Hyper virulent Core Genome Multilocus Sequence Type CT 11424 of Listeria monocytogenes isolate causing stillbirth in Bangladesh

Hyper virulent Core Genome Multilocus Sequence Type CT 11424 of Listeria monocytogenes isolate causing stillbirth in Bangladesh | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hyper virulent Core Genome Multilocus Sequence Type CT 11424 of Listeria monocytogenes isolate causing stillbirth in Bangladesh Muntasir Alam, Md Saiful Islam, M Ishrat Jahan, Arpita Shyama Deb, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4510467/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2025 Read the published version in BMC Microbiology → Version 1 posted 14 You are reading this latest preprint version Abstract Background Listeria monocytogenes ( Lm ) is a foodborne pathogen that can lead to severe pregnancy outcomes. This study reports the clinical and genomic characteristics of a Listeria -mediated stillbirth identified through the Child Health and Mortality Prevention Surveillance (CHAMPS) project in Bangladesh. The Lm -BD-CHAMPS-01 isolate was recovered from the blood and cerebrospinal fluid (CSF) of a male stillborn baby. Maternal history, clinical, and demographic data were collected by the CHAMPS surveillance platform. An expert panel evaluated all reports to determine the role of Lm infection in the causal chain of stillbirth. Genomic characterization included multilocus sequence typing (MLST), core genome MLST (cgMLST), serotyping, and the presence or absence of virulence and stress adaptation genes. Genetic divergence and phylogenetic analyses were conducted to determine the relationship with other reported isolates globally. Results The isolate Lm -BD-CHAMPS-01 was identified as a novel cgMLST CT11424. It belonged to ST 308, Serotype 4b, Clonal Complex 1, and Phylogenetic Lineage 1. Key Lm virulence genes facilitating the crossing of the placental barrier, including full-length inlA , LIPI-1, and LIPI-3, were detected. The isolate was closely related to clinical Lm isolates, as determined by Grapetree based on cgMLST. SNP-based phylogenetic analysis found Lm -BD-CHAMPS-01 to be the most distant from other CC1 isolates in the database. Possible sources of infection included the consumption of contaminated raw vegetables or exposure to pigeons. Conclusions The virulence profile of the first genome sequence of clinical Lm from Bangladesh, which also caused stillbirth, underscores the understudied nature of this pathogen in the region. Rural healthcare professionals should be aware of Lm infection risks during pregnancy. Pregnant women should be counseled on the dangers of consuming potentially contaminated raw food and exposure to animals or birds to prevent adverse pregnancy outcomes due to Lm infection. Listeria monocytogenes Stillbirth Bangladesh Pregnancy CHAMPS Mother-to-Child transmission Pigeon Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Listeria monocytogenes ( Lm ) infection in humans and ruminants leads to septicemia, gastroenteritis, and central nervous system (CNS) infections ( 1 ). Lm multiplies intracellularly and is capable of cell-to-cell transmission, avoiding the extracellular space ( 2 ). Lm is vertically transmitted from pregnant women to fetuses or neonates by crossing the placenta before delivery ( 3 ). The infection is 20 times more common in pregnancy than in non-pregnant women, and 27% of all infections with Lm occur in pregnant women. Lm infection during pregnancy can lead to fetal loss in 20% of cases ( 1 ). Listeriosis during pregnancy is often asymptomatic or presents with mild symptoms. Therefore, many Lm infections during pregnancy remain undiagnosed, especially in low- and middle-income countries like Bangladesh. Lm is commonly found in the environment ( 4 , 5 ). This pathogen has been reported in the feces of sheep/goats, cattle ( 6 ), dogs, rats ( 7 ), chickens, pigeons ( 8 , 9 ), and crows ( 10 ), as well as in cloacal swabs of chickens and gulls ( 11 ). These animals are potential sources of contamination for both raw and processed food consumed by humans. In developed nations, outbreaks of Lm infection have been linked to various food sources, including meat, fish, mixed dishes, vegetables, juices, and dairy products ( 4 ). Lm contamination in market and restaurant produce, such as cabbage, corn, carrots, lettuce, cucumbers, parsley, and salad mixes, is well reported ( 12 – 15 ). Lm enters food processing facilities through contaminated raw ingredients, making it a significant food safety concern. Certain strains can persist for extended periods, acting as constant sources of cross-contamination due to their ability to adhere to abiotic surfaces. Eradicating Lm , even with the most stringent safety protocols, can be challenging ( 16 ). Lm is classified into four distinct evolutionary lineages denoted I, II, III, and IV, with most isolates belonging to I and II ( 17 ). Serotypes 1/2b and 4b of lineage I are responsible for 95% of listeriosis cases in humans, with 4b being the predominant serotype among clinical isolates and outbreaks ( 18 ). Major clonal complexes (CC) CC1, CC2, CC4, and CC6 of lineage I are associated with clinical listeriosis ( 19 ). As of July 2023, there are few publications available regarding the detection of Lm from Bangladesh ( 20 – 22 ). Lm has been detected in goat/sheep slaughterhouse environments, sick goats, frozen shrimp, and locally manufactured cosmetics. The only reported clinical listeriosis case was from a female cancer patient in Bangladesh ( 23 ). All of these studies used culture-based detection except for the most recent one, which used molecular detection by PCR. We isolated Lm from postmortem specimens of a stillbirth collected through Child Health and Mortality Prevention Surveillance (CHAMPS) using both microbial culture and PCR ( 24 ). Here, we report on an Lm -mediated stillbirth as well as the only clinical Lm isolate reported from Bangladesh, using next-generation sequencing to understand the molecular characteristics and virulence factors of the Lm isolate. Methods Study settings The CHAMPS is a multi-country project operating in the Rajbari and Faridpur districts of Bangladesh. The project has implemented a minimally invasive tissue sampling (MITS) technique for collecting postmortem specimens from stillbirths and under-5 deaths in low-income settings. Clinical, demographic and laboratory diagnosis information of the deceased along with maternal information are reviewed by a panel of experts termed DeCoDe (Determination of Cause of Death) panel to identify the cause of death. More information regarding the CHAMPS protocols and methods can be found at ( 25 ). Isolation and identification of Lm Microbial culture was performed from postmortem blood and cerebrospinal fluid (CSF) specimens. The infectious agents were identified using the Vitek-2 system by Biomerieux (France). The antimicrobial sensitivity was determined using the Kirby-Bauer disk diffusion susceptibility test following CLSI guidelines ( 26 ). The isolate was named Lm -BD-CHAMPS-01. Additionally, Lm DNA was detected from the blood specimen using TaqMan Array Card based Real-time PCR system. Whole Genome Sequencing Lm -BD-CHAMPS-01 DNA was extracted from a pure culture using the DNeasy Blood and Tissue Kits with a gram-positive pretreatment (Qiagen, Germany). DNA library for whole genome sequencing was prepared from 1 ng of DNA with Nextera XT DNA Library Preparation Kit (Illumina Inc, USA). The sequencing procedure was carried out on the MiSeq platform (Illumina Inc, USA), employing the mid/high output reagent cartridge v2/3 (502 cycles) with standards 251-bp paired-end reads (Illumina Inc, United States). All experiments were performed following the manufacturer's guidelines. Genome assembly and characterization Genome assembly and annotation were performed using SPAdes version ( 27 ) and RAST server ( 28 ), respectively. More details of bioinformatics tools are available in supplementary data (Supplementary Table 1). The Bacterial Isolate Genome Sequence Database (BIGSdb) for the Lm database ( 29 ) was used to deduce the seven-gene MLST profile (ST), the cgMLST analysis using 1748 core genes (CT) and clonal complex in silico. Additionally, the BIGSdb server was also used to screen for the presence/absence of virulence genes, antimicrobial resistance determinants, stress survival islands and disinfectants resistance genes. Minimum spanning and SNP-based Phylogenetic analysis A Minimum spanning tree (MST) was constructed using GrapeTree software (Kruskal's algorithm) to visualize the core genomic relationships among all Lm isolates (n = 695) that had cgMLST profile ID (Supplementary Table 2), which were obtained from BIGSdb- Lm database ( 30 ). The tree was visualized using the GrapeTree server. For phylogenetic analysis, SNP-based phylogeny was constructed using the CFSAN pipeline ( 31 ). Lm sequences from evolutionary lineage 1 and human origin were selected. For comparison, 1 food isolate was also included (Supplementary Table 2). F2635_ Lm was used as a reference genome and Listeria innocua was used as an outlier. IQtree web server was used to build a maximum likelihood tree from the alignment file (without any insertion and deletion) using the TVMe + ASC substitution model. The draft genome sequence data are available in the Sequence Read Archive at National Center for Biotechnology Information with accession number SRR28678198. Results Clinical narrative CHAMPS project has been operating in Baliakandi, Bangladesh since September 2017. As of December 2023, a total of 848 MITS have been conducted from stillbirths and postmortem under-5 deaths to determine the cause of death. On 25 January 2022, Lm was isolated from one case (n = 1 of 848) during the study period. The 22-year-old pregnant woman from Baliakandi at 32-week gestational age was admitted to Bangabandhu Sheikh Mujib Medical College & Hospital (BSMMCH). The household reported maintaining a flock of five pigeons and a single hen. During pregnancy period, the mother reportedly consumed cow milk but boiled. Maternal complaints during pregnancy were severe headaches, blurring of vision, pre-eclampsia and anemia (Table 1 ). From the mother’s recall, she received a total of seven antenatal care visits during this pregnancy. Maternal complaints during admission were intrauterine fetal demise confirmed by ultrasonography, fever, burning sensation during micturition and lower abdominal pain for seven hours. The post-admission examination failed to detect fetal movement and heartbeat. A maternal complete urine examination provided normal findings. In the hospital, the mother was treated with oral ciprofloxacin and intravenous ceftriaxone. After a few hours of hospital admission, a male stillborn baby with breech presentation was delivered by normal vaginal delivery. Following CHAMPS protocol, the family was approached for consent to conduct MITS. The stillborn baby was macerated without any significant gross findings. Specimens were collected within an hour of delivery using MITS technique. Body weight during MITS was recorded at 1820 g, height 45 cm and head circumfluence of 29 cm. CHAMPS laboratory platform isolated Lm from both blood and CSF culture. Resistance was observed against cefotaxime and ceftriaxone (Supplementary Table 3). The DeCoDe panel determined the underlying cause of this antepartum stillbirth as intrauterine infection by Lm . The panel also stated maternal infection was the main condition affecting the fetus. To understand the virulence gene landscape and phylogeny of the Lm CHAMPS-BD-01, we used next-generation sequencing to analyze the draft genome sequence. Table 1 Clinical features available from the pregnant women and the stillborn infected with Lm -BD-CHAMPS-01 Maternal features Parameters Clinical symptoms Fever before delivery Yes Anemia Yes Headache Yes Dizziness Yes Pre-eclampsia Yes Malnutrition