Msr Genes Research Articles

ICESp1109, a novel hybrid Integrative Conjugative Element of macrolide-resistant Streptococcus pyogenes serotype M77 collected between 2003 and 2017 in Poland.

Genetic characterization of the antibiotic resistance determinants and associated mobile genetic elements (MGEs) among Streptococcus pyogenes [Group A streptococci (GAS)] clinical isolates of an M77 serotype collected in Poland between 2003 and 2017. The genomes of 136 M77 GAS isolates were sequenced using Illumina, and selected with long-read approach (Oxford Nanopore). Whole genome sequences were analyzed to determine the presence of macrolide resistance determinants, and their genetic context. The strains used in the study were collected in the two multicenter surveys from in- and outpatients. Sequencing data analysis revealed that all strains carried the tet(O) gene (100%, N=136). They were classified as a single sequence type ST63. For erythromycin resistance, the unique determinant was erm(TR) detected in 76.5% (N=104) isolates. A single appearance of tet(M) and erm(B) on Tn3872 was noticed. The mefA, mefE, and msr(D) genes were detected in neither of the genomes. This correlated with the detected strain phenotypes - 11 exhibited cMLSB, 93 - iMLSB, and no M phenotype.The erm(TR) gene was predominantly (N=74) found within a novel hybrid Integrative Conjugative Element composed of the ICESp1108-like sequence and ICESp2906 variant which was then named ICESp1109. However, in strains isolated before 2008, erm(TR) was located within ICESp2905 (N=27). The erm(TR) gene was detected within stand-alone ICESp1108-like sequences in 3 strains. Based on phylogenetic analysis results the clonal dissemination of the macrolide-resistant S. pyogenes M77/ST63 strain with hybrid ICESp1109 was observed between 2008 and 2017. ICESp1109 is the novel hybrid ICE in Gram-positive bacteria.

Genetic basis of macrolide resistance in porcine Pasteurella multocida isolates from the German national resistance monitoring program GERM-Vet 2008-2021.

To analyse porcine Pasteurella multocida isolates obtained from the national resistance monitoring program GERM-Vet 2008-2021 in Germany for phenotypic and genotypic macrolide resistance. The antimicrobial susceptibility of the isolates was determined by broth microdilution according to CLSI standards. Closed genomes were obtained by WGS via Illumina MiSeq and MinION platforms followed by a hybrid assembly. Of 1114 porcine P. multocida isolates, only four isolates (0.36%), one each from 2008, 2010, 2019 and 2021, exhibited resistance to at least one macrolide tested. The isolate from 2010 was only resistant to erythromycin and WGS analysis neither revealed a macrolide resistance gene nor a macrolide resistance-mediating mutation. The isolates from 2008 and 2019 were resistant to erythromycin, tilmicosin, tildipirosin, tulathromycin and gamithromycin and showed either only the A2058G mutation in all six 23S rRNA operons or the chromosomally located macrolide resistance genes msr(E) and mph(E), respectively. The isolate from 2021 was resistant to erythromycin, tulathromycin, gamithromycin and tylosin and carried a novel integrative and conjugative element of 64 966 bp, designated Tn7730, in its chromosomal DNA. It harboured the macrolide resistance genes mef(C), mph(G) and estT, the lincosamide resistance gene lnu(H), and the tetracycline resistance gene tet(Y), the last two were detected for the first time in P. multocida. Macrolide resistance in German porcine P. multocida can be due to resistance-mediating mutations or resistance genes. The presence of the novel Tn7730 carrying three different macrolide resistance genes is alarming and should be monitored.

Antimicrobial resistance determinants in the oropharyngeal microbiome of 'men having sex with men' attending an sexually transmitted infection clinic.

