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We assessed the characteristics of Neisseria meningitidis pharyngeal carriage in a cohort of ‘men having sex with men’, including patients with pharyngeal Neisseria gonorrhoeae infection. In the period 2017-2019, among all the oropharyngeal samples tested for gonorrhoea from MSM attending a STI Clinic in Bologna (Italy), we randomly selected 244 N. gonorrhoeae-positive samples and 403 negatives (n=647). Pharyngeal specimens were tested for N. meningitidis presence, by the detection of sodC gene. N. meningitidis-positive samples were further grouped by PCR tests for the major invasive genogroups (i.e., A, B, C, W, and Y). A molecular assay, targeting capsule transporter gene, was used to determine meningococcal capsular status. Overall, 75.8% (491/647) of samples tested positive for sodC gene, indicating a pharyngeal meningococcal carriage. Meningococcal colonisation was significantly more frequent in younger subjects (P=0.009), with no association with HIV infection. Non-groupable meningococci represented most of pharyngeal carriages (about 71%). The commonest N. meningitidis serogroup was B (23.6%), followed by C (2.1%), Y (1.8%) and W (1.1%). Meningococci were often characterized by the genetic potential of capsule production. Interestingly, a negative association between N. meningitidis and N. gonorrhoeae was found: pharyngeal gonorrhoea was significantly more present in patients without meningococcal carriage (P=0.03). Although preliminary, our data added knowledge on the epidemiology of meningococcal carriage in MSM communities at high risk of gonococcal infections, gaining new insights into the interactions/dynamics between N. meningitidis and N. gonorrhoeae.

The burden and epidemiology of anaerobic bacteremia is poorly described. The aim of the study was to characterize the incidence and evolution trends of anaerobic bacteremia in Europe and neighbouring countries in 2012 and 2018 both in terms of microbial epidemiology and antimicrobial resistance. Retrospective multicenter study: 17 laboratory hospitals from 12 countries Results data: total number of adult patients with anaerobes, patients’ age, gender and hospitalization ward, laboratory methods, identification of isolates and antimicrobial susceptibility results were analysed. Burden of anaerobic bacteremia • The mean frequency of patients with anaerobes in BCs was 5% of patients, with important variations according to centers (2% to 14%), probably reflecting their medical activity. The reported incidence moderately increased between 2012 and 2018. • These patients were frequently hospitalized in emergency wards and intensive care units (25% and 22% of isolates), followed by medicine, digestive surgery and oncology (6.7%) Epidemiology of anaerobes in blood cultures • 50% of anaerobic BCs were obtained from emergency and Intensive care units, demonstrating their clinical importance • 4 genera represented 75% of isolates : Bacteroides, Cutibacterium, Clostridium and Fusobacterium • Cutibacterium acnes frequency significantly varied among centers, suggesting variations in Laboratory management of BCs (incubation time). Antimicrobial resistance reported rates • Antimicrobial resistance rates varied according to genera and remained stable between 2012 and 2018. • Clindamycin resistance was the most frequently observed phenotype, predominating in Bacteroides spp, Clostridium spp and anaerobic Gram positive rods. • 10% of Bacteroides spp were resistant to piperacillin- tazobactam. Carbapenems and metronidazole resistance rate were below 5%. • Metronidazole resistance was frequently reported in anaerobic Gram Positive cocci

La disfunzione delle ghiandole di Meibomio (Meibomian Gland Dysfunction - MGD) è la forma più comune di blefarite posteriore, ed è riconosciuta come la principale causa della malattia dell’occhio secco (Dry Eye Disease – DED) nella forma iperevaporativa. Ciò è riconducibile ad insufficienza in qualità e/o quantità del meibum secreto, che contribuisce ad un inadeguato film lipidico, responsabile di una eccessiva evaporazione del film lacrimale. L’attività di ricerca di anni recenti, unita alla crescente potenzialità delle tecniche analitiche, ha evidenziato come il microbiota locale giochi un ruolo fondamentale in questo complesso sistema. E’ sorprendente come la superficie oculare, seppure esposta alla contaminazione dell’ambiente esterno, contenga microorganismi commensali limitati in numero ed in grado di diversità. Questo aspetto fisiologico è assai peculiare nell’organismo: a differenza di altri siti biologici, come ad esempio la superficie della mucosa orale, la congiuntiva normale, i margini palpebrali e le lacrime contengono una varietà di specie assai inferiori.

Background: Chlamydia trachomatis (CT) is the agent of the most common bacterial sexually transmitted infection. This microorganism can infect different epithelial targets, such as the genital tract and the rectal mucosa. In particular stressful conditions, as the deprivation of tryptophan induced by IFN-γ, CT fails to successfully replicate inside the epithelial cells and switches to a persistence state. This condition can be reverted by the presence of indole, used by CT for tryptophan re-synthesis. In vivo, CT can find high levels of indole in the intestinal tract, thanks to enterobacteria, as Escherichia coli. The aim of this study was to investigate the role played by indole in the rescue of a normal chlamydial cycle for two different CT serovars (D and L2), in an in-vitro model of intestinal cells pre-exposed to INF-γ. Materials/methods: Intestinal cells (i407), grown normally for 24 hours in individual tubes, were exposed to INF-γ (10 ng/ml) for 24 hours before chlamydial infection. Afterwards, cells were infected with a total of 2500 elementary bodies of CT serovar D or L2 and incubated for 48h. A state of persistence was then confirmed by the presence of ‘aberrant inclusions’ and by the inability of chlamydiae to normally replicate. Different doses of purified indole (1, 10 and 20 µM) or different amounts of filtered supernatants (5 and 10 µl, corresponding to 1.25 and 2.5 µM indole concentrations, respectively) collected from a broth culture of Escherichia coli, were added to cell cultures, in order to evaluate the restoration of a normal CT cycle. The determination of indole levels in E. coli supernatants was carried out by a biochemical approach (hydroxylamine test). Results: Both the purified indole and the E. coli supernatants significantly restored a normal chlamydial cycle, in a dose-dependent manner, compared to controls (P

