The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

Inactivation of the Response Regulator AgrA Has a Pleiotropic Effect on Biofilm Formation, Pathogenesis and Stress Response in Staphylococcus lugdunensis.

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus that emerges as an important opportunistic pathogen. However, little is known about the regulation underlying the transition from commensal to virulent state. Based on knowledge of S. aureus virulence, we suspected that the agr quorum sensing system may be an important determinant for the pathogenicity of S. lugdunensis. We investigated the functions of the transcriptional regulator AgrA using the agrA deletion mutant. AgrA played a role in cell pigmentation: ΔargA mutant colonies were white while the parental strains were slightly yellow. Compared with the wild-type strain, the ΔargA mutant was affected in its ability to form biofilm and was less able to survive in mice macrophages. Moreover, the growth of ΔagrA was significantly reduced by the addition of 10% NaCl or 0.4 mM H2O2 and its survival after 2 h in the presence of 1 mM H2O2 was more than 10-fold reduced. To explore the mechanisms involved beyond these phenotypes, the ΔagrA proteome and transcriptome were characterized by mass spectrometry and RNA-Seq. We found that AgrA controlled several virulence factors as well as stress-response factors, which are well correlated with the reduced resistance of the ΔagrA mutant to osmotic and oxidative stresses. These results were not the consequence of the deregulation of RNAIII of the agr system, since no phenotype or alteration of the proteomic profile has been observed for the ΔRNAIII mutant. Altogether, our results highlighted that the AgrA regulator of S. lugdunensis played a key role in its ability to become pathogenic. IMPORTANCE Although belonging to the natural human skin flora, Staphylococcus lugdunensis is recognized as a particularly aggressive and destructive pathogen. This study aimed to characterize the role of the response regulator AgrA, which is a component of the quorum-sensing agr system and known to be a major element in the regulation of pathogenicity and biofilm formation in Staphylococcus aureus. In the present study, we showed that, contrary to S. aureus, the agrA deletion mutant produced less biofilm. Inactivation of agrA conferred a white colony phenotype and impacted S. lugdunensis in its ability to survive in mice macrophages and to cope with osmotic and oxidative stresses. By global proteomic and transcriptomic approaches, we identified the AgrA regulon, bringing molecular bases underlying the observed phenotypes. Together, our data showed the importance of AgrA in the opportunistic pathogenic behavior of S. lugdunensis allowing it to be considered as an interesting therapeutic target.

Accurate detection of oxacillin-resistant Staphylococcus lugdunensis by use of agar dilution.

BACKGROUND/PURPOSE: Staphylococcus lugdunensis is a Gram-positive coagulase-negative bacterium and is recognized as a critical pathogenic species recently. Here, we aimed to evaluate the cefoxitin disk diffusion (CDD), oxacillin agar dilution (OAD), and mecA PCR for detecting oxacillin-resistant S. lugdunensis (ORSL) isolates. METHODS: Multilocus sequence typing (MLST) analysis was performed to determine the clonality of 117 S. lugdunensis isolates isolated between May 2009 and Jul 2014. CDD, OAD, and mecA PCR were used to identify oxacillin-resistant S. lugdunensis (ORSL). RESULTS: MLST results showed that the most common sequence type (ST) of our S. lugdunensis isolates was ST6 (35.9%) followed by ST3 (28.2%), ST27 (17.9%), and ST4 (6.8%). CDD and OAD showed that 39 and 43 isolates were ORSL, respectively. 4 ST3 CDD-susceptible S. lugdunensis (OSSL) isolates had MIC values ≥ 4 for oxacillin. mecA PCR results showed that 43 OAD-resistant S. lugdunensis and 3 OAD-susceptible ST27 S. lugdunensis had the mecA gene. Therefore, OAD was used as the gold standard to evaluate the performance of CDD and mecA PCR for identifying ORSL. The overall sensitivity, specificity, and accuracy of CCD for ORSL detection was 90.7%, 100%, and 96.8%, respectively. The sensitivity, specificity, and accuracy of mecA PCR for identifying ORSL was 100%, 95.9%, and 97.44%, respectively. CONCLUSION: Our results indicate that OAD shows higher accuracy for ORSL detection compared with CDD and mecA PCR.

