Canine Staphylococcaceae circulating in a Kenyan animal shelter.

Animal shelters, especially in resource-poor countries, bring together pets from different regions and with different backgrounds. The crowding of such animals often results in infectious diseases, such as respiratory infections. This study characterized Staphylococcaceae from diseased and apparently healthy dogs housed in an animal shelter in Kenya, to determine their antibiotic resistance profiles, their genetic relatedness, and the presence of dominant clones. Therefore, bacteria were collected from all 167 dogs present in the shelter in June 2015 and screened for Staphylococcaceae using standard cultivation techniques. In all, 92 strains were isolated from 85 dogs and subsequently sequenced by PacBio long-read sequencing. Strains encompassed nine validated species, while S. aureus (n = 47), S. pseudintermedius (n = 21), and Mammaliicoccus (M.) sciuri (n = 16) were the three most dominant species. Two S. aureus clones of ST15 (CC15) and ST1292 (CC1) were isolated from 7 and 37 dogs, respectively. All 92 strains isolated were tested for their antimicrobial susceptibility by determining the minimum inhibitory concentrations. In all, 86 strains had resistance-associated minimal inhibitory concentrations to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, kanamycin/gentamicin, or streptomycin. Many virulence-encoding genes were detected in the S. aureus strains, other Staphylococcaceae contained a different set of homologs of such genes. The presence of mobile genetic elements, such as plasmids and prophages, known to facilitate the dissemination of virulence- and resistance-encoding genes, was also assessed. The unsuspected high presence of two S. aureus clones in about 50% of dogs suggests dissemination within the shelter and a human source.IMPORTANCEMicrobiological data from sub-Saharan Africa are scarce compared to data from North America, Europe, or Asia, and data derived from dogs, the man's best friend, kept in sub-Saharan Africa are largely missing. This work presents data on Staphylococcaceae mainly isolated from the nasal cavity of dogs stationed at a Kenyan shelter in 2015. We characterized 92 strains isolated from 85 dogs, diseased and apparently healthy ones. The strains isolated covered nine validated species and we determined their phenotypic resistance and characterized their complete genomes. Interestingly, Staphylococcus aureus of two predominant genetic lineages, likely to be acquired from humans, colonized many dogs. We also detected 15 novel sequence types of Mammaliicoccus sciuri and S. pseudintermedius indicating sub-Saharan-specific phylogenetic lineages. The data presented are baseline data that guide antimicrobial treatment for dogs in the region.

Clinical significance of Nosocomiicoccus ampullae isolated from blood cultures.

IMPORTANCE: Nosocomiicoccus species are recently described as members of the Staphylococcaceae family. With their inclusion in commercial matrix-assisted laser desorption/ionization-time of flight mass spectrometry databases, Nosocomiicoccus species can now be identified when Gram-positive cocci in clusters are detected in positive blood cultures. However, their clinical significance is not known, making it difficult for the clinical microbiology laboratory to decide the extent of work-up. Based on our study, Nosocomiicoccus species demonstrate low pathogenicity and opportunistic potential. If isolated from a single blood culture set, limited work-up should be performed to an extent similar to other possible blood culture contaminants.

Phylogenomic and molecular markers based studies on Staphylococcaceae and Gemella species. Proposals for an emended family Staphylococcaceae and three new families (Abyssicoccaceae fam. nov., Salinicoccaceae fam. nov. and Gemellaceae fam. nov.) harboring four new genera, Lacicoccus gen. nov., Macrococcoides gen. nov., Gemelliphila gen. nov., and Phocicoccus gen. nov.

