Validation of an ampicillin selection protocol to enrich for mutants of Listeria monocytogenes unable to replicate on fresh produce.

Several outbreaks of listeriosis have implicated fresh produce but genetic factors required for growth of Listeria monocytogenes on produce remain poorly characterized. Based on the fact that ß-lactam antibiotics only kill bacterial cells that are growing, we hypothesized that ampicillin selection can enrich for L. monocytogenes mutants unable to grow on produce. For validation, we examined relative recovery of L. monocytogenes strain 2011L-2858 and its cold-sensitive mutant L1E4 following inoculation of cantaloupe rind fragments with 1:1 mixture of the strains and incubation at 4°C with or without ampicillin. Listeria monocytogenes from rind fragments inoculated with the mixed cultures and incubated in the presence of ampicillin were used to inoculate fresh rind fragments for a second round of enrichment. In the presence of ampicillin, the proportion of L1E4 increased from 55% on day 0 to 78% on day 14, with higher recovery (85% after 14 days) in the second round of enrichment. These data suggested that L1E4 was enriched on cantaloupe rind fragments while growing cells of the wildtype were killed by ampicillin. Application of this protocol to transposon mutant libraries from three L. monocytogenes strains yielded several mutants unable to grow on cantaloupe. Thus, ampicillin selection can facilitate discovery of genes essential for growth of L. monocytogenes on fresh produce.

Endoscopic Islet Autotransplantation Into Gastric Submucosa-1000-Day Follow-up of Patients.

BACKGROUND: Total pancreatectomy and autologous transplantation of pancreatic islets is a treatment option for patients with severe pain due to chronic pancreatitis. In the standard procedure, pancreatic islets are isolated and subsequently administered into the portal vein. In the case of patients with a history of thrombosis or at risk of thrombosis, this route of administration is not viable. Animal studies conducted in our department led to the development of a technique of endoscopic islets transplantation into the gastric submucosa. In 2013 and 2014, the first human autologous transplant procedures were performed. The objective of this study was to present the results of a 3-year follow-up of these patients. METHODS: Two pancreatectomies were performed in our department, the first in 2013 and another in 2014, along with subsequent autologous transplantation of pancreatic islets into the gastric submucosa. RESULTS: Both patients had been diagnosed previously with diabetes, and both had endogenous islet activity detected. Peptide C concentration after pancreatectomy and before pancreatic cell transplantation was 0.1 ng/mL. After the transplantation, peptide C concentrations for the 2 patients were 0.8 and 0.5 ng/mL on day 7, 1.2 and 0.6 ng/mL on day 30, 1.3 and 0.8 ng/mL on day 180, 1.1 and 0.7 ng/mL on day 360, and 3.0 and 0.6 ng/mL at 3 years, respectively, after transplantation. The pain symptoms resolved in both cases. CONCLUSION: Pancreatic islets may survive in the gastric wall. Endoscopic submucosal transplantation may present an alternative for the management of patients who cannot undergo a classic transplantation procedure.

Analysis of Distribution of Expanded- and Standard-Criteria Donors and Complications Among Polish Recipients by Kidney Donor Risk Index Value.

