Columbia Blood Agar Research Articles

Genomic characterization of Pantoea dispersa A003 isolated from a clinical patient

IntroductionPantoea dispersa is a Gram-negative bacterium generally considered as a plant pathogen and rarely causes human infections. To date, there have been 13 studies that have documented clinical infections linked to P. dispersa, with a primary emphasis on the initial identification of this pathogen. The genomic features and the mechanisms underlying the pathogenesis of P. dispersa remain largely uninvestigated. In the present study, we describe a clinical infection caused by P. dispersa and provide the first genomic analysis of this bacterium.MethodsThe bacterial strain designated A003 was isolated from blood samples. Preliminary identification of strain A003 was conducted using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) technology (VITEK® MS, bioMérieux, France). Biochemical and antimicrobial susceptibility assessments were carried out utilizing the VITEK-2 compact automated microbial analysis system (bioMérieux, France). Additionally, whole genome sequencing and subsequent analysis were performed to further elucidate the potential pathogenicity.ResultsThe bacterial isolate was cultured overnight at 35°C in a CO2-enriched environment on Columbia blood agar, resulting in the appearance of a white, smooth, Gram-negative rod-shaped bacterium with diameters ranging from 1 to 2 mm. Identification of strain A003 was achieved with a high confidence level of 99.9% as P. dispersa using MALDI-TOF mass spectrometry. The average nucleotide identity (ANI) value between strain A003 and the reference strain P. dispersa DSM 30073T was 98.08%, while the DNA–DNA hybridization (DDH) value was 84.10% (Formula 2) based on genome sequencing data, which further confirmed that A003 belonged to the P. dispersa species. Comprehensive analysis revealed the presence of 372 virulence factors, 15 antibiotic resistance genes, 222 carbohydrate-active enzymes, and 666 genes related to Type III secretion system (T3SS) effector proteins, as identified through the core databases of VFDB (Virulence Factors of Pathogenic Bacteria), CARD (Comprehensive Antibiotic Resistance Database), CAZY (Carbohydrate-Active enZYmes Database), and T3SS. The virulence factors included type IV pili (TFP), type VI secretion system, flagella, iron uptake system, and ompA, which are implicated in bacterial pathogenicity. The antibiotic resistance profile indicated resistance to fluoroquinolones, cephalosporins, penams, macrolides, and aminoglycosides, as annotated by the CARD database.ConclusionThe draft genome sequenced represents the inaugural genomic sequence of P. dispersa derived from clinical sources. This advancement may enhance clinical practitioners’ comprehension of the organism’s clinical attributes and serve as a foundational resource for future research into its virulence, antibiotic resistance, and host–pathogen interactions.

Incidence of erysipelas in waterfowl in Poland – clinical and pathological investigations

ABSTRACT 1. Erysipelas in geese and ducks is an infectious disease causing considerable economic losses in the poultry sector in Poland. The aim of this study was to determine the distribution of erysipelas cases in age groups of geese and in periods of the calendar year and to assess macroscopic lesions in birds infected with Erysipelothrix rhusiopathiae. 2. The study used 286 geese and 16 ducks delivered to the laboratory for diagnosis in the years 2019–2021. The birds came from 99 different farms. E. rhusiopathiae from clinical samples were cultured on Columbia blood agar and identified by real-time PCR. Pathological lesions were characterised during anatomopathological examination. 3. Distribution of erysipelas cases in age groups of geese and periods of the calendar year differed significantly from the assumed even distribution. Most cases were noted in August and September in birds aged between 11 and 13 weeks. Necropsy of birds infected with E. rhusiopathiae showed anatomopathological lesions characteristic of generalised infection, affecting multiple organs. 4. The uneven distribution of erysipelas cases in geese in different age groups and periods of the calendar year suggested that both the age of the birds and climatic conditions are predisposing factors for infection.

Characterization of Metronidazole, Clarithromycin and Amoxicillin Resistance Genes in Helicobacter pylori Isolated from Gastroenteritis Patients.