Phylogenetic Distribution of CRISPR-Cas Systems in Staphylococcus lugdunensis.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) genes (CRISPR-Cas) are present in many bacterial genomes with functions beyond adaptive immunity. We aimed to characterize the CRISPR-Cas system in the pathogenic Gram-positive bacterium Staphylococcus lugdunensis and determine its association with sequence types (STs) determined by multilocus sequence typing (MLST) and oxacillin susceptibility. Primers were designed to detect and sequence types IIIA and IIC CRISPR-Cas in 199 S. lugdunensis isolates. MLST and oxacillin susceptibility tests were also performed on the isolates. We found that 84 S. lugdunensis isolates had type IIIA CRISPR-Cas, while 46 had type IIC. The results showed a strong association between STs and CRISPR-Cas types. The ST1, ST6, ST12, and ST15 isolates had type IIIA CRISPR-Cas systems, and the ST4, ST27, and ST29 isolates had type IIC CRISPR-Cas. Interestingly, of 83 isolates containing type IIIA CRISPR-Cas, 17 (20.5%) were oxacillin-resistant S. lugdunensis (ORSL), and all of these ORSL isolates belonged to ST6 cluster 1. Moreover, spacers 23 and 21 were found in 16 and 17 ORSL isolates, respectively. In contrast, all 46 isolates with type IIC CRISPR-Cas were susceptible to oxacillin. Our results showed that 41.3% of CRISPR-Cas IIIA spacers were homologous to plasmids and 20.2% were homologous to phages. However, in type IIC CRISPR-Cas, 11.8% and 39.9% of spacers showed sequence homology with plasmids and phages, respectively. In conclusion, we found that the distribution and composition of the CRISPR-Cas system in S. lugdunensis was associated with STs and oxacillin susceptibility. IMPORTANCE CRISPR-Cas systems have been characterized as playing several biological roles in many bacterial genomes. Moreover, CRISPR-Cas systems are useful for epidemiological, diagnostic, and evolutionary studies of pathogenic bacteria. However, the characteristics of CRISPR-Cas systems in Staphylococcus lugdunensis have been rarely reported. In this study, we revealed that type IIIA CRISPR-Cas was dominant in S. lugdunensis isolates, followed by type IIC CRISPR-Cas. Moreover, the composition of CRISPR-Cas spacers was strongly associated with multilocus sequence typing and oxacillin susceptibility of S. lugdunensis. These results advance our understanding of the evolution of CRISPR-Cas systems; however, the biological functions of CRISPR-Cas systems in S. lugdunensis remain to be further characterized.

Cross-sectional study of Staphyloccus lugdunensis prevalence in cats.

Staphylococcus lugdunensis is a commensal bacterium in humans and other animals that can cause serious infections. The aim of this research was to estimate the frequency of S. lugdunensis in pet cats and to characterize the S. lugdunensis isolates obtained. The prevalence of S. lugdunensis was 0.77% (4/523) in healthy cats and 1.23% (1/81) in sick cats. The isolates (N = 5), which colonized conjunctival sacs, nares, and the anus, were almost fully phenotypically sensitive to antibiotics, but harbored resistance genes to four chemotherapeutic groups. Their sequence types (STs) included ST2, ST3, ST9, and ST15. There was detected a far lower prevalence of S. lugdunensis in pet cats than is reported in the human population. Nevertheless, the phenotypic and genotypic properties of S. lugdunensis isolates found in the current study were very similar to those described previously in isolates of human origin.

Epidemiology and antimicrobial susceptibility of Staphylococcus lugdunensis in a Greek tertiary-care hospital.

