Irene Rodríguez

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.

Salmonella enterica subsp. enterica producing VIM-1 carbapenemase isolated from livestock farms

Sir, Thirdand fourth-generation cephalosporins and carbapenems are ‘critically important’ antimicrobials as classified by the WHO (www.who.int). In fact, carbapenems are last-line clinical antibiotics against infections caused by multidrug-resistant Gram-negative bacteria. In contrast to cephalosporins, carbapenems are not hydrolysed by most b-lactamases, including AmpC b-lactamases and extended-spectrum b-lactamases (ESBLs). However, during the last decade the prevalence of carbapenem resistance in Enterobacteriaceae has increased worldwide. Whereas the increase in the prevalence of ESBL-producing Enterobacteriaceae isolated from livestock is becoming an important public health problem, the increasing prevalence of carbapenemases has only affected hospitals and the community. Recently, however, the occurrence of carbapenemase-carrying commensal Escherichia coli isolated from livestock and their environment has been reported, and this could be the beginning of a new era in the antibiotic resistance field. Within the national RESET project (www.reset-verbund.de) several longitudinal and cross-sectional studies, collecting potential ESBL-carrier organisms from German farms, have been performed (using MacConkey agar with 1 mg/L cefotaxime as the selective medium). From the 221 isolates collected during 2011, 3 of them were ascribed to Salmonella enterica subsp. enterica (Table 1). The three Salmonella isolates (R3, R25 and R27) were obtained from two pig-fattening farms (R25 was collected outside the farm) and one broiler farm (Table 1). The three farms were distributed in different locations in the same German federal region, and although there was no apparent link between them, a common source cannot be excluded. The three isolates were tested for their susceptibility to 35 antimicrobials, including b-lactams/b-lactamase inhibitors (Table 1), phenicols, aminoglycosides, quinolones/fluoroquinolones, tetracycline, folate pathway antagonists, lipopeptides and fosfomycin, as previously described. For the present study, tigecycline (15 mg) and nitrofurantoin (300 mg) were included as well. The presence of ESBLs, AmpC b-lactamases and/or carbapenemase-encoding genes, class 1 and 2 integrons and other resistance genes was screened by PCR/sequencing, as previously described (Table S1, available as Supplementary data at JAC Online). The MIC values for some carbapenemase producers can be lower than the currently recommended breakpoints, and the results of the carbapenem susceptibility tests can be influenced by the genetic background. The Salmonella isolates R3, R25 and R27 showed decreased susceptibility to these antimicrobials [non-wild-type by the EUCAST epidemiological cut-off (ECOFF), but susceptible or intermediate according to the CLSI clinical breakpoint; Table 1]. This ‘decreased susceptibility’ could be transformed to a competent E. coli recipient, but conjugation or mobilization under the conditions used was unsuccessful. The three isolates carried both the AmpC-encoding gene blaACC-1 and the carbapenemase gene blaVIM-1, like in the previously reported E. coli isolates R178 and R29. When Salmonella R3 and the control strain E. coli R178 were grown in liquid medium with carbapenems (Luria-Bertani broth with 16 mg/L imipenem or 8 mg/L ertapenem inoculated with 1:1000 overnight culture), both isolates grew well, showing full carbapenem resistance (clinical breakpoints, CLSI versus EUCAST: imipenem ≥4 versus .8 mg/L; and ertapenem ≥1 versus .1 mg/L). Several class 1 integrons (In31, In70, In71, In110 and In450), transposons (Tn3, Tn402 and Tn21) and plasmids (incompatibility groups IncHI2, IncN, IncI1 and IncW) carrying blaVIM genes have been described. Like in E. coli R178 and R29, in the three Salmonella isolates the blaVIM-1 gene was located on a class 1 integron (variable region with blaVIM-1-aacA4-aadA1 gene cassettes) harboured by an 300 kb IncHI2 plasmid (determined by S1-nuclease PFGE analysis, PCR-based replicon typing and Southern blot hybridization, as previously described; HI2 double plasmid sequence typing failed). The plasmid also carried blaACC-1, strA/B, catA1 and a trimethoprim resistance gene (not identified with the primers used; see Table S1, available as Supplementary data at JAC Online). The sequence of the complete integron of Salmonella R3 (5436 bp, including complete sul1 and orf5, obtained using as template the pRHR3 plasmid of this isolate; see Table S1, available as Supplementary data at JAC Online) was identical to the one from E. coli R178 (accession number HE663536) and was related to Tn402 (like in GQ422826). Salmonella R27 was isolated from the same farm as both E. coli R178 and R29 (Table 1). However, the IncHI2 plasmids harboured by these Salmonella and E. coli isolates were different in size and gene content ( 300 versus 220 kb and presence versus absence of chloramphenicoland trimethoprimresistance genes), suggesting different plasmid evolutions. The three isolates were classified as S. enterica group C, antigenic formula ‘6,7:–:– ’ (www.pasteur.fr) at the National Salmonella Reference Laboratory (NRL-Salm, BfR). The sequence type ST32 (http://mlst.ucc.ie/mlst/dbs/Senterica) and the PFGE patterns found (Figure S1, available as Supplementary data at JAC Online) are typical of Salmonella Infantis (6,7:r:1,5) and have also been detected in German isolates from humans, poultry/ poultry meat and pig/pork meat. Salmonella Infantis is among the top 10 Salmonella serovars implicated in human salmonellosis worldwide (ranking in third place in 2011 in Europe; www.ecdc. europa.eu). Isolates from this serovar also caused disease with Research letters

