The benzalkonium chloride resistant or sensitive phenotype of Listeria monocytogenes planktonic cells did not dictate the susceptibility of its biofilm counterparts

The main goal of this work was to approach food industry conditions in the comparison of the susceptibility of biofilms of Listeria monocytogenes to the biocides benzalkonium chloride (BAC) and peracetic acid (PAA). Twelve isolates of L. monocytogenes, including nine well characterized BAC resistant strains were used. Biofilms were produced on stainless steel coupons (SSC), at 11 °C (refrigeration temperature) or at 25 °C (room temperature), in culture media simulating clean (nutrient limiting) or soiled (nutrient rich) growth conditions. Neither different nutrient availability nor growth temperature showed significant effect (p > .05) on biofilm formation. PAA confirmed to be more effective than BAC in biofilm elimination. Biofilms formed under nutritional stress tended to differentiate more the response to BAC of the resistant or sensitive strains, but the resistant or sensitive phenotype of the planktonic cells did not dictate biofilm susceptibility.

Las cepas enterohemorrágicas de «Escherichia coli» como paradigma de patógenos emergentes. Lecciones de la gran epidemia de infección alimentaria centrada en Alemania en mayo y junio de 2011 / Enterohaemorrhagic strains of Escherichia coli as a paradigm of emerging pathogens. Lessons from the large outbreak of foodborne infections centred in Germany throughout May and June of 2011

Las cepas enterohemorrágicas de Escherichia coli (EHEC) son patógenos zoonóticos de transmisión alimentaria asociados a epidemias y a casos esporádicos de diarrea y colitis hemorrágica, cuadros que pueden complicarse y dar lugar al síndrome hemolítico urémico (HUS). La importancia de las cepas EHEC se debe a la gravedad del HUS, que es la causa más frecuente de insuficiencia renal aguda en los niños en América y Europa. E. coli O157:H7 es el serotipo fundamental del patotipo EHEC. Ya hace años se predijo que cepas EHEC de otros serotipos podrían convertirse en importantes patógenos transmitidos por alimentos, y desde entonces estos microorganismos se han relacionado con numerosos casos de enfermedad en todo el mundo. La incidencia de estos serotipos distintos del O157:H7sigue creciendo, lo que significa que pueden considerarse patógenos emergentes. Un ejemplo reciente es el gran brote de infecciones transmitidas por los alimentos y causadas por EHEC O104:H4, que se centró principalmente en Alemania durante mayo y junio de 2011. La cepa responsable del brote posee una combinación de factores de virulencia típicos de diferentes patotipos de E. coli, lo que corrobora que la plasticidad de los genomas bacterianos facilita la emergencia de nuevos patógenos especialmente virulentos. Las investigaciones epidemiológicas identificaron el origen del brote en una partida de semillas de alholva (fenogreco) importadas de Egipto en 2009. La dimensión internacional de esta epidemia ilustra la urgente necesidad de mejorar la vigilancia epidemiológica de las cepas EHEC(AU)

Enterohaemorrhagic strains of Escherichia coli (EHEC) are foodborne zoonotic pathogens associated with major outbreaks and sporadic cases of diarrhoea and haemorrhagic colitisor bloody diarrhea, which can progress to the hemolyticuremic syndrome (HUS). The importance of EHEC lies in the severity of HUS, which is the most frequent cause of acute renal failure in children in the Americas and Europe. It was predicted years ago that EHEC strains other than the prototypic O157:H7 serotype would emerge as significant foodborne pathogens. Since then, these microorganisms have been linked to numerous outbreaks and sporadic cases of disease around the world. The incidence of these serotypes continues to grow, which means they can be considered emerging pathogens. A recent example is the large outbreak of foodborne infections caused by EHEC O104:H4, which was mainly centred in Germany lasting throughout May and June of 2011. The outbreaks train shows a combination of virulence factors from different E. coli pathotypes, highlighting the way in which the plasticity of bacterial genomes facilitates the emergence of new highly virulent pathogens. Epidemiologic investigations traced the origin of the outbreak to fenugreek seeds imported from Egyptin 2009. The international dimension of the outbreak illustrated the urgent need for improving the epidemiologic surveillance of EHEC(AU)

Predominance and persistence of a single clone of Listeria ivanovii in a Manchego cheese factory over 6 months.

