Slawomir Owczarek

Molecular Characterization of Multidrug-Resistant Strains of Salmonella enterica Serotype Typhimurium and Monophasic Variant (S. 4,[5],12:i:–) Isolated from Human Infections in Italy

Salmonella enterica serovar Typhimurium (STM) represents the prevalent cause of foodborne gastroenteritis in Italy with the majority of isolates exhibiting multidrug resistance. A resistant pattern that includes ampicillin (A), streptomycin (S), sulfonamide (Su), and tetracycline (T) (ASSuT) but lacks resistance to chloramphenicol (C) has recently emerged in Italy among strains of STM and of its monophasic variant, S. enterica subspecies enterica serovar S. 4,[5],12:i:-. With the aim to evaluate their clonal relationships, 553 strains of STM and S. 4,[5],12:i:- with the ASSuT and ACSSuT resistance patterns isolated in Italy from human infections between 2003 and 2006 were characterized by pulsed-field gel electrophoresis (PFGE) according to the PulseNet-Europe protocol and nomenclature. Among both the STM and S. 4,[5],12:i:- ASSuT strains, the predominant PFGE profile was STYMXB.0079 (53.2-73.0% of strains, respectively), while the STM ACSSuT strains belonged to the STYMXB.0061 (37.2% of strains) and STYMXB.0067 (29.9% of strains). Bionumerics cluster analysis of the nonunique PFGE profiles showed that more than 90% of ASSuT and ACSSuT-resistant strains were included in two distinct clusters with a genetic homology of 73% each other, suggesting that the ASSuT-resistant strains belong to a same clonal lineage different from that of the ACSSuT strains. Phage typing showed that 23% of the ASSuT STM strains were not typeable and 22.3% were U302. The same phage types were observed among the ASSuT strains of S. 4,[5],12:i:-. A different figure was observed for the ACSSuT strains: the STM isolates mostly belonged to DT104 (70.2%), while none of the S. 4,[5],12:i:- strains belonged to this phage type. This study indicates that the tetra-resistant ASSuT strains of STM and S. 4,[5],12:i:-, increasingly isolated in Italy, belong to a same clonal lineage and that the S. 4,[5],12:i:- strains circulating in our country mainly derive from this STM clonal lineage.

Nucleotide sequence of the chromosomal region conferring multidrug resistance (R-type ASSuT) in Salmonella Typhimurium and monophasic Salmonella Typhimurium strains

OBJECTIVES The aim of this study was to sequence the chromosomal region conferring resistance to ampicillin, streptomycin, sulphonamides and tetracycline (R-type ASSuT) in a Salmonella Typhimurium (STM) monophasic strain (4,[5],12:i:-) belonging to the PFGE profile STYMXB.0079. The presence of this resistance region and the analysis of its genetic environment was investigated in a selection of strains. METHODS A Sau3A1 genomic library was used to determine the nucleotide sequence of the genomic resistance region. PCRs were performed on 10 epidemiologically unrelated Salmonella strains, both STM and monophasic STM, with R-type ASSuT and PFGE profile STYMXB.0079, in order to investigate the presence of the resistance genes, the left and right junctions and the internal regions of the resistance region, as well as the genetic environment. RESULTS The genomic resistance region consisted of two regions, resistance region 1 (RR1), conferring resistance to ampicillin, streptomycin and sulphonamides, and resistance region 2 (RR2), conferring tetracycline resistance. These resistance regions were both surrounded by IS26 elements and sequence comparative analysis showed 99% sequence identity with a region of plasmid pO111_1 from an Escherichia coli strain. All 10 strains were positive for the four resistance genes, the left and right junctions and the internal regions of RR1 and RR2. Concerning the genetic environment, all the strains lacked the STM1053-1997 and STM2694 genes, while only monophasic STM strains showed deletion of the fljA-fljB operon. CONCLUSIONS This study describes two resistance regions localized on the bacterial chromosome of a clonal lineage of STM and monophasic STM that are widespread in Italy.

Molecular characterisation of multidrug-resistant Salmonella enterica serotype Infantis from humans, animals and the environment in Italy.

