Analysis of Bacterial Culture of Fluid in the Surgical Area in Transanal Total Mesorectal Excision and Laparoscopic Total Mesorectal Excision.

Purpose: To investigate the clinical value of the bacterial culture of fluid in the surgical area in laparoscopic transanal total mesorectal excision (Lap-taTME) and laparoscopic total mesorectal excision (Lap-TME). Methods: Clinical data of 106 patients with rectal cancer who had undergone surgery were retrospectively collected, including 56 patients in the Lap-taTME group and 50 patients in the Lap-TME group. In the Lap-taTME group, the initial pelvic fluid, the rectal cavity fluid after purse-string suture, and the pelvic cavity fluid after anastomosis were collected and recorded as culture No. 1, No. 2, and No. 3, respectively. In the Lap-TME group, culture No. 1 and No. 3 were collected as done in the Lap-taTME group. The culture results and postoperative complications were statistically analyzed. Results: The positive rate of culture No. 1 was zero in both groups, and there were 6 cases (10.7%) with positive culture No. 2 in the Lap-taTME group. However, the number of patients with positive culture No. 3 (7, 12.5%) and cumulative positive culture cases (11, 19.6%) in the Lap-taTME group were significantly higher than those in the Lap-TME group (0) (all P < .05). Pelvic infection occurred in 4 (7.1%) of the 11 cases (19.6%) with positive culture in the Lap-taTME group, accounting for 36.4% (4/11). There were no significant intergroup differences in anastomotic leakage and pelvic infection (all P > .05). Conclusion: Positive bacterial culture of fluid during Lap-taTME indicates an increased risk of pelvic infection after operation. Lap-taTME is more prone to intraoperative contamination than Lap-TME but does not significantly increase the risk of postoperative pelvic infection.

An alternative peptone preparation using Hermetia illucens (Black soldier fly) hydrolysis: process optimization and performance evaluation.

Background: Hermetia illucens (HI), commonly known as the black soldier fly, has been recognized for its prowess in resource utilization and environmental protection because of its ability to transform organic waste into animal feed for livestock, poultry, and aquaculture. However, the potential of the black soldier fly's high protein content for more than cheap feedstock is still largely unexplored. Methods: This study innovatively explores the potential of H. illucens larvae (HIL) protein as a peptone substitute for microbial culture media. Four commercial proteases (alkaline protease, trypsin, trypsase, and papain) were explored to hydrolyze the defatted HIL, and the experimental conditions were optimized via response surface methodology experimental design. The hydrolysate of the defatted HIL was subsequently vacuum freeze-dried and deployed as a growth medium for three bacterial strains (Staphylococcus aureus, Bacillus subtilis, and Escherichia coli) to determine the growth kinetics between the HIL peptone and commercial peptone. Results: The optimal conditions were 1.70% w/w complex enzyme (alkaline protease: trypsin at 1:1 ratio) at pH 7.0 and 54 °C for a duration of 4 h. Under these conditions, the hydrolysis of defatted HIL yielded 19.25% ±0.49%. A growth kinetic analysis showed no significant difference in growth parameters (µmax, Xmax, and λ) between the HIL peptone and commercial peptone, demonstrating that the HIL hydrolysate could serve as an effective, low-cost alternative to commercial peptone. This study introduces an innovative approach to HIL protein resource utilization, broadening its application beyond its current use in animal feed.

Construction and characterisation of a structured, tuneable, and transparent 3D culture platform for soil bacteria.

We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the carbon catabolism of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms, Bacillus subtilis and Streptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as competition and warfare.

Tulathromycin metaphylaxis increases nasopharyngeal isolation of multidrug resistant Mannheimia haemolytica in stocker heifers.

