 women. The main outcome was the risk of premature birth, which was analysed using logistic regression with a random effect on mother ID to account for dependence of multiple pregnancies in the same mother. Significance testing was performed using Wald tests.

Plain water was given to the controls at the same restricted time (R-Water). Clock gene Per2 expression was measured by a bioluminescence reporter in cultured brain tissues. In SCN-intact rats, GDC-0068 ic50 MAO was induced by R-MAP and behavioral rhythms were phase-delayed from the restricted time under ad-MAP with relative coordination. Circadian Per2 rhythms in R-MAP rats were not affected in the SCN but were slightly phase-advanced in the

olfactory bulb (OB), caudate–putamen (CPU) and substantia nigra (SN) as compared with R-Water rats. Following SCN lesion, R-MAP-induced MAO phase-shifted more slowly and did not show a sign of relative coordination. In these rats, circadian Per2 rhythms were significantly phase-shifted in the OB and SN as compared with SCN-intact rats. These findings indicate that MAO was induced by MAP given at a restricted time of day in association with phase-shifts of the extra-SCN circadian oscillators in the brain dopaminergic areas. The findings also suggest that these extra-SCN oscillators are the components of MAO and receive dual regulation by MAO and the SCN circadian pacemaker. The circadian rhythms of physiology and behavior in mammals are controlled by a hierarchical multi-oscillator system, consisting

of a central circadian pacemaker in the suprachiasmatic nucleus (SCN) and peripheral oscillators in a variety of tissues and organs (Reppert & Weaver, 2002;

Mohawk et al., 2012). The SCN circadian pacemaker entrains to light–dark cycles (LD) and resets the peripheral oscillators. Intracellular Decitabine in vitro Astemizole mechanisms of the central and peripheral circadian oscillators are considered to be an autoregulatory molecular feedback loop involving several clock genes and their protein products. On the other hand, at least two oscillators in the circadian range are reported to be induced independent of the SCN circadian pacemaker (Honma & Honma, 2009). One is the methamphetamine (MAP)-induced oscillator (MAO) and the other is the food-entrainable oscillator (FEO). MAO is induced by chronic MAP treatment via drinking water (Honma et al., 1986a; Tataroglu et al., 2006) and desynchronises some extra-SCN oscillators in the brain as well as behavioral rhythm from the SCN circadian pacemaker (Masubuchi et al., 2000; Natsubori et al., 2013b). The MAP-induced behavioral rhythms are regarded as an animal model of the human sleep–wake cycle because they show characteristics specifically observed in the human sleep–wake cycle such as internal desynchronisation, circabidian (ca. 48 h) rhythms and non-photic entrainment. On the other hand, FEO is induced by restricted daily feeding (RF) and characterised by anticipatory activity prior to daily meals (Stephan et al., 1979).

Plain water was given to the controls at the same restricted time (R-Water). Clock gene Per2 expression was measured by a bioluminescence reporter in cultured brain tissues. In SCN-intact rats, selleck products MAO was induced by R-MAP and behavioral rhythms were phase-delayed from the restricted time under ad-MAP with relative coordination. Circadian Per2 rhythms in R-MAP rats were not affected in the SCN but were slightly phase-advanced in the

olfactory bulb (OB), caudate–putamen (CPU) and substantia nigra (SN) as compared with R-Water rats. Following SCN lesion, R-MAP-induced MAO phase-shifted more slowly and did not show a sign of relative coordination. In these rats, circadian Per2 rhythms were significantly phase-shifted in the OB and SN as compared with SCN-intact rats. These findings indicate that MAO was induced by MAP given at a restricted time of day in association with phase-shifts of the extra-SCN circadian oscillators in the brain dopaminergic areas. The findings also suggest that these extra-SCN oscillators are the components of MAO and receive dual regulation by MAO and the SCN circadian pacemaker. The circadian rhythms of physiology and behavior in mammals are controlled by a hierarchical multi-oscillator system, consisting

of a central circadian pacemaker in the suprachiasmatic nucleus (SCN) and peripheral oscillators in a variety of tissues and organs (Reppert & Weaver, 2002;

