M. Carmen Mendoza

Characterization of a Self-Transferable Plasmid from Salmonella enterica Serotype Typhimurium Clinical Isolates Carrying Two Integron-Borne Gene Cassettes Together with Virulence and Drug Resistance Genes

ABSTRACT An unusual self-transferable virulence-resistance plasmid (pUO-StVR2) was found in nine multidrug-resistant (ACSSuT phenotype) Salmonella enterica serotype Typhimurium clinical isolates that were assigned to four different phage types and a single and distinctive XbaI pulsed-field gel electrophoresis profile. pUO-StVR2 is an IncFII plasmid of about 140 kb in length carrying the spvA, spvB, and spvC (Salmonella plasmid virulence) and rck (resistance to complement killing) genes. It also carries the oxa1/aadA1a (ampicillin resistance and streptomycin-spectinomycin resistance) gene cassette configuration located within a class 1 integron with qacEΔ1/sul1 (ammonium antiseptics resistance and sulfadiazine resistance); the transposon genes merA, tnpA, and tnpR (mercury resistance, transposase, and resolvase of Tn21, respectively); and the catA1 (chloramphenicol resistance) and tet(B) (tetracycline resistance) genes. The insertion of resistance genes into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and presents a serious public health problem.

Extended-spectrum β-lactamases and AmpC β-lactamases in ceftiofur-resistant Salmonella enterica isolates from food and livestock obtained in Germany during 2003–07

OBJECTIVES Detection and characterization of extended-spectrum beta-lactamases (ESBLs) and AmpC-encoding genes was conducted in German Salmonella isolated from different sources from 2003 to 2007. METHODS Non-duplicate German isolates from the National Salmonella Reference Laboratory Collection (2003-07) with ceftiofur MICs of > or =4 mg/L were tested for beta-lactam/beta-lactamase inhibitor susceptibility, presence of ESBLs or AmpC-encoding genes, class 1 and 2 integrons, other resistance genes, and IS26 and ISEcp1 sequences by PCR/sequencing. The isoelectric point of the beta-lactamase was determined. Strains were analysed by PFGE and plasmid profiling. The bla genes were mapped by Southern-blot hybridization. Plasmids were characterized by rep-PCR typing. RESULTS Sixteen isolates (10 Salmonella Typhimurium, 2 Salmonella Anatum, 2 Salmonella Paratyphi B dT + , 1 Salmonella Infantis and 1 Salmonella London) carried bla(CTX-M) (15 bla(CTX-M-1) and one bla(CTX-M-15)) genes located on self-transferable IncB/O, IncI1 and/or IncN plasmids. Seven of the Salmonella Typhimurium isolates carried the SGI1-M variant. Six isolates (five Salmonella Agona and one Salmonella Kentucky) carried the bla(CMY-2) gene on IncI1 conjugative plasmids. bla(TEM-20) genes were detected in two Salmonella Paratyphi B dT+ isolates, and bla(TEM-52) in one Salmonella Paratyphi B dT+ and one Salmonella Virchow, located on IncI1 plasmids. All Salmonella Paratyphi isolates harboured a 2300 bp/dfrA1-sat2-aadA1 class 2 integron. CONCLUSIONS Among the 22 679 German Salmonella isolates investigated, the ESBL and AmpC beta-lactamase prevalence was still low; however, it is slowly increasing. Various beta-lactamase genes are linked to a variety of genetic elements capable of horizontal DNA transfer. Consequently, their dissemination is likely and demands adequate risk management strategies.

Characterization of pUO-StVR2, a Virulence-Resistance Plasmid Evolved from the pSLT Virulence Plasmid of Salmonella enterica Serovar Typhimurium

ABSTRACT pUO-StVR2 is a virulence-resistance plasmid which originated from pSLT of Salmonella enterica serovar Typhimurium through acquisition of a complex resistance island, flanked by regions that provide a toxin-antitoxin system and an iron uptake system. The presence of resistance and virulence determinants on the same plasmid allows coselection of both properties, potentially increasing health risks.