'Men having sex with men' (MSM) represent a key population with a significant prevalence of pharyngeal Neisseria gonorrhoeae (NG) infections and a high rate of antimicrobial resistance genes in the pharyngeal microbiome. As NG can acquire antibiotic resistance genes from other commensal oropharyngeal bacteria, monitoring the prevalence of these resistance determinants is critical to curtail the spread of NG-resistant strains. Here, we assessed the distribution of five resistance genes (pen (A), mtr (R), gyr (A), par (C), msr (D)) in the oropharynx of 164 MSM, attending an Outpatient clinic for STI screening. The most frequently detected resistance gene was msr (D) (88.4%), followed by gyr (A) (67.1%). The distribution of resistance genes was not influenced by pharyngeal gonorrhea nor by the HIV status, whereas a younger age was associated with mtr (R) presence (p = .008). Subjects using mouthwash exhibited significantly lower levels of mtr (R) (p = .0005). Smoking habit was associated with a higher prevalence of par (C) (p = .02). A noteworthy association was observed between the presence of msr (D) gene and the use of antibiotics (p = .014). Our findings reveal an enrichment of antimicrobial resistance genes in the oropharynx of MSM. These insights could aid in the development of screening programs and antimicrobial stewardship initiatives targeting populations at heightened risk of pharyngeal gonorrhea.

Gut diversity and the resistome as biomarkers of febrile neutropenia outcome in paediatric oncology patients undergoing hematopoietic stem cell transplantation

The gut microbiota of paediatric oncology patients undergoing a conditioning regimen before hematopoietic stem cell transplantation is recently considered to play role in febrile neutropenia. Disruption of commensal microbiota and evolution of opportune pathogens community carrying a plethora of antibiotic-resistance genes play crucial role. However, the impact, predictive role and association of patient´s gut resistome in the course of the therapy is still to be elucidated. We analysed gut microbiota composition and resistome of 18 paediatric oncology patients undergoing hematopoietic stem cell transplantation, including 12 patients developing febrile neutropenia, hospitalized at The Bone Marrow Transplantation Unit of the National Institute of Children´s disease in Slovak Republic and healthy individuals (n = 14). Gut microbiome of stool samples obtained in 3 time points, before hematopoietic stem cell transplantation (n = 16), one week after hematopoietic stem cell transplantation (n = 16) and four weeks after hematopoietic stem cell transplantation (n = 14) was investigated using shotgun metagenome sequencing and bioinformatical analysis. We identified significant decrease in alpha-diversity and nine antibiotic-resistance genes msr(C), dfrG, erm(T), VanHAX, erm(B), aac(6)-aph(2), aph(3)-III, ant(6)-Ia and aac(6)-Ii, one week after hematopoietic stem cell transplantation associated with febrile neutropenia. Multidrug-resistant opportune pathogens of ESKAPE, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli found in the gut carried the significant subset of patient’s resistome. Over 50% of patients treated with trimethoprim/sulfamethoxazole, piperacillin/tazobactam and amikacin carried antibiotic-resistance genes to applied treatment. The alpha diversity and the resistome of gut microbiota one week after hematopoietic stem cell transplantation is relevant predictor of febrile neutropenia outcome after hematopoietic stem cell transplantation. Furthermore, the interindividual diversity of multi-drug resistant opportunistic pathogens with variable portfolios of antibiotic-resistance genes indicates necessity of preventive, personalized approach.

Pan msr gene deleted strain of Salmonella Typhimurium suffers oxidative stress, depicts macromolecular damage and attenuated virulence

Salmonella encounters but survives host inflammatory response. To defend host-generated oxidants, Salmonella encodes primary antioxidants and protein repair enzymes. Methionine (Met) residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO) which compromises protein functions and subsequently cellular survival. However, by reducing Met-SO to Met, methionine sulfoxide reductases (Msrs) enhance cellular survival under stress conditions. Salmonella encodes five Msrs which are specific for particular Met-SO (free/protein bound), and ‘R’/‘S’ types. Earlier studies assessed the effect of deletions of one or two msrs on the stress physiology of S. Typhimurium. We generated a pan msr gene deletion (Δ5msr) strain in S. Typhimurium. The Δ5msr mutant strain shows an initial lag in in vitro growth. However, the Δ5msr mutant strain depicts very high sensitivity (p < 0.0001) to hypochlorous acid (HOCl), chloramine T (ChT) and superoxide-generating oxidant paraquat. Further, the Δ5msr mutant strain shows high levels of malondialdehyde (MDA), protein carbonyls, and protein aggregation. On the other side, the Δ5msr mutant strain exhibits lower levels of free amines. Further, the Δ5msr mutant strain is highly susceptible to neutrophils and shows defective fitness in the spleen and liver of mice. The results of the current study suggest that the deletions of all msrs render S. Typhimurium highly prone to oxidative stress and attenuate its virulence.