Mohawk et al., 2012). The SCN circadian pacemaker entrains to light–dark cycles (LD) and resets the peripheral oscillators. Intracellular Aloxistatin cell line MRIP mechanisms of the central and peripheral circadian oscillators are considered to be an autoregulatory molecular feedback loop involving several clock genes and their protein products. On the other hand, at least two oscillators in the circadian range are reported to be induced independent of the SCN circadian pacemaker (Honma & Honma, 2009). One is the methamphetamine (MAP)-induced oscillator (MAO) and the other is the food-entrainable oscillator (FEO). MAO is induced by chronic MAP treatment via drinking water (Honma et al., 1986a; Tataroglu et al., 2006) and desynchronises some extra-SCN oscillators in the brain as well as behavioral rhythm from the SCN circadian pacemaker (Masubuchi et al., 2000; Natsubori et al., 2013b). The MAP-induced behavioral rhythms are regarded as an animal model of the human sleep–wake cycle because they show characteristics specifically observed in the human sleep–wake cycle such as internal desynchronisation, circabidian (ca. 48 h) rhythms and non-photic entrainment. On the other hand, FEO is induced by restricted daily feeding (RF) and characterised by anticipatory activity prior to daily meals (Stephan et al., 1979).

A self-administered 29-item questionnaire comprising four sections was developed based on a literature learn more review, current national asthma guidelines[26] and the research experience of the investigators (Table 1).

As the guidelines do not articulate the specific elements of pharmacist delivered asthma interventions, the guidelines were used to identify all the potential activities/aspects of asthma management in which the pharmacist could engage or participate. Section 1 (role) covered pharmacists’ perceptions of their role in asthma management (items 1–10), with responses on a five-point Likert scale (0 = strongly disagree and 4 = strongly agree). Positive agreement to each item was indicated by a rating of 3 or 4. Section 2 (barriers) looked at pharmacists’ perceptions regarding barriers to the provision of pharmacy asthma management services (items 11–27); respondents were asked to indicate the extent to which each item impacts on their ability to provide specific

selleck inhibitor asthma counselling or services using a five-point Likert scale (0 = no impact to 4 = high impact). Section 3 (inter-professional contact) covered perceptions regarding inter-professional contact (items 28 and 29), with responses on a five-point Likert scale (0 = strongly disagree and 4 = strongly agree). Positive agreement to each item was indicated by a rating of 3 or 4. Section 4 (demographics) contained eight questions covering demographics: gender, age group, number of years since registration, position in the pharmacy, hours worked in the pharmacy/week, accreditation for Home Medicines Review

(HMR),[29] pharmacy location and postcode. Pharmacies were classified as metropolitan or regional based on the pharmacy postcode.[30] All data collected were de-identified and double-entered to ensure accuracy. Exploratory factor analysis was used to explore the linear relationships amongst the 10 items and the possibility of grouping related items together into a smaller number of factors.[31] Principal components analysis with varimax rotation was used to examine the factor structure. Factorability of the data set was assessed by the Kaiser–Meyer–Olkin (KMO) measure of sampling adequacy (index >0.6). Factor extraction was based on eigenvalues, the scree plot and the proportion of total variance explained. Items Megestrol Acetate that had poor factor loadings (<0.55) or cross loaded on two or more factors were removed. Internal consistency of the derived subscales was assessed by determining Cronbach's alpha coefficient (values >0.70 were sought).[32] Mean level of agreement scores to each factor for metropolitan versus regional pharmacists were compared using Mann–Whitney U tests. The proportion of pharmacists indicating a positive agreement to each individual item (i.e. a rating ≥3 on a five-point Likert scale from 0–4), each factor and all items was also calculated. Results were expressed as the proportion of pharmacists who rated any level of impact (i.e.

