Kåre Bergh

Identification of Virulence Genes Linked with Diarrhea Due to Atypical Enteropathogenic Escherichia coli by DNA Microarray Analysis and PCR

ABSTRACT The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood diarrhea is controversial. The aim of the present study was to search for genes linked with diarrhea in atypical EPEC strains from a case-control study among Norwegian children. Using DNA microarray analysis, genomic DNAs from strains isolated from children with (n = 37) and without (n = 20) diarrhea were hybridized against 242 different oligonucleotide probes specific for 182 virulence genes or markers from all known E. coli pathotypes. PCR was performed to test the strains for seven putative virulence genes not included in the microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest statistical association with diarrhea (P = 0.0008). Other OI-122 genes (set/ent, nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were also associated with diarrheal disease. The phylogenetic marker gene yjaA was negatively associated with diarrhea (P = 0.0004). Atypical EPEC strains could be classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In conclusion, using DNA microarray analysis to determine the virulence gene profile of atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Based on their composition of virulence genes, the majority of strains could be classified in two virulence groups, of which one was seen mainly in children with diarrhea.

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro

Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces. Methods Discs of titanium and steel were incubated in the presence of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Propionibacterium acnes in Mueller Hinton broth. Non-adherent bacteria were removed by repeated rinsing of the discs. 10 parallels of each disc were subjected to 1 of 4 methods for bacterial recovery: (A) sonication of the discs, (B) scraping of the discs using surgical blades followed by streaking of the blades onto agar plates, (C) scraping of the discs followed by vortex mixing of the surgical blades, and (D) scraping of the discs followed by sonication of the surgical blades. Quantitative bacterial cultures were performed for each sampling method. Results With the exception of S. epidermidis on steel, sonication efficiently and reliably dislodged biofilm bacteria. The scraping methods employed did not detach bacteria embedded in biofilm. Interpretation Scraping of metal surfaces is not an adequate method for sampling of biofilm bacteria in vitro.

Cancellous bone as an antibiotic carrier.

We compared the release characteristics of antibiotics from in vivo and in vitro processed morselized cancellous bone. The bone was impregnated with 7 antibiotics and compressed into a wire-mesh cylinder. In vitro, the bone was processed by daily transfer of the cylinder with its contents into test tubes with broth. The amount of antibiotic eluted from the bone was measured after 1, 3 and 7 days. In vivo, the cylinder was implanted intramuscularly in the interscapular region in rats. After 1, 3 and 7 days, the cylinder was removed and the amount of antibiotic eluted in broth was measured. The results showed that morselized cancellous bone can act as a carrier of antibiotics in vitro and in vivo. The elution profiles of netilmicin-, vancomycin-, clindamycin- and rifampicin-impregnated cancellous bone processed in vitro and in vivo were similar.

Activation of the complement system and adverse effects of biodegradable pins of poly-lactic acid (Biofix®) in osteochondritis dissecans

Biodegradable pins of polyglycolic acid (PGA) or polylactic acid (PLA) have been used in the treatment of fractures and osteotomies during the past 5 years. Adverse effects reported have included swelling at the implantation site and sinus formation, considered to represent nonspecific foreign-body reactions. Recent reports, however, have shown severe reactions after intraarticular fracture fixation. Reactions in 2 patients, treated with polylactic pins for osteochondritis dissecans (OCD) in our hospital, prompted the present clinical investigation and further evaluation of the complement-activating potential of polylactic pins. 10 knees underwent arthroscopic fixation of an OCD-lesion with Biofix (PLA) pins. Clinical follow-ups were carried out at 2, 6, and 12 weeks and at 6 and 12 months. Blood samples were collected from 5 patients 9-24 months postoperatively for biocompatibility tests. Quantification of human C5a des Arg was performed with a recently developed sandwich ELISA technique, using neoepitope-specific monoclonal antibodies. 6 knees developed diffuse swelling and a prolonged postoperative course. 2 patients had a particularly prolonged course which could not be attributed to infection. Levels of C5a des Arg in plasma incubated in the presence of polylactic acid were higher than in plasma incubated in the absence of PLA. The high frequency of long-term postoperative inflammatory signs in these knees treated for OCD and the demonstration of a complement activation potential of PLA pins warrant further studies on the biocompatibility of this material. Until more information is available, we do not recommend intraarticular use of PLA pins.

Adsorption and release of antibiotics from morselized cancellous bone. In vitro studies of 8 antibiotics.