Methicillin-Resistant Staphylococcusaureus Strains Isolated from Burned Patients in a Tunisian Hospital: Molecular Typing, Virulence Genes, and Antimicrobial Resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of a variety of infections in hospitals and the community. Their spread poses a serious public health problem worldwide. Nevertheless, in Tunisia and other African countries, very little molecular typing data on MRSA strains is currently available. In our study, a total of 64 MRSA isolates were isolated from clinical samples collected from burned patients hospitalized in the Traumatology and Burns Center of Ben Arous in Tunisia. The identification of the collection was based on conventional methods (phenotypic and molecular characterization). The characterization of the genetic support for methicillin resistance was performed by amplification of the mecA gene by polymerase chain reaction (PCR), which revealed that 78.12% of S. aureus harbors the gene. The resistance of all the collection to different antibiotic families was studied. Indeed, the analysis of strain antibiotic susceptibility confirmed their multi-resistant phenotype, with high resistance to ciprofloxacin, gentamicin, penicillin, erythromycin, and tetracycline. The resistance to the last three antibiotics was conferred by the blaZ gene (73.43%), the erm(C) gene (1.56%), the msr(A) gene (6.25%), and tet(M) gene (7.81%), respectively. The clonal diversity of these strains was studied by molecular typing of the accessory gene regulator (agr) system, characterization of the SCCmec type, and spa-typing. The results revealed the prevalence of agr types II and III groups, the SCCmec type III and II cassettes, and the dominance of spa type t233. The characterization of the eight enterotoxins genes, the Panton-Valentine leukocidin and the toxic shock syndrome toxin, was determined by PCR. The percentage of virulence genes detected was for enterotoxins (55%), tst (71.88%), leukocidin E/D (79.69%), and pvl (1.56%) factors. Furthermore, our results revealed that the majority of the strains harbor IEC complex genes (94%) with different types. Our findings highlighted the emergence of MRSA strains with a wide variety of toxins, leukocidin associated with resistance genes, and specific genetic determinants, which could constitute a risk of their spread in hospitals and the environment and complicate infection treatment.

Genome-Wide Identification and Characterization of the Msr Gene Family in Alfalfa under Abiotic Stress.

Alfalfa (Medicago sativa) is an important leguminous forage, known as the "The Queen of Forages". Abiotic stress seriously limits the growth and development of alfalfa, and improving the yield and quality has become an important research area. However, little is known about the Msr (methionine sulfoxide reductase) gene family in alfalfa. In this study, 15 Msr genes were identified through examining the genome of the alfalfa "Xinjiang DaYe". The MsMsr genes differ in gene structure and conserved protein motifs. Many cis-acting regulatory elements related to the stress response were found in the promoter regions of these genes. In addition, a transcriptional analysis and qRT-PCR (quantitative reverse transcription PCR) showed that MsMsr genes show expression changes in response to abiotic stress in various tissues. Overall, our results suggest that MsMsr genes play an important role in the response to abiotic stress for alfalfa.

Streptococcus pyogenes Φ1207.3 Is a Temperate Bacteriophage Carrying the Macrolide Resistance Gene Pair mef(A)-msr(D) and Capable of Lysogenizing Different Streptococci.

Streptococcus pyogenes prophage Φ1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if Φ1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the Φ1207.3-carrying lysogen were treated with mitomycin C to assess if Φ1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if Φ1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of Φ1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 × 104 and 6.06 × 104 excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 × 103 and 7.68 × 103 phage particles per milliliter of supernatant. The phage preparations of Φ1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 × 10-6 lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 103 phage particles. IMPORTANCE Bacteriophages play an important role in bacterial physiology and genome evolution. The widespread use of genome sequencing revealed that bacterial genomes can contain several different integrated temperate bacteriophages, which can constitute up to 20% of the genome. Most of these bacteriophages are only predicted in silico and are never shown to be functional. In fact, it is often difficult to induce the lytic cycle of temperate bacteriophages. In this work, we show that Φ1207.3, a peculiar bacteriophage originally from Streptococcus pyogenes, which can lysogenize different streptococci and carries the macrolide resistance mef(A)-msr(D) gene pair, is capable of producing mature virions, but only at a low level, while not being able to produce plaques. This temperate phage is probably a partially functional phage, which seems to have lost lytic characteristics to specialize in lysogenization. While we are not used to conceiving phages separately from lysis, this behavior could actually be more frequent than expected.