garvieae strains was PF-02341066 mouse determined using RAPD and REP-PCR with BOXA1R and (GTG)5 primers. These methods, which use short arbitrary primers or primers targeting short repetitive sequences interspersed throughout the genome are an established approach for delineation

of bacteria at the species and strain-level (Randazzo et al., 2009; Švec et al., 2010). The discriminatory power of these primer sets was similar, with 20 different profiles obtained by BOXA1R and (GTG)5 and 23 different profiles obtained by M13 for a collection of 49 strains. Although isolated at different times, some strains had identical fingerprints with all tested primers; on the contrary, most of the strains grouped at low similarity values. Independently from the primer used, the 49 strains grouped in two distinct selleck chemicals clusters, which we named AT and BT (Fig. 1): one cluster (AT) contained all meat isolates (with the exception of BOXA1R experiment where the meat isolate Sa113 showed a unique fingerprint at a very low similarity value), whereas the other cluster (BT) included all dairy isolates. Unexpectedly, four of 12 strains isolated from fish (V32, V63, Lg23, and V79), always grouped with dairy isolates, whereas the others grouped with meat isolates. Likewise, strains isolated from vegetables allocated between the two main groups. The cluster analysis resulting from the combined profiles of the three primer sets

employed, confirmed the existence of two major divisions, which were separated at a level of similarity of 0.13 (Fig. 1), and did not coincide with the ecological niche of isolation. In particular, the low correlation value between the two clusters suggested the existence of a marked genetic divergence. When we tested several genes belonging to the core genome of L. garvieae, we observed again that all bacterial isolates can be shared out between two clusters, which are correlated to a low similarity level. Specifically, on the basis of conserved regions identified by sequence comparison

of several housekeeping or functional genes in L. garvieae, we selected suitable primers to employ for PCR amplification (Table 2). The expected fragment length of the α-subunit of ATP synthase, elongation factor EF-Tu, D-alanine-D-alanyl carrier Edoxaban protein ligase, α-acetolactate synthase, glyceraldehyde-3-phosphate dehydrogenase, and galactose permease amplicons was observed for all the 49 strains studied. Restriction analysis of each of the loci tested produced one to seven different patterns consisting of one to seven bands, depending on locus, restriction enzyme and strain examined (Table 3). The cluster analysis resulting from the combined restriction profiles of the six amplicons reported in Fig. 2, revealed two distinct L. garvieae clusters at similarity level of approximately 0.12. Notably, the groups obtained were highly similar to PCR-fingerprinting clusters (AT and BT).

, 1998). Enteric septicemia of catfish

(ESC), caused by the bacterium E. ictaluri, is responsible for approximately 50% of economic losses to catfish farmers in the BGJ398 supplier United States (Klesius, 1993; Shoemaker et al., 2009). Edwardsiella ictaluri is a gram-negative enteric pathogen in catfish, and outbreaks of ESC are seasonal, occurring mainly in spring and fall with a temperature range of 22–28 °C (Tucker & Robinson, 1990). Ichthyophthiriasis is a major parasitic disease of freshwater fish worldwide, caused by a ciliated protozoan Ich. The parasite life cycle consists of an infective theront, a parasitic trophont, and a reproductive tomont (Hines & Spira, 1974; Matthews, 2005; Dickerson, 2006). Mature tomonts leave the fish host, attach to a substrate, and undergo multiple divisions to produce hundreds to thousands of infective theronts. Theronts swim actively in water in search of new fish hosts (Dickerson, 2006). The temperature ranges of ESC outbreaks overlap the optimum temperature window of Ich infection at 22–24 °C (Matthews, 2005; Dickerson, 2006). In 2002, 50.5% and 44.3% of all catfish operations (approximately 1000 total in the USA) had losses caused by ESC and by Ich (white spot), respectively (Hanson et al., 2008). The ability of parasites to enhance mortality because of bacterial diseases is presently receiving attention in aquaculture

GABA Receptor research. However, there is limited information on whether Ulixertinib molecular weight parasites act as vectors to transmit pathogenic bacteria in fish. To prevent and manage bacterial diseases in aquaculture, it is

important to understand the potential of parasites to vector bacteria in fish. Parasites may easily transmit pathogenic bacteria from one fish to another within high-density fish populations on farms. In this trial, we used Ich–E. ictaluri as a model to study the interaction between the parasite, the bacteria, and the fish host. This study tested the hypothesis that Ich can vector E. ictaluri into channel catfish, Ictalurus punctatus. We further established that the bacteria were associated with the surface of the parasite. The bacteria multiplied and were transferred as the parasite divided. Channel catfish (industry pool strain) were obtained from disease-free stock from the USDA-ARS Catfish Genetic Research Unit, Stoneville, MS, and reared to the experimental size in indoor tanks at the USDA, Aquatic Animal Health Research Unit, Auburn, AL. I. multifiliis (ARS 10-1 strain) originally isolated from infected tropical pet fish was maintained by serial transmission on channel catfish held in 50-L glass aquaria, and theronts were cultured as described by Xu et al. (2000). Edwardsiella ictaluri AL-93-58 was transformed with the pZsGreen vector (Clontech, Mountain View, CA) by Russo et al. (2009).