Background: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent the most common agents of sexually transmitted pharyngeal infections. These conditions are frequently asymptomatic, acting as important reservoir for further transmission. The aim of this study was to assess the prevalence and predictors of pharyngeal infections due to CT and NG in a cohort of patients attending a STI Clinic in the North of Italy. Materials/methods: From January 2016 to September 2018, all the consecutive patients attending the STI Outpatients Clinic of St. Orsola-Malpighi Hospital (Bologna) and reporting unsafe oral intercourses were considered eligible for the study. After a clinical examination, a pharyngeal swab for the molecular detection of CT and NG (Versant CT/GC DNA 1.0 Assay; Siemens) was collected from each patient. Moreover, on the basis of patients’ sexual behavior and symptoms, urine samples and/or ano-rectal swabs for CT/NG detection were collected. In case of a CT positivity, a molecular genotyping based on omp1 gene PCR, was performed. Results: During the study period, 893 patients (119 females and 774 MSM) were enrolled. Globally, a total of 134 cases of gonorrhoea (15%) and 34 chlamydial infections (3.8%) were detected in the pharyngeal site, with no significant differences between males and females. More than 90% of the pharyngeal infections were completely asymptomatic and the presence of symptoms was not a predictor of a pharyngeal infection (P=0.7). On the contrary, a history of sexual contacts with a partner positive for CT and/or NG (P

Background: Chlamydia trachomatis (CT) and Neisseria gonorroheae (NG) represent the most common agents of bacterial sexually transmitted infections (STIs), worldwide. Recently, nucleic acid amplification techniques (NAATs) have become the reference methods for CT/NG infection diagnosis, thanks to the excellent sensitivity and specificity. Moreover, the use of multiplex tests allows the detection of both pathogens from the same sample, leading to a more simplified syndromic management. The aim of this study was to assess the performance of BD ProbeTec CT/GC Qx DNA Amplified assay for CT and NG detection, by a comparison with another commercial NAAT. Materials/methods: From January 2017, a total of 352 consecutive uro-genital samples, submitted to the Microbiology Unit of S. Orsola-Malpighi Hospital in Bologna (Italy) for CT and NG detection, were selected. In particular, 26 swabs and 141 urines were collected from subjects attending different clinics in the Bologna city area, while 120 urines and 65 swabs from patients attending the STI Outpatients Clinic of the Hospital. All the samples were tested with a multiplex real-time PCR (Versant CT/GC DNA 1.0 Assay; Siemens), currently used in the diagnostic routine. Subsequently, all the specimens were tested also with the BD ProbeTec CT/GC Qx DNA Amplified assay run on BD Viper LT platform. Both the methods are characterized by highly automated systems for nucleic acids extraction and target detection. Results: When compared, the two methods showed excellent concordance values. In particular, a concordance of 98.4% and 98.8% was found for CT detection on urine samples and genital swabs,with Cohen k of 0.86 and 0.94, respectively. For NG infection, a 100%-concordance was found on urines and a 98.9% of concordance for the genital swabs, with Cohen k of 1 and 0.85, respectively. NG infection prevalence ranged from the 4% in the population attending the STI Clinic of the Hospital, to the 0.8% in the general population. CT infection prevalence reached the 9.1% in the high-risk subjects, being 2.4% in the general population. Conclusions: The BD assay and the Vyper system can represent an excellent choice for the molecular detection of CT and NG infection.

Background: The global spread of carbapenemase-producing Enterobacteriaceae is of great concern to health services worldwide. In particular, multi-drug resistant Klebsiella pneumoniae (KP) harbouring KPC enzymes has been causing epidemics of international proportions. The analysis of the metabolic profiles of KP strains could represent an intriguing approach to obtain useful information to set up new diagnostic methods and to develop new antimicrobial strategies. The aim of this study was to characterize the metabolic profiles of several KP strains, characterized by specific resistance pattern. Materials/methods: A total of 59 KP strains, isolated from clinical samples submitted to the Microbiology Unit of S.Orsola-Malpighi Hospital of Bologna (Italy) for diagnostic procedures, were included in the study. In particular, 27 carbapenemase-negative and 32 carbapenemase-positive strains were analyzed. For each strain, bacteria were grown overnight in Mueller-Hinton (MH) broth. Afterwards, the suspension was standardized (2.8 McF) and centrifuged to separate the cell pellet from the supernatant. The metabolomic analysis was performed by means of 1H-NMR spectroscopy analysis (Avance III Spectrometer; Bruker), starting from 700 μL of filtered supernatants (‘external metabolome’) and 100 μL of cell lysates (‘internal metabolome’). The same protocol was used to study the metabolic profile of 8 KP strains (4 wild-type and 4 KPCproducing) under ‘antibiotic-stress’, allowing the bacteria to grow in MH broth with a meropenem concentration corresponding to 1/8 of the MIC value.Results: A total of 44 and 32 molecules, mainly belonging to organic acids, amino acids and alcohols, were detected in the external and the internal metabolome, respectively. In basal conditions, we found 6 metabolites (acetate, isobutyrate, lysine, phenylacetate, hydroxybutyrate) whose concentration was significantly different between carbapenemase-positive and carbapenemase-negative KP strains. TheTable shows the molecules that differed between carbapenemase-positive and carbapenemase-negative strains under ‘meropenem-stress’.Conclusions: The metabolomic analysis allows to detect molecules that significantly differed between carbapenemase-positive and carbapenemase-negative KP strains, both in basal conditions and under ‘antibiotic stress’.

