Epidemiology of methicillin-resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated (HA), community-associated (CA) and livestock-associated (LA) infections. Recently, the discovery of human and bovine MRSA isolates carrying a new mecA gene homologue, mecA(LGA251) (now designated mecC), has caused concern because they are not detected by conventional, confirmatory tests for MRSA. Very little is known about their frequency, epidemiology and possible transmission between livestock and humans. In this study, the epidemiology of the mecC isolates in Denmark was investigated by screening the national collections of MRSA cases (from 1988 onwards) and S. aureus bacteraemia cases (from 1958 onwards). Isolates carrying mecC were only recovered infrequently before 2003 (n = 2) but now seem to be increasing, with 110 cases in 2003-2011. Clinical data on mecC-carrying MRSA demonstrated that mecC-MRSA were primarily community-acquired (CA-MRSA) and affected persons typically living in rural areas, being older than other CA-MRSA patients. Among 22 cases in Region Zealand, four reported contact with cattle and sheep. Two of these persons lived on farms with livestock positive for mecC-carrying MRSA, sharing spa type (t843), MLVA (MT429) and PFGE pattern with the human isolates. These observations indicate that mecC-carrying MRSA can be exchanged between humans and ruminants.

Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay.

Rapid molecular typing methods can be a valuable aid in the investigation of suspected outbreaks. We used a semi-automated repetitive sequence-based polymerase chain reaction (Rep-PCR) typing assay and pulsed field gel electrophoresis (PFGE) to investigate the relationship between local Klebsiella pneumoniae (K. pneumoniae) producing extended spectrum ß-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of bla (TEM), bla (SHV), and bla (CTX-M), and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and bla (CTX-M15), bla (SHV-28), and bla (TEM-1) positive by PCR. Ten out of 52 were ST16 and tested positive for bla (CTX-M15), bla (SHV-1), and bla (TEM-1). Isolates from previously recognized hospital outbreaks were also ST15 and PCR positive for bla (CTX-M15), bla (SHV-28), and bla (TEM-1), and typed within the main cluster by both Rep-PCR and PFGE. In conclusion, K. pneumoniae ST15 containing bla (CTX-M15) and bla (SHV-28) constitutes an epidemic clone in the Copenhagen area and this clone can be rapidly recognized by semi-automated Rep-PCR.

Pasteurella aerogenes isolated from ulcers or wounds in humans with occupational exposure to pigs: a report of 7 Danish cases.

Pasteurella aerogenes is rarely isolated from human specimens. The species is found in the digestive tract of pigs. From 1976 to 1994 7 strains were cultured in Denmark from wounds or ulcers. Five patients were bitten by pigs and 2 patients with ulcers were employed in pig farming. A mixture of bacterial species was often found. All 7 strains of P. aerogenes were susceptible to ampicillin, cephalosporins and ciprofloxacin. Ability to hydrolyse urea, to produce oxidase and catalase, to decarboxylate ornithine and to produce gas from glucose and inability to produce indole was characteristic for P. aerogenes. Most bite wounds were located on the lower lateral part of the thigh. Foul smelling pus and abscess formation was the rule. Incision, drainage and antibiotic treatment were usually necessary.

[Purulent arthritis and bursitis after local injection of depot steroids]. / Purulent arthritis og bursitis efter lokalinjektion af depotsteroid.

The occurrence of severe joint and bursal infections preceded by local steroid injection was investigated in a retrospective survey. Of 37 consecutive patients with pyogenic arthritis or bursitis admitted to two hospitals over a three-year-period, nine (95% confidence limits 12-41%) had received previous intraarticular or intrabursal injections of steroid. Staphylococcus aureus was cultured in all cases. This incidence rate of endemic iatrogenic infection is at least 60 times higher than earlier reported. The alarming frequency may be attributed to insufficient aseptic precautions and/or the injection of depot steroids into cavities with overlooked established infection. An increased awareness of this complication seems warranted. Meticulous hygienic measures should be emphasized in drug information inserts. Health authorities and quality control bodies should monitor and analyze the occurrence of such accidents.

Nosocomial epidemic of Serratia marcescens septicemia ascribed to contaminated blood transfusion bags.

Two cases of transfusion-related Serratia marcescens bacteremia prompted extensive epidemiologic investigations in three independent hospitals. Test tubes and plasma from donors whose blood was drawn into bags from a single production batch were cultured. Analysis of the ribotype of S. marcescens isolates was performed. For comparison, a strain from the production plant and eight other, unrelated bacteremia isolates were examined. In addition, a retrospective national survey was carried out. S. marcescens was cultured from 11 (0.73%) of 1515 blood units, and an additional (third) bacteremic patient was identified. The clinical isolates from three patients, the three units of blood transfused, and the plant-derived strain shared a unique ribotype. The incident is interpreted as a sporadic, bacterial contamination of blood bags with the S. marcescens epidemic strain, occurring during the manufacturing or packaging. A similar incident has not previously been reported. Attention is drawn to the possibility of significant contamination during the complex production of multiple-bag blood collection systems. Guidelines for improved registration and handling of transfusion complications in wards are suggested. Manufacturers should be encouraged to provide blood packs with sterile exteriors, in appropriate, single, outer packages.

