Impacts of a DUF2207 Family Protein on Streptococcus mutans Stress Tolerance Responses and Biofilm Formation.

Locus SMU.243 in Streptococcus mutans was annotated as a member of the DUF2207 family proteins highly conserved in all bacteria but with unknown function. To investigate its role in S. mutans physiology, a SMU.243-deficient mutant was constructed using allelic exchange mutagenesis, and the impacts of SMU.243 deletion on bacterial growth, stress tolerance response, and biofilm formation were analyzed. Compared to the wild-type UA159, S. mutans lacking SMU.243 displayed a reduced growth rate and a reduced overnight culture density (p < 0.01) when grown at low pH and in the presence of methyl viologen. Relative to the parent strain, the deficient mutant also had a reduced survival rate following incubation in a buffer of pH 2.8 (p < 0.01) and in a buffer containing hydrogen peroxide at 58 mM after 60 min (p < 0.001) and had a reduced capacity in biofilm formation especially in the presence of sucrose (p < 0.01). To study any ensuing functional/phenotypical links between SMU.243 and uppP, which is located immediately downstream of SMU.243 and encodes an undecaprenyl pyrophosphate phosphatase involved in recycling of carrier lipid undecaprenyl phosphate, a uppP deficient mutant was generated using allelic exchange mutagenesis. Unlike the SMU.243 mutant, deletion of uppP affected cell envelope biogenesis and caused major increases in susceptibility to bacitracin. In addition, two variant morphological mutants, one forming rough colonies and the other forming mucoid, smooth colonies, also emerged following the deletion of uppP. The results suggest that the SMU.243-encoded protein of the DUF2207 family in S. mutans plays an important role in stress tolerance response and biofilm formation, but unlike the downstream uppP, does not seem to be involved in cell envelope biogenesis, although the exact roles in S. mutans' physiology awaits further investigation.

Involvement of hpap2 and dgkA Genes in Colistin Resistance Mediated by mcr Determinants.

Plasmid-mediated colistin resistance (mcr) determinants are challenging the efficacy of polymyxins against Gram-negative pathogens. Among 10 mcr genes described so far, the major determinants mcr-1 and mcr-3 are found closely linked to hpap2 or dgkA genes, encoding a hypothetical phosphatidic acid phosphatase of type 2 (PAP2) and a diacylglycerol kinase, respectively, whose functions are still unknown. In this study, mcr-1, mcr-1-hpap2, mcr-3, and mcr-3-dgkA were expressed in Escherichia coli, and recombinant strains were analyzed to detect antimicrobial susceptibility and changes in the expression of genes involved in phospholipid metabolism. The mcr-1 or mcr-3 single genes were enough to drive growth on colistin selective media, although co-expression of linked genes conferred maximal antibiotic resistance. Expression of mcr determinants downregulated endogenous genes involved in lipopolysaccharide (LPS) modification or phospholipid recycling, although to different extents of repression: strong for arnB, ybjG, and pmrR; medium for eptA, lpxT, and dgkA; small for bacA and pgpB. Four of these genes (bacA, lpxT, pgpB, and ybjG) encode undecaprenyl pyrophosphate (UPP) phosphatases. In these conditions, cells presented resistance against bacitracin, an antibiotic that sequesters UPP from PAP2 enzymes. The hpap2 and dgkA genes might play a role in colistin resistance by compensating for phospholipid metabolism functions altered during LPS modification by colistin resistance determinants.

Identification and pathogenicity of an XDR Streptococcus suis isolate that harbours the phenicol-oxazolidinone resistance genes optrA and cfr, and the bacitracin resistance locus bcrABDR.

