VIM-1, VIM-34, and IMP-8 Carbapenemase-Producing Escherichia coli Strains Recovered from a Portuguese River

Abstract

The emergence of acquired carbapenemases is currently one of the most serious public health threats worldwide. These enzymes confer resistance to almost all ␤-lactams, including carbapenems leading to very few therapeutic options for treating patients infected by multidrug-resistant bacteria.Here we report the identification of four environmental carbapenem-resistant Escherichia coli strains, E61, E201, E202, and E203. These strains were recovered in February 2015, from the Ave river, in the north of Portugal. Water samples of 100 ml were filtered through 0.45-m-pore-size membrane filters (Millipore Corporation, USA), which were then placed on tryptone bile Xglucuronide agar (TBX) (BioKar Diagnostics, Beauvais, France) plates supplemented with imipenem (2 mg/liter). Strains growing on those selective plates were checked for carbapenemase activity by using the Carba NP test (1). Antimicrobial susceptibility testing was performed according to the standard disk diffusion method following CLSI recommendations (2) and using cation-adjusted Muller-Hinton plates (Bio-Rad, Cressier, Switzerland). The four isolates were resistant to all ␤-lactams, fluoroquinolones, and aminoglycosides (except amikacin), being susceptible only to tigecycline and colistin. Two out of the four isolates (E61 and E202) also remained susceptible to fosfomycin. MICs of carbapenems (imipenem, ertapenem, and meropenem) were performed using Etest strips (bioMérieux, La Balme-les-Grottes, France) ( Table 1). Molecular investigations were then performed by PCR using specific primers for carbapenemase genes (3) followed by sequencing (Microsynth, Balgach, Switzerland). The phylogenetic group was determined as previously described (4) and showed that isolates E201 and E203 belonged to group E, strain E61 belonged to group A, and strain E202 belonged to group F. Multilocus sequence typing performed as described previously (5) confirmed that isolates E201 and E203 belonged to the same sequence type, whereas the two other strains were clonally unrelated. PCR and sequencing analysis revealed that strains E201 and E203 harbored the bla IMP-8 gene and strains E61 and E202 harbored the bla VIM-1 and bla genes, respectively.Conjugation assays were performed in liquid medium using the azide-resistant E. coli J53 as the recipient strain. Transconjugants were selected onto Luria-Bertani agar plates supplemented with ertapenem (2 mg/liter) and azide (100 mg/liter) to detect the transfer of the carbapenemase genes. Transconjugants were obtained for three out of the four isolates, but not for strain E202 despite repeated attempts. Plasmid extractions were performed using the Kieser extraction method (6) using the environmental strains and transconjugants. Further analysis showed that the bla VIM-1 and bla IMP-8 genes were carried on a ca. 150-kb plasmid. Molecular typing of plasmids was performed using the PCR-based replicon typing (Diatheva, Fano, Italy) (7), revealing that all plasmids bearing the carbapenemase genes belonged to the IncFIB group.The failure to o...

Carbapenemase-producing enterobacteriaceae recovered from a Spanish river ecosystem

“…Surprisingly, in the present study, in which the isolates were recovered only one year later, no OXA-48 was detected and VIM-type carbapenemases were represented at a lower frequency than KPC-type enzymes. Previous results available in the literature also describe a major prevalence of KPC and VIM determinants in the aquatic environment [3,9,12,29]; whereas, to our knowledge, no report has detected an OXA-48-producing CPE in the same type of samples.…”

“…Some studies have been conducted in Portugal [9], US [10], Tunisia [11], India [12] and China [13,14], but none in Spain. Moreover, little attention has been directed towards the prevalence of PAS and PPS in carbapenemase-encoding plasmids.…”

The rapid spread of carbapenem-resistant Enterobacteriaceae

“…Two E. coli isolates expressing bla IMP-8 and two E. coli isolates expressing bla VIM-1 and bla VIM-34 were obtained in February 2015 from the Ave river in northern Portugal. bla IMP-8 and bla VIM-1 were found on IncFIB type plasmids capable of transconjugation while bla VIM-34 was suspected to be chromosomally integrated [Kieffer 2016]. As reported in 2016, water collected from Rio De Janeiro yielded many bla KPC producing Enterobacteriaceae as well as bla GES , bla OXA-48-like , and bla NDM genes amplified directly from aquatic microbial communities [de Araujo 2016].…”

Reusing Treated Wastewater: Consideration of the Safety Aspects Associated with Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes

“…A general pattern has been observed, in which ARB and ARGs emerge not long after new antibiotics are introduced to the market [13]. Correspondingly, ARGs associated with particularly life-threatening infections, encoding resistance to a wide range of antibiotics, have been detected in WWTPs or in the environment soon after the first reports of clinical cases [14,15]. Phenomena of proliferation, bioaccumulation, and transport have been observed for ARB and ARGs in human-impacted environments, bringing to light the potential for exposure to humans and the need to delineate possible health risks [11,16,17].…”