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A case of bacteremia with the fastidious bacteria Actinomyces urogenitalis following lengthy urinary retention is reported in a sixty-year-old man. In 2013, the first case of bacteremia due to A. urogenitalis was presented, secondary to a tubo-ovarian abscess following transvaginal oocyte retrieval. To the best of our knowledge, this is the first male bacteremic episode involving A. urogenitalis related to a urinary tract focus. The patient had no prior urogenital medical history. Extensive susceptibility testing was done on isolates from urinary and blood cultures. The organism exhibited fluoroquinolone resistance but was susceptible to most other antibiotics used in the treatment of urinary infections. Due to its unusual growth requirements infections with A. urogenitalis are most likely an underdiagnosed entity., (© 2019 APMIS. Published by John Wiley & Sons Ltd.)

The use of broad range PCR and DNA sequencing of bacterial 16S ribosomal RNA genes for routine diagnostics of bacterial infections was evaluated. Here, the results from more than 2600 analyses during a 6-year period (2003-2009) are presented. Almost half of the samples were from joints and bones, and the second most frequent origin of samples was from the central nervous system. Overall, 26% of all samples were positive for bacterial DNA and bacterial identification was obtained in 80% of the PCR-positive samples by subsequent DNA sequencing. Ambiguous species identification was noticed among non-haemolytic streptococci, especially within the mitis group. The data show that ribosomal PCR with subsequent DNA sequencing of the PCR product is a most valuable supplement to culture for identifying bacterial agents of both acute and prolonged infections. However, some bacteria, including non-haemolytic streptococci, may not be precisely identified., (© 2013 APMIS. Published by John Wiley & Sons Ltd.)

In the past decades efforts to further diminish the case-fatality rate from meningococcal disease have proven challenging due to the often rapid progression of the disease in patients. In this study our objective was to characterise a subset of Neisseria meningitidis isolates to establish which sequence types were associated with increased mortality in Denmark during the period 2000-2007. We designed a matched case control and performed serogrouping, serotyping, serosubtyping and multilocus sequence typing (MLST) on 100 isolates. The clonal complex ST-32/ET-5 was found in 36% of the isolates, followed by the ST-11/ET-37 complex (14%) and ST-41/44 complex/Lineage 3 (14%). Eight new sequence types were found. None of the clonal complexes were significantly associated with increased mortality. Phenotype B:15:P1.7,16 tended to be a better predictor of death than ST-32. Although the numbers were low, the present study indicates that phenotyping may be a better predictor of mortality than MLST, which suggests that each typing method has its advantages and disadvantages. If this notion can be confirmed by other studies, it may stimulate additional research regarding the pathogenesis of severe illness, for example, if certain surface molecules trigger a cytokine storm more than others.

The results of partial polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene and subsequent DNA sequencing of clinical samples from children are described. In 13 out of 62 samples, DNA from bacteria likely to be the cause of infection was identified. In the vast majority (11/13) of samples with significant pathogen culture the specimen had been negative. Antibiotics had been given in all cases except for three prior to sampling. PCR and subsequent DNA sequencing is a valuable supplementary tool for establishing the cause of bacterial infections in children when culture is negative.

Papanicolaou-stained cervicovaginal smears (Pap smears) are used to screen for cervical cancer. Since there is a lack of consensus in published reports respecting the efficacy of Pap-stained smears in BV diagnostics, there is a need to validate their use for diagnosis of BV. Slides from the international BV00 workshop were Pap stained and independently analyzed by four investigators under a phase-contrast microscope. All workshop slides--whether Pap-stained, Gram-stained or rehydrated air-dried smears--were scored according to the same Nugent classification. The diagnostic accuracy of Pap smears for diagnosis of BV had a sensitivity of 0.85 and a specificity of 0.92, with a positive and negative predictive value of 0.84 and 0.93, respectively. The interobserver weighted kappa index was 0.86 for Pap-stained smears compared to 0.81 for Gram-stained smears, and 0.70 for rehydrated air-dried smears using the mean Nugent score as the criterion standard. Provided that the samples are taken from equivalent locations (the vaginal fornix) and analyzed according to the same scoring criteria, there is no discernable difference in the diagnostic accuracy of the three smear-staining methods. The Pap-stained vaginal smears can be used as a wholly adequate alternative to Gram-stained smears for BV diagnosis.

