Genome analysis of Streptococcus spp. isolates from animals in pre-antibiotic era with respect to antibiotic susceptibility and virulence gene profiles.

Lyophilized Streptococcus spp. isolates (n = 50) from animal samples submitted to the diagnostic laboratory at the University of Connecticut in the 1940s were revivified to investigate the genetic characteristics using whole-genome sequencing (WGS). The Streptococcus spp. isolates were identified as follows; S. agalactiae (n = 14), S. dysgalactiae subsp. dysgalactiae (n = 10), S. dysgalactiae subsp. equisimils (n = 5), S. uberis (n = 8), S. pyogenes (n = 7), S. equi subsp. zooepidemicus (n = 4), S. oralis (n = 1), and S. pseudoporcinus (n = 1). We identified sequence types (ST) of S. agalactiae, S. dysgalactiae, S. uberis, S. pyogenes, and S. equi subsp. zooepidemicus and reported ten novel sequence types of those species. WGS analysis revealed that none of Streptococcus spp. carried antibiotic resistance genes. However, tetracycline resistance was observed in four out of 15 S. dysgalactiae isolates and in one out of four S. equi subsp. zooepidemicus isolate. This data highlights that antimicrobial resistance is pre-existed in nature before the use of antibiotics. The draft genome sequences of isolates from this study and 426 complete genome sequences of Streptococcus spp. downloaded from BV-BRC and NCBI GenBank database were analyzed for virulence gene profiles and phylogenetic relationships. Different Streptococcus species demonstrated distinct virulence gene profiles, with no time-related variations observed. Phylogenetic analysis revealed high genetic diversity of Streptococcus spp. isolates from the 1940s, and no clear spatio-temporal clustering patterns were observed among Streptococcus spp. analyzed in this study. This study provides an invaluable resource for studying the evolutionary aspects of antibiotic resistance acquisition and virulence in Streptococcus spp.

The complete coding sequence of a rabies lyssavirus (RABV) detected in an American black bear (Ursus americanus) in Connecticut, USA.

The complete coding sequence of a rabies lyssavirus (RABV) detected in a black bear (Ursus americanus) was generated. RNA extracted from brain tissues was amplified using reverse transcription followed by tiling PCR sequencing to obtain RABV whole viral genome. Sequencing was performed using an Illumina ISeq 100 instrument.

Suburban Population of Bobcats (Lynx rufus) in Connecticut, USA, Tested Negative for SARS-CoV-2, November 2021-February 2022.

A SARS-CoV-2 genomic and serologic survey was performed in a population of bobcats (Lynx rufus) inhabiting the state of Connecticut, USA. Wild animal populations are becoming established in densely populated cities with increased likelihood of direct or indirect contact with humans, as well as with household cats and dogs. Wild-caught bobcats (n=38) tested negative for SARS-CoV-2 genomic RNA by reverse-transcription quantitative PCR and for virus-neutralizing antibodies by ELISA, suggesting that either the species is not susceptible to SARS-CoV-2 or that the surveyed population has not yet been exposed to a source of infectious virus. However, this limited survey cannot rule out that human-to-bobcat or unknown reservoir-to-bobcat transmission of the virus occurs in nature.

Whole genome sequencing and phylogenetic analysis of West Nile viruses from animals in New England, United States, 2021.

West Nile virus is a mosquito-borne Flavivirus which is the leading cause of global arboviral encephalitis. We sequenced WNVs from an American crow found in Connecticut and an alpaca found in Massachusetts which were submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL). We report here the complete protein-coding sequences (CDS) of the WNVs (WNV 21-3957/USA CT/Crow/2021 and WNV 21-3782/USA MA/Alpaca/2021) and their phylogenetic relationship with other WNVs recovered from across the United States. In the phylogenetic analysis, the WNVs from this study belonged to the WNV lineage 1. The WNV 21-3957/USA CT/Crow/2021 clustered with WNVs from a mosquito and birds in New York during 2007-2013. Interestingly, the virus detected in the alpaca, WNV 21-3782/USA MA/Alpaca/2021 clustered with WNVs from mosquitos in New York, Texas, and Arizona during 2012-2016. The genetic differences between the viruses detected during the same season in an American crow and an alpaca suggest that vector-host feeding preferences are most likely driving viral transmission. The CDS of the WNVs and their phylogenetic relationships with other WNVs established in this study would be useful as reference data for future investigations on WNVs. Seasonal surveillance of WNV in birds and mammals and the genetic characterization of detected viruses are necessary to monitor patterns of disease presentations and viral evolution within a geographical area.

Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut, USA, 2018-2019.

