Diversity of bla genes and low incidence of CTX‐M in plasmid‐mediated AmpC‐producing Escherichia coli clinical isolates

The aim of this study was to characterize plasmid‐mediated AmpC (pAmpC)‐producing Escherichia coli clinical isolates. A total of 101 strains with AmpC‐susceptibility pattern were prospectively included. All isolates were tested by multiplex PCR to detect different bla genes. Phylogenetic groups were determined by a multiplex PCR assay. Antimicrobial susceptibility was tested by a microdilution commercial method. Presence of blapAmpC was detected in 79 (78.2%) of the strains; in these pAmpC‐producing isolates, blaTEM was detected in 41 (51.9%) strains, blaSHV in 5 (6.3%) strains, blaOXA in 3 (3.8%) strains, and blaCTX‐M in 3 (3.8%) strains. blaVIM and blaKPC were detected in one strain. Sixteen strains belonged to phylogroup A, 27 to B1, 20 to B2, and 16 to D. As conclusion, the majority of the strains of E. coli with AmpC‐susceptibility pattern are pAmpC positive, although the association of extended‐spectrum beta‐lactamases (ESBL) and pAmpC is unusual.     

Development and analysis of furazolidone‐resistant Escherichia coli mutants

Furazolidone‐resistant mutants were obtained from four clinical isolates of diarrhoeagenic Escherichia coli. The stability of the resistance and the frequency of mutation were established. The minimal inhibitory concentration of furazolidone, nitrofurantoin, nalidixic acid, ampicillin, chloramphenicol and tetracycline was established both in the presence and absence of the efflux pump inhibitor Phe‐Arg‐β‐Naphtylamyde. The presence of mutations in the nitroreductase genes nfsA and nfsB was analysed by PCR; sequencing and their enzymatic activity was assessed by a spectrophotometric assay. Alterations in outer membrane proteins were studied by SDS‐PAGE. The frequency of mutation ranged from <9.6 × 10^?10 to 9.59 × 10^?7. Neither an effect on efflux pumps inhibited by Phe‐Arg‐β‐Naphtylamyde nor cross‐resistance with the antibiotics studied was observed. Nineteen mutants (52.94%) presented mutations in the nitroreductase‐encoding genes: 17 in the nfsA gene (15 mutants with an internal stop codon, 2 with amino acid changes), 2 in the nfsB (all amino acid changes). Alterations in the outer membrane proteins OmpA and OmpW were also observed. Although more studies are necessary to find other resistance mechanisms, present data showed the low potential of selecting furazolidone‐resistant mutants, together with the lack of cross‐resistance with unrelated antimicrobial agents.     

Predominance of IncL/M and IncF plasmid types among CTX‐M‐ESBL‐producing Escherichia coli and Klebsiella pneumoniae in Bulgarian hospitals

Our objective was to investigate the plasmid replicon‐types involved in spread of ESBLs among Bulgarian Klebsiella pneumoniae and Escherichia coli. Sixty‐three isolates, with transferable beta‐lactam resistance determinants, collected between 2007 and 2009 in six medical institutions, were analysed with respect to their antimicrobial susceptibility, ESBL‐, RAPD‐, and plasmid replicon‐type. Phylogenetic typing and screening for the O25b‐ST131 lineage were carried out for E. coli. The predominant ESBLs were CTX‐M‐15 (81%) among E. coli and CTX‐M‐3 (58%) among K. pneumoniae. Other sporadically found ESBLs were SHV‐12 and TEM‐139, and for the first time in Bulgaria, CTX‐M‐1 and CTX‐M‐14. Replicon typing revealed that plasmids carrying bla_CTX‐M‐3 exclusively belonged to IncL/M‐type, while bla_CTX‐M‐15 was predominantly (94%) associated with IncF‐type plasmids. Among E. coli, 59% of the isolates were clonally related. Isolates of that cluster produced CTX‐M‐15, belonged to the O25b‐ST131 lineage, predominantly harboured plasmids with the FIA replicon, and were found in five centres. Among CTX‐M‐3‐producing K. pneumoniae, two prevailing RAPD‐types were found, one remained restricted to one centre and the second was found in three centres. The incompatibility groups IncN and IncA/C linked with bla_SHV‐12 respectively bla_TEM‐139 were found only once. To the best of our knowledge, this is the first detailed investigation of plasmids carrying ESBL genes among Bulgarian isolates demonstrating wide distribution of conjugative IncF plasmids among CTX‐M‐15‐producing E. coli and IncL/M plasmids among CTX‐M‐3 positive K. pneumoniae isolates.     

