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Background This study aimed to evaluate the impact of optimized diagnostic protocols on pathogen detection rates in abscess specimens. Methods Our retrospective study analyzed 1,297 abscess specimens collected between 2018 and 2024 using an enhanced diagnostic protocol combining four key methodologies: routine aerobic/anaerobic culture, gram-stain microscopy, acid-fast bacilli staining, and blood culture bottle enrichment techniques. Results The implementation of optimized diagnostic protocols significantly enhanced pathogen detection efficacy ( P  < 0.001, χ = 9.663), achieving an overall positivity rate of 81.9% (1,062/1,297)—a 20.1 percentage point improvement over conventional methods. Among culture-positive specimens, polymicrobial infections were identified in 27.6% of cases (293/1,062). A total of 1,651 microbial isolates were recovered, dominated by gram-negative bacteria (50.6%, 836/1,651) with  Escherichia coli  (55.0%),  Klebsiella pneumoniae  (23.0%), and  Acinetobacter baumannii  (4.0%) as predominant species. Gram-positive cocci accounted for 33.7% (557/1,651), primarily  Streptococcus  spp. (45.0%),  Staphylococcus aureus  (19.0%), and  Enterococcus faecium  (6.0%). Enhanced methodology detected 261 additional pathogens (20.1% of total yield), including anaerobes (33.7%), smear-positive organisms (32.2%), acid-fast bacilli (6.9%), and  Brucella melitensis  (1.5%). Anatomic distribution analysis revealed perianal abscesses (358 cases, 407 isolates) predominantly associated with  E. coli  (51.8%),  K. pneumoniae  (14.7%), and  Streptococcus  spp. (15.7%), followed by maxillofacial infections (244 cases, 297 isolates; 18.0%). Other significant sites included abdominal abscesses/peritonitis (17.0%), hepatic abscesses (5.2%), and Periappendicular abscess (5.5%). Conclusions Systematic optimization of diagnostic protocols significantly enhanced pathogen detection in abscess specimens, demonstrating substantial clinical utility for infectious disease management. These findings support the adoption of comprehensive, standardized approaches for abscess specimen processing. Highlights Adopting optimized standardized operating procedures, abscess specimens were detected positive accounting for 81.9%, and the positivity rate increased by 20.1%. Additional abscess specimens were detected positive, including anaerobic bacteria, smear test, and acid-fast bacteria accounting for 33.7%, 32.2%, and 6.9%, respectively.

Background There has been increasing interest in the use of newer, culture-independent techniques to study the airway microbiome of COPD patients. We investigated the relationships between the three common potentially pathogenic microorganisms (PPMs) Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis , as detected by quantitative PCR (qPCR), and inflammation and health status in stable patients in the London COPD cohort. Methods We prospectively collected sputum, serum and plasma samples for analysis of airway bacterial presence and load, and airway and systemic inflammation from 99 stable COPD patients between January 2011 and October 2012. Health status was measured with St George’s Respiratory Questionnaire and COPD Assessment Test. Results Airway inflammation and plasma fibrinogen, but not C-reactive protein, were greater in samples with PPM detection (p < 0.001, p = 0.049 and p = 0.261, respectively). Increasing total bacterial load was associated with increasing airway (p < 0.01) but not systemic inflammation (p > 0.05). Samples with high total bacterial loads had significantly higher airway inflammation than both samples without PPM detection and those with lower loads. Haemophilus influenzae presence was associated with significantly higher levels of airway but not systemic inflammation for all given pathogen loads (p < 0.05), and was significantly greater than with other PPMs. No association was observed between inflammation and health status (p > 0.05). Conclusions Airway and systemic inflammation, as measured by fibrinogen, is greater in stable COPD patients with PPMs detected using the culture-independent qPCR technique. The airway, but not systemic inflammatory response, appears to have a total pathogen-load threshold and appears attributable to Haemophilus influenzae . However, discordance between inflammation and health status was observed.

Introduction: Monitoring the microbial quality of water in dental unit waterlines is an important part of infection control measures carried out in dental clinics. Fungal contamination of such waterlines has not been extensively studied, compared with bacterial contamination. This study aimed at assessing the magnitude and risk factors for fungal contamination of dental unit waterlines. Methodology: This cross-sectional study included 82 dental units, randomly collected from 3 private clinics and 8 governmental hospitals in Alexandria, Egypt. A total of 204 water samples from dental unit waterlines output were membrane-filtered and cultured for fungal enumeration and species identification. The biofilm forming-ability was assessed for the most prevalent fungal species. The acceptability of samples was determined according to the Swedish drinking water guidelines. Results: The acceptability of samples was 89.7%. The most common mould was Aspergillus flavus, while Candida spp. was the most common yeast (10 isolates), with unusual predominance of Candida dubliniensis (9 isolates). All isolates of Aspergillus flavus and Candida dubliniensis were biofilm-formers. The risk factors for fungal contamination of dental unit waterlines included: dental specialty (p = 0.042), time of sample collection (p < 0.001), older age of dental unit (p < 0.001) and use of 5-15% of sodium hypochlorite. Conclusions: The presence of biofilm-forming fungi in dental unit waterlines is a potential hazard, even when samples have acceptable levels of fungal counts. Risk factors for contamination are numerous and should be addressed.

