Comprehensive genome data analysis establishes a triple whammy of carbapenemases, ICEs and multiple clinically relevant bacteria

Carbapenemases inactivate most β-lactam antibiotics, including carbapenems, and have frequently been reported among Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. Traditionally, the horizontal gene transfer of carbapenemase-encoding genes (CEGs) has been linked to plasmids. However, given that integrative and conjugative elements (ICEs) are possibly the most abundant conjugative elements among prokaryotes, we conducted an in silico analysis to ascertain the likely role of ICEs in the spread of CEGs among all bacterial genomes (n=182 663). We detected 17 520 CEGs, of which 66 were located within putative ICEs among several bacterial species (including clinically relevant bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli). Most CEGs detected within ICEs belong to the IMP, NDM and SPM metallo-beta-lactamase families, and the serine beta-lactamase KPC and GES families. Different mechanisms were likely responsible for acquisition of these genes. The majority of CEG-bearing ICEs belong to the MPFG, MPFT and MPFF classes and often encode resistance to other antibiotics (e.g. aminoglycosides and fluoroquinolones). This study provides a snapshot of the different CEGs associated with ICEs among available bacterial genomes and sheds light on the underappreciated contribution of ICEs to the spread of carbapenem resistance globally

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 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 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

Unveiling clones and integrons dynamics associated with carbapenemase-producing P. aeruginosa clinical isolates in a portuguese hospital

[Excerpt] Particular epidemic lineages (e.g. 235, 111, ST175 and 244) are associated with carbapenemase-producing P. aeruginosa (CPPA) isolates causing infections. Due to limited therapeutic options and possible carbapenemase spread, CPPA infections are of great concern. Nevertheless, the occurrence, genetic background and clonal dynamics over a long period of time of CPPA strains in Portuguese hospitals are unknown. The study aimed to assess the occurrence and the genetic background of CPPA isolates obtained from a Portuguese University Hospital. Carbapenem-resistant P. aeruginosa isolates obtained from different biological samples from inpatients attending the Hospital Geral de Santo António, Porto during 2006 (n=27) and 2011-3 (n=135) were included. Carbapenemase production was searched by Blue-Carba. Carbapenemase and associated integrons were characterized by PCRs and sequencing. Antimicrobial susceptibility was performed by disc diffusion, E-test and agar dilution methods. Clonality was assessed by MLST. The bla genes location was assessed by I-CeuI/ S1 PFGE and hybridization with specific probes. Plasmid analysis included identification of incompatibility groups by PCR and electrotransformation of P. aeruginosa PAO1. [...

Diversity of non-clinical Acinetobacter species in a sub-saharan Africa region: evidence of carbapenem-hydrolysing class D-β-lactamase producers

[Excerpt] Although Acinetobacter baumannii has been the main agent for healthcare infections, recent reports suggest that some Acinetobacter environmental species should be considered as a potential cause of disease. In Angola, there are no previous data on its environmental reservoirs and resistance features. We aimed to unveil the occurrence and diversity of Acinetobacter species and the presence of resistance mechanisms in different non-clinical settings in Angola

Environmental reservoirs of Acinetobacter-producing carbapenem-hydrolysing class D-beta-lactamases in Angolawith evidence of human exposure

Although Acinetobacter baumannii has been the main agent for healthcare infections, recent reports suggest that some Acinetobacter environmental species should be considered as a potential cause of disease. In Angola, an African country with an emerging economy, there are no previous data on Acinetobacter environmental reservoirs and resistance features. We aimed to investigate the occurrence and diversity of Acinetobacter species and the presence of resistance mechanisms in different non-clinical settings in Angola. Sixty-three samples collected from different sources in Benguela (2013) were included: faeces [healthy volunteers (n=8), wildlife (n=5) and healthy farm animals (n=10)]; water [rivers (n=3), wastewaters (n=5), treated (n=7) and untreated (n=5) drinking water for human/animal, water for farm (n=3) and wildlife (n=2) animals]; animal feed (n=3) and floor/walls farm samples (n=2). Identification was performed by MALDI-TOF MS. Presumptive Acinetobacter isolates were confirmed by rpoB partial gene sequencing. Antimicrobial susceptibility was assessed by disc diffusion/E-test methods. Carbapenemase activity was search by Blue-CARBA. Carbapenemase genes (blaNDM, blaIMP, blaVIM, blaOXA-51, blaOXA-23, blaOXA-58, blaOXA-24, blaKPC) and their genetic context were investigated by PCR and sequencing. Genetic location for carbapenemase genes was determined by I-CeuI and S1-hybridizations. Clonality was studied by ApaI-PFGE. Acinetobacter species were detected in 46% of samples and some harbored more than one species: faeces [healthy volunteers (n=8/63; A.baumannii, A.ursingii, A.junii, A.berezinae), healthy farm animals (n=7; A.genospecies 15TU, A.gerneri, A.baumannii, A.pittii, A.soli)], water [rivers (n=3; A.junii, A.towneri, A.johnsonii, A.baumannii, A.pittii, A.soli)], wastewater (n=4; A.junii, A.towneri, A.baumannii, A.pittii), treated drinking water for humans (n=1; A.johnsonii, A.baumannii, A.pittii) and untreated drinking water for humans/animals (n=6; A.junii, A.baumannii, A.pittii)]. Susceptibility to aminoglycosides and quinolones was variable. Eleven isolates had reduced susceptibility to carbapenems but only 4 presented carbapenemase activity: ANG 1-4. ANG1, identified as A.johnsonii, showed resistance to cefotaxime, cefepime and intermediate behaviour to ciprofloxacin, tetracycline, ceftriaxone, ceftazidime, imipenem (MIC=1.5mg/L) and meropenem (MIC=0.75mg/L), presenting blaOXA-58 followed by ISAba3 and associated with a~75 kb plasmid. ANG 2-4 were identified as A.towneri by MALDI-TOF MS but rpoB only confirmed the close relatedness to this species. ANG2-4 had MICs>ECOFF at least to one carbapenem and revealed blaOXA-23 preceded by ISAba1, being epidemiologically unrelated (ApaI-PFGE). The wide range of environments studied revealed a high diversity of Acinetobacter species. It is of note the frequent detection (36%) of A.baumannii, considered a hospital-adapted species. This work also describes the first environmental OXA-58-producing A.johnsonii isolate (domestic drinking water), and OXA-23-producing A.towneri (river and wastewater). These findings could suggest that human action might drive the spread of antibiotic resistance genes to geographic areas with low selection pressures (carbapenems are not approved in Angola), or that these regions might be at the origin of these genes. In any case, they could act as important reservoirs in the global epidemiological context. Keywords: Acinetobacter, Angola, Carbapenem-resistance.info:eu-repo/semantics/publishedVersio

Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020

Background: The pet industry is expanding worldwide, particularly raw meat -based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibioticresistant bacteria or zoonotic pathogens. Aim: We aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last -line antibiotics such as colistin. Methods: Fifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics. Results: Only RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella . One turkey -based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/ cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance ( bla TEM + strA-strB + sul2 + tet(B) ) and metal tolerance profiles ( pco + sil + ars ) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/ cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide. Conclusion: Some RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last -line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet -food industry and consumer levels, are needed to mitigate these public health risks