Characterization of CTX‐M‐producing Escherichia coli by repetitive sequence‐based PCR and real‐time PCR‐based replicon typing of CTX‐M‐15 plasmids

The emergence of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae is a major global concern. CTX‐M is the dominating ESBL type worldwide, and CTX‐M‐15 is the most widespread CTX‐M type. The dissemination of CTX‐M appears to be in part due to global spread of the Escherichia coli clone O25b‐ST131. However, the gene‐encoding CTX‐M is mainly located on mobile genetic elements, such as plasmids, that also promote the horizontal dissemination of the CTX‐M genes. In this study, 152 CTX‐M‐producing E. coli isolated in 1999–2008 in Örebro County, Sweden, were typed using a commercial repetitive sequence‐based PCR (the DiversiLab system), and the prevalence of ST131 was investigated by pabB PCR. Real‐time PCR‐based plasmid replicon typing was performed on 82 CTX‐M‐15‐producing E. coli isolates. In general, the CTX‐M‐producing E. coli population was genetically diverse; however, ST131 was highly prevalent (27%), and the dominating clone in our area. The bla_CTX‐M‐15 gene was mainly located on IncF plasmids (69%), but a relatively high proportion of IncI1 plasmids (29%) were also detected among E. coli with diverse rep‐PCR patterns, indicating that horizontal transmission of IncI1 plasmids carrying bla_CTX‐M‐15 may have occurred between different E. coli strains.     

Two plasmid‐encoded genes of enteropathogenic Escherichia coli strain K798 promote invasion and survival within HEp‐2 cells

Enteropathogenic Escherichia coli (EPEC) are considered to be extracellular pathogens, inducing attaching and effacing lesions following their attachment to the surface of eukaryotic cells; however, in vitro and in vivo invasion by EPEC has been reported in several studies. A cloned 4.6 kb fragment of EPEC plasmid pLV501 has been shown to facilitate invasion of E. coli K‐12, and here we further investigate the nature of this process. Two of the three complete open reading frames contained within the plasmid fragment have been cloned to E. coli, and in HEp‐2 adherence assays both tniA2 and pecM were shown to be expressed during the first 3 h of infection from a plac promoter. Escherichia coli transformants carrying pecM alone or in combination with tniA2 were able to both survive intracellularly and escape eukaryotic cells to re‐establish themselves within the medium, whereas those bacterial cells carrying tniA2 alone could not be isolated from within HEp‐2 cells after 24 h of infection, but were present in the previously sterile medium surrounding the cells. Bacteria carrying pecM and tniA2 adhered to HEp‐2 cells with sites of adhesion characterized by underlying actin polymerization. The invasive potential conferred by these genes may give EPEC strains a survival advantage during prolonged infection.     

The first human report of mobile colistin resistance gene,mcr‐1, in Finland

Colistin resistance mediated by mobile mcr‐1 gene has raised concern during the last years. After steep increase in mcr‐1 reports, other mcr‐gene variants (mcr‐2 to mcr‐5) have been revealed as well. In 2016, a clinical study was conducted on asymptomatic stool carriage of extended spectrum beta‐lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae among Finnish adults. All suspected ESBL producing bacterial isolates were first tested by phenotypic ESBL‐confirmation methods, and then further analyzed with whole genome sequencing to identify the resistance genes. We found one study subject carrying a colistin resistant E. coli with a transferrable mcr‐1 gene. This multi‐drug resistant isolate, although initially suspected to be an ESBL producer, did not carry any ESBL genes, but was proven to carry several other resistance genes by using whole genome sequencing. Sequence type was ST93. The mcr‐1 gene was connected to IncX4 plasmid which suggests that the colistin resistance gene locates in the respective plasmid. Here, we report the finding of a mcr‐1 harboring human E. coli isolate from Finland. Clinical antimicrobial resistance (AMR) rates are low in Finland, and mobile colistin resistance has not been reported previously. This highlights the importance of AMR surveillance also in populations with low levels of resistance.     

