Occurrence of beta-lactamases, namely GES-5 carbapenemase, among Gram-negative isolates from wastewater samples in Northern Portugal

Antimicrobial resistant pathogens are profoundly relevant to human health and many were the studies that focused on their spread. However, natural and human associated environmental reservoirs of resistance are yet poorly understood. The main goal of this study was to evaluate the main antibiotic resistance mechanisms in Gram-negative bacteria isolates from different wastewater environments. Water samples were collected from different environments within an urban water cycle in the region of Northern Portugal, which included treated and raw wastewater, water to the consumers and water surface. Screening of antimicrobial susceptibility of 48 Gram-negative isolates (20 Escherichia coli, 8 Citrobacter spp, 7 Klebsiella spp, 6 Kluyvera spp, 2 Enterobacter spp, 1 Hafnia alvei, 1 Pantoea agglomerans, 1Pseudomonas luteola, 1Roultella ornithinolytica, 1Serratia spp) was performed by disk diffusion method. Interpretative reading of susceptibilities allowed to direct the search for antibiotic resistant genes. PCR and sequencing were used to screen and identify bla and plasmid-mediated quinolone resistance (PMQRs) genes. All isolates were also screened for the presence of class 1 integrons. Overall, 29.2% of the isolates were multidrug resistant, suggesting a great diversity of resistance mechanisms. Noteworthy, 2 isolates showed non-susceptibility to carbapenems, which constitutes one of the last resorts on the antimicrobial therapy. Their phenotypic and molecular characterization revealed the expression of a chromosomal metalo-beta-lactamase in P. luteola and the presence of a GES-5 encoding gene in a Klebsiella pneumoniae isolate. Furthermore, we detected a vast variety of beta-lactamase encoding genes, specifically 12 blaTEM-1 with distinct promoters, 4 blaSHV (2 blaSHV-1 and 2 blaSHV-11), besides different chromossomal AmpC beta-lactamases, namely CMY-65. Class 1 integrons were detected among 6 of TEM-1-producing isolates. Together, these beta-lactamases explain the level of beta-lactam resistance. None PMQR genes were detected. In conclusion, this study provides the first description of a class A carbapenemase in an environmental setting in Portugal, in addition to several other beta-lactam resistance mechanisms. The study highlights the need of surveillance of these resistance mechanisms in environmental backgrounds, since it represents a liable reservoir of potential pathogenic resistant bacteria

Diversity of β-lactamase-encoding genes among Gram-negative isolates from water samples in Northern Portugal

