Eva M. Kulik

Antimicrobial susceptibility of periodontopathogenic bacteria

OBJECTIVES The aim of this study was to evaluate the resistance profiles of Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia/Prevotella nigrescens and to detect possible changes in antibiotic resistance over the time period of 1991-2005. METHODS A. actinomycetemcomitans (125 strains), P. gingivalis (152 strains) and P. intermedia/P. nigrescens (326 strains) isolated during the years 1991-2005 were tested for their susceptibility to amoxicillin/clavulanic acid, clindamycin, metronidazole, phenoxymethylpenicillin and tetracycline using the Etest. RESULTS No antibiotic resistance was detected in P. gingivalis, whereas a few isolates of P. intermedia were not susceptible to clindamycin (0.9%), phenoxymethylpenicillin (13.5%) or tetracycline (12.6%). Amoxicillin/clavulanic acid, tetracycline and metronidazole were the most effective antibiotics against A. actinomycetemcomitans with 0%, 0.8% and 20.8% non-susceptible isolates, respectively. However, 88% of the A. actinomycetemcomitans isolates were non-susceptible to phenoxymethylpenicillin and 88% to clindamycin. When strains isolated in the years 1991-94 were compared with those isolated in the years 2001-04, there was no statistically significant difference in the percentage of A. actinomycetemcomitans strains non-susceptible to clindamycin, metronidazole or phenoxymethylpenicillin, or in the percentage of P. intermedia strains non-susceptible to phenoxymethylpenicillin or tetracycline (P > 0.4 each). CONCLUSIONS Increasing antibiotic resistances in periodontopathogenic bacteria are not yet a problem in the Northern part of Switzerland.

Antimicrobial activity of Streptococcus salivarius K12 on bacteria involved in oral malodour

OBJECTIVE To investigate the antimicrobial activity of the bacteriocin-producing strain Streptococcus salivarius K12 against several bacteria involved in halitosis. DESIGN The inhibitory activity of S. salivarius K12 against Solobacterium moorei CCUG39336, four clinical S. moorei isolates, Atopobium parvulum ATCC33793 and Eubacterium sulci ATCC35585 was examined by a deferred antagonism test. Eubacterium saburreum ATCC33271 and Parvimonas micra ATCC33270, which have been tested in previous studies, served as positive controls, and the Gram-negative strain Bacteroides fragilis ZIB2800 served as a negative control. Additionally, the occurrence of resistance in S. moorei CCUG39336 to S. salivarius K12 was analysed by either direct plating or by passage of S. moorei CCUG39336 on chloroform-inactived S. salivarius K12-containing agar plates. RESULTS S. salivarius K12 suppressed the growth of all Gram-positive bacteria tested, but the extent to which the bacteria were inhibited varied. E. sulci ATCC35585 was the most sensitive strain, while all five S. moorei isolates were inhibited to a lesser extent. Natural resistance seems to be very low in S. moorei CCUG39336, and there was only a slight decrease in sensitivity after exposure to S. salivarius K12 over 10 passages. CONCLUSION Our studies demonstrate that S. salivarius K12 has antimicrobial activity against bacteria involved in halitosis. This strain might be an interesting and valuable candidate for the development of an antimicrobial therapy for halitosis.

The antimicrobial activity of alpha-bisabolol and tea tree oil against Solobacterium moorei, a Gram-positive bacterium associated with halitosis

OBJECTIVE To investigate the antimicrobial effect of alpha-bisabolol and tea tree oil alone and in combination against the halitosis-associated Gram-positive bacillus Solobacterium moorei. DESIGN The inhibitory activity of alpha-bisabolol and tea tree oil against the reference strain S. moorei CCUG39336 and four clinical S. moorei isolates was investigated by a direct exposure test. Additionally, the ability of alpha-bisabolol to increase the sensitivity of S. moorei was tested by pretreating the bacteria with sublethal concentrations prior to the administration of tea tree oil. RESULTS A dose-dependent killing was observed for the antimicrobial agents in a direct exposure test with the reference strain S. moorei CCUG39336. Concentrations of ≥0.5% tea tree oil caused decreases in viability of >5 log colony forming units/ml even after short incubation periods, while bacterial viability was less affected by alpha-bisabolol. The combination of 0.1% alpha-bisabolol plus 0.05% tea tree oil showed a synergistic effect on S. moorei strain CCUG39336 and on two of the four clinical S. moorei isolates tested. However, incubation of S. moorei with a sublethal concentration of 0.1% alpha-bisabolol for three days prior to the administration of 0.05% tea tree oil did not enhance the antibacterial effect of tea tree oil. CONCLUSION Halitosis-associated bacterium S. moorei is susceptible to the antimicrobial agents tea tree oil and alpha-bisabolol, suggesting that these compounds might be beneficial in oral healthcare products.