Helicobacter pylori (H. pylori)is a Gram-negative, microaerophilic, and spiral shape bacterium that resides inside the human stomach. The human stomach serves as its primary reservoir. Complaints about stomach complication due to H. pylori infections are reported in the majority of populations around the globe. Chronic gastritis and intestinal metaplasia of the gastric mucosa are major complications of a long-term H. pylori infections that can lead to gastric cancer in severe cases. This study aims to characterize H. pylori isolates from gastroenteritis patients and to determine the resistance of H. pylori to various antibiotics. In the current study, a total of (n = 80) gastric biopsy samples were randomly collected from gastroenteritis patients in brain-heart infusion broth. These were inoculated on Columbia blood agar supplemented with Helicobacter pylori selective supplement (DENT). After culturing, Microscopy and biochemical tests were performed. The susceptibility profile of H. pylori isolates was evaluated using the Kirby Bauer disk diffusion method. On the basis of the drug resistance profile, a total of (n = 20) isolates including (n = 10) from females and (n = 10) from males were selected for the detection and characterization of resistant genes. After confirmation of H. pylori using 16s rRNA, polymerase chain reaction (PCR) was done for the detection of resistance genes including Metronidazole resistance (rdxA gene), Clarithromycin resistance (23s rRNA gene) and Amoxicillin resistance (Penicillin-binding protein A1 (pbpA1) gene). In a total of (n = 80) samples, H. pylori was isolated from 72.5% (n = 58) samples. Among the positive patients, there were 62% (n = 36) of female positive patients while in males, its ratio was 38% (n = 22). It was more common in the age between 30-50 years 55.17% (n = 32). It has shown the highest resistance towards Metronidazole 90% (n = 52), and the lowest toward Levofloxacin 65% (n = 38). Metronidazole resistance gene (rdxA gene) was detected in (n = 13) isolates including (n = 9) isolates from females and (n = 4) from males. In the case of, the Clarithromycin resistance gene (23s rRNA) (n = 10) was positive for H. pylori including (n = 6) isolates from females and (n = 7) were positive for Amoxicillin (pbpA1 gene) including (n = 2) in female and (n = 5) from male patients. This study highlights the increasing incidence of H. pylori infections in both male and female patients. It also revealed the current status of antibiotic resistance and its resistance genes in patients facing gastrointestinal issues. Continuous surveillance of resistant clones will help in formulating strategies that can help in combating of resistant clone. It will also help clinician in proper prescription and management of H. pylori infections.

Advancements in Helicobacter pylori: A Novel Culture AA-Medium for Helicobacter pylori Detection

This research provides crucial insights into the detection and culturing of Helicobacter pylori, a significant pathogen associated with gastric diseases. New culture media were prepared as X1, X2 and X3 in comparison with Columbia blood agar. Culturing success varied between Columbia blood agar and the novel X1-media which named as AA-media, while it failed on X2-media and X3-media. 48.8% of the positive samples were successfully cultured on Columbia blood agar and AA-media. Remarkably, colonies on AA-media displayed larger and distinct features within the first 24 hours, a stage achieved on Columbia blood agar after 48 hours. This research underscores the importance of considering alternative culture media to improve culture processing and obtaining rapid and distinctive results for the accurate diagnosis of Helicobacter pylori-associated diseases.

Isolation and Identification of Morganella morganii from Rhesus Monkey (Macaca mulatta) in China

A bacterium was isolated and identified from the secretion of a rhesus monkey with endometritis. The morphological results showed that the strain exhibited round, convex, gray-white colonies with smooth surfaces and diameters ranging from 1 to 2 mm when cultured on Columbia blood agar at 37 °C for 24 h; on salmonella–shigella agar (S.S.) at 37 °C for 24 h, the colonies appeared round, flat, and translucent. Gram staining showed negative results with blunt ends and non-spore-forming characteristics. Molecular biology results showed that the 16S rRNA sequence of the strain revealed over 96.9% similarity with published sequences of M. morganii from different sources in the NCBI GenBank database. Morphological and molecular biology analysis confirmed that the strain (RM2023) isolated from cervical secretions of rhesus monkey was M. morganii. Drug sensitivity testing demonstrated that the isolated strain (RM2023) was sensitive to ceftriaxone, amikacin, gentamicin, cefazolin, cefuroxime, ceftazidime, levofloxacin, cotrimoxazole, norfloxacin, and tetracycline; moderately sensitive to ampicillin; and resistant to penicillin, vancomycin, ciprofloxacin, and clindamycin. The research findings provide valuable insights for disease prevention in rhesus monkeys and contribute to molecular epidemiological studies.