Staphylococcus lugdunensis is considered more pathogenic than other coagulase-negative Staphylococci (CoNS), with its virulence resembling that of Staphylococcus aureus. We report a retrospective study of all S. lugdunensis infection cases during a 3.5-year period in a large tertiary university hospital in Greece. S.lugdunensis was susceptible to most tested antibiotics, although a high resistance percentage was found to clindamycin (27%) and erythromycin (25%). The susceptibility rate to penicillin was 49%, much lower than previously reported elsewhere, indicating that penicillin may not be an optimal treatment choice for S. lugdunensis infections in our region.

Complete genome sequence of a methicillin-resistant Staphylococcus lugdunensis strain and characteristics of its staphylococcal cassette chromosome mec.

Symptoms of Staphylococcus lugdunensis infection are often similar to those of Staphylococcus aureus infection, including skin and soft-tissue lesions, bacteremia and infective endocarditis. Despite the severity of these infections, S. lugdunensis is regarded as a less important pathogen than drug-resistant S. aureus. To investigate its ability to cause infectious diseases, a methicillin-resistant S. lugdunensis (MRSL) strain JICS135 was isolated from a patient with bacteremia and subjected to whole genome sequencing. Similar to most strains of methicillin-resistant S. aureus (MRSA), this MRSL strain possessed the staphylococcal cassette chromosome mec (SCCmec) located close to the origin of replication. However, the SCCmec in this MRSL strain, with three ccr complexes, was structurally unique and currently untypable. Moreover, the SCCmec of this MRSL strain was found to carry two genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM)-like proteins accompanied by glycosyl transferases, one of which may have been derived from S. aureus and the other from S. epidermidis, indicating that this MRSL evolved to carry virulence factors from other staphylococci. The emergence of this strain, the first MRSL strain whose genome has been sequenced completely, may be of public concern.

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides, proteins, eDNA and their correlation with biofilm mass.

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R2 = 0.882), while the content of proteins turned out to be weakly (R2 = 0.465) and that of eDNA very weakly associated (R2 = 0.202) to the biofilm mass.

Evaluation of disc diffusion tests and agar screening for predicting mecA-mediated oxacillin resistance in Staphylococcus lugdunensis revealed a cefoxitin-susceptible, mecA-positive S. lugdunensis clonal complex 27 clone.

OBJECTIVES: This study evaluated disc diffusion tests and agar screening for detecting mecA-mediated oxacillin resistance in Staphylococcus lugdunensis (S. lugdunensis). METHODS: Staphylococcus lugdunensis isolates (n = 179) from diverse sources in Hong Kong during 1998-2018 were investigated by disc diffusion tests (cefoxitin and oxacillin) and inoculation onto oxacillin (1 µg/mL and 2 µg/mL) and chromID methicillin-resistant Staphylococcus aureus (MRSA) agars. The results were compared with mecA PCR as the reference. Isolates with discordant results were further tested by MIC and penicillin-binding protein 2a (PBP2a) assays. RESULTS: Cefoxitin and oxacillin zone diameters were not distributed in ways that allowed reliable division of the mecA-positive (n = 52) and mecA-negative (n = 127) isolates. On applying the 2019 Clinical Laboratory Standards Institute (CLSI) M100 breakpoints for cefoxitin disc results, there was 88% categorical agreement (CA) and 40% very major error (VME). Screening using 2 µg/mL oxacillin agar reliably differentiated mecA-positive and mecA-negative isolates (100% CA) without any major error (ME) or VME results. The performance of screening using 1 µg/mL oxacillin agar or ChromID MRSA agar was variable (74-89% CA, 0-38% ME and 0-37% VME). The mecA-positive isolates (n = 21) that could not be detected by the cefoxitin disc test were further characterised. The cefoxitin MIC for all 21 isolates was ≤4 µg/mL. Twenty isolates had an oxacillin MIC of 1-2 µg/mL and one had an oxacillin MIC of 4 µg/mL. All had positive PBP2a results and were typed as clonal cluster 27/SCCmec V. CONCLUSIONS: These findings highlight the need to evaluate phenotypic methods using mecA-positive S. lugdunensis with different oxacillin resistance phenotypes.

