Bifidobacteria Strain Typing by Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum, it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy

Classification of Salmonella enterica of the (Para-)Typhoid Fever Group by Fourier-Transform Infrared (FTIR) Spectroscopy

Typhoidal and para-typhoidal Salmonella are major causes of bacteraemia in resource-limited countries. Diagnostic alternatives to laborious and resource-demanding serotyping are essential. Fourier transform infrared spectroscopy (FTIRS) is a rapidly developing and simple bacterial typing technology. In this study, we assessed the discriminatory power of the FTIRS-based IR Biotyper (Bruker Daltonik GmbH, Bremen, Germany), for the rapid and reliable identification of biochemically confirmed typhoid and paratyphoid fever-associated Salmonella isolates. In total, 359 isolates, comprising 30 S. Typhi, 23 S. Paratyphi A, 23 S. Paratyphi B, and 7 S. Paratyphi C, respectively and other phylogenetically closely related Salmonella serovars belonging to the serogroups O:2, O:4, O:7 and O:9 were tested. The strains were derived from clinical, environmental and food samples collected at different European sites. Applying artificial neural networks, specific automated classifiers were built to discriminate typhoidal serovars from non-typhoidal serovars within each of the four serogroups. The accuracy of the classifiers was 99.9%, 87.0%, 99.5% and 99.0% for Salmonella Typhi, Salmonella Paratyphi A, B and Salmonella Paratyphi C, respectively. The IR Biotyper is a promising tool for fast and reliable detection of typhoidal Salmonella. Hence, IR biotyping may serve as a suitable alternative to conventional approaches for surveillance and diagnostic purposes

Arte e memoria culturale nell'età della Seconda Sofistica

Studio dei risvolti archeologici e storici del fenomeno culturale della c.d. Seconda Sofistica, delle forme di autorappresentazione, delle evergesie monumentali e delle dediche di statue e di altri monumenti nei santuari, città e praedia nel II-III sec. d.C

Il trattamento chirurgico nella sindrome di Budd-Chiari primitiva : una terapia in evoluzione

Background. Budd-Chiari syndrome (BCS) is an uncommon form of portal hypertension caused by obstruction of the hepatic venous outflow. Methods. From 1969 to 1997, 19 patients (7 men and 12 women, with a mean age of 37.6 years) affected with primary BCS were treated. In most of the cases no etiologic factors were identified; in the remaining cases the etiology was associated to polycythemia vera, use of oral contraceptives, presence of endoluminal membranes and repeated episodes of sepsis. Three patients with membranous occlusion of the major hepatic veins were treated by percutaneous placement of a self-expanding metallic stent, inserted by means of a transjugular or transhepatic approach. The remaining 16 patients underwent a side-to-side porto-caval shunt, which required the interposition of a graft in 5 cases. In 2 patients with a significant caval obstruction a metallic vascular stent was placed into the narrowed tract of inferior vena cava, before shunting, by means of a transfemoral venous approach. Results. One patient died within the first 30 postoperative days. The 18 survivors were followed for a mean of 66.7 months. The 5-year survival rate was 83%. Conclusions. Primary BCS requires different therapies depending on the stage of disease. The fulminant or chronic forms with irreversible hepatic damage, need a definitive treatment, such as orthotopic liver transplantation. On the contrary, in the acute or subacute forms, characterized by reversible hepatic injury, the porto-systemic shunt represents the most effective treatment. The patients with bad hepatic risk can be treated by using the new procedures of interventional radiology. In both cases preliminary caval stenting is necessary if the syndrome is complicated by a significant obstruction of the inferior vena cava

Outbreak of NDM-1-producing Enterobacteriaceae in northern Italy, July to August 2011.

