W.J.G. Melchers

Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe

Fifty verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and the E. coli attaching and effacing (eae) gene, and random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n = 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-located eae gene, which indicates its usefulness as virulence marker. RAPD-PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed.

The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca2+ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca2+ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca2+ into the stores. Measurement of intra-organelle-free Ca2+ in permeabilized cells revealed that the ability of 2B to reduce the Ca2+ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cell-free system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca2+-ATPase that transports Ca2+ from the cytosol into the stores. To investigate whether 2B specifically affects Ca2+ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H+ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.

Nosocomial outbreak of colonization and infection with Stenotrophomonas maltophilia in preterm infants associated with contaminated tap water

Between March and May 1996 Stenotrophomonas maltophilia was cultured from endotracheal aspirate samples from five preterm infants in a neonatal intensive care unit (NICU). Four infants were superficially colonized, but a fifth died due to S. maltophilia septicaemia. S. maltophilia was cultured from tap water from three outlets in the NICU including one with a previously unnoticed defective sink drain. Water from these outlets was used to wash the preterm infants. Environmental and clinical S. maltophilia isolates yielded identical banding patterns on random arbitrary polymorphic DNA (RAPD) PCR analysis. The outbreak was controlled by reinforcement of hand disinfection, limitation of the use of tap water for hand washing and by using sterile water to wash the preterm infants. We conclude that tap water should not be used for washing preterm infants in the NICU, unless steps are taken to prevent microbial growth in the outlets.

Multimerization reactions of coxsackievirus proteins 2B, 2C and 2BC: a mammalian two-hybrid analysis

Recently, homomultimerization and heteromultimerization reactions of the poliovirus P2 region proteins were investigated using a yeast two-hybrid approach (Cuconati et al., Journal of Virology 72, 1297-1307, 1998). In this study, we investigated multimerization reactions of the 2B, 2C and 2BC proteins of the closely related coxsackie B3 virus (CBV3) using a mammalian two-hybrid system. This system allows the characterization of protein:protein interactions within a cellular environment that more closely mimics the native protein environment. Homomultimerization reactions were observed with the 2BC protein and, albeit weakly, with the 2B protein, but not with the 2C protein. To identify the determinants involved in the 2BC and 2B homomultimerization reactions, several mutants containing deletions or point mutations in the 2B region were tested. Disruption of the hydrophobic character of either the cationic amphipathic alpha-helix or the second hydrophobic domain of the 2B protein disturbed both the 2BC:2BC and the 2B:2B homomultimerization reactions. Disruption of either the cationic or the amphipathic character of the alpha-helix or deletion of the N-terminal 30 amino acids of the 2B protein, however, had no effect on the 2BC and 2B homomultimerization reactions. Heteromultimerization reactions were observed between proteins 2BC and 2B, and also between proteins 2BC and 2C, but not between the 2B and 2C proteins. The 2BC:2B and 2BC:2C heteromultimerization reactions were also mediated by hydrophobic determinants located in the amphipathic alpha-helix and the second hydrophobic domain. The nature of the interactions and their implications for the virus life-cycle are discussed.

Phylogenetic relationships of five species of Aspergillus and related taxa as deduced by comparison of sequences of small subunit ribosomal RNA

The nucleotide sequences of the genes encoding the 18S rRNA of Aspergillus flavus, A. nidulans, A. terreus and A. niger were elucidated and aligned to the sequences of A. fumigatus. In addition, the 18S rRNA sequences of the V4-V9 region of morphologically similar filamentous fungi, e.g. Penicillium chrysogenum, P. marneffei and Paecilomyces variotii, were elucidated. Phylogenetic analysis and comparison showed a very close intergeneric relationship of the genus Aspergillus to species of the genera Paecilomyces and Penicillium. However, the sequenced Aspergillus species also showed a very close relationship to Eurotium rubrum and Monascus purpureus. Phylogenetic analysis of fungal 18S rRNA sequences divided the general Aspergillus, Penicillium and Paecilomyces into two coherent clusters and showed a close intergeneric relationship which is in keeping with the existing morphological and taxonomic classification.

Pseudo-outbreak of multiresistant Pseudomonas aeruginosa in a hematology unit.