Trends in viable microbial bioburden on surfaces within a paediatric bone marrow transplant unit

Despite their role being historically overlooked, environmental surfaces have been shown to play a key role in the transmission of pathogens causative of healthcare-associated infection. To guide infection prevention and control (IPC) interventions and inform clinical risk assessments, more needs to be known about microbial surface bioburdens. To identify the trends in culturable bacterial contamination across communal touch sites over time in a hospital setting. Swab samples were collected over nine weeks from 22 communal touch sites in a paediatric bone marrow transplant unit. Samples were cultured on Columbia blood agar and aerobic colony counts (ACC) per 100cm2 were established for each site. Individual colony morphologies were grouped and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or 16s rDNA sequencing. Highest mean counts were observed for sites associated with ward management activity and computer devices (3.29 and 2.97 ACC/100cm2 respectively). A nurses' station keyboard had high mean ACC/100cm2 counts (10.67) and diversity, while laundry controls had high mean ACC/100cm2 counts (4.70) and low diversity. Micrococcus luteus was identified in all sampling groups. Clinical staff usage sites were contaminated with similar proportions of skin and environmental flora (52.19-46.59% respectively), but sites associated with parental activities were predominantly contaminated by environmental microflora (86.53%). The trends observed suggest patterns in microbial loading based on site activities, surface types and user groups. Improved understanding of environmental surface contamination could help support results interpretation and IPC interventions, improving patient safety.

Comparison of culture-dependent and -independent bacterial detection results on nasal swabs in dogs with nasal discharge.

The role of bacterial communities in the pathophysiology of canine nasal disease is still unclear. How and when to treat dogs with suspected secondary bacterial rhinitis and on which test to rely before making a decision to treat with antimicrobials has not been established. The objective is to compare the results of bacterial identification using agar-plate cultures and 16S rRNA gene amplicon sequencing in dogs with nasal discharge suspected to be of bacterial origin. Twenty-nine client-owned dogs presented for investigation of nasal disease were included in the study. Paired swabs were collected from the same affected nasal cavity. One swab was streaked on 4 agar media (Columbia Blood Agar, MacConkey, Chapman and Edward's). The other swab was stored in a sterile cryotube at -80°. Extracted DNA underwent a polymerase chain reaction targeting the V1-V3 region of the 16S rRNA gene. At least one of the species detected by amplicon sequencing with a relative abundance of >10% was also identified by culture in 14 cases (48.3%), in association with marked predominance of one taxon (>80% relative abundance) in six of 14 cases. In 12 dogs (41.4%), the cultured isolates were rare or undetected components of the corresponding sequence libraries. A negative culture in the face of bacterial predominance (>50% relative abundance) of a potentially pathogenic bacteria detected by sequencing occurred in 17% (n=5) of cases; however, the use of other agar media may have decreased this percentage. Standard culture does not reliably predict the bacterial profile detected by 16S rRNA gene amplicon sequencing.

COMPARISON OF CULTIVATION TESTS TO DETECTION MASTITIS IN DAIRY COWS

Mastitis is an inflammation of the mammary gland which has a particular importance in ruminants. The purpose of this study was to evaluate and compare two methods for cultivation of udder pathogens: classical laboratory cultivation on Columbia agar with 5% sheep blood versus MicroMast rapid plates. The results allowed assessing the incidence of mastitis and the prevalence of the pathogens. During the study have been investigated 227 cows in a dairy farm localized in the east of Slovakia. Subsequently, 141 quarter milk samples from the positive cows with California mastitis test score 1-4 have been undergone the laboratory culture on Columbia agar and MicroMast test in accordance with their respective steps. The values obtained from these tests showing sensitivity of positive samples using the MicroMast test at the level of 64.5%, and sensitivity of cultures on Columbia blood agar at the level of 61.7%. After biochemical identification of cultured isolates, the Columbia blood agar and MicroMast test identified both as the main pathogen present Staphylococcus aureus (S. aureus) with results of 13.4% and 14.9%, respectively. On the base of results, both tests are comparable and therefore a test that speeds up testing is more useful in practice. It will be a relevant importance in the following decades to succeed in the development of tests with directly detection of udder pathogens.

Moraxella haemolytica sp. nov., isolated from a goat with respiratory disease.