Molecular Epidemiology, Antimicrobial Susceptibility, and Clinical Features of Methicillin-Resistant Staphylococcus aureus Bloodstream Infections over 30 Years in Barcelona, Spain (1990-2019).

Methicillin-resistant Staphylococcus aureus bloodstream infections (MRSA-BSI) are a significant cause of mortality. We analysed the evolution of the molecular and clinical epidemiology of MRSA-BSI (n = 784) in adult patients (Barcelona, 1990−2019). Isolates were tested for antimicrobial susceptibility and genotyped (PFGE), and a selection was sequenced (WGS) to characterise the pangenome and mechanisms underlying antimicrobial resistance. Increases in patient age (60 to 71 years), comorbidities (Charlson’s index > 2, 10% to 94%), community-onset healthcare-associated acquisition (9% to 60%), and 30-day mortality (28% to 36%) were observed during the 1990−1995 and 2014−2019 periods. The proportion of catheter-related BSIs fell from 57% to 20%. Current MRSA-BSIs are caused by CC5-IV and an upward trend of CC8-IV and CC22-IV clones. CC5 and CC8 had the lowest core genome proportions. Antimicrobial resistance rates fell, and only ciprofloxacin, tobramycin, and erythromycin remained high (>50%) due to GyrA/GrlA changes, the presence of aminoglycoside-modifying enzymes (AAC(6′)-Ie-APH(2″)-Ia and ANT(4′)-Ia), and mph(C)/msr(A) or erm (C) genes. Two CC22-IV strains showed daptomycin resistance (MprF substitutions). MRSA-BSI has become healthcare-associated, affecting elderly patients with comorbidities and causing high mortality rates. Clonal replacement with CC5-IV and CC8-IV clones resulted in lower antimicrobial resistance rates. The increased frequency of the successful CC22-IV, associated with daptomycin resistance, should be monitored.

Methionine sulfoxide reductase B5 plays vital roles in tomato fruit defense response against Botrytis cinerea induced by methyl jasmonate

A methionine sulfoxide reductase (Msr) gene is identified in tomato fruit, and its protein’s identity as MsrB5 was determined by phylogenetic analysis and multiple sequence alignment. Correspondingly, the role of SlMsrB5 in methyl jasmonate (MeJA)-mediated defense to B. cinerea in tomato fruit was investigated by using 0.05 mM MeJA treatment on SlMsrB5-silenced fruit during storage. The results showed that MeJA treatment upregulated total phenols and flavonoids, disease resistance enzymes activities, and expression of pathogenesis-related (PR) genes, including SlPR1/2a/2b/3a/3b/STH2 in tomato fruit. Additionally, MeJA inhibited the increase of hydrogen peroxide and superoxide anions by improving antioxidant enzyme activities and the ascorbic acid-glutathione (ASA-GSH) cycle. Similarly, MeJA induced the accumulation of jasmonic acid and the transcriptions of its signal transduction-related genes (SlCOI1 and SlMYC2), which could lead to the transcription of disease response-related marker genes such as SlPI I and SlPI II. These MeJA-regulated changes may contribute to improved disease resistance in tomato fruit. However, SlMsrB5 silence reduced the effect of MeJA on the above parameters and caused severer disease occurrence in the SlMsrB5-silenced + MeJA treated group than in MeJA treated group, probably because the low levels of SlMsrB5 transcription could not restore the activity of defense and antioxidant-related enzymes. Furthermore, correlation analysis revealed that the above effects regulated by MeJA were highly correlated with SlMsrB5 transcript levels. Therefore, these findings provide insight on SlMsrB5's role in MeJA-mediated tomato immune responses to B. cinerea by regulating defensive enzymes and genes, antioxidant capacity, and the JA signaling pathway.