Between July 2011 and August 2011, the New Delhi metallo-beta-lactamase 1 (NDM-1) gene was detected in Klebsiella pneumoniae and Escherichia coli isolates obtained from six patients hospitalised in four health- care facilities in northern Italy. The patient who had been hospitalised in New Delhi, India, from February to May 2011 and subsequently in the Bologna area, Italy, from May to July 2011, may have been the source of the outbreak. Our findings suggest ongoing spread of this carbapenem-resistance gene in Italy and high- light the need for intensive surveillance

DETERMINAZIONE DELLA PRODUZIONE DI CARBAPENEMASI IN ENTEROBATTERI CON RIDOTTA SENSIBILITA’ AI CARBAPENEMI MEDIANTE METODICA LC-MS (LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY)

INTRODUZIONE Le resistenza ai carbapenemi negli enterobatteri rappresenta ad oggi la principale problematica di antibiotico-resistenza. Negli ultimi anni nel contesto epidemiologico italiano si è osservata una rapida diffusione di microrganismi con queste caratteristiche, legata principalmente a Klebsiella pneumoniae produttrice di carbapenemasi KPC. Una rapida e corretta definizione della produzione di carbapenemasi riveste notevoli implicazioni sia da un punto di vista clinico che epidemiologico per la necessità di mettere in atto il più precocemente possibile una terapia antibiotica appropriata e le idonee misure preventive. In questo lavoro abbiamo valutato la performance analitica di un saggio di LC-MS (Liquid Chromatography Mass Spectrometry) nel determinare la produzione di carbapenemasi in isolati di enterobatteri. MATERIALI E METODI Sono stati selezionati per lo studio 48 isolati clinici (40 K.pneumoniae, 4 E.coli, 4 C.freundii) precedentemente caratterizzati: 20 ceppi produttori di carbapenemasi KPC, 10 di metallo-βlattamasi (MBL), 6 di OXA-48, 12 non produttori di carbapenemasi (6 con ridotta sensibilità ai carbapenemi da produzione di ESBL con deficit di porine e 6 wild-type). La metodica di valutazione della produzione di carbapenemasi prevede l’incubazione per 1 ora a 37°c dei ceppi in esame in presenza di meropenem ad una concentrazione di 0.05 mg/ml. In seguito si procede ad una centrifugazione ed il sovranatante viene utilizzato per il dosaggio del meropenem mediante LC-MS, metodica che coniuga la cromatografia liquida alla spettrometria di massa. L’analisi è stata eseguita su sistema HPLC Prominance UFLC-XR (Shimadzu) accoppiato a spettrometro di massa triplo quadrupolo API 4000 QTRAP (AB Sciex). Una curva di calibrazione permette di ottenere una quantificazione assoluta, mentre l’attività carbapenemasica è stata valutata in base al rapporto tra la concentrazione di meropenem nei campioni incubati con i ceppi e quella dell’antibiotico incubato in assenza di batteri (ratio). RISULTATI Tutti i ceppi produttori di KPC hanno evidenziato una rapida attività di degradazione del meropenem, determinando valori di ratio inferiori a 0.2 in 19 dei 20 campioni. Per gli isolati produttori di MBL i valori di ratio sono risultati tutti inferiori a 0.6, mentre per quelli positivi per OXA-48 in 2 casi i valori di ratio sono risultati sovrapponibili a quelli ottenuti per i ceppi produttori di ESBL. Complessivamente, utilizzando un cut-off di ratio ottimale identificato ad un valore di 0.75, la sensibilità del metodo nel determinare la produzione di carbapenemasi è risultata pari al 94.4%, la specificità del 100%. CONCLUSIONI La metodica di LC-MS valutata in questo studio ha dimostrato una elevata affidabilità diagnostica nella valutazione dell’attività carbapenemasica da parte di ceppi di enterobatteri. La sensibilità non ottimale ottenuta su isolati produttori di OXA-48 è probabilmente legata ad una più lenta attività enzimatica di questa specifica βlattamas

