Community-acquired bacteraemia by Klebsiella pneumoniae producing KPC-3 and resistant to ceftazidime/avibactam.

OBJECTIVES: To describe the clinical and microbiological features of a case of community-acquired infection by KPC-producing K. pneumoniae (KPCKP) resistant to ceftazidime/avibactam (CAZ-AVI). METHODS: Identification of microorganisms was performed with MALDI Biotyper CA System (BrukerDaltonics, Madrid, Spain). Antimicrobial susceptibility testing was performed using Sensitre EURGNCOL panels (Thermo Fisher Scientific, Madrid, Spain) and gradient strips (Etest, bioMérieux, Madrid, Spain) in the case of CAZ-AVI, using EUCAST breakpoints for interpretation. Whole genome sequencing of blood culture and rectal swab isolates was performed using the Illumina NovaSeq 6000 sequencing system, with 2 × 150-bp paired-end reads (Illumina, Inc.). RESULTS: Blood culture and rectal swab KPCKP isolates were resistant to carbapenems and to CAZ-AVI. The blood culture isolate showed susceptibility to trimethoprim-sulfamethoxazole (TMP-SMX), but the rectal swab culture isolate was resistant to this antibiotic. Both isolates belonged to clonal lineage ST512, harboured a single copy of blaKPC-3 gene, and showed 16 single nucleotide polymorphisms (SNP) between them and 38 SNPs with regard to the first KPC-3 producing K. pneumoniae isolated in our hospital in an initial outbreak in 2012. Genome-wide resistome analysis revealed the presence of a IncFIB(K) plasmid harbouring sul1 and dfrA12 genes only in the rectal swab culture isolate, which may explain its resistance to TMP-SMX. CONCLUSIONS: Resistance to ceftazidime-avibactam is an emerging nosocomial problem. This case shows that CAZ-AVI-resistant KPCKP strains may disseminate into the community and cause serious infections.

Clinical differences between invasive pulmonary infection by Scedosporium apiospermum and invasive pulmonary aspergillosis.

Invasive pulmonary infection by Scedosporium apiospermum (IPSA) and invasive pulmonary aspergillosis (IPA) are clinically similar. Our objective was to identify clinical parameters that may differentiate IPSA from IPA. Ours was a prospective cohort study that included patients with different degrees of immunosuppression and respiratory isolation of S. apiospermum (SCA). Episodes of invasive infection were classified according to the EORTC and MSG criteria. Clinical variables corresponding to patients with IPSA were compared with those collected from patients with a diagnosis of IPA during the same period. Twenty-seven patients with positive culture for SCA from respiratory samples were evaluated. Of the 27 positive cultures, nine were classified as IPSA. When compared with the 89 patients with IPA, patients with IPSA were most likely to have received prophylaxis with either aerosolised (14.6% vs. 66.7%; P < 0.001) or intravenous amphotericin B (AMB; 4.5% vs. 44.4%; P = 0.002), to have prior episode of acute rejection (19% vs. 66.7%; P = 0.005), to have a later onset of infection after transplantation (251 days vs. 404 days; P = 0.009), and to have higher CD4(+) lymphocyte count (207.6 vs. 289.4; P = 0.005). Late-onset disease after transplantation and prophylaxis with AMB are more frequent in patients with IPSA compared with IPA.

Structure of L-A virus: a specialized compartment for the transcription and replication of double-stranded RNA.

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment-the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-A resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10-15 A across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40-45 A, compared with 25-30 A in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.

Sharpening high resolution information in single particle electron cryomicroscopy.

Advances in single particle electron cryomicroscopy have made possible to elucidate routinely the structure of biological specimens at subnanometer resolution. At this resolution, secondary structure elements are discernable by their signature. However, identification and interpretation of high resolution structural features are hindered by the contrast loss caused by experimental and computational factors. This contrast loss is traditionally modeled by a Gaussian decay of structure factors with a temperature factor, or B-factor. Standard restoration procedures usually sharpen the experimental maps either by applying a Gaussian function with an inverse ad hoc B-factor, or according to the amplitude decay of a reference structure. EM-BFACTOR is a program that has been designed to widely facilitate the use of the novel method for objective B-factor determination and contrast restoration introduced by Rosenthal and Henderson [Rosenthal, P.B., Henderson, R., 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721-745]. The program has been developed to interact with the most common packages for single particle electron cryomicroscopy. This sharpening method has been further investigated via EM-BFACTOR, concluding that it helps to unravel the high resolution molecular features concealed in experimental density maps, thereby making them better suited for interpretation. Therefore, the method may facilitate the analysis of experimental data in high resolution single particle electron cryomicroscopy.

