Ângela Novais

Dissemination of Clonally Related Escherichia coli Strains Expressing Extended-Spectrum β-Lactamase CTX-M-15

E. coli ST131 and ST405 and multidrug-resistant IncFII plasmids may determine spread of this lactamase.

Extended-spectrum β-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/A, ST23 complex/A and ST131/B2

In this study, we investigated the population structure of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in Spain and determined possible associations between specific multilocus sequence typing (MLST) types and ESBL types. Ninety-two ESBL-producing E. coli isolates from 11 Spanish hospitals were studied. The predominant ESBLs in this collection were CTX-M-14 (45.7%), SHV-12 (21.7%) and CTX-M-9 (20.6%). Phylogenetic groups and MLST types were studied. Thirty-seven isolates (40.2%) belonged to phylogroup A, 26 (28.3%) to group B1, 13 (14.1%) to group B2 and 16 (17.4%) to group D. Fifty-six sequence types (STs) were identified, but ST131 (eight isolates) and ST167 (five isolates) were the most prevalent. The most common ST complexes were ST10 (13 isolates; 14.3%) and ST23 (10 isolates; 11%). Escherichia coli ST131 carried six different ESBLs (CTX-M-1, CTX-M-9, CTX-M-10, CTX-M-14, CTX-M-15 and SHV-12), E. coli ST10 complex carried five ESBLs and E. coli ST23 complex carried four ESBLs. A great diversity of MLST types was observed among Spanish ESBL-producing E. coli isolates.

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.

Evolutionary Trajectories of Beta-Lactamase CTX-M-1 Cluster Enzymes: Predicting Antibiotic Resistance

Extended-spectrum beta-lactamases (ESBL) constitute a key antibiotic-resistance mechanism affecting Gram-negative bacteria, and also an excellent model for studying evolution in real time. A shift in the epidemiology of ESBLs is being observed, which is characterized by the explosive diversification and increase in frequency of the CTX-M-type β-lactamases in different settings. This provides a unique opportunity for studying a protein evolutionary radiation by the sequential acquisition of specific mutations enhancing protein efficiency and fitness concomitantly. The existence of driver antibiotic molecules favoring protein divergence has been investigated by combining evolutionary analyses and experimental site-specific mutagenesis. Phylogenetic reconstruction with all the CTX-M variants described so far provided a hypothetical evolutionary scenario showing at least three diversification events. CTX-M-3 was likely the enzyme at the origin of the diversification in the CTX-M-1 cluster, which was coincident with positive selection acting on several amino acid positions. Sixty-three CTX-M-3 derivatives containing all combinations of mutations under positively selected positions were constructed, and their phenotypic efficiency was evaluated. The CTX-M-3 diversification process can only be explained in a complex selective landscape with at least two antibiotics (cefotaxime and ceftazidime), indicating the need to invoke mixtures of selective drivers in order to understand the final evolutionary outcome. Under this hypothesis, we found congruent results between the in silico and in vitro analyses of evolutionary trajectories. Three pathways driving the diversification of CTX-M-3 towards the most complex and efficient variants were identified. Whereas the P167S pathway has limited possibilities of further diversification, the D240G route shows a robust diversification network. In the third route, drift may have played a role in the early stages of CTX-M-3 evolution. Antimicrobial agents should not be considered only as selectors for efficient mechanisms of resistance but also as diversifying agents of the evolutionary trajectories. Different trajectories were identified using a combination of phylogenetic reconstructions and directed mutagenesis analyses, indicating that such an approach might be useful to fulfill the desirable goal of predicting evolutionary trajectories in antimicrobial resistance.

Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized patients: a successful story of international clones (ST15, ST147, ST336) and epidemic plasmids (IncR, IncFIIK).

