Culture Isolates Research Articles

Genetic diversity of Mycobacterium tuberculosis isolates from the central, eastern and southeastern Ethiopia

IntroductionThe population structure of Mycobacterium tuberculosis complex (MTBC) in Ethiopia is diverse but dominated by Euro-American (Lineage 4) and East-African-Indian (Lineage 3) lineages. The objective of this study was to describe the genetic diversity of MTBC isolates in Central, Eastern and Southeastern Ethiopia. MethodsA total of 223 MTBC culture isolates obtained from patients referred to Adama and Harar TB reference laboratories were spoligotyped. Demographic and clinical characteristics were collected. ResultsSix major lineages: Euro-American (Lineage 4), East-African-Indian (Lineage 3), East Asian (Lineage 2), Indo-Oceanic (Lineage 1), Mycobacterium africanum (Lineage 5 and Lineage 6) and Ethiopian (Lineage 7) were identified. The majority (94.6 %) of the isolates were Euro-American and East-African-Indian, with proportions of 75.3 % and 19.3 %, respectively. Overall, 77 different spoligotype patterns were identified of which 42 were registered in the SITVIT2 database. Of these, 27 spoligotypes were unique, while 15 were clustered with 2–49 isolates. SIT149/T3_ETH (n = 49), SIT53/T1 (n = 33), SIT21/CAS1_Kili (n = 24) and SIT41/Turkey (n = 11) were the dominant spoligotypes. A rare Beijing spoligotype pattern, SIT541, has also been identified in Eastern Ethiopia. The overall clustering rate of sub-lineages with known SIT was 71.3 %. Age group (25–34) was significantly associated with clustering. ConclusionWe found a heterogeneous population structure of MTBC dominated by T and CAS families, and the Euro-American lineage. The identification of the Beijing strain, particularly the rare SIT541 spoligotype in Eastern Ethiopia, warrants a heightened surveillance plan, as little is known about this genotype. A large-scale investigation utilizing a tool with superior discriminatory power, such as whole genome sequencing, is necessary to gain a thorough understanding of the genetic diversity of MTBC in the nation, which would help direct the overall control efforts.

Molecular Patterns of Resistance in Carbapenem Resistant Organisms Detected in Stool Surveillance Cultures in Patients Undergoing Hematopoietic Cell Transplant

Introduction Patients undergoing hematopoietic stem cell transplant (HSCT) are at high risk of life-threatening bacterial infections. Although the prevalence and pattern of resistance varies amongst centres and countries, there is a growing global problem of resistance to antibiotics. The increased use of broad-spectrum antibiotics and injudicious antibiotic policies have contributed to the selection of multidrug resistant(MDR) pathogens. Translocation of organisms which normally colonize the gut into the bloodstream during the febrile neutropenia has long been postulated as a pathogenic factor for life-threatening gram-negative infections. We routinely do stool surveillance culture of all patients admitted for autologous and allogenic stem cell transplant to guide us on empirical antibiotic usage. This analysis was conducted to evaluate the incidence of carbapenem resistant organisms (CROs) and their molecular pattern of resistance in stool surveillance cultures. Methods This is a single centre retrospective analysis of patients who underwent autologous and allogenic stem cell transplant for haematological malignancies from December 2021 to January 2023. In all patients stool surveillance cultures were sent at baseline during admission and then weekly thereafter till discharge . A blood Biofire syndromic multiplex PCR was performed in a fraction of stool isolates to identify the gene responsible for carbapenem resistance. Biofire syndromic multiplex PCR was performed only once for one patient on the first stool culture that grew a CRO. Biofire is a multiplex PCR that identifies Gram negative organisms including Acinetobacter Baumanni, Bacteroides Fragilis, Enterobacter cloacae, E. coli, Klebsiella, Proteus, Salmonella, Serratia, H. influenza, Neisseria meningitides, Pseudomonas aeruginosa, Stenotrophomonas maltophilia; it also identifies the gene responsible for resistance. The Carbapenem resistant genes included in the Biofire syndromic multiplex PCR were IMP Carbapenemase, KPC Carbapenamase, OXA-48, NDM, VIM, CTXM. Results Seventy-one patients underwent HSCT from December 2021 to January 2023. These included 39 allogenic and 32 autologous stem cell transplants. The median age of the cohort was 33 years (range 3-63 years) with 48 males and 23 females. The diagnoses were AML(n=8), ALL(n=14), CML(n=14), HL(n=10), Multiple Myeloma(n=19), Myelofibrosis(n=1), Non-Hodgkin Lymphoma (n=5). A total of 430 stool surveillance cultures from 71 patients were analysed. Of 430, 90 (20.9%) stool samples in 30 (42%) patients grew carbapenem resistant organism. Of these 90 CROs, twenty-three stool culture isolates belonging to 23 different patients were analysed for genotypic resistance. These 23 CROs included Escherichia coli 12 (52%) and Klebsiella pneumonia 11 (48%). On phenotypic sensitivity testing, the CROs were sensitive to Ceftriaxone-Sulbactam-EDTA (23/23, 100%), Ceftazidime-Avibactam (2/15, 13%), Colistin (23/23, 100%), Aztreonam (3/23, 13%), Tigecycline (17/23, 74%), Minocycline (9/23, 39%) and Aminoglycosides (11/23, 48%) shown in Figure 1a Resistance gene panel in these 23 stool isolates revealed NDM alone in 7 (31%), CTX-M along with NDM in 9 (39%), CTX-M along with OXA 48 like in 2(9%), CTX-M, OXA 48 along with NDM in 4 (17%) and NDM with OXA 48 in 1 (4%) (Figure 1b). Of the remaining 67 stools in which Biofire was not performed, Escherichia coli grew in 31 (46%) and Klebsiella pneumonia grew in 35 (52%) and Pseudomonas Aeruginosa in 1 (2%). On drug sensitivity testing, these CROs were sensitive to Ceftriaxone-Sulbactam-EDTA (65/67, 97%), Ceftazidime-Avibactam (4/15, 26%), Colistin (65/67, 97%), Aztreonam (7/65, 11%), Tigecycline (39/57, 68%), Minocycline (24 /62, 39%) and Aminoglycosides (33/67, 49%). Conclusion: About 40% of our patients were colonized with CROs at some point during their transplant. Molecular patterns identified NDM alone or in combination with other resistance genes in more than 90% of stools where molecular testing was performed. The antibiotics to which these MDR organisms were most susceptible included Ceftriaxone-sulbactam-EDTA, Colistin and tigecycline. Our study suggests that NDM is the most common resistance gene responsible for carbapenem resistance in our patients. Thus, antibiotics or antibiotic combinations that target NDM should be considered early in patients with MDR sepsis.