Isolation of Pasteurella caballi from an infected wound on a veterinary surgeon.

Comparison of phenotypical characters obtained from the type strain of Pasteurella caballi and a previously unclassified P.sp., isolated from an infected wound on a veterinary surgeon, allowed classification of the P.sp. with P. caballi. The P.sp. was isolated in a mixed culture with Escherichia coli and probably represents the first human isolate of P. caballi.

Susceptibility testing of Danish isolates of Capnocytophaga and CDC group DF-2 bacteria.

Twelve Capnocytophaga and seven DF-2 strains were tested for their susceptibility to 14 antimicrobial agents using an agar dilution and an agar diffusion method. Twenty-three other antibiotics were evaluated using the diffusion test only. All strains were fully susceptible to penicillin, ampicillin, cefuroxime, cefotaxime, erythromycin, clindamycin, chloramphenicol, doxycycline, rifamycin and ofloxacin using both methods. Clindamycin, rifamycin and cefotaxime were most active. Using agar dilution some strains were susceptible to gentamicin, but agar diffusion showed total resistance. One Capnocytophaga strain was susceptible and another moderately susceptible to metronidazole, other strains were resistant. The agar diffusion test showed that both Capnocytophaga and DF-2 were resistant to most other aminoglycosides, to fosfomycin, polymyxin and trimethoprim. All strains of both taxa were fully susceptible to piperacillin, cefoxitin, imipenem and fusidic acid and showed different susceptibilities to the other agents. Susceptibility testing by means of agar diffusion using an enriched chocolate agar and 5% CO2 atmosphere could be used to test Capnocytophaga and DF-2 strains and gives sufficient accuracy for routine use, when revised inhibition zone breakpoints are employed.

Evaluation of a conventional routine method for identification of clinical isolates of coagulase-negative Staphylococcus and Micrococcus species. Comparison with API-Staph and API-Staph-Ident.

A collection of 138 consecutive isolates from blood primarily identified as Gram-positive, cluster-forming, coagulase-negative cocci was examined by a conventional routine method for identification of clinical isolates of coagulase-negative Staphylococcus and Micrococcus species. The method was based on selected reactions from the Kloos & Schleifer scheme, utilizing the conventional media of Statens Seruminstitut. Double determinations for each isolate were performed by the conventional method. The results were compared with speciation by the commercial micromethods API-Staph and API-Staph-Ident. For control, 31 Staphylococcus and 13 Micrococcus reference strains were included. Of the 31 Staphylococcus spp. (reference strains), the conventional system, API-Staph, and API-Staph-Ident correctly identified 87%, 87% and 81%, respectively. Micrococcus spp. were only identified to genus level by the conventional method as well as by API-Staph. API-Staph-Ident is not designed for Micrococcus identification. Of 138 blood isolates, 121 belonged to the genus Staphylococcus while 17 were Micrococcus spp. S. epidermidis dominated with all three methods, constituting approx. 35% of the isolates tested. In only 57% of the isolates identification by all three methods agreed. The three methods were unable to put a name on 7.5% (conventional method), 10.7% (API-Staph) and 2.5% (API-Staph-Ident) of the isolates. Reproducibility was high with the conventional method (100% for the reference strains and 91% for blood culture isolates) as well as with API-Staph and API-Staph-Ident (88%/81% and 81%/81%, respectively). We concluded that our conventional system was able to identify most clinically significant staphylococcal species by means of relatively few tests with a high certainty and a high degree of reproducibility.

The ultrastructure of antibiotic-susceptible and multi-resistant strains of group JK diphtheroid rods isolated from clinical specimens.

Two antibiotic-susceptible and two multi-resistant strains of diphtheroid rods of the group JK, obtained from clinical specimens in Denmark and from CDC in the U.S. were studied. The cells of all four strains presented an ordinary Gram-positive cell wall and an additional surface layer. Septum formation in dividing cells appeared to result in a "snapping-like" dividing mechanism, thus corroborating the relationship of the JK cells to the genus Corynebacterium. A significantly increased thickness of the surface layer of the multi-resistant strains was observed when cells were treated with ruthenium red. It is suggested that such a structural difference on the exterior of the cell-wall among JK bacteria may affect the cell-wall's permeability to antibiotics.

