Improving Activity of New Arylurea Agents against Multidrug-Resistant and Biofilm-Producing Staphylococcus epidermidis.

Multidrug-resistant (MDR) strains of Staphylococcus epidermidis (S. epidermidis), prevalent in hospital environments, contribute to increased morbidity and mortality, especially among newborns, posing a critical concern for neonatal sepsis. In response to the pressing demand for novel antibacterial therapies, we present findings from synthetic chemistry and structure-activity relationship studies focused on arylsulfonamide/arylurea derivatives of aryloxy[1-(thien-2-yl)propyl]piperidines. Through bioisosteric replacement of the sulfonamide fragment with a urea moiety, compound 25 was identified, demonstrating potent bacteriostatic activity against clinical multidrug-resistant S. epidermidis strains (MIC50 and MIC90 = 1.6 and 3.125 µg/mL). Importantly, it showed activity against linezolid-resistant strains and exhibited selectivity over mammalian cells. Compound 25 displayed antibiofilm-forming properties against clinical S. epidermidis strains and demonstrated the capacity to eliminate existing biofilm layers. Additionally, it induced complete depolarization of the bacterial membrane in clinical S. epidermidis strains. In light of these findings, targeting bacterial cell membranes with compound 25 emerges as a promising strategy in the fight against multidrug-resistant S. epidermidis strains.

Staphylococcus aureus from Atopic Dermatitis Patients: Its Genetic Structure and Susceptibility to Phototreatment.

We characterized the population of Staphylococcus aureus from patients with atopic dermatitis (AD) in terms of (i) genetic diversity, (ii) presence and functionality of genes encoding important virulence factors: staphylococcal enterotoxins (sea, seb, sec, sed), toxic shock syndrome 1 toxin (tsst-1), and Panton-Valentine leukocidin (lukS/lukF-PV) by spa typing, PCR, drug resistance profile determination, and Western blot. We then subjected the studied population of S. aureus to photoinactivation based on a light-activated compound called rose bengal (RB) to verify photoinactivation as an approach to effectively kill toxin-producing S. aureus. We have obtained 43 different spa types that can be grouped into 12 clusters, indicating for the first-time clonal complex (CC) 7 as the most widespread. A total of 65% of the tested isolates had at least one gene encoding the tested virulence factor, but their distribution differed between the group of children and adults, and between patients with AD and the control group without atopy. We detected a 3.5% frequency of methicillin-resistant strains (MRSA) and no other multidrug resistance. Despite genetic diversity and production of various toxins, all isolates tested were effectively photoinactivated (bacterial cell viability reduction ≥ 3 log10 units) under safe conditions for the human keratinocyte cell line, which indicates that photoinactivation can be a good option in skin decolonization. IMPORTANCE Staphylococcus aureus massively colonizes the skin of patients with atopic dermatitis (AD). It is worth noting that the frequency of detection of multidrug-resistant S. aureus (MRSA) in AD patients is higher than the healthy population, which makes treatment much more difficult. Information about the specific genetic background of S. aureus accompanying and/or causing exacerbations of AD is of great importance from the point of view of epidemiological investigations and the development of possible treatment options.

Design and synthesis of novel arylurea derivatives of aryloxy(1-phenylpropyl) alicyclic diamines with antimicrobial activity against multidrug-resistant Gram-positive bacteria.

The alarming increase in the resistance of bacteria to the currently available antibiotics necessitates the development of new effective antimicrobial agents that are active against bacterial pathogens causing major public health problems. For this purpose, our in-house libraries were screened against a wide panel of clinically relevant Gram-positive and Gram-negative bacteria, based on which compound I was selected for further optimization. Synthetic efforts in a group of arylurea derivatives of aryloxy(1-phenylpropyl) alicyclic diamines, followed with an in vitro evaluation of the activity against multidrug-resistant strains identified compound 44 (1-(3-chlorophenyl)-3-(1-{3-phenyl-3-[3-(trifluoromethyl)phenoxy] propyl}piperidin-4-yl)urea). Compound 44 showed antibacterial activity against Gram-positive bacteria including fatal drug-resistant strains i.e., Staphylococcus aureus (methicillin-resistant, MRSA; vancomycin-intermediate, VISA) and Enterococcus faecium (vancomycin-resistant, VREfm) at low concentrations (0.78-3.125 µg/mL) comparable to last resort antibiotics (i.e., vancomycin and linezolid). It is also potent against biofilm-forming S. aureus and Staphylococcus epidermidis (including linezolid-resistant, LRSE) strains, but with no activity against Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). Compound 44 showed strong bactericidal properties against susceptible and drug-resistant Gram-positive bacteria. Depolarization of the bacterial cytoplasmic membrane induced by compound 44 suggests a dissipation of the bacterial membrane potential as its mechanism of antibacterial action. The high antimicrobial activity of compound 44, along with its selectivity over mammalian cells (lung MCR-5 and skin BJ fibroblast cell lines) and no hemolytic properties toward horse erythrocytes, proposes arylurea derivatives of aryloxy(1-phenylpropyl) alicyclic diamines for development of novel antibacterial agents.