INTRODUCTION: The approach toward transplanting kidneys from expanded-criteria donors (ECDs) in Poland is largely site-dependent. The Kidney Donor Risk Index (KDRI) allows for obtaining a more precise characteristic of ECDs and further stratification into "better" and "worse" quality grafts. METHODS: Comparison of the incidence of delayed graft function (DGF) and biopsy-proven acute rejection (BPAR), median of hospitalization time and median of estimated glomerular filtration rate (eGFR) at 1 year after transplantation among kidney graft recipients (n = 468), divided by donor status (ECD/standard-criteria donor [SCD]) and KDRI value (I: 0.67-1.2, II: 1.21-1.6, III: 1.61-2.0, IV: 2.01-3.48). RESULTS: ECD kidneys have been transplanted to 32.47% of recipients. There were no ECD recipients in KDRI compartment I, 16.55% in compartment II, 79.22% in compartment III, and 100% in IV. In KDRI compartment II, DGF was diagnosed in 34.9% of SCDs and 56% of ECDs (P = .003), BPAR occurred in 7.8% of SCDs and 16% of ECDs (P = .073), median hospital stay was 12 days for SCDs and ECDs (P = 1), and eGFR was 50.7 mL/min for SCDs and 49.4 mL/min for ECDs (P = .734). In KDRI compartment III, DGF was diagnosed in 43.8% of SCDs and 49.2% of ECDs (P = .139), BPAR occurred in 6.3% of SCDs and 31.7% of ECDs (P = .001), median hospital stay was 10 days for SCDs and 12 days for ECDs (P = .634), and eGFR was 49.5 mL/min for SCDs and 45.2 mL/min for ECDs (P = .382). Among ECD recipients, DGF was diagnosed in 56.0%, 49.2%, and 47.7% of patients for KDRI compartments II, III, and IV respectively (P = .776); BPAR occurred in 16% (compartment II), 31.7% (compartment III), and 23.1% (compartment IV) (P = .273); the median hospital stay was 12 days (compartment II), 12 days (compartment III), and 12.5 days (compartment IV) (P = 1); and eGFR was 49.5 mL/min (compartment II), 45.4 mL/min (compartment III), and 36.1 mL/min (compartment IV) (P = .002). CONCLUSION: Assessment using both the ECD and KDRI systems allows for a more precise evaluation of prognosis and predicting complications among recipients.

Islets Allotransplantation Into Gastric Submucosa in a Patient with Portal Hypertension: 4-year Follow-up.

BACKGROUND: Islets transplantation is an established treatment method for patients suffering from brittle diabetes with hypoglycemia unawareness. The standard implantation technique is through the portal vein into the liver. In case of liver diseases or portal hypertension, finding an extra-hepatic site is recommended. There have been attempts to perform islets transplantations into muscles and into the gastric submucosa. OBJECTIVE: The aim of this study is to show a 4-year follow-up of allotransplantation into gastric submucosa in a case of portal hypertension observed during the procedure of islets infusion. PATIENTS AND METHODS: A 36-year-old woman with complicated diabetes for over 30 years was selected to receive simultaneous islets and kidney transplantation. The patient underwent an unsuccessful simultaneous pancreas and kidney transplantation 2 years earlier in another transplantation center. The patient's daily insulin requirement was 60 IU, which corresponded to 1.15 IU/kg of body weight. The HbA1c level was 7.4%. C-peptide levels, both fasting and stimulated, were 0.01 ng/mL. On December 7, 2013, the patient received transplanted kidney and islets procured from the same donor. Only 124,000 islets equivalents (IEQ) were isolated (2400 IEQ/kg body weight). Islets were suspended in 300 mL of Ringer's solution along with albumin, antibiotics, and heparin. After infusing 100 mL of the islets suspension into the portal vein, pressure in portal vein increased from 5 mm Hg to 23 mm Hg. Despite stopping the infusion, pressure did not drop after 30 minutes. The decision was made to transplant the reminder of the islets (200 mL) into the gastric wall. RESULTS: No complications were observed after the procedure. Serum creatinine level was 1.6 mg/dL on day 10 and 1.5 mg/dL 4 years after the transplantation. Fasting C-peptide levels were 1.7, 0.65, 0.55, 0.69, 0.68, and 0.2 ng/mL at 1, 3, 6, 12, 18, and 36 months after the transplantation, respectively. HbA1c levels were 5.2, 6.4, 4.7, 5.2, and 5.9% at 3, 6, 12, 18, and 36 months, respectively. The patient's insulin requirement dropped to 15 U/day immediately after transplantation and equaled 20 and 27 U/day at 18 and 48 months after the simultaneous islet and kidney transplantation, respectively. CONCLUSION: Allotransplantation of islets into the gastric wall may be a safe alternative in cases of contraindications for transplantation into the portal vein.

National Transplants Registry in Poland: Early and Long-term Results of Organ Transplantations in the Years 1998 to 2014.