Use of temocillin for identification of class D carbapenemases

Objectives. Currently, the main difficulty in laboratory confirmation of carbapenemases production concerns the identification of class D enzymes (OXA-48, OXA-181 and OXA-181-like), harbored in Enterobacteriaceae. Nevertheless, determination of susceptibility to temocillin provides an important indication of their possible presence. Aims of the study are to evaluate the susceptibility to temocillin of Enterobacteriaceae strains with reduced susceptibility to carbapenems, and to correlate it with the mechanism of resistance defined by genotypic tests, in order to assess the performance of the temocillin-susceptibility test. Methods. 231 strains of Enterobacteriaceae with reduced susceptibility to carbapenems at routine analysis (Vitek2 - BioMérieux), were tested, selected on the basis of the results to disk diffusion synergy test for detection and typing of carbapenemases (DDST, Rosco). These strains, belonging to different genus and species (Klebsiella, Escherichia, Enterobacter, Citrobacter, Proteus), have also been molecularly characterized with Hyplex® PCR (Amplex), a multiplex-PCR for blaKPC, blaVIM, blaIMP, blaNDM and blaOXA-48; their susceptibility to temocillin has been evaluated by disk diffusion test. Results. The 231 tested strains, divided according to DDST, gave the following results: - 33 class A carbapenemase-producers: 33 resulted positive for blaKPC, 31 subsceptible to temocillin, 2 resistant; - 41 class B (MBL) producers: 34 resulted positive for blaVIM (12 susceptible to temocillin, 22 resistant), and 7 for blaNDM (6 susceptible to temocillin, 1 susceptible); - 147 negative to DDST: 2 resulted positive for blaOXA-48, both resistant to temocillin, while the other 145 were negative to multiplex PCR and temocillin-susceptible; - 10 AmpC-producers: all resulted negative to multiplex PCR and susceptible to temocillin. Conclusion. Susceptibility to temocillin proved to be the only phenotypic test that allows the identification of class D carbapenemases-producers strains if combined to DDST, even if this study suggest that temocillin resistance, not performed with DDST, does not provide a result specific for class D carbapenemases. However its introduction in the laboratory routine could allow the clinical microbiologist to provide a reliable result about presence/absence of all main known carbapenemases in strains with reduced susceptibility to carbapenems; particularly, a negative result to DDST, associated with susceptibility totemocillin, allows to exclude with very high probability the presence of any carbapenemases (negative predictive value of 100%)

DETERMINAZIONE DELLA PRODUZIONE DI CARBAPENEMASI IN ENTEROBATTERI CON RIDOTTA SENSIBILITA’ AI CARBAPENEMI MEDIANTE METODICA LC-MS (LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY)

