16S PCR Research Articles

Environmental Fungal Contamination Characterization of Three Inpatient Units Utilizing Optimized Detection Methods

Background: Environmental sampling and detection methods for fungi in healthcare settings are not well-established. We previously refined methods for fungal sampling and detection in a controlled laboratory environment and aimed to validate them in a real-world healthcare setting. Methods: We performed a microbiological analysis of air and surfaces in three inpatient units at a tertiary care center. Surface samples were obtained with foam sponges from 3 locations in patient rooms (Patient bedrails, bathroom floor, HVAC export) and 5 locations in units (HVAC exports 3x, clean linen storage, soiled linen storage). Air samples were taken with an active air sampler directly below HVAC exports. Sponges were processed using the stomacher technique. Samples underwent DNA extraction followed by qPCR with FungiQuant primers targeting the 18S rRNA gene. Amplicons from positive samples were sequenced (NextSeq 1000, 300bp PE) and SmartGene databases were used to interpret sequence data. For comparison to culture methods, samples were also plated onto Sabouraud and HardyCHROM Candida + auris medias. Fungal growth underwent DNA extraction, 18S PCR and Sanger sequencing for genus and species identification. Results: A total of 85 samples were obtained, from 15 patient rooms and three units resulting in 61 surface and 24 air samples. Patients in study rooms had a median age of 53, 9 (60%) were male, and no patients had an invasive fungal infection during their hospital encounter. 44 (53%) and 39 (46%) samples were positive for fungi via qPCR and culture, respectively. Of the 44 positive qPCR samples, microbiome analyses identified at least one fungi to the species, genus and family levels in 43 (98%), 28 (64%), 18 (41%) samples, respectively (Table 1). 114 total isolates were identified of which the most common were Mallassezia restricta (30 [26%]), Malassezia globose (29 [25%]), and Pennicillium paradoxum (4 [4%]). 39 genera were identified of which the most common were Mucor (19 [49%]) and Candida (8 [21%]). Of the 39 culture positive samples, 90 total isolates were recovered. The most common species were Paradendryphiella arenariae (19 [21%]), Aspergillus niger (12 [13%]) and Penicillium commune (12 [13%]). Conclusion: These results demonstrate the presence of diverse fungal species in both air and surface samples across inpatient units. Higher sensitivity was noted utilizing qPCR, however, identified genera and species were markedly different between qPCR and culture methods. Larger studies are needed to assess the efficacy of qPCR for fungal detection in the healthcare environment.

Synovial fluid alpha-defensins in Lyme arthritis-a useful marker.

Lyme arthritis, one of the possible late manifestations of Lyme borreliosis, predominantly affects the supporting joints and in adults most often occurs in the form of monoarthritis of the knee. Early diagnosis is based on clinical findings and serology. PCR detection of Borrelia in synovial fluid has become an integral part of the laboratory testing algorithm. The clinical presentation and inflammatory markers in Lyme arthritis can resemble septic arthritis. Determining the levels of alpha-defensins (human neutrophil peptide (HNP 1-3)) in synovial fluid by liquid chromatography is a highly sensitive method revealing the presence of inflammatory process. Between 2020 and 2022, we examined eleven patients with Lyme arthritis of the knee. We measured levels of HNP 1-3 from synovial fluid by HPLC in patients, and we compared it with the corresponding C-reactive protein (CRP) levels in paired serum samples. In patients diagnosed with Lyme arthritis, HNP 1-3 levels in synovial fluid ranged from 2.5 to 261mg/L, with a median of 46.5mg/L. Average serum CRP was 43mg/L. The results show that elevated HNP 1-3 can be consistent with not only septic arthritis or systemic disease, but also with Lyme arthritis, especially in patients with negative culture and 16S PCR from synovial fluid. Final diagnosis must be verified by examination for anti-Borrelia antibodies from serum and synovial fluid. The aim of this work is to introduce an HPLC method for the determination of alpha-defensins as one of the possible diagnostic markers.

