Serotype epidemiology and multidrug resistance patterns of Salmonella enterica infecting humans in Italy

A total of 734 unique enteropathogenic isolates, obtained exclusively from human patients, were received and characterized by the Microbiology and Virology Unit of the Padua Teaching Hospital. Samples were collected between January 2011 and December 2012, according to the current version of the Declaration of Helsinki. Putative Salmonella containing samples were plated on selective xylose lysine deoxycholate agar (XLD agar, Becton–Dickinson Italia, Milan, Italy) and colonies from pure cultures were characterized using a VITEK^® 2 automated system (bioMérieux, Marcy l’Étoile, France). The isolates identified as Salmonella spp. were subjected to serotyping by slide agglutination, according to the Kaufmann-white classification [13].

Antimicrobial susceptibility testing was performed on all confirmed Salmonella isolates, by the disc diffusion (Kirby-Bauer) method according to EUCAST criteria [14]. The following antibiotics (μg/disk) were tested on Mueller–Hinton agar (Difco Laboratories, Detroit, MI), according to Enter-Net specifications: nalidixic acid (30), ampicillin (10), cefotaxime (30), ciprofloxacin (5), chloramphenicol (30), gentamicin (10), kanamycin (30), streptomycin (10), sulphonamide (300), tetracycline (30), trimethoprim (5), ampicillin-clavulanic acid (20-10), cefalotin (30) and sulfamethoxazole-trimethoprim (23.75–1.25) (BD BB™ Sensi-Disk™, Becton–Dickinson Italia, Milan, Italy). Moreover, the disk diffusion test was performed to detect Extended-spectrum beta-lactamases (ESBL) (BD BB™ Sensi-Disk™, Becton–Dickinson Italia, Milan, Italy).

Colistin susceptibility was determined using Sensititre ARIS^® 2X (Trek Diagnostic Systems, Thermo Scientific Inc., Milan, Italy). Escherichia coli ATCC25922 and E. coli ATCC35218 were used as control strains.

Plasmid DNA was obtained from pure bacterial cultures performing phenol/chloroform extraction. Resistance genes (bla_TEM, bla_SHV, bla_CTX-M, and AmpC) were detected by PCR as previously described [15, 16]. Positive PCR products were sequenced in an ABI3130xl sequencer (Applied Biosystems, Thermo Scientific Inc., Milan, Italy) and sequences were aligned to those present in GenBank using BLAST (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi).

Plasmids containing PCR-confirmed resistance determinants were introduced into chemo-competent E. coli cells (SCSI, Agilent Technologies Italia, Milan, Italy). Transformants were plated on selective Mueller–Hinton (cefotaxime 2 mg/L or ampicillin 100 mg/L) (Becton–Dickinson & Co., DifcoTM) agar plates. The presence of plasmids was confirmed by PCR targeting the whole sequence of the bla _TEM/CTX/CMY gene; PCR products were treated with exosap and fully sequenced (ABI3130xl sequencer, Applied Biosystems). Microbial identification and antimicrobial susceptibility testing on transformants were performed at a VITEK^® two automated system (bioMérieux, Marcy l’Étoile, France).

For conjugation experiments, one colony of each donor (S. enterica serovars: Rissen, monophasic S. typhimurium 1,4 [5],12:i- and Paratyphi B) and the recipient strain E. coli J53 (F− met pro Azi^r Col^r) were cultured separately under weak agitation in LB broth at 37 °C. Conjugation was performed incubating different dilutions (from 1:100 to 1:10) of donor suspensions with overnight culture of the recipient strain under low agitation. Mating assays were carried out at room temperature from 1 h to overnight depending on the donor strain. The transconjugants were selected on agar plates containing colistin (8 mg/L) and cefotaxime (2 mg/L) or ampicillin (100 mg/L). Microbial identification and antimicrobial susceptibility testing on transformants and transconjugants were performed by VITEK^® two automated system (bioMérieux, Marcy l’Étoile, France).

All plasmids were analyzed by PCR-based replicon typing (PBRT) targeting the replicons of the major plasmid families occurring in Enterobacteriaceae [17]. Each vector was assigned to a specific Inc group. Plasmid typing was also performed in the obtained transformants and transconjugants.

Bacterial total DNA was obtained by heat shock from pure culture suspension. PmrA and pmrB genes, the major regulators of LPS modifications in S. enterica, were amplified as previously described [18]. PCR amplicons were sequenced by end-primers in an ABI3130xl sequencer (Applied Biosystems, Thermo Scientific Inc., Milan, Italy) and the obtained sequences were compared to the corresponding sequences in the NCBI database (http://​www.​ncbi.​nlm.​nih.​gov/​nucleotide).

The screening of 734 enteropathogenic human specimens yielded identification of 350 S. enterica isolates. On the basis of the Kauffmann-White serotyping, three samples were positive for typhoidal Salmonella strains: two isolates were typed as Salmonella typhi, whereas one belonged to the Paratyphi B serovar. The remaining 99 % samples belonged to different non-typhoidal serovars (NTS). The most common serotypes in the North-eastern part of Italy were monophasic S. typhimurium 1,4 [5],12:i- and S. typhimurium, which were retrieved in approximately 29.5 and 26.5 % samples, respectively (Table 1). The remaining samples belonged predominantly to S. enteritidis, S. panama and S. napoli; other NTS were detected to a minor extent (Table 1).

