Fighting Antimicrobial Resistance and Healthcare-Associated Infections in EU-JAMRAI: The One-Health Response from Italy.

INTRODUCTION: Antimicrobial resistance (AMR) is a serious health threat, and it has high priority among the European public health agenda. The development and implementation of the National Action Plans (NAPs) with a One-Health perspective to fight AMR was supported in 2017 by the European Union (EU) through a Joint Action on Antimicrobial Resistance and Healthcare Associated Infections (EU-JAMRAI). The Italian National Institute of Health (Istituto Superiore di Sanità), supported by the University of Udine, and the University of Foggia were among the 44 partners involved. This paper describes the results of EU-JAMRAI relevant to Italy and its impact on national policies. METHODS: The activities involved national and international experts who worked in groups, either in virtual or face-to-face meetings. Country-to-country visits were organized to assess and compare the national strategies to counteract AMR and to exchange best practices. In addition, qualitative research methods, particularly focus groups (FGs) and structured interviews, were carried out to collect information and opinions from the experts. RESULTS: The Italian team of experts from the Ministry of Health and the University of Foggia visited the Netherlands and hosted the Polish expert team in Italy. In two FG, stakeholders' opinions from different organizations were collected and analyzed to identify critical areas and provide recommendations to ensure implementation of the NAP and effective One-Health integration. In addition, attitudes of medical professionals toward antimicrobial stewardship were evaluated in a medium/large Italian hospital. Strengths were identified in the multidisciplinary approach and the hospital management's proactive involvement. As for the veterinary sector, Italy was among the 10 EU countries that did not have any national AMR surveillance in place for animal bacterial pathogens. Consequently, a European surveillance system was proposed with the adhesion of Italy. Regarding research and innovation to fight AMR and healthcare-associated infection, Italy worked with the other European partners to identify national research gaps and opportunities. As a result, recommendations were issued to the authorities to promote research and innovation with a One-Health approach. CONCLUSIONS: The Italian participation in the EU JAMRAI provided experience and examples to the Italian government for implementing the NAP and planning the roadmap to fight AMR and helped point out the system's criticalities. It also supported the promotion of the One-Health integrated vision that was included in the updated NAP.

Group B streptococci (GBS) strains evading molecular diagnostics showed novel chromosomal deletions encompassing the CAMP-factor (cfb) encoding gene.

A highly conserved fragment adjacent to the cfb gene encoding the CAMP factor is the target of PCR-based molecular diagnostic systems for the identification of S. agalactiae (group B streptococci (GBS)). Six PCR-negative, culture-positive GBS strains were whole genome sequenced to assess why they escaped molecular diagnostics. GBS strains did not constitute a clonal cluster and presented variably sized chromosomal deletions (from 7 to 33 kb) which always included the cfb gene, a finding never described before. GBS strains that escape molecular diagnostics are considered rare; however, they can cause false-negative results using molecular diagnostics alone, affecting medical decisions.

Neonatal invasive disease caused by Streptococcus agalactiae in Europe: the DEVANI multi-center study.

PURPOSE: Group B streptococcus (GBS) remains a leading cause of invasive disease, mainly sepsis and meningitis, in infants < 3 months of age and of mortality among neonates. This study, a major component of the European DEVANI project (Design of a Vaccine Against Neonatal Infections) describes clinical and important microbiological characteristics of neonatal GBS diseases. It quantifies the rate of antenatal screening and intrapartum antibiotic prophylaxis among cases and identifies risk factors associated with an adverse outcome. METHODS: Clinical and microbiological data from 153 invasive neonatal cases (82 early-onset [EOD], 71 late-onset disease [LOD] cases) were collected in eight European countries from mid-2008 to end-2010. RESULTS: Respiratory distress was the most frequent clinical sign at onset of EOD, while meningitis is found in > 30% of LOD. The study revealed that 59% of mothers of EOD cases had not received antenatal screening, whilst GBS was detected in 48.5% of screened cases. Meningitis was associated with an adverse outcome in LOD cases, while prematurity and the presence of cardiocirculatory symptoms were associated with an adverse outcome in EOD cases. Capsular-polysaccharide type III was the most frequent in both EOD and LOD cases with regional differences in the clonal complex distribution. CONCLUSIONS: Standardizing recommendations related to neonatal GBS disease and increasing compliance might improve clinical care and the prevention of GBS EOD. But even full adherence to antenatal screening would miss a relevant number of EOD cases, thus, the most promising prophylactic approach against GBS EOD and LOD would be a vaccine for maternal immunization.

