A Survey Assessment of Neurosurgeons' Interest in Osteopathic Medicine and Its Integration Into Their Practice.

INTRODUCTION: Osteopathic manipulative medicine (OMM) encompasses techniques guided by the tenets of osteopathy aimed at facilitating the body's natural self-healing capabilities as a treatment option for injury or illness. This approach recognizes the interrelationship of structure and function in promoting overall health. The clinical applications of OMM have been highly researched throughout different subspecialties of medicine; however, there is a notable lack of osteopathic-based research targeted toward neurosurgical patient populations. METHODS: This cross-sectional descriptive study was conducted via a survey generated using SurveyMonkey (SurveyMonkey, San Mateo, CA, USA; accessed at www.surveymonkey.com). Subjects for this survey were gathered using a convenience sampling method in which emails of all neurosurgeons listed in the "Member Directory" on the American Association of Neurological Surgeons website were compiled into a mailing list. The survey was sent to all 6,503 emails collected, and the responses were recorded over the next month. The responses for each survey question were averaged and, when appropriate, compared using a two-tailed T-test, with statistical significance defined as a p<0.05. Where applicable, simple linear regression analysis was used to assess correlations between survey data. The measured outcomes included neurosurgeons' (1) knowledge of and (2) attitudes toward OMM. RESULTS: Both MD and DO neurosurgeons reported using OMM (or referring their patients for OMM) less than once per year. In comparison to their MD colleagues, neurosurgeons carrying a DO degree ranked their familiarity with the tenets of osteopathic medicine (p<0.0001) and their knowledge of the applications of OMM in their practice (p=0.0018) significantly higher. Greater reported familiarity with the tenets of osteopathic medicine and applications of OMM showed a positive correlation with neurosurgeons' comfort in recommending OMM as a nonsurgical, preoperative treatment option, as a post-surgical, rehabilitative treatment option, and as a pain management option (p<0.0001 for all). There was a clear interest in seeing further osteopathic-based neurosurgery research by both MD and DO neurosurgeons, as well as a trend of interest in incorporating OMM into their practice if shown to be clinically beneficial. CONCLUSIONS: Both MD and DO neurosurgeons are interested in seeing more research into the applications of OMM in their patient populations and, most importantly, are likely to integrate OMM into their practice if presented with research detailing clinical benefits to their patients. This study highlights the clinical interest of neurosurgeons in further research into the applications of OMM specific to the field of neurosurgery.

The implementation and utility of clinical exome sequencing in a South African infant cohort.

Genetic disorders are significant contributors to infant hospitalization and mortality globally. The early diagnosis of these conditions in infants remains a considerable challenge. Clinical exome sequencing (CES) has shown to be a successful tool for the early diagnosis of genetic conditions, however, its utility in African infant populations has not been investigated. The impact of the under-representation of African genomic data, the cost of testing, and genomic workforce shortages, need to be investigated and evidence-based implementation strategies accounting for locally available genetics expertise and diagnostic infrastructure need to be developed. We evaluated the diagnostic utility of singleton CES in a cohort of 32 ill, South African infants from two State hospitals in Johannesburg, South Africa. We analysed the data using a series of filtering approaches, including a curated virtual gene panel consisting of genes implicated in neonatal-and early childhood-onset conditions and genes with known founder and common variants in African populations. We reported a diagnostic yield of 22% and identified seven pathogenic variants in the NPHS1, COL2A1, OCRL, SHOC2, TPRV4, MTM1 and STAC3 genes. This study demonstrates the utility value of CES in the South African State healthcare setting, providing a diagnosis to patients who would otherwise not receive one and allowing for directed management. We anticipate an increase in the diagnostic yield of our workflow with further refinement of the study inclusion criteria. This study highlights important considerations for the implementation of genomic medicine in under-resourced settings and in under-represented African populations where variant interpretation remains a challenge.

Chimeric DNA byproducts in strand displacement amplification using the T7 replisome.

Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation.

Factors Affecting Compliance With Myringotomy Tube Follow-up Care.