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018-2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Dog in Connecticut in February 2021.

We report the first detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a 3-month-old dog in Connecticut that died suddenly and was submitted to the state veterinary diagnostic laboratory for postmortem examination. Viral RNA was detected in multiple organs of the dog by reverse transcription real time-PCR (RT-qPCR). Negative and positive sense strands of viral RNA were visualized by in situ hybridization using RNAscope technology. Complete genome sequencing and phylogenetic analysis of the hCoV-19/USA/CT-CVMDL-Dog-1/2021 (CT_Dog/2021) virus were conducted to identify the origin and lineage of the virus. The CT_Dog/2021 virus belonged to the GH/B1.2. genetic lineage and was genetically similar to SARS-CoV-2 identified in humans in the U.S. during the winter of 2020-2021. However, it was not related to other SARS-CoV-2 variants identified from companion animals in the U.S. It contained both the D614G in spike and P323L in nsp12 substitutions, which have become the dominant mutations in the United States. The continued sporadic detections of SARS-CoV-2 in companion animals warrant public health concerns about the zoonotic potential of SARS-CoV-2 and enhance our collective understanding of the epidemiology of the virus.

Genomic Features of Salmonella enterica Subspecies houtenae Serotype 45:g,z51:- Isolated from Multiple Abdominal Abscesses of an African Fat-Tailed Gecko, United States, 2020.

Salmonella enterica subsp. houtenae (S. houtenae) is a common subspecies in reptiles and has been implicated as a source of serious and life-threatening diseases in humans. Although occurrence and significance of S. houtenae infections have been extensively studied, the genetic features of S. houtenae have remained unknown due to a lack of available high-quality genome sequences. We obtained the complete genome sequence of S. houtenae 45:g,z51:- strain 20-369 isolated from multiple abdominal abscesses of an African fat-tailed gecko (Hemitheconyx caudicinctus) using Nanopore and Illumina sequencing technologies and generated the 4.65Mbp complete genome sequence of the S. houtenae str. 20-369. We annotated and analyzed the genome sequence with the aim to gain a deeper understanding of the genome characteristics associated with its pathogenicity. Overall, this study found several interesting genomic features such as pseudogene formation, virulence gene profile, and novel genomic islands. This study provides basis for an understanding possible genetic mechanism underlying pathogenicity of S. houtenae 45:g,z51:- as well as a high-quality genome reference for future comparison studies.

Complete mitochondrial genome of Asian longhorned tick, Haemaphysalis longicornis, Neumann, 1901 (Acari: Ixodida: Ixodidae) identified in the United States.

Haemaphysalis longicornis (Ixodida: Ixodidae), the Asian longhorned tick, which is native to temperate East Asia, has been recently detected in the northeastern region of the United States, drawing concerns about its potential impact on the US animal and public health sectors. Knowledge about the genetic features of H. longicornis found in the US is limited. Therefore, we sequenced the complete mitochondrial genome (mt-genome) from two H. longicornis ticks recently collected in the State of New York, USA, in 2020. These ticks were morphologically identified and tested for tick-borne pathogens at the Connecticut Veterinary Medical Diagnostic Laboratory (Storrs, CT). The mt-genome was 14,694 bp in length and encoded 37 genes, including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Phylogenetic analysis showed that the mt-genome clustered with those of other H. longicornis identified in China. The mt-genome sequence was 99.7% identical to a H. longicornis mt-genome (GenBank: MK439888) collected in China. The cox1 gene haplotype in these ticks belonged to the H1 type, which is the dominant haplotype present in central NJ and Staten Island, NY. The complete mt-genome data are needed to provide insights into genetic changes and phylogenetic studies of H. longicornis ticks.

Genetic features of Salmonella enterica subspecies diarizonae serovar 61:k:1,5 isolated from abortion cases in sheep, United States, 2020.

Salmonella enterica subspecies diarizonae serovar 61:(k):1, 5, (7) (sheep associated S. diarizonae, SASd) is the most common Salmonella serotype identified in sheep flocks. Despite the involvement with animal and human infections, there is limited information regarding virulence profiles of SASds and their antibiotic resistance gene complement, particularly for those circulating in the U.S. In this study, we genetically characterized three SASds, 20-265, 20-269, and 20-312, isolated from sheep placental tissues during an abortion storm affecting a flock in Connecticut during 2020. SASds were the only bacteria isolated from analyzed sheep tissues. The isolates were sensitive to all the antibiotics tested, but all these SASd isolates carry the aminoglycoside resistance gene, aac(6')-Iaa, and a chromosomal substitution in the parC gene. The proportion of pseudogenes (5.3-5.5%) was similar among the isolates, and these SASds carry IncX1 type plasmids. Comparing with the SASds isolates from Enterobase, the three isolates showed an identical genomic virulence profile carrying virulence genes in the conserved set of other SASd isolates except for steC, iagB, iacP, sseI, and slrP genes. In the SNP-based phylogenetic analysis, SASd sequences were grouped into group A-C, and the group C was further subdivided into subgroup C1-C6. The three isolates clustered with other SASd isolates from the U.S. and Canada in subgroup C6. SASd isolates in the identical phylogenetic groups tended to have similar geographical origin. The results of our study did not provide conclusive evidence about which are the genetic traits that trigger SASds to become virulent in sheep, but our data will provide a point for comparative studies of this Salmonella serovar.