Investigation of Macrolide Resistance Genotypes of Pasteurella multocida Isolates from Cattle and Small Ruminants.

Macrolides are commonly used to control respiratory tract infections in ruminants, but the susceptibility of Pasteurella multocida strains has shown a decrease to macrolide antibiotics in the last decade. In this work we assessed the prevalence of macrolide resistance of 100 P. multocida isolates from ruminant hosts and studied the resistance genotypes with newly designed PCRs. Susceptibility to erythromycin and tilmicosin was tested using minimal inhibitory concentration (MIC) test strips. A newly designed PCR was used for the detection of macAB genes, and a PCR plus restriction enzyme-based technique was developed for detecting a 23S rRNA gene mutation at position 2059. Five bovine isolates with notably increased MICs (≥256 μg/mL for erythromycin and ≥32 μg/mL for tilmicosin) carried resistance genes msr(E) and mph(E) or the A2059G point mutation in the 23S rRNA gene. Over 73% strains from small ruminants and all bovine isolates were MacAB PCR positive. Bovine strains were less sensitive to macrolide antibiotics than isolates from small ruminants, and an increase in the prevalence of macrolide resistance in bovine P. multocida isolates has also been observed over time.

Identification and characterisation of a novel multidrug-resistant streptococcus, Streptococcus toyakuensis sp. nov., from a blood sample

In 2018, we isolated multidrug-resistant α-haemolytic streptococci TP1632 from the blood of a 34-year-old patient with bacteraemia. This study aimed to characterise α-haemolytic streptococci TP1632 and elucidate its multidrug resistance mechanisms. TP1632 was characterised by whole genome sequencing and biochemical testing. Based on the genome sequence, digital DNA-DNA hybridization (dDDH) and average nucleotide identity based on BLAST (ANIb) values were calculated. In addition, antimicrobial-resistance mechanisms were evaluated by ResFinder and transformation assay using Streptococcus mitis. TP1632 showed resistance to β-lactams, macrolides, and quinolones. Genomic analysis revealed that dDDH and ANIb values between TP1632 and S. mitis were 56.3% and 93.63%, respectively, indicating TP1632 as the novel species. TP1632 exhibited macrolide resistance genes such as mef(A), msr(D), and erm(B) and tetracycline resistance gene tet(M). In addition, amino acids at position 81 in GyrA and 79 in ParC were tyrosine and isoleucine, respectively. When penicillin-binding proteins of TP1632 were introduced into S. mitis, recombinants showed β-lactam resistance. Thus, acquired resistance genes and low affinities towards antimicrobial agents contribute to multidrug resistance. Our findings indicated that multidrug-resistant TP1632 is a novel species in the genus Streptococcus. Furthermore, antimicrobial resistance of this strain could transfer horizontally among α-haemolytic streptococci. These data indicated the risk of emergence of multidrug-resistant streptococci not only as a pathogen but also a reservoir of antimicrobial resistance. This isolate was proposed as a novel species, Streptococcus toyakuensis sp. nov. The type strain is TP1632T (= JCM 34623T=CCUG 75492T).

A greater effect on clarithromycin resistance of mef(A)-associated msr(D) than mef(E)-associated msr(D) in Streptococcus pyogenes.

The mef(A)- and its subclass mef(E) systems had long been considered to constitute one of the primary macrolide-resistant mechanisms in Streptococcus pyogenes. However, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A)/mef(E) genes plays a predominant role in these systems. In previous studies, furthermore, mef(A)-associated msr(D)10-85 of an S. pyogenes strain (10-85) exhibited a greater increase in clarithromycin minimum inhibitory concentration (MIC) than mef(E)-associated msr(D)13-O-10 of another strain (13-O-10). Both msr(D) genes encode 487 amino acid residues, 13 amino acid residues of which are different from each other. In this study, we performed mutational analysis of the msr(D) genes and showed that a single-nucleotide polymorphism to cause a substitution of Asp238 with Gly is mainly associated with the greater increase in clarithromycin MIC by the msr(D)10-85 than by the msr(D)13-O-10 allele. In addition, another substitution of Ser with Arg at codon 194 is partially associated with the greater increase by the msr(D)10-85 than by the msr(D)13-O-10 allele.