Optical density (OD) measurements of microbial growth are one of the most common techniques used in microbiology, with applications ranging from studies of antibiotic efficacy to investigations of growth under different nutritional or stress environments, to characterization of different mutant strains, including those harbouring synthetic circuits. OD measurements are performed under the assumption that the OD value obtained is proportional to the cell number, i.e. the concentration of the sample. However, the assumption holds true in a limited range of conditions, and calibration techniques that determine that range are currently missing. Here we present a set of calibration procedures and considerations that are necessary to successfully estimate the cell concentration from OD measurements.

To compare bacterial contamination of military-approved uniforms and hospital-provided scrubs donned by nursing staff in an inpatient setting. Randomized experimental crossover study. Large academic military medical center. Inpatient units were randomized to predetermine the order of uniform sampling. Participants included nursing staff who provided direct patient care across 7 eligible inpatient units. Sampling of 6 designated sites on the uniform was completed on arrival to work, at ~4 hours into their shift, and at the 8-hour time point, for a total of 18 samples. Sampling of each participant occurred on 2 separate occasions, once in a military-approved uniform, and once in hospital-provided scrubs. After 24 hours of incubation, a colony-counting machine was used to calculate the total colony-forming units (CFU) of the sample. Across all time points, military-approved uniforms demonstrated a 2-fold bacterial increase at the abdominal site and 3-fold increases at the sleeve cuff and waist pocket regions compared to the same regions on hospital-provided scrubs. Nurses should be aware that bacteria are present at much higher levels on their personal military uniforms compared to hospital-provided scrubs. Additional research is needed to determine whether these findings are a function of wear, laundering, or environmental factors. Nurses should adhere to daily uniform washing to reduce bacterial load and minimize risk of nosocomial infections to the patients they care for.

A study was conducted to test the effects of Jerusalem artichoke inulin (JA) or chicory inulin (CH) in snack bars on composition of faecal microbiota, concentration of faecal SCFA, bowel habit and gastrointestinal symptoms. Forty-five volunteers participated in a double-blind, randomized, placebo-controlled, parallel-group study. At the end of a 7 d run-in period, subjects were randomly assigned to three groups of fifteen subjects each, consuming either snack bars with CH or JA, or snack bars without fructans (placebo); for 7 d (adaptation period), they ingested one snack bar per day (7·7 g fructan/d) and continued for 14 d with two snack bars per day. The composition of the microbiota was monitored weekly. The consumption of CH or JA increased counts of bifidobacteria (+1·2 log10 in 21 d) and reduced Bacteroides/Prevotella in number and the Clostridium histolyticum/C. lituseburense group in frequency at the end of intervention (P < 0·05). No changes in concentration of faecal SCFA were observed. Consumption of snack bars resulted in a slight increase in stool frequency. Stool consistency was slightly affected in subjects consuming two snack bars containing CH or JA per day (P < 0·05). Consumption of CH or JA resulted in mild and sometimes moderate flatulence in a few subjects compared to placebo (P < 0·05). No structural differences were detected between CH and JA before and after processing. In conclusion, adaptation on increased doses of CH or JA in bakery products stimulates the growth of bifidobacteria and may contribute to the suppression of potential pathogenic bacteria.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. The disease causes diarrhea, reduced milk production, poor reproductivity, emaciation, and eventually death. Culture on Herrold's egg yolk agar is considered to be the definitive test for diagnosis of Johne's in cattle. This method has moderate sensitivity (30 to 50%) and is 100% specific; however, it can take up to 16 wk due to the slow growth of MAP. Currently, serum ELISA is used to screen herds for Johne's disease, but positive tests must be confirmed culturally or by PCR. The current research sought to evaluate an in-house direct fecal PCR procedure and directly compare it to ELISA using culture as the gold standard. Serum and fecal samples were collected from cows (n=250) with unknown Johne's status. Fecal samples were processed for culture on Herrold's egg yolk agar and direct PCR. Serum samples were tested using the Parachek serum ELISA. Overall, 67/250 [26.8%, 95% confidence interval (CI) 21.4 to 32.8] animals were culturally confirmed to be shedding MAP. The PCR and ELISA detected 74/250 (29.6%, 95% CI 24 to 35.7) and 25/250 (10%, 95% CI 6.6 to 14.4), respectively. Culture and PCR were able to detect more positive animals than ELISA. Overall, direct fecal PCR was 70.2% sensitive and 85.3% specific when using culture as the gold standard. The ELISA method was 31.3% sensitive and 97.8% specific. When culture reported <10 cfu, the sensitivity and specificity of PCR and ELISA were 57.1 and 85.3%, and 4.8 and 97.8%, respectively. When culture reported 10 to <40 cfu, the sensitivity of PCR and ELISA were 75 and 50%, respectively. When culture reported ≥40 cfu, the sensitivity of PCR and ELISA were 100 and 88.2%, respectively. Specificity could not be calculated at these levels because there were no negative samples. The direct PCR outperformed the ELISA in detecting animals potentially infected with MAP and was not significantly different when compared with culture. The direct fecal PCR method described here provides faster results than traditional culture and is more sensitive than ELISA at detecting animals suspected of Johne's disease. These data support the use of PCR as an alternative method for screening herds for prevalence and diagnosis of Johne's disease.

