High oral carriage of multidrug resistant Gram-negative bacilli in adolescents: the SOPKARD-Junior study.

Introduction: The colonization of the oral cavity by potentially pathogenic antimicrobial-resistant bacteria in adolescents and its consequences is very poorly understood. The present study focused on the occurrence of oral colonization by Gram-negative bacilli (GNB) and their multidrug resistance, including the production of extended-spectrum ß-lactamases (ESBLs) and carbapenemases, among healthy adolescents and risk factors associated with GNB colonization. Materials and methods: This study was conducted as part of "A program for the early detection of risk factors for lifestyle diseases SOPKARD-Junior" (SOPKARD-Junior). Oral samples were collected from 182 adolescents from four public elementary schools in Sopot, Poland, aged 13-14 years. Bacterial strains were identified by the MALDI-TOF MS method. Screening of antimicrobial resistance was performed using a disk diffusion method. The NG-Test® CARBA-5 was used to detect and differentiate the five most widely distributed carbapenemases. Demographic and clinical data were collected and statistical analysis of risk factors was performed. Results: A total of 68 out of 182 (37.4%) healthy adolescents was documented oral colonization with Gram-negative bacilli, including 50/182 (27.5%) multidrug resistant (MDR-GNB) strains. Over 60% of oral carriage concerned three main genera Enterobacter spp., Pseudomonas spp., and Serratia spp., which were detected in 22.1%, 19.1%, and 19.1% of participants, respectively. Citrobacter spp., Escherichia coli, Klebsiella spp., Hafnia spp., Aeromonas spp., Acinetobacter spp., and Stenotrophomonas spp. were also isolated. The antimicrobial resistance to ampicillin (100%), ceftazidime (69.1%), meropenem (60.3%), gentamycin (60.3%), piperacillin/tazobactam (52.9%), and piperacillin (45.6%) were the most common. Among 73.5% GNB strains multidrug resistance was observed, including all Pseudomonas spp. strains. Among MDR-GNB, 30.4% were resistant to four groups of antibiotics, half of the MDR Pseudomonas spp. strains were resistant to 10 groups of antibiotics. Extended-spectrum ß-lactamases were produced by Enterobacter cloacae, Klebsiella spp., and Serratia spp. (7.4%). Colonization by ESBLs-positive GNB strains was significantly associated with recurrent respiratory infections, nasal congestion, and bronchitis (p<0.05). Conclusion: Our study revealed high oral carriage of multi-drug resistant Gram-negative bacilli in healthy adolescents and the association of ESBL-producing strains with respiratory infections. Further studies on oral colonization with GNB are necessary due to the possibility of distinct infections and the acquisition of antibiotic resistance by resident microbiota.

Staphylococcus aureus Isolated from the Oral Cavity: Phage Susceptibility in Relation to Antibiotic Resistance.

Nowadays, research on bacteriophage therapy and its potential use in combination with antibiotics has been gaining momentum. One hundred and ten oral Staphylococcus aureus isolates were phage-typed and their antibiotic resistance was determined by standard and molecular methods. The prevalence of MSSA and MRSA strains was 89.1% and 10.9%, respectively. Nearly all (91.8%) analyzed isolates, whether MSSA or MRSA, were susceptible to the phages used from the international set. The highest lytic activity showed phages 79 and 52 A from lytic group I. The predominant phage groups were mixed, the I+III group and a mixed group containing phages from at least three various lytic groups. S. aureus strains sensitive to phage group I were usually resistant to penicillin and susceptible to ciprofloxacin, whereas the strains typeable with group V or group V with the 95 phage were susceptible to most antibiotics. Epidemic CA-MRSA strains (SCCmecIV) of phage type 80/81 carried Panton-Valentine leucocidin genes. Considering the high sensitivity of oral S. aureus to the analyzed phages and the promising results of phage therapies reported by other authors, phage cocktails or phage-antibiotic combinations may potentially find applications in both the prevention and eradication of staphylococcal infections.

Distribution and antibiotic-resistance of different Staphylococcus species identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) isolated from the oral cavity.

