Genomic Epidemiology of C2/H30Rx and C1-M27 Subclades of Escherichia coli ST131 Isolates from Clinical Blood Samples in Hungary.

Extended-spectrum ß-lactamase-producing Escherichia coli ST131 has become widespread worldwide. This study aims to characterize the virulome, resistome, and population structure of E. coli ST131 isolates from clinical blood samples in Hungary. A total of 30 C2/H30Rx and 33 C1-M27 ST131 isolates were selected for Illumina MiSeq sequencing and 30 isolates for MinION sequencing, followed by hybrid de novo assembly. Five C2/H30Rx and one C1-M27 cluster were identified. C1-M27 isolates harbored the F1:A2:B20 plasmid in 93.9% of cases. Long-read sequencing revealed that blaCTX-M-27 was on plasmids. Among the C2/H30Rx isolates, only six isolates carried the C2-associated F2:A1:B- plasmid type. Of 19 hybrid-assembled C2/H30Rx genomes, the blaCTX-M-15 gene was located on plasmid only in one isolate, while in the other isolates, ISEcp1 or IS26-mediated chromosomal integration of blaCTX-M-15 was detected in unique variations. In one isolate a part of F2:A1:B- plasmid integrated into the chromosome. These results suggest that CTX-M-15-producing C2/H30Rx and CTX-M-27-producing C1-M27 subclades may have emerged and spread in different ways in Hungary. While blaCTX-M-27 was carried mainly on the C1/H30R-associated F1:A2:B20 plasmid, the IncF-like plasmids of C2/H30Rx or its composite transposons have been incorporated into the chromosome through convergent evolutionary processes.

Carbapenem-resistant Escherichia coli in Black-headed gulls, the Danube, and human clinical samples: A One Health comparison of contemporary isolates.

OBJECTIVES: Our aim was to characterize and compare contemporary carbapenem-resistant Enterobacterales (CRE) isolates from gulls, the River Danube, and humans in Hungary, Budapest. METHODS: Multiresistant Enterobacterales were sought for in 227 gull faecal and 24 Danube water samples from 2019 to 2020. Eosin-methylene blue agar containing 2 mg/L cefotaxime and Colilert-test containing 10 mg/L cefotaxime were used for gull and water samples, respectively. Isolates were characterized by polymerase chain reactions (PCRs); acquired carbapenemase producers were further analysed by whole-genome sequencing, together with 21 Hungarian human CR Escherichia coli (CREc) isolates. RESULTS: Gull and water samples exhibited a CRE prevalence of 7.4% (9/122) and 6.7% (7/105), none and 5/12 water samples yielded CRE from 2019 and 2020, respectively; CRE were found only in samples taken downstream of Budapest. The dominant species was Escherichia coli and the most prevalent carbapenemase was blaNDM-1. High-risk CREc clones were found both in gulls (ST224, ST372, ST744) and the Danube (ST10, ST354, ST410); the closest associations were between ST410 from humans and the Danube, among ST1437 among gulls, and between ST1437 in gulls and the Danube (46, 0, and 22-24 allelic distances, respectively). Direct links between human and gull isolates were not demonstrated. CONCLUSION: The study demonstrates potential epidemiological links among humans, a river crossing a city, and urbanised birds, suggesting a local transmission network. Water bodies receiving influent wastewater, together with animals using such habitats, may serve as a local reservoir system for CRE, highlighting the importance of One Health in CRE transmission, even in a country with a low CRE prevalence in humans.

Occurrence and Characteristics of Staphylococcus aureus in a Hungarian Dairy Farm during a Control Program.

