Short-read whole genome sequencing for determination of antimicrobial resistance mechanisms and capsular serotypes of current invasive Streptococcus agalactiae recovered in the USA.

OBJECTIVES: Our objective was to evaluate and exploit a whole genome sequence (WGS) bioinformatics pipeline for predicting antimicrobial resistance and capsular serotypes from invasive group B streptococci (iGBS). METHODS: For 1975 iGBS recovered during 2015 from CDC's Active Bacterial Core surveillance, we compared pipeline predictions with broth dilution testing. Fifty-six isolates from earlier surveillance were included for testing ß-lactams. Conventional serotyping was compared to WGS-based assignments for 302 isolates. RESULTS: All 28 isolates with reduced susceptibility to ß-lactam antibiotics harboured one of 19 rare PBP2x types. Resistances to erythromycin/clindamycin (808/1975 isolates, 41.0%), erythromycin (235/1975, 11.9%) and lincosamide/streptogramin A/pleuromutilins (56/1975, 2.8%) were predicted by the presence of erm-methylase, mef and lsa determinants, respectively (41 of 56 lsa gene-positive isolates also contained lnu, erm and/or mef genes). Presence of both erm and lsa determinants (25 isolates) predicted non-susceptibility to quinupristin/dalfopristin. Most isolates (1680/1975, 85.1%) were tet gene-positive, although 41/1565 (2.6%) tetM-positive isolates were tetracycline-susceptible. All 53 fluoroquinolone-resistant isolates contained ParC and/or GyrA substitutions. Resistances to rifampin (eight isolates), trimethoprim, chloramphenicol and vancomycin (two isolates each) were predicted by the pipeline. Resistance to macrolides/lincosamides without pipeline prediction was rare and correlated to divergent resistance genes or rRNA A2062G substitution. A selection of 267 isolates assigned WGS-based serotypes were also conventionally serotyped. Of these, 246 (92.1%) were in agreement, with the remaining 21 (7.8%) conventionally non-serotypeable. For 32 of 1975 isolates (1.6%), WGS-based serotypes could not be assigned. CONCLUSION: The WGS-based assignment of iGBS resistance features and serotypes is an accurate substitute for phenotypic testing.

Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States.

Our whole genome sequence (WGS) pipeline was assessed for accurate prediction of antimicrobial phenotypes. For 2316 invasive pneumococcal isolates recovered during 2015 we compared WGS pipeline data to broth dilution testing (BDT) for 18 antimicrobials. For 11 antimicrobials categorical discrepancies were assigned when WGS-predicted MICs and BDT MICs predicted different categorizations for susceptibility, intermediate resistance or resistance, ranging from 0.9% (tetracycline) to 2.9% (amoxicillin). For ß-lactam antibiotics, the occurrence of at least four-fold differences in MIC ranged from 0.2% (meropenem) to 1.0% (penicillin), although phenotypic retesting resolved 25%-78% of these discrepancies. Non-susceptibility to penicillin, predicted by penicillin-binding protein types, was 2.7% (non-meningitis criteria) and 23.8% (meningitis criteria). Other common resistance determinants included mef (475 isolates), ermB (191 isolates), ermB + mef (48 isolates), tetM (261 isolates) and cat (51 isolates). Additional accessory resistance genes (tetS, tet32, aphA-3, sat4) were rarely detected (one to three isolates). Rare core genome mutations conferring erythromycin-resistance included a two-codon rplD insertion (rplD69-KG-70) and the 23S rRNA A2061G substitution (six isolates). Intermediate cotrimoxazole-resistance was associated with one or two codon insertions within folP (238 isolates) or the folA I100L substitution (38 isolates), whereas full cotrimoxazole-resistance was attributed to alterations in both genes (172 isolates). The two levofloxacin-resistant isolates contained parC and/or gyrA mutations. Of 11 remaining isolates with moderately elevated MICs to both ciprofloxacin and levofloxacin, seven contained parC or gyrA mutations. The two rifampin-resistant isolates contained rpoB mutations. WGS-based antimicrobial phenotype prediction was an informative alternative to BDT for invasive pneumococci.

Strain features and distributions in pneumococci from children with invasive disease before and after 13-valent conjugate vaccine implementation in the USA.

