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This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018–2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018–2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Gardnerella vaginalis (GV) is an anaerobic bacterial species involved in the pathogenesis of bacterial vaginosis (BV), a condition of vaginal dysbiosis associated with adverse pregnancy outcomes. GV strains are categorized into four clades, characterized by a different ability to produce virulence factors, such as sialidase. We investigated the distribution of GV clades and sialidase genes in the vaginal ecosystem of a cohort of pregnant women, assessing the correlations between GV clades and the whole vaginal microbiome. A total of 61 Caucasian pregnant women were enrolled. Their vaginal swabs, collected both at the first and third trimester of pregnancy, were used for (i) evaluation of the vaginal status by Nugent score, (ii) vaginal microbiome profiling by 16S rRNA sequencing, (iii) detection and quantification of GV clades and sialidase A gene by qPCR assays. DNA of at least one GV clade was detected in most vaginal swabs, with clade 4 being the most common one. GV clade 2, together with the presence of multiple clades (>2 simultaneously), were significantly associated with a BV condition. Significantly higher GV loads and sialidase gene levels were found in BV cases, compared to the healthy status. Clade 2 was related to the major shifts in the vaginal microbial composition, with a decrease in Lactobacillus and an increase in several BV-related taxa. As the number of GV clades detected simultaneously increased, a group of BV-associated bacteria tended to increase as well, while Bifidobacterium tended to decrease. A negative correlation between sialidase gene levels and Lactobacillus, and a positive correlation with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia were observed. Our results added knowledge about the interactions of GV clades with the inhabitants of the vaginal microbiome, possibly helping to predict the severity of BV and opening new perspectives for the prevention of pregnancy-related complications.

Purpose: Intrapartum antibiotic prophylaxis (IAP) prevents group B streptococcus (GBS) early-onset disease (EOD). No European study evaluates the relative impact of risk factors (RFs) for EOD after...

During pregnancy, the vaginal microbiome plays an important role in both maternal and neonatal health outcomes. Throughout pregnancy, the vaginal microbial composition undergoes significant changes, including a decrease in overall diversity and enrichment with Lactobacillus spp. In turn, the modifications in the microbial profiles are associated with shifts in the composition of vaginal metabolites. In this study, we characterized the vaginal metabolic profiles throughout pregnancy at two different gestational ages, correlating them with a microscopic evaluation of the vaginal bacterial composition. A total of 67 Caucasian pregnant women presenting to the Family Advisory Health Centres of Ravenna (Italy) were enrolled and a vaginal swab was collected at gestational ages 9–13 weeks (first trimester) and 20–24 weeks (second trimester). The composition of the vaginal microbiome was assessed by Nugent score and women were divided in ‘H’ (normal lactobacilli-dominated microbiota), ‘I’ (intermediate microbiota), and ‘BV’ (bacterial vaginosis) groups. Starting from the cell-free supernatants of the vaginal swabs, a metabolomic analysis was performed by means of a 1H-NMR spectroscopy. From the first to the second trimester, a greater number of women showed a normal lactobacilli-dominated microbiota, with a reduction of cases of dysbiosis. These microbial shifts were associated with profound changes in the vaginal metabolic profiles. Over the weeks, a significant reduction in the levels of BV-associated metabolites (e.g. acetate, propionate, tyramine, methylamine, putrescine) was observed. At the same time, the vaginal metabolome was characterized by higher concentrations of lactate and of several amino acids (e.g. tryptophan, threonine, isoleucine, leucine), typically found in healthy vaginal conditions. Over time, the vaginal metabolome became less diverse and more homogeneous: in the second trimester, women with BV showed metabolic profiles more similar to the healthy/intermediate groups, compared to the first trimester. Our data could help unravel the role of vaginal metabolites in the pathophysiology of pregnancy.