ROLE OF THE MOLECULAR TESTS FOR PATIENTS WITH INFECTIVE ENDOCARDITIS UNDERGOING CARDIAC SURGERY: AN EXPANDING HORIZON

Abstract Background Molecular tests have been proven to be a highly sensible tool to identify the pathogen of infective endocarditis (IE). However, their usefulness in the management of both blood culture positive and valve culture negative IE remains unclear. Methods We prospectively enrolled 100 consecutive patients with IE who underwent cardiac surgery between April 2020 and June 2023. Molecular testing (broad–range PCR 16S rRNA) was systematically performed and results were collected together with those of preoperative blood culture, valve culture and histopathological analysis of the specimen. Results Blood culture, valve culture and molecular testing were positive in 83%, 47% and 76% of patients, respectively. Both the sensitivity of valve culture and 16S rRNA PCR decreased significantly with increasing duration of preoperative antibiotic therapy. In 7% of cases, molecular test was the only positive test, allowing an etiological diagnosis. In 33% of cases, valve culture was negative but molecular test was positive. In the majority of these patients, histopathological analysis documented acute inflammation. Finally, molecular test clarified discrepancies between the results of blood cultures and those of valve cultures in 12.5% of patients. Conclusions The molecular tests showed high sensitivity. Although it decreased as the duration of preoperative antibiotic therapy increased, it was still higher than that of valve culture 28 days after the start of treatment. In addition to patients without an etiological diagnosis, the molecular test proved useful in other circumstances: 1) when the valve culture was negative because, together to the results of the histopathological analysis, it allowed to identify those patients who could benefit from a prolongation of antibiotic treatment; 2) in cases of discordance between the results of blood cultures and those of valve cultures, because it allowed the type of antibiotic treatment to be directed. Figure 1: Usefulness of molecular tests (PCR) according to the results of blood and valve cultures. Figure 2: Valve cultures are of paramount importance in the management of antibiotic therapy (ABT). However, their sensibility is low and the risk of under–treatment exists. Molecular tests may help targeting the duration of ABT but they could be persistently positive leading to an over–treatment. Figure 3: Flow–chart showing the possible role of molecular test in the management of patients with infective endocarditis.

296 Effects of dietary antimicrobials on the ruminant gastrointestinal tract microbiome

Abstract Monensin, tylosin, and chlortetracycline (CTC) are antimicrobials frequently employed in high grain feedlot cattle diets to inhibit liver abscesses and enhance growth. However, increased public concerns regarding antibiotic resistance have created a pressing need to develop alternative strategies. A more mechanistic understanding of how antimicrobial drugs effect the microbiome in the ruminant gastrointestinal tract (GIT) may help develop effective alternatives. Therefore, the objective of this study was to determine changes in the GIT microbiome of grain-fed feedlot steers after antimicrobial drug administration. Thirty individually housed Angus x Simmental steers [body weight (BW) = 355 ± 16.8 kg] previously fed a 56% forage diet were immediately transitioned to a 93% concentrate diet and assigned to one of three treatments (10 steers/treatment): 1) control, without antibiotics, 2) 75 mg of tylosin and 200 mg of monensin per animal per day (TYLMON), and 3) 70 mg of CTC per animal per day. Fecal samples were collected on d 0, 7, 14, 28, 56, 112, and at slaughter and digesta were obtained from the rumen, jejunum, and cecum at slaughter. DNA was extracted from samples, amplified via 16S PCR, and sequenced. Jejunal samples had the least alpha diversity compared with ruminal, cecal, or fecal samples (P < 0.001). There were no differences in measures of alpha diversity in the ruminal, jejunal, or cecal digesta at slaughter (P ≥ 0.10). However, overall measures of alpha diversity (Shannon entropy, observed features) in feces were greater in CTC-fed steers (P ≤ 0.009) as well as in TYLMON-fed steers (P ≤ 0.04) compared with control steers. Measures of alpha diversity for all treatments decreased in feces from d 0 to 7, then increased from d 7 to slaughter (time effect, (P ≤ 0.001). There were no differences in fecal alpha diversity between treatment groups on d 0 (P ≥ 0.50), but by d 28 both TYLMON and CTC treated steers had greater fecal alpha diversity compared with the control steers (P ≤ 0.05). With regard to differential abundance, Phascolarctobacterium and Prevotellaceae UCG-003 were more abundant in feces of control steers relative to antimicrobial treated steers (P < 0.05). Bifidobacterium, Dorea, CAG-56, and Erysipelotrichaceae UCG-003, were the 4 genera found to be in the greatest abundance in the feces of TYLMON-fed relative to control steers (P < 0.05). Akkermansia, Succinivibrio, and Turicbacter were the 3 genera found to be in the greatest abundance in the feces of CTC-fed compared with control steers (P < 0.05). Taken together, these data suggest that differences in the microbiome associated with dietary antimicrobial treatment occur to a greater extent in the lower sections of the GIT, and that these changes in the microbiome occur after 28 d of feeding.

First Report of Bacterial Blight of Pennycress Caused by Xanthomonas campestris in Wisconsin.

