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Introduction

Meningitis is one of the major causes of mortality and morbidity in neonates. Together with group B streptococci, neonatal meningitis-causing Escherichia coli (NMEC) is the leading cause of this disease worldwide(1). Mortality rates can reach up to 40%, while survivors often suffer with severe neurological sequelae and may develop serious neurological disorders(2). Pathogenesis of NMEC meningitis is complex, encompassing the need for bacterial translocation to the bloodstream, and crossing of the blood-brain barrier(1). To accomplish this, the NMEC are equipped with a wide variety of virulence traits such as adhesins, iron acquisition systems, invasins, toxins, and serum resistance genes. These factors can be encoded by bacterial chromosome or are carried via plasmids(1,3). One of the most common virulence factors in NMEC strains is K1 antigen. It is a homopolymer of N-acetylneuraminic (sialic) acid and functions as a molecular mimicry(4).

The aim of this study was molecular typing of four NMEC isolates. Three strains were isolated from two diseased neonates and one isolate originated from the surface in the neonatal unit of the same hospital.

Materials and Methods

Isolation and cultivation of bacterial strains

Strains used in this study were isolated in the Neonatal Department of Trnava University Hospital (Slovakia). KMB- 1033 and KMB-1034 were isolated from Patient 1, KMB-1032 was isolated from Patient 2., and KMB-1031 strain was isolated from the surface swab of the hospital environment. Strains were grown on Columbia agar 5% Sheep Blood or in Luria-Bertani (LB) medium.

DNA isolation and NGS library preparation

Nucleic acid isolation was performed by Higher PurityTM Bacterial Genomic DNA Isolation Kit (CanvaxBiotech). DNA concentration was measured by Qubit (Invitrogen). Genomic libraries for sequencing were prepared by Nextera XT DNA Prep Kit (Illumina), purified on AMPure XP magnetic beads (Beckman Coulter Life Sciences) and reviewed by chip electrophoresis. Sequencing was performed on NextSeq 500TM platform using 2x150bp read lengths (Illumina) at the Comenius University Science Park.

Bioinformatics and genome analysis

Raw data was assembled into contigs by SPAdes. Assembled genomes were annotated and analysed using the PAT- RIC (https://www.patricbrc.org/), RAST (https://rast.nmpdr. org/) and CGE (https://www.genomicepidemiology.org/) online platforms.

Results and discussion

All four tested strains were identified as Escherichia coli based on biochemical EnteroTest 24 + latex agglutination of liquor, which is positive in E. coli K1 samples, and verified by PCR and MALDI-TOF methods.

Using whole genome sequencing, we obtained the average of 86 contigs with 76 times coverage per genome. All four strains were positive for K1 capsule and belonged to the MLST sequence type ST-95 which is very common among NMEC strains(5,6). They were distinguishable only by cgML- ST. Furthermore, the KMB-1031 strain isolated from environmental swab also showed difference in serotype and fimH fimbrial adhesin (Figure 1). To further distinguish the highly similar genomes, whole genome SNP analysis was applied, which confirmed strain separation into two clusters. Strains isolated from patients differed from each other in 111 to 128 SNPs. However, the environmental KMB-1031 strain was less relative to the other strains and differed in 2830 to 2865 SNPs. Our isolates were further compared with three reference NMEC genomes and we found out that the clinical strains were highly relative to E. coli S88 (Figure 1). This strain is a typical representative of the O45:H7:K1 serotype, frequently found in NMEC in Spain and reported to account for one-third of all E. coli neonatal meningitis cases in France in 2009(6,7). The O1:H7:K1 serotype, seen in the strain isolated from the surface swab (KMB-1031), was the most prevalent NMEC clonal group in France between 2001 and 2013 and in the USA(5) as well.

We compared proteomes of all four strains with S88 as the reference and observed high similarity between S88 and KMB-1032, KMB-1033 and KMB-1034 strains, while lower similarity to KMB-1031, which was anticipated based on previous analyses (Figure 2). Main differences were seen in regions encoding for prophages, amino-acid biosynthesis, colanic acid biosynthesis, formate and nitrate metabolism, type 1 fimbriae as well as hypothetical proteins. These differences suggest that the strain isolated from the surface swab was not the cause of the meningitis outbreak in the neonatal department.

Based on the CARD database, TEM-1 and EC-5 genes which confer resistance to penicillins and cephalosporins were detected in all four strains(8). This was consistent with the ampicillin resistance (>32 mg/l) detected in KMB-1031, KMB-1033 and KMB-1034 strains. Furthermore, E. coli KMB- 1031 showed resistance against ampicillin + sulbactam (>32 mg/l) which may be caused by the fact that sulbactam is a relatively weak inhibitor of TEM-1, or possibly a result of TEM-1 hyperproduction(9).

Presence of plasmids was detected by selection of contigs with higher coverage. In all four strains, we detected a large (~135 kb) plasmid similar to previously published pRK100 and pCERC5 plasmids belonging to the ColV group with RepFIB and RepFII origins(10). We observed similarity in the whole plasmid sequence with the only difference that unlike pRK100 and pCERC5, plasmids from our strains did not harbour the tetA gene (data not shown). We observed presence of twelve virulence genes (cia, cvaC, iroN, iss, mchF, traT, etsC, hlyF, ompT, iucC, sitA and iutA) on the pRK100-like plasmid found in our strains. No differences in the presence of virulence factors localised on the pRK100-like plasmids were detected between the patient and environmental strains. The IncFIB plasmids are very prominent among ExPEC strains and other studies(11,12) have also described an association between these plasmids and genes encoding for siderophores and serum survival determinants in NMEC.

Conclusion

This paper describes an analysis of three clinical NMEC strains and one environmental swab isolate. All four strains were characterised as ST-95 possessing O45:H7:K1 and O1:H7:K1 serotypes which both belong to widely spread NMEC types in many countries. Virulence factors found in these strains could help identify specific set of genes for distinguishing NMEC from other ExPEC. Furthermore, this paper contributed to the monitoring of transmission of epidemics in a neonatal medical facility.

Acknowledgement

This paper was supported by the Research and Development Operational Programme, funded by the ERDF (SmartFarm, ITMS 313011W112).

Poďakovanie

Tento príspevok je výsledkom realizácie projektu SmartFarm (ITMS 313011W112) financovaného z Operačného programu Výskum a vývoj Európskeho fondu regionálneho rozvoja.

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