In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. ...Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.
•ColE-type plasmid with blaOXA-232 increased persister formation rate against ciprofloxacin and amikacin.•Plasmids with deletion of hypothetical gene, vbhA, and MOB decreased persister ...formation.•Particularly, vbhA-encoding antitoxin significantly effected persister formation against ciprofloxacin.
Bacterial persister cells are a sub-population of cells that are tolerant to high concentrations of antibiotics. In this study, we investigated the effect of plasmids bearing carbapenemase genes on persister cell formation.
Three plasmids, IncX3-type plasmid with blaNDM-1, IncN-type plasmid with blaKPC-2, and ColE-type plasmid with blaOXA-232, were transformed into Escherichia coli MG1655. For the ColE-type plasmid (pM5_OXA232), gene-deletion plasmids were constructed and transformed into the MG1655. Persister assays were performed against ciprofloxacin and amikacin, and expression levels of relA and spoT were measured for the wild-type E. coli and all transformants.
Unlike the other two plasmids, transformation of ColE-type plasmid (pM5_OXA232) caused a significant increase in the formation of persister cells. Compared with transformants that harboured intact pM5_OXA232, transformants that harboured plasmids with deletions of gene(s), vbhA, hypothetical gene, or a mobile gene cassette showed decreased persister cell formation. Expression levels of relA and spoT exhibited patterns similar to those of persister cell formation rates, particularly against ciprofloxacin.
In this study, we showed that a small ColE-type plasmid bearing blaOXA-232 has an effect on persister cell formation, possibly contributing to the dissemination of low-level carbapanemase.
Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of ...colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.
Pneumococcal β-lactam resistance was first detected in Iceland in the late 1980s, and subsequently peaked at almost 25% of clinical isolates in the mid-1990s largely due to the spread of the ...internationally-disseminated multidrug-resistant PMEN2 (or Spain6B-2) clone of Streptococcus pneumoniae.
Whole genome sequencing of an international collection of 189 isolates estimated that PMEN2 emerged around the late 1960s, developing resistance through multiple homologous recombinations and the acquisition of a Tn5253-type integrative and conjugative element (ICE). Two distinct clades entered Iceland in the 1980s, one of which had acquired a macrolide resistance cassette and was estimated to have risen sharply in its prevalence by coalescent analysis. Transmission within the island appeared to mainly emanate from Reykjavík and the Southern Peninsular, with evolution of the bacteria effectively clonal, mainly due to a prophage disrupting a gene necessary for genetic transformation in many isolates. A subsequent decline in PMEN2's prevalence in Iceland coincided with a nationwide campaign that reduced dispensing of antibiotics to children in an attempt to limit its spread. Specific mutations causing inactivation or loss of ICE-borne resistance genes were identified from the genome sequences of isolates that reverted to drug susceptible phenotypes around this time. Phylogenetic analysis revealed some of these occurred on multiple occasions in parallel, suggesting they may have been at least temporarily advantageous. However, alteration of 'core' sequences associated with resistance was precluded by the absence of any substantial homologous recombination events.
PMEN2's clonal evolution was successful over the short-term in a limited geographical region, but its inability to alter major antigens or 'core' gene sequences associated with resistance may have prevented persistence over longer timespans.
Transformation efficiency is very low in hypermucoviscous Kllebsiella pneumoniae owing to high amount of capsular polysaccharide. Here, we propose a method to improve the transformation in ...hypermucoviscous K. pneumoniae using citric acid. An increased number of recovered transformants was observed in the strains cultured in the presence of citric acid.
•Transformation in hypermucoviscous Klebsiella pneumoniae strains have frequently resulted in very low efficiency of transformation, because of the high amount of capsular polysaccharides.•We show that electroporation method using citric acid for transforming hypermucoviscous K. pneumoniae is efficient by reducing the amount of capsular polysaccharide.•The time constant value, an indicator of transformation efficiency was optimized when K. pneumoniae is treated citric acid.
In this study, the whole genome sequences of two Streptococcus pneumoniae clinical isolates from South Korea were determined and compared. They were found to be the same serotype (11 A) and ...multilocus sequence typing analysis showed that they are single‐locus variants (SLVs; ST8279 and ST166) of each other, differing at one allele (aroE). However, the ST8279 strain is extensively drug‐resistant (XDR) whereas the ST166 strain is not. The genome of the XDR strain is very similar in structure to that of two previously reported genomes, AP200 (11 A:ST62) and 70585 (5:ST5803); however, some regions were inverted and there were some exogenous regions in the ST8279 strain. It was found that 6,502 single nucleotide polymorphisms are dispersed across the genome between the two serotype 11 A ST8279 and ST166 strains. Many of them are located in genes associated with antibiotic resistance. In addition, many amino acid differences were also identified in genes involved in DNA repair (mutL, uvrA and uvrC) and recombination (recU, recR and recA). On the basis of these results, it was inferred that the XDR strain did not evolve from its SLV via a single recombination event involving a large portion of the genome including the aroE gene. Rather, the strain likely evolved through many point mutations and recombination events involving small portions of the genome.