In an attempt to study the diversity and persistence of molecular subtypes of pathogenic Listeria spp. in a cheese factory at the La Mancha region of Spain, 43 samples were taken from incoming raw milk (cow's, ewe's, goat's and mixed species) and from certain food-contact and environmental surfaces before and/or after sanitation. Of these samples, 12 contained pathogenic Listeria. From the chromogenic agar plates corresponding to those, 46 phosphatidylinositol-specific phospholipase C-positive isolates were randomly taken for further analysis, including biochemical tests and pulsed-field gel electrophoresis (PFGE). They coincided in identifying all the 46 as Listeria ivanovii subsp. ivanovii, apparently a single PFGE type. Both ewe's and goat's raw milk batches from asymptomatic animals tested along the 6-month period persistently carried the same strain, which was also obtained from inner surfaces of raw milk truck tanks and the milk dump tank at the cheese factory. Biofilm-forming abilities of this L. ivanovii clone and interference against L. monocytogenes Scott A reference strain were tested, but failed to account for the clone's apparent pervasive presence.

Different contamination patterns of lineage I and II strains of Listeria monocytogenes in a Spanish broiler abattoir.

The purpose of this study was to determine whether genetically similar or diverse strains of Listeria monocytogenes colonize the environment and carcasses in a single Spanish broiler abattoir over time. The study was composed of 5 surveys over a 1.5-yr period and included the monitoring of cleaning and disinfection procedures. Overall, a total of 212 samples were tested for the presence of L. monocytogenes, and 31% of the samples were found to be positive. Listeria monocytogenes was isolated from carcasses and product contact and noncontact sites in the evisceration and carcass classification areas of the abattoir. A total of 132 L. monocytogenes isolates were characterized by PCR-based serotyping and pulsed-field gel electrophoresis (PFGE) restriction analysis with the endonucleases ApaI and AscI. Molecular serotyping showed that L. monocytogenes isolates were of serotypes 1/2a and 1/2b. Isolates of serotype 1/2b (89.4%) were contaminating carcasses as well as environmental product contact and noncontact sites, whereas isolates of serotype 1/2a (10.6%) were recovered only from environmental product noncontact sites. A relatively low genetic diversity was found in this group of L. monocytogenes isolates from the abbatoir; only 14 different PFGE types (A1 to A14) were obtained. Nine pulsotypes belonging to serotype 1/2b (lineage I) were grouped in only one PFGE genetic cluster, whereas 5 pulsotypes belonging to serotype 1/2a (lineage II) were grouped into 4 PFGE genetic clusters. Two genetically related pulsotypes of serotype 1/2b (A1 and A2, 64.4% of the isolates) predominated and persisted in the abattoir. Our study indicated that a few strains of L. monocytogenes lineage I that were genetically very closely related might be specifically adapted to colonizing the evisceration zone of the abattoir and were predominant on carcasses over 1 yr. On the other hand, a genetically diverse group of lineage II strains were present in the abattoir environment, but never contaminated carcasses.

Evaluation of ALOA plating medium for its suitability to recover high pressure-injured Listeria monocytogenes from ground chicken meat.

AIMS: To evaluate a chromogenic plating medium for the isolation of sublethally injured cells of Listeria monocytogenes from processed foods. METHODS AND RESULTS: The inactivation of L. monocytogenes at pressures up to 400 MPa and 12 degrees C in ground chicken meat was employed to examine the recovery of high-pressure injured cells. Before and after different repair incubation periods at 30 degrees C in a nonselective broth, samples were plated onto a selective and differential agar [Agar Listeria according to Ottaviani and Agosti (ALOA)] and in the same medium supplemented with 4% sodium chloride (ALOA-S), and incubated at 37 degrees C. Sublethally injured cells were able to grow when directly plated onto the ALOA medium, without a previous repair incubation period. However, only uninjured cells grew on the ALOA-S medium. CONCLUSIONS: Sublethally injured cells of L. monocytogenes can be quantified by subtracting counts on ALOA-S medium from counts on ALOA medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Possible applications include direct enumeration on ALOA of stressed cells of L. monocytogenes in foods with more than 100 colony forming units per gram.

[Foodborne Listeria monocytogenes: are all the isolates equally virulent?]. / Listeria monocytogenes en alimentos: son todos los aislamientos igual de virulentos?

