Genotypic characterization of Staphylococcus chromogenes and Staphylococcus simulans from Swedish cases of bovine subclinical mastitis.

Staphylococcus chromogenes and Staphylococcus simulans are commonly found in intramammary infections (IMI) associated with bovine subclinical mastitis, but little is known about genotypic variation and relatedness within species. This includes knowledge about genes encoding antimicrobial resistance (AMR) and potential virulence factors (pVF). The aim of this study was therefore to investigate these aspects by whole-genome sequencing of milk isolates from Swedish dairy cows with subclinical mastitis in an observational study. We also wanted to study if specific genotypes were associated with persistent IMI and the inflammatory response at udder quarter level. In total, 105 and 118 isolates of S. chromogenes and S. simulans, respectively, were included. Isolates were characterized using a 7-locus multilocus sequence typing (7-MLST), core genome analysis and in-silico analysis of AMR and pVF genes. Forty-seven sequence types (ST) and 7 core genome clusters of S. chromogenes were identified, and the most common ST were ST-6 and ST-109, both belonging to cluster VII. A 7-locus MLST scheme for S. simulans was not available, but 3 core genome clusters and 5 subclusters were described. Overall, substantial variation in ST and clusters among cows and herds were found in both species. Some ST of S. chromogenes were found in several herds, indicating spread between herds. Moreover, within-herd spread of the same genotype was observed for both species. Only a few AMR genes [blaZ, strpS194, vga(A)] were detected in a limited number of isolates, with the exception of blaZ coding for ß-lactamase, which was identified in 22% of the isolates of S. chromogenes with ST-19, ST-102, and ST-103 more commonly carrying this gene compared with other ST. However, the blaZ gene was not identified in S. simulans. The average total number of pVF detected per isolate was similar in S. chromogenes (n = 30) and S. simulans (n = 33), but some variation in total numbers and presence of specific pVF or functional groups of pVF, was shown between ST/clusters within species. Differences in inflammatory response and potentially in persistent IMI at udder quarter level were found between S. chromogenes subtypes but not between S. simulans subtypes. In conclusion, the results from the present study generates new insight into the epidemiology of bovine S. chromogenes and S. simulans IMI, which can have implications for future prevention and antimicrobial treatment of infections related to these species.

Intramammary infections and risk factors in freshly calved heifers in Swedish dairy herds.

Subclinical mastitis can be common among freshly calved heifers (FCH), but the prevalence differs between herds, possibly due to variation in risk factors. The aims of this observational study were to identify differences in occurrence of intramammary infection (IMI) in FCH between herds with documented good or poorer first-parity udder health based on cow somatic cell count (CSCC) in early lactation, and to study herd differences in animal factors important for udder health, such as udder and hock skin lesions and animal cleanliness. Three groups of herds were included: those with high proportions of FCH with low CSCC (≤75,000 cells/mL) at the first 2 milk recordings after calving (LL), herds with high proportions of FCH with high CSCC (>100,000 cells/mL) at the first and low CSCC at the second recording (HL), and herds with high proportions of FCH with high CSCC at both recordings (HH). Thirty-nine herds (13 LL, 11 HL, 15 HH) were visited 3 times during a 12-mo period for observation of cleanliness and hock lesions, and sampling of udder and teat skin using swab cloths of milk-fed calves, early-pregnant heifers, and late-pregnant heifers. In 25 (9 LL, 9 HL, 7 HH) udder quarter samples from colostrum and milk on d 3 to 4 after calving were taken by the farmers from FCH during one year. The farmers also provided information on calving (individual or group), use of restraint and oxytocin at milking, and presence of teat and udder skin lesions. Bacterial growth in swab samples and quarter samples was investigated by culturing, and a selection of isolates was genotyped using whole-genome sequencing. Cleanliness, hock and udder skin lesions other than udder-thigh dermatitis, and growth of bacteria in swab samples did not differ between herd groups. It was more common that FCH from LL herds, compared with FCH in HH and HL herds, calved in a group of animals. Use of restraint at milking was more common in LL herds than in HH herds, whereas presence of udder-thigh dermatitis was lowest in LL herds. Specific infection was found in 14% of 5,593 quarter samples from 722 FCH. The most common IMI was Staphylococcus chromogenes. Growth of Staphylococcus simulans was more common in HH than in LL and HL herds. In colostrum samples, Staphylococcus haemolyticus was more common in HL and HH than in LL herds. The proportion of quarters with the same specific infection at both samplings was higher in HH than in LL herds and tended to be higher in HH than in HL herds. The proportion of quarters with Staph. chromogenes IMI at both samplings tended to differ between herd groups and was highest in HH herds. Whole-genome sequencing found the same sequence type of Staph. chromogenes and Staphylococcus aureus in both samples in almost all quarters with the same infection at both samplings. The differences in IMI between herd groups were in line with the higher somatic cell count in HH herds. The reasons for the predominance of Staph. chromogenes IMI in FCH need further studies.