Prevalence data were collected from official microbiological records monitoring four selected foodborne pathogens (Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Campylobacter jejuni) in raw milk sold by self-service vending machines in seven Italian regions (60,907 samples from 1,239 vending machines) from 2008 to 2011. Data from samples analyzed by both culture-based and real-time PCR methods were collected in one region. One hundred raw milk consumers in four regions were interviewed while purchasing raw milk from vending machines. One hundred seventy-eight of 60,907 samples were positive for one of the four foodborne pathogens investigated: 18 samples were positive for Salmonella, 83 for L. monocytogenes, 24 for E. coli O157:H7, and 53 for C. jejuni in the seven regions investigated. No significant differences in prevalence were found among regions, but a significant increase in C. jejuni prevalence was observed over the years of the study. A comparison of the two analysis methods revealed that real-time PCR was 2.71 to 9.40 times more sensitive than the culture-based method. Data on consumer habits revealed that some behaviors may enhance the risk of infection linked to raw milk consumption: 37% of consumers did not boil milk before consumption, 93% never used an insulated bag to transport raw milk home, and raw milk was consumed by children younger than 5 years of age. These results emphasize that end-product controls alone are not sufficient to guarantee an adequate level of consumer protection. The beta distribution of positive samples in this study and the data on raw milk consumer habits will be useful for the development of a national quantitative risk assessment of Salmonella, L. monocytogenes, E. coli O157, and C. jejuni infection associated with raw milk consumption.

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin–producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121,and O145 from ground beef enrichments. A post–immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111,O121, and O145 from raw ground beef.

The U.S. Department of Agriculture, Food Safety Inspection Service has determined that six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) are adulterants in raw beef. Isolate and phenotypic discrimination of non-O157 STEC is problematic due to the lack of suitable agar media. The lack of distinct phenotypic color variation among non-O157serogroups cultured on chromogenic agar poses a challenge in selecting colonies for confirmation. In this study, visible and near-infrared hyperspectral imaging and chemometrics were used to detect and classify non-O157 STEC serogroups grown on Rainbow agar O157. The method was first developed by building spectral libraries for each serogroup obtained from ground-truth regions of interest representing the true identity of each pixel and thus each pure culture colony in the hyperspectral agar-plate image. The spectral library for the pure-culture non-O157 STEC consisted of 2,171 colonies, with spectra derived from 124,347 of pixels. The classification models for each serogroup were developed with a k nearest-neighbor classifier. The overall classification training accuracy at the colony level was 99%. The classifier was validated with ground beef enrichments artificially inoculated with 10, 50, and 100 CFU/ml STEC. The validation ground-truth regions of interest of the STEC target colonies consisted of 606 colonies, with 3,030 pixels of spectra. The overall classification accuracy was 98%. The average specificity of the method was 98% due to the low false-positive rate of 1.2%. The sensitivity ranged from 78 to 100% due to the false-negative rates of 22, 7, and 8% for O145, O45, and O26, respectively. This study showed the potential of visible and near-infrared hyperspectral imaging for detecting and classifying colonies of the six non-O157 STEC serogroups. The technique needs to be validated with bacterial cultures directly extracted from meat products and positive identification of colonies by using confirmatory tests such as latex agglutination tests or PCR.