Frequent colonization of betahaemolytic streptococci at various body sites including the perineum and anal canal during erysipelas and cellulitis.

Background: Erysipelas and cellulitis are usually caused by betahaemolytic streptococci but the aetiology is often difficult to verify in clinical practice. Methods: Patients with erysipelas or cellulitis were analysed for betahaemolytic streptococci in samples from multiple body sites, including the perineum and the anal canal, during the acute episode and at follow up. Healthy control persons were sampled from the same sites. Results: Betahaemolytic streptococci group A, C or G were identified in 23/28 (82%) patients, most commonly group G. A wound or ulcer, present in 16/28 (57%), was colonized in 8/16 (50%). The perineum and anal canal were colonized in 11/28 (39%) and 10/28 (36%), respectively. At follow-up after about 4 weeks, only 4/28 (14%) were colonized (p<.001). In 39 healthy control persons, no betahaemolytic streptococci group A were found, groups C or G were found in 4/39 (10%). Group B streptococci were more often identified in controls, than in patients,12/39 (31%). Conclusions: Acute episodes of erysipelas or cellulitis are associated with colonization of betahaemolytic streptococci at multiple sites including the perineum and anal canal, in particular serogroup G. This may be important for choice of primary antibiotic therapy and possibilities for prevention of relapses.

Reactive arthritis in relation to internal derangements of the temporomandibular joint: a case control study.

The aim of this study was to find out if reactive arthritis was involved in the aetiology of chronic closed lock of the temporomandibular joint (TMJ) by looking for bacterial antigens in the synovial membrane of the TMJ, and by studying the antibody serology and carriage of human leucocyte antigen (HLA) B27 in patients with chronic closed lock. Patients with reciprocal clicking and healthy subjects acted as controls. We studied a total of 43 consecutive patients, 15 with chronic closed lock, 13 with reciprocal clicking, and 15 healthy controls with no internal derangements of the TMJ. Venous blood samples were collected from all subjects for measurement of concentrations of HLA tissue antigen and serology against Chlamydia trachomatis, Yersinia enterocolitica, Salmonella spp., Campylobacter jejuni, and Mycoplasma pneumoniae. Samples of synovial tissue from patients with closed lock and reciprocal clicking were obtained during discectomy and divided into two pieces, the first of which was tested by strand displacement amplification for the presence of C trachomatis, and the second of which was analysed for the presence of species-specific bacterial DNA using 16s rRNA pan-polymerase chain reaction (PCR). There were no significant differences between the groups in the incidence of antibodies against M pneumoniae, Salmonella spp. or Y enterocolitica. No patient had antibodies towards C trachomatis or C jejuni. We found no bacterial DNA in the synovial fluid from any patient. The HLA B27 antigen was present in 2/15 subjects in both the closed lock and control groups, and none in the reciprocal clicking group. In conclusion, reactive arthritis does not seem to be the mechanism of internal derangement of the TMJ.

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance.

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding.

Interplay of efflux, impermeability, and AmpC activity contributes to cefuroxime resistance in clinical, non-ESBL-producing isolates of Escherichia coli.

Cefuroxime resistance in Escherichia coli strains susceptible to extended-spectrum cephalosporins is not uncommon, but the resistance mechanisms have so far not been elucidated. Therefore, 14 clinical non-extended-spectrum beta-lactamase isolates of E. coli were examined, 11 of which were cefuroxime resistant. Quantitative RT-PCR was used to examine the transcription levels of the genes acrA (encoding AcrA, part of the AcrAB-TolC efflux pump system) and ompF (encoding the porin OmpF). Isoelectric focusing was used for detection of beta-lactamases, and a spectrophotometric assay was used to measure AmpC activity. Among the 11 cefuroxime-resistant isolates, 7 had increased acrA transcription (from 2.4 to 38 times the ATCC strain), 3 isolates had very low ompF transcription levels (

Cefuroxime non-susceptibility in multidrug-resistant Klebsiella pneumoniae overexpressing ramA and acrA and expressing ompK35 at reduced levels.

OBJECTIVES: The aims were to study if efflux and down-regulation of porins contribute to cefuroxime resistance in Klebsiella pneumoniae and to co-resistance to unrelated antibiotics. METHODS: Ten cefuroxime-non-susceptible but cefotaxime-susceptible blood culture isolates of K. pneumoniae and one multiply antibiotic-resistant (MAR) laboratory strain (selected by chloramphenicol) were examined. Transcription of the genes acrA, ompK35, ramA, marA and soxS was determined with quantitative RT-PCR. RESULTS: All clinical isolates and the MAR laboratory strain had similar antibiograms with non-susceptibility to cefuroxime, tigecycline, chloramphenicol and nalidixic acid. Phenylalanine arginine beta-naphthylamide (PAbetaN) increased susceptibility to tigecycline, chloramphenicol and nalidixic acid, but not to cefuroxime. Increased acrA transcription and decreased ompK35 transcription was seen in all strains. Increased ramA transcription was seen in all strains except one clinical isolate. CONCLUSIONS: This multidrug-resistant phenotype of K. pneumoniae is associated with increased acrA and ramA transcription and decreased ompK35 transcription. Since the cefuroxime resistance was not reversed by PAbetaN, it was probably attributable to decreased levels of OmpK35, rather than to efflux.

