Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Peptoniphilus faecalis sp. nov., isolated from swine faeces.

An obligately anaerobic, Gram-positive, non-motile, coccus-shaped bacterial strain designated AGMB00490T 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 10418T (=KCTC 5947T, 97.22 % 16S rRNA gene sequence similarity). Whole genome sequence analysis determined that the DNA G+C content of strain AGMB00490T 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 AGMB00490T 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 C16 : 0 and C18 : 1 ω9c. Strain AGMB00490T produced acetic acid as a major end product of metabolism. Accordingly, phylogenetic, physiologic and chemotaxonomic analyses revealed that strain AGMB00490T represents a novel species for which the name Peptoniphilus faecalis sp. nov. is proposed. The type strain is AGMB00490T (=KCTC 15944T=NBRC 114159T).

How is the microbial diagnosis of bacterial vertebral osteomyelitis performed? An 11-year retrospective study.

Vertebral osteomyelitis (VOM) is often diagnosed with delays, resulting in poorer outcomes. Microbial documentation is particularly challenging and obtained using blood cultures (BCs) and vertebral biopsies (VBs; CT-guided or surgical). We retrospectively analysed VOM cases in a tertiary reference centre between 2004 and 2015, focusing on how and how quickly microbiological diagnosis was performed. Among 220 VOM, 88.2% had documentation, including Gram-positive cocci (GPC) (70.6%), Gram-negative rods (GNR) (9.3%), anaerobes (3.6%), polybacterial infections (6.7%) and tuberculosis (9.8%). BCs were performed in 98.2% and positive in 59.3%, identifying most GPC (80.3%) and half of GNR (54.6%). VBs were performed in fewer cases (37.7%), but were more frequently positive (68.8% for CT-guided and 81.0% for surgical biopsies). They documented all anaerobes (100.0%), most M. tuberculosis (84.2%) and polybacterial infections (76.9%), and GNR (45.4%). Extra-vertebral samples highly contributed to tuberculosis diagnosis (52.6%, and 15.8% as the only positive sample). Documentations most often followed radiological diagnosis (53.4%). They were obtained earlier by BCs than by VB after first clinical symptoms (median of 14 versus 51 days). Antibiotic treatments were mostly initiated after samplings (88.0%). BCs allow the documentation of most VOM and should be performed without delay in case of clinical or radiological suspicion; however, they may miss 1 out of 5 GPC and 1 out of 2 GNR. VBs have a higher positivity rate and should be rapidly performed if negative BCs. It is likely that delayed and missed diagnoses result from the insufficient use of VB.

Risk for infective endocarditis in bacteremia with Gram positive cocci.

PURPOSE: Infective endocarditis (IE) is a serious complication of bacteremia and is most often caused by Gram positive cocci. We investigated the prevalence of IE in patients where echocardiography was encouraged when bacteremia with Gram positive cocci was present. METHODS: The study included patients with Gram positive cocci bacteremia hospitalized at two Danish hospitals between March and December 2016. Information concerning echocardiography, demographics and bacterial species was collected from the patients' medical files. Patients without echocardiography were followed for 6 months in order to confirm or reject possible IE. RESULTS: The study included 585 patients with Gram positive cocci bacteremia, and echocardiography was performed in 414 (71%) of them. The prevalence of IE in patients with high risk bacteremia, i.e. Staphylococcus aureus, non-beta-hemolytic streptococci, Enterococcus faecalis, and coagulase-negative staphylococci was 16%. Patients with Enterococcus faecalis had the highest prevalence of IE (33%) followed by non-beta-hemolytic streptococci (23%) and Staphylococcus aureus (12%). Among low risk bacteremia the prevalence of IE was 1%. The mean age of patients with IE was 74 years (SD 12.9) and 71% were male. CONCLUSION: These findings strongly support routine echocardiography in patients with high risk bacteremia and non-performance of echocardiography in patients with low risk bacteremia.

Short-course antibiotic regimen compared to conventional antibiotic treatment for gram-positive cocci infective endocarditis: randomized clinical trial (SATIE).