Integron-Related Resistance in New Emerged Staphylococcus lugdunensis Infection in Burn Patients.

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus species that may cause various infections with unusual severity. In spite of the administration of various antibiotics, infections caused by such bacteria are become resistant significantly. Transmission of antibiotic resistance genes, especially by Integron structures, exacerbates the prevalence of resistant strains. To investigate the antibiotic susceptibility pattern of S. lugdunensis as a new emergence in burns, the presence of integron classes (I, II, III) was performed in recent study. Sampling for this study was carried out over a period of 13 months (January 2017 to February 2018) from Amir-Al-Momenin burn center of southwest of Iran, affiliated with Shiraz University of Medical Sciences. Twenty-eight isolates of S. lugdunensis were confirmed by phenotypic tests. The presence of integron classes was evaluated by PCR technique and specific primers. The majority of studied infectious agents were seen in ICU with 28.57%. The prevalence of class I and II integrons was 7 (25.00%) and 2 (7.14%), respectively, in S. lugdunensis isolates, whereas no integron III was found. No significant association was seen between antibiotic resistance and the present integrons (P > .05). Since the prevalence of S. lugdunensis strains as a new emergence infection is increasing in clinical settings especially burns, preventing drug resistance in these isolates is inevitable. So knowing the epidemiology pattern of new emerging infections and their resistant pattern is very helpful in infection control and save hospitalized patients life.

Characterization of a novel, type II staphylococcal cassette chromosome mec element from an endemic oxacillin-resistant Staphylococcus lugdunensis clone in a hospital setting.

BACKGROUND: Staphylococcus lugdunensis is a significant pathogen that causes community-acquired and nosocomial infections. The high prevalence of oxacillin-resistant S. lugdunensis (ORSL) is of major concern. Resistance to ß-lactams is caused by acquisition of the staphylococcal cassette chromosome mec (SCCmec) element. The cassette is highly diverse, both structurally and genetically, among CoNS. Isolates carrying SCCmec II-ST6 are the major persistent clones in hospitals. OBJECTIVES: To investigate the structure and evolutionary origin of a novel type II SCCmec element in an endemic ST6 S. lugdunensis clone. METHODS: The structure of the SCCmec II element carried by ST6 strain CGMH-SL118 was determined by WGS and compared with those reported previously. RESULTS: A novel 39 kb SCCmec element, SCCmecCGMH-SL118, with a unique mosaic structure comprising 41 ORFs integrated into the 3' end of the rlmH gene, was observed. Some regions of SCCmecCGMH-SL118 were homologous to SCCmec IIa of the prototype MRSA strain N315. The structure of SCCmecCGMH-SL118 was similar to that of SCCmec IIb of the MRSA strain, JCSC3063, mainly lacking the aminoglycoside resistance determinant pUB110 in the J3 region but containing the insertion sequence IS256 in the J2 region. Notably, SCCmecCGMH-SL118 deletions in the J1 region compared with SCCmec types IIa and IIb, and a high homology to SCCmec elements of Staphylococcus aureus JCSC4610 and Staphylococcus haemolyticus strain 621 were found. CONCLUSIONS: The genetic diversity of the type II SCCmec element in ORSL suggests that CoNS is a potential reservoir for interspecies transfer of SCCmec to S. aureus in hospitals.

Staphylococcus lugdunensis: antimicrobial susceptibility and optimal treatment options.