Background: Chlamydia trachomatis (CT) represents the agent of the most common bacterial sexually transmitted infection worldwide. Although several in-vitro studies have investigated the metabolic adaptation of CT to host cells, no data are available about the metabolic profile that can be found in human biological fluids during an ongoing CT infection. The aim of this study was to compare the urine metabolome in women with or without Chlamydia urethritis. Materials/methods: From January to July 2016, all the pre-menopausal non-pregnant Caucasian women attending the STI Outpatients Clinic of Sant’Orsola-Malpighi Hospital in Bologna (Italy) and meeting one of the following criteria were enrolled: presence of uro-genital symptoms and/or presence of risk factors for CT infection. After a clinical examination, an urine sample was collected from each woman and used for CT and Neisseria gonorrhoeae molecular detection (Versant CT/GC DNA 1.0 Assay; Siemens Healthineers). Moreover, 1 mL of urine specimen was subjected to the metabolomic analysis, by means of a proton-based nuclear magnetic resonance (1H-NMR) spectroscopy. In case of a CT positive result, CT molecular genotyping was performed by omp1 gene semi-nested PCR followed by RFLP analysis. A written consent was obtained by all the patients and the study protocol was approved by the Ethics committee of the Hospital. Results: During the study period, a total of 119 subjects were enrolled: 98 women were negative for a CT infection, whereas 21 were assigned to the CT-positive group. We were able to identify 3 metabolites whose concentrations were significantly higher in the urine samples of CT-infected women: sucrose (P=0.0005), mannitol (P=0.003) and lactate (P=0.002). These results were comparable for all the CT serovars detected (D, E, F, G, K). Conclusions: Since the presence of sugars can increase the stability of chlamydial proteins, higher levels of sucrose and mannitol in the urethral lumen could have favoured CT infection acquisition or persistence. Lactate could result from CT-induced immunopathology, as a product of the inflammatory microenvironment. Further studies are needed to understand the potential role of these metabolites in the pathogenesis of CT infection, as well as their diagnostic/prognostic use.

INTRODUZIONE: Gardnerella vaginalis (GV) è un batterio anaerobio facoltativo, pleomorfico, Gram-variabile. GV è la specie batterica più comunemente ritrovata a livello vaginale in corso vaginosi batterica (BV), condizione caratterizzata da una deplezione di lattobacilli e un’eccessiva crescita di anaerobi. Nonostante sia la specie più diffusa e più virulenta trovata in BV, GV fa parte del microbiota vaginale in una proporzione significativa di donne sane. Oltre a questo aspetto, è meno noto il ruolo di GV in altre condizioni cliniche che interessano il tratto genitale delle donne, come la candidosi vulvo-vaginale e le infezioni da Chlamydia trachomatis (CT). Lo scopo di questo studio è stato valutare e confrontare la distribuzione di GV nel tratto vaginale in 4 diversi gruppi: donne sane, con BV, con candidosi o con infezione da CT. METODI: A partire da luglio 2016 sono state arruolate tutte le donne in età fertile afferenti all’ambulatorio MTS del Policlinico Sant’Orsola-Malpighi di Bologna e che soddisfacevano uno dei seguenti criteri: presenza di sintomi vulvo-vaginali (es: perdite vaginali, sanguinamento anomalo, prurito) o presenza di fattori di rischio per l'infezione da CT (età

INTRODUZIONE: Mycoplasma hominis (MH), Ureaplasma parvum (UP) e Ureaplasma urealyticum (UU), sono membri della classe dei Mollicutes, che possono essere frequentemente ritrovati nel tratto urogenitale di soggetti sani, come comuni microrganismi commensali. Il loro eventuale ruolo patologico è piuttosto controverso e molti aspetti della loro fisio-patologia devono ancora essere chiariti. I farmaci antimicrobici usati per il trattamento dei Mollicutes comprendono i chinolonici, i macrolidi e le tetracicline, anche se, a causa dell'eccessivo e improprio uso di farmaci, stanno emergendo sempre più resistenze. Lo scopo di questo studio è stato quello di valutare la prevalenza e il profilo di resistenza di MH e UU/UP isolati da campioni genitali, in un periodo di due anni. METODI: Sono stati raccolti ed analizzati i dati relativi agli esami colturali per ureaplasmi/micoplasmi a partire da campioni urogenitali, presso la U.O. Microbiologia del Policlinico Sant’Orsola di Bologna, nel periodo gennaio 2015-dicembre 2016. La coltura, l’identificazione differenziale di Ureaplasma (UU/UP) e MH e le prove di suscettibilità antimicrobica sono state eseguite con un kit commerciale (Mycoview, Zeakon Diagnostics) che permette di testare i seguenti antimicrobici: azitromicina, roxitromicina, josamicina, minociclina, doxiciclina, ofloxacina e ciprofloxacina. RISULTATI: Nel periodo di studio, sono stati testati complessivamente 4660 campioni (di cui 84,4% provenienti da donne) ed è stata rilevata una positività per Mollicutes pari al 30,9%. E’ stata ritrovata una percentuale di positività quasi doppia nelle donne rispetto agli uomini (33,3% vs 17,8%). In generale, la positività per Mollicutes tendeva a diminuire con l’aumentare dell’età dei soggetti. Gli ureaplasmi sono risultati le specie più comunemente isolate (prevalenza del 24,2%), nel 6,5% dei casi si è osservata la crescita sia di ureaplasmi che di MH, mentre MH è stato isolato solo nel 3,9% dei campioni. I ceppi di ureaplasma hanno mostrato livelli significativi di resistenza ai chinoloni, in particolare alla ciprofloxacina (77%), mentre i ceppi di MH erano resistenti ad azitromicina e a roxitromicina in circa il 90% dei casi. In caso del contemporaneo ritrovamento di ureaplasmi e MH, il livello di resistenza agli antimicrobici era significativamente maggiore rispetto ai casi di infezioni singole. Rispetto al 2015, nel 2016 si è assistito a un aumento significativo della prevalenza delle infezioni da MH e a un incremento del tasso di resistenza ad azitromicina e roxitromicina (P