Water has been recognized as a reservoir for antibiotic resistance genes (ARG), where the presence of mobile genetic elements, including plasmids, favors their dissemination. It is noteworthy that non- pathogenic environmental organisms, where plasmids encoding multiple ARG are prevalent, can provide resistance to most classes of antimicrobials including :-lactams, aminoglycosides, chloramphenicol, trimethoprim, streptomycin, fosfomycin, quinolones, among others. The main goal of this study was to evaluate the presence of ARGs, related with :-lactam and quinolone resistance, in Gram-negative bacteria isolates from surface and raw and treated waste water environments. Water samples were collected from different environments within an urban water cycle in the region of Northern Portugal, which included treated and raw wastewater, water to the consumers and water surface. Screening of antimicrobial susceptibility of 56 Gram-negative isolates (20 Escherichia coli, 8 Citrobacter spp., 7 Klebsiella spp., 6 Kluyvera spp., 4 Sphingomonas panni, 2 Enterobacter spp., 1 Acinetobacter johnsonii, 3 Aeromonas veronii, 1 Hafnia alvei, 1 Pantoea agglomerans, 1 Roultella ornithinolytica, 1 Serratia sp., 1 Stenotrophomonas maltophilia), identified by 16S rRNA gene sequencing analysis using universal primers, was performed by disk diffusion method. Interpretative reading of susceptibilities allowed to direct the search for antibiotic resistant genes. PCR and sequencing were used to screen and identify beta-lactamase- and plasmid- mediated quinolone resistance (PMQRs)-encoding genes. All isolates were also screened for the presence of class 1 integrons. PCR-based replicon typing (PBRT) was used to type the resistance plasmids of the blaGES-5- producing isolate among the major incompatibility (Inc) groups, specifically FIA, FIB, FIC, HI1, HI2, I1-I , L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. Multilocus sequence typing (MLST) of the GES-5 K. pneumoniae-producing isolate was performed according to the Institute Pasteur scheme (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). Overall, 16/56 isolates were multidrug-resistant (MDR), i.e. presenting a reduced susceptibility to 3 or more structurally unrelated antibiotics, suggesting a great diversity of resistance mechanisms. Noteworthy, 10 isolates (4 S. panni, 1 A. johnsonii, 3 A. veronii, 1 K. pneumoniae, and 1 S. maltophilia) showed nonsusceptibility to carbapenems, which constitutes one of the last resorts on the antimicrobial therapy. Their phenotypic and molecular characterization revealed the expression of several enzymes: the naturally occurring carbapenemase in one S. maltophilia, ImiS in three A. veronii, both MBLs, and OXA-type carbapenemase in one A. johnsonii, responsible for their intrinsic resistance; the class A GES-5-producing K. pneumoniae isolate belonged to a novel MLST sequence type, the ST961 (18-22-18-90-142-13-179). PBRT of the plasmid-carrying blaGES-5 gene showed that it did not belong to any of the Inc groups tested. No carbapenemases were found in the 4 S. panni isolates. The :-lactam resistance, carbapenem susceptibility, found in 33 isolates was justified by the presence of various Class A (12 blaTEM-1 with distinct promoters, 6 blaSHV) and different Class C :-lactamase-encoding genes (blaCMY, blaACC, blaACT), some here firstly described: blaCMY-65 (JF780936), blaCMY-89 (HE819403), blaCMY-90 (HE819404), blaACT-13 (HE819402) and blaACC-5 (HE819401). Class 1 integrons were detected among 6 of TEM- 1-producing isolates. Together, the beta-lactamases identified explain the level of beta-lactam resistance. Besides quinolone resistance detected, none PMQR were identified, suggesting chromosomal alterations in the quinolone resistance-determining region. This study identified ARGs related not only to commonly used antibiotics, but also to carbapenems, providing, at our knowledge, the first description of a GES-5-producing Enterobacteriaceae recovered in an environmental setting. The study highlights the need of surveillance of these antibiotic resistance mechanisms in environmental backgrounds, since it represents a liable reservoir of potential pathogenic resistant bacteria. Worryingly, recent studies demonstrated that while the WWTP reduced the bacterial load, the treatment is inefficient to remove antibiotic resistant bacteria

Two Novel CMY-2-Type β-Lactamases Encountered in Clinical Escherichia Coli Isolates

BACKGROUND: Chromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level

First comparative genomic characterization of the MSSA ST398 lineage detected in aquaculture and other reservoirs

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage.VS has her Ph.D. fellowship granted by FCT (Fundação para a Ciência e a Tecnologia) with the reference SFRH/BD/133100/2017 co-financed by European Social Fund and the Operational Program for Human Capital (POCH), Portugal. This work was financial supported with funding from FCT/MCTES (UIDB/00211/2020) through national funds.info:eu-repo/semantics/publishedVersio

First description of food-borne Salmonella enterica resistance regions R1 and R3 associated with IS26 elements