The interaction was labile to oxidants, such as diamide Alisertib chemical structure and menadione. Based on these data, NCgl0899 was named spiA (stress protein interacting with WhcA). Physical association and dissociation of the purified His6–WhcA and GST–SpiA fusion proteins, as assayed by in vitro pull-down experiments, were consistent with in vivo results. These data indicated that the

interaction between WhcA and SpiA is not only specific but also modulated by the redox status of the cell and the functionality of the WhcA protein is probably modulated by the SpiA protein. Corynebacterium glutamicum is a Gram-positive bacteria that belongs to the order Actinomycetales, which also includes the genera Mycobacterium and Streptomyces (Ventura et al., 2007). Corynebacterium glutamicum is a remarkable organism and is capable of producing a variety of amino acids and nucleotides in large quantities (Leuchtenberger et al., 2005). Because of the industrial importance of this organism, its relevant genetic and biochemical features have been extensively characterized. Accordingly, strategies that C. glutamicum cells adopt in response to cellular stresses have attracted scientific interests in recent years. WhiB-like genes are a class of genes that perform diverse cellular processes, such as cell division, differentiation, pathogenesis, starvation survival, and stress

response (Gomez, 2000; Steyn et al., 2002; this website Kim et al., 2005; Geiman et al., 2006; Raghunand & Bishai, 2006; Singh et al., 2007; Choi et al., 2009). The whiB gene, which was originally identified and characterized in Streptomyces coelicolor, is a developmental regulatory gene that is essential for the sporulation of aerial hyphae (Davis & Chater, 1992). The whiB homologues are only found in the order Actinomycetales. Seven whiB homologues have been identified in the Mycobacterium tuberculosis

genome and at least six are present in S. coelicolor (Soliveri et al., 2000), whereas only four are found in C. glutamicum (Kim et al., 2005). The WhiB-like over proteins have four conserved cysteine residues that bind to a redox-sensitive Fe–S cluster (Jakimowicz et al., 2005; Alam et al., 2007; Singh et al., 2007; Crack et al., 2009; Smith et al., 2010), which plays a critical role in controlling protein function. In general, the cluster loss reaction followed by oxidation of the coordinating cysteine thiols that form disulfide bridges is important for activity. For example, S. coelicolor WhiD loses its Fe–S cluster upon exposure to oxygen (O2) and the apo-WhiD may play important roles in cell physiology (Crack et al., 2009). Some WhiB-like proteins may function as transcription factors, as suggested by the presence of predicted helix–turn–helix DNA-binding motif. Recently, the M. tuberculosis WhiB1 protein in its apo-form was shown to have DNA-binding activity (Smith et al., 2010).

Hyphomicrobium sulfonivorans S1T was grown in a batch culture on dimethylsulfone as described previously by Boden et al. (2011) and R. sulfidophilum was grown photoorganoautotrophically on DMS according to McDevitt et al. (2002). Sagittula stellata was grown in

steady-state chemostats at a range of dilution rates (D) between 0.01 and 0.15 h−1 on fructose (12 mM) and between 0.01 and 0.10 h−1 on succinate (2 mM) with or without the addition of DMS (1 mM). Kinetic parameters were determined as described previously (Boden et al., 2010). Five volume changes at each steady state occurred before the kinetic parameters were determined. Cells were harvested for enzyme assays by centrifugation at 13 000 g for 30 min at 4 °C. learn more this website Cells were washed and resuspended in 50 mM PIPES-HCl, pH 7.4, containing 50 mM magnesium sulfate. If not used immediately, cells were snap-frozen in liquid nitrogen and stored at −80 °C. Spectrophotometric enzyme assays were routinely conducted at 30 °C in an Ultrospec 3100pro UV/Visible Spectrophotometer

