Microbiological Identification Research Articles

Accelerated Bacterial Identification with MALDI-TOF MS Leads to Fewer Diagnostic Tests and Cost Savings

Introduction: Rapid microbiology reporting can enhance both clinical and economic outcomes. Material and Methods: This three-year, quasi-experimental study, single-group pretest–posttest study, conducted at a university medical center, aimed to evaluate the clinical and economic impact of rapid microbiological identification reporting using MALDI-TOF MS. A total of 363 consecutive hospitalized patients with bacterial infections were evaluated, comparing a historical control group (CG, n = 183) with an intervention group (IG, n = 180). In the CG, microbiological information (bacterial identification and antibiotic susceptibility) was provided between 18:00 and 22:00 h, while in the IG, bacterial identification was reported between 12:00 and 14:00 h, and antibiotic susceptibility was reported between 18:00 and 22:00 h. Results: The IG demonstrated a significant reduction in the number of patients undergoing Microbiology (p = 0.01), Biochemistry (p = 0.05), C-Reactive Protein (p = 0.02), Radiological Tests (p = 0.05), Computed Tomography Tests (p = 0.04), and Pathology (p = 0.01). However, no statistically significant reduction was observed in economic costs related to microbiological testing (p = 0.76) or antibiotic consumption (p = 0.59). The timely reporting of microbiological identification to clinicians resulted in fewer patients undergoing additional diagnostic tests, ultimately contributing to reduced healthcare resource utilization without adversely affecting clinical outcomes.

Oat Okara Fermentation: New Insights into the Microbiological and Metabolomic Characterization

The importance of the valorization of industrial by-products has led to increasing research into their reuse. In this research, the innovative by-product okara oat flour, derived from the vegetable beverage industry, was studied. Oat okara sourdough was also produced and evaluated. The microbiological identification and typing involved bacterial and yeast isolates from both flour and sourdough. Untargeted metabolomics allowed the identification of biomarkers of fermented flour, such as phenolic classes, post-fermentation metabolites, fatty acids, and amino acids. The microorganisms most found were Weissella confusa, Enterococcus faecium, Pediococcus pentosaceus, and Pichia kudriavzevii, while Saccharomyces cerevisiae appeared only at the end of the sourdough’s back-slopping. Untargeted metabolomics identified a total of 539 metabolites, including phenolic compounds, lipids, amino acids, and organic acids. An increase in polyphenols released from the food matrix was detected, likely because of the higher bio-accessibility of phenolic metabolites promoted by microbial fermentation. Fermentation led to an increase in isoferulic acid, p-coumaric acid, sinapic acid, and a decrease in amino acids, which can be attributed to the metabolism of lactic acid bacteria. Some key markers of the fermentation process of both lactic acid bacteria and yeast were also measured, including organic acids (lactate, succinate, and propionate derivatives) and flavor compounds (e.g., diacetyl). Two bioactive compounds, such as gamma-aminobutyric acid and 3-phenyl-lactic acid had accumulated at the end of fermentation. Taken together, our findings showed that oat okara flour can be considered an excellent raw material for formulating more sustainable and functional foods due to fermentation promoted by autochthonous microbiota.

Whole genome sequencing reveals antibiotic resistance pattern and virulence factors in Klebsiella quasipneumoniae subsp. Similipneumoniae from Hospital wastewater in South-West, Nigeria

Klebsiella quasipneumoniae is a distinct species from K. pneumoniae, even though it is sometimes mistaken phenotypically for the latter in clinical situations. K. quasipneumoniae is a pathogen and this study aims at understanding the genomic antibiotic resistance and virulence characteristics of Klebsiella quasipneumoniae subsp. similipneumoniae (B105 strain) isolated from tertiary hospital wastewater and the potential risks associated with its environmental spread.The Illumina platform was used for whole-genome sequencing (WGS), the generated raw reads (de novo) was assembled using RAPT NCBI, while other standardized bioinformatics tools were utilized to validate and examine the landscape of the genome's antibiotic resistance and virulence factors. The K. quasipneumoniae subsp. similipneumoniae (B105 strain), belonged to sequence type 1422 and was resistant to ampicillin, amoxicillin-clavulanic acid, ceftazidime, cefepime, meropenem, tetracycline, but susceptible to gentamicin. The annotated genome acknowledged the presence of blaOKP-B-2, ompK 36, fosA5, oqxAB, virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, efllux pump (acrA, acrB) adherence, (mrkC, mrkD, and fimD) and two plasmids replicon IncFIB(K) and IncR. The study resonates the inadequacy of conventional microbiological identification methods to distinguish K. pneumoniae and K. quasipneumoniae and at the same time heightens the importance of using a genomic platform to extol the identity of K. quasipneumoniae subsp. similipneumoniae strain. Furthermore, the peculiarities of the acquired antimicrobial resistance and virulence genes, in this strain, are a potential risk to the environment.

