Caterina Graziani

Escherichia coli of human and avian origin: detection of clonal groups associated with fluoroquinolone and multidrug resistance in Italy

OBJECTIVES Poultry have been suggested as a reservoir for fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli (ExPEC). Our aim was to investigate whether genotypes associated with ciprofloxacin and multidrug resistance were shared among human and avian E. coli. METHODS We compared 277 human ExPEC isolates from urinary tract infection (UTI) and sepsis (142 susceptible and 135 ciprofloxacin resistant) and 101 avian isolates (68 susceptible and 33 ciprofloxacin resistant) by antimicrobial resistance phenotype, phylogenetic group and multilocus sequence type (ST). RESULTS Most ciprofloxacin-resistant isolates from both human and avian sources were multidrug resistant. Human and avian isolates strongly differed in phylogenetic group assignment (B2 and A predominated among human and avian isolates, respectively), but a shift towards group A associated with ciprofloxacin resistance was observed among human isolates (8/100, 8.0% versus 17/87, 19.5%, P =0.021 for UTI and 5/42, 11.9% versus 15/48, 31.3%, P = 0.028 for sepsis). Heterogeneity of ST clones was observed, with ST131 strongly predominant in human ciprofloxacin-resistant strains (58/135, 43.0%), but not in avian strains. However, two major ST clonal complexes (CCs; CC10 and CC23, both belonging to group A) associated with ciprofloxacin resistance and multiresistance were shared by human and avian isolates. CONCLUSIONS The major human and avian E. coli ST clones associated with multidrug resistance were identified. A subset of ST clones belonging to CC10 and CC23 poses a potential zoonotic risk.

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.

Evidence for a Second Genomic Island Conferring Multidrug Resistance in a Clonal Group of Strains of Salmonella enterica Serovar Typhimurium and its Monophasic Variant Circulating in Italy, Denmark, and the United Kingdom

ABSTRACT During the 2000s, a new clonal group with resistances to ampicillin, streptomycin, sulfonamides, and tetracycline (ASSuT) emerged in Italy among strains of Salmonella enterica serovar Typhimurium and its monophasic variant, Salmonella enterica subspecies enterica serovar 4,[5],12:i:−. The PulseNet Europe database allowed us to identify ASSuT strains of both S. Typhimurium and its monophasic variant, isolated in Denmark and the United Kingdom, with the same or very closely related pulsed-field gel electrophoresis (PFGE) patterns as the Italian strains, suggesting that the ASSuT clonal group is circulating in different European countries. With the aim of analyzing the molecular basis of antibiotic resistance, resistance genes were identified and their localization was investigated in 66 ASSuT strains and, as controls, in 11 strains with different resistance patterns and PFGE profiles, belonging both to S. Typhimurium and to its monophasic variant, isolated from humans in Italy, Denmark, and the United Kingdom. All the ASSuT strains were positive for the following resistance genes: blaTEM-1, strA-strB, sul2, and tet(B). A localization experiment demonstrated that the ASSuT resistance genes are chromosomally located. This study confirms that a multidrug-resistant clonal group, ASSuT, of S. Typhimurium and its monophasic variant has emerged and is circulating in Italy, Denmark, and the United Kingdom. Moreover, the results of this work demonstrate that the multidrug resistance in this clonal group of Salmonella strains is conferred by a new genomic island.

First Report of Plasmid-Mediated Quinolone Resistance Determinant qnrS1 in an Escherichia coli Strain of Animal Origin in Italy

ABSTRACT A qnrS1-positive strain of Escherichia coli was detected among 73 poultry isolates showing ciprofloxacin MICs of ≥0.125 μg/ml. The qnrS1 gene was associated with a Tn3-like transposon, as previously described to occur in a Salmonella enterica serovar Infantis strain of animal origin, but the plasmid scaffold carrying this element resembled that of a plasmid previously identified in Salmonella enterica serovar Dublin. These elements suggest genetic exchanges among Salmonella and E. coli and a potential animal reservoir for the qnr genes.

IncI1 plasmids associated with the spread of CMY‐2, CTX‐M‐1 and SHV‐12 in Escherichia coli of animal and human origin

Fourteen plasmids carrying blaCTX -M-1, blaSHV -12 or blaCMY -2 genes from Escherichia coli of both avian and human origin were analysed. IncI1 plasmids were largely predominant. Plasmid mutilocus sequence typing and comparative analysis revealed that the blaCMY -2 -ST12-IncI1 plasmids from avian E. coli were identical to those previously found in Salmonella from humans, but different to those associated with human E. coli. The IncI1-ST3 plasmids carrying blaCTX -M-1 or blaSHV -12 were related to those previously identified in avian E. coli, but different to those identified in human E. coli. Overall, no plasmids shared by E. coli of both origin (human/avian) were identified; however, further investigations are needed.