European Multicentre Tics in Children Studies (EMTICS): protocol for two cohort studies to assess risk factors for tic onset and exacerbation in children and adolescents.

Genetic predisposition, autoimmunity and environmental factors [e.g. pre- and perinatal difficulties, Group A Streptococcal (GAS) and other infections, stress-inducing events] might interact to create a neurobiological vulnerability to the development of tics and associated behaviours. However, the existing evidence for this relies primarily on small prospective or larger retrospective population-based studies, and is therefore still inconclusive. This article describes the design and methodology of the EMTICS study, a longitudinal observational European multicentre study involving 16 clinical centres, with the following objectives: (1) to investigate the association of environmental factors (GAS exposure and psychosocial stress, primarily) with the onset and course of tics and/or obsessive-compulsive symptoms through the prospective observation of at-risk individuals (ONSET cohort: 260 children aged 3-10 years who are tic-free at study entry and have a first-degree relative with a chronic tic disorder) and affected individuals (COURSE cohort: 715 youth aged 3-16 years with a tic disorder); (2) to characterise the immune response to microbial antigens and the host's immune response regulation in association with onset and exacerbations of tics; (3) to increase knowledge of the human gene pathways influencing the pathogenesis of tic disorders; and (4) to develop prediction models for the risk of onset and exacerbations of tic disorders. The EMTICS study is, to our knowledge, the largest prospective cohort assessment of the contribution of different genetic and environmental factors to the risk of developing tics in putatively predisposed individuals and to the risk of exacerbating tics in young individuals with chronic tic disorders.

Group B streptococcus late-onset disease,contaminated breast milk and mothers persistently GBS negative: report of 3cases.

BACKGROUND: Human milk is fundamental for its nutritional properties and to protect newborns, but it is not sterile and can sometime transmit bacteria. Few anecdotal cases suggest that breast milk could be a possible source of group B Streptococcus (GBS) late onset disease, although the pathogenesis is not entirely understood. CASE PRESENTATION: We report 3 cases of GBS late onset disease in full-term newborns. Fresh breast milk cultures yielded GBS, but mothers of neonates had no signs of mastitis and remained persistently GBS negative at rectovaginal site. CONCLUSIONS: Breast milk containing group B Streptococcus can be a risk factor for late onset disease. The persistent negative maternal GBS status supports the assumption that newborns, colonised in the throat, could be the initial source of GBS, while the mammary gland could act as a GBS replication site. It is unclear whether a low bacterial load may represent only contamination rather than true milk infection.

The Protective Value of Maternal Group B Streptococcus Antibodies: Quantitative and Functional Analysis of Naturally Acquired Responses to Capsular Polysaccharides and Pilus Proteins in European Maternal Sera.