An aerobic, haemolytic, Gram-negative and rod-shaped bacterial strain ZY171148T was isolated from the lung of a dead goat with respiratory disease in Southwest China. The strain grew at 24-39°C, at pH 6.0-9.0 and in the presence of 0.5-2.0% (w/v) NaCl. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain belongs to the genus Moraxella. The nucleotide sequence similarity analysis of the 16S rRNA gene showed that the strain has the highest similarity of 98.1% to Moraxella (M.) caprae ATCC 700019T. Phylogenomic analysis of 800 single-copy protein sequences indicated that the strain is a member of the genus Moraxella and forms a separated branch on the Moraxella phylogenetic tree. The strain exhibited the highest orthologous average nucleotide identity (OrthoANI) and average amino acid identity (AAI) values of 77.0 and 77.9% to M. nasibovis CCUG 75921T and M. ovis CCUG 354T, respectively. The strain shared the highest digital DNA-DNA hybridization (dDDH) value of 26.2% to M. osloensis CCUG 350T. The genome G + C content of strain ZY171148T was 42.6mol%. The strain had C18:1 ω9c (41.7%), C18:0 (11.2%), C16:0 (14.1%) and C12:0 3OH (9.7%) as the predominant fatty acids and CoQ-8 as the major respiratory quinone. The strain contained phosphatidylglycerol, phosphatidylethanolamine, cardiolipin, dilysocardiolipin, monolysocardiolipin and phosphatidic acid as the major polar lipids. β-haemolysis was observed on Columbia blood agar. All results confirmed that strain ZY171148T represents a novel species of the genus Moraxella, for which the name Moraxella haemolytica sp. nov. is proposed, with strain ZY171148T = CCTCC AB 2021471T = CCUG 75920T as the type strain.

Surgical helmet systems in total joint arthroplasty: assessment of hood sterility and donning technique

BackgroundThe incidence of prosthetic joint infection (PJI) is increasing, coincident with the rising volume of joint arthroplasty being performed. With recent controversy regarding the efficacy of surgical helmet systems (SHS) in preventing infection, the focus has turned to the correct donning techniques and usage of surgical hoods. The aim of this study was to compare the bacterial contamination of the operating surgeon’s gloves after two common donning techniques of SHS hoods. We also evaluated the baseline sterility of the SHS hoods at the beginning of the procedure.MethodsThe bacterial contamination rate was quantified using colony-forming units (CFUs), with 50 trials performed per donning technique. Samples were cultured on 5% Columbia blood agar in ambient air at 37 °C for 48 h and all subsequent bacterial growth was identified using a MALDI-TOF mass spectrometer. In Group 1, the operating surgeon donned their colleague’s hood. In Group 2, the operating surgeon had their hood applied by a non-scrubbed colleague. After each trial, the operating surgeon immediately inoculated their gloves onto an agar plate. The immediate sterility of 50 SHS hoods was assessed at two separate zones—the screen (Zone 1) and the neckline (Zone 2).ResultsThere was no significant difference in contamination rates between the two techniques (3% vs. 2%, P = 0.99) or between right and left glove contamination rates. Immediately after donning, 6/50 (12%) of SHS hoods cultured an organism. Contamination rates at both the face shield and neckline zones were equivalent. The majority of bacteria cultured were Bacillus species.DiscussionWe found no significant difference in the operating surgeon’s glove contamination using two common SHS hood-donning techniques when they were performed under laminar airflow with late fan activation. We suggest the SHS hood should not be assumed to be completely sterile and that gloves are changed if it is touched intraoperatively.

168 Characterization of the Culturable Fraction of Bovine Ocular Microbiota and Its in Vitro Antimicrobial Activities Against Pinkeye Pathogens

