Nocardioides agri sp. nov., isolated from garden soil.

A novel bacterial strain, designated as MAH-18T, was isolated from soil sampled in a flower garden. Cells of strain MAH-18T were Gram-stain-positive, aerobic, motile, and rod-shaped. The colonies were beige in colour, smooth, and spherical when grown on Reasoner's 2A agar medium. Strain MAH-18T grew at 20-40 °C, pH 6.0-8.0, and 0-1.0 % NaCl. Cells were able to hydrolyse aesculin, gelatin, and Tween 20. According to the 16S rRNA gene sequence comparisons, the isolate was determined to be a member of the genus Nocardioides and most closely related to Nocardioides pyridinolyticus OS4T (97.9 %), Nocardioides hankookensis DS-30T (97.9 %), Nocardioides aquiterrae GW-9T (97.6 %), Nocardioides soli mbc-2T (97.5 %), Nocardioides conyzicola HWE 2-02T (97.4 %), and Nocardioides mangrovi GBK3QG-3T (96.3 %). Strain MAH-18T has a draft genome size of 4 788 325 bp (eight contigs), 4572 protein-coding genes, 46 tRNA, and three rRNA genes. The average nucleotide identity and digital DNA-DNA hybridization values between strain MAH-18T and the closest type strains were 81.5-83.4 % and 24.4-25.8 %, respectively. In silico genome mining revealed several biosynthetic gene clusters in the genome of the novel strain MAH-18T. The G+C content of the genomic DNA of strain was 72.2 mol% and the predominant isoprenoid quinone was MK-8 (H4). The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and unknown phospholipids. The major cellular fatty acids were determined to be C16:0 iso and C17 : 1 ω6c. The DNA-DNA hybridization results and phenotypic, genotypic, and chemotaxonomic data demonstrated that strain MAH-18T represents a novel species, for which the name Nocardioides agri sp. nov. is proposed, with MAH-18T as the type strain (=KACC 19744T=CGMCC 1.13656T).

Antibiotic Stewardship in the Management of Infected Diabetic Foot Ulcer Disease in Less Developed Countries.

BACKGROUND: Diabetic foot ulcers in developing countries often become infected. The healthcare systems are often not equipped to conduct the culture and the sensitivity tests required for prescribing a targeted antibiotic treatment for diabetic foot infection (DFI). METHODS: We evaluate antibiotic stewardship programmes for DFIs, at every level of health care, with an emphasis on resource-poor settings such as in Africa. RESULTS: The management of DFI very often is adapted to the financial and practical realities of the resource-poor regions. The application of the point-of-care Gram stain of deep tissue samples is efficient, rapid, low cost and ubiquitously available. Upon the identification of the predominant pathogen in the Gram stain, a semi-quantitative preemptive antibiotic treatment can be started in accordance with the World Health Organization Aware, Watch and Restrict Essential Medicine List. This list is catered to every country and is a powerful tool. However, some basic knowledge of the local microbiological epidemiology is necessary to choose the most appropriate agent. We report our experience on using the rapidly available Gram stain for narrowing the preemptive choice of listed antibiotic agents, as an economic tool for antibiotic stewardship in DFIs. CONCLUSIONS: In the practical and resource-saving management of DFI, the 'therapeutic' use of Gram stains is not common in resource-rich countries but should be added to the arsenal of the general efforts for antibiotic stewardship.

Evaluation of Gram Stain-Guided Antibiotic Therapy for Methicillin-Resistant Staphylococcus aureus Pneumonia in Intensive Care Unit Patients.