In this study, we assessed the presence of IS26 in food-borne ASSuT-type Salmonella enterica isolates. A new genetic region (R3) wasdescribed, that included a C14 caspase gene between IS26 elements. R3 was present in two Salmonella Rissen isolates from a swine carcass anda meat handler, collected at the same abattoir. Furthermore, a new rearrangement of resistance region R1, harboring the blaTEM-1 gene flanked byIS26 elements, was identified in Salmonella Typhimurium and Salmonella 4,[5],12:i:-, from different samples. This study highlights the zoonoticpotential of Salmonella spp. isolates and the possible role of IS26 in the mobilization of resistance genes.In this study, we assessed the presence of IS26 in food-borne ASSuT-type Salmonella enterica isolates. A new genetic region (R3) wasdescribed, that included a C14 caspase gene between IS26 elements. R3 was present in two Salmonella Rissen isolates from a swine carcass anda meat handler, collected at the same abattoir. Furthermore, a new rearrangement of resistance region R1, harboring the blaTEM-1 gene flanked byIS26 elements, was identified in Salmonella Typhimurium and Salmonella 4,[5],12:i:-, from different samples. This study highlights the zoonoticpotential of Salmonella spp. isolates and the possible role of IS26 in the mobilization of resistance genes

Feasibility study of an intensive multi-strategy rehabilitation program for Parkinson disease

Poster presented at the 19th International Congress of Parkinson’s Disease and Movement Disorders (MDS Congress 2015). San Diego, 14-18 June 2015

Snapshot of resistome, virulome and mobilome in aquaculture

Aquaculture environments can be hotspots for resistance genes through the surrounding environment. Our objective was to study the resistome, virulome and mobilome of Gram-negative bacteria isolated in seabream and bivalve molluscs, using a WGS approach. Sixty-six Gram-negative strains (Aeromonadaceae, Enterobacteriaceae, Hafniaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Vibrionaceae, and Yersiniaceae families) were selected for genomic characterization. The species and MLST were determined, and antibiotic/disinfectants/heavy metals resistance genes, virulence determinants, MGE, and pathogenicity to humans were investigated. Our study revealed new sequence-types (e.g. Aeromonas spp. ST879, ST880, ST881, ST882, ST883, ST887, ST888; Shewanella spp. ST40, ST57, ST58, ST60, ST61, ST62; Vibrio spp. ST206, ST205). >140 different genes were identified in the resistome of seabream and bivalve molluscs, encompassing genes associated with β-lactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, trimethoprim, phenicols, macrolides and fosfomycin resistance. Disinfectant resistance genes qacE-type, sitABCD-type and formA-type were found. Heavy metals resistance genes mdt, acr and sil stood out as the most frequent. Most resistance genes were associated with antibiotics/disinfectants/heavy metals commonly used in aquaculture settings. We also identified 25 different genes related with increased virulence, namely associated with adherence, colonization, toxins production, red blood cell lysis, iron metabolism, escape from the immune system of the host. Furthermore, 74.2 % of the strains analysed were considered pathogenic to humans. We investigated the genetic environment of several antibiotic resistance genes, including blaTEM-1B, blaFOX-18, aph(3″)-Ib, dfrA-type, aadA1, catA1-type, tet(A)/(E), qnrB19 and sul1/2. Our analysis also focused on identifying MGE in proximity to these genes (e.g. IntI1, plasmids and TnAs), which could potentially facilitate the spread of resistance among bacteria across different environments. This study provides a comprehensive examination of the diversity of resistance genes that can be transferred to both humans and the environment, with the recognition that aquaculture and the broader environment play crucial roles as intermediaries within this complex transmission network.Highlights: - New STs (17) and possible evolutionary relationships with other STs were identified. - Over 140 resistance genes provided a snapshot of the aquaculture resistome. - Many resistance genes found are common to those of clinical isolates (e.g. qnrB19). - Many ARGs detected (e.g. sul) are associated to antibiotics used in aquaculture. - Several (74.2 %) strains studied were classified as pathogenic to human.V.S. has a Ph.D. fellowship granted by the FCT (Fundação para a Ciência e a Tecnologia) with the reference SFRH/BD/133100/2017 cofinanced by European Social Fund and the Operational Program for Human Capital (POCH), Portugal. This work was supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme (WORLDCOM project), and by FCT/MCTES [UIDB/ 00211/2020] through national funds.info:eu-repo/semantics/publishedVersio

Serotypes and Antibiotic Susceptibility of Streptococcus pneumoniae Isolates from Invasive Pneumococcal Disease and Asymptomatic Carriage in a Pre-vaccination Period, in Algeria

