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Background/purpose: Ceftaroline, with a unique activity against methicillin-resistant Staphylococcus aureus (MRSA), was not launched in Taiwan before 2019. The in vitro susceptibility data of ceftaroline against important Taiwanese pathogens are lacking., Methods: The in vitro susceptibility of ceftaroline against important pathogens collected from 2012 through 2018 were extracted from the Antimicrobial Testing Leadership and Surveillance program. Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) to ceftaroline against all isolates., Results: During the study period, the in vitro data regarding isolates of S. aureus (n = 2049), Staphylococcus epidermidis (n = 185), Streptococcus pneumoniae (n = 334), Streptococcus pyogenes (n = 170), Haemophilus influenzae (n = 75), Haemophilus parainfluenzae (n = 10) and Klebsiella pneumoniae (n = 680) regardless of hospital sites of collection were analyzed. Among the S. aureus isolates studied, 19.4% showed MICs of 1 mg/L to ceftaroline, and 4.4% showed in vitro susceptible-dose dependent to ceftaroline (all MICs, 2 mg/L). Most of other Gram-positive cocci, all H. influenzae and H. parainfluenzae isolates were susceptible to ceftaroline. By contrast, about one-third (35.9%) of K. pneumoniae isolates, irrespective of infection sources, exhibited non-susceptibility to ceftaroline (MIC range, 0.015-256 mg/L; MIC 50 and MIC 90 values, 0.12 and 256 mg/L, respectively)., Conclusions: From the pharmacodynamic perspectives, the ceftaroline dosage of 600 mg as a 2-h intravenous infusion every 8 h is effective against all S. aureus and other Gram-positive isolates regardless of acquisition sites in Taiwan. Before ceftaroline is prescribed in treatment of the patient with Gram-negative infection, a cautious evaluation about patient's healthcare-associated factor is warranted., Competing Interests: Declaration of Competing Interest We declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

An obligately anaerobic, Gram-positive, non-motile, coccus-shaped bacterial strain designated AGMB00490 T was isolated from swine faeces. 16S rRNA gene sequence-based phylogenetic analysis indicated that the isolate belongs to the genus Peptoniphilus and that the most closely related species is Peptoniphilus gorbachii WAL 10418 T (=KCTC 5947 T , 97.22 % 16S rRNA gene sequence similarity). Whole genome sequence analysis determined that the DNA G+C content of strain AGMB00490 T was 31.2 mol% and moreover that the genome size and numbers of tRNA and rRNA genes were 2 129 517 bp, 34 and 10, respectively. Strain AGMB00490 T was negative for oxidase and urease; positive for catalase, indole production, arginine arylamidase, leucine arylamidase, tyrosine arylamidase and histidine arylamidase; and weakly positive for phenylalanine arylamidase and glycine arylamidase. The major cellular fatty acids (>10 %) of the isolate were determined to be C 16 : 0 and C 18 : 1  ω9 c . Strain AGMB00490 T produced acetic acid as a major end product of metabolism. Accordingly, phylogenetic, physiologic and chemotaxonomic analyses revealed that strain AGMB00490 T represents a novel species for which the name Peptoniphilus faecalis sp. nov. is proposed. The type strain is AGMB00490 T (=KCTC 15944 T =NBRC 114159 T ).

We report a case of surgical site infection after total hip prosthesis replacement due to an ofloxacin-resistant Peptoniphilus isolate belonging to an unknown species for which the name 'Peptoniphilus nemausus' sp. nov. is proposed. Follow-up was favourable under clindamycin and rifampin for 3 months in this patient whom had a Proteus mirabilis infection treated by fluoroquinolone., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction. Among the causative agents of bloodstream infections (BSIs), methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus (VRE) are the key causative pathogens. Their rapid detection directly from Gram-positive cocci-positive blood culture specimens will promote timely treatment and help to implement effective infection control measures. Aim. We aim to develop a PCR-dipstick technique for the rapid detection of MRSA and VRE directly from positive blood culture specimens. Methodology. PCR-dipstick is a PCR-based multiplex detection technique where DNA-DNA hybridization is employed, and the results are interpreted with the naked eye. It was designed to target three drug resistance genes: mecA in MRSA and vanA / vanB in VRE from positive blood culture specimens. A total of 120 clinical isolates were used to evaluate the sensitivity and specificity of PCR-dipstick. Then, PCR-dipstick was examined for MRSA and VRE detection directly from positive blood cultures. Results. PCR-dipstick showed 100 % sensitivity and specificity in detecting mecA , vanA and vanB genes directly from bacterial colonies in comparison with multiplex PCR for genomic DNA followed by agarose gel electrophoresis. Further, it could differentially detect multiple resistant genes in pooled bacterial colonies ( n =10). Ultimately, PCR-dipstick could detect MRSA and VRE in positive blood cultures in ~3 h. Conclusion. The results of the current study substantiate that PCR-dipstick can be used as an efficient detection system for MRSA and VRE directly from Gram-positive cocci-positive blood cultures. Its affordability and rapidity indicate that PCR-dipstick can be an effective tool for controlling nosocomial pathogens.

Objective: Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) is widely used for fast identification of bacteria from blood cultures (BC). We compared the performance of two procedures, one including a pre-enrichment step in brain heart infusion and the other a direct method using vacutainer separator gel tubes (DI), for identification of bacteria from blood cultures by MALDI-TOF MS., Methods: We first prepared a training set of 20 simulated bacteremia specimens, including 10 Gram-negative and 10 Gram-positive species. A total of 145 non-consecutive BCs flagged as positive (68 Gram-negative rods, and 77 Gram-positive cocci) were prospectively analyzed (validation set)., Results: A total of 82% and 49% of isolates were correctly identified to the species level by the respective methods., Conclusions: The pre-enrichment method outperformed the DI method for identification of virtually all bacterial species included in the panels., (©The Author 2019. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).)

