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.

A new concept and a comprehensive evaluation of SYSMEX UF-1000i flow cytometer to identify culture-negative urine specimens in patients with UTI.

Urinary tract infections (UTIs) are among the most common bacterial infections in men and urine culture is gold standard for diagnosis. Considering the high prevalence of culture-negative specimens, any method that identifies such specimens is of interest. The aim was to evaluate a new screening concept for flow cytometry analysis (FCA). The outcomes were evaluated against urine culture, uropathogen species and three conventional screening methods. A prospective, consecutive study examined 1,312 urine specimens, collected during January and February 2012. The specimens were analyzed using the Sysmex UF1000i FCA. Based on the FCA data culture negative specimens were identified in a new model by use of linear discriminant analysis (FCA-LDA). In total 1,312 patients were included. In- and outpatients represented 19.6% and 79.4%, respectively; 68.3% of the specimens originated from women. Of the 610 culture-positive specimens, Escherichia coli represented 64%, enterococci 8% and Klebsiella spp. 7%. Screening with FCA-LDA at 95% sensitivity identified 42% (552/1312) as culture negative specimens when UTI was defined according to European guidelines. The proposed screening method was either superior or similar in comparison to the three conventional screening methods. In conclusion, the proposed/suggested and new FCA-LDA screening method was superior or similar to three conventional screening methods. We recommend the proposed screening method to be used in clinic to exclude culture negative specimens, to reduce workload, costs and the turnaround time. In addition, the FCA data may add information that enhance handling and support diagnosis of patients with suspected UTI pending urine culture [corrected].

Coagulase-negative staphylococci: update on the molecular epidemiology and clinical presentation, with a focus on Staphylococcus epidermidis and Staphylococcus saprophyticus.

Coagulase-negative staphylococci (CoNS), originally described as ubiquitous commensals of the healthy human skin and mucosa, have emerged as important opportunistic pathogens primarily causing healthcare-associated infections in patients with indwelling medical devices. Recent studies, utilizing new molecular typing methods, particularly on Staphylococcus epidermidis, have increased our understanding of the mechanisms that contribute to the evolutionary success of these extremely versatile microorganisms. In the following mini-review, we summarize recent research in this area focusing on the molecular methods and epidemiology of S. epidermidis and S. saprophyticus.

Molecular epidemiology of meticillin-resistant coagulase-negative staphylococci in a Swedish county hospital: evidence of intra- and interhospital clonal spread.

During a 10-month period in 2003, consecutive routine clinical cultures from patients treated in 15 different ward units in a Swedish county hospital were screened for the presence of meticillin-resistant coagulase-negative staphylococci (CNS). Genetic similarity between isolates was analysed using pulsed-field gel electrophoresis (PFGE). The results were compared with multi-drug-resistant Staphylococcus epidermidis isolated previously at the tertiary referral hospital. In total, 428 isolates of CNS were identified, of which 188 (44%) were meticillin resistant. Three clusters (Groups A, B and C) of S. epidermidis, each consisting of more than 10 isolates, with a PFGE-DNA similarity of >90% were identified. The strains in Groups A and B (N=15 and N=13, respectively), which were generally resistant to gentamicin, co-trimoxazole and clindamycin, originated from 24 patients, of whom 21 had been treated in the intensive care unit (ICU) before sampling. The third cluster, Group C, involved 14 isolates from 14 patients. Only two of these patients had stayed at the ICU, and all for less than one day. Isolates in Group C were less resistant than those in Groups A and B. Isolates belonging to Group A showed an identical PFGE profile compared with multi-drug-resistant S. epidermidis isolated from patients at the referral hospital. This study demonstrated the persistence and spread of meticillin-resistant clones of CNS within the county hospital, especially in the ICU, and possible interhospital spread of a multi-drug-resistant clone between the county and referral hospitals.

A preliminary evaluation of a new selective agar supplemented with desferrioxamine for detection of methicillin-resistant Staphylococcus aureus.

