Safety and Efficacy of Prolonged Use of Dalbavancin in Bone and Joint Infections.

Dalbavancin is a lipoglycopeptide with potent activity against Gram-positive microorganisms, a long half-life, a favorable safety profile, and a high concentration in bone, which makes it an interesting alternative for treatment of osteoarticular infections. We performed a multicentric retrospective study of all patients with an osteoarticular infection (septic arthritis, spondylodiscitis, osteomyelitis, or orthopedic implant-related infection) treated with at least one dose of dalbavancin between 2016 and 2017 in 30 institutions in Spain. In order to evaluate the response, patients with or without an orthopedic implant were separated. A total of 64 patients were included. Staphylococcus epidermidis and Staphylococcus aureus were the most frequent microorganisms. The reasons for switching to dalbavancin were simplification (53.1%), adverse events (25%), or failure (21.9%). There were 7 adverse events, and no patient had to discontinue dalbavancin. In 45 cases, infection was related to an orthopedic implant. The implant material was retained in 23 cases, including that in 15 (65.2%) patients that were classified as cured and 8 (34.8%) that presented improvement. In 21 cases, the implants were removed, including those in 16 (76.2%) cases that were considered successes, 4 (19%) cases were considered improved, and 1 (4.8%) case that was considered a failure. Among the 19 cases without implants, 14 (73.7%) were considered cured, 3 (15.8%) were considered improved, and 2 (10.5%) were considered failures. The results show that dalbavancin is a well-tolerated antibiotic, even when >2 doses are administered, and is associated with a high cure rate. These are preliminary data with a short follow-up; therefore, it is necessary to gain more experience and, in the future, to establish the most appropriate dose and frequency.

Antifungal stewardship in a tertiary-care institution: a bedside intervention.

Antifungal stewardship (AFS) programmes are needed in tertiary-care hospitals. Our aim is to describe a bedside non-restrictive AFS programme, and to evaluate its economic impact. During the first year of the AFS a bundle of non-interventional measures were implemented. During the second year an infectious diseases specialist visited 453 patients receiving candins, liposomal amphotericin B, voriconazole or posaconazole. Monthly costs were studied with an interrupted time series (ITS) analysis. The main prescribing departments were haematology (35%), medical departments (23%), and intensive care units (20%). Reasons to start antifungal therapy were: targeted therapy (36%), prophylaxis (32%), empirical therapy (20%) and pre-emptive therapy (12%). At the initial visit, diagnostic advice was provided in 40% of cases. The most common therapeutic recommendations were to de-escalate the antifungal drug (17%) or to suspend it (7%). Annual total antifungal expenditure was reduced from US$3.8 million to US$2.9 million over the first 2 years, generating net savings of US$407,663 and US$824,458 per year after considering the cost of additional staff required. The ITS analyses showed a significant economic impact after the first 12 months of the intervention (p 0.042 at month 13), which was enhanced in the following 24 months (p 0.006 at month 35). The number of defined daily doses decreased from 66.4 to 54.8 per 1000 patient-days. Incidence of candidaemia was reduced from 1.49 to 1.14 (p 0.08) and related mortality was reduced from 28% to 16% (p 0.1). A collaborative and non-compulsory AFS program based on bedside intervention is an efficacious and cost-effective approach that optimizes the use of AF drugs.

Espondilodiscitis por Fusobacterium nucleatum: nueva forma de diagnóstico / Spondylodiscitis due to Fusobacterium nucleatum: New diagnostic method

Fusobacterium spp. son bacterias anaerobias Gram negativas. La osteomielitis vertebral por dichas bacterias es muy rara, de hecho, sólo podemos encontrar 11 casos en la literatura. Se presenta un caso de un varón de 46 años con dolor lumbar irradiado a la pierna derecha, de varias semanas de evolución y que no respondió al tratamiento con AINEs. Para el diagnóstico se utiliza la RMN, una biopsia con drenaje de la colección y una PCR universal seguida de secuenciación de ADNr 16S, con la que se obtuvo el diagnóstico microbiológico del paciente, identificando un Fusobacterium nucleatum como responsable. Posteriormente se pautó clindamicina como tratamiento final. En conclusión, la espondilodiscitis por Fusobacterium spp. es una entidad rara y su diagnóstico es a menudo difícil, tanto por las características clínicas como por la dificultad de obtener el diagnóstico microbiológico apropiado. La biopsia vertebral y las técnicas moleculares microbiológicas como la PCR ADNr Universal, son esenciales para la identificación del organismo y permiten la determinación de un diagnóstico y un tratamiento antibiótico apropiados.