Background: Despite high negative predictive values (NPVs) seen with methicillin-resistant Staphylococcus aureus (MRSA) nares polymerase chain reaction (PCR) assays, utilization of both respiratory sample Gram stain and MRSA nares PCR in patients with pneumonia may contribute to overuse of laboratory resources. The purpose of this study was to evaluate if a Gram stain demonstrating no Gram-positive organisms from a respiratory sample is sufficient to allow for de-escalation of vancomycin therapy. Methods: This single center study retrospectively identified intensive care unit (ICU) patients started on vancomycin for presumed pneumonia at University of Wisconsin (UW) Health in Madison, WI between August 2022 and March 2023. Patients with respiratory sample demonstrating no Gram-positives on Gram stain met inclusion criteria if the sample was ordered within 24 h of vancomycin initiation. The primary outcome was NPV of respiratory sample Gram stain demonstrating no Gram-positive organisms with respect to MRSA detection of the respiratory culture. Secondary outcomes included the NPV of combined MRSA nares PCR plus respiratory sample Gram stain, and difference in time to event in patients that had both a respiratory sample and MRSA nares PCR ordered. Results: A total of 370 patients were screened for study eligibility; of which 99 patients met inclusion criteria. NPV of respiratory sample Gram stain was 99% for MRSA culture. The combined NPV of respiratory sample Gram stain plus MRSA nares PCR was 98.9% for MRSA culture (n = 88). Respiratory sample was ordered 2.3 h faster compared to MRSA nares PCR (4.3 vs 6.6 h, P = .036). Respiratory sample Gram stain resulted 4.5 h faster compared to MRSA nares PCR (10.7 vs 15.2 h, P = .002). Conclusion: Respiratory sample Gram stains demonstrating no Gram-positive organisms may be used to de-escalate vancomycin and deprioritize the use of MRSA nares PCR.

Q-Scoring System for the Evaluation of the Superficial Wound Swab Samples: A Clinical Microbiological Aspect.

Purpose: Wound swab cultures are frequently requested from patients suspected of having a wound infection. The quality of the sample should also be evaluated by performing a Gram-stained microscopic examination. "Q-scoring system" is not widely used and the literature on the subject is limited. Methods: A total of 4648 wound swab samples were evaluated. Samples with a Q-score of "0" were considered as "poor quality samples", and those with a score of " ≥ 1" were classified as "good quality samples". Microorganisms grown in the culture of samples that scored above one were identified by mass spectrometry, and antimicrobial susceptibility testing was performed. Results: Gram stain results were found to be consistent with the culture result in 57.10% (n = 1078) of and inconsistent with the culture result in 42.90% (n = 813) of the samples. The number of samples with Q-scores one, two, and three among the 813 samples was 62, 29, and 722, respectively. The value observed in Q3 was found to be statistically significantly higher than the values observed in Q1 and Q2 (p < 0.05). Samples sent from surgical departments (61.92%) with a Q-score of ≥ 1, were statistically significant compared to internal medicine departments (p < 0.0001). There was no significant difference between samples sent from intensive care units and those sent from other inpatient services. For both groups with Q-scores ≥ 1 and "0" similar microorganisms were identified. Conclusion: As a conclusion, the Q-scoring system will provide a common language between the laboratory and the clinic, especially by standardizing the evaluation of wound swab samples.

Diagnostic Accuracy of Point-of-Care Gram Stains in Obstructive Pyelonephritis due to Ureteral Stones.

Background: The diagnostic utility of point-of-care (POC) Gram stains for obstructive pyelonephritis with hydronephrosis is not well established. The current study aimed to assess the diagnostic accuracy of urine Gram stains in patients with obstructive pyelonephritis due to ureteral stones. Methods: A retrospective observational study was conducted on patients with obstructive pyelonephritis admitted to our hospital between January 2011 and December 2021. The diagnostic accuracy of Gram stains was evaluated based on the severity of hydronephrosis, including Gram stains performed by both trained physicians and microbiological technicians. Results: After analyzing 210 patients, POC Gram stains of bladder urine presented a sensitivity, specificity, positive predictive value, and negative predictive value of 86.8%, 81.8%, 93.7%, and 66.7%, respectively, for gram-negative rods and 65.7%, 83.4%, 48.9%, and 91.0%, respectively, for gram-positive cocci. The agreement between POC Gram stains and urine culture was good for gram-negative rods, with a kappa (κ) coefficient of 0.637 and agreement rate of 85.6%, and moderate for gram-positive cocci, with a κ coefficient of 0.435 and agreement rate of 80%. The agreement between POC Gram stains and bladder urine culture results for gram-negative rods was higher in the mild hydronephrosis group (κ coefficient = 0.677) than in the severe hydronephrosis group (κ coefficient = 0.466). Discrepancies in Gram stain results between physicians and technicians were observed in 21 of 180 cases (11.7%). Conclusions: POC Gram stains for gram-negative rods may be a useful diagnostic tool for obstructive pyelonephritis, particularly in cases of mild hydronephrosis.