BACKGROUND: Most serious complications of infective endocarditis (IE) appear in the so-called "critical phase" of the disease, which represents the first days after diagnosis. The majority of patients overcoming the acute phase has a favorable outcome, yet they remain hospitalized for a long period of time mainly to complete antibiotic therapy. The major hypothesis of this trial is that in patients with clinically stable IE and adequate response to antibiotic treatment, without signs of persistent infection, periannular complications or metastatic foci, a shorter antibiotic time period would be as efficient and safe as the classic 4 to 6 weeks antibiotic regimen. METHODS: Multicenter, prospective, randomized, controlled open-label, phase IV clinical trial with a non-inferiority design to evaluate the efficacy of a short course (2 weeks) of parenteral antibiotic therapy compared with conventional antibiotic therapy (4-6 weeks). SAMPLE: patients with IE caused by gram-positive cocci, having received at least 10 days of conventional antibiotic treatment, and at least 7 days after surgery when indicated, without clinical, analytical, microbiological or echocardiographic signs of persistent infection. Estimated sample size: 298 patients. INTERVENTION: Control group: standard duration antibiotic therapy, (4 to 6 weeks) according to ESC guidelines recommendations. Experimental group: short-course antibiotic therapy for 2 weeks. The incidence of the primary composite endpoint of all-cause mortality, unplanned cardiac surgery, symptomatic embolisms and relapses within 6 months after the inclusion in the study will be prospectively registered and compared. CONCLUSIONS: SATIE will investigate whether a two weeks short-course of intravenous antibiotics in patients with IE caused by gram-positive cocci, without signs of persistent infection, is not inferior in safety and efficacy to conventional antibiotic treatment (4-6 weeks). TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04222257 (January 7, 2020). EudraCT 2019-003358-10.

Comparison of breakthrough Gram-positive cocci infection during vancomycin vs teicoplanin therapy in patients receiving haematopoietic stem cell transplantation.

WHAT IS KNOWN AND OBJECTIVE: Our previous report indicated that teicoplanin (TEIC) caused fewer adverse effects than vancomycin (VCM) in patients with febrile neutropenia (FN) receiving haematopoietic stem cell transplantation (HSCT). However, we observed breakthrough methicillin-resistant-Staphylococcus haemolyticus (MR-S haemolyticus) infection during TEIC therapy in these patients. In this study, we sought to compare the incidence of breakthrough Gram-positive cocci (GPC) infection during VCM and TEIC therapy in this population. METHODS: A single-centre, retrospective cohort study was conducted. Patients who had received HSCT and were administered VCM (n = 19) or TEIC (n = 38) for FN from 1 September 2011 to 31 August 2019 were enrolled. We compared the incidence of breakthrough GPC infection between the VCM and TEIC groups. RESULTS: Breakthrough GPC infection during glycopeptide therapy in febrile neutropenic patients received HSCT was observed in three patients (7.9%) in the TEIC group but in none of patients (0%) in the VCM group. MR-S haemolyticus with low glycopeptide susceptibility (TEIC MIC = 2-8 µg/mL, VCM MIC = 2-4 µg/mL) was isolated from blood cultures in all patients with breakthrough GPC infections. All breakthrough infections were cured by changing from TEIC to daptomycin (DAP). WHAT IS NEW AND CONCLUSION: The incidence of breakthrough GPC infection during glycopeptide therapy in febrile neutropenic HSCT patients was higher in the TEIC group than in the VCM group. MR-S haemolyticus with low glycopeptide susceptibility was isolated from all patients with breakthrough GPC infection and successfully treated with DAP.

In Vitro Antimicrobial Activity of Diacerein on 76 Isolates of Gram-Positive Cocci from Bacterial Keratitis Patients and In Vivo Study of Diacerein Eye Drops on Staphylococcus aureus Keratitis in Mice.

Bacterial keratitis is an aggressive infectious corneal disease. With the continuing rise in antibiotic resistance and a decline in the discovery of new antibiotics, new antimicrobial drugs are now required. In the present study, we determined the antibacterial activity of diacerein, an anti-inflammatory drug, against 76 Gram-positive cocci isolated from bacterial keratitis patients in vitro and anti-Staphylococcus aureus activity in a mouse bacterial keratitis model in vivo The MICs of diacerein were tested using the broth microdilution method in vitro A BALB/c Staphylococcus aureus keratitis animal model was selected and the corneal clinical observation, viable bacteria, and hematoxylin-eosin and Gram staining of infected corneas were measured to evaluate the antibacterial efficacy of diacerein eye drops in vivo An in vivo eye irritation study was carried out by a modified Draize test in rabbits. Our in vitro results showed that diacerein possesses satisfactory antibacterial activity against the majority of Gram-positive cocci (60/76), including all 57 tested Staphylococcus spp. and 3 Enterococcus spp. The in vivo experiment showed that diacerein eye drops reduced bacterial load and improved ocular clinical scores after topical administration of diacerein drops on infected corneas. The ocular irritation test revealed that diacerein eye drop had excellent ocular tolerance. These results indicated that diacerein possesses in vivo anti-Staphylococcus aureus activity. We suggest that diacerein is a possible topically administered drug for Staphylococcus aureus-infected patients, especially those with ocular surface inflammatory disorders.