Yes Burning sensation during micturition Yes BP systolic, mmHg 90 BP diastolic, mmHg 60 Pulse (bpm) 108 Laboratory diagnosis Hemoglobin (g/dL) 9.8 Random Blood Sugar (mmol/L) 4.9 Thyroid-stimulating hormone (µIU/ml) 3.1 HBsAg Negative Urine Routine Examination Normal Maternal Medications during pregnancy Medications Indication Iron Anemia Misoprostol Medical induction of labour Paracetamol Fever Antibiotics Ceftriaxone, Ciprofloxacin Stillborn features Maceration Level Level 1 - Maceration Anthropomorphic measurements during MITS Weight (g) 1820 Height or Length (cm) 45 MUAC (cm)* 7.5 Head Circumference (cm) 29 Right Leg Length (cm) 6 Right Foot Length (cm) 6.5 *MUAC = Mid upper arm circumfluence Lm -BD-CHAMPS-01 is a novel cgMLST type of Listeria monocytogenes The Lm -BD-CHAMPS-01 genome had 13 contigs, a genome size of 2932029 bases, and a 37% GC content. The genome coverage was 127x, with an N50 of 1491565 and an L50 of 1. Prokka annotation found 2873 CDS, 5 rRNA, 1 repeat region, 56 tRNA, and 1 tmRNA (Fig. 1 ). MLST and K-mer dependent analysis confirmed the sequence as Lm . Using the Bigsdb tool, the isolate was identified as ST 308 (MLST), CC1 (clonal complex), lineage I, sub-lineage SL150, and PCR serogroup 4b. cgMLST analysis declared the isolates as a novel type, and a new cgMLST profile identification number, CT 11424, was provided. Virulence and stress-associated genes Lm -BD-CHAMPS-01 was found to contain 65 virulence genes. Genes lap , dltA , fbpA , inlJ , lapB , actA and inlF were found which are known to aid bacterial adhesion. Invasion-related genes inlA , inlB , inlE , vip , ant , iap , lgt , and lepA without any stop codon (full length) were also identified. The isolate harbored several genes to survive inside intracellular environment including llo (Listeriolysin O), plcA , plcB (enhance phagosomal membrane disruption), prsA2 , lsp , svpA . For intracellular growth hpt , IpA1 and oppA genes (survival inside macrophage) were detected. Lm 4b serotype-specific gene cassette gltA-gltB was present. These genes were found arranged inside Listeria pathogenicity island (LIPI-1) 1 ( hly, mpl, actA, plcB, plcA, prfA ) and LIPI-3 ( llsA , llsG , llsH , llsX , llsB , llsY , llsD , llsP )( 32 ). Gene synteny of these two pathogenicity islands was similar when compared with the F2635 reference strain (Supplementary Fig. 1). Listeria genomic island locus, LGI-2_ LM OSA2310 and LGI-2_ LM OSA2320 were found Lm -BD-CHAMPS-01. Other stress adaptation genes include SigmaB operon, gadD2 and gadT2 (stress adaptation), cspB gene (cold adaptation), betL (salt tolerance), gtcA (rhamnose operon gene) and 29 motility-related genes. The presence or absence of genes was illustrated using the isolates included in the phylogenetic analysis (Fig. 2 ). The analysis showed that Lm -BD-CHAMPS-01 has the same virulence and stress adaptation profile as the all CC1 isolates, except one Lm from Chile. It also contains multidrug resistance transporters mdrM . The isolate contains one intact phage (PHAGE_Lister_vB_ Lm oS_293; GenBank accession number NC_02892). The intact phage is 42.4kb long and has 64 total proteins. From this draft genome of Lm -BD-CHAMPS-01, we were unable to find any plasmid. Genetic relatedness and phylogenetic analysis of Lm isolate We calculated the MST, to visualize the genomic relatedness of Lm -BD-CHAMPS-01 in comparison to cgMLSTs of Lm from different sources. Analysis was performed with the representative sequence of each 695 cgMLST available in Bigsdb (Fig. 3 ). Among 1748 core genes, 1746 were detected from the Lm -BD-CHAMPS-01; only two hypothetical genes were missing. In MST, the isolate was related to clinical origin cgMLST CT 5635 with 484 allelic differences (Fig. 3 ). Lm CT 5635 was located in the center of the cluster. Most genetically adjacent of Lm CT5635, was from a food source (CT 2830, 64 allelic difference) and animal feed (CT6145, 23 allelic difference). The other connected clinical isolate was CT2872 with 29 allelic differences. To investigate the evolutionary origin of Lm -BD-CHAMPS-01 phylogenetic analysis was performed with selected sequences of lineage I from global isolates (Fig. 4 ). The log-likelihood of the tree was − 360.7421. The Lm -BD-CHAMPS-01 diverged from Lm reported from the food source of the USA in 1985. Indian Lm isolates reported from 2004 to 2014 all are phylogenetically similar and this cluster was adjacent to Lm -BD-CHAMPS-01. Among all the CC1 isolates, Lm -BD-CHAMPS-01 was the most distant in evolution, acquiring more changes in it (most distant on the tree scale at 0.63). Discussion Here we report a highly virulent Lm strain causing stillbirth in Bangladesh. Phylogenetic analysis based on cgMLST identified the Lm as a novel cgMLSTCT 11424. The clinical narrative aligns with the genomic landscape showing all major virulence genes required for crossing the placental barrier through inter-cellular movement. Listeriosis is a rare illness usually linked with consumption of raw unpasteurized milk or raw food causing severe consequences during pregnancy. Globally, reporting of Lm outbreak is uncommon, reports of at least two cases from same place are considered as outbreak ( 33 ). A major reason for unreported listeriosis is the lack of severe symptoms in non-pregnant individuals. As a result, laboratory diagnosis is limited especially in low- and middle-income countries. However, during pregnancy, Lm invades the placenta where inflammatory and immune-regulatory T-cell response is modulated to promote maternal tolerance to the semi-allogeneic fetus. This leads to increased host susceptibility to Lm infection. However, Lm infection triggers innate immune response which ultimately triggers host immune response in the placenta leading to fetal damage or demise ( 34 ). The Lm -BD-CHAMPS-01 isolate is a virulent strain indicated by the presence of listeriolysin O-expressing gene llo ( 35 ). In addition, it possesses of all genes required for intracellular survival and intercellular movement to cross the placental barrier. The presence of full-length lap ( Listeria adhesion protein) mediates adhesion of with host cell and gene inlA (internalin genes) facilitates its ability to cross the intestinal as well as placental barrier ( 36 , 37 ). In mice models, it has been shown that the inlF gene enhances the early stage of infection by modulating host inflammatory responses ( 38 ). Recently, it has been reported that the actA gene enhances the shedding of Lm several by magnitudes which leads to better transmission from host to environment ( 39 ). It also expresses multidrug resistance transporter mdrM which triggers host immunity leading to activation of the placental immune system against the fetus. It expresses CspB protein which controls stress adaptation and virulence phenotypes such as oxidative stress, biofilm production, hemolysis, cell evasion and intracellular growth ( 40 ). Although highly virulent, the Lm -BD-CHAMPS-01 remains sensitive to a wide range of antibiotics. Resistance was only observed for those against which Lm possessed natural resistance ( 41 ). Lm -BD-CHAMPS-01 also belongs to lineage I and serotype 4b which is one of the predominant serotypes reported to cause listeriosis in humans and ruminants. Lm strains from lineage I and serotype 4b have shown the lowest diversity among the other lineages; and lowest levels of recombination among the lineages indicating genomic stability ( 41 ). This is also evident from the detection of similar isolates from 2004 to 2014 in India, which shares a border with Bangladesh. In addition, the possible lack of mobile genetic elements like plasmids indicates the outbreaks by Lm -BD-CHAMPS-01 like isolates are treatable with available antibiotics. The prophage PHAGE_Lister_vB_ Lm oS_293 has been reported recently from South Africa from both pathogenic Lm and less-pathogenic L. innocua ( 42 , 43 ). However, the exact role of this prophage in Lm virulence or evolution is yet to be discovered. The ST308 was reported previously from Chinese fresh aquatic products (2011–2016) and confiscated foods from non-European passengers (2012–2013) at a Spanish airport ( 44 , 45 ). Due to the lack of reporting from Bangladesh and the low evolutionary rate of Lm isolates, it can be assumed that the isolate Lm -BD-CHAMPS-01 has been also circulating in the population for a long time. The antibiotic sensitivity pattern and health care-seeking approach (7 visits) of the pregnant women discussed here showed that the antenatal care system was unable to identify the infection. Timely diagnosis could have prevented this stillbirth using common antibiotics such as ampicillin. This indicates the importance of microbiological diagnosis during antenatal care. Lm serotype 4b has been reported from an outbreak of urban poultry flocks in US ( 46 ). The same serotype has been also reported from fecal samples of hen in Germany ( 47 ). Although out of scope of this analysis, the presence of domestic pigeon and hen in the household could be a potential source of Lm infecting the pregnant women. In summary, the Lm -BD-CHAMPS-01 isolate is a genetically stable and highly invasive strain likely circulating at least in the community of our study area. Circulation of such virulent strains indicates the necessity to consider Lm infection as a notifiable disease for diagnostic facilities. Backyard poultry along with free living birds could be investigated as a potential carrier of Lm in Bangladeshi households. Dietary guidelines and exposure to animals adapted for local food habit and local culture should be developed considering potential Lm infection during pregnancy. Conclusions This study presents the identification and characterization of a highly virulent Listeria monocytogenes strain, Lm -BD-CHAMPS-01, causing stillbirth in Bangladesh. Our findings highlight the presence of key virulence genes that enable the pathogen to cross the placental barrier, posing significant risks during pregnancy. The genomic stability and evolutionary insights suggest this strain's prolonged history within the region. Importantly, the sensitivity of Lm -BD-CHAMPS-01 to common antibiotics underscores the potential for prevention and treatment through timely diagnosis and quality antenatal care. The findings also suggest that backyard poultry, such as hens, and free-living birds, such as pigeons, could be potential reservoirs, necessitating further investigation. The development of locally adapted dietary and animal exposure guidelines to prevent Lm infections during pregnancy will be useful. This report contributes valuable insights to the field of public health microbiology as well as maternal and neonatal health. The findings suggest advocacy for enhanced surveillance and preventive measures against Listeria infections. Abbreviations BD – Bangladesh CC- Clonal Complex cgMLST - core genome Multilocus sequence typing CHAMPS - Child health and mortality prevention surveillance CLSI - Clinical and laboratory standards institute CNS- Central Nervous System CSF – Cerebrospinal fluid DeCoDe - Determination of cause of death LIPI - Listeria pathogenicity island Lm - Listeria monocytogenes MITS - Minimally invasive tissue sampling MLST – Multilocus sequence typing MST - Minimum spanning tree Declarations Ethics approval and consent to participate: The CHAMPS study was conducted following the approval of the research review committee (icddr,b protocol number PR-16082) and the ethical review committee of icddr,b. All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the legal guardians or family members of all stillbirths or deceased children included in the study. The consent process ensured that participants were fully informed about the purpose, procedures, risks, and benefits of the study, and their participation was entirely voluntary. Written consent forms were signed by the legal guardians or family members before any study-related