Is it possible to perform bacterial identification and antimicrobial susceptibility testing with a positive blood culture bottle for quick diagnosis of bloodstream infections?

Abstract INTRODUCTION: Bloodstream infections can be fatal, and timely identification of the etiologic agent is important for treatment. METHODOLOGY: An alternative method, consisting of direct identification and susceptibility testing of blood culture bottles using the automated VITEK 2® system, was assessed. RESULTS: All 37 of the Gram-negative bacilli (GNB) identifications and 57.1% of the 28 Gram-positive cocci (GPC) identifications matched those obtained with standard methods. In susceptibility testing, the agreement was greater than 90%. CONCLUSIONS: This alternative methodology may assist in the early identification and susceptibility testing of GNB. Further research is necessary to develop appropriate methods for GPC.

Filifactor alocis has virulence attributes that can enhance its persistence under oxidative stress conditions and mediate invasion of epithelial cells by porphyromonas gingivalis.

Filifactor alocis, a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of F. alocis and its ability to interact with Porphyromonas gingivalis W83, several clinical isolates of F. alocis were characterized. F. alocis showed nongingipain protease and sialidase activities. In silico analysis revealed the molecular relatedness of several virulence factors from F. alocis and P. gingivalis. In contrast to P. gingivalis, F. alocis was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with P. gingivalis or F. alocis monocultures. In those epithelial cells, endocytic vesicle-mediated internalization was observed only during coculture. The F. alocis clinical isolate had an increased invasive capacity in coculture with P. gingivalis compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that F. alocis has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease.

Central line-associated bloodstream infection in hospitalized children with peripherally inserted central venous catheters: extending risk analyses outside the intensive care unit.

BACKGROUND: Increasingly, peripherally inserted central venous catheters (PICCs) are placed for prolonged intravenous access. Few data exist regarding risk factors for central line-associated bloodstream infection (CLABSI) complicating PICCs in hospitalized children, especially children hospitalized outside the intensive care unit (ICU). METHODS: We identified all children with a PICC inserted at The Johns Hopkins Hospital (Baltimore, MD) from 1 January 2003 through 31 December 2009 and used Poisson regression models to identify risk factors for PICC-associated CLABSIs. RESULTS: A total of 2592 PICCs were placed in 1819 children. One hundred sixteen CLABSIs occurred over 44,972 catheter-days (incidence rate [IR], 2.58 cases per 1000 catheter-days; 95% confidence interval [CI], 2.07-3.00 cases per 1000 catheter-days). Independent predictors of CLABSI in the entire cohort included PICC dwell time of > 21 days (IR ratio [IRR], 1.53; 95% CI, 1.05-2.26), parenteral nutrition as indication for insertion (IRR, 2.24; 95% CI, 1.31-3.84), prior PICC-associated CLABSI (IRR, 2.48; 95% CI, 1.18-5.25), underlying metabolic condition (IRR, 2.07; 95% CI, 1.14-3.74), and pediatric ICU exposure during hospitalization (IRR, 1.80; 95% CI, 1.18-2.75). Risk factors for CLABSI in children without PICU exposure included younger age, underlying malignancy and metabolic conditions, PICCs inserted in the lower extremity, and a prior PICC-associated CLABSI. CONCLUSIONS: Prolonged catheter dwell time, pediatric ICU exposure, and administration of parenteral nutrition as the indication for PICC insertion are important predictors of PICC-associated CLABSI in hospitalized children. A careful assessment of these risk factors may be important for future success in preventing CLABSIs in hospitalized children with PICCs.

Comprehensive detection of causative pathogens using real-time PCR to diagnose pediatric community-acquired pneumonia.