In May 2023, pennycress (Thlaspi arvense, L.) lines undergoing seed production in the Walnut Street Greenhouse at the University of Wisconsin-Madison displayed symptoms of chlorosis and black necrotic leaf spots (Fig. S1-A). Lesions eventually enlarged to 1-2 cm in diameter, became necrotic, and coalesced to cover a substantial portion of leaves. Symptoms were observed in ~30% of the pennycress lines adversely affecting overall growth and reproduction. Symptomatic leaves were surface sterilized for 30 seconds in 0.75% sodium hypochlorite, rinsed in sterile deionized water, and bacteria were isolated using three-phase streaking of symptomatic tissue onto KB medium (King et al., 1954). Single colonies of three isolates (creamy white to yellow) from this initial isolation were streaked onto KB medium to obtain pure cultures. Individual colonies were transferred for growth overnight in nutrient broth (Difco) and an equal amount of the broth was added to 30% glycerol in deionized (di) water and stored at -80 °C. To validate Koch's Postulates, bacteria were grown from these stocks on Yeast Dextrose Calcium Carbonate medium (Wilson et al., 1967) and were used to inoculate 5-week-old pennycress plants in the greenhouse. The bacteria were grown for 48 hours at 26°C, suspended in 300 ml of 0.05 M PBS buffer (pH=7.2) for inoculum preparation. Plants were inoculated with three bacterial isolates (approx. 108 CFU/ml) by piercing the mid veins or hydathodes with a sterilized toothpick dipped in the suspension. Inoculated plants were then enclosed in clear plastic bags for 24-48 hours and maintained in the greenhouse at a constant temperature of 26°C with a 16-hour photoperiod. After seven days, water-soaked lesions appeared on the inoculated leaves, eventually developing into the characteristic black spots (Fig. S1-B). DNA from the original isolates was extracted, and 16S PCR and sequencing of the positive bands was done. The negative control only produced brown spots at the site of inoculation (Fig. S1-C). The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT (Eden et al., 1991; Weisburg et al., 1991). A BLAST analysis showed that the isolates had an E value of 0.0 to the genus Xanthomonas as well as 100% identity. Amplification and sequencing of the bacterium using gyrB amplicons revealed a 99-100% pairwise match with Xc. To enhance taxonomy resolution and confirm the identity of these isolates, the complete genomes of three samples were sequenced using NextSeq2000 Illumina platform (NCBI bioproject ID PRJNA1040293). Average Nucleotide Identity (ANI) analysis was conducted with representative strains from the Xc species (Dubrow et al., 2022), using PanExplorer (Dereeper et al., 2020) featuring integrated FastANI module (Jain et al., 2018). The isolates genomes exhibited over 98% identity and clustered with that of Xc pv. incanae and Xc pv. barbarae (Fig S2). Further work will be required to identify the pathovar of Xc identified in this study through phenotypic host range assay. This marks the first documented case of Xc in pennycress in the Midwestern US. Given the potential use of pennycress as a cover crop in the region, further investigations are warranted to assess its economic impact on production and develop management strategies.

Diagnosis of external ventricular drainage related infections with real-time 16S PCR and third-generation 16S sequencing

Objective Investigate the performance of real-time 16S PCR and third-generation 16S sequencing in the diagnosis of external ventricular drain related infections (EVDRI). Methods Subjects with suspected EVDRI were prospectively included at Uppsala University Hospital. Subjects were included into three groups: subjects with negative CSF culture with and without antibiotic treatment and subjects with positive CSF culture, respectively. CSF was analysed with real-time 16S PCR and third-generation 16S sequencing. Real-time 16S PCR positivity/negativity and number of 16S sequence reads were compared between groups. For culture positive subjects, species identification in third-generation sequencing and routine culture was compared. Results 84 subjects were included. There were 18, 44 and 22 subjects in the three groups. Real-time PCR was positive in 17 of 22 subjects in the culture positive group and negative in 61 of the 62 subjects in the two culture negative groups. The sensitivity and specificity for real-time 16S PCR compared to culture was estimated to 77% and 98%, respectively. Species identification in 16S sequencing and culture was concordant in 20 of 22 subjects. The number of 16S sequence reads were significantly higher in the culture positive group than in both culture negative groups (p < 0.001). There was no significant difference in number of 16S sequences between the two culture negative groups. Conclusions Real-time 16S PCR predict culture results with sufficient reliability. Third-generation 16S sequencing could enhance sensitivity and species identification in diagnostics of EVD-related infections. False negative culture results appear to be uncommon in patients with suspected EVDRI.

Microbiota in adult perianal abscess revealed by metagenomic next-generation sequencing.