procedures were initiated. No animal was included in this investigation. Consent for publication: Not applicable Availability of data and materials: The draft genome sequence data are available in the Sequence Read Archive at National Center for Biotechnology Information (NCBI) with accession number SRR28678198. Competing interests: The authors declare no competing interests. Funding: The study was performed as part of the Child Health and Mortality Prevention Surveillance study, funded by the Bill and Melinda Gates Foundation (grant ID OPP1126780). Authors' contributions: M.A. drafted the manuscript and guided the overall analysis. M.S.I., M.I.J., and A.S.D. generated the whole genome sequencing data, conducted the bioinformatics analysis and generated the figures/tables. A.R., K.M.I., and M.Z.H. were responsible for collecting and analyzing the clinical data and generated the tables. A.I.C. gathered the demographic information. D.A. isolated the bacteria. S.E.A. and E.S.G. provided overall supervision for the CHAMPS project, secured funding, reviewed the manuscript. M.R. supervised the laboratory work and reviewed the manuscript. Acknowledgements: We acknowledge the contribution of the International Centre for Diarrheal Diseases Research, Bangladesh (icddr,b), for organizational support with the work. icddr,b is grateful to the Governments of Bangladesh, and Canada for providing core/unrestricted support. Authors' information: 1 icddr,b, Mohakhali, Dhaka-1212, 2 Infectious Disease Epidemiology, Johns Hopkins University, Baltimore, MD, USA, 3 Division of Genomics & Transcriptomics, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan. References Allerberger F, Huhulescu S. Pregnancy related listeriosis: treatment and control. Expert Rev Anti Infect Ther. 2015 Mar 4;13(3):395–403. https://doi.org/10.1586/14787210. 2015.1003809. Corr SC, O’Neill LAJ. Listeria monocytogenes infection in the face of innate immunity. Cell Microbiol. 2009 May;11(5):703–9. https://doi.org/10.1111/j.1462-5822.2009.01294.x. Charlier C, Perrodeau É, Leclercq A, Cazenave B, Pilmis B, Henry B, et al. Clinical features and prognostic factors of listeriosis: the MONALISA national prospective cohort study. Lancet Infect Dis. 2017 May;17(5):510–9. https://doi.org/10.1016/S1473-3099(16)30521-7. Grigore-Gurgu L, Bucur FI, Mihalache OA, Nicolau AI. Comprehensive Review on the Biocontrol of Listeria monocytogenes in Food Products. Foods (Basel, Switzerland). 2024 Feb 28;13(5). https://doi.org/10.3390/foods13050734. Schoder D, Guldimann C, Märtlbauer E. Asymptomatic Carriage of Listeria monocytogenes by Animals and Humans and Its Impact on the Food Chain. Foods (Basel, Switzerland). 2022 Nov 1;11(21). https://doi.org/10.3390/foods11213472. Obaidat MM, Stringer AP. Prevalence, molecular characterization, and antimicrobial resistance profiles of Listeria monocytogenes, Salmonella enterica , and Escherichia coli O157:H7 on dairy cattle farms in Jordan. J Dairy Sci. 2019 Oct;102(10):8710–20. https://doi.org/10.3168/jds.2019-16461. Iida T, Kanzaki M, Maruyama T, Inoue S, Kaneuchi C. Prevalence of Listeria monocytogenes in intestinal contents of healthy animals in Japan. J Vet Med Sci. 1991 Oct;53(5):873–5. https://doi.org/10.1292/jvms.53.873. Hellström S, Kiviniemi K, Autio T, Korkeala H. Listeria monocytogenes is common in wild birds in Helsinki region and genotypes are frequently similar with those found along the food chain. J Appl Microbiol . 2008 Mar;104(3):883–8. https://doi.org/10.1111/j.1365-2672.2007.03604.x. Weber A, Potel J, Schäfer-Schmidt R. The occurrence of Listeria monocytogenes in fecal samples of pigeons. Berl Munch Tierarztl Wochenschr. 1995 Jan;108(1):26–7. Yoshida T, Sugimoto T, Sato M, Hirai K. Incidence of Listeria monocytogenes in wild animals in Japan. J Vet Med Sci. 2000 Jun;62(6):673–5.https://doi.org/10.1292/jvms. 62.673. Pohjola L, Nykäsenoja S, Kivistö R, Soveri T, Huovilainen A, Hänninen ML, et al. Zoonotic Public Health Hazards in Backyard Chickens. Zoonoses Public Health. 2016 Aug;63(5):420–30. https://doi.org/10.1111/zph.12247. Sy K V, Murray MB, Harrison MD, Beuchat LR. Evaluation of gaseous chlorine dioxide as a sanitizer for killing Salmonella, Escherichia coli O157:H7, Listeria monocytogenes , and yeasts and molds on fresh and fresh-cut produce. J Food Prot. 2005 Jun;68(6):1176–87. https://doi.org/10.4315/0362-028X-68.6.1176. Zhu Q, Gooneratne R, Hussain MA. Listeria monocytogenes in Fresh Produce: Outbreaks, Prevalence and Contamination Levels. Foods (Basel, Switzerland). 2017 Mar 9;6(3). https://doi.org/10.3390/foods6030021. Skalina L, Nikolajeva V. Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads. Int J Food Microbiol. 2010 Dec 15;144(2):317–21.https://doi.org/ 10.1016/j.ijfoodmicro.2010.10.001. Hou W, Ma Y, Zhang C, Zhao W, Zhao S, Wang P, et al. Investigation on the inactivation effect and mechanism of Listeria monocytogenes in fresh-cut cucumber during storage by ultrasound combined with sodium hypochlorite. Ultrason Sonochem. 2023 Dec;101:106706.https://doi.org/10.1016/j.ultsonch.2023.106706. Stessl B, Fricker M, Fox E, Karpiskova R, Demnerova K, Jordan K, et al. Collaborative survey on the colonization of different types of cheese-processing facilities with Listeria monocytogenes. Foodborne Pathog Dis. 2014 Jan;11(1):8–14. https://doi.org/10.1089/ fpd.2013.1578. Orsi RH, Bakker HC den, Wiedmann M. Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics. Int J Med Microbiol. 2011 Feb;301(2):79–96. https://doi.org/10.1016/j.ijmm.2010.05.002. Vines * , Bala Swaminathan A. Identification and Characterization of Nucleotide Sequence Differences in Three Virulence-Associated Genes of Listeria monocytogenes Strains Representing Clinically Important Serotypes. Curr Microbiol. 1998 May 1;36(5):309–18. https://doi.org/10.1007/s002849900315. Chenal-Francisque V, Lopez J, Cantinelli T, Caro V, Tran C, Leclercq A, et al. Worldwide Distribution of Major Clones of Listeria monocytogenes . Emerg Infect Dis. 2011 Jun;17(6):1110–2. https://doi.org/10.3201/eid1706.101778. Noor R, Hasan MF, Rahman MM. Molecular characterization of the virulent microorganisms along with their drug-resistance traits associated with the export quality frozen shrimps in Bangladesh. Springerplus. 2014 Dec 26;3(1):469. https://doi.org/10. 1186/2193-1801-3-469. Paul P, Faruque MR, Rahman MK, Das P, Chowdhury MYE. Study on bacterial pathogens through multiplex polymerase chain reaction system and their antimicrobial resistance pattern in goats presumed with fever and/or diarrhea. Vet World. 2021 May 6;1080–92. https://doi.org/10.14202/vetworld.2021.1080-1092. Nusrat N, Ahmad Zahra M, Ahmed A, Haque F. Assessment of potential pathogenic bacterial load and multidrug resistance in locally manufactured cosmetics commonly used in Dhaka metropolis. Sci Rep. 2023 May 13;13(1):7787. https://doi.org/10.1038/s41598-023-34782-9. Nahid MA, Sadique T, Mazumder R, Abdullah A, Sami AB, Rahaman MA, et al. Listeria monocytogenes infection in a 56-year-old female cancer patient: a case report. JMM Case Reports. 2015 Aug 1;2(4).https://doi.org/10.1099/jmmcr.0.000076. Sultana N, Pervin M, Sultana S, Islam M, Mostaree M, Khan MAHNA. Pathological study and molecular detection of zoonotic diseases in small ruminants at slaughter houses in Mymensingh, Bangladesh. Vet World. 2022 Sep 5;2119–30. https://doi.org/10.14202 /vetworld.2022.2119-2130. CHAMPS - We Build Knowledge to Save Children’s Lives. https://champshealth.org/.Accessed 23May 2024. Clinical & Laboratory Standards Institute: CLSI Guidelines. https://clsi.org/. Accessed 23 May 2024. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes De Novo Assembler. Curr Protoc Bioinforma. 2020 Jun 19;70(1). https://doi.org/10.1002/cpbi.102. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008 Dec 8;9(1):75. https://doi.org/10.1186/1471-2164-9-75. Listeria monocytogenes .https://bigsdb.pasteur.fr/listeria/. Accessed 23May 2024. Zhou Z, Alikhan NF, Sergeant MJ, Luhmann N, Vaz C, Francisco AP, et al. GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens. Genome Res. 2018 Sep;28(9):1395–404. https://doi.org/10.1101/216788. Bertels F, Silander OK, Pachkov M, Rainey PB, van Nimwegen E. Automated Reconstruction of Whole-Genome Phylogenies from Short-Sequence Reads. Mol Biol Evol. 2014 May;31(5):1077–88.https://doi.org/10.1093/molbev/msu088. Wiktorczyk-Kapischke N, Skowron K, Wałecka-Zacharska E. Genomic and pathogenicity islands of Listeria monocytogenes —overview of selected aspects. Front Mol Biosci. 2023 Jun 14;10. https://doi.org/10.3389/fmolb.2023.1161486. Listeria Outbreaks. https://www.cdc.gov/listeria/outbreaks/index.html. Accessed 23May 2024. Agbayani G, Wachholz K, Murphy SP, Sad S, Krishnan L. Type I interferons differentially modulate maternal host immunity to infection by Listeria monocytogenes and Salmonella enterica serovar Typhimurium during pregnancy. Am J Reprod Immunol. 2019 Jan 4;81(1). https://doi.org/10.1111/aji.13068. Cossart P, Vicente MF, Mengaud J, Baquero F, Perez-Diaz JC, Berche P. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect Immun. 1989 Nov;57(11):3629–36. https://doi.org/10.1128/iai.57 .11.3629-3636.1989. Drolia R, Bhunia AK. Crossing the Intestinal Barrier via Listeria Adhesion Protein and Internalin A. Trends Microbiol. 2019 May;27(5):408–25. https://doi.org/10.1016/j.tim. 2018.12.007. Burkholder KM, Bhunia AK. Listeria monocytogenes Uses Listeria Adhesion Protein (LAP) To Promote Bacterial Transepithelial Translocation and Induces Expression of LAP Receptor Hsp60. Infect Immun. 2010 Dec;78(12):5062–73. https://doi.org/10.1128/iai. 00516-10. Ling Z, Zhao D, Xie X, Yao H, Wang Y, Kong S, et al. inlF Enhances Listeria monocytogenes Early-Stage Infection by Inhibiting the Inflammatory Response. Front Cell Infect Microbiol. 2022 Feb 10;11. https://doi.org/10.3389/fcimb.2021.748461. Travier L, Guadagnini S, Gouin E, Dufour A, Chenal-Francisque V, Cossart P, et al. ActA Promotes Listeria monocytogenes Aggregation, Intestinal Colonization and Carriage. Monack DM, editor. PLoS Pathog. 2013 Jan 31;9(1):e1003131. https://doi.org/10. 1371/journal.ppat.1003131. Muchaamba F, Stephan R, Tasara T. Listeria monocytogenes Cold Shock Proteins: Small Proteins with A Huge Impact. Microorganisms. 2021 May 14;9(5):1061. https://doi. org/10.3390/microorganisms9051061. Luque-Sastre L, Arroyo C, Fox EM, McMahon BJ, Bai L, Li F, et al. Antimicrobial Resistance in Listeria Species. Aarestrup FM, Schwarz S, Shen J, Cavaco L, editors. Microbiol Spectr. 2018 Jul 27;6(4). https://doi.org/10.1128/microbiolspec.arba-0031-2017. Matle, Pierneef, Mbatha, Magwedere, Madoroba. Genomic Diversity of Common Sequence Types of Listeria monocytogenes Isolated from Ready-to-Eat Products of Animal Origin in South Africa. Genes (Basel). 2019 Dec 4;10(12):1007. https://doi.org/ 10.3390/genes10121007. Mafuna T, Matle I, Magwedere K, Pierneef RE, Reva ON. Comparative Genomics of Listeria Species Recovered from Meat and Food Processing Facilities. Denes TG, editor. Microbiol Spectr. 2022 Oct 26;10(5). https://doi.org/10.1128/spectrum.01189-22. Rodríguez-Lázaro D, Ariza-Miguel J, Diez–Valcarce M, Stessl B, Beutlich J, Fernández-Natal I, et al. Identification and molecular characterization of pathogenic bacteria in foods confiscated from non-EU flights passengers at one Spanish airport. Int J Food Microbiol. 2015 Sep;209:20–5. https://doi.org/10.1016/j.ijfoodmicro.2014.10.016. Chen M, Cheng J, Wu Q, Zhang J, Chen Y, Xue L, et al. Occurrence, Antibiotic Resistance, and Population Diversity of Listeria monocytogenes Isolated From Fresh Aquatic Products in China. Front Microbiol. 2018 Sep 19;9. https://doi.org/10.3389/ fmicb.2018.02215. Crespo R, Garner MM, Hopkins SG, Shah DH. Outbreak of Listeria monocytogenes in an urban poultry flock. BMC Vet Res. 2013 Oct 11;9:204.https://doi.org/10.1186/1746-6148-9-204 Weber A, Potel J, Schäfer-Schmidt R, Prell A, Datzmann C. Studies on the occurrence of Listeria monocytogenes in fecal samples of domestic and companion animals. Zentralbl Hyg Umweltmed. 