We have developed a real-time reverse transcription-PCR (RT-PCR) method to detect 13 respiratory viruses: influenza virus A and B; respiratory syncytial virus (RSV) subgroup A and B; parainfluenza virus (PIV) 1, 2, and 3; adenovirus; rhinovirus (RV); enterovirus; coronavirus (OC43); human metapneumovirus (hMPV); and human bocavirus (HBoV). The new method for detection of these viruses was applied simultaneously with real-time PCR for the detection of six bacterial pathogens in clinical samples from 1700 pediatric patients with community-acquired pneumonia (CAP). Of all the patients, 32.5% were suspected to have single bacterial infections; 1.9%, multiple bacterial infections; 15.2%, coinfections of bacteria and viruses; 25.8%, single viral infections; and 2.1%, multiple viral infections. In the remaining 22.6%, the etiology was unknown. The breakdown of suspected causative pathogens was as follows: 24.4% were Streptococcus pneumoniae, 14.8% were Mycoplasma pneumoniae, 11.3% were Haemophilus influenzae, and 1.4% were Chlamydophila pneumoniae. The breakdown of viruses was as follows: 14.5% were RV, 9.4% were RSV, 7.4% were hMPV, 7.2% were PIV, and 2.9% were HBoV. The new method will contribute to advances in the accuracy of diagnosis and should also result in the appropriate use of antimicrobials.

Mediastinitis after cardiac surgery in Madani Heart Center, Tabriz, Iran.

Post-sternotomy infections are a kind of nosocomial infection involving the mediastinum space and the sternum, with a high morbidity and mortality rate. The present study was carried out to identify the incidence of mediastinitis following cardiac surgery and the most common risk factors. Cardic patients undergoing surgery were studied for suspicious mediastinitis infection at the Madani Heart Center, Tabriz, Iran from 2004 to 2006. The most common isolated agents included the coagulase-negative staphylococci, Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacter spp. Incidence of postoperative mediastinitis after cardiac surgery was 1.2%. The mortality rate of postoperative mediastinitis was high (34.3%). Wound infection, especially mediastinitis following cardiac surgery, is rare but could be life-threatening. The most important step in the management of wound infections is prevention, and preventive measures could be strengthened by identifying the risk factors.

Biological activities of novel gyrase inhibitors of the aminocoumarin class.

Thirty-one aminocoumarin antibiotics derived from mutasynthesis experiments were investigated for their biological activities. Their inhibitory activities toward Escherichia coli DNA gyrase were determined in two different in vitro assays: an ATPase assay and a DNA supercoiling assay. The assays gave a similar rank order of the activities of the compounds tested, although the absolute 50% inhibitory concentrations (IC(50)s) obtained in each assay were different. To confirm that the compounds also acted as gyrase inhibitors in vivo, reporter gene assays were carried out with E. coli by using gyrA and sulA promoter fusions with the luxCDABE operon. A strong induction of both promoters was observed for those compounds that showed gyrase inhibitory activity in the biochemical assays. Compounds carrying analogs of the prenylated benzoyl moiety (ring A) of clorobiocin that were structurally very different showed high levels of activity both in the biochemical assay and in the reporter gene assay, indicating that the structure of this moiety can be varied considerably without a loss of affinity for bacterial gyrase. The experimentally determined IC(50)s were compared to the binding energies calculated in silico, which indicated that a shift of the pyrrole carboxylic acid moiety from the O-3'' to the O-2'' position of the deoxysugar moiety has a significant impact on the binding mode of the compounds. The aminocoumarin compounds were also investigated for their MICs against different bacterial pathogens. Several compounds showed high levels of activity against staphylococci, including a methicillin-resistant Staphylococcus aureus strain. However, they showed only poor activities against gram-negative strains.

The recent evolution of therapeutic weapons against resistant Gram-positive microorganisms