The microbiota of perianal abscesses is scarcely investigated. Identifying causative bacteria is essential to develop antibiotic therapy. However, culture-based methods and molecular diagnostics through 16S PCR technology are often hampered by the polymicrobial nature of perianal abscesses. We sought to characterize the microbiota composition of perianal abscesses via metagenomic next-generation sequencing (mNGS). Fourteen patients suffering from perianal abscesses between March 2023 and August 2023 underwent retrospective assessment. Information from medical records was used, including clinical information, laboratory data, and culture and mNGS results. Forty bacterial taxa were identified from perianal abscesses through mNGS, with Bilophila wadsworthia (71.4%), Bacteroides fragilis (57.1%), and Escherichia coli (50.0%) representing the most prevalent species. mNGS identified an increased number of bacterial taxa, with an average of 6.1 compared to a traditional culture-based method which only detected an average of 1.1 in culture-positive perianal abscess patients, predominantly E. coli (75.0%), revealing the polymicrobial nature of perianal abscesses. Our study demonstrates that a more diverse bacterial profile is detected by mNGS in perianal abscesses, and that Bilophila wadsworthia is the most prevalent microorganism, potentially serving as a potential biomarker for perianal abscess.IMPORTANCEAccurately, identifying the bacteria causing perianal abscesses is crucial for effective antibiotic therapy. However, traditional culture-based methods and 16S PCR technology often struggle with the polymicrobial nature of these abscesses. This study employed metagenomic next-generation sequencing (mNGS) to comprehensively analyze the microbiota composition. Results revealed 40 bacterial taxa, with Bilophila wadsworthia (71.4%), Bacteroides fragilis (57.1%), and Escherichia coli (50.0%) being the most prevalent species. Compared to the culture-based approach, mNGS detected a significantly higher number of bacterial taxa (average 6.1 vs 1.1), highlighting the complex nature of perianal abscesses. Notably, Bilophila wadsworthia emerged as a potential biomarker for these abscesses. This research emphasizes the importance of mNGS in understanding perianal abscesses and suggests its potential for improving diagnostic accuracy and guiding targeted antibiotic therapy in the future.

Amplifying PCR productivity and environmental sustainability through shortened cycling protocols

Since its inception in the 1980s, advancements in PCR technology using improved thermal cyclers, engineered DNA polymerases and commercial master mixes, have led to increased PCR productivity. Despite these advancements, PCR cycling protocols have largely remained unchanged over the same period. This study aimed to systemically evaluate the effect of reduced PCR cycling parameters on amplicon production. The 1466bp fragment from the 16S rRNA gene present in low-, medium- and high-CG bacteria was amplified using three commercially available PCR master mixes. The shortest cycling parameters required to successfully amplify the 16S fragment from all bacteria and master mixes comprised 30-cycles of 5 s denaturation, 25 s annealing, and 25 s extension. While all produced an amplicon with sufficient yield to enable downstream sequence analysis, the PCRBIO Ultra Mix in conjunction with the shortened parameters was found to achieve the highest amplicon yield across low-, medium- and high CG bacteria. Comparing the run times to that of a typical 16S PCR protocol, the shortened cycling parameters reduced the program duration by 46 % and consumed 50 % less electricity, translating into increased productivity and helping to improve laboratory environmental sustainability.

Fewer culturable Lactobacillaceae species identified in faecal samples of pigs performing manipulative behaviour

Manipulative behaviour that consists of touching or close contact with ears or tails of pen mates is common in pigs and can become damaging. Manipulative behaviour was analysed from video recordings of 45-day-old pigs, and 15 manipulator-control pairs (n = 30) were formed. Controls neither received nor performed manipulative behaviour. Rectal faecal samples of manipulators and controls were compared. 16S PCR was used to identify Lactobacillaceae species and 16S amplicon sequencing to determine faecal microbiota composition. Seven culturable Lactobacillaceae species were identified in control pigs and four in manipulator pigs. Manipulators (p = 0.02) and females (p = 0.005) expressed higher Lactobacillus amylovorus, and a significant interaction was seen (sex * status: p = 0.005) with this sex difference being more marked in controls. Females (p = 0.08) and manipulator pigs (p = 0.07) tended to express higher total Lactobacillaceae. A tendency for an interaction was seen in Limosilactobacillus reuteri (sex * status: p = 0.09). Results suggest a link between observed low diversity in Lactobacillaceae and the development of manipulative behaviour.

LATE DIAGNOSIS OF Cutibacterium acnes (FORMERLY Propionibacterium acnes ) ENDOPHTHALMITIS AND THE IMPORTANCE OF DUAL TESTING WITH BACTERIAL CULTURE AND POLYMERASE CHAIN REACTION.

