Rapid increase in multidrug-resistant enteric bacilli blood stream infection after prostate biopsy - A 10-year population-based cohort study.

BACKGROUND: Bloodstream infection following a transrectal prostate biopsy is a well-known and feared complication. Previous studies have shown an increase in multi-resistant bacterial infections as a consequence of higher usage of antibiotics in investigated populations. Our aim was to analyze bacterial resistance patterns in positive blood cultures, after prostate biopsies in Stockholm, Sweden, where the use of antibiotics has been low and decreasing during the last 10 years. METHODS: From the three pathology laboratories in Stockholm, reports of prostate examinations were retrieved (n = 56,076) from 2003 to 2012. By linking men to the National Patient Register all but prostate core biopsies were excluded (n = 12,024). Prostate biopsies in men younger than 30 years of age were excluded (n = 5) leaving 44,047 biopsies for analysis. From laboratory information systems data regarding blood cultures were retrieved. Proportions of blood cultures within 30 days by year were calculated. Crude and adjusted logistic regression models were used to estimate ORs. RESULTS: In total, 44,047 prostate biopsies were performed in 32,916 men over 10 years. On 620 occasions a blood culture was drawn within 30 days of the biopsy; 266 of these were positive. The proportions with positive blood cultures in 2003 and 2012 were 0.38 and 1.14%, respectively. The proportion of multidrug-resistant bacteria increased significantly during the study. In the crude and the adjusted analysis, the year of biopsy and Charlson Comorbidity Index were associated with the risk of having a positive blood culture. CONCLUSION: Multidrug-resistant enteric bacilli are becoming a problem in Sweden, despite low antimicrobial use. Men need to be informed about the increasing risks of infectious complications of transrectal prostate biopsy. One out of 50 men undergoing a prostate biopsy will develop symptoms suggestive of a bloodstream infection after the biopsy and one in 100 men will have a positive blood culture.

Tolerability and performance of BIP endotracheal tubes with noble metal alloy coating--a randomized clinical evaluation study.

BACKGROUND: Hospital acquired infections worsen the outcome of patients treated in intensive care units and are costly. Coatings with silver or metal alloys may reduce or alter the formation of biofilm on invasive medical devices. An endotracheal tube (ETT) is used to connect the patient to a ventilator and coated tubes have been tested in relation to bacterial colonization and respiratory infection. In the present study, we aimed to evaluate and compare a coated and uncoated ETT for patient symptoms and local tracheal tolerability during short term clinical use. Degree of bacterial colonization was also described. METHODS: A silver-palladium-gold alloy coating ('Bactiguard®'Infection Protection, BIP) has been extensively used on urinary tract catheters and lately also on central venous catheters. We performed a randomised, single-blinded, controlled, first in man, post Conformité Européenne (EC) certification and CE marking study, focused on Bactiguard® coated ETTs (BIP ETT). Thirty patients at a tertiary university hospital scheduled for upper abdominal elective surgery with an expected duration of anaesthesia of at least 3 h were randomised; BIP ETT (n = 20) or standard ETT (n = 10). The tolerability was assessed with a modified version of Quality of Life Head and Neck Module, QLQ-H&N35 and by inspection of the tracheal mucosa with a fibre-optic bronchoscope before intubation and at extubation. Adverse Events (AE) and bacterial adherence were also studied. Statistical evaluations were carried out with the Fisher's Exact Test, the Clopper-Pearson method, as well as a Proportional Odds Model. RESULTS: Differences between groups were identified in 2 of 8 patient related symptoms with regard to tolerability by QLQ-H&N35 (cough, p = 0.022 and dry mouth, p = 0.014 in the treatment group.). No mucosal damage was identified with bronchoscopy. A low level of bacterial colonization with normal flora, equal between groups, was seen after short-term of intubation (median 5 h). No serious Adverse Events related to the use of an ETT were observed. The results should be treated with caution due to statistical confounders, a small study size and large inter-individual variability in bacterial adhesion. CONCLUSIONS: The new device BIP ETT is well tolerated and has good clinical performance during short-term intubation. Studies with larger sample sizes and longer intubation periods (>24 h) in the ICU-setting are needed and can now be planned in order to identify possible differences in clinical outcomes. TRIAL REGISTRATION: Registered in ClinicalTrials.gov, REGISTRATION NUMBER: NCT01682486 , Date of Registration: August, 30, 2012.

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.

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.

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.

DNA extraction and PCR assays for detection of Toxoplasma gondii.

