Trends in hospital acquired New Delhi metallo-beta-lactamase-producing Enterobacterales in Tuscany (Italy) from 2019 to 2021: impact of the COVID-19 pandemic.

OBJECTIVES: In Tuscany, Italy, New Delhi metallo-beta-lactamase-producing carbapenem-resistant Enterobacterales (NDM-CRE) in hospitalized patients has increasingly been observed since 2018, leading in 2019 to the implementation of enhanced control measures successfully reducing transmission. We describe the NDM-CRE epidemiology during the COVID-19 pandemic in Tuscany. METHODS: Data on NDM-CRE patients hospitalized in five Tuscan hospitals were collected from January 2019 to December 2021. Weekly rates of NDM-CRE cases on hospital days in medical and critical-care wards were calculated. In March-December 2020, NDM-CRE rates were stratified by COVID-19 diagnosis. Multi-variate regression analysis was performed to assess outcomes' differences among two periods analysed and between COVID-19 populations. RESULTS: Since March 2020, an increase in NDM-CRE cases was observed, associated with COVID-19 admissions. COVID-19 patients differed significantly from non-COVID-19 ones by several variables, including patient features (age, Charlson index) and clinical history and outcomes (NDM-CRE infection/colonization, intensive care unit stay, length of stay, mortality). During the pandemic, we observed a higher rate of NDM-CRE cases per hospital day in both non-COVID-19 patients (273/100,000) and COVID-19 patients (370/100,00) when compared with pre-pandemic period cases (187/100,00). CONCLUSIONS: Our data suggest a resurgence in NDM-CRE spread among hospitalized patients in Tuscany during the COVID-19 pandemic, as well as a change in patients' case-mix. The observed increase in hospital transmission of NDM-CRE could be related to changes in infection prevention and control procedures, aimed mainly at COVID-19 management, leading to new challenges in hospital preparedness and crisis management planning.

Evaluation of a modified cleaning procedure in the prevention of carbapenem-resistant Acinetobacter baumannii clonal spread in a burn intensive care unit using a high-sensitivity luminometer.

BACKGROUND: Enhanced environmental cleaning practices are among the most accepted measures for controlling the spread of carbapenem-resistant Acinetobacter baumannii (CR-Ab). AIM: To evaluate the impact of heightened cleaning on an ongoing CR-Ab outbreak in a burn intensive care unit (BICU) of an Italian teaching hospital, where chlorhexidine-60% isopropyl alcohol was applied as a complementary disinfectant on high-touch surfaces. METHODS: Compliance with the microbial limit proposed for the BICU by AFNOR-NF-S90-351 (20 colony-forming units/100cm2) was assessed by plate count, and compared with the results obtained with intracellular adenosine triphosphate (ATP) detection. Genotyping was performed using pulsed-field gel electrophoresis. FINDINGS: During the standard cleaning regimen, three out of 23 samples (13%) gave results over the AFNOR limit and five (21.7%) showed unacceptable ATP levels with 100 relative light units/100cm2 as the benchmark limit (sensibility 86.4%, specificity 92.2%). Following improvement of the cleaning procedure, only two samples out of 50 (4%) did not satisfy the microbiological criteria and seven (14%) exceeded the ATP limit. In a successive phase, eight of 30 samples collected showed unacceptable results (27%). CONCLUSIONS: Adding chlorhexidine-60% isopropyl alcohol as complementary disinfectant proved to be effective for reducing environmental microbial contamination, ATP levels and CR-Ab infection/colonization in patients admitted to the BICU. Real-time monitoring by ATP assay was useful for managing the cleaning schedule and reducing hospital infections, although the calculated values must be interpreted as cleanliness indicators rather than risk indicators.

Saponin promotes rapid identification and antimicrobial susceptibility profiling of Gram-positive and Gram-negative bacteria in blood cultures with the Vitek 2 system.