Bovine respiratory disease (BRD) is a leading cause of disease in feedlot and stocker calves with Mannheimia haemolytica (MH) as one of the most common etiologies. One of the most effective means of controlling BRD is through metaphylaxis, which involves administering antimicrobials to all animals at high risk of developing BRD. However, increasing prevalence of multidrug resistant (MDR) MH may reduce efficacy of metaphylaxis due to decreased susceptibility to drugs used for metaphylaxis. Primarily, this study aimed to determine the effect of tulathromycin metaphylaxis and subsequent BRD treatment on antimicrobial resistance (AMR) in MH isolated from stocker calves. Secondary objectives included evaluating the effect of metaphylaxis and treatment for BRD on animal health and comparing the genetic relationship of MH isolated. Crossbred beef heifers (n = 331, mean weight = 232, SD = 17.8 kg) at high risk for BRD were randomly assigned to receive tulathromycin metaphylaxis (META, n = 167) or not (NO META, n = 164). Nasopharyngeal swabs were collected for MH isolation, antimicrobial susceptibility testing and whole genome sequencing at arrival and 3 (WK3) and 10 (WK10) weeks later. Mixed-effects logistic regression was used to identify risk factors for isolation of MH and MDR MH (resistant to ≥3 antimicrobial drug classes) at 3 and 10 weeks, BRD morbidity, and crude mortality. Animals in the META group had higher odds of isolation of MDR MH at 3 weeks [OR (95% CI) = 13.08 (5-30.9), p < 0.0001] and 10 weeks [OR (95% CI) = 5.92 (1.34-26.14), p = 0.019] after arrival. There was no difference in risk of isolation of any MH (resistant or susceptible) between META and NO META groups at all timepoints. Animals in the NO META group had 3 times higher odds of being treated for BRD [WK3: OR (95% CI) = 3.07 (1.70-5.52), p = 0.0002; WK10: OR (95% CI) = 2.76 (1.59-4.80), p = 0.0002]. Antimicrobial resistance genes found within isolates were associated with integrative conjugative element (ICE) genes. Tulathromycin metaphylaxis increased risk of isolation of MDR MH and in this population, the increase in MDR MH appeared to be associated with ICE containing antimicrobial resistance genes for multiple antimicrobial classes. This may have important implications for future efficacy of antimicrobials for control and treatment of BRD.

Informed interpretation of metagenomic data by StrainPhlAn enables strain retention analyses of the upper airway microbiome.

IMPORTANCE: The usage of 16S rRNA gene sequencing has become the state-of-the-art method for the characterization of the microbiota in health and respiratory disease. The method is reliable for low biomass samples due to prior amplification of the 16S rRNA gene but has limitations as species and certainly strain identification is not possible. However, the usage of metagenomic tools for the analyses of microbiome data from low biomass samples is not straight forward, and careful optimization is needed. In this work, we show that by validating StrainPhlAn 3 results with the data from bacterial cultures, the strain-level tracking of the respiratory microbiome is feasible despite the high content of host DNA being present when parameters are carefully optimized to fit low biomass microbiomes. This work further proposes that strain retention analyses are feasible, at least for more abundant species. This will help to better understand the longitudinal dynamics of the upper respiratory microbiome during health and disease.

Alterations to the bovine bacterial ocular surface microbiome in the context of infectious bovine keratoconjunctivitis.

BACKGROUND: Infectious bovine keratoconjunctivitis (IBK) is a common cause of morbidity in cattle, resulting in significant economic losses. This study aimed to characterize the bovine bacterial ocular surface microbiome (OSM) through conjunctival swab samples from Normal eyes and eyes with naturally acquired, active IBK across populations of cattle using a three-part approach, including bacterial culture, relative abundance (RA, 16 S rRNA gene sequencing), and semi-quantitative random forest modeling (real-time polymerase chain reaction (RT-PCR)). RESULTS: Conjunctival swab samples were obtained from eyes individually classified as Normal (n = 376) or IBK (n = 228) based on clinical signs. Cattle unaffected by IBK and the unaffected eye in cattle with contralateral IBK were used to obtain Normal eye samples. Moraxella bovis was cultured from similar proportions of IBK (7/228, 3.07%) and Normal eyes (1/159, 0.63%) (p = 0.1481). Moraxella bovoculi was cultured more frequently (p < 0.0001) in IBK (59/228, 25.88%) than Normal (7/159, 4.40%) eyes. RA (via 16 S rRNA gene sequencing) of Actinobacteriota was significantly higher in Normal eyes (p = 0.0045). Corynebacterium variabile and Corynebacterium stationis (Actinobacteriota) were detected at significantly higher RA (p = 0.0008, p = 0.0025 respectively) in Normal eyes. Rothia nasimurium (Actinobacteriota) was detected at significantly higher RA in IBK eyes (p < 0.0001). Alpha-diversity index was not significantly different between IBK and Normal eyes (p > 0.05). Alpha-diversity indices for geographic location (p < 0.001), age (p < 0.0001), sex (p < 0.05) and breed (p < 0.01) and beta-diversity indices for geographic location (p < 0.001), disease status (p < 0.01), age (p < 0.001), sex (p < 0.001) and breed (p < 0.001) were significantly different between groups. Modeling of RT-PCR values reliably categorized the microbiome of IBK and Normal eyes; primers for Moraxella bovoculi, Moraxella bovis, and Staphylococcus spp. were consistently the most significant canonical variables in these models. CONCLUSIONS: The results provide further evidence that multiple elements of the bovine bacterial OSM are altered in the context of IBK, indicating the involvement of a variety of bacteria in addition to Moraxella bovis, including Moraxella bovoculi and R. nasimurium, among others. Actinobacteriota RA is altered in IBK, providing possible opportunities for novel therapeutic interventions. While RT-PCR modeling provided limited further support for the involvement of Moraxella bovis in IBK, this was not overtly reflected in culture or RA results. Results also highlight the influence of geographic location and breed type (dairy or beef) on the bovine bacterial OSM. RT-PCR modeling reliably categorized samples as IBK or Normal.

