Characterisation of Cronobacter strains isolated from hospitalised adult patients.

Bacteria belonging to the genus Cronobacter are opportunistic pathogens known for causing rare but serious infections in neonates, including meningitis, necrotising enterocolitis and sepsis. Cronobacter infections occur also in adult populations, however, they generally have milder manifestations and their prevalence is uncertain. In this study, the presence of Cronobacter strains from adult patients in the University Hospital in Bratislava was investigated and overall 18 confirmed isolates from 321 patients (5.3%) were recovered. No Cronobacter positive sample was detected in 215 sputum samples from outpatients. The highest occurrence of Cronobacter strains was observed from stroke patients and this may be associated with an abnormal swallowing ability. The isolated strains belonged to the species Cronobacter sakazakii and Cronobacter malonaticus. In silico genotyping (MLST, CRISPR-cas array profiling) of whole genome sequences assigned the strains to three different MLST clones. The majority (12/18) of the isolated strains were sequence type ST513 or single locus variants ST514 and ST515, thereby being members of C. sakazakii pathovar clonal complex CC4. However, according to core genome MLST analysis the ST513-ST515 strains created a unique cluster substantially different from other CC4 strains. The isolated strains were susceptible to 18 tested antibiotics. All strains possess a genomic island encoding for increased thermal tolerance. As Cronobacter strains are frequently present in dried foods of plant origin, spread of a specific clone within a hospital may be caused by food transmission and may be facilitated by its tolerance to environmental stresses such as desiccation and temperature.

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order.

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.

The molecular characterisation of Escherichia coli K1 isolated from neonatal nasogastric feeding tubes.

BACKGROUND: The most common cause of Gram-negative bacterial neonatal meningitis is E. coli K1. It has a mortality rate of 10-15 %, and neurological sequelae in 30-50 % of cases. Infections can be attributable to nosocomial sources, however the pre-colonisation of enteral feeding tubes has not been considered as a specific risk factor. METHODS: Thirty E. coli strains, which had been isolated in an earlier study, from the residual lumen liquid and biofilms of neonatal nasogastric feeding tubes were genotyped using pulsed-field gel electrophoresis, and 7-loci multilocus sequence typing. Potential pathogenicity and biofilm associated traits were determined using specific PCR probes, genome analysis, and in vitro tissue culture assays. RESULTS: The E. coli strains clustered into five pulsotypes, which were genotyped as sequence types (ST) 95, 73, 127, 394 and 2076 (Achman scheme). The extra-intestinal pathogenic E. coli (ExPEC) phylogenetic group B2 ST95 serotype O1:K1:NM strains had been isolated over a 2 week period from 11 neonates who were on different feeding regimes. The E. coli K1 ST95 strains encoded for various virulence traits associated with neonatal meningitis and extracellular matrix formation. These strains attached and invaded intestinal, and both human and rat brain cell lines, and persisted for 48 h in U937 macrophages. E. coli STs 73, 394 and 2076 also persisted in macrophages and invaded Caco-2 and human brain cells, but only ST394 invaded rat brain cells. E. coli ST127 was notable as it did not invade any cell lines. CONCLUSIONS: Routes by which E. coli K1 can be disseminated within a neonatal intensive care unit are uncertain, however the colonisation of neonatal enteral feeding tubes may be one reservoir source which could constitute a serious health risk to neonates following ingestion.

Population Genome Programs across the Middle East and North Africa: Successes, Challenges, and Future Directions.

In this review, we discuss the current state of population genome programs (PGPs) conducted in the Middle East and North African (MENA) region. This region has high prevalence of genetic diseases and significant health challenges as well as being a significantly underrepresented population in public genetic databases. The majority of ongoing PGPs represent regions in Europe, North and South America, South Asia, Australia, and Africa, with little to no descriptive information highlighted only on the MENA Region when it comes to genome programs databases, outcomes, or the challenges that MENA region countries may face establishing their own national programs. This review has identified 6 PGPs currently underway in the MENA region, namely in the Kingdom of Saudi Arabia, Qatar, Egypt, the United Arab Emirates, Bahrain, and Iran. Due to the rapidly growing involvement of the MENA region in national-scale genomic data collection, an increase in representation in public genetic databases is to be expected to occur in the near future. Whilst significant progress is being made in some MENA countries, future initiatives as well as ongoing programs will be facing several challenges related to collaboration, finance, infrastructure and institutional data access, data analysis, sustainability, health records, and biobanks. The review also reiterates the need for ensuring ethical and regulated genomic initiatives which can drive developments in personalized medicine treatments to improve patient prognosis and quality of life.

Durability of antibodies post vaccination with two doses of inactivated BBIBP-CorV vaccine.