1995 Dec;198(2):117–23 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.docx Supplementarytable1.xlsx Supplementarytable2.xlsx Supplementarytable3.xlsx Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 08 Jul, 2024 Reviews received at journal 29 Jun, 2024 Reviews received at journal 27 Jun, 2024 Reviews received at journal 27 Jun, 2024 Reviewers agreed at journal 22 Jun, 2024 Reviewers agreed at journal 19 Jun, 2024 Reviewers agreed at journal 18 Jun, 2024 Reviewers agreed at journal 17 Jun, 2024 Reviewers agreed at journal 17 Jun, 2024 Reviewers invited by journal 17 Jun, 2024 Editor invited by journal 17 Jun, 2024 Editor assigned by journal 14 Jun, 2024 Submission checks completed at journal 14 Jun, 2024 First submitted to journal 31 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4510467","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":320769650,"identity":"b7d33b4a-9d68-4f37-a4fc-865cb3d1d906","order_by":0,"name":"Muntasir Alam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYPACCQZ+ZiDJw3CAKOWMDSAtks0kamFgMDhArBbdGbnHH/z4Y5FvfJz54Y23e+4w8Esfv4BXi9mNvMTG3jYJy22H2Ywt5zx7xiDZl1NAQEuOYQNvg4SB2WEeNmmeA4cZDM7wJBDU0vjnj4SBcTMpWoCKJQwMmOFa2A/g13LmjeFs2TYJAwmwXw4845Hs4cGrg8HseI7Bxzd/6gz4+w8/vPHmwB05fh72B/j1oAOgFTwGpGkBAlJtGQWjYBSMguEOACfORiRGzPDZAAAAAElFTkSuQmCC","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":true,"prefix":"","firstName":"Muntasir","middleName":"","lastName":"Alam","suffix":""},{"id":320769651,"identity":"24ba3db8-6741-43dc-9649-bec63bb09d79","order_by":1,"name":"Md Saiful Islam","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Saiful","lastName":"Islam","suffix":""},{"id":320769652,"identity":"741d1a09-e3b4-416e-8be2-04e96f265e01","order_by":2,"name":"M Ishrat Jahan","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"M","middleName":"Ishrat","lastName":"Jahan","suffix":""},{"id":320769653,"identity":"a077dc96-5ae3-4603-acc4-f6f142ae9cbd","order_by":3,"name":"Arpita Shyama Deb","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Arpita","middleName":"Shyama","lastName":"Deb","suffix":""},{"id":320769654,"identity":"988e00ae-6f78-4ac0-b45c-8e602eb48304","order_by":4,"name":"Afruna Rahman","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Afruna","middleName":"","lastName":"Rahman","suffix":""},{"id":320769655,"identity":"4a07e50e-791f-44a9-9603-d3f6f560b8f1","order_by":5,"name":"Atique Iqbal Chowdhury","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Atique","middleName":"Iqbal","lastName":"Chowdhury","suffix":""},{"id":320769656,"identity":"b924029e-745b-41ec-ab72-1e637d4030d1","order_by":6,"name":"Kazi Munisul Islam","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Kazi","middleName":"Munisul","lastName":"Islam","suffix":""},{"id":320769657,"identity":"8e9d8f83-de2b-46de-82d9-0d481e65b8ff","order_by":7,"name":"Mohammad Zahid Hossain","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Zahid","lastName":"Hossain","suffix":""},{"id":320769658,"identity":"eef4795d-1613-410b-94c0-343fe8836ce2","order_by":8,"name":"Dilruba Ahmed","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Dilruba","middleName":"","lastName":"Ahmed","suffix":""},{"id":320769659,"identity":"b5cb23ea-37e5-469a-9833-61ee7dd11313","order_by":9,"name":"Shams El Arifeen","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Shams","middleName":"El","lastName":"Arifeen","suffix":""},{"id":320769660,"identity":"c12d6c33-88f9-40d3-b072-68be3074afee","order_by":10,"name":"Emily S Gurley","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"S","lastName":"Gurley","suffix":""},{"id":320769661,"identity":"85d5d208-4c94-444c-90dc-f6ff188da085","order_by":11,"name":"Mustafizur Rahman","email":"","orcid":"","institution":"International Centre for Diarrhoeal Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Mustafizur","middleName":"","lastName":"Rahman","suffix":""}],"badges":[],"createdAt":"2024-05-31 17:32:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4510467/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4510467/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-024-03650-5","type":"published","date":"2025-02-03T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60200430,"identity":"705ffe17-fabd-4460-9ac0-ac1a00a36ac0","added_by":"auto","created_at":"2024-07-13 02:33:03","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":667716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic features of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLm\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-BD-CHAMPS-01. \u003c/strong\u003eThe Circular presentation of annotated genetic features of genome of \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 was generated using Prokka.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/2c84cacec4b4da0b8d0cc91a.jpeg"},{"id":60200432,"identity":"1e17da0f-0ed6-4e4c-98b0-81ad8d09e211","added_by":"auto","created_at":"2024-07-13 02:33:03","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1059151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePresence and absence of virulence genes in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Lm\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-BD-CHAMPS-01.\u003c/strong\u003e \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 was compared with clonal complexes from lineage I and II (x-axis). The y-axis represents the function of virulence genes. The data for the presence/absence of virulence genes was generated using BIGSdb server.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/364843a346536574b776ed8f.jpeg"},{"id":60200433,"identity":"02975dbe-a8ca-4f01-a23a-2a11bd3b71b6","added_by":"auto","created_at":"2024-07-13 02:33:04","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":279846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMinimum Spanning tree demonstrating the divergence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLm\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-BD-CHAMPS-01 with other \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLm\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esequences. \u003c/strong\u003eThe MST was built with 695 \u003cem\u003eLm\u003c/em\u003ecgMLST sequences available in the BIGSdb-LM database. \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 resides within the circle in the left figure. Zooming in on the circle shows the location of \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 (CT11424) with respect to the nearest isolate. \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 is 484 alleles different from the closest isolate (CT5635), which is a clinical isolate.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/5df0a8d1e2566617689bb5cf.jpeg"},{"id":60200436,"identity":"d3af831b-6c7a-440a-b181-966d8925d965","added_by":"auto","created_at":"2024-07-13 02:33:04","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":283197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSNP based Phylogenetic analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLm\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-BD-CHAMPS-01. \u003c/strong\u003eSNP based maximum likelihood tree indicating \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 (CC1 Bangladesh 2022 Human) is the most distant among all the CC1. \u003cem\u003eListeria innocua\u003c/em\u003e was used as an outlier. The tree was visualized in iTOL.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/fb7fd03751453fcc00407ee8.jpeg"},{"id":75930547,"identity":"2956fa42-a121-40f3-85b9-7468793f99c5","added_by":"auto","created_at":"2025-02-10 16:13:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3329555,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/a9c44be4-18a0-42b6-9c83-a320a543edd1.pdf"},{"id":60201006,"identity":"d8dac804-a70d-40cf-881e-869f68797aa4","added_by":"auto","created_at":"2024-07-13 02:41:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":293479,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/707bd18493e230d8346b6dd4.docx"},{"id":60200434,"identity":"d1735636-2cd7-4575-bf52-3b0b3f877438","added_by":"auto","created_at":"2024-07-13 02:33:04","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":74205,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/d9b5db6b4be01f563b69198b.xlsx"},{"id":60200435,"identity":"cf820ab9-57ed-4ca9-8014-e06fec287645","added_by":"auto","created_at":"2024-07-13 02:33:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12724,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/8bdff0769a01225a738e51cd.xlsx"},{"id":60201007,"identity":"77d6c1b9-b7ed-4e2f-a5d5-04421296f93d","added_by":"auto","created_at":"2024-07-13 02:41:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8796,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4510467/v1/f5dbd7616e96dc71f6678b92.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hyper virulent Core Genome Multilocus Sequence Type CT 11424 of Listeria monocytogenes isolate causing stillbirth in Bangladesh","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eListeria monocytogenes\u003c/em\u003e (\u003cem\u003eLm\u003c/em\u003e) infection in humans and ruminants leads to septicemia, gastroenteritis, and central nervous system (CNS) infections (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). \u003cem\u003eLm\u003c/em\u003e multiplies intracellularly and is capable of cell-to-cell transmission, avoiding the extracellular space (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). \u003cem\u003eLm\u003c/em\u003e is vertically transmitted from pregnant women to fetuses or neonates by crossing the placenta before delivery (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The infection is 20 times more common in pregnancy than in non-pregnant women, and 27% of all infections with \u003cem\u003eLm\u003c/em\u003e occur in pregnant women. \u003cem\u003eLm\u003c/em\u003e infection during pregnancy can lead to fetal loss in 20% of cases (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Listeriosis during pregnancy is often asymptomatic or presents with mild symptoms. Therefore, many \u003cem\u003eLm\u003c/em\u003e infections during pregnancy remain undiagnosed, especially in low- and middle-income countries like Bangladesh.