Beatriz Guerra

Acquired Antibiotic Resistance Genes: An Overview

In this review an overview is given on antibiotic resistance (AR) mechanisms with special attentions to the AR genes described so far preceded by a short introduction on the discovery and mode of action of the different classes of antibiotics. As this review is only dealing with acquired resistance, attention is also paid to mobile genetic elements such as plasmids, transposons, and integrons, which are associated with AR genes, and involved in the dispersal of antimicrobial determinants between different bacteria.

Emergence of human pandemic O25:H4-ST131 CTX-M-15 extended-spectrum-β-lactamase-producing Escherichia coli among companion animals

OBJECTIVES In view of the intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15 extended-spectrum beta-lactamase (ESBL) in human clinical settings it would be of great interest to explore its existence in animals to unravel a possible reservoir function and the origin and transmission of this group of multiresistant strains. METHODS A total of 177 clinical phenotypically ESBL-producing E. coli isolates, mainly obtained from companion animals with urinary tract infections, wound infections and diarrhoea, were collected in a veterinary diagnostic laboratory covering a European-wide service area. They were screened for molecular subtype O25b and multilocus sequence type 131. O25b-ST131 isolates were subsequently tested for ESBL types, and phenotypic and genotypic resistance determinants. Further characterization of the strains was performed by PFGE and virulence gene typing. RESULTS Ten (5.6%) of 177 phenotypically ESBL-producing E. coli isolates, nine strains from dogs and one strain from a horse, were allocated to the B2-O25b-ST131 lineage. Nine of these isolates harboured a CTX-M-15-type beta-lactamase enzyme while one strain possessed an SHV-12-type ESBL. Macrorestriction analysis revealed a cluster formation of six of the animal CTX-M-15-type ESBL-producing strains from five different European countries together with a human control strain constituting a group of clonally related strains at a similarity value of 87.0%. CONCLUSIONS Our findings demonstrate that the group of clonally related human B2-O25:H4-ST131 CTX-M-15-type ESBL-producing E. coli strains is present in companion animals from various European countries. This highlights the possibility of inter-species transmission of these multiresistant strains from human to animal and vice versa.

Antimicrobial Resistance and Spread of Class 1 Integrons among Salmonella Serotypes

ABSTRACT The resistance profiles, for 15 antimicrobial agents, of 333Salmonella strains representing the most frequent nontyphoidal serotypes, isolated between 1989 and 1998 in a Spanish region, and 9 reference strains were analyzed. All strains were susceptible to amikacin, ceftazidime, ciprofloxacin, and imipenem, and 31% were susceptible to all antimicrobials tested. The most frequent types of resistance were to sulfadiazine, tetracycline, streptomycin, spectinomycin, ampicillin, and chloramphenicol (ranging from 46 to 22%); 13% were resistant to these six drugs. This multidrug resistance pattern was found alone or together with other resistance types within serotypes Typhimurium (45%), Panama (23%), and Virchow (4%). Each isolate was also screened for the presence of class 1 integrons and selected resistance genes therein; seven variable regions which carried one (aadA1a, aadA2, orpse-1) or two (dfrA14-aadA1a,dfrA1-aadA1a, oxa1-aadA1a, orsat1-aadA1a) resistance genes were found in integrons.

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.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

ABSTRACT A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6′)-Ie-aph(2″)-Ia and/or ant(4′)-Ia but also to aph(3′)-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Carbapenemase-producing Enterobacteriaceae and non-Enterobacteriaceae from animals and the environment: an emerging public health risk of our own making?

Acquired carbapenemases pose one of the most pressing public health threats relating to antibiotic resistance. In most countries, the number of carbapenemase-producing bacteria from human clinical specimens is rising, and the epidemiological status of these multiresistant bacteria is progressively worsening. Furthermore, there is a growing number of reports of carbapenemases found either in bacteria isolated from non-human sources or in Salmonella enterica subsp. enterica, a zoonotic species. However, carbapenemases are not yet systematically sought in bacteria from non-human sources, reports of them are largely observational, and there is limited investigation of carbapenemase-positive bacteria in animals and possible links with people who may have acted as potential sources. Active surveillance and monitoring for carbapenem-resistant bacteria in the food chain and other non-human sources is urgently needed, with an enhanced and rigorous follow-up of all positive results. The carbapenems are currently our last good defence against multiresistant Gram-negative bacteria. Our ability to limit the rise and spread of carbapenemase producers, which occur only at basal levels in many countries at present, should serve as a key performance indicator for the success or failure of the efforts that have been called for by international organizations and governments to reduce the impact of antibiotic resistance.

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 and Localization of Drug Resistance Determinants in Multidrug-Resistant, Integron-Carrying Salmonella enterica Serotype Typhimurium Strains

The genetic background of the antimicrobial resistance of 10 selected multiresistant Salmonella serotype Typhimurium (S. Typhimurium) strains (including the emerging monophasic variant [4,5,12:i:- ]) was investigated. All strains shared class 1 integrons (with seven types of variable regions) and belonged to different lineages (L1-L6) according to their phage types, DNA polymorphisms by XbaI-pulsed-field gel electrophoresis (PFGE), integrons, and/or resistance patterns. The strains were screened for the presence and localization (chromosomal or plasmid) of 32 DNA sequences representing integron-, Tn21-like transposon-, resistance-, and virulence-plasmid genes. Strains belonging to lineage L1 (definitive phage type DT104) carried the 90-kb Salmonella virulence plasmid together with the complete or partial chromosomally located Salmonella Genomic Island 1 (SGI1). All strains belonging to the other five lineages carried their resistance determinants on various resistance plasmids. Two of these strains showed complex plasmid profiles, which included a 95 kb virulence plasmid together with two or four resistance plasmids. Two strains carried a resistance plasmid that lacked the virulence-plasmid-encoding sequences. The remaining two strains carried two different hybrid virulence-resistance plasmids. Twenty-three of the DNA sequences could be assigned to distinct XbaI genomic restriction patterns (PFGE profiles). In this way, the influence of the resistance and virulence plasmids on the PFGE profiles was determined, and several groups of resistance genes could be identified. The data obtained represent a useful epidemiological tool for tracing the emergence and distribution of multiresistant S. Typhimurium worldwide.

Virulotyping and antimicrobial resistance typing of Salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

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