Implications of <Emphasis Type="Italic">stx</Emphasis> loss for clinical diagnostics of Shiga toxin-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The dynamics related to the loss of stx genes from Shiga toxin-producing Escherichia coli remain unclear. Current diagnostic procedures have shortcomings in the detection and identification of STEC. This is partly owing to the fact that stx genes may be lost during an infection or in the laboratory. The aim of the present study was to provide new insight into in vivo and in vitro stx loss in order to improve diagnostic procedures. Results from the study support the theory that loss of stx is a strain-related phenomenon and not induced by patient factors. It was observed that one strain could lose stx both in vivo and in vitro. Whole genome comparison of stx-positive and stx-negative isolates from the same patient revealed that different genomic rearrangements, such as complete or partial loss of the parent prophage, may be factors in the loss of stx. Of diagnostic interest, it was shown that patients can be co-infected with different E. coli pathotypes. Therefore, identification of eae-positive, but stx-negative isolates should not be interpreted as “Shiga toxin-lost” E. coli without further testing. Growth and recovery of STEC were supported by different selective agar media for different strains, arguing for inclusion of several media in STEC diagnostics.     

Increasing incidence of carbapenemase-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in Belgian hospitals

Carbapenemase-producing Enterobacteriaceae are increasingly reported worldwide. The aim of the study was to determine the incidence and molecular epidemiology of carbapenemase-producing (CP) Escherichia coli and Klebsiella pneumoniae (CP-E/K) in Belgium. Eleven hospital-based laboratories collected carbapenem non-susceptible (CNS) isolates of E. coli and K. pneumoniae detected in clinical specimens from January 2013 to December 2014. All CNS strains were tested for carbapenemase production and typed by multilocus sequence typing (MLST) for a 6-month period as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae in Europe (EuSCAPE) structured survey. In addition, an equal number of carbapenem-susceptible isolates collected were preserved as a control group for risk factor analysis. The overall incidence rate of CP-E/K isolates in hospitals increased from 0.124 in 2013 to 0.223 per 1000 admissions in 2014. From November 2013 to April 2014, 30 CP K. pneumoniae [OXA-48 (n?=?16), KPC (n?=?13), OXA-427 (n?=?1)] and five CP E. coli [OXA-48 (n?=?3), NDM (n?=?1), OXA-427 (n?=?1)] isolates were detected in ten hospitals. The 16 OXA-48-producing K. pneumoniae strains were distributed into eight sequence types (STs), while the 13 KPC-producing K. pneumoniae clustered into three STs dominated by ST512 (n?=?7) and ST101 (n?=?5). Compared to controls, we observed among CP-E/K carriers significantly higher proportion of males, respiratory origins, previous hospitalization, nosocomial setting, and a significantly lower proportion of bloodstream infections. Our study confirms the rapid spread of CP-E/K in Belgian hospitals and the urgent need for a well-structured and coordinated national surveillance plan in order to limit their dissemination.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Enzyme-Linked Immunosorbent Assay for Detection of Shiga Toxin-Producing <Emphasis Type="Italic">Escherichia coli</Emphasis> Infection by Antibodies to <Emphasis Type="Italic">Escherichia coli</Emphasis> Secreted Protein B in Children with Hemolytic Uremic Syndrome

In order to detect immunoglobulin (Ig)A and IgG antibodies to Escherichia coli-secreted protein B in sera of children infected with Shiga toxin-producing Escherichia coli, an enzyme-linked immunosorbent assay was developed. The assay was tested using acute sera from 40 children with diarrhea-associated hemolytic uremic syndrome compared with 238 sera obtained from pediatric controls. Two cut-off values were used for children <5 (n=27) or 5 (n=13) years of age. Among the younger patients, 24 of 27 had IgA antibodies to Escherichia coli-secreted protein B (sensitivity, 89%; specificity, 98%) and 22 of 27 had IgG antibodies (sensitivity, 82%; specificity, 94%). Among the older patients, 13 of 13 had IgA antibodies (sensitivity, 100%; specificity, 96%) and 11 of 13 had IgG antibodies (sensitivity, 85%; specificity, 96%). This enzyme-linked immunosorbent assay detects Shiga-toxin-producing Escherichia coli independent of serogroup and could serve as a complementary assay for detection of infection.     