OBJECTIVE: Myringotomy and tube insertion is a commonly practiced procedure within pediatric otolaryngology. Though relatively safe, follow-up appointments are critical in preventing further complications and monitoring for improvement. This study sought to evaluate the factors associated with compliance of post-myringotomy follow-up visits in an urban safety-net tertiary care setting. METHODS: This study is a retrospective chart review conducted in outpatient otolaryngology clinic at an urban, safety-net, tertiary-care, academic medical center. All patients from ages 0 to 18 who received myringotomy and tube placement between February 3, 2012, to May 30, 2018 at the aforementioned clinic were included. RESULTS: A total of 806 patients had myringotomy tubes placed during this period; 190 patients were excluded due to no visits being scheduled within 1 and 6 month visit windows post-operatively, leaving 616 patients included for analysis. Of 616 patients, 574 patients were seen for the 1-month visit, (42 patients did not have follow-up visits within the 1-month window), and 356 patients were examined for the 6-month visit (260 patients did not schedule follow-up visits within the 6-month window). For the 1-month follow-up visits post-procedure, only race/ethnicity type "Other" was associated with lower no-show rates (OR = 0.330, 95% CI: 0.093-0.968). With the 6-month follow-up visits, having private insurance (OR = 0.446, 95% CI: 0.229-0.867) and not having a 1-month visit scheduled (OR = 0.404, 95% CI: 0.174-0.937) predicted lower no-show rates. CONCLUSION: No meaningful factors studied were significantly associated with compliance of short-term, 1-month visits post-myringotomy. Compliance of longer-term, 6-month post-operative visits was associated with insurance type and previous visit status.

Replacement of the heme axial lysine as a test of conformational adaptability in the truncated hemoglobin THB1.

Amino acid replacement is a useful strategy to assess the roles of axial heme ligands in the function of native heme proteins. THB1, the protein product of the Chlamydomonas reinhardtii THB1 gene, is a group 1 truncated hemoglobin that uses a lysine residue in the E helix (Lys53, at position E10 by reference to myoglobin) as an iron ligand at neutral pH. Phylogenetic evidence shows that many homologous proteins have a histidine, methionine or arginine at the same position. In THB1, these amino acids would each be expected to convey distinct reactive properties if replacing the native lysine as an axial ligand. To explore the ability of the group 1 truncated Hb fold to support alternative ligation schemes and distal pocket conformations, the properties of the THB1 variants K53A as a control, K53H, K53M, and K53R were investigated by electronic absorption, EPR, and NMR spectroscopies. We found that His53 is capable of heme ligation in both the Fe(III) and Fe(II) states, that Met53 can coordinate only in the Fe(II) state, and that Arg53 stabilizes a hydroxide ligand in the Fe(III) state. The data illustrate that the group 1 truncated Hb fold can tolerate diverse rearrangement of the heme environment and has a strong tendency to use two protein side chains as iron ligands despite accompanying structural perturbations. Access to various redox pairs and different responses to pH make this protein an excellent test case for energetic and dynamic studies of heme ligation.

Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

Heme ligation in hemoglobin is typically assumed by the "proximal" histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two α-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a "distal" histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has been replaced with a leucine, though expected to bind a water molecule and yield a high-spin iron complex at neutral pH, has low-spin spectral properties. Here, we applied nuclear magnetic resonance and electronic absorption spectroscopic methods to GlbN modified with heme and amino acid replacements to identify the distal ligand in H46L GlbN. We found that His117, a residue located in the C-terminal portion of the protein and on the proximal side of the heme, is responsible for the formation of an alternative bis-histidine complex. Simultaneous coordination by His70 and His117 situates the heme in a binding site different from the canonical site. This new holoprotein form is achieved with only local conformational changes. Heme affinity in the alternative site is weaker than in the normal site, likely because of strained coordination and a reduced number of specific heme-protein interactions. The observation of an unconventional heme binding site has important implications for the interpretation of mutagenesis results and globin homology modeling.

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii.

BACKGROUND: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. METHODS: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. RESULTS: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment. CONCLUSIONS: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. GENERAL SIGNIFICANCE: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.

Histidine-Lysine Axial Ligand Switching in a Hemoglobin: A Role for Heme Propionates.

