Teresa Ribeiro

Phylogeny and Comparative Genomics Unveil Independent Diversification Trajectories of qnrB and Genetic Platforms within Particular Citrobacter Species

ABSTRACT To gain insights into the diversification trajectories of qnrB genes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose, Citrobacter sp. isolates (n = 21) and genome or plasmid sequences (n = 56) available in public databases harboring complete or truncated qnrB genes were analyzed. Citrobacter species identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates, the location of qnrB genes, and the genetic surroundings of qnrB genes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification of Citrobacter isolates was achieved using leuS and recN gene sequences, and isolates characterized in this study were diverse and harbored chromosomal qnrB genes. Phylogenetic analysis of all known qnrB genes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprising pspF and sapA and varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of these qnrB clusters, and the reliable identification of all Citrobacter isolates revealed that each platform evolved in different recognizable (Citrobacter freundii, C. braakii, C. werkmanii, and C. pasteurii) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome of Citrobacter spp. and in different plasmids of Enterobacteriaceae. Our data corroborate Citrobacter as the origin of qnrB and further suggest divergent evolution of closely related qnrB genes/platforms in particular Citrobacter spp., which were delineated using particular genotypic markers.

Long-term dissemination of acquired AmpC β-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 β-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 blaqAmpC 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 β-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.

Filling the map for antimicrobial resistance in sub-Saharan Africa: ampicillin-resistant Enterococcus from non-clinical sources in Angola