OBJECTIVEnTo describe the investigation of a pseudo-outbreak of multiresistant Pseudomonas aeruginosa fecal colonization in a hematology unit.nnnDESIGNnRetrospective chart review; prospective environmental sampling and observation of stool culture technique; genotyping by random arbitrary primer polymorphic DNA polymerase chain reaction (RAPD-PCR).nnnSETTINGnAn academic tertiary-care hospital.nnnPATIENTSnBetween August and October 1994, P aeruginosa resistant to imipenem, ceftazidime, ciprofloxacin, and all aminoglycosides was isolated from surveillance stool cultures from 10 neutropenic patients cared for in the hematology unit. P aeruginosa, with an identical susceptibility pattern, was isolated from three patients admitted to the same unit in the year before the outbreak. Two months before the outbreak, 12 healthcare workers had been added to the staff.nnnRESULTSnObservation of stool sampling techniques as performed by healthcare workers revealed that samples for surveillance cultures were taken from feces in the toilet. When the proper sampling technique was used, P aeruginosa was not isolated from stool samples from 8 of 10 patients with previously positive cultures. P aeruginosa also was isolated from two wash basins, toilet flushing water, and a toilet brush. Genotyping by RAPD-PCR showed that the isolate from the toilet flushing water was identical to the P aeruginosa strains of eight patients from the outbreak.nnnCONCLUSIONSnThis pseudo-outbreak emphasizes the importance of proper sampling techniques and that periodic observation may be necessary to verify proper sampling techniques.

Voriconazole-Susceptible and Voriconazole-Resistant Aspergillus fumigatus Coinfection

Azole resistance is an increasing problem in Aspergillus fumigatus infection (1). Two mutations, TR34/L98H and TR46/Y121F/T289A, are frequently recovered from isolates of patients with azoleresistant invasive aspergillosis and are believed to originate from the environment (2, 3). In regions with these environmental mutations, azole-resistant Aspergillus diseases may develop in patients not previously treated with azoles, and mortality rates are very high (4–6). We review the clinical course of three patients with proven invasive aspergillosis resulting from voriconazolesusceptible and voriconazole-resistant A. fumigatus strains. We hypothesized that in regions with TR34/L98H and TR46/Y121F/ T289A environmental mutations, individual pulmonary lesions may arise from A. fumigatus strains with different azole resistance profiles.

Polymerase chain reaction as a diagnostic tool for invasive aspergillosis: evaluation in bronchoalveolar lavage fluid from low risk patients

An Aspergillus genus-specific polymerase chain reaction (PCR), which has been developed for the diagnosis of invasive aspergillosis (IA) in high risk patients9, was evaluated in 72 bronchoalveolar lavage (BAL) samples obtained from 70 non-neutropenic, low risk patients in order to establish the rate of detectable colonization with Aspergillus species of the respiratory tract. A positive amplification was found in 11 out of 72 samples (15%). Risk factors for colonization, e.g. corticosteroid therapy and cigarette smoking, were present in 64% of the evaluable cases, and a significantly higher rate of colonization was found when risk factors were present (P < 0.05). The low rate of detectable colonization justifies further evaluation of this PCR assay in the diagnosis of IA.

A Novel Environmental Azole Resistance Mutation in Aspergillus fumigatus and a Possible Role of Sexual Reproduction in Its Emergence

ABSTRACT This study investigated the dynamics of Aspergillus fumigatus azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR46/Y121F/T289A genotype, 2% (1/45) harbored the TR46/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR463/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR463 mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR46 repeats. Importantly, in the laboratory, we recovered the TR463 mutation from a sexual cross between two TR46 isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases. IMPORTANCE Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in Aspergillus fumigatus. In this article, we show the dominance of azole-resistant A. fumigatus in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant A. fumigatus isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of Aspergillus diseases. Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in Aspergillus fumigatus. In this article, we show the dominance of azole-resistant A. fumigatus in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant A. fumigatus isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of Aspergillus diseases.

Multicenter Comparison of Molecular Methods for Detection of Legionella spp. in Sputum Samples

ABSTRACT Legionellosis can be diagnosed by PCR using sputum samples. In this report, the methods of nine laboratories for 12 sputum samples with Legionella pneumophila and Legionella longbeachae are compared. We conclude that (i) liquefaction prevents PCR inhibition, (ii) the employed mip gene PCRs detected L. pneumophila only, and (iii) the 16S rRNA gene PCR detected both Legionella species and is preferred for the diagnosis of legionellosis.