Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.

Neovascularisation is not an innocent bystander in recurrence after great saphenous vein surgery.

INTRODUCTION: This study sought to establish the causes of failure of great saphenous vein surgery among patients in whom there was confidence that accurate saphenofemoral ligation had been carried and the great saphenous vein had been stripped at least to knee level. METHODS: This was an observational study of 100 limbs (66 patients) operated on by a single surgeon 5-22 years previously (mean: 12 years). The index operation was primary in 54 limbs and for recurrence in 46 limbs. Thirty-two patients were studied having been re-referred for recurrence while 34 were recalled for review. All were examined clinically and with duplex ultrasonography, and all completed questionnaires (Aberdeen varicose vein questionnaire [AVVQ] and EQ-5D-3L). In order to measure the extent of visible recurrence, a scoring system similar to that in the venous clinical severity score was used but with a range of 0-8. RESULTS: There were visible varicosities in 77 of the 100 limbs. Duplex ultrasonography showed that recurrent saphenofemoral incompetence (SFI) was present in 27% of the limbs. This was judged to result from neovascularisation and was the most common source of reflux. AVVQ scores for the 27 limbs with recurrent SFI (median: 34, interquartile range [IQR]: 20-42) were higher than for the 73 with no recurrent SFI (median: 17, IQR: 11-29), which was a significant difference (Mann-Whitney U test, p<0.01). Analysing clinical scores with chi-squared tests indicated fewer visible varicosities in limbs without recurrent SFI than in those with recurrent SFI (p<0.01). CONCLUSIONS: Neovascularisation remains poorly understood but it cannot be considered an innocent bystander.

Multiple-locus variable-number tandem-repeat analysis of Francisella tularensis from Quebec, Canada.

UNLABELLED: Francisella tularensis is ubiquitous in the Northern Hemisphere. Yet, little is known about the disease and its ecology within Canada as few serological studies have shown exposure to the disease and fewer case studies have been reported. This report is the first to describe the molecular subtyping of F. tularensis isolates within eastern Canada using multiple-locus variable-number tandem-repeat analysis. From 1998 to 2011, a total of 73 specimens were isolated from unique human and animal sources. As expected, F. tularensis subsp. tularensis AI and F. tularensis subsp. holarctica subtypes were observed, corresponding to the known geographical division within this species. The majority of human isolates (78%) and all animal (hare) isolates were of the more virulent, AI type. Half of the B isolates were isolated from patients living in a region of Quebec where muskrat densities are known to be high. A relatively high level of marker diversity was found, suggestive of multiple introductions of the organism to the region, or more likely ongoing endemicity. There was no evidence of ongoing outbreaks or transmission, and the bulk of cases were likely due to interaction between human activity and the environment (e.g. hunting/trapping activities). SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals the diversity of Francisella tularensis in eastern Canada using multiple-locus variable-number tandem-repeat analysis. It was initiated to further the understanding of the species within North America as previous studies elucidating the diversity and phylogeography of the species have consisted mostly of specimens from the United States. Type A tularaemia, the most life-threatening subtype of the species and a Category A biothreat agent, is restricted to North America, and this study serves to broaden the knowledge of the epidemiology and diversity of the organism.

Two novel toxin variants revealed by whole-genome sequencing of 175 Clostridium botulinum type E strains.

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay.

Development of real-time PCR assays for detection of the Streptococcus milleri group from cystic fibrosis clinical specimens by targeting the cpn60 and 16S rRNA genes.

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.

Treatment of varicose veins: the present and the future--a questionnaire survey.

INTRODUCTION: The treatment options for varicose veins have increased over the last few years. Despite a lack of randomised trials comparing the various modalities, many surgeons are changing their practice. The aim of this study was to assess the current practice of surgeons in Great Britain and Ireland. MATERIALS AND METHODS: A postal questionnaire survey was sent to surgical members of the Vascular Society of Great Britain and Ireland and the Venous Forum of The Royal Society of Medicine. Of 561 questionnaires sent, 349 were returned completed (62%). RESULTS: The types of varicose vein treatments offered by each surgeon varied widely in both NHS and private practice. The vast majority (96%) offered conventional surgery (CS) on the NHS. Foam sclerotherapy (FS) endovenous laser (EVL) and radiofrequency ablation (RF) were more likely to be offered in private practice than in NHS practice. Overall, 38% of respondents for NHS practice and 45% of respondents for private practice offered two or more modalities. Of the respondents who were not yet performing FS, EVL, or RF, 19% were considering or had undertaken training in FS, 26% in EVL and 9% in RF. When asked to consider future practice, 70% surgeons felt that surgery would remain the most commonly used treatment. This was followed by FS (17%), EVL (11%) and RF (2%). CONCLUSIONS: Over one-third of respondents are now offering more than one treatment modality for the treatment of varicose veins. Whilst there is movement towards endovascular treatments, the problem of cost has yet to be solved. At present, surgery remains the most popular modality in both the NHS and private practice; however, improved outcomes and patient preference may lead to a change in practice.