Evaluation of rapid polymerase chain reaction-based organism identification of gram-positive cocci for patients with a single positive blood culture.

For patients with a single-positive blood culture growing gram-positive cocci, organism identification can provide supportive information for differentiating contamination from infection. We investigated the effect of a rapid blood culture identification panel (BCID) on vancomycin-prescribing patterns and patient outcomes for single positive blood culture (PBC) growing gram-positive cocci. Adult patients with single-positive blood culture growing gram-positive cocci with conventional organism identification (pre-BCID) were compared with organism identification by BCID (post-BCID). Antimicrobial Stewardship Program (ASP) review of PBC was performed in both study groups. Vancomycin prescribing patterns were studied. Secondary endpoints were the incidence of nephrotoxicity, length of stay (LOS), readmission rate, mortality, and hospital costs. A total of 188 patients (86 pre-BCID, 102 post-BCID) were included. Organism identification was known 21 h sooner in the post-BCID group (P < 0.001). Coagulase-negative staphylococci were the most commonly isolated organisms (73%). In patients where vancomycin was deemed unnecessary (n = 133), vancomycin use (51% pre-BCID vs 36% post-BCID; P = 0.09) and time from culture positivity to vancomycin discontinuation (1.5 vs. 1.7 days; P = 0.92) did not differ between groups. We found no differences in the development of nephrotoxicity, LOS, readmission, mortality, or hospital costs. Earlier identification of single positive blood culture growing gram-positive cocci did not significantly influence prescribing patterns of vancomycin. However, baseline antimicrobial stewardship review of single positive blood culture growing gram-positive cocci may have lessened the opportunity for detectable differences. Larger studies, accounting for the impact of ASP intervention, should be performed to determine the value of each individual component.

Multiplex PCR of bronchoalveolar lavage fluid in children enhances the rate of pathogen detection.

BACKGROUND: Culturing of bronchoalveolar lavage (BAL) fluid is a commonly used method for pathogen detection in pneumonia. However, the sensitivity is low, especially in patients pre-treated with anti-infective agents. The early detection of a pathogen is crucial for the outcome of respiratory tract infections. For bloodstream infections, a multiplex polymerase chain reaction (PCR) assay (SeptiFast®, SF) is available for improved pathogen detection from blood. OBJECTIVE: The aim of the present study was to determine whether the SF assay is applicable to the BAL of children with pulmonary infections and whether the frequency of pathogen detection is enhanced by the use of this multiplex PCR method. METHODS: We investigated 70 BAL samples of 70 children simultaneously by culture and multiplex PCR. The frequency of pathogen detection was compared. RESULTS: Pathogens were detected more frequently by SF than by culture (83% vs. 31%; p < 0.001). This advantage was shown for immunocompetent patients (p = 0.001) as well as for immunocompromised patients (p = 0.003). The majority (38/44; 86%) of the Gram positive cocci were only detected by SF. Fungal organisms were detected in 7/70 patients (10%) by SF and in 2/70 (3%) by culture (p = 0.125). CONCLUSION: Compared to conventional culture, the use of the SF assay on the BAL of children with pneumonia increases pathogen detection rates and therefore adds important information to guide anti-infective therapy.

Implementation of Rapid Diagnostic Testing without Active Stewardship Team Notification for Gram-Positive Blood Cultures in a Community Teaching Hospital.

In community hospitals, antimicrobial stewardship team notification of rapid diagnostic testing (RDT) results may not be feasible. A retrospective quasi-experimental study was conducted evaluating 252 adult inpatients with blood cultures positive for Gram-positive cocci in clusters (pre-RDT, n = 143; post-RDT, n = 109). The median time to appropriate therapy was significantly shorter in the post-RDT group (15 versus 0 h, P < 0.001), and the mean length of stay for patients with coagulase-negative staphylococcus was significantly shorter (10.5 versus 7.7 days; P = 0.015).

Activity of dalbavancin and comparator agents against Gram-positive cocci from clinical infections in the USA and Europe 2015-16.