During 2005-2006, Salmonella enterica serotype Infantis strains isolated from human and non-human sources and resistant to ampicillin (A), chloramphenicol (C), streptomycin (S), sulphonamide (Su), tetracycline (T), kanamycin (K) and trimethoprim/sulfamethoxazole (Sxt) emerged in Italy. The aim of this study was to analyse the molecular basis of antibiotic resistance and to evaluate the clonal origin of multiresistant S. Infantis strains isolated from different sources. Seventy S. Infantis strains, susceptible or resistant to antimicrobial drugs, were chosen for this study. Polymerase chain reaction (PCR) and conjugation experiments were performed to identify and localise the resistance genes in multidrug-resistant strains. PCR-based replicon typing was carried out for characterisation of conjugative plasmids. All strains were tested by pulsed-field gel electrophoresis (PFGE) according to the PulseNet protocol, and cluster analysis was performed using BioNumerics software. Strains with resistance (R)-type ACSSuTKSxt harboured bla(TEM-1), strA-B, sul2, tet(B), catA1 and aphA-1 resistance genes as well as a 2.2-kb class 1 integron containing folA, catB3, aadA4 and sul1 gene cassettes. A unique plasmid, belonging to the HI1 incompatibility group, harboured all the resistance genes. Cluster analysis showed that all ACSSuTKSxt-resistant strains belonged to a large cluster (A) with >90% genetic similarity. The presence of a plasmid harbouring all the resistance gene cassettes as well as molecular typing by PFGE demonstrated the circulation of a cluster of S. Infantis R-type ACSSuTKSxt during 2005-2006 in Italy. The presence of a plasmid conferring multidrug resistance could have facilitated the spread of a group of similar isolates through a variety of sources.

Characterization of the Plasmid-Borne Quinolone Resistance Gene qnrB19 in Salmonella enterica Serovar Typhimurium

ABSTRACT A qnrB19 gene variant, carried by an IncL/M-like plasmid, was detected in a multidrug Salmonella enterica serovar Typhimurium human strain with reduced susceptibility to ciprofloxacin. The genetic environment around the gene was fully sequenced (20 kb). A large gene cluster, containing the aph, qnrB19, and blaSHV-12-like resistance genes, is inserted inside a Tn3 transposon.

Distribution of Salmonella enterica isolates from human cases in Italy, 1980 to 2011

We describe trends of Salmonella enterica serovars isolated from humans in Italy from January 1980 to December 2011. A total of 229,279 Salmonella isolates were reported during this period. Serovars Enteritidis, Typhimurium, Infantis, Derby, 4,[5],12,:i:-, and Napoli accounted for 135,783 (59%) of these isolates. Temporal trends from 2000 to 2011 varied by serovar: Enteritidis and Infantis decreased significantly (with a mean of -3.0% and -2.8% isolates per year, respectively, p<0.001); Typhimurium remained stable; while 4,[5],12:i:-, Derby and Napoli increased significantly (+66.4%, p<0.001; +8.1%, p<0.001; and +28.2%, p<0.05, respectively). Since 2000, Enteritidis fell consistently below Typhimurium, which is the most reported serovar in Italy in contrast to the international situation where Enteritidis still ranks at the top despite its significant decrease. Most serovars showed a marked seasonality, increasing over the summer months and peaking in August/September. Typhimurium, 4,[5],12:i:-, and Napoli were most likely to be isolated from children, whereas Enteritidis, Derby, and Infantis from adults. We conclude that the applied control measures are not equally efficient against the considered Salmonella serovars and that sources of infection other than those of Enteritidis (laying hens and eggs) have become increasingly important. Further investigations on the emerging serovars and on the causes related to their emergence are needed to define and implement newly tailored control measures.

Emergence of Ciprofloxacin-Resistant Salmonella enterica Serovar Typhi in Italy.