In Algeria, few data is available concerning the distribution of pneumococcal serotypes and respective antibiotic resistance for the current pre-vaccination period, which is a public health concern. We identified the most frequent Streptococcus pneumoniae serogroup/types implicated in invasive pneumococcal disease (IPD; n = 80) and carriage (n = 138) in Algerian children younger than 5 years old. Serogroup/types of 78 IPD isolates were identified by capsular typing using a sequential multiplex PCR. Overall, serotypes 14, 19F, 6B, 23F, 18C, 1, 5, 7F, 19A, and 3 (55% of PCV7 serotypes, 71.3% of PCV10, and 90% of PCV13) were identified. Additionally, 7.5% of the non-vaccine serotypes 6C, 9N/L, 20, 24F, 35B, and 35F, were observed. In the case of S. pneumoniae asymptomatic children carriers, the most common serogroup/types were 6B, 14, 19F, 23F, 4, 9V/A, 1, 19A, 6A, and 3 (42.7% of PCV7 serotypes, 44.2% of PCV10, and 58% of PCV13). For 6.1% of the cases co-colonization was detected. Serotypes 14, 1, 5, and 19A were more implicated in IPD (p 2μg/ml). Resistance to cefotaxime was higher in isolates from meningitis (40.5%); however, resistance to erythromycin and co-trimoxazole (>40%) was more pronounced in no-meningeal forms. Overall, our results showed that PCV13 conjugate vaccine would cover up to 90% of the circulating isolates associated with IPD in Algeria, highlighting the importance of monitoring the frequency of S. pneumoniae serogroups/types during pre- and post-vaccination periods

Draft Genomic Analysis of an Avian Multidrug Resistant Morganella morganii Isolate Carrying qnrD1

Morganella morganii is a commensal bacterium and opportunistic pathogen often present in the gut of humans and animals. We report the 4.3 Mbp draft genome sequence of a M. morganii isolated in association with an Escherichia coli from broilers in Portugal that showed macroscopic lesions consistent with colisepticemia. The analysis of the genome matched the multidrug resistance phenotype and enabled the identification of several clinically important and potentially mobile acquired antibiotic resistance genes, including the plasmid-mediated quinolone resistance determinant qnrD1. Mobile genetic elements, prophages, and pathogenicity factors were also detected, improving our understanding toward this human and animal opportunistic pathogen

Staphylococcus aureus and Methicillin-resistant coagulase-negative staphylococci in nostrils and buccal mucosa of healthy camels used for recreational purposes

Several different species of animals host staphylococci as normal microbiota. These animals can be a source of staphylococci zoonotic infections. People with routine or occupational exposure to infected/colonized animals are at risk of a potential transmission. Therefore, we aimed to investigate the presence of S. aureus and other staphylococci in camels used for recreational purposes as well as their antimicrobial resistance, virulence factors and genetic lineages. A total of 172 samples were collected from 86 healthy camels (nose and mouth) from different farms located in the Canary Islands, Spain. Antimicrobial susceptibility testing was performed against 14 antimicrobial agents. The presence of virulence genes was studied by PCR. Multilocus sequence typing, spa typing and agr typing were performed in all S. aureus isolates. From the 86 camels tested, 42 staphylococci were isolated, of which there were 11 S. aureus, 13 S. lentus, 12 S. sciuri, 3 S. xylosus, S. epidermidis, S. hominis and S. chromogenes. Staphylococci isolates were resistant to penicillin, ciprofloxacin, clindamycin and fusidic acid. All S. aureus isolates harbored the hla, hlb and hld virulence genes. S. aureus isolates were ascribed to three sequence types (STs) and three spa types. All S. aureus isolates belonged to agr type III. Camels from Gran Canaria used in recreational purposes have a moderate prevalence of S. aureus and other coagulase-negative staphylococci. Nevertheless, S. aureus isolates are susceptible to almost all antibiotics tested.Na publicação: Newton Verbisck