Changing Strategies for the Detection of Bacteria in Platelet Components in Ireland: From Primary and Secondary Culture (2010-2020) to Large Volume Delayed Sampling (2020-2023).

Bacterial contamination of platelet components (PC) poses the greatest microbial risk to recipients, as bacteria can multiply over the course of PC storage at room temperature. Between 2010 and 2020, the Irish Blood Transfusion Service (IBTS) screened over 170,000 buffy coat-derived pooled (BCDP) and single-donor apheresis platelets (SDAPs) with the BACT/ALERT 3D microbial detection system (Biomerieux, L'Etoile, France), using a two-step screening protocol which incorporated primary and secondary cultures. Although the protocol was successful in averting septic transfusion reactions (STRs), testing large sample volumes at later time points was reported to improve detection of bacterial contamination. A modified large-volume delayed sampling (LVDS)-type protocol was adopted in 2020, which in the case of SDAP was applied to collections rather than individual splits (2020-2023, 44,642 PC screened). Rates of bacterial contamination for BCDP were 0.125% on Day-2, 0.043% on Day-4 vs. 0.191% in the post-LVDS period. SDAP contamination rates in the pre-LVDS period were 0.065% on Day-1, 0.017% on Day-4 vs. 0.072% in the post-LVDS period. Confirmed STRs were absent, and the interdiction rate for possibly contaminated SDAP was over 70%. In the post-LVDS period, BCDPs had a higher total positivity rate than SDAPs, 0.191% (1:525) versus 0.072% (1:1385), respectively, (chi-squared 12.124, 1 df, p = 0.0005). The majority of organisms detected were skin-flora-type, low pathogenicity organisms, including coagulase-negative staphylococci and Cutibacterium acnes, with little change in the frequency of clinically significant organisms identified over time. Both protocols prevented the issue of potentially harmful components contaminated (rarely) with a range of pathogenic bacteria, including Escherichia coli, Serratia marcesens, Staphylococcus aureus, and streptococci. Culture positivity of outdates post-LVDS whereby 100% of expired platelets are retested provides a residual risk estimate of 0.06% (95% CI 0.016-0.150). However, bacterial contamination rates in expired platelets did not demonstrate a statistically significant difference between the pre-LVDS 0.100% (CI 0.033-0.234) and post-LVDS 0.059% (0.016-0.150) periods (chi-squared = 0.651, 1 df, p = 0.42).

Culturing the Plastisphere: comparing methods to isolate culturable bacteria colonising microplastics.

Microplastics quickly become colonised by diverse microbial communities, known as the Plastisphere. There is growing concern that microplastics may support the enrichment and spread of pathogenic or antimicrobial resistant microorganisms, although research to support the unique role of microplastics in comparison to control particles remains inconclusive. Limitations to this research include the microbiological methods available for isolating adhered microbes. Culture-based methods provide some of the most established, accessible and cost-effective microbiological protocols, which could be extremely useful in helping to address some of the remaining key questions in Plastisphere research. Previous works have successfully cultured bacteria from plastics, but these have not yet been reviewed, nor compared in efficiency. In this study, we compared four common biofilm extraction methods (swabbing, sonication, vortexing, sonication followed by vortexing) to extract and culture a mixed community of bacteria from both microplastic (polyethylene, polypropylene and polystyrene) and control (wood and glass) particles. Biofilm extraction efficiency and viability of bacterial suspension was determined by comparing CFU/mL of four different groups of bacteria. This was verified against optical density and 16S rRNA qPCR. Overall, we found that all tested methods were able to remove biofilms, but to varying efficiencies. Sonicating particles with glass beads for 15 min, followed by vortexing for a further minute, generated the highest yield and therefore greatest removal efficiency of culturable, biofilm-forming bacteria.