In 2018, the Clinical and Laboratory Standards Institute (CLSI) revised ciprofloxacin (CIP)-susceptible breakpoint for
from ≤1 μg/mL to ≤0.25 μg/mL, based on pharmacokinetic-pharmacodynamic (PK-PD) ...analysis. However, clinical data supporting the lowered CIP breakpoint are insufficient. This retrospective cohort study evaluated the clinical outcomes of patients with bacteremic urinary tract infections (UTIs) caused by
, which were previously CIP-susceptible and changed to non-susceptible. Bacteremic UTIs caused by
with CIP minimal inhibitory concentration (MIC) ≤ 1 μg/mL were screened, and then patients treated with CIP as a definitive treatment were finally included. Patients in CIP-non-susceptible group (MIC = 0.5 or 1 μg/mL) were compared with patients in CIP-susceptible group (MIC ≤ 0.25 μg/mL). Primary endpoints were recurrence of UTIs within 4 weeks and 90 days. A total of 334 patients were evaluated, including 282 of CIP-susceptible and 52 of CIP-non-susceptible. There were no significant differences in clinical outcomes between two groups. In multivariate analysis, CIP non-susceptibility was not associated with recurrence of UTIs. CIP non-susceptibility based on a revised CIP breakpoint, which was formerly susceptible, was not associated with poor clinical outcomes in bacteremic UTI patients were treated with CIP, similar to those of the susceptible group. Further evaluation is needed to guide the selection of definitive antibiotics for UTIs.
Methicillin-resistant Staphylococcus aureus (MRSA) is highly prevalent in hospitals in many Asian countries. Recent emergence of community-associated (CA) MRSA worldwide has added another serious ...concern to the epidemiology of S. aureus infections. To understand the changing epidemiology of S. aureus infections in Asian countries, we performed a prospective, multinational surveillance study with molecular typing analysis.
We evaluated the prevalence of methicillin resistance in S. aureus isolates in CA and healthcare-associated (HA) infections, and performed molecular characterization and antimicrobial susceptibility tests of MRSA isolates.
MRSA accounted for 25.5% of CA S. aureus infections and 67.4% of HA infections. Predominant clones of CA-MRSA isolates were ST59-MRSA-SCCmec type IV-spa type t437, ST30-MRSA-SCCmec type IV-spa type t019 and ST72-MRSA-SCCmec type IV-spa type t324. Previously established nosocomial MRSA strains including sequence type (ST) 239 and ST5 clones were found among CA-MRSA isolates from patients without any risk factors for HA-MRSA infection. CA-MRSA clones such as ST59, ST30 and ST72 were also isolated from patients with HA infections.
Our findings confirmed that MRSA infections in the community have been increasing in Asian countries. Data also suggest that various MRSA clones have spread between the community and hospitals as well as between countries.
Abstract
Objectives
Bacteria that acquire plasmids incur a biological cost. Despite this fact, clinical Enterobacteriaceae isolates commonly contain multiple co-existing plasmids harbouring ...carbapenemase genes.
Methods
Six different plasmids carrying blaNDM-1, blaNDM-5, blaCTX-M-15, blaKPC-2, blaOXA-181 and blaOXA-232 genes were obtained from Klebsiella pneumoniae and Escherichia coli clinical isolates. Using the E. coli DH5α strain as recipient, 14 transconjugants with diverse plasmid combinations (single or double plasmids) were generated. For each of these, the effects of plasmid carriage on the bacterial host were investigated using in vitro and in vivo competition assays; additionally, the effects were investigated in the context of biofilm formation, serum resistance and survival inside macrophages. Transcriptomic changes in single- and double-plasmid recipients were also investigated.
Results
Increased in vitro and in vivo competitiveness was observed when two plasmids carrying blaNDM-1 and blaOXA-232 were co-introduced into the host bacteria. However, DH5α::pNDM5 + pOXA232 and other double-plasmid recipients did not show such competitiveness. DH5α::pNDM5 + pOXA181 did not show any fitness cost compared with a plasmid-free host and single-plasmid transconjugants, while both the double-plasmid recipients with pCTXM15 or pKPC2 exhibited a fitness burden. The double-plasmid recipient DH5α::pNDM1 + pOXA232 also exhibited increased biofilm formation, serum resistance and survival inside macrophages. Transcriptomic analysis revealed that the genes of DH5α::pNDM1 + pOXA232 involved in metabolic pathways, transport and stress response were up-regulated, while those involved in translation were down-regulated.
Conclusions
Our study suggests that bacterial strains can gain fitness through the acquisition of multiple plasmids harbouring antibiotic resistance genes, which may be mediated by transcriptomic changes in the chromosomal genes of the bacterial host.
Abstract The characteristics of 218 Klebsiella pneumoniae isolates from patients with hospital-acquired pneumonia in nine Asian countries were investigated. In total, 92 isolates (42.2%) produced ...extended-spectrum β-lactamases (ESBLs), amongst which 67 (72.8%) possessed CTX-M ESBL genes; CTX-M-15 was the major ESBL (55 isolates; 59.8%). Multilocus sequence typing (MLST) and plasmid replicon typing were performed to investigate the genetic backgrounds of the 55 CTX-M-15-producing K. pneumoniae isolates. Twenty-five sequence types (STs) were identified. Clonal complex 11 (CC11) including ST11 was the most prevalent clone (20 isolates; 36.4%) and was distributed in all Asian countries except Taiwan. ST15 was the next most frequently identified clone (8 isolates; 14.5%). An IncFIIA-type plasmid was predominantly associated with blaCTX-M-15 (45 isolates; 81.8%). However, another plasmid type (IncA/C) was also identified and replicon types of seven isolates could not be determined. The high prevalence of CTX-M-15 amongst K. pneumoniae isolates in Asian countries may be due both to the acquisition of plasmids carrying blaCTX-M-15 and the spread of certain clones such as ST11 and ST15.
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