INTRODUZIONE: Negli ultimi anni si è assistito ad un notevole aumento dei fenomeni di resistenza ai farmaci carbapenemici in ceppi di Klebsiella pneumoniae (KP), per lo più mediati dalla produzione di carbapenemasi di tipo KPC. Lo studio del profilo metabolico di questi microorganismi rappresenta un approccio innovativo in grado di fornire informazioni utili per la messa a punto di nuove metodiche diagnostiche e lo sviluppo di nuovi agenti terapeutici. Lo scopo del presente lavoro è stato quello di valutare il profilo metabolico di diversi ceppi di KP, caratterizzati da specifici fenotipi di resistenza, mediante spettrometria di risonanza magnetica nucleare. METODI: Sono stati inclusi nello studio 59 ceppi di KP, di cui 27 non-produttori di carbapenemasi (16 wild-type e 11 produttori di ESBL) e 32 produttori di carbapenemasi (24 KPC, 4 MBL e 4 OXA-48). I ceppi sono stati isolati da vari campioni clinici provenienti da pazienti ricoverati presso il Policlinico Sant’Orsola-Malpighi di Bologna. Tramite micro-diluizione in brodo secondo linee guida EUCAST, sono state calcolate le minime concentrazioni inibenti (MIC) del meropenem. Dopo una crescita O/N in brodo Mueller-Hinton (MH), la sospensione batterica di ogni ceppo è stata standardizzata a 2.8 McF ed è stata centrifugata al fine di separare il pellet batterico dal surnatante. Il surnatante è stato filtrato e congelato, fino al momento dell’analisi metabolica, mentre i metaboliti intracellulari sono stati estratti processando il pellet con metanolo freddo. L’analisi metabolomica è stata condotta a partire da 700 μL di surnatante e da 100 μL di lisato intracellulare, tramite spettroscopia di risonanza magnetica nucleare (1H-NMR), con spettrometro AVANCE III (Bruker). Lo stesso protocollo è stato inoltre utilizzato per studiare il profilo metabolico di 8 ceppi di KP (4 wild-type e 4 KPC produttori) in condizioni di ‘stress’, facendo crescere i ceppi in presenza di meropenem ad una concentrazione pari a 1/8 della MIC. RISULTATI: I risultati preliminari evidenziano la presenza di differenze nel profilo metabolico fra ceppi produttori e non produttori di carbapenemasi. Per i ceppi non-carbapenemasi produttori le MIC del meropenem erano comprese tra 0.008 e 0.25 mg/L, mentre per i ceppi produttori variavano tra 0.5 e 512 mg/L. In caso di ‘stress’ indotto da concentrazioni subletali di meropenem, emerge una maggior ‘perturbazione metabolica’ nei ceppi KPC-produttori. CONCLUSIONI: Lo studio metabolomico permetterà di ampliare la conoscenza delle basi fisiologiche della cellula batterica e dei meccanismi di virulenza associati. Inoltre, l’identificazione di biomarker metabolici specifici potrebbe aprire nuovi scenari per l’approccio diagnostico e terapeutico delle infezioni da ceppi di KP produttori di carbapenemasi

INTRODUZIONE: Chlamydia trachomatis (CT) e Neisseria gonorrhoeae (NG) rappresentano gli agenti eziologici delle più comuni infezioni batteriche a trasmissione sessuale al mondo. Nelle donne, le infezioni urogenitali da CT e GC sono spesso asintomatiche e possono provocare severe complicanze come la malattia infiammatoria pelvica e infertilità. Grazie alle ottime performance in termini di sensibilità e specificità e alla elevata automazione, i test di amplificazione degli acidi nucleici (NAATs) rappresentano i test di riferimento per la diagnosi di infezione da CT e GC. Lo scopo del presente studio è stato quello di valutare la performance del test Aptima Combo 2 per la diagnosi di infezione da CT e GC in un gruppo selezionato di donne in età fertile. Inoltre è stata valutata l’adeguatezza del sistema di raccolta con tampone E-Swab (Copan) per tale sistema NAAT. METODI: Attualmente, per le donne afferenti all’ambulatorio MTS del Policlinico Sant’Orsola-Malpighi di Bologna che presentano sintomi uro-genitali o che riportano rapporti sessuali non protetti, viene effettuata una visita clinica e vengono raccolti un campione di urina e un tampone vaginale per la ricerca di CT e GC tramite una duplex real-time PCR (Versant CT/GC DNA 1.0 assay; Siemens). A partire da gennaio 2016, sono state selezionate 100 pazienti e i loro campioni (urine e tamponi vaginali) sono stati testati in parallelo con il test Aptima Combo 2 su sistema Panther (Hologic). In particolare, a partire da campioni precedentemente congelati, 2 mL di urine e 100 μL del secreto vaginale raccolto in E-Swab sono stati trasferiti nei sistemi di raccolta dedicati per i test Aptima. Infine, è stato eseguito un confronto fra i due sistemi NAAT, stratificato per il tipo di campione analizzato e per singolo analita. RISULTATI: Nel gruppo di donne selezionate sono state incluse 25 pazienti positive per CT, 4 per GC, con un caso di co-infezione da entrambi i microorganismi. Con il test NAAT utilizzato di routine, tutti i casi di positività sono stati riscontrati contemporaneamente sia sul campione di urine che sul tampone vaginale, con l’eccezione di 3 infezioni da CT e una da GC, riscontrate solo su uno dei due campioni. Dal confronto con il test Aptima, è stata rilevata un’ottima concordanza, con valori di k di Cohen variabili tra 0.85 e 1.0 a seconda dell’analita e del materiale preso in considerazione. CONCLUSIONI: Il nostro studio conferma le ottime performance diagnostiche dei test Aptima Combo 2 e VERSANT CT/GC DNA 1.0 Assay, entrambi altamente automatizzati e di facile impego per gli utilizzatori. Da questo studio si è visto che è possibile analizzare con Aptima Combo 2 su sistema Panther anche campioni raccolti in E-Swab, potendo così procedere contemporaneamente a diagnosi molecolare e colturale nei casi di sospette infezioni gonococciche