Priming effect with photoinactivation against extensively drug-resistant Enterobacter cloacae and Klebsiella pneumoniae.

In this study, we present antimicrobial blue light (aBL) and antimicrobial photoinactivation with green light in the presence of Rose Bengal (aPDI) to modulate the susceptibility of extensively drug-resistant (XDR) Enterobacter cloacae and Klebsiella pneumoniae clinical isolates to antimicrobials. This process can be considered a photodynamic priming tool that influences other therapeutic options, such as antibiotics. The current study evaluated the different environments to estimate the most effective priming conditions by testing a broad spectrum of antimicrobials (including antimicrobials with different targets and mechanisms of action). The susceptibility of the E. cloacae and K. pneumoniae clinical isolates to various antibiotics after aBL and green light (with rose bengal) as aPDI treatment was examined with multiple methods of synergy testing (e.g., diffusion methods, checkerboard assay, postantibiotic effect), and most effective photoinactivation conditions were implemented for each environment. When Enterobacteriaceae were exposed to aBL, the most efficient reduction in survival rate under TSB conditions was observed. Similar results were observed when rose bengal, as a photosensitizer, was present during the exposure to green light in PBS. aBL and aPDI led to an increased susceptibility of K. pneumoniae and E. cloacae isolates to chloramphenicol and colistin or fosfomycin and colistin antibiotics, respectively. However, among the 4 tested isolates, we observed synergies between different antimicrobial agents and photoinactivation conditions. Thus, it may suggest that the sensitization process may be considered a strain dependent priming tool.

MRSA surveillance programmes worldwide: moving towards a harmonised international approach.

Multinational surveillance programmes for methicillin-resistant Staphylococcus aureus (MRSA) are dependent on national structures for data collection. This study aimed to capture the diversity of national MRSA surveillance programmes and to propose a framework for harmonisation of MRSA surveillance. The International Society of Antimicrobial Chemotherapy (ISAC) MRSA Working Group conducted a structured survey on MRSA surveillance programmes and organised a webinar to discuss the programmes' strengths and challenges as well as guidelines for harmonisation. Completed surveys represented 24 MRSA surveillance programmes in 16 countries. Several countries reported separate epidemiological and microbiological surveillance. Informing clinicians and national policy-makers were the most common purposes of surveillance. Surveillance of bloodstream infections (BSIs) was present in all programmes. Other invasive infections were often included. Three countries reported active surveillance of MRSA carriage. Methodology and reporting of antimicrobial susceptibility, virulence factors, molecular genotyping and epidemiological metadata varied greatly. Current MRSA surveillance programmes rely upon heterogeneous data collection systems, which hampers international epidemiological monitoring and research. To harmonise MRSA surveillance, we suggest improving the integration of microbiological and epidemiological data, implementation of central biobanks for MRSA isolate collection, and inclusion of a representative sample of skin and soft-tissue infection cases in addition to all BSI cases.

Evaluation of Biofilm Formation and Prevalence of Multidrug-Resistant Strains of Staphylococcus epidermidis Isolated from Neonates with Sepsis in Southern Poland.