Staphylococcus lugdunensis is a coagulase-negative staphylococcus (CoNS) with unusual pathogenicity resembling that of S. aureus. Unlike other CoNS, S. lugdunensis remains susceptible to most antibiotics. The resistance to penicillin varies widely (range, 15-87% worldwide), whereas methicillin resistance is still rare. We aimed to evaluate treatment options for infections caused by S. lugdunensis and more specifically to investigate whether penicillin G could be a better treatment choice than oxacillin. Susceptibility testing was performed using the disc diffusion method for penicillin G, cefoxitin, trimethoprim/sulfamethoxazole, erythromycin, clindamycin, gentamicin, norfloxacin, fusidic acid, rifampicin, and fosfomycin. Isolates susceptible to penicillin G were further tested with a gradient test for penicillin G and oxacillin. Of the 540 clinical isolates tested, 74.6% were susceptible to penicillin G. Among these penicillin-susceptible isolates, the MIC50 and MIC90 values for penicillin G were threefold lower than that for oxacillin. A majority of the isolates were susceptible to all other antibiotics tested. Breakpoints for fosfomycin have not yet been defined, and so no conclusions could be drawn. Two isolates were resistant to cefoxitin and carried the mecA gene; whole-genome sequencing revealed that both harbored the SCCmec element type IVa(2B). S. lugdunensis isolated in Sweden were susceptible to most tested antibiotics. Penicillin G may be a more optimal treatment choice than oxacillin. Although carriage of the mecA gene is rare among S. lugdunensis, it does occur.

Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk.

The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.

In vitro activity of tedizolid against clinical isolates of Staphylococcus lugdunensis and Staphylococcus haemolyticus from Europe and the United States.

Staphylococcus lugdunensis and Staphylococcus haemolyticus are unique among CoNS in that the former often causes aggressive disease, while the latter consistently exhibits high rates of multidrug resistance. We evaluated the in vitro susceptibility of contemporary (2012-2013) isolates from both pathogens to tedizolid and comparators, using standard methodology. Results were interpreted using CLSI and EUCAST breakpoints. Overall, 106 S. lugdunensis and 103 S. haemolyticus isolates were collected from 51 medical centers in the United States and 30 centers in 18 European countries. Tedizolid showed good activity against S. lugdunensis (MIC50/MIC90: 0.12/0.12 mg/L) and S. haemolyticus (MIC50/MIC90: 0.12/0.12 mg/L), inhibiting all isolates at MIC ≤0.25 mg/L. Based on the EUCAST breakpoint for staphylococci and when substituting the CLSI breakpoint for Staphylococcus aureus, all isolates were tedizolid susceptible. All isolates were also susceptible to linezolid, but the in vitro potency of tedizolid was 4-fold greater than that of linezolid against both S. lugdunensis and S. haemolyticus, based on MIC90 values. S. lugdunensis exhibited ≥99% susceptibility to vancomycin, teicoplanin, gentamicin, levofloxacin, and trimethoprim-sulfamethoxazole; 7% of isolates were resistant to tetracycline, 11% to clindamycin, and 2% were methicillin-resistant. S. haemolyticus exhibited high rates of resistance to commonly used anti-staphylococcal agents: 71% of isolates were resistant to methicillin, 36%-37% to clindamycin, and 30%-50% to gentamicin. These in vitro findings suggest that tedizolid could be an alternative treatment option for infections due to these medically important CoNS pathogens. Additional clinical evaluation and continued surveillance of tedizolid in vitro activity against S. lugdunensis and S. haemolyticus are warranted.

In vitro antibacterial and anticancer properties of Helicteres hirsuta Lour. leaf and stem extracts and their fractions.