Can bone SPECT/CT determine optimal sites for microbiological identification in post-traumatic or chronic osteomyelitis of extremities?

IntroductionAccurate microbiological identification is crucial when managing chronic osteomyelitis (COM) and post-traumatic osteomyelitis (PTO). Although bone single photon emission computed tomography/computed tomography (SPECT/CT) has helped in localizing osteomyelitis lesions, its effectiveness in guiding microbiological sampling remains unclear. This study aimed to determine whether bone SPECT/CT can improve microbiological identification rates in COM or PTO of the extremities. Patients and methodsFrom February 2020 to August 2024, 53 patients with suspected COM or PTO in the extremities were retrospectively analyzed. All patients underwent bone SPECT/CT, followed by microbiological sampling during surgery. Tissue samples were taken from the areas of high SPECT/CT uptake or based on intraoperative findings where no uptake was observed. Microorganism identification rates were analyzed, including a sub-group analysis based on antibiotic discontinuation. ResultsOf the 53 patients, 42 had positive bone SPECT/CT scan findings, with pathogen identification in 30 patients (71.4 %). In contrast, pathogen identification occurred in one out of twelve patients (9.1 %) with negative findings (odds ratio 25, p< 0.001). Bone SPECT/CT demonstrated a sensitivity of 96.8 % and an overall accuracy of 75.5 %. When antibiotics had been discontinued for ≥2 weeks, the pathogen identification rate increased to 90 %, compared with 50 % for <2 weeks of discontinuation (odds ratio 10.0, p= 0.006). In a sub-group of 30 patients with adequate antibiotic discontinuation duration, a positive bone SPECT/CT scan yielded a pathogen identification rate of 90.1 % (odds ratio 60.0, p= 0.001). ConclusionBone SPECT/CT effectively identifies optimal sites for microbiological sampling in COM and PTO of the extremities, particularly when antibiotics have been discontinued for ≥2 weeks, enhancing pathogen detection rates.

Genotypic and phenotypic characterization of the first Latin America isolates of Corynebacterium rouxii, a recently described member of the Corynebacterium diphtheriae complex reported in Europe.

The genus Corynebacterium is the largest genera among corynebacteria and has a range of species widely spread in ecological niches, some with epidemic potential and capable of causing fatal diseases. In recent years, due to the reclassifications and discoveries of new potentially toxin-producing species, microbiological identification and epidemiological control have been compromised, becoming possible only with sequencing techniques. Two bacterial strains isolated from a cat were identified by MALDI-TOF mass spectrometry as Corynebacterium diphtheriae and sent to the collaborating center of the Brazilian Ministry of Health for molecular identification and determination of toxigenicity potential, which were initially performed by multiplex PCR method. In addition, the antimicrobial susceptibility profile was determined according to BrCAST. Finally, for the final identification at the species level and effective epidemiological monitoring, the sequencing of the 16S rRNA and rpoB housekeeping genes was carried out. The isolates were identified as nontoxigenic C. diphtheriae strains by mPCR. Both strains were found susceptible to all antimicrobial agents. Although the identification at the species level was not possible through similarity analysis of S rRNA and rpoB housekeeping genes, the phylogenetic analysis showed that the isolates belonged to the species Corynebacterium rouxii with a high value of reliability. This is the first report of the isolation of C. rouxii in Latin America. Molecular identification, whether by the MALDI-TOF mass spectrometry or PCR techniques, does not discriminate C. rouxii from C. diphtheriae, requiring gene sequencing and phylogenetic analysis for correct identification at the species level.

A rare case report of tissue infection caused by Pantoea piersonii (basionym Kalamiella piersonii).