Identical genotypes of community-associated MRSA (ST59) and livestock-associated MRSA (ST9) in humans and pigs in rural China.

This study investigated the prevalence of MRSA in samples taken in households, with and without backyard pigs in villages in a rural area of Shandong Province, China. Community-associated MRSA and livestock-associated MRSA, belonging to ST59 and ST9, respectively, were identified in both humans and pigs. The genotypic and phenotypic comparison of isolates indicates that bidirectional transmission of MRSA has occurred between humans and pigs in the villages.

Udder infections with Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis at calving in dairy herds with suboptimal udder health.

Udder infections with Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis are common causes of bovine mastitis. To study these pathogens in early lactation, a 12-mo longitudinal, observational study was carried out in 13 herds with suboptimal udder health. The aims of the study were to investigate the occurrence of these pathogens and to identify if presence of the 3 pathogens, and of genotypes within the pathogens, differed with respect to herd, season, and parity. Quarter milk samples, collected at calving and 4 d in milk (DIM), were cultured for the 3 pathogens. Genotyping of staphylococcal and streptococcal isolates was performed using spa typing and pulsed-field gel electrophoresis, respectively. For each of the 3 pathogens, cows with an udder infection at calving or 4 DIM were allocated to 1 of 4 infection types: cleared (pathogen present only at calving), persistent (pathogen present in the same quarter at calving and 4 DIM), new (pathogen present only at 4 DIM), or cleared/new (pathogen present in 1 quarter at calving and in another quarter at 4 DIM). Associations between season or parity and overall occurrence of pathogens or infection types were determined using univariable mixed-effect logistic-regression models and the Fisher's exact test, respectively. The most commonly occurring pathogen was Staph. aureus, followed by Strep. dysgalactiae and Strep. uberis. Persistent infections were the most common infection type among Staph. aureus-infected cows, whereas cleared infections were the most common among Strep. dysgalactiae- and Strep. uberis-positive cows. The proportion of cows with persistent Staph. aureus infections and the proportion of cows having a Strep. uberis infection at calving or 4 DIM were higher in the multiparous cows than in primiparous cows. Infections with Strep. dysgalactiae were less common during the early housing season than during the late housing or pasture seasons, whereas persistent Strep. uberis infections were less common during the pasture season than during the late housing season. The relative occurrence of the 3 pathogens, infection types of each pathogen, and genotype diversity of each pathogen throughout the year or in different seasons and parities varied among the herds, indicating that underlying factors predisposing for udder infections at calving differ between herds. Genotyping of bacterial isolates gave important insight into how such infection patterns differed within and between herds. These findings emphasize the need to choose preventive strategies for each individual herd.

Staphylococcus pseudintermedius can be misdiagnosed as Staphylococcus aureus in humans with dog bite wounds.

The purpose of this study was to investigate whether S. pseudintermedius is misdiagnosed as S. aureus by clinical laboratories when isolated from humans with dog bite wounds. In addition, we attempted to determine whether S. pseudintermedius isolates related to dog bite wounds share phenotypic and genotypic traits. S. pseudintermedius was identified by PCR targeting the nuc gene. Isolates were tested for antibiotic susceptibility using VetMIC GP-mo microdilution panels. The occurrence of genes encoding leukocidins, exfoliatins, pyrogenic toxin superantigens and enterotoxins was determined by PCR. The relatedness of S. pseudintermedius isolates was investigated using Multi Locus Sequence Typing (MLST). Out of 101 isolates defined as S. aureus by human clinical microbiology laboratories, 13 isolates were re-identified as S. pseudintermedius and one isolate was confirmed to carry the mecA gene, i.e. methicillin-resistant (MRSP). The MRSP isolate was also defined as multi-resistant. Two methicillin-susceptible S. pseudintermedius isolates were also multi-resistant and five were susceptible to all antibiotics tested. With the exception of three S. pseudintermedius isolates belonging to multi locus sequence type (MLST) 158, all the isolates belonged to unique STs. All isolates contained lukS/F-I, siet and se-int, and expA were identified in two isolates and expB and sec canine-sel in one isolate respectively. S. pseudintermedius is frequently misdiagnosed as S. aureus from humans with dog bite wounds showing that it can act as an opportunistic pathogen in humans. No common phenotypic and genotypic traits shared by the S. pseudintermedius isolates could be identified.

Characterization of plasmid-mediated AmpC-producing E. coli from Swedish broilers and association with human clinical isolates.

A selection of plasmid-mediated AmpC-producing Escherichia coli isolates carrying blaCMY-2 from Swedish broilers were characterized to establish their relatedness to and a possible overlap with human clinical E. coli isolates. The results showed diversity among the E. coli isolated from broilers, indicating that the spread in the population was not due to one strain. However, only one type of plasmid belonging to replicon type incK was identified. Furthermore, there were no indications of spread of blaCMY-2 E. coli isolates from broilers to human clinical settings, although Swedish broilers may be a source of blaCMY-2 and/or the plasmid carrying blaCMY-2 .

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012.

Genotyping of important medical or veterinary prokaryotes has become a very important tool during the last decades. Rapid development of fragment-separation and sequencing technologies has made many new genotyping strategies possible. Among these new methods is multilocus variable-number tandem repeat analysis (MLVA). Here we present an update on the use of MLVA in eight European countries (Denmark, France, Germany, Ireland, Italy, the Netherlands, Norway and Sweden). Researchers in Europe have been active in developing and implementing a large array of different assays. MLVA has been used as a typing tool in several contexts, from aiding in resolving outbreaks of foodborne bacteria to typing organisms that may pose a bioterrorist threat, as well as in scientific studies.

Misuse of antimicrobials and selection of methicillin-resistant Staphylococcus pseudintermedius strains in breeding kennels: genetic characterization of bacteria after a two-year interval.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains have been isolated from dogs with increasing frequency; prolonged or excessive use of antimicrobials is associated with the selection of MRSP, and misuse of antimicrobials is frequent in breeding kennels. This study was carried out in two breeding kennels (A and B) in which we had isolated MRSP in 2008: the aim was to assess colonization of previously positive bitches and of other bitches sharing the same environment and to assess the genetic profile of both the old and the new strains [spa typing, staphylococcal cassette chromosome mec (SCCmec) typing and Pulsed-field Gel Electrophoresis (PFGE)]. Six animals from Kennel A (two from 2008) and eight from Kennel B (one from 2008) were tested: 16 MRSP strains were isolated only from bitches housed in Kennel B. Old and new isolates were mecA positive, resulted spa type t02 and carried SSCmec II-III. PGFE showed that all isolates were related and belonged to the main clone lineage dominating in Europe, ST71-J-t02-II-III. Kennels A and B differ in the use of antimicrobials, which has been reduced over time in Kennel A, while has remained excessive in Kennel B, where many agents belonging to different classes (third-generation cephalosporins, fluoroquinolones, macrolids) are administered to dogs, without veterinary supervision, especially around parturition. Misuse of antimicrobials is the key factor for the selection of MRSP strains in healthy dogs and for their persistence over time. Dog breeders should be aware that infections caused by multiresistant bacteria have very limited therapeutical options and represent a huge challenge for animal health.

