Impact of oral amoxicillin and amoxicillin/clavulanic acid treatment on bacterial diversity and ß-lactam resistance in the canine faecal microbiota.

BACKGROUND: Aminopenicillins with or without a ß-lactamase inhibitor are widely used in both human and veterinary medicine. However, little is known about their differential impact on the gut microbiota and development of antimicrobial resistance. OBJECTIVES: To investigate changes in the faecal microbiota of dogs treated with amoxicillin or amoxicillin/clavulanic acid. METHODS: Faeces collected from 42 dogs (21 per treatment group) immediately before, during and 1 week after termination of oral treatment with amoxicillin or amoxicillin/clavulanic acid were analysed by culture and 16S rRNA gene sequence analysis. RESULTS: In both groups, bacterial counts on ampicillin selective agar revealed an increase in the proportion of ampicillin-resistant Escherichia coli during treatment, and an increased occurrence and proportion of ampicillin-resistant enterococci during and after treatment. 16S rRNA gene analysis showed reductions in microbial richness and diversity during treatment followed by a return to pre-treatment conditions approximately 1 week after cessation of amoxicillin or amoxicillin/clavulanic acid treatment. While no significant differences were observed between the effects of amoxicillin and amoxicillin/clavulanic acid on microbial richness and diversity, treatment with amoxicillin/clavulanic acid reduced the abundance of taxa that are considered part of the beneficial microbiota (such as Roseburia, Dialister and Lachnospiraceae) and enriched Escherichia, although the latter result was not corroborated by phenotypic counts. CONCLUSIONS: Our results suggest a limited effect of clavulanic acid on selection of antimicrobial resistance and microbial richness when administered orally in combination with amoxicillin. However, combination with this ß-lactamase inhibitor appears to broaden the spectrum of amoxicillin, with potential negative consequences on gut health.

Chromobacterium spp. harbour Ambler class A ß-lactamases showing high identity with KPC.

OBJECTIVES: The origin of KPC is unknown. The aim of this study was to detect progenitors of KPC in silico and to functionally verify their ß-lactam hydrolysis activity. METHODS: The sequence of KPC-2 was used to mine the NCBI protein sequence database. The best non-KPC hits were analysed by amino acid (aa) alignment and phylogenetic tree construction. Genes encoding KPC-2 homologues were expressed in Escherichia coli. The carbapenemase activities of the recombinant strains were characterized by the CarbaNP test and UV spectrophotometry and MICs of selected ß-lactams were determined. RESULTS: Genes encoding the closest KPC-2 homologues were identified on the chromosome of Chromobacterium piscinae strain ND17 (CRP-1, 76% aa identity), Chromobacterium sp. C-61 (CRS-1, 70% aa identity) and Chromobacterium haemolyticum DSM19808 (CRH-1, 69% aa identity). All three Chromobacterium ß-lactamases were phylogenetically more related to KPC than to other Ambler class A ß-lactamases. The 27 bp region preceding the start codon of blaCRP-1 displayed high nucleotide identity to the corresponding region upstream from blaKPC (74%). Heterologous expression of blaCRP-1 and to a lesser extent of blaCRH-1 in E. coli significantly increased the MICs of meropenem and most cephalosporins. The CarbaNP test was positive for both recombinant strains, but spectrophotometric analysis confirmed higher carbapenemase activity for CRP-1-producing clones. CONCLUSIONS: The recovery of three class A ß-lactamases with up to 76% aa identity to KPC from distinct Chromobacterium species is highly indicative of the role played by this genus in the evolution of KPC.

The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing ß-Lactamases of Potential Clinical Relevance.

The origin of carbapenem-hydrolyzing metallo-ß-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacterium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemase production was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA ß-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.

Biochemical Characterization of CPS-1, a Subclass B3 Metallo-ß-Lactamase from a Chryseobacterium piscium Soil Isolate.

CPS-1 is a subclass B3 metallo-ß-lactamase from a Chryseobacterium piscium isolate collected from soil, showing 68% amino acid identity to the GOB-1 enzyme. CPS-1 was overproduced in Escherichia coli Rosetta (DE3), purified by chromatography, and biochemically characterized. This enzyme exhibits a broad-spectrum substrate profile, including penicillins, cephalosporins, and carbapenems, which overall resembles those of L1, GOB-1, and acquired subclass B3 enzymes AIM-1 and SMB-1.

vanO, a new glycopeptide resistance operon in environmental Rhodococcus equi isolates.

We describe here the sequence and gene organization of a new glycopeptide resistance operon (vanO) in Rhodococcus equi from soil. The vanO operon has low homology to enterococcal van operons and harbors a vanHOX cluster transcribed in the direction opposite that of the vanS-vanR regulatory system and composed of three open reading frames with unknown function. This finding has clinical interest, since glycopeptides are used to treat R. equi infections and resistance has been reported in clinical isolates.

New insights into Staphylococcus aureus stress tolerance and virulence regulation from an analysis of the role of the ClpP protease in the strains Newman, COL, and SA564.

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.