The hemoglobin of Synechococcus sp. PCC 7002, GlbN, is a monomeric group I truncated protein (TrHb1) that coordinates the heme iron with two histidine ligands at neutral pH. One of these is the distal histidine (His46), a residue that can be displaced by dioxygen and other small molecules. Here, we show with mutagenesis, electronic absorption spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy that at high pH and exclusively in the ferrous state, Lys42 competes with His46 for the iron coordination site. When b heme is originally present, the population of the lysine-bound species remains too small for detailed characterization; however, the population can be increased significantly by using dimethyl-esterified heme. Electronic absorption and NMR spectroscopies showed that the reversible ligand switching process occurs with an apparent pKa of 9.3 and a Lys-ligated population of ∼60% at the basic pH limit in the modified holoprotein. The switching rate, which is slow on the chemical shift time scale, was estimated to be 20-30 s-1 by NMR exchange spectroscopy. Lys42-His46 competition and attendant conformational rearrangement appeared to be related to weakened bis-histidine ligation and enhanced backbone dynamics in the ferrous protein. The pH- and redox-dependent ligand exchange process observed in GlbN illustrates the structural plasticity allowed by the TrHb1 fold and demonstrates the importance of electrostatic interactions at the heme periphery for achieving axial ligand selection. An analogy is drawn to the alkaline transition of cytochrome c, in which Lys-Met competition is detected at alkaline pH, but, in contrast to GlbN, in the ferric state only.

Covalent attachment of the heme to Synechococcus hemoglobin alters its reactivity toward nitric oxide.

The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. GlbN contains a b heme, which can be modified under certain reducing conditions. The modified protein (GlbN-A) has one heme-histidine C-N linkage similar to the C-S linkage of cytochrome c. No clear functional role has been assigned to this modification. Here, optical absorbance and NMR spectroscopies were used to compare the reactivity of GlbN and GlbN-A toward nitric oxide (NO). Both forms of the protein are capable of NO dioxygenase activity and both undergo heme bleaching after multiple NO challenges. GlbN and GlbN-A bind NO in the ferric state and form diamagnetic complexes (FeIII-NO) that resist reductive nitrosylation to the paramagnetic FeII-NO forms. Dithionite reduction of FeIII-NO GlbN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected FeII-NO complex, NO binding to FeII GlbN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining FeIII GlbN, 15N-labeled nitrite, and excess dithionite resulted in the formation of FeII-H15NO GlbN. Dithionite-mediated HNO production was also observed for the related GlbN from Synechocystis sp. PCC 6803. Although ferrous GlbN-A appeared capable of trapping preformed HNO, the histidine-heme post-translational modification extinguished the NO reduction chemistry associated with GlbN. Overall, the results suggest a role for the covalent modification in FeII GlbNs: protection from NO-mediated heme loss and prevention of HNO formation.

Characterization of THB1, a Chlamydomonas reinhardtii truncated hemoglobin: linkage to nitrogen metabolism and identification of lysine as the distal heme ligand.

The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(•)) in either the ferric or ferrous state and has efficient NO(•) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.

Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection.

We describe the success of adjunctive bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection in the context of bilateral ureteric stents and bladder ulceration, after repeated failure of antibiotics alone. No bacteriophage-resistant bacteria arose, and the kinetics of bacteriophage and bacteria in urine suggest self-sustaining and self-limiting infection.

The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women.

BACKGROUND: The problems of adherence to energy restriction in humans are well known. OBJECTIVE: To compare the feasibility and effectiveness of intermittent continuous energy (IER) with continuous energy restriction (CER) for weight loss, insulin sensitivity and other metabolic disease risk markers. DESIGN: Randomized comparison of a 25% energy restriction as IER (∼ 2710 kJ/day for 2 days/week) or CER (∼ 6276 kJ/day for 7 days/week) in 107 overweight or obese (mean (± s.d.) body mass index 30.6 (± 5.1) kg m(-2)) premenopausal women observed over a period of 6 months. Weight, anthropometry, biomarkers for breast cancer, diabetes, cardiovascular disease and dementia risk; insulin resistance (HOMA), oxidative stress markers, leptin, adiponectin, insulin-like growth factor (IGF)-1 and IGF binding proteins 1 and 2, androgens, prolactin, inflammatory markers (high sensitivity C-reactive protein and sialic acid), lipids, blood pressure and brain-derived neurotrophic factor were assessed at baseline and after 1, 3 and 6 months. RESULTS: Last observation carried forward analysis showed that IER and CER are equally effective for weight loss: mean (95% confidence interval ) weight change for IER was -6.4 (-7.9 to -4.8) kg vs -5.6 (-6.9 to -4.4) kg for CER (P-value for difference between groups = 0.4). Both groups experienced comparable reductions in leptin, free androgen index, high-sensitivity C-reactive protein, total and LDL cholesterol, triglycerides, blood pressure and increases in sex hormone binding globulin, IGF binding proteins 1 and 2. Reductions in fasting insulin and insulin resistance were modest in both groups, but greater with IER than with CER; difference between groups for fasting insulin was -1.2 (-1.4 to -1.0) µU ml(-1) and for insulin resistance was -1.2 (-1.5 to -1.0) µU mmol(-1) l(-1) (both P = 0.04). CONCLUSION: IER is as effective as CER with regard to weight loss, insulin sensitivity and other health biomarkers, and may be offered as an alternative equivalent to CER for weight loss and reducing disease risk.