Multiresistant Gram-positive cocci, including Staphylococcus aureus, the group of coagulase-negative staphylococci, Enterococcus faecalis and Enterococcus faecium, as well as Streptococcus pneumoniae and other streptococci, represent emerging pathogens. This issue is especially concerning in the setting of immunocompromised, hospitalized patients, in particular when surgery, invasive procedures, or prosthetic implants are carried out, patients are admitted in intensive care units, or underlying chronic disorders and immunodeficiency are of concern, and broad-spectrum antibiotics are widely used in the environment; moreover, a community spread of resistant Gram-positive cocci has been recognized during recent years. The spectrum of antimicrobials available for an effective management of these relevant infections is significantly threatened by the emerging of methicillin-resistant and more recently glycopeptide-resistant strains. The streptogramine association represented by quinupristin/dalfopristin, the oxazolidinone derivative linezolid, and the recently licensed daptomycin and tigecycline, together with a number of glycopeptides, fluoroquinolones, cephalosporins, and other experimental compounds, represent an effective response. It is due to the innovative mechanisms of action of these compounds, their maintained or enhanced activity against multiresistant pathogens, their effective pharmacokinetic/pharmacodynamic properties, their frequent possibility of synergistic activity with other compounds effective against Gram-positive pathogens, and a diffuse potential for a safe and easy administration, also to compromised patients.

Howardella ureilytica gen. nov., sp. nov., a Gram-positive, coccoid-shaped bacterium from a sheep rumen.

An unidentified obligately anaerobic, fastidious, Gram-positive, non-motile, non-spore-forming, non-fermentative coccoid-shaped bacterium (designated strain GPC 589(T)) was isolated from the rumen fluid of a sheep. The major fatty acid constituents (>5 %) were C(16 : 0) (29.2 %), C(18 : 0) (40.7 %) and an unidentified compound (19.7 %) with an equivalent chain-length of 13.523. The G+C content of the DNA was 34 mol%. The organism was strongly ureolytic and generated ATP through the hydrolysis of urea. Comparative 16S rRNA gene sequence analysis demonstrated that strain GPC 589(T) was far removed, phylogenetically, from the ruminococci and related Gram-positive anaerobic cocci but exhibited a phylogenetic association with Clostridium rRNA cluster XIVa [as defined by Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). Int J Syst Bacteriol 44, 812-826]. Sequence divergence values of 12.5 % or more were observed between strain GPC 589(T) and all other recognized species within this and related rRNA clostridial clusters. Phylogenetic analysis showed that strain GPC 589(T) represents a new genus within cluster XIVa. On the basis of both phylogenetic and phenotypic evidence, it is proposed that strain GPC 589(T) should be classified as representing a new genus and novel species, Howardella ureilytica gen. nov., sp. nov. The type strain is strain GPC 589(T) (=DSM 15118(T)=JCM 13267(T)).

Planococcus donghaensis sp. nov., a starch-degrading bacterium isolated from the East Sea, South Korea.

A novel Gram-positive, aerobic bacterium, strain JH1T, was isolated from deep-sea sediment of the East Sea, South Korea, and identified by methods of polyphasic taxonomy. The strain was oxidase-positive, motile and coccus-shaped. The genomic DNA G+C content of strain JH1T was 47 mol%. The major fatty acid of strain JH1T was anteiso-C15:0 and the predominant menaquinones were MK-7 and MK-8. Similarity of the 16S rRNA gene sequence (1452 nt) of strain JH1T to those of species of the genera Planococcus and Planomicrobium was 96.0-98.2%. The signature nucleotides in the 16S rRNA gene sequence were compared with those of previously studied type strains of species in the genera Planococcus and Planomicrobium, and suggested that strain JH1T belongs to the genus Planococcus. In addition, phylogenetic analysis showed that strain JH1T was located within the cluster comprising Planococcus antarcticus and Planococcus kocurii. DNA-DNA hybridization showed that it had 9.3% genomic relatedness with Planococcus antarcticus DSM 14505T and 22.9% with Planococcus kocurii DSM 20747T. On the basis of the phenotypic, phylogenetic and genomic data, a novel species of the genus Planococcus, Planococcus donghaensis sp. nov., is proposed, with type strain JH1T (=KCTC 13050T=LMG 23779T).

Humicoccus flavidus gen. nov., sp. nov., isolated from soil.