Listeria monocytogenes is a foodborne human pathogen responsible for invasive infections presenting overall a high mortality. Despite the ubiquity of the microorganism, the actual disease rate is quite low and the disease is most often associated with an underlying predisposition. Foodborne and environmental isolates were traditionally considered of similar pathogenicity compared to clinical isolates. But the analysis of mutations in the genes encoding specific virulence factors (internalin, hemolysin, phospholipases, surface protein ActA and regulator protein PrfA), quantitative studies with cell cultures and population genetics have raised considerable concerns about virulence differences among L. monocytogenes strains. Despite this great step forward, there is not a single marker available to test the virulence of field isolates of this species. In the future, the combination of different molecular markers will probably allow the screening of food contamination by only the virulent clones of L. monocytogenes, thus improving the prevention of foodborne human listeriosis.

Sequencing and heterologous expression in Saccharomyces cerevisiae of a Cryptococcus neoformans cDNA encoding a plasma membrane H(+)-ATPase.

A cDNA containing an open reading frame encoding a putative plasma membrane H(+)-ATPase in the human pathogenic basidiomycetous yeast Cryptococcus neoformans was cloned and sequenced by means of PCR and cDNA library hybridization. The cloned cDNA is 3475 bp in length, containing a 2994 bp open reading frame encoding a polypeptide of 997 amino acids. As in the case of another basidiomycetous fungus (Uromyces fabae), the deduced amino acid sequence of CnPMA1 was found to be more homologous to those of P-type H(+)-ATPases from higher plants than to those from ascomycetous fungi. In order to prove the sequenced cDNA corresponds to a H(+)-ATPase, it was expressed in Saccharomyces cerevisiae and found to functionally replace its own H(+)-ATPase. Kinetic studies of CnPMA1 compared to ScPMA1 show differences in V(max) values and H(+)-pumping in reconstituted vesicles. The pH optimum and K(m) values are similar in both enzymes.

Electrotransformation of the human pathogenic fungus Scedosporium prolificans mediated by repetitive rDNA sequences.

The regions encoding the 5.8S rRNA and the flanking internal transcribed spacers (ITSI and ITSII) from two isolates of the human pathogenic fungus Scedosporium prolificans and one isolate of the taxonomically related species Pseudallescheria boydii (S. apiospermum) were sequenced. The sequences of the two S. prolificans isolates were identical. However, there were minor differences between both species. Phylogenetic analysis of known fungal sequences confirmed a close relationship between S. prolificans and P. boydii. An attempt was made to transform S. prolificans by electroporation using a plasmid vector, pMLF2, bearing the Escherichia coli hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans promoter and terminator sequences. To increase transformation efficiency, the sequenced ribosomal cluster of S. prolificans was used to construct a new vector for homologous recombination.

Comparative in-vitro activity of voriconazole (UK-109,496) and six other antifungal agents against clinical isolates of Scedosporium prolificans and Scedosporium apiospermum.

We report the in-vitro susceptibility of 27 clinical isolates of Scedosporium apiospermum and 43 of Scedosporium prolificans. S. apiospermum was resistant to fluconazole and flucytosine, with variable susceptibility to amphotericin B, itraconazole, ketoconazole and susceptible to miconazole. Voriconazole was much more active than fluconazole and flucytosine, more active than amphotericin B, itraconazole and ketoconazole and was as active as miconazole against S. apiospermum isolates. Voriconazole and the other six antifungal agents showed low activity against S. prolificans isolates.

Comparison of the in-vitro activity of voriconazole (UK-109,496), itraconazole and amphotericin B against clinical isolates of Aspergillus fumigatus.

Voriconazole was compared with amphotericin B and itraconazole by a modification of the NCCLS microdilution reference method for yeasts against 62 clinical isolates of Aspergillus fumigatus. MICs of voriconazole were slightly lower than those of amphotericin B and itraconazole. The MIC of voriconazole at which 90% of isolates were inhibited was 1 mg/L and the MIC range was 0.25-2 mg/L. Voriconazole is a new antifungal agent with potential for use in the treatment of A. fumigatus infections.

Change in fluconazole susceptibility patterns and genetic relationship among oral Candida albicans isolates.