BackgroundMost studies regarding late-onset sepsis (LOS) address selected populations (i.e., neonates with low birth weight or extremely preterm neonates). Studying all age groups is more suitable to assess the burden of single pathogens and their clinical relevance.MethodsThis is a retrospective regional study involving paediatric departments and NICUs in Emilia-Romagna (Italy). Regional laboratory databases were searched from 2009 to 2012. Records of infants (aged 4 to 90 days) with a positive blood or cerebrospinal fluid (CSF) culture were retrospectively reviewed and analysed according to acquisition mode (whether hospital- or community-acquired).ResultsDuring the study period, there were 146,682 live births (LBs), with 296 patients experiencing 331 episodes of LOS (incidence rate: 2.3/1000 LBs). Brain lesions upon discharge from the hospital were found in 12.3% (40/296) of cases, with death occurring in 7.1% (23/296; 0.14/1000 LBs). With respect to full-term neonates, extremely preterm or extremely low birth weight neonates had very high risk of LOS and related mortality (> 100- and > 800-fold higher respectively). Hospital-acquired LOS (n = 209) was significantly associated with very low birth weight, extremely preterm birth, pneumonia, mechanical ventilation, and death (p< 0.01). At multivariate logistic regression analysis, catecholamine support (OR = 3.2), central venous line before LOS (OR = 14.9), and meningitis (OR = 44.7) were associated with brain lesions or death in hospital-acquired LOS (area under the ROC curve 0.81, H-L p = 0.41). Commonly identified pathogens included coagulase-negative staphylococci (CoNS n = 71, 21.4%), Escherichia coli (n = 50, 15.1%), Staphylococcus aureus (n = 41, 12.4%) and Enterobacteriaceae (n = 41, 12.4%). Group B streptococcus was the predominant cause of meningitis (16 of 38 cases, 42%). Most pathogens were sensitive to first line antibiotics.ConclusionsThis study provides the first Italian data regarding late-onset sepsis (LOS) in all gestational age groups. Compared to full-term neonates, very high rates of LOS and mortality occurred in neonates with a lower birth weight and gestational age. Group B streptococcus was the leading cause of meningitis. Excluding CoNS, the predominant pathogens were Escherichia coli and Staphylococcus aureus. Neonates with hospital-acquired LOS had a worse outcome. Antibiotic associations, recommended for empirical treatment of hospital- or community-acquired LOS, were adequate.

Objectives The early detection of bacteraemia and fungemia is of paramount importance to guide antimicrobial therapy in septic patients. In this study the ‘time to detection' (TTD) value for the new blood culture system BacT/ALERT VIRTUO (VIRTUO) was evaluated in 1462 positive clinical bottles and compared with the TTD for 1601 positive clinical bottles incubated in the BacT/ALERT 3D system (BTA-3D). Methods The most representative microorganisms isolated from bottles incubated in both blood culture systems were divided into eight categories (in order of frequency): coagulase-negative staphylococci (CoNS), Escherichia coli, Enterobacteriaceae (other than E. coli ), Staphylococcus aureus, Enterococcus spp , viridans group streptococci, Pseudomonas aeruginosa , and Candida spp . Results The comparison of TTD values for the two blood culture systems strongly indicated that growth of the first five groups listed above was detected earlier with VIRTUO than with BTA-3D ( p Conclusions The new VIRTUO blood culture system can reduce the TTD for more than 75% of isolated microorganisms.

Objectives: To start a surveillance program to investigate the possible diffusion of mobilized colistin resistance genes in Enterobacteriaceae strains isolated in the Unit of Microbiology of the Great Romagna Hub Laboratory. Methods: All the colistin resistant Enterobacteriaceae, isolated from August 1st 2016 to July 31st 2017, were prospectively evaluated for mcr-1 and mcr-2. Backdated survey of mcr-3, mcr-4 and mcr-5 was performed on the same group of isolates. Species identification was achieved by Vitek MS and the antibiotic susceptibility testing was performed both with Vitek-2 and Sensititre systems. Colistin resistant isolates were screened by PCR for the presence of the plasmid-mediated colistin resistance genes and amplicons were verified by sequencing. All mcr-1 positive isolates were subjected to MLST analysis. Results: Over the total of 19053 isolates belonging to Enterobacteriaceae, 90 were colistin resistant. The presence of mcr-1 was detected in 26 Escherichia coli. The overall prevalence of mcr-1 was 0.14%. The mcr-1 positive E. coli strains were assigned to 13 distinct sequence types (STs) according to MLST. Conclusions: The prospective epidemiological survey carried out in our study gave a glimpse of the plasmid-mediated colistin resistance dissemination in Romagna. Since the prevalence rate of carbapenem resistant Enterobacteriaceae (CRE) in some hospital wards in our area is alarming, we underline the importance of a Surveillance Program to monitor the spread of the plasmid-mediated colistin resistance genes into MDR Gram-negative bacteria. Keywords: Laboratory Surveillance, Escherichia coli, mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, Antimicrobial resistance

