Dynamics of clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings over time.

OBJECTIVES: The objective of this work was to provide detailed molecular data on clinically acquired AmpC (qAmpC)-producing Enterobacteriaceae from two different periods (2002-2008 and 2010-2013) in order to clarify the contribution of clonal and plasmid genetic platforms for the current epidemiological scenario concerning extended-spectrum beta-lactams resistance. METHODS: We analysed 1246 Enterobacteriaceae non-susceptible to third-generation cephalosporins from two hospitals and one community laboratory between 2010 and 2013. Bacterial identification, antibiotic susceptibility, identification of qAmpC and plasmid-mediated quinolone resistance genes, clonal (pulsed-field gel electrophoresis (PFGE), Multilocus sequence typing (MLST)) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed by standard methods. Whole-genome sequencing (WGS) was performed in two ST11-Klebsiella pneumoniae isolates harbouring DHA-1. RESULTS: The occurrence of qAmpC was lower (2.6%) than that observed in a previous survey (7.4%), and varied slightly over time. Isolates produced DHA-1 (53%), CMY-2 (44%) or DHA-6 (3%), but significant epidemiological changes were observed in the two surveys. While DHA-1 persisted in different institutions by selection of a worldwide epidemic IncR plasmid in an ST11 harbouring KL105, CMY-2 rates increased over time linked to IncI1 plasmids (instead of IncK or IncA/C2) in multiple Escherichia coli clones. CONCLUSIONS: The higher frequency of DHA-1 qAmpC in these species contrasts with the scenario in most European countries. Furthermore, the different genetic backgrounds associated with either extended-spectrum ß-lactamases (ESBLs) or acquired AmpC ß-lactamases (qAmpC) in our country might have contributed to their differential expansion.

Different Escherichia coli B2-ST131 clades (B and C) producing extended-spectrum ß-lactamases (ESBL) colonizing residents of Portuguese nursing homes.

ESBL-producing Enterobacteriaceae and particularly Escherichia coli ST131 isolates producing CTX-M enzymes are commonly found colonizing the intestine of nursing home (NH) residents, but ST131 subclonal structure has been scarcely explored in this vulnerable population. Our goal was to perform a pilot study to assess the faecal carriage rate and epidemiological features of ESBL- and/or carbapenemase-producing Enterobacteriaceae (ESBL-E and CPE, respectively) among NH residents. For this purpose, faecal samples from residents at 4 different NHs in the North of Portugal (representing 9·5% of the residents' population, July 2014) were screened for ESBL-E and/or CPE by phenotypic and genotypic methods. Clonal structure and plasmid typing of ESBL-producing E. coli (ESBL-Ec) was performed by PCR and sequencing. Four ESBL-Ec isolates (2 CTX-M-15/2 CTX-M-14) were found in 20% of the samples, all belonging to the pandemic clonal lineage B2-ST131-O25b:H4. Two different clades were identified, the C2/H30-Rx-virotype C producing CTX-M-15 and an atypical B/H22-like-virotype D5 (producing CTX-M-14 and fluoroquinolone-resistant), firstly described in Portugal. This pilot study highlights the role of NH residents as a source of different ST131 clades, besides emphasizing the importance of E. coli B2-ST131 subtyping in different clinical settings, and understanding the transmission dynamics of the different variants.

High occurrence and unusual serotype diversity of non-typhoidal Salmonella in non-clinical niches, Angola.

Non-typhoidal Salmonella is an important burden, particularly in developing countries of the African region. We report for the first time in Angola, a sub-Saharan African country with commercial/travel relationships with Europe, an unexpectedly high occurrence of Salmonella (n = 12/63, 19%) from a high diversity of sources, particularly farm and wild animals. The detection of diverse serotypes (n = 12), involving putative new S. enterica subsp. salamae serotypes, is also of note, reinforcing the need for a comprehensive surveillance in Angola critical to identify animal/food/environmental sources of salmonellosis with impact on animal health, local people, tourists and exported products.

Elucidating constraints for differentiation of major human Klebsiella pneumoniae clones using MALDI-TOF MS.