Sir, A recent compilation of antimicrobial resistance (AMR) data from sub-Saharan Africa (sSA) by Leopold et al. did not consider the role of non-clinical contexts for the dispersion of AMR. Also, among the microorganisms presented they did not include Enterococcus spp., opportunistic bacteria associated with community and hospital infections worldwide, mainly in individuals with underlying risk factors often identified in sSA (e.g. immunocompromised). – 6 AMR rates of Enterococcus from diverse sSA environments and hosts were variable among countries, but often included resistance to b-lactams, widely used due to their low cost and ready availability. – 8 Ampicillin resistance (AmpR) is considered a marker of worldwide Enterococcus faecium (Efm) nosocomial epidemic clones [Bayesian Analysis of Population Structure (BAPS) subgroups 2.1a and 3.3a], although in sSA the genetic background of AmpR-Efm is barely known in clinical or community settings (http://pubmlst.org/). Among the countries pointed out by Leopold et al. with very limited AMR data is Angola, an emergent economy with a population of 24 million, recently leaving almost four decades of civil war and still with a precarious health system (http://www.who. int/countryfocus/cooperation_strategy/ccs_ago_en.pdf?ua=1). As part of a surveillance project on the occurrence of Grampositive and Gram-negative bacteria resistant to key antibiotics used to treat human infections, we searched for AmpR-Efm in humans, animals and the environment from Benguela province (June 2013). Samples (n1⁄462) from different regions of Benguela province, hosts and environments were collected from: (i) healthy humans who did not take antibiotics for 3 months before the sampling (rectal swabs; n1⁄420); (ii) a wild park (free grey monkeys-n1⁄42 and springboks-n1⁄43 fresh faeces; water from animal drinkers and a cistern supplying the drinkers-n1⁄42); (iii) 4 farm production animals and their environment [rectal swabs of healthy chickens-n1⁄45 (pools of 13 animals), of cows-n1⁄43 (pools of 9 animals) and of pigs-n1⁄42 (pools of 6 animals); swabs from facility walls/floor-n1⁄42; water from animal drinkers-n1⁄43]; (iv) rivers serving inhabitants of Lobito and Catumbela cities-n1⁄43, and treated-n1⁄47 and untreated-n1⁄45 (4 boreholes and 1 fountain) human drinking waters; and (v) residual waters from two points of a Benguela wastewater treatment station or from urban/hospital sewer lines of Benguela, Damba-Maria and Catumbela cities-n1⁄45. Waters were filtered (100 mL) and then these and all other samples were enriched in peptone water+8 mg/L ampicillin. A 0.1 mL aliquot was plated in Slanetz–Bartley agar+8 mg/L ampicillin and different bacteria morphotypes per plate were selected. Resistance to 12 antibiotics was evaluated by disc diffusion/Etest following EUCAST guidelines or, when not possible, CLSI guidelines. Species, AMR genes [tet(MLOSK), erm(B), aadE and pbp5] and virulence factors (IS16, esp, hyl and acm) were searched by PCR and clonality by MLST. A high percentage (10%, n1⁄46/62) of samples (1 from hospital+community residual water, 2 from chicken farm facilities, 2 from chicken pool faeces and 1 from pig pool faeces) carried AmpR isolates (n1⁄48), which were identified as Efm with variable ampicillin MICs (12 to .256 mg/L) (http://www.eucast.org/ fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_5. 0_Breakpoint_Table_01.pdf) and belonging to ST610 (BAPS 2.3a) and ST245, ST650 and ST971 (all BAPS 2.1b), the latter described here for the first time (Table 1). Recently, BAPS 2.1b was positively associated with animal-origin ampicillin-susceptible Efm, contrasting with our AmpR isolates. Also, Efm (AmpR or unknown phenotype) from different STs of BAPS 2.1b and 2.3a were previously linked to human infections in sSA and other African regions (e.g. Tanzania and Algeria) (http://pubmlst.org/). Amino-acid sequence analysis of PBP5 from four AmpR-Efm showed three different sequences, corresponding mostly to the AmpR consensus region previously described (Table S1, available as Supplementary data at JAC Online; GenBank accession numbers KR140010, KR140011, KR140012 and KR140013). Two sequences were new, while a chicken farm facility Efm PBP5 was previously identified in 19 Efm from Europe/the USA/Israel (bloodstream infections-n1⁄410 and unknown origin-n1⁄49) belonging to hospital-associated clonal lineages (BAPS 2.1a and 3.3a; 1992– 2006) (Table S1). All but one of our AmpR-Efm were MDR. They were resistant to tetracycline [n1⁄47; tet(M), n1⁄46 and tet(L), n1⁄43], high concentrations of streptomycin (n1⁄46; aadE negative), quinupristin/dalfopristin (n1⁄45), erythromycin [n1⁄45; erm(B)], ciprofloxacin (n1⁄44) and nitrofurantoin (n1⁄44). Among virulence genes, acm was identified in five isolates of chicken and chicken farm facilities origins. In summary, the occurrence of MDR AmpR-Efm outside of an Angolan hospital context suggests b-lactam selective pressure, supporting the data for other countries and pathogens. This is of concern as b-lactams are critical in the treatment of diverse enterococcal infections in sSA, where antibiotics such as glycopeptides are not readily available. The results of this study (Efm of BAPS groups linked to sSA acute infections and PBP5 type

Citrobacter portucalensis sp. nov., isolated from an aquatic sample

A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter, and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter. Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6 % and 58.4 % identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter. The G+C content of strain A60T is 52.0 %. The results obtained support the description of a novel species within the genus Citrobacter, for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).

Citrobacter europaeus sp. nov., a novel Citrobacter species isolated from water and human faecal samples.

Strains 97/79T and A121, recovered respectively from human faeces and well water, were compared to currently known species of the genus Citrobacter using genotypic and phenotypic approaches. Multilocus sequence analysis based on housekeeping genes fusA, leuS, pyrG, rpoB and recN, showed that the two strains formed a distinct phylogenetic lineage within the genus Citrobacter. Average nucleotide identity (ANI) between strains 97/79T and A121 was 99.2 %, whereas ANI values of strain 97/79T with the type strains of closely related species of the genus Citrobacter, C. werkmanii, C. braakii, C. freundii, C. youngae and C. pasteurii, were all below 93.0 %. The ability to metabolize different compounds also discriminated strains 97/79T and A121 from other species of the genus Citrobacter. Based on these results, strains 97/79T and A121 represent a novel species of the genus Citrobacter, for which the name Citrobacter europaeus sp. nov. is proposed, with strain 97/79T (=CIP 106467T=DSM 103031T) as the type strain. The DNA G+C content of strain 97/79T is 52.0 %.

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.