Gut microbiome development affects infant health and postnatal physiology. The gut microbe assemblages of preterm infants have been reported to be different from that of healthy term infants. However, the patterns of ecosystem development and inter-individual differences remain poorly understood. We investigated hospitalised preterm infant gut microbiota development using 16S rRNA gene amplicons and the metabolic profiles of 268 stool samples obtained from 17 intensive care and 42 term infants to elucidate the dynamics and equilibria of the developing microbiota. Infant gut microbiota were predominated by Gram-positive cocci, Enterobacteriaceae or Bifidobacteriaceae , which showed sequential transitions to Bifidobacteriaceae -dominated microbiota. In neonatal intensive care unit preterm infants (NICU preterm infants), Staphylococcaceae abundance was higher immediately after birth than in healthy term infants, and Bifidobacteriaceae colonisation tended to be delayed. No specific NICU-cared infant enterotype-like cluster was observed, suggesting that the constrained environment only affected the pace of transition, but not infant gut microbiota equilibrium. Moreover, infants with Bifidobacteriaceae -dominated microbiota showed higher acetate concentrations and lower pH, which have been associated with host health. Our data provides an in-depth understanding of gut microbiota development in NICU preterm infants and complements earlier studies. Understanding the patterns and inter-individual differences of the preterm infant gut ecosystem is the first step towards controlling the risk of diseases in premature infants by targeting intestinal microbiota.

Objectives: Gram-positive, anaerobic cocci (GPAC) can cause infections in humans. Only a few cases of bacteraemia with GPAC have been reported. We describe the clinical and microbiological characteristics of GPAC bacteraemia., Methods: A retrospective population-based study of GPAC bacteraemia 2012-2016 in southern Sweden was performed. GPAC were identified using matrix-associated laser desorption ionization time-of-flight mass spectrometry or 16S rRNA gene sequencing. Etests were used to determine antibiotic susceptibilities. Data on patient and infection characteristics, treatment, and outcome were collected from the medical records., Results: A total of 226 episodes of GPAC bacteraemia in adults were studied; this corresponds to an annual incidence of 3.4 cases per 100,000 persons per year. The bacteria identified were Anaerococcus spp. (n = 43), Atopobium spp. (n = 7), Blautia spp. (n = 1), Finegoldia spp. (n = 15), Parvimonas spp. (n = 100), Peptoniphilus spp. (n = 52), Peptostreptococcus spp. (n = 2), and Ruminococcus spp. (n = 9) of which 200 isolates were identified to the species level. Resistance to imipenem and piperacillin was not identified, whereas resistance among the 229 isolates to penicillin was detected in four, to metronidazole in six, and clindamycin in 16 isolates. The median age of patients was 73 years (55-83, IQR), 57% were male and comorbidities were common. Fifty-one per cent of infections were polymicrobial. In 60% of cases a focus of infection was identified. Forty per cent of patients had either organ dysfunction or shock. The 30-day mortality was 11%, and nosocomial infections were over-represented among the deceased., Conclusions: GPAC bacteraemia is much more common than previously reported. GPAC-bacteraemia is a condition with significant mortality mainly affecting elderly persons with comorbidities., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Dissemination of pathogenic multiresistant bacteria is of public health concern. Reliable data can be difficult to obtain, especially in developing countries. This work aimed to characterize the skin and oropharyngeal microbiota, as well as their antimicrobial resistance profiles, of East-Timor populations to identify potentially pathogenic Gram-positive cocci. In order to assess the prevalence of pathogenic bacteria in East-Timor, the oropharyngeal and skin microbiota of 74 individuals was characterized. Gram-positive cocci were evaluated and their antimicrobial resistance profiles were determined. A total of 228 oropharyngeal and 278 skin samples were obtained. The population consisted of 36.5% of asymptomatic carriers of Gram-positive cocci. Kocuria rosea (n = 7, 19.4%), Staphylococcus spp. (n = 6, 16.7%), and Micrococcus luteus (n = 6, 16.7%) were isolated, among others. Antimicrobial resistance levels ranged between 0% and 36.1%, and a multiresistance profile was observed in one third of the isolates. Gram-positive cocci colonization was associated with age group. Prevalence of multiresistant isolates was higher in males who were sampled at the refugee camp. Results show that the prevalence of antimicrobial resistance on East Timor may be underestimated. This study represents the first step toward the full characterization of potential pathogenic Gram-positive cocci present in the populations from East Timor.

Strain Marseille-P2341 T , a nonmotile, nonspore-forming, Gram-stain-positive anaerobic coccus, was isolated in the vaginal specimen of a patient with bacterial vaginosis using culturomics. Its growth occurred at temperatures ranging from 25 to 42°C, with pH between 6.5 and 8.5, and at NaCl concentrations lower than 5%. The major fatty acids were C 18:1n9 (27.7%) and C 16:0 (24.4%). Its genome is 1,671,491 bp long with 49.48 mol% of G+C content. It is composed of 1,501 genes: 1,446 were protein-coding genes and 55 were RNAs. Strain Marseille-P2341 T shared 97.3% of 16S rRNA gene sequence similarity with Murdochiella asaccharolytica, the phylogenetically closest species. These results enabled the classification of strain Marseille-P2341 T as a new species of the genus Murdochiella for which we proposed the name Murdochiella vaginalis sp. nov. The type strain is strain Marseille-P2341 T (=DSM 102237, =CSUR P2341)., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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.

Finegoldia magna, a Gram-positive anaerobic coccus, is an opportunistic pathogen, associated with medical device-related infections. F. magna is the only described species of the genus Finegoldia. We report the analysis of 17 genomes of Finegoldia isolates. Phylogenomic analyses showed that the Finegoldia population can be divided into two distinct clades, with an average nucleotide identity of 90.7%. One clade contains strains of F. magna, whereas the other clade includes more heterogeneous strains, hereafter tentatively named "Finegoldia nericia". The latter species appears to be more abundant in the human microbiome. Surface structure differences between strains of F. magna and "F. nericia" were detected by microscopy. Strain-specific heterogeneity is high and previously identified host-interacting factors are present only in subsets of "F. nericia" and F. magna strains. However, all genomes encode multiple host factor-binding proteins such as albumin-, collagen-, and immunoglobulin-binding proteins, and two to four copies of CAMP (Christie-Atkins-Munch-Petersen) factors; in accordance, most strains show a positive CAMP reaction for co-hemolysis. Our work sheds new light of the genus Finegoldia and its ability to bind host components. Future research should explore if the genomic differences identified here affect the potential of different Finegoldia species and strains to cause opportunistic infections.