The aim of this study was to characterize by a multi-level approach extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eight ESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitals located in the North (A) or Centre (B, C) regions during two time periods (2006-7 and 2010) were analyzed. Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL characterization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis. Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2, -5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local and temporal variations. The increase of CTX-M-15 and diverse SHV ESBL-types observed in Hospital A was associated with the amplification of multidrug-resistant (MDR) K. pneumoniae epidemic clones (ST15, ST147, ST336). SHV-12 and TEM-type ESBLs were mostly identified in diverse isolates of different Enterobacteriaceae species in Hospitals B and C in 2006-7. Particular plasmid types were linked to blaCTX-M-15 (IncR or non-typeable plasmids), blaSHV-12 (IncR or IncHI2), blaSHV-28/-55/-106 (IncFIIK1 or IncFIIK5), blaTEM-10 (IncL/M) or blaTEM-24 (IncA/C), mostly in epidemic clones. In our country, the amplification of CTX-M-15 and diverse SHV-type ESBL among non-E. coli Enterobacteriaceae is linked to international MDR K. pneumoniae clones (ST15, ST147, ST336) and plasmid types (IncR, IncFIIK). Furthermore, we highlight the potential of IncFIIK plasmids (here firstly associated with blaSHV-2/-28/-55/-106) to disseminate as antibiotic resistance plasmids.

Characterization of Globally Spread Escherichia coli ST131 Isolates (1991 to 2010)

ABSTRACT The characterization of a broad representative sample of ST131 Escherichia coli isolates from different origins and settings (1991 to 2010) revealed that this clonal group has likely diversified recently and that the expansion of particular variants has probably been favored by the capture of diverse, multidrug-resistant IncFII plasmids (pC15-1a, pEK499, pKF3-140-like). The low ability to adhere and to grow as biofilm that was detected in this study suggests unknown mechanisms for the persistence of this clonal group which need to be further explored.

International Spread and Persistence of TEM-24 Is Caused by the Confluence of Highly Penetrating Enterobacteriaceae Clones and an IncA/C2 Plasmid Containing Tn1696::Tn1 and IS5075-Tn21

ABSTRACT TEM-24 remains one of the most widespread TEM-type extended-spectrum β-lactamases (ESBLs) among Enterobacteriaceae. To analyze the reasons influencing its spread and persistence, a multilevel population genetics study was carried out on 28 representative TEM-24 producers from Belgium, France, Portugal, and Spain (13 Enterobacter aerogenes isolates, 6 Escherichia coli isolates, 6 Klebsiella pneumoniae isolates, 2 Proteus mirabilis isolates, and 1 Klebsiella oxytoca isolate, from 1998 to 2004). Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] and E. coli phylogroups) and antibiotic susceptibility were determined by standard procedures. Plasmid analysis included determination of the incompatibility group (by PCR, hybridization, and/or sequencing) and comparison of restriction fragment length polymorphism (RFLP) patterns. Characterization of genetic elements conferring antibiotic resistance included integrons (classes 1, 2, and 3) and transposons (Tn3, Tn21, and Tn402). Similar PFGE patterns were identified among E. aerogenes, K. pneumoniae, and P. mirabilis isolates, while E. coli strains were diverse (phylogenetic groups A, B2, and D). Highly related 180-kb IncA/C2 plasmids conferring resistance to kanamycin, tobramycin, chloramphenicol, trimethoprim, and sulfonamides were identified. Each plasmid contained defective In0-Tn402 (dfrA1-aadA1, aacA4, or aacA4-aacC1-orfE-aadA2-cmlA1) and In4-Tn402 (aacA4 or dfrA1-aadA1) variants. These integrons were located within Tn21, Tn1696, or hybrids of these transposons, with IS5075 interrupting their IRtnp and IRmer. In all cases, blaTEM-24 was part of an IS5075-ΔTn1 transposon within tnp1696, mimicking other genetic elements containing blaTEM-2 and blaTEM-3 variants. The international dissemination of TEM-24 is fuelled by an IncA/C2 plasmid acquired by different enterobacterial clones which seem to evolve by gaining diverse genetic elements. This work highlights the risks of a confluence between highly penetrating clones and highly promiscuous plasmids in the spread of antibiotic resistance, and it contributes to the elucidation of the origin and evolution of TEM-2 ESBL derivatives.

MALDI-TOF mass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131) and D (ST69, ST405, ST393).