Experience gained by basic surgical trainees in varicose vein surgery over a 15 year period - has it changed?

OBJECTIVE: The aim of this study was to see if the training provided for Basic Surgical Trainees (BST's) by one consultant vascular surgeon has changed over a 15-year period. METHOD: From a computerized database we have a 15-year record of varicose vein operations identifying the first and second surgeon. We have analysed cases involving the BST and those in which the consultant operated alone. In such instances training opportunities were lost. RESULTS: No change was found in the total number of cases performed, the number of operations carried by the BST as the first surgeon, or the cases performed by the consultant operating alone. The number of operations performed by a BST annually correlated positively with the total number of cases. A BST was named as first surgeon in 39% of cases (632/1622). CONCLUSION: This study has shown no definite evidence to support the view that training in varicose vein surgery has deteriorated.

Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease.

Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His(6) tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His(6)-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H(465) with G(465) or A(465), E(377) with A(377) or D(377), or H(380) with P(380) or A(380). Mutagenesis of H(465), E(377), or H(380) resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H(380) was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil alpha-1 proteinase inhibitor, alpha(2)-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.

An extracellular zinc metalloprotease gene of Burkholderia cepacia.

Burkholderia cepacia produces at least one extracellular zinc metalloprotease that may be involved in virulence. A B. cepacia zinc metalloprotease gene was cloned using a Burkholderia pseudomallei zinc metalloprotease gene as a probe. The predicted amino acid sequences of these B. cepacia and a B. pseudomallei extracellular zinc metalloproteases indicate that they are similar to the thermolysin-like family of metalloproteases (M4 family of metalloendopeptidases) and they are likely to be secreted via the general secretory pathway. zmpA isogenic mutants were constructed in B. cepacia genomovar III strains Pc715j and K56-2 by insertional inactivation of the zmpA genes. The zmpA mutants produced less protease than the parent strains. The B. cepacia strain K56-2 zmpA mutant was significantly less virulent than its parent strain in a chronic respiratory infection model; however, there was no difference between the virulence of B. cepacia strain Pc715j and a Pc715j zmpA mutant. The results indicate that this extracellular zinc metalloprotease may play a greater role in virulence in some strains of B. cepacia.

Identification of a siderophore receptor required for ferric ornibactin uptake in Burkholderia cepacia.

Ornibactins are linear hydroxamate siderophores produced by Burkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads. The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms. The orbA precursor was predicted to be a protein with a molecular mass of 81 kDa. An orbA mutant was constructed and demonstrated to be unable to take up (59)Fe-ornibactins or to grow in medium supplemented with ornibactins. Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant. When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron. The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B. cepacia respiratory infections.

Complications of varicose vein surgery.

A retrospective review was carried out of patients who had undergone surgery for varicose veins over an 8 year period between 1985 and 1993. We wished to determine the incidence of various complications so that the risks of surgery could be openly discussed with patients. A total of 973 limbs were operated upon in 599 patients (413 F, 186 M; mean age 49 years). All patients were under the care of a single consultant vascular surgeon who was present at 92% of operations and all patients were reviewed postoperatively. There was no perioperative mortality. Wound complications (haematoma, cellulitis or abscess) occurred in 2.8% of limbs and minor neurological disturbance (numbness or tingling) in 6.6%. Leakage of lymph from the groin occurred in five patients, all of whom had undergone exploration for groin recurrence. Major complications included three cases of deep venous thrombosis (0.5%), one pulmonary embolus, and one foot-drop. There was one major vascular injury, the common femoral vein being damaged in a patient having a third operation on the groin for persistent recurrence. Vein patch repair was performed and patency was maintained. The overall incidence of major complications was 0.8%. Minor complications occurred in 17% of patients. It is unlikely that major complications can be eliminated. In this retrospective review there will be some under-reporting, but we are confident that this is restricted to minor complications.

Reasons to strip the long saphenous vein.

Whether the long saphenous vein (LSV) should be stripped or not remains contentious. Varicography in patients with recurrent varicose veins frequently demonstrates an intact LSV. A series of 41 varicograms have been reviewed to find how often an unstripped LSV is associated with recurrence. Thirty-two patients, 22 female and 10 male, of mean age 49 years (33-78 years) were investigated. Forty-one limbs had previously had high saphenous ligation, yet still had clinical and ultrasound evidence of superficial venous reflux from a source above the knee. The mean interval between first and the last operations was 16 years (2-33 years). An intact LSV in some part of the thigh was present in 22/41 limbs (54%). Midthigh perforator incompetence (MTPI) via the unstripped LSV was present in 14/41 limbs (34%) and MTPI direct from femoral vein to a superficial varix occurred in 6/41 limbs (15%). The commonest problem is a residual communication at the groin found at operation in 80 p. cent of limbs. Accurate saphenofemoral ligation is crucial but we suggest that recurrence from MTPI via the intact LSV, and the possibility of recanalisation from the upper end of the intact LSV to the femoral vein are arguments for continued stripping.