Appropriateness of Empirical Antibiotic Therapy in Hospitalized Patients with Bacterial Infection: A Retrospective Cohort Study.

Objective: The incidence of inappropriate and excessive empirical antibiotic therapy is unclear. The aim of this study was to determine the prevalence of different empirical antibiotic therapy prescriptions, related factors, and outcomes in hospitalized patients with bacterial infection. Methods: A retrospective cohort study was performed and patients with bacterial infection who were admitted between October 1, 2019, and September 30, 2020, were included. Multivariable analysis was performed by the logistic regression model. Results: A total of 536 (42.6%) of the 1257 included patients received inappropriate empirical antibiotic therapy (IEAT), and 368 (29.3%) patients received appropriate but unnecessarily broad-spectrum empirical antibiotic therapy (AUEAT). MDRO (adjusted OR 2.932 [95% CI 2.201~3.905]; p < 0.001) and fever on admission (adjusted OR 0.592 [95% CI 0.415~0.844]; p = 0.004) were correlates of IEAT; sepsis (adjusted OR 2.342 [95% CI 1.371~3.999]; p = 0.002), age (adjusted OR 1.019 [95% CI 1.008~1.030]; p < 0.001), MDRO (adjusted OR 0.664 [95% CI 0.469~0.941]; p = 0.021), and urinary tract infection (adjusted OR 0.352 [95% CI 0.203~0.611]; p < 0.001) were correlates of AUEAT. Patients who received AUEAT were more likely to have a poor prognosis (63 [17.8%] vs 101 [27.4%]; p = 0.002). Both IEAT (median [IQR], 24,971 [13,135-70,155] vs 31,489 [14,894-101,082] CNY; p = 0.007) and AUEAT (median [IQR], 24,971 [13,135-70,155] vs 30,960 [16,475-90,881] CNY; p = 0.002) increased hospital costs. 45.3% (570/1257) of patients were infected with MDRO and 62.9% of them received IEAT. Conclusion: Inappropriate and excessive empirical antibiotic use was widely prevalent among hospitalized patients. Either inappropriate or excessive use of antibiotics may increase the burden of healthcare costs, the latter of which may be associated with poor prognosis. Clinicians need to be more judicious in choosing antibiotic(s). The MDRO epidemic was severe, especially in patients who received IEAT. It is imperative to take effective measures to improve the current situation of antibiotic abuse and antimicrobial resistance.

Technical Note: Use of a Novel Bacterial Culture Sampling Device for Pooled Whole Blood Platelets.

OBJECTIVE: Transfusion associated sepsis is a serious risk after platelet transfusion. Although platelet culture can be performed to avoid such risk, culture results are often available after transfusion due to the 4-hour shelf-life after pooling. To decrease such risk, we implemented a needleless closed system device to culture for contamination before pooling and release for transfusion. METHODS: We customized a needleless device to permit sterile sampling of whole blood platelets without retrograde or cross-contamination. Then aliquots of platelets were injected into culture media for detection of aerobic organisms and cultured for 24 hours but released for transfusion after 12 hours of negative culture. RESULTS: In a period of two years, we used this device in 5,741 whole blood derived pooled platelets and only 24 units tested positive (0.4%) but none of initial positive was later confirmed. There were 11 Staphylococcus and 9 Bacillus species identified. All but one of the positive units were discarded; there was no clinical impact in the patient who received the positive unit. CONCLUSION: This device allows for sampling of whole blood derived platelets before pooling, warranting a transfusion of a culture negative unit after 12 hours of negative culture, consequently reducing transfusion of bacterially contaminated whole blood derived pooled platelets.

Parapharyngeal Abscess: Antibiotic-only or Open Surgical Drainage?