Characterization of carbapenem-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> from a healthcare region in Hong Kong

Carbapenem-resistant Enterobacteriaceae represents a major public health issue. This study investigated the clonality and resistance mechanisms of 92 carbapenem-resistant E. coli (n?=?21) and K. pneumoniae (n?=?71) isolates collected consecutively from clinical specimens and patients at high risk of carriage between 2010 and 2012 in a healthcare region in Hong Kong. Combined disk tests (CDTs) and the Carba NP test were used for phenotypic detection of carbapenemases. PCR assays were used to detect carbapenemase genes. All isolates were intermediate or resistant to at least one carbapenem. Nine (9.8 %) isolates were genotypic carbapenemase producers and included six K. pneumoniae (one ST1306/bla _IMP-4, one ST889/bla _IMP-4, two ST11/bla _KPC-2, one ST258/bla _KPC-2, one ST483/bla _NDM-1) and three E. coli (one ST131/bla _IMP-4, two ST744/ bla _NDM-1) isolates. All nine isolates carrying carbapenemase genes could be detected by the CDTs and the Carba NP test. PCR identified bla _CTX-M and bla _AmpC alone or in combination in 77.8 % (7/9) and 96.4 % (80/83) of the carbapenemase-producers and non-producers, respectively. Porin loss was detected in 22.2 % (2/9) and 59.0 % (49/83) of the carbapenemase-producers and non-producers, respectively. Overall, the E. coli clones were diverse (14 different STs), but 36.6 % (26/71) of the K. pneumoniae isolates belonged to ST11. In conclusion, the prevalence of carbapenemases among carbapenem-nonsusceptible E. coli and K. pneumoniae remained low in Hong Kong. Porin loss combined with AmpC and/or CTX-M type ESBL was the major mechanism of carbapenem resistance in the study population.     

Effect of chemotype on <Emphasis Type="Italic">Escherichia coli</Emphasis> interactions with bactericidal proteins

We studied the effects of bactericidal proteins (lysozyme and lactoferrin) on endotoxin release from cell wall and inhibition of the growth of Escherichia coli colonies of different chemotypes. The structure of LPS core was found to be essential for the mechanisms of the interactions of the studied proteins with the cell wall. Cell viability after contact with cationic proteins is determined not by the amount of released LPS, but by the mechanism of damage to the cell wall. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 191–193, August, 2007     

How heterologously expressed <Emphasis Type="Italic">Escherichia coli</Emphasis> genes contribute to understanding DNA repair processes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

DNA-damaging agents constantly challenge cellular DNA; and efficient DNA repair is therefore essential to maintain genome stability and cell viability. Several DNA repair mechanisms have evolved and these have been shown to be highly conserved from bacteria to man. DNA repair studies were originally initiated in very simple organisms such as Escherichia coli and Saccharomyces cerevisiae, bacteria being the best understood organism to date. As a consequence, bacterial DNA repair genes encoding proteins with well characterized functions have been transferred into higher organisms in order to increase repair capacity, or to complement repair defects, in heterologous cells. While indicating the contribution of these repair functions to protection against the genotoxic effects of DNA-damaging agents, heterologous expression studies also highlighted the role of the DNA lesions that are substrates for such processes. In addition, bacterial DNA repair-like functions could be identified in higher organisms using this approach. We heterologously expressed three well characterized E. coli repair genes in S. cerevisiae cells of different genetic backgrounds: (1) the ada gene encoding O⁶-methylguanine DNA-methyltransferase, a protein involved in the repair of alkylation damage to DNA, (2) the recA gene encoding the main recombinase in E. coli and (3) the nth gene, the product of which (endonuclease III) is responsible for the repair of oxidative base damage. Here, we summarize our results and indicate the possible implications they have for a better understanding of particular DNA repair processes in S. cerevisiae.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Development of <Emphasis Type="Italic">T</Emphasis><Subscript><Emphasis Type="Italic">m</Emphasis></Subscript>-shift genotyping method for detection of cat-derived <Emphasis Type="Italic">Giardia lamblia</Emphasis>

To develop T _m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5′-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T _m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T _m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10^?5 and 4.88 × 10^?5 ng/μL samples of assemblage A and F standard plasmids. The T _m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T _m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T _m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.     

Characteristics of Emergence of Mutants Resistant to Nalidixic Acid and Novobiocin in <Emphasis Type="Italic">E. coli</Emphasis> Strains with <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">lexA</Emphasis> Mutations

The main problem of the modern medical microbiology is the widespread resistance of bacteria to many antibiotics used in therapy. Resistance to antibiotics is mainly developed due to mutations in the bacterial genome. One of the proposed mechanisms for the occurrence of mutations is the functioning of the inducible SOS response system, the proteins of which are synthesized in the cell affected by antibiotics. The RecA and LexA proteins encoded by the corresponding genes are regulators of the SOS response in bacteria. The effect of the recA13 and lexA1 mutations on bacterial resistance to nalidixic acid and novobiocin and on the rates of antibiotic resistance development was studied. The SOS response system was shown to play a minor role in the development of the bacterial resistance to quinolones and aminocoumarins, as well as in mutagenesis, during application of these antibiotics.     