Successful Intra- but Not Inter-species Recombination of msr(D) in Neisseria subflava.

Resistance acquisition via natural transformation is a common process in the Neisseria genus. Transformation has played an important role in the emergence of resistance to many antimicrobials in Neisseria gonorrhoeae and Neisseria meningitidis. In a previous study, we found that currently circulating isolates of Neisseria subflava had acquired an msr(D) gene that has been found to result in macrolide resistance in other bacteria but never found in Neisseria species before. To determine if this resistance mechanism is transferable among Neisseria species, we assessed if we could transform the msr(D) gene into other commensal and pathogenic Neisseria under low dose azithromycin pressure. Intraspecies recombination in commensal N. subflava was confirmed with PCR and resulted in high-level macrolide resistance. Whole-genome sequencing of these transformed strains identified the complete uptake of the msr(D) integration fragment. Sequence analysis showed that a large fragment of DNA (5 and 12 kb) was transferred through a single horizontal gene transfer event. Furthermore, uptake of the msr(D) gene had no apparent fitness cost. Interspecies transformation of msr(D) from N. subflava to N. gonorrhoeae was, however, not successful.

Impact of intra-partum azithromycin on carriage of group A streptococcus in the Gambia: a posthoc analysis of a double-blind randomized placebo-controlled trial

BackgroundGroup A Streptococcus (GAS) is a major human pathogen and an important cause of maternal and neonatal sepsis. Asymptomatic bacterial colonization is considered a necessary step towards sepsis. Intra-partum azithromycin may reduce GAS carriage.MethodsA posthoc analysis of a double-blind, placebo-controlled randomized-trial was performed to determine the impact of 2 g oral dose of intra-partum azithromycin on maternal and neonatal GAS carriage and antibiotic resistance. Following screening, 829 mothers were randomized who delivered 843 babies. GAS was determined by obtaining samples from the maternal and newborn nasopharynx, maternal vaginal tract and breastmilk. Whole Genome Sequencing (WGS) of GAS isolates was performed using the Illumina Miseq platform.ResultsGAS carriage was lower in the nasopharynx of both mothers and babies and breast milk among participants in the azithromycin arm. No differences in GAS carriage were found between groups in the vaginal tract. The occurrence of azithromycin-resistant GAS was similar in both arms, except for a higher prevalence in the vaginal tract among women in the azithromycin arm. WGS revealed all macrolide-resistant vaginal tract isolates from the azithromycin arm were Streptococcus dysgalactiae subspecies equisimilis expressing Lancefield group A carbohydrate (SDSE(A)) harbouring macrolide resistant genes msr(D) and mef(A). Ten of the 45 GAS isolates (22.2%) were SDSE(A).ConclusionsOral intra-partum azithromycin reduced GAS carriage among Gambian mothers and neonates however carriage in the maternal vaginal tract was not affected by the intervention due to azithromycin resistant SDSE(A). SDSE(A) resistance must be closely monitored to fully assess the public health impact of intrapartum azithromycin on GAS.Trial registration ClinicalTrials.gov Identifier NCT01800942

Distribution and Clonal Diversity of Staphylococcus aureus and Other Staphylococci in Surface Waters: Detection of ST425-t742 and ST130-t843 mecC-Positive MRSA Strains.