Fusobacterium spp. are Gram negative anaerobe bacteria. Vertebral osteomyelitis caused by these bacteria is very unusual; in fact, we could only find 11 cases in the literature. We report the case of a male, 46 year-old patient who had had lumbar pain for several weeks that irradiated to the right leg, and did not respond to NSAID treatment. The work-up included MRI, biopsy with draining of the collection and a universal PCR followed by 16S rDNA sequencing. The latter was used to make the microbiologic diagnosis, which identified Fusobacterium nucleatum as the causative agent. Final treatment consisted of clindamycin. In conclusion, spondylodiscitis due to Fusobacterium spp. is a rare and difficult to diagnose entity, due both to its clinical characteristics and to the difficulty in making the right microbiologic diagnosis. Vertebral biopsy and molecular microbiologic techniques such as Universal PCR rDNa, are essential to identifying the organism, making the diagnosis and prescribing appropriate antibiotic therapy.

Gram-negative prosthetic joint infection: outcome of a debridement, antibiotics and implant retention approach. A large multicentre study.

We aim to evaluate the epidemiology and outcome of gram-negative prosthetic joint infection (GN-PJI) treated with debridement, antibiotics and implant retention (DAIR), identify factors predictive of failure, and determine the impact of ciprofloxacin use on prognosis. We performed a retrospective, multicentre, observational study of GN-PJI diagnosed from 2003 through to 2010 in 16 Spanish hospitals. We define failure as persistence or reappearance of the inflammatory joint signs during follow-up, leading to unplanned surgery or repeat debridement>30 days from the index surgery related death, or suppressive antimicrobial therapy. Parameters predicting failure were analysed with a Cox regression model. A total of 242 patients (33% men; median age 76 years, interquartile range (IQR) 68-81) with 242 episodes of GN-PJI were studied. The implants included 150 (62%) hip, 85 (35%) knee, five (2%) shoulder and two (1%) elbow prostheses. There were 189 (78%) acute infections. Causative microorganisms were Enterobacteriaceae in 78%, Pseudomonas spp. in 20%, and other gram-negative bacilli in 2%. Overall, 19% of isolates were ciprofloxacin resistant. DAIR was used in 174 (72%) cases, with an overall success rate of 68%, which increased to 79% after a median of 25 months' follow-up in ciprofloxacin-susceptible GN-PJIs treated with ciprofloxacin. Ciprofloxacin treatment exhibited an independent protective effect (adjusted hazard ratio (aHR) 0.23; 95% CI, 0.13-0.40; p<0.001), whereas chronic renal impairment predicted failure (aHR, 2.56; 95% CI, 1.14-5.77; p 0.0232). Our results confirm a 79% success rate in ciprofloxacin-susceptible GN-PJI treated with debridement, ciprofloxacin and implant retention. New therapeutic strategies are needed for ciprofloxacin-resistant PJI.

[Spondylodiscitis due to Fusobacterium nucleatum: new diagnostic method]. / Espondilodiscitis por Fusobacterium nucleatum: nueva forma de diagnóstico.

Fusobacterium spp. are Gram negative anaerobe bacteria. Vertebral osteomyelitis caused by these bacteria is very unusual; in fact, we could only find 11 cases in the literature. We report the case of a male, 46 year-old patient who had had lumbar pain for several weeks that irradiated to the right leg, and did not respond to NSAID treatment. The work-up included MRI, biopsy with draining of the collection and a universal PCR followed by 16S rDNA sequencing. The latter was used to make the microbiologic diagnosis, which identified Fusobacterium nucleatum as the causative agent. Final treatment consisted of clindamycin. In conclusion, spondylodiscitis due to Fusobacterium spp. is a rare and difficult to diagnose entity, due both to its clinical characteristics and to the difficulty in making the right microbiologic diagnosis. Vertebral biopsy and molecular microbiologic techniques such as Universal PCR rDNa, are essential to identifying the organism, making the diagnosis and prescribing appropriate antibiotic therapy.