Performance evaluation of machine-assisted interpretation of Gram stains from positive blood cultures.

Manual microscopy of Gram stains from positive blood cultures (PBCs) is crucial for diagnosing bloodstream infections but remains labor intensive, time consuming, and subjective. This study aimed to evaluate a scan and analysis system that combines fully automated digital microscopy with deep convolutional neural networks (CNNs) to assist the interpretation of Gram stains from PBCs for routine laboratory use. The CNN was trained to classify images of Gram stains based on staining and morphology into seven different classes: background/false-positive, Gram-positive cocci in clusters (GPCCL), Gram-positive cocci in pairs (GPCP), Gram-positive cocci in chains (GPCC), rod-shaped bacilli (RSB), yeasts, and polymicrobial specimens. A total of 1,555 Gram-stained slides of PBCs were scanned, pre-classified, and reviewed by medical professionals. The results of assisted Gram stain interpretation were compared to those of manual microscopy and cultural species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparison of assisted Gram stain interpretation and manual microscopy yielded positive/negative percent agreement values of 95.8%/98.0% (GPCCL), 87.6%/99.3% (GPCP/GPCC), 97.4%/97.8% (RSB), 83.3%/99.3% (yeasts), and 87.0%/98.5% (negative/false positive). The assisted Gram stain interpretation, when compared to MALDI-TOF MS species identification, also yielded similar results. During the analytical performance study, assisted interpretation showed excellent reproducibility and repeatability. Any microorganism in PBCs should be detectable at the determined limit of detection of 105 CFU/mL. Although the CNN-based interpretation of Gram stains from PBCs is not yet ready for clinical implementation, it has potential for future integration and advancement.

Occurrence of Macrorhabdus ornithogaster in exotic birds.

Avian gastric yeast (Macrorhabdus ornithogaster) is a microorganism that infects aviary birds worldwide, both captive and wild. A total number of 352 birds, belonging to 18 avian species, were examined from 2019 to 2022 for M. ornithogaster, using fecal smears of live birds or cytological samples of the proventriculus taken at necropsy. These cytological samples were taken from birds that died from different causes. Some of the birds exhibited symptoms such as lethargy, regurgitation, weight loss and anorexia. Faecal samples were collected from all the birds and analysed for gastric yeast using a direct smear and Gram-staining method. The microorganism was diagnosed most frequently in budgerigars (55.5%), the African gray parrot (33.3%), and nymphs (34.3%). The prevalence of M. ornithogaster in canaries was 10%. The infection was detected in 31% of the examined birds, which shows that the occurrence of M. ornithogaster in exotic birds is common. No clinical signs were observed in the vast majority of birds that tested positive for gastric yeast.

Competency Assessment on Gram Stain Examination and Interpretation Among Medical Laboratory Professionals Working in Selected Hospitals of Addis Ababa, Ethiopia.

Background: Laboratory test results are the cornerstone for patient diagnosis and treatment. Gram staining is a classic laboratory test method used to differentiate between bacteria. Competence assessment can help identify gaps and provide suggestions to academics, researchers, and policymakers to address competency gaps. In Ethiopia, there is no evidence of competency assessment by medical laboratory professionals using the Gram-staining technique. Objective: To assess the competency of medical laboratory professionals on Gram stain examination and interpretation in selected hospitals of Addis Ababa, Ethiopia. Methods: A cross-sectional study was conducted to assess the competency of medical laboratory professionals on Gram stain examination and interpretation from September 2015 to December 2017. Results: Of 190 participants, 55 (28.9%) participants scored low knowledge, 131 (68.9%) scored medium knowledge, and only 4 (2.1%) respondents scored high knowledge. From the study variables, education level, supervision by regional or federal government bodies, and training about Gram staining were significantly associated with the knowledge level of study participants. Forty eight (25.3%), 78 (41%), and 64 (33.7%) participants scored low, medium, and high skill level, respectively, from a total of 190 participants. From skill level analysis, hospital type, microscope type, and availability of health information resources were significantly associated with skill levels. There were 44 observations (4%) with major errors and 321 observations (28%) with very major errors from all 1140 observations. Of all observations, 321 (28.2%) reported without grading, 39 observations (3.4%) reported gram-positive bacteria as gram-negative bacteria, and 15 observations (1.4%) reported gram-negative bacteria as gram-positive bacteria. Conclusion: The current study found that most medical laboratory professionals work without supervision or refresher training in Gram stain examination and interpretation. Hence, medical laboratory professionals' knowledge and skill levels are unsatisfactory. Regular competence assessments, training, and follow-up are necessary to improve the professional competence in medical laboratories.

