Aránzazu Valverde

Dramatic Increase in Prevalence of Fecal Carriage of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae during Nonoutbreak Situations in Spain

ABSTRACT The occurrence of extended-spectrum β-lactamase (ESBL)-producing isolates has increased worldwide. Fecal carriage of ESBL-producing isolates has mainly been detected in nosocomial outbreaks, and few studies have evaluated fecal carriage during nonoutbreak situations and among patients in the community. We have studied the prevalence of ESBLs in 1,239 fecal samples from 849 patients (64.1% of whom were ambulatory) in 1991 and have compared the prevalence data with those obtained in 2003 for 400 fecal samples from 386 patients (75.9% of whom were ambulatory) and 108 samples from independent healthy volunteers. Samples were diluted in saline and cultured in two MacConkey agar plates supplemented with ceftazidime (1 μg/ml) and cefotaxime (1 μg/ml), respectively. Colonies were screened (by the double-disk synergy test) for ESBL production. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis with XbaI digestion; and the ESBLs of all ESBL-producing isolates were characterized by isoelectric focusing, PCR, and sequencing. The rates of fecal carriage of ESBL-producing isolates increased significantly (P < 0.001) in both hospitalized patients and outpatients, from 0.3 and 0.7%, respectively, in 1991, to 11.8 and 5.5%, respectively, in 2003. The rate of occurrence of ESBL-producing isolates among healthy volunteers was 3.7%. All ESBL-producing isolates recovered in 2003 were nonepidemic clones of Escherichia coli. ESBL characterization revealed an increasing diversity of ESBL types: TEM-4 and CTX-M-10 were the only enzymes detected in 1991, whereas TEM-4, TEM-52, SHV-12, CTX-M-9, CTX-M-10, CTX-M-14, and a CTX-M-2-like enzyme were recovered in 2003. The ESBL-producing isolates recovered from outpatients in 2003 corresponded to a CTX-M-9-type cluster (62.5%) and SHV-12 (31.2%), whereas TEM-4 was detected only in hospitalized patients. The frequencies of coresistance in isolates recovered in 2003 were as follows: sulfonamide, 75%; tetracycline, 64.3%; streptomycin, 57.1%; quinolones, 53.5%; and trimethoprim, 50%. The increased prevalence of fecal carriage of ESBL-producing isolates during nonoutbreak situations in hospitalized patients and the establishment of these isolates in the community with coresistance to non-β-lactam antibiotics, including quinolones, represent an opportunity for these isolates to become endemic.

High Rate of Intestinal Colonization with Extended-Spectrum-β-Lactamase-Producing Organisms in Household Contacts of Infected Community Patients

ABSTRACT Fecal carriage of extended-spectrum-β-lactamase (ESBL)-producing organisms was detected in 70% of index cases of patients (n = 40) with community-acquired infections due to ESBL producers and reached 16.7% in household contacts (n = 54). A total of 66% of ESBL-producing organisms from index cases were indistinguishable from isolates from household contacts by pulsed-field gel electrophoresis. Patients with community infections and members of their households represent a reservoir for ESBL producers, increasing dispersal of resistance in healthy people.

Antibiotic Coresistance in Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae and In Vitro Activity of Tigecycline

ABSTRACT The spread of extended-spectrum-β-lactamase (ESBL)-producing organisms, particularly those harboring the CTX-M-type enzymes, both in the hospital and in the community, is difficult to discontinue due to the successful mobilization and evolution of the genetic elements harboring ESBL genes and coresistance rates in these isolates. The activities of tigecycline against 285 non-clonally related isolates (172 from Escherichia coli, 84 from Klebsiella spp., 20 from Enterobacter spp., 5 from Salmonella spp., and 4 from Citrobacter spp.) expressing well-characterized ESBLs and recovered in our hospital and its community area of influence were comparatively assessed (CLSI microdilution). Susceptibility rates for meropenem, imipenem, tigecycline, amikacin, and piperacillin-tazobactam were 100%, 100%, 97.5%, 93.3%, and 93%, respectively. Tigecycline (mode MIC, 0.5 μg/ml; MIC90, 1 μg/ml) was 4- to 256-fold more active than doxycycline and minocycline (mode MIC range, 2 to 128 μg/ml). CTX-Ms were the most frequent ESBLs (61.4%), 65.8% in community and 58.6% in nosocomial isolates. CTX-M-9 (22%), CTX-M-14 (15.8%), and CTX-M-10 (14%) were the most represented derivatives. SHV and TEM variants constituted 22.8% and 15.8% of the ESBLs, respectively. Overall coresistance rates were as follows: gentamicin, 27.4%; tobramycin, 27.4%; amikacin, 6.7%; and chloramphenicol, 29.1%. Sulfonamide (61.7%), trimethoprim (52.3%), streptomycin (50.5%), and ciprofloxacin (37.2%) resistance levels were significantly (P < 0.001) associated with CTX-M-9 producers. No tigecycline resistance was observed, although seven Klebsiella pneumoniae isolates exhibited intermediate MICs (4 μg/ml). Tigecycline, lacking cross-resistance with other compounds, could represent an opportunity to reduce the intensity of selection for ESBL-producing organisms derived from the use of other antimicrobial agents. However, this in vitro promise requires support from clinical studies.