The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem?

Underdiagnosis of Clostridium difficile infection (CDI) because of lack of clinical suspicion or the use of non-sensitive diagnostic techniques is a known problem whose real magnitude has not yet been quantified. In order to estimate the extent of this underdiagnosis, we performed C. difficile cultures on all unformed stool specimens sent-irrespective of the type of request-to a series of laboratories in Spain on a single day. The specimens were cultured, and isolates were characterized at a central reference laboratory. A total of 807 specimens from 730 patients aged ≥ 2 years were selected from 118 laboratories covering 75.4% of the Spanish population. The estimated rate of hospital-acquired CDI was 2.4 episodes per 1000 admissions or 3.8 episodes per 10,000 patient-days. Only half of the episodes occurred in patients hospitalized for >2 days. Two of every three episodes went undiagnosed or were misdiagnosed, owing to non-sensitive diagnostic tests (19.0%) or lack of clinical suspicion and request (47.6%; mostly young people or non-hospitalized patients). The main ribotypes were 014/020 (20.5%), 001 (18.2%), and 126/078 (18.2%). No ribotype 027 strains were detected. Strains were fully susceptible to metronidazole and vancomycin. CDI was underdiagnosed in diarrhoeic stools in a high proportion of episodes, owing to the use of non-sensitive techniques or lack of clinical suspicion, particularly in people aged <65 years or patients with community-acquired diarrhoea. C. difficile toxins should be routinely sought in unformed stools of any origin sent for microbiological diagnosis. The ribotype 027 clone has not yet disseminated in Spain.

Role of universal 16S rRNA gene PCR and sequencing in diagnosis of prosthetic joint infection.

The etiological diagnosis of prosthetic joint infection (PJI) requires the isolation of microorganisms from periprosthetic samples. Microbiological cultures often yield false-positive and false-negative results. 16S rRNA gene PCR combined with sequencing (16SPCR) has proven useful for diagnosing various infections. We performed a prospective study to compare the utility of this approach with that of culture to diagnose PJI using intraoperative periprosthetic samples. We analyzed 176 samples from 40 patients with PJI and 321 samples from 82 noninfected patients using conventional culture and 16SPCR. Three statistical studies were undertaken following a previously validated mathematical model: sample-to-sample analysis, calculation of the number of samples to be studied, and calculation of the number of positive samples necessary to diagnose PJI. When only the number of positive samples is taken into consideration, a 16SPCR-positive result in one sample has good specificity and positive predictive value for PJI (specificity, 96.3%; positive predictive value, 91.7%; and likelihood ratio [LR], 22), while 3 positive cultures with the same microorganism are necessary to achieve similar specificity. The best combination of results for 16SPCR was observed when 5 samples were studied and the same microorganism was detected in 2 of them (sensitivity, 94%; specificity, 100%; and LR, 69.62). The results for 5 samples with 2 positive cultures were 96% and 82%, respectively, and the likelihood ratio was 1.06. 16SPCR is more specific and has a better positive predictive value than culture for diagnosis of PJI. A positive 16SPCR result is largely suggestive of PJI, even when few samples are analyzed; however, culture is generally more sensitive.

Laboratory diagnosis of Clostridium difficile infection in Spain: a population-based survey.

A survey of laboratory diagnosis of Clostridium difficile infection (CDI) was conducted in 103 Spanish hospitals. A mean of 23.2 stool specimens/1000 admissions were processed to detect CDI. Overall, 35.9% of the laboratories specifically selected stool specimens for diagnostic C. difficile toxin testing. The most commonly used criteria were loose or watery stools, previous antibiotic therapy and nosocomial diarrhoea. Most laboratories (95.1%) processed the stool specimens in house, mainly five to seven days/week. All laboratories except one detected toxins directly in stool specimens, and 94.9% used enzyme immunoassays (EIAs). Only 16.3% of the laboratories used toxigenic culture as a diagnostic tool, and most of them used EIAs to detect toxins in isolates. The most common diagnostic strategy was toxin detection in stool specimens using EIA alone (79.6%), and the second most common strategy was the combination of toxin detection in stool specimens and isolates (10.2%). The estimated annual incidence of CDI was 1.71 cases/1000 admissions, and this was significantly higher in large hospitals. CDI is underdiagnosed in Spain because most laboratories use EIAs performed directly on stool specimens as the only diagnostic procedure. A national laboratory survey of diagnostic methods and testing protocols for the diagnosis of CDI is simple and inexpensive, and could act as the first step towards implementing national standardized criteria for optimal diagnosis of CDI.

Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral.

AIMS: The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral. METHODS AND RESULTS: The occurrence of sublethal injury in the outer membrane was measured using selective recovery media containing bile salts. Loss of membrane integrity was measured by the increased uptake of the fluorescent dye propidium iodide (PI). PEF caused nonpermanent and permanent envelope permeabilization of Ent. sakazakii at pH 4·0. After PEF, most surviving cells showed transient cell permeabilization and sublethal injury in their outer membranes. The simultaneous application of a mild PEF treatment (100 pulses, 25 kV cm(-1) ) and 200 µl l(-1) of citral to cells suspended in pH 4·0 buffer at a final concentration of 10(7) cells per ml showed an outstanding synergistic lethal effect, causing the inactivation of more than two extra log(10) cycles. CONCLUSIONS: Our results confirm that the detection of sublethal injury in the outer membrane after PEF may contribute to the identification of the treatment conditions under which PEF may act synergistically with hydrophobic compounds such as citral. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the mechanism of microbial inactivation by PEF will aid the establishment of successful combined preservation treatments.

sigB absence decreased Listeria monocytogenes EGD-e heat resistance but not its Pulsed Electric Fields resistance.

This study shows the behaviour of Listeria monocytogenes under heat and Pulsed Electric Fields (PEF) treatments, as well as the influence of sigB in bacterial resistance and recovery. Absence of the sigB gene resulted in a decreased heat tolerance, showing that sigma(B) influences L. monocytogenes heat survival. Heat treatments at 60 degrees C (40s) caused sublethal membrane injuries in 99.99% of survivors. The repair of heat damage required energy production and lipid, protein, and RNA synthesis, and it lasted 6h. Furthermore, deletion of sigB did not affect the heat injuries repair. PEF resistance at pH 4.0 and 7.0 was not influenced by sigB. Sublethal damage after PEF treatments was only detected when PEF-treated cells had previously been heat-shocked (45 degrees C/1h). The membrane repair only required energy production, and it was independent of sigB. Although both heat and PEF treatments have an effect on cellular membrane, the repair of the sublethal damages suggests different membrane targets, and thus we propose a different mechanism of inactivation by these food preservation technologies.

Effect of citral on the thermal inactivation of Escherichia coli O157:H7 in citrate phosphate buffer and apple juice.

Inactivation and sublethal injury of Escherichia coli O157:H7 cells induced by heat in citrate phosphate buffer and apple juice (both at pH 3.8) were studied, and the effect of a combined preservation treatment using citral and heat treatments was determined. Heat resistance of E. coli O157:H7 was similar in both treatment media; after 27 min at 54°C, 3 log units of the initial cell population was inactivated in both treatment media. However, under less harsh conditions a protective effect of apple juice was found. Whereas inactivation followed linear kinetics in the citrate phosphate buffer, when cells were treated in apple juice the survival curves were concave downward. Heat treatment caused a great degree of sublethal injury; 4 min at 54°C inactivated less than 0.5 log CFU/ml but sublethally injured more than 3 log CFU/ml. The addition of 18 and 200 ppm of citral to the treatment medium acted synergistically with heat at 54°C to inactivate 3 × 10(4) and 3 × 10(7) CFU/ml, respectively. Addition of citral thus reduced the time needed to inactivate 1 log unit of the initial E. coli O157:H7 population from 8.9 to 1.7 min. These results indicate that a combined process of heat and citral can inactivate E. coli O157:H7 cells and reduce their potential negative effects.

Organic acids make Escherichia coli more resistant to pulsed electric fields at acid pH.