Staphylococcus epidermidis strains play an important role in nosocomial infections, especially in the ones associated with biofilm formation on medical devices. The paper was aimed at analyzing the mechanisms of antibiotic resistance and confirming the biofilm-forming ability among S. epidermidis strains isolated from the blood of hospitalized newborns. Genetic analysis of resistance mechanism determinants included multiplex PCR detection of mecA, ermA, ermB, ermC, msrA, and mef genes. Biofilm analysis comprised phenotypic and genotypic methods including Christensen and Freeman methods and PCR detection of the icaADB gene complex. Among the tested S. epidermidis strains, 89% of the isolates were resistant to methicillin, 67%-to erythromycin, 53%-to clindamycin, 63%-to gentamicin, and 23%-to teicoplanin, while all the strains were susceptible to vancomycin and linezolid. The mecA gene was detected in 89% of the isolates, the ermC gene was the most common and present among 56% of the strains, while the msrA gene was observed in 11% isolates. Eighty-five percent of the strains were described as biofilm-positive by phenotypic methods and carried the icaADB gene cluster. Multidrug resistance and the biofilm-forming ability in most of the strains tested may contribute to antimicrobial therapy failure (p < 0.05).

Staphylococcus saprophyticus From Clinical and Environmental Origins Have Distinct Biofilm Composition.

Biofilm formation has been shown to be critical to the success of uropathogens. Although Staphylococcus saprophyticus is a common cause of urinary tract infections, its biofilm production capacity, composition, genetic basis, and origin are poorly understood. We investigated biofilm formation in a large and diverse collection of S. saprophyticus (n = 422). Biofilm matrix composition was assessed in representative strains (n = 63) belonging to two main S. saprophyticus lineages (G and S) recovered from human infection, colonization, and food-related environment using biofilm detachment approach. To identify factors that could be associated with biofilm formation and structure variation, we used a pangenome-wide association study approach. Almost all the isolates (91%; n = 384/422) produced biofilm. Among the 63 representative strains, we identified eight biofilm matrix phenotypes, but the most common were composed of protein or protein-extracellular DNA (eDNA)-polysaccharides (38%, 24/63 each). Biofilms containing protein-eDNA-polysaccharides were linked to lineage G and environmental isolates, whereas protein-based biofilms were produced by lineage S and infection isolates (p < 0.05). Putative biofilm-associated genes, namely, aas, atl, ebpS, uafA, sasF, sasD, sdrH, splE, sdrE, sdrC, sraP, and ica genes, were found with different frequencies (3-100%), but there was no correlation between their presence and biofilm production or matrix types. Notably, icaC_1 was ubiquitous in the collection, while icaR was lineage G-associated, and only four strains carried a complete ica gene cluster (icaADBCR) except one that was without icaR. We provided evidence, using a comparative genomic approach, that the complete icaADBCR cluster was acquired multiple times by S. saprophyticus and originated from other coagulase-negative staphylococci. Overall, the composition of S. saprophyticus biofilms was distinct in environmental and clinical isolates, suggesting that modulation of biofilm structure could be a key step in the pathogenicity of these bacteria. Moreover, biofilm production in S. saprophyticus is ica-independent, and the complete icaADBCR was acquired from other staphylococci.

Staphylococcus saprophyticus Causing Infections in Humans Is Associated with High Resistance to Heavy Metals.

Staphylococcus saprophyticus is a common pathogen of the urinary tract, a heavy metal-rich environment, but information regarding its heavy metal resistance is unknown. We investigated 422 S. saprophyticus isolates from human infection and colonization/contamination, animals, and environmental sources for resistance to copper, zinc, arsenic, and cadmium using the agar dilution method. To identify the genes associated with metal resistance and assess possible links to pathogenicity, we accessed the whole-genome sequence of all isolates and used in silico and pangenome-wide association approaches. The MIC values for copper and zinc were uniformly high (1,600 mg/liter). Genes encoding copper efflux pumps (copA, copB, copZ, mco, and csoR) and zinc transporters (zinT, czrAB, znuBC, and zur) were abundant in the population (20 to 100%). Arsenic and cadmium showed various susceptibility levels. Genes encoding the ars operon (arsRDABC), an ABC transporter and a two-component permease, were linked to resistance to arsenic (MICs ≥ 1,600 mg/liter; 14% [58/422]; P < 0.05). At least three cad genes (cadA or cadC and cadD-cadX or czrC) and genes encoding multidrug efflux pumps and hyperosmoregulation in acidified conditions were associated with resistance to cadmium (MICs ≥ 200 mg/liter; 20% [85/422]; P < 0.05). These resistance genes were frequently carried by mobile genetic elements. Resistance to arsenic and cadmium were linked to human infection and a clonal lineage originating in animals (P < 0.05). Altogether, S. saprophyticus was highly resistant to heavy metals and accumulated multiple metal resistance determinants. The highest arsenic and cadmium resistance levels were associated with infection, suggesting resistance to these metals is relevant for S. saprophyticus pathogenicity.