In this research, our aim was to assess the occurrence of Staphylococcus aureus in a Hungarian large-scale dairy farm during the S. aureus control program conducted in the course of our studies. Furthermore, the phenotypic and genotypic properties of the isolates (type of haemolysis, antibiotic susceptibility, staphylococcal enterotoxin (SE) gene carrying ability and spa type) were determined. S. aureus was detected in all bulk tank milk samples collected during this study. Two different spa types were identified among the 17 strains isolated in the farm. A total of 14 of the 17 studied strains (82%) showed ß-haemolysis on blood agar, 2/17 strains (12%) expressed double zone and 1/17 strains (6%) showed weak ß-haemolysis. All strains were susceptible to most antibiotics tested (cefoxitin, chloramphenicol, clindamycin, erythromycin, gentamicin, tetracycline and trimethoprim/sulphamethoxazole), but all strains were resistant to penicillin G. A total of 11 of the 17 strains (65%) were found to harbour seg, sei, selm, seln, selo genes; 4/17 strains (24%) harboured sei, selm, seln, selo genes and 2/17 strains (11%) harboured sei gene. Since the new SEs/SEls can also cause foodborne outbreaks potentially and all strains were found to be resistant to penicillin G, it is essential to decrease and keep the prevalence of S. aureus low in the dairy farm and the implementation of the S. aureus control program is also highly justified. The results showed that the S. aureus count decreased by the end of our studies, so the control program was proved to be effective.

Transmissible silver resistance readily evolves in high-risk clone isolates of Klebsiella pneumoniae.

Silver is used extensively in both hospitals and outpatient clinics as a disinfectant coating agent on various devices. Resistance to silver was recently reported as an emerging problem in Enterobacteriaceae. Multidrug-resistant high-risk clones of Klebsiella pneumoniae are common causes of serious healthcare-associated infections worldwide posing a serious threat to patients. In this study, we investigated the capacity of both high-risk (CG14/15 and CG258) and minor clone strains of K. pneumoniae to develop resistance to silver. Resistance was induced in vitro in silver-susceptible but otherwise multidrug-resistant clinical isolates. Genetic alterations in the silver-resistant derivative strains with regard to the silver-susceptible isolates were investigated by whole-genome sequencing. The transferability of high-level resistance to silver was also tested. We demonstrated that the high-level resistance to silver can quickly evolve as a consequence of a single-point mutation either in the cusS gene of the chromosomally encoded CusCFBARS efflux system and/or in the silS gene of the plasmid-encoded Copper Homeostasis and Silver Resistance Island (CHASRI) coding also for a metallic efflux. The minimal inhibitory concentrations (MICs) of the strains increased from 4 mg/L (23.5 µM) AgNO3 to >8,500 mg/L (>50,000 µM) AgNO3 during induction. Harboring the CHASRI proved an important selective asset for K. pneumoniae when exposed to silver. Successful conjugation experiments using Escherichia coli K12 J5-3Rif as recipient showed that high-level silver resistance can transmit between strains of high-risk clones of K. pneumoniae (ST15 and ST11) and isolates from additional species of Enterobacteriaceae. The lack of fitness cost associated with the carriage of the CHASRI in a silver-free environment and the presence of the RelEB toxin-antitoxin system on the conjugative plasmids could advance the dissemination of silver resistance. Our results show that multidrug-resistant high-risk clones of K. pneumoniae are capable of evolving and transmitting high-level resistance to silver. This observation should warrant a more judicious use of silver coated-devices to prevent the extensive dissemination of silver resistance.

Phagocytosis and intracellular killing of heterogeneous vancomycin-intermediate Staphylococcus aureus strains.

Risk factors for invasive infections by heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) may involve resistance to opsonophagocytosis and bacterial killing. hVISA strains typically have a thickened cell wall with altered peptidoglycan cross-linking. To determine whether hVISA may be endowed with an increased resistance to phagocytosis, this study assessed the characteristics of uptake and killing by granulocytes of three hVISA strains. All isolates were analysed by multilocus sequence typing and staphylococcal chromosome cassette mec typing. One of the strains belonged to the Hungarian meticillin-resistant S. aureus (MRSA) clone ST239-MRSA-III and the other two to the New York/Japan MRSA clone ST5-MRSA-II. In the presence of 10 % normal serum, the extent of phagocytosis and killing by blood granulocytes was equivalent for hVISA, MRSA and meticillin-sensitive S. aureus (MSSA) strains. Using granulocytes and serum from one patient who survived hVISA infection, the rate of phagocytosis and killing was also found to be comparable to that by control cells in the presence of 10 % serum. However, phagocytosis and killing of hVISA and MRSA (ATCC 25923) strains by normal granulocytes was markedly decreased in the presence of low concentrations (1 and 2.5 %) of serum from the patient who survived hVISA infection compared with that found with normal human serum. These data suggest that hVISA and MRSA isolates may be more resistant to opsonophagocytosis and bacterial killing than MSSA isolates, at least in some cases.