In 2019, Pantoea piersonii was initially isolated from the interior surfaces of the International Space Station. This microorganism is a species within the genus Pantoea in the family Erwiniaceae, belonging to the order Enterobacterales. Recent literature has documented four cases of its isolation. Despite initial predictions suggesting the non-pathogenicity of P. piersonii strains, evidence from observed cases indicates potential pathogenicity. According to documented evidence in the literature, this microorganism is capable of causing severe and life-threatening conditions, including sepsis. Traditional tests, as well as automated systems, may fail to provide complete differentiation due to these similarities. While MALDI-TOF MS is a valuable tool for identification in clinical diagnostic microbiology, sequencing may be necessary for precise identification. To determine the antibiotic susceptibility profile, various methods can be utilized, including minimum inhibitory concentration determination, disk diffusion testing (Kirby-Bauer test), genotypic resistance assays (PCR and sequencing), and automated systems. The literature reports a limited number of cases associating P. piersonii with human infection. This study contributes to this body of knowledge by reporting a novel case in which P. piersonii was isolated from a tissue sample for the first time. In this case report, the patient achieved recovery following the administration of appropriate antibiotic treatment based on the diagnosis. It underscores the need for precise identification and understanding of its pathogenicity.

A-282 Purpura fulminans caused by Haemophilus influenzae: case report of unusual etiology

Abstract Background Fulminant purpura is a clinical syndrome characterized by a purplish, ischemic-looking skin rash, commonly accompanied by systemic symptoms ranging from acute febrile syndrome to septic shock. Due to high lethality of possible infectious etiologies, immediate recognition and early initiation of empirical antimicrobial therapy are crucial to ensure better outcomes. Interdisciplinary collaboration among healthcare teams, infectious disease specialists and microbiologists is essential to ensure accurate and early microbiological diagnosis. Methods This work aims to report a case of Haemophilus influenzae-induced purpura fulminans that occurred in Fortaleza-CE, a coastal city in northeastern Brazil, highlighting clinical recognition and microbiological identification, based on review of medical records. Results Case report A 59-year-old man, originally from Germany, was on vacation in Brazil. He presented to emergency medical service with an acute onset of fever, abdominal pain and diarrhea, progressing to decreased level of consciousness and purplish ischemic skin lesions with hemorrhagic bullae on trunk and extremities (Figure 1), with no history of trauma. Upon admission, the blood exams showed bicytopenia - 1900 leukocytes/µL (86% segmented) and 51,000 platelets/µL - and very high C-reactive protein (41.58 mg/dL). Although empirical antimicrobial therapy with vancomycin and piperacillin-tazobactam was initiated, patient progressed to septic shock, hepatic dysfunction, requiring mechanical ventilation, vasopressor support, and renal replacement therapy. Blood cultures from hospital admission were positive after about 14 hours and allowed isolation of small and translucent colonies in Chocolate PVX plate Agar (BioMerieux™). Maldi tof analysis by Vitek MS identified H. influenzae (99.9 % accuracy). Despite prolonged hospitalization due to multiple organ dysfunctions, patient had a positive outcome. Conclusions This case illustrates the importance of immediate recognition of the syndrome coupled with assertive empirical treatment and early microbiological diagnosis for a positive clinical outcome, even in a severe presentation. Multiprofessional approach is essential for accurate and early diagnosis.

Utilidad del PCR múltiplex de hemocultivo para el diagnóstico microbiológico de infecciones nosocomiales del sistema nervioso central en pacientes críticos

Background and objectiveTo determine the usefulness of a molecular biology technique for nucleic acid amplification using real-time polymerase chain reaction (PCR) originally designed for microbiological diagnosis in blood cultures, when applied to cerebrospinal fluid (CSF) samples. Materials and methodsRetrospective, observational, single-center study, carried out between January 2017 and February 2024 in a 28-bed multipurpose critical care unit. All patients with suspected postneurosurgical infection and CSF cytochemical alterations were included. In addition to the microbiological study of the CSF by traditional methods, the BioFire® Blood Culture Identification2 (BCID) panel was applied directly to CSF samples. Traditional culture methods were considered as the reference standard for microbiological identification. The main variable of interest was the percentage of concordance of microbial identification between traditional cultures and BCID. Results29 patients were included. The diagnosis of central nervous system infection was confirmed in 14 cases (48.2%), 7 post-neurosurgical meningitis, 6 infections associated with ventriculostomy and 1subdural empyema. The most frequently found microorganisms were Acinetobacter baumannii and Klebsiella pneumoniae. No discordances were found between PCR and cultures, so both the positive (14/14) and negative identification concordance (15/15) were 100% between both methods. ConclusionsIn patients with suspected post-neurosurgical infection and compatible CSF alterations, multiplex PCR blood cultures applied directly to CSF samples could be considered a useful and accurate tool for rapid microbiological diagnosis in infections of the central nervous system.