The aim of the present study was to evaluate the performance of two new selective screening agars, Colombia agar supplemented with 1000 mg/L desferrioxamine, 5 mg/L amphotericin B, 16 mg/L polymyxin B, and 2 mg/L methicillin (CMDAP agar) or 0.5 mg/L oxacillin (CODAP agar), for detection of methicillin-resistant Staphylococcus aureus (MRSA). Both the CMDAP and the CODAP agar effectively inhibited growth of 151 isolates of coagulase-negative staphylococci (CoNS), 45 of Enterobacteriaceae and six Candida spp. examined. The sensitivity and specificity of the CMDAP and CODAP agars for detection of MRSA was calculated by comparing the growth of 52 MRSA with the inhibition of 74 mecA negative S. aureus and of 151 CoNS. The performance of the new agars was compared with four previously described MRSA screening agars. The sensitivity and specificity for detection of MRSA after incubation at 35 degrees C for 24 h was 0.94 and 0.91, respectively, for the CMDAP agar, 0.60 and 0.90 for the CODAP agar, 0.98 and 0.57 for methicillin aztreonam mannitol salt agar (MAMSA), 0.23 and 0.84 for oxacillin mannitol salt agar (OMSA), 0.48 and 0.76 for oxacillin Mueller-Hinton agar (OMHA) and 0.75 and 0.77 for lithium oxacillin mannitol salt agar (LOMSA). Agars supplemented with desferrioxamine, CMDAP and CODAP, were more specific for detecting MRSA compared with agars not supplemented with desferrioxamine. The detection rate was higher for agars supplemented with methicillin than for agars supplemented with oxacillin.

Mueller-Hinton agar is superior to PDM blood agar for detection of methicillin-resistant Staphylococcus aureus.

The aim of this study was to compare the expression of oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) on Paper Disc Method agar supplemented with 5% defibrinated blood (PDM blood agar) and Mueller-Hinton agar supplemented with 2% NaCl (MH NaCl agar) using different susceptibility tests. Fifty mecA-containing isolates of S. aureus, exhibiting 46 different pulsed-field gel electrophoresis patterns, were comparatively tested using the E test, the single disk diffusion test, and the multipoint inoculation technique, under various culture conditions. The E test incubated at 35 degrees C for 24 h (breakpoint of resistance > or = 2.0 mg/L) detected 94% of the isolates on MH NaCl agar, compared with 28% for PDM blood agar (P < 0.05). The disk diffusion test (breakpoint < 10 mm in diameter) under these incubation conditions detected resistance in 100% of the isolates on MH NaCl agar and in 80% of the isolates on PDM blood agar (P < 0.05). The multipoint technique (breakpoint > or = 1 mg/L), applied at 35 degrees C for 24 h, detected 100% on MH NaCl agar and 46% on PDM blood agar (P < 0.05). Irrespective of the method of susceptibility testing evaluated, MH NaCl agar was superior to PDM blood agar for the detection of oxacillin resistance in mecA-containing S. aureus.

Bactericidal effect of pulsed 1,064 nm Nd:YAG laser light on Staphylococcus epidermidis is of photothermal origin: an in vitro study.

BACKGROUND AND OBJECTIVE: The aim was to investigate the bactericidal effect of the 1,064 nm Nd:YAG laser on Staphylococcus epidermidis. MATERIAL AND METHODS: S. epidermidis was inoculated on agar plates and then exposed to pulsed laser light in three different modes: with an uninterrupted train of pulses, or with two different repeated cycles of fractionated trains of pulses. The agar temperature was measured directly after uninterrupted radiation. RESULTS: A bacterial growth inhibition area of 0.3 cm(2) and maximum temperature of approximately 80 degrees C was observed after uninterrupted radiation at 2,000 J cm(-2). The corresponding figures after an exposure of 5,000 J cm(-2) were 0.9 cm(2) and 100 degrees C, respectively. No bacterial inhibition was observed after exposure to repeated cycles of 20 seconds of radiation followed by 60 seconds of rest. CONCLUSION: The antimicrobial effect of the 1,064 nm Nd:YAG laser light is caused by a photothermal rather than a photochemical effect.

Brain abscess caused by methicillin-resistant Staphylococcus aureus.

A Swedish tourist was admitted to a Cuban hospital due to epileptic seizures caused by brain tumors. Upon return to Sweden and admission to our hospital, methicillin-resistant Staphylococcus aureus (MRSA) was isolated. He was later considered to be free of MRSA but then developed a brain abscess from which MRSA was isolated.

Characterization of Actinomyces isolates from infected root canals of teeth: description of Actinomyces radicidentis sp. nov.

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733.

Clonal spread of staphylococci among patients with peritonitis associated with continuous ambulatory peritoneal dialysis.