Foodborne Origin and Local and Global Spread of Staphylococcus saprophyticus Causing Human Urinary Tract Infections.

Staphylococcus saprophyticus is a primary cause of community-acquired urinary tract infections (UTIs) in young women. S. saprophyticus colonizes humans and animals but basic features of its molecular epidemiology are undetermined. We conducted a phylogenomic analysis of 321 S. saprophyticus isolates collected from human UTIs worldwide during 1997-2017 and 232 isolates from human UTIs and the pig-processing chain in a confined region during 2016-2017. We found epidemiologic and genomic evidence that the meat-production chain is a major source of S. saprophyticus causing human UTIs; human microbiota is another possible origin. Pathogenic S. saprophyticus belonged to 2 lineages with distinctive genetic features that are globally and locally disseminated. Pangenome-wide approaches identified a strong association between pathogenicity and antimicrobial resistance, phages, platelet binding proteins, and an increased recombination rate. Our study provides insight into the origin, transmission, and population structure of pathogenic S. saprophyticus and identifies putative new virulence factors.

A Kayvirus Distant Homolog of Staphylococcal Virulence Determinants and VISA Biomarker Is a Phage Lytic Enzyme.

Staphylococcal bacteriophages of the Kayvirus genus are candidates for therapeutic applications. One of their proteins, Tgl, is slightly similar to two staphylococcal virulence factors, secreted autolysins of lytic transglycosylase motifs IsaA and SceD. We show that Tgl is a lytic enzyme secreted by the bacterial transport system and localizes to cell peripheries like IsaA and SceD. It causes lysis of E. coli cells expressing the cloned tgl gene, but could be overproduced when depleted of signal peptide. S. aureus cells producing Tgl lysed in the presence of nisin, which mimics the action of phage holin. In vitro, Tgl protein was able to destroy S. aureus cell walls. The production of Tgl decreased S. aureus tolerance to vancomycin, unlike the production of SceD, which is associated with decreased sensitivity to vancomycin. In the genomes of kayviruses, the tgl gene is located a few genes away from the lysK gene, encoding the major endolysin. While lysK is a late phage gene, tgl can be transcribed by a host RNA polymerase, like phage early genes. Taken together, our data indicate that tgl belongs to the kayvirus lytic module and encodes an additional endolysin that can act in concert with LysK in cell lysis.

Transient Silencing of Antibiotic Resistance by Mutation Represents a Significant Potential Source of Unanticipated Therapeutic Failure.

Sporadic literature reports describe isolates of pathogenic bacteria that harbor an antibiotic resistance determinant but remain susceptible to the corresponding antibiotic as a consequence of a genetic defect. Such strains represent a source from which antibiotic resistance may reemerge to cause treatment failure in patients. Here, we report a systematic investigation into the prevalence and nature of this phenomenon, which we term silencing of antibiotic resistance by mutation (SARM). Instances of SARM were detected among 1,470 Staphylococcus aureus isolates through side-by-side comparison of antibiotic resistance genotype (as determined by whole-genome sequencing) versus phenotype (as assessed through susceptibility testing). Of the isolates analyzed, 152 (10.3%) harbored a silenced resistance gene, including 46 (3.1%) that exhibited SARM to currently deployed antistaphylococcal drugs. SARM resulted from diverse mutational events but most commonly through frameshift mutation of resistance determinants as a result of point deletion in poly(A) tracts. The majority (∼90%) of SARM strains reverted to antibiotic resistance at frequencies of ≥10-9; thus, while appearing antibiotic sensitive in the clinical microbiology laboratory, most S. aureus isolates exhibiting SARM will revert to antibiotic resistance at frequencies achievable in patients. In view of its prevalence in a major pathogen, SARM represents a significant potential threat to the therapeutic efficacy of antibiotics.IMPORTANCE Antibiotic resistance hinders the treatment of bacterial infection. To guide effective therapy, clinical microbiology laboratories routinely perform susceptibility testing to determine the antibiotic sensitivity of an infecting pathogen. This approach relies on the assumption that it can reliably distinguish bacteria capable of expressing antibiotic resistance in patients, an idea challenged by the present study. We report that the important human pathogen Staphylococcus aureus frequently carries antibiotic resistance genes that have become inactivated ("silenced") by mutation, leading strains to appear antibiotic sensitive. However, resistance can rapidly reemerge in most such cases, at frequencies readily achievable in infected patients. Silent antibiotic resistance is therefore prevalent, transient, and evades routine detection, rendering it a significant potential threat to antibacterial chemotherapy.