The family Staphylococcacae and genus Gemella contain several organisms of clinical or biotechnological importance. We report here comprehensive phylogenomic and comparative analyses on 112 available genomes from species in these taxa to clarify their evolutionary relationships and classification. In a phylogenomic tree based on 678 core proteins, Gemella species were separated from Staphylococcacae by a long branch indicating that they constitute a distinct family (Gemellaceae fam. nov.). In this tree, Staphylococcacae species formed two main clades, one encompassing the genera Aliicoccus, Jeotgalicoccus, Nosocomiicoccus and Salinicoccus (Family "Salinicoccaceae"), while the other clade consisted of the genera Macrococcus, Mammaliicoccus and Staphylococcus (Family Staphylococcaceae emend.). In this tree, species from the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus each formed two distinct clades. Two species clades for these genera are also observed in 16S rRNA gene trees and supported by average amino acid identity analysis. We also report here detailed analyses on protein sequences from Staphylococcaceae and Gemella genomes to identify conserved signature indels (CSIs) which are specific for different genus and family-level clades. These analyses have identified 120 novel CSIs robustly demarcating different proposed families and genera. The identified CSIs provide independent evidence that the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus consist of two distinct clades, which can be reliably distinguished based on multiple exclusively shared CSIs. We are proposing transfers of the species from the novel clades of the above four genera into the genera Gemelliphila gen. nov., Phocicoccus gen. nov., Macrococcoides gen. nov. and Lacicoccus gen. nov., respectively. The identified CSIs also provide strong evidence for division of Staphylococcaceae into an emended family Staphylococcaceae and two new families, Abyssicoccaceae fam. nov. and Salinicoccaceae fam. nov. All of these families can be reliably demarcated based on several exclusively shared CSIs.

The emergence of novel macrolide resistance island in Macrococcus caseolyticus and Staphylococcus aureus of food origin.

Food-derived Staphylococcaceae species with severe antimicrobial resistance, especially Staphylococcus aureus, is a major threat to public health. Macrococcus caseolyticus (M. caseolyticus) is a member of the Staphylococcaceae family which plays a vital role in fermented products and disease causation in animals. In our previous study, several Staphylococcus aureus antibiotic-resistant island msr (SaRImsr) were found in multidrug-resistant S. aureus. In this study, novel SaRImsr, SaRImsr-III emerged from S. aureus. Another novel SaRImsr-like further emerged in M. caseolyticus from food. These isolates' prevalence and genetic environment were investigated and characterized to understand the distribution and transmission of these novel SaRImsr strains. All SaRImsr-positive S. aureus isolates exhibited a multidrug resistance (MDR) phenotype, within which a series of antimicrobial resistance genes (ARGs) and virulence factor genes (VFs) were identified. In addition, three SaRImsr types, SaRImsr-I (15.1 kb), SaRImsr-II (16-17 kb), and SaRImsr-III (18 kb) carrying mef(D)-msr(F), were identified in these isolates' chromosomes. SaRImsr-(I-III) contains a site-specific integrase gene int and operon mef(D)-msr(F). SaRImsr-III has an additional orf3-orf4-IS30 arrangement downstream of mef(D) and msr(F). Moreover, the SaRImsr-like and macrolide-resistant transposon Tn6776 forming a novel mosaic structure coexisted in one M. caseolyticus isolate. Within this mosaic structure, the macrolide-resistant genes mef(D)-msr(F) were absent in SaRImsr-like, whereas an operon, mef(F)-msr(G), was identified in Tn6776. The SaRImsr-(I-III) and SaRImsr-like structure were inserted into the rpsI gene encoding the 30S ribosomal protein S9 in the chromosome. Excision and cyclisation of SaRImsr-III, SaRImsr-like, operon mef(D)-msr(F), and orf3-orf4-IS30 arrangements were confirmed using two-step PCR. This study is the first to report MDR S. aureus harbouring novel SaRImsr-III and M. caseolyticus containing novel mosaic structures isolated from retail foods. Similar SaRImsr-type resistant islands' occurrence and propagation in Staphylococcaceae species require continuous monitoring and investigation.

Genomic Characterization and Antimicrobial Susceptibility of Dromedary-Associated Staphylococcaceae from the Horn of Africa.