INTRODUZIONE: Chlamydia trachomatis e Neisseria gonorroheae sono i principali patogeni ad eziologia batterica coinvolti nelle infezioni sessualmente trasmesse. Sebbene siano microorganismi dalle caratteristiche molto diverse, portano a manifestazioni cliniche simili e spesso confondenti. Negli ultimi anni, per la diagnosi di laboratorio di tali batteri, si è ampiamente diffuso l’uso di metodiche di amplificazione degli acidi nucleici, caratterizzate da sensibilità e specificità maggiori rispetto alle metodiche tradizionali di coltura e/o identificazione di antigeni. In particolare, si è affermato l’uso di test multiplex, che permettono di determinare parallelamente più patogeni a partire dallo stesso campione biologico. Lo scopo di questo studio è stato quello di valutare le performance del BD ProbeTec CT/GC Qx DNA Amplified assay su piattaforma BD Viper LT per la diagnosi delle infezioni da clamidia e gonococco, in comparazione ai dati ottenuti con il test VERSANT CT/GC 1.0 (Real-time PCR) (Siemens). METODI: Sono stati analizzati, in maniera retrospettiva, 352 campioni progressivi pervenuti al laboratorio di Microbiologia del Policlinico Sant’Orsola-Malpighi di Bologna per la ricerca di acidi nucleici di C. trachomatis e N. gonorrhoeae. In totale, 232 campioni erano stati raccolti da soggetti recatisi in poliambulatori presenti sul territorio comunale e provinciale di Bologna (26 tamponi e 141 urine), mentre 120 urine e 65 tamponi provenivano da pazienti dell’ambulatorio di Malattie a Trasmissione Sessuale (MTS) del Policlinico. I campioni sono stati testati con il kit VERSANT CT/GC 1.0, attualmente utilizzato nella routine diagnostica e successivamente con il BD ProbeTec CT/GC Qx DNA Amplified assay su piattaforma BD Viper LT. Le due metodiche si avvalgono di sistemi altamente automatizzati per le fasi di estrazione, allestimento piastre e rivelazione. RISULTATI: Le due metodiche hanno mostrato ottimi valori di concordanza tra loro; in particolare, la concordanza è stata del 98.4% per la determinazione delle infezioni da clamidia sulle urine e del 98.9% sui tamponi. Invece, la concordanza per le infezioni da gonococco è stata del 100% sulle urine e del 98.9% sui tamponi. La prevalenza di casi da N. gonorrhoeae nella popolazione afferente all’ambulatorio MTS è risultata del 4%, mentre è stata pari allo 0.8% nella popolazione a minor rischio di infezioni sessualmente trasmesse. Infine, la prevalenza di C. trachomatis nella popolazione a più alto rischio è risultata pari al 9.1%, mentre nella la popolazione generale è stata del 2.4%. CONCLUSIONI: Il nostro studio conferma le ottime performance diagnostiche dei test BD ProbeTec CT/GC Qx DNA Amplified assay su piattaforma BD Viper LT e VERSANT CT/GC DNA 1.0 Assay, entrambi altamente automatizzati e di facile impego per gli utilizzatori.