Management of multidrug resistant Gram-negative bacilli infections in solid organ transplant recipients: SET/GESITRA-SEIMC/REIPI recommendations.

Solid organ transplant (SOT) recipients are especially at risk of developing infections by multidrug resistant (MDR) Gram-negative bacilli (GNB), as they are frequently exposed to antibiotics and the healthcare setting, and are regulary subject to invasive procedures. Nevertheless, no recommendations concerning prevention and treatment are available. A panel of experts revised the available evidence; this document summarizes their recommendations: (1) it is important to characterize the isolate's phenotypic and genotypic resistance profile; (2) overall, donor colonization should not constitute a contraindication to transplantation, although active infected kidney and lung grafts should be avoided; (3) recipient colonization is associated with an increased risk of infection, but is not a contraindication to transplantation; (4) different surgical prophylaxis regimens are not recommended for patients colonized with carbapenem-resistant GNB; (5) timely detection of carriers, contact isolation precautions, hand hygiene compliance and antibiotic control policies are important preventive measures; (6) there is not sufficient data to recommend intestinal decolonization; (7) colonized lung transplant recipients could benefit from prophylactic inhaled antibiotics, specially for Pseudomonas aeruginosa; (8) colonized SOT recipients should receive an empirical treatment which includes active antibiotics, and directed therapy should be adjusted according to susceptibility study results and the severity of the infection.

Cholinergic system, rearing environment and trajectory learning during aging in mice.

Three, 12- and 20-month-old C57BL6/J mice, reared in standard conditions or in enriched environments, were administered subcutaneously either scopolamine hydrobromide, 0.6 or 1.2 mg kg(-1), or physiological saline (control mice) 15 min before testing their abilities to find an invisible platform in a modified version of the Morris water maze, the starting point being kept unchanged throughout the experiment to allow the aged animals to solve the task. The results demonstrated that: 1) All control mice, whatever their age, were able to learn the platform location, but the number of trials needed to reach the learning criterion (3 consecutive trials in less than 8 s) increased with age; 2) All the scopolamine-treated mice, whatever their age, were also able to learn the platform location. However, compared to age-matched controls, the number of trials needed to reach the learning criterion was greater; 3) Rearing the animals in an enriched environment antagonized the effect of scopolamine, but only in the youngest (3 month-old) mice. All control and scopolamine-treated mice, whatever their age and their rearing environment, remembered, 7 days later, the platform location.

Seasonal variation of the impact of a stressful procedure on open field behaviour and blood corticosterone in laboratory mice.

Behavioural and hormonal seasonal changes are well documented in various vertebrate species living in their natural environment but circannual variations that may occur in laboratory animals reared in standard conditions are poorly investigated. This study shows that, in laboratory mice, the effects of stress on behavioural inhibition, investigatory behaviour and blood concentration of corticosterone are seasonally dependent. No consistency was observed between the reactivity of biological structures controlling the hormonal response to stress and the behavioural activities investigated at every period of the year. During the spring time, stress, which elicited a decrease of investigatory behaviour (estimated by the walking time in an open field), increased behavioural inhibition (estimated by the percentage of walking in the central area of the open field) as well as the blood corticosterone concentration in laboratory mice. In autumn, stress had no significant effect on behaviour despite the great hormonal concentration increase. The results reveal that, at certain period of the year, a stressful procedure is unable to affect behavioural parameters in laboratory mice which were maintained in constant 12-h dark/12-h light cycle. The report constitutes a novel piece of information suggesting a potential role of the endogenous biological clock in the modulation of stress response in mammals.

Management of cytomegalovirus infection in solid organ transplant recipients: SET/GESITRA-SEIMC/REIPI recommendations.

Cytomegalovirus (CMV) infection remains a major complication of solid organ transplantation. Because of management of CMV is variable among transplant centers, in 2011 the Spanish Transplantation Infection Study Group (GESITRA) of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) developed consensus guidelines for the prevention and treatment of CMV infection in solid organ transplant recipients. Since then, new publications have clarified or questioned the aspects covered in the previous document. For that reason, a panel of experts revised the evidence on CMV management, including immunological monitoring, diagnostics, prevention, vaccines, indirect effects, treatment, drug resistance, immunotherapy, investigational drugs, and pediatric issues. This document summarizes the recommendations.