Members of the Staphylococcaceae family, particularly those of the genus Staphylococcus, encompass important human and animal pathogens. We collected and characterized Staphylococcaceae strains from apparently healthy and diseased camels (n = 84) and cattle (n = 7) in Somalia and Kenya. We phenotypically characterized the strains, including their antimicrobial inhibitory concentrations. Then, we sequenced their genomes using long-read sequencing, closed their genomes, and subsequently compared and mapped their virulence- and resistance-associated gene pools. Genome-based phylogenetics revealed 13 known Staphylococcaceae and at least two novel species. East African strains of different species encompassed novel sequence types and phylogenetically distant clades. About one-third of the strains had non-wild-type MICs. They were resistant to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, gentamicin, or streptomycin, encoded by tet(K), blaZ/blaARL, mecA/mecA1, msrA/mphC, salA, dfrG, aacA-aphD, and str, respectively. We identified the first methicillin- and multidrug-resistant camel S. epidermidis strain of sequence type (ST) 1136 in East Africa. The pool of virulence-encoding genes was largest in the S. aureus strains, as expected, although other rather commensal strains contained distinct virulence-encoding genes. We identified toxin-antitoxin (TA) systems such as the hicA/hicB and abiEii/abiEi families, reported here for the first time for certain species of Staphylococcaceae. All strains contained at least one intact prophage sequence, mainly belonging to the Siphoviridae family. We pinpointed potential horizontal gene transfers between camel and cattle strains and also across distinct Staphylococcaceae clades and species. IMPORTANCE Camels are a high value and crucial livestock species in arid and semiarid regions of Africa and gain importance giving the impact of climate change on traditional livestock species. Our current knowledge with respect to Staphylococcaceae infecting camels is very limited compared to that for other livestock species. Better knowledge will foster the development of specific diagnostic assays, guide promising antimicrobial treatment options, and inform about potential zoonotic risks. We characterized 84 Staphylococcaceae strains isolated from camels with respect to their antimicrobial resistance and virulence traits. We detected potentially novel Staphylococcus species, resistances to different classes of antimicrobials, and the first camel multidrug-resistant S. epidermidis strain of sequence type 1136.

Evaluation of the Occurrence of Staphylococcaceae with Reduced Susceptibility to Cefoxitin in Wild Ungulates in Brandenburg, Germany, Based on Land Use-Related Factors.

Interactions between natural and human-used environments have a significant influence on the spread of antimicrobial resistance in wild ecosystems. Despite current knowledge, fundamental questions about the degree of impact of land use-related factors on the spread of antimicrobial-resistant staphylococci in European wild game animal populations have not yet been answered with certainty. In this study, we evaluated the occurrence of Staphylococcaceae showing reduced susceptibility to cefoxitin in nasal swabs of fallow deer (Dama dama), red deer (Cervus elaphus), roe deer (Capreolus capreolus), and wild boar (Sus scrofa) hunted in Brandenburg, Germany. Evaluations were focused on the use of open-source data regarding the extent as well as the degree of land use, especially for settlement or animal husbandry. Results showed that the detection rate of Staphylococcaceae showing a non-wild-type phenotype for cefoxitin differed between animal species of the studied hunting districts. Statistical analyses of results combined with data on land use features revealed that a high density of cattle or poultry in a county may be associated with an increased detection rate in roe deer or wild boar, respectively. Furthermore, positive correlations were determined between the prevalence of non-wild-type Staphylococcaceae in roe deer or fallow deer and the proportional extent of surface water bodies in the corresponding area. The presented approach establishes a general basis for a risk-oriented assessment of the effects of human activities on the epidemiology of transmissible microorganisms in the human-animal-environment interface, including antimicrobial-resistant bacteria. IMPORTANCE Intensive research regarding the impact of land use-related factors on the prevalence and distribution of antimicrobial-resistant Staphylococcaceae in game ungulate populations is necessary for adequately determining risks related to interactions between wild animals, domestic animals, and humans in common geographic locations. This systematic approach for the analysis of the observations in specific hunting districts of Brandenburg, Germany, adds an innovative value to the research strategy of antimicrobial resistance in wild game animals, which is in accordance with current recommendations worldwide. Thus, results and information obtained in this study build a relevant foundation for future risk assessment regarding the safety of game products. Furthermore, the data generated represent an important basis for improving existing guidelines in land use practices and hunting practices. The use of existing open source data collections provided by official governmental and nongovernmental entities increases not only the impact but also the applicability and comparability of information beyond the regional level.

A prevalence and molecular characterization of novel pathogenic strains of Macrococcus caseolyticus isolated from external wounds of donkeys in Khartoum State -Sudan.

A pathogenic strain of Macrococcus caseolyticus (M. caseolyticus) was isolated from wounds infection during an investigation on donkeys in Khartoum State. (122) samples were collected from external wounds (head, abdomen, back and leg) during different seasons. One isolate (124B) was identified using whole-genome sequence analysis. RAST software identified 31 virulent genes of disease and defense, including methicillin-resistant genes, TatR family and ANT(4')-Ib. Plasmid rep22 was identified by PlasmidFindet-2.0 Server and a CRISPR. MILST-2.0 predicted many novel alleles. NCBI notated the genome as a novel M. caseolyticus strain (DaniaSudan). The MLST-tree-V1 revealed that DaniaSudan and KM0211a strains were interrelated. Strain DaniaSudan was resistant to ciprofloxacin, ceftazidime, erythromycin, oxacillin, clindamycin and kanamycin. Mice modeling showed bacteremia and many clinical signs (swelling, allergy, wounds, and hair loss). Enlargement, hyperemia, adhesions and abscesses were observed in many organs.Constructive conclusionThe prevalence of the strain was 4.73%, with significant differences between collection seasons and locations of wounds. A highly significant association between doses (105 CFU/ml, 102 CFU/ml, Intra-peritoneum and sub-cutaneous) and swelling, developing of allergy and loss of hair (p = 0.001, p = 0.000 and p = 0.005) respectively were seen.This result represents the first report of pathogenic strains of M. caseolyticus worldwide.