For detection of Toxoplasma gondii we compared the sensitivity of two different DNA extraction methods and three different PCR assays. Sensitivities of DNA extraction by QIAamp DNA mini Kit or MagNa pure followed by PCR, nested PCR and oligochromatography or Light Cycler PCR using either SYBR green chemistry or TaqMan probe were compared. No significant difference between extraction methods was found using pure T. gondii tachyzoites. Spiked blood samples, 10(4) to 10 parasites per sample, generated no difference in sensitivity between the two DNA extraction methods when analysed by nested PCR detected by oligochromatography or analysed by Light Cycler PCR TaqMan. In spiked blood samples Light Cycler PCR SYBR green was unable to detect the parasite and a reduction in sensitivity was observed with the TaqMan assay. Conventional PCR was more sensitive when DNA was extracted from the spiked samples using the QIAamp DNA mini Kit. Conventional and nested PCR were found to be more sensitive than Light Cycler PCR TaqMan using the QIAamp DNA mini Kit. It was not possible to use Light Cycler PCR SYBR green in blood samples. Conventional PCR was more sensitive for detection of T. gondii in spiked blood samples using QIAamp DNA mini Kit DNA extraction, suggesting that the choice of DNA extraction method may affect PCR assays differently.

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.

Ciprofloxacin Affects Host Cells by Suppressing Expression of the Endogenous Antimicrobial Peptides Cathelicidins and Beta-Defensin-3 in Colon Epithelia.

Antibiotics exert several effects on host cells including regulation of immune components. Antimicrobial peptides (AMPs), e.g., cathelicidins and defensins display multiple functions in innate immunity. In colonic mucosa, cathelicidins are induced by butyrate, a bacterial fermentation product. Here, we investigated the effect of antibiotics on butyrate-induced expression of cathelicidins and beta-defensins in colon epithelial cells. Real-time PCR analysis revealed that ciprofloxacin and clindamycin reduce butyrate-induced transcription of the human cathelicidin LL-37 in the colonic epithelial cell line HT-29. Suppression of LL-37 peptide/protein by ciprofloxacin was confirmed by Western blot analysis. Immunohistochemical analysis demonstrated that ciprofloxacin suppresses the rabbit cathelicidin CAP-18 in rectal epithelia of healthy and butyrate-treated Shigella-infected rabbits. Ciprofloxacin also down-regulated butyrate-induced transcription of the human beta-defensin-3 in HT-29 cells. Microarray analysis of HT-29 cells revealed upregulation by butyrate with subsequent down-regulation by ciprofloxacin of additional genes encoding immune factors. Dephosphorylation of histone H3, an epigenetic event provided a possible mechanism of the suppressive effect of ciprofloxacin. Furthermore, LL-37 peptide inhibited Clostridium difficile growth in vitro. In conclusion, ciprofloxacin and clindamycin exert immunomodulatory function by down-regulating AMPs and other immune components in colonic epithelial cells. Suppression of AMPs may contribute to the overgrowth of C. difficile, causing antibiotic-associated diarrhea.

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.

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.

Bacteraemia in patients with hidradenitis suppurativa undergoing carbon dioxide laser surgery: detection and quantification of bacteria by lysis-filtration.

BACKGROUND: Hidradenitis suppurativa (HS) is a cicatrising and persistent disease of apocrine gland-bearing areas in adults. The severity of this condition varies from a few suppurating lesions to widespread, disabling disease. The aetiology is obscure, but suggested contributory factors include a genetic predisposition, comedones occluding the pilosebaceous apparatus, bacterial infections, and hormonal factors. Treatment consists mainly of surgery, while medical therapies serve principally as adjunct therapy. OBJECTIVES: The aim of the study was to determine the number and type of bacteria circulating in the bloodstream in patients with HS undergoing surgical treatment with a carbon dioxide laser stripping-secondary intention technique. METHODS: Twenty-one patients (20 females and 1 male, mean age 36, range 20-55 years) were included in the study. One blood sample (8.3 ml) was taken before surgery, one during the operation and the last one 10 min after surgery. Five healthy persons (all females, mean age 36, range 23-48 years) not undergoing any operation were used as the controls. The blood was cultured by a lysis-filtration technique which had been shown to be very sensitive. Since the filter catches the microorganisms and colonies are formed during culturing, the number of bacteria in the samples is easily determined. RESULTS: In 6 patients, all samples were negative, which indicates that the method of surgery itself caused no spread of bacteria from the lesions. Bacterial growth in the first blood sample was found in 9 patients, from the second sample in 10 and from the third one in 6. In 1 patient, bacteria were detected in three samples. At least 12 bacterial species were identified. The dominating bacteria were coagulase-negative staphylococci of which most were subtyped as Staphylococcus warneri. Among the anaerobic microorganisms, Propionibacterium acnes and P.granulosum were the most frequently isolated bacteria. The bacterial findings in the blood samples accord well with the results from a previous study in which cultures were taken from the deep parts of the HS lesions. In the 5 controls, no microbial growth was detected. CONCLUSION: The carbon dioxide laser stripping technique caused no additional spread of bacteria into the bloodstream. The evaluation of cultures containing microorganisms from normal skin flora is controversial. Since the bacteria encountered in this study are in close agreement with the findings in cultures from the deeper parts of HS lesions they seem to be relevant. The growth of bacteria in the first blood sample taken before surgery may indicate that some of these patients have bacteria continuously circulating in their blood.