Abstract Pinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is considered one of the most important ocular diseases in cattle, causing an estimated $226 million in annual losses to U.S. cow-calf producers. While Moraxella bovis is considered the principal IBK pathogen, Moraxella bovoculi has also been associated with the pathogenesis of IBK. Thus, these Moraxella species are often the target for antibiotic and/or vaccine-based treatment and prevention strategies. Despite extensive use of antibiotics, IBK infections are challenging to treat, highlighting the increased need for an antibiotic-alternative approach to control IBK. Recent evidence suggests that the microbial community associated with the bovine oculus is important for eye health, and that the ocular microbiota can be a target for enhancing disease resistance against IBK in cattle. The objectives of this study were to characterize the ocular microbiota of healthy and IBK infected cattle using sequencing and culturing techniques, and to isolate and screen commensal ocular isolates for their antimicrobial activity against M. bovis and M. bovoculi. Ocular swabs (n = 64) were collected using eSwabs from cattle diagnosed with IBK or without IBK (healthy control) before antibiotic treatment from beef herds across North Dakota. Swabs were plated onto four different agar plates (blood, Columbia blood, MRS and multi-sliced agar plates), and cultured aerobically and anaerobically. A total of 658 bacterial isolates were obtained via culturing and cryopreserved. Taxonomic identification was performed on a subset of banked isolates (n = 277) by near full-length sequencing of the 16S rRNA gene. These isolates taxonomically belong to 6 different bacterial phyla: Bacillus (42%), Firmicutes (27%), Actinomycetota (13%), Pseudomonadota (11%), Actinobacteria (5%), and Proteobacteria (2%). A total of 55 different genera were identified with Bacillus (25%), Streptococcus (13%), Staphylococcus (10%), Moraxella (6%), Macrococcus (5%), Paenibacillus (3%), Caldibacillus (3%), and Rummeliibacillus (3%) being the most dominant genera. Next, a subset of these commensal isolates (n 53) were screened for their growth inhibition against M. bovis and M. bovoculi using an agar slab method. Among the screened isolates, 6 isolates showed inhibition against M. bovis and M. bovoculi with zones of inhibition ranging from 12 mm to 28 mm. Ocular isolates identified as Weizmannia coagulans (43Y-MRSC), Lentillactobacillus buchneri (23D-MRSA) and Paenibacillus polymyxa (785-WCF) exhibited relatively strong inhibition against both Moraxella isolates. Scanning electron microscopy revealed noticeable structural and morphological damages in M. bovis and M. bovoculi cells due to Weizmannia coagulans culture-supernatant treatment. Overall, our results indicate that ocular mucosa of beef cattle is inhabited by relatively rich and diverse culturable bacterial communities, and that some ocular commensals can inhibit IBK pathogens. This suggests the potential for the development of bacterial therapeutics based on commensal ocular bacteria to mitigate IBK in cattle.

Corynebacterium lipophilum sp. nov., a lipophilic bacterium isolated from clinical breast specimens and emended description of the species Corynebacterium pilbarense.

Two isolates (MC-18T and MC-17D), representing the Gram-stain-positive, facultatively anaerobic, irregular rod-shaped, non-motile, and non-spore-forming actinobacteria, were isolated from clinical breast specimens in Guangzhou, China. The growth of the isolates is enhanced by supplementing 1% Tween-80 on Luria Bertani agar. Optimal growth of the isolates was observed at 37°C, pH 7-8, and with 1% (w/v) NaCl on Columbia blood agar. Pairwise comparison of the 16S rRNA gene sequences revealed that isolates MC-18T and MC-17D shared the highest sequence similarities with Corynebacterium liangguodongii 2184T (96.9%), which were lower than the threshold value for species delineation (98.65%). Phylogenetic dendrograms based on the 16S rRNA gene, rpoB gene, and core genomes indicated that two isolates formed a distinct lineage within the genus Corynebacterium. The estimated dDDH, ANIb, ANIm, and AAI values between strain MC-18T and its closely related strains were below the threshold values generally considered for recognizing a new species. The genome DNA G + C contents of both the isolates MC-18T and MC-17D are 60.6%. The two isolates have virulence-related genes of the VF classes of adhesion and antiphagocytosis, and also contain the antimicrobial resistance genes ErmX, mtrA, rpoB2, and RbpA. The major fatty acids (> 10%) of isolates MC-18T and MC-17D were C16:0, C18:1 ω9c, C18:0 and summed feature 5 (anteiso-C18:0 and/or C18:2 ω6,9c). The main respiratory quinone of strain MC-18T was MK-8(H2), and the polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, three unidentified glycolipids, an unidentified aminolipid, and four unidentified phosphoglycolipids. The two isolates lack mycolic acids in the cell envelope. Based on the above findings, the two isolates are considered to represent a novel species of the genus Corynebacterium, for which the name Corynebacterium lipophilum sp. nov. is proposed, with isolate MC-18T (= NBRC 115144T = CCTCC AB 2020201T) as the type strain. An emended description of the Corynebacterium pilbarense is also provided.

