Detection of Carbapenemase Production in a Collection of Enterobacteriaceae with Characterized Resistance Mechanisms from Clinical and Environmental Origins by Use of Both Carba NP and Blue-Carba Tests.

Rapid-screening methods to confirm the presence of resistance mechanisms in multidrug-resistant bacteria are currently recommended. Carba NP and Blue-Carba tests were evaluated in carbapenemase-producing Enterobacteriaceae from hospital (n = 102) and environmental (n = 57) origins for detecting the different molecular classes among them. Both methods showed to be fast and cost-effective, with high sensitivity (98% to 100%) and specificity (100%), and may be easily introduced in the routine laboratory.

Antibiotic-resistant Klebsiella pneumoniae and Escherichia coli high-risk clones and an IncFII(k) mosaic plasmid hosting Tn1 (blaTEM-4) in isolates from 1990 to 2004.

We describe the genetic background of bla(TEM-4) and the complete sequence of pRYC11::bla(TEM-4), a mosaic plasmid that is highly similar to pKpQIL-like variants, predominant among TEM-4 producers in a Spanish hospital (1990 to 2004), which belong to Klebsiella pneumoniae and Escherichia coli high-risk clones responsible for the current spread of different antibiotic resistance genes. Predominant populations of plasmids and host adapted clonal lineages seem to have greatly contributed to the spread of resistance to extended-spectrum cephalosporins.

Detection of virulence-associated genes characteristic of intestinal Escherichia coli pathotypes, including the enterohemorrhagic/enteroaggregative O104:H4, in bovines from Germany and Spain.

Cattle are reservoirs of enterohemorrhagic Escherichia coli; however, their role in the epidemiology of other pathogenic E. coli remains undefined. A new set of quantitative real-time PCR assays for the direct detection and quantification of nine virulence-associated genes (VAGs) characteristic of the most important human E. coli pathotypes and four serotype-related genes (wzxO104 , fliCH4 , rbfO157 , fliCH7 ) that can be used as a surveillance tool for detection of pathogenic strains was developed. A total of 970 cattle fecal samples were collected in slaughterhouses in Germany and Spain, pooled into 134 samples and analyzed with this tool. stx1, eae and invA were more prevalent in Spanish samples whereas bfpA, stx2, ehxA, elt, est and the rbfO157 /fliCH7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzxO104 /fliCH4 ) were simultaneously detected in six fecal pools from one German abattoir located near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzxO104 /fliCH4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection by this direct approach of VAGs from a novel human pathogenic E. coli strain in cattle samples implies that the E. coli gene pool in these animals can be implicated in de novo formation of such highly-virulent strains. The application of this set of qPCRs in surveillance studies could be an efficient early-warning tool for the emergence of zoonotic E. coli in livestock.

Rapid detection of ß-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae by use of the new chromogenic ßLacta test.

The chromogenic ßLacta test developed for the rapid detection of ß-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae revealed good performance with extended-spectrum ß-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.

Multiclonal dispersal of KPC genes following the emergence of non-ST258 KPC-producing Klebsiella pneumoniae clones in Madrid, Spain.

OBJECTIVES: To analyse the ongoing epidemiology of KPC-producing Enterobacteriaceae after a non-ST258 KPC-3-producing Klebsiella pneumoniae outbreak in a university hospital in Madrid, Spain. METHODS: Enterobacterial isolates (one per patient based on bacterial identification and typing patterns) with carbapenem MICs higher than the EUCAST epidemiological cut-off values, a positive modified Hodge test and carbapenem/boronic acid combination disc test results were studied (16 March 2010 to 31 January 2012) and compared with KPC-producing isolates previously described in our institution (September 2009 to February 2010). The bacterial population structure (PFGE and multilocus sequence typing), carbapenemase genes and KPC plasmids were studied. Patients' clinical records were reviewed. RESULTS: Twenty-four KPC-producing Enterobacteriaceae (20 K. pneumoniae, 2 Escherichia coli and 2 Enterobacter cloacae) from 23 patients (13 males, median age 72 years) were studied. Most KPC-producer strains were considered as colonizers. Six K. pneumoniae clones (ST11, ST20, ST384, ST454, ST659 and ST971), two E. coli clones (ST224 and ST357) and one E. cloacae clone were identified. blaKPC-3 was located on IncFIIK plasmids of ∼100 kb (E. coli ST357 and K. pneumoniae ST384, ST454, ST659 and ST971 clones) and on IncN plasmids of ∼40 kb (K. pneumoniae ST11 clone). Non-typeable plasmids of ∼20 kb containing blaKPC-2 were detected in scarcely represented clones (K. pneumoniae ST20, E. coli ST224 and E. cloacae). CONCLUSIONS: During the 2 year period following the emergence of non-ST258 KPC-3-producing K. pneumoniae isolates in our institution, the blaKPC-3 and blaKPC-2 genes efficiently penetrated other Enterobacteriaceae lineages. Non-ST258 K. pneumoniae isolates were mostly responsible for the dissemination of KPC enzymes, producing a complex epidemiological picture.