Reviving Sternheimer stain: A single-center retrospective study to detect the diagnostic utility of urinary tract infections in the emergency department.

INTRODUCTION: Qualitative urinalysis using the Sternheimer stain is a common method in Japan for identifying bacteriuria, but there is a lack of studies examining its test characteristics. In this study, we aimed to investigate the sensitivity and specificity of the Sternheimer stain for urine culture results and compare it with the sensitivity and specificity of the Gram stain. Our goal was to determine the usefulness of the Sternheimer stain in identifying bacteriuria. PATIENTS AND METHODS: Among 986 patients aged 16 years or older from whom samples for both urinalysis and urine culture were obtained at the emergency room of Tenri Hospital from January 2019 to December 2019, 342 patients with pyuria, defined as the presence of 10 or more white cells per cubic millimeter in a urine specimen, who had not received prior antimicrobial therapy were included. Urine cultures were used for comparison to determine the sensitivity and specificity of Sternheimer and Gram stain in this patient group. A positive Sternheimer stain result was defined as bacteriuria ≥ (1+), and that of Gram stain was defined as ≥ 1/1 field of high-power ( × 1000) oil immersion. RESULTS: Using urine culture results for comparison, the sensitivity of Sternheimer stain was 92.2%, the specificity was 48.5%, the positive likelihood ratio was 1.79, and the negative likelihood ratio was 0.16. DISCUSSION: Sternheimer stain is a rapid and useful method to exclude bacteriuria in a group of patients with pyuria in the emergency department.

The accuracy and diagnostic value of gram staining joint aspirates in suspected joint infections.

BACKGROUND: Septic arthritis is a debilitating condition with prolonged treatment and adverse outcomes. A gram stain is often performed from the joint aspirate sample, followed by a definitive culture. In our study, we assessed the accuracy of gram staining for suspected septic arthritis and explored factors associated with positive culture growth and false negatives in the gram stain. METHODS: We retrospectively reviewed joint aspirates performed from 2015-2021 at a major trauma centre. Aspirates not cultured for septic arthritis were excluded. Data collected included aspirate site, gram stain and culture result delay, patient demographics, orthopaedic/rheumatological history, and comorbidities. Outcomes measured were gram stain sensitivity and specificity. Factors influencing positive cultures and false negative gram stain results were analysed using logistic regression. RESULTS: Of 408 joint aspirates meeting the criteria, 37 did not undergo initial gram staining. Gram stain sensitivity was 30.4%, specificity was 97.6%. The delay from aspirate to definitive gram stain and culture results was 1.1 and 5.4 days, respectively Logistic regression identified that prosthetic joint(p = 0.007), past joint infections(p = 0.006), arthritis(p < 0.001), hypertension(p = 0.007), diabetes(p = 0.019) were positively associated with positive cultures. Past joint infections(p = 0.004) were positively associated with false negative gram stain results. Patients on antibiotics during the aspirate had a higher risk of false negative gram stain results (OR = 5.538, 95%CI, 2.802-10.948; p < 0.001). CONCLUSIONS: In conclusion, the initial gram stain has limited sensitivity and caution should be exercised when interpreting negative results. Vigilance is crucial when the highlighted comorbidities or antibiotic use are present, to assess patients with potential joint infections.

Diagnostic value of dual-fluorescence staining in bacterial vaginosis.