Diagnostic tools for identification of bacteria have developed dramatically in the last decade. Sequencing of genes coding for rRNA has led to revolutionary insights into the phylogeny and taxonomy of bacteria, and to new demands on the service provided by national reference laboratories for identification of bacteria. At the Danish Reference Laboratory for Identification of Bacteria, partial 16S rDNA sequencing has been used since 2001 to identify "difficult" strains submitted for taxonomic elucidation. Experiences relating to phenotypic as well as 16S rDNA sequencing of the first 175 strains examined are presented. Approximately 2/3 of the strains were Gram-positive and 1/3 Gram-negative. One fifth of the strains were anaerobic, while 4/5 were either facultatively anaerobic or aerobic. Methodological agreement was seen for most strains at species and/or genus level. Methodological disagreement was relatively rare. In 1/6 of the strains valuable information was obtained from sequencing results, while for some strains identification was based primarily on the phenotypic results. Only a few strains could not be clearly identified by either method. A very large number of strains representing taxons ranging from facultatively anaerobic to aerobic and anaerobic species and genera, Gram-positive as well as Gram-negative, were successfully examined. Of the submitted strains many have only rarely been encountered as human pathogens. Thus, genotypic identification may result in recognition of hitherto seldom recognized or unrecognized bacteria as human pathogens, which will lead to a better understanding of the nature of human infections. It is self evident that we should focus on slowly growing, fastidious or 'difficult' organisms when using sequencing for national reference purposes. Short sequences (450-650 base pairs) seem sufficient for most identifications. Molecular bacterial identification is a powerful tool for national reference laboratories, enhancing both the speed and validity of examinations performed.

A polyphasic characterization of Aerococcus urinae is presented. In this study the intraspecies relationships between 26 strains of varying geographical origin were examined by phenotypic tests, ribotyping and multilocus enzyme electrophoresis. The results demonstrated two main phenotypic patterns that could be distinguished in tests for hydrolysis of aesculin, and acid production from amygdalin and salicin. Strains were either negative (n=19) or positive (n=6) in these tests. One strain had a deviating pattern. Heterogeneity within the 19 pattern I strains was demonstrated especially by phenotypic tests (acid production from ribose, mannitol, sorbitol, sucrose and D-arabitol) and by multilocus enzyme electrophoresis. However, DNA sequence analysis of the 16S rRNA (n=7) and gyrB genes (n=3) from strains representing the two main patterns showed no variation in sequences among strains. Comparison of A. urinae and representatives of related taxa by 16S rDNA sequence analysis showed that the taxon is related to, but distinct from, other Aerococcus spp.

An international workshop on vaginal smear-based diagnosis of bacterial vaginosis was organized where 13 investigators scoring 258 slides with smears from vaginal fluid. Interobserver reproducibility of interpretations of Nugent scores, Hay/Ison scores and wet smear scores for the diagnosis of bacterial vaginosis was shown to be high. Detailed analysis of individual scoring results however indicated that basic standards of quality control to ensure robust individual readings of slides must be adhered to.

A 38-year-old man with a history of alcohol abuse developed rapidly fulminating septicaemia and died. Selenomonas sputigena and Acinetobacter calcoaceticus were isolated from a blood culture. Selenomonas sputigena is a motile anaerobic gram-negative rod rarely associated with systemic disease. Difficulties in isolation and taxonomic identification are discussed.

Branhamella catarrhalis isolates from lower respiratory tract specimens collected in 1983/84 (n = 50) and 1988 (n = 30) were examined for beta-lactamase production. The percent of beta-lactamase-producing strains increased from 25% to 63% from 1983/84 to 1988. beta-lactamases from 30 strains could be typed, and of these 28 were of the BRO-1 type and two the BRO-2 type. The two beta-lactamase inhibitors, clavulanic acid and brobactam were very active against beta-lactamase extracts whether of the BRO-1 or the BRO-2 type. Susceptibility to a number of antimicrobial agents or combinations of agents was determined by a plate dilution method (MICs) and by a tablet diffusion method. The penicillin tablet (Rosco Neo-Sensitabs) was found to be useful in discriminating between beta-lactamase-producing and non-beta-lactamase- producing strains of B. catarrhalis.