2732. Peri-Operative Antibiotic Management for Lung Transplant Recipients with Cystic Fibrosis

Abstract Background Lung transplant (LTx) recipients universally receive peri- and post-transplant antibiotics. Approaches to antibiotics for candidates with cystic fibrosis (CF) are institution specific. Little has been published about these practices. Routine LTx perioperative prophylaxis at our institution is 96 hours of vancomycin and cefepime with antibiotics then adjusted for positive intraoperative donor and recipient cultures. In recipients with CF, perioperative prophylaxis is determined pre-LTx to cover colonizing organisms isolated in culture from the 2 years before LTx, adjusted for intraoperative cultures, and administered for 14-21 days post-LTx. Our study aims to add to the current literature by describing our center’s experience with antibiotic management and outcomes in lung transplant recipients with CF to help guide best practices for prophylaxis strategies for this patient population. Methods We performed a retrospective chart review of patients with CF who underwent LTx from 3/1/2015-11/18/21 at our center and were colonized pre-LTx with MRSA, Pseudomonas aeruginosa, or Burkholderia cepacia complex. Peri- and post-operative antibiotic regimens, donor and recipient culture results, changes in antibiotic therapy post-LTx, and duration of antibiotic therapy were documented. Outcomes included rehospitalization, isolation in culture of the prophylaxis-targeted organisms post-LTx, and mortality, within 6 months of LTx. Results Nineteen incidents of LTx were included in this study. Pre-LTx colonizing organisms and antibiotics administered are outlined in table 1. Prophylaxis-targeted organisms were infrequently isolated in culture after LTx (table 2), with 7/19 (36.8%) developing post-transplant infection with prior colonizing organisms. Four (21.1%) recipients developed post-LTx Clostridioides difficile infection. One (5.3%) recipient died within 6 months post-LTx. Conclusion LTx recipients with CF who received targeted perioperative prophylaxis for pre-LTx colonizing organisms infrequently developed post-LTx culture positivity with the same bacteria including none during the antimicrobial prophylaxis period. Future prospective studies are needed to determine optimal antibiotic duration of peri-LTx antibiotics in this patient population. Disclosures All Authors: No reported disclosures

2241. Antibiotic Use Before and After Hospital Admission Among Hospitalized Patients with Acute Febrile Illness in Bangladesh