Virulotyping of Salmonella enterica Serovar Napoli Strains Isolated in Italy from Human and Nonhuman Sources

Salmonella enterica serovar Napoli is an emerging serovar in Italy, France, and Switzerland, but little is known about its pathogenicity to humans. A collection of 112 strains of Salmonella Napoli isolated in Italy from human cases, foods of animal origin, and the environment have been characterized by the detection of a set of virulence genes, pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility. All the strains examined were susceptible to all the antimicrobials tested. The Salmonella pathogenicity islands genes ssaQ, mgtC spi_4D, and sopB were present from 75.0% to 100% of the tested strains. Only one human and four environmental strains showed the avrA gene. The phage-related sopE1 gene was present in 93% of the strains, whereas sodC1 and gipA genes were only in four and two environmental strains, respectively. The bcfC fimbrial gene was present in all the animal/food strains, in the 71.4% of environmental strains, and in 46.8% of the human strains, respectively. Overall, we observed 10 distinct virulence profiles (VP), but VP1-2-3 included 99 out of 112 strains. PFGE showed that 103 out of 111 isolates were grouped in four major clusters and three minor clusters, whereas two strains were totally unrelated. The most represented PFGE clusters mainly correlated with the virulotypes are VP1, VP2, and VP3. Salmonella Napoli shows an array of virulence genes similar to other serovars considered of public health importance and confirming its capability to cause infection in human. Concerning possible source of infection or reservoirs, the results did not point out any, but our hypothesis is that the environment can act as the main reservoir for Salmonella Napoli, and from there it can spill over to animals and humans. Further studies are needed to increase the knowledge on the ecology of Salmonella Napoli serovar and on the main risk factors for human infection.

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.

Phylogenetic Background and Virulence Genotype of Ciprofloxacin-Susceptible and Ciprofloxacin-Resistant Escherichia coli Strains of Human and Avian Origin

BACKGROUND Previous studies have suggested that fluoroquinolone-resistant strains of Escherichia coli that infect humans probably emerged as a consequence of using fluoroquinolones in poultry. This study aims to provide further insight into the possible avian origin of fluoroquinolone-resistant extraintestinal pathogenic E. coli (ExPEC) strains that infect humans. METHODS We compared the phylogenetic backgrounds, virulence gene profiles, and genetic relatedness of 125 ExPEC strains recovered from humans (61 were ciprofloxacin susceptible and 64 were ciprofloxacin resistant) and 113 E. coli strains recovered from poultry (47 were ciprofloxacin susceptible and 66 were ciprofloxacin resistant). RESULTS Ciprofloxacin-resistant strains of both human and avian origin harbored fewer virulence genes than did ciprofloxacin-susceptible strains, but ciprofloxacin-resistant strains from humans were found to be clearly distinct from ciprofloxacin-resistant avian strains, based on their phylogenetic backgrounds and virulence gene profiles. The phylogenetic background of ciprofloxacin-susceptible and ciprofloxacin-resistant strains of human origin was not different, and no shift from the phylogenetic group B2 toward other groups was detected in association with ciprofloxacin resistance. No genetic relatedness was observed among human and avian strains that belonged to the major virulence profile (traT-iucD-iutA). CONCLUSIONS Our results did not support the hypothesis of an avian origin for the ciprofloxacin-resistant human ExPEC strains analyzed. Nevertheless, prudent use of fluoroquinolones in both human and veterinary medicine is recommended.

Diversity of Salmonella spp. serovars isolated from the intestines of water buffalo calves with gastroenteritis

BackgroundSalmonellosis in water buffalo (Bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. Several Salmonella serovars seem to be able to infect water buffalo, but Salmonella isolates collected from this animal species have been poorly characterized. In the present study, the prevalence of Salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a polyphasic characterization of isolated strains of S. Typhimurium was performed.ResultsThe microbiological analysis of the intestinal contents obtained from 248 water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of Salmonella spp. (25%), characterized by different serovars, most frequently Typhimurium (21%), Muenster (11%), and Give (11%). The 13 S. Typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis, such as Cryptosporidium spp., Rotavirus or Clostridium perfringens. Other Salmonella isolates were mostly isolated from minor intestinal lesions, and often (78% of cases) isolated with other microorganisms, mainly toxinogenic Escherichia coli (35%), Cryptosporidium spp. (20%) and Rotavirus (10%). The S. Typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (PCR) detection of 24 virulence genes. The isolates exhibited nine different phage types and 10 different genetic profiles. Three monophasic S. Typhimurium (B:4,12:i:-) isolates were also found and characterized, displaying three different phage types and three different virulotypes. The molecular characterization was extended to the 7 S. Muenster and 7 S. Give isolates collected, indicating the existence of different virulotypes also within these serovars. Three representative strains of S. Typhimurium were tested in vivo in a mouse model of mixed infection. The most pathogenic strain was characterized by a high number of virulence factors and the presence of the locus agfA, coding for a thin aggregative fimbria.ConclusionsThese results provide evidence that Salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly S. Typhimurium. Moreover, the variety in the number and distribution of different virulence markers among the collected S. Typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes.

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.