Dissemination and Persistence of blaCTX-M-9 Are Linked to Class 1 Integrons Containing CR1 Associated with Defective Transposon Derivatives from Tn402 Located in Early Antibiotic Resistance Plasmids of IncHI2, IncP1-α, and IncFI Groups

ABSTRACT This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing blaCTX-M-9, and its association with Tn402, Tn21, and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 (n = 26), IncP-1α (n = 10), IncFI (n = 4), and IncI (n = 1) groups. Genetic platforms containing blaCTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn402 derivatives. They were associated with Tn21 sequences when located in IncP-1α or IncHI2 plasmids. Our study identified blaCTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn402 derivatives, often to Tn21, highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of blaCTX-M-9 on broad-host-range IncP-1α plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae.

Spread of blaCTX-M-14 Is Driven Mainly by IncK Plasmids Disseminated among Escherichia coli Phylogroups A, B1, and D in Spain

ABSTRACT Since its first description in 2000, CTX-M-14 has become one of the most widespread extended-spectrum β-lactamases in Spain. In the present Escherichia coli multilevel population genetic study involving the characterization of phylogroups, clones, plasmids, and genetic platforms, 61 isolates from 16 hospitalized patients and 40 outpatients and healthy volunteers recovered from 2000 to 2005 were analyzed. Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] type, phylogenetic group, multilocus sequence type [MLST]) was established by standard methods. Analysis of transferred plasmids (I-CeuI; S1 nuclease; restriction fragment length polymorphism analysis; and analysis of RNA interference, replicase, and relaxase) was performed by PCR, sequencing, and hybridization. The genetic environment of blaCTX-M-14 was characterized by PCR on the basis of known associated structures (ISEcp1, IS903, ISCR1). The isolates were mainly recovered from patients in the community (73.8%; 45/61) with urinary tract infections (62.2%; 28/45). They were clonally unrelated by PFGE and corresponded to phylogenetic groups A (36.1%), D (34.4%), and B1 (29.5%). MLST revealed a high degree of sequence type (ST) diversity among phylogroup D isolates and the overrepresentation of the ST10 complex among phylogroup A isolates and ST359/ST155 among phylogroup B1 isolates. Two variants of blaCTX-M-14 previously designated blaCTX-M-14a (n = 59/61) and blaCTX-M-14b (n = 2/61) were detected. blaCTX-M-14a was associated with either ISEcp1 within IncK plasmids (n = 27), ISCR1 linked to an IncHI2 plasmid (n = 1), or ISCR1 linked to IncI-like plasmids (n = 3). The blaCTX-M-14b identified was associated with an ISCR1 element located in an IncHI2 plasmid (n = 1) or with ISEcp1 located in IncK (n = 1). The CTX-M-14-producing E. coli isolates in our geographic area are frequent causes of community-acquired urinary tract infections. The increase in the incidence of such isolates is mostly due to the dissemination of IncK plasmids among E. coli isolates of phylogroups A, B1, and D.

CTX-M-10 Linked to a Phage-Related Element Is Widely Disseminated among Enterobacteriaceae in a Spanish Hospital

ABSTRACT CTX-M-10 has been widely disseminated among multiple clones of several species of Enterobacteriaceae, harboring seemingly different plasmids, for over a decade in Ramón y Cajal University Hospital, Madrid, Spain. Cloning and sequencing of a 12.2-kb DNA fragment from plasmid pRYCE21 from Klebsiella pneumoniae strain KP4aC revealed a novel phage-related element immediately upstream of blaCTX-M-10 conserved among different CTX-M-10-producing strains. This is the first report showing an extended-spectrum-β-lactamase gene linked to a phage-related element.