Occurrence of class 1 integrons in uropathogenic fluoroquinolone‐resistant clinical Escherichia coli isolates from Jamaica

Quinolone resistance is generally caused by chromosomal mutations, but has been more recently found associated with the plasmid‐mediated qnr genes. The objective of this study was to screen and analyse polymorphisms of integrons in clinical isolates of Escherichia coli in Jamaica. Previous studies in Jamaica identified fluoroquinolone resistance in predominantly uropathogenic E. coli clinical isolates: 45% harbouring qnrA, qnrB and/or qnrS, and 17% were (Extended‐spectrum beta‐lactamase) ESBL‐producers. These isolates were analysed for the presence and variation of class 1 and 2 integrase genes, 5′‐ and 3′‐ conserved segments and the Orf513 recombinase gene by primer‐specific polymerase chain reaction (PCR) and restriction fragment‐length polymorphism (RFLP). Results indicated integron‐encoded integrases in 93% of isolates primarily harbouring class 1 integrase genes; four of 58 isolates carried both classes. The Orf513 and 5′‐ and 3′‐conserved segment (CS) regions were identified in 83% and 55% of the isolates respectively. RFLP evaluation of the 5′‐ and 3′‐CS regions in int1‐positive strains yielded two main types. The reduced diversity, but wide dispersion of class 1 integrons harbouring qnr genes may give rise to the conservation of the mobile genetic elements in which they are carried.     

Clonal dissemination of multilocus sequence type ST15 KPC‐2‐producing Klebsiella pneumoniae in Bulgaria

A total of 36 consecutive clinical and two fecal‐screening carbapenem‐resistant Klebsiella pneumoniae isolates from two Bulgarian university hospitals (Varna and Pleven) were investigated. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta‐lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and multilocus sequence typing (MLST). Most of the isolates demonstrated multidrug resistance profile. Amikacin and tigecycline retained good activity with susceptibility rates of 95 and 87%, respectively. The resistance rate to colistin was 63%. Six RAPD‐ and MLST‐types were identified: the dominating MLST‐type was ST15 (27 isolates), followed by ST76 (six isolates), and ST1350 (two isolates). ST101, ST258, and ST151 were detected once. All except one of the K. pneumoniae produced KPC‐2, mostly in combination with CTX‐M‐15, while for one isolate (ST101) the enzymes OXA‐48 and CTX‐M‐14 were found. All KPC‐2‐producing transconjugants revealed the presence of IncFII plasmid. The OXA‐48‐ and CTX‐M‐14‐producing isolate showed the presence of L/M replicon type. The dissemination of KPC‐2‐producing K.pneumoniae in Bulgaria is mainly due to the sustained spread of successful ST15 clone and to a lesser extent of ST76 clone. This is the first report of OXA‐48 producing ST101 K. pneumoniae in Bulgaria.     

Survival in the environment is a possible key factor for the expansion of Escherichia coli strains producing extended‐spectrum β‐lactamases

Acquired resistance to cephalosporins in Enterobacteriaceae is a global problem. After an outbreak at Uppsala University Hospital of extended‐spectrum β‐lactamase (ESBL)‐positive Klebsiella pneumoniae producing CTX‐M‐15, there was a shift from AmpC to ESBL production among Escherichia coli isolates. To explore the basis for this epidemiological shift, 46 E. coli isolates (ESBLs, n = 23; AmpC, n = 23) were characterized with regard to genetic relatedness, β‐lactamase, replicon and integron types, antibiotic resistance profiles, and genes encoding virulence factors. In addition, the survival in the environment and on hospital‐associated materials was analysed. CTX‐M‐15 was the most frequent ESBL (78%). Only three (13%) of the AmpC enzymes were harboured on plasmids (CMY‐2, DHA‐1). Independent of plasmid‐mediated beta‐lactamase, IncF plasmids predominated and only class I integrons were detected. The ESBL producers carried more virulence genes (p = 0.04), exhibited a broader resistance phenotype (p = 0.01) and survived significantly longer (p = 0.03) on different materials than the AmpC‐producing isolates. In conclusion, ESBL‐producing isolates had properties which are likely to augment their competitiveness. Apart from antibiotic resistance and virulence factors, extended survival in the environment could be a selective trait for successful ESBL‐producing E. coli strains.     