A Gram-positive, non-motile, spherical, non-spore-forming bacterial strain, DS-52(T), was isolated from soil from Dokdo, Korea, and its taxonomic position was investigated by using a polyphasic approach. It grew optimally at 25 degrees C and pH 6.0-7.0. Strain DS-52(T) had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and galactose, mannose, xylose and rhamnose as whole-cell sugars. It contained MK-8(H(4)) and MK-9(H(4)) as the predominant menaquinones and anteiso-C(15 : 0), iso-C(15 : 0) and C(17 : 0) as major fatty acids. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyldimethylethanolamine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DS-52(T) is most closely related to the genus Nakamurella of the suborder Frankineae. Strain DS-52(T) exhibited 16S rRNA gene sequence similarity values of 96.5 % to Nakamurella multipartita JCM 9543(T) and 92.0-93.9 % to other members of the suborder Frankineae. The diagnostic diamino acid type and polar lipid profile of strain DS-52(T) were the same as those of the genus Nakamurella. However, strain DS-52(T) could be clearly distinguished from the genus Nakamurella by differences in predominant menaquinones, major fatty acids and cell-wall sugars. Accordingly, based on combined phenotypic, chemotaxonomic and phylogenetic data, strain DS-52(T) (=KCTC 19127(T)=CIP 108919(T)) is proposed as the type strain of a novel species in a new genus, Humicoccus flavidus gen. nov., sp. nov.

Multiple-stress tolerance of ionizing radiation-resistant bacterial isolates obtained from various habitats: correlation between stresses.

Isolation of five ionizing radiation (IR)-resistant bacteria by screening of isolates from various habitats classified as common and stressed is reported. IR-resistant isolates exhibited varying degrees of resistance to gamma-radiation and were classified as highly and moderately radiation resistant. Resistance to ultraviolet (UV) radiation correlated well with gamma-radiation resistance, whereas a comparable desiccation resistance for all the highly and moderately radiation-resistant isolates was observed. However, salt tolerance failed to correlate with IR resistance, indicating a divergent evolution of the salt tolerance and radiation resistance. Characterization of isolates by the amplified rDNA restriction analysis profiling attested to the clustering of these isolates with their stress phenotype. 16S rRNA gene-based analysis of the isolates showed that the bacteria with similar-resistance physiologies clustered together and belonged to related genera. Hydrogen peroxide resistance and mitomycin survival patterns of the isolates indicated the roles of oxidative-stress tolerance in desiccation survival and recombination repair in higher radiation resistance, respectively.

In vitro activities of the lipopeptides palmitoyl (Pal)-Lys-Lys-NH(2) and Pal-Lys-Lys alone and in combination with antimicrobial agents against multiresistant gram-positive cocci.

The in vitro activities of the lipopeptides palmitoyl (Pal)-Lys-Lys-NH(2) and Pal-Lys-Lys against gram-positive cocci were investigated. Enterococci and streptococci demonstrated higher susceptibilities than staphylococci and Rhodococcus equi. A positive interaction was shown when the lipopeptides were combined with beta-lactams and vancomycin. These results suggest that lipopeptides are promising candidates for antimicrobial therapy for infections caused by gram-positive organisms.

Bacteremia after diagnostic conventional laparoscopy and minilaparoscopy: a prospective study in 100 patients.

BACKGROUND/GOALS: Diagnostic laparoscopy under sedoanalgesia is a valuable tool in the work-up of liver diseases and is helpful as a staging procedure. The rate of bacteremia caused by this procedure is unknown, in particular when performed as minilaparoscopy. STUDY: A 100 consecutive patients having undergone diagnostic laparoscopy carried out either conventionally (group I, n=50) or as minilaparoscopy (group II, n=50) were prospectively enrolled in this study. Blood cultures were drawn before and within 5 minutes after the procedure. Risk factors for bacteremia were evaluated. RESULTS: Bacterial growth occurred in 4 blood cultures drawn immediately after laparoscopy. No patient developed fever or other signs of infection in the follow-up. Risk factors predisposing to bacteremia could not be identified. CONCLUSIONS: Conventional diagnostic laparoscopy under sedoanalgesia and minilaparoscopy are associated with a low rate of bacteremia as in diagnostic upper endoscopy.

