Identification Of Gram-negative Bacteria Research Articles

Isolation and Identification of Gram Negative Bacteria in The Lumen Fluid of Colorectal Cancer Patients

Colorectal cancer is one of the major health problems in Indonesia and the third most common cause of death worldwide. Colorectal cancer is a cancer of the colon and the rectum. There is considerable research evidence demonstrating the important role of complex gut microbiota communities in developing intestinal tumorigenesis. This study aims to isolate and identify gram-negative bacteria found in the colonic lumen fluid of colorectal cancer patients using the gram-staining method and biochemical test. From microscopic identification results, macroscopic gram staining, motility test, Simon citrates agar (SCA), and triple sugar iron agar (TSIA) tests against H1 and H2 isolates described gram-negative stem bacteria included in the Enterobacteriaceae family belonging to the species Shigella sp.

Isolation and Identification of Gram Negative Bacteria from Chicken Meat collected from Retail Meat Shops of Kathmandu Valley

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Isolation and Identification of Gram-Negative Bacteria on Cattle Farms Indicated by Mastitis

Mastitis is one of the causes of diseases that can reduce livestock production. Mastitis can be caused by various types of bacteria that cause a decrease in livestock production that is difficult to cure with antibiotics has been reported resistance. This study aims to determine gram-negative bacteria that can be the cause of mastitis in cows. The study used samples taken from folk farms by means of livestock udder swabs. Bacterial isolation is carried out by planting in Mannitol Salt Agar (MSA) media and identifying by Gram staining and biochemical tests (maltose and lactose). The data analysis used is descriptive analysis, namely the types of bacteria as the causative agents of mastitis. The results of the study obtained bacteria that cause mastitis of gram-negative species Klebsiella sp and Enterobacter sp yang classified as Gram-negative bacteria. It can be concluded that in cows indicated mastitis there are Gram-negative bacteria, these are morphologically identified, namely Klebsiella sp and Enterobacter sp.

A Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Direct-from-Urine-Specimen Diagnostic for Gram-Negative Pathogens.

Urinary tract infections (UTIs) pose a major public health burden. The vast majority of UTIs are caused by Gram-negative bacteria. Current culture-based pathogen identification methods may require up to 24 to 48 h of incubation. In this study, we developed and evaluated a method for Gram-negative pathogen identification direct from urine, without culture, via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in approximately 1 h. Urine samples were collected (n = 137) from the University of Maryland Medical Center clinical microbiology laboratory. To identify bacteria direct from urine, two methods were evaluated. First, 1 μL of urine was directly spotted onto the MALDI target plate, and second, 1 mL of urine was centrifuged at 8,000 rpm for 5 min before processing using the fast lipid analysis technique (FLAT). Mass spectra were acquired on the Bruker MALDI Biotyper sirius system in the negative-ion mode. Results were compared to those of standard culture methods. When 1 μL of urine was directly spotted, positive agreement was 81.5% (101/124) and, after centrifugation, 94.4% (117/124) relative to that of standard culture methods. Negative agreement for both methods was 100% (13/13). The time to results for both of the specimen preparation methods using the FLAT extraction protocol was approximately 1 h, with minimal hands-on time required (<5 min). The ability to rapidly identify pathogens directly from urine, without the need for culture, allows for faster turnaround times and, potentially, improved patient outcomes. Overall, the FLAT extraction protocol, in combination with lipid A identification, provides a reproducible and accurate method to rapidly identify urinary pathogens. IMPORTANCE This study describes and evaluates a direct-from-urine extraction method that allows identification of Gram-negative bacteria via MALDI-TOF MS within 1 h. Currently, identification of urinary pathogens requires 24 h of culture prior to identification. While this method may not replace culture, we demonstrate its utility in screening for common urinary pathogens. By providing identifications in under 1 h, clinicians can potentially treat patients sooner with more-targeted antimicrobial therapy. In turn, earlier treatment can improve patient outcome and antimicrobial stewardship. Furthermore, MADLI-TOF MS is a readily available, easy-to-use diagnostic tool in clinical laboratories, making implementation of this method possible.