Abstract Background The inappropriate use of antibiotics is common in Bangladesh, contributing to the significant burden of antimicrobial resistance. The WHO has categorized Access, Watch, and Reserve groups of antibiotics (AWaRe classification) to promote optimal use. We explored antibiotic use among hospitalized patients with acute febrile illness (AFI) at five tertiary-level hospitals in Bangladesh. Methods From September 2021 to February 2023, 1544 patients were randomly selected among all hospitalized patients with measured fever (≥100.4°F) or a history of fever in the past 14 days. Blood and urine culture isolates were analyzed for antibiotic sensitivity using the VITEK-2 system. Results Of enrolled AFI patients, 835 (54%) reported the use of antibiotics prior to hospitalization, with 79% of reported antibiotics from the Watch group, 16% from the Access group, and none from the Reserve group. Five percent of the antibiotics prescribed were not recommended by WHO for clinical practice. The top five frequently reported antibiotics before hospitalization were cefixime (24%), azithromycin (19%), ceftriaxone (16%), amoxicillin (6%), and cefuroxime (5%). Bacterial infection was diagnosed among 205 patients (13% of all enrollees) through Rapid diagnostic tests, culture and Rt-PCR. In-hospital, 1385 (90%) patients received at least one antibiotic. The most frequently prescribed antibiotics were from the Watch group (84%), followed by the Access group (15%) and none from the Reserve group. The top five antibiotics prescribed in-hospital were ceftriaxone (64%), amoxiclav (7%), ceftazidime (3.4%), clarithromycin (2.8%), and moxifloxacin (2.6%). Antibiotic susceptibility testing (AST) results from blood and urine culture isolates showed high resistance pattern to ceftriaxone (45% and 56%) and ceftazidime (63% and 50%). Of blood culture isolates 80% were sensitive to amoxiclav, while 46% of urine culture isolates were resistant to amoxiclav. At least one bacterial organism was identified in only 13% of enrolled patients. List of antibiotics that were reported to be received by the enrolled AFI patients before Hospitalization Before hospitalization, 132 patients reported receiving antibiotics from Access group, while 662 patients received antibiotics from WATCH group List of antibiotics received by the enrolled AFI patients during hospitalization During hospitalization, 211 patients received antibiotics from Access group and 1169 patients received antibiotics from Watch Group List of 235 bactrial organisms which were identified from clinical samples of 205 AFI patients (some patient had co-infection) Conclusion In conclusion, antibiotics were used in nine of ten patients. The frequent and inappropriate use of Watch group antibiotics in outpatient settings likely contributes to decreased effectiveness and demonstrates the need for improved antimicrobial stewardship. Disclosures All Authors: No reported disclosures

Identification of Microbial Populations Present in Agricultural and Nonarable Soils in the Talas Valley, Northern Kyrgyzstan, in Autumn

Soil bacterial and fungal communities were investigated in relation to soil type and farm management practices after vegetation harvesting in autumn. Soils from fields cultivated with Phaseolus vulgaris (bean) and Pyrus comminus (pear) and nonarable, natural areas were studied. Microbial diversity was analysed using cultivation-dependent methods (isolation of pure cultures) and cultivation-independent methods (direct extraction of DNA from soil, followed by PCR amplification of the 16S rRNA and 18S rRNA genes). The use of cultivation-dependent methods revealed that there were no differences in the biodiversity of the soil bacterial and fungal communities between fields cultivated with bean plants and pear trees. However, the use of cultivation-independent methods showed that there were clear soil and crop type-specific effects on the composition of the soil bacterial and fungal communities. The density of the bacterial population was two times higher in northern mountain-valley serozem (NMVS) soil samples than in light chestnut (LC) soil samples. In contrast, the densities of the fungal communities were almost equal in the studied soil types. The density of the actinomycetes community was almost two times higher in LC soil than in NMVS soil under bean plants. The Shannon index values showed that the bacterial biodiversity in the NMVS soil samples was greater than that in the LC soil samples. Soils under fallow appeared to have diverse bacterial communities that mainly consisted of local autochthonous microflora and a small amount of zymogenic microflora (since fresh plant residue does not enter the soil). The Shannon index results revealed two interesting facts: (1) the soil bacterial community was highly diverse in soils that supported bean plants and (2) the soil fungal biodiversity was high under pear trees in both soil types.

981. Sink drain environmental hygiene intervention successfully reduces burden of AROs

Abstract Background Healthcare associated infections (HAIs) and outbreaks due to hospital sinks have been noted for many years, however, there remains no standardized environmental hygiene protocol to combat these HAIs. The goal of this study was to evaluate the impact of a sink environmental hygiene intervention on gram negative microbial load. Methods We implemented our intervention across 18 rooms in two ICUs at Barnes Jewish Hospital in St. Louis, USA (Figure 1). At low frequency (1x/week) and high frequency (5x/week) intervals, we wiped sink surfaces with 10% bleach wipes and pumped (Foam-It Pump, FOAMit, Grand Rapids, MI) hydrogen-peroxide based foam (Virasept, Ecolab, St. Paul, MN) into sink drains, allowing a contact time of 3 minutes before rinsing. Control rooms received no intervention. We used E-swabs (Copan, Murrieta, CA) to swab sink drains and surrounding surfaces during a pre-intervention, two intervention, and two post-intervention periods. Swabs and water samples were selectively cultured for AROs, and isolates were identified using MALDI-TOF MS. We also obtained information on HAIs that occurred in the ICUs during the study period. Study design overview. We tested 2 frequencies of a disinfectant foam pump and bleach wipe intervention in 18 rooms across a SCTO ICU and SICU at Barnes Jewish Hospital. We sampled multiple surfaces including sink drains across a baseline phase (Phase 1), intervention phase (Phase 2), post-intervention phase (Phase 3), a second intervention phase (Phase 4), and another post intervention phase (Phase 5). Results Sink drains yielded the most unique AROs compared to other surfaces. The most identified AROs were P. aeruginosa and S. maltophilia (Figure 2). The intervention reduced total microbial and gram-negative burden at both frequencies, with the high frequency reducing the fraction of sink drains yielding gram-negatives by >80% (p< 0.05, Figure 3). The low and high frequencies reduced the fraction of sink drains yielding P. aeruginosa (62-82% and 81-100%, respectively) or S. maltophilia (44-68% and 72-94%, respectively); for many weeks neither was recovered (Figure 4). The number of HAIs (n=69) recorded was insufficient to comment on impacts on HAIs. In-room sink drains yielded the most AROs. a) Average weekly fraction of rooms with growth on BAP from each site. Average weekly b) unique ARO isolates c) fraction of rooms yielding P. aeruginosa and d) fraction of rooms yielding S. maltophilia. Permutation test, BH corrected. Both interventions reduced the fraction of rooms with culturable growth. Average weekly fraction of rooms in each intervention group that yielded culturable growth on a) BAP and b) MAC plates. Dunn’s test with BH correction was used to evaluate differences between groups in Phase 1 (baseline). Changes within groups versus Phase 1 were tested using permutation tests and BH corrected. Significant changes are denoted above the phase that has changed versus Phase 1 as follows: *: p ≤ 0.05, **: p ≤ 0.01. Interventions reduced the fraction of in-room sink drains yielding ARO isolates. a) Weekly counts per sink drain of unique AROs. Fraction of rooms yielding b) P. aeruginosa and c) S. maltophilia from in-room sink drain. Both interventions significantly reduced culturable ARO isolates, and frequently resulted in no P. aeruginosa or S. maltophilia collected for multiple weeks. Significant changes are denoted above the phase that has changed versus Phase 1 as follows: *: p ≤ 0.05, **: p ≤ 0.01. Conclusion As antibiotic resistance continues to rise, preventing HAIs caused by AROs becomes increasingly important. There are no standardized sink drain environmental hygiene protocols, even as outbreaks tied to sink drain colonization are regularly reported. This protocol effectively reduced ARO colonization of sinks drains. While larger studies are needed to observe effects of this intervention on HAIs, this study provides a promising tool for combatting sink drain-associated HAIs. Disclosures Carey-Ann Burnham, PhD, Pattern Bioscience: Stocks/Bonds