INTRODUZIONE: Il microbiota vaginale comprende numerose specie batteriche, fra cui microorganismi della classe dei Mollicutes. I più importanti Mollicutes che si ritrovano nel tratto genitale femminile sono rappresentati da Ureaplasma urealyticum (UU), Ureaplasma parvum (UP), Mycoplasma hominis (MH) e Mycoplasma genitalium (MG). Nonostante numerosi studi abbiano indagato il loro ruolo fisio-patologico nel microambiente vaginale, molti aspetti rimangono ancora da chiarire. L’obiettivo del presente studio è stato quello di valutare la presenza di micoplasmi e ureaplasmi nel microbiota vaginale in diverse condizioni cliniche: donne ‘sane’, donne con vulvo-vaginite da Candida, donne con infezione da Chlamydia trachomatis (CT) e donne con vaginosi batterica. METODI: Tramite saggi di PCR real-time quantitativi, sono stati valutati sia la presenza che il rispettivo ‘bacterial load’ dei principali Mollicutes (UU, UP, MH, MG) nel microbiota vaginale di donne caucasiche in età riproduttiva, afferenti all’ambulatorio MTS del Policlinico Sant’Orsola-Malpighi di Bologna. I criteri di inclusione comprendevano la presenza di sintomi uro-genitali o la presenza di fattori di rischio per infezioni a trasmissione sessuale. La recente assunzione di antibiotici, la gravidanza, un BMI > 30 e l’uso di farmaci anti-concezionali rappresentavano i principali criteri di esclusione. Per ogni paziente, dopo la visita clinica, è stato raccolto un tampone vaginale per la diagnosi molecolare di infezione da CT (Versant CT/GC DNA 1.0; Siemens) e per l’esecuzione di indagini microscopiche e colturali. In base ai risultati ottenuti, le donne sono state suddivise in quattro gruppi: 29 donne ‘sane’ (assenza di sintomi urogenitali e negatività alle principali indagini microbiologiche), 25 donne con infezione da C. trachomatis (positività per CT al test Versant CT/GC DNA 1.0 Assay su tampone vaginale), 23 donne con vulvo-vaginite da Candida (presenza di lieviti all’esame microscopico e/o colturale) e 21 donne con vaginosi batterica (presenza di almeno 3 dei 4 criteri di Amsel e score di Nugent > 7). RISULTATI: Complessivamente, le percentuali di positività per i diversi Mollicutes nella coorte delle 98 donne sono state le seguenti: 67.3% per UP, 14.3% per MH, 9.2% per UU e 3.1% per MG. Con l’eccezione di un caso, non è stata mai individuata una contemporanea positività per UU e UP. MH ha mostrato una significativa maggior prevalenza e più elevati ‘bacterial loads’ nelle donne con vaginosi batterica rispetto agli altri gruppi (P < 0.05). Sia dal punto di vista qualitativo che quantitativo, le donne con infezione da Candida e da CT hanno mostrato un pattern vaginale per Mollicutes simile a quello delle donne sane. CONCLUSIONI: I Mollicutes, in particolare UP, possono essere frequentemente ritrovati nel microbiota vaginale di donne ‘sane’. Sebbene non sempre presente, MH è fortemente associato alla condizione di vaginosi batterica

We studied prevalence of Chlamydia trachomatis (CT) urogenital infection and distribution of different genotypes in a non-selected STD population of 1625 patients, evaluating presence of coinfections with other sexually transmitted diseases. Each patient was bled in order to perform serological tests for syphilis and HIV, then urethral or endocervical swabs were obtained for the detection of CT and Neisseria gonorrhoeae by culture. DNA extracted from remnant positive swabs was amplified by omp1 Nested PCR and products were sequenced. Total prevalence of CT infection was 6.3% (103/1625), with strong differences between men and women (11.4% vs 3.9%, P

In 1998 a network of 20 regional tuberculosis (TB) laboratories (the Italian Multicentre Study on Resistance to Antituberculosis drugs (SMIRA) network) was established in Italy to implement proficiency testing and to monitor the prevalence of drug resistance nationwide. The network managed 30% of all TB cases reported in Italy each year. The aim of the present report is to describe: 1) the accuracy of drug-susceptibility testing in the network; 2) the prevalence of drug resistance for the period 1998-2000. Data were collected from the network laboratories. Sensitivity to streptomycin and ethambutol increased from the first survey (1998-1999) to the second survey (2000) from 87.7 to 91.9%. Specificity, predictive values for resistance and susceptibility, efficiency and reproducibility were consistent in both surveys. In previously untreated cases, the prevalence of multidrug-resistance was the same in both surveys (1.2%), while a slight decrease from the first to the second survey was observed for monoresistance to rifampicin (from 0.8 to 0.4%) and isoniazid (from 2.9 to 2%). The significant association found between isoniazid resistance and immigration is a useful indicator for both clinicians managing individual tuberculosis cases and public health services planning control strategies.

(1) Dipartimento di Biologia Animale e dell'Uomo, Università di Roma La Sapienza, Via A. Borelli 50, 00161 Roma.

An Italian HIV-positive man having sex with men (MSM) attended the STIs Outpatients Clinic of Sant'Orsola Hospital in Bologna complaining of anal pain and constipation. According to patient's sexual history and repertoires, NAAT testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) was performed. Pharyngeal and anal swabs resulted positive for CT DNA detection and the following molecular genotyping identified a L2 serovar, coming to the final diagnosis of pharyngeal and rectal lymphogranuloma venereum (LGV) infection. After an antibiotic therapy with doxycycline 100 mg twice a day for 3 weeks, the patient completely recovered and the test of cure was negative for LGV infection.

We evaluated the analytical performance of a liquid chromatography-tandem mass spectrometry assay to detect carbapenemase activity in a group of carbapenemase-producing Enterobacteriaceae by meropenem hydrolysis. This one-hour method showed a sensitivity of 94% and a specificity of 100%, representing a rapid and reliable option compared to conventional phenotypic assays.

Although far less common now than in the past, syphilis continues to pose a danger to public health and should not be overlooked. In this study, we evaluated the presence and characteristics of syphilis in a group of patients attending an STI Clinic in the North of Italy. A retrospective study was carried out, analysing the data from the 5609 subjects who attended the STI Clinic of St. Orsola-Malpighi Hospital (Bologna) for syphilis screening from January 2016 to December 2017. Globally, 692 patients (12.3%) were found positive for treponemal tests, with a significant difference between males and females (16.6% vs 4.1%; P

Chlamydia trachomatis (CT) is the causative agent of the most common bacterial sexually transmitted infection (STI) worldwide. CT molecular typing plays a fundamental role in finding associations between serovars and epidemiological features and can be crucial for therapeutic appropriateness. The aim of this study was to assess CT infection prevalence and serovar distribution in a high density urban area in the North of Italy, by comparing different groups of subjects divided on the basis of care providers they referred to. From January 2011 to May 2014, data about all the urogenital and extra-genital samples (anorectal and/or pharyngeal swabs) submitted to the Microbiology of St. Orsola Hospital in Bologna for CT detection were collected. The specimens were obtained from three different groups of patients: subjects attending the STI Outpatients Clinic of the Hospital, patients attending gynecological clinics or people referring to general practitioners. All the samples were processed by a real-time PCR (Versant CT/GC DNA 1.0 Assay; Siemens) and in case of positivity, molecular genotyping based on RFLP analysis was performed. A P < 0.05 was considered statistically significant. Overall CT infection prevalence was 8.1% with significant differences between sub-groups (P