Background: Many infections due to Gram-positive cocci (GPC; staphylococci, streptococci and enterococci) regularly involve prolonged systemic antibiotic use. Dalbavancin has demonstrated activity against GPC isolates and has been approved for the treatment of acute bacterial skin and skin structure infections (ABSSSIs) in adults. Objectives: To evaluate the activity of dalbavancin against GPC isolated from a variety of infection types in the USA and Europe. Methods: A total of 14 319 Staphylococcus aureus, 1992 CoNS, 3269 ß-haemolytic streptococci (BHS), 2071 Enterococcus faecalis, 936 Enterococcus faecium, 71 Enterococcus gallinarum/casseliflavus and other Enterococcus spp., 3487 Streptococcus pneumoniae and 1063 viridans group streptococci (VGS) causing clinical infections were consecutively collected (2015-16) and tested for susceptibility by broth microdilution methods. Results: All S. aureus (36.4% MRSA) isolates were susceptible to dalbavancin, teicoplanin and vancomycin, while daptomycin and linezolid showed susceptibility rates of >99.9% (according to CLSI criteria). Dalbavancin MIC results were at least 16-fold lower than these comparators against all S. aureus. Dalbavancin was the most potent agent against CoNS [62.2%/59.6% methicillin-resistant (CLSI/EUCAST)], followed by daptomycin, linezolid and vancomycin. All vancomycin-susceptible E. faecalis isolates were inhibited by dalbavancin at ≤0.25 mg/L (FDA susceptible breakpoint). Dalbavancin was very active against BHS (MIC90 0.03 mg/L) and the most active agent against S. pneumoniae and VGS (highest MIC 0.25 mg/L). Ceftriaxone, daptomycin, levofloxacin and vancomycin were also active (93.5%-100.0% susceptible) against VGS, whereas clindamycin, erythromycin, penicillin and tetracycline had lower activity. Conclusions: Dalbavancin appears to be a viable candidate for treating serious infections caused by GPC.

Urinary tract infections following radical cystectomy and urinary diversion: a review of 1133 patients.

OBJECTIVE: To investigate the incidence and microbiology of urinary tract infection (UTI) within 90 days following radical cystectomy (RC) and urinary diversion. METHODS: We reviewed 1133 patients who underwent RC for bladder cancer at our institution between 2003 and 2013; 815 patients (72%) underwent orthotopic diversion, 274 (24%) ileal conduit, and 44 (4%) continent cutaneous diversion. 90-day postoperative UTI incidence, culture results, antibiotic sensitivity/resistance and treatment were recorded through retrospective review. Fisher's exact test, Kruskal-Wallis test, and multivariable analysis were performed. RESULTS: A total of 151 urinary tract infections were recorded in 123 patients (11%) during the first 90 days postoperatively. 21/123 (17%) had multiple infections and 25 (20%) had urosepsis in this time span. Gram-negative rods were the most common etiology (54% of positive cultures). 52% of UTI episodes led to readmission. There was no significant difference in UTI rate, etiologic microbiology (Gram-negative rods, Gram-positive cocci, fungi), or antibiotic sensitivity and resistance patterns between diversion groups. Resistance to quinolones was evident in 87.5% of Gram-positive and 35% of Gram-negative bacteria. In multivariable analysis, Charlson Comorbidity Index > 2 was associated with higher 90-day UTI rate (OR = 1.8, 95% CI 1.1-2.9, p = 0.05) and Candida UTI (OR 5.6, 95% CI 1.6-26.5, p = 0.04). CONCLUSIONS: UTI is a common complication and cause of readmission following radical cystectomy and urinary diversion. These infections are commonly caused by Gram-negative rods. High comorbidity index is an independent risk factor for postoperative UTI, but diversion type is not.

Validation of blood culture gram staining for the detection of Staphylococcus aureus by the 'oozing sign' surrounding clustered gram-positive cocci: a prospective observational study.

BACKGROUND: Staphylococcus aureus bacteraemia is a common and significant infection, associated with high rates of mortality. Therefore, early identification is important for the initiation of appropriate treatment. The objective of this study was to evaluate the accuracy of blood culture Gram staining along with the finding of an 'oozing sign' to diagnose either Staphylococcus aureus or coagulase-negative staphylococci. METHODS: This single-centre, prospective observational study was performed from May 2017 to November 2017. We used routine blood culture bottles (BacT/ALERT FA and BacT/ALERT SN; bioMérieux, Inc., Durham, NC). Bacterial species were identified and the minimum inhibitory concentration was determined by using the MicroScan WalkAway 96 SI system (Beckman Coulter, Tokyo, Japan). Bottles showing growth were removed, and Gram staining was performed. RESULTS: A total of 118 samples, including 55 aerobic and 63 anaerobic bottle samples, were analysed. The overall sensitivity of Gram staining was 78.7% (95% CI: 65.8-94.3%), and the specificity was 95.0% (95% CI: 84.7-98.4%). CONCLUSION: The 'oozing sign' observed in Gram staining may be useful for the rapid prediction of S. aureus in BacT/ALERT blood culture bottles.