The effect of second-generation pneumococcal conjugate vaccines on invasive pneumococcal disease (IPD) strain distributions have not yet been well described. We analysed IPD isolates recovered from children aged <5 years through Active Bacterial Core surveillance before (2008-2009; n = 828) and after (2011-2013; n = 600) 13-valent pneumococcal conjugate vaccine (PCV13) implementation. We employed conventional testing, PCR/electrospray ionization mass spectrometry and whole genome sequence (WGS) analysis to identify serotypes, resistance features, genotypes, and pilus types. PCV13, licensed in February 2010, effectively targeted all major 19A and 7F genotypes, and decreased antimicrobial resistance, primarily owing to removal of the 19A/ST320 complex. The strain complex contributing most to the remaining ß-lactam resistance during 2011-2013 was 35B/ST558. Significant emergence of non-vaccine clonal complexes was not evident. Because of the removal of vaccine serotype strains, positivity for one or both pilus types (PI-1 and PI-2) decreased in the post-PCV13 years 2011-2013 relative to 2008-2009 (decreases of 32-55% for PI-1, and >95% for PI-2 and combined PI-1 + PI-2). ß-Lactam susceptibility phenotypes correlated consistently with transpeptidase region sequence combinations of the three major penicillin-binding proteins (PBPs) determined through WGS analysis. Other major resistance features were predictable by DNA signatures from WGS analysis. Multilocus sequence data combined with PBP combinations identified progeny, serotype donors and recipient strains in serotype switch events. PCV13 decreased the frequency of all PCV13 serotype clones and concurrently decreased the frequency of strain subsets with resistance and/or adherence features conducive to successful carriage. Our results serve as a reference describing key features of current paediatric IPD strains in the USA after PCV13 implementation.

Haemopoietic stem cell transplantation in Australia and New Zealand, 1992-2001: progress report from the Australasian Bone Marrow Transplant Recipient Registry.

BACKGROUND: Bone marrow and blood stem cell transplantation is now used as curative therapy for a range of haematological malignancies and other conditions. The Australasian Bone Marrow Transplant Recipient Registry (ABMTRR) has recorded transplant activity in Australia since 1992; transplant centres in New Zealand have corresponded with the Registry since 1998. AIM: To describe allogeneic and autologous bone marrow and blood stem cell transplantation activity and outcomes in Australia and New Zealand from 1992 to 2001. METHODS: Each haemopoietic stem cell transplant centre in Australia and New Zealand contributes information to the Registry via a single information form compiled when a transplant is performed. An annual follow-up request is then sent from the Registry to the contributing centre at the anniversary of each individual transplant. RESULTS: Haemopoietic stem cell transplants in Australia have increased in number from 478 in 1992 to 937 in 2001, whereas in New Zealand the number has grown from 91 in 1998 to 105 in 2001, mainly as a result of an increase in autologous blood stem cell transplants. The number of hospitals contributing to the ABMTRR has grown from 20 in 1992 to 37 in 2001. The most common indication for autologous transplantation in 2001 was non-Hodgkin's lymphoma, whereas for allogeneic transplants it was acute myeloid leukaemia. The 9-year actuarial disease-free survival probability for patients aged 16 and above between 1992 and 2000 was 37% for autologous, 39% for allogeneic related donor and 30% for allogeneic unrelated donor transplants. Recurrence of the underlying disease was the main cause of death post-transplant after both allogeneic (26.3% of deaths in the first year and 68.0% of deaths in the second year) and autologous transplants (59.0% and 86.2%). Treatment-related mortality was 16.9% after allogeneic transplantation and 2.1% after autologous transplantation in 2000. CONCLUSIONS: The ABMTRR provides a comprehensive source of information on the use of bone marrow transplant, and allows for continuing analysis of changes in the application of this high-cost technology and the outcome of patients undergoing these procedures. Registry data provide a means for directing future clinical research into perceived areas of priority for improvement of outcome, such as the reduction in the risk of disease recurrence post-transplant.

Assessment of replication and virulence of attenuated pseudorabies virus in swine.

A nonclinical study was conducted to characterize the replication behavior of a modified live gE-deleted pseudorabies virus (PRV MS+1) in swine and potential for reversion to virulence after animal passages. Two to 3 week-old weaned pigs, negative for PRV, were maintained in isolation and challenged by intranasal instillation. For the first passage, 6 pigs were given 1 mL of PRV MS+1 (10(7.3)TCID(50)/mL) and 2 were necropsied at 3, 4 and 5 days post-inoculation (PI). Brain and secondary lymphoid tissues were collected, homogenized and the supernatants individually pooled for virus isolation, and PRV was recovered from each sample. No clinical signs of PRV infection were observed, but each pig had a nasal swab suspect or positive for PRV. For the second passage, 5 pigs were given 1 mL of the homogenate of mixed tissues from 1 animal in the previous passage (PRV at 10(1.9) TCID(50)/mL). At 5 days PI, all pigs were necropsied, and PRV was not recovered from their tissue homogenates or nasal swabs, and no clinical signs were observed. During a second attempt at a second passage, tissue homogenates from all pigs in the first passage (PRV at approximately 10(1.7)TCID50(50)/mL) were pooled and used to inoculate 15 pigs with 2 mL for 3 consecutive days. Ten pigs were monitored for clinical signs and seroconversion through 21 days PI, and 5 pigs were necropsied at 5 days PI. No clinical signs or PRV antibodies were detected in the 10 monitored pigs, and no PRV was recovered from the homogenates or nasal swabs of the 5 necropsied pigs. Thus, no evidence of reversion to virulence was demonstrated in pigs given the attenuated PRV.