Legionnaires' disease (LD) refers to a serious form of acute pneumonia caused by Legionella species. LD can be difficult to diagnose because the signs and symptoms are nonspecific, and therefore a rapid laboratory diagnosis is of paramount importance. In this study, a recently introduced immunochromatographic test (Immunocatch Legionella; Eiken Chemical Co., Ltd.) for Legionella pneumophila (serogroup 1) urinary antigen detection was compared with the Sofia Legionella fluorescent immunoassay (FIA) (Quidel) (routinely used in our laboratory) and with the widely used BinaxNOW Legionella assay (Alere). A total of 248 urine samples (60 frozen and 188 fresh) were evaluated. All of the samples were collected from patients with high clinical suspicion of Legionnaires' disease. The three assays were performed simultaneously according to the manufacturers' instructions. A total of 180 concordant negative and 66 concordant positive results were obtained. Only 2 discrepant results were registered. The sensitivity and specificity of Immunocatch compared with Sofia were, respectively, 98.5% and 99.4%. Cohen's kappa coefficient and overall percent agreement between Immunocatch and Sofia were also calculated and resulted in, respectively, 0.97 and 99.2%. These performances suggest that the Immunocatch test is a useful tool for Legionella pneumophila (serogroup 1) urinary antigen detection.

Objective This study aimed to assess the levels of microbial contamination in semen samples before and after the micro swim-up (MSU) procedure in intra-cytoplasmic sperm injection (ICSI). The new method is an upgrade to the classic wash swim-up procedure. Methods Semen analysis and microbiological tests were carried out before and after the MSU procedure. A total of twenty semen samples were analyzed. Results Pathogens were observed in semen samples only before MSU and never after ICSI. Microbiological tests revealed a large prevalence of gram-positive cocci [Staphylococcus spp. (n=16, 80%) and viridans streptococci (n=10, 50%)]. The results of this study indicate that direct MSU in ICSI improved the ICSI workflow. Conclusion The new workflow is faster and more affordable, and is likely to prevent infection problems that could arise from the normal microbial flora of the semen.

A new immunochromatographic test (bioNexia Legionella ; bioMérieux) for the detection of Legionella pneumophila urinary antigen was evaluated in 255 urine samples. The results were compared with those obtained by the BinaxNOW and Sofia Legionella tests. The novel test compared well with those currently in use.

Bloodstream infection (BSI) and associated sepsis represent a major source of mortality in industrialized countries. Prompt treatment with targeted antibiotics affects both the financial impact and the clinical outcome of BSI: every hour gained in initiating the correct antimicrobial therapy significantly increases the probability of patient survival. However, the current standard-of-care, which depends on blood culture-based diagnosis, are often unable to provide such a fast response. Fast and sensitive molecular techniques for the detection of sepsis-related pathogens from primary blood samples are strongly needed. The aim of this study was to assess the usefulness of the IRIDICA BAC BSI Assay, a PCR/ESI-MS-based technology for the early diagnosis of bloodstream infections from primary blood samples in critical patients. This evaluation has been performed by comparison with the traditional culture-based methods. The study was performed on a total of 300 prospective whole blood specimens obtained from patients suspected of sepsis, admitted to enrolling ER units from The Greater Romagna Area. The overall concordance between the two techniques was of 86%, with a calculated sensitivity of 76% and an assay specificity of 90%. The clinical significance of discrepant results was evaluated reviewing the patients' clinical records and the results of additional relevant microbiological tests. The data here obtained support the ability of the IRIDICA BAC BSI Assay to identify a broad range of bacteria directly from primary whole blood samples, within eight hours. This might allow a timely administration of a suitable treatment.

