Sonication protocols and their contributions to the microbiological diagnosis of implant-associated infections: a review of the current scenario.

Addressing the existing problem in the microbiological diagnosis of infections associated with implants and the current debate about the real power of precision of sonicated fluid culture (SFC), the objective of this review is to describe the methodology and analyze and compare the results obtained in current studies on the subject. Furthermore, the present study also discusses and suggests the best parameters for performing sonication. A search was carried out for recent studies in the literature (2019-2023) that addressed this research topic. As a result, different sonication protocols were adopted in the studies analyzed, as expected, and consequently, there was significant variability between the results obtained regarding the sensitivity and specificity of the technique in relation to the traditional culture method (periprosthetic tissue culture - PTC). Coagulase-negative Staphylococcus (CoNS) and Staphylococcus aureus were identified as the main etiological agents by SFC and PTC, with SFC being important for the identification of pathogens of low virulence that are difficult to detect. Compared to chemical biofilm displacement methods, EDTA and DTT, SFC also produced variable results. In this context, this review provided an overview of the most current scenarios on the topic and theoretical support to improve sonication performance, especially with regard to sensitivity and specificity, by scoring the best parameters from various aspects, including sample collection, storage conditions, cultivation methods, microorganism identification techniques (both phenotypic and molecular) and the cutoff point for colony forming unit (CFU) counts. This study demonstrated the need for standardization of the technique and provided a theoretical basis for a sonication protocol that aims to achieve the highest levels of sensitivity and specificity for the reliable microbiological diagnosis of infections associated with implants and prosthetic devices, such as prosthetic joint infections (PJIs). However, practical application and additional complementary studies are still needed.

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken.

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host's physiology, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken's GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken's GIT.

Evaluation of the utility and cost of secondary confirmatory testing for Neisseria gonorrhoeae identification from culture.

Current guideline recommends the use of two identification methods for Neisseria gonorrhoeae. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is now used for primary identification and may be sufficient for definitive identification of N. gonorrhoeae. The performance of three secondary tests (BactiCard, RapID NH and NET test) were compared using 45 bacterial isolates, including 37 Neisseria species. These secondary tests demonstrated diminished specificity (67% - 88%) for N. gonorrhoeae compared with MALDI-TOF. Additionally, data from six clinical microbiology laboratories was used to compare confirmatory test costs and the agreement of results with MALDI-TOF. Discrepancies were documented for 9.4% of isolates, though all isolates (n= 288) identified by MALDI-TOF as N. gonorrhoeae were confirmed by the reference laboratory. These data demonstrate that MALDI-TOF alone is sufficient for N. gonorrhoeae identification, as secondary did not add diagnostic value but do add costs to the testing process.

A forced aeration system for microbial culture of multiple shaken vessels suppresses volatilization.

Shake-flask culture, an aerobic submerged culture, has been used in various applications involving cell cultivation. However, it is not designed for forced aeration. Hence, this study aimed to develop a small-scale submerged shaking culture system enabling forced aeration into the medium. A forced aeration control system for multiple vessels allows shaking, suppresses volatilization, and is attachable externally to existing shaking tables. Using a specially developed plug, medium volatilization was reduced to less than 10%, even after 45 h of continuous aeration (~ 60 mL/min of dry air) in a 50 mL working volume. Escherichia coli IFO3301 cultivation with aeration was completed within a shorter period than that without aeration, with a 35% reduction in the time-to-reach maximum bacterial concentration (26.5 g-dry cell/L) and a 1.25-fold increase in maximum concentration. The maximum bacterial concentration achieved with aeration was identical to that obtained using the Erlenmeyer flask, with a 65% reduction in the time required to reach it.

Identification of the Optimal Cultivation Period Required to Isolate Representatives of Mycobacterium abscessus Complex Isolated from Patients with Cystic Fibrosis.