Topology of the components of the DNA packaging machinery in the phage phi29 prohead.

Chromosome condensation inside dsDNA viral particles is a complex process requiring the coordinated action of several viral components. The similarity of the process in different viral systems has led to the suggestion that there is a common underlying mechanism for DNA packaging, in which the portal vertex or connector plays a key role. We have studied the topology of the packaging machinery using a number of antibodies directed against different domains of the connector. The charged amino-terminal, the carboxyl-terminal, and the RNA binding domain are accessible areas in the connector assembled into the prohead, while the domains corresponding to the 12 large appendages of the connector are buried inside the prohead. Furthermore, while the antibodies against the carboxyl and amino-terminal do not affect the packaging reaction, incubation of proheads with antibodies against the RNA binding domain abolishes the packaging activity. The comparison of the three-dimensional reconstructions of bacteriophage phi29 proheads with proheads devoid of their specific pRNA by RNase treatment shows that this treatment removes structural elements of the distal vertex of the portal structure, suggesting that the pRNA required for packaging is located at the open gate of the channel in the narrow side of the connector.

Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of asymmetric Gag dimers.

The primary functions of most virus capsids are to protect the viral genome in the extra-cellular milieu and deliver it to the host. In contrast, the capsids of fungal viruses, like the cores of all other known double stranded RNA viruses, are not involved in host recognition but do shield their genomes, and they also carry out transcription and replication. Nascent (+) strands are extruded from transcribing virions. The capsids of the yeast virus L-A are composed of Gag (capsid protein; 76 kDa), with a few molecules of Gag-Pol (170 kDa). Analysis of these 420 A diameter shells and those of the fungal P4 virus by cryo-electron microscopy and image reconstruction shows that they share the same novel icosahedral structure. Both capsids consist of 60 equivalent Gag dimers, whose two subunits occupy non-equivalent bonding environments. Stoichiometry data on other double-stranded RNA viruses indicate that the 120-subunit structure is widespread, implying that this molecular architecture has features that are particularly favorable to the design of a capsid that is also a biosynthetic compartment.

Effects of systemic lupus erythematosus on spatial cognition and cerebral regional metabolic reactivity in BxSB lupus-prone mice.

Brain-reactive auto-antibodies appear as key elements in the progressive CNS disturbances associated with systemic lupus erythematosus. The BxSB lupus prone mice are a model of this pathology, in which a gene located on the Y chromosome provokes a sex specific morbidity in males. This study was aimed to establish and characterize the relationships between behavioral disorders, neurological deficiencies and the aged-related immunological perturbations in this murine model. For this purpose, spatial and motor abilities were evaluated in male and female mice at six and 26 weeks of age. The results showed that the older males were greatly altered in their spatial abilities while the young ones and the females, whatever their age, were not. None of the animals had motor skill and motor learning disabilities. These spatial alterations were associated with modifications of basal neuronal activity measured by the cytochrome oxidase histochemical method in several areas directly or indirectly involved in spatial behavior, such as the hippocampus, the amygdala, the parietal and perirhinal cortex. Immunological study allowed us to correlate the behavioral abnormalities to the appearance of antibodies reactivities against cellular and nuclear components.

Spatial and motor abilities during the course of autoimmune disease in (NZW x BXSB)F1 lupus-prone mice.

In the systemic autoimmune/inflammatory lupus erythematosus disease, the involvement of the central nervous system is well recognized and frequently includes deficits in neurological function, cognition, and affect. The (NZW x BXSB)F1 lupus-prone mice are model of this pathology, in which a gene located on the Y chromosome provokes a sex specific morbidity in males. The present study examines whether autoimmune (NZW x BXSB)F1 mice develop impairments in learning and memory that correlate with severity of lupus-like disease. For this purpose, spatial and motor abilities were evaluated in 6- and 20-week-old male and female mice, and the immune status of these behaviorally tested mice was assessed by the presence of anti-nuclear antibodies (ANAbs) in the serum. The results showed that none of the animals had motor skill and motor learning disabilities, but that the older males were greatly impaired in their spatial abilities while the young ones and the females, whatever their age, were not. Besides, the ANAbs levels were similar and low in the young males, the young females and the old females, and very much higher in the old males, showing that spatial alterations were correlated to the anti-nuclear antibodies level.