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLm\u003c/em\u003e is commonly found in the environment (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). This pathogen has been reported in the feces of sheep/goats, cattle (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), dogs, rats (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), chickens, pigeons (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), and crows (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), as well as in cloacal swabs of chickens and gulls (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These animals are potential sources of contamination for both raw and processed food consumed by humans. In developed nations, outbreaks of \u003cem\u003eLm\u003c/em\u003e infection have been linked to various food sources, including meat, fish, mixed dishes, vegetables, juices, and dairy products (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). \u003cem\u003eLm\u003c/em\u003e contamination in market and restaurant produce, such as cabbage, corn, carrots, lettuce, cucumbers, parsley, and salad mixes, is well reported (\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). \u003cem\u003eLm\u003c/em\u003e enters food processing facilities through contaminated raw ingredients, making it a significant food safety concern. Certain strains can persist for extended periods, acting as constant sources of cross-contamination due to their ability to adhere to abiotic surfaces. Eradicating \u003cem\u003eLm\u003c/em\u003e, even with the most stringent safety protocols, can be challenging (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLm\u003c/em\u003e is classified into four distinct evolutionary lineages denoted I, II, III, and IV, with most isolates belonging to I and II (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Serotypes 1/2b and 4b of lineage I are responsible for 95% of listeriosis cases in humans, with 4b being the predominant serotype among clinical isolates and outbreaks (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Major clonal complexes (CC) CC1, CC2, CC4, and CC6 of lineage I are associated with clinical listeriosis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). As of July 2023, there are few publications available regarding the detection of \u003cem\u003eLm\u003c/em\u003e from Bangladesh (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). \u003cem\u003eLm\u003c/em\u003e has been detected in goat/sheep slaughterhouse environments, sick goats, frozen shrimp, and locally manufactured cosmetics. The only reported clinical listeriosis case was from a female cancer patient in Bangladesh (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). All of these studies used culture-based detection except for the most recent one, which used molecular detection by PCR. We isolated \u003cem\u003eLm\u003c/em\u003e from postmortem specimens of a stillbirth collected through Child Health and Mortality Prevention Surveillance (CHAMPS) using both microbial culture and PCR (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Here, we report on an \u003cem\u003eLm\u003c/em\u003e-mediated stillbirth as well as the only clinical \u003cem\u003eLm\u003c/em\u003e isolate reported from Bangladesh, using next-generation sequencing to understand the molecular characteristics and virulence factors of the \u003cem\u003eLm\u003c/em\u003e isolate.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy settings\u003c/h2\u003e \u003cp\u003eThe CHAMPS is a multi-country project operating in the Rajbari and Faridpur districts of Bangladesh. The project has implemented a minimally invasive tissue sampling (MITS) technique for collecting postmortem specimens from stillbirths and under-5 deaths in low-income settings. Clinical, demographic and laboratory diagnosis information of the deceased along with maternal information are reviewed by a panel of experts termed DeCoDe (Determination of Cause of Death) panel to identify the cause of death. More information regarding the CHAMPS protocols and methods can be found at (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation and identification of\u003c/b\u003e \u003cb\u003eLm\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMicrobial culture was performed from postmortem blood and cerebrospinal fluid (CSF) specimens. The infectious agents were identified using the Vitek-2 system by Biomerieux (France). The antimicrobial sensitivity was determined using the Kirby-Bauer disk diffusion susceptibility test following CLSI guidelines (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The isolate was named \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01. Additionally, \u003cem\u003eLm\u003c/em\u003e DNA was detected from the blood specimen using TaqMan Array Card based Real-time PCR system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWhole Genome Sequencing\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 DNA was extracted from a pure culture using the DNeasy Blood and Tissue Kits with a gram-positive pretreatment (Qiagen, Germany). DNA library for whole genome sequencing was prepared from 1 ng of DNA with Nextera XT DNA Library Preparation Kit (Illumina Inc, USA). The sequencing procedure was carried out on the MiSeq platform (Illumina Inc, USA), employing the mid/high output reagent cartridge v2/3 (502 cycles) with standards 251-bp paired-end reads (Illumina Inc, United States). All experiments were performed following the manufacturer's guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGenome assembly and characterization\u003c/h2\u003e \u003cp\u003eGenome assembly and annotation were performed using SPAdes version (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and RAST server (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), respectively. More details of bioinformatics tools are available in supplementary data (Supplementary Table\u0026nbsp;1). The Bacterial Isolate Genome Sequence Database (BIGSdb) for the \u003cem\u003eLm\u003c/em\u003e database (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) was used to deduce the seven-gene MLST profile (ST), the cgMLST analysis using 1748 core genes (CT) and clonal complex in silico. Additionally, the BIGSdb server was also used to screen for the presence/absence of virulence genes, antimicrobial resistance determinants, stress survival islands and disinfectants resistance genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMinimum spanning and SNP-based Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eA Minimum spanning tree (MST) was constructed using GrapeTree software (Kruskal's algorithm) to visualize the core genomic relationships among all \u003cem\u003eLm\u003c/em\u003e isolates (n\u0026thinsp;=\u0026thinsp;695) that had cgMLST profile ID (Supplementary Table\u0026nbsp;2), which were obtained from BIGSdb-\u003cem\u003eLm\u003c/em\u003e database (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The tree was visualized using the GrapeTree server. For phylogenetic analysis, SNP-based phylogeny was constructed using the CFSAN pipeline (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). \u003cem\u003eLm\u003c/em\u003e sequences from evolutionary lineage 1 and human origin were selected. For comparison, 1 food isolate was also included (Supplementary Table\u0026nbsp;2). F2635_\u003cem\u003eLm\u003c/em\u003e was used as a reference genome and \u003cem\u003eListeria innocua\u003c/em\u003e was used as an outlier. IQtree web server was used to build a maximum likelihood tree from the alignment file (without any insertion and deletion) using the TVMe\u0026thinsp;+\u0026thinsp;ASC substitution model. The draft genome sequence data are available in the Sequence Read Archive at National Center for Biotechnology Information with accession number SRR28678198.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eClinical narrative\u003c/h2\u003e \u003cp\u003eCHAMPS project has been operating in Baliakandi, Bangladesh since September 2017. As of December 2023, a total of 848 MITS have been conducted from stillbirths and postmortem under-5 deaths to determine the cause of death. On 25 January 2022, \u003cem\u003eLm\u003c/em\u003e was isolated from one case (n\u0026thinsp;=\u0026thinsp;1 of 848) during the study period. The 22-year-old pregnant woman from Baliakandi at 32-week gestational age was admitted to Bangabandhu Sheikh Mujib Medical College \u0026amp; Hospital (BSMMCH). The household reported maintaining a flock of five pigeons and a single hen. During pregnancy period, the mother reportedly consumed cow milk but boiled. Maternal complaints during pregnancy were severe headaches, blurring of vision, pre-eclampsia and anemia (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). From the mother\u0026rsquo;s recall, she received a total of seven antenatal care visits during this pregnancy. Maternal complaints during admission were intrauterine fetal demise confirmed by ultrasonography, fever, burning sensation during micturition and lower abdominal pain for seven hours. The post-admission examination failed to detect fetal movement and heartbeat. A maternal complete urine examination provided normal findings. In the hospital, the mother was treated with oral ciprofloxacin and intravenous ceftriaxone. After a few hours of hospital admission, a male stillborn baby with breech presentation was delivered by normal vaginal delivery. Following CHAMPS protocol, the family was approached for consent to conduct MITS. The stillborn baby was macerated without any significant gross findings. Specimens were collected within an hour of delivery using MITS technique. Body weight during MITS was recorded at 1820 g, height 45 cm and head circumfluence of 29 cm. CHAMPS laboratory platform isolated \u003cem\u003eLm\u003c/em\u003e from both blood and CSF culture. Resistance was observed against cefotaxime and ceftriaxone (Supplementary Table\u0026nbsp;3). The DeCoDe panel determined the underlying cause of this antepartum stillbirth as intrauterine infection by \u003cem\u003eLm\u003c/em\u003e. The panel also stated maternal infection was the main condition affecting the fetus. To understand the virulence gene landscape and phylogeny of the \u003cem\u003eLm\u003c/em\u003e CHAMPS-BD-01, we used next-generation sequencing to analyze the draft genome sequence.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical features available from the pregnant women and the stillborn infected with \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal features\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eClinical symptoms\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFever before delivery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeadache\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDizziness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePre-eclampsia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalnutrition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBurning sensation during micturition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP systolic, mmHg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP diastolic, mmHg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePulse (bpm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLaboratory diagnosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRandom Blood Sugar (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThyroid-stimulating hormone (\u0026micro;IU/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBsAg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrine Routine Examination\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMaternal Medications during pregnancy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedications\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eIndication\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIron\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnemia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMisoprostol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedical induction of labour\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParacetamol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFever\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAntibiotics\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftriaxone, Ciprofloxacin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStillborn features\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaceration Level\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLevel 1 - Maceration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnthropomorphic measurements during MITS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1820\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeight or Length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMUAC (cm)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHead Circumference (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRight Leg Length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRight Foot Length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e*MUAC\u0026thinsp;=\u0026thinsp;Mid upper arm circumfluence\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLm\u003c/b\u003e \u003cb\u003e-BD-CHAMPS-01 is a novel cgMLST type of\u003c/b\u003e \u003cb\u003eListeria monocytogenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 genome had 13 contigs, a genome size of 2932029 bases, and a 37% GC content. The genome coverage was 127x, with an N50 of 1491565 and an L50 of 1. Prokka annotation found 2873 CDS, 5 rRNA, 1 repeat region, 56 tRNA, and 1 tmRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). MLST and K-mer dependent analysis confirmed the sequence as \u003cem\u003eLm\u003c/em\u003e. Using the Bigsdb tool, the isolate was identified as ST 308 (MLST), CC1 (clonal complex), lineage I, sub-lineage SL150, and PCR serogroup 4b. cgMLST analysis declared the isolates as a novel type, and a new cgMLST profile identification number, CT 11424, was provided.