The bla and mec families of ß-lactam resistance genes in the genera Macrococcus, Mammaliicoccus and Staphylococcus: an in-depth analysis with emphasis on Macrococcus.

ß-Lactamases (Bla) and low-affinity penicillin-binding proteins (PBP2A) are responsible for ß-lactam resistance in the genera Macrococcus, Mammaliicoccus and Staphylococcus. These resistance mechanisms are in most species acquired through mobile genetic elements that carry a blaZ-like ß-lactamase gene for penicillin resistance and/or a mec gene (mecA, mecB, mecC,mecD) encoding a PBP2A for resistance to virtually all classes of ß-lactams. The mecA and mecC genes can be acquired through staphylococcal cassette chromosome mec (SCCmec) elements in Staphylococcus and Mammaliicoccus. The mecB and mecD genes are found in Macrococcus on SCCmec elements, as well as on unrelated mecD-carrying Macrococcus resistance islands (McRImecD) and large mecB-carrying plasmids. This review provides a phylogenetic overview of Macrococcus, Mammaliicoccus and Staphylococcus species and an in-depth analysis of the genetic structures carrying bla and mec genes in these genera. Native bla genes were detected in species belonging to the novobiocin-resistant Staphylococcus saprophyticus group and Mammaliicoccus. The evolutionary relatedness between Macrococcus and Mammaliicoccus is illustrated on the basis of a similar set of intrinsic PBPs, especially, the presence of a second class A PBP. The review further focuses on macrococcal elements carrying mecB and mecD, and compares them with structures present in Staphylococcus and Mammaliicoccus. It also discusses the different recombinases (ccr of SCCmec) and integrases (int of McRI) that contribute to the mobility of methicillin resistance genes, revealing Macrococcus as an important source for mobilization of antibiotic resistance genes within the family of Staphylococcaceae.

Occurrence of emerging multiresistant pathogens in the production chain of artisanal goat coalho cheese in Brazil.

Sanitary-hygienic failures in cheese making can pose health risks to consumers. This study aimed to identify multiresistant pathogens in different production stages of artisanal goat coalho cheese in Brazil and characterize their phenotypic and genotypic resistance. Eleven properties in the state of Pernambuco, Brazil, participated in the study. Samples were obtained from different stages of production and the humans involved. The samples obtained were submitted to microbiological culture, then all the isolated microorganisms were submitted to the Matrix Associated Laser Desorption-Ionization - Time of Flight technique for the microbiological identification of the species. Subsequently, Staphylococcus spp., Enterococcus spp. and Macrococcus caseolyticus were subjected to polymerase chain reaction to search for resistance genes and disc diffusion technique to evaluate the resistance profile. A total of 111 isolates were obtained and 31 species were identified, with the frequency of Staphylococcus spp. (62.20%; 69/111), Enterococcus spp. (11.60%; 13/111), Macrococcus caseolyticus (10%; 11/111), Bacillus spp. (3.60%; 4/111), Enterobacter spp. (3.60%; 4/111), Aureobasidium pullulans (1.80%; 2/111), Corynebacterium camporealensis (1.80%; 2/111), Issatchenkia occidentalis (1.80%; 2/111), Kocuria kristinae (1.80%; 2/111), Aerococcus viridans (0.90%; 1/111) and Filifactor villosus (0.90%; 1/111). Phenotypic and genotypic resistance was also detected with the occurrence of 15.90% (7/44) of the mecA gene, 4% (1/25) vanA, and 4% (1/25) vanB in Staphylococcus spp. and 20% (2/10) vanB in and Enterococcus spp. Emerging multiresistant pathogens are present in the production chain of artisanal goat cheese and humans, who exert an important role in disseminating these bacteria with imminent risks to human health.