Evaluation of local and systemic immune responses induced by intramuscular injection of a Mycoplasma hyopneumoniae bacterin to pigs.

OBJECTIVE: To evaluate immune responses induced by administration of Mycoplasma hyopneumoniae bacterin to pigs. Animals-60 healthy 7- to 10-day-old cross-bred boars. PROCEDURE: Pigs were assigned to 1 of 4 pig groups (15 pigs/group): vaccinated, challenged; vaccinated, nonchallenged; nonvaccinated, challenged; nonvaccinated, nonchallenged. Vaccinated pigs received IM injections of a mycoplasma bacterin on days 0 and 14, whereas nonvaccinated pigs received saline (0.9% NaCl) solution. Pigs in the challenged groups were inoculated intratracheally with M hyopneumoniae on day 42. Pigs were euthanatized and necropsied 41, 44, 48, and 70 days after the first vaccination, and proportion of lung surface with pneumonic lesions was determined. Percentage of lymphocyte subpopulations and number of interferon-gamma (IFN-gamma) secreting lymphocytes in blood and tissues, cytokine and antibody concentrations in bronchoalveolar lavage (BAL) fluid, and serum antibody concentrations were determined. RESULTS: Vaccination against and infection with M hyopneumoniae induced a local mucosal immune response in the respiratory tract of pigs. Proportion of lung surface with pneumonic lesions in vaccinated challenged pigs was reduced on day 70, compared with nonvaccinated challenged pigs. Vaccination stimulated the production of M hyopneumoniae-specific IFN-gamma secreting blood lymphocytes. Tumor necrosis factor-alpha concentration in BAL fluid on day 70 was increased in nonvaccinated challenged pigs, compared with vaccinated challenged pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination against M hyopneumoniae induced local, mucosal, humoral, and cellular immune responses. Moreover, vaccination reduced the severity of lung lesions in challenged pigs, suggesting that mucosal antibodies, mediation of the inflammatory response, and cell-mediated immune responses are important for control of mycoplasmal pneumonia in pigs.

Estimation of the rate of oxygen consumption of the common eider duck (Somateria mollissima), with some measurements of heart rate during voluntary dives.

The relationship between heart rate (f(H)) and rate of oxygen consumption (V(O2)) was established for a marine diving bird, the common eider duck (Somateria mollissima), during steady-state swimming and running exercise. Both variables increased exponentially with speed during swimming and in a linear fashion during running. Eleven linear regressions of V(O2) (ml kg(-1 )min(-1)) on f(H) (beats min(-1)) were obtained: five by swimming and six by running the birds. The common regression was described by V(O2)=10.1 + 0.15f(H) (r(2)=0.46, N=272, P<0.0001). The accuracy of this relationship for predicting mean V(O2) was determined for a group of six birds by recording f(H) continuously over a 2-day period and comparing estimated V(O2) obtained using the common regression with (i) V(O2) estimated using the doubly labelled water technique (DLW) and (ii) V(O2) measured using respirometry. A two-pool model produced the most accurate estimated V(O2) using DLW. Because of individual variability within mean values of V(O2) estimated using both techniques, there was no significant difference between mean V(O2) estimated using f(H) or DLW and measured V(O2) values (P>0.2), although individual errors were substantially less when f(H) was used rather than DLW to estimate V(O2). Both techniques are, however, only suitable for estimating mean V(O2) for a group of animals, not for individuals. Heart rate and behaviour were monitored during a bout of 63 voluntary dives by one female bird in an indoor tank 1.7 m deep. Tachycardia occurred both in anticipation of and following each dive. Heart rate decreased before submersion but was above resting values for the whole of the dive cycle. Mean f(H) at mean dive duration was significantly greater than f(H) while swimming at maximum sustainable surface speeds. Heart rate was used to estimate mean V(O2) during the dive cycle and to predict aerobic dive limit (ADL) for shallow dives.