Comparison of Media for the Selective Culture of Enteroinvasive <Emphasis Type="Italic">Escherichia coli</Emphasis>

MacConkey, eosine-methylene blue, deoxycholate-citrate, salmonella-shigella, and xylose-lysine-deoxycholate agars were compared for their ability to support the growth and to facilitate the recovery of enteroinvasive Escherichia coli strains from artificially contaminated as well as from clinical faecal samples. When grown as pure cultures, the 78 enteroinvasive Escherichia coli strains, as a group, exhibited the same growth characteristics as did Shigella isolates (n=59), i.e. both organisms grew more weakly than did Salmonella strains (n=22) on the various selective plates but 4- to 10-fold better than normal Escherichia coli isolates (n=53). Xylose-lysine-deoxycholate and deoxycholate-citrate plates were more effective in recovering enteroinvasive Escherichia coli from faecal samples than was salmonella-shigella agar. Likewise, xylose-lysine-deoxycholate agar, similar to the differentiating MacConkey and eosine-methylene blue agars, was less inhibitory for defined "sensitive" strains than were the selective media tested. Preincubating clinical faecal samples in selenite F or in gram-negative broth did not influence the recovery of enteroinvasive Escherichia coli significantly. These data show that the use of xylose-lysine-deoxycholate, in combination with MacConkey or eosine-methylene blue agar, provides the best chance for recovery of enteroinvasive Escherichia coli when randomly selecting colonies from faecal cultures for subsequent molecular or immunological identification assays. Electronic Publication     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

Droplet-based non-faradaic impedance sensors for assessment of susceptibility of <Emphasis Type="Italic">Escherichia coli</Emphasis> to ampicillin in 60 min

Direct antibiotic susceptibility tests (AST) are essential for rapid detection of bacterial infection and administration of appropriate antibiotics. Conventional AST systems are usually slow as they rely on cell growth for an indirect assessment of antibiotics’ effectiveness. Therefore, a faster method is desirable, especially for emergency cases. In this work, we studied the performance of label-free, droplet-based impedance sensors for rapid characterization of the effects of ampicillin (Amp) on Escherichia coli. Ampicillin damages cell wall integrity and makes cells permeable (leaky). The leakage results in significant increase of the electrical conductance measured directly by the microfabricated sensing unit. We studied the conductance signal as a function of both antibiotic treatment time and dosage and demonstrated susceptibility testing within 60 min. These findings demonstrate the potential of droplet-based electrical chips for the realization of electrical antibiotic susceptibility testing (e-AST) for early-stage diagnostic/treatment, and consequently, preventing antibiotic misuse/overuse.     

Selective activity of extracts of <Emphasis Type="Italic">Margaritaria discoidea</Emphasis> and <Emphasis Type="Italic">Homalium africanum</Emphasis> on <Emphasis Type="Italic">Onchocerca ochengi</Emphasis>

Background  

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.     

Evaluation of the BactiCard <Emphasis Type="Italic">Neisseria</Emphasis> for Identification of Pathogenic <Emphasis Type="Italic">Neisseria</Emphasis> Species and <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis>

The BactiCard Neisseria (Remel, USA) is a chromogenic enzyme substrate system for identifying Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Moraxella catarrhalis. The identification system consists of a card with four test circles impregnated with chromogenic substrates for indoxyl butyrate esterase (IB), prolyl aminopeptidase (PRO), γ-glutamyl aminopeptidase (GLUT), and ?-galactosidase (BGAL). These substrates permit the identification of Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica, respectively. After hydration of the circles with buffer, colonies from growth on selective media or a subculture are applied to the four circles. IB and BGAL reactions are read for a blue-green color after 2 and 15 min, respectively. PRO and GLUT reactions are read at 15 min for a red color after addition of a developer reagent. Identifications obtained with the BactiCard Neisseria were compared with those obtained using conventional procedures for 558 isolates in a blinded fashion. The BactiCard Neisseria identified 100% of 254 Neisseria gonorrhoeae, 100% of 125 Neisseria meningitidis, 53 (98.2%) of 54 Neisseria lactamica, and 123 (98.4%) of 125 Moraxella catarrhalis isolates. The BactiCard Neisseria is an accurate and rapid system for identification of these microorganisms in the clinical laboratory. Electronic Publication     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.