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eVirulence and stress-associated genes\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 was found to contain 65 virulence genes. Genes \u003cem\u003elap\u003c/em\u003e, \u003cem\u003edltA\u003c/em\u003e, \u003cem\u003efbpA\u003c/em\u003e, \u003cem\u003einlJ\u003c/em\u003e, \u003cem\u003elapB\u003c/em\u003e, \u003cem\u003eactA\u003c/em\u003e and \u003cem\u003einlF\u003c/em\u003e were found which are known to aid bacterial adhesion. Invasion-related genes \u003cem\u003einlA\u003c/em\u003e, \u003cem\u003einlB\u003c/em\u003e, \u003cem\u003einlE\u003c/em\u003e, \u003cem\u003evip\u003c/em\u003e, \u003cem\u003eant\u003c/em\u003e, \u003cem\u003eiap\u003c/em\u003e, \u003cem\u003elgt\u003c/em\u003e, and \u003cem\u003elepA\u003c/em\u003e without any stop codon (full length) were also identified. The isolate harbored several genes to survive inside intracellular environment including \u003cem\u003ello\u003c/em\u003e (Listeriolysin O), \u003cem\u003eplcA\u003c/em\u003e, \u003cem\u003eplcB\u003c/em\u003e (enhance phagosomal membrane disruption), \u003cem\u003eprsA2\u003c/em\u003e, \u003cem\u003elsp\u003c/em\u003e, \u003cem\u003esvpA\u003c/em\u003e. For intracellular growth \u003cem\u003ehpt\u003c/em\u003e, \u003cem\u003eIpA1\u003c/em\u003e and \u003cem\u003eoppA\u003c/em\u003e genes (survival inside macrophage) were detected. \u003cem\u003eLm\u003c/em\u003e 4b serotype-specific gene cassette \u003cem\u003egltA-gltB\u003c/em\u003e was present. These genes were found arranged inside \u003cem\u003eListeria\u003c/em\u003e pathogenicity island (LIPI-1) 1 (\u003cem\u003ehly, mpl, actA, plcB, plcA, prfA\u003c/em\u003e) and LIPI-3 (\u003cem\u003ellsA\u003c/em\u003e, \u003cem\u003ellsG\u003c/em\u003e, \u003cem\u003ellsH\u003c/em\u003e, \u003cem\u003ellsX\u003c/em\u003e, \u003cem\u003ellsB\u003c/em\u003e, \u003cem\u003ellsY\u003c/em\u003e, \u003cem\u003ellsD\u003c/em\u003e, \u003cem\u003ellsP\u003c/em\u003e)(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Gene synteny of these two pathogenicity islands was similar when compared with the F2635 reference strain (Supplementary Fig.\u0026nbsp;1). Listeria genomic island locus, LGI-2_\u003cem\u003eLM\u003c/em\u003eOSA2310 and LGI-2_\u003cem\u003eLM\u003c/em\u003eOSA2320 were found \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01. Other stress adaptation genes include \u003cem\u003eSigmaB\u003c/em\u003e operon, \u003cem\u003egadD2\u003c/em\u003e and \u003cem\u003egadT2\u003c/em\u003e (stress adaptation), \u003cem\u003ecspB\u003c/em\u003e gene (cold adaptation), \u003cem\u003ebetL\u003c/em\u003e (salt tolerance), \u003cem\u003egtcA\u003c/em\u003e (rhamnose operon gene) and 29 motility-related genes. The presence or absence of genes was illustrated using the isolates included in the phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The analysis showed that \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 has the same virulence and stress adaptation profile as the all CC1 isolates, except one \u003cem\u003eLm\u003c/em\u003e from Chile. It also contains multidrug resistance transporters \u003cem\u003emdrM\u003c/em\u003e. The isolate contains one intact phage (PHAGE_Lister_vB_\u003cem\u003eLm\u003c/em\u003eoS_293; GenBank accession number NC_02892). The intact phage is 42.4kb long and has 64 total proteins. From this draft genome of \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01, we were unable to find any plasmid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic relatedness and phylogenetic analysis of\u003c/b\u003e \u003cb\u003eLm\u003c/b\u003e \u003cb\u003eisolate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe calculated the MST, to visualize the genomic relatedness of \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 in comparison to cgMLSTs of \u003cem\u003eLm\u003c/em\u003e from different sources. Analysis was performed with the representative sequence of each 695 cgMLST available in Bigsdb (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among 1748 core genes, 1746 were detected from the \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01; only two hypothetical genes were missing. In MST, the isolate was related to clinical origin cgMLST CT 5635 with 484 allelic differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eLm\u003c/em\u003e CT 5635 was located in the center of the cluster. Most genetically adjacent of \u003cem\u003eLm\u003c/em\u003e CT5635, was from a food source (CT 2830, 64 allelic difference) and animal feed (CT6145, 23 allelic difference). The other connected clinical isolate was CT2872 with 29 allelic differences.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the evolutionary origin of \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 phylogenetic analysis was performed with selected sequences of lineage I from global isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The log-likelihood of the tree was \u0026minus;\u0026thinsp;360.7421. The \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 diverged from \u003cem\u003eLm\u003c/em\u003e reported from the food source of the USA in 1985. Indian \u003cem\u003eLm\u003c/em\u003e isolates reported from 2004 to 2014 all are phylogenetically similar and this cluster was adjacent to \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01. Among all the CC1 isolates, \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 was the most distant in evolution, acquiring more changes in it (most distant on the tree scale at 0.63).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we report a highly virulent \u003cem\u003eLm\u003c/em\u003e strain causing stillbirth in Bangladesh. Phylogenetic analysis based on cgMLST identified the \u003cem\u003eLm\u003c/em\u003e as a novel cgMLSTCT 11424. The clinical narrative aligns with the genomic landscape showing all major virulence genes required for crossing the placental barrier through inter-cellular movement.\u003c/p\u003e \u003cp\u003eListeriosis is a rare illness usually linked with consumption of raw unpasteurized milk or raw food causing severe consequences during pregnancy. Globally, reporting of \u003cem\u003eLm\u003c/em\u003e outbreak is uncommon, reports of at least two cases from same place are considered as outbreak (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). A major reason for unreported listeriosis is the lack of severe symptoms in non-pregnant individuals. As a result, laboratory diagnosis is limited especially in low- and middle-income countries. However, during pregnancy, \u003cem\u003eLm\u003c/em\u003e invades the placenta where inflammatory and immune-regulatory T-cell response is modulated to promote maternal tolerance to the semi-allogeneic fetus. This leads to increased host susceptibility to \u003cem\u003eLm\u003c/em\u003e infection. However, \u003cem\u003eLm\u003c/em\u003e infection triggers innate immune response which ultimately triggers host immune response in the placenta leading to fetal damage or demise (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 isolate is a virulent strain indicated by the presence of listeriolysin O-expressing gene \u003cem\u003ello\u003c/em\u003e (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In addition, it possesses of all genes required for intracellular survival and intercellular movement to cross the placental barrier. The presence of full-length \u003cem\u003elap\u003c/em\u003e (\u003cem\u003eListeria\u003c/em\u003e adhesion protein) mediates adhesion of with host cell and gene \u003cem\u003einlA\u003c/em\u003e (internalin genes) facilitates its ability to cross the intestinal as well as placental barrier (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In mice models, it has been shown that the \u003cem\u003einlF\u003c/em\u003e gene enhances the early stage of infection by modulating host inflammatory responses (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Recently, it has been reported that the \u003cem\u003eactA\u003c/em\u003e gene enhances the shedding of \u003cem\u003eLm\u003c/em\u003e several by magnitudes which leads to better transmission from host to environment (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). It also expresses multidrug resistance transporter \u003cem\u003emdrM\u003c/em\u003e which triggers host immunity leading to activation of the placental immune system against the fetus. It expresses CspB protein which controls stress adaptation and virulence phenotypes such as oxidative stress, biofilm production, hemolysis, cell evasion and intracellular growth (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Although highly virulent, the \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 remains sensitive to a wide range of antibiotics. Resistance was only observed for those against which \u003cem\u003eLm\u003c/em\u003e possessed natural resistance (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 also belongs to lineage I and serotype 4b which is one of the predominant serotypes reported to cause listeriosis in humans and ruminants. \u003cem\u003eLm\u003c/em\u003e strains from lineage I and serotype 4b have shown the lowest diversity among the other lineages; and lowest levels of recombination among the lineages indicating genomic stability (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). This is also evident from the detection of similar isolates from 2004 to 2014 in India, which shares a border with Bangladesh. In addition, the possible lack of mobile genetic elements like plasmids indicates the outbreaks by \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 like isolates are treatable with available antibiotics. The prophage PHAGE_Lister_vB_\u003cem\u003eLm\u003c/em\u003eoS_293 has been reported recently from South Africa from both pathogenic \u003cem\u003eLm\u003c/em\u003e and less-pathogenic \u003cem\u003eL. innocua\u003c/em\u003e (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). However, the exact role of this prophage in \u003cem\u003eLm\u003c/em\u003e virulence or evolution is yet to be discovered.\u003c/p\u003e \u003cp\u003eThe ST308 was reported previously from Chinese fresh aquatic products (2011\u0026ndash;2016) and confiscated foods from non-European passengers (2012\u0026ndash;2013) at a Spanish airport (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Due to the lack of reporting from Bangladesh and the low evolutionary rate of \u003cem\u003eLm\u003c/em\u003e isolates, it can be assumed that the isolate \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 has been also circulating in the population for a long time. The antibiotic sensitivity pattern and health care-seeking approach (7 visits) of the pregnant women discussed here showed that the antenatal care system was unable to identify the infection. Timely diagnosis could have prevented this stillbirth using common antibiotics such as ampicillin. This indicates the importance of microbiological diagnosis during antenatal care. \u003cem\u003eLm\u003c/em\u003e serotype 4b has been reported from an outbreak of urban poultry flocks in US (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The same serotype has been also reported from fecal samples of hen in Germany (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Although out of scope of this analysis, the presence of domestic pigeon and hen in the household could be a potential source of \u003cem\u003eLm\u003c/em\u003e infecting the pregnant women.