Bacterial Detection and Recovery From Poultry Litter

The level of pathogens in poultry litter used for raising broiler chickens is critical to the overall health of a broiler chicken flock and food safety. Therefore, it is imperative that methods used for determining bacterial concentration in litter are accurate and reproducible across studies. In this perspective, we discuss the shortcomings associated with current methods used for bacterial quantification and detection from litter and assess the efficacy of one method for pathogen and commensal (Campylobacter, Salmonella, Escherichia coli, and Enterococcus spp.) recovery. The limit of quantitation and detection for this method differed between pathogens, and the recovery rate (∼138–208%) was higher for Salmonella, E. coli, and Enterococcus compared to Campylobacter (24%). Our results suggest that pathogen recovery from litter is highly variable and pathogen concentrations need to be reported in dry weight before comparisons can be made between studies.

Blood Culture Testing Outcomes among Non-Malarial Febrile Children at Antimicrobial Resistance Surveillance Sites in Uganda, 2017–2018

Blood culture (BC) processes are critical to the utility of diagnostic testing, bloodstream infection (BSI) management, and antimicrobial resistance (AMR) surveillance. While Uganda has established BC guidelines, often laboratory practice does not meet the desired standards. This compromises pathogen recovery, reliability of antimicrobial susceptibility testing, and diagnostic test utility. This study assessed laboratory BC process outcomes among non-malarial febrile children below five years of age at five AMR surveillance sites in Uganda between 2017 and 2018. Secondary BC testing data was reviewed against established standards. Overall, 959 BC specimens were processed. Of these, 91% were from female patients, neonates, infants, and young children (1–48 months). A total of 37 AMR priority pathogens were identified; Staphylococcus aureus was predominant (54%), followed by Escherichia coli (19%). The diagnostic yield was low (4.9%). Only 6.3% of isolates were identified. AST was performed on 70% (18/26) of identified AMR priority isolates, and only 40% of these tests adhered to recommended standards. Interventions are needed to improve laboratory BC practices for effective patient management through targeted antimicrobial therapy and AMR surveillance in Uganda. Further research on process documentation, diagnostic yield, and a review of patient outcomes for all hospitalized febrile patients is needed.

Impact of delays to incubation and storage temperature on blood culture results: a multi-centre study

Background Blood cultures are one of the most important tests performed by microbiology laboratories. Many hospitals, particularly in low and middle-income countries, lack either microbiology services or staff to provide 24 h services resulting in delays to blood culture incubation. There is insufficient guidance on how to transport/store blood cultures if delays before incubation are unavoidable, particularly if ambient temperatures are high. This study set out to address this knowledge gap. Methods In three South East Asian countries, four different blood culture systems (two manual and two automated) were used to test blood cultures spiked with five common bacterial pathogens. Prior to incubation the spiked blood culture bottles were stored at different temperatures (25 °C, in a cool-box at ambient temperature, or at 40 °C) for different lengths of time (0 h, 6 h, 12 h or 24 h). The impacts of these different storage conditions on positive blood culture yield and on time to positivity were examined. Results There was no significant loss in yield when blood cultures were stored < 24 h at 25 °C, however, storage for 24 h at 40 °C decreased yields and longer storage times increased times to detection. Conclusion Blood cultures should be incubated with minimal delay to maximize pathogen recovery and timely result reporting, however, this study provides some reassurance that unavoidable delays can be managed to minimize negative impacts. If delays to incubation ≥ 12 h are unavoidable, transportation at a temperature not exceeding 25 °C, and blind sub-cultures prior to incubation should be considered.

332. Spinal Infections: Clinical and Microbiological Characteristics in our Urban Referral Health Center

Background There has been an increasing trend in spinal infections (SI) in the U.S. over recent years. We sought to characterize the clinical and microbiological characteristics of SI at our hospital. Methods We conducted a retrospective review of SI over a 3-year period (2016 - 2019) utilizing ICD codes for data retrieval. Search terms included vertebral osteomyelitis, discitis, and epidural abscess. SPSS was used to compute the data. Results Of the initially screened 254 patients, 166 were included for analysis. Pertinent demographics were: mean age 59 years, male (61.4%), obese (44.5%), diabetic (25%), and drug-users (20%). Lumbosacral involvement was most common (69.8%); epidural abscess was present in 51.8% of patients. 15.7% had existing hardware. Overall, 79.5% (132/166) of cases had a positive culture from at least one site: blood 56.6% (94/166), CT-guided 83.5% (56/67), and surgical 51.1% (24/47). Of those patients with negative blood cultures, 22% (16/72) had pathogen recovery by CT-guided methods and 33% (24/72) from surgical specimens. S aureus was the most common pathogen isolated at 53.7% (71/132): MSSA comprised 38.6% (51/132) and MRSA 15.2% (20/132). The mean CRP (8.46 vs 15.83 mg/dL; P&lt; 0.001), and WBC (9.08 vs 13.18 k/mcL; P&lt; 0.001) were higher in culture-positive as compared to culture-negative cases. Mean ESR and temperature more than 100.4 oF did not differ significantly between these two groups. The 8-week median recurrence rate was 11.4%, of which nearly half had index S aureus bacteremia. Frequency of organisms isolated Association of mean inflammatory markers with positive cultures Conclusion Our study affirmed that S aureus is the most common cause of SI, of which MSSA was predominant. Epidural abscess was encountered in a substantial fraction of our case population. Leukocytosis and elevated CRP tended to predict culture-positive infection, whereas ESR and fever did not. As recommended in the IDSA Vertebral Osteomyelitis guidelines, blood cultures were obtained in all cases, which yielded positive results in more than half of patients. Pathogen recovery was further improved to nearly 80% with supplemental deep tissue sampling, thus highlighting the opportunity to enhance microbiological diagnosis at our institution. Disclosures All Authors: No reported disclosures