The establishment of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) in routine microbial identification boosted many developments towards high-throughput applications, including bacterial typing. However, results are still controversial for different bacterial species. We aim to evaluate the suitability of MALDI-TOF MS for typing clinically relevant multidrug resistant (MDR) Klebsiella pneumoniae subsp. pneumoniae clones using routine conditions and a previously validated chemometric analysis workflow. Mass spectra of 83 K. pneumoniae clinical isolates representing major human MDR clones [11 sequence types (STs), 22 PFGE-types] recovered in Portugal and Spain during outbreaks and non-outbreak situations (2003-2012) were obtained from cell extracts (CE) and intact cells (IC), and analysed with different chemometric tools. We observed a highly consistent peak pattern among isolates from different clones either with CE or IC, suggesting a high degree of conservation of biomolecules analysed (a large part corresponding to ribosomal proteins). Moreover, the low degree of agreement between MALDI-TOF MS and other methods (from 34.9 % to 43.4 % of correct assignments for CE and from 40.8 % to 70.1 % for IC) corroborates the low discriminatory potential of the technique at infraspecies level. Our results suggest a low discriminatory power of MALDI-TOF MS for clinically relevant MDR K. pneumoniae clones and highlight the need of developing tools for high-resolution typing in this species.

Atypical epidemiology of CTX-M-15 among Enterobacteriaceae from a high diversity of non-clinical niches in Angola.

OBJECTIVES: The objective of this study was to investigate the distribution and molecular epidemiology of ESBLs, acquired AmpCs and carbapenemases in Enterobacteriaceae from non-clinical niches in Angola, an under-researched sub-Saharan country. METHODS: Eighty-one samples were recovered from healthy persons (n = 18), healthy animals (n = 33) and their environments (n = 10) or aquatic settings (n = 20) in south Angola (2013). Samples were plated onto CHROMagar™ Orientation with/without antibiotics. Standard methods were used for bacterial identification, characterization of bla genes, antibiotic susceptibility testing and conjugation assays. Clonal analysis (XbaI-PFGE, MLST and Escherichia coli phylogroups), location of bla and plasmid characterization (S1-PFGE, I-CeuI-PFGE, replicon typing and hybridization) were also performed. RESULTS: ESBLs (almost exclusively CTX-M-15, 98%) were detected in 21% (45/216) of the isolates, recovered from diverse non-clinical niches and belonging to different Enterobacteriaceae species (mainly E. coli). Acquired AmpCs or carbapenemases were not found. The pandemic B2-ST131 E. coli clone was not identified, but some widespread clonal complexes (CCs) from A (CC10 and CC168), B1 (CC156) or D (CC38) phylogroups were detected. blaCTX-M-15 was variably identified on typeable (29%; 100-335 kb; IncFII, IncFIIK6, IncHI2 and IncY) or non-typeable (16%; 70-330 kb) plasmids or on the chromosome (14%), while for 41% of the isolates its specific location was not determined. CONCLUSIONS: This study reports, for the first time in Angola, an unexpected high occurrence of CTX-M-15 in diverse non-clinical niches and Enterobacteriaceae species, and uncovers novel plasmid replicons in under-researched geographical regions. The diffusion of blaCTX-M-15 through such a high diversity of genetic backgrounds (clones, typeable/non-typeable plasmids and genetic environments) unveils an extraordinary ability for blaCTX-M-15 acquisition and mobilization favoured by unrecognized ecological factors.

Salmonellosis: the role of poultry meat.

Salmonellosis remains one of the most frequent food-borne zoonoses, constituting a worldwide major public health concern. Currently, at a global level, the main sources of infection for humans include meat products, including the consumption of contaminated poultry meat, in spite of the success of Salmonella control measures implemented in food-animal production of industrialized countries. In recent years, a shift in Salmonella serotypes related to poultry and poultry production has been reported in diverse geographical regions, being particularly associated with the spread of certain well-adapted clones. Moreover, antimicrobial resistance in non-typhoidal Salmonella is considered one of the major public health threats related with food-animal production, including the poultry production chain and poultry meat, which is an additional concern in the management of salmonellosis. The circulation of the same multidrug-resistant Salmonella clones and/or identical mobile genetic elements encoding antibiotic resistance genes from poultry to humans highlights this scenario. The purpose of this review was to provide an overview of the role of poultry meat on salmonellosis at a global scale and the main problems that could hinder the success of Salmonella control measures at animal production level. With the increasing globalization of foodstuffs like poultry meat, new problems and challenges might arise regarding salmonellosis control, making new integrated intervention strategies necessary along the food chain.