In117, an Unusual In0-Like Class 1 Integron Containing CR1 and blaCTX-M-2 and Associated with a Tn21-Like Element

ABSTRACT An unusual In0-like class 1 integron containing a common region that includes the putative recombinase gene named orf513 (CR1) and blaCTX-M-2 was characterized from Escherichia coli. The integron contained an unusual gene cassette array, estX-aadA1, embedded between the 5′-conserved segment (5′-CS) and 3′-CS1 regions and was flanked by mer-Tn21 sequences downstream of the tni truncated module. This element constitutes one of the few examples of CR1-bearing class 1 integrons that has been fully characterized.

Staphylococcus aureus Carrying mecC Gene in Animals and Urban Wastewater, Spain

To the Editor: A new methicillin resistance mechanism gene, a divergent mecA homologue named mecC (formerly mecALGA251), was recently described in Staphylococcus aureus (1). Methicillin-resistant S. aureus (MRSA) isolates carrying mecC have been recovered from humans, ruminants, pets, and other animals such as rats, seals, and guinea pigs (1–3). It has been suggested that mecC-carrying MRSA isolates might not be detected by using MRSA selective media (4). For mecC-carrying S. aureus isolates, cefoxitin MICs of 4–64 mg/L have been demonstrated (1–2,4), values that would normally include susceptible isolates, according to the epidemiologic cutoff value established by the European Committee on Antibiotic Susceptibility Testing (EUCAST; www.eucast.org). mecC-carrying S. aureus isolates have been classified as heteroresistant (5), and MICs can be affected by the drug-susceptibility testing method used (1,5). These observations led us to retrospectively investigate the presence of mecC gene in a set of 361 mecA-negative S. aureus isolates collected during 2009–2012 (Table), independently of their susceptibility to cefoxitin. Isolates were recovered from healthy carriers in livestock (n = 39), from wild animals (n = 254), and from wastewater (effluents) from an urban sewage plant (n = 68). Specific amplification of the mecC gene was performed as described (6). The mecC-carrying S. aureus isolates were tested by broth microdilution using Microtiter EUST plates (Trek Diagnostic Systems, East Grinstead, UK) for susceptibility to benzylpenicillin, cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, florfenicol, fusidic acid, gentamicin, kanamycin, linezolid, mupirocin, rifampin, sulfamethoxazole, streptomycin, quinupristin-dalfopristin, tetracycline, thiamulin, trimethoprim, and vancomycin. Additionally, susceptibility to oxacillin was determined by using microScan Gram Positive Combo panel 37 (Siemens, Erlangen, Germany). MICs were interpreted according to EUCAST epidemiologic cutoff values. Table Testing of Staphylococcus aureus isolates for presence of methicillin resistance mechanism gene mecC, Spain* mecC was detected in a total of 4 isolates from wild boar (n = 1), fallow deer (n = 2), and urban wastewater (n = 1); these isolates represent 1% of the 361 tested isolates. The 3 isolates recovered from animals were susceptible to all antimicrobial drugs tested other than β-lactams and to oxacillin (MICs 0.5–1 mg/L) but were resistant to penicillin (MICs 0.5–2 mg/L). Two of the isolates were resistant to cefoxitin (MICs 8 and 16 mg/L) and the third was susceptible (MIC 4 mg/L). The wastewater isolate was resistant to penicillin (MIC 2 mg/L) and erythromycin (MIC 16 mg/L) and susceptible to all other antimicrobial drugs tested, including cefoxitin (MIC 4 mg/L) and oxacillin (MIC ≤0.25 mg/L). Previous studies have described mecC-positive isolates as susceptible to all antimicrobial drugs tested except β-lactams (2,3), although sporadic resistance to fluoroquinolones has been found (4,7). We additionally found erythromycin resistance in 1 mecC-carrying S. aureus isolate. For the 4 mecC-carrying S. aureus isolates we detected, MICs of oxacillin were interpreted as susceptible, and 2 isolates were susceptible to cefoxitin according to EUCAST guidelines, findings that agree with previous reports (1–2,4). Thus, mecC presence is not always linked to resistance phenotypes for cefoxitin or oxacillin; such unclear findings could hinder the detection of mecC-carrying isolates. We further characterized the 4 mecC-carrying S. aureus isolates by spa typing and detection of Panton-Valentin leukocidin (PVL) toxin genes (6,8). Multilocus sequence typing (MLST) was performed according to Enright et al. (9) by using self-designed primers arc (down 5′-CGATTTGTTGTTGATTAGGTTC-3′), tpi (up 5′-CATTAGCAGATTTAGGCGTTA-3′), and yqiL (down 5′-GATTGGYTCACCTTTRCGTTG-3′). All 4 isolates were PVL negative. The 3 animal isolates were assigned to a new spa type (t11212) and to clonal complex (CC) 425 and sequence type (ST) 425 (Table). ST425 has been previously associated with mecC-carrying S. aureus isolates in cattle and humans (1–2); the animals we sampled were from a game estate and may have had contact with cattle and with urban wastewater. The wastewater isolate was assigned to spa type t843 and to a new allelic profile, ST2676, in CC130 (Table). ST2676 represents a single-locus variant of ST130 carrying a different allele for the gene aroE. MRSA isolates of CC130 have been associated with humans and animals (1–4,6). This result indicates that mecC-carrying S. aureus isolates can be found in urban wastewater, which may act as an environmental reservoir, as has been demonstrated for mecA-carrying S. aureus (10). In conclusion, we detected the methicillin resistance mechanism gene mecC in nonclinical S. aureus isolates from animals and urban wastewater in Spain. Although our data indicate that the frequency of this resistance mechanism is low, this gene appears to be expanding to new areas. Prospective studies should be performed to evaluate epidemiologic changes and to analyze the genetic lineages that carry this resistance mechanism.