Staphylococcus aureus is a leading cause of community-acquired infections and healthcare-associated infections. Epidemiological data are useful for understanding the dynamics of the diffusion of this pathogen, and to plan control activities and monitor their efficacy.S. aureus isolates were collected in 13 public hospital laboratories of Emilia-Romagna (northern Italy region) during February-March 2009; phenotypic and molecular characterizations of these isolates were performed.The study sample included 267 isolates, 57 from blood, 81 from respiratory tract, and 129 from wounds; 106 (40%) were methicillin-resistant S. aureus (MRSA). MRSA showed a limited number of circulating clones with 2 predominant spa types--t008 and t041--accounting for 36% and 27% of MRSA isolates, respectively. The t041 type had a higher prevalence of antimicrobial resistance compared to other spa types and accounted for most of the retrieved hetero-vancomycin-intermediate S. aureus (h-VISA), while t008 was more frequently detected in non-hospital isolates. A higher degree of genetic diversity was observed in methicillin-susceptible S. aureus (MSSA), with no predominant clones and low prevalence of antimicrobial resistance. The occurrence of community-acquired MRSA infection appears to be rare in Emilia-Romagna.In contrast to previous studies reporting Italian data, t008 was the most frequent spa type among MRSA isolates in Emilia-Romagna. The prevalence of antimicrobial resistance of different MRSA spa types could influence their ability to cause infections with hospital onset. The presence of only 2 major MRSA clones circulating in Emilia-Romagna increases the chances that a regional strategy aimed at MRSA prevention will be effective.

To determine factors influencing intrapartum antibiotic prophylaxis (IAP) failure in the prevention of group B streptococcus (GBS) early-onset disease (EOD).GBS EOD case is defined as isolation of GBS from a normally sterile body site (e.g. blood or cerebrospinal fluid) in infants aged ≤7 days. During a consecutive 93-month period, GBS EOD cases and care data were reviewed.Seventy-nine GBS EOD cases were registered; 67 infants were born to women who received no i.v. antibiotics during labor. The 12 EOD cases exposed to IAP were more likely to be associated with emergency caesarean section (p = 0.0015), maternal obstetric risk factors (ORFs) (p = 0.0061), particularly intrapartum fever (p = 0.0002), and to present with signs of illness at birth (p = 0.0015). Correct dosages, agents, and timing were registered in three cases only; of which two were associated with intrapartum fever.ORFs, emergency caesarean section, and signs of illness at birth are significantly associated with GBS EOD in infants exposed to IAP. This study also suggests that recommended IAP agents, dosages, and timing are infrequently associated with EOD. Strict protocol adherence is recommended in all cases.

Small natural or synthetic sponges are commonly used in daily hygiene and in removing make-up. In our study we try to assess the role of sponges as reservoirs and vehicles in the transmission of potentially pathogenic bacterial species. We demonstrate that numerous Gram-negative and Gram-positive bacterial species are detectable in sponges after normal use by the healthy population. The most common bacteria to be found are Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli in both cellulose and nylon sponges; this observation supports the idea that the environmental factors (humidity, temperature) favour bacterial growth more than the different materials the sponges are made of. Sponges should be restricted to personal use, regularly washed, completely dried and kept in a dry place.

Department of Infectious Parasitic and ImmuneMediated Diseases, Istituto Superiore di Sanita, Rome, Italy (M L Ricci BiolD, S Fontana PhD, F Pinci BiolD, M Scaturro PhD); Azienda Unita Sanitaria Locale, Forli, Italy (E Fiumana MD); Azienda Unita Sanitaria Locale, Cesena, Italy (M F Pedna BiolD); Agenzia Regionale Prevenzione e Ambiente, Bologna, Italy (M A Bucci Sabattini BiolD); and “G B Morgagni-Pierantoni” Hospital, Department of Emergency Anaesthesia and Intensive Care Unit, Forli, Italy (P Farolfi MD)