Characterization of the emerging clinically-relevant multidrug-resistant Salmonella enterica serotype 4,[5],12:i:- (monophasic variant of S. Typhimurium) clones.

To better understand the recent success/emergence of Salmonella enterica serotype 4,[5],12:i:- we characterized the population diversity, fljAB deletion patterns, antibiotic resistance features and associated genetic elements of a comprehensive collection obtained in the last decade from Portugal (2002-2010). One hundred thirty-one isolates from human clinical specimens, food, environment and piggeries, verified by PCR as S. 4,[5],12:i:-, were studied for clonality (Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing), antibiotic resistance by phenotypic (disk diffusion and/or agar dilution) and genotypic (PCR/Restriction Fragment Length Polymorphism and sequencing, genomic location) methods and fljAB-deletions (PCR). Plasmid analysis included determination of size, content and characterization of the incompatibility group (PCR-Based Replicon Typing and I-CeuI/S1-hybridization). Results showed three multidrug-resistant (MDR) clones circulating and causing infections, associated with particular phenotypic and genotypic features. Most of the isolates belonged to the widespread European (ASSuT phenotype, RR1-RR2 resistance regions, ST34) and Spanish (carrying a sul3-type III integron within IncA/C plasmids, ST19) clones circulating in Europe. A third clone, here designated Southern European clone (carrying a sul3-type I integron within IncR plasmids, ST19), presents a fljAB region different from the previous clones and similar to the US strains, despite differences in the MDR mobile genetic platforms. The success of S. 4,[5],12:i:- might be related to the selective advantage offered by MDR profiles associated with stable genetic elements, also carrying virulence features, along with well adapted clones to the animal food production and causing human infections.

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.

Discrimination of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex species by Fourier transform infrared spectroscopy.

The main goal of this work was to assess the ability of Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) to discriminate between the species of the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex, i.e. A. baumannii, A. nosocomialis, A. pittii, A. calcoaceticus, genomic species "Between 1 and 3" and genomic species "Close to 13TU". A total of 122 clinical isolates of the Acb complex previously identified by rpoB sequencing were studied. FTIR-ATR spectra was analysed by partial least squares discriminant analysis (PLSDA) and the model scores were presented in a dendrogram form. This spectroscopic technique proved to be effective in the discrimination of the Acb complex species, with sensitivities from 90 to 100%. Moreover, a flowchart aiming to help with species identification was developed and tested with 100% correct predictions for A. baumannii, A. nosocomialis and A. pittii test isolates. This rapid, low cost and environmentally friendly technique proved to be a reliable alternative for the identification of these closely related Acinetobacter species that share many clinical and epidemiological characteristics and are often difficult to distinguish. Its validation towards application on a routine basis could revolutionise high-throughput bacterial identification.

Long-term dissemination of acquired AmpC ß-lactamases among Klebsiella spp. and Escherichia coli in Portuguese clinical settings.

We investigated the occurrence, diversity and molecular epidemiology of genes coding for acquired AmpC ß-lactamases (qAmpC) among clinical isolates of Enterobacteriaceae lacking inducible chromosomal AmpCs in Portugal. A total of 675 isolates non-susceptible to broad-spectrum cephalosporins obtained from four hospitals and three community laboratories during a 7-year period (2002-2008) were analysed. The presence of genes coding for qAmpC was investigated by phenotypic criteria, polymerase chain reaction (PCR) and sequencing. Bacterial identification, antibiotic susceptibility testing, conjugation assays and clonal analysis were performed by standard procedures. The presence of bla(qAmpC) genes was detected in 50 % (50/100; 41 Klebsiella pneumoniae, 5 Escherichia coli, 4 Klebsiella oxytoca) of the presumptive qAmpC producers. DHA-1, detected in those species, was the most prevalent qAmpC (94 %, 47/50), being identified since 2003 and throughout the studied period in different institutions. Despite the high clonal diversity observed, three DHA-1-producing Klebsiella spp. clones were more frequently identified. CMY-2 (6 %, 3/50) was observed in B1-E. coli clones. Conjugative transfer was only observed in one (2 %) CMY-2-producing isolate. Most qAmpC producers (94 %, 47/50) co-expressed SHV-type and/or OXA-1 or CTX-M-32 extended-spectrum ß-lactamases (ESBLs). To the authors' knowledge, this is the first description of the molecular epidemiology and the long-term dissemination of qAmpC-producing Enterobacteriaceae in Portuguese clinical settings, highlighting an evolution towards a more complex epidemiological situation regarding cephalosporin resistance in Portugal.