Helicteres hirsuta Lour. (H. hirsuta) has been considered as a herbal medicine for the treatment of malaria and diabetes but limited studies have been conducted on its anticancer and antibacterial properties. In this study, the in vitro antibacterial and anticancer properties of the leaf and stem extracts and their two sub-fractions (aqueous and saponin-enriched butanol fractions) prepared from H. hirsuta were elucidated. MTT and CCK-8 assays were employed to assess their in vitro anticancer properties against various cancer cell lines. The antibacterial activity was assessed using the disc diffusion method and minimum inhibitory concentration (MIC) values were determined. The results revealed that the saponin-enriched fractions from H. hirsuta leaves and stems showed the highest antibacterial activity against E. coli (MIC values of 2.50 and 5.00 mg/mL, respectively) and S. lugdunensis (MIC values of 0.35 and 0.50 mg/mL, respectively). Importantly, these saponin-enriched fractions possessed strong anticancer activity in vitro towards a range of cancer cell lines including MIA PaCa-2 (pancreas); A2780 (ovarian); H460 (lung); A431 (skin); Du145 (prostate); HT29 (colon); MCF-7 (breast); SJ-G2, U87, SMA (glioblastoma) and BE2-C (neuroblastoma) at low doses (GI50 values of 0.36-11.17 µg/mL). They especially revealed potent anti-pancreatic cancer activity in vitro against MIA PaCa-2, BxPC-3 and CFPAC-1 cells with IC50 values of 1.80-6.43 µg/mL. This finding provides scientific evidence of the cytotoxic activity of the extracts prepared from H. hirsuta leaves and stems, and suggests further studies to isolate active compounds for development of new anticancer agents from these plant extracts.

Characterization of two novel variants of staphylococcal cassette chromosome mec elements in oxacillin-resistant Staphylococcus lugdunensis.

OBJECTIVES: Staphylococcus lugdunensis, a species of CoNS, has become an important hospital pathogen because of increasing resistance to ß-lactam antibiotics such as methicillin and oxacillin. Methicillin resistance is mainly due to the acquisition of the staphylococcal cassette chromosome (SCC) mec (SCCmec). Little is known about the structure of SCCmec in methicillin- or oxacillin-resistant CoNS. METHODS: WGS was performed to determine the structure of SCCmec elements of two clinical S. lugdunensis isolates: CMUH-22 and CMUH-25. RESULTS: These elements were found to be flanked by DRs and IRs with unique mosaic structures and a common integration site in the 3' end of the rlmH gene. The sequences of the regions located between rlmH and the ISSau4-like transposase genes of both elements were similar to those of SCCmec Vt of Staphylococcus aureus PM1. The SCCmec (type V, 5C2&4) of CMUH-25 harboured a novel ccrC complex and a C2-like mec complex in opposite orientations, similar to the type V SCCmec of S. aureus WIS. The sequences of the ccrA4B4 genes and J1 and J2 regions of CMUH-25 were similar to those of the SCC element of Staphylococcus haemolyticus NCTC 11042. In contrast, portions of the sequence of the J1 region of type Vt (5C2) SCCmec in strain CMUH-22 were highly similar to portions of those of Staphylococcus epidermidis RP62A and the composite SCCmec type V of S. aureus WAMRSA40. CONCLUSIONS: These observations suggest that the SCCmec elements of CMUH-25 and CMUH-22 evolved separately and assembled through different recombination events.

Efficient Killing of Planktonic and Biofilm-Embedded Coagulase-Negative Staphylococci by Bactericidal Protein P128.

Coagulase-negative staphylococci (CoNS) are the major causative agents of foreign-body-related infections, including catheter-related bloodstream infections. Because of the involvement of biofilms, foreign-body-related infections are difficult to treat. P128, a chimeric recombinant phage-derived ectolysin, has been shown to possess bactericidal activity on strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). We tested the killing potential of P128 on three clinically significant species of CoNS, S. epidermidis, S. haemolyticus, and S. lugdunensis, under a variety of physiological conditions representing growing and nongrowing states. The MIC90 and minimum bactericidal concentration at which 90% of strains tested are killed (MBC90) of P128 on 62 clinical strains of CoNS were found to be 16 and 32 µg/ml (0.58 and 1.16 µM), respectively, demonstrating the bactericidal nature of P128 on CoNS strains. Serum showed a potentiating effect on P128 inhibition, as indicated by 4- to 32-fold lower MIC values observed in serum. P128 caused a rapid loss of viability in all CoNS strains tested. Persisters of CoNS that were enriched in the presence of vancomycin or daptomycin were killed by P128 at 1× the MIC in a rapid manner. Low concentrations of P128 caused a 2- to 5-log reduction in CFU in stationary-phase or poorly metabolizing CoNS cultures. P128 at low concentrations eliminated CoNS biofilms in microtiter plates and on the surface of catheters. Combinations of P128 and standard-of-care (SoC) antibiotics were highly synergistic in inhibiting growth in preformed biofilms. Potent activity on planktonic cells, persisters, and biofilms of CoNS suggests that P128 is a promising candidate for the clinical development of treatments for foreign-body-related and other CoNS infections.