Microbiological profile and prevalence of histamine-producing bacteria in fresh sardines stored at different temperatures.

The European pilchard (Sardina pilchardus) is an important fish species for the Moroccan economy in terms of production and export. Biogenic amine histamine is a metabolite produced in the flesh of some fish species after death due to the decarboxylation of free histidine by histaminogenic bacteria. Failure to control the histamine risk in European pilchard may lead to public health and socioeconomic issues. This study aimed to estimate the prevalence of histaminogenic bacteria in association with histamine levels and the growth of microflora in Moroccan sardines (European pilchard). We conducted the study by monitoring Moroccan sardines of histamine content and microbiological profile (aerobic plate count [APC], coliforms, and thermo-tolerant coliforms [TTC]) during 6 days of storage at three different temperatures (0°C, 10°C, and ambient temperature [22°C]). The histamine assay was performed using a spectrofluorometric method, and the microbiological identification of histamine-producing bacteria was performed using a combination of biochemical and molecular tests. The histamine content in European pilchard stored at 0°C was negligible. However, high concentrations were observed at 10°C and 22°C. The microbiological profile showed a positive association between microflora counts and histamine content according to storage time. At 0°C, a moderate increase in the APC, a decrease in coliforms, and an absence of TTC were observed. The rapid proliferation of all microflora was observed at 10°C, whereas at 22°C, the proliferation was almost exponential. Bacterial identification revealed the exclusive presence of species belonging to the Enterobacteriaceae family at varying frequencies depending on storage temperature. Morganella morganii and Proteus mirabilis had the highest histamine induction rates in L-histidine-supplemented broth, with 1600 and 255 parts per million (ppm), respectively, after 48-h incubation at 35°C. Klebsiella ozaenae could produce 136 ppm and Serratia plymuthica 115 ppm. Reverse transcription polymerase chain reaction showed positive results for the presence of genes associated with histidine decarboxylase. The hdc genes of M. morganii, P. mirabilis, and K. ozaenae were successfully amplified and exhibited strong similarity with the reference gene of M. morganii. This study describes for the first time the hdc gene in bacteria that form histamine in Moroccan sardines. The results also confirm that respect for the cold chain integrity is a crucial factor in histamine management. This information should help stakeholders in the implementation of sound strategies for managing the hazards associated with seafood and their products.

Predicting Pseudomonas aeruginosa drug resistance using artificial intelligence and clinical MALDI-TOF mass spectra.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used in clinical microbiology laboratories for bacterial identification but its use for detection of antimicrobial resistance (AMR) remains limited. Here, we used MALDI-TOF MS with artificial intelligence (AI) approaches to successfully predict AMR in Pseudomonas aeruginosa, a priority pathogen with complex AMR mechanisms. The highest performance was achieved for modern β-lactam/β-lactamase inhibitor drugs, namely, ceftazidime/avibactam and ceftolozane/tazobactam. For these drugs, the model demonstrated area under the receiver operating characteristic curve (AUROC) of 0.869 and 0.856, specificity of 0.925 and 0.897, and sensitivity of 0.731 and 0.714, respectively. As part of this work, we developed dynamic binning, a feature engineering technique that effectively reduces the high-dimensional feature set and has wide-ranging applicability to MALDI-TOF MS data. Compared to conventional feature engineering approaches, the dynamic binning method yielded highest performance in 7 of 10 antimicrobials. Moreover, we showcased the efficacy of transfer learning in enhancing the AUROC performance for 8 of 11 antimicrobials. By assessing the contribution of features to the model's prediction, we identified proteins that may contribute to AMR mechanisms. Our findings demonstrate the potential of combining AI with MALDI-TOF MS as a rapid AMR diagnostic tool for Pseudomonas aeruginosa.IMPORTANCEPseudomonas aeruginosa is a key bacterial pathogen that causes significant global morbidity and mortality. Antimicrobial resistance (AMR) emerges rapidly in P. aeruginosa and is driven by complex mechanisms. Drug-resistant P. aeruginosa is a major challenge in clinical settings due to limited treatment options. Early detection of AMR can guide antibiotic choices, improve patient outcomes, and avoid unnecessary antibiotic use. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid species identification in clinical microbiology. In this study, we repurposed mass spectra generated by MALDI-TOF and used them as inputs for artificial intelligence approaches to successfully predict AMR in P. aeruginosa for multiple key antibiotic classes. This work represents an important advance toward using MALDI-TOF as a rapid AMR diagnostic for P. aeruginosa in clinical settings.