The purpose of this study was to describe two cases of Cutibacterium acnes endophthalmitis that reinforce the importance of performing both bacterial culture and 16S polymerase chain reaction when the causative pathogen is unclear or difficult to culture, such as C. acnes . A case of C. acnes endophthalmitis complicated by subbuckle scleral perforation is illustrated with intraoperative photography. This is a two-case series. Case 1 describes a case of C. acnes endophthalmitis in a longstanding pseudophakic patient after multiple vitrectomies for recurrent retinal detachment, complicated by subbuckle scleral perforation. Bacterial culture revealed C. acnes while 16S PCR was negative. Conversely, Case 2 demonstrates a case of chronic endophthalmitis diagnosed one year after cataract surgery. PCR (with repeat tap for confirmation) revealed C. acnes with a negative culture. When the causative pathogen of endophthalmitis is unclear, dual testing of microbial culture and C. acnes 16S PCR improves the diagnostic yield of investigations for fastidious pathogens. C. acnes can present as an indolent or virulent endophthalmitis.

Microbiological profile of peri-implantitis: Analyses of peri-implant microbiome.

To characterize the microbiome composition in peri-implant pocket of peri-implantitis and peri-implant sulcus controls using 16S rRNA gene sequencing. In this controlled clinical cross-sectional study, 23 subjects with control implants (n = 14) and diseased implants (peri-implantitis, n = 21) were included. The peri-implant pocket/sulcus was sampled and used to extract DNA and amplify the 16S rRNA gene using universal primers targeting the V3-V4 regions. The resulting 16S PCR amplicons were sequenced on Illumina MiSeq, and the sequences were processed using DADA2 and the Human Oral Microbiome Database (HOMD) as references. Alpha and Beta diversity, as well as core microbiome and differential abundance analyses, were performed using the MicrobiomeAnalyst workflow. There were no significant differences in microbial diversity between control implants and implants with peri-implantitis (Shannon p = 0.82). Overall bacterial community structure assessed through beta diversity analysis was also not significantly different between the two groups (p = 0.18). However, high levels of Gram-negative bacteria were detected in peri-implant pockets compared to the control sulcus. Abundant species in peri-implantitis were Capnocytophaga leadbetteri, Treponema maltophilum, Peptostreptococcus, Neisseria, P. gingivalis, and Porphyromonas endodontali, Lactococcus lactis and Filifactor alocis (p < 0.05). Gram-positive bacteria such as Streptococcus salivaris, Prevotella melaninogenica, L. wadei, and Actinomyces spp. serve were more abundant in peri-implant control sulcus. Peri-implant sulcus in control implants harbors predominantly Gram-positive bacteria, whereas pockets of implants with peri-implantitis harbor predominantly Gram-negative bacteria.

Bacterial microbiota management in free-living amoebae (Heterolobosea lineage) isolated from water: The impact of amoebae identity, grazing conditions, and passage number

Free-living amoebae (FLA) are ubiquitous protozoa mainly found in aquatic environments. They are well-known reservoirs and vectors for the transmission of amoeba-resistant bacteria (ARB), most of which are pathogenic to humans. Yet, the natural bacterial microbiota associated with FLA remains largely unknown. Herein, we characterized the natural bacterial microbiota of different FLA species isolated from recreational waters in Guadeloupe.Monoxenic cultures of Naegleria australiensis, Naegleria sp. WTP3, Paravahlkampfia ustiana and Vahlkampfia sp. AK-2007 (Heterolobosea lineage) were cultivated under different grazing conditions, during successive passages. The whole bacterial microbiota of the waters and the amoebal cysts was characterized using 16S rRNA gene metabarcoding. The culturable subset of ARB was analyzed by mass spectrometry (MALDI-TOF MS), conventional 16S PCR, and disk diffusion method (to assess bacterial antibiotic resistance). Transmission electron microscopy was used to locate the ARB inside the amoebae.According to alpha and beta-diversity analyses, FLA bacterial microbiota were significantly different from the ones of their habitat. While Vogesella and Aquabacterium genera were detected in water, the most common ARB belonged to Pseudomonas, Bosea, and Escherichia/Shigella genera. The different FLA species showed both temporary and permanent associations with differentially bacterial taxa, suggesting host specificity. These associations depend on the number of passages and grazing conditions. Additionally, Naegleria, Vahlkampfia and Paravahlkampfia cysts were shown to naturally harbor viable bacteria of the Acinetobacter, Escherichia, Enterobacter, Pseudomonas and Microbacterium genera, all being pathogenic to humans. To our knowledge, this is the first time Paravahlkampfia and Vahlkampfia have been demonstrated as hosts of pathogenic ARB in water.Globally, the persistence of these ARB inside resistant cysts represents a potential health risk. To ensure the continued safety of recreational waters, it is crucial to (i) regularly control both the amoebae and their ARB and (ii) improve knowledge on amoebae-bacteria interactions to establish better water management protocols.