In developed countries, typhoid fever is often associated with persons who travel to endemic areas or immigrate from them. Typhoid fever is a systemic infection caused by Salmonella enterica serovar Typhi. Because of the emergence of antimicrobial resistance to standard first-line drugs, fluoroquinolones are the drugs of choice. Resistance to ciprofloxacin by this Salmonella serovar represents an emerging public health issue. Two S. enterica ser. Typhi strains resistant to ciprofloxacin (CIP) were reported to the Italian surveillance system for foodborne and waterborne diseases (EnterNet-Italia) in 2013. The strains were isolated from two Italian tourists upon their arrival from India. A retrospective analysis of 17 other S. enterica ser. Typhi strains isolated in Italy during 2011–2013 was performed to determine their resistance to CIP. For this purpose, we assayed for susceptibility to antimicrobial agents and conducted PCR and nucleotide sequence analyses. Moreover, all strains were typed using pulsed-field gel electrophoresis to evaluate possible clonal relationships. Sixty-eight percent of the S. enterica ser. Typhi strains were resistant to CIP (MICs, 0.125–16 mg/L), and all isolates were negative for determinants of plasmid-mediated quinolone resistance. Analysis of sequences encoding DNA gyrase and topoisomerase IV subunits revealed mutations in gyrA, gyrB, and parC. Thirteen different clonal groups were detected, and the two CIP-resistant strains isolated from the individuals who visited India exhibited the same PFGE pattern. Because of these findings, the emergence of CIP-resistant S. enterica ser. Typhi isolates in Italy deserves attention, and monitoring antibiotic susceptibility is important for efficiently managing cases of typhoid fever.

Salmonella enterica Serovar Napoli Infection in Italy from 2000 to 2013: Spatial and Spatio-Temporal Analysis of Cases Distribution and the Effect of Human and Animal Density on the Risk of Infection

Background Salmonella Napoli is uncommon in Europe. In Italy however, it has been growing in importance since 2000. To date, no risk factors have been identified to account for its rise. This study aims at describing the epidemiology, spatial and spatio-temporal patterns of S. Napoli in Italy from 2000 to 2013, and to explore the role of several environmental correlates, namely urbanization, altitude and number of livestock farms, on the risk of S. Napoli infection among humans. Method Data were obtained from Enter-Net Italy, a network of diagnostic laboratories. The data were aggregated at the municipality level. Descriptive epidemiology, multivariate regression models, spatial and spatio-temporal analyses were performed on the number of cases and incidence rates. Results S. Napoli showed an expanding trend at the national level, and an increasing number of cases. Compared to the other main serovars in Italy, the risk of S. Napoli infection was higher in the age group <1 year, and lower in the other age groups. Although urbanization and the number of farms were associated with the risk of S. Napoli infection to some extent, their role in the epidemiology of the disease remains inconclusive. S. Napoli cases showed a positive global spatial autocorrelation as well as a significant spatio-temporal interaction. Twenty-four spatial and spatio-temporal clusters were identified, seven purely spatial and 17 spatio-temporal, mainly in north-western Italy. Most of the clusters were in areas characterized by urban and industrial settlements surrounded by agricultural land and an abundance of freshwater bodies. Conclusions Our results point to the presence, in a number of areas in Italy, of a Salmonella of public health concern originating in the environment. This highlights the increasing relevance of environmental, non-food-related sources of human exposure to enteric pathogens.

First isolation of Salmonella enterica serovar Napoli from wild birds in Italy

Salmonella enterica serovar Napoli (S. Napoli) is an emerging serovar in Italy. It accounts for 2-4% of all serovars isolated from human infections. The zoonotic origin of this serovar is still unknown and this makes difficult to apply any control intervention. We report here the isolation of S. Napoli from a river nightingale (Cettia cetti, Temminck 1820) which represents the first description of this serovar from wild birds. This finding adds knowledge to the ecology of S. Napoli and addresses further studies aimed to assess the epidemiologic link between S. Napoli isolated from wild birds, food, environmental sources and human infections.

Emergence of quinolone-resistant Shigella flexneri in Italy (March 2017)

In March 2017, a 45-year-old Italian man who has sex with men was admitted to the Infectious Diseases Department of Trieste Hospital (northeast Italy), because of fever, abdominal pain and dysentery. The patient had neither foreign travel history nor sexual contact with non-Italian partners. Stool cultures grew multidrug-resistant Shigella flexneri (resistant to ampicillin, chloramphenicol, streptomycin, tetracycline, trimethoprim, amoxicillin/clavulanic acid and ciprofloxacin) and whole genome sequencing detailed the resistance features. The phylogenetic analysis showed that the strain was unrelated to any previously reported strain. The patient was treated successfully with ceftriaxone. We hereby report the first case of locally-acquired, multidrug-resistant S. flexneri infection in Italy and also the emergence of a new clone.