The effect of malalignment on proximal tibial strain in fixed-bearing unicompartmental knee arthroplasty: A comparison between metal-backed and all-polyethylene components using a validated finite element model.

OBJECTIVES: Elevated proximal tibial bone strain may cause unexplained pain, an important cause of unicompartmental knee arthroplasty (UKA) revision. This study investigates the effect of tibial component alignment in metal-backed (MB) and all-polyethylene (AP) fixed-bearing medial UKAs on bone strain, using an experimentally validated finite element model (FEM). METHODS: A previously experimentally validated FEM of a composite tibia implanted with a cemented fixed-bearing UKA (MB and AP) was used. Standard alignment (medial proximal tibial angle 90°, 6° posterior slope), coronal malalignment (3°, 5°, 10° varus; 3°, 5° valgus), and sagittal malalignment (0°, 3°, 6°, 9°, 12°) were analyzed. The primary outcome measure was the volume of compressively overstrained cancellous bone (VOCB) < -3000 µÎµ. The secondary outcome measure was maximum von Mises stress in cortical bone (MSCB) over a medial region of interest. RESULTS: Varus malalignment decreased VOCB but increased MSCB in both implants, more so in the AP implant. Varus malalignment of 10° reduced the VOCB by 10% and 3% in AP and MB implants but increased the MSCB by 14% and 13%, respectively. Valgus malalignment of 5° increased the VOCB by 8% and 4% in AP and MB implants, with reductions in MSCB of 7% and 10%, respectively. Sagittal malalignment displayed negligible effects. Well-aligned AP implants displayed greater VOCB than malaligned MB implants. CONCLUSION: All-polyethylene implants are more sensitive to coronal plane malalignments than MB implants are; varus malalignment reduced cancellous bone strain but increased anteromedial cortical bone stress. Sagittal plane malalignment has a negligible effect on bone strain.Cite this article: I. Danese, P. Pankaj, C. E. H. Scott. The effect of malalignment on proximal tibial strain in fixed-bearing unicompartmental knee arthroplasty: A comparison between metal-backed and all-polyethylene components using a validated finite element model. Bone Joint Res 2019;8:55-64. DOI: 10.1302/2046-3758.82.BJR-2018-0186.R2.

Risk factors for infection with carbapenem-resistant Klebsiella pneumoniae after liver transplantation: the importance of pre- and posttransplant colonization.

Improved understanding of risk factors associated with carbapenem-resistant-Klebsiella pneumoniae (CR-KP) infection after liver transplantation (LT) can aid development of effective preventive strategies. We performed a prospective cohort study of all adult patients undergoing LT at our hospital during 30-month period to define risk factors associated with CR-KP infection. All patients were screened for CR-KP carriage by rectal swabs before and after LT. No therapy was administered to decolonize or treat asymptomatic CR-KP carriers. All patients were monitored up to 180 days after LT. Of 237 transplant patients screened, 41 were identified as CR-KP carriers (11 at LT, 30 after LT), and 20 developed CR-KP infection (18 bloodstream-infection, 2 pneumonia) a median of 41.5 days after LT. CR-KP infection rates among patients non-colonized, colonized at LT, and colonized after LT were 2%, 18.2% and 46.7% (p < 0.001). Independent risk factors for CR-KP infection identified by multivariate analysis, included: renal-replacement-therapy; mechanical ventilation > 48 h; HCV recurrence, and colonization at any time with CR-KP. Based on these four variables, we developed a risk score that effectively discriminated patients at low versus higher risk for CR-KP infection (AUC 0.93, 95% CI 0.86-1.00, p < 0.001). Our results may help to design preventive strategies for LT recipients in CR-KP endemic areas.

FtcR is a new master regulator of the flagellar system of Brucella melitensis 16M with homologs in Rhizobiaceae.