One hundred and seven Streptococcus suis isolates were collected from healthy pigs or asymptomatic carriers in Jiangsu, China in 2016-2017. Thirty-eight percent of the isolates were linezolid-resistant and all carried the optrA gene. Among them, one isolate, SFJ44, was resistant to all 20 of the antibiotics tested, except for ceftiofur, and thus exhibited an extensively-drug-resistant phenotype. This isolate carried the optrA gene and the bacitracin resistance locus bcrABDR on an antibiotic-resistance-associated genomic island (ARGI1), and harboured the resistance genes cfr, aadE, sat4, spw-like, aphA3, mef(A), msr(D), erm(A)-like, erm(B), tetAB(P)', tet(M) and catQ on ARGI2∼4. The IS1216E-bcrABDR-ISEnfa1 segment showed >99.9% sequence identity to corresponding sequences from other species. The cfr gene was located on ARGI4, and two IS6 family insertion sequences, IS1216E and ISTeha2, were found upstream and downstream of cfr-ΔISEnfa5, respectively. A circular intermediate of bcrABDR-ISEnfa1 was detected, suggesting the role of ISEnfa1 in dissemination of bcrABDR. Other antibiotic resistance genes might be acquired from different Gram-positive pathogens. Infection of zebrafish showed that SFJ44 exhibited a virulence level comparable to serotype 2 hypervirulent strain SC070731, highlighting the need for surveillance of the pathogenicity of multi-drug-resistant S. suis isolates. This is the first report of the co-existence of optrA and cfr, and of the bcrABDR locus in streptococci. As it has been suggested that S. suis may act as an antibiotic resistance reservoir contributing to the spread of resistance genes to major streptococcal pathogens, the potential dissemination of these resistance genes among Gram-positive bacteria is of concern and routine surveillance should be strengthened.

Metagenomic insights into the abundance and composition of resistance genes in aquatic environments: Influence of stratification and geography.

A global survey was performed with 122 aquatic metagenomic DNA datasets (92 lake water and 30 seawater) obtained from the Sequence Read Archive (SRA). Antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) were derived from the dataset sequences via bioinformatic analysis. The relative abundances of ARGs and MRGs in lake samples were in the ranges ND (not detected)-1.34 × 100 and 1.22 × 10-3-1.98 × 10-1 copies per 16S rRNA, which were higher than those in seawater samples. Among ARGs, multidrug resistance genes and bacitracin resistance genes had high relative abundances in both lake and sea water samples. Multi-metal resistance genes, mercury resistance genes and copper resistance genes had the greatest relative abundance for MRGs. No significant difference was found between epilimnion and hypolimnion in abundance or the Shannon diversity index for ARGs and MRGs. Principal coordinates analysis and permutational multivariate analysis of variance (PERMANOVA) test showed that stratification and geography had significant influence on the composition of ARGs and MRGs in lakes (p < 0.05, PERMANOVA). Coastal seawater samples had significantly greater relative abundance and a higher Shannon index for both ARGs and MRGs than deep ocean and Antarctic seawater samples (p < 0.05, Kruskal-Wallis one-way ANOVA), suggesting that human activity may exert more selective pressure on ARGs and MRGs in coastal areas than those in deep ocean and Antarctic seawater.

Antibiotic resistance plasmids and mobile genetic elements of Clostridium perfringens.

Clostridium perfringens is an anaerobic bacterium that is a major human and animal pathogen. The key features of C. perfringens-mediated infections are that disease pathogenesis involves the production of protein toxins and that disease epidemiology generally involves the production of environmentally resistant endospores. Many of the toxins involved in these diseases are encoded on conjugative plasmids that are closely related to the paradigm tetracycline resistance plasmid pCW3. This plasmid encodes the Tet(P) tetracycline resistance determinant, and the tcp locus, which mediates conjugative transfer and is also present on the toxin plasmids. In addition to being directly responsible for the widely dispersed distribution of the Tet(P) determinant, which is not located on a transposable genetic element, this family of conjugative plasmids facilitates the spread of other mobile resistance elements. These elements include the chloramphenicol resistance integrative mobilisable elements typified by Tn4451, the bacitracin resistance integrative conjugative element typified by ICECp1, and the lincomycin resistance transferable insertion sequence typified by tISCpe8. Each of these elements are found on conjugative plasmids that are closely related to pCW3, providing evidence that this large plasmid family has a key role in the distribution of antibiotic resistance genes in C. perfringens.