BACKGROUND: The use of antibiotics in dentistry is associated with the emergence and spread of antibiotic-resistant microorganisms, including commensal staphylococci. METHODS: A total of 367 oral samples were collected, from which staphylococci were isolated and identified by using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). The antibiotic susceptibility of the isolates was determined and molecular characteristics for methicillin-resistant staphylococci was performed. RESULTS: A total of 103 coagulase-negative staphylococci (CoNS), among them S. warneri, S. haemolyticus, S. saprophyticus, S. pasteuri, S. epidermidis, S. hominis, S. xylosus, S. equorum, S. kloosii, S. succinus, S. cohnii, and S. simulans, were confirmed by MALDI-TOF. Resistance to most tested antibiotics was statistically higher in CoNS than in S. aureus isolates (P-value < 0.05). CoNS isolates showed high resistance to penicillin (S. saprophyticus 88.9%), erythromycin (S. haemolyticus 84.6%), fusidic acid (S. saprophyticus 77.8%), co-trimoxazole (S. epidermidis 71.4%), gentamicin (S. warneri 63.8%), and tetracycline (S. saprophyticus 55.6%). Multidrug resistance was largely observed, especially among S. haemolyticus and S. saprophyticus species. Methicillin-resistance in S. haemolyticus (38.5%), S. saprophyticus (22.2%) and S. aureus (13.5%) was associated with the presence of the mecA gene and SCCmec type IV or V. CONCLUSION: Coagulase-negative staphylococci, especially S. haemolyticus and S. saprophyticus, seem to be a reservoir of methicillin resistance and multidrug resistance in the oral cavity.

Novel staphylococci nucH taxonomical marker used in identification of human-associated Staphylococcus succinus subsp. casei.

The aim of our study was to assess the sequencing of unique nucH gene fragment based on performed bioinformatics analysis as a novel diagnostic method for the identification of difficult to identify staphylococcal human pathogenic strains. Initially, PCR-RFLP-rrn analysis specific to the spacers between 16SrDNA and 23SrDNA followed by HhaI restriction analysis was performed. Further, sequencing of nucH and 16S rDNA genes fragments was carried out. Blast analysis from the NCBI showed 99% similarity of nucH gene fragment with reference genomic DNA for S. succinus with the accession no. CP018199. This result was also confirmed by MALDI-TOF analysis. Sequencing analysis of 16S rDNA gene fragment allowed for 100% identification of two strains isolated from human samples as Staphylococus succinus subsp. casei. Sequencing of identified unique nucH gene fragment seems to be a promising diagnostic assay for the identification of Staphylococcus species. Based on our results, we can assume that probably other Staphylococcus species originated from different clinical samples could be identified using nucH gene sequencing method we developed. However, an extension of the genetic databases with a substantially bigger number of reference staphylococcal species for nucH gene is needed to make this method better than widely used standard 16S rDNA sequencing assay. To the best of our knowledge, it is the second published isolation of S. succinus subsp. casei from human clinical specimens. Moreover, possibility of decreasing the number of dimensions from multi-PCR-bands results using ribotyping analysis is also described.

Presence of egc-positive major clones ST 45, 30 and 22 among methicillin-resistant and methicillin-susceptible oral Staphylococcus aureus strains.