BACKGROUND: In 2006, the National Transplants Registry, administered by the national transplant organization, was introduced in Poland for monitoring the results of organ transplantations. Statistical analysis is published yearly in Poltransplant Bulletin and publicly available on the website and reported to European institutions. The Transplants Registry cooperates with another registers functioning on-line, based on the web-net tool www.rejestry.net. We present the formal analysis of data collected for the years 1998 to 2014. METHODS: Analysis covered the total number of organ transplantations in every transplant center; outcomes after 3 months and 1, 3, and 5 years; and if data were available after 10, 15, and 20 years from transplantation. Results presented are real, not extrapolated. RESULTS: Some examples are as follows. The total number of deceased kidney transplantations was 15,009; 1-year recipient survival was 95%, graft survival was 88% (data completeness of 81%), 5-year recipient survival was 87%, and graft survival was 74% (data completeness of 82%). The total number of deceased liver transplantations was 3143; 1-year recipient survival was 84%, graft survival was 81% (data completeness of 99%), 5-year recipient survival was 73%, and graft survival was 89% (data completeness of 99%). CONCLUSIONS: The National Transplant Registry is an important tool for quality and safety systems in the transplantation field on the national level. Nowadays, the Registry efficiently and effectively fulfills its tasks related to collecting records of all performed transplantations. Monitoring function for graft and recipient survival is also satisfied. Collected numbers are an important and unique source of information to be used by transplant institutions and referred to in the literature.

Renal Transplantation Using Kidneys Procured From Elderly Donors Older Than 70 Years.

AIM: A major problem for the transplant society is a shortage of organs for transplantation compared with the number of patients on the waiting list. This study aimed to assess the results of the transplantation of kidneys procured from older donors. PATIENTS AND METHODS: A total of 27 kidneys procured from donors age 70 years or older were transplanted between January 1, 2010, and April 25, 2015. These represented only 4.1% of the 657 kidneys transplanted from deceased donors during this period at the same center. RESULTS: Delayed graft function (DGF) in the recipients of kidneys procured from donors age 70 or older occurred in 46.1% of patients, whereas the recipients of kidneys from younger donors showed DGF at a frequency of 32.7% (P = NS). The annual and 3-year survival rates of kidneys in the study group were 85% and 80%, respectively, and in the control group were 92.5% and 88.6%, respectively (P = NS). According to the Polish National Organ Procurement Organization (Poltransplant), the annual survival rate of a transplanted kidney in Poland stands at 89%, whereas the 3-year survival rate is 82%. We detected no significant posttransplantation differences in the serum creatinine concentration and in the estimated glomerular filtration rate between the study and control groups. The donor age and donor creatinine were the variables independently associated with DGF. CONCLUSIONS: The results of transplantation of kidneys from elderly donors were comparable to those of transplantation from younger donors. Kidneys harvested from elderly donors should be used for a transplant after a preliminary assessment.

Rapid Identification and Classification of Listeria spp. and Serotype Assignment of Listeria monocytogenes Using Fourier Transform-Infrared Spectroscopy and Artificial Neural Network Analysis.

The use of Fourier Transform-Infrared Spectroscopy (FT-IR) in conjunction with Artificial Neural Network software NeuroDeveloper™ was examined for the rapid identification and classification of Listeria species and serotyping of Listeria monocytogenes. A spectral library was created for 245 strains of Listeria spp. to give a biochemical fingerprint from which identification of unknown samples were made. This technology was able to accurately distinguish the Listeria species with 99.03% accuracy. Eleven serotypes of Listeria monocytogenes including 1/2a, 1/2b, and 4b were identified with 96.58% accuracy. In addition, motile and non-motile forms of Listeria were used to create a more robust model for identification. FT-IR coupled with NeuroDeveloper™ appear to be a more accurate and economic choice for rapid identification of pathogenic Listeria spp. than current methods.

Clinical implication of intra-amniotic sludge on ultrasound in patients with cervical cerclage.