Clinical Characteristics, Serotypes and Antimicrobial Resistance of Invasive Salmonella Infections in HIV-Infected Patients in Hangzhou, China, 2012-2023.

Developing countries, invasive Salmonella infections can cause considerable morbidity and mortality. There is a relative lack of data on coinfection with Salmonella in HIV-infected patients in Hangzhou, China. In this study, we manually collected case data of patients aged >18 years with HIV combined with invasive Salmonella infections admitted to Xixi Hospital in Hangzhou from January 2012 to August 2023 by logging into the Hospital Information System, and identified 26 strains of invasive Salmonella using a fully automated microbiological identification system and mass spectrometer. Serotypes were determined using Salmonella diagnostic sera based on the White-Kauffmann-Le Minor scheme. Drug sensitivity tests were performed using the automated instrumental method of the MIC method. A total of 26 HIV-infected patients with invasive Salmonella coinfections were identified over 11 years; Twenty-five of the 26 patients (96.2%) were males, with a mean age of 33.5 years (26.75, 46.75). The most common type of infection was bloodstream infection (92.3%). One patient also had concomitant meningitis and osteoarthritis, followed by pneumonia (7.7%). The presence of multiple bacterial infections or even multiple opportunistic pathogens was clearly established in 7 (26.9%) patients. Three (11.6%) patients were automatically discharged from the hospital with deterioration of their condition, and one (3.8%) patient died. Salmonella enteritidis was the most common serotype in 6 patients (23.2%), and Salmonella Dublin was the most common serotype in 6 patients (23.2%). Drug sensitivity results revealed multidrug resistance in a total of 8 (30.8%) patients. The clinical presentation of invasive Salmonella infection in HIV patients is nonspecific and easily masked by other mixed infections. A CD4+ count <100 cells/µL and comorbid intestinal lesions may be important susceptibility factors. Salmonella has a high rate of resistance to common antibiotics, and the risk of multidrug resistance should not be ignored.

The antibiotic resistance profiles of Pseudomonas aeruginosa in the Asia Cornea Society Infectious Keratitis Study

PurposeTo describe the prevalence and antibiotic resistance profiles of Pseudomonasaeruginosa isolated from the Asia Cornea Society Infectious Keratitis Study (ACSIKS).MethodsAll bacterial isolates from ACSIKS underwent repeat microbiological identification in a central repository in Singapore. Minimum inhibitory concentration (MIC) determination was conducted for isolates of P. aeruginosa against thirteen antibiotics from 6 different classes, and categorized based on Clinical Laboratory Standard Institutes’ reference ranges. The percentage rates of resistance (non-susceptibility) to each antibiotic included isolates of both intermediate and complete resistance. Multi-drug resistance (MDR) was defined as non-susceptibility to at least one agent in three or more antimicrobial classes.ResultsOf the 1493 unique bacterial specimens obtained from ACSIKS, 319 isolates were of P. aeruginosa. The majority of isolates were from centers in India (n = 118, 37%), Singapore (n = 90, 28.2%), Hong Kong (n = 31, 9.7%) and Thailand (n = 30, 9.4%). The cumulative antibiotic resistance rate was the greatest for polymyxin B (100%), ciprofloxacin (17.6%) and moxifloxacin (16.9%), and lowest for cefepime (11.6%) and amikacin (13.5%). Isolates from India demonstrated the highest antibiotic resistance rates of all the centers, and included moxifloxacin (47.5%) and ciprofloxacin (39.8%). Forty-eight of the 59 MDR isolates also originated from India. Antibiotic resistance rates were significantly lower in the other ACSIKS centers, and were typically less than 10%.ConclusionsThe antibiotic resistance profiles of P. aeruginosa varied between different countries. While it was low for most countries, substantial antibiotic resistance and a significant number of multi-drug resistant isolates were noted in the centers from India.