The rapid identification and antimicrobial susceptibility testing (AST) of bacteria in clinical blood cultures is crucial to optimise antimicrobial therapy. A previous study involving small sample numbers revealed that the addition of saponin to blood cultures, further referred to as the new method, shortened considerably the turn-around time for the identification and AST of Gram-positive cocci as compared to the current method involving an overnight subculture. Here, we extend previous results and compare the identification and AST of blood cultures containing Gram-negative bacilli by the new and current methods. The identification and AST of 121 Gram-positive and 109 Gram-negative bacteria in clinical monomicrobial blood cultures by the new and current methods and, in the case of Gram-negative bacilli, by direct (no additions) inoculation into an automated system (rapid method) was assessed using the Vitek 2 system. Discrepancies between the results obtained with the different methods were solved by manual methods. The new method correctly identified 88 % of Gram-positive and 98 % of Gram-negative bacteria, and the rapid method correctly identified 94 % of Gram-negative bacteria. The AST for all antimicrobials by the new method were concordant with the current method for 55 % and correct for an additional 9 % of Gram-positive bacteria, and concordant with the current method for 62 % and correct for an additional 21 % of Gram-negative bacilli. The AST by the rapid method was concordant with the current method for 62 % and correct for an additional 12 % of Gram-negative bacilli. Together, saponin-treated monomicrobial blood cultures allow rapid and reliable identification and AST of Gram-positive and Gram-negative bacteria.

Evidence for a role of frataxin in pancreatic islets isolated from multi-organ donors with and without type 2 diabetes mellitus.

Frataxin (FXN) is a mitochondrial protein involved in iron metabolism and in the modulation of reactive oxygen and/or nitrogen species production. No information is currently available as for the role of frataxin in isolated human pancreatic islets. We studied islets from pancreases of multi-organ donors with (T2DM) and without (Ctrl) Type 2 diabetes mellitus. In these islets, we determined FXN gene and protein expression by qualitative and quantitative Real-Time RT-PCR, nitrotyrosine concentration, and insulin release in response to glucose stimulation (SI). FXN gene and protein were expressed in human islets, though the level of expression was much lower in T2DM islets. The latter also had lower insulin release and higher concentration of nitrotyrosine. A positive correlation was apparent between SI and FXN gene expression, while a negative correlation was found between nitrotyrosine islet concentration and FXN expression. Transfection of Ctrl islets with siRNA FXN caused reduction of FXN expression, increase of nitrotyrosine concentration, and reduction of insulin release. In conclusion, in human pancreatic islets FXN contributes to regulation of oxidative stress and insulin release in response to glucose. In islets from T2DM patients FXN expression is reduced while oxidative stress is increased and insulin release in response to glucose impaired.

Rapid identification and antimicrobial susceptibility profiling of Gram-positive cocci in blood cultures with the Vitek 2 system.

Rapid identification and antimicrobial susceptibility profiling of the bacteria in blood cultures can result in clinical and financial benefits. Addition of saponin to the fluid from blood culture bottles promotes the recovery of the bacteria and thus may shorten the turnaround time of the microbiological analyses. In this study we compared the identification and susceptibility profiles of saponin-treated and untreated (standard method) blood cultures monomicrobial for Gram-positive cocci using Vitek 2. We concordantly identified 49 (89%) of 55 monobacterial cultures using the results with the standard method as reference. Complete categorical agreement between the susceptibility profiles with the new and the standard method was found for 26 (53%) of 49 isolates, while discrepancies were seen for 23 (47%) cultures. E-tests indicated that the new method resulted in a correct susceptibility profile for 8 (35%) of these 23 blood cultures. Therefore, 34 (69%) of 49 cultures showed a concordant/correct susceptibility profile for all antimicrobials with an overall error rate of 2.3%. Thus, addition of saponin to the fluid from blood culture bottles of the Bactec 9240 leads to the rapid (results available >or=12 hours earlier) and reliable identification and susceptibility profiling of Gram-positive cocci in blood cultures with Vitek 2.

Rapid identification and antimicrobial susceptibility testing of Gram-positive cocci in blood cultures by direct inoculation into the BD Phoenix system.

Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections are essential for the selection of appropriate antimicrobial therapy. To speed up the identification and AST of the causative agent, the fluid from blood culture bottles of a Bactec 9240 instrument (Becton Dickinson) containing Gram-positive cocci was mixed with saponin. After a 15-min incubation, the bacteria were harvested and transferred to the appropriate panel of a BD Phoenix automated microbiology system (Becton Dickinson) for identification and AST. With this approach (referred to as the direct method), we concordantly/correctly identified 56 (82%) of 68 monomicrobial cultures using the results obtained with the method currently used in our laboratory (current method) as comparator. Two (3%) isolates could not be identified and ten (15%) were misidentified. Complete agreement, concerning clinical susceptibility categories and MIC values, between the AST results determined with the direct method and the current method was found for 32 (55%) of 58 isolates. The E-test indicated that the direct method yielded a correct susceptibility profile for 13 of the remaining 26 blood culture isolates. Therefore, a concordant/correct susceptibility profile (with all antimicrobial agents tested) was obtained for 45 (77%) of 58 cultures. The overall error rate amounted to 1.9%, with the majority (1.3%) of errors being minor. Importantly, the results obtained with the direct method were available 12-24h earlier than those obtained with the current method.

Efficacy of Chromogenic Candida Agar for isolation and presumptive identification of pathogenic yeast species.

Chromogenic Candida Agar is a novel differential culture medium that is claimed to facilitate isolation and identification of Candida albicans, Candida tropicalis and Candida krusei. The performance of this medium was evaluated for presumptive identification of 521 yeast strains, representing 23 different species, for detection of specimens containing yeast mixtures, and for direct isolation of yeast from blood cultures. All yeasts grew well on the medium following a 48-h incubation period at 37 degrees C, and distinctive colonies were produced by C. albicans, C. tropicalis, C. krusei, Candida guilliermondii, Saccharomyces cerevisiae, Trichosporon mucoides and Geotrichum capitatum. The sensitivity and specificity of the medium exceeded 99.4% for each of these species. The medium provided some indication of the presence of Candida dubliniensis and Candida pulcherrima, and allowed the identification of polyfungal samples in 89.4% of the yeast mixtures. Finally, direct isolation on the medium from blood cultures that were positive for yeast according to Gram's stain (n = 42) showed that the expected colour and morphology of each species were not altered in the presence of blood.

Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa.

AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.

Evaluation of a novel tuberculosis complex-specific 34 kDa protein in the serological diagnosis of tuberculosis.

Tuberculosis (TB) serological testing with antigen complexes, although very sensitive, is not always as specific due to reactive serum antibodies in patients with inactive TB or nontuberculous infections. Since the use of recombinant M. tuberculosis proteins may enhance specificity, this study was designed to evaluate a novel 34 kDa tuberculosis complex-specific protein as a component of an antigen panel of recombinant proteins. Seventy patients with active TB (41 positive and 29 negative for acid-fast bacilli (AFB) in sputum) were evaluated, in comparison with 30 tuberculin purified protein derivative skin test positive (PPD+) and 30 PPD- normals, 20 subjects with inactive TB and 20 PPD+ subjects with nontuberculous pneumonia as controls. Serum antibody levels were quantified using enzyme linked immunosorbent assay (ELISA) tests with MS2-34, a fusion protein comprising the NH2-terminal 16 kDa of the 34 kDa protein, a recombinant 38 kDa protein (p38), and PPD. Using MS2-34 and p38 as an antigen panel in active TB patients yielded higher sensitivity and negative predictive value (sensitivity 86%; negative predictive value 91%) than using PPD (sensitivity 66%; negative predictive value 81%). Importantly, the MS2-34+p38 panel yielded a higher sensitivity (83%) than PPD (66%) in the subset of AFB- active TB patients. Thus, this novel protein increases sensitivity and specificity of serological testing for TB when used in panels of recombinant proteins.

Molecular diagnosis of tuberculosis.