Natural aquatic environments represent one of the most important vehicles of bacterial dissemination. Therefore, we aimed to isolate staphylococci from surface waters and to investigate the presence of antimicrobial resistance genes and virulence factors as well as the genetic lineages of all Staphylococcus aureus isolates. Staphylococci were recovered from water samples collected from 78 surface waters, including rivers, streams, irrigation ditches, dams, lakes, and fountains. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Multilocus sequence typing and spa-typing were performed in all S. aureus isolates. From the 78 water samples, 33 S. aureus, one S. pseudintermedius, and 51 coagulase-negative staphylococci (CoNS) were identified. Among the S. aureus isolates, four MRSA were identified, and all harbored the mecC gene. Fourteen S. aureus were susceptible to all antimicrobials tested and the remaining showed resistance to penicillin, erythromycin and/or tetracycline encoded by the blaZ, ermT, msr(A/B), tetL, and vgaA genes. Regarding the clonal lineages, one mecC-MRSA isolate belonged to spa-type t843 and sequence type (ST) 130 and the other three to t742 and ST425. The remaining S. aureus were ascribed 14 spa-types and 17 sequence types. Eleven species of CoNS were isolated: S. sciuri, S. lentus, S. xylosus, S. epidermidis, S. cohnii spp. urealyticus, S. vitulinus, S. caprae, S. carnosus spp. Carnosus, S. equorum, S. simulans, and S. succinus. Thirteen CoNS isolates had a multidrug resistance profile and carried the following genes: mecA, msr(A/B), mph(C), aph(3′)-IIIa, aac(6′)-Ie–aph(2′’)-Ia, dfrA, fusB, catpC221, and tetK. A high diversity of staphylococci was isolated from surface waters including mecCMRSA strains and isolates presenting multidrug-resistance profiles. Studies on the prevalence of antibiotic-resistant staphylococci in surface waters are still very scarce but extremely important to estimate the contribution of the aquatic environment in the spread of these bacteria.

Antimicrobial resistance and genetic diversity of Enterococcus faecalis from yolk sac infections in broiler chicks

Despite restrictions on the use of antibiotics in poultry, the percentage of multidrug resistant bacteria, isolated from both adult birds and chicks, remains high. These bacteria can spread between countries via hatching eggs or chicks. Antibiotic resistant bacteria can also pose a threat to hatchery and farm workers or to consumers of poultry. The aim of the study was to perform a phenotypic and genotypic analysis of the drug resistance of E. faecalis isolates from yolk sac infections in broiler chicks from Poland and the Netherlands and to determine their genetic diversity. The tests revealed resistance to antibiotics from category D, that is, tetracycline (69.7%); category C – lincomycin (98.7%), erythromycin (51.3%), aminoglycosides (high-level streptomycin and kanamycin resistance – 10.5% and 3.95%, respectively), and chloramphenicol (7.9%); and category B – ciprofloxacin (25% with resistance or intermediate resistance). No resistance to penicillin, ampicillin, high-level gentamicin, tigecycline, or linezolid was noted. Various combinations of the erm(B), tet(M), tet(L), tet(O), ant(6)-Ia, aph(3′)-IIIa, ant(4′)-Ia, cat, and msr(A/B) genes were detected in all isolates (irrespective of the drug-resistance phenotype). Among isolates that carried the tet(M) and/or the tet(L) gene, 28% also had the Int-Tn gene, in contrast with isolates possessing tet(O). There were 28 sequence types and 43 PFGE restriction patterns. About 60% of isolates were of sequences types ST59, ST16, ST116, ST282, ST36, and ST82. Nine new sequence types were shown (ST836-ST844). In conclusion, broiler chicks can be a source of drug-resistant sequence types of E. faecalis that are potentially hazardous for people and animals. Restrictive programs for antibiotic use in broiler breeding flocks should be developed to decrease drug resistance in day-old chicks and reduce economic losses during rearing.

Antimicrobial Susceptibility of Lactic Acid Bacteria Strains of Potential Use as Feed Additives - The Basic Safety and Usefulness Criterion.

The spread of resistance to antibiotics is a major health concern worldwide due to the increasing rate of isolation of multidrug resistant pathogens hampering the treatment of infections. The food chain has been recognized as one of the key routes of antibiotic resistant bacteria transmission between animals and humans. Considering that lactic acid bacteria (LAB) could act as a reservoir of transferable antibiotic resistance genes, LAB strains intended to be used as feed additives should be monitored for their safety. Sixty-five LAB strains which might be potentially used as probiotic feed additives or silage inoculants, were assessed for susceptibility to eight clinically relevant antimicrobials by a minimum inhibitory concentration determination. Among antimicrobial resistant strains, a prevalence of selected genes associated with the acquired resistance was investigated. Nineteen LAB strains displayed phenotypic resistance to one antibiotic, and 15 strains were resistant to more than one of the tested antibiotics. The resistance to aminoglycosides and tetracyclines were the most prevalent and were found in 37 and 26% of the studied strains, respectively. Phenotypic resistance to other antimicrobials was found in single strains. Determinants related to resistance phenotypes were detected in 15 strains as follows, the aph(3″)-IIIa gene in 9 strains, the lnu(A) gene in three strains, the str(A)-str(B), erm(B), msr(C), and tet(M) genes in two strains and the tet(K) gene in one strain. The nucleotide sequences of the detected genes revealed homology to the sequences of the transmissible resistance genes found in lactic acid bacteria as well as pathogenic bacteria. Our study highlights that LAB may be a reservoir of antimicrobial resistance determinants, thus, the first and key step in considering the usefulness of LAB strains as feed additives should be an assessment of their antibiotic resistance. This safety criterion should always precede more complex studies, such as an assessment of adaptability of a strain or its beneficial effect on a host. These results would help in the selection of the best LAB strains for use as feed additives. Importantly, presented data can be useful for revising the current microbiological cut-off values within the genus Lactobacillus and Pediococcus.