Effects of different pretreatment methods on microbial recovery of infected tissues

This study aimed to evaluate the effects of different pretreatment methods on the degree of microbial recovery in infected tissues. Standard strains of Staphylococcus aureus (SA), Escherichia coli (EC) and Candida albicans (CA) were used to construct single-surface, full-surface, and internal infection models in sterile pork tissue. Manual milling (MM), mechanical homogenization (MH), ultrasonic lysis (UL), dithiothreitol (DTT), and direct culture (DC) were used to pretreat infection tissues, the ability of the different pretreatment methods to achieve pathogen recovery in the different bacterial infection models was compared. At the same time, periprosthetic tissues collected from periprosthetic joint infection (PJI) were pretreated with the same methods. We showed that regardless of whether the single-surface or full-surface infection model was used in SA, EC, and CA infection, the microbial acquisition in the MH group was significantly higher than that in the MM (P <0.01) and UL groups (P <0.01). In the internal infection model, the microbial acquisition of the MH group was significantly higher than that of the MM (P <0.01), UL (P <0.01), DTT (P <0.01), and DC groups (P <0.01). In the PJI cases, the number of bacterial colonies obtained by MH was significantly higher than that obtained by other pretreatment methods (P = 0.004). The effects of MH and DTT in microbial recovery were significantly better than that of DC, UL and MM, and these methods can be used to process multiple tissue samples at the same time, which can further improve the efficiency of clinical microbial diagnosis.

847. The Effect of Antimicrobial Administration on Blood Culture Positivity in Patients with Severe Manifestations of Sepsis

Background Current guidelines recommend obtaining blood cultures prior to antimicrobial therapy in patients with sepsis. Administering antimicrobials immediately without waiting for blood cultures could potentially decrease time to treatment and improve outcomes, but it is unclear the degree to which this strategy impacts diagnostic yield. Methods We performed a patient-level, single-arm, diagnostic trial. Seven urban emergency departments affiliated with academic medical centers across Canada and the United States participated in the study. Adults ≥18 years of age presenting to the emergency department with evidence of severe manifestations of sepsis, including a systolic blood pressure <90 mmHg and/or a serum lactate ≥4 mmol/L were included. Study participants had 2 sets of blood cultures drawn prior to and immediately following antimicrobial administration. The primary outcome was the difference in blood culture pathogen recovery rates before and after administration of antimicrobial therapy. Results Of the 3,164 participants screened, 325 were included in the study (mean age, 65.6 years; 63.0% men) and had repeat blood cultures drawn after the initiation of antimicrobial therapy (median time of 70 minutes, IQR 50 to 110 minutes). Pre-antimicrobial blood cultures were positive for one or more microbial pathogens in 102/325 (31.4%) patients. Fifty-four participants (52.9%) had matching blood culture results after initiation of antimicrobial treatment. The absolute difference in pathogen recovery rates was 14.5% ([95% CI 8.0 to 21.0%]; P < 0.0001) between pre- and post-antimicrobial blood cultures. Results were consistent in an analysis of the per-protocol population (absolute difference, 13.3% [95% CI 6.1 to 20.4%]; P < 0.0001). Including the results of other microbiological cultures done as part of routine care, microbial pathogens were recovered in 69 of 102 (67.7%) participants (absolute difference, 10.2% [95% CI 3.4 to 16.8%]; P < 0.0001). Conclusion Among patients with severe manifestations of sepsis, the administration of empiric antimicrobial therapy significantly reduces the yield of pathogen recovery when blood cultures are drawn shortly after treatment initiation. Disclosures All Authors: No reported Disclosures.