BACKGROUND: Peritonitis is the most important complication of continuous ambulatory peritoneal dialysis (CAPD). Coagulase-negative staphylococci (CNS) are the most common causes of peritonitis, only limited information is available regarding the distribution and epidemiology of different CNS species associated with CAPD peritonitis. METHODS: CNS isolated from dialysis effluent from CAPD patients with peritonitis was identified by species and further analyzed with pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 216 microorganisms (206 bacteria and 10 Candida species) were isolated from 196 consecutive culture-positive CAPD samples obtained from 75 patients. One hundred and twenty-one (56%) isolates represented staphylococci. The four most frequently isolated staphylococcal species were Staphylococcus epidermidis (70 isolates), Staphylococcus aureus (31 isolates), Staphylococcus hemolyticus (10 isolates), and Staphylococcus hominis (4 isolates). PFGE analysis revealed the clonal spread among patients of three different clones of S. epidermidis and one clone of S. aureus among the investigated patients. Indistinguishable isolates of either S. epidermidis, S. hominis, or S. aureus were also isolated in repeated samples from several patients. CONCLUSION: PFGE is a useful method for the epidemiological evaluation of staphylococci-associated CAPD infections and should replace older and less accurate methods, such as antibiotic sensitivity patterns. We recommend that CNS isolates from patients with CAPD-associated peritonitis should be saved for future investigations and typing, which would aid in the management of this patient category.

Antibiotic susceptibility of staphylococci isolated in blood cultures in relation to antibiotic consumption in hospital wards.

A total of 510 isolates of Micrococcaceae, 500 of staphylococci and 10 micrococci, detected in 485 (3.3%) of 14,860 consecutive blood cultures obtained from patients at a Swedish university hospital and 2 local hospitals were identified to species level and investigated for antibiotic susceptibility. The 5 most frequently isolated species were Staphylococcus epidermidis (54.8%), S. aureus (28.0%), S. hominis (3.4%), S. warneri (3.2%) and S. haemolyticus (2.8%). All isolates of S. aureus were oxacillin sensitive. Great diversity in antibiotic resistance among coagulase negative staphylococci between hospitals and different ward units in the university hospital was observed. The frequency of antimicrobial resistance among S. epidermidis correlated with the antibiotic consumption at different ward units, in particular for ciprofloxacin (p < 0.001) and co-trimoxazole (p < 0.004). The study emphasizes the importance of monitoring antibiotic consumption and resistance patterns of nosocomial staphylococci in order to avoid emergence and spread of multi-resistant bacteria within the hospital environment.

Trovafloxacin susceptibility of aerobic clinical bacterial isolates from Sweden.

Trovafloxacin susceptibility was studied in aerobic clinical isolates of bacterial pathogens from 5 microbiology laboratories in Sweden. Trovafloxacin and ciprofloxacin minimum inhibitory concentration (MIC) determinations were performed on 474 clinical isolates. Disk diffusion tests using trovafloxacin and ciprofloxacin 10 microg disks were performed on a total of 7142 clinical isolates (trovafloxacin). Susceptibility interpretations for trovafloxacin and ciprofloxacin were determined from MIC values and disk diffusion tests using species-related MIC-limits and zone diameter breakpoints. Eight of 12 gram-positive species groups were fully susceptible to trovafloxacin as judged by MIC tests. Trovafloxacin gave MIC50 values of 0.032 mg/l for S. aureus, 1.0 mg/l for MRSA, 0.064 mg/l for coagulase negative staphylococci, 1.0 mg/l for MRSE, 0.064 mg/l for S. saprophyticus, 0.125 mg/l for group A and group B streptococci, 0.064 mg/l for group C and G streptococci and S. pneumoniae, 0.25 mg/l for E. faecalis, and 16.0 mg/l for E. faecium. These MIC values were 4-16-fold lower than those of ciprofloxacin. Both MIC and disk tests showed similar levels of susceptibility among gram-negative isolates for trovafloxacin and ciprofloxacin. For most gram-negative species the trovafloxacin MIC50 values were similar to or slightly higher than those for ciprofloxacin. Trovafloxacin MIC values were much lower for Acinetobacter strains, but higher for P. mirabilis compared with ciprofloxacin. The favourable susceptibility levels of Swedish aerobic pathogens to trovafloxacin emphasize the potential of this drug for the treatment of serious infections.

Calibration of disk diffusion antibiotic susceptibility testing: species-related trovafloxacin interpretive zone breakpoints and selection of disk potency.

International comparisons of antibiotic susceptibility require the use of common minimum inhibitory concentration (MIC) limits. Disk diffusion test results are not directly suitable for such comparisons, since different standards are often used and zone breakpoints issued might reflect different MIC limits. We have used single strain regression analysis (SRA) for the calibration of the disk test, both according to species and individual laboratory, and for quality control of trovafloxacin disk diffusion tests in 5 laboratories in Sweden. Preliminary controls using histogram analysis including subtraction histograms of reference strains revealed marked differences between different laboratories. SRA was performed on 4 reference strains, S. aureus, E. faecalis, E. coli and P. aeruginosa, using disks containing 1, 3, 10, 30 and 100 microg trovafloxacin. The results using SRA showed a difference between laboratories using Biodisk PDM medium, which produced smaller zones, and those using Oxoid IsoSensitest. Species-related regression lines for laboratories using either medium were calculated and corresponding interpretive zone breakpoints determined for MIC limits. Rational criteria for the selection of a suitable disk content of an antibiotic were also defined and applied to trovafloxacin. The 10 microg disk selected by NCCLS (National Committee for Clinical Laboratory Standards) proved optimal.