Spread of an OmpK36-modified ST15 Klebsiella pneumoniae variant during an outbreak involving multiple carbapenem-resistant Enterobacteriaceae species and clones.

We aim to characterise multiple ertapenem-resistant (ERT-R, n = 15) Enterobacteriaceae isolates identified as presumptive carbapenemase producers in a Portuguese hospital in a short period of time (March-July 2010). Antibiotic susceptibility patterns, ß-lactamases, genetic relatedness [pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST)], plasmid content and major enterobacterial porins were investigated. Ertapenem resistance was associated with deficiencies in major porins and, in some cases, extended-spectrum ß-lactamase (ESBL) or AmpC ß-lactamase production among outbreak and non-outbreak clones. Most isolates (n = 8) corresponded to two ERT-R Klebsiella pneumoniae ST15 PFGE-types: (i) a sporadic variant (Kp-A-ERT, n = 1) presenting a premature stop codon in ompK36 and (ii) an epidemic variant (Kp-B-ERT, n = 7) exhibiting a new OmpK36 porin variant, which differed additionally in plasmid and antibiotic susceptibility profiles. ST14 (n = 1) and ST45 (n = 1) K. pneumoniae, ST131 (n = 1) and ST354 (n = 1) Escherichia coli, Enterobacter asburiae (n = 1), Enterobacter cloacae (n = 1) and Enterobacter aerogenes (n = 1) ERT-R clones were also sporadically detected. Porin changes in these isolates included non-sense mutations [ompK35, ompK36, ompF; minimum inhibitory concentration (MIC) = 4-32 mg/l], IS-mediated porin disruptions (ompK36, ompC; MIC = 12->32 mg/l) or alterations in the L3 loop (ompK36; MIC = 4-16 mg/l). We describe, for the first time in Portugal, the simultaneous emergence of multiple ERT-R Enterobacteriaceae species and clones in a short period of time. Moreover, our results support that a CTX-M-15-producing ST15 K. pneumoniae with an OmpK36-modified porin might successfully spread in the nosocomial setting.

Understanding the dynamics of imipenem-resistant Acinetobacter baumannii lineages within Portugal.

A recent collection of 213 imipenem-resistant Acinetobacter baumannii (IRAB) clinical isolates was characterized for the presence of acquired carbapenem-hydrolysing class D ß-lactamases (CHDLs) and clonality. A population structure analysis of IRAB was also conducted, with five molecular typing methods. Three main clusters, each one associated with a specific CHDL, were observed with multilocus sequence typing. Overall, our results suggest a switch in the dominant clone, with sequence type (ST) 92, carrying bla(OXA-23) (63.4%), replacing the closely related ST98, carrying bla(OXA-24/40) (22%). In addition, ST103, an independent lineage, was associated with bla(OXA-58) -carrying isolates (14.6%).

Optimization of processing conditions for the quantification of enterococci biofilms using microtitre-plates.

We performed a comparison of four selected protocols from the literature using microtitre-plate and colorimetric biomass assay for evaluation of enterococci biofilm formation ability and optimized a protocol after the identification and stringent performance of biofilm formation steps. The optimized protocol uses a dynamic model that provided a greater discrimination of enterococci biofilm formation ability, and could better simulate in vivo real conditions. Moreover different biofilm quantification approaches, such as the colorimetric biomass (crystal violet), the resazurin and CFU's assays could be used with the optimized protocol, with adequate reproducibility. This study also recognizes that parameters such as the biofilm formation index (BFi), the cut-off values and the Z' factor provide greater accuracy, possibility of inter and intra laboratory comparison and quality evaluation of the biofilm screening assays, respectively.

Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues.