565. Antimicrobial susceptibility profiles of Acinetobacter species in blood: 10-year experience from a major United States reference laboratory

Abstract Background Antimicrobial resistance (AMR) is a threat to healthcare, and infections with antimicrobial resistant organisms carry significant morbidity and mortality. Owing to high rates of AMR, the Centers for Disease Control and Prevention listed Acinetobacter baumannii as a top priority pathogen for research and development of new antimicrobial agents. Antimicrobial susceptibility testing is challenging for this organism, and susceptibility profiles are variable, leading to need of a potential guidance document with assistance in selecting effective empiric therapeutic regimens. Methods We retrospectively reviewed the microbiology and antimicrobial susceptibility testing (AST) results for Acinetobacter spp. from blood culture isolates submitted to Mayo Clinic Laboratories from December 2012 to 2022. AST was performed using agar dilution and interpretations following Clinical & Laboratory Standards Institute (CLSI) guidelines. Isolates were identified by MALDI mass spectrometry and/or 16S sequencing, as available (Figure 1).Figure 1:Isolate identification Isolate identification description. Isolates were analyzed based on this classification used in out laboratory. Results 804 blood culture isolates were included. A. baumannii/calcoaceticus complex (27%) was most frequently isolated, followed by A. lwoffii (22%), and showed the highest resistance across all antimicrobials (73% susceptible or below) (Figure 2). A. baumannii/calcoaceticus complex showed rising resistance rates over time from 2012 to 2022 for meropenem, minocycline and ampicillin-sulbactam (Figure 3). Cefiderocol non-susceptible rate was 35% for A. baumannii/calcoaceticus complex.Figure 2:Antimicrobial susceptibility profiles for Acinetobacter spp. Description of susceptibility profiles for Acinetobacter spp. isolates, based on the classification used by our laboratory. Antimicrobials are presented at the top and isolates on the left. Number of isolates analyzed are presented in the table with the percentage of the isolates susceptible to each antibiotic class in parenthesis.Figure 3:Acinetobacter baumannii/calcoaceticus complex isolates and resistance, trend Trend of isolates per year and resistance percentage to meropenem, colistin, minocycline and amp-sulb. Number of isolates per year at the base of each bar; percentage of resistance for antimicrobials expressed on lines. Amp-sulb = ampicillin-sulbactam. Conclusion A. baumannii/calcoaceticus complex showed high levels of resistance ( >75%) across multiple antimicrobials and increasing rates of resistance over time from 2012 to 2022. A high number of cefiderocol non-susceptible organisms is also concerning (Figure 2). Disclosures Audrey N. Schuetz, MD, Merck: Advisor/Consultant