We studied the basic release patterns of antibiotics from cancellous bone in vitro. Antibiotic-impregnated bone was compressed into a wire-mesh cylinder and the release of antibiotic was assessed by two different in vitro methods: agar diffusion and broth elution. The zones of inhibition were measured on seeded agar and the amounts of antibiotics released in elution tubes were assessed by a bioassay. The study continued for 21 days with daily transfer of the cylinders. The results indicated that benzylpenicillin, dicloxacillin, cephalotin, netilmicin, clindamycin, vancomycin, ciprofloxacin and rifampicin were adsorbed to cancellous bone in vitro. Compared to broth elution, agar diffusion showed a prolonged period of release, owing to the small amounts of antibiotic leaking out of the cylinder into the agar. The betalactams had antibacterial activity in broth for a shorter time than the other antibiotics. The release patterns of the betalactams were similar, in spite of their differences in thermal stability. Only rifampicin showed a concentration higher than MIC for longer than 21 days.

Phylogenetic Backgrounds and Virulence Profiles of Atypical Enteropathogenic Escherichia coli Strains from a Case-Control Study Using Multilocus Sequence Typing and DNA Microarray Analysis

ABSTRACT Atypical enteropathogenetic Escherichia coli (EPEC) strains are frequently detected in children with diarrhea but are also a common finding in healthy children. The aim of this study was to compare the phylogenetic ancestry and virulence characteristics of atypical (eae positive, stx and bfpA negative) EPEC strains from Norwegian children with (n = 37) or without (n = 19) diarrhea and to search for an association between phylogenetic ancestry and diarrhea. The strains were classified in phylogenetic groups by phylogenetic marker genes and in sequence types (STs) by multilocus sequence typing. Phylogenetic ancestry was compared to virulence characteristics based on DNA microarray analysis. Serotyping and pulsed-field gel electrophoresis (PFGE) were also performed. All four phylogenetic groups, 26 different STs, and 20 different clonal groups were represented among the 56 atypical EPEC strains. The strains were separated into three clusters by overall virulence gene profile; one large cluster with A, B1, and D strains and two clusters with group B2 strains. There was considerable heterogeneity in the PFGE profiles and serotypes, and almost half of the strains were O nontypeable. The efa1/lifA gene, previously shown to be statistically linked with diarrhea in this strain collection (J. E. Afset et al., J. Clin. Microbiol. 44:3703-3711, 2006), was present in 8 of 26 STs. The two phylogenetic groups B1 and D were weakly associated with diarrhea (P = 0.06 and P = 0.09, respectively). In contrast, group B2 was isolated most frequently from healthy controls (P = 0.05). In conclusion, the atypical EPEC strains were heterogeneous both phylogenetically and by virulence profile. Phylogenetic ancestry was less useful as a predictor of diarrhea than were specific virulence genes.

Cortical allograft as a vehicle for antibiotic delivery

Background Infection can be a devastating complication after implantation of a cortical bone allograft. The allograft could act as a vehicle for local antibiotic prophylaxis. Material and methods We studied the release of antibiotics in vitro from cortical bone allografts impregnated with antibiotics for different periods of time. We also studied whether cortical allografts impregnated with antibiotics could eradicate Staphylococcus aureus from an experimentally infected graft in vivo. In the in vitro study, pieces of cortical bone were impregnated with netilmicin, vancomycin, ciprofloxacin and rifampicin for 1 h, 10 h and 100 h. The antibiotics were eluted into phosphate-buffered saline (PBS) for 7 days, with daily transfer of the bone into fresh PBS. In the in vivo study, cortical allografts impregnated with antibiotics were placed in rats intramuscularly. 10 μL of an S. aureus suspension (0.6 × 105 CFU) was placed in the intramedullary cavity. After 15 days, the allografts were removed and examined for bacterial growth. Results The amount of antibiotics released in vitro was influenced by the time used for antibiotic impregnation of the bone. Allografts impregnated with netilmicin, vancomycin and rifampicin effectively eradicated perioperative contamination with S. aureus in vivo. Interpretation This study shows that a cortical bone allograft would be an effective vehicle for local antibiotic delivery.