Helcococcus ovis in a patient with an artificial eye: a case report and literature review.

BACKGROUND: Helcococcus ovis, belonging to the genus of Helcococcu in Peptostreptococcaceae, is one kind of facultative anaerobic and gram-positive cocci, which was first isolated from a mixed infection in sheep in 1999. To our knowledge, it's known as an invasive pathogen in animals, and never been reported as a human pathogen in published literature. The aims of this work are to describe the first report of H. ovis which was recovered from the artificial eye of human case and perform a literature review. CASE PRESENTATION: A 26 year-old man reporting pyogenic infection with an artificial eye attended ophthalmic ward in Tongji hospital. After physical examination, clinical and laboratory investigations, the diagnosis of eye infection caused by Helcococcus ovis and Staphylococcus aureus was established. Receiving a medico-surgical approach, the patient was successfully treated. The treatment consisted in intravenous cefotaxime and ornidazole, levofloxacin eye drops during two weeks and removing of right artificial eye with debridement. CONCLUSIONS: We describe here the first known case of H. ovis which was recovered from human artificial eye. This report different from previous data found in the literature emphasizes the invasive potential of this bacterial species as a pathogen in human. Prospectively, the application of next generation sequencing tools would contribute to a more accurate classification of clinical strains.

Sustained impact of a rapid microarray-based assay with antimicrobial stewardship interventions on optimizing therapy in patients with Gram-positive bacteraemia.

OBJECTIVES: To compare antibiotic optimization and outcomes of patients before implementation of the Verigene Gram-Positive Blood Culture (Verigene BC-GP) nucleic acid microarray assay to after implementation with antimicrobial stewardship (AS) interventions and after discontinuation of AS interventions. METHODS: A retrospective pre-post-post quasi-experimental study was conducted to compare the three periods. AS interventions consisted of real-time guidance to clinicians on antibiotic selection. The primary outcome was median time from Gram stain to optimal therapy. Secondary outcomes included median time to effective therapy, median duration of therapy for contaminant organisms, median length of stay after blood cultures were collected, and all-cause in-hospital mortality. RESULTS: Out of a total of 923 patients, 390 (125 baseline, 134 intervention, 131 post-intervention) who were not on optimal therapy at the time of Gram stain or had contaminated blood cultures were assessed. Compared with baseline, only the median time to optimal therapy for MSSA bacteraemia was reduced in both the intervention and post-intervention periods (17 versus 17 versus 50 h; P < 0.001), respectively. In an analysis adjusted for baseline differences among the groups using quantile regression models, use of the Verigene BC-GP assay in both periods significantly reduced time to optimal therapy by 14-22 h in patients who would achieve optimal therapy at ≥ 26 h without the assay. There were no differences in in-hospital mortality or hospital length of stay between study periods. CONCLUSIONS: A real-time AS intervention implemented alongside introduction of the Verigene BC-GP assay led to improvements in antibiotic therapy for patients with bacteraemia due to Gram-positive cocci, even after the AS intervention was discontinued.

Duodenoscope reprocessing surveillance with adenosine triphosphate testing and terminal cultures: a clinical pilot study.

BACKGROUND AND AIMS: Recent reports of infectious outbreaks linked to duodenoscopes have led to proposals for duodenoscope surveillance culturing, which has inherent limitations. We aimed to assess the feasibility of real-time adenosine triphosphate (ATP) testing after manual cleaning and its ability to predict reprocessing adequacy, as determined by terminal duodenoscope cultures. METHODS: Clinically used duodenoscopes underwent reprocessing per current guidelines. After manual cleaning, ATP samples were obtained from the elevator, within the proximal biopsy port, and by flushing of the biopsy channel. After high-level disinfection (HLD), aerobic cultures of the elevator and biopsy channel were obtained using sterile technique. Duodenoscopes with any ATP sample ≥200 relative light units underwent repeated cycles of cleaning, ATP testing, HLD, and terminal culturing. RESULTS: Twenty clinically used duodenoscopes were included; 18 underwent a second reprocessing cycle, and 6 underwent a third reprocessing cycle because of detection of high ATP. After the initial reprocessing cycle, 12 of 20 (60%) duodenoscopes had positive culture results, most commonly yielding gram-negative bacilli (GNB, n = 11 from 9 duodenoscopes), and catalase-positive gram-positive cocci (CP-GPC, n = 7 from 7 duodenoscopes), suggesting staphylococcal organisms. Ambient environmental controls also showed GNB and CP-GPC growth. The overall sensitivity and specificity of ATP testing compared with terminal cultures were 30% and 53%, respectively. CONCLUSIONS: ATP sampling appears to correlate poorly with terminal culture results and cannot be recommended as a surrogate for terminal cultures. The performance and interpretation of cultures remains complicated by the potential recovery of environmental contaminants.