A molecular biological approach, based on preproregion homology in the precursors of several diverse antibacterial peptides, was used to clone a pig bone marrow cDNA encoding a novel 167-residue polypeptide. The preproregion of this polypeptide is highly similar to corresponding regions in congeners from pig, cattle and rabbit. It is followed by a unique, cationic, 37-residue sequence, which was predicted to have a high propensity for an α-helical conformation. A peptide, termed PMAP-37, corresponding to this sequence, was chemically synthesized and shown to undergo a transition from a random coil to an ordered, mainly helical, conformation on addition of trifluoroethanol. This behaviour is typical of an amphipathic α helix, a structure common to several membrane-active, antimicrobial peptides. In vitro experiments showed that PMAP-37 strongly inhibits the growth of several strains of Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging over 1–4 μM, and permeabilizes the inner membrane of Escherichia coll. Interestingly, the 15–32 stretch of PMAP-37 show a remarkable similarity to N-terminal stretches in cecropins B and A from Drosophila melanogaster and Cecropia hyalophora, respectively. This affords an uncommon example of sequence convergence.

ObjectivesLymphogranuloma venereum (LGV) is an STI caused by Chlamydia trachomatis serovars L1-L3. In Europe, the current epidemic is caused mainly by L2b genovariant, although increasing cases associated with other L2 variants have been reported. Here, we assessed the distribution of rectal LGV genovariants among men having sex with men (MSM) in Italy.MethodsFrom 2016 to 2020, all the anorectal swabs collected from MSM attending the STI Clinic of St. Orsola-Malpighi Hospital in Bologna and positive for C. trachomatis were stored. LGV infection was confirmed by a pmpH PCR, and, subsequently, a fragment of the ompA gene was amplified and sequenced. Sequences were aligned to reference strains representing different LGV variants.ResultsLGV cases accounted for one-third of all chlamydial rectal infections with a total prevalence of 4.1% (76/1852). Total number of LGV cases per year remained constant. LGV was mainly found in symptomatic patients (>65%), older than 30 years, with a high burden of other STIs (63.7% HIV-positive, 35.5% with concurrent rectal gonorrhoea, 19.7% with early syphilis). A decreasing trend in HIV-LGV co-infection was noticed over time. Three main LGV genovariants were detected (L2f, 46.1%; L2b, 23.0%; L2-L2b/D-Da, 16.9%), together with other known L2b variants (mainly L2bV2 and L2bV4). Two novel L2b ompA variants with non-synonymous single-nucleotide polymorphisms were found. Over time, the percentage of L2f cases dropped gradually, with a significant increase in L2-L2b/D-Da cases (p=0.04).ConclusionsIn our area, LGV is endemic among MSM with different circulating genovariants. Active surveillance and genotyping programmes are needed to reduce re-establishing of LGV infection.

The oropharynx represents a crucial site for the emergence of multi-drug resistance in Neisseria gonorrhoeae. The mosaic penA alleles, associated with decreased susceptibility to cephalosporins, have emerged by DNA recombination with partial penA genes, particularly those from commensal pharyngeal Neisseria species. Here, we investigated the prevalence of the mosaic structure of the penA gene in the oropharynx of men who have sex with men testing negative for pharyngeal gonorrhoea. From January 2016 to June 2018, 351 gonorrhoea-negative men who have sex with men attending a sexually transmitted infection clinic in Italy were enrolled. Pharyngeal swabs underwent a real-time polymerase chain reaction (PCR) for the detection of the mosaic penA gene. In case of positivity, PCR products were sequenced and searched against several sequences of Neisseria strains. Overall, 31 patients (8.8%) were found positive for the presence of the mosaic penA gene. The positivity was significantly associated with previous cases of pharyngeal gonorrhoea (relative risk [RR]: 3.56, 95% confidence interval 1.44–8.80) and with recent exposure to beta-lactams (RR: 4.29, 95% confidence interval 2.20–8.38). All penA-positive samples showed a high relatedness (90–99%) with mosaic-positive Neisseria strains. Our data underline that commensal Neisseria species of the oropharynx may be a significant reservoir for genetic material conferring antimicrobial resistance in N. gonorrhoeae.

Candida spp. are commensal organisms of the skin, mucous membranes, gastrointestinal tract, blood, and vagina of animals and humans. In recent decades, the incidence of human fungal infections has increased, with Candida spp. (mainly C. albicans) infections being the most frequent, and the treatment of fungal infections is still a clinical challenge. Colonization of the human gastrointestinal tract by Candida spp. is significant because infections (e.g., candidemia and vulvovaginal candidiasis) frequently arise from commensal microorganisms. The aim of this study was to test in vitro the antifungal activity and the eventual synergistic effect of five pure components of essential oils: cinnamaldehyde, α-pinene, limonene, eucalyptol, and eugenol. These compounds were tested on 18 Candida strains (15 C. albicans, 2 C. glabrata, and 1 C. lusitaniae) derived from a culture collection of vaginal clinical strains. Methods: Fungistatic activity was evaluated using the disk diffusion method. For fungicidal activity, microdilution and time–kill curve protocols were set up. The checkerboard method was chosen to evaluate a possible synergistic effect of these compounds when mixed. Results: Cinnamaldehyde and eugenol gave the best results, inhibiting all the Candida strains and showing a highly additive effect (FICI 0.625). The cinnamaldehyde inhibition zone (IZ), MIC, and MFC means were 69 mm, 50.05 mg/L, and 109.26 mg/L respectively. Cinnamaldehyde led to the total loss of viable Candida cells within 4 h. Eugenol IZ, MIC, and MFC means were 35.2 mm, 455.42 mg/L, and 690.09 mg/L, respectively. Eugenol led to the total loss of viable fungal cells within 1 h. Treatment with α-pinene inhibited 88.9% of Candida strains, with an IZ mean of 21.2 mm, a MIC mean of 195.41 mg/L, and a MFC mean of 251.27 mg/L; this compound led to the total loss of viable fungal cells only after 24 h. Limonene inhibited only 33.3% of the tested strains and eucalyptol did not produce an inhibition halo, so these compounds were not tested further. Conclusions: These characteristics, together with the well-known safety of cinnamaldehyde and eugenol for human use, make these two natural compounds the perfect candidates for the treatment of candidiasis. This was a pilot study, the purpose of which was to evaluate the best composition of a mixture to be used against intestinal and vulvovaginal candidiasis; in vivo studies are needed to confirm these very encouraging results.