European external quality assessments for identification, molecular typing and characterization of Staphylococcus aureus.

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols. Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed. Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes. Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives.

Complete Genome Sequences of Two Novel Staphylococcus aureus Podoviruses of Potential Therapeutic Use, vB_SauP_phiAGO1.3 and vB_SauP_phiAGO1.9.

Here, we report the genome sequences of two Staphylococcus aureus phages belonging to the family Podoviridae and subfamily Picovirinae, vB_SauP_phiAGO1.3 and vB_SauP_phiAGO1.9, which were isolated from Warsaw sewage. Analysis of their genomes provides valuable information about the diversity of phages belonging to the genus Rosenblumvirus and their genes that undergo evolutionary adaptation to cells of different host strains.

Emergence and spread of worldwide Staphylococcus aureus clones among cystic fibrosis patients.

BACKGROUND: The aim of this study was to assess the relatedness of molecular types of Staphylococcus aureus isolates colonizing cystic fibrosis (CF) patients with their antimicrobial resistance and prevalence of toxin genes. METHODS: A total of 215 isolates from the airways of 107 patients with CF were tested for spa and SCCmec type, antimicrobial resistance and carriage of toxin genes. RESULTS: t015, t084, t091, t700 and t002 were the largest group (approximately 25%) among all 69 identified spa types. Five new spa types, t14286, t14287, t14288, t14289 and t14290, were identified and registered. Isolates from CF patients were clustered into 11 multi-locus sequence typing clonal complexes, with CC30, CC22, CC97, CC45, CC15 and CC5 being the most frequent ones. Twelve (5.6%) methicillin-resistant S. aureus (MRSA) isolates and 102 (47.7%) multidrug-resistant isolates were identified, along with three SCCmec types (I, III and V). All isolates (both MRSA and methicillin-sensitive S. aureus) were Panton-Valentine leucocidin-negative, and 56.7% harbored egc genes. This was the first study documenting the presence of ST398-V-t571 livestock-associated MRSA in a European patient with CF. CONCLUSION: These findings imply that individuals with CF can also be colonized with animal-related ST398 MRSA, and justify constant monitoring of staphylococcal colonization and identification of epidemic S. aureus clones in this group.

The Ability of Lytic Staphylococcal Podovirus vB_SauP_phiAGO1.3 to Coexist in Equilibrium With Its Host Facilitates the Selection of Host Mutants of Attenuated Virulence but Does Not Preclude the Phage Antistaphylococcal Activity in a Nematode Infection Model.

Phage vB_SauP_phiAGO1.3 (phiAGO1.3) is a polyvalent Staphylococcus lytic podovirus with a 17.6-kb genome (Gozdek et al., 2018). It can infect most of the Staphylococcus aureus human isolates of dominant clonal complexes. We show that a major factor contributing to the wide host range of phiAGO1.3 is a lack or sparcity of target sites for certain restriction-modification systems of types I and II in its genome. Phage phiAGO1.3 requires for adsorption ß-O-GlcNAcylated cell wall teichoic acid, which is also essential for the expression of methicillin resistance. Under certain conditions an exposure of S. aureus to phiAGO1.3 can lead to the establishment of a mixed population in which the bacteria and phages remain in equilibrium over multiple generations. This is reminiscent of the so called phage carrier state enabling the co-existence of phage-resistant and phage-sensitive cells supporting a continuous growth of the bacterial and phage populations. The stable co-existence of bacteria and phage favors the emergence of phage-resistant variants of the bacterium. All phiAGO1.3-resistant cells isolated from the phage-carrier-state cultures contained a mutation inactivating the two-component regulatory system ArlRS, essential for efficient expression of numerous S. aureus virulence-associated traits. Moreover, the mutants were unaffected in their susceptibility to infection with an unrelated, polyvalent S. aureus phage of the genus Kayvirus. The ability of phiAGO1.3 to establish phage-carrier-state cultures did not preclude its antistaphylococcal activity in vivo in an S. aureus nematode infection model. Taken together our results suggest that phiAGO1.3 could be suitable for the therapeutic application in humans and animals, alone or in cocktails with Kayvirus phages. It might be especially useful in the treatment of infections with the majority of methicillin-resistant S. aureus strains.