Biodegradation of benzene and its derivatives by a psychrotolerant and moderately haloalkaliphilic Planococcus sp. strain ZD22.

The potential for biodegradation of aromatic hydrocarbons simultaneously at low temperatures and under saline and alkaline conditions is not well understood, but such biodegradation would be useful for remediation of polluted sites. A psychrotolerant, moderately haloalkaliphilic pure culture using benzene as a sole source of carbon and energy was isolated by selective enrichment from alkaline and saline soils in the vicinity of the Daqing oil field in China. An analysis of the 16S rDNA gene sequence and morphological and physiological characteristics showed that this strain is a member of the genus Planococcus, and it was designated as strain ZD22. Strain ZD22 could grow at temperatures between 2 and 36 degrees C (pH 7.5-11) and salt concentrations from 0.5 to 25%. Its optimal conditions for biodegradation of benzene were 20 degrees C (pH 9.5) and 10% salt concentration. Strain ZD22 not only utilized benzene, toluene, ethylbenzene and o-xylene, but also degraded chlorobenzene, bromobenzene, iodobenzene and fluorobenzene. The kinetic model of strain ZD22 for benzene was solved to obtain mumax=0.34 h-1, Ks=0.041 mM, n=1.21, Sm=10.2 mM. To our knowledge, this is the first report of biodegradation of benzene and its derivatives simultaneously under multiple extreme conditions.

Quadrisphaera granulorum gen. nov., sp. nov., a Gram-positive polyphosphate-accumulating coccus in tetrads or aggregates isolated from aerobic granules.

A Gram-positive bacterium, designated strain AG019(T), was isolated by micromanipulation from aerobic granules obtained from a laboratory-scale sequencing batch reactor. This isolate grew axenically as cocci clustered predominantly in tetrads, and was morphologically similar to the dominant organisms observed in the biomass. The morphology also resembled that of the tetrad-forming organisms commonly seen in activated sludge samples. Strain AG019(T) was found to be an oxidase-negative, catalase-positive, non-motile aerobe that does not reduce nitrate and grows at temperatures between 15 and 40 degrees C, with an optimum at 37 degrees C. The pH range for growth was 5.0-9.0, with an optimum at pH 7.5. Strain AG019(T) contained a peptidoglycan with directly cross-linked meso-diaminopimelic acid (type A1gamma) and lacked mycolic acids. The G+C content of the DNA was 75 mol%. Menaquinone MK-8(H(2)) was the major isoprenoid quinone. The bacterium stained positively for intracellular polyphosphate granules but not for poly-beta-hydroxyalkanoates. It produced capsular material and showed autoaggregation ability. Phenotypic and 16S rRNA gene analyses showed that the bacterium differed sufficiently from its closest phylogenetic relatives, namely members of the suborder Frankineae, which includes the genera Geodermatophilus, Blastococcus, Frankia, Sporichthya, Acidothermus and Microsphaera, that it is proposed that it be placed in a novel genus, Quadrisphaera, as Quadrisphaera granulorum gen. nov., sp. nov. The type strain is AG019(T) (=ATCC BAA-1104(T)=DSM 44889(T)).

Tetragenococcus koreensis sp. nov., a novel rhamnolipid-producing bacterium.

A Gram-positive, non-motile, non-spore-forming coccus (strain JS(T)) was isolated from kimchi (a traditional Korean food) and investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between strain JS(T) and its closest relative, Tetragenococcus halophilus IAM 1676(T), was 98.1%. The level of DNA-DNA relatedness between the two strains was 9.7%. Strain JS(T) had a DNA G+C content of 38.3% and a cellular fatty acid profile containing 16:0, 18:1 and cyclo fatty acids. Phylogenetic data and genomic and phenotypic features demonstrated that strain JS(T) represents a novel species, for which the name Tetragenococcus koreensis sp. nov. is proposed. The type strain is JS(T) (=KCTC 3924(T)=DSM 16501(T)=LMG 22864(T)).

Atopococcus tabaci gen. nov., sp. nov., a novel Gram-positive, catalase-negative, coccus-shaped bacterium isolated from tobacco.