Molecular Identification of Marine Symbiont Bacteria of Holothuria scabra from Tanjung Tiram Beach and its Potential as a Methicilin-Resistant Staphylococcus aureus Antibacterial agent

The resistance of Staphylococcus aureus may occur to many types of antibiotics, especially in the cases of noncompliance in using antibiotics, which is likely to allow the evolution of Multi-Drug Resistant (MDR) bacteria. Holothuria scabra are marine invertebrates that were reported being capable of producing secondary metabolites as antibacterial MDR. The purpose of the study was to identify the Holothuria scabra symbiont bacteria found in Tanjung Tiram, southern Konawe, South Sulewesi, which has the ability to produce antibacterial compounds against MRSA (Methicillin-Resistant Staphylococcus aureus). The stages of this research began with the isolation of bacteria, antibacterial activity test, DNA extraction, amplification by PCR 16S rDNA and sequencing. The results of the study showed that 9 isolates of bacteria were isolated from Holothuria scabra, those are HS1, HS2, HS3, HS4, HS5, HS6, HS7, HS8 and HS9. Among them, 2 isolates showed highest activity against MRSA test bacteria. The highest activities were displayed by HS2 and HS3 isolates code with the largest inhibition zone were 35mm and 36mm. The molecular identification of HS2 isolate has 72% homology with Streptomyces albus, while HS3 isolate has 97% homology with Streptomyces albus.

First report of Enterobacter sp. causing bacterial wilt on patchouli in Aceh, Indonesia

Abstract. Zulfadli, Wasistha NI, Oktarina H, Khairan, Sriwati R. 2023. First report of Enterobacter sp. causing bacterial wilt on patchouli in Aceh, Indonesia. Biodiversitas 24: 3814-3820. Patchouli (Pogostemon cablin Benth), is an essential commodity in Indonesia, with Aceh Jaya being one of its main producing regions. Several incidences of wilt disease symptoms have been reported in patchouli plantations in Ranto Sabon, Sampoianet Sub-district, Aceh Jaya District. This study aimed to isolate and identified the bacterial pathogen responsible for wilt disease in patchouli in Aceh Jaya. Various tests, including morphology characterization and molecular characteristics using 16s PCR primer, hypersensitive test, and pathogenicity test were conducted to identify the cause of disease. The isolation result revealed that Enterobacter sp. was responsible for invading the patchouli plant. Further testing was carried out on various culture media including Nutrient Broth, Tryptic Soy, Kings B, Sulfide Indole Motility, Sucrose Potato Agar, and Yeast Potato Agar. The highest colony density was found in Tryptic Soy media (2.002) after 120 hours of inoculation. The results of hypersensitivity and pathogenicity tests showed that Enterobacter sp. caused symptoms similar to those of natural infection. Among all the media, Sucrose Potato Agar media had the fastest incubation period, with 7 and 48 days after inoculation. Therefore, this is the first study to report the presence of Enterobacter sp. associated with bacterial wilt in patchouli in Aceh.

P.147 Exploring the bacterial hypothesis of low back pain: a prospective cohort study

Background: Occult bacterial infection is a proposed etiology of low back pain (LBP). However, a causative link between LBP and bacteria remains unconfirmed. Herein, we determined the incidence of occult discitis in patients receiving surgery for LDH. Methods: Study Design: prospective cohort study. Inclusion criteria: consecutive adult patients undergoing discectomy for symptomatic LDH. Exclusion criteria: prior epidural steroid use, prior spinal surgery, and antibiotic use within 2 weeks of surgery. Tissue samples: Four nuclear tissue and ligamentum flavum (control) samples were obtained per patient using stringent aseptic protocol. Samples underwent 16S-PCR and culturing. Results: Eighty-one patients were enrolled (mean age 43.3±13.3 years). All (100%) of tissue samples were negative by 16S PCR and no virulent species were detected. Nuclear and ligament cultures were both negative in 51 (62.9%) cases. Cultures were positive for nuclear tissue only, ligament only, or both in 14.8%, 12.3%, and 9.9% of cases, respectively. Fifteen of 20 (75%) disc positive samples grew a single colony of an indolent species. Conclusions: The findings of this prospective cohort study of consecutive patients receiving surgery for LDH do not support the theory of occult discitis. All samples were 16S-PCR negative, and most cultures were negative or grew a single colony suggestive of contamination.

First Report of 'Candidatus Phytoplasma solani' (16SrXII-A) Associated with Fox Nut (Euryale ferox Salisb.) Stolbur Disease in India.

Fox nut also known as Gorgon nut, Makhana (Euryale ferox Salisb.) is a high value aquatic crop belonging to the family Nymphaeaceae. In India, it is generally grown in flood prone areas of North Bihar, lower Assam, parts of West Bengal, Odisha, Manipur and Tripura (Jana et al., 2018). India contributes nearly 70-80% of the global fox nut production. During September 2021, a phytoplasma like symptom was noticed on fox nut leaves at Basudeopur Farm of Research Centre for Makhana, Darbhanga, Bihar, India (23° 9' N and 65° 53' E). The characteristic symptom was that some portion of leaf lamina deformed along the veins with wrinkled and raised overgrowth or hypertrophy. The veins were thickened and reddened in the infected leaf area. The infection occurs in petiole as well as in flower stalk. The disease incidence was found as high as 30% which caused severe yield loss which was calculated to be 40% in that particular field. Total of 20 sampled fox nut plants, 10 symptomatic and 10 asymptomatic ones, were collected and tested for the presence of phytoplasma. A nested PCR assay using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (J. Jović et al. 2011) amplified the expected ~1.2-kb 16S rDNA fragment in all 10 symptomatic samples. No amplification was detected from asymptomatic samples. One of the ten 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced and was deposited in NCBI under the Accession no.OL873590. BLAST analysis showed that the sequence of the PCR 16S rDNA product was 100% identical to several GenBank sequences of Ca. P. solani (16SrXII Stolbur group) viz. KF907506. Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup. Identification of 'Ca. P. solani' was conducted following the STOL11 stolbur-specific protocol (Radonjić et al. 2009). Sequencing of tuf gene (Elongation factor Tu) was performed by using tuf marker genes (Cvrković et al. 2014) from 10 symptomatic and 10 asymptomatic samples. Sequence of the amplified gene (896 bp) was deposited in GenBank under Accession number OM174272. The presence of 'Ca. P. solani' was detected in all symptomatic samples, while all control plants tested negative. The RFLP analysis of tuf gene nested PCR products using HpaII endonuclease (Fermentas) revealed uniform tuf-b type in all positive samples. Nucleotide blast analyses showed that the tuf gene was 100% identical to STOL11 strain of C. P. solani subgroup 16SrXII-A (Accession No JQ797670). For developing a suitable management strategy, identification of the vector is essential. Leaf hoppers visiting the infected plants as well as nearby crop fields will be tested for presence of the phytoplasma. To the best of our knowledge, this is the first report of Candidatus Phytoplasma solani' (16SrXII-A) infecting Fox nut (Euryale ferox Salisb.) in India. References Cvrković et al. 2014. Plant Pathol. 63:42. https://doi.org/10.1111/ppa.12080 Jana, B. R., et al. 2018. Int. J. Curr. Microbiol. App. Sci. 7(12): 578-587. https://doi.org/10.20546/ijcmas.2018.712.072 Jović, J. et al. 2011. B. Insectol. 64:S83. ISI Radonjić, S. et al. 2009. J. Phytopathol. 157:682. https://doi.org/10.1111/j.1439- 0434.2009.01560.

Direct and culture-enriched 16S rRNA sequencing of cecal content of healthy horses and horses with typhlocolitis.

Next generation sequencing has demonstrated that alpha diversity of the fecal microbiota is significantly altered in horses with typhlocolitis. The objective of this study was to evaluate the bacterial composition of the cecum content of horses with and without typhlocolitis through direct and culture-enriched 16S gene sequencing of six healthy horses and six horses with acute typhlocolitis; a case-control study design. Cecal content was collected after euthanasia. An aliquot was used for direct 16S gene sequencing. Another was serially diluted with brain heart infusion (BHI) and plated onto five different agar media. All culture medias, except for MacConkey, were incubated anaerobically. Bacterial colonies were harvested in bulk and used for DNA extraction, 16S PCR amplification, and sequenced using the Illumina MiSeq platform. Predominant phyla in healthy and diseased horses were Firmicutes, followed by Bacteroidetes in all cultured medias, except for MacConkey agar, in which Proteobacteria was the dominant phylum. Greater bacterial richness was identified in sequenced cecal contents as compared to cultured plates (P < 0.05). Culture-enriched molecular profiling combined with 16S rRNA gene sequencing offer an alternative method for the study of the gut microbiota of horses. For direct cecum content 16S gene amplification, the alpha diversity indices were lower in diarrheic horses compared to healthy horses (P < 0.05). A higher relative abundance of Fusobacteriota was found in 2/6 samples from diarrheic horses. The role of Fusobacteriota in equine colitis deserves investigation.

Detection of Babesia conradae in Coyotes (Canis latrans) and Coyote-Hunting Greyhound Dogs (Canis familiaris).

Babesia conradae is a small piroplasm previously detected in coyote-hunting Greyhound dogs in California and Oklahoma. In dogs, B. conradae causes clinical signs similar to other tick-borne illnesses, and if not treated it can lead to acute kidney injury and other life-threating complications. To date, the life cycle of this apicomplexan parasite has not been fully described, but suggestions of direct contact or tick transmission have been proposed. The purpose of this study was to test coyote tissue samples from coyotes hunted by Greyhound dogs with a history of B. conradae infection to determine if this parasite is present in the coyote population in Northwestern Oklahoma. The analyzed tissue samples included liver, lung and tongue samples collected by hunters. DNA was isolated from these tissues and assessed by RT-PCR of the 18S rRNA and PCR of the COX1 genes for B. conradae. A total of 66 dogs and 38 coyotes were tested, and the results demonstrated the presence of B. conradae DNA in 21 dogs (31.8%) and 4 coyotes (10.5%). These results indicate that B. conradae is present in the dog and coyote population from the same area and that direct contact with coyotes may increase the risk of infection in dogs. Further studies are required to test possible modes of transmission, including direct bite, tick or vertical transmission.

Comparative analysis of diagnostic methods for the detection of Cryptococcus neoformans meningitis.

Cryptococcosis is a devastating opportunistic infection in immunocompromised individuals, primarily in people living with HIV/AIDS. This study evaluated a protocol for the early diagnosis of meningitis due to C. neoformans, utilizing established molecular techniques from serum and CSF samples. The 18S and 5.8S (rDNA-ITS) sequence-specific nested PCR assays were compared with direct India ink staining and the latex agglutination test for detection of C. neoformans in serum and cerebrospinal fluid (CSF) from 49 Brazilian suspected meningitis patients. Results were validated with samples obtained from 10 patients negative for cryptococcosis and HIV, and by analysis of standard C. neoformans strains. The 5.8S DNA-ITS PCR was more sensitive (89-100%) and specific (100%) than the 18S rDNA PCR and conventional tests (India ink staining and latex agglutination) for identification of C. neoformans. While the 18S PCR exhibited a sensitivity (72%) similar to that of the latex agglutination assay in serum samples, it was superior to the latex agglutination assay when testing CSF, with a sensitivity of 84%. However, the latex agglutination was superior to the 18SrDNA PCR in specificity in CSF (92%). The 5.8S DNA-ITS PCR yielded the highest levels of accuracy (96-100%) of any test for detection (serological and mycological) of C. neoformans in both serum and CSF. Use of the nested 5.8S PCR was superior to other techniques for the diagnosis of cryptococcosis. The possibility of using serum, a non-invasively collected material, in a targeted 5.8S PCR analysis to identify Cryptococcus spp. is recommended, especially in immunosuppressed patients. Our results indicate that nested 5.8S PCR can increase the diagnostic capability of cryptococcosis, and we suggest its use to monitor patients in the future.

Prevalence of three Mycoplasma sp. by multiplex PCR in cattle with and without respiratory disease in central Mexico.

This study aimed to identify Mycoplasma bovis, Myc. dispar, and Myc. bovirhinis, which are involved in bovine respiratory disease through a multiplex PCR as an alternative to culture's features that hamper Mycoplasma isolation. Nasal swabs were taken from 335 cattle with and without respiratory disease background (RDB) from dairy herds in the central region of Mexico. Each sample was divided in two; the first part was processed for the direct DNA extraction of the nasal swab and the second for Mycoplasma isolation, culture, and then the multiplex PCR was performed. In the nasal swabs, Myc. bovis was identified in 21.1%; Myc. dispar, in 11.8%; and Myc. bovirhinis, in 10.8% in cattle with RDB. Isolates were identified as Myc. bovis, 20.1%; Myc. dispar, 11.8%; and Myc. bovirhinis, 6.1%. There is a strong correlation between the presence of Mycoplasma identified by PCR and the clinical history of the disease (ρ < 0.0000). In animals without RDB, Myc. bovirhinis was the only species detected in 6.1% of the samples processed directly for multiplex PCR, and in 2% of the isolates. There is an excellent correlation (kappa 0.803) between the isolation and the 16S PCR and a high correlation (kappa 0.75) between the isolation and the multiplex PCR. Therefore, we conclude that the PCR multiplex test is highly sensitive and may be used for the diagnosis and surveillance of the three species in biological samples and mycoplasma isolates.