Malocclusions in primary and early mixed dentition in very preterm children.

Objective: To compare the prevalence of malocclusions in the primary and early mixed dentition of very preterm and full-term children.Material and methods: Study subjects consisted of 205 very preterm (90 girls and 115 boys), and 205 age- and gender-matched full-term children. Data were collected from the register of Turku University Hospital (children born before the 37th week of pregnancy with a birth weight of less than 1500 g, and all infants born before the 32nd week of pregnancy) and from public health centre dental registers.Results: In primary dentition, case children had a higher odds of dental crowding (OR = 2.94, 95% CI 1.17-7.35, p = .021), a tendency toward increased overbite (OR = 1.55, 95% CI 0.93-2.59, p = .096), and a lower odds of increased overjet (OR = 0.19, 95% CI 0.07-0.57, p = .003) compared to control children. In early mixed dentition, there were no statistically significant differences in occlusal traits; however, case children were significantly more likely to have received orthodontic treatment (OR = 2.80, 95% CI 1.50-5.23, p = .001) compared to controls.Conclusions: The results indicate that in primary dentition, the prevalence of malocclusion varies between very preterm and full-term children. In early mixed dentition, the distribution of occlusal traits is more similar.

"It's a gut feeling" - Escherichia coli biofilm formation in the gastrointestinal tract environment.

Escherichia coli can commonly be found, either as a commensal, probiotic or a pathogen, in the human gastrointestinal (GI) tract. Biofilm formation and its regulation is surprisingly variable, although distinct regulatory pattern of red, dry and rough (rdar) biofilm formation arise in certain pathovars and even clones. In the GI tract, environmental conditions, signals from the host and from commensal bacteria contribute to shape E. coli biofilm formation within the multi-faceted multicellular communities in a complex and integrated fashion. Although some major regulatory networks, adhesion factors and extracellular matrix components constituting E. coli biofilms have been recognized, these processes have mainly been characterized in vitro and in the context of interaction of E. coli strains with intestinal epithelial cells. However, direct observation of E. coli cells in situ, and the vast number of genes encoding surface appendages on the core or accessory genome of E. coli suggests the complexity of the biofilm process to be far from being fully understood. In this review, we summarize biofilm formation mechanisms of commensal, probiotic and pathogenic E. coli in the context of the gastrointestinal tract.

Antimicrobial activity of HL-60 cells compared to primary blood-derived neutrophils against Staphylococcus aureus.

BACKGROUND: The human leukemia cell line HL-60 is considered an alternative cell culture model to study neutrophil differentiation and migration. The aim of this study was to characterize the suitability of HL-60 cells differentiated to neutrophil-like cells (nHL-60) as substitute for blood-derived human neutrophils to investigate the interaction of neutrophils with Staphylococcus aureus. METHODS: For this purpose, antimicrobial activity, bacterial uptake, production of reactive oxygen species and the release of neutrophil extracellular traps (NETs) by nHL-60 cells were analyzed and compared to primary blood-derived neutrophils using Staphylococcus aureus as important human and animal pathogen. RESULTS: Overall, the antimicrobial activities of nHL-60 cells were distinctly lower compared to blood-derived neutrophils. Furthermore, production of reactive oxygen species as well as NET formation was clearly impaired in nHL-60 cells. CONCLUSION: This study indicates that HL-60 cells are of limited usage as an alternative model to study antimicrobial functions of neutrophils against Staphylococcus aureus.

Controlled Evaluation of the New BacT/Alert Virtuo Blood Culture System for Detection and Time to Detection of Bacteria and Yeasts.

We compared the newly approved BacT/Alert Virtuo blood culture system to the BacT/Alert 3D system using 115 clinical bacterial and fungal isolates in 784 simulated blood culture bottles. The time to detection was reduced by roughly 20% in the Virtuo system (P< 0.0001) while the detection rate did not differ.

Microbiological diagnosis of Eggerthella lenta blood culture isolates in a Swedish tertiary hospital: Rapid identification and antimicrobial susceptibility profile.

Eggerthella lenta is a Gram-positive anaerobic bacillus. Improved diagnostics and increased awareness of rare pathogens have revealed its potential to cause serious invasive infections. In this study, 18 clinical E. lenta isolates derived from positive blood cultures were included. Underlying problems of the patients were in the majority of cases related to the gastrointestinal tract. The performance of two MALDI-TOF MS systems, i.e. Bruker and Vitek MS, in identification of E. lenta was analyzed. In addition, the minimal inhibitory concentrations for clinically relevant antimicrobial agents were determined by routine procedures using E-test. 17 of the 18 E. lenta isolates investigated in this study were correctly identified to species level by the Bruker MS system, while the Vitek MS system identified all 18 isolates. Antimicrobial sensitivity towards the tested agents was in general good. However, high resistance rates were observed for penicillin G and piperacillin-tazobactam based on EUCAST breakpoints.

Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli.

BACKGROUND: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the ß-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. RESULTS: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. CONCLUSIONS: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.

Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37.

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.

Identification of microorganisms directly from blood culture bottles with polymicrobial growth: comparison of FilmArray and direct MALDI-TOF MS.