Livestock-associated Staphylococcus aureus on Polish pig farms.

BACKGROUND: Livestock-associated Staphylococcus aureus (LA-SA) draws increasing attention due to its particular ability to colonize farm animals and be transmitted to people, which in turn leads to its spread in the environment. The aim of the study was to determine the dissemination of LA-SA on pig farms selected throughout Poland, characterize the population structure of identified S. aureus, and assess the prevalence of LA-SA carriage amongst farmers and veterinarians being in contact with pigs. METHODS AND FINDINGS: The study was conducted on 123 pig farms (89 farrow-to-finish and 34 nucleus herds), located in 15 out of 16 provinces of Poland. Human and pig nasal swabs, as well as dust samples were analyzed. S. aureus was detected on 79 (64.2%) farms from 14 provinces. Amongst these farms LA-SA-positive farms dominated (71/79, 89.9%, 95% CI [81.0%, 95.5%]). The prevalence of LA-MRSA-positive farms was lower than LA-MSSA-positive (36.6% of LA-SA-positive farms, 95% CI [25.5%, 48.9%] vs. 74.6%, 95% CI [62.9%, 84.2%]). In total, 190 S. aureus isolates were identified: 72 (38%) MRSA and 118 (62%) methicillin-susceptible S. aureus (MSSA), of which 174 (92%) isolates were classified to three livestock-associated lineages: CC398 (73%), CC9 (13%), and CC30/ST433 (6%). All CC398 isolates belonged to the animal clade. Four LA-MRSA clones were detected: ST433-IVa(2B) clone (n = 8, 11%), described to the best of our knowledge for the first time, and three ST398 clones (n = 64, 89%) with the most prevalent being ST398-V(5C2&5)c, followed by ST398-V(5C2), and ST398-IVa(2B). Nasal carriage of LA-SA by pig farmers was estimated at 13.2% (38/283), CC398 carriage at 12.7% (36/283) and ST398-MRSA carriage at 3.2% (9/283), whereas by veterinarians at 21.1% (8/38), 18.4% (7/38) and 10.5% (4/38), respectively. CONCLUSIONS: The prevalence of LA-MRSA-positive pig farms in Poland has increased considerably since 2008, when the first MRSA EU baseline survey was conducted in Europe. On Polish pig farms CC398 of the animal clade predominates, this being also reflected in the prevalence of CC398 nasal carriage in farmers and veterinarians. However, finding a new ST433-IVa(2B) clone provides evidence for the continuing evolution of LA-MRSA and argues for further monitoring of S. aureus in farm animals.

KAEA (SUDPRO), a member of the ubiquitous KEOPS/EKC protein complex, regulates the arginine catabolic pathway and the expression of several other genes in Aspergillus nidulans.

The kaeA(KAE1) (suDpro) gene, which was identified in Aspergillus nidulans as a suppressor of proline auxotrophic mutations, encodes the orthologue of Saccharomyces cerevisiae Kae1p, a member of the evolutionarily conserved KEOPS/EKC (Kinase, Endopeptidase and Other Proteins of Small size/Endopeptidase-like and Kinase associated to transcribed Chromatin) complex. In yeast, this complex has been shown to be involved in tRNA modification, transcription, and genome maintenance. In A. nidulans, mutations in kaeA result in several phenotypic effects, the derepression of arginine catabolism genes, and changes in the expression levels of several others, including genes involved in amino acid and siderophore metabolism, sulfate transport, carbon/energy metabolism, translation, and transcription regulation, such as rcoA(TUP1), which encodes the global transcriptional corepressor.

