The metabolic impact of extracellular nitrite on aerobic metabolism of Paracoccus denitrificans.

Nitrite, in equilibrium with free nitrous acid (FNA), can inhibit both aerobic and anaerobic growth of microbial communities through bactericidal activities that have considerable potential for control of microbial growth in a range of water systems. There has been much focus on the effect of nitrite/FNA on anaerobic metabolism and so, to enhance understanding of the metabolic impact of nitrite/FNA on aerobic metabolism, a study was undertaken with a model denitrifying bacterium Paracoccus denitrificans PD1222. Extracellular nitrite inhibits aerobic growth of P. denitrificans in a pH-dependent manner that is likely to be a result of both nitrite and free nitrous acid (pKa = 3.25) and subsequent reactive nitrogen oxides generated from the intracellular passage of FNA into P. denitrificans. Increased expression of a gene encoding a flavohemoglobin protein (Fhp) (Pden_1689) was observed in response to extracellular nitrite. Construction and analysis of a deletion mutant established Fhp to be involved in endowing nitrite/FNA resistance at high extracellular nitrite concentrations. Global transcriptional analysis confirmed nitrite-dependent expression of fhp and indicated that P. denitrificans expressed a number of stress response systems associated with protein, DNA and lipid repair. It is therefore suggested that nitrite causes a pH-dependent stress response that is due to the production of associated reactive nitrogen species, such as nitric oxide from the internalisation of FNA.

Mechanisms of Bacterial Extracellular Electron Exchange.

The biochemical mechanisms by which microbes interact with extracellular soluble metal ions and insoluble redox-active minerals have been the focus of intense research over the last three decades. The process presents two challenges to the microorganism. Firstly, electrons have to be transported at the cell surface, which in Gram-negative bacteria presents an additional problem of electron transfer across the ~6nm of the outer membrane. Secondly, the electrons must be transferred to or from the terminal electron acceptors or donors. This review covers the known mechanisms that bacteria use to transport electrons across the cell envelope to external electron donors/acceptors. In Gram-negative bacteria, electron transfer across the outer membrane involves the use of an outer membrane ß-barrel and cytochrome. These can be in the form of a porin-cytochrome protein, such as Cyc2 of Acidithiobacillus ferrooxidans, or a multiprotein porin-cytochrome complex like MtrCAB of Shewanella oneidensis MR-1. For mineral-respiring organisms, there is the additional challenge of transferring the electrons from the cell to mineral surface. For the strict anaerobe Geobacter sulfurreducens this requires electron transfer through conductive pili to associated cytochrome OmcS that directly reduces Fe(III)oxides, while the facultative anaerobe S. oneidensis MR-1 accomplishes mineral reduction through direct membrane contact, contact through filamentous extensions and soluble flavin shuttles, all of which require the outer membrane cytochromes MtrC and OmcA in addition to secreted flavin.

FASTER (Face, Arm, Speech, Time, Emergency Response): experience of Central Coast Stroke Services implementation of a pre-hospital notification system for expedient management of acute stroke.

Despite benefit in acute ischaemic stroke, less than 3% of patients receive tissue plasminogen activator (tPA) in Australia. The FASTER (Face, Arm, Speech, Time, Emergency Response) protocol was constructed to reduce pre-hospital and Emergency Department (ED) delays and improve access to thrombolysis. This study aimed to determine if introduction of the FASTER protocol increases use of tPA using a prospective pre- and post-intervention cohort design in a metropolitan hospital. A pre-hospital assessment tool was used by ambulance services to screen potential tPA candidates. The acute stroke team was contacted, hospital bypass allowed, triage and CT radiology alerted, and the patient rapidly assessed on arrival to ED. Data were collected prospectively during the first 6 months of the new pathway and compared to a 6-month period 12 months prior to protocol initiation. In the 6 months following protocol introduction, 115 patients presented within 24 hours of onset of an ischaemic stroke: 22 (19%) received thrombolysis, significantly greater than five (7%) of 67 patients over the control period, p=0.03. Overall, 42 patients were referred via the FASTER pathway, with 21 of these receiving tPA (50%). One inpatient stroke was also treated. Only two referrals (<5%) were stroke mimics. Introduction of the FASTER pathway also significantly reduced time to thrombolysis and time to admission to the stroke unit. Therefore, fast-track referral of potential tPA patients involving the ambulance services and streamlined hospital assessment is effective and efficient in improving patient access to thrombolysis.

Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans.

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer-monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

Workload and short-term outcome of babies weighing 2,500 grams or more at birth admitted to the paediatric unit of the Rotunda Hospital.

BACKGROUND: Audit is important in ensuring adequate use of resources and maintaining optimum standards of care. Most of the emphasis in neonatal audit is focused on very low birth weight infants. However, term and near-term infants account for a significant proportion of the workload in neonatal units and warrant regular audit. In addition, audit of these infants may be useful as a marker of the organisation of the perinatal service. METHODS: A retrospective audit was performed of all infants with birth weights greater than or equal to 2,500 grams admitted to the neonatal department in the first week of life over a two-year period, examining mode of delivery, level of care, duration of stay, diagnosis and short-term outcome. RESULTS: Eight hundred and seventy infants were admitted greater than or equal to 2,500 grams birth weight, 54% of all neonatal admissions, during the study period. Six hundred and eighty seven of these infants were admitted in the first week of life and were included in the study; this was 5.8% of infants born with a birth weight 2500 grams or more. Infants born by caesarean section were twice as likely to require admission (9.8%) compared with infants born by vaginal delivery (4.5%). The median length of stay was 3 days (3 hours to 45 days). One hundred and six (15.4%) infants required level 1 or level 2 care. One hundred and eleven infants received normal care, only. Most of these infants were admitted for maternal or social reasons. Other common reasons for admission were jaundice, respiratory disease, neonatal abstinence syndrome and congenital abnormality. Forty-one infants required transfer to another hospital, most commonly for surgical or cardiac conditions. Six infants died after admission. However, only one normally formed infant delivered in our hospital died prior to discharge or transfer. One infant was born at home and four infants who had a lethal congenital abnormality are known to have died following transfer. CONCLUSION: Term and near-term infants account for a significant proportion of neonatal admissions and deserve regular audit. Many admissions are potentially avoidable. Survival for infants weighing 2,500 grams or greater is excellent. Only one normally formed infant died following admission during the study period. The number of "social" admissions of "well" infants highlighted by this study reflects poorly on the services available for well infant whose mothers are unable to care for them for whatever reason. We recommend regular audit of these infants in order to ensure efficient use of neonatal resources and to ensure optimum levels of neonatal intensive care.

The value of the perinatal and neonatal autopsy.

The postmortem historically has been considered a valuable diagnostic exercise which contributes to medical knowledge. Despite this, there has been a significant reduction in autopsy rates throughout the developed world. This audit was a retrospective study of autopsy reports of stillbirths and neonates [corrected] greater than 500 grams over a five year period from 1995 to 1999. The audit was performed to assess the impact of autopsy on the current practice of perinatal medicine. The audit compared the final pathological diagnosis to the clinical diagnosis. The pathological diagnosis was categorised as (I) diagnostic, (II) confirmative, (III) unexplained. The recurrence risk estimates as a result of necropsy were also identified, as were any additional findings that were felt to be relevant but did not belong to any of the above categories. A total of 262 perinatal deaths (including stillbirths) and neonatal deaths greater than 500 grams birth weight were recorded during this period. The autopsy rate was 81%. The 213 autopsies performed were assessed, of which 76 (36%) were found to be diagnostic, 108 (51%) confirmatory, and 29 (13%) were unexplained or revealed no new findings. Change in recurrence risk estimates was identified in 24 (11%) and additional relevant information was obtained in 38 (18%). There were a number of cases where an unexpected diagnosis was made as a result of autopsy; these diagnoses included a respiratory chain disorder in a twenty nine week infant, and an occult necrotising enterocolitis presenting with severe haemolysis post transfusion in a preterm infant. The perinatal post mortem examination remains an indispensable part of clinical management. It contributes to medical education and quality assurance. It can aid in the identification of inheritable diseases and provide information for accurate parental counseling.

