The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?

The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.     

A clinical strain of Escherichia coli possessing CMY-2 plasmid-mediated amp C beta-lactamase: an emerging concern in pediatrics?

A 5-year-old child was colonized by an isolate of Escherichia coli that transferred resistance to third-generation cephalosporins and cefoxitin. This resistance phenotype was encoded on a >75-kb plasmid pLRM 22. The transferable plasmid contained both blaCMY-2 and blaTEM-1b. Increasing reports of CMY-2 beta-lactamase in clinical isolates in children raise concerns about the empiric use of third-generation cephalosporins in this patient group.     

Extended-spectrum beta-lactamase-producing Escherichia coli infections in children: Are community-acquired strains different from nosocomial strains?

Infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are an important cause of morbidity and mortality, especially in children. We compared 58 epidemiologically unrelated ESBL-producing E. coli strains that caused infections. They were isolated between 2008 and 2012 in two Parisian pediatric hospitals and grouped according to their origin into either community-acquired (CA) (n = 37) or nosocomially acquired (NA) (n = 21) strains. Molecular characteristics of the ESBLs, phylogenetic traits of the strains including their belonging to clone O25b-ST131, prevalence of associated virulence genes, growth capacities in different media, metabolic phenotype and biofilm formation abilities were studied. ESBL type, associated resistance and distribution of phylogenetic groups were similar in the CA and NA groups. More than 60% of the B2 phylogroup strains in both groups belonged to the ST131 clone. Interestingly, CA strains possessed more genes encoding virulence factors and the distribution of these genes differed significantly between the two groups: fyuA, hlyC, papC and papGII were more frequent in the CA group, whereas iroN was more frequent in the NA group. CA strains also showed enhanced growth capacities in Luria Bertani rich medium. They tended to produce more biofilm but the difference was not significant. This study confirms the wide spread of clone ST131 among infected children, regardless of whether their infections were community- or nosocomially acquired. It highlights genotypic and phenotypic differences according to the origin of the strains that could indicate adaptability of these multi-resistant bacteria to specific environmental and host factors.     

Detection of β-lactamase and urovirulence genes in Escherichia coli serogroups isolated from urinary tract infection in cats

Sixty-five Escherichia coli isolates were recovered from cats with urinary tract infection and their serogroups were determined. The isolates were screened for the presence of β-lactamase (bla _TEM) and urovirulence (cnf1, cdtB, afaIB-C, papE-F, sfa/focD-E and iutA) genes. Serogroup determination of 65 uropathogenic E. coli isolates showed that 23 (35.38%) were O-typeable and belonged to 11 different O serogroups including: O1, O2, O4, O6, O8, O11, O25, O49, O75, O115 and O125. Fourteen (21.54%) isolates were positive for the bla _TEM gene. The bla _TEM-positive isolates were distributed in both typeable (five) and non-typeable (nine) serogroups. Eleven bla _TEM-positive isolates showed combination patterns of virulence genes, whereas the combination of cnf1-hlyA-papE-F-sfa/focDE was the most prevalent pattern. Polymerase chain reaction tests revealed that 38 isolates (58.46%) contained at least two urovirulence genes. Among the examined isolates, 56.92% had cnf1, 52.30% hlyA, 50.76% sfa/focD-E, 47.69% papE-F, 13.84% iutA and 6.15% afaIB-C. None of the isolates contained the cdtB gene. Eight different combinations of virulence genes were detected. The combination pattern of cnf1-hlyA-papE-F-sfa/focD-E was the most prevalent (30.77%), whereas the positive isolates fell into O2 (three), O6 (two), and O49 (one) serogroup and 14 isolates were O-non-typeable. In conclusion, feline E. coli isolates from urinary tract infections belonged to several serogroups and may possess β-lactamase and urovirulence coding genes.     

High prevalence of blaCTX-M extended-spectrum β-lactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in China

As a cause of community-acquired infections, extended-spectrum β-lactamase (ESBL)-producing Escherichia coli constitute an emerging public-health concern. Few data on the molecular epidemiology of ESBL-producing E. coli isolates from pets are available in China. Detection and characterization of ESBL genes (bla_CTX-M, bla_SHV and bla_TEM) was conducted among 240 E. coli isolates recovered from healthy and sick pets in South China from 2007 to 2008. The clonal relatedness of ESBL-producing E. coli isolates was assessed by pulsed field gel electrophoresis. ESBL-encoding genes were identified in 97 (40.4%) of the 240 isolates and 96 (40.0%) of them harbored CTX-M. The most common CTX-M types were CTX-M-14 (n = 45) and CTX-M-55 (n = 24). The recently reported CTX-M-64 was identified in three isolates. Isolates producing CTX-M-27, -15, -65, -24, -3 and -9 were also identified. Ten isolates carried two or three CTX-M types, with the combination of CTX-M-14 and CTX-M-55 being the most frequent (n = 6). ISEcp1 was identified in the upstream region of 93 out of the 107 bla_CTX-M genes (86.9%). The sequence of the spacer region (45 bp) between ISEcp1 and the start codon of all bla_CTX-M-55 genes (except four) was identical to that of bla_CTX-M-64. No major clonal relatedness was observed among these CTX-M producers. It is suggested that the horizontal transfer of bla_CTX-M genes, mediated by mobile elements, contributes to their dissemination among E. coli isolates from pets. Our finding of high prevalence of ESBL in E. coli of companion animal origin illustrates the importance of molecular surveillance in tracking CTX-M-producing E. coli strains in pets.     