BACKGROUND: Group B Streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. A vaccine targeting pregnant women could protect infants through placentally transferred antibodies. The association between GBS maternal antibody concentrations and the risk of neonatal infection has been investigated in US and African populations. Here we studied naturally acquired immunoglobulin G (IgG) responses to GBS capsular polysaccharides (CPS) and pilus proteins in European pregnant women. METHODS: Maternal sera were prospectively collected in 8 EU countries from 473 GBS non-colonized and 984 colonized pregnant women who delivered healthy neonates and from 153 mothers of infants with GBS disease. GBS strains from these colonized women and infected infants were obtained in parallel and their capsular and pilus types were identified by serological and molecular methods. Maternal serum concentrations of IgG anti- Ia, -Ib, -III and -V polysaccharides and anti-BP-1, -AP1-2a and -BP-2b pilus proteins were determined by enzyme-linked immunosorbent assay. Antibody functional activity was quantified by Opsonophagocytic Killing Assay. RESULTS: Antibody levels against CPS and pilus proteins were significantly higher in GBS colonized women delivering healthy babies than in mothers of neonates with GBS disease or non-colonized women. Moreover, maternal anti-capsular IgG concentrations showed a significant correlation with functional titers measured by Opsonophagocytic Killing Assay. CONCLUSIONS: Maternal anti-capsular IgG concentrations above 1 µg/mL mediated GBS killing in vitro and were predicted to respectively reduce by 81% (95% confidence interval, 40%-100%) and 78% (45%-100%) the risk of GBS Ia and III early-onset disease in Europe.

A fatal case of streptococcal toxic shock syndrome caused by Streptococcus suis carrying tet (40) and tet (O/W/32/O), Italy.

We report the first human fatal case of streptococcal toxic shock syndrome (STSS) caused by Streptococcus suis serotype 2 carrying the tetracycline efflux tet (40) gene and the tetracycline ribosomal protection tet (O/W/32/O) gene. The patient was splenectomized. The case was characterized by multi-organ dysfunction and disseminated intravascular coagulopathy, in accordance with the clinical parameters of STSS. More investigations are needed to improve the epidemiology and the pathogenesis of S. suis in human infection.

Matrix-assisted laser desorption ionization-time of flight and comparative genomic analysis of M-18 group a Streptococcus strains associated with an acute rheumatic fever outbreak in northeast Italy in 2012 and 2013.

Acute rheumatic fever (ARF) is a postsuppurative sequela caused by Streptococcus pyogenes infections affecting school-age children. We describe here the occurrence of an ARF outbreak that occurred in Bologna province, northeastern Italy, between November 2012 and May 2013. Molecular analysis revealed that ARF-related group A Streptococcus (GAS) strains belonged to the M-18 serotype, including subtypes emm18.29 and emm18.32. All M-18 GAS strains shared the same antigenic profile, including SpeA, SpeB, SpeC, SpeL, SpeM, and SmeZ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed that M-18 GAS strains grouped separately from other serotypes, suggesting a different S. pyogenes lineage. Single nucleotide polymorphisms and phylogenetic analysis based on whole-genome sequencing showed that emm18.29 and emm18.32 GAS strains clustered in two distinct groups, highlighting genetic variations between these subtypes. Comparative analysis revealed a similar genome architecture between emm18.29 and emm18.32 strains that differed from noninvasive emm18.0 strains. The major sources of differences between M-18 genomes were attributable to the prophage elements. Prophage regions contained several virulence factors that could have contributed to the pathogenic potential of emm18.29 and emm18.32 strains. Notably, phage ΦSPBO.1 carried erythrogenic toxin A gene (speA1) in six ARF-related M-18 GAS strains but not in emm18.0 strains. In addition, a phage-encoded hyaluronidase gene (hylP.2) presented different variants among M-18 GAS strains by showing internal deletions located in the α-helical and TSßH regions. In conclusion, our study yielded insights into the genome structure of M-18 GAS strains responsible for the ARF outbreak in Italy, thus expanding our knowledge of this serotype.

Emergence of High-Level Gentamicin Resistance in Streptococcus agalactiae Hypervirulent Serotype IV ST1010 (CC452) Strains by Acquisition of a Novel Integrative and Conjugative Element.

Streptococcus agalactiae (group B streptococci, GBS) is responsible for severe infections in both neonates and adults. Currently, empiric antimicrobial therapy for sepsis and meningitis is the combined use of penicillin and gentamicin due to the enhanced bactericidal activity. However, high-level gentamicin resistance (HLGR) abrogates the synergism. The rate of HLGR was investigated within a dataset of 433 GBS strains collected from cases of invasive disease in both adults and neonates as well as from pregnant carriers. GBS isolates (n = 20, 4.6%) presented with HLGR (gentamicin MIC breakpoint >1024 mg/L) that was differently diffused between strains from adults or neonates (5.2% vs. 2.8%). Notably, 70% of HLGR GBS strains (14 isolates) were serotype IV. Serotype IV HLGR-GBS isolates were susceptible to all antibiotics tested, exhibited the alpha-C/HvgA/PI-2b virulence string, and belonged to sequence type 1010 (clonal complex (CC) 452). The mobile element that harbored the HLGR aac(6')-aph(2)″ gene is a novel integrative and conjugative element (ICE) about 45 kb long, derived from GBS 515 ICE tRNALys. The clonal expansion of this HLGR hypervirulent serotype IV GBS CC452 sublineage may pose a threat to the management of infections caused by this strain type.

Forensic Microbiology: When, Where and How.

Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.

Serotype IV and invasive group B Streptococcus disease in neonates, Minnesota, USA, 2000-2010.

Group B Streptococcus (GBS) is a major cause of invasive disease in neonates in the United States. Surveillance of invasive GBS disease in Minnesota, USA, during 2000-2010 yielded 449 isolates from 449 infants; 257 had early-onset (EO) disease (by age 6 days) and 192 late-onset (LO) disease (180 at age 7-89 days, 12 at age 90-180 days). Isolates were characterized by capsular polysaccharide serotype and surface-protein profile; types III and Ia predominated. However, because previously uncommon serotype IV constitutes 5/31 EO isolates in 2010, twelve type IV isolates collected during 2000-2010 were studied further. By pulsed-field gel electrophoresis, they were classified into 3 profiles; by multilocus sequence typing, representative isolates included new sequence type 468. Resistance to clindamycin or erythromycin was detected in 4/5 serotype IV isolates. Emergence of serotype IV GBS in Minnesota highlights the need for serotype prevalence monitoring to detect trends that could affect prevention strategies.

Interspecies mobilization of an ermT-carrying plasmid of Streptococcus dysgalactiae subsp. equisimilis by a coresident ICE of the ICESa2603 family.

OBJECTIVES: The recently documented presence of almost identical, small, non-self-transmissible, erm(T)-carrying plasmids in clonally unrelated erythromycin-resistant isolates of Streptococcus pyogenes and Streptococcus agalactiae suggests that these plasmids somehow circulate in the streptococcal population. The objective of this study was to characterize the erm(T)-carrying genetic element in a clinical isolate of Streptococcus dysgalactiae subsp. equisimilis (Sde5580) and to provide a possible explanation for the spread of erm(T)-carrying plasmids in streptococci. METHODS: The erm(T)-carrying element of Sde5580 was investigated by plasmid analysis, PCR experiments and sequencing. Transfer and retransfer experiments were performed using S. pyogenes, S. agalactiae and Streptococcus suis strains as recipients and by selection in the presence of suitable drug concentrations. Transconjugants were analysed by SmaI-macrorestriction analysis. Genetic studies also included PCR-restriction fragment length polymorphism analysis using HindIII endonuclease. RESULTS: Sde5580 contained two mobile genetic elements: a 4950 bp erm(T)-carrying plasmid (p5580) almost identical to the non-self-transmissible erm(T)-carrying plasmids of S. pyogenes and S. agalactiae mentioned above, and an ~63 kb cadC/cadA-carrying integrative and conjugative element (ICESde3396-like) of the ICESa2603 family. p5580 was transferable at high frequency to the recipients of all three species through in trans mobilization by the coresident ICESde3396-like element. p5580 and ICESde3396-like were able to be transferred either separately or together. CONCLUSIONS: This is the first evidence of horizontal transfer of an erm(T)-carrying plasmid between streptococci. In trans mobilization by coresident ICEs may be one mechanism for the spread of erm(T)-carrying plasmids in the streptococcal population.

Group B streptococcal colonization in 160 mother-baby pairs: a prospective cohort study.

OBJECTIVES: To identify the source of postnatal colonization with group B Streptococcus (GBS) and to evaluate the impact of intrapartum antibiotic prophylaxis (IAP) administration in newborn infant transmission. STUDY DESIGN: A prospective, longitudinal study evaluated GBS colonization in 160 mother-baby pairs. Specimens were collected from the time of delivery to 8 weeks post-partum, from rectum, vagina, and milk of mothers, and from throat and rectum of neonates. Women were grouped according to their GBS status at discharge from the hospital: culture-positive carriers (n = 83), culture-negative carriers (n = 26), and noncarriers (n = 51). Newborns were considered colonized if GBS was yielded from at least 1 site. RESULTS: A total of 35 (21.9%) neonates were colonized; 30 were born to culture-positive carriers, 2 to culture-negative carriers, and 3 to noncarriers. Infants of culture-positive carriers exposed to IAP were less likely to be colonized (15/57 vs 15/26, P = .01), or heavily colonized, (7/57 vs 9/26, P = .04). Of all newborns, those exposed to IAP and discharged GBS-free from hospital, often became colonized subsequently (12/57 vs 1/26, P = .09). Molecular typing analysis (available for 30 of 32 carrier mothers and their infants) confirmed an identical strain of GBS in all mother-baby pairs. Six of 83 culture-positive carrier mothers had a positive milk culture. Their respective neonates all were heavily colonized. CONCLUSIONS: Newborns exposed to IAP and GBS-free at hospital discharge subsequently acquire GBS from their mothers. Culture-positive milk is associated with heavy neonatal colonization.

Group A streptococcal meningitis: a case report.

Streptococcus pyogenes (Group A streptococcus, GAS) is a rare cause of bacterial meningitis, accounting for less than 1% of cases. GAS meningitis has rarely been reported in children, and is associated with a high (46%) rate of morbidity and a high (10-17%) case fatality rate. This paper describes a case of meningitis caused by GAS in a previously healthy child; M protein genotyping demonstrated an emm type 12. Although not common, GAS meningitis must be considered in children vaccinated for other invasive pathogens. Continuous monitoring of the molecular epidemiology of circulating invasive GAS strains is of crucial importance for planning intervention policies.

Early Warning Systems for Emerging Profiles of Antimicrobial Resistance in Italy: A National Survey.

Antimicrobial resistance (AMR) national surveillance systems in Italy lack alert systems for timely detection of emerging profiles of AMR with potential relevance to public health. Furthermore, the existence of early warning systems (EWS) at subnational level is unclear. This study aims at mapping and characterizing EWS for microbiological threats available at regional level in Italy, focusing on emerging AMR, and at outlining potential barriers and facilitators to their development/implementation. To this end, a three-section, web-based survey was developed and administered to all Italian regional AMR representatives from June to August 2022. Twenty out of twenty-one regions and autonomous provinces (95.2%) responded to the survey. Among these, nine (45%) reported the implementation of EWS for microbiological threats at regional level, three (15%) reported that EWS are in the process of being developed, and eight (40%) reported that EWS are not currently available. EWS characteristics varied widely among the identified systems concerning both AMR profiles reported and data flow: the microorganisms most frequently included were extensively drug-resistant (XDR) Enterobacterales, with the lack of a dedicated regional IT platform reported in most cases. The results of this study depict a highly heterogeneous scenario and suggest that more efforts aimed at strengthening national AMR surveillance systems are needed.

Lumbar Puncture and Meningitis in Infants with Proven Early- or Late-Onset Sepsis: An Italian Prospective Multicenter Observational Study.

Background: To evaluate the rates of lumbar puncture (LP) in infants with culture-proven sepsis. Study design: We prospectively enrolled 400 infants with early- or late-onset sepsis due to Group B streptococcus (GBS) or Eschericha coli, diagnosed within 90 days of life. Rates of LP and potential variables associated with LP performance were evaluated. Moreover, cerebrospinal fluid (CSF) characteristics and results of the molecular analysis were investigated. Results: LP was performed in 228/400 (57.0%) infants; 123/228 LPs (53.9%) were performed after antibiotic initiation, hampering the ability to identify the pathogen in the CSF culture. However, polymerase chain reaction increased the probability of positive results of CSF analysis compared to microbiological culture (28/79, 35.4% vs. 14/79, 17.7%, p = 0.001). Severe clinical presentation and GBS infection were associated with higher LP rates. The rate of meningitis was 28.5% (65/228). Conclusions: Rates of LP are low in culture-proven neonatal sepsis and antibiotics are frequently given before LP is carried out. Thus meningitis may be underestimated, and the chances of giving an effective therapy to the newborn are reduced. LP should be performed before the start of antibiotics when there is a clinical suspicion of infection.

Timing of Symptoms of Early-Onset Sepsis after Intrapartum Antibiotic Prophylaxis: Can It Inform the Neonatal Management?

The effectiveness of "inadequate" intrapartum antibiotic prophylaxis (IAP administered < 4 h prior to delivery) in preventing early-onset sepsis (EOS) is debated. Italian prospective surveillance cohort data (2003-2022) were used to study the type and duration of IAP according to the timing of symptoms onset of group B streptococcus (GBS) and E. coli culture-confirmed EOS cases. IAP was defined "active" when the pathogen yielded in cultures was susceptible. We identified 263 EOS cases (GBS = 191; E. coli = 72). Among GBS EOS, 25% had received IAP (always active when beta-lactams were administered). Most IAP-exposed neonates with GBS were symptomatic at birth (67%) or remained asymptomatic (25%), regardless of IAP duration. Among E. coli EOS, 60% were IAP-exposed. However, IAP was active in only 8% of cases, and these newborns remained asymptomatic or presented with symptoms prior to 6 h of life. In contrast, most newborns exposed to an "inactive" IAP (52%) developed symptoms from 1 to >48 h of life. The key element to define IAP "adequate" seems the pathogen's antimicrobial susceptibility rather than its duration. Newborns exposed to an active antimicrobial (as frequently occurs with GBS infections), who remain asymptomatic in the first 6 h of life, are likely uninfected. Because E. coli isolates are often unsusceptible to beta-lactam antibiotics, IAP-exposed neonates frequently develop symptoms of EOS after birth, up to 48 h of life and beyond.

Escherichia coli Is Overtaking Group B Streptococcus in Early-Onset Neonatal Sepsis.

The widespread use of intrapartum antibiotic prophylaxis (IAP) to prevent group B streptococcus (GBS) early-onset sepsis (EOS) is changing the epidemiology of EOS. Italian prospective area-based surveillance data (from 1 January 2016 to 31 December 2020) were used, from which we identified 64 cases of culture-proven EOS (E. coli, n = 39; GBS, n = 25) among 159,898 live births (annual incidence rates of 0.24 and 0.16 per 1000, respectively). Approximately 10% of E. coli isolates were resistant to both gentamicin and ampicillin. Five neonates died; among them, four were born very pre-term (E. coli, n = 3; GBS, n = 1) and one was born full-term (E. coli, n = 1). After adjustment for gestational age, IAP-exposed neonates had ≥95% lower risk of death, as compared to IAP-unexposed neonates, both in the whole cohort (OR 0.04, 95% CI 0.00-0.70; p = 0.03) and in the E. coli EOS cohort (OR 0.05, 95% CI 0.00-0.88; p = 0.04). In multi-variable logistic regression analysis, IAP was inversely associated with severe disease (OR = 0.12, 95% CI 0.02-0.76; p = 0.03). E. coli is now the leading pathogen in neonatal EOS, and its incidence is close to that of GBS in full-term neonates. IAP reduces the risk of severe disease and death. Importantly, approximately 10% of E. coli isolates causing EOS were found to be resistant to typical first-line antibiotics.