Ixodes scapularis microbiome correlates with life stage, not the presence of human pathogens, in ticks submitted for diagnostic testing.

Ticks are globally distributed arthropods and a public health concern due to the many human pathogens they carry and transmit, including the causative agent of Lyme disease, Borrelia burgdorferi. As tick species' ranges increase, so do the number of reported tick related illnesses. The microbiome is a critical part of understanding arthropod biology, and the microbiome of pathogen vectors may provide critical insight into disease transmission and management. Yet we lack a comprehensive understanding of the microbiome of wild ticks, including what effect the presence of multiple tick-borne pathogens (TBPs) has on the microbiome. In this study we chose samples based on life stage (adult or nymph) and which TBPs were present. We used DNA from previously extracted Ixodes scapularis ticks that tested positive for zero, one, two or three common TBPs (B. burgdorferi, B. miyamotoi, Anaplasma phagocytophilum, Babesia microti). We produced 16S rRNA amplicon data for the whole tick microbiome and compared samples across TBPs status, single vs multiple coinfections, and life stages. Focusing on samples with a single TBP, we found no significant differences in microbiome diversity in ticks that were infected with B. burgdorferi and ticks with no TBPs. When comparing multiple TBPs, we found no significant difference in both alpha and beta diversity between ticks with a single TBP and ticks with multiple TBPs. Removal of TBPs from the microbiome did not alter alpha or beta diversity results. Life stage significantly correlated to variation in beta diversity and nymphs had higher alpha diversity than adult ticks. Rickettsia, a common tick endosymbiont, was the most abundant genus. This study confirms that the wild tick microbiome is highly influenced by life stage and much less by the presence of human pathogenic bacteria.

Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin-1 and Their Association among Bacteremic and Infective Endocarditis Patients in Egypt.

PURPOSE: Infective endocarditis (IE) is a major complication in patients with bacteremia of Staphylococcus (S.) aureus infection. Our aim was to determine the association of the major Staphylococcal superantigens (SAgs), including Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1), among hospitalized patients diagnosed with bacteremia and those with IE. METHODS: This study was conducted on 88 patients; of these, 84 (95.5%) had two positive blood cultures. Eighteen out of the 84 patients (21.4%) were diagnosed based on the modified Duke criteria by a cardiologist to have IE. The recovered isolates were screened phenotypically using ELISA followed by molecular analysis of sea, seb, sec, sed, see, and tsst-1, the major SAg coding genes, and the obtained findings were statistically analyzed. RESULTS: Phenotypic screening for SE production of 26 selected Staphylococci (15 isolated from the IE patients (10 S. aureus and 5 coagulase negative staphylococci (CoNS)) and 11 from bacteremic patients (10 S. aureus and 1 CoNS)) using ELISA revealed that 12/26 (46%) isolates were SE producers. PCR analysis showed that 19 (73%) isolates were PCR positive for SAg genes with the highest prevalence of the sea gene (79%), followed by seb (63%) and tsst-1 (21%). The least frequent gene was sed (5.3%). Statistical correlations between bacteremic and IE isolates with respect to prevalence of SAgs showed no significant difference (P value = 0.139, effect size = 0.572) indicating no specific association between any of the detected SAgs and IE. CONCLUSION: There is high prevalence of SEs among clinical isolates of Staphylococci recovered from patients suffering bacteremia and those with IE. No significant difference was found among Staphylococcal isolates recovered from patients with bacteremia or IE regarding both phenotypic and genotypic detection of the tested SAgs.

Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout.

Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.

Canine circoviral hemorrhagic enteritis in a dog in Connecticut.