Pseudomonas aeruginosa utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii.

Pseudomonas aeruginosa is a free-living and common environmental bacterium. It is an opportunistic and nosocomial pathogen causing serious human health problems. To overcome its predators, such as macrophages and environmental phagocytes, it utilises different survival strategies, such as the formation of microcolonies and the production of toxins mediated by a type III secretion system (TTSS). The aim of this study was to examine interaction of TTSS effector proteins of P. aeruginosa PA103 with Acanthamoeba castellanii by co-cultivation, viable count, eosin staining, electron microscopy, apoptosis assay, and statistical analysis. The results showed that P. aeruginosa PA103 induced necrosis and apoptosis to kill A. castellanii by the effects of TTSS effector proteins ExoU, ExoS, ExoT, and ExoY. In comparison, Acanthamoeba cultured alone and co-cultured with P. aeruginosa PA103 lacking the known four TTSS effector proteins were not killed. The results are consistent with P. aeruginosa being a strict extracellular bacterium that needs TTSS to survive in the environment, because the TTSS effector proteins are able to kill its eukaryotic predators, such as Acanthamoeba.

Role of outer membrane protein OprD and penicillin-binding proteins in resistance of Pseudomonas aeruginosa to imipenem and meropenem.

The main mechanism of imipenem resistance in Pseudomonas aeruginosa is via downregulation of the gene for OprD porin. In a previous study, it was shown that the level of resistance did not parallel with the degree of downregulation of the porin gene, thus arguing for the existence of other resistance mechanisms. Penicillin-binding protein (PBP) 2 and PBP3 are involved in carbapenem resistance in Escherichia coli. The genes for PBPs were sequenced in three imipenem-resistant clinical strains and these strains were conjugated with two susceptible P. aeruginosa PA0 strains, selecting for auxotrophic markers. In all the clinical and resistant isolates there was no obvious elevation of AmpC cephalosporinase. The active sites of PBP1b (ponB), PBP2 (pbpA), PBP3 (pbpB) and PBP6 (dacC) had no mutations in any of the examined strains. Production of oprD mRNA was significantly lower in clinical strains and transconjugants after selection for the proB marker (PA4565 at 5113kb). The clinical strains had alterations in OprD that were not found in transconjugants. Our findings suggest that PBPs do not play a role in imipenem resistance in the clinical strains examined here, but that a regulatory gene for oprD contributing to carbapenem resistance is located close to the proB gene.

Alterations of porin, pumps, and penicillin-binding proteins in carbapenem resistant clinical isolates of Pseudomonas aeruginosa.

Carbapenem-resistant clinical isolates of Pseudomonas aeruginosa from Sweden and Norway (n=27) were characterized regarding transcription of genes encoding OprD, efflux pumps MexAB-OprM and MexCD-OprJ, as well as penicillin-binding proteins (PBP) 2 and 3. Quantification of mRNA was performed with real-time RT-PCR. Levels of mRNA in clinical isolates were compared to ATCC 27853 and average transcription levels of four carbapenem-susceptible clinical isolates of P. aeruginosa. Sequencing of oprD, pbp 2, and pbp 3 was performed in selected isolates. An efflux inhibition assay was performed with Phe-Arg-beta-naphtylamide. Most carbapenem-resistant isolates had decreased levels of oprD mRNA. Among six isolates with normal amount of oprD mRNA, half of them had significant OprD sequence alterations. Increased transcription of mexB was observed in 16 of 23 meropenem-resistant isolates, and one isolate showed mexD hyperproduction. Decreased amount of pbp 2 and pbp 3 was found in two and three isolates, respectively. Sequencing of pbp 2 and 3 revealed no amino acid changes potentially leading to conformational changes. In conclusion, OprD changes were the predominant mechanism of high-level carbapenem resistance, and increased transcription of mexB was often present in meropenem-resistant isolates. Reduced transcription of pbp 2 and 3 may contribute to the carbapenem resistance.

Identification of species of viridans group streptococci in clinical blood culture isolates by sequence analysis of the RNase P RNA gene, rnpB.

OBJECTIVES: Viridans group streptococci (VGS) cause severe diseases such as infective endocarditis and septicaemia. Genetically, VGS species are very close to each other and it is difficult to identify them to species level with conventional methods. The aims of the present study were to use sequence analysis of the RNase P RNA gene (rnpB) to identify VGS species in clinical blood culture isolates, and to compare the results with the API 20 Strep system that is based on phenotypical characteristics. METHODS: Strains from patients with septicaemia or endocarditis were analysed with PCR amplification and sequence analysis of the rnpB gene. Clinical data were registered as well. RESULTS: One hundred and thirty two VGS clinical blood culture isolates from patients with septicaemia (n=95) or infective endocarditis (n=36) were analysed; all but one were identified by rnpB. Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii strains were most common in the patients with infective endocarditis. In the isolates from patients with haematological diseases, Streptococcus mitis and S. oralis dominated. In addition in 76 of the isolates it was possible to compare the results from rnpB analysis and the API 20 Strep system. In 39/76 (51%) of the isolates the results were concordant to species level; in 55 isolates there were no results from API 20 Strep. CONCLUSION: Sequence analysis of the RNase P RNA gene (rnpB) showed that almost all isolates could be identified. This could be of importance for evaluation of the portal of entry in patients with septicaemia or infective endocarditis.

Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain.

A highly discriminative and objective genetic characterization of N. gonorrhoeae, which increases our knowledge of strain populations in different geographic areas, is crucial for the development of improved control measures. In the present study, conventional phenotypic characterization and genetic characterization by means of pulsed-field gel electrophoresis (PFGE), sequencing of the entire porB gene, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and pyrosequencing of a quinolone resistance determining region (QRDR) of the gyrA gene of Swedish ciprofloxacin-resistant N. gonorrhoeae serovar IB-10 isolates (n=45) were performed. The genetic characterization identified one widely spread ciprofloxacin-resistant N. gonorrhoeae ST147 strain. In addition, isolates with slightly different genetic characteristics, which presumably reflect the ongoing evolution only, were also identified. All the isolates contained single nucleotide polymorphisms in QRDR of the gyrA gene that are highly correlated with ciprofloxacin resistance. Consequently, comprehensive characterization identified the first confirmed large domestic transmission, mainly among young heterosexuals, of one ciprofloxacin-resistant N. gonorrhoeae strain in Swedish society during 2002-2003. In conclusion, a precise, i.e. genetic, characterization for identification of individual strains is a very valuable support to the crucial active surveillance of the epidemiological characteristics and the antibiotic susceptibility of N. gonorrhoeae in the effective treatment of gonorrhoea.

The epidemiology of gonorrhea among men who have sex with men in Stockholm, Sweden, 1990-2004.

OBJECTIVES: The objectives of this study were to analyze the spread of gonorrhea in men who have sex with men (MSM) in Stockholm regarding serovars, HIV status, and site of infection and to compare the distribution of serovars among HIV-positive and HIV-negative MSM. STUDY DESIGN: Clinical and epidemiologic data were collected for all MSM diagnosed with gonorrhea in 1990 to 2004 at a clinic primarily serving MSM. Neisseria gonorrhoeae strains were serotyped. RESULTS: A total of 1,039 isolates from 840 gonorrhea episodes in 721 patients were included. A sharp increase was seen during the 2000s. Ten percent of the cases were HIV-positive. The proportion of pharyngeal infections increased significantly (P <0.001) from 15% to 38% during the last 7 years. A great variation of serovars (n = 66) was observed, but only 5 were present >10 years. There was a significant difference (P = 0.001) in distribution of serovars correlated to HIV status. CONCLUSION: Gonorrhea is a marker for HIV infection in MSM, but the increase in gonorrhea may be associated with genital-oral sexual practice rather than with high-risk sexual practice.

Viridans group streptococci in blood culture isolates in a Swedish university hospital: antibiotic susceptibility and identification of erythromycin resistance genes.

One hundred and twenty-nine isolates of viridans group streptococci in blood cultures from patients with septicaemia or endocarditis isolated between 1998 and 2003 were tested for antibiotic susceptibility to penicillin, ciprofloxacin, clindamycin, dalbavancin, daptomycin, erythromycin, linezolid, tigecycline, trimethoprim/sulphamethoxazole and vancomycin. Reduced susceptibility to penicillin (minimum inhibitory concentration (MIC) > or =0.25 microg/mL) was found in 18% of the isolates, and 4% of the strains were resistant to penicillin (MIC> or =4.0 microg/mL). Nineteen percent of the isolates had reduced susceptibility to erythromycin (MIC> or =0.5 microg/mL), among which ermB and mefA were found in 40% and 80%, respectively. Strains sequenced as Streptococcus mitis by rnpB had a high degree of non-susceptibility to erythromycin (32%) and penicillin (21%). The level of penicillin resistance in this Swedish study was lower compared with studies from other countries where the antibiotic pressure might be higher than in Sweden. Susceptibility to newer antibiotics was high; all strains were susceptible to dalbavancin, daptomycin, linezolid and vancomycin.

Transformation of ciprofloxacin-resistant Neisseria gonorrhoeae gyrA, parE and porB1b genes.

In several transformation experiments, we have shown that introduction of an alteration in GyrA at position 95 of a ciprofloxacin-susceptible Neisseria gonorrhoeae strain (minimum inhibitory concentration (MIC) 0.008 mg/L) increases the MIC to 0.064 mg/L. Two alterations (positions 91 and 95) increase the MIC to 0.125-0.25 mg/L. Transformants with ciprofloxacin MICs of 0.5-16 mg/L were obtained from a moderately ciprofloxacin-resistant strain (MIC 0.25 mg/L). These transformants had alterations in the gene for PorB1b and probably other genes. In one transformant, an alteration in ParE was also introduced, which probably contributed to ciprofloxacin resistance. The ciprofloxacin-resistant transformants had the donor porB1b sequence, and most of them also had altered serovars. We conclude that alterations in N. gonorrhoeae PorB1b could be involved in ciprofloxacin resistance.

Gram-negative bacteria from patients seeking medical advice in Stockholm after the tsunami catastrophe.

Microbiological cultures from 229 patients seeking medical advice in Stockholm after the tsunami catastrophe of December 2004 were analysed at the Clinical Microbiology Laboratory, Karolinska University Hospital, Stockholm, Sweden. Gram-negative bacilli were the most common findings from wound cultures. Common human pathogens such as Escherichia coli, Proteus species, Klebsiella spp., and Pseudomonas aeruginosa were isolated. More rare species of Gram-negative bacilli, e.g. Myroides odoratus, Sphingomonas paucimobilis and Bergeyella zoohelcum were also isolated. Resistance towards ordinary antibiotics was more extensive compared to our Swedish reference material for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Acinetobacter spp., but not for Pseudomonas aeruginosa, probably reflecting that the resistant isolates were nosocomially acquired in Asia.

Cat bite wound infections: a prospective clinical and microbiological study at three emergency wards in Stockholm, Sweden.

OBJECTIVE: The rate of infection following cat bites appears to be greater than that from dog bites. To study the clinical picture, complications and microbiology (in humans and cats), this prospective study was performed. METHODS: A prospective study with patients with clinical symptoms of infection due to cat bites from three emergency wards during two years in Stockholm, Sweden. Aerobic and anaerobic cultures from the wounds were performed as well as cultures from the biting cat's mouth. Clinical data and complications were registered. RESULTS: Seventy-nine episodes in 78 patients with infective cat bites were included. Pasteurella multocida was isolated in 70% of the patients; in addition anaerobic pathogens were isolated in 16% concurrently with P. multocida, while Staphylococcus aureus was isolated in only two patients. Pasteurella spp. was also isolated from 80% of the pharynx of the biting cats. The dominating symptoms of infection were erythema, pain and oedema, often emerging as early as 3h after the bite. Complications such as tendosynovitis, arthritis, abscesses and septicaemia occurred in 18% of the patients. No patient died due to the infection. The majority of the patients received penicillin or amoxicillin as antibiotic treatment. CONCLUSIONS: P. multocida was the dominating pathogen among patients with infected cat bites and antibiotic treatment should cover P. multocida.

Molecular epidemiology of Neisseria gonorrhoeae- identification of the first presumed Swedish transmission chain of an azithromycin-resistant strain.

In the present study, 10 azithromycin-resistant Neisseria gonorrhoeae isolates from 6 Swedish male patients in 2004, 3 sporadic Swedish azithromycin-resistant N. gonorrhoeae isolates from recent years and one Swedish N. gonorrhoeae isolate from 2003 that was susceptible to azithromycin but assigned the same serological variant (serovar), i.e. IB-37, as the isolates from 2004 were included. The isolates were characterized phenotypically using antibiograms and serovar determination and genetically with pulsed-field gel electrophoresis (PFGE), entire porB gene sequencing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). The epidemiological information and the results of the thorough phenotypic characterisation and genetic characterisation identified the first presumed domestic transmission of one azithromycin-resistant N. gonorrhoeae strain in Sweden in 2004. This stresses the need for continuous surveillance of the antibiotic susceptibility of N. gonorrhoeae in order to identify emergence of new resistance, monitor the changing patterns of the susceptibility, and be able to update treatment recommendations on a regular basis.

Pyrosequencing of the DNA gyrase gene in Neisseria species: effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae.

The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.

Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing.

Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.