Recovery of Gram-Negative Bacteria from Aerobic Blood Culture Bottles Containing Antibiotic Binding Resins after Exposure to β-Lactam and Fluoroquinolone Concentrations

ABSTRACT Blood culture bottles containing antibiotic binding resins are routinely used to minimize artificial sterilization in the presence of antibiotics. However, the resin binding kinetics can differ between antibiotics and concentrations. This study assessed the impact of clinically meaningful peak, midpoint, and trough concentrations of meropenem, imipenem, cefepime, cefazolin, levofloxacin, and piperacillin-tazobactam on the recovery of Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae from resin-containing BacT/Alert FA Plus and Bactec Aerobic/F blood culture bottles. P. aeruginosa-inoculated bottles alarmed positive in 4/20 (20%), 16/20 (80%), and 20/20 (100%) of those with peak, midpoint, and trough concentrations of antipseudomonal agents, respectively (P ≤ 0.001). E. coli was recovered from 8/24 (33%), 11/24 (46%), and 14/24 (58%) of bottles with peak, midpoint, and trough concentrations, respectively (P = 0.221). K. pneumoniae was recovered from 8/16 (50%) at all concentrations of the studied antibiotics (P = 1.0). BacT/Alert and Bactec bottles inoculated with antibiotics and P. aeruginosa had similar times to detection (TTD) (P = 0.352); however, antibiotic-containing BacT/Alert bottles had a shorter TTD compared with antibiotic-containing Bactec bottles for E. coli (P = 0.026) and K. pneumoniae (P ≤ 0.001). Pathogen recovery in BacT/Alert FA Plus and Bactec Aerobic/F blood culture bottles containing antibiotic binding resins was greatly reduced in the presence of antibiotics, especially at higher concentrations. These data support the practice of drawing blood cultures immediately before an antibiotic dose to maximize the chances of pathogen recovery.

Molecular and Pathogenic Characteristics of Paecilomyces formosus, a New Causal Agent of Oak Tree Dieback in Iran

Oak dieback is one of the most important diseases that presently affects the Kermanshah oak forests (West Iran). During the period from 2013 to 2015, oak trees exhibiting branch dieback were sampled, and fungal colonies resembling those of the Paecilomyces sp. were obtained from diseased tissues. Based on morphology, physiology, and phylogeny of the internal transcribed spacer rDNA, all isolates were identified as Paecilomyces formosus. Pathogenicity tests in vivo were made on 2-year-old seedlings, potted Q. brantii plants, and excised branch sections under controlled conditions. Inoculated seedlings produced characteristic symptoms, and subsequent pathogen recovery confirmed pathogenicity. We determined these to be thermophilic fungi, which may be related to warming and drought. This is the first report of Paecilomyces formosus as the causal agent of Q. brantii dieback in Iran and the world.

Inoculation Method Impacts Symptom Development Associated with Diaporthe aspalathi, D. caulivora, and D. longicolla on Soybean (Glycine max)

One hundred fifty-two Diaporthe isolates were recovered from symptomatic soybean (Glycine max) stems sampled from the U.S. states of Iowa, Indiana, Kentucky, Michigan, and South Dakota. Using morphology and DNA sequencing, isolates were identified as D. aspalathi (8.6%), D. caulivora (24.3%), and D. longicolla (67.1%). Aggressiveness of five isolates each of the three pathogens was studied on cultivars Hawkeye (D. caulivora and D. longicolla) and Bragg (D. aspalathi) using toothpick, stem-wound, mycelium contact, and spore injection inoculation methods in the greenhouse. For D. aspalathi, methods significantly affected disease severity (P < 0.001) and pathogen recovery (P < 0.001). The relative treatment effects (RTE) of stem-wound and toothpick methods were significantly greater than for the other methods. For D. caulivora and D. longicolla, a significant isolate × method interaction affected disease severity (P < 0.05) and pathogen recovery (P < 0.001). Significant differences in RTEs were observed among D. caulivora and D. longicolla isolates only when the stem-wound and toothpick methods were used. Our study has determined that the stem-wound and toothpick methods are reliable to evaluate the three pathogens; however, the significant isolate × method interactions for D. caulivora and D. longicolla indicate that multiple isolates should also be considered for future pathogenicity studies.

Methyl Bromide Fumigation to Eliminate Thousand Cankers Disease Causal Agents from Black Walnut

Phytosanitary treatments for logs and barked wood products are needed to mitigate the spread of thousand cankers disease through the movement of these commodities. The disease threatens eastern black walnut (Juglans nigra L.) populations in the United States. It is caused by repeated attacks by the walnut twig beetle (Pityophthorus juglandis Blackman) and subsequent canker development caused by the fungal pathogen Geosmithia morbida M. Kolařík et al. Methyl bromide (MB) fumigations were evaluated for efficacy against P. juglandis and G. morbida in J. nigra bolts. Fumigation with 82 mg/L MB for 24 h at 4.5° C eliminated P. juglandis in J. nigra, but was ineffective against G. morbida. Subsequent experiments focused on eliminating G. morbida, but results were inconclusive because of low rates of pathogen recovery from naturally infested control bolts. Final experiments used J. nigra bolts artificially inoculated with G. morbida. Fumigations with 240 and 320 mg/L MB for 72 h at 10° C were effective in eliminating G. morbida from J. nigra bolts. Results confirm that the USDA fumigation treatment schedule for logs with the oak wilt pathogen will also mitigate the risk of spreading the thousand cankers disease vector and pathogen by movement of walnut bolts and wood products.