Microbiological Landscape of Intensive Care Unit: A Retrospective Analysis of Bacterial Pathogens and Their Implications

Introduction: The intensive care unit (ICU) is a high-risk environment for hospital-acquired infections (HAIs) due to the complex interplay of patient vulnerability, invasive procedures, and the selective pressure of antimicrobial use. Understanding the local epidemiology of bacterial pathogens is crucial for effective infection prevention and control strategies. This study aimed to characterize the bacterial profile in an ICU setting, providing insights into the prevalent pathogens and their potential implications for patient care. Methods: A retrospective cross-sectional study was conducted, encompassing patients admitted to the ICU of Bangli Hospital Bali in 2023 and 2024. Data on patient demographics, sample sources, and microbiological findings were collected from medical records. Samples included blood cultures, sputum cultures (from both endotracheal tubes and spontaneous expectoration), wound swabs, and other sterile site cultures. Microbiological identification was performed using standard laboratory techniques. Results: A total of 219 patients were included in the study. The most common sample source was blood culture (42.9%), followed by sputum culture from endotracheal tubes (39.3%). A significant proportion of cultures showed no growth (34.7%), highlighting the challenges in identifying causative pathogens in the ICU. Among the identified pathogens, Staphylococcus spp. was predominant (15.5%), followed by Stenotrophomonas maltophilia (9.6%) and coagulase-negative staphylococci (8.7%). The distribution of pathogens varied across sample sources, with Staphylococcus spp. being prevalent in sputum cultures from endotracheal tubes and Klebsiella pneumoniae in sputum cultures. Conclusion: This study underscores the dynamic nature of the microbiological landscape in the ICU. The predominance of Staphylococcus spp., Stenotrophomonas maltophilia, and other opportunistic pathogens emphasizes the need for robust infection prevention and control measures. Further research is warranted to explore the impact of these pathogens on patient outcomes and to optimize antimicrobial stewardship in this critical care setting.

Microbiological identification of some culturable native strains associated with the digestive tract in Panaque cochliodon

Objective. To characterize by microbiology the native strains associated to the intestinal tract of the species Panaque cochliodon. Materials and methods. Three adult specimens captured in the Magdalena River that were transported and sacrificed under animal welfare regulations were used. Dissection of the intestinal tract was performed, obtaining samples for microbiological isolation, using culture media, purifying the microorganisms, performing metabolic biochemical tests and API 20E (Biomeriux) tests for their identification. Results. Information on the microbial population structure was obtained, reporting the phylum Proteobacteria with the species: Pantoea sp, Erwinia sp, Providencia stuarti, Providencia alcalifaciens, Serratia ficaria, Citrobacter koseri and the phylum Firmicutes with the species: Bacillus sphaericus, Bacillus subtilis, Bacillus thurigiensis, Bacillus mycoides, Bacillus coagulans and Bacillus circulans. Conclusion. The predominant culturable microorganisms in P. cohliodon belong to the phylum Proteobacteria and the phylum Firmicutes.

Broad-range amplification and sequencing of the rpoB gene: a novel assay for bacterial identification in clinical microbiology.

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

Hospital environment microbiome

The aim of the review is to give a brief description of the biodiversity and structure of the hospital environment microbiome based on molecular genetic research methods. Until a certain time, studies of the hospital environment microbiota for the purposes of epidemiological surveillance and control of healthcare-associated infections (HAIs) were based on routine microbiological identification of clinically relevant bacterial taxa. Discovery of DNA, the development of sequencing technologies, PCR and cloning techniques enabled the investigation of microbial communities using cultivation-independent, DNA and RNA-based approaches. At the current level of knowledge, the hospital environment can be considered as a superorganism with its own microbiome. Multiomic technologies, including meta-transcriptomic, meta-proteomic and metabolomic approaches, provide detailed information about microbial activity in the environment. Now it has been established that there is a stable core of the hospital microbiome where the vast majority of microorganisms are necessary for the functioning of the hospital ecosystem and are not classified as human pathogens. The hospital microbiome has a homogeneous structure composed by a massive dominance of a few taxa and microbial network with low connectivity forming a clustered topology. A keystone species is a taxon whose importance for maintaining community structure is relatively higher than others and its identification is of paramount importance. Due to the lack of knowledge of the hospital environment microbiome by molecular genetic technologies, there is no single shared point of view on the microbial diversity in different healthcare facilities. But there is no doubt that molecular genetic technologies will shed light on the evolution of hospital strains and determine which indicators are the most informative for monitoring and prognosis of HAIs.