Boar Semen Contamination: Identification of Gram-Negative Bacteria and Antimicrobial Resistance Profile

Simple SummaryBoar semen can contain many bacterial species, some of which can have a negative impact upon the quality of the semen, as well as on the sows’ reproductive capacity. Semen contamination may occur at time of collection or during semen processing. The aim of this study was to identify gram-negative bacteria that appear in boar semen and to establish models of antimicrobial resistance of isolated gram-negative bacteria. Semen doses examined contained bacterial species with a known negative effect on sows’ reproductive tracts (Pseudomonas, Enterobacter, Klebsiella, E. coli), and more than half of these isolates were resistance to gentamycin (56.52%) and penicillin (58.69%) antimicrobials commonly used in boar semen extenders. This work proved the presence of pathogenic multiple resistant bacteria in semen, and therefore, we highly recommend periodic microbiological screening of bacteriospermia in boars to avoid the use of low-quality semen in the pig industry.Bacterial contamination of boar semen occurs with some frequency in artificial insemination centers and may have a negative effect on the quality of the semen as well as on the sows’ reproductive capacity. Normally, the source of bacterial contamination in pig seminal doses is the own boar. However, distilled water or laboratory equipment used to elaborate the seminal doses can be an important source of bacterial contamination. This study focused on the identification of gram-negative bacteria in boar semen, and impact on the quality of ejaculates obtained from boar, as well as on the establishment of antimicrobial resistance patterns of isolated gram-negative bacteria. Semen samples were collected from 96 boars, ranging in age from 12–36 month, from three artificial insemination centers from the North-West of Romania. Bacterial species were identified by two methods: matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and API 20 E (BioMerieux, France). The main bacteria isolated from the doses diluted semen were gram-negative bacteria (47.91%), with a majority of the contaminant bacteria belonging to the family Enterobacteriaceae: Seratia marcescens 19.56%, Proteus mirabilis 15.21% and Escherichia coli 10.86% and to the family Pseudomonaceae: Ralstonia picketii 17.39%, Burkholderia cepacia 10.86%, Pseudomonas aeruginosa 8.69%, and Pseudomonas fluorescens 4.34%, respectively. More than half of these isolates (56.52%) were resistant to gentamycin and 58.69% were resistant to penicillin. These antibiotics are very frequently added in sperm diluent in the centers for the processing of sperm from boars in Romania. Regular monitoring for bacterial contamination is an important aspect of a control program.

Veterinary clinic surfaces as reservoirs of multi-drug- and biocide-resistant Gram-negative bacteria.

This cross-sectional study was carried out to determine the common Gram-negative bacteria (GNB) contaminating veterinary clinic environments, and to evaluate the susceptibility of the isolates to commonly used antibiotics and biocides. A total of 62 swab samples were collected from different frequently touched surfaces in the 4 veterinary clinics visited. The samples were processed for isolation and identification of GNB using standard microbiological procedures. The susceptibility of the isolates to disinfectants and antibiotics was determined using agar dilution and disc diffusion techniques, respectively. A total of 114 GNB were isolated from the 4 clinics with isolation rates of 21.9, 22.8, 23.7 and 31.6% in clinics A, B, C and D, respectively. The surfaces of treatment tables were more contaminated (16.7 %) than receptionist/clinician desks (15.8%), weighing balances (10.5 %), door handles (7.9 %), drip stands (7.9 %), handwashing basins (7.0 %) and client chairs (7.0%). The surface-contaminating isolates were distributed into 20 genera, with members of Enterobacteriaceae predominating (n=97). Fifty-nine per cent of the isolates were resistant to the disinfectant Septol, while 5.3 and 0.9% were resistant to Purit and Dettol disinfectants, respectively. Multiple drug resistance was observed among 99% of the isolates with approximately 100% resistance to beta-lactams. Phenotypic expression of extended-spectrum (3.5 %) and AmpC beta-lactamase (38.6 %) production was detected. These findings highlight the role of clinic environments in serving as reservoirs for potential pathogens and sources for the spread of multi-drug resistant GNB.