Background: The present investigation was an endeavor into the elucidation of the disease-causing pathogen of streptococcosis in Nile tilapia (Oreochromis niloticus) in Egypt affecting adult fish cultured and wild fish in the Nile river. Fish were obtained from commercial fishermen, collected as part of their routine fishing activities. The researchers observed the routine fishing process and selected fish for use in the study, at the point of purchase from the fisherman., Results: Diseased fish showed exophthalmia with accumulation of purulent and haemorrhagic fluid around eyes, and ventral petechial haemorrhages. The Post mortem examination revealed, abdominal fat haemorrhage, pericarditis and enlargement of the liver, spleen and kidney. Gram-stained smears revealed the presence of Gram-positive cocci, β-hemolytic, oxidase and catalase negative. Analysis of the 16S rRNA gene confirmed that the 17 tilapia isolates studied were 6/17 Enterococcus faecalis, 2/17 Enterococcus gallinarum, 3/17 Streptococcus pluranimalium, 2/17 Aerococcus viridans, 1/17 isolate of each Streptococcus dysgalactiae, Streptococcus anginosus, Lactococcus garvieae and Granulicetella elegans/Leuconostoc mesenteroides cremoris. It should be noted that there was no mixed infection. Multiple resistance was observed and the most frequent antibiotic combination was penicillin, ampicillin, vancomycin, chloramphenicol, rifampicin, ofloxacin, clindamycin, erythromycin and tetracycline representing eight classes., Conclusions: Consequently, we concluded that Streptococcus species are an emerging pathogen for Nile tilapia aquaculture in Egypt and to be considered as a new candidate in the warm water fish diseases in Egypt with special reference to L. garvieae, S. dysgalactiae in addition to L. mesenteroides cremoris which was not reported before from tilapia and taking into consideration their zoonotic implications for public health.

Introduction: In the structure of tuberculosis morbidity, urogenital tuberculosis (UGT) ranks second or third only to pulmonary tuberculosis. Every fourth patient has the stage of cavern formation at first presentation. Seventy five percent of nephrotuberculosis cases co-occur with nonspecific pyelonephritis, which excludes "sterile pyuria". We aimed to determine the microbial spectrum of urine and gonadal appendages in patients with suspected UGT and to investigate the susceptibility of the isolated microorganisms to the main antibacterial drugs., Material and Methods: The study investigated the spectrum of pathogenic microflora isolated from patients admitted to the urogenital clinic of the TB Research Institute from January 1, 2012 to September 30, 2016 to rule out UGT. Microorganism species were identified by time-of-flight mass spectrometry using the microbiological analyzer Phoenix 100 (Becton Dickinson, USA). Antibiotic resistance was determined using the Phoenix 100 microbiological analyzer (Becton Dickinson, USA)., Results: A total of 3004 bacteriological tests for nonspecific microflora were performed during the analyzed period. In the structure of pathogens, E. coli comprised about half of the cases thus confirming its role as the leading uropathogen. Only piperacillin/tazobactam and nitrofurantoin provided relatively plausible antimicrobial resistance rates, for other antibacterial drugs the rates of susceptible strains progressively decreased and by 2016 dropped to 40% and lower., Conclusion: Our findings cannot be extrapolated to the entire population since patients presenting for differential diagnosis receive multiple courses of treatment with various antibacterial drugs, which led to the selection of resistant strains. The findings emphasize the need for a strict selection of antibiotics for urogenital infection empirical therapy. In the absence of response, the patient should be immediately examined for tuberculosis using molecular and genetic diagnostic techniques.

A study was conducted to evaluate Sensititre ® Automated Reading and Incubation System 2x System (ARIS), API ® (API), and Bruker MALDI-TOF MS (MALDI) bacterial species identification systems using 132 diverse bacterial isolates from bovine milk samples and bulk tank milk received at the Penn State Animal Diagnostic Laboratory. The results were compared with 16S rRNA gene sequence analysis, which served as the reference method for species identification. The ARIS, API, and MALDI identified 0%, 40%, and 33.4% of species classified as Gram-positive rod isolates belonging to genera Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, and Corynebacterium, respectively. It was observed that 76.5%, 93.9%, and 96.9% of catalase-negative, Gram-positive cocci (n = 33; Aerococcus, Enterococcus, Lactococcus, Streptococcus) were correctly identified to the species level by ARIS, API, and MALDI, respectively, while 33.4%, 84.5%, and 97.7% of catalase-positive, Gram-positive cocci (n = 45; Kocuria, Staphylococcus) were correctly identified to their species by ARIS, API, and MALDI, respectively. A total of 48 isolates (Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea, Pasteurella, Providencia, Pseduomonas, Serratia) of Gram-negative bacteria were examined, of which 85.4%, 93.7%, and 95.8% of the isolates were correctly identified to the species level by ARIS, API, and MALDI, respectively. In our laboratory, the MALDI had the least costs associated with consumables and reagents compared to ARIS, API, and 16S rRNA identification methods. Identification of bacterial species was accomplished in <2 h using MALDI and 24 h for ARIS, API, and 16S rRNA identification systems.

Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a rapid diagnostic assay that can identify certain organisms growing in blood cultures 30-90 min from the time of positive Gram-stain. Existing studies have demonstrated a clinical utility with this assay when antibiotic stewardship programs assist clinicians with interpreting the results. However, the benefit of these rapid assays in the absence of concomitant antibiotic stewardship involvement is unclear. In this randomized study of 220 patients with enterococcal or streptococcal bacteremia, we found that PNA FISH, in the absence of concomitant input from an antibiotic stewardship program, had no impact on time to effective or optimal therapy, length of hospital stay, or in-hospital mortality. Our results suggest that in the absence of guidance from an antibiotic stewardship program, the clinical benefits of rapid diagnostic microbiological tools may be reduced., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A novel Gram-stain-positive, coccus-shaped, obligately anaerobic bacterium was isolated from a faecal sample obtained from an individual in a traditional community located off the southern coast of Peru. Comparative 16S rRNA gene sequence analysis showed the novel bacterium belonged to the genus Peptoniphilus but showed no particular relationship with any species, demonstrating less than 91 % 16S rRNA gene sequence similarity with all members of the genus. The major cellular fatty acids of the novel isolate were determined to be C10 : 0, C14 : 0, C16 : 0, C18 : 1ω9c and C18 : 2ω6,9c/anteiso-C18 : 0. The DNA G+C content was 34.4 mol%. End-products of metabolism from peptone-yeast-glucose broth (PYG) were determined to be acetate and butyrate. Based on the phenotypic, chemotaxonomic and phylogenetic results, the organism represents a novel species of the genus Peptoniphilus, for which the name Peptoniphilus catoniae sp. nov. is proposed. The type strain is M6.X2DT ( = DSM 29874T = CCUG 66798T).