OBJECTIVE: The aim of this study was to investigate the epidemiology of bacterial vaginosis (BV) in Shanghai, China, and to explore the value of a dual-fluorescence staining method in the diagnosis of BV. METHODS: Specimens were collected from women with vaginitis at the Obstetrics and Gynecology Hospital of Fudan University from January 2020 to December 2021, and the proportions of various vaginitis types (such as Candida vaginitis, Trichomonas, and bacterial vaginitis) were analyzed statistically. To explore the diagnostic value of dual-fluorescence staining for BV, we first executed a dual-fluorescence staining method to analyze the vaginal secretions of 265 patients, then confirmed our diagnoses by consulting clinical physicians and by using Nugent scoring of Gram staining. RESULTS: There were 16,905 patients who were diagnosed with vaginitis over the previous 2 years, with a median age of 32 (minimum age of 9 years and maximum of 84 years). Of these patients, we noted 10,887 cases (64.40%) of BV. Our staining results revealed that the dual-fluorescence method was consistent with Gram staining in the diagnosis of BV, with a P value of less than .001 using a χ 2 test and a consistency kappa value of 0.896. Compared with Gram staining, the dual-fluorescence staining method required an acceptable time (2.2 min vs 2.5 min, respectively) and exhibited different visual effects (green and yellow vs purple and red, respectively). CONCLUSION: Dual-fluorescence staining for the detection of bacterial diseases of the vagina exhibited acceptable consistency with Gram staining and performed well with respect to dyeing time, stability, and the interpretation of results. We argue that this method should be used in outpatient services.

The diagnostic accuracy of Gram stain on formalin-fixed paraffin-embedded sections of skin tissue in the diagnosis of bacterial skin infection.

BACKGROUND AND OBJECTIVE: To evaluate the sensitivity, specificity, and likelihood ratios of Gram stain on formalin-fixed, paraffin-embedded (GS-FFPE) sections of skin in diagnosing bacterial skin infection. METHODS: We reviewed a retrospective series of skin specimens reported at our institution wherein histopathological assessment included Gram stain and fresh tissue was concurrently submitted for microscopy and culture. The clinicopathological correlation was the reference standard, whereby the presence of infection was deduced from the final diagnosis in each patient's case notes. RESULTS: Our sample included 168 cases (105 positive for infection). GS-FFPE showed a sensitivity of 0.43 (95% confidence interval 0.29, 0.57), a specificity of 0.98 (0.95, 1.01), a positive likelihood ratio of 21.50 (19.76, 23.24), and a negative likelihood ratio of 0.58 (0.41, 0.75). CONCLUSIONS: GS-FFPE has poor sensitivity, and a negative result should not be used as evidence to exclude infection. In contrast, it has excellent specificity and, unless the pretest probability of infection is very low, a positive result would make infection much more likely. The value of the GS-FFPE lies in cases where sterile tissue was not submitted for microbiological studies, or sterile tissue culture was negative, and there is at least a low-to-moderate pretest probability of infection.

Sputum Gram stain of hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae has virulence genes relevant to capsule overproduction, which could lead to the finding of thick transparent area ("halo") in Gram stain. In sputum Gram stain, observation of a large Gram-negative rod with a thick capsule may not only indicate that it is K. pneumoniae but also that it is a highly virulent strain.

The diagnostic utility of microscopic quality assessment of sputum samples in the era of rapid syndromic PCR testing.