OBJECTIVE: The aim of this study was to determine whether intra-amniotic (IA) sludge, a sonographic finding of hyperechoic matter in the amniotic fluid close to the internal cervical os, is associated with preterm delivery in patients with cervical cerclage. METHODS: A retrospective cohort study of patients who had undergone McDonald cerclage between January 1997 and December 2004 was conducted. Transvaginal ultrasound examinations had been performed at 14-28 weeks of gestation, and the ultrasound images were assessed by three reviewers (blinded to patient outcome) to determine the presence or absence of IA sludge. The primary outcome studied was the gestational age at delivery. RESULTS: A total of 177 patients who had undergone cervical cerclage, and for whom adequate records were available, were identified. Sixty had sonographic evidence of IA sludge (Group 1) and 117 had absence of IA sludge (Group 2). There was no significant difference in the mean gestational age at delivery between the two groups (36.4 ± 4.0 vs. 36.8 ± 2.9 weeks, P = 0.53), and no statistical difference in the rate of preterm delivery at < 28 (6.7% vs. 1.7%, P = 0.18), < 30 (6.7% vs. 3.4%, P = 0.45), < 32 (8.3% vs. 6.8%, P = 0.77) or < 36 (16.7% vs. 19.7%, P = 0.69) weeks of gestation. CONCLUSION: Intra-amniotic sludge on ultrasound is not associated with an increased risk of preterm delivery in patients with cervical cerclage.

Subtyping Listeria monocytogenes from bulk tank milk using automated repetitive element-based PCR.

Sixty-one Listeria monocytogenes strains from raw milk were analyzed with an automated repetitive element-based PCR (rep-PCR) system to examine the utility of this system for serotype grouping and to determine whether specific regional relationships could be identified. Results of the similarity analysis revealed two primary clusters of L. monocytogenes isolates. Cluster 2 exclusively contained serogroup 1/2a isolates; however, two 1/2a isolates were also found in cluster 1. Isolates of serogroups 1/2b, 4b, 3b, and 4c were also in cluster 1. Clusters 1 and 2 were separated at a relative similarity of 86%. Listeria species other than L. monocytogenes (L. ivanovii, L. seeligeri, L. welshimeri, L. grayi, and L. innocua) had similarity scores of less than 80% in pairwise comparisons with the L. monocytogenes isolates. Thus, this method may be useful for species identification once an isolate is characterized as Listeria. When rep-PCR fingerprints of the L. monocytogenes 1/2a isolates were compared, there was no apparent regional grouping. However, discrimination between isolates suggests that the rep-PCR assay might be useful for tracking L. monocytogenes 1/2a and for tracking isolates across regions or within smaller ecological niches. The automated rep-PCR method could not discriminate between serotypes 1/2b and 4b but may be useful for discriminating between 1/2a and other serotypes and for tracking isolates within serotype 1/2a.

Prevalence of Salmonellae, Listeria monocytogenes, and fecal coliforms in bulk tank milk on US dairies.

The objective of this study was to determine the prevalence of Salmonella, Listeria monocytogenes, and fecal coliforms in bulk tank milk in the United States. As part of the NAHMS Dairy 2002 survey, 861 bulk tank milk samples were collected from farms in 21 states. Milk was directly plated on selective agars for direct bacterial enumeration and was enriched in selective broths to increase detection sensitivity. Somatic cell counts (SCC) and standard plate counts (SPC) were also determined. Coliforms were detected in 95% (818 of 860) of the samples, and the average SCC was 295,000 cells/mL. Twenty-two samples (2.6%) were culture-positive for Salmonella, and 9 serotypes were identified: Montevideo (n = 7), Newport (n = 4), Muenster (n = 2), Meleagridis (n = 2), Cerro (n = 2), 44:Z36 (Z38) (n = 2), Dublin (n = 1), Anatum (n = 1), and 9, 12:nonmo-tile (n = 1). Listeria monocytogenes was isolated from 56 (6.5%) samples, and serotyping of these isolates yielded 5 serotypes (1/2a, 1/2b, 3b, 4b, and 4c). Of the L. monocytogenes isolates, 93% were serotypes 1/2a, 1/2b, and 4b, the most common human clinical isolates. Regional differences in L. monocytogenes and Salmonella prevalence were observed, but more studies are needed to determine the validity of these differences. There were no apparent relationships between SCC or SPC and incidence of Salmonella or L. monocytogenes. Although the prevalence of L. monocytogenes and Salmonella was low, these pathogens represent a potential risk to consumers of raw milk and raw milk products.