Rapid and sensitive tools for the diagnosis of tuberculosis are needed, due to the increased incidence of tuberculosis epidemics and the length of time required by classical diagnostic tests, especially among human immunodeficiency virus (HIV)-infected patients. In this context, the recent advances in cloning and characterization of M. tuberculosis genes has allowed the application of basic molecular biology techniques to the examination of clinical samples, such as sputum and bronchoalveolar lavage (BAL), for the molecular diagnosis of tuberculous infection. By using the polymerase chain reaction (PCR) for the amplification of mycobacterial nucleic acids and nonradiometric revelation techniques, the time required for the identification of mycobacteria has been considerably shortened (24-48 h), in comparison to the time required by microbiological tests. When PCR technique is performed by experienced laboratory personnel using controlled protocols, false-negative (caused primarily by endogenous polymerase inhibitors) and false-positive results (due to contamination) can generally be avoided, achieving sensitivity and specificity close to 100%. In the clinical practice, the use of molecular testing for the diagnosis of tuberculosis, in combination with "classic" diagnostic tools, can greatly enhance the diagnostic ability of pulmonary clinicians, particularly in paucibacillary infections and in patients with atypical presentation, such as immunodeficient individuals.

Transformation of mycobacterial species using hygromycin resistance as selectable marker.

Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials (Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [10(3)-10(5) transformants (micrograms DNA)-1] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG. The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed.

First characterization in Italy of clinical isolates of mutans streptococci by using specific monoclonal antibodies.

The aim of this investigation was to gain further insight into the prevalence of different serotypes of mutans streptococci in the Italian population by using specific monoclonal antibodies in an enzyme immunoassay. Isolates from dental plaque samples, collected from an adult population living in Pisa (Italy), were identified as mutans streptococci on the basis of their morphological and biochemical properties, and were then serotyped. The results show that 77.5% of the strains isolated belonged to serotype c or f (i.e., S. mutans), 15.9% were serotype e (i.e., S. mutans) and only two strains (1.4%) belonged to serotype g (i.e., S. sobrinus). These data are partially in agreement with other studies in Europe and in the U.S.A. The distribution pattern of the various serotypes turned out to be substantially similar among the different groups of patients, subdivided on the basis of their caries status, indicating that none of the serotypes was particularly associated with dental caries.

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

The activity of three enzymes involved in the salvage pathway of purine nucleosides--purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)--was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.

Epidemiological survey of Streptococcus mutans in a group of adult patients living in Pisa (Italy).

An epidemiological investigation was carried out to evaluate the prevalence of Streptococcus mutans in a group of 134 adult patients. Markedly higher frequency of isolation was observed in caries-active subjects than in caries-inactive or caries-free subjects, indicating a significant association between the prevalence of the microorganism and the caries status. Moreover, the presence of the microorganism appeared to have a significant association with the extent of caries experience evaluated by the DMF score. These findings are in agreement with those reported previously for school children in other areas of Italy. Isolation of S. mutans was compared among patients groups with different caries activity in relation to culture times of dental plaque samples in a transport medium (Colorimetric Broth Medium). S. mutans was most frequently isolated from caries-active subjects when the medium was incubated for 48 h after inoculation with dental plaque samples.

Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues.

Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate--200 mM 2-mercaptoethanol--0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process.

Structural and stereospecific requirements for the nucleoside-triggered germination of Bacillus cereus spores.

A selection of adenosine analogues was tested for their ability to trigger germination of Bacillus cereus NCIB 8122 spores. The germination-inducing activity was governed by the structural properties of the sugar rather than the base moieties of the nucleosides. Among the sugar-modified analogues, only those containing a 2'-deoxy-D-ribose moiety promoted spore germination. Requirements for a specific molecular structure of the base were not clearly identified, although the highest activity was observed when substituents were inserted at position 6 of the purine ring. All the base-modified analogues, even those such as coformycin and 2'-deoxycoformycin with an expanded base ring, retained the germination-inducing activity of adenosine. However, of the two 2'-deoxycoformycin diastereoisomers characterized by an asymmetric carbon atom at position 8 of the homopurine ring, only the 8S-isomer induced germination, thus indicating that stereospecific configuration of the inducer, at least in the case of 2'-deoxycoformycin, appears to be essential for the initiation of spore germination. The differences in the germination-inducing activity of the various analogues tested were not affected significantly by spore activation at different temperatures, although the higher the activation temperature, the lower was the concentration of each analogue required for maximum germination.