The flagellar regulon of Brucella melitensis 16M contains 31 genes clustered in three loci on the small chromosome. These genes encode a polar sheathed flagellum that is transiently expressed during vegetative growth and required for persistent infection in a mouse model. By following the expression of three flagellar genes (fliF, flgE, and fliC, corresponding to the MS ring, hook, and filament monomer, respectively), we identified a new regulator gene, ftcR (flagellar two-component regulator). Inactivation of ftcR led to a decrease in flagellar gene expression and to impaired Brucella virulence. FtcR has a two-component response regulator domain as well a DNA binding domain and is encoded in the first flagellar locus of B. melitensis. Both the ftcR sequence and its genomic context are conserved in other related alpha-proteobacteria. During vegetative growth in rich medium, ftcR expression showed a peak during the early exponential phase that paralleled fliF gene expression. VjbR, a quorum-sensing regulator of the LuxR family, was previously found to control fliF and flgE gene expression. Here, we provide some new elements suggesting that the effect of VjbR on these flagellar genes is mediated by FtcR. We found that ftcR expression is partially under the control of VjbR and that the expression in trans of ftcR in a vjbR mutant restored the production of the hook protein (FlgE). Finally, FtcR binds directly to the upstream region of the fliF gene. Therefore, our data support the role of FtcR as a flagellar master regulator in B. melitensis and perhaps in other related alpha-proteobacteria.

The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection.

Persistence infection is the keystone of the ruminant and human diseases called brucellosis and Malta fever, respectively, and is linked to the intracellular tropism of Brucella spp. While described as non-motile, Brucella spp. have all the genes except the chemotactic system, necessary to assemble a functional flagellum. We undertook to determine whether these genes are expressed and are playing a role in some step of the disease process. We demonstrated that in the early log phase of a growth curve in 2YT nutrient broth, Brucella melitensis expresses genes corresponding to the basal (MS ring) and the distal (hook and filament) parts of the flagellar apparatus. Under these conditions, a polar and sheathed flagellar structure is visible by transmission electron microscopy (TEM). We evaluated the effect of mutations in flagellar genes of B. melitensis encoding various parts of the structure, MS ring, P ring, motor protein, secretion apparatus, hook and filament. None of these mutants gave a discernible phenotype as compared with the wild-type strain in cellular models of infection. In contrast, all these mutants were unable to establish a chronic infection in mice infected via the intraperitoneal route, raising the question of the biological role(s) of this flagellar appendage.

Fun stories about Brucella: the "furtive nasty bug".

Although Brucella is responsible for one of the major worldwide zoonosis, our understanding of its pathogenesis remains in its infancy. In this paper, we summarize some of the research in progress in our laboratory that we think could contribute to a better understanding of the Brucella molecular virulence mechanisms and their regulation.

Identification of Brucella spp. genes involved in intracellular trafficking.

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis. We also examined the intracellular fate of three virB mutants (virB2, virB4 and virB9) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago-lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61beta protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.

Genetic organisation of the lipopolysaccharide O-antigen biosynthesis region of brucella melitensis 16M (wbk).

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause a zoonotic world-wide disease. As in other Gram-negative bacteria, its S-LPS (smooth lipopolysaccharide) is a major determinant of virulence. The Brucella melitensis 16M LPS O-antigen is a homopolymer of 4-formamido-4,6, dideoxymannose. In this study, the previously cloned 14-kb wbk gene cluster was sequenced, and seven open reading frames (ORFs) as well as four insertion sequences were identified. Six of the seven ORFs are homologous to LPS biosynthesis genes from other organisms. The gmd, per and wbkC gene products are predicted to be involved in 4-formamido-4,6,dideoxymannose synthesis. By deletion experiments, we demonstrated that the putative formyltransferase WbkC is absolutely required for the O-side-chain production. The wbkA gene product is similar to several mannosyltransferases and is probably involved in the polymerisation of the B. melitensis O-side-chain. We also identified two genes (wzm and wzt) encoding proteins with high similarity to several two-component ABC (ATP-binding cassette) transporters. Their implication in O-antigen translocation across the inner membrane was confirmed by gene replacement. Finally, no function has been assigned to the wbkB gene either by homology search or functionally, because deletion of wbkB did not interfere with the O-antigen structure. The seven ORFs have a low G + C content, indicating that they might have been acquired by lateral transfer from a progenitor with more A + T rich DNA.

Identification and characterization of in vivo attenuated mutants of Brucella melitensis.

Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.

Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages.

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described. Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined. A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella. One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet. Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7. The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp. The survival of B. melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined. The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages.