Genomic, phenotypic and demographic characterization of Mycobacterium tuberculosis in Israel in 2021.

According to World Health Organization WHO, Tuberculosis (TB) is the second cause of death from infectious disease worldwide. During 2021, 10.6 million people were infected with TB, and 1.6 million people died. TB is caused by pathogens belonging to the Mycobacterium tuberculosis complex (MTBC), mainly by Mycobacterium tuberculosis (M.tb). Members of this complex are acid-fast bacilli, which can cause intrapulmonary and extra pulmonary TB, and can be divided into various lineages, based on genomic markers. The main public health threat comes from drug resistant M.tb strains, which are responsible for about 25% of TB death and treatment failure worldwide. Treating drug resistant TB patients significantly raises the costs of TB treatment. This study provides an overview of the demographic and drug susceptibility characteristics of newly diagnosed TB patients in Israel in 2021. The State of Israel has a very low level of TB endemicity and is at a pre-elimination phase. Notably, only 11.7% of the newly diagnosed TB patients were born in Israel. In this report, of the 154 new laboratory-confirmed TB patients, 66.7% had pulmonary TB, while 16% had extrapulmonary TB. Males accounted for 52% of the patients, with the most prevalent age group being 21-40. Most patients were citizens of Israel (53.9%), while 37.7% had no Israeli citizenship. Among non-citizens, there was a predominance of males and patients aged 21-40. The susceptibility profile showed a high resistance rate to streptomycin (18.2%) and to a lower extent to isoniazid (13.6%), pyrazinamide (8.4%), rifampicin (7.8%), and ethambutol (3.2%). Only 2 cases of XDR-TB and 10 MDR-TB strains were detected in Israel in 2021, with both XDR strains and 5 out of 10 MDR strains belonging to the Beijing lineage. Most of Beijing isolates were resistant to at least one tested drug. Genomic sequencing of 134 out of 156 strains and bioinformatics analysis using the MTBseq program and WHO mutation catalogue shows a good match with only 9 discrepancies between phenotypic and genotypic susceptibility profiles in first line drugs. The most common lineage is Delhi-Cas (23%) followed by the Beijing lineage (17%). Most patients from the Delhi-Cas lineage were born in Africa, while patients with Beijing isolates were born in different countries. Minimum spanning tree analysis identified 15 clusters. The study highlights the need for ongoing surveillance of TB using molecular and phenotypic tools to further decreasing the spreading level of the disease and develop effective treatment strategies.

Prolonged survival of a patient with active MDR-TB HIV co-morbidity: insights from a Mycobacterium tuberculosis strain with a unique genomic deletion.

Coinfection of HIV and multidrug-resistant tuberculosis (MDR-TB) presents significant challenges in terms of the treatment and prognosis of tuberculosis, leading to complexities in managing the disease and impacting the overall outcome for TB patients. This study presents a remarkable case of a patient with MDR-TB and HIV coinfection who survived for over 8 years, despite poor treatment adherence and comorbidities. Whole genome sequencing (WGS) of the infecting Mycobacterium tuberculosis (Mtb) strain revealed a unique genomic deletion, spanning 18 genes, including key genes involved in hypoxia response, intracellular survival, immunodominant antigens, and dormancy. This deletion, that we have called "Del-X," potentially exerts a profound influence on the bacterial physiology and its virulence. Only few similar deletions were detected in other non-related Mtb genomes worldwide. In vivo evolution analysis identified drug resistance and metabolic adaptation mutations and their temporal dynamics during the patient's treatment course.

Challenging diagnosis of Mycolicibacterium cosmeticum/canariasense infection: A case report and literature review.

We present the case of an immunocompromised child with Mycolicibacterium cosmeticum/ canariasense infection. Our case highlights the difficulty in adequate speciation. Most isolates described in the literature were identified using 16 s-rRNA PCR, which if performed on our sample would at best be inconclusive. Misidentifications could have a real impact on the body of evidence collected on these isolates thus far.

Type VII secretion system and its effect on group B Streptococcus virulence in isolates obtained from newborns with early onset disease and colonized pregnant women.

Introduction: GBS may cause a devastating disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to better understand T7SS contribution to virulence in these different clinical scenarios. Methods: GBS genomes [N=33, 17 EOD isolates (serotype III/ST17) and 16 colonizing isolates (12 serotype VI/ST1, one serotype VI/ST19, one serotype VI/ST6, and two serotype 3/ST19)] were analyzed for presence of T7SS genes and genes encoding WXG100 proteins. We also perform bioinformatic analysis. Galleria mellonella larvae were used to compare virulence between colonizing, EOD, and mutant EOD isolates. The EOD isolate number 118659 (III/ST17) was used for knocking out the essC gene encoding a membrane-bound ATPase, considered the driver of T7SS. Results: Most GBS T7SS loci encoded core component genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030)].Bioinformatic analysis indicated that based on sequence type (ST) the clinicalGBS isolates encode at least three distinct subtypes of T7SS machinery. In all ST1isolates we identified two copies of esxA gene (encoding putative WXG100proteins), when only 23.5% of the ST17 isolates harbored the esxA gene. Five ST17isolates encoded two copies of the essC gene. Orphaned WXG100 molecule(SAG0230), distinct from T7SS locus, were found in all tested strains, except inST17 strains where the locus was found in only 23.5% of the isolates. In ST6 andST19 isolates most of the structure T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella modelcompared to colonizing isolates. The 118659DessC strain was attenuated in itskilling ability, and the larvae were more effective in eradicating 118659DessC. Conclusions: We demonstrated that T7SS plays a role during infection. Knocking out the essC gene, considered the driver of T7SS, decreased the virulence of ST17 responsible for EOD, causing them to be less virulent comparable to the virulence observed in colonizing isolates.

Mpox proctitis as a likely predisposing factor for chlamydial perihepatitis in a male patient.

Perihepatitis (Fitz-Hugh-Curtis syndrome) is a rare complication of sexually transmitted infections, mostly seen in women. Only 12 male cases have been reported to date, of which Chlamydia trachomatis was confirmed in 2. We report a case of chlamydial perihepatitis in a male patient, occurring 1 month after Mpox and associated with the unusual LGV ST23 strain. Our case suggests that rectal Mpox lesions may facilitate chlamydial dissemination.

Group B streptococcus virulence factors associated with different clinical syndromes: Asymptomatic carriage in pregnant women and early-onset disease in the newborn.

Background: Group B streptococcus (GBS) harbors many virulence factors but there is limited data regarding their importance in colonization in pregnancy and early-onset disease (EOD) in the newborn. We hypothesized that colonization and EOD are associated with different distribution and expression of virulence factors. Methods: We studied 36 GBS EOD and 234 GBS isolates collected during routine screening. Virulence genes (pilus-like structures-PI-1, PI-2a, PI-2b; rib and hvgA) presence and expression were identified by PCR and qRT-PCR. Whole genome sequencing (WGS) and comparative genomic analyses were used to compare coding sequences (CDSs) of colonizing and EOD isolates. Results: Serotype III (ST17) was significantly associated with EOD and serotype VI (ST1) with colonization. hvgA and rib genes were more prevalent among EOD isolates (58.3 and 77.8%, respectively; p < 0.01). The pilus loci PI-2b and PI-2a were more prevalent among EOD isolates (61.1%, p < 0.01), while the pilus loci PI-2a and PI-1 among colonizing isolates (89.7 and 93.1% vs. 55.6 and 69.4%, p < 0.01). qRT PCR analysis revealed that hvgA was barely expressed in colonizing isolates, even though the gene was detected. Expression of the rib gene and PI-2b was two-fold higher in EOD isolates compared to colonizing isolates. Transcription of PI-2a was three-fold higher in colonizing isolates compared to EOD isolates. ST17 isolates (associated with EOD) had a smaller genome size compared ST1 and the genome was more conserved relative to the reference strain and ST17 isolates. In a multivariate logistic regression analysis virulence factors independently associated with EOD were serotype 3, and PI-1 and PI-2a was protective. Conclusion: There was a significant difference in the distribution of hvg A, rib, and PI genes among EOD (serotype III/ST17) and colonizing (serotype VI/ST1) isolates suggesting an association between invasive disease and these virulence factors. Further study is needed to understand the contribution of these genes to GBS virulence.

Prevalence and Molecular Characterization of Extended-Spectrum ß-Lactamase Producing Enterobacterales in Healthy Community Dogs in Israel.

BACKGROUND: antimicrobial resistance is a global problem in human and veterinary medicine. We aimed to investigate the extended spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) gut colonization in healthy community dogs in Israel. METHODS: Rectal swabs were sampled from 145 healthy dogs, enriched, plated on selective plates, sub-cultured to obtain pure cultures, and ESBL production was confirmed. Bacterial species and antibiotic susceptibility profiles were identified. WGS was performed on all of the ESBL-PE isolates and their resistomes were identified in silico. Owners' questionnaires were collected for risk factor analysis. RESULTS: ESBL-PE gut colonization rate was 6.2% (n = 9/145, 95% CI 2.9-11.5). Overall, ten isolates were detected (one dog had two isolates); the main species was Escherichia coli (eight isolates), belonging to diverse phylogenetic groups-B1, A and C. Two isolates were identified as Citrobacter braakii, and C. portucalensis. A phylogenetic analysis indicated that all of the isolates were genetically unrelated and sporadic. The isolates possessed diverse ESBL genes and antibiotic-resistance gene content, suggesting independent ESBL spread. In a multivariable risk factor analysis, coprophagia was identified as a risk factor for ESBL-PE gut colonization (p = 0.048, aOR = 4.408, 95% CI 1.014-19.169). CONCLUSIONS: healthy community dogs may be colonized with ESBL-PE MDR strains, some of which were previously reported in humans, that carry wide and diverse resistomes and may serve as a possible source for AMR.

Emergence of Mycobacterium canariasense infections in central Israel.

In 2018, Mycobacterium canariasense bloodstream infection was diagnosed in Israel. Further investigation had identified additional five cases in three medical centers, including isolates from blood (1), cornea (1), and sputum (3). Isolates were susceptible to all the antimicrobial tested. All but one isolate was related by whole-genome phylogeny.

Triple site sexually transmitted infection testing as a crucial component of surveillance for men who have sex with men: A prospective cohort study.

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections are common among men who have sex with men (MSM). Many oropharyngeal and anorectal infections remain asymptomatic. We aimed to evaluate triple-site screening following PrEP introduction. We enrolled a prospective cohort study including 210 asymptomatic MSM during 2019-2020, analyzed by groups: HIV positive (HIV+), HIV-uninfected using PrEP (HIV-/PrEP+), or HIV-uninfected not using PrEP (HIV-/PrEP-). A self-administered questionnaire captured demographic information and sexual risk-taking behaviors. CT/NG testing results were compared between study groups and predictors of infection were evaluated. We included 59 HIV+, 70 HIV-/PrEP+, and 81 HIV-/PrEP- subjects. 30% (n = 62) of participants tested positive for CT/NG. HIV-/PrEP+ group had highest proportion of infections (n = 33, 47%) followed by HIV-/PrEP- (n = 16, 22%) and HIV+ (n=13, 20%; p < .001). Importantly, 98% (80/82) of pharyngeal/anorectal CT/NG infections were missed in genitourinary tract screening alone. PrEP use and previous syphilis infection were the strongest risk factor for CT/NG. Extra-genital asymptomatic CT/NG infections were prevalent among MSM. These data highlight the importance of routine extra-genital CT/NG testing in asymptomatic sexually active MSM. The study describes the consequences for three-site testing lack of implementation in the PrEP era.

Mycobacterium intracellulare subsp. chimaera from Cardio Surgery Heating-Cooling Units and from Clinical Samples in Israel Are Genetically Unrelated.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens that cause illness primarily in the elderly, in the immunocompromised or in patients with underlying lung disease. Since 2013, a global outbreak of NTM infection related to heater-cooler units (HCU) used in cardio-thoracic surgery has been identified. This outbreak was caused by a single strain of Mycobacterium intracellulare subsp. chimaera. In order to estimate the prevalence of this outbreak strain in Israel, we sampled Mycobacterium intracellulare subsp. chimaera from several HCU machines in Israel, as well as from patients, sequenced their genomes and compared them to the outbreak strain. The presence of mixed mycobacteria species in the samples complicated the analysis of obtained sequences. By applying a metagenomic binning strategy, we were able to obtain, and characterize, genomes of single strains from the mixed samples. Mycobacterium intracellulare subsp. chimaera strains were compared to each other and to previously reported genomes from other countries. The strain causing the outbreak related to the HCU machines was identified in several such machines in Israel but not in any clinical sample.

Transcontinental Dissemination of the L2b/D-Da Recombinant Chlamydia trachomatis Lymphogranuloma venereum (LGV) Strain: Need of Broad Multi-Country Molecular Surveillance.

Previously, we identified a Chlamydia trachomatis lymphogranuloma venereum (LGV) recombinant strain possessing a non-LGV ompA genotype. Here, culture-independent genome sequencing confirms its circulation in Europe, Middle East, and North America, and unveils emergence of antibiotic resistance. Broad surveillance is needed.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA.

BACKGROUND: Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom's MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom's MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom's MLST scheme in clinical samples with low concentrations of Chlamydia DNA. RESULTS: In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom's MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. CONCLUSIONS: The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene.

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Group B Streptococcus serotypes associated with different clinical syndromes: Asymptomatic carriage in pregnant women, intrauterine fetal death, and early onset disease in the newborn.

OBJECTIVES: To study Group B Streptococcus (GBS) isolates associated with different clinical syndromes: asymptomatic carriage in pregnant women, intrauterine fetal death (IUFD), and early onset disease (EOD) in the newborn. METHODS: GBS isolates were collected from asymptomatic pregnant women admitted for labor, IUFD cases, and neonates with EOD. Serotypes and antibiotic susceptibilities were determined. Multilocus sequence typing (MLST) was performed to assess genetic epidemiology. RESULTS: GBS carriage rate was 26.1% (280/1074). The dominant serotype among asymptomatic pregnant women was VI [98/240 women (40.8%)], followed by serotypes III, V and IV in 42/240 (17.5%), 30/240 (12.5%) and 28/240 (11.7%) women, respectively. The dominant serotype in IUFD cases was serotype VI [10/13 (76.9%)]. In contrast the prevalent serotype among EOD cases was III [16/19 (84.2%)]. ST-1 was associated with IUFD [7/13 (53.8%)], ST-17 was associated with serotype III and EOD in the newborn 14/19 (73.7%)]. Erythromycin and clindamycin resistance reached 36.8%, 7.7% and 20.0%among EOD, vaginal carriage and IUFD, respectively. CONCLUSIONS: Serotypes VI and ST-1 were dominant among asymptomatic pregnant women and in IUFD cases while EOD was associated with serotype III and ST-17. Invasive mechanisms thus may differ between IUFD and EOD in the newborn and virulence may be related to capsule serotype. Resistance rates to erythromycin and clindamycin were high in EOD cases.

Extended spectrum ß lactamase-producing Enterobacteriaceae shedding by race horses in Ontario, Canada.

BACKGROUND: We aimed to investigate the prevalence, molecular epidemiology and prevalence factors for Extended Spectrum ß-Lactamase-producing Enterobacteriaceae (ESBL-E) shedding by race horses. A cross-sectional study was performed involving fecal samples collected from 169 Thoroughbred horses that were housed at a large racing facility in Ontario, Canada. Samples were enriched, plated on selective plates, sub-cultured to obtain pure cultures and ESBL production was confirmed. Bacterial species were identified and antibiotic susceptibility profiles were assessed. E. coli sequence types (ST) and ESBL genes were determined using multilocus sequence type (MLST) and sequencing. Whole genome sequencing was performed to isolates harboring CTX-M-1 gene. Medical records were reviewed and associations were investigated. RESULTS: Adult horses (n = 169), originating from 16 different barns, were sampled. ESBL-E shedding rate was 12% (n = 21/169, 95% CI 8-18%); 22 ESBL-E isolates were molecularly studied (one horse had two isolates). The main species was E. coli (91%) and the major ESBL gene was CTX-M-1 (54.5%). Ten different E. coli STs were identified. Sixty-four percent of total isolates were defined as multi-drug resistant. ESBL-E shedding horses originated from 8/16 different barns; whereas 48% (10/21) of them originated from one specific barn. Overall, antibiotic treatment in the previous month was found as a prevalence factor for ESBL-E shedding (p = 0.016, prevalence OR = 27.72, 95% CI 1.845-416.555). CONCLUSIONS: Our findings demonstrate the potential diverse reservoir of ESBL-E in Thoroughbred race horses. Multi-drug resistant bacteria should be further investigated to improve antibiotic treatment regimens and equine welfare.