Repeated stress alters caffeine action on motor coordination in C57Bl6/J male mice.

This study was aimed to evaluate the effects of stress on caffeine action on motor coordination in mice. For 6 consecutive days, the mice were subjected to three different stressors. Saline or caffeine (30, 60 or 120 mg kg(-1)) was i.p. administered after the last stressful experience, then the animals were behaviorally tested in the holeboard. Their stumbling frequency was compared to that of unstressed mice injected with either saline or caffeine. (1) There was a strong trend for stress to impair motor coordination. (2) In unstressed mice, caffeine induced a linear dose-dependent increase of stumbling frequency. (3) Stress decreased the stumbling frequency induced by the highest dose of caffeine. The results are discussed in terms of interaction of stress and caffeine on dopaminergic and GABAergic systems.

Effects of prenatal stress on anxiety and social interactions in adult rats.

Deficits in social behavior are found in several neuro-psychiatric disorders with a presumed developmental origin. The aim of the present study is to determine if prenatal stress at a given day of gestation alters social behavior in adult offspring. Pregnant rats were exposed to an acute stress (presence of a cat) either at the 10th (S10), the 14th (S14) or the 19th (S19) gestational day. When adult, their offsprings were studied in anxiety, neophobic and social behaviors. The results showed that S10 and S19 rats were more anxious and less aggressive than control rats, while the anxious and aggressive behavior of S14 rats was similar to that of the control ones. It is suggested that day 14 of pregnancy is a hyposensitive period to stressful agents due to an important plasticity of the developing gross nervous structures.

Administration of 17alpha-ethinylestradiol during pregnancy elicits modifications of maternal behavior and emotional alteration of the offspring in the rat.

Intraperitoneal administration of 17alpha-ethinylestradiol (15 microg.kg(-1)) in pregnant rats, every day from day 9 to day 14 of pregnancy, elicited a high percentage of abortions. Quantification of maternal behavior showed that treated dams took better care of their pups than control dams, injected with the vehicle only, did. Postnatal reflexes, which reflect maturational rate, were established more promptly in the offspring of treated dams than in the offspring of control dams. However, when adult, the rats born from treated dams developed anxiety- and depressive-like behaviors. All these results are explained by the effects of the exogenous estrogen on the developing brain of the fetuses.

Motor skills and motor learning in Lurcher mutant mice during aging.

Motor learning abilities on the rotorod and motor skills (muscular strength, motor coordination, static and dynamic equilibrium) were investigated in three-, nine-, 15- and 21-month-old Lurcher and control mice. Animals were subjected to motor training on the rotorod before being subjected to motor skills tests. The results showed that control mice exhibited decrease of muscular strength and specific equilibrium impairments in static conditions with age, but were still able to learn the motor task on the rotorod even in old age. These results suggest that, in control mice, efficiency of the reactive mechanisms, which are sustained by the lower transcerebellar loop (cerebello-rubro-olivo-cerebellar loop), decreased with age, while the efficiency of the proactive adjustments, which are sustained by the upper transcerebellar loop (cerebello-thalamo-cortico-ponto-cerebellar loop), did not. In spite of their motor deficits, Lurcher mutants were able to learn the motor task at three months, but exhibited severe motor learning deficits as soon as nine months. Such a deficit seems to be associated with dynamic equilibrium impairments, which also appeared at nine months in these mutants. By two months of age, degeneration of the cerebellar cortex and the olivocerebellar pathway in Lurcher mice has disrupted both lower and upper transcerebellar loops. Disruption of the lower loop could well explain precocious static equilibrium deficits. However, in spite of disruption of the upper loop, motor learning and dynamic equilibrium were preserved in young mutant mice, suggesting that either deep cerebellar nuclei and/or other motor structures involved in proactive mechanisms needed to maintain dynamic equilibrium and to learn motor tasks, such as the striatopallidal system, are sufficient. The fact that, in Lurcher mutant mice, motor learning decreased by the age of nine months suggests that the above-mentioned structures are less efficient, likely due to degeneration resulting from precocious and focused neurodegeneration of the cerebellar cortex. From this behavioral approach of motor skills and motor learning during aging in Lurcher mutant mice, we postulated the differential involvement of two transcerebellar systems in equilibrium maintenance and motor learning. Moreover, in these mutants, we showed that motor learning abilities decreased with age, suggesting that the precocious degeneration of the cerebellar Purkinje cells had long-term effects on motor structures which are not primarily affected. Thus, from these results, Lurcher mutant mice therefore appear to be a good model to study the pathological evolution of progressive neurodegeneration in the central nervous system during aging.