A 5-mo-old Bassett Hound-Labrador Retriever cross was autopsied following a bout of lethargy, inappetence, and bleeding gums. Mucous membranes were white, and the small intestine was blue-black; the colon contained black feces. The spleen was swollen, and multiple lymph nodes were enlarged and hemorrhagic. Microscopically, the small intestine had focal crypt cell necrosis and circumferential transmural vasculitis, the latter the cause of infarction and the blue-black coloration. Lymphocytes were necrotic in spleen and lymph nodes, and erythrophagocytosis was present in some nodes. Vasculitis was present in brain, meninges, lung, liver, and kidneys. Electron microscopy revealed aggregates of 15-18 nm round viral particles in damaged crypt cells and in the endothelium of small blood vessels. Electron-dense intracytoplasmic inclusions consisting of paracrystalline-arrayed virus were demonstrated in macrophages in medullary lymph node sinuses. These virions were identified as circovirus, which was confirmed by real-time PCR and sequencing.

Phylogenetic analysis of Infectious Bursal Disease viruses according to newly proposed model of classification into geno-groups.

BACKGROUND: Infectious bursal disease virus (IBDV) is the causative agent of Infectious Bursal Disease (IBD), the disease causes immunosuppression which leads to secondary infections among rearing poultry flocks. Characterization of the virus is important for its control and eradication. The circulating IBDVs are classified on the basis of their antigenic and pathogenic properties. The virus is categorised as classical, variant and very virulent IBDV (vvIBDV). IBDV is a non-envelop, icosahedral double stranded virus. Viral protein 2 (VP2) is the major structural protein of capsid that determines the host-pathogen relationship. The aim of this study was to characterise the IBD virus of Pak-Asian region. METHODOLOGY: IBDV suspected flocks were examined in Punjab, Pakistan from 2014-2018. Two hundred and fifty samples were collected with complete history of the disease. The suspected samples were collected from broiler, layer and rural poultry farms. RNA was extracted and hyper-variable region of VP2 gene was amplified using specific primers. Nucleotide sequence of the VP2 gene was determined and its Amino Acid sequence was deduced. Moreover, phylogenetic analysis was also performed. RESULTS: The current classifications based in a hyper-variable region of the capsid protein VP2 (hvVP2), classification of IBDVs is split into newly proposed geno-groups according to Jackwood group. Among these prevailing, some IBDVs are limited geographically whereas, others are reported cosmopolitan. Genetic alterations are continuously playing role in evolution of new strains of the virus. CONCLUSION: During this study it was found that isolates of IBDV fall in first three geno-groups. Most of the geno-groups are prevalent around the world, whereas the mutated and re-assorted ones are confined in particular areas of the globe.

A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus.

Infectious bronchitis virus (IBV) affects poultry respiratory, renal and reproductive systems. Currently the efficacy of available live attenuated or killed vaccines against IBV has been challenged. We designed a novel IBV vaccine alternative using a highly innovative platform called Self-Assembling Protein Nanoparticle (SAPN). In this vaccine, B cell epitopes derived from the second heptad repeat (HR2) region of IBV spike proteins were repetitively presented in its native trimeric conformation. In addition, flagellin was co-displayed in the SAPN to achieve a self-adjuvanted effect. Three groups of chickens were immunized at four weeks of age with the vaccine prototype, IBV-Flagellin-SAPN, a negative-control construct Flagellin-SAPN or a buffer control. The immunized chickens were challenged with 5x10(4.7) EID50 IBV M41 strain. High antibody responses were detected in chickens immunized with IBV-Flagellin-SAPN. In ex vivo proliferation tests, peripheral mononuclear cells (PBMCs) derived from IBV-Flagellin-SAPN immunized chickens had a significantly higher stimulation index than that of PBMCs from chickens receiving Flagellin-SAPN. Chickens immunized with IBV-Flagellin-SAPN had a significant reduction of tracheal virus shedding and lesser tracheal lesion scores than did negative control chickens. The data demonstrated that the IBV-Flagellin-SAPN holds promise as a vaccine for IBV.

High Prevalence of blaNDM-1, blaVIM, qacE, and qacEΔ1 Genes and Their Association with Decreased Susceptibility to Antibiotics and Common Hospital Biocides in Clinical Isolates of Acinetobacter baumannii.