Genomic Comparison of Two O111:H? Enterohemorrhagic Escherichia coli Isolates from a Historic Hemolytic-Uremic Syndrome Outbreak in Australia

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhea and hemolytic-uremic syndrome (HUS) worldwide. Australia''s worst outbreak of HUS occurred in Adelaide in 1995 and was one of the first major HUS outbreaks attributed to a non-O157 Shiga-toxigenic E. coli (STEC) strain. Molecular analyses conducted at the time suggested that the outbreak was caused by an O111:H⁻ clone, with strains from later in the outbreak harboring an extra copy of the genes encoding the potent Shiga toxin 2 (Stx2). Two decades later, we have used next-generation sequencing to compare two isolates from early and late in this important outbreak. We analyzed genetic content, single-nucleotide polymorphisms (SNPs), and prophage insertion sites; for the latter, we demonstrate how paired-end sequence data can be leveraged to identify such insertion sites. The two strains are genetically identical except for six SNP differences and the presence of not one but two additional Stx2-converting prophages in the later isolate. Isolates from later in the outbreak were associated with higher levels of morbidity, suggesting that the presence of the additional Stx2-converting prophages is significant in terms of the virulence of this clone.     

Heterozygous missense variant in EIF6 gene: A novel form of Shwachman–Diamond syndrome?

Shwachman–Diamond syndrome (SDS) is a rare multisystem ribosomal biogenesis disorder characterized by exocrine pancreatic insufficiency, hematologic abnormalities and bony abnormalities. About 90% of patients have biallelic mutations in SBDS gene. Three additional genes—EFL1, DNAJC21 and SRP54 have been reported in association with a SDS phenotype. However, the cause remains unknown for ~10% of patients. Herein, we report a 6‐year‐old Chinese boy, who presented in the neonatal period with pancytopenia, liver transaminitis with hepatosplenomegaly and developmental delay, and subsequently developed pancreatic insufficiency complicated by malabsorption and poor growth. Exome sequencing identified a novel de novo heterozygous variant in EIF6 (c.182G>T, p.Arg61Leu). EIF6 protein inhibits ribosomal maturation and is removed in the late steps of ribosomal maturation by SBDS and EFL1 protein. Given the interaction of EIF6 with SBDS and EFL1, we postulate heterozygous variants in EIF6 as a novel cause of Shwachman–Diamond‐like phenotype. We compared the phenotype of our patient with those in patients with mutation in SBDS, EFL1, DNAJC21, and SRP54 genes to support this association. Identification of more cases of this novel phenotype would strengthen the association with the genetic etiology.     

The Tol–Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate

Chromate is a toxic metal that enters bacteria by using oxyanion importers. Here, we show that each mutant of the Tol–Pal system of Escherichia coli exhibited increased chromate resistance. This system, which spans the cell envelope, plays a major role in envelope integrity and septation. The ΔtolQR mutant accumulated three-fold less chromate than the wild-type. Addition of phosphate but not sulfate to rich medium drastically reduced chromate toxicity and import in the wild-type strain. Furthermore, the intracellular concentration of free inorganic phosphate was significantly reduced for the ΔtolR mutant in comparison to the wild-type strain. Moreover, extracellular labeled phosphate was significantly less incorporated into the ΔtolR mutant. Finally, two distinct TolQR mutant complexes, specifically affected in Tol–Pal energization without affecting the TolQRA complex structure, did not complement the ΔtolQR mutant for inorganic phosphate accumulation. We thus propose that, while the Pst system is well known to import inorganic phosphate, the Tol–Pal system participates to phosphate uptake in particular at medium to high extracellular phosphate concentrations. Since mutations disabling the Tol–Pal system lead to pleiotropic effects, chromate resistance and reduced inorganic phosphate import could occur from an indirect effect of mutations in components of the Tol–Pal system.     

Hydrogen peroxide induces protection against lethal effects of cumene hydroperoxide in Escherichia coli cells: an Ahp dependent and OxyR independent system?

Pretreatment with 2.5 mM H₂O₂ protects bacterial cells against cumene hydroperoxide killing. This response is independent of the OxyR system, but possibly involves the participation of Ahp protein, since ahp mutants are not protected. Treatment of bacterial cells with high H₂O₂ concentrations caused an alteration on the electrophoretic profile of the smaller subunit (22-kDa) of Ahp. This alteration does not require novel gene products and is not dependent on the OxyR protein. In this way, we propose that the modification of the 22-kDa subunit of Ahp by high H₂O₂ concentration may be responsible for the protection against the lethal effects of cumene hydroperoxide.     