Bone and Joint Infections in Children With Sickle Cell Disease in French Guiana: A 13-Year Retrospective Multicenter Review.

Sickle cell disease (SCD) is a genetic disorder with a high infectious morbidity and mortality and a heterogeneous distribution in France. One of the challenges is to differentiate a bone and joint infection (BJI) from a vaso-occlusive crisis. This challenge is particularly prevalent in French Guiana, an overseas territory with the highest incidence of SCD in France. The aim of this study was to describe the epidemiology of BJI in children with SCD in French Guiana. This was a retrospective multicentric descriptive study of SCD patients living in French Guiana aged under 18 and diagnosed with a BJI between 2010 and 2022. These BJI were divided into 2 groups: those with microbiological documentation (d-BJI) and those without microbiological identification (ud-BJI). A total of 53 episodes of BJI in 42 patients (mean age 7.2 years) were reported. Clinical symptoms on arrival were comparable between the d-BJI and ud-BJI groups. Patients in the d-BJI group had longer average hospital stays (40.4 days vs. 16.8 days, P = 0.01) and Salmonella spp. were the most identified bacteria (n = 8/13). White blood cell count was greater in the d-BJI group (30.3 G/L vs. 18.G/L, P = 0.01) and a collection was more frequently identified on imaging (11/13 vs. 16/40, P = 0.01) in this group. Initial in-hospital antibiotic therapy was longer in the d-BJI group (17.2 days vs. 12.8, P = 0.02), as were infection-related complications (9/13 vs. 12/40 P = 0.01). BJI in children with SCD is not sufficiently microbiologically documented. Progress must be made to improve the documentation of BJI.

Histologic Features of Mycobacterial Spindle Cell Pseudotumors: A Multi-institutional Clinicopathologic Analysis of 14 Cases.

Mycobacterial spindle cell pseudotumors (MSPs) are a rare and diagnostically challenging manifestation of non-tuberculous mycobacterial (NTM) infections. Proper recognition of these pseudotumors is important because they are treatable and benign. In this study, we evaluated the morphologic patterns of MSPs to improve their pathologic identification. Clinical and morphologic features of 14 MSPs were analyzed. Histologic factors evaluated included the architectural growth pattern of spindled or epithelioid macrophages, granulomas and their location within the lesion, neutrophilic microabscesses, multinucleated giant cells, necrosis, and effacement of background tissue. The composition of inflammatory infiltrates, organism density by acid-fast staining, and stromal changes were also assessed. In addition, 8 of 14 cases underwent molecular microbiology identification by a clinical amplicon-sequencing assay for non-tuberculous mycobacteria. MSP sites included 2 bowel, 10 lymph nodes, 1 liver, and 1 extremity. Cases with available clinical history (n=10) all occurred in immunocompromised patients. All demonstrated effacement of normal structures with spindled cells arranged in a storiform or fascicular architectural pattern. In addition, all cases showed lymphocytic inflammation, with prominent concurrent neutrophilic inflammation in 50% (7/14) of cases. Other morphologic findings included foamy histiocytes (64%, 9/14), peripherally situated granulomas (21%, 3/14), and neutrophilic microabscesses (21%, 3/14). All tested cases were positive for NTM by PCR methods. Mycobacterium avium was the most commonly isolated pathogen (6/8). Mycobacterial spindle cell pseudotumors show predominantly spindled morphology that may be mistaken as a neoplasm. Surgical pathologists who evaluate lymph nodes, soft tissue, and gastrointestinal tissues should be aware of this spindled tumefactive phenomenon in the setting of immunocompromised patients. Recognition of key morphologic features of neutrophilic inflammation, peripheral granulomas, or foamy histiocytes within a spindled lesion can help guide the pathologist to a correct diagnosis of an inflammatory process secondary to infection rather than a spindle cell neoplasm. Accurate diagnosis to facilitate appropriate antimicrobial and/or surgical therapy requires a comprehensive evaluation combining clinical, histopathologic, and microbiological findings.