Escherichia coli producing VIM-1 carbapenemase isolated on a pig farm

and the source from which the isolate was obtained. Further target gene mutations were detected in single isolates: (i) a mutation that resulted in the Glu471Asp exchange in GrlB was present in an avian ST5 MRSA; (ii) a mutation at codon 517 in the gyrB gene (resulting in an Arg to Lys exchange) was found in the ST1791 MRSA of turkey meat origin; and (iii) the ST2269 isolate had an additional grlA mutation at codon 84 that caused a Glu to Asp exchange, and a gyrA mutation that resulted in a Glu88Asp exchange. The GrlA alterations Ser80Leu and Glu84Asp and the GyrA exchange Glu88Asp have not been identified so far in S. aureus. The role in fluoroquinolone resistance of the Glu422Asp exchange in GrlB needs further investigation as the corresponding mutation was present along with other mutations in staphylococci that varied in their enrofloxacin MICs between 1 and 8 mg/L, but it was also the only mutation detected in two porcine MRSA isolates with an enrofloxacin MIC of 1 mg/L. All but one of the MRSA and MSSA isolates investigated in this study showed one of two types of point mutations (A and B in Figure S1, available as Supplementary data at JAC Online) in the norA promoter region; these, however, affected neither the 235 and 210 positions, nor the norA-associated ribosome binding site. For one avian MSSA isolate, no norA-specific PCR product could be obtained in repeated attempts. The results of this study show that increased MICs of enrofloxacin among MRSA and MSSA isolates from diseased foodproducing animals or food of animal origin is mainly mediated by grlA and/or gyrA mutations, which—aside from the four novel mutations detected during this study—correspond to those reported previously in S. aureus isolates of different origin. 2

Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany.

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla CTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla CTX-M-group-1 and bla OXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, bla OXA-1-like and bla CTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

Diversity of STs, plasmids and ESBL genes among Escherichia coli from humans, animals and food in Germany, the Netherlands and the UK

OBJECTIVES This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes. METHODS A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST. RESULTS The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons. CONCLUSIONS ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts.

Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n=356) obtained in Germany, The Netherlands and the UK (2005-2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. β-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 β-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.

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.

blaCTX-M-15-carrying Escherichia coli and Salmonella isolates from livestock and food in Germany

OBJECTIVES The characterization of CTX-M-₁₅ β-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. METHODS E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to third-generation cephalosporins, originating from routine and monitoring submissions (2003-12) to the Federal Institute for Risk Assessment and different national targeted studies (2011-12), were examined for the presence of blaCTX-M-₁₅ genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-₁₅ were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. RESULTS Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-₁₅ producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-₁₅ genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. CONCLUSIONS The prevalence of CTX-M-₁₅-producing isolates from non-human sources in Germany is still low. The blaCTX-M-₁₅ gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-₁₅ gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-₁₅-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance.

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.

A Predominant Multidrug-Resistant Salmonella enterica Serovar Saintpaul Clonal Line in German Turkey and Related Food Products

ABSTRACT Recently, Salmonella enterica subsp. enterica serovar Saintpaul has increasingly been observed in several countries, including Germany. However, the pathogenic potential and epidemiology of this serovar are not very well known. This study describes biological attributes of S. Saintpaul isolates obtained from turkeys in Germany based on characterization of their pheno- and genotypic properties. Fifty-five S. Saintpaul isolates from German turkeys and turkey-derived food products isolated from 2000 to 2007 were analyzed by using antimicrobial agent, organic solvent, and disinfectant susceptibility tests, isoelectric focusing, detection of resistance determinants, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and hybridization experiments. These isolates were compared to an outgroup consisting of 24 S. Saintpaul isolates obtained from humans and chickens in Germany and from poultry and poultry products (including turkeys) in Netherlands. A common core resistance pattern was detected for 27 German turkey and turkey product isolates. This pattern included resistance (full or intermediate) to ampicillin, amoxicillin-clavulanic acid, gentamicin, kanamycin, nalidixic acid, streptomycin, spectinomycin, and sulfamethoxazole and intermediate resistance or decreased susceptibility to ciprofloxacin (MIC, 2 or 1 μg/ml, respectively) and several third-generation cephalosporins (including ceftiofur and cefoxitin [MIC, 4 to 2 and 16 to 2 μg/ml, respectively]). These isolates had the same core resistance genotype, with blaTEM-1, aadB, aadA2, sul1, a Ser83→Glu83 mutation in the gyrA gene, and a chromosomal class 1 integron carrying the aadB-aadA2 gene cassette. Their XbaI, BlnI, and combined XbaI-BlnI PFGE patterns revealed levels of genetic similarity of 93, 75, and 90%, respectively. This study revealed that a multiresistant S. Saintpaul clonal line is widespread in turkeys and turkey products in Germany and was also detected among German human fecal and Dutch poultry isolates.