Typing of human isolates of Streptococcus agalactiae (group B streptococcus, GBS) strains from Zimbabwe

Serotyping and genotyping are important tools in epidemiological studies of group B streptococcal (GBS) infections, which are important diseases in man, particularly in newborns. In the present study, 241 GBS isolates from Zimbabwe, comprising 124 carrier isolates from pregnant women and 117 isolates from patients hospitalised for various diseases, were serotyped. Antibodies specific for the capsular polysaccharide antigens (CPAs) Ia, Ib and II-V and antibodies specific for the surface-localised proteins, c(alpha), c(beta), R1, R3 and R4 were used for serotyping. Strains of the CPA types Ia (17%), III (47.7%) and V (23.2%) predominated. Of the various protein antigens, c(alpha) and R4 were expressed with highest frequency, c(alpha) by 100% of the CPA type Ia strains and R4 by 92% of the CPA type III strains. The R3 protein occurred frequently (24%), especially in type V strains (84%). A total of 25 serovariants was detected in the strain collection with the variants Ia/c(alpha) (16%), III/R4 (43.5%) and V/c(alpha), R3 (14.1%) occurring with the highest frequency. Serotype and subtype distribution of the carrier isolates were essentially similar to those of the disease-associated isolates. Genomic heterogeneity was demonstrated by pulsed-field gel electrophoresis of type III/R4 and type V/c(alpha), R3 isolates, but to a much lesser extent than recorded with Norwegian strains. These results demonstrate that many variants of GBS occur in the Zimbabwean population. The data obtained may assist in the formulation of a possible future GBS vaccine for Zimbabwe and perhaps for other African countries.

A comprehensive microbiological evaluation of fifty-four patients undergoing revision surgery due to prosthetic joint loosening.

The diagnosis of a chronic prosthetic joint infection (PJI) is challenging, and no consensus exists regarding how best to define the criteria required for microbiological identification. A general view is that culture of periprosthetic biopsies suffers from inadequate sensitivity. Recently, molecular analyses have been employed in some studies but the specificity of molecular analyses has been questioned, mainly due to contamination issues. In a prospective study of 54 patients undergoing revision surgery due to prosthetic joint loosening, we focused on two aspects of microbiological diagnosis of chronic PJI. First, by collecting diagnostic specimens in a highly standardized manner, we aimed at investigating the adequacy of various specimens by performing quantitative bacteriological culture. Second, we designed and performed real-time 16S rRNA gene PCR analysis with particular emphasis on minimizing the risk of false-positive PCR results. The specimens analysed included synovial fluid, periprosthetic biopsies from the joint capsule and the interface membrane, and specimens from the surface of the explanted prosthesis rendered accessible by scraping and sonication. No antibiotics were given prior to specimen collection. Based on five diagnostic criteria recently suggested, we identified 18 PJIs, all of which fulfilled the criterion of ≥2 positive cultures of periprosthetic specimens. The rate of culture-positive biopsies from the interface membrane was higher compared to specimens from the joint capsule and synovial fluid, and the interface membrane contained a higher bacterial load. Interpretational criteria were applied to differentiate a true-positive PCR from potential bacterial DNA contamination derived from the reagents used for DNA extraction and amplification. The strategy to minimize the risk of false-positive PCR results was successful as only two PCR results were false-positive out of 216 negative periprosthetic specimens. Although the PCR assays themselves were very sensitive, three patients with low bacterial numbers in periprosthetic specimens tested negative by real-time PCR. This overall lowered sensitivity is most likely due to the reduced specimen volume used for PCR analysis compared to culture and may also be due to interference from human DNA present in tissue specimens. According to the protocol in the present study, 16S rRNA gene real-time PCR did not identify more cases of septic prosthetic loosening than did culture of adequate periprosthetic biopsies.

Release of netilmicin and vancomycin from cancellous bone

First, we studied the effect of the following variables used for netilmicin- and vancomycin-impregnation of cancellous bone: a) antibiotic concentration of the impregnation fluid, b) time used for impregnation, c) pH of the impregnation fluid, d) the degree of bone morselizing and e) antibiotic combination. An increase in the antibiotic concentration of the impregnation fluid increased the amount of antibiotics released from bone. In addition, the amount of vancomycin eluted was also dependent on the time used for impregnation. The fraction of the total amount of netilmicin and vancomycin released after 24 h was 80% and 30%, respectively. More netilmicin and vancomycin were eluted from bone impregnated with antibiotics at pH 7 than the amount eluted from bone impregnated at pH 3. More netilmicin was eluted from fine morselized bone than from coarse morselized bone. By combining netilmicin and vancomycin in the impregnation fluid, the release of vancomycin was reduced. Secondly, we analyzed if the release of antibiotics from bone was complete: 99.9% of the total amount of netilmicin adsorbed to the bone was released by elution during 6 weeks. Finally, after implantation of netilmicin-impregnated bone in rabbit femur condyle, we measured netilmicin and vancomycin in serum: peak serum values of netilmicin were 4.2 (3.7-4.7) mg/L 2-3 h postoperatively.