The diagnosis of urinary tract infection (UTI) by urine culture is a time-consuming and costly procedure. Usage of a screening method, to identify negative samples, would therefore affect time-to-diagnosis and laboratory cost positively. Urine flow cytometers are able to identify particles in urine. Together with the introduction of a cut-off value, which determines if a urine sample is subsequently cultured or not, the number of cultures can be reduced, while maintaining a low level of false negatives and a high negative predictive value. Recently, Sysmex developed additional software for their urine flow cytometers. Besides measuring the number of bacteria present in urine, information is given on bacterial morphology, which may guide the physician in the choice of antibiotic. In this study, we evaluated this software update. The UF1000i classifies bacteria into two categories: 'rods' and 'cocci/mixed'. Compared to the actual morphology of the bacterial pathogen found, the 'rods' category scores reasonably well with 91% chance of classifying rod-shaped bacteria correctly. The 'cocci/mixed' category underperforms, with only 29% of spherical-shaped bacteria (cocci) classified as such. In its current version, the bacterial morphology software does not classify bacteria, according to their morphology, well enough to be of clinical use in this study population., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media., Results: Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0)., Conclusions: The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

We evaluated the performance of two MALDI-TOF MS systems for the identification of clinically important Gram-positive cocci. Vitek MS and Microflex LT correctly identified 97.2% and 94.7%, respectively. Both systems offer reliable and rapid identification of clinically important Gram-positive cocci isolated in clinical laboratories, including staphylococci, streptococci, and enterococci. Expanding the databases, especially of coagulase-negative staphylococci and viridans streptococci, would enhance performance., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Periodontitis is a highly prevalent polymicrobial disease worldwide, yet the synergistic pattern of the multiple oral pathogens involved is still poorly characterized. Here, saliva, supragingival and subgingival plaque samples from periodontitis patients and periodontally healthy volunteers were collected and profiled with 16S rRNA gene pyrosequencing. Different oral habitats harbored significantly different microbiota, and segregation of microbiota composition between periodontitis and health was observed as well. Two-step redundancy analysis identified twenty-one OTUs, including Porphyromonas gingivalis, Tannerella forsythia and Filifactor alocis, as potential pathogens that were significantly associated with periodontitis and with two periodontitis diagnostic parameters (pocket depth and attachment loss) in both saliva and supragingival plaque habitats. Interestingly, pairwise correlation analysis among the 21 OTUs revealed that Filifactor alocis was positively correlated with seven other putative pathogens (R > 0.6, P < 0.05), forming a co-occurrence group that was remarkably enriched in all three habitats of periodontitis patients. This bacterial cluster showed a higher diagnostic value for periodontitis than did any individual potential pathogens, especially in saliva. Thus, our study identified a potential synergistic ecological pattern involving eight co-infecting pathogens across various oral habitats, providing a new framework for understanding the etiology of periodontitis and developing new diagnoses and therapies.

Heifer mastitis is a well-known problem, with several pathogens being involved. Several generic risk factors associated with the likelihood of intramammary infections (IMI) in fresh dairy heifers have been identified before. Yet, a need exists to identify pathogen group-specific factors, as the effect of (groups of) pathogens on udder health and milk yield is different. The aim of the present study was to identify pathogen group-specific risk factors for IMI in heifers participating in a prepartum antimicrobial treatment trial, allowing us to test the hypothesis that different factors are of importance between treated and untreated control heifers as well. Data from a clinical trial in which end-term heifers were treated systemically (over 3 consecutive days) 2 wk before calving with penethamate hydriodide (n=76) or remained untreated (n=73), were available. Several potential risk factors at the herd, heifer, and quarter level were recorded in the first 3 d in milk. Quarters from untreated heifers supplemented with ≥4 mg of selenium/d prepartum were significantly less likely to be infected with coagulase-negative staphylococci (CNS), whereas quarters were more likely to be infected with CNS when assistance during calving was needed. Udder edema before calving significantly decreased the odds of IMI with major pathogens. In treated heifers, no factors were detected that were associated with the likelihood of CNS IMI, whereas quarters from heifers were significantly more likely to be infected with major pathogens when they were housed in the calving pen more than 1 d and when they had been in contact with the lactating cows before calving. The risk factors for IMI that were identified in treated heifers were different than those in untreated heifers, independent of the pathogen group that was considered. It looks as if prepartum treatment not only changed the likelihood of infection, but also the factors that were associated with infection. However, except for treated heifers with an IMI with major pathogens, only a small proportion of the variation could be explained in the final models. Therefore, factors other than those that were studied could explain the likelihood of infection., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A species of a previously unknown Gram-positive-staining, anaerobic, coccus-shaped bacterium recovered from a swine manure storage tank was characterized using phenotypic, chemotaxonomic, and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies and biochemical characteristics demonstrated that this organism is genotypically and phenotypically distinct, and represents a previously unknown sub-line within the order Clostridiales, within the phylum Firmicutes. Pairwise sequence analysis demonstrated that the novel organism clustered within the genus Peptoniphilus, most closely related to Peptoniphilus methioninivorax sharing a 16S rRNA gene sequence similarity of 95.5%. The major long-chain fatty acids were found to be C14:0 (22.4%), C16:0 (15.6%), C16:1ω7c (11.3%) and C16 : 0 ALDE (10.1%) and the DNA G +C content was 31.8 mol%. Based upon the phenotypic and phylogenetic findings presented, a novel species Peptoniphilus stercorisuis sp. nov. is proposed. The type strain is SF-S1(T) ( = DSM 27563(T) = NBRC 109839(T)). In addition, it is proposed to accommodate the genera Peptoniphilus, Anaerococcus, Anaerosphaera, Finegoldia, Gallicola, Helcococcus, Murdochiella and Parvimonas in a new family of the order Clostridiales, for which the name Peptoniphilaceae fam. nov. is proposed; the type genus of the family is Peptoniphilus.