A novel Gram-positive, aerobic, catalase-negative, coccus-shaped organism originating from tobacco was characterized using phenotypic and molecular taxonomic methods. The organism contained a cell wall murein based on L-lysine (variation A4alpha, type L-lysine-L-glutamic acid), synthesized long-chain cellular fatty acids of the straight-chain saturated and monounsaturated types (with C(16:1)omega9, C(16:0) and C(18:1)omega9 predominating) and possessed a DNA G+C content of 46 mol%. Based on morphological, biochemical and chemical characteristics, the coccus-shaped organism did not conform to any presently recognized taxon. Comparative 16S rRNA gene sequencing studies confirmed the distinctiveness of the unknown coccus, with the bacterium displaying sequence divergence values of greater than 7% with other recognized Gram-positive taxa. Treeing analysis reinforced its distinctiveness, with the unidentified organism forming a relatively long subline branching at the periphery of an rRNA gene sequence cluster which encompasses the genera Alloiococcus, Allofustis, Alkalibacterium, Atopostipes, Dolosigranulum and Marinilactibacillus. Based on phenotypic and molecular phylogenetic evidence, it is proposed that the unknown organism from tobacco be classified as a new genus and species, Atopococcus tabaci gen. nov., sp. nov. The type strain of Atopococcus tabaci is CCUG 48253(T) (=CIP 108502(T)).

Planococcus stackebrandtii sp. nov., isolated from a cold desert of the Himalayas, India.

The taxonomic position of a bacterium isolated from a cold desert of the Himalayas, India, was analysed by using a polyphasic approach. The isolated strain, designated K22-03T, had phenotypic characteristics that matched those of the genus Planococcus and it represents a novel species. The almost-complete 16S rRNA gene sequence (1464 bases) of the novel strain was compared with those of previously studied Planococcus type strains and confirmed that the strain belongs to the genus Planococcus. 16S rRNA gene sequence analysis indicated that strain K22-03T differs from all other species of Planococcus by at least 2.5 %. DNA-DNA hybridization showed that it had low genomic relatedness with Planomicrobium mcmeekinii (MTCC 3704T, 23 %), Planococcus psychrophilus (MTCC 3812T, 61 %), Planococcus antarcticus (MTCC 3854T, 45 %) and Planomicrobium okeanokoites (MTCC 3703T, 51 %), the four species with which it was most closely related based on 16S rRNA gene sequence analysis (97-97.5 % similarity). Therefore, strain K22-03T should be recognized as a novel species, for which the name Planococcus stackebrandtii sp. nov. is proposed. The type strain is K22-03T (=MTCC 6226T=DSM 16419T=JCM 12481T).

Planococcus rifietensis sp. nov, isolated from algal mat collected from a sulfurous spring in Campania (Italy).

The taxomony of strain M8, isolated from algal mat formed at the origin of a sulfurous spring in Rifieto (Savignano Irpino, Campania, Italy), was investigated in a polyphasic approach. The morphological, physiological and genetic characteristics were compared with of Planococcus and Planomicrobium species. The isolate grew optimally at pH 9.0, 1.8 M NaCl at 37 degrees C. The cells were Gram-positive cocci that form pairs, tetrads and aggregates of several cells. The isolate was aerobic/microaerophilic and accumulated glycine-betaine, as a major osmolyte, with minor components glutamate and an unknown compound. M8 was able to hydrolyse X-Glc (5-bromo-4-chloro-3-indoyl beta-d-glucopyranoside). The polar lipid profile consisted of phosphatidylglycerol and diphosphatidylglycerol as major components, and phosphocholine as a minor compound. MK8 was the only quinone found and the fatty acid composition was dominated by branched acids, mainly aiC15:0. The G+C content of DNA was 47.9% and its phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus Planococcus. The DNA/DNA similarity of M8 to the type species Planococcus citreus was less than 55%. For this reason and for physiological and chemotaxonomic features, it is proposed to create a new species Planococcus rifietensis sp. nov.