BACKGROUND: Breakthrough infections post-COVID-19 vaccination occur with the emerging variants of the SARS-CoV virus which might be either due to the newer variants escaping immune response or the waning of antibodies over time. However, there is lack of long-term follow-up evidence on the waning of immune response following inactivated COVID-19 vaccine. METHODS: A retrospective, observational study was conducted on serum samples of individuals who had received two doses of BBIBP-CorV vaccine. Individual's antibody responses were evaluated based on IgG anti-S and neutralizing antibodies measurements. Antibody samples were categorized into four groups, defined by the time interval from the individual's receipt of the BBIBP-CorV vaccine: <30 days, 30-90 days, 91-180 days and >180 days. RESULTS: A total of 6668 serum samples from inactivated BBIBP-CorV vaccine recipients were analyzed for IgG anti-S and neutralizing antibodies. 571 (8.6%) samples were tested during the first 29 days interval post vaccination, 3642 (54.6%) were tested during 30-90 days interval, 2173 (32.6%) samples were tested during 91 to 180 days interval and 282(4.2%) were tested at >180 days interval post vaccination. We found that more than 50% of the individuals had antibody titers below the average cut-off range at the 91-180 days interval post vaccination. Older age (>60 years), male gender, chronic kidney disease, hypertension, immunodeficiencies and increased interval post vaccination emerged as independent risk factors associated with lower immune response. CONCLUSION: Inactivated BBIBP-CorV vaccine recipients, based on age, gender and associated comorbid conditions might need booster doses at an earlier interval than the currently followed six months interval.

Genomic epidemiology of SARS-CoV-2 in the UAE reveals novel virus mutation, patterns of co-infection and tissue specific host immune response.

To unravel the source of SARS-CoV-2 introduction and the pattern of its spreading and evolution in the United Arab Emirates, we conducted meta-transcriptome sequencing of 1067 nasopharyngeal swab samples collected between May 9th and Jun 29th, 2020 during the first peak of the local COVID-19 epidemic. We identified global clade distribution and eleven novel genetic variants that were almost absent in the rest of the world and that defined five subclades specific to the UAE viral population. Cross-settlement human-to-human transmission was related to the local business activity. Perhaps surprisingly, at least 5% of the population were co-infected by SARS-CoV-2 of multiple clades within the same host. We also discovered an enrichment of cytosine-to-uracil mutation among the viral population collected from the nasopharynx, that is different from the adenosine-to-inosine change previously reported in the bronchoalveolar lavage fluid samples and a previously unidentified upregulation of APOBEC4 expression in nasopharynx among infected patients, indicating the innate immune host response mediated by ADAR and APOBEC gene families could be tissue-specific. The genomic epidemiological and molecular biological knowledge reported here provides new insights for the SARS-CoV-2 evolution and transmission and points out future direction on host-pathogen interaction investigation.

DNA-Sequence Based Typing of the Cronobacter Genus Using MLST, CRISPR-cas Array and Capsular Profiling.

The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST), capsular profiling of the K-antigen and colanic acid (CA) biosynthesis regions, and CRISPR-cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC) 1 and 4, sequence types (ST) 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis, C. dublinensis and C. universalis. The majority of CRISPR-cas types across the genus was the I-E (Ecoli) type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo) type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors.

Rapid in situ imaging and whole genome sequencing of biofilm in neonatal feeding tubes: A clinical proof of concept.

The bacterial flora of nasogastric feeding tubes and faecal samples were analysed for a low-birth weight (725 g) neonate EGA 25 weeks in intensive care. Samples were collected at age 6 and 8 weeks of life. Optical coherence tomography (OCT) was used to visualise bacterial biofilms inside the nasogastric feeding tubes. The biofilm was heterogeneously distributed along the tube lumen wall, and had a depth of up to 500 µm. The bacterial biofilm and faecal samples included Enterococcus faecalis and Enterobacter hormaechei. Representative strains, recovered from both feeding tubes and faecal samples, were whole genome sequenced using Illumina, Mi-Seq, which revealed indistinguishable strains, each with less than 28 SNP differences, of E. faecalis and E. hormaechei. The E. faecalis strains were from two sequence types (ST191 and ST211) and encoded for a number of traits related to biofilm formation (BopD), adherence (Epb pili), virulence (cps loci, gelatinase, SprE) and antibiotic resistances (IsaA, tetM). The E. hormaechei were all ST106, and encoded for blaACT-15 ß-lactamase and fosfomycin resistance (fosA). This proof of concept study demonstrates that bacterial flora within the neonatal feeding tubes may influence the bacterial colonisation of the intestinal tract and can be visualised non-destructively using OCT.

CRISPR-cas loci profiling of Cronobacter sakazakii pathovars.

AIM: Cronobacter sakazakii sequence types 1, 4, 8 and 12 are associated with outbreaks of neonatal meningitis and necrotizing enterocolitis infections. However clonality results in strains which are indistinguishable using conventional methods. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)-cas loci profiling for epidemiological investigations. MATERIALS & METHODS: Seventy whole genomes of C. sakazakii strains from four clonal complexes which were widely distributed temporally, geographically and origin of source were profiled. RESULTS & CONCLUSION: All strains encoded the same type I-E subtype CRISPR-cas system with a total of 12 different CRISPR spacer arrays. This study demonstrated the greater discriminatory power of CRISPR spacer array profiling compared with multilocus sequence typing, which will be of use in source attribution during Cronobacter outbreak investigations.

Draft Genome Sequence of an Enterobacter Species Associated with Illnesses and Powdered Infant Formula.

This is the first report of the draft genome sequence of an Enterobacter species that may have been transmitted from powdered infant formula (PIF) to infants, resulting in illness. Enterobacter spp. are currently permitted in PIF, but the transmission of this strain indicates that the microbiological criteria for PIF may need revision.