Stationary growth phase cells of Escherichiacoli were more pulsed electric fields (PEF) resistant in citrate-phosphate McIlvaine buffer at pH 4.0 than at pH 7.0. The greater PEF resistance was also confirmed in fruit juices of similar acid pH. In this work we studied whether the higher PEF resistance of E. coli at acid pH was due to the low pH itself or to the interaction of the components of the treatment medium with the cells. The protective effect on E. coli cells was due to the presence of organic acids such as citric, acetic, lactic or malic at pH 4.0. The protective effect of citric acid at pH 4.0 depended on its concentration. A linear relationship was observed between the Log(10) of the citric acid concentration and the degree of inactivation. Organic acids contained in laboratory treatment media (citrate-phosphate buffer) or in fruit juices did not sensitize E. coli cells to PEF but, on the contrary, they induced a protective effect that made E. coli cells more resistant at pH 4.0 than at neutral pH. This work could be useful for improving food preservation by PEF technology and it contributes to the knowledge of the mechanism of microbial inactivation by PEF.

Inactivation of Escherichia coli by citral.

AIMS: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. METHODS AND RESULTS: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 microl l(-1) of citral at pH 4.0 for 24 h at 20 degrees C caused the inactivation of more than 5 log(10) cycles of cells starting at an inoculum size of 10(6) or 10(7) CFU ml(-1), whereas increasing the cell concentration to 10(9) CFU ml(-1) caused <1 log(10) cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4.0 than pH 7.0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild-type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. CONCLUSIONS: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

Comparison of three commercial methods for rapid detection of Clostridium difficile toxins A and B from fecal specimens.

Three rapid enzyme immunoassays (X/pect Clostridium difficile Toxin A/B test, Wampole Tox A/B Quik Chek, and ImmunoCard Toxins A&B) were compared for the diagnosis of Clostridium difficile infection. Of the 367 stool specimens tested, 102 (27.8%) were positive for toxigenic C. difficile when a combination of direct cytotoxicity assay and cytotoxic culture was used as the gold standard. Sensitivity/specificity values were 49.0%/95.8%, 54.9%/95.5%, and 66.7%/95.1%, respectively. The median times to test five stool specimens were 28, 30, and 24 min, respectively. The ImmunoCard test was the quickest and most sensitive test of the three enzyme immunoassays evaluated.

Metronidazole resistance in Clostridium difficile is heterogeneous.

At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-mug metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-microg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-microg metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.

Recovery of Saccharomyces cerevisiae sublethally injured cells after Pulsed Electric Fields.

The objective was to investigate the influence of the recovery liquid medium on the repair of Saccharomyces cerevisiae sublethally injured cells after Pulsed Electric Fields (PEF) in media of different pH. Sublethal injury was detected in the yeast S. cerevisiae after 50 pulses at 12.0 kV cm(-1) in both pH 4.0 and 7.0, by using a selective medium plating technique. PEF treatments cause a repairable sublethal injury in S. cerevisiae. Injured cells showed their maximum repair capacity when suspended in Sabouraud Broth compared to Peptone Water or citrate-phosphate buffer of pH 4.0. The extent to which cells repair their injuries depended on the treatment medium pH, and on the nature of the storage medium. No repair was detected when the recovery liquid medium was citrate-phosphate buffer of pH 7.0. Acid conditions favour repair and survival of PEF-treated S. cerevisiae cells. This work contributes to the description of the mechanisms of PEF injury and inactivation, which would be useful in defining adequate PEF treatments, alone or in combination with additional hurdles, to assure food stability.

Effect of environmental factors and cell physiological state on Pulsed Electric Fields resistance and repair capacity of various strains of Escherichia coli.