Bloodstream infections (BSIs) are related to high mortality and morbidity. Rapid administration of effective antimicrobial treatment is crucial for patient survival. Recently developed rapid methods to identify pathogens directly from blood culture bottles speed up diagnosis of BSIs. The present study compares the performance of two rapid identification methods, FilmArray and direct MALDI-TOF MS, on identifying microorganisms directly from positive blood culture bottles with polymicrobial growth. FilmArray and direct MALDI-TOF MS were performed directly on positive clinical and simulated polymicrobial blood culture bottles. Assay results were compared with standard culture methods. In total, 110 polymicrobial blood culture samples, of which 96 samples contained two microorganisms while 14 samples contained three microorganisms, were studied. FilmArray was able to identify 215/234 (92.0%) of isolates detected by the standard culture method and successfully identified all microorganisms in 88/110 (80.0%) of blood culture bottles. In contrast, direct MALDI-TOF MS was only able to identify 65/234 (27.8%) of isolates and managed to identify all microoganisms in 2/110 (2.1%) of blood culture bottles. FilmArray is a rapid method for direct identification of polymicrobial blood culture samples that can complement the conventional identification methods. Direct MALDI-TOF MS has low performance with polymicrobial samples.

Molecular basis of resistance to macrolides and lincosamides among staphylococci and streptococci from various animal sources collected in the resistance monitoring program BfT-GermVet.

In this study, erythromycin- and/or clindamycin-resistant isolates among 248 coagulase-positive and coagulase-variable staphylococci and 500 streptococci, collected all over Germany during 2004-2006 in the resistance monitoring program BfT-GermVet, were investigated for their genetic basis of macrolide and/or lincosamide resistance. Staphylococci were sampled from various disease conditions of dogs/cats or pigs, whereas streptococci were from dogs/cats, pigs or horses. Resistant staphylococci were further identified biochemically to species and subspecies level and tested for the resistance genes erm(A), erm(B), erm(C), erm(TR), msr(A), msr(D), mef(A), mph(C), lnu(A), lnu(B) and lnu(C). The methylase genes erm(A), erm(B) and erm(C) were detected in staphylococci, alone or in different combinations. The erm(B) gene was the predominant gene in Staphylococcus intermedius and streptococci. The efflux gene msr(A) and the genes mph(C) and lnu(A) coding for inactivating enzymes were detected in single staphylococcal isolates. The efflux genes mef(A) and msr(D) were detected in three streptococci, in one of them together with the erm(B) gene. The lnu(B) gene was detected in seven porcine streptococcal isolates with reduced susceptibility to clindamycin. These data confirm that high-level resistance to erythromycin and clindamycin in staphylococci and streptococci was mainly due to rRNA methylases. The lnu(B) gene was detected for the first time in streptococci of animal origin.

Vitamin D-deficient mice have more invasive urinary tract infection.

Vitamin D deficiency is a common health problem with consequences not limited to bone and calcium hemostasis. Low levels have also been linked to tuberculosis and other respiratory infections as well as autoimmune diseases. We have previously shown that supplementation with vitamin D can induce the antimicrobial peptide cathelicidin during ex vivo infection of human urinary bladder. In rodents, however, cathelicidin expression is not linked to vitamin D and therefore this vitamin D-related effect fighting bacterial invasion is not relevant. To determine if vitamin D had further protective mechanisms during urinary tract infections, we therefore used a mouse model. In vitamin D-deficient mice, we detected more intracellular bacterial communities in the urinary bladder, higher degree of bacterial spread to the upper urinary tract and a skewed cytokine response. Furthermore, we show that the vitamin D receptor was upregulated in the urinary bladder and translocated into the cell nucleus after E. coli infection. This study supports a more general role for vitamin D as a local immune response mediator in the urinary tract.

Draft Genome Sequences of Semiconstitutive Red, Dry, and Rough Biofilm-Forming Commensal and Uropathogenic Escherichia coli Isolates.

Strains of Escherichia coli exhibit diverse biofilm formation capabilities. E. coli K-12 expresses the red, dry, and rough (rdar) morphotype below 30°C, whereas clinical isolates frequently display the rdar morphotype semiconstitutively. We sequenced the genomes of eight E. coli strains to subsequently investigate the molecular basis of semiconstitutive rdar morphotype expression.

Broad-Range Detection of Microorganisms Directly from Bronchoalveolar Lavage Specimens by PCR/Electrospray Ionization-Mass Spectrometry.