(Amersham). Each reaction was conducted in a sevenfold replicate against a blank. Cell-free extracts were prepared by three passages through a French pressure cell (120 MPa), with debris removed by centrifugation (13 000 g, 30 min, 4 °C). Protein was quantified using the method of Bradford (1976). DMS dehydrogenase Smoothened activity was assayed using a modification of the method of McDevitt et al. (2002). Two milliliters of 500 mM Tris-HCl, pH 8.0, 300 μL of 35 mM phenazine methosulfate and 300 μL of 100 μM 2,6-dichlorophenolindophenol (DCPIP) were placed in a 3 mL modified Thunberg cell (Baumberger, 1933) and degassed by bubbling with oxygen-free nitrogen for 10 min before adding 100 μL cell-free extract (containing 5–10-mg protein). Sixty microliters of 100 mM DMS solution in ethanol was placed in the bulb of the side-arm and the cell was assembled. The cell was evacuated on ice for 10 min before sealing and the reaction was initiated by pouring

the contents of the side-arm into the main chamber. The A600 nm was monitored and the rate of reduction of DCPIP was determined using the millimolar extinction coefficient for the oxidized form of 21.5 mM−1 cm−1. Cell-free extracts prepared from R. sulfidophilum SH1 grown photoorganoautotrophically with DMS as an energy source were used as a positive control (McDevitt et al., 2002). An alternative assay was performed using 300 μL of 3 mM potassium ferricyanide in place of DCPIP solution and reduction was monitored at 420 nm with a millimolar extinction coefficient of 1.0 mM−1 cm−1. DMSO reductase was assayed in the same way using a reaction mixture comprising 150 μL of 1.0 M Tris-HCl, pH 7.6, 20 μL of cell-free extract and 1.03 mL of MilliQ water in the main chamber of the cell. These were degassed in situ before adding 1.

The study was carried out in 1999–2000 and had an overall IR of 778/100 000 PYO, which is very similar to our estimates. The study included few HIV-infected individuals and did not report on IRs according to HIV transmission group. A follow-up study from 2000 to 2006 by the same group [24] also identified HIV infection as a significant risk factor for SAB. However, in that study the relative risk conferred by HIV infection decreased from 23.7 to 17.1 over the two study periods, suggesting a similar decline in IR to that reported in the present study. Interestingly, they found that HCV infection was associated with an increased risk of SAB but were unable

to attribute this to liver disease or IDU. Our study did not address VX-770 concentration HCV infection per se but, as more than 90% of HIV-infected IDUs are or previously have been infected with HCV, the markedly increased IR among IDUs would suggest that HCV infection may be a marker of www.selleckchem.com/products/icg-001.html IDU. Our study provides new information as we report specific IRs and their changes over time according to HIV transmission group. Over the last decade, the degree of immune deficiency in HIV-infected individuals has diminished as a result of increased coverage of HAART [25]. The incidence of bacterial BSIs has similarly decreased [26,27]. In our study, lack of HAART was associated with a 2-fold increased

risk of SAB and, correspondingly, individuals who were virologically nonsuppressed were at an increased risk. MSM acquired SAB at a old lower CD4 cell count and

had a higher rate of HA SAB, indicating that these cases are probably caused by intravascular devices related to therapy for AIDS-associated diseases, as described previously [4,10,12]. IDUs predominantly acquired CA SAB at higher CD4 cell counts, suggesting that these cases are likely to be related to repeated injections. Further reductions in SAB IRs can be expected to be achieved by reducing immunodeficiency via increased HAART coverage, reducing the proportion of late presenters and encouraging sterile injecting methods among IDUs. Several studies have reported an increased risk of MRSA colonization and infection in HIV-infected individuals compared with the general population [28–32]. The prevalence of MRSA in Denmark is low [16] and, correspondingly, rates were low among HIV-infected individuals and comparable to those in the general population. The strengths of our study include the long study period, the population-based design, the use of nationwide cohorts of HIV-infected individuals, the nationwide registration of SAB and complete data on immigration, emigration and death. There was no evidence of outbreaks or common source infections among HIV-infected individuals during the study period based on phage types (data not shown). However, the study has some limitations. Of 15 clinical microbiological departments in Denmark, one department irregularly contributed with isolates; however, this laboratory covers only 6% of the Danish population [17].