Neonatal transportation: the effects of a national neonatal transportation programme.

BACKGROUND: The transport of critically ill newborns by specialised transport teams has been shown to be associated with a significant improvement in their clinical condition on arrival at the receiving hospital. AIM: To determine if the National Neonatal Transport Programme introduced in 2001 improved clinical condition of newborns at the end of transfer. METHODS: A retrospective study of all 176 patients transported by the National Neonatal Transport Programme between March 2001 and March 2002. RESULTS: Before transfer, 17% of patients were hypothermic, 2% hypoglycaemic and 11% acidotic as were 7%, 3% and 5% respectively at the end of transfer. A review of 172 neonatal transports between 1987 and 1989 revealed that 21% of patients were hypothermic, 13% hypoglycaemic and 20% acidotic at the end of transfer. CONCLUSIONS: The National Neonatal Transport Programme has resulted in improved clinical condition of newborns at the end of transfer when compared to their condition before transfer and compared to outcomes prior to the introduction of the programme.

Outcome of infants born to hepatitis C infected women.

BACKGROUND: Hepatitis C virus (HCV) can be transmitted vertically from mother to infant, either late in pregnancy or at delivery. AIMS: To determine the outcome of infants born to HCV infected women, to characterise epidemiology and to design an appropriate infant monitoring schedule. METHODS: Three hundred and fourteen infants, born to 296 HCV positive women between 1994 and 1999 were monitored for a median of 18 months (range 1-52). RESULTS: Forty per cent of infants were small for age and 46% had neonatal abstinence syndrome (NAS). Of 173 infants of defined status, 11 were infected (vertical transmission rate [VTR] 6.4%, 95% CI 2.8-10). Infected infants were diagnosed at a median of three months (range 0.5-10). Liver transaminases elevation was documented in 8% of uninfected infants. A negative HCV PCR test before one month of age did not exclude infection but all infected patients had detectable HCV RNA when next tested (range 2-10 months). CONCLUSIONS: 94% of infants born to HCV antibody positive women are not HIV infected. Liver transaminase elevation in exposed infants is not always indicative of infection. A minimum monitoring schedule of testing (PCR and antibody) at six to eight weeks, six and 18 months allows early diagnosis while detecting late seroconversions.

Formation of a tight 1:1 complex of Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein: evidence for long-range interactions between the Fe protein binding sites during catalytic hydrogen evolution.

It has been well documented that the combination of the MoFe protein of Azotobacter vinelandii nitrogenase (Av1) with the Fe protein (Cp2) from Clostridium pasteurianum nitrogenase produces an inactive, stable complex. However, we report that this heterologous nitrogenase has a low level of activity for H(2) evolution, with a specific activity of 12 nmol min(-)(1) mg(-)(1) of Av1. This activity does not arise from contaminating hydrogenase since it required the presence of both Cp2 and Av1 and showed saturation kinetics when increasing amounts of Cp2 were added to the assay. Incubation of the two proteins at a 4:1 Cp2:Av1 ratio in the absence of MgATP followed by analytical gel filtration showed, surprisingly, that the stoichiometry of the isolated complex was Av1.Cp2 instead of Av1.(Cp2)(2) as determined previously. The presence of MgATP in the elution buffer did not change the elution profile of the complex. The hydrodynamic radius of the isolated complex determined by dynamic light scattering was 5.93 +/- 0.14 nm, intermediate between Av1 and a stable 2:1 nitrogenase complex, consistent with a 1:1 assignment for the Av1.Cp2 complex. When assayed with Av2, the isolated Av1.Cp2 complex showed full half-site reactivity with a specific activity of 750 nmol of C(2)H(2) reduced min(-)(1) mg(-)(1) of Av1. The EPR spectrum of the isolated complex showed the Cp2 to be oxidized and the Av1 to retain the S = (3)/(2) signal characteristic of FeMoco. In the presence of MgATP, under turnover conditions at a 2:1 ratio of Cp2:Av1, the [4Fe-4S] center of Cp2 was protected from the chelator 2,2'-bipyridyl. This is consistent with the formation of a tight 2:1 complex of Av1.(Cp2)(2) which is more stable than the homologous Cp nitrogenase. Assuming that the Lowe-Thorneley model for nitrogenase applies and that a rate-limiting dissociation of the complex is required for H(2) evolution, then with a rate of 0.032 s(-)(1) the 1:1 complex is too stable to be involved in catalysis. The differences in the stability of the 2:1 and 1:1 complexes indicate cooperativity between the Fe protein binding sites of Av1, which structural data show to be separated by 105 A. On the basis of these observations, we propose a model for nitrogenase catalysis in which the stable 1:1 complex formed between oxidized Fe protein and the one-electron-reduced MoFe protein plays an essential role. In this scheme, the two Fe protein binding sites of the MoFe protein alternately bind and release Fe protein in a shuttle mechanism associated with long-range conformational changes in the MoFe protein.