BACKGROUND: In patients with cystic fibrosis (CF), representatives of the fast-growing Mycobacterium abscessus complex (MABSc) are often distinguished, but the culture of the material taken from such patients increases the growth time. We analyzed the terms of cultivation of MABSc representatives on dense nutrient media and also evaluated the productivity of a modified nutrient medium based on agar for the isolation of Burkholderia cepacia complex (BCC). METHODS: Sixty-four strains of MABSc isolated from patients with CF and suspected tuberculosis were analyzed. The material from the patients was cultured on a universal chromogenic medium, 5% blood agar, yolk-salt agar, selective medium for isolation of BCC, and Löwenstein-Jensen medium. The cultures were incubated for 5 days (37°C, aerobic conditions), after for 23 days (28°C, aerobic conditions). The productivity of the developed nutrient medium was evaluated by the number of cells that gave visible growth after culturing 0.1 mL of a bacterial suspension of 103 CFU/mL. RESULTS: 76.8% of the strains grew in a 2-week period, and 23.2% of the strains were obtained at a later date from 18 to 28 days (average: 21.23 days). The modified medium with a concentration of 240 mg of iron (III) polymaltose hydroxide proved to be the most optimal for the isolation of MABSc. CONCLUSION: When using a chromogenic medium for culture material from patients with CF, it is necessary to extend incubation up to 28 days to increase the probability of MABSc isolation. The modified BCC medium showed a good selectivity result but required further investigation.

Development and evaluation of an artificial intelligence for bacterial growth monitoring in clinical bacteriology.

In clinical bacteriology laboratories, reading and processing of sterile plates remain a significant part of the routine workload (30%-40% of the plates). Here, an algorithm was developed for bacterial growth detection starting with any type of specimens and using the most common media in bacteriology. The growth prediction performance of the algorithm for automatic processing of sterile plates was evaluated not only at 18-24 h and 48 h but also at earlier timepoints toward the development of an early growth monitoring system. A total of 3,844 plates inoculated with representative clinical specimens were used. The plates were imaged 15 times, and two different microbiologists read the images randomly and independently, creating 99,944 human ground truths. The algorithm was able, at 48 h, to discriminate growth from no growth with a sensitivity of 99.80% (five false-negative [FN] plates out of 3,844) and a specificity of 91.97%. At 24 h, sensitivity and specificity reached 99.08% and 93.37%, respectively. Interestingly, during human truth reading, growth was reported as early as 4 h, while at 6 h, half of the positive plates were already showing some growth. In this context, automated early growth monitoring in case of normally sterile samples is envisioned to provide added value to the microbiologists, enabling them to prioritize reading and to communicate early detection of bacterial growth to the clinicians.

Feasibility and utility of a combined nasogastric-tube-and-string-test device for bacteriologic confirmation of pulmonary tuberculosis in young children.

For microbiological confirmation of pediatric pulmonary tuberculosis (PTB), gastric aspirates (GA) are often operationally unfeasible without hospitalization, and the encapsulated orogastric string test is not easily swallowed in young children. The Combined-NasoGastric-Tube-and-String-Test (CNGTST) enables dual collection of GA and string specimens. In a prospective cohort study in Kenya, we examined its feasibility in children under five with presumptive PTB and compared the bacteriological yield of string to GA. Paired GA and string samples were successfully collected in 95.6 % (281/294) of children. Mycobacterium tuberculosis was isolated from 7.0 % (38/541) of GA and 4.3 % (23/541) of string samples, diagnosing 8.2 % (23/281) of children using GA and 5.3 % (15/281) using string. The CNGTST was feasible in nearly all children. Yield from string was two-thirds that of GA despite a half-hour median dwelling time. In settings where the feasibility of hospitalisation for GA is uncertain, the string component can be used to confirm PTB.

Automated detection of methicillin-resistant Staphylococcus aureus with the MRSA CHROM imaging application on BD Kiestra Total Lab Automation System.

The virulence of methicillin-resistant Staphylococcus aureus (MRSA) and its potentially fatal outcome necessitate rapid and accurate detection of patients colonized with MRSA in healthcare settings. Using the BD Kiestra Total Lab Automation (TLA) System in conjunction with the MRSA Application (MRSA App), an imaging application that uses artificial intelligence to interpret colorimetric information (mauve-colored colonies) indicative of MRSA pathogen presence on CHROMagar chromogenic media, anterior nares specimens from three sites were evaluated for the presence of mauve-colored colonies. Results obtained with the MRSA App were compared to manual reading of agar plate images by proficient laboratory technologists. Of 1,593 specimens evaluated, 1,545 (96.98%) were concordant between MRSA App and laboratory technologist reading for the detection of MRSA growth [sensitivity 98.15% (95% CI, 96.03, 99.32) and specificity 96.69% (95% CI, 95.55, 97.60)]. This multi-site study is the first evaluation of the MRSA App in conjunction with the BD Kiestra TLA System. Using the MRSA App, our results showed 98.15% sensitivity and 96.69% specificity for the detection of MRSA from anterior nares specimens. The MRSA App, used in conjunction with laboratory automation, provides an opportunity to improve laboratory efficiency by reducing laboratory technologists' labor associated with the review and interpretation of cultures.