Methicillin-resistant Staphylococcus aureus (MRSA) in municipal wastewater: an uncharted threat?

AIMS: (i) To cultivate methicillin-resistant Staphylococcus aureus (MRSA) from a full-scale wastewater treatment plant (WWTP), (ii) To characterize the indigenous MRSA-flora, (iii) To investigate how the treatment process affects clonal distribution and (iv) To examine the genetic relation between MRSA from wastewater and clinical MRSA. METHODS: Wastewater samples were collected during 2 months at four key sites in the WWTP. MRSA isolates were characterized using spa typing, antibiograms, SSCmec typing and detection of Panton-Valentine leukocidin (PVL). CONCLUSIONS: MRSA could be isolated on all sampling occasions, but only from inlet and activated sludge. The number of isolates and diversity of MRSA were reduced by the treatment process, but there are indications that the process was selected for strains with more extensive antibiotic resistance and PVL+ strains. The wastewater MRSA-flora had a close genetic relationship to clinical isolates, most likely reflecting carriage in the community. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that MRSA survives in wastewater and that the WWTP may be a potential reservoir for MRSA.

Attempts to improve absolute quantification of cerebral blood flow in dynamic susceptibility contrast magnetic resonance imaging: a simplified T1-weighted steady-state cerebral blood volume approach.

BACKGROUND: Attempts to retrieve absolute values of cerebral blood flow (CBF) by dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) have typically resulted in overestimations. PURPOSE: To improve DSC-MRI CBF estimates by calibrating the DSC-MRI-based cerebral blood volume (CBV) with a corresponding T1-weighted (T1W) steady-state (ss) CBV estimate. MATERIAL AND METHODS: 17 volunteers were investigated by DSC-MRI and 133Xe SPECT. Steady-state CBV calculation, assuming no water exchange, was accomplished using signal values from blood and tissue, before and after contrast agent, obtained by T1W spin-echo imaging. Using steady-state and DSC-MRI CBV estimates, a calibration factor K = CBV(ss)/CBV(DSC) was obtained for each individual. Average whole-brain CBF(DSC) was calculated, and the corrected MRI-based CBF estimate was given by CBF(ss) = K x CBF(DSC). RESULTS: Average whole-brain SPECT CBF was 40.1+/-6.9 ml/min x 100 g, while the corresponding uncorrected DSC-MRI-based value was 69.2+/-13.8 ml/min x 100 g. After correction with the calibration factor, a CBF(ss) of 42.7+/-14.0 ml/min x 100 g was obtained. The linear fit to CBF(ss)-versus-CBF(SPECT) data was close to proportionality (R = 0.52). CONCLUSION: Calibration by steady-state CBV reduced the population average CBF to a reasonable level, and a modest linear correlation with the reference 133Xe SPECT technique was observed. Possible explanations for the limited accuracy are, for example, large-vessel partial-volume effects, low post-contrast signal enhancement in T1W images, and water-exchange effects.

Can the average glandular dose in routine digital mammography screening be reduced? A pilot study using revised image quality criteria.