An observational study of farmer-reported clinical mastitis in New Zealand dairy ewes

ABSTRACT Aims To describe the incidence, aetiology, treatment, and outcomes of farmer-reported clinical mastitis on New Zealand dairy sheep farms. Methods A prospective cohort study was conducted on 20 spring-lambing New Zealand sheep milking farms over the 2022–2023 season. Clinical mastitis was defined as a change in the appearance of milk and/or signs of inflammation in the gland. Farmers were required to report all cases of clinical mastitis and collect information on affected ewes’ demographics, clinical features, treatments (where applicable), and outcomes. Milk samples from mastitic glands were submitted for microbiological culture and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF). Results Partial or complete clinical mastitis data were available for 236 cases from 221 ewes on 18/20 study farms. Clinical mastitis was diagnosed in 0–6% of ewes at the farm level, with an overall incidence of 1.8 (95% CI = 1.0–3.2)% using the study data, or 2.3 (95% CI = 1.6–3.3)% using the study data and farmer estimates that included unreported cases. Cases occurred mostly in early lactation, with 59% detected during the lambing period (August–October), at a median of 7 (IQR 3, 40) days in milk. The majority of cases featured clots in the milk (59%), swelling (55%), and unevenness (71%) of the glands. Pyrexia (rectal temperature ≥ 40.0°C) was diagnosed in 25% of cases and depression (lethargy, inappetence, or inability to stand) in 26% of cases. Treatment was given to 46% of cases, with tylosin being the most commonly used treatment (50% of treated cases). The most common outcome was immediate drying off to be culled without treatment (32%), followed by still milking and recovered but with lasting problems (25%). Nearly half of all the milk samples submitted were culture negative. Streptococcus uberis (14%), non-aureus staphylococci (12%), and Staphylococcus aureus (11%) were the most common isolates, found on 12, 8 and 8 of the 16 farms with microbiological data, respectively. Conclusions Clinical mastitis affected up to 6% of ewes at the farm level. Systemic signs were observed in one quarter of affected ewes, suggesting a role for supportive treatment. Clinical mastitis can be severe and challenging to fully resolve in New Zealand dairy sheep. Clinical relevance This is the first systematic study of clinical mastitis in New Zealand dairy ewes. It provides baseline information specific to New Zealand conditions for farmers, veterinarians, and other advisors to guide the management of mastitis for the relatively new dairy sheep industry in New Zealand.

Utility of a multiplex pathogen detection system directly from respiratory specimens for treatment and diagnostic stewardship.

The availability of syndrome-based panels for various ailments has widened the scope of diagnostics in many clinical settings. These panels can detect a multitude of pathogens responsible for a particular condition, which can lead to a timely diagnosis and better treatment outcomes. In contrast to traditional identification methods based on pathogen growth on culture, syndrome-based panels offer a quicker diagnosis, which can be especially beneficial in situations requiring urgent care, such as intensive care units. One such panel is the Biofire Filmarray Pneumonia plus Panel (BFP), which we have compared against microbiological culture and identification. The lower respiratory samples from patients were tested with BFP, culture, and identification with culture considered the gold standard. The phenotypic antibiotic susceptibility results (Vitek 2) were compared with the antimicrobial resistance (AMR) genes detected in BFP. Statistical analysis was carried out using GraphPad 7.0 and MS Excel (Microsoft Inc.). The results showed a positive percent agreement of 100% and a negative percent agreement of 47.8% with an overall agreement of 76.72% compared to culture. BFP was better at identifying fastidious bacteria, and the agreement with culture was higher for high bacterial identification numbers (107 and 106). There was also a correlation between the number of pathogens detected and growth in culture. Carbapenemase genes were detected in around 80% of phenotypically resistant samples and correlated with in-house PCR 60% of the time. Hence, BFP results need to be interpreted with caution especially when multiple pathogens are detected. Similarly, the presence or absence of AMR genes should be used to guide the therapy while being watchful of unusual resistance or susceptibility. The cost constraints and low throughput call for patient selection criteria and prioritization in emergency or resource-limited conditions.IMPORTANCEApplication of syndrome-based panels in clinical microbiology is of huge support in infectious conditions requiring urgent interventions, such as pneumonia. Interpreting the results requires caution; hence, we have compared the results obtained from Biofire Filmarray Pneumonia plus Panel with standard microbiological methods.