Background: The routine diagnosis of Streptococcus spp. and other mastitis associated gram-positive, catalase-negative cocci is still based upon biochemical tests and serological methods, which frequently provide ambiguous identification results. We therefore aimed to establish an accurate identification system for differential diagnosis of mastitis associated Streptococcus spp. and related species using biophysical techniques such as Fourier-transform infrared (FTIR) spectroscopy and MALDI - TOF/MS., Results: Based on a panel of 210 isolates from cases of bovine mastitis, an unsupervised FTIR spectral reference library was established and an artificial neural network (ANN) - assisted identification system was developed. All bacterial isolates were previously identified by species-specific PCR and/or 16S rRNA gene sequence analysis. An overall identification rate of 100% at species level for 173 strains unknown to the ANN and the library was achieved by combining ANN and the spectral database, thus demonstrating the suitability of our FTIR identification system for routine diagnosis. In addition, we investigated the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of mastitis associated Streptococcus spp. and related bacteria. Using the Microflex LT System, MALDI Biotyper software™ (V3.3) we achieved an accuracy rate of 95.2%. A blind study, including 21 clinical samples from dairy cows, revealed a 100% correct species identification rate for FTIR and 90.5% for MALDI-TOF MS, indicating that these techniques are valuable tools for diagnosis., Conclusions: This study clearly demonstrates that FTIR spectroscopy as well as MALDI-TOF MS can significantly improve and facilitate the identification and differentiation of mastitis associated Streptococcus spp. and related species. Although the FTIR identification system turned out being slightly superior to MALDI-TOF MS in terms of identification on species level, both methods offer interesting alternatives to conventional methods currently used in mastitis diagnosis as both of them provide high accuracy at low operating costs once the instrument is acquired.

Introduction: The genera Enterococcus, Staphylococcus and Streptococcus are recognized as important Gram-positive human pathogens. The aim of this study was to evaluate the performance of Vitek 2 in identifying Gram-positive cocci and their antimicrobial susceptibilities., Methods: One hundred four isolates were analyzed to determine the accuracy of the automated system for identifying the bacteria and their susceptibility to oxacillin and vancomycin., Results: The system correctly identified 77.9% and 97.1% of the isolates at the species and genus levels, respectively. Additionally, 81.8% of the Vitek 2 results agreed with the known antimicrobial susceptibility profiles., Conclusion: Vitek 2 correctly identified the commonly isolated strains; however, the limitations of the method may lead to ambiguous findings.

A novel non-sporulating, non-motile, catalase-positive, oxidase-negative, facultatively anaerobic, Gram-positive coccus, designated strain JSM 078151(T), was isolated from an intertidal sediment sample collected from Naozhou Island in the South China Sea, China. Growth was found to occur in the presence of 0-15 % (w/v) NaCl (optimum 0.5-3 % (w/v) NaCl), at pH 6.5-10.5 (optimum pH 7.0-8.0) and at 5-35 °C (optimum 25-30 °C). The peptidoglycan type was determined to be A3a, containing lysine, glutamic acid and alanine. The major cellular fatty acid identified was anteiso-C15:0 and the predominant menaquinones are MK-7 and MK-8. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, glycolipid and one unidentified phospholipid. The genomic DNA G+C content of strain JSM 078151(T) was determined to be 55.2 mol%. A phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 078151(T) should be assigned to the genus Rothia, and was most closely related to Rothia nasimurium CCUG 35957(T) (98.3 % sequence similarity), followed by Rothia amarae J18(T) (97.5 %) and Rothia terrae L-143(T) (97.3 %). A combination of phylogenetic analysis, DNA-DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the suggestion that strain JSM 078151(T) represents a novel species of the genus Rothia, for which the name Rothia marina sp. nov. is proposed. The type strain is JSM 078151(T) (= DSM 21080(T) = KCTC 19432(T)).

Few labs isolate and perform susceptibility tests on anaerobes; therefore, we studied the differences between 1185 anaerobes isolated from complicated intra-abdominal infections and 470 isolated from moderate to severe diabetic foot infections. They differed markedly in the distribution of species, including Bacteroides fragilis and anaerobic Gram-positive cocci, as well as in resistance patterns, especially to fluoroquinolones., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Among the Gram-positive anaerobic bacteria associated with clinical infections, the Gram-positive anaerobic cocci (GPAC) are the most prominent and account for approximately 25-30% of all isolated anaerobic bacteria from clinical specimens. Still, routine culture and identification of these slowly growing anaerobes to the species level has been limited in the diagnostic laboratory, mainly due to the requirement of prolonged incubation times and time-consuming phenotypic identification. In addition, GPAC are mostly isolated from polymicrobial infections with known pathogens and therefore their relevance has often been overlooked. However, through improvements in diagnostic and in particular molecular techniques, the isolation and identification of individual genera and species of GPAC associated with specific infections have been enhanced. Furthermore, the taxonomy of GPAC has undergone considerable changes over the years, mainly due to the development of molecular identification methods. Existing species have been renamed and novel species have been added, resulting in changes of the nomenclature. As the abundance and significance of GPAC in clinical infections grow, knowledge of virulence factors and antibiotic resistance patterns of different species becomes more important. The present review describes recent advances of GPAC and what is known of the biology and pathogenic effects of Anaerococcus, Finegoldia, Parvimonas, Peptoniphilus and Peptostreptococcus, the most important GPAC genera isolated from human infections., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.

In recent years, the Infectious Diseases Society of America has highlighted a faction of antibiotic-resistant bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) - acronymically dubbed 'the ESKAPE pathogens' - capable of 'escaping' the biocidal action of antibiotics and mutually representing new paradigms in pathogenesis, transmission and resistance. This review aims to consolidate clinically relevant background information on the ESKAPE pathogens and provide a contemporary summary of bacterial resistance, alongside pertinent microbiological considerations necessary to face the mounting threat of antimicrobial resistance.