The effects of sodium sulfite on Helicobacter pylori by establishing a hypoxic environment

Helicobacter pylori (H. pylori) is an obligate microaerobion and does not survive in low oxygen. Sodium sulfite (SS) reacts and consume oxygen in solutions. The present study aimed to investigate the effects of SS on H. pylori. The effects of SS on oxygen concentrations in solutions and on H. pylori in vivo and in vitro were examined, and the mechanisms involved were explored. The results showed that SS decreased the oxygen concentration in water and artificial gastric juice. In Columbia blood agar and special peptone broth, SS concentration-dependently inhibited the proliferation of H. pylori ATCC43504 and Sydney strain-1 in Columbia blood agar or special peptone broth, and dose-dependently decreased the number of H. pylori in Mongolian gerbils and Kunming mouse infection models. The H. pylori was relapsed in 2 weeks withdrawal and the recurrence in the SS group was lower than that in the positive triple drug group. These effects were superior to positive triple drugs. After SS treatments, the cell membrane and cytoplasm structure of H. pylori were disrupted. SS-induced oxygen-free environment initially blocked aerobic respiration, triggered oxidative stress, disturbed energy production. In conclusion, SS consumes oxygen and creates an oxygen-free environment in which H. pylori does not survive. The present study provides a new strategy and perspective for the clinical treatment of H. pylori infectious disease.

2024. Human Factor Considerations in Support of a Flexible IV Connector Safeguard to Prevent Central Line Associated Blood Stream Infections (CLABSI)

Abstract Background One of the sources of central line infections is contaminated and poorly disinfected IV connectors(CON). A recent study found 23% of IV-line CONs were contaminated with bacteria, despite strict infection control guidelines that include the use of alcohol-lined caps (ALC). Contamination (CONT) occurs after protective ALC has been removed in preparation for an infusion, and nurses perform their multiple tasks. These include purging air bubbles from the IV line, setting pumps, verifying medications, and keeping an eye on the patient. It is not surprising that nurses admitted that sterilization of IV CONs was not prior to 3% - 4% of infusions. Schielded IV connector Methods A flexible rounded shield extending above a standard needleless CON to protect it from CONT was designed and tested (Pat pend). A total of 4, 2 ICU Medical Inc. needleless CONs, one shielded and one unshielded, and two Komashine Inc. CONs, also one shielded and one unshielded were disinfected with 70% alcohol and their lines taped to the abdomen of 2 volunteers wearing loose shirts over the connectors. The CONs remained on the volunteers for 3 hours, in 2 sessions. All 16 CON ends were cultured on Columbia blood agar, and plates held 48 h. Readings were evaluated by statistical analysis of 16 predictions (coinflips). Results It was confirmed that the tested shielded CONs were easy to sterilize, connect to infusion lines and that there was ample space left to screw on and off alcohol caps. Colonies were absent from the protected connector ends, whereas all the unshielded ends grew several colonies. The probability of observing the projected efficacy results, 8 unshielded with subsequent bacterial growth, and eight shielded, remaining sterile, i.e. not growing bacteria is < 0.00002. The lack of contamination on the shielded ends proved that the air currents generated by the movements of the volunteers, between their skin and their skirts did not transport bacteria to the shielded connector ends. Conclusion A properly designed flexible contamination shield will prevent the proportion of line infections that are caused by human factors. Pls, submit severe adverse event reports (prolongation of hospitalization or death) caused by contemporary "defective" IV connectors to the FDA to change their faulty design. Disclosures All Authors: No reported disclosures.

Determinants of Infection as Genetic Indicators in Cows Mastitis

The resistance to antimicrobial substances severely impacts public health and the abuse of antibiotics leads to antimicrobial resistance (AMR) or the antibiotic "resistome" (Wright, 2007). Bovine mastitis is largely diagnosed in dairy farms and is caused by a variety of pathogens including Streptococcus spp., Staphylococcus spp. and Escherichia coli. AMR gene expression testing in bacteria involved in mastitis in dairy cows was performed. Milk samples were subjected to the California Mastitis Test. Positive samples were transferred using eSwab, cultured on Columbia blood agar and on MacConkey agar. The Qiagen DNeasy kit was used for DNA extraction and qPCRs were run using an Agilent thermocycler. In most of the samples tested (n = 42, from three different lactating farms), the presence of ampC (36 out of 42; 85.7%) and blaZ (95.2%), correlated with confirmed resistance to beta-lactam and cephalosporin antibiotics. A variable presence of other tested AMR genes was detected, including ermB, resistance to lincosamide, macrolide (35.7%), ermC (28.6%), erythromycin resistance, mecA,methicillinresistance (42.9%), and tetK, tetracyclineresistance (78.6%). The phenomenon of antimicrobial resistance is present in dairy farms in West Romania. Multiple AMR genes were detected intested samples, with the highest resistance observed to beta-lactam antibiotics and cephalosporins.

