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.

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.

Quantitative study about the role of environmental conditions in the survival capability of multidrug-resistant bacteria.

BACKGROUND: Healthcare-associated infections (HAIs) caused by multidrug-resistant bacteria (MDRB) are of global concern and hospital textiles can contribute to their transmission. MDRB are able to survive on textiles for more than enough time to spread in the environment. Some studies summarized the effect of environmental factors on the duration of bacterial survival, but it remained an open question how these factors influence the quantity of surviving bacteria in a period of a few days, which is relevant from the perspective of HAIs. Investigating this effect can contribute to better understand the spread of MDRB and the emergence of hospital outbreaks. METHODS: We investigated quantitatively the survival capability of 15 vancomycin-resistant Enterococcus faecium (VRE), 15 methicillin-resistant Staphylococcus aureus (MRSA), 15 multidrug-resistant Acinetobacter baumannii (MACI) and 15 multidrug-resistant Klebsiella pneumoniae (MRKP) in five environmental conditions using the plate count method. We examined the role of nutrients, textile types, temperature and level of relative humidity on bacterial survival after 1-7days of incubation. RESULTS: Each bacterial group showed higher survival capability on 100% cotton towel than on 100% cotton sheet (P<0.01). MRSAs and VREs showed higher (P<0.01), MACIs showed lower (P=0.02) CFU/swatch values on 100% polyester sheet than on cotton sheet. The survival capability of MRKPs and MRSAs was higher inoculated in nutrient broth than in saline solution (P<0.01). Each bacterial group showed lower survival capability (P<0.01) at body condition (T=35°C, Rh=83%) than at control (T=25°C, Rh=52%). CONCLUSIONS: Towels proved to be excellent conditions for each bacteria to survive, however chemical composition of the textiles affected differently the survival of Gram-positive and Gram-negative bacteria. These findings could be useful in searching for the source of outbreaks. Organic contamination of the textiles can increase the survival of desiccation-sensitive bacteria, therefore nutrient-rich inoculating medium is recommended in survival studies.