Objectives: Vulvo-vaginal atrophy (VVA) is a highly prevalent chronic condition affecting the lives of postmenopausal women. Ospemifene and systemic hormone therapy (HT) improve vaginal health. This study aims to characterize the vaginal metabolic profile of women with VVA at baseline and after ospemifene and systemic HT. Study design: Sixty postmenopausal women, 32 of whom were affected by VVA, were consecutively enrolled. The vaginal metabolic profile of women with and without VVA at baseline and after three months of ospemifene or HT treatment was assessed. Main outcome measures: The following parameters were evaluated: (i) the Vaginal Health Index; (ii) the Vaginal Maturation Index; and (iii) vaginal metabolic profile, by means of 1H NMR spectroscopy. Results: The vaginal metabolome of postmenopausal women with VVA was different from that of postmenopausal women without VVA, including a more profound decrease in the levels of lactate and several amino acids, typically found in eubiosis, together with an enrichment of molecules derived from anaerobes and gut microbes. After 3 months, ospemifene and HT had modified the vaginal metabolome of the women with VVA, specifically by increasing the levels of beneficial molecules (e.g., lactate, leucine, glycine) and reducing those involved in dysbiosis (e.g., formate). HT improved the vaginal metabolome to a lesser extent. Conclusions: The vaginal metabolic profile of postmenopausal women with VVA differs from that of postmenopausal women without VVA. Our preliminary data show that both ospemifene and HT treatment increase the levels of molecules beneficial for vaginal health and reduce the levels of those involved in dysbiosis. HT improves the vaginal metabolic profile to a lower extent than ospemifene over the course of three months.

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Lactobacilli are the dominant members of the healthy human vaginal microbiota and represent the first defense line from pathogen infection, including vulvovaginal candidiasis. Biofilm is the predominant microbial growth form in nature, and the formation of biofilms inside the human body has important implications in health and disease. In particular, the formation of biofilm by members of the human resident microbiota is desirable, as it can improve microbial persistence and influence functionality. In the present study, we investigated the capability of 16 vaginal Lactobacillus strains (belonging to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus vaginalis, and Lactobacillus plantarum species) to form biofilms, and we correlated their mode of growth to anti-Candida activity. L. plantarum strains were the best biofilm producers, and high variability was registered in the level of biofilm formation among L. crispatus and L. gasseri strains. Culture supernatants derived from Lactobacillus biofilm and planktonic growth were tested toward a panel of Candida clinical isolates (Candida albicans, Candida glabrata, Candida lusitaniae, Candida tropicalis, Candida krusei, and Candida parapsilosis) and their metabolome assessed by 1H-NMR. L. crispatus and L. plantarum strains exhibited the best fungistatic profile, and biofilms enhanced their anti-Candida activity; on the contrary, L. gasseri strains were more effective when grown in a planktonic mode. Biofilm/planktonic mode of growth also affects Lactobacillus metabolism, mainly influencing nitrogen and amino acid pathways, and anti-Candida activity is instead strictly related to carbohydrate metabolism. The present study underlined the strict interdependence between microbial mode of growth, metabolism, and functional properties. Biofilm formation by members of the healthy human microbiota represents a crucial issue in the field of microbial physiology and host–microbiota interactions, beyond supporting the development of new antimycotic strategies based on probiotics grown in adherence.

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Staphylococcus aureus is the major causative agent of skin and soft tissue infections, whose prevention and treatment have become more difficult due to the emergence of antibiotic-resistant strains. In this regard, the development of an effective treatment represents a challenge that can be overcome by delivering new antibiofilm agents with appropriate nanocarriers. In this study, a biosurfactant (BS) isolated from Lactobacillus gasseri BC9 and subsequently loaded in liposomes (LP), was evaluated for its ability to prevent the development and to eradicate the biofilm of different methicillin resistant S. aureus (MRSA) strains. BS from L. gasseri BC9 was not cytotoxic and was able to prevent formation and to eradicate the biofilm of different MRSA strains. BS loaded liposomes (BS-LP) presented a mean diameter (lower than 200 nm) suitable for topical administration and a low polydispersity index (lower than 0.2) that were maintained over time for up 28 days. Notably, BS-LP showed higher ability than free BS to inhibit S. aureus biofilm formation and eradication. BS-LP were loaded in lyophilized matrices able to quickly dissolve (dissolution time lower than 5 s) upon contact with exudate, thus allowing vesicle reconstitution. In conclusion, in this work, we demonstrated the antibiofilm activity of Lactobacillus-derived BS and BS-LP against clinically relevant MRSA strains. Furthermore, the affordable production of lyophilized matrices containing BS-LP for local prevention of cutaneous infections was established.