A case of Haemophilus parainfluenzae bacteremia without known infectious focus is reported. Phenotypically, the isolated strain is a typical H. parainfluenzae except for its ability to produce indole and beta-lactamase. Beta-lactamase producing H. parainfluenzae organisms are encountered occasionally, but to the best of our knowledge this is the first reported blood culture isolate with this ability. We propose a new biotype (IV) of H. parainfluenzae to accommodate strains that are indole, urease and ornithine decarboxylase positive.

A two-year survey at our hospital revealed 19 patients suspected of pulmonary infection from whom Neisseria meningitidis was isolated from lower respiratory tract specimens, in 17 cases in pure culture. Of the isolates, 16, two and one belonged to serogroup B, C and 29E, respectively. All except three patients suffered from chronic pulmonary disease, notably chronic bronchitis. It was found likely or possible that N. meningitidis was the causative agent in 12 patients with symptoms of acute bronchitis. Results of tests for complement-fixing antibodies against N. meningitidis (MAT) were generally not found to be useful when assessing the possible causative role of N. meningitidis in non-pneumonic pulmonary infections.

A case of Branhamella catarrhalis bacteremia without known infectious focus in a granulocytopenic patient is reported. There were no unusual clinical features of bacteremia, such as purpura, in this case which distinguished it from bacteremia due to other bacteria. The isolated strain was beta-lactamase producing. The patient recovered uneventfully on antibiotics; at the same time, however, the bone marrow was also normalized.

Decreased susceptibility in vitro to erythromycin has been demonstrated for few C. trachomatis isolates outside Scandinavia, making local susceptibility-screening indicated. Eleven recent isolates of C. trachomatis found in a Danish hospital have been examined for susceptibility, expressed as minimal inhibitory concentration (MIC) to antibacterial agents commonly used in genito-urinary infections. Full susceptibility to doxycycline and erythromycin was demonstrated. Clindamycin and ampicillin showed moderate activity, and sulfamethizole had a MIC value in the border area of what is needed for therapeutic effect in non-urinary infections. C. trachomatis, being a major pathogen in pelvic inflammatory disease, makes combination chemotherapy desirable in order to protect against resistance development, to obtain synergistic effect and to ensure effect in infections of mixed etiology - provided antagonism could not be anticipated. In three checkerboard trials, with the combinations doxycycline plus ampicillin, erythromycin plus sulfamethizole and ampicillin plus sulfamethizole, using MIC as end-point, neither synergism nor antagonism could be demonstrated in the concentration range from 1/8 to 4 times the MIC values of each drug.

A three-month survey revealed 29 patients at our hospital with symptoms of acute pulmonary infection, from whom Branhamella catarrhalis was isolated from lower respiratory tract specimens, in 18 cases in pure culture. Approximately 2% of all respiratory tract specimens examined during the period yielded growth of B. catarrhalis. All except one patient suffered from chronic pulmonary disease, notably chronic bronchitis. A phenotypic comparison was made between 55 strains of B. catarrhalis, of which 50 were recent isolates from lower respiratory tract specimens, and 23 Neisseria strains representing Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria cinerea, Neisseria flavescens, Neisseria mucosa, Neisseria pharyngis, and Neisseria lactamica. The morphology of B. catarrhalis colonies is very characteristic, and when the diagnosis is suspected, testing for the ability to hydrolyze tributyrin may confirm it within hours. Ability to produce deoxyribonuclease is another property which differentiates B. catarrhalis from the Neisseria species. Otherwise, the combination of nitrate reduction and failure to produce acid from glucose, maltose, and sucrose establishes the diagnosis.

Aerococci are only occasionally isolated from urinary tract specimens. A 4-month' survey revealed 29 patients suspected of urinary tract infection (UTI), from whom an Aerococcus-like organism (ALO) was isolated in significant numbers from urinary tract specimens and singly in 11 cases. Approximately 0.8% of all urinary tract specimens examined during the period yielded growth of ALO. The median age of patients was 75 years. Half of the patients were found to have conditions predisposing to UTI. 9 of 11 patients without indwelling catheter, having ALO isolated in pure culture from the urine, showed one or more typical signs of UTI (fever, dysuria and/or pollakisuria and pyuria). A phenotypic comparison was made between 29 strains of ALO and 6 related organisms, including Aerococcus viridans, Streptococcus faecium, Streptococcus sanguis, Streptococcus bovis, Streptococcus Group Q and Streptococcus Group R. ALO and A. viridans showed many similarities, but, owing to characteristic differences with regard to colony morphology, biochemical reactions and antibiotic susceptibility pattern, the strains were easily separated.