INTRODUZIONE Le resistenza ai carbapenemi negli enterobatteri rappresenta ad oggi la principale problematica di antibiotico-resistenza. Negli ultimi anni nel contesto epidemiologico italiano si è osservata una rapida diffusione di microrganismi con queste caratteristiche, legata principalmente a Klebsiella pneumoniae produttrice di carbapenemasi KPC. Una rapida e corretta definizione della produzione di carbapenemasi riveste notevoli implicazioni sia da un punto di vista clinico che epidemiologico per la necessità di mettere in atto il più precocemente possibile una terapia antibiotica appropriata e le idonee misure preventive. In questo lavoro abbiamo valutato la performance analitica di un saggio di LC-MS (Liquid Chromatography Mass Spectrometry) nel determinare la produzione di carbapenemasi in isolati di enterobatteri. MATERIALI E METODI Sono stati selezionati per lo studio 48 isolati clinici (40 K.pneumoniae, 4 E.coli, 4 C.freundii) precedentemente caratterizzati: 20 ceppi produttori di carbapenemasi KPC, 10 di metallo-βlattamasi (MBL), 6 di OXA-48, 12 non produttori di carbapenemasi (6 con ridotta sensibilità ai carbapenemi da produzione di ESBL con deficit di porine e 6 wild-type). La metodica di valutazione della produzione di carbapenemasi prevede l’incubazione per 1 ora a 37°c dei ceppi in esame in presenza di meropenem ad una concentrazione di 0.05 mg/ml. In seguito si procede ad una centrifugazione ed il sovranatante viene utilizzato per il dosaggio del meropenem mediante LC-MS, metodica che coniuga la cromatografia liquida alla spettrometria di massa. L’analisi è stata eseguita su sistema HPLC Prominance UFLC-XR (Shimadzu) accoppiato a spettrometro di massa triplo quadrupolo API 4000 QTRAP (AB Sciex). Una curva di calibrazione permette di ottenere una quantificazione assoluta, mentre l’attività carbapenemasica è stata valutata in base al rapporto tra la concentrazione di meropenem nei campioni incubati con i ceppi e quella dell’antibiotico incubato in assenza di batteri (ratio). RISULTATI Tutti i ceppi produttori di KPC hanno evidenziato una rapida attività di degradazione del meropenem, determinando valori di ratio inferiori a 0.2 in 19 dei 20 campioni. Per gli isolati produttori di MBL i valori di ratio sono risultati tutti inferiori a 0.6, mentre per quelli positivi per OXA-48 in 2 casi i valori di ratio sono risultati sovrapponibili a quelli ottenuti per i ceppi produttori di ESBL. Complessivamente, utilizzando un cut-off di ratio ottimale identificato ad un valore di 0.75, la sensibilità del metodo nel determinare la produzione di carbapenemasi è risultata pari al 94.4%, la specificità del 100%. CONCLUSIONI La metodica di LC-MS valutata in questo studio ha dimostrato una elevata affidabilità diagnostica nella valutazione dell’attività carbapenemasica da parte di ceppi di enterobatteri. La sensibilità non ottimale ottenuta su isolati produttori di OXA-48 è probabilmente legata ad una più lenta attività enzimatica di questa specifica βlattamas

Rapid communications Outbreak of NDM-1-producing Enterobacteriaceae in

metallo-beta-lactamase 1 (NDM-1) gene was detected in Klebsiella pneumoniae and Escherichia coli isolates obtained from six patients hospitalised in four healthcare facilities in northern Italy. The patient who had been hospitalised in New Delhi, India, from February to May 2011 and subsequently in the Bologna area, Italy, from May to July 2011, may have been the source of the outbreak. Our findings suggest ongoing spread of this carbapenem-resistance gene in Italy and highlight the need for intensive surveillance. Outbreak description On 2 July 2011, we isolated Klebsiella pneumoniae harbouring the New Delhi metallo-beta-lactamase gene (bla), cultured from the urine of a patient (Patient ND

[Role of surgical therapy in the treatment of refractory ascites]

In 5-10% of cases ascites is not controlled by medical therapy and is defined refractory. These patients may be submitted to one of the four following surgical options: portal-systemic shunt, peritoneo-venous shunt, transjugular intrahepatic portal-systemic shunt, orthotopic liver transplantation. Although the portal-systemic shunt is efficient in clearing ascites, it does not improve the survival, which depends on liver function, and it is complicated by an important incidence of encephalopathy. Since the patients with refractory ascites and good hepatic risk are not usually many, it is possible to understand why derivative surgery has been disappointing with this indication. Although the peritoneo-venous shunt is associated with a significant rate of valve obstruction, it is an easy, effective and not expensive treatment. So, till now, it has been considered the first choice procedure of refractory ascites, if any situations, determinating the onset of postoperative complications, are not present. Recently a new method has been introduced in the therapy of portal hypertension, the transjugular intrahepatic portal-systemic shunt. This is a bloodless portal-systemic derivation and so it has caused great enthusiasm even if the available data are insufficient to give a definitive opinion on its role in management of ascites. Certainly the liver transplantation, which presents the great advantage to treat both the cirrhosis and its complications, seems to be the most rational therapy for these patients. However, at least for this moment, the well-known absence of organ donors makes still actual the palliative surgical measures