The oral cavity may comprise a significant reservoir for Staphylococcus aureus but the data on molecular epidemiology and clonal distribution of oral strains are really scarce. This study aimed to evaluate the clonal relatedness in S. aureus isolated from oral cavity and their relationship with carriage of virulence genes, and antimicrobial resistance profiles. A total of 139 oral S. aureus isolates were obtained from 2327 analysed oral samples of dental patients. Antimicrobial susceptibility testing was performed. Isolates were characterized using protein A gene (spa) typing, spa-CC clonal complexes, toxin genes and SCCmec typing for MRSA. High resistance rates for penicillin, tetracycline and gentamicin were detected, respectively 58.3%, 42.4%, and 35.2%. Twelve (8.6%) S. aureus isolates were identified as MRSA. All of MRSA isolates were mecA-positive and mecC-negative. SCCmec IV was the most common type (66.7%), which was typical for community-acquired MRSA (CA-MRSA). Overall, the enterotoxin gene cluster (egc) was the most frequent detected virulence factor (44.9%), both in MSSA and MRSA isolates. Presence of genes encoding for the enterotoxins (sea, seb, sec, seh, sek), exfoliative toxin A (eta), and toxic shock syndrome toxin-1 (tst) was also observed. Strains carrying lukS-PV/lukF-PV genes belonged to SCCmecV- spa type t437. The most prevalent spa types were t091, t015, t084, t002, t571, and t026 among all 57 identified. Spa types, including 3 new ones, grouped in 6 different spa-CC clonal complexes, with four major dominated; CC45, CC30, CC5, and CC15. This study demonstrated that both methicillin-susceptible and methicillin-resistant major European clones of S. aureus could be isolated from the oral cavity of dental patients, with the emergence of PVL-positive CA-MRSA strains. The oral cavity should be considered as a possible source of toxigenic egc-positive S. aureus strains, in terms of potential risk of cross-infection and dissemination to other body sites.

Denture stomatitis associated with small-colony variants of Staphylococcus aureus: a case report.

BACKGROUND: The small-colony variants (SCVs) of Staphylococcus aureus were isolated from persistent and recurrent infections, especially after placement of medical devices having direct contact with human tissues. The emergence of SCVs is a survival strategy of S. aureus which enables them to hide inside host's cells and induces a less severe immune response than to wild-type S. aureus. However, contrary to other medical devices, dental prosthesis as a surface potentially colonized by SCVs of S. aureus has not been examined thus far. We reported the first case of SCVs - S. aureus infection in denture wearer. CASE PRESENTATION: A 62-year-old woman with a complete removable acrylic denture presented extensive elevated erythematous lesions on the palate, compatible with denture stomatitis. The patient had a history of arterial hypertension, cigarette smoking and wearing denture at night. The fungal colonies, identified as Candida albicans, were cultured on Sabouraud agar. From three swabs (from hard palate mucosa, denture surface and angular cheilitis lesions) were cultured of pinpoint, clear, non-pigmented, and non-haemolytic colonies on Columbia agar. The small colonies turned out to be Gram-positive cocci, catalase-, Pastorex Staph Plus -, and clumping factor-positive, and oxidase-negative. Suspected phenotypically SCVs forms were definitively identified as S. aureus based on PCR amplification of species specific nuc and coa genes. Methicillin-resistance was verified by mecA gene detection. The isolates turned out to be susceptible to methicillin (MSSA) and resistant to gentamicin. The isolate was identified as menadione-auxotrophic variant. CONCLUSIONS: This case demonstrated that oral cavity in denture wearers may be a reservoir of small-colony variants of S. aureus, besides C. albicans. The prevalence of these bacteria and their role in the pathogenesis of oral diseases are not understood. Due to problems with their detection and identification, the true prevalence of oral SCVs may be underestimated.

Do the oral Staphylococcus aureus strains from denture wearers have a greater pathogenicity potential?

We used flow cytometry to compare the phagocytic activity of monocytes against Staphylococcus aureus strains (both biofilm and planktonic cells) isolated from denture wearers and non-wearers. Staphylococcal strains were cultured in Brain Heart Infusion broth in both planktonic and biofilm form and were stained with a fluorescent reporter (propidium iodide) and incubated with monocytes. The fluorescence of the monocytes containing phagocytized bacteria was determined by flow cytometry and normalized to that of the bacterial strains used in the experiment. Staphylococcal strains from denture wearers caused greater activation of monocytes but were less prone to phagocytosis. The percentage of monocytes containing bacterial cells after exposition to staphylococcal strains varied from 2.7% to 81.4% for planktonic cells. For biofilm-released cells, this value ranged from 0.6% to 36.2%. The effectiveness of phagocytosis, estimated based on an increase in monocyte fluorescence, amounted to 32.4 and 71 FL2 units for the biofilm and planktonic cells, respectively. The lesser efficiency of phagocytosis against biofilm S. aureus in denture wearers suggests that they might have been colonized with the strains which were less prone to eradication than those from non-wearers.