The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia.

BACKGROUND: Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly. OBJECTIVE: To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates. METHOD: 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2. RESULT: Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages. CONCLUSION: The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment.

Application of BactTiter-Glo ATP bioluminescence assay for Mycobacterium tuberculosis detection.

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a global health threat, necessitating faster and more accessible diagnostic methods. This study investigates critical parameters in the application of a commercial ATP bioluminescence assay for the detection of MTB. METHOD: Our objective was to optimize the ATP bioluminescence protocol using BacTiter-Glo™ for MTB, investigating the impact of varying volumes of MTB suspension and reagent on assay sensitivity, evaluating ATP extraction methods, establishing calibration curves, and elucidating strain-specific responses to antimicrobial agents. RESULTS: ATP extraction methods showed no significant improvement over controls. Calibration curves revealed a linear correlation between relative light units (RLU) and colony-forming units (CFU/mL), establishing low detection limits. Antimicrobial testing demonstrated strain-specific responses aligning with susceptibility and resistance patterns. CONCLUSION: Our findings contribute to refining ATP bioluminescence protocols for enhanced MTB detection and susceptibility testing. Further refinements and validation efforts are warranted, holding promise for more efficient diagnostic platforms in the future.

Comparison of bacteriological culture method and multiplex real-time PCR for detection of mastitis.

This study includes the evaluation of multiplex real-time PCR (rPCR) kit, which was developed to provide rapid diagnosis of mastitis infections, by working with milk samples of 2 different sources of mastitis and comparing the results with the classical bacteriological culture method (BC). A total of 273 bacteria were isolated in 226 samples (47.88%) out of 472 samples by BC. These were 139 (50.91%) Staphylococcus spp., 61 (22.34%) Streptococcus spp., 15 (5.49%) E. coli, 8 (2.93%) Enterococcus spp., 50 (18.31%) other bacteria. When we look at the multiplex rPCR results; 1052 positive were obtained for the gene regions of 14 different bacteria, 1 yeast, and 1 ß-lactamase gene examined in 472 samples. While no searched gene region was found by rPCR in 78 (16.5%) of the 472 samples studied, at least 1 gene was detected in 394 (83.5%) samples. These 1052 positive samples by rPCR were; 263 (28.43%) Staphylococcus spp., 51 (5.51%) S. aureus, 57 (6.16%) Enterococcus spp., 49 (5.29%) C. bovis, 16 (1.73%) S. dysgalactiae, 84 (9.08%) S. agalactiae, 71 (7.67%) S. uberis, 73 (7.89%) E. coli, 14 (1.51%) Prototheca spp., 39 (4.21%) T. pyogenes/P. indolicus, 5 (0.54%) S. marcescens, 15 (1.62%) K. oxytoca/pneumonia, 117 (12.64%) Mycoplasma spp., 31 (3.35%) M. bovis, 40 (4.32%) yeast, and 127 samples (26.90%) were ß-lactamase positive. When the antibiotic resistance of the isolates was evaluated, 78 (31.96%) tetracycline, 72 (29.5%) penicillin, and 60 (24.59%) clindamycin resistance were observed predominantly in Gram-positive isolates, while 6 (23.07%) tigecycline, 6 (23.07%) netilmicin, 6 (23.07%) pipercillin resistance was found in gram-negative isolates. While a bacteria and/or yeast gene was found by rPCR in 187 of 246 (76.01%) samples with no bacterial growth, a bacterium was isolated with BC in only 20 (8.84%) samples whose gene region was not found by rPCR. As a result, the multiplex rPCR system used in the diagnosis of mastitis has been found to be quite reliable as it can detect a large number of bacteria in a very short time compared to classical methods. Therefore, we advise the use of rPCR and/or culture for confirmation of clinical signs in mastitis and at routine mastitis surveillance.