Rapid Identification and Antimicrobial Susceptibility of Gram Negative Bacteria Detected in Positive Blood Culture Bottles

Abstract Background Blood stream infection (BSI) is one of the most serious situations in infectious disease .Accurate and timely identification of the causative agent and determination of its antimicrobial susceptibility profile are essential for guiding targeted and effective antimicrobial treatment. Current methods involve culturing of blood in a liquid medium and subsequently subculturing of signal positive bottles on solid media in order to obtain isolated colonies that can be further used in identification and susceptibility testing of the isolates. The standard method requires additional 48-72hours following the appearance of a positive signal in order to provide a reliable patient report. On the other hand, rapid identification of the causative agent and determination of its susceptibility profile by direct inoculation of the biochemical test media with the blood-broth mixtures from signal positive bottles and performing primary susceptibility testing might help reducing the time needed for provision of results compared to the standard isolated-colony based method and hence would help the rapid initiation of effective and targeted antimicrobial therapy and reduce the bacteremia-related morbidity and mortality. Objective The aim of the present study was to determine the accuracy and precision of the non-standard methods (direct identification and susceptibility testing using the blood/broth mixture) by comparing its results to those of the standard isolate-based identification and susceptibility testing methods. Material and method The study included 52 signal blood culture bottles yielding gram negative isolates. Bottles were selected amongst blood culture bottles submitted to the Main Microbiology laboratory, Ain Shams University hospital, for culture and antimicrobial susceptibility testing during the period between May 2018 and October 2018. a portion of the blood-broth mixture was aspirated from positive blood culture bottles, after being well mixed, and was subcultured onto agar media for the isolation of the causative agent and subsequently determination of its antimicrobial susceptibility profile was performed. Another part of the aspirated blood-broth mixture was diluted with sterile saline, its turbidity was adjusted against a 0.5McFarland standard and was used to inoculate directly the biochemical test media panel used for the identification of gram negative organisms as well as to perform direct (primary) antimicrobial susceptibility. Results The present study revealed there was 100% categorical agreement between the results of the direct biochemical inoculation method and those of the standard isolate-based inoculation method regarding the identification of the causative agent. The results of the direct biochemical identification method were also consistent giving rise to a 100% withinrun precision categorical agreement and a 100% between-run precision categorical agreement. The overall categorical agreement between the results of the standard isolate-based AST method and the results of the direct (primary susceptibility) AST method was 96.3% for the signal blood culture media. The major error rate was 0.5% whereas the minor error rate was 3.3% . Consistent results were also obtained for the AST done directly from the signal blood culture bottles since the between-run and within-run precision categorical agreement were 96.3% and 98.6%, respectively. Conclusion the overall performance of the AST done directly from positive blood culture bottles fulfilled the acceptable performance criteria specified in the Cumitech 31A so the direct method can be used for the earlier determination of AST and identification of Gram negative bacteria and thus to reduce the time for early initiation of appropriate antibiotic

Prevalence and Antibiogram of Extended-Spectrum Beta-Lactamase Producing Gram-negative Bacteria Isolated from Septicemic Neonates in a Tertiary Care Hospital