Background: It remains important to have a thorough knowledge of the micro-flora harboring the white coats of doctors to minimize cross-contamination and improve patient safety by reducing the risk of nosocomial infections. This study presents the microbiological analysis of the white coats in clinical departments of a dental college and hospital., Materials and Methods: The swabs for the study were taken from the white coats of undergraduate students posted in various clinical departments, interns, and the post-graduate students. The microbial contamination was studied by observing and recording the colony morphology on the culture plates, Gram's staining with light microscopic screening of the slides, and the biochemical characterization of the isolates using standard microbiology protocols., Results: Microbiological analysis of swabs taken from the white coats in the dental operatory showed that 100% coats had bacterial contamination. Out of 30 swabs collected, 46 cultures were obtained. 50% cultures showed Gram-positive cocci, making it the major microbial group contaminating the white coats in the dental operatory., Conclusion: The presented study highlights the fact that the white coats are a potential source of cross infection. The results of this study mandate a strict audit process and protocols to be set in place for preventing cross-contamination from the white coats in a dental operatory.

Infective endocarditis (IE) is traditionally diagnosed by microbiological analysis of blood cultures, following which therapeutic antibiotics are chosen based on antimicrobial sensitivity tests. However, such conventional techniques do not always lead to an accurate etiological diagnosis. Recently, PCR analysis of the 16S rRNA gene has been employed to identify organisms isolated from excised heart valves. In this study, we analyzed 19 valve samples from patients with confirmed IE, as identified by Duke's criteria. Using broad-range PCR amplification, followed by direct gene sequencing, pathological agents were identified in all samples. Although blood cultures yielded negative results in 4 cases, PCR analysis of valve samples showed positive identification of causative organisms. In 3 cases, there was a difference between blood culture and PCR in identification of pathological agents, which are likely to be misidentified by the conventional method based on the phenotypic database. Postoperative antibiotics were chosen considering the severity of lesions and the results of PCR, Gram staining, and valve cultures. All patients were cured without relapse. The broad-range PCR method was therefore beneficial for the management of IE because it enabled us to identify pathogens directly from the site of infection, even organisms that were difficult to culture or likely to be misidentified by the conventional culture method. Identification of the agents provided precise knowledge of the microbiological spectrum involved in the cases of IE.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry with a Bruker Daltonics microflex LT system was applied to 90 well-characterized catalase-negative, Gram-positive cocci not belonging to the streptococci or enterococci. Biotyper version 2.0.43.1 software was used singly or in combination with a database extension generated in this study with 51 collection strains from 16 genera. Most strains were identified by using both databases individually, and some were identified only by applying the combined database. Thus, the methodology is very useful and the generated database extension was helpful.

The aim of this study was to compare the ecology of catalase-positive cocci (CPC) present in traditional fermented sausages produced using different breeds of pork, each of which was raised in two different environments and processed using two different technologies. Semi-quantitative molecular methods were used to determine bacterial identities. Almost all fermentations were characterised by a significant increase in CPC during the first few days of fermentation, reaching values of 10(5)-10(6) cfu g(-1) within 3 days. Staphylococcus xylosus and Staphylococcus equorum species, which were detected over the course of fermentation, were found to be the predominant population in all the monitored fermentation. Staphylococcus haemolyticus, Staphylococcus lentus, Micrococcus luteus, Macrococcus caseolyticus and Staphylococcus succinus were also present, but their concentrations were found to vary under the different experimental conditions. Using cluster analysis, we concluded that a plant-specific CPC ecology existed. In addition, the breed of pork used for production was found to influence the presence of some CPC species. However, from this study, it was not possible to reach the same conclusion regarding the breeding system used., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Rapid and accurate identification of bacterial pathogens grown in blood cultures of patients with sepsis is crucial for prompt initiation of appropriate therapy in order to decrease related morbidity and mortality rates. Although current automated blood culture systems led to a significant improvement in bacterial detection time, more rapid identification systems are still needed to optimise the establishment of treatment. Novel genotype technology which is developed for the rapid diagnosis of sepsis, is a molecular genetic assay based on DNA multiplex amplification with biotinylated primers followed by hybridization to membrane bound probes. The aim of this study was to evaluate the performance of "Genotype® BC gram-positive” test for the identification of gram-positive cocci grown in blood cultures and rapid detection of mecA and van genes. This test uses DNA.STRIP® technology which includes a panel of probes for identification of 17 gram-positive bacterial species and is able to determinate the methicillin and vancomycin resistance mediating genes (mecA and vanA, vanB, vanC1, vanC2/C3) simultaneously, in a single test run. A total of 55 positive blood cultures from BACTECTM Plus/F (Becton Dickinson, USA) aerobic and pediatric blood culture vials were included in the study. The isolates which exhibit gram-positive coccus morphology by Gram staining were identified by Genotype ® BC gram-positive test (Hain Life Science, Germany). All of the samples were also identified with the use of Phoenix PMIC/ID Panel (Becton Dickinson, USA) and antibiotic susceptibilities were determined. Of the 55 blood culture isolates, 17 were identified as Staphylococcus epidermidis [all were methicillin-resistant (MR)], 9 were S.aureus (one was MR), 18 were S.hominis (10 were MR), 4 were E.faecalis, 3 were E. faecium (one was vanconycin-resistant), 2 were S.saprophyticus (one was MR), 1 was S.warneri and 1 was S.haemolyticus, by Phoenix automated system. Genotype® BC gram-positive test results revealed consistency with Phoenix system regarding bacterial identification in 46 (83.6%) of the samples. The two bacteria identified as S.saprophyticus by the Phoenix system could not be identified by the Genotype® BC test since this species were not included in the identification panel of the system, however, mecA gene were detected in these two samples by Genotype® BC test. Genotype® BC test detected mecA gene in five samples which were not detected as methicillin resistant by the Phoenix system. Besides polymicrobial growth was determined in five samples by Genotype ® BC test, but not by the automated system. One E.faecium isolate with vanA gene was correctly identified by Genotype® BC test. In conclusion, Genotype® BC gram-positive test is a fast and reliable test for the identification of the most important gram-positive pathogens and mecA and van genes directly from positive blood culture bottles. This test was also found superior than the automated Phoenix system regarding the detection of polymicrobial growth. These data indicated that, routine use of DNA strip technology-based assay would be useful for clinical diagnosis in patients with sepsis.

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.