The objective of this study was to evaluate the susceptibility of metallo-ß-lactamase (MBL)-producing Acinetobacter baumannii (A. baumannii) clinical isolates to biocides. We also determined the prevalence and correlation of efflux pump genes, class 1 integron and MBL encoding genes. In addition, blaVIM, blaNDM-1, qacE and qacEΔ1 nucleotide sequence analysis was performed and compared to sequences retrieved from GenBank at the National Center for Biotechnology Information database. A. baumannii had a resistance rate to carbapenem of 71.4% and 39.3% and was found to be a MBL producer. The minimum inhibitory concentrations (MICs) of chlorhexidine and cetrimide were higher than the recommended concentrations for disinfection in 54.5% and 77.3% of MBL-positive isolates respectively and their MICs were significantly higher among qac gene-positive isolates. Coexistence of qac genes was detected in 68.1% and 50% of the isolates with blaVIM and blaNDM-1 respectively. There was a significant correlation between the presence of qac genes and MBL-encoding blaVIM and blaNDM-1 genes. Each of the blaNDM-1, blaVIM, qacE and qacEΔ1 DNA sequences showed homology with each other and with similar sequences reported from other countries. The high incidence of Verona integron-encoded metallo-ß-lactamases (VIM) and New-Delhi-metallo-ß-lactamase (NDM) and qac genes in A.baumannii highlights emerging therapeutic challenges for being readily transferable between clinically relevant bacteria. In addition reduced susceptibility to chlorhexidine and cetrimide and the potential for cross resistance to some antibiotics necessitates the urgent need for healthcare facilities to periodically evaluate biocides efficacy, to address the issue of antiseptic resistance and to initiate a "biocidal stewardship".

ELISA Serology for Antibodies Against Chlamydia trachomatis in Crohn's Disease.

BACKGROUND: Recently we reported IgA anti-Chlamydia antibodies in patients with Crohn's disease (CD), in particular in four patients from a single family of six with CD. METHODS: We studied sera from four cohorts from the north of France. These were identified as: EPIMAD (80 pediatric onset CD and 20 pediatric onset ulcerative colitis), MINOTOR (148 adult onset sporadic CD and 50 adult onset ulcerative colitis), Grande Famillies (50) and matched controls for the Grande Famillies cohort (49). Sera were tested using commercial anti-Chlamydia trachomatis (LGV2:434) IgG and IgA human enzyme-linked immunosorbent assay (ELISA) kits. Cutoff for positivity was 11.0 standard units. RESULTS: Patients with sporadic CD, unaffected first degree relatives from multiplex families and ulcerative colitis patients had no greater serologic reactivity than controls. However, multiplex families' patients had twice as many positives as the other groups: for IgG 20% vs. 8%; for IgA 20% vs. 10%. CONCLUSIONS: Though not attaining statistical significance, the data showed that familial CD patients had greater exposure to C. trachomatis than sporadic CD patients, supporting our earlier results from one family from the north of France. More specific serologic tests based on outer membrane proteins will need to be employed against the various Chlamydia species with zoonotic potential.

Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge.

Current influenza vaccines should be improved by the addition of universal influenza vaccine antigens in order to protect against multiple virus strains. We used our self-assembling protein nanoparticles (SAPNs) to display the two conserved influenza antigens M2e and Helix C in their native oligomerization states. To further improve the immunogenicity of the SAPNs, we designed and incorporated the TLR5 agonist flagellin into the SAPNs to generate self-adjuvanted SAPNs. We demonstrate that addition of flagellin does not affect the ability of SAPNs to self-assemble and that they are able to stimulate TLR5 in a dose-dependent manner. Chickens vaccinated with the self-adjuvanted SAPNs induce significantly higher levels of antibodies than those with unadjuvanted SAPNs and show higher cross-neutralizing activity compared to a commercial inactivated virus vaccine. Upon immunization with self-adjuvanted SAPNs, mice were completely protected against a lethal challenge. Thus, we have generated a self-adjuvanted SAPN with a great potential as a universal influenza vaccine.

Unexpectedly high proportion of ancestral Manu genotype Mycobacterium tuberculosis strains cultured from tuberculosis patients in Egypt.

Tuberculosis is one of the important public health problems in Egypt. However, limited information on the Mycobacterium tuberculosis genotypes circulating in Egypt is available. A total of 151 M. tuberculosis strains were characterized by spoligotyping. The results revealed that 74.8% of M. tuberculosis isolates grouped into 13 different clusters, while 25.2% had unique spoligotype patterns. Comparison with an international spoligotyping database (the SITVIT2 database) showed that types SIT53 (T1 variant) and SIT54 (Manu2 variant) were the most common types between cluster groups. In addition, new shared types SIT2977, SIT2978, and SIT2979 were observed. The results identified for the first time an unusually high proportion of ancestral Manu strains of M. tuberculosis from patients in Egypt. The percentage of the Manu clade in this study (27.15%) was significantly higher than its overall representation of 0.4% in the SITVIT2 database. We show that in Egypt tuberculosis is caused by a predominant M. tuberculosis genotype belonging to the ancestral Manu lineage which could be a missing link in the split between ancestral and modern tubercle bacilli during the evolution of M. tuberculosis.