Macrococcus armenti sp. nov., a novel bacterium isolated from the skin and nasal cavities of healthy pigs and calves.

Gram-positive coccoid bacteria were isolated from the nasal cavities of pigs and calves as well as from axillar and inguinal skin regions of pigs. Phylogenetic analysis of seven strains based on complete genome, 16S rRNA, hsp60, dnaJ, rpoB and sodA gene sequences and MALDI-TOF MS profiles revealed that they belonged to the genus Macrococcus with the closest relatedness to Macrococcus canis, Macrococcus caseolyticus subsp. caseolyticus and Macrococcus caseolyticus subsp. hominis. DNA relatedness of the type strain JEK37T with the type strains of M. canis, M. caseolyticus subsp. caseolyticus and M. caseolyticus subsp. hominis was 23.4, 23.1 and 23.0 % by digital DNA-DNA hybridization and 80.39, 80.45 and 80.87 % by average nucleotide identity (ANI) calculations, confirming that they do not belong to the same species. The DNA G+C content of JEK37T was 35.65 mol%. The novel strains can be differentiated from M. canis KM 45013T by the ability to fermentate d-ribose and by the absence of DNAase production and haemolysis, from M. caseolyticus subsp. caseolyticus CCUG 15606T by the ability to fermentate sucrose and from both species by the inability to grow in 9 and 12% NaCl. They differ from M. caseolyiticus subsp. hominis by the presence of α-glucosidase. The most common fatty acids of JEK37T were C14 : 0, C18 : 1 ω9c and C18 : 0. Known polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, aminolipid, aminoglycolipid, aminophospholipid, glycolipid and phospholipid. Cell-wall peptidoglycan of JEK37T was of type A3α l-Lys-Gly2-L-Ser-Gly (similar to A11.3) and the respiratory quinolone was menaquinone 6. Based on their genotypic and chemotaxonomic characteristics, these strains represent a novel species of the genus Macrococcus, for which we propose the name Macrococcus armenti sp. nov. The type strain is JEK37T (=DSM 112712T=CCOS 1982T).

Identification and characterization of a novel GNAT superfamily Nα -acetyltransferase from Salinicoccus halodurans H3B36.

Nα -acetyl-α-lysine was found as a new type of compatible solutes that acted as an organic cytoprotectant in the strain of Salinicoccus halodurans H3B36. A novel lysine Nα -acetyltransferase gene (shkat), encoding an enzyme that catalysed the acetylation of lysine exclusively at α position, was identified from this moderate halophilic strain and expressed in Escherichia coli. Sequence analysis indicated ShKAT contained a highly conserved pyrophosphate-binding loop (Arg-Gly-Asn-Gly-Asn-Gly), which was a signature of the GNAT superfamily. ShKAT exclusively recognized free amino acids as substrate, including lysine and other basic amino acids. The enzyme showed a wide range of optimal pH value and was tolerant to high-alkali and high-salinity conditions. As a new member of the GNAT superfamily, the ShKAT was the first enzyme recognized free lysine as substrate. We believe this work gives an expanded perspective of the GNAT superfamily, and reveals great potential of the shkat gene to be applied in genetic engineering for resisting extreme conditions.

Methicillin-resistant Macrococcus canis in a human wound.

A hemolytic Macrococcus canis strain (LI021) was isolated for the first time from a human skin infection. The complete genome of LI021 consisting of a 2,216,765-bp circular chromosome was obtained by de novo hybrid assembly of Illumina and Oxford Nanopore technology reads. Strain LI021 belonged to the new sequence type ST75 and was resistant to ß-lactam antibiotics due to the presence of a methicillin resistance gene mecB. The mecB gene as well as putative hemolysin genes hlgB and hlgC were located on a novel composite pseudo (Ψ) SCCmec island. These findings show that a methicillin-resistant M. canis may be associated with human infection and indicate that this bacterium should be considered by human diagnostic laboratories.

Effect of enterocins against methicillin-resistant animal-derived staphylococci.