IncA/C plasmids mediate antimicrobial resistance linked to virulence genes in the Spanish clone of the emerging Salmonella enterica serotype 4,[5],12:i:−

OBJECTIVES To broaden knowledge of the molecular bases and genetics of multidrug resistance in clinical isolates of Salmonella enterica serotype 4,5,12:i:- belonging to the Spanish clone. METHODS The relatedness of the isolates was determined by phage typing and XbaI-PFGE. Resistance genes, integrons and transposable elements were identified by PCR amplification and sequencing. Plasmids were characterized by alkaline lysis, S1-PFGE, conjugation, replicon typing and Southern blot hybridization. RESULTS The isolates were closely related and resistant to five to seven antimicrobials (ampicillin, chloramphenicol, gentamicin, streptomycin/spectinomycin, sulphonamides, trimethoprim and tetracycline, arranged in different combinations). Most of the responsible genes were provided by a conventional class 1 integron with the dfrA12-orfF-aadA2 variable region, an atypical class 1 integron containing sul3 next to the estX-psp-aadA2-cmlA1-aadA1 variable region and a truncated Tn1721 transposon carrying tet(A). A defective Tn21 with the mer operon and ISVsa3 associated with sul2 were also detected. All resistance genes and mobile genetic elements were located on large, non-conjugative and highly variable plasmids carrying one (A/C) or two (A/C and N) replicons, as well as virulence genes of pSLT. CONCLUSIONS IncA/C plasmids are responsible for multidrug resistance in an increasing number of relevant human and animal bacterial pathogens, and hence are regarded as an important threat to public health. Those found in the Spanish clone of Salmonella 4,5,12:i:- constitute a relevant example of short-term evolution, and could have been involved in the successful adaptation of this pathogen.

Molecular epidemiology of Salmonella serotype Enteritidis. Relationships between food, water and pathogenic strains

A molecular epidemiology study of Salmonella serotype Enteritidis was carried out by ribotyping performed with a mixture of PstI and SphI (PS ribotyping), and randomly amplified polymorphic DNA (RAPD) typing conducted with the OPB17 primer. A series, including 38 food and 25 water strains, which were epidemiologically unrelated and collected in Spain over 1985-1996, was differentiated into 20 PS ribotypes [discrimination index (DI) = 0.67], RAPD types (DI = 0.28), and by combining both methods into 23 genomic groups (DI = 0.76). With ribotyping data from the strains tested in this and in a previous work, including clinical and reference strains, a similarity dendrogram was traced and the subsequent branches and groupings were correlated with RAPD types, phage types and sources of origin. At a similarity level of 55%, a major cluster (grouping five subclusters and three single branches) and two minor clusters were revealed. Results supported the fact that organisms representing, at least, 40 genomic groups are currently circulating in Spain, but that only the organisms of five groups predominate and these fall into a single subcluster or lineage. Organisms of these five groups could be considered endemic, associated with food-borne human infections and, for epidemiological purposes, can be differentiated by phage typing. The most frequent phage types can be subdivided into genomic groups. Organisms of the prevalent genomic groups and several less frequent ones were mainly associated with poultry transmission and gastroenteritis as the major clinical forms, while organisms of another two frequent groups were mainly associated with extra-intestinal infections, and organisms of four infrequent groups were only collected from sewage or environmental waters.

Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004

In this study, 119 multidrug-resistant isolates of non-typhoidal Salmonella enterica serovars collected in Spain (2002-2004) were screened for integrons. Among the isolates, 73.1% contained class 1 integrons, however classes 2 and 3 were not detected. Integrons containing gene cassettes were found in S. Enteritidis (16/32), S. Typhimurium biphasic (18/32) and monophasic [4,5,12:i:-] (11/19), S. Virchow (17/18) and S. Brandenburg (8/8), but not in S. Hadar (0/10). Ten complete and four incomplete gene cassettes, combined in 10 variable regions, were identified, one of which (2100 bp/dfrA1-597 bp-aadA24) was a new description. Most integrons mapped on plasmids of ca. 40-340 kb. Exceptions were 1000 bp/aadA2 and 1200 bp/bla PSE-1 found on the chromosome of biphasic S. Typhimurium, probably as part of SGI1-like structures.