First Emergence of Resistance to Macrolides and Tetracycline Identified in Mannheimia haemolytica and Pasteurella multocida Isolates from Beef Feedlots in Australia.

Bovine respiratory disease (BRD) causes high morbidity and mortality in beef cattle worldwide. Antimicrobial resistance (AMR) monitoring of BRD pathogens is critical to promote appropriate antimicrobial stewardship in veterinary medicine for optimal treatment and control. Here, the susceptibility of Mannheimia haemolytica and Pasteurella multicoda isolates obtained from BRD clinical cases (deep lung swabs at post-mortem) among feedlots in four Australian states (2014–2019) was determined for 19 antimicrobial agents. The M. haemolytica isolates were pan-susceptible to all tested agents apart from a single macrolide-resistant isolate (1/88; 1.1%) from New South Wales (NSW). Much higher frequencies of P. multocida isolates were resistant to tetracycline (18/140; 12.9%), tilmicosin (19/140; 13.6%), tulathromycin/gamithromycin (17/140; 12.1%), and ampicillin/penicillin (6/140; 4.6%). Five P. multocida isolates (3.6%), all obtained from NSW in 2019, exhibited dual resistance to macrolides and tetracycline, and a further two Queensland isolates from 2019 (1.4%) exhibited a multidrug-resistant phenotype to ampicillin/penicillin, tetracycline, and tilmicosin. Random-amplified polymorphic DNA (RAPD) typing identified a high degree of genetic homogeneity among the M. haemolytica isolates, whereas P. multocida isolates were more heterogeneous. Illumina whole genome sequencing identified the genes msr(E) and mph(E)encoding macrolide resistance, tet(R)-tet(H) or tet(Y) encoding tetracycline resistance, and blaROB-1 encoding ampicillin/penicillin resistance in all isolates exhibiting a corresponding resistant phenotype. The exception was the tilmicosin-resistant, tulathromycin/gamithromycin-susceptible phenotype identified in two Queensland isolates, the genetic basis of which could not be determined. These results confirm the first emergence of AMR in M. haemolytica and P. multocida from BRD cases in Australia, which should be closely monitored.

Distribution of methionine sulfoxide reductases in fungi and conservation of the free-methionine-R-sulfoxide reductase in multicellular eukaryotes

Methionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases named methionine sulfoxide reductase (Msr) enzymes to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity and are found in almost all organisms. Another type of thiol-oxidoreductase, the free-methionine-R-sulfoxide reductase (fRMsr), identified so far in prokaryotes and a few unicellular eukaryotes, reduces the R MetO diastereomer of the free amino acid. Moreover, some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. All these Msrs play important roles in the protection of organisms against oxidative stress. Fungi are heterotrophic eukaryotes that colonize all niches on Earth and play fundamental functions, in organic matter recycling, as symbionts, or as pathogens of numerous organisms. However, our knowledge on fungal Msrs is still limited. Here, we performed a survey of msr genes in almost 700 genomes across the fungal kingdom. We show that most fungi possess one gene coding for each type of methionine sulfoxide reductase: MsrA, MsrB, and fRMsr. However, several fungi living in anaerobic environments or as obligate intracellular parasites were devoid of msr genes. Sequence inspection and phylogenetic analyses allowed us to identify non-canonical sequences with potentially novel enzymatic properties. Finaly, we identified several ocurences of msr horizontal gene transfer from bacteria to fungi.