Objective:Many problems of parapharyngeal abscess (PPA), such as etiology, predisposing factors, and therapeutic methods, are still controversial. We aim to investigate the characteristics of PPA to better understand the therapeutic effects of the disease. Methods: We retrospectively collated the medical record reviews of 49 PPA patients who were treated as PPA inpatients when a patient was hospitalized and diagnosed with PPA, and empiric antibiotics were used. Only if the drug treatment was ineffective, the abscess was large, or the disease continued to progress, and surgical treatment was adopted. Results: In total, 49 patients who met the research criteria were identified. Streptococcus was the most common organism in PPA patients. The morbidity of diabetes in PPA patients was higher than the prevalence of diabetes in the overall population. Interestingly, the length of hospital stay was shorter in the antibiotic-only group than in the surgery group (P < 0.05). Furthermore, the duration from onset to treatment in the antibiotic-only group was shorter than in the surgery group. Conclusion: Our treatment protocol is effective. Antibiotic-only method is also recommended for the PPA which was effective for the empiric antibiotics and localized. Early diagnosis and treatment of PPA could ultimately reduce the severity of PPA.

Diagnostic and Management Guidelines of Thyroid Tuberculosis: Our Experience and Systematic Review.

Objective: Thyroid tuberculosis has non-specific clinical presentation, difficult diagnosis and specific medical management. The aim of this article is to present and share a review of the English-language literature on thyroid tuberculosis in order to gain a better understanding of diagnostic methods and provide guidelines for its management and to present our experience of three cases. Methods: The systematic search of the literature was performed on Pubmed and Medline from 1950 to 2019 according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement. Results: We retrieved 13 manuscripts meeting our criteria from the search. There were 7 case series, and 6 manuscripts with review of the literature. Conclusion: Direct histopathological demonstration is the best diagnostic modality. FNAC is the study of choice and PCR assay increases its sensitivity. The standard short course ATT for 6 months is recommended for isolated thyroid TB and for widespread disease, 12 months therapy is recommended. Surgery is reserved for failure of medical therapy and abscess formation.

Synovial sepsis diagnostics and antimicrobial resistance: a one-health perspective.

This article, as part of the Currents in One Health series, reviews the current state of diagnostics for synovial sepsis. Synovial sepsis is a condition that affects veterinary and human medicine and requires coordinated efforts from both parties, as well as environmental considerations to accurately diagnose and preserve effective treatments. The article discusses best practices to identify the causative agent in septic synovitis, trends in bacterial identification and antimicrobial resistance patterns across common bacterial species, and a one-health perspective to optimize diagnostics across species. Antimicrobial resistance is a challenge facing both human and veterinary medicine and requires mindful and attentive prescribing to reduce the development of antimicrobial resistance and preserve antimicrobials for future application. The current standard of care for bacterial identification in veterinary practice is culture and antimicrobial susceptibility; however, positive culture rates from synovial sepsis cases often remain < 50%. Recent developments in advanced bacterial identification present opportunities for improved bacterial identification in synovial sepsis. Increased bacterial isolation will also help guide empirical antimicrobial therapy. Utilizing information and recommendations from both the human and veterinary literature will improve timely and accurate bacterial identification and therefore rapid and effective treatment of synovial sepsis across species and limit the development of antimicrobial resistance.

Association of the Bacteria of the Vermiform Appendix and the Peritoneal Cavity with Complicated Acute Appendicitis in Children.

BACKGROUND: Primary infection has been questioned as the pathogenetic cause of acute appendicitis. We attempted to identify the bacteria involved and to investigate if their species, types, or combinations affected the severity of acute appendicitis in children. METHODS: Samples from both the appendiceal lumen and the peritoneal cavity of 72 children who underwent appendectomy were collected to perform bacterial culture analysis. The outcomes were studied to identify if and how they were associated with the severity of the disease. Regression analysis was performed to identify any risk factors associated with complicated appendicitis. RESULTS: Escherichia coli, Pseudomonas aeruginosa, and Streptococcus species were the most common pathogens found in the study population. The same microorganisms, either combined or separate, were the most common in the appendiceal lumen and the peritoneal cavity of patients with complicated appendicitis. Gram-negative bacteria and polymicrobial cultures in the peritoneal fluid and in the appendiceal lumen were associated with complicated appendicitis. Polymicrobial cultures in the peritoneal cavity presented a four times higher risk of complicated appendicitis. CONCLUSIONS: Polymicrobial presentation and Gram-negative bacteria are associated with complicated appendicitis. Antibiotic regimens should target the combinations of the most frequently identified pathogens, speculating the value of early antipseudomonal intervention.