Strain NRRL B-23883(T) was isolated from retail ground beef as part of a study on the genetic diversity of Clostridium perfringens. The strain was found to be a strictly anaerobic, Gram-positive coccus that was able to utilize peptone as a sole carbon source. Analysis of the 16S rRNA gene sequence revealed that the strain was closely related to species within the genera Peptoniphilus and Anaerosphaera, but it was substantially different from the closest recognized species by nearly 10 % sequence divergence. The strain was also found to be closely related (>99 % sequence similarity) to an uncultured bacterial strain that was sequenced from a 16S rRNA gene clone library constructed to characterize the bacterial community of faeces from a captive spotted hyena. Strain NRRL B-23883(T) shared the peptidoglycan type A4β, l-Orn-d-Glu with members of the genus Peptoniphilus. Further phenotypic analysis revealed that strain NRRL B-23883(T) was able to utilize glycyl l-methionine as a sole carbon source, in contrast to other species of the genus Peptoniphilus. Therefore, it is proposed that the isolate represents a novel species, Peptoniphilus methioninivorax sp. nov.; the type strain is NRRL B-23883(T) ( = DSM 22461(T)).

We evaluated Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 305 clinical isolates of staphylococci, streptococci, and related genera by comparing direct colony testing with preparatory extraction. Isolates were previously identified by use of phenotypic testing and/or 16S rRNA gene sequencing. Manufacturer-specified score cutoffs for genus- and species-level identification were used. After excluding 7 isolates not present in the Biotyper library, the Biotyper correctly identified 284 (95%) and 207 (69%) isolates to the genus and species levels, respectively, using extraction. By using direct colony testing, the Biotyper identified 168 (56%) and 60 (20%) isolates to the genus and species levels, respectively. Overall, more isolates were identified to the genus and species levels with preparatory extraction than with direct colony testing (P < 0.0001). The analysis was repeated after dividing the isolates into two subgroups, staphylococci, streptococci, and enterococci (n = 217) and "related genera" (n = 81). For the former subgroup, the extraction method resulted in the identification of 213 (98%) and 171 (79%) isolates to the genus and species levels, respectively, whereas the direct colony method identified 136 (63%) and 56 (26%) isolates to the genus and species levels, respectively. In contrast, for the subgroup of related genera, the extraction method identified 71 (88%) and 36 (44%) isolates to the genus and species levels, respectively, while the direct colony method identified 32 (40%) and 4 (5%) isolates to the genus and species levels, respectively. For both subgroups, preparatory extraction was superior to direct colony testing for the identification of isolates to the genus and species levels (P < 0.0001). Preparatory extraction is needed for the identification of a substantial proportion of Gram-positive cocci using the Biotyper method according to manufacturer-specified score cutoffs.

Bacterial infections are the main cause of death within the first year after liver transplantation, and the increased incidence of multidrug-resistant gram-positive pathogens has created a major challenge in the treatment of these patients. Linezolid, the first US Food & Drug Administration-approved oxazolidinone, offers a valuable novel treatment option for serious gram-positive infections. Linezolid is relatively non-toxic but prolonged treatment with linezolid was associated with thrombocytopenia. Here we report on the experience of linezolid treatment in adult liver transplant patients, who are at an increased risk for thrombocytopenia because of hypersplenism. From November 2003 until December 2009, we evaluated the clinical course of 46 liver transplant patients (27 male/19 female) in our surgical intensive care unit. For proven or probable gram-positive infection, all patients received linezolid 600 mg intravenously every 12 h. On clinical improvement, treatment was changed to oral linezolid 600 mg twice daily. Treatment duration was 11 ± 7 days. Treatment indications were pneumonia (n = 8), blood stream infection (n = 30), and surgical site/abdominal infection (n = 3). Clinical cure was achieved in 43 out of 46 patients. During the course of treatment, no cases of severe thrombocytopenia occurred and a statistically significant platelet count increase was seen from day 1 (110 ± 73/nL) to day 7 (165 ± 116/nL) and day 14 (180 ± 140/nL). We did not observe any further adverse events, especially no severe neurological complications (e.g., serotonin syndrome) or signs of lactate acidosis. Two patients died from uncontrolled vancomycin-resistant Enterococcus faecium sepsis with septic shock and one due to uncontrolled methicillin-resistant Staphylococcus aureus pneumonia. These deaths were considered to be unrelated to linezolid treatment, and linezolid was regarded as the optimal treatment choice in these patients. A subgroup analysis of patients treated for >14 days revealed no statistically significant differences when compared with patients on shorter treatment. In particular, no cases of thrombocytopenia occurred during longer treatment. In conclusion, linezolid is a safe and effective treatment for adult liver transplant patients with gram-positive infections., (© 2011 John Wiley & Sons A/S.)

As peri-implantitis is an emerging problem, the development of validated animal models is mandatory. The aim of this pilot study was to provide a first step in describing the normal oral flora of minipigs. In five minipigs, samples of the oral flora were collected with sterile cotton swabs from the buccal gingiva of the lower jaw. Two swabs per animal were collected, followed by bacterial isolation under both aerobe and anaerobe conditions. Microbiological analyses included biochemical tests, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rDNA gene sequence analysis. A total of 61 taxa were detected, 14-21 different bacterial taxa from each minipig. Among the Gram-positive cocci, mainly staphylococcal and streptococcal species were identified. Different Actinomyces species were the most abundant taxa in the group of Gram-positive rods. Among the anaerobic bacteria, the Gram-negative genera Fusobacterium, Bacteroides and Prevotella were the most often observed taxa. This is the first study which begins to describe the normal oral flora in minipigs in cultures to allow for the detection of a broad spectrum. Several bacterial species identified are different from human ones. No specific species for peri-implantitis could be detected in that healthy sample.

Sixty-eight (68) patients with serious upper extremity suppurative infections, presenting within a period of fifteen (15) months, were prospectively studied clinically, Gram stain of aspirates/pus were performed, specimen cultured, planted, and where indicated glucose levels and haemoglobin genotype determined. Half of the patients had hand infections. Staphylococcus aureus was isolated from thirty-nine (39) patients. Gram Negative bacilli, including Salmonella were more isolated from patients with diabetes mellitus or Hgb SS or SC. The Gram stain results correlated with the culture result 90%. When Gram Positive cocci were demonstrated in the primary microscopic examination, cultures were not mandatory. When no organism was demonstrated on primary Gram stain or the patient was diabetic or a sickler, cultures of the specimens were done. The Gram stain, well performed, remains a useful, inexpensive, technologically appropriate laboratory test for abetting decision making in patients with upper extremity suppurative infections. Organisms encountered in this study included: Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhi, Proteus mirabilis, Pseudomonas aeruginosa, and Coliforms.