Placenta growth factor is not an early marker for the development of severe preeclampsia.

OBJECTIVE: Our purpose was to determine whether plasma concentrations of placenta growth factor may be used as a marker for women who ultimately have severe preeclampsia. STUDY DESIGN: We performed a nested case-control study to compare plasma concentrations of placenta growth factor in women with severe preeclampsia with the concentrations in normotensive pregnant control subjects. Plasma samples were collected at <20 weeks' gestation and again in the third trimester. Twenty-two women who ultimately had severe preeclampsia were matched for gestational age at delivery with 22 normotensive control subjects. Placenta growth factor concentrations were measured by a specific antigen capture enzyme-linked immunosorbent assay. Comparisons were made by using the Mann-Whitney U test for nonparametric data such as placenta growth factor concentrations. The Student t test was used for parametric data. RESULTS: A total of 880 pregnant women were screened. Severe preeclampsia developed in 22, for an incidence of 2.5%. As expected, women with severe preeclampsia had significantly higher systolic and diastolic blood pressures, and their infants had lower birth weights. Placental weights at delivery were similar between those with severe preeclampsia and control subjects (659 vs 699 g; P =.51). During the third trimester, the median placenta growth factor concentrations were significantly lower in women with severe preeclampsia than in normotensive control subjects (125 vs 449 pg/mL; P =.003). When samples drawn at <20 weeks' gestation were compared, there was no difference between the group with severe preeclampsia and those who remained normotensive (98.8 vs 56.34 pg/mL; P =.15). CONCLUSION: During the third trimester, patients with severe preeclampsia have decreased maternal concentrations of placenta growth factor. This difference is not seen earlier in pregnancy. Lower concentrations of placenta growth factor may be a result of severe preeclampsia rather than a causal factor. Placenta growth factor is not a good marker for the subsequent development of severe preeclampsia.

Configuration specificity in bisection acuity.

Crucial for the perception of form are the spatial relationships between the elements of a visual stimulus. To investigate the mechanisms involved in coding the distance between visual stimuli, thresholds for detecting whether a central marker accurately bisects a spatial interval were compared for a variety of configurations. Thresholds are best when all three members of the bisection configuration are identical. Performance is impaired, often by as much as a factor of two, when the outer delimiters of the spatial interval differ from the central marker in either length, orientation or contrast polarity. Illusory contours act poorly as borders for bisection by a central line. Disparity thresholds are not affected by orientation differences between test and flanking lines. Because in peripheral vision bisection acuity improves with practice, transfer of training between configurations can be used to gauge overlap of neural processing mechanisms. Transfer is complete only between patterns where all markers are similar, reduced when the outer markers differ by 20 degrees in orientation and absent when they are orthogonal. The dependence of bisection discrimination on similarity between the elements of the stimulus demonstrates that the encoding of spatial location and spatial extent are coupled to the coding of other stimulus properties.

TPN update: making each visit count.

Caring for patients on TPN is a challenge for the home care nurse. These patients may present with either a short- or long-term need for TPN but in either case, the impact on daily life related to administration and monitoring of this complex infusion therapy is significant for both patients and their caregivers. The risks and complications of TPN can be serious. Nurses must possess expert knowledge of TPN administration and a high level of competence in assessment and monitoring of patients on TPN. Patients and caregivers will learn technical procedures in the home that produce anxiety in graduate nurses! The nurse must also possess skill and confidence in teaching patients to effectively administer, manage, and incorporate TPN infusion therapy into their lives.

A common step for changing cell shape in fruiting body and starvation-independent sporulation of Myxococcus xanthus.