16S rRNA-Based Microbiota Profiling Assists Conventional Culture Analysis of Airway Samples from Pediatric Cystic Fibrosis Patients.

16S-based sequencing provides broader information on the respiratory microbial community than conventional culturing. However, it (often) lacks species- and strain-level information. To overcome this issue, we used 16S rRNA-based sequencing results from 246 nasopharyngeal samples obtained from 20 infants with cystic fibrosis (CF) and 43 healthy infants, which were all 0 to 6 months old, and compared them to both standard (blind) diagnostic culturing and a 16S-sequencing-informed "targeted" reculturing approach. Using routine culturing, we almost uniquely detected Moraxella catarrhalis, Staphylococcus aureus, and Haemophilus influenzae (42%, 38%, and 33% of samples, respectively). Using the targeted reculturing approach, we were able to reculture 47% of the top-5 operational taxonomical units (OTUs) in the sequencing profiles. In total, we identified 60 species from 30 genera with a median of 3 species per sample (range, 1 to 8). We also identified up to 10 species per identified genus. The success of reculturing the top-5 genera present from the sequencing profile depended on the genus. In the case of Corynebacterium being in the top 5, we recultured them in 79% of samples, whereas for Staphylococcus, this value was only 25%. The success of reculturing was also correlated with the relative abundance of those genera in the corresponding sequencing profile. In conclusion, revisiting samples using 16S-based sequencing profiles to guide a targeted culturing approach led to the detection of more potential pathogens per sample than conventional culturing and may therefore be useful in the identification and, consequently, treatment of bacteria considered relevant for the deterioration or exacerbation of disease in patients like those with CF. IMPORTANCE Early and effective treatment of pulmonary infections in cystic fibrosis is vital to prevent chronic lung damage. Although microbial diagnostics and treatment decisions are still based on conventional culture methods, research is gradually focusing more on microbiome and metagenomic-based approaches. This study compared the results of both methods and proposed a way to combine the best of both worlds. Many species can relatively easily be recultured based on the 16S-based sequencing profile, and it provides more in-depth information about the microbial composition of a sample than that obtained through routine (blind) diagnostic culturing. Still, well-known pathogens can be missed by both routine diagnostic culture methods as well as by targeted reculture methods, sometimes even when they are highly abundant, which may be a consequence of either sample storage conditions or antibiotic treatment at the time of sampling.

Antibiograms of field and hospital acquired equine neonatal bacterial fluid cultures in the Midwestern United States: 149 samples (2007-2018).

BACKGROUND: Contemporary data reflecting local pathogens and their antibiograms is necessary to select empirical antimicrobial therapy for equine neonates. HYPOTHESIS/OBJECTIVES: Describe bacterial isolates associated with equine neonatal infection and their antibiograms in the Midwestern United States. An increase in gram-positive infection and antibiotic resistance compared to previous literature was expected. ANIMALS: Data from 149 fluid samples from 133 foals <30 days of age submitted for bacterial culture between January 2007 and December 2018. METHODS: A retrospective evaluation of equine neonatal fluid cultures was performed. Fluid submission type, bacterial culture and antibiogram, empirical antibiotic treatment, and foal outcome was included. Isolate susceptibility to individual antimicrobials and combination protocols relevant to equine practice were recorded. The effect of recorded variables on foal survival was evaluated using Fisher's exact or chi-squared tests. RESULTS: Ninety bacterial isolates (78 aerobes and 12 anaerobes) were identified and gram-positive organisms predominated (n = 50/90, 56%). Greater than 70% of aerobic isolates were susceptible to ampicillin, ceftiofur, chloramphenicol, trimethoprim/sulfamethoxazole, and all penicillin/aminopenicillin and aminoglycoside combinations. Seventy-seven (n = 81/105) percent of foals survived. Survival was associated with a negative fluid culture and was not associated with empirical antimicrobial choice. CONCLUSIONS AND CLINICAL IMPORTANCE: Gram positive and anaerobic isolates associated with equine neonatal fluid cultures exceed that of previous reports. Historical empirical antimicrobial choices for equine neonatal infection in the Midwestern United States are supported by local antibiogram results.