OBJECTIVE: To assess the genetic homogeneity or heterogeneity within each set of Candida albicans isolates colonizing/infecting the oral cavities of HIV-infected patients undergoing azole therapy when changes in susceptibility to fluconazole were detected. DESIGN: Fourteen HIV-positive patients suffering recurrent episodes of oral candidosis were prospectively followed from the first episode to the isolation of strains with decreased susceptibility to fluconazole. The strains of C. albicans isolated either from episodes or controls throughout the prospective study were analysed. METHODS: Electrophoretic karyotyping and hybridization with the repeated sequence probe 27A were used to delineate sequential isolates. In vitro susceptibility tests to fluconazole and ketoconazole were also performed. The results obtained by DNA fingerprinting with the probe combined with computer-assisted analysis were used to assess the genetic relationships amongst the strains. In addition, comparison with the genetic relatedness of a group of geographically unrelated strains was made. RESULTS: Isogenic populations of sequential isolates were observed only in two patients; 12 patients harboured heterogenic populations over time, although in 11 patients there was a predominant strain that was isolated more than once, and only one of these patients carried strains with a similarity index less than 80%. With the exception of two patients, each patient carried a major strain that became less susceptible to fluconazole. The similarity index for the unrelated strains was 59%. CONCLUSIONS: HIV-infected patients may carry a mixed population of strains, but the strains tend to be related to each other. The strains were maintained throughout the course of infection and at least one developed secondary resistance to fluconazole.

Standardization of antifungal susceptibility testing and clinical relevance.

Standardization of antifungal susceptibility testing became essential to overcome the problem of interlaboratory variability and to determine the clinical relevance of in vitro data. This evolving process began for the yeasts and consequently broth macrodilution and microdilution methods (NCCLS M27 document) have been developed. These tests may not be useful for testing all organism-drug combinations or be the most convenient techniques for routine use in the clinical laboratory. Different alternatives to the NCCLS methods are currently under investigation. The identification of standard guidelines for antifungal susceptibility testing has reduced interlaboratory variability and further progress has been achieved with the determination of tentative interpretive breakpoints for certain drug-yeast combinations. However, these breakpoints are not adequate for interpretations of MICs for fungi-drug combinations beyond the setting for which they were determined. The NCCLS Subcommittee has also generated data for proposed testing guidelines for the moulds.

Molecular tracking of Candida albicans in a neonatal intensive care unit: long-term colonizations versus catheter-related infections.

Nosocomial neonatal candidiasis is a major problem in infants requiring intensive therapy. The subjects of this retrospective study were nine preterm infants admitted to the neonatal intensive care unit of the Hospital Central de Asturias between March 1993 and August 1994. The infants were infected with or colonized by Candida albicans. Five patients developed C. albicans bloodstream infections. A total of 36 isolates (including isolates from catheters and parenteral nutrition) were examined for molecular relatedness by PCR fingerprinting and restriction fragment length polymorphism (RFLP) analysis. The core sequence of phage M13 was used as a single primer in the PCR-based fingerprinting procedure, and RFLP analysis was performed with C. albicans-specific DNA probe 27A. Both techniques were evaluated with a panel of eight C. albicans reference strains, and each technique showed eight different patterns. With the 36 isolates from neonates, each technique enabled us to identify by PCR and RFLP analysis seven and six different patterns, respectively. The combination of these two methods (composite DNA type) identified eight different profiles. A strain with one of these profiles was present in three patients and in their respective catheters. Patients infected with or colonized by this isolate profile were clustered in time. Among the other patients, each patient was infected over time and at multiple anatomic sites with a C. albicans strain with a distinct DNA type. We conclude that C. albicans was most commonly producing long-term colonizations, although horizontal transmission probably due to catheters also occurred.

Use of random amplification of polymorphic DNA (RAPD) and PCR-fingerprinting for genotyping a Scedosporium prolificans (inflatum) outbreak in four leukemic patients.

Four isolates of the pathogenic fungus Scedosporium prolificans (inflatum), causing a previously reported nosocomial outbreak in four leukemic patients, were typed by random amplification of polymorphic DNA (RAPD) with two different 10-mer primers and PCR-fingerprinting with the core sequence of phage M13 as a single primer. Both techniques allowed 10 additional clinical isolates of Scedosporium prolificans from different areas of Spain, including Scedosporium prolificans NCPF 2884, to be classified into 10 different molecular types. The four outbreak isolates consisted of three molecular types with two patients sharing a similar strain, and the remaining two patients infected by two different strains.

Deep infections caused by Scedosporium prolificans. A report on 16 cases in Spain and a review of the literature. Scedosporium Prolificans Spanish Study Group.