Background: In recent years, Extended-spectrum beta-lactamase (ESBL) producing microorganisms have complicated treatment of infections due to resistance of ESBL producing strains to a wide range of antimicrobials.&#x0D; Objective: Target of this study was to determine the prevalence of ESBL producing gramnegative bacteria in neonatal sepsis cases and to reveal the antimicrobial susceptibility pattern of those isolated ESBL producers.&#x0D; Materials and Methods: This cross sectional study was carried out in Dhaka Medical College Hospital (DMCH) over a period of 12 months from January to December in 2016. Following isolation and identification of gram-negative bacteria from blood samples of suspected septicemic neonates, antimicrobial susceptibility test was performed by Kirby Bauer disk-diffusion method and ESBL producers were detected by Double Disk Synergy (DDS) test.&#x0D; Results: Among 52 Gram-negative bacteria isolated from 106 blood samples, 34.61% ESBL producers were detected and Enterobacter spp. (45%) was predominant followed by Klebsiella pneumoniae (33.33%). None of the ESBL producers was resistant to colistin and tigecycline. All ESBL producing Acinetobacter baumannii, 77.78% and 66.67% of ESBL producing Enterobacter spp and Klebsiella spp. respectively showed resistance to meropenem. All ESBL producers were resistant to piperacillintazobactam.&#x0D; Conclusion: Appropriate measures should be taken to prevent the spread of ESBL producing strains by combining strategies for infection prevention, control and rational use of antibiotics.&#x0D; KYAMC Journal Vol. 11, No.-4, January 2021, Page 171-175

Source and antibiotic susceptibility of gram-negative bacteria causing superficial incisional surgical site infections

BackgroundSurgical site infections (SSIs) are a major public health problem worldwide and the second-most frequently reported type of nosocomial infection. The presence of Gram-negative bacteria (GNB) in the hospital environment is significantly associated with SSIs. As such, this study aimed to explore the rate, antibiotic profile and common sources of bacteria in SSI. MethodsA cross-sectional study was conducted between August 2018 and July 2019 in three surgical centers in Kalar city, Kurdistan region, Iraq. A total of 512 patients, who underwent surgery were followed up with for superficial incisional SSI. Pre-, intra- and postoperative swabs were collected from patients and the surrounding hospital environment and processed for isolation and identification of GNB by microbiological and molecular methods. The isolates were typed by sequencing and tested for antibiotic resistance using the disc-diffusion technique. ResultsSSI developed in 113 (22.07%) of the patients studied. GNB were involved in 48 (9.38%) cases; among these, Klebsiella spp. were the predominant cause 21/48 (43.75%), followed by Escherichia coli 14/48 (29.17%), Pseudomonas aeruginosa 7/48 (14.58%), Enterobacter spp. 3/48 (6.25%) and A. baumannii 2/48 (4.17%). Citrobacter koseri showed the lowest rate of infection 1/48 (2.08%). Sequencing analysis and the antibacterial resistance profile revealed that 25 (74.28%) of the 48 SSI isolates were from the hospital environment, whereas the rest were exogenous, with an undetermined source. The involved GNB were highly resistant to most antibiotics tested. ConclusionSSIs caused by GNB were mostly exogenous or from the hospital environment, not from endogenous sources. All of the bacterial isolates detected from SSI patients were multidrug resistant.

Isolation and Identification of Gram-Negative Bacteria from Oral Cancer Site Infections and Study of Their Antibiotic Resistance Pattern

Cancer is one of the non-communicable lethal diseases which affects millions of people worldwide and is responsible for the majority of global deaths. Among the types of cancers occurring in humans, oral cancer has a higher incident rate which is significantly alarming. Oral cancer sites are associated with increased risk of metastasis, along with making the cancer site susceptible to infection by a vast number of opportunistic pathogens. Gram-negative opportunistic bacilli can dwell in these cancer ulcer sites since the patient becomes immune compressed due to radiotherapy and chemotherapy treatments, which prolong the infection. The present study was undertaken to find the most prevalent gram-negative bacilli in oral cavity cancer sites. Swab samples from 50 oral cancer patients were taken to check the presence of the opportunistic organisms. While the control group was set with people with no cancer, only 4 bacteria were isolated from 40 healthy volunteers. Among the isolates from both test group and control group, the most prevalent organism was Klebsiella species 37(45%), followed by Pseudomonas species 29 (34.5%) and Proteus species 8(9.5%). The least prevalent was Escherichia coli 5(6%). The isolates were all taken for antibiotic sensitivity testing (AST) against 15 antibiotics from 11 different groups used in hospitals. Results of the AST were as follows: 100% of the isolates from cancer patients were resistant to vancomycin, clindamycin, amoxicillin, penicillin G, and metronidazole. It was followed by azithromycin, where 86.25% of the isolates were resistant to it. The least resistance was seen against aminoglycoside antibiotic group, amikacin (6.25%), then gentamicin having a resistance percentage of 7.5. A substantial amount of qualitative data on medical history, clinical examination, and treatment, etc. was documented and presented in this study.&#x0D; Bangladesh J Microbiol, Volume 36 Number 2 December 2019, pp 85-90