Comparison of blood pressure measured at the arm, ankle and calf.

SUMMARY: The suitability of alternative sites for non-invasive blood pressure (NIBP) measurement was investigated in 100 awake healthy volunteers. The calf and the ankle were chosen for comparison with the arm, and the results analysed subjected to Bland-Altman analysis. Discomfort was graded using a Visual Analogue Scale. There was a poor agreement between the different sites with respect to systolic blood pressure: the agreement was closer for diastolic and mean measurements. The mean blood pressure calf measurement was on average 4 mmHg (95% limits of agreement -12 to 20), higher than the arm. The ankle was 8 mmHg higher (-8 to 24) than the arm. ANOVA demonstrated a statistically significant difference in the discomfort scores between the sites (p < 0.001). The calf demonstrated the highest discomfort score and the ankle the lowest. We suggest that the ankle should be considered in preference to the calf as an alternative site for NIBP measurement if use of an arm is undesirable or impossible.

Penile size and penile enlargement surgery: a review.

Penile size is a considerable concern for men of all ages. Herein, we review the data on penile size and conditions that will result in penile shortening. Penile augmentation procedures are discussed, including indications, procedures and complications of penile lengthening procedures, penile girth enhancement procedures and penile skin reconstruction.

Relationship of tibial speed of sound and lower limb length to nutrient intake in preterm infants.

BACKGROUND: Metabolic bone disease of prematurity is characterised by impaired postnatal mineralisation of the rapidly growing infant skeleton. OBJECTIVE: To longitudinally evaluate postnatal changes in tibial speed of sound (tSOS; which reflects cortical thickness and bone mineral density) and lower limb length (LLL; a measure of tibial growth) in very low birthweight preterm infants receiving contemporary neonatal care. METHODS: tSOS and LLL were measured using a quantitative ultrasound device and an electronic neonatal knemometer, respectively, in the same limb, weekly, for a median period of four weeks (3-16 weeks) in 84 preterm infants (median gestation 26.8 weeks (range 23-35.2 weeks) and median birth weight 869.5 g (range 418-1481 g)). RESULTS: Initial tSOS and LLL were correlated with gestation (r = 0.42, p<0.001; r = 0.76, p<0.001, respectively) and birth weight (r = 0.23, p = 0.038; r = 0.93, p<0.001, respectively). Postnatally, tSOS decreased (r = -0.15, p = 0.011) whereas LLL increased (r = 0.96, p<0.001) with age. The rate of postnatal change in LLL, but not in tSOS, was positively influenced by intake of calcium (p = 0.03), phosphorus (p = 0.01) and vitamin D (p = 0.03). CONCLUSIONS: The postnatal decline in tSOS, which is probably due to cortical thinning secondary to endocortical bone loss, and increase in LLL provide new insight into the development of long bones in preterm infants.

The role of preoperative templating in total knee arthroplasty: comparison of three prostheses.