There is a need for tools that in a simple way can be used for the evaluation of image quality related to clinical requirements in mammography. The aim of this work was to adjust the present European image quality criteria to be relevant also for digital mammography images, and to use as simple and as few criteria as possible. A pilot evaluation of the new set of criteria was made with mammograms of 28 women from a General Electric Senographe 2000D full-field digital mammography system. One breast was exposed using the standard automatic exposure mode, the other using about half of that absorbed dose. Three experienced radiologists evaluated the images using visual grading analysis technique. The results indicate that the new quality criteria can be used for the evaluation of image quality related to clinical requirements in digital mammography in a simple way. The results also suggest that absorbed doses for the mammography system used may be substantially reduced.

Clinical evaluation of a new set of image quality criteria for mammography.

The European Commission (EC) quality criteria for screen-film mammography are used as a tool to assess image quality. A new set of criteria was developed and initially tested in a previous study. In the present study, these criteria are further evaluated using screen-film mammograms that have been digitised, manipulated to simulate different image quality levels and reprinted on film. Expert radiologists have evaluated these manipulated images using both the original (EC) and the new criteria. A comparison of three different simulated dose levels reveals that the new criteria yield a larger separation of image criteria scores than the old ones. These results indicate that the new set of image quality criteria has a higher discriminative power than the old set and thus seems to be more suitable for evaluation of image quality in mammography.

Threshold pixel size for shape determination of microcalcifications in digital mammography: a pilot study.

The effect of pixel size on shape determination in screening digital mammography systems was studied using a shape identification task as the measured outcome. Ten microcalcifications on screen-films were digitised to a range of pixel sizes (2.5-200 microm) and extracted from computed radiography (CR) images (50 microm) acquired under equivalent imaging conditions. Fifteen observers attempted to identify the shape of each microcalcification at each pixel size. The results were collated to provide a fraction of correct responses vs. pixel size curve for each microcalcification. Averaging over all shapes, pixel values >100 microm lead to a significant decrease in shape determination ability (p < 0.01) for digitised screen-film. For CR images, half the shapes were not properly identified. Hence, although 20-100 microm was sufficient for microcalcification shape determination for digitised screen-film images, 50 microm was only borderline sufficient for the CR digital images.

Delayed chemotherapy-induced nausea is augmented by high levels of endogenous noradrenaline.

The relation between pretreatment night-time urinary catecholamine excretion and chemotherapy-induced nausea and vomiting was studied. The first cohort included 17 women and three men with various cancer forms receiving low or moderately emetogenic chemotherapy. The second cohort included 42 women receiving cisplatinum (50 mg m-2) for ovarian cancer and ondansetron as an antiemetic (8 mg i.v. x 3 at chemotherapy and 8 mg p.o. x 3 for 5 days). Relatively higher noradrenaline, but not adrenaline, excretion was associated with an increased intensity of delayed nausea following treatment. Vomiting was not consistently related to the excretion of either catecholamine. The results indicate that noradrenaline modulates delayed nausea resulting from chemotherapy.

Effects of isoprenaline and 1-methyl-3-isobutylxanthine on cyclic nucleotides and cyclic AMP-dependent protein kinase in rabbit colon smooth muscle.

From a homogenate of rabbit colon a crude protein kinase was isolated. The phosphorylation of histone by this enzyme was maximal at 20 mM NaF. Higher concentrations inhibited the incorporation of 32P. After chromatography on a DEAE-cellulose column the crude protein kinase was partly purified. Cyclic AMP and cGMP stimulated the protein kinases eluted at 0.09 M-NaCl (Type I) and at approximately 0.2M-NaCl (Type II). The main peak was of type II. Mix (1-methyl-3-isobutylxanthine) relaxed the rabbit colon muscle. The increase in the protein kinase activity was closely correlated to the changes of the cAMP level. The cGMP content was also increased. Isoprenaline increased the cAMP level and the protein kinase activity of the colon muscle. Isoprenaline had no effect on the cGMP level. It is suggested that relaxing drugs which increase the cAMP level of rabbit colon muscle may activate protein kinase. This enzyme might be an important factor of the cAMP mediated relaxing mechanism.