Population analysis and epidemiological features of inhibitor-resistant-TEM-beta-lactamase-producing Escherichia coli isolates from both community and hospital settings in Madrid, Spain.

Punctual mutations in the TEM-1 or TEM-2 gene may lead to inhibitor-resistant-TEM (IRT) beta-lactamases with resistance to beta-lactam-beta-lactamase inhibitor combinations and susceptibility to cephalosporins. The aim of this work was to analyze the current epidemiology of IRT beta-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 beta-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.

Early OXA-48-Producing Enterobacterales Isolates Recovered in a Spanish Hospital Reveal a Complex Introduction Dominated by Sequence Type 11 (ST11) and ST405 Klebsiella pneumoniae Clones.

Carbapenemase-producing Enterobacterales (CPE) have become an important public health concern. In our hospital, VIM enzymes were first detected in 2005, Klebsiella pneumoniae carbapenemase (KPC) enzymes in 2009, and OXA-48 enzymes in 2012. We assess the population biology of the first OXA-48-producing Enterobacterales isolates recovered in our hospital (2012 to 2013) where infections by other carbapenemases had been endemic for several years. Over a 21-month period, 71 isolates (61 Klebsiella pneumoniae, 5 Escherichia coli, 2 Klebsiella aerogenes, and 1 each of Enterobacter cloacae, Klebsiella oxytoca, and Citrobacter amalonaticus) recovered from clinical and surveillance specimens from 57 patients (22.8% nonhospitalized) were investigated for OXA-48-like-producing enzymes. Analyses for characterization and determination of the location of the blaOXA-48 gene, plasmid transferability, sequence, and clonal relatedness were performed. Most of the isolates also coproduced CTX-M-15 (57/71, 80.3%) and/or VIM-1 (7/71, 9.8%). K. pneumoniae was predominantly identified as sequence type 11 (ST11) (63.4%) and ST405 (9.8%) and E. coli as ST540, ST1406, ST3163, and ST4301. The blaOXA-48 gene was part of Tn1999.2 located at the tir gene of plasmids (ca. ≥50 kb) of the IncL/M group, also carrying blaVIM-1 and blaCTX-M-15 genes. We selected one ST11 K. pneumoniae isolate for whole-genome sequencing in which we studied the plasmid containing the blaOXA-48 gene. This plasmid was compared with indexed plasmids existing in NCBI database by the use of BRIG and MAUVE. Our data suggest a rapid spread of blaOXA-48 genes between commonly isolated high-risk clones of Enterobacterales species, frequently associated with antibiotic resistance. Moreover, the emergence of the multiresistant ST11 K. pneumoniae clone among nonhospitalized patients emphasizes the difficulty of preventing its dissemination into the community.IMPORTANCE We present results of microbiological analysis of the first Enterobacterales isolates that were isolated in 2012 in our institution expressing OXA-48 carbapenemase. This enzyme confers resistance to carbapenems, an important group of antibiotics widely used in the hospitals. OXA-48 carbapenemase is currently present in many parts of the world, but it is found particularly frequently in the Mediterranean area. It was disseminated at the Ramón y Cajal Hospital and found to be associated with a particular Klebsiella pneumoniae strain, so-called high-risk clone ST11, which was previously found in our institution in association with other enzymes such as CTX-M-15, VIM-1, and KPC-3. This clone might have acquired a plasmid harboring the blaOXA-48 gene. Our results point out the importance of local epidemiology in the dissemination and maintenance of multidrug-resistant bacteria.

Spread of bla(CTX-M-14) is driven mainly by IncK plasmids disseminated among Escherichia coli phylogroups A, B1, and D in Spain.

Since its first description in 2000, CTX-M-14 has become one of the most widespread extended-spectrum beta-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 bla(CTX-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 bla(CTX-M-14) previously designated bla(CTX-M-14a) (n = 59/61) and bla(CTX-M-14b) (n = 2/61) were detected. bla(CTX-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 bla(CTX-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.

High rate of intestinal colonization with extended-spectrum-beta-lactamase-producing organisms in household contacts of infected community patients.