Syntheses of Novel 4-Substituted N-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamide Derivatives with Potential Antifungal Activity.

Candidiasis represent a serious threat for patients with altered immune responses. Therefore, we have undertaken the synthesis of compounds comprising a pyridine-3-sulfonamide scaffold and known antifungally active 1,2,4-triazole substituents. Thus a series of novel 4-substituted N-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamides have been synthesized by multistep reactions starting from 4-chloropyridine-3-sulfonamide via N'-cyano-N-[(4-substitutedpyridin-3-yl)sulfonyl]carbamimidothioates which were further converted with hydrazine hydrate to the corresponding 1,2,4-triazole derivatives 26-36. The final compounds were evaluated for antifungal activity against strains of the genera Candida, Geotrichum, Rhodotorula, and Saccharomycess isolated from patients with mycosis. Many of them show greater efficacy than fluconazole, mostly towards Candida albicans and Rhodotorula mucilaginosa species, with MIC values ≤ 25 µg/mL. A docking study of the most active compounds 26, 34 and 35 was performed showing the potential mode of binding to Candida albicans lanosterol 14α-demethylase. Also in vitro cytotoxicity of selected compounds have been evaluated on the NCI-60 cell line panel.

N-Substituted N'-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidines--Antibacterial, Cytotoxic Activities and Some Structure-Activity Relationships.

A series of N-substituted N'-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidine derivatives bearing sulfonamide moiety have been screened in vitro for antibacterial activity against isolates from patients with infections of oral cavity, respiratory tract and intestinal tract. The majority of compounds exhibited good antibacterial potency. 1-[4-Chloro-5-methyl-2-(4-trifluoromethylbenzylthio)benzenesulfonyl]-3-(3-sulfamoylphenyl)guanidine (13) showed very strong activity, with MIC ≤ 6.2 µg/ml against eleven bacteria strains belonged to Gram-positive anaerobes and aerobes. Furthermore, compound 13 exhibited promising activity toward highly resistant microorganisms such as methicillin-resistant Staphylococcus aureus and Enterococcusfaecalis. It was found that Parvimonas micra, Finegoldia magna, Peptostreptococcus anaerobius, Propionibacterium acnes showed the highest susceptibility toward the investigated guanidines.

Synthesis and anti-yeast evaluation of novel 2-alkylthio-4-chloro-5-methyl-N-[imino-(1-oxo-(1H)-phthalazin-2-yl)methyl]benzenesulfonamide derivatives.

Pathogenic fungi are one of the main causes of hospital-related infections. Since conventional antifungals have become less effective because of the increasing fungal resistance to the standard drugs, the need for new agents is becoming urgent. Herein we report a synthesis of a series of novel N-[imino-(1-oxo-(1H)-phthalazin-2-yl)methyl]-benzenesulfonamide derivatives with in vitro activity against yeast-like fungi isolated from the oral cavity and respiratory tract of patients with candidiasis. These compounds were synthesized by the one-step or two-step reactions of 1-(2-alkylthiobenzensulfonyl)-2-aminoguanidines with the appropriate ortho-carbonyl benzoic acids. The biological study revealed that new derivatives have shown significant growth-inhibitory activity, superior or comparable, than those of the reference drug fluconazole. The most promising activities were observed against Candida albicans, with inhibition at least 1-3 (12.5%-37.5%) of the eight tested strains at the low MIC level of ≤6.2-25 µg/mL.

Synthesis and antibacterial activity of novel 4-chloro-2-mercaptobenzenesulfonamide derivatives.

Few series of novel 4-chloro-2-mercaptobenzenesulfonamides have been synthesized by the reactions of N-(benzenesulfonyl)cyanamide potassium salts 7-15 with corresponding hydrazinecarbodithioic acid esters, 1-substituted carbothioic acid hydrazides, methyl 3-aminothiophene-2-carboxylate, methyl 2-aminobenzoate, 2-aminophenol or 2-aminothiophenol. The synthesized compounds (16-49) were screened in vitro for their antibacterial activity. Some of the tested compounds 16, 17, 23, 24, 31, 32 and 48 showed the promising activity against many of anaerobic Gram-positive bacteria strains.