Incidence and outcomes of multidrug-resistant gram-negative bacteria infections in intensive care unit from Nepal- a prospective cohort study

BackgroundInfections caused by multi-drug resistant gram-negative bacterial infections are the principle threats to the critically ill patients of intensive care units. Increasing reports of these infections from the Nepalese intensive care unit underline the clinical importance of these pathogens. However, the impact of these infections on the patient’s clinical outcome has not yet been clearly evaluated. The objective of our study was to determine the incidence and associated clinical outcome of multi-drug resistant gram-negative bacterial infections in intensive care unit from a tertiary care center of Nepal.MethodsA prospective cohort study was conducted among adult patients admitted in intensive care unit of B. P Koirala Institute of Health Sciences from July to December 2017. Patients infected with multi-drug resistant gram-negative bacteria, non-multi-drug resistant gram-negative bacteria and those without infection were included. Identification of gram-negative bacteria and their antibiotic susceptibility pattern was performed with standard microbiological methods. Demographic, clinical profiles and outcomes (in-hospital-mortality, intensive care unit and hospital length of stay) were documented.ResultsThe incidence rate of multi-drug resistant gram-negative bacteria infections was 47 per 100 admitted patients (64/137) with 128 episodes. Acinetobacter species (41%, 52/128) was the commonest followed by Klebsiella pneumoniae (28%, 36/128) and Pseudomonas spp (21%, 27/128). Patients with multi-drug resistant gram-negative bacteria in comparison to non-multi-drug resistant gram-negative bacteria had high healthcare-associated infections (95%, 61/64 versus 20%, 2/10; p = < 0.001). In-hospital-mortality was 38% (24/64), 20% (2/10) and 10% (4/41) in multi-drug resistant, non-multi-drug resistant and uninfected group respectively (p = 0.007). After adjustment for independent risk factors, compared to uninfected patients, the odds ratio (CI) for in-hospital-mortality in multi-drug resistant and non-multi-drug resistant group was (4.7[1.4–15.5], p = 0.01) and 2.60 [0.38–17.8], p = 0.32) respectively. Multi-drug resistant patients also had longer intensive care unit and hospital stay, however, it was statistically insignificant.ConclusionThe incidence of multi-drug resistant gram-negative bacterial infections was remarkably high in our intensive care unit and showed a significant association with healthcare-associated infections and in-hospital-mortality.

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Comparison of biomarker based Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and conventional methods in the identification of clinically relevant bacteria and yeast

BackgroundMALDI-TOF MS is an analytical method that has recently become integral in the identification of microorganisms in clinical laboratories. It relies on databases that majorly employ pattern recognition or fingerprinting. Biomarker based databases have also been developed and there is optimism that these may be superior to pattern recognition based databases. This study compared the performance of ribosomal biomarker based MALDI-TOF MS and conventional methods in the identification of selected bacteria and yeast.MethodsThe study was a cross sectional study identifying clinically relevant bacteria and yeast isolated from varied clinical specimens submitted to a clinical laboratory. The identification of bacteria using conventional Vitek 2™ automated system, serotyping and MALDI-TOF MS was performed as per standard operating procedures. Comparison of sensitivities were then carried out using Pearson Chi-Square test and p-value of <0.05 was considered statistically significant. Secondary outcomes analyzed included the major and minor error rates.ResultsOf the 383 isolates MALDI-TOF MS and conventional methods identified 97.6 and 95.7% (p = 0.231) to the genus level and 97.4 and 88.0% (p = 0.000) to the species level respectively. Biomarker based MALDI-TOF MS was significantly superior to Vitek 2™ in the identification of Gram negative bacteria and Gram positive bacteria to the species level. For the Gram positive bacteria, significant difference was observed in the identification of Coagulase negative Staphylococci (p = 0.000) and Enterococcus (p = 0.008). Significant difference was also observed between serotyping and MALDI-TOF MS (p = 0.005) and this was attributed to the lack of identification of Shigella species by MALDI-TOF MS. There was no significant difference observed in the identification of yeast however some species of Candida were unidentified by MALDI-TOF MS.ConclusionBiomarker based MALDI-TOF MS had good performance in a clinical laboratory setting with high sensitivities in the identification of clinically relevant microorganisms.