Virulence markers of enteroaggregative Escherichia coli isolated from children and adults with diarrhea in Brasília,Brazil

Escherichia coli strains isolated from sporadic cases of acute diarrhea in children and adults and from children without diarrhea were investigated for the presence of the pAA plasmid. Strains harboring the pAA plasmid were isolated at similar frequencies from children with (19.6%) and without (10.8%) diarrhea and from adults with diarrhea (11.8%). The genotypic and phenotypic virulence markers of these strains were further analyzed. Most of the strains were positive for EAST1 (73%), and this toxin was detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.05). Likewise, pic sequences were detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.005) and controls (P < 0.025). Furthermore, the association of pAA positivity (pAA(+)) and pic positivity (pic(+)) was more frequently found for strains from children with diarrhea than for strains from controls, indicating that pAA(+) pic(+) strains may represent a subset of pAA(+) strains associated with disease in children. Most of the strains (82.5%) adhered to cells presenting the typical aggregative pattern. The frequency of occurrence of enteropathogenic E. coli (EPEC) serogroups in the strains from children with diarrhea was very high (56%), while none of the strains from adults with diarrhea belonged to EPEC serogroups. Extraintestinal virulence markers were very commonly found in strains from adults with diarrhea. The frequencies of occurrence of the adhesins AFA and SFA were significantly higher in strains from adults with diarrhea than in strains from children with diarrhea. More than one extraintestinal virulence marker was found in 58% of the strains from adults with diarrhea but in only 7.7% of the strains from children with diarrhea. Our results show that pAA(+) strains isolated from children and adults with diarrhea present very different profiles when enteroaggregative E. coli virulence markers, serotypes, and extraintestinal virulence markers are considered.     

Molecular characterization of vancomycin-resistant enterococci and extended-spectrum β-lactamase-containing Escherichia coli isolates in wild birds from the Azores Archipelago

To study the prevalence of vancomycin-resistant enterococci (VRE) and extended-spectrum β-lactamase (ESBL)-containing Escherichia coli isolates, and the mechanisms of resistance implicated, 220 faecal samples from wild birds were collected between 2006 and 2010 in the Azores Archipelago. Samples were spread on Slanetz–Bartley agar plates supplemented with 4 mg/l vancomycin and on Levine agar plates supplemented with 2 mg/l cefotaxime for VRE and ESBL-containing E. coli isolation, respectively. vanA-containing enterococcal isolates (four Enterococcus faecium and two Enterococcus durans) and vanC-1 Enterococcus gallinarum isolates were detected in six and seven faecal samples, respectively. VRE isolates showed ampicillin (n=11), ciprofloxacin (n=9), tetracycline (n=6), erythromycin (n=5), quinupristin/dalfopristin (n=3) and high-level kanamycin resistance (n=1). The tet(L) and/or tet(M) gene was found in all tetracycline-resistant isolates and the erm(B) gene in all erythromycin-resistant isolates. Three vanA-containing E. faecium and two E. gallinarum presented specific sequences of the Tn5397 transposon. Four VRE isolates harboured the ace virulence gene. One faecal sample revealed one ESBL-containing E. coli isolate that belongs to the A phylogenetic group, showed a phenotype of resistance to β-lactams and tetracycline, and harboured the bla _CTX-M-14, bla _SHV-12 and the tet(A) genes. To our knowledge, this is the first study to focus on defining the prevalence of VRE and/or ESBL-containing E. coli strains in wild birds from the Azores. The data recovered are essential to improve knowledge about the dissemination of resistant strains through wild ecosystems and their possible implications by transferring these resistances to other animals or to humans.     

A novel mutation of Twinkle in Perrault syndrome: A not rare diagnosis?

Perrault syndrome is a rare disorder characterized by ovarian dysgenesis, bilateral sensorineural hearing loss and associated with mutations in six mitochondrial proteins. Additional neurological features were also described. Herein, we report on a 27-year-old woman with Perrault syndrome (PS), moderate ataxia and axonal sensory-motor peripheral neuropathy in whom we identified compound heterozygous mutations in the TWNK gene (p.Val507Ile and the novel p.Phe248Ser variant). Fewer than 30 patients with PS have been reported worldwide. Neurological involvement is more frequently associated with mutations in TWNK and indicates possible genotype–phenotype correlations. TWNK mutations should be searched in patients with sensory ataxia, early onset bilateral sensorineural hearing loss, and ovarian dysfunction in women.     

DNA synthesized in Escherichia coli K12 (λ) after infection with an rII mutant of bacteriophage T4

Deoxyribonucleic acid (DNA) synthesized in Escherichia coli K12 (λ) after abortive infection with rII mutants of phage T 4 was found to contain 5-hydroxymethylcytosine without any cytosine, as characteristic of phage DNA. It appears that physiological defect associated with the rII mutation is not a failure to form deoxycytidylate hydroxymethylase.