Antimicrobial susceptibility testing showed that 38.4 % of isolates were susceptible to all classes of antibiotics. The two Thyphi serovar isolates belonged to this fully susceptible group. The remaining Salmonella isolates displayed different antimicrobial resistance profiles: (i) 12.1 % exhibited reduced susceptibility or resistance to two classes of antimicrobials; (ii) 13.4 % were classified as multidrug-resistant (MDR), i.e. they showed resistance against three or more non-structurally related antimicrobials; (iii) 36.1 % were resistant to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT resistant pattern). The resistance rate to quinolones and fluoroquinolones (i.e., nalidixic acid and ciprofloxacin) and third generation cephalosporins (i.e., cefotaxime, ceftazidime and ceftriaxone) was relatively low among the isolates. Fluoroquinolones and third generation cephalosporins are the antibiotics most widely used in the treatment of both human and animal infections. In detail, the isolates resistant to nalidixic acid and/or ciprofloxacin were 6 and 0.5 %, respectively. Reduced susceptibility to third generation cephalosporins was detected in 0.9 % isolates. Notably, 0.5 % isolates displayed a resistance phenotype against fourth generation cephalosporins (i.e., cefepime) as well. As reported in other countries, all isolates were fully susceptible to imipenem and meropenem [19, 20].

The presence of extended spectrum beta lactamases (ESBL) and acquired class C cephalosporinases (AmpC) was also investigated by double disk synergy assays. Three isolates displayed an ESBL/AmpC phenotype. These were further characterized at the molecular level to detect plasmid-borne resistance determinants and additional resistance mechanisms.

The three isolates that possessed an ESBL/AmpC phenotype belonged to the following serovars: S. paratyphi B, S. rissen and monophasic S. typhimurium 1,4 [5],12:i-. Characterization of resistance determinants was performed on total bacterial DNA extracted as previously described [16, 21]. The major resistance determinants, i.e., bla _TEM, bla _SHV, plasmidic AmpC genes and bla _CTX-M-type, were amplified and sequenced. Genotypic analysis indicated that S. paratyphi B harbored bla _TEM-52, S. rissen was positive for bla _CMY-2, while monophasic S. typhimurium 1,4 [5],12:i- encoded bla _CTX-M-15.

The detected molecular determinants belonged to Ambler Class A and C, which have been previously reported within mobile elements in other members of the Enterobacteriaceae family, responsible for human outbreaks [22]. Therefore, the possible spreading in Salmonella of these resistance determinants through highly diffusible plasmid was investigated.

Plasmid content of the three isolates was analyzed by multiple techniques. Plasmid DNA extractions were analyzed by PCR: the efficient amplification of the selected resistance genes indicated that all determinants conferred plasmid-mediated resistance. For epidemiological tracing purposes, backbones were characterized by PCR amplification of incompatibility groups to determine plasmid replicon typing (PBRT) [17]. This analysis indicated that bla _CMY-2 was embedded in an IncA/C plasmid and bla _TEM-52 in an IncI1-Iγ plasmid. Plasmid analysis of the bla _CTX-M-15-positive monophasic S. typhimurium 1,4 [5],12:i- isolate displayed a double replicon profile, i.e. IncFIIs and IncFIA, indicating the presence of more than one vector or of a backbone with a multi-replicon status.

To confirm plasmid-embedding of the resistance determinants, IncI and IncF plasmids were transformed into E. coli and transformants were analyzed for replicon typing and for the presence of bla_CTX-M-15 and bla_TEM-52. Transformation experiments demonstrated that bla_CTX-M-15 was contained in the IncFIA backbone, while no transformation of the IncFIIs plasmid and bla_CMY-2 determinant was obtained. Plasmid size or degradation might account for the absence of transformants.

Many antibiotic resistance plasmids can promote horizontal dissemination of genetic traits among bacteria of different genera through the conjugation process [11]. For this reason, transferability of the resistance determinants was assessed by mating experiments. All three plasmids conjugated successfully: PBRT, along with the presence of the three resistance determinants, was confirmed by PCR in the recipient E. coli J53 strain. The straightforward horizontal transfer of the three backbones suggested that both clonal spread and horizontal plasmid transfer may have contributed to Salmonella resistance dissemination in Salmonella isolates (Table 2).

Because of the recent uprising rate of colistin resistance reported in Enterobacteriaceae [23, 24], susceptibility to colistin was also evaluated in all Salmonella isolates. Indeed, two isolates of S. enterica serotype Enteritidis, displayed MIC₉₀ higher than 4 μg/mL and were thus considered resistant according to EUCAST breakpoints [14]. As mutations in the pmrAB operon were demonstrated to be involved in Salmonella resistance to colistin and other polymixins [18], pmrA and pmrB genes were amplified and sequenced. PmrA and pmrB nucleotide sequences were identical to those reported for colistin-susceptible isolates: no polymorphic positions with synonymic amino acid substitution, or point nucleotide mutation were detected, suggesting the presence of other resistance mechanisms responsible for the observed reduced susceptibility to colistin [25].