Prevalence and clinical significance of early high Endotoxin Activity in septic shock: An observational study

PurposeTo measure the prevalence of elevated Endotoxin Activity (EA) in a large cohort of patients with Septic Shock (SS), and to assess its value as an early indicator of Gram-Negative (GN) infection, disease severity, and patient risk. Materials and methodsAdult patients were enrolled in this observational study if an EA determination was obtained within 24-h from SS onset. Demographic, clinical, and microbiological data were collected. In-hospital follow-up was also conducted. ResultsA high prevalence of endotoxemia was observed in the 107 subjects included, with 82% of patients showing either intermediate (≥0.4 units), or high (≥0.6) EA. Patients with positive cultures for GNs showed a higher mean EA (0.63±0.18 vs. 0.53±0.22; p<0.05). However, the test showed poor accuracy in the identification of GN bacteria as SS causative agents. Significantly higher lactate concentration (p=0.006), SOFA (p=0.04) and inotropic score (p=0.006) were observed in patients with endotoxemia. However, higher EA levels neither influenced mortality, nor length of stay. ConclusionsEarly after SS onset, patients showed a high prevalence of endotoxemia, particularly those infected with GN bacteria. The EA assay might be a useful marker of disease severity. The complexity of such patients, however, limits EA accuracy in identifying GN sepsis and predicting outcome.

Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system Clin-TOF-II MS in identification of gram-negative bacteria

Objective To evaluate the performance of domestic matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system Clin-TOF-Ⅱ MS with BioExplorer V2.3 database (Clin-TOF MS system) on gram-negative bacteria identification. Methods This was a methodological comparison study. A total of 1 025 gram-negative strains of 32 genus, 56 species or species complex were included in this study from 1999 to 2000 and 2014 to 2016 in Peking Union Medical College Hospital.The Bruker Biotyper MS system (Bruker MS system), Bruker Autoflex Speed with Biotyper v3.1 database were used as control. Identification by both MALDI-TOF MS systems were parallel conducted by direct smear method. The 16S rDNA sequencing based was performed when either MALDI-TOF MS system gave unbelievable result or results from two systems were not consistent. Results Amongst the isolates studied, 98.05% (1 005/1 025) was correctly identified to species or species complex level by Clin-TOF MS system. Comparatively, 99.22% (1 017/1 025) was correctly identified by Bruker MS system. There were 17 isolates just identified to genus level and 2 isolates were no identification by Clin-TOF MS system, meanwhile 1 Pseudomonas monteilii misidentified as P. putida. There were only two 2 isolates identified to genus level and 3 isolates were no identification by Bruker MS system. But it misidentified all 3 Aeromonas hydrophila (2 isolates as A. caviae and 1 isolate as A. media). It′s noted that both MS systems identified 1 Chryseobacterium gleum and 1 C. bernardetii to genus level. Conclusion The capability of domestic Clin-TOF MS system was good on gram-negative bacteria.(Chin J Lab Med, 2017, 40: 41-45) Key words: Gram-negative bacteria; Spectrometry, mass, matrix-assisted laser desorption-ionization

Supramolecular Conjugated Polymer Materials for in Situ Pathogen Detection.