Diurnal rhythm returns to normal after elimination of portacaval shunting.

Previous studies showed that portacaval shunting causes metabolic and behavioral changes in rats. Most metabolic changes reversed within 1-2 wk after restoration of normal circulation. However, the rate of cerebral glucose metabolism (CMRGlc) remained depressed in some areas. The question arose whether complete recovery was possible. Therefore, a long-term behavioral study was undertaken to determine the time course of recovery. Diurnal activity was monitored for 48 h each week over a period of 14 wk: 2 wk before shunting, 6 wk after shunting, and 6 wk after restoration of normal hepatic circulation. Nighttime activity was depressed within 1 wk of shunting and did not change. Normal circulation to the liver was reestablished after 6 wk. The diurnal cycle was normal 3 wk later. Thus, although recovery of the diurnal rhythm is possible, the relatively long period necessary suggests the correction of a significant structural or chemical abnormality. A study of CMRGlc was made using the behavioral study as an index of the time necessary for recovery. CMRGlc returned to normal throughout the brain 6 wk after cessation of shunting except in the hippocampus and amygdala (7-8% decrease).

Penetration of glutamate into brain of 7-day-old rats.

The permeability of the blood-brain barrier to glutamate was measured by quantitative autoradiography in brains of 7-day-old rats (average plasma glutamate 114 microM) and rats injected subcutaneously with glutamate (average plasma glutamate 2,670 microM). Measurements of glutamate permeability were initiated by the injection of [14C]glutamate into the inferior vena cava and the 7-day-old rats sacrificed at 1 minute to avoid the accumulation of [14C]glutamate metabolites in plasma. Glutamate entered the brain at a slow rate, with an average permeability-surface area product of 12 microl x min(-1) x g(-1), except in those areas known to have fenestrated capillaries. Thus, glutamate readily entered and accumulated in circumventricular organs where the radioactivity was localized. Although three areas with a blood-brain barrier, the cerebral cortex, the hypothalamus and the midbrain, of 7-day-old rats had permeabilities similar to adult rats, the other areas of the brain with a blood-brain barrier had a permeability about 1.5-1.9 times that of adult rats. The greater permeability of the brain of 7-day-old rats may reflect the degree of immaturity of the blood-brain barrier.

Reversal of portacaval shunting normalizes brain energy consumption in most brain structures.

The cerebral metabolic rate of glucose consumption (CMRGlc) was measured throughout brains of rats with 1) portacaval shunts created for 2 wk, followed by restoration of normal blood circulation for 2 wk; 2) portacaval shunts created for 2 wk, followed by a sham operation and 2 wk of recovery; 3) two sham operations, each with 2 wk of recovery times. Portacaval-shunted rats had diminished CMRGlc (decreases of 7-23%) throughout the brain in agreement with previous studies. After restoration of normal liver blood flow, the CMRGlc of most structures returned to near-normal values, although a few structures, notably the hippocampus, remained 11-13% lower. These data suggest that the consequences of portacaval shunting to brain energy metabolism can be markedly improved, if not completely reversed, by restoring the normal pattern of blood flow to an otherwise healthy liver. Other metabolic and physical data collected (liver weight, liver-to-body weight, plasma ammonia) returned to normal except plasma glucose concentrations, which remained lower by 11%, suggesting a persistent, albeit mild, defect in glucose homeostasis.

Eliminating metabolic abnormalities of portacaval shunting by restoring normal liver blood flow.

Portacaval shunting causes a variety of anatomic, metabolic, and physiological changes. However, it has not been determined whether, and to what degree, these changes are permanent after a sustained period of shunting. We prepared three groups of rats for study of the recovery process. One group had side-to-side shunts for 3 wk, one group had side-to-side shunts for 2 wk followed by the restoration of normal liver circulation for 1 wk, and one group (control) had sham operations. Side-to-side shunting causes liver atrophy, increased plasma ammonia, altered plasma and brain amino acid spectra, decreased plasma glucose, and increased transport of neutral amino acids across the blood-brain barrier. After restoration of the normal pattern of liver circulation by shunt repair, the liver regained its normal size within 1 day. All abnormalities associated with liver dysfunction disappeared with the exception of plasma glucose, which remained approximately 15% lower than control values.

Comparison of the metabolic disturbances caused by end-to-side and side-to-side portacaval shunts.