Hepatitis C infection in an Irish antenatal population.

BACKGROUND: Hepatitis C infection (HCV) has an estimated seroprevalence of 1-2% in women of child-bearing age and vertical transmission rate of 5-15%. AIMS: To characterise the current trends of HCV in an Irish antenatal population. METHODS: Infants of HCV seropositive women, born 1994 to 1999, were referred to the Paediatric Infectious Diseases service. Maternal details were collected retrospectively. RESULTS: 296 HCV seropositive women were studied. 244 (82%) were infected through intravenous drug use (IVDU), 25 (8%) through heterosexual contact and 13 (7%) via blood products. Nine women had no identifiable risk factors. Coinfection with other blood borne viruses was uncommon (4.7% HIV, 3.4% hepatitis B). Of 84 women tested for HCV-RNA, 46 (55%) were positive. Eighty three (26%) delivered prematurely; the caesarean section rate was 11%. CONCLUSIONS: HCV is increasingly detected in antenatal clinics. Heterosexual contact is a mode of spread. Maternal HCV viraemia can be variable in pregnancy. Further study of HCV in pregnancy is needed to define the impact of pregnancy on HCV, accurately predict infant outcome and selectively target interventions to women at greatest risk of transmission.

Klebsiella pneumoniae nitrogenase: formation and stability of putative beryllium fluoride-ADP transition state complexes.

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1.{Kp2.(MgADP.BeFx)2}2. At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1.Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations of the components allowing constants for their formation of 2.1 x 10(6) and 5.6 x 10(5) M(-1) to be calculated for the 1:1 and 2:1 complexes, respectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.

Strategies for reduction of neonatal mortality.

Infant mortality rates in developed countries have shown significant decreases in recent years. Two-thirds of infant mortality still occurs in the neonatal period and our aim in this study was to review the causes of these neonatal deaths and see where further improvements may be possible. A 6-yr review of all neonatal deaths of live-born infants over 500 g birthweight from 1991 to 1996 was made. The 1989 amended Wigglesworth classification was used to categorize cause of death and other perinatal variables were also recorded. Results show there were 34,375 births and 153 neonatal deaths. Classification of these deaths by Wigglesworth found 78 (51 per cent) due to congenital malformations, 58 (38 per cent) due to prematurity, 6 (4 per cent) due to asphyxia and 11 (7 per cent) due to specific other causes. The corrected neonatal mortality was 2.18. Neural tube defects alone accounted for 10 per cent of the total neonatal mortality. Fifty-five out of 58 infants who died due to prematurity had birthweight < 1000 g and survival rates in this group compared well to international standards. We conclude that a reduction in neonatal mortality is possible but is most likely to result from community focused measures such as increased use of pre- and peri-conceptional folate.

The Marshall-Smith syndrome: a review of the laryngeal complications.

UNLABELLED: The Marshall-Smith syndrome is characterised by a triad of facial dysmorphism, failure to thrive and accelerated osseous maturation. We report a further case of this rare syndrome with the unusual but previously reported complication of laryngeal hypoplasia and review the associated laryngeal anomalies that have been reported to date. CONCLUSION: Severe airway obstruction due to congenital anomalies must be excluded in any dysmorphic child presenting with respiratory distress at birth. Rapid airway assessment will enable early and appropriate intervention and may be important when deciding on the long-term plan for the infant.