Reliable, quick and low-cost methods are needed for the early detection of multidrug-resistant and highly virulent high-risk B2 and D Escherichia coli clones or clonal complexes (HiRCC). Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) seems to have a good discriminatory potential at different subspecies levels, but it was never evaluated for the discrimination of E. coli clones. We assessed the potential of MALDI-TOF MS coupled to multivariate data analysis to discriminate representative E. coli B2 and D HiRCC. Seventy-three E. coli isolates from B2 (including ST131 and B2 non-ST131 clones) and D (ST69, ST393, ST405) with variable pulsed-field gel electrophoresis (PFGE) patterns, origins and dates (1980–2010) were tested. MS spectra were acquired from independent extracts obtained from different plate cultures in two different Microflex LT MALDI-TOF devices (Bruker) after a standard extraction procedure. MALDI-TOF MS fingerprinting analysis revealed a good discriminatory ability between the four HiRCC analysed (ST131, ST69, ST405, ST393) and between B2 ST131 and other B2 non-ST131 isolates. Clusters defined by MALDI-TOF MS were consistent with the clonal complexes assigned by multilocus sequence typing (MLST), although differences were detected regarding the composition of clusters obtained by the comparison of PFGE profiles. We demonstrate, for the first time, that characteristic mass fingerprints of different E. coli HiRCC are sufficiently discriminatory and robust to enable their differentiation by MALDI-TOF MS, which might represent a promising tool for the optimisation of infection control, individual patient management and large-scale epidemiological studies of public health relevance. The good correlation between phenotypic and genotypic features further corroborates phylogenetic relationships delineated by MLST.

In Vitro Selection of Variants Resistant to β-Lactams plus β-Lactamase Inhibitors in CTX-M β-Lactamases: Predicting the In Vivo Scenario?

ABSTRACT CTX-M β-lactamases are the most prevalent group of enzymes within the extended-spectrum β-lactamases (ESBL). The therapeutic options for CTX-M-carrying isolates are scarce, forcing the reexamination of the therapeutic possibilities of β-lactams plus β-lactamase inhibitors (BBLIs). Inhibitor-resistant CTX-M β-lactamases (IR-CTX-M) have not hitherto been described in natural isolates. In this study, 168 cultures of the hypermutagenic Escherichia coli GB20 strain carrying plasmid pBGS18 with different blaCTX-M genes were submitted to parallel experimental evolution assays in the presence of increasing concentrations of a combination of amoxicillin and clavulanate. Fourteen CTX-M β-lactamases belonging to the three most representative clusters (CTX-M-1, -2, and -9) and the two main phenotypes (cefotaxime resistance and cefotaxime-ceftazidime resistance) were studied. Three types of IR-CTX-M mutants were detected, having mutations S130G, K234R, and S237G, which are associated with different resistance patterns. The most frequently recovered mutation was S130G, which conferred the highest resistance levels to BBLIs (reaching 12 μg/ml for amoxicillin-clavulanate and 96 μg/ml for piperacillin-tazobactam when acquired by CTX-M-1 cluster enzymes). The S130G change also provided a clear antagonistic pleiotropy effect, strongly decreasing the enzymes activity against all cephalosporins tested. A double mutation, S130G L169S, partially restored the resistance against cephalosporins. A complex pattern observed in CTX-M-58, carrying P167S and S130G or K234R changes, conferred ESBL and IR phenotypes simultaneously. The K234R and S237G changes had a smaller effect in providing inhibitor resistance. In summary, IR-CTX-M enzymes might evolve under exposure to BBLIs, and the probability is higher for enzymes belonging to the CTX-M-1 cluster. However, this process could be delayed by antagonistic pleiotropy.

Role of Common blaOXA-24/OXA-40-Carrying Platforms and Plasmids in the Spread of OXA-24/OXA-40 among Acinetobacter Species Clinical Isolates

ABSTRACT The spread of OXA-24/OXA-40 (OXA-24/40)-producing Acinetobacter spp. in the Iberian Peninsula has been strongly influenced by clonal expansion, but the role of horizontal gene transfer has scarcely been explored. blaOXA-24/40-carrying plasmids and genetic environments were characterized in representative (n = 15) Acinetobacter species clinical isolates (obtained between 2001 and 2007) by Acinetobacter baumannii PCR-based replicon typing, sequencing, hybridization, and restriction fragment length polymorphism. Besides the identification of blaOXA-24/40 within the chromosomes of some isolates, the circulation of common blaOXA-24/40-carrying plasmids (30-kb repA_AB; 10-kb aci2) and genetic backbones among Acinetobacter spp. was demonstrated.