Acquired carbapenemases are emerging resistance determinants in Gram-negative pathogens, including Enterobacteriaceae, Pseudomonas aeruginosa and other Gram-negative non-fermenters. A consistent number of acquired carbapenemases have been identified during the past few years, belonging to either molecular class B (metallo-beta-lactamases) or molecular classes A and D (serine carbapenemases), and genes encoding these enzymes are associated with mobile genetic elements that allow their rapid dissemination in the clinical setting. Therefore, detection and surveillance of carbapenemase-producing organisms have become matters of major importance for the selection of appropriate therapeutic schemes and the implementation of infection control measures. As carbapenemase production cannot be simply inferred from the resistance profile, criteria must be established for which isolates should be suspected and screened for carbapenemase production, and for which tests (phenotypic and/or genotypic) should be adopted for confirmation of the resistance mechanism. Moreover, strategies should be devised for surveillance of carbapenemase producers in order to enable the implementation of effective surveillance programmes. The above issues are addressed in this article, as a follow-up to an expert meeting on acquired carbapenemases that was recently organized by the ESCMID Study Group for Antibiotic Resistance Surveillance.

Emergence and spread of vancomycin resistance among enterococci in Europe.

Nowadays, six types of acquired vancomycin resistance in enterococci are known; however, only VanA and to a lesser extent VanB are widely prevalent. Various genes encode acquired vancomycin resistance and these are typically associated with mobile genetic elements which allow resistance to spread clonally and laterally. The major reservoir of acquired vancomycin resistance is Enterococcus faecium; vancomycin-resistant Enterococcus faecalis are still rare. Population analysis of E. faecium has revealed a distinct subpopulation of hospital-acquired strain types, which can be differentiated by molecular typing methods (MLVA, MLST) from human commensal and animal strains. Hospital-acquired E. faecium have additional genomic content (accessory genome) including several factors known or supposed to be virulence-associated. Acquired ampicillin resistance is a major phenotypic marker of hospital-acquired E. faecium in Europe and experience has shown that it often precedes increasing rates of VRE with a delay of several years. Several factors are known to promote VRE colonisation and transmission; however, despite having populations with similar predispositions and preconditions, rates of VRE vary all over Europe.

Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe.

Extended-spectrum beta-lactamases (ESBLs) represent a major threat among resistant bacterial isolates. The first types described were derivatives of the TEM-1, TEM-2 and SHV-1 enzymes during the 1980s in Europe, mainly in Klebsiella pneumoniae associated with nosocomial outbreaks. Nowadays, they are mostly found among Escherichia coli isolates in community-acquired infections, with an increasing occurrence of CTX-M enzymes. The prevalence of ESBLs in Europe is higher than in the USA but lower than in Asia and South America. However, important differences among European countries have been observed. Spread of mobile genetic elements, mainly epidemic plasmids, and the dispersion of specific clones have been responsible for the increase in ESBL-producing isolates, such as those with TEM-4, TEM-24, TEM-52, SHV-12, CTX-M-9, CTX-M-14, CTX-M-3, CTX-M-15 and CTX-M-32 enzymes.

Illegal use of nitrofurans in food animals: Contribution to human salmonellosis?

Recent observations in Portugal of a remarkable incidence (65%) of Salmonella isolates from several sources (predominantly human and poultry) with decreased susceptibility to nitrofurantoin (MIC > or =64 mg/L), mostly comprising serogroup D isolates of Salmonella Enteritidis belonging to different phage types, suggest that illegal use of nitrofurans, especially in the poultry industry, might have contributed to the selection and prevalence of S. Enteritidis in food animals, and consequently to human salmonellosis in Portugal. Indiscriminate use of nitrofurans might also be implicated in the emergence of two multiresistant Salmonella Typhimurium clones disseminated throughout the country.

Antimicrobial resistance among faecal enterococci from healthy individuals in Portugal.

Analysis of 247 faecal enterococcal isolates from 99 healthy Portuguese individuals during 2001 revealed the presence of enterococci resistant to vancomycin (5%) and highly resistant to streptomycin (52%), kanamycin (40%) or gentamicin (11%). Most isolates were also resistant to tetracycline, erythromycin, ciprofloxacin and quinupristin-dalfopristin. The vanA (two Tn1546 types), vanC1, erm(B), aac(6')-aph(2'')-Ia, aph(3')-IIIa, vat(E) and vat(D) genes were detected. Enterococcus faecalis and Enterococcus faecium isolates with high-level resistance to gentamicin were related to Portuguese poultry isolates described previously. E. faecium isolates that were highly resistant to vancomycin or gentamicin harboured different housekeeping purK alleles associated previously with different hosts.