This prospective study assessed the value of initial microscopy evaluation of sputum samples submitted for rapid syndromic PCR-based testing. Bacterial detections by the BioFire FilmArray Pneumonia Panel plus in 126 high- and 108 low-quality sputum samples, based on initial microscopy evaluation in samples from patients with lower respiratory tract infections were compared. We found that high-quality samples had a higher proportion of bacterial detections compared to low-quality samples (P = 0.013). This included a higher proportion of detections of bacteria deemed clinically relevant by predefined criteria (70% and 55%, P = 0.016), as well as a higher proportion of detections of Haemophilus influenzae (36% and 20%, P = 0.010). High-quality samples also had more detections of bacteria with high semi-quantitative values. The study found no significant difference between high- and low-quality samples in the proportions of samples with a single species of bacteria detected, samples with a bacteria treated by the clinician, samples with detection of a proven etiology of community-acquired pneumonia by predefined criteria, the number of bacterial species detected, or the detection of Streptococcus pneumoniae, Moraxella catarrhalis, or Staphylococcus aureus. The results showed that 40% (95% CI 35%-47%) of the bacterial detections would have been missed if only high-quality samples were analyzed. This included 41% (27%-56%) of detections of S. pneumoniae, 33% (23%-45%) of detections of H. influenzae, 42% (28%-58%) of detections of S. aureus, and 37% (23%-54%) of detections of M. catarrhalis. These findings suggest that all sputum samples submitted for rapid syndromic PCR testing should be analyzed, regardless of initial microscopy quality assessment. (This study has been registered at ClinicalTrials.gov under registration no. NCT04660084.) IMPORTANCE Microscopic quality assessment of sputum samples was originally designed for sputum culture, and its applicability in today's workflow, which includes syndromic PCR testing, may differ. Addressing this crucial gap, our study emphasizes the need to optimize the use and workflow of syndromic PCR panels, like the BioFire FilmArray Pneumonia plus (FAP plus), in microbiology laboratories. These advanced PCR-based tests offer rapid and comprehensive pathogen detection for respiratory infections, yet their full potential remains uncertain. By comparing bacterial detections in high- and low-quality sputum samples, we underscore the importance of including low-quality samples in testing. Our findings reveal a significant proportion of potentially clinically relevant bacterial detections that would have been missed if only high-quality samples were analyzed. These insights support the efficient implementation of syndromic PCR panels, ultimately enhancing patient care and outcomes.

Simple and Rapid Discrimination of Methicillin-Resistant Staphylococcus aureus Based on Gram Staining and Machine Vision.

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinical threat with high morbidity and mortality. Here, we describe a new simple, rapid identification method for MRSA using oxacillin sodium salt, a cell wall synthesis inhibitor, combined with Gram staining and machine vision (MV) analysis. Gram staining classifies bacteria as positive (purple) or negative (pink) according to the cell wall structure and chemical composition. In the presence of oxacillin, the integrity of the cell wall for methicillin-susceptible S. aureus (MSSA) was destroyed immediately and appeared Gram negative. In contrast, MRSA was relatively stable and appeared Gram positive. This color change can be detected by MV. The feasibility of this method was demonstrated in 150 images of the staining results for 50 clinical S. aureus strains. Based on effective feature extraction and machine learning, the accuracies of the linear linear discriminant analysis (LDA) model and nonlinear artificial neural network (ANN) model for MRSA identification were 96.7% and 97.3%, respectively. Combined with MV analysis, this simple strategy improved the detection efficiency and significantly shortened the time needed to detect antibiotic resistance. The whole process can be completed within 1 h. Unlike the traditional antibiotic susceptibility test, overnight incubation is avoided. This new strategy could be used for other bacteria and represents a new rapid method for detection of clinical antibiotic resistance. IMPORTANCE Oxacillin sodium salt destroys the integrity of the cell wall of MSSA immediately, appearing Gram negative, whereas MRSA is relatively stable and still appears Gram positive. This color change can be detected by microscopic examination and MV analysis. This new strategy has significantly reduced the time to detect resistance. The results show that using oxacillin sodium salt combined with Gram staining and MV analysis is a new, simple and rapid method for identification of MRSA.

The role of gram stain in reducing broad-spectrum antibiotic use: A systematic literature review and meta-analysis.