Cationic poly(fluorene-co-phenylene) derivative (PFP-NMe3+) forms a supramolecular complex with cucurbit[7]uril (CB[7]), which could be reversibly disassembled by amantadine (AD) to release PFP-NMe3+ due to the formation of more stable CB[7]/AD complex. The cationic PFP-NMe3+ is an amphiphilic structure and could bind to negatively charged membrane of pathogen by multivalent interactions. Upon the formation of PFP-NMe3+/CB[7] complex, the CB[7] could bury the side-chain alkyl groups and decreases the hydrophobic interactions of PFP-NMe3+ on the surface of pathogens; thus, PFP-NMe3+ exhibits different interaction modes with pathogens before and after assembly with CB[7]. The PFP-NMe3+/CB[7] supramolecular complex could be explored as optical sensor for simple, rapid, and in situ detection and discrimination of multiple pathogens by taking advantage of optical signal changes of PFP-NMe3+/CB[7] complex before and after disassembly by AD on the pathogen surfaces. The new sensor can realize in situ detection and identification of Gram-negative bacteria (E. coli, P. aeruginosa), Gram-positive bacteria (B. subtilis, S. aureus, E. faecalis), and the fungi (C. albicans, S. cerecisiae) and can also discriminate different strains of the same species. Blend samples of these pathogens could be identified successfully as well. In comparison with conventional blood culture-based pathogen assay methods that require at least for 24 h, the PFP-NMe3+/CB[7] complex only needs 2 h (including pathogen culture, pathogen harvest by centrifuging, and optical assay procedures) to stratify diverse pathogen types and also does not require specific biomarkers or cell labeling.

Direct identification and susceptibility testing of positive blood cultures using high speed cold centrifugation and Vitek II system.

Compared to routine isolated colony-based methods, direct testing of bacterial pellets from positive blood cultures reduces turnaround time for reporting of antibiotic susceptibility. The aim of this study was to compare the accuracy, and precision, of a rapid method for direct identification and susceptibility testing of blood cultures with the routine method used in our laboratory, using Vitek 2. A total of 60 isolates were evaluated using the candidate and the routine method. The candidate method had 100% accuracy for the identification of Gram negative bacteria, Staphylococcus and Enterococcus, 50% for Streptococcus and 33.3% for Corynebacterium species. Susceptibility testing of Gram negative isolates yielded 98-100% essential agreement. For Staphylococcus and Enterococcus isolates, essential agreement was 100% for 17 antibiotics except for moxifloxacin. Direct testing of blood culture samples with Vitek 2 produced reliable identification and susceptibility results 18-24h sooner for aerobic/anaerobic facultative Gram-negative bacteria and Gram-positive Staphylococcus and Enterococcus strains.

Rapid identification of gram-negative bacteria and corresponding resistance determinants from blood cultures by BC-GN assay

Objective To evaluate the performance and clinical utility of Verigene gram-negative blood culture (BC-GN) assay for a rapid identification of gram-negative bacteria and resistance genes. Methods Non-repetitive blood culture samples containing gram-negative bacteria were collected from inpatients in the Second Affiliated Hospital of Zhejiang University School of Medicine from June to October, 2013. BC-GN assay was performed to identify the species and genetic resistance determinants of gram-negative bacteria directly from the positive blood culture bottles. VITEK MS and the VITEK 2 Compact were used for species identification and antimicrobial susceptibility test, the results of which were considered as gold standards. The resistance genes were further validated by PCR amplification and sequencing analysis. A comparison of the results and time between the BC-GN assay and routine methods was conducted. Results The detection range of BC-GN assay almost covered all of the common gram-negative bacteria. BC-GN assay showed an advantage of high accuracy in the identification of Escherichia coli (13/13), Klebsiella pneumoniae (19/24), Klebsiella oxytoca (9/9), Pseudomonas aeruginosa (39/39), Serratia marcescens (4/5), Enterobacter spp. (6/8), Citrobacter spp. (11/11), Proteus spp. (6/6) and Acinetobacter spp. (24/24) with an overall accuracy of 94.24% for the identification of mono-microbial blood culture samples. Moreover, BC-GN assay accurately identified all of the bacteria and resistance genes from the two multi-microbial samples. Species identification and resistance profiles could be 42 hours earlier available by using BC-GN assay than those by using routine methods. Conclusion BC-GN assay could simultaneously and accurately identify bacteria and resistance determinants from blood cultures within 2 hours. More time for clinically effective therapy could be achieved by using BC-GN assay for the reduction of mortality associated with bloodstream infection. Key words: Bloodstream infection; Gram-negative; Antimicrobial resistance; Rapid identification