S2.5a Magnusiomyces spp.

S2.5 Rare yeasts, September 21, 2022, 3:00 PM - 4:30 PM Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and the determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available. We, therefore, determined the epidemiology and susceptibility of Magnusiomyces isolates from bloodstream infections (BSI) isolated in Germany and Austria from 2001 to 2020. A total of 34 Magnusiomyces BSI were analyzed; isolates were identified by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antifungal susceptibility was determined by EUCAST broth microdilution and gradient tests. Of the 34 isolates, M. clavatus was more common (n = 24) than M. capitatus (n = 10). BSI by Magnusiomyces spp. were more common in men (62%) and mostly occurred in patients with hemato-oncological malignancies (79%). The highest in vitro antifungal activity against M. clavatus/M. capitatus was observed for voriconazole (MIC50, 0.03/0.125 mg/l), followed by posaconazole (MIC50, 0.125/0.25 mg/l). M. clavatus isolates showed overall lower MICs than M. capitatus. With the exception of amphotericin B, a low essential agreement between gradient test and microdilution was recorded for all antifungals (0%-70%). Both species showed distinct morphologic traits on ChromAgar Orientation medium and Columbia blood agar, which can be used for differentiation if no MALDI-TOF MS or molecular identification is available. In conclusion, most BSI were caused by M. clavatus. The lowest MICs were recorded for voriconazole. Gradient tests demonstrated unacceptably low agreement and should preferably not be used for susceptibility testing of Magnusiomyces spp.

Epidemiological Evaluation of Associated Risk Factors of Helicobacter pylori Infection Using Noninvasive Methods: A Case Study of Peptic Ulcer Patients in Lafia, Nigeria.

Background: Due to its persistent nature, ulcers brought on by Helicobacter pylori have been a significant public health concern. This study looked at how specific risk factors affected the prevalence of Helicobacter pylori infection among peptic ulcer patients visiting medical institutions in Lafia, Nigeria. Methods: The blood and stool samples of 180 individuals (71 men and 109 women) were collected randomly, checked for H. pylori using test strips for H. pylori antibodies and antigens (Azure Biotech Inc.), and the feces also were grown on Columbia blood agar base (TITAN Biotech Ltd). Using a standardized questionnaire, some participant risk data was also gathered.Results: A positive culture method (CM) test result was obtained from 14/71 (19.72%) of the 71 male patients and 37/109 (33.94%) of the 109 female subjects out of a total of 210 subjects. For CM alone, it was discovered that female participants had a considerably greater incidence of Helicobacter pylori infection than their male counterparts (p=0.038). Nevertheless, all analytical techniques discovered no evidence of a significant difference between age groups (p>0.05). Only the blood antibody (BAB) approach showed a substantially greater prevalence (p=0.021) in married patients, with 79/116 (68.10%) reactive instances. The presence of drinking water source (p < 0.001, 0.001, 0.002 using CM, BAB, and SAG, respectively) and number of occupants per room (p < 0.001, 0.001, 0.002 using CM, BAB, and SAG, respectively) as potential risk factors for H. pylori infection was also demonstrated.Conclusion: The majority of risk factors that were taken into consideration for this study demonstrated a strong correlation with Helicobacter pylori infection in Lafia, Nigeria.