An inexpensive and reliable method for routine identification of staphylococcal species.

The aim of this study was to develop a simple, reliable, and inexpensive in-house system for routine species identification of staphylococci in clinical practice. The system combines 15 key tests (including carbohydrate fermentation) performed in micro-well strips and antimicrobial disk diffusion susceptibility tests performed on standardised paper disk method antibiotic sensitivity medium agar. Twenty-eight staphylococcal reference strains belonging to 18 different species were correctly identified using this in-house system. A total of 291 clinical staphylococci isolates were evaluated with the in-house system and a conventional identification scheme. The in-house system identified 281 (96.6%) of these 291 isolates. Eleven different species were recognised. The five species most frequently identified were Staphylococcus epidermidis (48.6%), Staphylococcus aureus (27.8%), Staphylococcus haemolyticus (8.2%), Staphylococcus hominis (5.7%), and Staphylococcus warneri (5.3%). There was an agreement of 86.3% between the species identification obtained with the in-house system and the conventional identification scheme. All coagulase-negative isolates initially identified as species other than Staphylococcus epidermidis as well as indistinctly identified isolates were also evaluated with a commercial identification system. The agreement between species identification obtained with the in-house system and the commercial system for 101 identified isolates was 73%. Several isolates that were difficult to distinguish with the conventional scheme and/or the commercial system were identified with the aid of the antimicrobial susceptibility test included in the in-house system. The described test scheme should be of value for identification of clinically significant staphylococci species.

A case of Plesiomonas shigelloides cellulitis and bacteraemia from northern Europe.

Bacteremia caused by Plesiomonas shigelloides is a rare event, often associated with consumption of seafood and fresh or estuarine water in temperate or tropical climates. Most patients have showed underlying health disorders. Here we present a case of P. shigelloides septicaemia and cellulitis of the left hand associated with fish handling in Northern Sweden (65 degrees latitude north). The patient, who suffered from multiple myeloma, recovered uneventfully after initial treatment with intravenous cefuroxime followed by a course of oral ciprofloxacin. P. shigelloides seems to be ubiquitous in freshwater world-wide and may cause invasive infections also in cold climate areas.

Leuconostoc spp. septicaemia in a child with short bowel syndrome.

Septicaemia caused by the vancomycin-resistant Gram-positive bacteria Leuconostoc spp. is uncommon. We report a case of Leuconostoc spp. septicaemia in a child with short bowel syndrome fed through a central venous catheter and a gastrostomy. Leuconostoc spp. were isolated from several blood cultures. Despite several courses of antibiotics the fever continued and her condition deteriorated. After removal of the thrombotized central venous catheter her condition improved. Leuconostoc spp. was isolated from the thrombotic masses.

Anaerobic phagocytosis, killing, and degradation of Streptococcus pneumoniae by human peripheral blood leukocytes.

Encapsulated Streptococcus pneumoniae of serotypes 2, 9N, 14, 21, and 23F and an unencapsulated variant of type 2 pneumococci were efficiently phagocytosed by both aerobically and anaerobically incubated human leukocytes. In the presence of O2, the pneumococci rapidly lost their viability, whereas during anaerobiosis, killing was considerably delayed. Type 14 pneumococci radiolabeled with [14C]choline or [14C]ethanolamine for cell wall teichoic acid, [14C]uracil for nucleic acids, or [14C]arachidonic acid for unsaturated cytoplasmic membrane lipids were used in studies of the fate of bacterial macromolecules after phagocytosis. The degradation of teichoic acid, RNA, and DNA during anaerobiosis approached that recorded in air at 60 min of incubation (45 to 70% and 55 to 75%, respectively). In contrast, the marked loss of [14C]arachidonic acid from pneumococcal membrane lipids observed in aerobic leukocytes did not occur during anaerobic incubation. Hence, lipid peroxidation could be involved in the rapid aerobic leukocyte killing of pneumococci, whereas a different leukocyte function of as yet unknown nature appears to be responsible for the killing seen in anaerobiosis. Autolysis-resistant type 14 pneumococci were obtained by substituting ethanolamine for choline in a defined culture medium. Differences between such bacteria and normal (autolytic) pneumococci in their killing and degradation by leukocytes were not detected in either the presence or the absence of O2. The aerobic and anaerobic handling of phagocytosed pneumococci by human blood leukocytes thus proceeded independently of the bacterial autolytic system.