In vitro fluoroquinolone-resistant mutants of Salmonella enterica serotype Enteritidis : analysis of mechanisms involved in resistance

This study analysed the mechanisms involved in the acquisition of resistance to quinolones in mutants obtained in vitro of Salmonella enterica serotype Enteritidis. Two nalidixic acid-resistant (minimal inhibitory concentrations, MIC>256 mg/l), ciprofloxacin-susceptible (MIC 0.5 mg/l) clinical isolates of Salmonella Enteritidis with a mutation at amino acid codon Ser-83 of the gyrA gene were grown on plates containing increasing concentrations of ciprofloxacin. The increase in MIC to ciprofloxacin, sparfloxacin and trovafloxacin was totally or partially associated with over-expression of an AcrAB-like efflux pump. In addition, unidentified mechanism(s) may have been involved in the increased MIC to these antimicrobials. This study demonstrated that AcrAB-like efflux pumps appear to play a relevant role in the increase in MIC to some quinolones although, other, as yet undefined, mechanisms may be involved.

Diversity of Plasmids Encoding Virulence and Resistance Functions in Salmonella enterica subsp. enterica Serovar Typhimurium Monophasic Variant 4,[5],12:i:- Strains Circulating in Europe

Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3′ conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and bla TEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

Large Conjugative Plasmids from Clinical Strains of Salmonella enterica Serovar Virchow Contain a Class 2 Integron in Addition to Class 1 Integrons and Several Non-Integron-Associated Drug Resistance Determinants

ABSTRACT Two large conjugative resistance (R) plasmids from clinical strains of Salmonella enterica serovar Virchow carried a class 2 integron with the 5′ conserved sequence (5′CS)-dfrA1-sat1-aadA1-3′CS gene array, which is associated with defective Tn7 transposons. In addition, each contained a different class 1 integron (with 5′CS-aadA1-3′CS or 5′CS-sat-smr-aadA1-3′CS gene arrays) linked to Tn21-Tn9 sequences, and several non-integron-associated R determinants. An intact copy of Tn7 (including the class 2 integron) was present in the chromosome of each strain.

Population structure and exotoxin gene content of methicillin-susceptible Staphylococcus aureus from Spanish healthy carriers

The population structure of 111 methicillin-susceptible Staphylococcus aureus (MSSA), recovered in Spain from healthy and risk-free carriers was investigated using pulsed-field gel electrophoresis (PFGE), spa (staphylococcal protein A) typing, multi locus sequence typing (MLST) and the accessory gene regulator (agr). Results from the different techniques were highly concordant, and revealed twelve clonal complexes (CCs): CC30 (27%), CC5 (18.9%), CC45 (16.2%), CC15 (11.7%), CC25 (8.1%), CC1, CC9 (3.6% each), CC59, CC97 and CC121 (2.7% each), CC72 (1.8%) and CC8 (0.9%). Isolates with genetic backgrounds of hospital-acquired MSSA were detected and, consistent with the ability of diverse MSSA to act as recipients of the SCCmec cassette, a MSSA isolate from a healthy carrier shared the ST, spa-type and agr-type of a MRSA clone recovered in a hospital of the same region. All except two fragments of the PGFE-profiles of these isolates were identical, and the differential fragment of the MRSA carried mecA. Analyses of the exotoxin gene content of the nasal isolates revealed an increase in the number of exotoxin genes over time. This, together with the detection of lukPV and the high frequency of tst, exfoliatin and enterotoxin genes, is worrisome and requires further surveillance.