\u003c/p\u003e \u003cp\u003eIn summary, the \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 isolate is a genetically stable and highly invasive strain likely circulating at least in the community of our study area. Circulation of such virulent strains indicates the necessity to consider \u003cem\u003eLm\u003c/em\u003e infection as a notifiable disease for diagnostic facilities. Backyard poultry along with free living birds could be investigated as a potential carrier of \u003cem\u003eLm\u003c/em\u003e in Bangladeshi households. Dietary guidelines and exposure to animals adapted for local food habit and local culture should be developed considering potential \u003cem\u003eLm\u003c/em\u003e infection during pregnancy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study presents the identification and characterization of a highly virulent \u003cem\u003eListeria monocytogenes\u003c/em\u003e strain, \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01, causing stillbirth in Bangladesh. Our findings highlight the presence of key virulence genes that enable the pathogen to cross the placental barrier, posing significant risks during pregnancy. The genomic stability and evolutionary insights suggest this strain's prolonged history within the region. Importantly, the sensitivity of \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 to common antibiotics underscores the potential for prevention and treatment through timely diagnosis and quality antenatal care. The findings also suggest that backyard poultry, such as hens, and free-living birds, such as pigeons, could be potential reservoirs, necessitating further investigation. The development of locally adapted dietary and animal exposure guidelines to prevent \u003cem\u003eLm\u003c/em\u003e infections during pregnancy will be useful. This report contributes valuable insights to the field of public health microbiology as well as maternal and neonatal health. The findings suggest advocacy for enhanced surveillance and preventive measures against \u003cem\u003eListeria\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBD \u0026ndash; Bangladesh\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCC- Clonal Complex\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ecgMLST - core genome Multilocus sequence typing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCHAMPS -\u0026nbsp;Child health and mortality prevention surveillance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCLSI -\u0026nbsp;Clinical and laboratory standards institute\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCNS- Central Nervous System\u003c/p\u003e\n\u003cp\u003eCSF \u0026ndash; Cerebrospinal fluid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDeCoDe -\u0026nbsp;Determination of cause of death\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLIPI -\u0026nbsp;\u003cem\u003eListeria\u003c/em\u003e pathogenicity island\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLm\u003c/em\u003e - \u003cem\u003eListeria monocytogenes\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMITS -\u0026nbsp;Minimally invasive tissue sampling\u003c/p\u003e\n\u003cp\u003eMLST \u0026ndash; Multilocus sequence typing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMST - Minimum spanning tree\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThe CHAMPS study was conducted following the approval of the research review committee (icddr,b protocol number PR-16082) and the ethical review committee of icddr,b. All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the legal guardians or family members of all stillbirths or deceased children included in the study. The consent process ensured that participants were fully informed about the purpose, procedures, risks, and benefits of the study, and their participation was entirely voluntary. Written consent forms were signed by the legal guardians or family members before any study-related procedures were initiated. No animal was included in this investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe draft genome sequence data are available in the Sequence Read Archive at National Center for Biotechnology Information (NCBI) with accession number SRR28678198.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe study was performed as part of the Child Health and Mortality Prevention Surveillance study, funded by the Bill and Melinda Gates Foundation (grant ID OPP1126780).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u0026nbsp;\u003c/strong\u003eM.A. drafted the manuscript and guided the overall analysis. M.S.I., M.I.J., and A.S.D. generated the whole genome sequencing data, conducted the bioinformatics analysis and generated the figures/tables. A.R., K.M.I., and M.Z.H. were responsible for collecting and analyzing the clinical data and generated the tables. A.I.C. gathered the demographic information. D.A. isolated the bacteria. S.E.A. and E.S.G. provided overall supervision for the CHAMPS project, secured funding, reviewed the manuscript. M.R. supervised the laboratory work and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe acknowledge the contribution of the International Centre for Diarrheal Diseases Research, Bangladesh (icddr,b), for organizational support with the work. icddr,b is grateful to the Governments of Bangladesh, and Canada for providing core/unrestricted support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' information:\u003c/strong\u003e\u003csup\u003e1\u003c/sup\u003eicddr,b, Mohakhali, Dhaka-1212, \u003csup\u003e2\u003c/sup\u003eInfectious Disease Epidemiology, Johns Hopkins University, Baltimore, MD, USA, \u003csup\u003e3\u003c/sup\u003eDivision of Genomics \u0026amp; Transcriptomics, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllerberger F, Huhulescu S. Pregnancy related listeriosis: treatment and control. Expert Rev Anti Infect Ther. 2015 Mar 4;13(3):395\u0026ndash;403. https://doi.org/10.1586/14787210.\u003cbr\u003e 2015.1003809. \u003c/li\u003e\n\u003cli\u003eCorr SC, O\u0026rsquo;Neill LAJ. \u003cem\u003eListeria monocytogenes\u003c/em\u003e infection in the face of innate immunity. Cell Microbiol. 2009 May;11(5):703\u0026ndash;9. https://doi.org/10.1111/j.1462-5822.2009.01294.x.\u003c/li\u003e\n\u003cli\u003eCharlier C, Perrodeau \u0026Eacute;, Leclercq A, Cazenave B, Pilmis B, Henry B, et al. Clinical features and prognostic factors of listeriosis: the MONALISA national prospective cohort study. Lancet Infect Dis. 2017 May;17(5):510\u0026ndash;9. https://doi.org/10.1016/S1473-3099(16)30521-7.\u003c/li\u003e\n\u003cli\u003eGrigore-Gurgu L, Bucur FI, Mihalache OA, Nicolau AI. Comprehensive Review on the Biocontrol of \u003cem\u003eListeria monocytogenes\u003c/em\u003e in Food Products. Foods (Basel, Switzerland). 2024 Feb 28;13(5). https://doi.org/10.3390/foods13050734.\u003c/li\u003e\n\u003cli\u003eSchoder D, Guldimann C, M\u0026auml;rtlbauer E. Asymptomatic Carriage of \u003cem\u003eListeria monocytogenes \u003c/em\u003eby Animals and Humans and Its Impact on the Food Chain. Foods (Basel, Switzerland). 2022 Nov 1;11(21). https://doi.org/10.3390/foods11213472.\u003c/li\u003e\n\u003cli\u003eObaidat MM, Stringer AP. Prevalence, molecular characterization, and antimicrobial resistance profiles of \u003cem\u003eListeria monocytogenes, Salmonella enterica\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e O157:H7 on dairy cattle farms in Jordan. J Dairy Sci. 2019 Oct;102(10):8710\u0026ndash;20. https://doi.org/10.3168/jds.2019-16461.\u003c/li\u003e\n\u003cli\u003eIida T, Kanzaki M, Maruyama T, Inoue S, Kaneuchi C. Prevalence of \u003cem\u003eListeria monocytogenes\u003c/em\u003e in intestinal contents of healthy animals in Japan. J Vet Med Sci. 1991 Oct;53(5):873\u0026ndash;5. https://doi.org/10.1292/jvms.53.873.\u003c/li\u003e\n\u003cli\u003eHellstr\u0026ouml;m S, Kiviniemi K, Autio T, Korkeala H. \u003cem\u003eListeria monocytogenes\u003c/em\u003e is common in wild birds in Helsinki region and genotypes are frequently similar with those found along the food chain. J Appl Microbiol . 2008 Mar;104(3):883\u0026ndash;8. https://doi.org/10.1111/j.1365-2672.2007.03604.x.\u003c/li\u003e\n\u003cli\u003eWeber A, Potel J, Sch\u0026auml;fer-Schmidt R. The occurrence of \u003cem\u003eListeria monocytogenes\u003c/em\u003e in fecal samples of pigeons. Berl Munch Tierarztl Wochenschr. 1995 Jan;108(1):26\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eYoshida T, Sugimoto T, Sato M, Hirai K. Incidence of \u003cem\u003eListeria monocytogenes\u003c/em\u003e in wild animals in Japan. J Vet Med Sci. 2000 Jun;62(6):673\u0026ndash;5.https://doi.org/10.1292/jvms.\u003cbr\u003e 62.673.\u003c/li\u003e\n\u003cli\u003ePohjola L, Nyk\u0026auml;senoja S, Kivist\u0026ouml; R, Soveri T, Huovilainen A, H\u0026auml;nninen ML, et al. Zoonotic Public Health Hazards in Backyard Chickens. Zoonoses Public Health. 2016 Aug;63(5):420\u0026ndash;30. https://doi.org/10.1111/zph.12247.\u003c/li\u003e\n\u003cli\u003eSy K V, Murray MB, Harrison MD, Beuchat LR. Evaluation of gaseous chlorine dioxide as a sanitizer for killing \u003cem\u003eSalmonella, Escherichia coli\u003c/em\u003e O157:H7, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, and yeasts and molds on fresh and fresh-cut produce. J Food Prot. 2005 Jun;68(6):1176\u0026ndash;87. https://doi.org/10.4315/0362-028X-68.6.1176.\u003c/li\u003e\n\u003cli\u003eZhu Q, Gooneratne R, Hussain MA. \u003cem\u003eListeria monocytogenes\u003c/em\u003e in Fresh Produce: Outbreaks, Prevalence and Contamination Levels. Foods (Basel, Switzerland). 2017 Mar 9;6(3). https://doi.org/10.3390/foods6030021.\u003c/li\u003e\n\u003cli\u003eSkalina L, Nikolajeva V. Growth potential of \u003cem\u003eListeria monocytogenes\u003c/em\u003e strains in mixed ready-to-eat salads. Int J Food Microbiol. 2010 Dec 15;144(2):317\u0026ndash;21.https://doi.org/\u003cbr\u003e 10.1016/j.ijfoodmicro.2010.10.001.\u003c/li\u003e\n\u003cli\u003eHou W, Ma Y, Zhang C, Zhao W, Zhao S, Wang P, et al. Investigation on the inactivation effect and mechanism of \u003cem\u003eListeria monocytogenes\u003c/em\u003e in fresh-cut cucumber during storage by ultrasound combined with sodium hypochlorite. Ultrason Sonochem. 2023 Dec;101:106706.https://doi.org/10.1016/j.ultsonch.2023.106706.\u003c/li\u003e\n\u003cli\u003eStessl B, Fricker M, Fox E, Karpiskova R, Demnerova K, Jordan K, et al. Collaborative survey on the colonization of different types of cheese-processing facilities with \u003cem\u003eListeria monocytogenes. \u003c/em\u003eFoodborne Pathog Dis. 2014 Jan;11(1):8\u0026ndash;14. https://doi.org/10.1089/\u003cbr\u003e fpd.2013.1578.\u003c/li\u003e\n\u003cli\u003eOrsi RH, Bakker HC den, Wiedmann M. \u003cem\u003eListeria monocytogenes\u003c/em\u003e lineages: Genomics, evolution, ecology, and phenotypic characteristics. Int J Med Microbiol. 2011 Feb;301(2):79\u0026ndash;96. https://doi.org/10.1016/j.ijmm.2010.05.002.\u003c/li\u003e\n\u003cli\u003eVines * , Bala Swaminathan A. Identification and Characterization of Nucleotide Sequence Differences in Three Virulence-Associated Genes of \u003cem\u003eListeria monocytogenes\u003c/em\u003e Strains Representing Clinically Important Serotypes. Curr Microbiol. 1998 May 1;36(5):309\u0026ndash;18. https://doi.org/10.1007/s002849900315.\u003c/li\u003e\n\u003cli\u003eChenal-Francisque V, Lopez J, Cantinelli T, Caro V, Tran C, Leclercq A, et al. Worldwide Distribution of Major Clones of \u003cem\u003eListeria monocytogenes\u003c/em\u003e. Emerg Infect Dis. 2011 Jun;17(6):1110\u0026ndash;2. https://doi.org/10.3201/eid1706.101778.\u003c/li\u003e\n\u003cli\u003eNoor R, Hasan MF, Rahman MM. Molecular characterization of the virulent microorganisms along with their drug-resistance traits associated with the export quality frozen shrimps in Bangladesh. Springerplus. 2014 Dec 26;3(1):469. https://doi.org/10.\u003cbr\u003e 1186/2193-1801-3-469.