Portacaval shunting causes liver atrophy, hyperammonemia, and hepatic encephalopathy. A fundamental question is whether the changes, especially those to brain, are permanent. To answer this, it is necessary to have a model whereby a portacaval shunt can be created for a period of time and then the normal pattern of circulation to the liver restored at will. An end-to-side shunt, the most extensively studied model of liver dysfunction, is permanent. However, a side-to-side shunt can be constructed that results in a somewhat different pattern of circulation but with the potential to be reversed. The purpose of the present study was to compare the severity of the metabolic disturbances caused by the two models. Rats with an end-to-side shunt, a side-to-side shunt, or sham operation were prepared and studied after 14-19 days. Both models of shunting caused the same degree of liver atrophy, hyperammonemia, and indistinguishable disturbances in the amino acid content of plasma and brain. Furthermore, both models produced the same degree of cerebral depression as measured by glucose consumption.

Regional brain glutamate transport in rats at normal and raised concentrations of circulating glutamate.

The permeability of the blood-brain barrier to glutamate was measured by quantitative autoradiography in brains of control rats (average plasma glutamate concentration of 95 microns) and rats infused with glutamate (average plasma glutamate concentration of 837 microns). Measurements of glutamate permeability were initiated by the injection of [14C]glutamate and stopped at 1 min to avoid the accumulation of [14C]glutamate metabolites. Glutamate entered the brain at a slow rate, with an average permeability-surface area product of 7 microliters.min-g-1, except in those areas known to have fenestrated capillaries. Glutamate accumulated in the choroid plexus of ventricles, but did not seem to enter the cerebrospinal fluid in detectable amounts regardless of the circulating concentration. Glutamate accumulated in circumventricular organs, such as the median eminence, where the radioactivity was localized without detectable spread. Infusion of glutamate to create high plasma concentrations did not result in greater spread of [14C]glutamate beyond the immediate vicinity of the circumventricular organs.

Optimizing the measurement of regional cerebral glucose consumption with [6-14C]glucose.

[6-14C]Glucose is used to trace the cerebral metabolic rate of glucose (CMRGlc) in vivo in experiments lasting 5-10 min. Initially 14C is trapped in intermediary metabolite pools. Subsequently 14C is lost as a function of time and metabolic rate, primarily as 14CO2. Experiments were designed to evaluate the rate of 14C lost as 14CO2 or as [14C]lactate from brain labeled with [6-14C]glucose during times up to 15 min. CMRGlc was measured during 5, 7.5, 10 and 15 min in 60 brain areas. At longer times the loss of 14C was reflected by lower apparent values of brain CMRGlc. Arteriovenous measurements across brain revealed no significant loss of [14C]lactate in normal rats or rats with bicuculline-induced seizures. It was concluded that the primary form in which 14C was lost was as 14CO2. As expected, the rate of 14CO2 loss was greater in structures with high metabolic rates. The data were analyzed to determine the parameters necessary to rectify the data so that uniform values of CMRGlc were obtained up to 15 min. Tables were made to predict the degree of 14C loss as well as the 14C-metabolites/[6-14C]glucose ratio as a function of time and metabolic rate. These tables can be used to plan the maximum and minimum experimental times for optimal results.

In vivo and in vitro evaluation of the alkylating agent carmethizole.

Carmethizole, a novel bis-carbamate alkylating agent, was evaluated in vitro for potential mechanisms of interaction with DNA and in vivo for spectrum and degree of antitumor activity. In vitro, the concentration of carmethizole required to produce a 50% reduction in clonogenic cell survival was identical in O6-alkylguanine DNA alkyltransferase-positive and -negative human cell lines. The CHO cell line UV4, hypersensitive to mono- and bifunctional alkylating agents, was 37-fold more sensitive to carmethizole than normal cells. The UV5 cell line, which is not hypersensitive to cross-linkers, was 13-fold more sensitive to carmethizole than normal cells. Alkaline elution studies in L1210 cells exposed to carmethizole showed the presence of DNA-protein and DNA-DNA cross-links but not DNA strand breaks. These data suggested that the interaction of carmethizole with DNA produces monoadducts, DNA-protein, and DNA-DNA interstrand cross-links at several sites. In vivo, carmethizole was not cross-resistant with 1,3-bis(2-chloroethyl)-1-nitrosourea or Cytoxan as determined by testing against P388 leukemias resistant to the latter 2 agents. Carmethizole activity was similar to that of melphalan across the murine solid tumor panel, which consisted of B16 melanoma; colon adenocarcinomas 11a, 26, and 36; and the KHT sarcoma. Carmethizole, Cytoxan, and melphalan were all active and had comparable activity against the HCT-8 and MX-1 human tumor xenografts. The in vivo spectrum of activity and efficacy of carmethizole was similar to that of melphalan.