Characterization and susceptibility of streptococci and enterococci isolated from Nile tilapia (Oreochromis niloticus) showing septicaemia in aquaculture and wild sites in Egypt.

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, ß-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.

Rapid differentiation of cocci/mixed bacteria from rods in voided urine culture of women with uncomplicated urinary tract infections.

OBJECTIVES: To evaluate the ability of laser flow cytometry to predict cocci/mixed growth in the pre-analytical phase of urine specimens. METHODS: We retrospectively reviewed urine samples from women with uncomplicated urinary tract infections from urologic clinics for study. Urine analyses were performed with laser flow cytometry (UF1000i, Sysmex, Kobe, Japan) and then diagrams were generated (forward scatter vs. fluorescent light scatter). Each specimen (bacteria count >357 BACT/µL) was classified as either cocci bacteria or rods/mixed growth according to the diagrams. Standard urine cultures were performed, and the agreement between cultures and the UF1000i interpretations was analyzed with kappa statistics. RESULTS: Finally, 491 specimens met the criteria for analysis. Among the 376 specimens with single bacteria growth, there were 26 gram-positive cocci (13 Streptococci spp., 7 Staphylococci spp., 6 Enterococci spp.), 1 gram-positive rods (Corynebacterium spp.), and 349 gram-negative rods (273 Escherichia coli, 33 Klebsiella spp., 29 Proteus spp., 6 Citrobacter spp., 4 Enterobacter spp., 3 Pseudomonas spp., and 1 Providencia spp.). There were 115 specimens with two bacteria species or more that were regarded as mixed growth. Agreement of rods or cocci/mixed growth between the laser flow cytometry and urine cultures yielded a kappa value of 0.58. The positive and negative predictive rate of the UF1000i for cocci/mixed growth in voided urine culture was 81.8% and 84.7%, respectively. CONCLUSIONS: Through laser flow cytometry, we can predict growth of cocci/mixed growth in the pre-analytical phase of urine culture, thus avoiding unnecessary urine culture and waiting time.

The Association between Positive Tracheal Aspirate Cultures and Adverse Pulmonary Outcomes in Preterm Infants with Severe Bronchopulmonary Dysplasia.

Objective Bacterial colonization of the airway may contribute to the development of bronchopulmonary dysplasia. Whether airway colonization increases risk for later adverse respiratory outcomes is less clear. We described tracheal aspirate culture results obtained from preterm infants receiving mechanical ventilation at 36 weeks postmenstrual age (PMA) and evaluated the association between bacteria type and the risk for prolonged supplemental oxygen use. Study Design We conducted a retrospective, single-center cohort study comparing infants (1) with and without a tracheal aspirate culture that grew a Gram-negative rod (GNR) and (2) with and without a culture that grew a Gram-positive cocci (GPC). Results Among 121 infants, 65 (53.7%) and 51 (42.2%) had a tracheal aspirate culture that grew a potentially pathogenic GNR and GPC prior to 36 weeks PMA, respectively. GNR were associated with increased risk for death or use of supplemental oxygen at discharge (adjusted odds ratio [aOR], 6.2; 95% confidence interval [CI], 1.8-21.1), and use of supplemental oxygen at discharge among survivors (aOR, 5.5; 95% CI, 1.6-19.0). GPC did not affect the risk for any study outcomes. Conclusion GNR but not GPC in the airways of preterm infants receiving mechanical ventilation at 36 weeks PMA is associated with increased risk for prolonged supplemental oxygen use.

Evaluation of Three Bacterial Identification Systems for Species Identification of Bacteria Isolated from Bovine Mastitis and Bulk Tank Milk Samples.

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.