2809. In Vitro Synergy of Antibiotic Combinations Against Enterococcus gallinarum

Abstract Background Enterococci are an important cause of endocarditis, particularly involving prosthetic valves. The prevalence of E. gallinarum has been increasing in recent years. Unlike E. faecalis, no information is available regarding the bactericidal activity of ampicillin alone or combined with gentamicin or ceftriaxone against E. gallinarum. Methods Ten blood culture isolates of E. gallinarum were studied. The isolates came from unique patients at a single hospital. MICs of ampicillin, ceftriaxone and gentamicin were done by the CLSI microdilution method. ATCC strains E. faecalis 29212 and P. aeruginosa 27853 were used as controls. Time-kill studies were performed in CA-MHB for ampicillin 10 mg/L, ceftriaxone 10 mg/L, gentamicin at 0.25 x MIC up to a maximum of 4 mg/L, ampicillin + ceftriaxone, and ampicillin + gentamicin. Bactericidal activity was defined as a 1,000-fold decrease in the log10 cfu/mL at 24 h. Synergy was defined as a 100-fold decrease in the log10 cfu/ml at 24 h in a combination compared to each single agent. Results The MICs of ampicillin ranged from 1 to 8 mg/L, gentamicin from 2 to 16 mg/L, and ceftriaxone were all ≥ 32 mg/L. Results of the time-kill studies are summarized in the Table. Ampicillin alone was bactericidal against a single isolate. For that isolate, testing of ampicillin at 0.25 x MIC with gentamicin demonstrated synergy. The combination of ampicillin + ceftriaxone was synergistic against a single isolate, but was otherwise similar to ampicillin alone. Ampicillin + gentamicin was bactericidal against all isolates and was synergistic against all but one isolate. Conclusion Ampicillin alone was generally not bactericidal against E. gallinarum but had synergistic bactericidal activity when combined with gentamicin, similar to the findings with E. faecalis. However, unlike E. faecalis, ampicillin + ceftriaxone was not bactericidal against most isolates of E. gallinarum. These findings support the suggestion that ampicillin + gentamicin might be the optimal treatment for endovascular infections caused by E. gallinarum. Disclosures All Authors: No reported disclosures

Invasive non-typhoidal Salmonella from stool samples of healthy human carriers are genetically similar to blood culture isolates: a report from the Democratic Republic of the Congo

Invasive non-typhoidal Salmonella from stool samples of healthy human carriers are genetically similar to blood culture isolates: a report from the Democratic Republic of the Congo

Molecular Detection of Multidrug Resistance and Characterizations of Mutations in Mycobacterium Tuberculosis Using Polycarbonate Track-Etched Membrane Based DNA Bio-Chip.

With the widespread use of rifampicin (RMP) and isoniazid (INH), multidrug resistance (MDR) in Mycobacterium tuberculosis (M.tb) poses a threat to the success of tuberculosis (TB) control programs. We have developed a new polycarbonate track-etched membranes(PC-TEM) based DNA bio-chip designed for rapid detection of mutations conferring MDR inM.tbculture isolates. Bio-chips were designed to contain 14 specific probes for wild type and mutated allele of selected codons within 80bprifampicin resistance determining region of rpoB gene, katGgene andmabA-inhAregulatory region. RMP-resistance-associated gene mutation pointsrpoB 516, 526, 531and533, and the INH-resistance-associated gene mutation pointskatG315andinhA-15were targeted. Bio-chip signal was detected using enhanced chemiluminescence. A total of 50 culture isolates that were sensitive or resistant to RMP and/or INH were analyzed by bio-chip. The results of culture-based drug susceptibility testing (DST)were used as the gold standard and gene sequencing was performed to resolve the discordance. Amongst 50 culture isolates, we have detected 18 MDR, 9 RMP mono-resistant, 6 INH mono-resistant, and 17 fully susceptible isolates. The developed DNA bio-chip has a sensitivity of 90% for RMP and MDR and 100% for INH resistance. The bio-chip has a specificity of 100% for RMP and MDR and 88.8% for INH detection. The identification of mutations using the DNA bio-chip was 100% concordant with the sequencing data for the probes covered by the bio-chip. The detection ofrpoB, katGand inhA gene mutation points by a DNA bio-chip may be used as a rapid, accurate, and economical, clinical detection method for MDR detection in M.tb. This is very valuable for the control of TB epidemics.

Fungal communities and their correlation with the sensory quality of rice wine from the Xiaogan and Dazhou regions in China

In this study, we performed a comparative analysis of fungal communities and cultivable yeast isolates in rice wine from the Xiaogan and Dazhou regions using Illumina MiSeq high-throughput sequencing technology and traditional culture-dependent methods. And we further explored the correlation between the sensory quality and fungal communities in rice wine. The results demonstrated no significant difference (P > 0.05) in the abundance and diversity of fungal communities and cultivable yeast isolates between Xiaogan and Dazhou rice wine. The dominant fungal genera were Rhizopus and Saccharomyces, with Saccharomyces cerevisiae accounting for the greatest proportion of yeast isolates. The rice wine samples could be divided into two types based on the composition of fungal communities. Type 1 mainly contained Rhizopus and Saccharomyces, while type 2 mainly contained Rhizopus. Properties such as lightness, blueness-yellowness, methane levels, ethanol levels, organic sulfide content, and soluble solids content accounted for the main differences between the two types of rice wine. In addition, Candida and Clavispora showed a significant positive correlation (P < 0.05) with the aromatic substances in rice wine. The findings of the study enrich our understanding of traditional rice wine, providing meaningful information for the improvement and development of the traditional rice wine industry.

Characterization of the Cassava Mycobiome in Symptomatic Leaf Tissues Displaying Cassava Superelongation Disease.