Fecal carriage of extended-spectrum-beta-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.

Complex molecular epidemiology of extended-spectrum beta-lactamases in Klebsiella pneumoniae: a long-term perspective from a single institution in Madrid.

OBJECTIVES: To analyse all extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates recovered from 2001 to 2004 in our institution and to compare this period with that of 1989 to 2000. METHODS: All K. pneumoniae isolates recovered during the studied period were screened for ESBL production. One isolate per patient was selected for ESBL characterization and for population structure, including phylogenetic groups, and plasmid analysis. RESULTS: Ninety-three (3.2% mean) ESBL-producing K. pneumoniae isolates recovered from 61 patients (26%, medical wards; 18%, surgical wards; 25%, ICU; and 31%, outpatients) were identified. Outpatients significantly increased (P < 0.01) when compared with 1989-2000 (7%). The number of different ESBLs increased with persistence of previously identified enzymes (TEM-4, SHV-2, CTX-M-9 and CTX-M-10) and emergence of new ESBLs (TEM-110, SHV-11, SHV-12, CTX-M-14 and CTX-M-15). A polyclonal structure, including epidemic clones with specific ESBLs (TEM-4, SHV-12 and CTX-M-15), was observed. Phylogenetic analysis showed that most isolates (74.6%) belonged to KpI-type with a clear relationship between KpIII-type and CTX-M-10 producers. Persistence of specific plasmids associated with specific ESBLs (TEM-4, SHV-12, CTX-M-10 and CTX-M-15) was observed. Co-resistance analysis revealed an increment in resistance to trimethoprim (41.5% versus 10.3%), sulphonamide (54.7% versus 29.3%) and nalidixic acid (34.0% versus 6.9%) when compared with 1989-2000. CONCLUSIONS: K. pneumoniae is still an important ESBL producer in our institution with a complex epidemiology. The main features were a few outbreaks with persistence of specific plasmids, emergence of new enzymes and an increment in community isolates. These results should be taken into account for the implementation of epidemiological containment measures.

CHROMagar mSuperCARBA performance in carbapenem-resistant Enterobacteriaceae isolates characterized at molecular level and routine surveillance rectal swab specimens.

Performance of the CHROMagar mSuperCARBA media was assessed in both well-characterized carbapenem-resistant Enterobacteriaceae (n=52) and routine surveillance rectal swab specimens (n=211). Limit of detection ranged between 101 and 102CFU/mL except for OXA-48 producers with low-carbapenem MICs (106CFU/mL). High sensitivity (100%) and specificity (100%) were obtained with rectal swabs.

Insights into a Novel blaKPC-2-Encoding IncP-6 Plasmid Reveal Carbapenem-Resistance Circulation in Several Enterobacteriaceae Species from Wastewater and a Hospital Source in Spain.

Untreated wastewater, particularly from hospitals and other healthcare facilities, is considered to be a reservoir for multidrug-resistant bacteria. However, its role in the spread of antibiotic resistances in the human population remains poorly investigated. We used whole genome sequencing to analyze 25 KPC-2-producing Enterobacteriaceae isolates from sewage water collected during a 3-year period and three clinical Citrobacter freundii isolates from a tertiary hospital in the same collection area in Spain. We detected a common, recently described, IncP-6 plasmid carrying the gene blaKPC-2 in 21 isolates from both sources. The plasmid was present in diverse environmental bacterial species of opportunistic pathogens such as C. freundii, Enterobacter cloacae, Klebsiella oxytoca, and Raoultella ornithinolytica. The 40,186 bp IncP-6 plasmid encoded 52 coding sequences and was composed of three uniquely combined regions that were derived from other plasmids recently reported in different countries of South America. The region harboring the carbapenem resistance gene (14 kb) contained a Tn3 transposon disrupted by an ISApu-flanked element and the core sequence composed by ISKpn6/blaKPC-2/ΔblaTEM-1/ISKpn27. We document here the presence of a novel promiscuous blaKPC-2 plasmid circulating in environmental bacteria in wastewater and human populations.

High clonal diversity in a non-outbreak situation of clinical ESBL-producing Klebsiella pneumoniae isolates in the first national surveillance program in Cuba.