Antibiotic resistance and molecular epidemiology of the beta‐lactamase‐producing Haemophilus influenzae isolated in Chongqing,China

This study aimed to determine the antibiotic resistance and molecular epidemiology of Haemophilus influenzae isolated from children with acute respiratory infection in Chongqing, China. To this end, 1967 H. influenzae isolates from 2006 to 2009 were analysed regarding β‐lactamase production and antibiotic resistance. Ninety‐nine β‐lactamase‐producing H. influenzae isolates from 2010 were analysed for antibiotic resistance and promoter regions of bla_TEM‐1. β‐lactamase production was found in 35.8% (705/1967) of the strains. All ninety‐nine β‐lactamase‐producing strains from 2010 were of the TEM‐1 type as determined by PCR but did not produce the predicted 1075 bp product. According to PCR‐SSCP and DNA sequencing, the promoter regions of bla_TEM‐1 were categorized into 6 genotypes as SSCP1 (Pdel), SSCP2 (Pa/Pb), SSCP3 (P4), SSCP4 (Prpt.b), SSCP5 (2Prpt) and SSCP6 (P3.b). The Pdel, Pa/Pb and Prpt.b were common promoters of bla_TEM‐1 for H. influenzae isolated from children in Chongqing. Strains with Prpt.b were more resistant to ampicillin (AMP) than strains with Pdel, Pa/Pb and P4 (p < 0.05). Therefore, bla_TEM‐1 β‐lactamase is the main mechanism for resistance of H. influenzae to ampicillin in Chongqing. Furthermore, the Prpt.b promoters may be related to the high resistance of H. influenzae to AMP.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Molecular characterization of CTX‐M‐15‐producing clinical isolates of Escherichia coli reveals the spread of multidrug‐resistant ST131 (O25:H4) and ST964 (O102:H6) strains in Norway

Nationwide, CTX‐M‐producing clinical Escherichia coli isolates from the Norwegian ESBL study in 2003 (n=45) were characterized on strain and plasmid levels. Bla_CTX‐M allele typing, characterization of the genetic environment, phylogenetic groups, pulsed field gel electrophoresis (PFGE), serotyping and multilocus sequence typing were performed. Plasmid analysis included S1‐nuclease‐PFGE, polymerase chain reaction‐based replicon typing, plasmid transfer and multidrug resistance profiling. Bla_CTX‐M‐15 (n=23; 51%) and bla_CTX‐M‐14 (n=11; 24%) were the major alleles of which 18 (78%) and 6 (55%), respectively, were linked to ISEcp1. Thirty‐two isolates were of phylogenetic groups B2 and D. Isolates were of 29 different XbaI‐PFGE‐types including six regional clusters. Twenty‐three different O:H serotypes were found, dominated by O25:H4 (n=9, 20%) and O102:H6 (n=9, 20%). Nineteen different STs were identified, where ST131 (n=9, 20%) and ST964 (n=7, 16%) were dominant. Bla_CTX‐M was found on ≥100 kb plasmids (39/45) of 10 different replicons dominated by IncFII (n=39, 87%), FIB (n=20, 44%) and FIA (n=19, 42%). Thirty‐nine isolates (87%) displayed co‐resistance to other classes of antibiotics. A transferable CTX‐M phenotype was observed in 9/14 isolates. This study reveals that the majority of CTX‐M‐15‐expressing strains in Norway are part of the global spread of multidrug‐resistant ST131 and ST‐complex 405, associated with ISEcp1 on transferrable IncFII plasmids.     

Carbapenemases‐producing Klebsiella pneumoniae in hospitals of two regions of Southern Italy

Carbapenem‐resistant Klebsiella pneumoniae infections are reported with increasing frequency elsewhere in the world, representing a worrying phenomenon for global health. In Italy, there are hotspot data on the diffusion and type of carbapenemase‐producing Enterobacteriaceae and K. pneumoniae in particular, with very few data coming from Apulia and Basilicata, two regions of Southern Italy. This study was aimed at characterizing by phenotypic and genotypic methods carbapenem‐resistant K. pneumoniae isolated from several Hospitals of Apulia and Basilicata, Southern Italy. Antibiotic susceptibility was also evaluated. The relatedness of carbapenemase‐producing K. pneumoniae strains was established by pulsed‐field gel electrophoresis (PFGE). Among the 150 K. pneumoniae carbapenemase producers, KPC‐3 genotype was the most predominant (95%), followed by VIM‐1 (5%). No other genotypes were found and no co‐presence of two carbapenemase genes was found. A full concordance between results obtained by both the phenotypic and the genotypic tests was observed. All strains were resistant to β‐lactam antibiotics including carbapenems, and among antibiotics tested, only tetracycline and gentamycin showed low percentage of resistance (18% and 15%, respectively). Resistance to colistin was detected in 17.3% of strains studied. The analysis of PFGE profiles of the carbapenemases‐positive strains shows that one group (B) of the five (A to E) main groups identified was the most prevalent and detected in almost all the hospitals considered, while the other groups were randomly distributed. Three different sequence types (ST 307, ST 258, and ST 512) were detected with the majority of isolates belonging to the ST 512. Our results demonstrated the wide diffusion of K. pneumoniae KPC‐3 in the area considered, the good concordance between phenotypic and genotypic tests. Gentamicin and colistin had a good activity against these strains.     