Antimicrobial resistance and virulence characterization of Staphylococcus aureus and coagulase-negative staphylococci from imported beef meat.

BACKGROUND: The objectives of this study were to characterize the diversity and magnitude of antimicrobial resistance among Staphylococcus species recovered from imported beef meat sold in the Egyptian market and the potential mechanisms underlying the antimicrobial resistance phenotypes including harboring of resistance genes (mecA, cfr, gyrA, gyrB, and grlA) and biofilm formation. RESULTS: The resistance gene mecA was detected in 50% of methicillin-resistant non-Staphylococcus aureus isolates (4/8). Interestingly, our results showed that: (i) resistance genes mecA, gyrA, gyrB, grlA, and cfr were absent in Staphylococcus hominis and Staphylococcus hemolyticus isolates, although S. hominis was phenotypically resistant to methicillin (MR-non-S. aureus) while S. hemolyticus was resistant to vancomycin only; (ii) S. aureus isolates did not carry the mecA gene (100%) and were phenotypically characterized as methicillin- susceptible S. aureus (MSS); and (iii) the resistance gene mecA was present in one isolate (1/3) of Staphylococcus lugdunensis that was phenotypically characterized as methicillin-susceptible non-S. aureus (MSNSA). CONCLUSIONS: Our findings highlight the potential risk for consumers, in the absence of actionable risk management information systems, of imported foods and advice a strict implementation of international standards by different venues such as CODEX to avoid the increase in prevalence of coagulase positive and coagulase negative Staphylococcus isolates and their antibiotic resistance genes in imported beef meat at the Egyptian market.

Clinical and Microbiological Aspects of ß-Lactam Resistance in Staphylococcus lugdunensis.

Antimicrobial susceptibility results from broth microdilution MIC testing of 993 Staphylococcus lugdunensis isolates recovered from patients at a tertiary care medical center from 2008 to 2015 were reviewed. Ninety-two oxacillin-susceptible isolates were selected to assess the accuracy of penicillin MIC testing, the penicillin disk diffusion test, and three ß-lactamase tests, including the cefoxitin-induced nitrocefin test, penicillin cloverleaf assay, and penicillin disk zone edge test. The results of all phenotypic tests were compared to the results of blaZ PCR. The medical records of 62 patients from whom S. lugdunensis was isolated, including 31 penicillin-susceptible and 31 penicillin-resistant strains, were retrospectively reviewed to evaluate the clinical significance of S. lugdunensis isolation, the antimicrobial agents prescribed, if any, and the clinical outcome. MIC testing revealed that 517/993 (52.1%) isolates were susceptible to penicillin and 946/993 (95.3%) were susceptible to oxacillin. The induced nitrocefin test was 100% sensitive and specific for the detection of ß-lactamase compared to the blaZ PCR results, whereas the penicillin disk zone edge and cloverleaf tests showed sensitivities of 100% but specificities of only 9.1% and 89.1%, respectively. The penicillin MIC test had 100% categorical agreement with blaZ PCR, while penicillin disk diffusion yielded one major error. Only 3/31 patients with penicillin-susceptible isolates were treated with a penicillin family antimicrobial. The majority of cases were treated with other ß-lactams, trimethoprim-sulfamethoxazole, or vancomycin. These data indicate that nearly all isolates of S. lugdunensis are susceptible to narrow-spectrum antimicrobial agents. Clinical laboratories in areas with resistance levels similar to those described here can help promote the use of these agents versus vancomycin by effectively designing their antimicrobial susceptibility reports to convey this message.