A simple method for the isolation of cold-tolerant Clostridium spp. from meat samples

Pure isolates of Clostridium spp. are required when further investigations are needed, such as the characterisation of the colonies, sporulation and germination, biochemical analysis, growth rate and growth conditions, toxin production, spoilage potential and genomic profile. However, the isolation of cold-tolerant Clostridium spp. from suspicious meat samples is challenging due to their slow growth rates and overgrowth with less fastidious meat microbiota. Therefore, this study aimed to establish a practical method for the isolation of psychrophilic and psychrotolerant Clostridium spp. Primarily, meat drip samples (n = 3), enriched in Peptone Yeast Glucose Starch (PYGS) broth, were heated with different temperatures and durations, before they were subcultured on Columbia blood agar (CBA). The treatment procedure of heating samples at 80 °C for 5 min was evaluated as effective and was further validated using pure cultures of lactic acid bacteria (n = 5), bacteria belonging to the family of Enterobacteriaceae (n = 5), and cold-tolerant Clostridium spp. (n = 34). Almost all other meat microbiota was inactivated by heating at 80 °C for 5 min, while 28 strains of cold-tolerant Clostridium spp. survived. Finally, the treatment procedure was applied with drip of meat samples (n = 41) previously tested positive for 49 cold-tolerant Clostridium spp. using specific multiplex qPCR. All meat drip samples were enriched in PYGS broth by incubating them at 4 °C for 3 weeks, to ensure growth of Clostridium spp. before the enrichments were proceeded to heat treatment and to culturing on CBA. A total of 35 (71 %) from 49 Clostridium spp. strains were isolated using this culturing procedure. The accuracy of the recovery rates obtained from two replicates was 68 %. The method of detection and isolation applied in this study is easy and resulted in a high isolation rate of cold-tolerant Clostridium spp.

In vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure) against polymicrobial communities and individual bacterial strains derived from diabetic foot infections.

Introduction. Diabetic foot infection (DFI) is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. DFIs are often complicated by ischaemia in the affected limb, the presence of polymicrobial biofilms and increasingly the occurrence of antibiotic resistant bacteria.Hypothesis/Gap statement. Antibiotic loaded beads could inhibit the growth of polymicrobial DFI communities with differing compositions in vitro.Aim. This study investigates the in vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure, Biocomposites Ltd., UK) against polymicrobial DFI communities and individual bacterial strains derived from DFIs.Methodology. Debrided tissue obtained from the base of infected diabetic foot ulcers was homogenised and spread over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates. Calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar and following incubation, zones of inhibition (ZOI) were measured. For individual bacterial strains isolated from the infected tissue, calcium sulfate beads containing vancomycin, gentamicin, flucloxacillin or rifampicin and beads containing a combination of vancomycin and gentamicin or flucloxacillin and rifampicin were tested for their ability to inhibit growth.Results. Calcium sulfate beads loaded with a combination of vancomycin and gentamicin were able to inhibit bacterial growth from all polymicrobial tissue homogenates tested, with ZOI diameters ranging from 15 to 40 mm. In the case of individual bacterial strains, beads containing combinations of vancomycin and gentamicin or flucloxacillin and rifampicin were able to produce ZOI with Gram-positive facultatitive anaerobic strains such as Staphylococcus aureus and Enterococcus faecalis, Gram-negative facultative anaerobic strains such as Pseudomonas aeruginosa and obligate anaerobic strains such as Finegoldia magna even where acquired resistance to one of the antibiotics in the combination was evidenced.Conclusion. The local use of calcium sulfate beads containing a combination of two antibiotics demonstrated high efficacy against polymicrobial DFI communities and individual DFI bacterial strains in in vitro zone of inhibition tests. These results show promise for clinical application, but further research and clinical studies are required.

Identification of a novel drug-resistant community-acquired Nocardia spp. in a patient with bronchiectasis

ABSTRACT A previously unknown Nocardia species was isolated from the lung tissue and bronchoalveolar lavage fluid (BALF) of a 58-year-old woman with bronchiectasis and recurrent pneumonia. This Nocardia (GZ2020T), which grew readily in Columbia blood agar and could induce pneumonia in a mouse model, represents a novel Nocardia species, and its closest known relatives are Nocardia anaemiae NBRC 100462T, Nocardia pseudovaccinii NBRC 100343T and Nocardia vinacea NBRC 16497T. However, unlike all previously known species, GZ2020T is the first genus of Nocardia spp. that is not susceptible to multiple drugs but does show susceptibility to linezolid and moxifloxacin, and thus, GZ2020T potentially represents a substantial health threat to patients with bronchiectasis and immunocompromised individuals. Although the original pathogen source and method of spreading remain uncertain, a mode of transmission from the environment to humans could exist. Vigilance with respect to its spread in the population and the transmission of antibiotic resistance genes in the environment should be maintained.