Role of the inferior olivary complex in motor skills and motor learning in the adult rat.

The inferior olivary complex of adult rats was chemically destroyed using intraperitoneal injection of 3-acetylpyridine. Animals were submitted to different motor tasks: hanging test, equilibrium test and motor co-ordination test. The different scores show that 3-acetylpyridine-treated rats had motor co-ordination and static equilibrium deficiencies, whereas their rod suspension capabilities were intact. Animals were also trained on an unrotated rod or on a rod rotating at 5, 10 or 20 r.p.m. 3-Acetylpyridine-treated rats were able to maintain their equilibrium on the unrotated rod and at 5 r.p.m. Moreover, after motor training at 5 r.p.m., rats were able to improve their motor skills and reached the same score as controls. Despite their good motor skills, animals were unable to maintain their equilibrium when rotated at 10 and 20 r.p.m. These results suggest that the inferior olivary complex is needed for motor learning involving the temporal organization of movement.

Transgenic mice with neuronal overexpression of bcl-2 gene present navigation disabilities in a water task.

In the CNS, Bcl-2 is an antiapoptotic gene involved in the regulation of neuronal death. Transgenic mice overexpressing the human gene Bcl-2 (Hu-bcl-2 mice) showed delayed acquisition in two tasks requiring them to find a hidden platform starting from either a random or a constant starting location. The same mice were not deficient in another task requiring them to find a visible platform suggesting that the delay observed was not due to motor, visual or motivational deficits in the water. The delay observed in Hu-bcl-2 mice was more important in the random starting test in which the allocentric demand for navigation was stronger. The results suggested that allocentric navigation is particularly sensitive to abnormal CNS maturation following the overexpression of the bcl-2 gene. The specific deficits (motor learning, fear-related behavior and allocentric navigation) observed in Hu-bcl-2 mice suggest that the regulation of developmental neuronal death is crucial for multisensorial learning and emotional behavior.

Properties of a folate binding protein (FBP) isolated from porcine kidney.

A specific high-affinity folate binding protein (FBP) that binds folic acid and folic acid derivatives and that was previously identified in porcine kidney has been purified 50,000-fold using the technique of affinity chromatography. The FBP had a molecular weight of 38,500 daltons and did not appear to aggregate in solution, as has been reported to be the case with folate binding protein from milk. At pH 7.6, the Ka was at least 5 X 10(12)M-1. At pH values greater than 9.5 or less than 5, the binding dramatically decreased. The specificity was determined by an isotopic dilution technique using [3H]folic acid and folic acid analogs and derivatives. The FBP reacted more rapidly with unsubstituted folates, and the number of glutamic acid moieties (N greater than or equal to 1) did not influence binding. Binding of folic acid to the FBP was unaffected by a variety of anions and cations, and 8 M urea, but was disrupted by 6 M guanidine hydrochloride. Proteolytic enzymes irreversibly destroyed binding affinity, but RNase, DNase, phospholipase and neuraminidase had no effect.

Delayed spontaneous alternation in intact and cerebellectomized control and lurcher mutant mice: differential role of cerebellar cortex and deep cerebellar nuclei.

Lurcher mutant (+/Lc) mice exhibit a massive loss of neurons in the cerebellar cortex and in the inferior olivary nucleus while deep cerebellar nuclei are essentially intact. To discriminate the respective participation of the cerebellar cortex and deep structures in learning and memory, the authors subjected 3- to 6-month-old +/Lc mice to a delayed spontaneous alternation task to test their working and long-term spatial memories. Results show that wild type (+/+) mice alternated above chance even after a 1-hr delay between the forced and choice trials, whereas in +/Lc mice, long-term memory was impaired. Cerebellectomized +/+ mice behave as +/Lc mice (working memory was preserved but long-term memory was not), whereas in the cerebellectomized +/Lc mice, both working and long-term memories were altered. These results are discussed in terms of relationships between the cerebellum and the hippocampus.