The occurence and spread of animal-derived methicillin resistant staphylococci (MRS) worldwide is a current problem, especially due to their increasing incidence in food animals and their products, with possible contamination of food consumers and handlers. Staphylococci isolated from animals (n = 123) were identified with MALDI-TOF mass spectrometry and screened for methicillin/oxacillin/cefoxitin resistance (MR) using the disk diffusion method. Twenty-three phenotypically MRS strains were analysed using PBP2' Latex Agglutination Test Kit to confirm the phenotypic MR and PCR was performed for mecA gene detection; mecA gene positive strains were furtherly confirmed by means of sequencing. The susceptibility of MRS to 11 partially-purified enterocins (Ent) produced by E. faecium, E. durans and E. mundtii strains of animal, feed/food and environmental origin was checked using agar spot tests. Out of 23 MRS, PBP testing confirmed MR in 17 strains. Three Staphylococcus epidermidis and one S. vitulinus were mecA positive. The majority of MRS, including two mecA gene-positive strains S. epidermidis R44/1 and P3/Tr2a, were susceptible to the tested enterocins, mainly to Ent7420, EntA(P)/EK13, Ent412, Ent55 and Ent9296 (in the range 100 - 12,800 AU/mL). The most susceptible strains appeared to be the mecA gene-positive S. epidermidis SE R44/1 and SE P3/Tr2a strains, inhibited by eight enterocins out of 11 tested (100-200 AU/mL). Only four strains (including mecA gene positive S. epidermidis SE P3/Tr1 and S. vitulinus SV K12PL/1) were resistant to the tested antimicrobial substances. These results indicate that the enterocins used offer a promising option for prevention and treatment of bacterial infection caused by MRS in animals.

Unexpected Reactions of α,ß-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases.

CYP152 peroxygenases catalyze decarboxylation and hydroxylation of fatty acids using H2 O2 as cofactor. To understand the molecular basis for the chemo- and regioselectivity of these unique P450 enzymes, we analyze the activities of three CYP152 peroxygenases (OleTJE , P450SPα , P450BSß ) towards cis- and trans-dodecenoic acids as substrate probes. The unexpected 6S-hydroxylation of the trans-isomer and 4R-hydroxylation of the cis-isomer by OleTJE , and molecular docking results suggest that the unprecedented selectivity is due to OleTJE 's preference of C2-C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450SPα and P450BSß regardless of the cis/trans-configuration of substrates. The combined H218 O2 tracing experiments, MD simulations, and QM/MM calculations unravel an unusual mechanism for Compound I-mediated aldehyde formation in which the active site water derived from H2 O2 activation is involved in the generation of a four-membered ring lactone intermediate. These findings provide new insights into the unusual mechanisms of CYP152 peroxygenases.

Nicotine concentration and mentholation affect bacterial community diversity in SPECTRUM research cigarettes.

Previous studies have characterized bacterial communities in menthol versus non-menthol cigarettes. However, these studies evaluated commercial cigarettes, for which levels of chemical constituents are largely unknown, and therefore, could not assess the impact of varying nicotine and menthol concentrations on tobacco bacterial communities. To address this knowledge gap, we performed time-series experiments using SPECTRUM research cigarettes with varying nicotine and menthol levels. Cigarettes were incubated under three storage conditions for 14 days. Cigarette tobacco was then sub-sampled (n = 288), DNA extracted, and subjected to PCR amplification of the V3V4 region of the 16S rRNA gene, followed by Illumina HiSeq sequencing. Sequences were analyzed using QIIME and R. Incubation under varying conditions did not affect bacterial diversity. However, significant differences in bacterial communities were observed across varying nicotine concentrations in menthol and non-menthol products. For example, Pseudomonas spp. was negatively correlated with nicotine concentrations in menthol cigarettes. A significantly higher relative abundance of P. veronii and P. viridiflava was observed in menthols versus non-menthols, while a significantly higher relative abundance of Bacillus foraminis and B. coagulans was found in non-menthols versus menthols. Additional bacteria (e.g., Staphylococcus spp., Jeotgalicoccus psychrophilus, and B. flexus) significantly changed in relative abundance between days 0 and 14. Our findings demonstrate that nicotine and menthol levels have a significant impact on the relative abundance of potential bacterial pathogens present in cigarettes. Future work is needed to demonstrate whether these tobacco-associated bacteria could be transferred to users while smoking, ultimately contributing to adverse respiratory impacts. KEY POINTS: • Varying nicotine levels changes bacterial composition of research cigarettes. • Mentholation affects the tobacco bacterial microbiome. • SPECTRUM research cigarettes are dominated by Pseudomonas and Bacillus.

Methicillin-Resistant Staphylococci and Macrococci at the Interface of Human and Animal Health.

The global impact of methicillin-resistant Staphylococcus aureus (MRSA) clonal lineages on human and animal health continues, even considering the decreasing MRSA rates in some parts of the world [...].