This work summarized the results obtained in an institutional Klebsiella pneumoniae surveillance program recently implemented in Cuba. Eighteen hospitals from five regions provided a total of 228 K. pneumoniae isolates (164 from admitted patients, four from hospital environmental sources, and 60 isolates from community patients). The genetic relationship was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. Antimicrobial susceptibility was determined by the agar dilution method, and bla(ESBL) genes were sequenced. Fifty four K. pneumoniae isolates were extended-spectrum ß-lactamases (ESBL)-producers (23.6%), mostly due to the CTX-M-15 enzyme (79.6%). ESBL isolates were grouped in 27 different sequence types (STs), being the most prevalent ST15 (15%), ST152 (13%), and both ST48 and ST147 (11%, respectively). Community-acquired criteria could be demonstrated in 60 patients (26%) corresponding to urological (33%), wound (27%), respiratory (27%), and otic (13%) infections. Population structure analysis showed that our isolates corresponded to a highly polyclonal population with 10 nonpreviously described STs, demonstrating the importance of local epidemiological studies. We report the first data of the population structure of ESBL-producing K. pneumoniae isolates obtained in a national multicenter surveillance Cuban program. Results showed that a highly polyclonal ESBL-producer K. pneumoniae population was mainly due to CTX-M-15 carriage, whereas carbapenemases production was not present.

Assessment of prevalence and changing epidemiology of extended-spectrum beta-lactamase-producing Enterobacteriaceae fecal carriers using a chromogenic medium.

Five hundred fecal samples from 462 patients (68.4% ambulatory) (February-April, 2007) from Madrid (Spain) were screened for extended-spectrum beta-lactamase (ESBL) producers using ceftazidime and cefotaxime (1 mg/L) MacConkey (MAC) agar plates and a chromogenic media (chromID ESBL; bioMérieux, Marcy-l'Etoile, France). bla(ESBL), qnr, aac(6')Ib-cr, and 16S rRNA methylase genes were assessed. A prevalence of 8.2% of ESBL fecal carriers was observed (8.9% hospitalized, 7.9% nonhospitalized patients), higher than that previously observed (1991, 0.6%; 2003, 7.0%). Sensitivity, specificity, and positive and negative predicted values were 100%, 94.8%, 63%, and 100% for chromID ESBL and 87.8%, 89.8%, 43.4%, and 98.9% for MAC, respectively. ESBL distribution was as follows: CTX-M-9-group, 40% (mainly CTX-M-14); CTX-M-1-group, 26.6% (mainly CTX-M-15); SHV-type, 29% (mainly SHV-12); and TEM-type, 4.4%. These enzymes were found in pulsed-field gel electrophoresis nonclonally related Escherichia coli and Klebsiella pneumoniae isolates. Transferable quinolone resistance was confirmed in CTX-M-9 (qnrS1), CTX-M-15 [aac(6')Ib-cr, qnrS1], and SHV-12 (qnrB7, qnrS1) producers but not 16S rRNA methylase genes. The chromID ESBL medium was reliable to screen ESBL fecal carriers with a general decrease in the laboratory workload. Time-to-time monitoring of ESBL fecal carriers is useful to ascertain current trend of ESBL epidemiology.

Differences in biofilm development and antibiotic susceptibility among Streptococcus pneumoniae isolates from cystic fibrosis samples and blood cultures.

OBJECTIVES: To compare the capability of biofilm development between Streptococcus pneumoniae isolates from cystic fibrosis (CF) respiratory samples and those from non-CF blood cultures. Antibiotic susceptibility of biofilm-forming isolates, as well as differences between antibiotic susceptibility of sessile cells [minimum biofilm inhibitory concentration (MBIC)] and their planktonic counterparts (conventional MIC), were also assessed. METHODS: Biofilm formation was performed using a microtitre method in 20 CF and 22 non-CF blood culture S. pneumoniae isolates. RESULTS AND CONCLUSIONS: Biofilm formation occurs more frequently among S. pneumoniae isolates from CF (80%) than among non-CF blood culture isolates (50%) (P = 0.04). Moreover MBICs were significantly higher than conventional planktonic MICs among CF but not among non-CF blood isolates, suggesting a high adaptability of CF strains to form biofilms in adverse conditions.

Real-time PCR for the detection and quantification of geodermatophilaceae from stone samples and identification of new members of the genus blastococcus.

Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus.

In117, an unusual In0-like class 1 integron containing CR1 and bla(CTX-M-2) and associated with a Tn21-like element.

An unusual In0-like class 1 integron containing a common region that includes the putative recombinase gene named orf513 (CR1) and bla(CTX-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.

Antibiotic coresistance in extended-spectrum-beta-lactamase-producing Enterobacteriaceae and in vitro activity of tigecycline.

The spread of extended-spectrum-beta-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 microg/ml; MIC(90), 1 microg/ml) was 4- to 256-fold more active than doxycycline and minocycline (mode MIC range, 2 to 128 microg/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 mug/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.