Myxococcus xanthus can sporulate in either of two ways: at the end of the program of fruiting body development or after exposure of growing cells to certain reagents such as concentrated glycerol. Fruiting body sporulation requires starvation, while glycerol sporulation requires rapid growth, and since the two types of spores are structurally somewhat different, it has generally been assumed that the two processes are different. However, a Tn5 Lac insertion mutation, Omega7536, has been isolated which simultaneously blocks the development of fruiting body spores as well as glycerol-induced spores. Both sporulation pathways are blocked in the mutant within the process that converts a rod-shaped cell into a spherical spore. The Omega7536 locus is expressed at the time of cell shape change appropriate to each process, early after glycerol induction and late after starvation induction. On the C-signal response pathway, it is possible to identify positions for the normal function of the Omega7536 locus and for the inducing stimulus from glycerol that are unique and consistent with the observations. Although the two sporulation pathways differ in certain respects, it is shown that they share at least one step for changing a rod-shaped cell into a spherical spore.

A sigma(54) activator protein necessary for spore differentiation within the fruiting body of Myxococcus xanthus.

Insertion of an internal DNA fragment into the act1 gene, which encodes one of several sigma(54)-activator proteins in Myxococcus xanthus, produced a mutant defective in fruiting body development. While fruiting-body aggregation appears normal in the mutant, it fails to sporulate (<10(-6) the wild-type number of viable spores). The A and C intercellular signals, which are required for sporulation, are produced by the mutant. But, while it produces A-factor at levels as high as that of the wild type, the mutant produces much less C-signal than normal, as measured either by C-factor bioassay or by the total amount of C-factor protein detected with specific antibody. Expression of three C-factor-dependent reporters is altered in the mutant: the level of expression of Omega4414 is about 15% of normal, and Omega4459 and Omega4403 have alterations in their time course. Finally, the methylation of FrzCD protein is below normal in the mutant. It is proposed that Act1 protein responds to C-signal reception by increasing the expression of the csgA gene. This C-signal-dependent increase constitutes a positive feedback in the wild type. The act1 mutant, unable to raise the level of csgA expression, carries out only those developmental steps for which a low level of C-signaling is adequate.

A clinical pathway for deep vein thrombosis.

The use of low molecular weight heparin to treat deep vein thrombosis at home represents a relatively new patient population for home care agencies. Use of a clinical pathway provides a framework for defining expected outcomes of care and direction for patient assessment, care, monitoring, and documentation. Implementation and evaluation of a clinical pathway are described.

Targeted mutagenesis of sigma54 activator proteins in Myxococcus xanthus.

Myxococcus xanthus DNA segments related to the highly conserved central sequence of sigma54 activator proteins have been investigated. A genetic technique designed to inactivate a gene that encodes such an activator by inserting a plasmid-borne internal fragment of the putative gene has been tested. When the internal fragment inserted by homologous recombination into the corresponding chromosomal locus, the expected duplication of the gene was observed by Southern hybridization. The single restriction fragment characteristic of each segment was replaced in the insertion strains by two hybridizing fragments, and one of these fragments hybridized with the kanamycin resistance gene of the plasmid vector. The combined molecular weights of the two fragments from the insertion strains were equal to the molecular weight of the original fragment plus the expected molecular weight contributed by the vector. In the duplication, one copy is expected to have an N-terminal deletion and the other copy is expected to have a C-terminal deletion. In most cases, the net result should be loss of activator function. If an activator is essential for vegetative growth, then it should not be possible to obtain the insertion strain by plasmid integration. Indeed, integrants for three of the segments were not obtained in repeated trials; however, a plausible explanation for these results other than lethality can be offered. Of the seven insertions validated by Southern hybridization, four strains exhibited defects in the development of fruiting bodies. One of these failed to develop in submerged culture, though it developed normally on agar. The other three showed arrested development of fruiting bodies, each at a morphologically different stage of aggregation. One of the mutants may be defective in the reception pathway of A-signal.

Managed Care: Effects on Home Health Care Providers and Stroke Survivors.

The advent of managed care has changed the delivery of home health care services for stroke survivors and their families. Because clinicians are providing fewer visits, they need to focus their assessment and diagnoses, interventions, and evaluation steps to offer the most effective, efficient, and economic care possible.