Population Structure and Oxacillin Resistance of Staphylococcus aureus from Pigs and Pork Meat in South-West of Poland.

The genotypes and oxacillin resistance of 420 S. aureus isolates from pigs (n = 203) and pork (n = 217) were analyzed. Among 18 spa types detected in S. aureus from pig t011, t021, t034, t091, t318, t337, and t1334 were the most frequent. Among 30 spa types found in S. aureus isolates from pork t084, t091, t499, t4309, t12954, and t13074 were dominant. The animal S. aureus isolates were clustered into MLST clonal complexes CC7, CC9, CC15, CC30, and CC398 and meat-derived isolates to CC1, CC7, and CC15. Thirty-six MRSA were isolated exclusively from pigs. All MRSA were classified to spa t011 SCCmecV. BORSA phenotype was found in 14% S. aureus isolates from pigs and 10% isolates from pork meat. spa t034 dominated among BORSA from pigs and t091 among meat-derived BORSA. This is the first report on spa types and oxacillin resistance of S. aureus strains from pigs and pork meat in Poland. Besides S. aureus CC9, CC30, and CC398 known to be distributed in pigs, the occurrence of genotype belonging to CC7 in this species has been reported for the first time. To our knowledge it is also the first report concerning CC398 BORSA isolates from pigs and pork meat.

Mupirocin-induced mutations in ileS in various genetic backgrounds of methicillin-resistant Staphylococcus aureus.

Topical mupirocin is widely used for the decolonization of methicillin-resistant Staphylococcus aureus (MRSA) carriers. We evaluated the capacity of various MRSA clonotypes to develop mutations in the ileS gene associated with low-level mupirocin resistance. Twenty-four mupirocin-sensitive MRSA isolates from a variety of genotypes (determined by a multilocus variable-number tandem-repeat assay) were selected. Mupirocin MICs were determined by Etest. The isolates were then incubated in subinhibitory concentrations of mupirocin for 7 to 14 days. Repeat MIC determinations and sequencing of the ileS gene were then performed. Doubling times of isolates exposed to mupirocin and of unexposed isolates were compared. We found that exposure to mupirocin led to rapid induction of low-level resistance (MICs of 8 to 24 µg/ml) in 11 of 24 (46%) MRSA isolates. This phenomenon was observed in strains with diverse genetic backgrounds. Various mutations were detected in 18 of 24 (75%) MRSA isolates. Acquisition of mutations appeared to be a stepwise process during prolonged incubation with the drug. Among the five isolates exhibiting low-level resistance and the highest MICs, four tested sensitive after incubation in the absence of mupirocin but there was no reversion to the susceptible wild-type primary sequence. Resistance was not associated with significant fitness cost, suggesting that MRSA strains with low-level mupirocin resistance may have a selective advantage in facilities where mupirocin is commonly used. Our findings emphasize the importance of the judicious use of this topical agent and the need to closely monitor for the emergence of resistance.

Antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus: An international survey.

The antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) isolates reported from 17 countries in the Americas, Europe and, Australia-Asia were analysed. Among a total of 3236 non-duplicate isolates, the lowest susceptibility was observed to erythromycin in all regions. Susceptibility to ciprofloxacin showed large variation (25%, 75% and 84% in the Americas, Europe and Australia-Asia, respectively). Two vancomycin-intermediate PVL-positive MRSA isolates were reported, one from Hong Kong and the other from The Netherlands. Resistance to trimethoprim/sulfamethoxazole and linezolid was <1%. Among 1798 MRSA isolates from 13 countries that were tested for the requested 10 non-ß-lactam antibiotics, 49.4% were multisusceptible. However, multiresistant isolates (resistant to at least three classes of non-ß-lactam antibiotics) were reported from all regions. Sequence type 30 (ST30) was reported worldwide, whereas ST80 and ST93 were exclusive to Europe and Australia, respectively. USA300 and related clones (ST8) are progressively replacing the ST80 clone in several European countries. Eight major clusters were discriminated by multilocus variable-number tandem repeat assay (MLVA), showing a certain geographic specificity. PVL-positive MRSA isolates frequently remain multisusceptible to non-ß-lactam agents, but multiresistance is already prevalent in all regions. Surveillance of MRSA susceptibility patterns should be monitored to provide clinicians with the most current information regarding changes in resistance patterns.