Population Analysis and Epidemiological Features of Inhibitor-Resistant-TEM-β-Lactamase-Producing Escherichia coli Isolates from both Community and Hospital Settings in Madrid, Spain

ABSTRACT Punctual mutations in the TEM-1 or TEM-2 gene may lead to inhibitor-resistant-TEM (IRT) β-lactamases with resistance to β-lactam-β-lactamase inhibitor combinations and susceptibility to cephalosporins. The aim of this work was to analyze the current epidemiology of IRT β-lactamases in contemporary clinical Escherichia coli. Isolates were prospectively collected in our hospital (2007 and 2008) from both outpatients (59.8%) and hospitalized patients (40.2%). The genetic relationships of the isolates were determined by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and phylogenetic group analysis. IRT genes were sequenced and located by hybridization, and the incompatibility group of the plasmids was determined. From a total of 3,556 E. coli isolates recovered during the study period, 152 (4.3%) showed reduced susceptibility to amoxicillin-clavulanate, with 18 of them producing IRT enzymes (0.5%). These were mostly recovered from urine (77.8%). A high degree of IRT diversity was detected (TEM-30, -32, -33, -34, -36, -37, -40, and -54), and the isolates were clonally unrelated but were mostly associated with phylogenetic group B2 (55.5%). In 12 out of 16 (75%) isolates, the bla IRT gene was plasmid located and transferred by conjugation in 9 of them, whereas chromosomal localization was demonstrated in 4 isolates (25%). The sizes of the plasmids ranged from 40 kb (IncN) to 100 kb (IncFII, IncFI/FIIA), and they showed different restriction patterns by restriction fragment length polymorphism analysis. Unlike extended-spectrum β-lactamase producers, the frequency of IRT producers remains low in both community and hospital settings, with most of them causing urinary tract infections. Although bla IRT genes are mainly associated with plasmids, they can be also located in the chromosome. Despite this situation, clonal expansion and/or gene dispersion was not observed, denoting the independent emergence of these enzymes.

Rapid Detection of β-Lactamase-Hydrolyzing Extended-Spectrum Cephalosporins in Enterobacteriaceae by Use of the New Chromogenic βLacta Test

ABSTRACT The chromogenic βLacta test developed for the rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae revealed good performance with extended-spectrum β-lactamase (ESBL) producers (97.5% true-positive results). However, false-negative results occurred with chromosomal AmpC hyperproducers and plasmid AmpC producers, whereas uninterpretable results were mostly due to VIM-1 carbapenemase producers and possibly low levels of expressed ESBLs.