Abstract 2011: Feasibility of serum proteomic companion diagnostic (CDx) test development on the microflex LT platform

Abstract Clinical oncology assays using mass spectrometry have exclusively been run on instrument platforms designed for use in research laboratories. Here, we report on the use of a bench-top MALDI-TOF mass spectrometer (MS) to run a serum proteomic Laboratory Developed Test (LDT), VeriStrat®. A number of previous studies have shown that this assay measures a serum proteomic fingerprint using MALDI-TOF MS which, following spectral acquisition and data processing, classifies advanced non-small cell lung cancer (NSCLC) patients into treatment categories. An extension of VeriStrat, BDX004, is currently under development as part of a global, randomized, double-blind Phase 2 clinical study, FOCAL. BDX004 will be used prospectively to select previously untreated, EGFR mutation-positive patients with advanced NSCLC to receive one of two treatments: (1) the combination of ficlatuzumab (a humanized monoclonal antibody against hepatocyte growth factor) and an EGFR tyrosine kinase inhibitor (TKI), or (2) an EGFR TKI with placebo. For the development of BDX004 as a companion diagnostic (CDx), we have included the microflex™ LT (LT), a component of Bruker's MALDI Biotyper CA system, which was granted FDA clearance for the identification of Gram negative bacteria. In the present study, we evaluated the LT platform through comparative studies with other MALDI-TOF instruments (autoflex™ III and autoflex SPEED) utilizing samples from patients previously diagnosed with NSCLC. In summary, we found that under common laboratory procedures the LT produces results equivalent to research grade MALDI-TOF MS instrumentation. In initial studies, the microflex LT showed similar resolution (R &amp;gt; 600 at 6632.1 m/z) and sensitivity (S/N ≥ 50 with 500 fmoles BSA in 100 laser shots) performance parameters as the autoflex series research instruments (R &amp;gt; 800 at 6632.1 m/z and S/N ≥ 50 with 500 fmoles BSA in 250 laser shots). The LT was qualified with several independent sample sets [n = 4 (8 replicates; 2000 spectra/replicate); n = 67 (3 replicates; 2000 spectra/replicate) and n = 20 (3 replicates; 2000 spectra/replicate)]. In these studies, the LT achieved 100%, &amp;gt;97%, and 100% concordance with reference data sets previously acquired on the autoflex platforms. As expected, due to the lower laser repetition rate of the LT's nitrogen laser, the acquisition speed was slower on the LT as compared to the autoflex instruments by a factor of two. However, the slower acquisition speed is partially compensated for with increased sensitivity, which is particularly evident in the reference sample mass spectra which have signal-to-noise ratios ∼1.5 greater than those acquired on the autoflex platform. Based on these results, the microflex LT appears to be suitable for measurement of serum proteomic profiles in NSCLC and suggests that the inclusion of the microflex LT in CDx test development has the potential to expand the utility of MALDI-based testing in the clinic. Citation Format: Nicholas Dupuis, Jamie Chang, Maximillian Steers, Zhigang Weng, Jeno Gyuris, Gary Pestano. Feasibility of serum proteomic companion diagnostic (CDx) test development on the microflex LT platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2011. doi:10.1158/1538-7445.AM2015-2011

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry