Ettore Amato

Methicillin-Resistant Staphylococcus aureus in Raw Milk: Prevalence, SCCmec Typing, Enterotoxin Characterization, and Antimicrobial Resistance Patterns.

Staphylococcus aureus is a known major cause of foodborne illnesses, and raw milk and dairy products are often contaminated by enterotoxigenic and antimicrobial-resistant S. aureus strains. In the present study, 35 S. aureus strains were isolated from 383 raw milk samples collected from various dairy herds in the province of Milan (northern Italy). The isolates were characterized based on their antimicrobial susceptibility patterns and the presence of genes encoding staphylococcal enterotoxins (sea, seb, sec, sed, and see). About half (45.7%) of the strains were enterotoxigenic, and 37.1% were resistant to at least one of the antimicrobial drugs tested. Seven (20%) of 35 isolates were identified as methicillin-resistant S. aureus (MRSA), and SCCmec typing performed with a multiplex PCR assay revealed the presence of gene cassettes IV and V, typical of community-acquired MRSA, and I and II, characteristic of health care-associated MRSA. The MRSA strains were evaluated for the presence of the Panton-Valentine leukocidin gene, but this gene was not found. The results of the present study revealed the presence of toxin-producing S. aureus and MRSA strains in raw milk. MRSA and enterotoxigenic S. aureus in dairy farms are an important risk factor for the spread of staphylococcal infections; therefore, further studies are needed to find strategies for monitoring and controlling the presence of S. aureus, especially MRSA, in dairy products.

A Multischool Outbreak Due to Salmonella enterica serovar Napoli Associated with Elevated Rates of Hospitalizations and Bacteremia, Milan, Italy, 2014

A multischool outbreak of salmonellosis caused by Salmonella enterica serovar Napoli was investigated in the province of Milan from October to November 2014, following an increase in school absenteeism coinciding with two positive cases. Epidemiological studies detected 47 cases in four primary schools: 46 children and 1 adult woman (51.4% males and 48.6% females, median age 8.9). From these, 14 cases (29.8%) were severe and resulted in hospitalization, including 6 children (12.8%) who developed an invasive salmonellosis. The epidemic curve revealed an abnormally long incubation period, peaking 1 week after the first confirmed case. Twenty-five available isolates were typed by pulsed-field gel electrophoresis showing an identical pattern. The isolate belongs to ST474, an ST composed exclusively of Salmonella Napoli human strains isolated in France and Italy. Antibiotic resistance analysis showed resistance to aminoglycosides, correlating with the presence of the aminoglycoside resistance gene aadA25 in its genome. Trace-back investigations strongly suggested contaminated ham as the most likely food vehicle, which was delivered by a common food center on 21 October. Nevertheless, this ingredient could not be retrospectively investigated since it was no longer available at the repository. This represents the largest Salmonella Napoli outbreak ever reported in Italy and provides a unique scenario for studying the outcome of salmonellosis caused by this emerging and potentially invasive nontyphoidal serotype.

Diversity and persistence of Listeria monocytogenes within the Gorgonzola PDO production chain and comparison with clinical isolates from the same area

Listeria monocytogenes causes invasive syndromes with high fatality rates in specific population groups. Cheeses have been commonly implicated in outbreaks worldwide. Gorgonzola is a cheese only produced in Northwestern Italy (it is the third Italian cheese in terms of production and export) and L. monocytogenes is frequently isolated from the production chain. The aims of this study were to assess the distribution of L. monocytogenes Virulence Types (VTs) in isolates collected in Gorgonzola processing plants and to determine the presence of Epidemic Clones (ECs). Fifty-Six L. monocytogenes strains collected between 2004 and 2016 from cheese and environmental samples were subtyped with Multi-Virulence-Locus Sequence Typing (MVLST) and compared to previously typed strains. Most isolates (n=50) belonged to two new VTs (VT113 and VT114). The remaining isolates belonged to previously identified VTs: VT14-ECVIII (milk chocolate outbreak, 1994, USA) and VT80 (ricotta salata outbreak, 2012, USA). VT14, VT80 and VT113 were shared with isolates from apparently sporadic human cases in the same geographical area and temporal period (Piedmont and Lombardy, 2005-2016). The overall L. monocytogenes population appears to be homogeneous and may be characteristic of Gorgonzola production. Nevertheless, the detection in cheese and environmental samples of VTs observed in clinical isolates or outbreak related strains (VT80, VT14) contributed to better describe the current scenario and pointed out the need for increased surveillance.

Epidemiology and Molecular Typing of Pregnancy-Associated Listeriosis Cases in Lombardy, Italy, over a 10-Year Period (2005–2014)

In developed countries, pregnancy-related listeriosis accounts for 20–43% of total invasive listeriosis. This work describes the first pregnancy-related listeriosis survey in Italy based on two data sources, that is, mandatory notification system and regional laboratory-based network. Out of 610 listeriosis cases reported over a 10-year period, 40 were pregnancy-related (6.6%). Among these, 29 pregnancy-related isolates were available and have been analysed with serotyping, Pulsed-Field Gel Electrophoresis, and Multi-Virulence-Locus Sequence Typing. No maternal fatality was recorded, but 11 (29.7%) pregnancies resulted in a foetal death, a miscarriage, or a birth of a foetus dying immediately after birth. The average incidence of pregnancy-related listeriosis was 4.3 cases per 100000 births, and the proportion of pregnancy-associated listeriosis among ethnic minorities was significantly higher compared to the general population (30.0% versus 3.5%, P < 0.01). L. monocytogenes isolates belonged to serotypes 1/2a, 1/2b, and 4b, with the latter significantly more prevalent among pregnancy-related isolates. Twenty different pulsotypes were distinguished and 16 out of the 29 isolates were classified into seven clusters. A total of 16 virulence types (VTs) were identified. Five VTs accounted for 45% of the total cases and coincided with those of previously described Epidemic Clones (ECs) of L. monocytogenes.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Napoli Strain SN310, Cause of a Multischool Outbreak in Milan, Italy, in 2014

ABSTRACT We report the draft genome sequence of Salmonella enterica subsp. enterica serovar Napoli strain SN310, isolated from a stool sample of an affected pupil during a multischool outbreak in 2014 in Milan, Italy. This represents the first reported draft genome sequence of the emerging serovar Napoli.

Antimicrobial susceptibility of Listeria monocytogenes isolates from human cases in northern Italy, 2008–2010: MIC determination according to EUCAST broth microdilution method

Abstract Susceptibility of 96 Listeria monocytogenes human isolates collected in northern Italy between 2008 and 2010, to 15 antimicrobials, was investigated. Minimum inhibitory concentration (MIC) was evaluated by means of the standardized broth microdilution method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) international guidelines. All L. monocytogenes human isolates were susceptible to penicillin G (MIC90≤0·06 μg/ml), meropenem (MIC90≤0·06 μg/ml), and erythromycin (MIC90 = 0·12 μg/ml). Susceptibility to the other tested antimicrobials could not be interpreted due to the lack of breakpoint values although two (2%) isolates were shown to have tetracycline MICs above EUCAST epidemiological cut-off values (ECOFF). Bactericidal activity for amoxicillin, gentamicin, and levofloxacin was generally observed at concentrations 2–4 times higher than MIC values. Though L. monocytogenes human strains, isolated in the north of Italy, appear to be susceptible to most antimicrobial agents used in human therapy, this study provides new data for epidemiological surveillance and clinical breakpoints definition.

Draft Genome Sequences of 510 Listeria monocytogenes Strains from Food Isolates and Human Listeriosis Cases from Northern Italy

ABSTRACT Listeriosis outbreaks are frequently multistate/multicountry outbreaks, underlining the importance of molecular typing data for several diverse and well-characterized isolates. Large-scale whole-genome sequencing studies on Listeria monocytogenes isolates from non-U.S. locations have been limited. Herein, we describe the draft genome sequences of 510 L. monocytogenes isolates from northern Italy from different sources.

Letter to the editor of infection in response to de Francesco et al., a cluster of invasive listeriosis in Brescia, Italy

Poliambulanza). Specifically, as shown in Fig. 1, we found that two isolates (strains 262 and 263) collected from patients hospitalized at Spedali Civili match with two isolates (strains 251 and 272) collected from pregnancyrelated cases at Fondazione Poliambulanza. These observations lead us to reflect on two aspects, one methodological and the other relating to the epidemiological findings. Regarding the methodological aspect, the serotype 1/2a of the reported cases cannot confirm that the isolates belong to the same cluster, given the low discriminatory power of serotyping and considering that the identified serotype is the most common in Italy, especially in Lombardy [2]. Furthermore, the molecular method (REP-PCR) chosen for the analysis does not ensure the maximum discriminating power. Despite some studies showing a certain correlation between the REP-PCR and PFGE [3], the latter is still today considered the gold standard subtyping method for investigating the molecular epidemiology of foodborne pathogens [4]. Even considering that the REP-PCR is simpler and less expensive, we argue that this molecular technique could be used as a first screening test, but the interpretation of the results must be carefully done and any similarities between the isolates should be confirmed by PFGE, MLST or other more updated molecular techniques (e.g., whole-genome sequencing, WGS) [5]. Regarding the epidemiological aspect, it is well known that listeriosis is often associated with the consumption of food with a long shelf life and wide commercial distribution. Thus, a hospital laboratory may not be appropriate to bring out the correlations between cases, more easily visible from a Regional Reference Laboratory, connected to the National Reference Centre (Istituto Superiore di Sanità) and the Istituto Zooprofilattico Sperimentale, which carries out the surveillance in the food chain. To the Editor,