Scedosporium prolificans, a mold morphologically similar to Scedosporium apiospermum, may cause asymptomatic colonization or localized or disseminated infection following trauma, surgery, and immunosuppression. S. prolificans is normally resistant to available antifungal agents, and prognosis depends largely on the host's immune status, extent of infection, and feasibility of surgical debridement. We report on 16 patients with deep S. prolificans infections, focusing on predisposing factors, clinical characteristics, outcome, postmortem findings, and antifungal susceptibility testing to 6 antifungal agents. Between 1989 and 1994, 16 cases of deep infections by S. prolificans were documented in 6 clinical centers in Spain (15 adults and 1 child: male/female = 0.77). Fifteen patients had underlying hematologic malignancy (14 with neutropenia) and 1 had a prosthetic cardiac valve. Syndromes included disseminated infection in 14 patients (1 with prosthetic valve endocarditis) and fungal pneumonia and meningoencephalitis in 1 patient each. S. prolificans was isolated from 2 specimens in 14 patients and from 1 specimen in 2 patients (blood, n = 12; respiratory tract, n = 4; CNS, n = 4; and skin biopsy, n = 3). Antifungal susceptibility testing by a micromethod with RPMI-2% glucose medium was performed in 8 isolates, all of which were resistant to amphotericin B, flucytosine, ketoconazole, fluconazole, itraconazole, and miconazole. All patients received antifungal therapy (amphotericin B, n = 9; amphotericin B+ flucytosine, n = 1; amphotericin B+ itraconazole, n = 2; liposomal amphotericin B+ itraconazole, n = 1; amphotericin B+ fluconazole, n = 1 and 2 underwent surgical procedures. Two patients survived coinciding with hematologic recovery and 14 (87.5%) patients died in a median time of 4 days after the first positive culture (range, 0-60 d). Necropsy was performed in 10 patients, and disseminated infection was found in 9. In conclusion, S. prolificans is an emerging multiresistant fungal pathogen that may cause asymptomatic colonization, localized infection related to trauma or surgery, and rapidly fatal disseminated infection in immunocompromised hosts, particularly those with neutropenia. This mycosis underscores the urgent need for new antifungal agents.

Comparison of four molecular typing methods for evaluating genetic diversity among Candida albicans isolates from human immunodeficiency virus-positive patients with oral candidiasis.

Candida albicans strain delineation by karyotyping. NotI restriction pattern analysis, hybridization with specific probe 27A, and PCR fingerprinting with the phage M13 core sequence were performed with 30 isolates from the oral cavities of 30 human immunodeficiency virus (HIV)-infected patients and 8 reference strains. Within the panel of clinical isolates, 20 were geographically related, although 10 isolates were susceptible to fluconazole and 10 isolates were resistant to fluconazole. The remaining isolates used in this study were fluconazole resistant and geographically unrelated. A composite DNA type was defined for each of the strains as the combination of types obtained by the four molecular methods. By this procedure, a great diversity of DNA types was found among isolates from the oropharynges of HIV-infected individuals with oral candidiasis. This diversity was not reduced when isolates were evaluated on the basis of whether they came from the same geographical locale and whether they were fluconazole resistant. These data refute the idea of a clonal origin for fluconazole-resistant strains among HIV-positive patients. Karyotyping was the least discriminatory method, yielding 19 DNA types among the 38 strains analyzed. Conversely, hybridization with the 27A probe showed a unique DNA pattern for each of the strains examined in this study. Our results demonstrate that at least two different molecular methods are needed for Candida albicans typing and that there is a great deal of strain variation within the species, irrespective of place of origin or antifungal resistance patterns.

Patterns of fluconazole susceptibility in isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis due to Candida albicans.

We evaluated 119 episodes of oropharyngeal candidiasis due to C. albicans to study the patterns of fluconazole susceptibility of the isolates and the characteristics of the patients and to confirm the correlation between fluconazole susceptibility of isolates and therapeutic outcome. Sixty-one isolates were considered susceptible to fluconazole (MICs, < or = 0.5 microg/mL), 33 were intermediate (MICs, 1.0-8.0 microg/mL), and 25 were resistant (MICs, > or = 16.0 microg/mL). Patients infected with resistant strains had significantly lower CD4+ cell counts and a less recent AIDS diagnosis than patients infected with intermediate or susceptible strains. Previous fluconazole therapy and prophylaxis were significantly more frequent for patients infected with resistant and intermediate strains (P < .001). Decreased susceptibility to ketoconazole and itraconazole was observed in resistant and intermediate strains. Fluconazole treatment was ineffective for patients infected with resistant isolates; however, high doses of ketoconazole or itraconazole were successful for nine (81%) of them. Different patterns of fluconazole susceptibility among C. albicans strains are correlated with patients' characteristics and with therapeutic outcomes.