Group JK diphtheroid bacteremia. The successive isolation of an antibiotic-susceptible and an antigenically different multi-resistant strain.

A 71-year-old man with a permanent, subcutaneously implanted, intra-cardial pacemaker suffered from prolonged bacteremia with an antibiotic-susceptible group JK diphtheroid rod. He died in spite of the formation of specific serum antibody and parenteral treatment with ampicillin, cephradine and gentamicin. A second multi-resistant, but otherwise similar group JK strain was isolated post-mortem from the aseptically removed pacemaker electrode tip. The susceptible and the multi-resistant strains differed antigenically in crossed immunoelectrophoresis assays, and fatty acid isomer patterns were dissimilar. The theory that a multi-resistant group JK clone emerged by simple mutation in susceptible, indigenous group JK skin flora is rejected. The concept of major structural differences among group JK bacteria, possibly affecting cell-wall permeability, is supported. Crossed immunoelectrophoresis is suggested as a means for strain comparison in epidemiological surveys. Vancomycin is regarded as the antibiotic of choice for the treatment of potentially fatal, deep-seated infections.

The cultivation and rapid enzyme identification of DF-2.

The investigation of two clinical isolates and two reference strains of DF-2 showed that supplementary cysteine and incubation in a humid atmosphere were important growth-promoting factors for these fastidious, gram-negative bacteria. Broth-base media with phenol red indicator were proven to be satisfactory for carbohydrate fermentation tests. Two four-hour enzyme assays (API ZYM and Rosco Diagnostic Tablets) were used to compare the enzymatic activity of DF-2 with that of 27 species of other non-enterobacterial organisms. The Rosco assay revealed that only the DF-2 strains had a positive alpha-fucosidase reaction, suggesting that this character may provide the means for rapid characterization and identification of these bacteria and also be of value for taxonomic classification. The incongruent results of the API ZYM assay seem to be due to the different substrates of the two assay systems.

Rapid identification of Capnocytophaga isolated from septicemic patients.

Four Capnocytophaga strains from blood cultures of immunocompromised patients with malignant disease and the type strains of three Capnocytophaga species were examined and compared to strains representing five other genera that are hard to differentiate from Capnocytophaga. With three rapid identification methods, negative catalase and oxidase reactions and positive ONPG assay, Capnocytophaga was easily separated from Eikenella corrodens, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, and CDC group DF-2. Haemophilus aphrophilus was excluded by leucine, valine and cystine arylamidase and alpha-glucosidase reactions (API ZYM). Further confirmatory reactions constituted gelatin hydrolysis, haemin requirement, and carbohydrate and esculin breakdown. Although rapid identification of Capnocytophaga to the genus level was feasible, differentiation on a species level proved impossible.

Six cases of acute appendicitis with secondary peritonitis caused by Streptococcus pneumoniae.

In six previously healthy children and adults with typical acute appendicitis, Streptococcus pneumoniae was isolated from peritoneal swabs or periappendicular pus in pure culture (four patients) or together with intestinal flora. Pneumococci recovered by abdominal paracentesis are not pathognomonic of socalled primary or spontaneous peritonitis.

Recognition of coagulase-negative Staphylococcus aureus strains by primary polymyxin susceptibility testing.

A prospective study evaluating the usefulness of polymyxin susceptibility testing in unveiling coagulase-negative S. aureus strains was undertaken. During a six months study period, 14 staphylococcal isolates from four patients were initially found to lack coagulase activity and to be polymyxin resistant; in comparison, approximately 1500 ordinary, coagulase-positive, polymyxin resistant S. aureus isolates were found during the same period. One isolate from each of the four patients together with a previously isolated coagulase-negative S. aureus strain from our own collection were further characterized. Two of the strains turned out to show delayed coagulase activity on retesting, while the other three strains failed to produce clotting in any coagulase assay. Judged by thermostable nuclease activity, phage typing and biochemical profiles these three coagulase-negative strains are bona fide S. aureus strains. The polymyxin test thus appears to be useful in disclosing S. aureus variants not identified as S. aureus by routinely performed coagulase assays.

Polymyxin susceptibility in staphylococci differentiating coagulase-positive and coagulase-negative strains.

47 staphylococcal reference strains representing 13 species were tested for polymyxin sensitivity using tablet and disc diffusion methods. Corresponding MIC and IC50 values were determined with a plate dilution assay. Coagulase-positive strains were found to be more resistant towards polymyxin, and could thereby be separated from coagulase-negative strains. Furthermore, 100 recently isolated staphylococci from clinical specimens were tested, and again the coagulase-positive strains could be identified by their smaller tablet inhibition zones. We conclude that the polymyxin test is an aid in the fast differentiation of staphylococci in laboratories performing primary sensitivity tests.