Templating of preoperative radiographs is routinely recommended prior to knee arthroplasty. We performed this study to assess the reproducibility and accuracy of the templates for three commonly used knee implants (PFC, Kinemax, Scorpio). Six lower limb surgeons templated 10 patients for each of the three designs. The inter- and intra-observer reliability and accuracy was calculated. There was marked variation in the reliability of the templating with the tibial insert scoring better than the femoral and the Kinemax being the most reproducible of the three. In general, the intra-observer scores (kappa=0.57-0.81) were better than the inter-observer ones (kappa=0.21-0.60). The Scorpio was the most accurately templated of the three implants, with the percentage correlating with what was actually implanted ranging from 55% to 62% for the femur and 72% to 75% for the tibia, with no templated sizes more than one size different from the actual implant. The other implants ranged from 38% to 42% for the femur and 53% to 58% for the tibia with both having up to 3% more than 1 size difference from the actual implant. We believe that the use of templating in total knee arthroplasty should be interpreted with caution and we urge the development of more accurate prosthesis sizing techniques.

Living with discordance: knowledge, challenges, and prevention strategies of HIV-discordant couples in Uganda.

Prevalence of HIV-discordance among couples in sub-Saharan Africa is high. Negative partners are at high risk of HIV infection but few HIV/AIDS service providers have developed effective counseling messages for HIV-discordant couples. To identify clients' explanations for discordance, challenges, and prevention strategies, 24 in-depth interviews and 4 focus group discussions were conducted with 32 female and 35 male members of HIV-discordant couples who sought HIV voluntary counseling and testing (VCT) in Uganda. In addition, counselor explanations for discordance were gathered from 62 counselor trainers during 3 interactive workshops. Misconceptions about discordance were widespread among clients and counselors. Common explanations included: the concept of a hidden infection not detectable by HIV tests, belief in immunity, the thought that gentle sex protected HIV-negative partners, and belief in protection by God. Such explanations for discordance reinforced denial of HIV risk for the negative partner within discordant couples and potentially increased transmission risk. Couples identified negotiation of sexual relations as their most formidable challenge. Prevention strategies included condom use, abstinence and separation of beds, contractual agreements for outside sexual partners, and relationship cessation. Discordant couples represent a critical risk group and improved counseling protocols that clearly explain discordance, emphasize high risk of transmission, and support risk reduction are urgently needed.

Multiplex PCR for screening of integrons in bacterial lysates.

Bacterial integrons are a useful PCR amplification target in epidemiological surveys of bacterial antibiotic resistance, and a variety of primers have been published. We describe multiplex PCR methodology to test for classes 1, 2 and 3 integron-associated integrases in boiled lysates of Gram-negative bacteria. We report on performance in Acinetobacter spp. (n=50), Enterobacteriaceae (n=76), Pseudomonas aeruginosa (n=15), Bacteroidesspp. (n=69), and in undifferentiated mixed cultures derived from perineal swabs (n=50) and endotracheal aspirates (n=8). This method achieved 100% sensitivity and specificity in simple lysates made from a range of bacteria, without requiring DNA extraction, and is recommended as an efficient screening tool for surveys of integron cassettes.

Limited diversity among human isolates of Bartonella henselae.

A study of 59 isolates of Bartonella henselae reveals relatively limited diversity among those of human origin (n = 28). Either of two distinct alleles of both gltA and 16S ribosomal DNA (rDNA) was found in all isolates, with a high level of congruity between 16S and gltA inheritance among proven human pathogens. Human isolates from all over Eastern Australia were most commonly 16S rDNA (Bergmans) type I, with the same gltA allele as the type strain (Houston-1). Comparable feline isolates were more commonly 16S type II, with less congruity of inheritance between 16S and gltA alleles. Previously described arbitrarily primed PCR and EagI-HhaI infrequent restriction site PCR fingerprinting techniques separated Bartonella species effectively but lacked discriminating power within B. henselae. Examination of the 16-23S intergenic spacer region revealed for several strains several point mutations as well as a repeat sequence of unknown significance which is readily detected by HaeIII restriction fragment length polymorphism analysis. The bacteriophage-associated papA gene was present in all isolates. Enterobacterial repetitive intergenic consensus PCR proved to be a useful and robust typing tool and clearly separated human isolates (including imported strains) from the majority of feline isolates. Our data are consistent with published evidence and with previous suggestions of intragenomic rearrangements in the type strain and suggest that human isolates come from a limited subset of B. henselae strains. They strengthen arguments for careful exploration of genotype-phenotype relationships and for the development of a multilocus enzyme electrophoresis and multilocus sequence typing-based approach to the phylogeny of B. henselae.