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.

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.

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.

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.

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.

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.

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.

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.

Carbapenem resistance mechanisms in Pseudomonas aeruginosa: alterations of porin OprD and efflux proteins do not fully explain resistance patterns observed in clinical isolates.

Imipenem resistance in Pseudomonas aeruginosa is considered to be associated with loss of the porin OprD combined with activity of chromosomal beta-lactamase (AmpC), while overexpression of multidrug efflux pumps is considered to confer meropenem resistance. Carbapenem resistance can also result from production of metallo-beta-lactamases. Transcription of oprD and efflux pump genes mexB, mexY and mexF was analysed in 23 clinical isolates of P. aeruginosa by quantitative RT-PCR. oprD was sequenced in all, and mexR, regulator of efflux pump MexAB-OprM, in selected isolates. Four isolates that were imipenem susceptible had significant reduction of oprD mRNA and presence of oprD mutations causing frameshift or translational stop. In strains only resistant to imipenem no significant difference in transcription of oprD was observed between low-level and high-level resistant isolates. The differences could not be explained by either pattern of oprD mutations. Increased transcription of mexB generally correlated well with meropenem resistance. One high-level meropenem-resistant isolate showed no significant change in mexB mRNA, but sequencing confirmed presence of a nalB mutation. Furthermore, one meropenem-susceptible isolate showed significant increase in mexB transcription, but no mexR mutations. In summary, our findings indicate that the resistance patterns observed cannot be fully explained by the currently described carbapenem resistance mechanisms.

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.

Verification of clinical samples, positive in AMPLICOR Neisseria gonorrhoeae polymerase chain reaction, by 16S rRNA and gyrA compared with culture.

We compared 956 samples for AMPLICOR Neisseria gonorrhoeae polymerase chain reaction (PCR) (Roche) with species verification using the 16S rRNA gene to verification using gyrA gene. Control was the culture method. The gyrA verification uses pyrosequencing of the quinolone resistance-determining region of gyrA. Of 52 samples with optical density >/=0.2 in PCR, 27 were negative in culture, two samples from pharynx were false negative in culture and four samples from pharynx were false positives in verification with 16S rRNA. Twenty-five samples showed growth of gonococci, 18 of the corresponding PCR samples were verified by both methods; three urine samples were positive only in gyrA ; and one pharynx specimen was positive only in 16S rRNA. Three samples were lost. We conclude that AMPLICOR N. gonorrhoeae PCR with verification in gyrA gene can be considered as a diagnostic tool in populations with low prevalence of gonorrhoea and that pharynx specimens should not be analysed by PCR.

DNA gyrase gene in Neisseria gonorrhoeae as indicator for resistance to ciprofloxacin and species verification.

We have identified a unique region of eight amino acids in the quinolone resistance-determining region in the gyrA gene in Neisseria gonorrhoeae as an indicator of resistance to fluoroquinolones. We sequenced that region by the Pyrosequencing technology in 46 N. gonorrhoeae strains and 11 urine samples positive in AMPLICOR N. gonorrhoeae polymerase chain reaction (Roche Diagnostics), with corresponding isolates of N. gonorrhoeae. The results showed that 28 samples with minimum inhibitory concentration (MIC) of ciprofloxacin >1 mg/L had mutations in positions 91 and 95. Fifteen samples with MIC 0.125-1.0 mg/L had either one or both of the mutations. The 14 susceptible samples had no mutations. The target region also discriminates N. gonorrhoeae from other species of Neisseria. Our conclusion is that gyrA is an indicator of resistance to ciprofloxacin in N. gonorrhoeae and sequencing by Pyrosequencing technology is a suitable tool for analysis of DNA in urine samples.

[Rare bacteria species found in wounds of tsunami patients. Predominance of gram-negative rods, increased antibiotic resistance]. / Ovanliga bakteriefynd i sår hos tsunamipatienter. Dominans av gramnegativa stavar, ökad andel antibiotikaresistens.

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

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.

Mutations in gyrA, gyrB, parC, and parE in quinolone-resistant strains of Neisseria gonorrhoeae.

Mutations in the genes for the subunits GyrA and ParC of the target enzymes DNA gyrase and topoisomerase IV are important mechanisms of resistance in quinolone-resistant bacteria, including Neisseria gonorrhoeae. The target enzymes also consist of the subunits GyrB and ParE, respectively, though their role in quinolone-resistance has not been fully investigated. We sequenced the quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE in 25 ciprofloxacin-resistant strains from Bangladesh (MIC 4-->32 mg/l) and 5 susceptible strains of N. gonorrhoeae. All the resistant strains had three or four mutations. Two of these were at positions 91 and 95 of gyrA. Fourteen strains had an additional mutation in parC at position 91, and 17 strains had an additional mutation in parE in position 439. No alterations were found in gyrB. The five susceptible strains had identical DNA sequences. Data indicate that the mutations detected in the QRDR of gyrA and parC may be important in the development of quinolone resistance. According to transformation experiments we assume that the alteration in parE is not related to a high degree of quinolone resistance. There was no correlation between ciprofloxacin MICs and pattern or number of mutations in the target genes.

Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains.

We have investigated the occurrence of mutations in topoisomerase II (DNA gyrase) subunit B(gyrB) and topoisomerase IV subunit E(parE) and the hyperexpression of genes for four efflux pump proteins in 20 previously described, fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa. Amino acid alterations were found in GyrB in five strains and in ParE in three strains with MIC of norfloxacin > or = 8 mg/L, and it is likely that some of the alterations contribute to the quinolone resistance exhibited by these strains. Seventeen of the 20 strains overproduced mRNA for one or more pump proteins (MexB, MexD, MexF, or MexY), which caused multidrug resistance phenotype in more than half of strains. Two strains were hypermutable and one of them was highly resistant, but the other strain was only moderately resistant.

Cytokines in periradicular lesions: the effect of linezolid treatment.

OBJECTIVE: The purpose of this study was to quantify the cytokines interleukin (IL)-1 receptor antagonist (RA), IL-6, and transforming growth factor beta(1) found in extracts of inflammatory periapical tissues and to study the effect of a new antibiotic (linezolid) on the levels of these cytokines. STUDY DESIGN: Twenty-two patients with root-filled teeth with persisting periapical pathoses were randomly divided into a control group or an antibiotic group. One tooth from each of the patients was resected at the root-end, and the periapical tissue was collected. IL-1RA, IL-6, and TGF-beta(1) were quantified in tissue extracts through the use of enzyme-linked immunosorbent assays. RESULTS: Measurable amounts of all 3 cytokines were found in all extracts. A statistically significant reduction in IL-1RA per milliliter was observed in the linezolid group in comparison with the control group (P <.05). The difference in variation of IL-1RA in the treatment and control groups was also highly statistically significant (P <.001). CONCLUSIONS: Although drawn from a limited sample size, the results indicate that IL-1RA is a sensitive indicator of the effect of antibacterial treatment on the immune response in periapical inflammatory tissues.