Draft Genome Sequences of Cronobacter muytjensii Cr150, Cronobacter turicensis Cr170, and Cronobacter sakazakii Cr611.

The sequencing and bioinformatics analyses of isolates Cr150, Cr170, and Cr611 from powdered infant formula indicate that the three strains represent new members in the Cronobacter muytjensii, Cronobacter turicensis, and Cronobacter sakazakii groups, respectively.

The role of O-polysaccharide chain and complement resistance of Escherichia coli in mammary virulence.

Mastitis, inflammation of the mammary gland, is a common disease of dairy animals. The disease is caused by bacterial infection ascending through the teat canal and mammary pathogenic Escherichia coli (MPEC) are common etiology. In the first phase of infection, virulence mechanisms, designated as niche factors, enable MPEC bacteria to resist innate antimicrobial mechanisms, replicate in milk, and to colonize the mammary gland. Next, massive replication of colonizing bacteria culminates in a large biomass of microbe-associated molecular patterns (MAMPs) recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) mediating inflammatory signaling in mammary alveolar epithelial cells (MAEs) and macrophages. Bacterial lipopolysaccharides (LPSs), the prototypical class of MAMPs are sufficient to elicit mammary inflammation mediated by TLR4 signaling and activation of nuclear factor kB (NF-kB), the master regulator of inflammation. Using in vivo mastitis model, in low and high complements mice, and in vitro NF-kB luminescence reporter system in MAEs, we have found that the smooth configuration of LPS O-polysaccharides in MPEC enables the colonizing organisms to evade the host immune response by reducing inflammatory response and conferring resistance to complement. Screening a collection of MPEC field strains, we also found that all strains were complement resistant and 94% (45/48) were smooth. These results indicate that the structure of LPS O-polysaccharides chain is important for the pathogenesis of MPEC mastitis and provides protection against complement-mediated killing. Furthermore, we demonstrate a role for complement, a key component of innate immunity, in host-microbe interactions of the mammary gland.

Delftibactin-A, a Non-ribosomal Peptide With Broad Antimicrobial Activity.

The rapid emergence of drug resistant bacteria is occurring worldwide, outpacing the development of new antibiotics. It is known that some of the main sources of antibiotics are the bacteria themselves, many of which are secondary metabolites of Gram positive bacteria. Siderophores, which are secondary metabolites, function as natural chelators (e.g., iron). They are produced and secreted by many bacteria and have been experimented on as "carriers" of several types of antibiotics that pass the cell membrane of challenging Gram negative bacteria. Delftibactin A is a non-ribosomal peptide (NRP), which is known to detoxify gold in Delftia spp. and form gold nuggets, and is considered to be a siderophore. In this study we demonstrate that the supernatant from novel environmental isolates of Delftia spp. have antimicrobial activity. We characterized the active fraction and identified delftibactin A as a compound with antimicrobial activity. Delftibactin A exhibits potent antimicrobial activity against Gram positive multi drug resistant (MDR) bacteria like Methicillin-resistant Staphylococcus aureus (MRSA), and Vancomycin resistant Enterococcus (VRE), and also against the Gram negative pathogens Acinetobacter baumannii and Klebsiella pneumoniae. We discovered that the production of delftibactin A is greatly influenced by temperature. Furthermore, we have demonstrated the possibility of utilizing delftibactin A as a siderophore carrier of toxic metals such as gallium into Gram negative bacteria. These findings expose new opportunities of yet unexploited natural products such as delftibactin A, which have been known for other bacterial uses, as potent factors in the battle against MDR bacteria.

Draft Genome Sequence of Cronobacter sakazakii Cr268, Isolated from Infant Powder Formula.

Cronobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in premature infants. Strain Cr268 was isolated from imported powdered infant formula in 2009 during routine microbial examination according to ISO-22964 ("Microbiology of the food chain-horizontal method for the detection of Cronobacter spp."). Isolate Cr268 was confirmed to be C. sakazakii by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and standard biochemical analysis. Here, we announce its genome, which represents a new member in the C. sakazakii group.