The clinical demand on rapid microbiological diagnostic is constantly increasing. PCR coupled to electrospray ionization-mass spectrometry, PCR/ESI-MS, offers detection and identification of over 750 bacteria and Candida species directly from clinical specimens within 6 hours. In this study, we investigated the clinical performance of the IRIDICA BAC LRT Assay for detection of bacterial pathogens in 121 bronchoalveolar lavage (BAL) samples that were received consecutively at our bacterial laboratory for BAL culture. Commensal or pathogenic microorganisms were detected in 118/121 (98%) BAL samples by PCR/ESI-MS, while in 104/121 (86%) samples by routine culture (P<0.01). Detection of potentially pathogenic microorganisms by PCR/ESI-MS was evaluated in comparison with conventional culture-based or molecular methods. The agreement between positive findings was overall good. Most Staphylococcus aureus-positive PCR/ESI-MS results were confirmed by culture or species-specific PCR (27/33, 82%). The identity of Streptococcus pneumoniae could however be confirmed for only 6/17 (35%) PCR/ESI-MS-positive samples. Non-cultivable and fastidious pathogens, which were not covered by standard culture procedures were readily detected by PCR/ESI-MS, including Legionella pneumophila, Bordetella pertussis, Norcadia species and Mycoplasma pneumoniae. In conclusion, PCR/ESI-MS detected a broad range of potential pathogens with equal or superior sensitivity compared to conventional methods within few hours directly from BAL samples. This novel method might thus provide a relevant tool for diagnostics in critically ill patients.

Alterations of c-di-GMP turnover proteins modulate semi-constitutive rdar biofilm formation in commensal and uropathogenic Escherichia coli.

Agar plate-based biofilm of enterobacteria like Escherichia coli is characterized by expression of the extracellular matrix components amyloid curli and cellulose exopolysaccharide, which can be visually enhanced upon addition of the dye Congo Red, resulting in a red, dry, and rough (rdar) colony morphology. Expression of the rdar morphotype depends on the transcriptional regulator CsgD and occurs predominantly at ambient temperature in model strains. In contrast, commensal and pathogenic isolates frequently express the csgD-dependent rdar morphotype semi-constitutively, also at human host body temperature. To unravel the molecular basis of temperature-independent rdar morphotype expression, biofilm components and c-di-GMP turnover proteins of seven commensal and uropathogenic E. coli isolates were analyzed. A diversity within the c-di-GMP signaling network was uncovered which suggests alteration of activity of the trigger phosphodiesterase YciR to contribute to (up)regulation of csgD expression and consequently semi-constitutive rdar morphotype development.

Identification of microorganisms grown on chromogenic media by MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and chromogenic media are widely used in clinical microbiology laboratories to facilitate the rapid selection and identification of pathogens. The aim of this study was to evaluate whether usage of chromogenic media limits the diagnostic performance of MALDI-TOF MS for microbial identification. A total of 386 microorganisms collected and analyzed at five laboratories were included. Isolates were cultured on relevant chromogenic media and non-selective agar plates in parallel and identified using the Bruker MALDI-TOF MS. Among the tested isolates, no misidentification was recorded and there was no medium-related difference in the identification level. However, score values were overall slightly but significantly lower for isolates grown on chromogenic media. In conclusion, the use of chromogenic culture media tested here had no relevant impact on MALDI-TOF MS performance for diagnostic purposes.

Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene.

An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.

Novel Strategies in the Prevention and Treatment of Urinary Tract Infections.

Urinary tract infections are one of the most common bacterial infections, especially in women and children, frequently treated with antibiotics. The alarming increase in antibiotic resistance is a global threat to future treatment of infections. Therefore, alternative strategies are urgently needed. The innate immune system plays a fundamental role in protecting the urinary tract from infections. Antimicrobial peptides form an important part of the innate immunity. They are produced by epithelial cells and neutrophils and defend the urinary tract against invading bacteria. Since efficient resistance mechanisms have not evolved among bacterial pathogens, much effort has been put into exploring the role of antimicrobial peptides and possibilities to utilize them in clinical practice. Here, we describe the impact of antimicrobial peptides in the urinary tract and ways to enhance the production by hormones like vitamin D and estrogen. We also discuss the potential of medicinal herbs to be used in the prophylaxis and the treatment of urinary tract infections.

Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens.

Virulence factors of uropathogenic E. coli and their interaction with the host.

Urinary tract infections (UTIs) belong to the most common infectious diseases worldwide. The most frequently isolated pathogen from uncomplicated UTIs is Escherichia coli. To establish infection in the urinary tract, E. coli has to overcome several defence strategies of the host, including the urine flow, exfoliation of urothelial cells, endogenous antimicrobial factors and invading neutrophils. Thus, uropathogenic E. coli (UPEC) harbour a number of virulence and fitness factors enabling the bacterium to resist and overcome these different defence mechanisms. There is no particular factor which allows the identification of UPEC among the commensal faecal flora apart from the ability to enter the urinary tract and cause an infection. Many of potential virulence or fitness factors occur moreover with high redundancy. Fimbriae are inevitable for adherence to and invasion into the host cells; the type 1 pilus is an established virulence factor in UPEC and indispensable for successful infection of the urinary tract. Flagella and toxins promote bacterial dissemination, while different iron-acquisition systems allow bacterial survival in the iron-limited environment of the urinary tract. The immune response to UPEC is primarily mediated by toll-like receptors recognising lipopolysaccharide, flagella and other structures on the bacterial surface. UPEC have the capacity to subvert this immune response of the host by means of actively impacting on pro-inflammatory signalling pathways, or by physical masking of immunogenic structures. The large repertoire of bacterial virulence and fitness factors in combination with host-related differences results in a complex interaction between host and pathogen in the urinary tract.