The aim was to determine the resistance variation of four strains of Escherichia coli to Pulsed Electric Fields (PEF), the role of the sigma factor RpoS in PEF resistance, as well as the influence of several environmental factors and the cell physiological state on the PEF resistance and repair capacity. The rpoS null mutant, E. coli BJ4L1, exhibited decreased PEF resistance as compared with its wild-type parent, BJ4. W3110 and O157:H7 were the most PEF-resistant strains: whereas 2 and more than 3 Log10 cycles of BJ4 and BJ4L1 cells, respectively, were inactivated after 50 pulses at 35 kV/cm, only 0.5 Log10 cycle of inactivation of W3110 and O157:H7 was attained. A different pattern was observed and the resistance variation among strains was largely reduced, when selective recovery media were used. At exponential growth phase, the resistance of the four strains was lower, and more than 4 Log10 cycles of inactivation of all strains tested were attained at 30 kV/cm. Previous heat and cold shock treatments scarcely influenced cell PEF resistance. PEF survival increased with the reduction in water activity of the treatment medium to 0.94: the occurrence of sublethally injured cells was negligible, and less than 1 Log10 cycle of inactivation was attained at 35 kV/cm. PEF-treated cells were sensitive to a subsequent storage at pH 4.0 or in the presence of sorbic acid, attaining a final inactivation of 4-5 Log10 cycles after 24 hour-incubation. In conclusion, the work confirms the role of rpoS in PEF resistance. E. coli strains exhibit large differences in PEF resistance. These differences were less important when cells were recovered under selective conditions. Both resistance variation among strains and occurrence of sublethal damage were noticeably influenced by the environmental factors tested.

Biosynthetic requirements for the repair of sublethally injured Saccharomyces cerevisiae cells after pulsed electric fields.

AIMS: The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Saccharomyces cerevisiae cells after exposure to pulsed electric fields (PEF). METHODS AND RESULTS: The partial loss of the integrity and functionality of the cytoplasmic membrane was assessed by adding sodium chloride to the recovery medium. More than 2 log(10) cycles of survivors were sublethally injured after PEF. Repair of sublethal membrane damages occurred when survivors to PEF were incubated in Sabouraud Broth for 4 h at room temperature. The addition of inhibitors, such as chloramphenicol, rifampicin, 5-fluorocytosine, nalidixic acid, cycloheximide, cerulenin, miconazol and sodium azide to the liquid repair medium showed that the repair of PEF-injured cells required energy and protein synthesis. The extent of the sublethal damages was greater in PEF-treated cells at pH 4.0 than at pH 7.0. CONCLUSIONS: This work confirms that membrane damage is an important event in the PEF-inactivation of yeast. The mechanism of yeast inactivation by PEF seems to differ from that of bacteria, as the repair of sublethal damages requires protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the damages inflicted by PEF leads to a better description of the mechanism of yeast inactivation.

Relationship between sublethal injury and inactivation of yeast cells by the combination of sorbic acid and pulsed electric fields.

The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log10 5 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.

Reduction in liver-related hospital admissions and deaths in HIV-infected patients since the year 2002.

Since the advent of highly active antiretroviral therapy (HAART), complications of chronic liver disease (CLD) have emerged as one of the leading causes of hospital admission and death among HIV-infected patients with chronic hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections. The impact of CLD on hospital admissions and deaths in HIV-infected patients attended at one reference HIV hospital in Madrid during the last 9 years was analysed. All clinical charts from January 1996 to December 2004 were retrospectively examined. Demographics, discharge diagnosis, complications during inhospital stay and causes of death were recorded. A total of 2527 hospital admissions in 2008 distinct HIV-infected persons were recorded. Overall, 84% were iv drug users; mean age was 37 years and the mean CD4 count was 224 cells/muL. Both mean age and CD4 count significantly increased during the study period (P < 0.01). Overall, 42% of hospitalized patients were on antiretroviral therapy. Decompensated CLD was the cause of admission and/or developed during hospitalization in 345 patients (14%). Admissions caused by decompensated CLD increased significantly from 9.1% (30/329) in 1996 to 26% (78/294) in 2002. A significant steady decline occurred since then, being 11% (29/253) in the year 2004. Similarly, inhospital liver-related deaths were 9% (5/54) in 1996, peaked to 59% (10/17) in 2001 and declined to 20% (3/15) in the year 2004. Chronic hepatitis C was responsible for admissions and/or deaths in 73.5% of CLD cases. In conclusion, the rate of liver-related hospital admissions and deaths among HIV-infected patients peaked in the year 2002 and has steadily declined since then. A slower progression to liver cirrhosis in patients on HAART, avoidance of hepatotoxic antiretroviral drugs and more frequent use of anti-HCV therapy in HIV/HCV-coinfected patients could account for this benefit.