Superelongation disease (SED) is a fungal disease that affects cassava in the Caribbean. The symptoms include the appearance of dry necrotic spots and lesions on the leaves, which may severely affect the plant yield. However, the primary causal pathogen is difficult to culture and isolate in the lab because of its slow growth and potential contamination from faster-growing organisms. In addition, the leaf symptoms can be confused with those caused by other pathogens that produce similar necrotic spots and scab-like lesions. There is also little or no information on the contribution of endophytes, if any, to disease symptoms in cassava, a plant where the disease is prevalent. Therefore, this study aimed to characterize the fungal communities in cassava associated with SED symptoms by analyzing gross fungal morphology and performing metagenomics profiling. First, several individual pathogenic fungi were isolated and cultured from diseased cassava leaf tissues from seven locations in Barbados (BB). Both culture isolation and molecular community analyses showed the presence of several other fungi in the disease microenvironment of symptomatic cassava leaves. These included Fusarium, Colletotrichum, and Alternaria species and the suspected species Elsinoë brasiliensis synonym Sphaceloma manihoticola. Additionally, a community analysis using ITS2 amplicon sequencing of 21 symptomatic leaf tissues from BB, St. Vincent and the Grenadines (SVG), Trinidad and Tobago (TT), and Jamaica (JA) revealed that the disease symptoms of superelongation may also result from the interactions of fungal communities in the mycobiome, including Elsinoë species and other fungi such as Colletotrichum, Cercospora, Alternaria, and Fusarium. Therefore, we suggest that examining the pathobiome concept in SED in the future is necessary.

TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease

Effective control and monitoring the spread of foot-and-mouth disease (FMD) relies upon rapid and accurate laboratory detection of FMD virus (FMDV). Therefore, in this report, a multiplex TaqMan probe-based one-step RT-qPCR assay simultaneously targeting FMDV 5′UTR and 3Dpol regions, and 18S rRNA housekeeping gene (as an internal control) in a single reaction tube was developed and evaluated. The multiplex one-step RT-qPCR assay specifically detected viral genome in both FMDV-infected cell culture suspensions and clinical samples collected from known-FMD infected animals. The assay could detect FMDV RNA in the archived FMDV cell culture isolates (n = 120) collected during the last two decades in India. In addition, the new assay could also detect viral RNA in the FMD suspected clinical samples (n = 740) collected from various field outbreaks. At a cut-off Ct-value of &amp;lt;38, the assay could detect at least 20 and 10 copies of FMDV 3Dpol and 5′UTR genes, respectively. Further, the multiplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.3% to 3.03% for FMDV-3Dpol gene target, and from 1.44% to 4.69% for 5′UTR gene target. In addition, it was found that the new assay could be used to detect viral genome in a variety of samples (epithelium, saliva, OPF, milk and blood) without any significance difference in the detection limit of the assay. Hence, the multiplex one-step RT-qPCR assay could be considered a valuable tool for the detection of FMDV in India.

Strain-resolved metagenomic analysis of the gut as a reservoir for bloodstream infection pathogens among premature infants in Singapore

BackgroundGut dysbiosis contributes to the high risk of bloodstream infection (BSI) among premature infants. Most prior studies of the premature infant gut microbiota were conducted in Western countries and prior to development of current tools for strain-resolved analysis.MethodsWe performed metagenomic sequencing of weekly fecal samples from 75 premature infants at a single hospital in Singapore. We evaluated associations between clinical factors and gut microbiota composition using PERMANOVA and mixed effects linear regression. We used inStrain to perform strain-level analyses evaluating for gut colonization by BSI-causing strains.ResultsMedian (interquartile range) gestation was 27 (25, 29) weeks, and 63% of infants were born via Cesarean section. Antibiotic exposures (PERMANOVA; R2 = 0.017, p = 0.001) and postnatal age (R2 = 0.015, p = 0.001) accounted for the largest amount of variability in gut microbiota composition. Increasing postnatal age was associated with higher relative abundances of several common pathogens (Enterococcus faecalis: p < 0.0001; Escherichia coli: p < 0.0001; Klebsiella aerogenes: p < 0.0001; Klebsiella pneumoniae: p < 0.0001). Antibiotic exposures were generally associated with lower relative abundances of both frequently beneficial bacteria (e.g., Bifidobacterium species) and common enteric pathogens (e.g., Enterobacter, Klebsiella species). We identified strains identical to the blood culture isolate in fecal samples from 12 of 16 (75%) infants who developed BSI, including all infections caused by typical enteric bacteria.ConclusionsAntibiotic exposures were the dominant modifiable factor affecting gut microbiota composition in a large cohort of premature infants from South-East Asia. Strain-resolved analyses indicate that the gut is an important reservoir for organisms causing BSI among premature infants.