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.

Brazil is a country with continental proportions with high geographic and economic diversity. Despite its medical centers of excellence, antimicrobial resistance poses a major therapeutic challenge. Rates of methicillin-resistant Staphylococcus aureus are up to 60% and are related to an endemic Brazilian clone. Local resistance to vancomycin in Enterococci was first related to Enterococcus faecalis, which differs from European and American epidemiology. Also, local Klebsiella pneumoniae and Escherichia coli isolates producing extended-spectrum β-lactamases have a much higher prevalence (40%-50% and 10%-18%, respectively). Carbapenem resistance among the enterobacteriaceae group is becoming a major problem, and K. pneumoniae carbapenemase isolates have been reported in different states. Among nonfermenters, carbapenem resistance is strongly related to SPM-1 (Pseudomonasaeruginosa) and OXA-23 (Acinetobacter baumannii complex) enzymes, and a colistin-only susceptible phenotype has also emerged in these isolates, which is worrisome. Local actions without loosing the global resistance perspective will demand multidisciplinary actions, new policies, and political engagement.

The susceptibility of 14 species of 115 Gram-positive anaerobic cocci (GPAC) was determined for 14 antibiotics. To assure correct identification, strains were genotypically identified by fluorescence in situ hybridization and sequencing. Susceptibility differences (MIC₅₀ and MIC₉₀) for penicillin G, clindamycin, tigecycline, levofloxacin, amoxicillin-clavulanic acid, cefoxitin, ertapenem, meropenem, metronidazole, and doxycycline were found for the three clinically most relevant GPAC species: Finegoldia magna, Parvimonas micra, and Peptoniphilus harei.

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Bacterial and fungal infections are the leading cause of mortality in liver transplant (LT) recipients. Few studies have examined the incidence of culture-positive preservation fluid (PF) and the outcome of related recipients. The aim of this study was to determine the incidence and the microbiologic findings of PF positive cultures, and to evaluate the impact on morbidity and mortality of LT recipients. A retrospective analysis of PF cultures performed after 477 LTs from cadaveric grafts between January 2001 and February 2008 was conducted. Forty-five (9.5%) PFs were found to be positive with 1 or 2 pathogens. The demographic profiles of recipients of PF with positive or negative cultures were similar. Enterobacteriaceae species were the most frequent organisms (n = 30), followed by Staphylococcus aureus (n = 5), coagulase-negative staphylococci (n = 5), enterococci (n = 4), and yeasts (n = 3). Mortality rate at 1 month was not significantly different in recipients with positive or sterile PF cultures (88.1% vs. 87.7%, respectively). The rate of bacteremia among LT recipients with positive or negative PF cultures was not statistically different. Systemic infections caused by the pathogen cultured from the PF occurred in 8 (18%) of the 45 recipients, including bacteremia (4/8) or intra-abdominal sepsis (5/8). Causative organisms were Enterobacteriaceae species (n = 5), Candida species (n = 2), and Enterococcus faecium (n = 1). Among the 8 patients who developed infection with the PF organism, 4 (50%) died in the intensive care unit (ICU) vs. an ICU mortality rate of 8% (3/37) in those who did not develop infection with the PF organism (P < 0.05). Infection occurred less frequently in recipients who received antimicrobial therapy with activity against the PF isolate than in those without appropriate treatment (41% vs. 3.8%, P < 0.005). Those who develop infection with organisms recovered from PF cultures appear to have high early mortality rates; therefore, appropriate antimicrobial therapy against organisms cultured from PF should be given., (© 2010 John Wiley & Sons A/S.)

In cytology, automating the feature extraction process yields an objective, quantitative, detailed and reproducible computation of cell morphofunctional characteristics and allows the analysis of a large quantity of images. The objective of the present study is to develop an automatic tool to identify and classify the different types of cocci bacterial cells in digital microscopic cell images. Geometric features are used to identify the arrangement of cocci bacterial cells, namely cocci, diplococci, streptococci, tetrad, sarcinae and staphylococci using 3&sigma;, K-NN and Neural network classifiers. The current methods rely on the subjective reading of profiles by a human expert based on the various manual staining methods. In this paper, we propose a method for cocci bacterial cell classification by segmenting digital bacterial cell images and extracting geometric and statistical features for cell classification. The experimental results are compared with the manual results obtained by microbiology expert and other methods in the literature.

Linezolid is the only oxazolidinone agent approved for clinical use and has been administered to millions of patients over nearly a decade, becoming an important therapeutic alternative for infections caused by multidrug-resistant (MDR) Gram-positive pathogens. Resistance is due to mutations in 23S rRNA and the ribosomal proteins L3 and L4 and, more recently, the mobile cfr gene (causes resistance to several antimicrobial classes). Using reference broth microdilution methods in a central reference laboratory design, MIC values were obtained during the 2009 Zyvox® Annual Appraisal of Potency and Spectrum program (5754 sampled strains from 22 countries), and the following MIC(90) values were obtained for the leading pathogen or species groups: Staphylococcus aureus (2 μg/mL), coagulase-negative staphylococci (CoNS; 1 μg/mL), Enterococcus spp. (2 μg/mL), and 3 groups of streptococci (1 μg/mL) including Streptococcus pneumoniae. Linezolid-resistant strains (8 or 0.14% overall) that were not Staphylococcus aureus were observed in 5 nations and included CoNS (0.48%) having the mobile cfr gene. The results of this study demonstrate that linezolid continues to be effective in vitro against MDR pathogens, and the resistance rates appear stable., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Although the pathogenesis of Streptococcus oralis may be different from that of other viridans group streptococci, S. oralis shares a high degree of DNA sequence similarity with these streptococci. As a result, discrimination of S. oralis from its close relatives has long been considered difficult. This study was conducted to find specific genes that allow for the in vitro identification of S. oralis, but not other oral commensals. Four hundred ninety S. oralis clones obtained by suppressive subtractive hybridization were used for Southern hybridization, and positive clones were sequenced. Of 5 S. oralis-specific clones, newly designed primer sets based on the glucosyltransferase regulatory gene amplified genomic DNA only from S. oralis strains, but not from any of the other 125 strains tested. Our findings may be useful for the future development of efficient diagnostic tools for the rapid identification and differentiation of S. oralis from other oral streptococci strains.