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium Gordonia alkanivorans S7.

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40 degrees C. K(m) values for NO(3)(-) (110 microM) and for ClO(3)(-) (138 microM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.

Mechanism of aniline degradation by yeast strain Candida methanosorbosa BP-6.

The ability of some bacteria and filamentous fungi to degrade aniline and its derivatives was reported earlier in the literature. However, there was no information about the biodegradation of aniline by yeast strains. The present work is focused on yeast strain Candida methanosorbosa BP-6 which was isolated from the wastewater pool of the old dye factory "Boruta" in Zgierz by enrichment technique and identified by standard microbiological methods. We have found that strain C. methanosorbosa BP-6 readily grows in the presence of aniline and can degrade this substrate. Relatively good separation of peaks corresponding to aniline and its biodegradation intermediates allowed us their identification and quantification by HPLC methodology. We have found that major intermediates of this degradation are: catechol, cis,cis-muconic acid, muconolactone, 3-oxoadipate enol-lactone, 3-oxoadipic acid and succinic acid. Our results provide strong evidence that biodegradation of aniline by the yeast strain C. methanosorbosa BP-6 proceeds according to the intradiolic pathway.

Studies on pyrazine derivatives. XLVII. Synthesis and antibacterial activity of novel pyrazine derivatives with amidoxime moiety.

The new pyrazine derivatives exhibiting an antibacterial activity have been synthesized. Initial amidoxime 1 was obtained in the reaction of pyrazinecarbonitrile with hydroxylamine. Upon treatment of amidoxime with methyl iodide O-methyl derivative 2 was formed. Both amidoximes were transformed into imidoyl chlorides 3, 4. Then the chloride atom in those derivatives was substituted with various secondary amines giving appropriate oximes 5-18 and O-methyl-oximes 19 and 20. The obtained compounds were tested in vitro for their tuberculostatic activity. The inhibiting concentration (MIC) values were within 25-100 microg/mL. Their activity towards 25 strains of anaerobic and 25 strains of aerobic bacteria was also studied. Three compounds exhibited activity against both types of bacteria.

[The occurrence of microorganisms in intraoral abscesses]. / Wystepowanie drobnoustrojów w ropniach wewnatrzustnych.

The aim of this study was to assess the occurrence of microorganisms in 39 intraoral abscesses. The samples were place in transport medium. The materials were inoculated on adequate enrichment and selective media and cultivated under anaerobic and aerobic conditions. The microorganisms were found in all samples (100%), Anaerobic bacteria most frequently were recovered. The predominant anaerobes were from genus Prevotella, Bacteroides, Fusobacterium and Peptostreptococcus. Among aerobic bacteria, the most frequent were gram-positive cocci. The microaerophilic bacteria and fungi most rarely were isolated from pus samples.

[Incidence of anaerobic bacteria in respiratory tract infections]. / Wystepowanie bakterii beztlenowych w zakazeniach ukladu oddechowego.

Anaerobic bacteria are predominant components of normal oral cavity, upper respiratory tract, gastrointestinal, genital and skin flora. They are involved in infections such as pneumonia, aspiration pneumonia, lung abscess and empyema. Laboratory diagnosis of anaerobic infections is based on recovering the etiological agents from clinical materials. Appropriatte specimens include: pus, purulent fluid, biopsy specimen of lung, transtracheal aspirates and bronchoalveolar lavage (BAL). Lower respiratory infections are usually either polymicrobial or mixed anaerobic-aerobic infections. Peptostreptococcus, Fusobacterium, Prevotella and Bacteroides are the most common anaerobes. Anaerobic bacteria are susceptible to metronidazole, tinidazole (exception of Gram-positive rods), amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, imipenem and clindamycin. Treatment includes an antibiotics regimen with an agent active against anaerobic and aerobic bacteria (therapy with 2 or 3 antimicrobial drugs).