New Antibiotic Binding Resin Bottles Collected from Children Enhances Rapid Microbial Identification.

BACKGROUND: Bloodstream infections (BSI) represent a common cause of sepsis and mortality in children. Blood culture (BC) is the gold standard for diagnosis of BSI. The low sensitivity of BC in the pediatric population is usually due to the small volume of blood used for inoculation and to the antibiotics used before sampling. Here, we explore the ways to effectively reduce antibiotic activity to maximize the chances of pathogen recovery, and to enhance the growth of microorganisms in lower blood volume and bacterial counts. METHODS: The recovery of common pathogens causing blood stream infections was analyzed after exposure to cefo-perazone/sulbactam, vancomycin, and caspofung by using resin-containing or not BacT/Alert PF Plus and BD FX400 peds plus pediatric bottles. The microbial growth in the resin-containing bottles was assessed using 0.5 colony-forming units (CFU) bacterial inoculum to mimic the bacteremia/fungemia condition. The usefulness of a diagnosis to confirm or exclude BSI was evaluated by lower than recommended blood culture sampling (102 CFU/mL, 0.3 mL). RESULTS: Staphylococcus aureus (S. aureus), and Candida glabrata (C. glabrata) were recovered from 100% of two types of resin-containing bottles in the presence of a sufficient antibiotic dose, while Escherichia coli (E. coli) was not restored to 100% in BD FX400 peds plus pediatric bottles. The shorter TTD for S. aureus, C. glabrata, and E. coli were observed in antibiotic-containing BacT/Alert PF Plus bottles. Both the PF Plus and BD resin test bottles showed consistently good TTD performances to Gram-negative, Gram-positive, and yeast species in low inoculum levels, with the exception of S. aureus. The lower volume of blood inoculated into culture bottles hardly affected the growth of most bacteria, but optimized PF Plus resin-bottles accelerated the detection of infectious agents, especially S. aureus, Streptococcus pneumoniae, and C. glabrata. CONCLUSIONS: It is possible to enhance recovery from antibiotic-containing pediatric bottles and shorten TTD for the identification of pathogens by using the BacT/Alert blood culture system combination with new resin-containing media.

Validation of rapid detection methods for Salmonella enterica in green chile.

The objective of this study is to validate the US Food and Drug Administration (FDA) rea-time polymerase chain reaction (qPCR) assay, the Neogen Amplified Nucleic Single Temperature Reaction (ANSR) assay, and the Vitek ImmunoDiagnostic Assay System (VIDAS) SLM procedure against the FDA cultural procedure for Salmonella detection in green chile pepper. Green chile was artificially contaminated with Salmonella according to the FDA guidelines (FDA. Guidelines for the Validation of Microbiological Methods for the FDA Foods Program, 3rd Edition. 2019. www.fda.gov/media/83812/download?attachment (17 March 2024, date last accessed)) at a fractional recovery level (where 50%-25% tests positive and at a level +1 log greater for each organism tested). Enriched samples were tested directly by the ANSR Salmonella test and by qPCR, and were subcultured into Rappaport-Vassiliadis and tetrathionate brilliant green broth for cultural detection and qPCR. For the VIDAS-SLM assay, the selective enrichments were further cultured in M broth before testing. Presumptive salmonellae were confirmed with biochemical tests, serology, and qPCR. All three rapid assays were compared favorably with the FDA-BAM (Bacteriological Analytical Manual) method. No significant differences at P < .05 were found between the procedures using McNemar's χ2 test. The three procedures were found to be rapid and reliable alternatives to cultural detection of Salmonella enterica in green chile.

Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME).

Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.

Use of a chromogenic medium with and without selective enrichment to screen for carbapenemase-producing Enterobacterales (CPE) from canine and feline fecal specimens during an outbreak of NDM-5-producing Escherichia coli.

Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Descriptions of CPE outbreaks in veterinary hospitals suggest the need for screening strategies for CPE from companion animals. Our aim was to optimize a chromogenic agar method with and without selective enrichment to isolate CPE from companion animal feces in an ongoing outbreak of New Delhi metallo-ß-lactamse-5 Escherichia coli. A limit of detection (LOD) assay for spiked canine and feline feces was performed for both methods using a carbapenamase-producing E. coli (24213-18); the LOD (1.5 × 103 cfu/g of feces) was equivalent to that reported for human fecal specimens. We screened 1,247 companion animal fecal specimens for carriage of CPE by 1) direct plating to chromogenic agar and 2) plating to chromogenic agar following selective enrichment. Twenty-one specimens were positive for CPE by both direct culture and enrichment culture. No specimens were positive with selective enrichment and negative by direct culture. A selective enrichment step did not result in any increased recovery of CPE from companion animals, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. It is important to continue to validate methods for the detection of CPE in companion animals as outbreaks become more common in veterinary facilities.

The operational and financial impact of adding anaerobic screening of platelets.

BACKGROUND AND OBJECTIVES: We evaluated the operational and safety impact of implementing anaerobic culture screening of apheresis and pooled platelets at the American Red Cross on the already established use of the aerobic culture screening of each donation performed no sooner than 24 h following collection. MATERIALS AND METHODS: Platelets were screened for bacterial contamination with the BACT/ALERT 3D® (bioMérieux, Durham, NC) microbial detection testing system. The addition of anaerobic culture to the already existing aerobic culture resulted in sampling an additional 8-10 mL from each donation. RESULTS: Implementation of anaerobic testing resulted in an approximate 3.5-fold increased rate of False Positive BACT/ALERT alarms. There was a modest increase in the rate of True Positive alarms of 1.4-fold with increased detection of Klebsiella and Propionibacterium species, including Cutibacterium acnes. In addition, there was an approximate 3.5-fold increase rate of False Positives and a 13.5-fold increase rate of Indeterminates, the majority (~57%) were due to Cutibacterium acnes. The combined costs and lost revenue associated with adding anaerobic screening increased by ~$1,000,000/year due to testing cost and product discards. CONCLUSION: The addition of anaerobic culture to aerobic culture to the original donation (without the introduction of sampling delay) resulted in a significant increase in the rate of alerts. The 40% increased rate of True Positive alarms may have modestly improved platelet safety. However, there was a disproportionate increase in the rate of False Positive and Indeterminate bacterial culture alarms, which added substantial cost and overall loss of platelet products.

Evaluation of PhenoMATRIX and PhenoMATRIX PLUS for the screening of MRSA from nasal and inguinal/perineal swabs using chromogenic media.

The objective of this study was to assess the clinical performances of PhenoMATRIX and PhenoMATRIX PLUS for the screening of methicillin-resistant Staphylococcus aureus (MRSA) from nasal and inguinal/perineal ESwabs using chromogenic media. The automated performances were compared to the manual reading. Additionally, we evaluated PhenoMATRIX PLUS for the automatic release of the negative results to the Laboratory Information System (LIS) and the automatic discharge of the negative plates from the incubators. A total of 6,771 non-duplicate specimens were used by PhenoMATRIX as a machine learning model. The validation of these settings was performed on 17,223 non-duplicate specimens. The MRSA positivity rate was 5% (866/17,223). Validated settings were then used by PhenoMATRIX PLUS on another 1,409 non-duplicate specimens. The sensitivities of PhenoMATRIX and PhenoMATRIX PLUS were 99.8% [95% confidence interval (CI), 99.2%-99.9%] and 100% (95% CI, 92.1%-100%), respectively. The specificities of PhenoMATRIX and PhenoMATRIX PLUS were 99.1% (95% CI, 99.0%-99.2%) and 95.2% (95% CI, 93.8%-96.1%), respectively. All the 1,297 MRSA-negative specimens analyzed by PhenoMATRIX PLUS were automatically released and sent to the LIS immediately after availability of the culture image on the WASPLab (100% accuracy). All negative media plates were automatically discarded. PhenoMATRIX PLUS decreases the time spent by technologists on negative plates and ensures optimal usage of the incubators' capacity.

Sensitive detection of viable Cronobacter sakazakii by bioluminescent reporter phage emitting stable signals with truncated holin.

As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs-specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health.