Antimicrobial resistance profiles of bacterial isolates from clinical specimens referred to Ethiopian Public Health Institute: analysis of 5-year data

BackgroundAntimicrobial resistance is one of the common global public health problems. The emergence of antimicrobial resistance is multifactorial, and tackling its development is challenging. Consequently, infections caused by resistant bacteria are unresponsive to conventional drugs, resulting in prolonged and severe illnesses, higher mortality rates, and considerable healthcare costs. Therefore, understanding the antimicrobial resistance profiles of bacterial pathogens is essential to optimize treatments and reduce the risks associated with infections. This study aimed to determine the antimicrobial resistance patterns of bacterial isolates from different clinical specimens at the Ethiopian Public Health Institute (EPHI).Materials and methodsThe retrospective cross-sectional study was conducted on the bacterial culture and antibiotic susceptibility reports of different clinical specimens referred to the Bacteriology Laboratory of EPHI from September 2015 to August 2019. Standard bacteriological techniques were used for the isolation and identification of the bacteria. Data were extracted from 840 patients’ records, which included the type of clinical sample cultured, the name of the bacteria, the representations of the antibiotics used for susceptibility testing, and the susceptibility results. Descriptive statistics were used to describe the bacterial isolates and the antimicrobial resistance profiles.ResultsEight types of clinical specimens were analyzed for bacterial isolates and urine specimens were the most analyzed. Ten different genera of bacteria were identified by culture. Almost all the isolates were gram-negative bacteria, while only one species of gram-positive (Staphylococcus aureus) was reported. Antibiotic sensitivity patterns were tested on 840 culture isolates. Escherichia coli strains revealed more than 57% resistance to seventeen antibiotics. Klebsiella pneumoniae showed nearly 70% or greater resistance rates for 17 of the antibiotics used. The overall detected multidrug resistance (MDR) was 64.29%. The highest MDR was reported in Acinetobacter strains (84%) followed by K. pneumoniae (80%).ConclusionsThe multidrug resistance rates found in this study were alarming. Strengthening antimicrobial resistance surveillance at the national level is mandatory, and antimicrobial sensitivity testing should be accessible at local diagnostic centers.

What did we learn about tocilizumab use against COVID-19? A single-center observational study from an intensive care unit in Serbia.

Selection of effective and safe therapy for management of patients with coronavirus disease is challenging. Tocilizumab (TZB) has emerged as a potential treatment option for COVID-19. Several aspects regarding Tocilizumab treatment remain uncertain, such as the optimal timing for its administration and the safety profile, including the potential risk of infections. The aim of the study is to present the clinical characteristics of patients with COVID-19 following the application of Tocilizumab. This is a retrospective analysis of 121 patients with severe forms of COVID-19 previously treated with Tocilizumab was conducted. All patients were admitted to intensive care units (ICUs). Of 121 patients, the majority were men 72 (59.5%) with a median age at presentation of 65 ± 13 years. Only 9 (7.43%) patients were without comorbidities, while the other 112 (92.55%) had two or more comorbidities. Almost all of the 120 patients (99.2%) needed oxygen therapy, such as nasal cannulas in 110 (90.9%) patients, high flow nasal catheter (HFNC) in 4 (3.3%) patients, and continuous positive airway pressure (CPAP) in 5 (4.1%) patients while 1 patient was intubated at the time of hospital admission. The average time from Tocilizumab application to admission to the ICU was 3 days. During clinical deterioration, almost half 57 (47.1%) of the patients were intubated, and 52 (82.5%) of these intubated patients (p < 0.001) had lethal outcomes. The most significant predictors for a lethal outcome according to multivariate analysis were diabetes mellitus (p < 0.001) followed by a subsequent elevation in C-reactive protein levels (CRP; p < 0.002) and ferritin (p < 0.013) after Tocilizumab application. Bloodstream infections were found in 20 (16.5%) patients, most frequently with Gram-negative pathogens like Acinetobacter spp. as in 12 (18.6%) patients, Klebsiella spp. in 6 (8%) patients, and Pseudomonas spp. in 2 (3.2%) patients. Urine culture isolates were found in 9 (7.43%) patients, with Candida spp. being most frequently isolated in 7 (5.8%) patients, followed by Klebsiella spp. and Pseudomonas spp. in 1 patient each (0.8%). Significantly lower survival was seen in patients with proven infection. The benefit of tocilizumab was not found in our study. The high mortality rate among intubated patients after Tocilizumab use suggests appropriate patient selection and monitoring and emphasizes the risk of superinfections. Diabetes mellitus, increased levels of CRP, and ferritin were identified as the most significant predictors of poor outcomes in contrast to increased levels of IL-6.

An Alternative Rapid Confirmation Method for Identifying Listeria monocytogenes from a Variety of 125 g Food Samples Within Two Days of a PCR Presumptive Positive

Cultural confirmation following detection of a Listeria monocytogenespresumptive positive can take 3–7 days to finalize; this uncertainty is a point of frustration for food producers needing to make time-sensitive disposition decisions. To address the demand for shortened time-to-results, an alternative L. monocytogenes confirmation method consisting of two components, (i) a secondary screen using a different rapid method, and (ii) concurrent cultural isolation followed by next-day colony identification was evaluated. For the study, four food matrices (hot dogs, peanut butter, frozen vegetables, and multicomponent frozen meals) were inoculated with low levels (0.36–1.39 MPN/125 g) of L. monocytogenes per the AOAC guidelines for a matrix study. Analyses were performed on 125 g test portions and started with a PCR primary screen (Bio-Rad iQ-Check Listeria monocytogenes II). Next, all enriched food samples underwent a secondary screen by bioMérieux’s GENE-UP LMO2 Real-Time PCR and VIDAS LMX ELFA along with streaking onto RAPID’L.mono Agar. Presumptive positive L. monocytogenes colonies were identified utilizing a high throughput rapid identification method (Hygiena’s BAX System L. monocytogenes Real-Time PCR assay, Neogen’s ANSR isothermal nucleic acid amplification assay, and Bruker’s MALDI Biotyper). Importantly, this study evaluated multiple commercially available options for the secondary screen (n = 2) and rapid identification (n = 3) to allow for easy adoption by testing laboratories. Overall, there was no statistically significant difference (p ≤ 0.05) between the number of L. monocytogenes-positive 125 g samples obtained by the cultural reference method and the alternative confirmation methods (regardless of which method combinations were evaluated). Additionally, this study supports that, when both the primary and secondary screen methods yield a positive result, the sample could be considered a confirmed positive for L. monocytogenes.

Fusariosis in Mexico: A 10-year retrospective series.

Fusarium species represent an opportunistic fungal pathogen. The data in Mexico about Fusarium infections in humans are scarce. Here, we present a retrospective series of patients with a confirmed diagnosis of fusariosis in eight different hospitals in Mexico from January 2010 to December 2019. The diagnosis of proven fusariosis was made according to the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORT/MSG) criteria. A total of 49 cases were identified in our series. Most patients had burn injuries (49%), and 37% had hematological malignancies. Most patients had fire injuries (40%), followed by electric injuries (8%), febrile neutropenia (10%), and pancytopenia (6%). Patients had skin and soft tissue involvement in 49%, followed by blood culture isolation and biopsies from different sites of the body (lung, sinuses, bone tissue, and eyes). Febrile neutropenia (10%) and fungemia (8%) were the most common clinical syndromes in immunosuppressed patients. Most patients received monotherapy (67%), where voriconazole was used in 30% of the cases, followed by conventional amphotericin B (16%), and lipidic formulations of amphotericin B in 10% (either liposomal amphotericin B or amphotericin B lipid complex). Combination therapy was used in 20% of the cases, and the most common combination therapy was triazole plus any lipidic formulation of amphotericin B (10%). Mortality related to Fusarium infection occurred in 22% of patients. Fusariosis is a serious threat. Burn injuries and hematologic malignancies represent the most common causes of infection in this small series from Mexico.

The relevance of coagulase-negative Staphylococcus isolates in blood culture in the context of a tertiary neonatal unit from East India

Background: Coagulase-negative Staphylococcus (CoNS) are known commensals and often contaminate neonatal blood cultures. Their unique ability to form biofilms, however, helps them evade immune mechanisms and antibiotics and cause neonatal sepsis (NS) in hospitalized neonates. True or probable Coagulase-negative Staphylococcus blood stream infection (CoNS BSI) must be differentiated from contaminants so that antibiotics are used judiciously and hospital stay is minimized. Aims and Objectives: The primary objective of the study was to estimate the proportion of NS and contaminants among CoNS-positive blood cultures in the index neonatal unit and the host and health-care variables associated with CoNS BSI. The secondary objective was to estimate the susceptibility of CoNS isolates to antibiotics used. Materials and Methods: This was a retrospective study from digital records, from January 2018 to December 2022. Results: 25% of CoNS isolates were associated with NS (health-care infections) and 75% were contaminants. Over 90% of CoNS BSI was associated with central lines (CLs) and prolonged hospital stay. All isolates were resistant to oxacillin while resistance to gentamicin rose annually to over 68%. Susceptibility to linezolid and vancomycin was present, but a few strains were resistant to them. Conclusion: CoNS were an important cause of NS in the index hospital. Prolonged hospital stays and CLs were associated with increased incidence of CoNS sepsis and must be minimized where possible. Antibiotic resistance was high, and reserve drugs could also become ineffective.

Characterization of Human Adenoviruses of Medical Importance: Isolation of Infectious Virus from Clinical Specimens and Molecular Typing.

Human adenoviruses (HAdVs) constitute a group of ubiquitous viruses that currently comprises 51 well-defined serotypes and more than 113 genotypes classified into seven species, HAdV-A through HAdV-G. The members of these species differ considerably in their genomic characteristics and also in their tropism and pathogenicity. Virus isolation in cell culture remains critical for the preservation and comprehensive characterization of viruses of biomedical interest but has been almost completely abandoned by diagnostic laboratories. Currently, the most frequently used approach for the detection of HAdV in clinical specimens is real-time qPCR targeting a region of the hexon gene, conserved among all genotypes described to the present. In the absence of typing, the detection of an HAdV in association with disease provides limited information. Molecular typing is therefore highly desirable and required in the epidemiological investigation of HAdV-associated disease. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Virus isolation from plasma and whole blood Alternate Protocol 1: Virus isolation from stool Alternate Protocol 2: Virus isolation from respiratory specimens and urine Alternate Protocol 3: Virus isolation from tissue specimens Support Protocol: Inoculation of shell vials Basic Protocol 2: Extraction of highly pure viral genomic DNA from infected cells Basic Protocol 3: Molecular detection of human adenovirus by real-time PCR Basic Protocol 4: Molecular typing for basic identification of species and hexon type Basic Protocol 5: Typing human adenoviruses by next-generation whole-genome sequencing and analysis.