Surfactant therapy in respiratory distress syndrome: the effect of a learning curve in improving outcome.

A retrospective study of all premature neonates who received artificial surfactant (Curosurf) at the Rotunda Hospital was performed. The period from October 1990 to June 1992 (n = 48) was compared with the initial experience from June 1987 to January 1988 (n = 15). In the initial period mortality rate was 67% in surfactant treated infants, and use of surfactant was not associated with an improvement in outcome compared with the previous six years. In the more recent period overall mortality was 21%. Overall survival in normally formed very low birthweight infants improved from 59% in 1986 to 86% in 1991-1992. Improvement in survival rates was most noticeable in infants with birthweight 750-999 grams, with survival increasing from 44% (before introduction of surfactant treatment) to 91% (in 1991-1992). It is probable that a certain level of experience with use of surfactant is required before optimal effects can be obtained.

Isolated single umbilical artery--the case for routine renal screening.

To determine the incidence of silent renal anomalies in infants with isolated single umbilical artery (SUA), all infants with SUA and without other obvious congenital anomalies, identified over a six year period, were screened using renal ultrasonography. Over 35,000 placentas were examined. An isolated single umbilical artery was identified in 112 (0.32%). Nineteen infants had abnormal renal imaging. Eight of 112 (7.1%) had significant persisting abnormalities. Vesicoureteric reflux was found in five infants (4.5%). It is recommended that renal ultrasonography be performed for all infants with isolated SUA.

Alcohol and cigarette use in a pregnant Irish population.

One hundred women were selected at random and interviewed. All were postnatal. The object was to establish the level of alcohol and cigarette consumption and the level of knowledge to potential adverse effects. Of the 100 women interviewed 89% drank prior to pregnancy, six drank between 100-120 grams/week and 19 drank > 120 grams/week. 11 women stopped drinking when they became pregnant. In the group which drank 100-120 grams/week, 66% decreased their alcohol consumption considerably ie > 100 grams/week while pregnant, while in the group which drank > 120 grams/week only 15% decreased their alcohol consumption. 38 women binged on at least one occasion while 21 said they had binged on at least one occasion during the first trimester. 58% of women were aware of the harmful effects of alcohol during pregnancy. They compared with 93% who were aware of the harmful effects of smoking during pregnancy. Only 11% of women said a doctor had mentioned alcohol as harmful, while 57% said that a doctor had mentioned the hazard of smoking in pregnancy. The overall results show a general ignorance to the effects of alcohol consumption in pregnancy compared to the level of knowledge about smoking. The results also highlight the fact that doctors do not make patients aware of the effects of alcohol in pregnancy while they make an effort to educate people about the problems of smoking during pregnancy.

Blood cultures and antibiotic use in a neonatal intensive care unit.

A review of the duration of antibiotic courses in our Neonatal Intensive Care Unit (N.I.C.U.) has shown that in a significant number of cases there was non compliance with the antibiotic policy. An audit of neonatal sepsis and antibiotic usage over a six month period was performed in the N.I.C.U. at the Rotunda Hospital. Three hundred and forty-nine of the 3,163 infants born during this time were admitted to the N.I.C.U. One hundred and fifty-two infants had one or more episodes of suspected sepsis. In 168 instances this preceded a course of antibiotic therapy lasting longer than 48 hours. In 56 (33%) the infant was clinically septic and in 16 (9.5%) of those there was bacteriological proven sepsis. There were 112 episodes (60%) where there was no strong clinical evidence of sepsis and a negative investigation for sepsis where antibiotics were continued for more than 48 hours. The possible reasons why antibiotics were not stopped include the non specific nature of signs of sepsis in neonates, physicians' reluctance to discontinue antibiotics once started, and the logistic difficulty of obtaining routine culture results at weekends. The study emphasizes the need for regular surveillance of antibiotic usage in a N.I.C.U.