Characterization of the Shiga toxin-producing Escherichia coli O26 isolated from human in Poland between 1996 and 2014.

Shiga toxin-producing Escherichia coli (STEC) O26 infections can be comparable with STEC O157 infections in severity of the acute haemolytic-uremic syndrome HUS and long-term sequelae. Among O26 STEC isolates, highly virulent clone O26:H11/H- Sequence Type 29 (ST 29) emerged in Germany in mid-1990s and spread to European countries. However, up to date, no STEC O26:H11/H- belonging to ST29 has been documented in Poland. In this study, we determined the relationship and clonal structure, stx genotypes, plasmid gene profiles and antimicrobial resistance of nine human STEC O26:H11/H- strains from human patients in Poland between 1996 and 2014. Of the 9 human STEC O26:H11/H- strains, two belonged to ST29 and were isolated from two children with HUS and renal failure with sepsis respectively. These strains showed the molecular characteristics of the emerging human-pathogenic ST29 clone (stx1-, stx2a+, eae+, ehxA+, etpD+, katP-, espP-). The remaining STEC O26:H11/H- strains examined in this study, belonged to ST21, with plasmid genes profiles frequently reported in ST21 strains in Europe. STEC O26 infections with serious human health consequences highlight the need of continuous surveillance of non-O157 STEC and implementation of the diagnostic approaches focused on their detection. Significance and impact of the study: These study provides the first data on the occurrence of emerging Shiga toxin-producing Escherichia coli O26:H11 ST 29 clone in human patients in Poland. Those strains show the molecular characteristics of highly virulent new ST29 pathotype (stx1-, stx2a+, eae+ ehxA+, etpD+, katP-, espP-). These results demonstrated prompt efforts to implement diagnostic approaches detection of those pathogen in the European countries.

Characteristics of multidrug-resistant hypervirulent Klebsiella pneumoniae strains ST29 and K212 harbouring tmexC2-tmexD2-toprJ2.

OBJECTIVES: This study aimed to characterize a tigecycline-resistant hypervirulent Klebsiella pneumoniae (HvKP) strain, identified as KLZT, which carries the tigecycline resistance gene cluster tmexC2-tmexD2-toprJ2 belonging to ST29 and serotype K212. METHODS: Antimicrobial susceptibility and virulence phenotypes were assessed, followed by whole-genome sequencing (WGS) using PacBio II and MiSeq sequencers. Genome annotation was carried out using the RAST server and bioinformatics analysis revealed the genetic characteristics of this strain. RESULTS: Antimicrobial and virulence phenotype testing indicated that K. pneumoniae strain KLZT could be considered as a multidrug-resistant HvKP. WGS analysis showed that KLZT has a single 5,536,506-bp chromosome containing three plasmids 290,963 bp (pKLZT-1), 199,302 bp (pKLZT-2), and 4820 bp (pKLZT-3) in size, and also includes the ST29 and K212 serotypes. Four (blaSHV-187, oqxA, oqxB, and fosA6) and six resistance genes (tmexC2-tmxeD2-toprJ2, blaOXA-1, aac(6')-Ib-cr, catB3, arr-3, and blaLEN27) were identified from chromosomal and plasmid pKLZT-1, respectively. Gene-based analysis of the resistance genes of plasmid pKLZT-1 showed that the tigecycline resistance gene cluster-carrying region was flanked by umuC and umuD (umuD-hps-IS5-tmexC2-tmexD2-toprJ2-umuC), as well as other resistance genes and virulence factors (ureB, ureC, and ureG), which were carried by IS5075-Tn3-intI1 -aac(6')-Ib-cr-blaOXA-1-catB3-arr-3-blaLEN27-Tn3-ISkpn26-ureBCG-IS5075. CONCLUSIONS: WGS has revealed that a multidrug-resistant strain, HvKP KLZT, belonging to ST29 with capsular serotype K212, contains a multidrug-resistance plasmid.

Emergence of multidrug-resistant Staphylococcus haemolyticus in neonatal intensive care unit in Southern France, a genomic study.

An emergence of multidrug-resistant (MDR) Staphylococcus haemolyticus has been observed in the neonatal intensive care unit (NICU) of Nîmes University Hospital in southern France. A case-control analysis was conducted on 96 neonates, to identify risk factors associated with S. haemolyticus infection, focusing on clinical outcomes. Forty-eight MDR S. haemolyticus strains, isolated from neonates between October 2019 and July 2022, were investigated using routine in vitro procedures and whole-genome sequencing. Additionally, five S. haemolyticus isolates from adult patients were sequenced to identify clusters circulating within the hospital environment. The incidence of neonatal S. haemolyticus was significantly associated with low birth weight, lower gestational age, and central catheter use (p < 0.001). Sepsis was the most frequent clinical manifestation in this series (20/46, 43.5%) and was associated with five deaths. Based on whole-genome analysis, three S. haemolyticus genotypes were predicted: ST1 (6/53, 11%), ST25 (3/53, 5.7%), and ST29 (44/53, 83%), which included the subcluster II-A, predominantly emerging in the neonatal department. All strains were profiled in silico to be resistant to methicillin, erythromycin, aminoglycosides, and fluoroquinolones, consistent with in vitro antibiotic susceptibility tests. Moreover, in silico prediction of biofilm formation and virulence-encoding genes supported the association of ST29 with severe clinical outcomes, while the persistence in the NICU could be explained by the presence of antiseptic and heavy metal resistance-encoding genes. The clonality of S. haemolyticus ST29 subcluster II-A isolates confirms healthcare transmission causing severe infections. Based on these results, reinforced hygiene measures are necessary to eradicate the nosocomial transmission of MDR strains.