The number of studies that verify whether Gram stain can help to reduce the use of broad-spectrum antibiotics is relatively limited compared to those evaluating its concordance with culture test results. Thereby, we aimed to evaluate the effectiveness of Gram staining in the reduction of broad-spectrum antibiotics and its impact on clinical outcomes. We systematically reviewed studies having used Gram stain to guide antibiotic selection and evaluated performance measures between 1996 and 2022. We extracted available data on broad-spectrum antibiotic use as a primary outcome of the studies in view of an exploratory meta-analysis designed to estimate the clinical effect of Gram stain. We also evaluated the clinical response and coverage rates of the initial antibiotic therapy. One randomized study and four non-randomized studies were eligible, all of which were conducted in tertiary care hospitals in Japan. Gram stain was associated with reduced broad-spectrum antibiotic use, including antipseudomonal antibiotics (odds ratio [OR], 0.05; 95% confidence interval [CI], 0.01-0.34), anti-methicillin-resistant Staphylococcus aureus antibiotics (OR, 0.21; 95% CI, 0.07-0.63), and carbapenems (OR, 0.07; 95% CI, 0.02-0.19), without impairing clinical outcomes, including clinical response rate (OR, 1.48; 95% CI, 0.95-2.31) and coverage rate of initial antibiotic therapy (OR, 0.70; 95% CI, 0.40-1.22) using random-effects models in our meta-analysis. In conclusion, Gram stain may be useful in guiding initial antibiotic selection without apparent adverse clinical outcomes. However, currently available studies evaluating the clinical usefulness of Gram stain are limited to specific clinical settings.

Bacterial Burden, Not Local Immune Response, Differs Between Acute and Chronic Peri-Prosthetic Joint Infections.

Background: Conducting gram stains in peri-prosthetic joint infections (PJI) is known to have poor sensitivity. However, the aims of this study were to use gram stain results of acute and chronic PJI to determine differences with respect to bacterial burden and levels of local innate immunologic response. Patients and Methods: Patients with acute and chronic PJI from January 1, 2016 and December 31, 2020 were identified by use of Current Procedural Terminology codes. Manual review of medical records for infecting organisms and gram stain results for stained bacteria and for local tissue inflammation (amount of polymorphonuclear leukocytes seen on high powered microscopic fields) were recorded. Statistical comparisons between acute (n = 70) and chronic (n = 134) PJI were analyzed with respect to gram stain sensitivity and amount of local tissue inflammation. Results: The ability to identify stained bacteria was statistically significantly higher in the acute cohort (61.4%) than the chronic cohort (9.7%; p < 0.0001). Interestingly, the amount of local inflammation was similar for acute and chronic PJI except in the subgroup analysis with chronic polymicrobial (p = 0.0229) and chronic culture negative (p = 0.0001) PJI. Conclusions: This study shows that both acute and chronic PJI had similar levels of local inflammation seen on gram stains, despite higher bacterial burdens in acute infections. This suggests that innate immune responses, and thus likelihood of infection eradication, is not solely dependent on bacterial burden. These findings should spearhead further research evaluating the different immunologic responses that occur in acute and chronic PJI to improve diagnostics, therapeutics, and infection-free implant survival.

Can Gram staining be a guiding tool for optimizing initial antimicrobial agents in bacterial infections?

The emergence of multidrug-resistant organisms poses a significant threat to global public health, making the optimization of antimicrobial use crucial. Antimicrobial therapy is often initiated in emergency rooms (ERs) and intensive care units (ICUs), where patients are at high risk of infection. Prompt antimicrobial selection is essential in these facilities, and point-of-care testing can guide the appropriate initial antimicrobial therapy. Gram staining, a quick and inexpensive method, was previously used for point-of-care testing by physicians in the 1980s but was discontinued in 1988 in the United States. However, in Japan, the clinical practice of Gram stain-based antimicrobial therapy by physicians has continued in a limited number of hospitals. Several studies undertaken in Japan have shown that Gram staining carried out by trained physicians can reduce the overuse of broad-spectrum antimicrobial agents in ERs and ICUs without worsening patients' outcomes. Gram stain-based antimicrobial therapy reduced unnecessary use of carbapenems in the ER. Furthermore, Gram staining has been shown to significantly reduce the overuse of broad-spectrum antimicrobials without worsening clinical cure and mortality for patients with ventilator-associated pneumonia in the ICU. The classic technique of Gram staining has regained its usefulness through persistent clinical practice in Japan. It is hoped that Japanese researchers in this field will demonstrate to the world the efficacy of the classic technique of Gram staining in addressing this critical problem. Gram staining carried out by trained physicians could serve as a valuable means of optimizing antimicrobial treatment in ERs and ICUs.