Technological Applications of Macrococcus caseolyticus and its Impact on Food Safety.

Macrococcus spp. are Gram-positive cocci that belong to the Staphylococcaceae family; they are closely related to staphylococci, but, unlike staphylococci, they are not considered as human pathogens. Macrococcus spp. are recognized as relevant veterinary pathogens, and their presence has been reported in food products of animal origin. Macrococcus caseolyticus, the most studied species of the Macrococcus genus, is associated with the development of aroma and flavor in fermented foods and is, thus, used as starter cultures in fermentations. However, certain important issues regarding food safety must be taken into account when employing these microorganisms in fermentations. Recent studies have reported the presence of genes associated with resistance to methicillin and other antibiotics in M. caseolyticus. This can be harmful to human health as these genes can be transferred to other bacteria present in the food, mainly staphylococcal species. This work, therefore, aims to highlight the importance of a more critical view on the presence of macrococci in foods and the possible indirect risks to human health.

The novel macrolide resistance genes mef(F) and msr(G) are located on a plasmid in Macrococcus canis and a transposon in Macrococcus caseolyticus.

OBJECTIVES: To analyse macrolide resistance in a Macrococcus canis strain isolated from a dog with an ear infection, and determine whether the resistance mechanism is also present in other bacteria, and associated with mobile genetic elements. METHODS: The whole genome of M. canis Epi0082 was sequenced using PacBio and Illumina technologies. Novel macrolide resistance determinants were identified through bioinformatic analysis, and functionality was demonstrated by expression in Staphylococcus aureus. Mobile genetic elements containing the novel genes were analysed in silico for strain Epi0082 as well as in other bacterial strains deposited in GenBank. RESULTS: M. canis Epi0082 contained a 3212 bp operon with the novel macrolide resistance genes mef(F) and msr(G) encoding a efflux protein and an ABC-F ribosomal protection protein, respectively. Cloning in S. aureus confirmed that both genes individually confer resistance to the 14- and 15-membered ring macrolides erythromycin and azithromycin, but not the 16-membered ring macrolide tylosin. A reduced susceptibility to the streptogramin B pristinamycin IA was additionally observed when msr(G) was expressed in S. aureus under erythromycin induction. Epi0082 carried the mef(F)-msr(G) operon together with the chloramphenicol resistance gene fexB in a novel 39 302 bp plasmid pMiCAN82a. The mef(F)-msr(G) operon was also found in macrolide-resistant Macrococcus caseolyticus strains in the GenBank database, but was situated in the chromosome as part of a novel 13 820 bp or 13 894 bp transposon Tn6776. CONCLUSIONS: The identification of mef(F) and msr(G) on different mobile genetic elements in Macrococcus species indicates that these genes hold potential for further dissemination of resistance to the clinically important macrolides in the bacterial population.

How external perturbations affect the chemoselectivity of substrate activation by cytochrome P450 OleTJE.

Cytochrome P450 enzymes are versatile biocatalysts found in most forms of life. Generally, the cytochrome P450s react with dioxygen and hence are haem-based mono-oxygenases; however, in specific isozymes, H2O2 rather than O2 is used and these P450s act as peroxygenases. The P450 OleTJE is a peroxygenase that binds long to medium chain fatty acids and converts them to a range of products originating from Cα-hydroxylation, Cß-hydroxylation, Cα-Cß desaturation and decarboxylation of the substrate. There is still controversy regarding the details of the reaction mechanism of P450 OleTJE; how the products are formed and whether the product distributions can be influenced by external perturbations. To gain further insights into the structure and reactivity of P450 OleTJE, we set up a range of large active site model complexes as well as full enzymatic structures and did a combination of density functional theory studies and quantum mechanics/molecular mechanics calculations. In particular, the work focused on the mechanisms leading to these products under various reaction conditions. Thus, for a small cluster model, we find a highly selective Cα-hydroxylation pathway that is preferred over Cß-H hydrogen atom abstraction by at least 10 kcal mol-1. Introduction of polar residues to the model, such as an active site protonated histidine residue or through external electric field effects, lowers the Cß-H hydrogen atom abstraction barriers are lowered, while a full QM/MM model brings the Cα-H and Cß-H hydrogen atom abstraction barriers within 1 kcal mol-1. Our studies; therefore, implicate that environmental effects in the second-coordination sphere can direct and guide selectivities in enzymatic reaction mechanisms.