\u003c/li\u003e\n\u003cli\u003ePaul P, Faruque MR, Rahman MK, Das P, Chowdhury MYE. Study on bacterial pathogens through multiplex polymerase chain reaction system and their antimicrobial resistance pattern in goats presumed with fever and/or diarrhea. Vet World. 2021 May 6;1080\u0026ndash;92. https://doi.org/10.14202/vetworld.2021.1080-1092.\u003c/li\u003e\n\u003cli\u003eNusrat N, Ahmad Zahra M, Ahmed A, Haque F. Assessment of potential pathogenic bacterial load and multidrug resistance in locally manufactured cosmetics commonly used in Dhaka metropolis. Sci Rep. 2023 May 13;13(1):7787. https://doi.org/10.1038/s41598-023-34782-9.\u003c/li\u003e\n\u003cli\u003eNahid MA, Sadique T, Mazumder R, Abdullah A, Sami AB, Rahaman MA, et al.\u003cem\u003e Listeria monocytogenes \u003c/em\u003einfection in a 56-year-old female cancer patient: a case report. JMM Case Reports. 2015 Aug 1;2(4).https://doi.org/10.1099/jmmcr.0.000076.\u003c/li\u003e\n\u003cli\u003eSultana N, Pervin M, Sultana S, Islam M, Mostaree M, Khan MAHNA. Pathological study and molecular detection of zoonotic diseases in small ruminants at slaughter houses in Mymensingh, Bangladesh. Vet World. 2022 Sep 5;2119\u0026ndash;30. https://doi.org/10.14202\u003cbr\u003e /vetworld.2022.2119-2130.\u003c/li\u003e\n\u003cli\u003eCHAMPS - We Build Knowledge to Save Children\u0026rsquo;s Lives. https://champshealth.org/.Accessed 23May 2024.\u003c/li\u003e\n\u003cli\u003eClinical \u0026amp; Laboratory Standards Institute: CLSI Guidelines. https://clsi.org/. Accessed 23 May 2024.\u003c/li\u003e\n\u003cli\u003ePrjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes De Novo Assembler. Curr Protoc Bioinforma. 2020 Jun 19;70(1). https://doi.org/10.1002/cpbi.102.\u003c/li\u003e\n\u003cli\u003eAziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008 Dec 8;9(1):75. https://doi.org/10.1186/1471-2164-9-75.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eListeria monocytogenes\u003c/em\u003e.https://bigsdb.pasteur.fr/listeria/. Accessed 23May 2024.\u003c/li\u003e\n\u003cli\u003eZhou Z, Alikhan NF, Sergeant MJ, Luhmann N, Vaz C, Francisco AP, et al. GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens. Genome Res. 2018 Sep;28(9):1395\u0026ndash;404. https://doi.org/10.1101/216788.\u003c/li\u003e\n\u003cli\u003eBertels F, Silander OK, Pachkov M, Rainey PB, van Nimwegen E. Automated Reconstruction of Whole-Genome Phylogenies from Short-Sequence Reads. Mol Biol Evol. 2014 May;31(5):1077\u0026ndash;88.https://doi.org/10.1093/molbev/msu088.\u003c/li\u003e\n\u003cli\u003eWiktorczyk-Kapischke N, Skowron K, Wałecka-Zacharska E. Genomic and pathogenicity islands of \u003cem\u003eListeria monocytogenes\u003c/em\u003e\u0026mdash;overview of selected aspects. Front Mol Biosci. 2023 Jun 14;10. https://doi.org/10.3389/fmolb.2023.1161486.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eListeria\u003c/em\u003e Outbreaks. https://www.cdc.gov/listeria/outbreaks/index.html. Accessed 23May 2024.\u003c/li\u003e\n\u003cli\u003eAgbayani G, Wachholz K, Murphy SP, Sad S, Krishnan L. Type I interferons differentially modulate maternal host immunity to infection by \u003cem\u003eListeria monocytogenes\u003c/em\u003e and \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium during pregnancy. Am J Reprod Immunol. 2019 Jan 4;81(1). https://doi.org/10.1111/aji.13068.\u003c/li\u003e\n\u003cli\u003eCossart P, Vicente MF, Mengaud J, Baquero F, Perez-Diaz JC, Berche P. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect Immun. 1989 Nov;57(11):3629\u0026ndash;36. https://doi.org/10.1128/iai.57\u003cbr\u003e .11.3629-3636.1989.\u003c/li\u003e\n\u003cli\u003eDrolia R, Bhunia AK. Crossing the Intestinal Barrier via \u003cem\u003eListeria\u003c/em\u003e Adhesion Protein and Internalin A. Trends Microbiol. 2019 May;27(5):408\u0026ndash;25. https://doi.org/10.1016/j.tim.\u003cbr\u003e 2018.12.007.\u003c/li\u003e\n\u003cli\u003eBurkholder KM, Bhunia AK. Listeria monocytogenes Uses Listeria Adhesion Protein (LAP) To Promote Bacterial Transepithelial Translocation and Induces Expression of LAP Receptor Hsp60. Infect Immun. 2010 Dec;78(12):5062\u0026ndash;73. https://doi.org/10.1128/iai.\u003cbr\u003e 00516-10.\u003c/li\u003e\n\u003cli\u003eLing Z, Zhao D, Xie X, Yao H, Wang Y, Kong S, et al. inlF Enhances Listeria monocytogenes Early-Stage Infection by Inhibiting the Inflammatory Response. Front Cell Infect Microbiol. 2022 Feb 10;11. https://doi.org/10.3389/fcimb.2021.748461.\u003c/li\u003e\n\u003cli\u003eTravier L, Guadagnini S, Gouin E, Dufour A, Chenal-Francisque V, Cossart P, et al. ActA Promotes Listeria monocytogenes Aggregation, Intestinal Colonization and Carriage. Monack DM, editor. PLoS Pathog. 2013 Jan 31;9(1):e1003131. https://doi.org/10.\u003cbr\u003e 1371/journal.ppat.1003131.\u003c/li\u003e\n\u003cli\u003eMuchaamba F, Stephan R, Tasara T. Listeria monocytogenes Cold Shock Proteins: Small Proteins with A Huge Impact. Microorganisms. 2021 May 14;9(5):1061. https://doi.\u003cbr\u003e org/10.3390/microorganisms9051061.\u003c/li\u003e\n\u003cli\u003eLuque-Sastre L, Arroyo C, Fox EM, McMahon BJ, Bai L, Li F, et al. Antimicrobial Resistance in Listeria Species. Aarestrup FM, Schwarz S, Shen J, Cavaco L, editors. Microbiol Spectr. 2018 Jul 27;6(4). https://doi.org/10.1128/microbiolspec.arba-0031-2017.\u003c/li\u003e\n\u003cli\u003eMatle, Pierneef, Mbatha, Magwedere, Madoroba. Genomic Diversity of Common Sequence Types of Listeria monocytogenes Isolated from Ready-to-Eat Products of Animal Origin in South Africa. Genes (Basel). 2019 Dec 4;10(12):1007. https://doi.org/\u003cbr\u003e 10.3390/genes10121007.\u003c/li\u003e\n\u003cli\u003eMafuna T, Matle I, Magwedere K, Pierneef RE, Reva ON. Comparative Genomics of Listeria Species Recovered from Meat and Food Processing Facilities. Denes TG, editor. Microbiol Spectr. 2022 Oct 26;10(5). https://doi.org/10.1128/spectrum.01189-22.\u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez-L\u0026aacute;zaro D, Ariza-Miguel J, Diez\u0026ndash;Valcarce M, Stessl B, Beutlich J, Fern\u0026aacute;ndez-Natal I, et al. Identification and molecular characterization of pathogenic bacteria in foods confiscated from non-EU flights passengers at one Spanish airport. Int J Food Microbiol. 2015 Sep;209:20\u0026ndash;5. https://doi.org/10.1016/j.ijfoodmicro.2014.10.016.\u003c/li\u003e\n\u003cli\u003eChen M, Cheng J, Wu Q, Zhang J, Chen Y, Xue L, et al. Occurrence, Antibiotic Resistance, and Population Diversity of Listeria monocytogenes Isolated From Fresh Aquatic Products in China. Front Microbiol. 2018 Sep 19;9. https://doi.org/10.3389/\u003cbr\u003e fmicb.2018.02215.\u003c/li\u003e\n\u003cli\u003eCrespo R, Garner MM, Hopkins SG, Shah DH. Outbreak of Listeria monocytogenes in an urban poultry flock. BMC Vet Res. 2013 Oct 11;9:204.https://doi.org/10.1186/1746-6148-9-204\u003c/li\u003e\n\u003cli\u003eWeber A, Potel J, Sch\u0026auml;fer-Schmidt R, Prell A, Datzmann C. Studies on the occurrence of Listeria monocytogenes in fecal samples of domestic and companion animals. Zentralbl Hyg Umweltmed. 1995 Dec;198(2):117\u0026ndash;23\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Listeria monocytogenes, Stillbirth, Bangladesh, Pregnancy, CHAMPS, Mother-to-Child transmission, Pigeon","lastPublishedDoi":"10.21203/rs.3.rs-4510467/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4510467/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eListeria monocytogenes\u003c/em\u003e (\u003cem\u003eLm\u003c/em\u003e) is a foodborne pathogen that can lead to severe pregnancy outcomes. This study reports the clinical and genomic characteristics of a \u003cem\u003eListeria\u003c/em\u003e-mediated stillbirth identified through the Child Health and Mortality Prevention Surveillance (CHAMPS) project in Bangladesh. The \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 isolate was recovered from the blood and cerebrospinal fluid (CSF) of a male stillborn baby. Maternal history, clinical, and demographic data were collected by the CHAMPS surveillance platform. An expert panel evaluated all reports to determine the role of \u003cem\u003eLm\u003c/em\u003e infection in the causal chain of stillbirth. Genomic characterization included multilocus sequence typing (MLST), core genome MLST (cgMLST), serotyping, and the presence or absence of virulence and stress adaptation genes. Genetic divergence and phylogenetic analyses were conducted to determine the relationship with other reported isolates globally.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe isolate \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 was identified as a novel cgMLST CT11424. It belonged to ST 308, Serotype 4b, Clonal Complex 1, and Phylogenetic Lineage 1. Key \u003cem\u003eLm\u003c/em\u003e virulence genes facilitating the crossing of the placental barrier, including full-length \u003cem\u003einlA\u003c/em\u003e, LIPI-1, and LIPI-3, were detected. The isolate was closely related to clinical \u003cem\u003eLm\u003c/em\u003e isolates, as determined by Grapetree based on cgMLST. SNP-based phylogenetic analysis found \u003cem\u003eLm\u003c/em\u003e-BD-CHAMPS-01 to be the most distant from other CC1 isolates in the database. Possible sources of infection included the consumption of contaminated raw vegetables or exposure to pigeons.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe virulence profile of the first genome sequence of clinical \u003cem\u003eLm\u003c/em\u003e from Bangladesh, which also caused stillbirth, underscores the understudied nature of this pathogen in the region. Rural healthcare professionals should be aware of \u003cem\u003eLm\u003c/em\u003e infection risks during pregnancy. Pregnant women should be counseled on the dangers of consuming potentially contaminated raw food and exposure to animals or birds to prevent adverse pregnancy outcomes due to \u003cem\u003eLm\u003c/em\u003e infection.\u003c/p\u003e","manuscriptTitle":"Hyper virulent Core Genome Multilocus Sequence Type CT 11424 of Listeria monocytogenes isolate causing stillbirth in Bangladesh","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-13 02:32:58","doi":"10.21203/rs.3.rs-4510467/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T10:04:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-29T19:41:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-27T17:00:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-27T11:26:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324139365611174317834614414452481121652","date":"2024-06-22T11:08:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171927694045423918987789700675635536054","date":"2024-06-19T13:33:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117395170413660972217123649189080092735","date":"2024-06-19T03:58:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287586790033233793806823259071202704809","date":"2024-06-17T17:38:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64326218517134321881736265328772861083","date":"2024-06-17T10:50:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-17T10:40:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-17T10:36:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-14T15:41:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-14T15:41:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2024-05-31T17:31:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b51faa3-74b1-4ced-bbb6-cfcd913ad3e4","owner":[],"postedDate":"July 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:05:30+00:00","versionOfRecord":{"articleIdentity":"rs-4510467","link":"https://doi.org/10.1186/s12866-024-03650-5","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-02-03 15:58:12","publishedOnDateReadable":"February 3rd, 2025"},"versionCreatedAt":"2024-07-13 02:32:58","video":"","vorDoi":"10.1186/s12866-024-03650-5","vorDoiUrl":"https://doi.org/10.1186/s12866-024-03650-5","workflowStages":[]},"version":"v1","identity":"rs-4510467","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4510467","identity":"rs-4510467","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}