Amomum tsao‐ko fruit extract suppresses lipopolysaccharide‐induced inducible nitric oxide synthase by inducing heme oxygenase‐1 in macrophages and in septic mice

Amomum tsao‐ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti‐inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao‐ko (AOM) in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS‐induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase‐1 (HO‐1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO‐1) and silencing HO‐1 using siRNA prevented the AOM‐mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF‐E2‐related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N‐acetyl‐l ‐cysteine (NAC; a ROS scavenger) diminished the AOM‐induced phosphorylation of ERK and JNK and AOM‐induced HO‐1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM‐induced signalling of HO‐1 expression. In LPS‐induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO‐1 expression and survival rates. These results indicate that AOM strongly inhibits LPS‐induced NO production by activating the ROS/MAPKs/Nrf2‐mediated HO‐1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.     

Tandem pore domain K+‐channel TASK‐3 (KCNK9) and idiopathic absence epilepsies

Recently, the gene coding for the tandem pore domain K⁺‐channel TASK‐3 (KCNK9) has been localized to the chromosomal region 8q24. Because mutations in ion channel genes have been recognized as an important factor in the etiology of abnormal neuronal excitability, TASK‐3 is an interesting candidate gene for epilepsies linked to 8q24. We therefore performed a mutation analysis of the TASK‐3 gene in 65 patients with childhood and juvenile absence epilepsy. Only one silent nucleotide exchange (636C/T) was detected in exon 2 of the TASK‐3 coding region. No evidence for an allelic association was found between the exon 2 polymorphism and absence epilepsy. Accordingly, genetic variation of the TASK‐3 coding region does not play a major role in the etiology of idiopathic absence epilepsies. © 2002 Wiley‐Liss, Inc.     

Super‐resolution imaging reveals changes in Escherichia coli SSB localization in response to DNA damage

The E. coli single‐stranded DNA‐binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with 100–150 associated with the genome at any one time, either at DNA replication forks or at sites of repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes. To visualize SSB localization and to ascertain potential spatiotemporal changes in response to DNA damage, SSB‐GFP chimeras were visualized using a novel, super‐resolution microscope optimized for the study of prokaryotic cells. In the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events.     

HIV‐specific T‐cell responses detected in the genital tract of chronically HIV‐infected women are largely monofunctional

HIV‐specific T cells that produce interferon‐γ (IFN‐γ) are present in the genital tract of HIV‐infected women although these do not provide protection against genital HIV shedding. Because polyfunctional HIV‐specific T cells have been implicated in better HIV control than those with a single function, this study aimed to investigate whether polyfunctional T cells were present at the female genital mucosa. Cervical cytobrush‐derived T cells were obtained from chronically HIV‐infected women and compared with blood. CD3⁺ T cells from both compartments were expanded with Dynal anti‐CD3/CD28 expander beads for 14 days and flow cytometry was used to evaluate four T‐cell functions (CD107a, IFN‐γ, tumour necrosis factor‐α and macrophage inflammatory protein‐1β) from 16 women. The majority of Gag‐specific T‐cell responses in the female genital tract were monofunctional, although low frequencies of HIV Gag‐specific polyfunctional CD8⁺ T cells were detected at the cervix in 81·3% (13/16) of women. The ability of CD8⁺ T cells at both the cervix and in blood to express CD107a and to exhibit polyfunctional responses (two or more functions) following Gag stimulation was inversely associated with plasma viral load and positively associated with blood CD4 counts, suggesting that clinical status impacted on the functionality of HIV‐specific T cells at the mucosa, in a similar way to blood. HIV Gag‐specific cervical T cells were largely monofunctional. Polyfunctional T cells were detected at the cervix in women with high blood CD4 count and low plasma viral load but these did not protect from HIV genital shedding.