The effect of ligation of the inferior thyroid artery upon thyroid remnant function.

The cause of hypothyroidism after subtotal thyroidectomy for primary thyrotoxicosis is not precisely understood. Activity is not just related to the size of the thyroid remnants, and ischemia of the remnants has been suggested as a factor which might contribute to the development of hypothyroidism. A prospective study has been carried out to look for evidence that ischemia is implicated. In 55 patients undergoing subtotal thyroidectomy for primary thyrotoxicosis, the inferior thyroid artery (ITA) was ligated on one side but preserved on the opposite side. All of the patients had postoperative isotope scans. We predicted that, if ITA ligation produced significant ischemia of the remnant, then the remnant with the intact ITA ought to show greater activity on the postoperative scan. This was not the case. The remnant with the intact ITA was dominant in only 15 of 55 patients, the remnant with the ligated ITA was dominant in the same proportion, 15 of 55 patients, while in the remaining 25 patients, activity was equally distributed between the two remnants. The results of this study provides no evidence to support the contention that nonligation of the inferior thyroid arteries will reduce ischemia of the thyroid remnants and thereby discourage postoperative hypothyroidism.

Isoenzymes of urinary N-acetyl-beta-D-glucosaminidase (NAG) in patients with renal transplants.

Renal transplant recipients were divided into five categories according to their clinical course from transplantation to their discharge from hospital. Total N-acetyl-beta-D-glucosaminidase (NAG) activity in urine was determined using a chromogenic substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The isoenzyme composition of the urine of each patient was determined by semi-automated DEAE-cellulose chromatography. Although raised NAG activity was found in stable transplant patients compared to controls, the level of activity was constant and no change in the isoenzyme profile was found. In reversible rejection there was a marked increase in the intermediate forms, particularly I2 and a concomitant fall in the relative amount of the A-form present but the profile became normal when the patient stabilised. Much more complex patterns were observed in patients who did not respond to treatment. Both the B and I forms were elevated with a fall in the A-form and in one case excretion of the serum As form was observed. The intermediate forms were always increased in rejection.

Bile peritonitis after removal of T-tubes from the common bile duct.

Bile peritonitis can occur when a T-tube is electively removed from the common bile duct, but this is regarded as a rare complication. Plastic T-tubes are known to increase the risk and should not be used. Latex rubber T-tubes are preferred but the complication can still occur. We present three patients with this complication despite the use of a latex T-tube. A questionnaire was sent to 107 surgeons in the South East Thames Region. The replies showed that the complication is far more common than generally realized. Based on these replies the risk of bile peritonitis each time a latex T-tube is electively removed from the common bile duct is calculated to be 0.84 per cent or 1 in every 119 explorations.

The penetration of ceftazidime into peritoneal fluid in patients undergoing elective abdominal surgery.

Using paper discs we have made repeated observations during laparotomy in 31 patients after giving 1 g of ceftazidime intravenously. Samples of serum were obtained simultaneously. Rapid transfer of antibiotic occurred. Peak concentration in peritoneal fluid occurred within 10 minutes of injection (66.7 mg/l, S.E. +/- 10.6), the concurrent mean serum level being 106.0 mg/l (S.E. +/- 10.7). Thereafter levels in serum and peritoneal fluid fell roughly in parallel.

Ruptured abdominal aortic aneurysm treated by ligation of the aorta and an axillobifemoral bypass.

Although a few elderly patients do survive operation for a ruptured aortic aneurysm mortality in patients over 70 years of age is very high. An alternative operation to conventional Dacron replacement of the aorta has been devised. This involves occlusion of the infrarenal aorta and iliac arteries with large plastic clips and insertion of a Dacron axillobifemoral graft. Eight patients with a mean age of 71 years were so treated. Half of them left hospital alive. We consider the results justify further use of this operation in selected patients.

The use of varicography to identify the sources of incompetence in recurrent varicose veins.

We have studied patients with recurrent varicose veins which were incompletely controlled by a thigh tourniquet. We used varicography, (a phlebogram via the varices), to detect sites of incompetence. Thirty patients (mean age 46 years) were investigated, 38 limbs being subjected to varicography and surgery. A primary operation had been performed between 3 months and 30 years earlier. A non-thrombogenic contrast medium, sodium meglumine ioxaglate 320 (Hexabix 320) was used. Metal markers were placed alongside the limb to identify the site of perforating veins on the phlebograms. The principal value of the technique was in the identification of mid-thigh perforator incompetence (MTPI) as we cannot diagnose this accurately by clinical or Doppler-ultrasound examination. Varicography demonstrated MTPI in 15/38 limbs (39%) and in only one thigh was this not confirmed at exploration. Varicography can demonstrate short saphenous incompetence and this was mainly of value in 3 patients who had previously undergone attempted short saphenous ligation; in all 3 the short saphenous vein was present and had not been ligated. The technique was less useful in demonstrating recurrence in the groin. Overall varicography influenced the operation performed in 17/38 limbs (45%), its main value being in the diagnosis of MTPI.