Antimicrobial Susceptibility of Bacteria Isolated from Freshwater Mussels in the Wildcat Creek Watershed, Indiana, United States.

Antimicrobial resistance (AMR) is a global health crisis that threatens the health of humans and animals. The spread of resistance among species may occur through our shared environment. Prevention of AMR requires integrated monitoring systems, and these systems must account for the presence of AMR in the environment in order to be effective. The purpose of this study was to establish and pilot a set of procedures for utilizing freshwater mussels as a means of surveillance for microbes with AMR in Indiana waterways. One hundred and eighty freshwater mussels were sampled from three sites along the Wildcat Creek watershed in north-central Indiana. Specimens were evaluated for the presence of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species), Escherichia coli, Campylobacter, and Salmonella species, and the isolates were tested for antimicrobial resistance. A total of 24 bacterial isolates were obtained from tissue homogenates of freshwater mussels collected at a site directly downstream from Kokomo, Indiana. Of these, 17 were Enterobacter spp., five were Escherichia coli, one was Pseudomonas aeruginosa, and one was Klebsiella pneumoniae. All isolates were resistant to three or more antimicrobial drug classes. Further work is necessary to determine the source of the bacterial species found in the mussels.

Clinical Diagnostics of Bacterial Infections and Their Resistance to Antibiotics-Current State and Whole Genome Sequencing Implementation Perspectives.

Antimicrobial resistance (AMR), defined as the ability of microorganisms to withstand antimicrobial treatment, is responsible for millions of deaths annually. The rapid spread of AMR across continents warrants systematic changes in healthcare routines and protocols. One of the fundamental issues with AMR spread is the lack of rapid diagnostic tools for pathogen identification and AMR detection. Resistance profile identification often depends on pathogen culturing and thus may last up to several days. This contributes to the misuse of antibiotics for viral infection, the use of inappropriate antibiotics, the overuse of broad-spectrum antibiotics, or delayed infection treatment. Current DNA sequencing technologies offer the potential to develop rapid infection and AMR diagnostic tools that can provide information in a few hours rather than days. However, these techniques commonly require advanced bioinformatics knowledge and, at present, are not suited for routine lab use. In this review, we give an overview of the AMR burden on healthcare, describe current pathogen identification and AMR screening methods, and provide perspectives on how DNA sequencing may be used for rapid diagnostics. Additionally, we discuss the common steps used for DNA data analysis, currently available pipelines, and tools for analysis. Direct, culture-independent sequencing has the potential to complement current culture-based methods in routine clinical settings. However, there is a need for a minimum set of standards in terms of evaluating the results generated. Additionally, we discuss the use of machine learning algorithms regarding pathogen phenotype detection (resistance/susceptibility to an antibiotic).

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

OBJECTIVES: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). PROCEDURE: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. RESULTS: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. CONCLUSION: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.

Assessment of Omitting MacConkey Agar as a Primary Inoculating Medium for MALDI-TOF MS-Based Bacterial Identification from Urine, Blood, and Respiratory Samples.

OBJECTIVE: MacConkey agar (MAC) is commonly used as a primary medium for conventional bacterial identification in clinical microbiology laboratories. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized microbial identification and is considered a reliable identification tool. While conventional identification methods rely on colony characteristics, MALDI-TOF MS requires a pure isolate on a solid medium. METHODS: This study investigated whether MAC can be omitted as a routine inoculation medium for urine, lower respiratory tract (LRT), and positive blood culture samples. The study included 462 clinical samples. Among these, 221 were urine samples, 141 were positive blood cultures, and the remaining 100 were LRT samples. The samples were inoculated on blood agar (BA) and MAC for the control group and on BA only for the experimental group, followed by incubation and identification with MALDI-TOF MS. RESULTS: The BA only group showed the same microbial identification using MALDI-TOF MS as the control BA and MAC groups for blood and LRT specimens. For urine samples, 99.1% (219/221) of the samples produced the same identification results for the two groups. The cause of discrepant results for two urine specimens was due to Proteus species overgrowth on BA, which hindered non-Proteus spp. identification for the BA-only group. CONCLUSION: Our results may indicate that omitting MAC has little or no impact on the recovery of organisms present in culture. However, due to possible Proteus spp. overgrowth, caution should be exercised in the decision to omit MAC from the primary inoculating medium, which necessitates further studies in other centers with a larger sample size.