Evaluation of cross-protection between S. Pneumoniae serotypes 35B and 29 in a mouse model.

Pneumococcal conjugate vaccines (PCVs) have reduced vaccine-type pneumococcal disease but in turn have also resulted in replacement with non-vaccine serotypes. One such serotype, 35B, a multidrug resistant type, has been associated with an increase in disease. Mice were immunized intramuscularly with monovalent pneumococcal polysaccharide 35B conjugated to CRM197 containing aluminum phosphate adjuvant on days 0, 14, and 28. Pneumococcal enzyme-linked immunosorbent assay, opsonophagocytic killing assays, and competition OPA were performed for STs 35B and 29 to measure serotype-specific binding and functional antibodies. On day 52, mice were intratracheally challenged with S. pneumoniae ST29 to evaluate cross-protection. 35B-CRM197 immunized mice had binding and functional antibodies to both PnPs 35B and 29. 35B-CRM197 immunized mice were 100% protected from IT challenge with S. pneumoniae ST29 as compared to 30% survival in the naïve group. Future vaccines containing polysaccharide 35B, such as the investigational 21-valent PCV, V116, may provide cross protection against the non-vaccine serotype 29 due to structural similarity.

Covert dissemination of pLVPK-like virulence plasmid in ST29-K54 Klebsiella pneumoniae: emergence of low virulence phenotype strains.

This study aimed to explore the epidemic, clinical characteristics, and molecular and virulence attributes of Klebsiella pneumoniae serotype K54 (K54-Kp). A retrospective study was conducted on 328 strains of Klebsiella pneumoniae screened in a Chinese hospital from January 2016 to December 2019. The virulence genes and antibiotic resistance genes (ARGs) were detected by PCR, and a drug sensitivity test was adopted to detect drug resistance. Multilocus sequence typing (MLST) and PFGE were performed to determine the clonal correlation between isolates. Biofilm formation assay, serum complement-mediated killing, and Galleria mellonella infection were used to characterize the virulence potential. Our results showed that thirty strains of K54-Kp were screened from 328 strains of bacteria, with an annual detection rate of 2.29%. K54-Kp had a high resistance rate to antibiotics commonly used in the clinic, and patients with hepatobiliary diseases were prone to K54-Kp infection. MLST typing showed 10 sequence typing, mainly ST29 (11/30), which concentrated in the B2 cluster. K54-Kp primarily carried virulence genes of aerobactin, silS, allS, wcaG, wabG, and mrkD, among which the terW gene was closely related to ST29 (p<0.05). The strains infected by the bloodstream had strong biofilm formation ability (p<0.05). Most strains were sensitive to serum. Still, the virulence of pLVPK-like virulence plasmid in ST29-K54 Klebsiella pneumoniae was lower than that of ST11 type and NTUH-K2044 in the Galleria mellonella model. Therefore, these findings supply a foundation to roundly comprehend K54-Kp, and clinicians should strengthen supervision and attention.

Plasmid-Mediated Transmission of KPC-2 Carbapenemase in Enterobacteriaceae in Critically Ill Patients.

Carbapenem-resistant Enterobacteriaceae (CRE) cause health care-associated infections worldwide, and they are of severe concern due to limited treatment options. We report an outbreak of KPC-2-producing CRE that was caused by horizontal transmission of a promiscuous plasmid across different genera of bacteria and hospitals in Germany. Eleven isolates (8 Citrobacter freundii, 2 Klebsiella oxytoca, and 1 Escherichia coli) were obtained from seven critically ill patients during the six months of the outbreak in 2016. One patient developed a CRE infection while the other six patients were CRE-colonized. Three patients died in the course of the hospital stay. Six of the seven patients carried the same C. freundii clone; one K. oxytoca clone was found in two patients, and one patient carried E. coli and C. freundii. Molecular analysis confirmed the presence of a conjugative, bla KPC-2-carrying 70 kb-IncN plasmid in C. freundii and E. coli and an 80 kb-IncN plasmid in K. oxytoca. All transconjugants harbored either the 70 or 80 kb plasmid with bla KPC-2, embedded within transposon variant Tn4401g. Whole genome sequencing and downstream bioinformatics analyses of all plasmid sequences showed an almost perfect match when compared to a bla KPC-2-carrying plasmid of a large outbreak in another German hospital two years earlier. Differences in plasmid sizes and open reading frames point to the presence of inserted mobile genetic elements. There are few outbreak reports worldwide on the transmission of bla KPC-2-carrying plasmids across different bacterial genera. Our data suggest a regional and supraregional spread of bla KPC-2-carrying IncN-plasmids harboring the Tn4401g isoform in Germany.