Complete Genomic Nucleotide Sequence of the Temperate Bacteriophage AaΦ23 of Actinobacillus actinomycetemcomitans

The entire double-stranded DNA genome of the Actinobacillus actinomycetemcomitans bacteriophage Aa Phi 23 was sequenced. Linear DNA contained in the phage particles is circularly permuted and terminally redundant. Therefore, the physical map of the phage genome is circular. Its size is 43,033 bp with an overall molar G+C content of 42.5 mol%. Sixty-six potential open reading frames (ORFs) were identified, including an ORF resulting from a translational frameshift. A putative function could be assigned to 23 of them. Twenty-three other ORFs share homologies only with hypothetical proteins present in several bacteria or bacteriophages, and 20 ORFs seem to be specific for phage Aa Phi 23. The organization of the phage genome and several genetic functions share extensive similarities to that of the lambdoid phages. However, Aa Phi 23 encodes a DNA adenine methylase, and the DNA packaging strategy is more closely related to the P22 system. The attachment sites of Aa Phi 23 (attP) and several A. actinomycetemcomitans hosts (attB) are 49 bp long.

Transduction of antibiotic resistance markers among Actinobacillus actinomycetemcomitans strains by temperate bacteriophages Aaφ23

Abstract.Actinobacillus actinomycetemcomitans (Aa) strain ST1 carries the tetracycline (Tc) resistance transposon Tn916 and the Aaφ ST1 prophage, which is closely related to temperate bacteriophage Aaφ23. High titre phage preparations were obtained from this strain by mitomycin C induction and were used to transduce the TcR determinant to the TcS recipient strains ZIB1001 and ZIB1015 (MIC 2 μg Tc/ml). TcR transductants (MIC ≥ 32 μg Tc/ml) were detected at frequencies of 3 × 10−6 to 5 × 10−8 per pfu. All TcR transductants examined contained the entire Tn916 inserted at several different locations within the Aa genome. They appear to have resulted from generalized transduction. In addition both bacteriophages, Aaφ23 and AaφST1, were capable of transducing the chloramphenicol (Cm) resistance marker of plasmid pKT210 (transduction frequencies of 2 × 10−5 to 3 × 10−7 per pfu) to the recipient strain ZIB1001 (MIC 8 μg Cm/ml). Eleven CmR ZIB1001 transductants (MIC ≥ 100 μg Cm/ml) studied carried a plasmid indistinguishable from pKT210 by restriction analyses. In view of the high prevalence of this phage family, and the increasing use of tetracycline in periodontitis therapy, these findings may have clinical importance.

The type IC hsd loci of the enterobacteria are flanked by DNA with high homology to the phage P1 genome: implications for the evolution and spread of DNA restriction systems

EcoR124I, EcoDXXI and EcoprrI are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while EcoprrI is chromosomally encoded. The enzymes are coded by three genes, hsdR, hsdM and hsdS. Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac, the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For EcoDXXI and EcoprrI the P1 homology extends for thousands of base pairs while for EcoR124I an IS1 insertion and an associated deletion have removed most of the P1‐homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.

Volatile compounds of Salvadora persica inhibit the growth of oral Candida species.

OBJECTIVE The antibacterial effect of Salvadora persica has been demonstrated both in vitro and in vivo. However, data on its possible antifungal effect is scarce. Therefore, the aim of the present study was to investigate the antifungal effect of solid or pulverized S. persica on clinically important oral Candida species in vitro. DESIGN The antifungal activity of S. persica was examined against reference strains and clinical isolates of oral Candida species by two different methods. In an agar diffusion test, solid as well as pulverized pieces of S. persica were tested. Mounting the S. persica test specimens inside the lid tested growth inhibition by volatile compounds. RESULTS S. persica exhibited antifungal activity against all Candida species tested. In particular, the volatile compounds of solid test specimens demonstrated strong growth inhibition, whereas pulverized S. persica revealed no antifungal activity. Parameters such as storage and incubation time as well as the diameter of the sticks influenced the growth inhibition. CONCLUSIONS Volatile compounds of S. persica have antifungal activity against oral Candida species. Storage time after harvesting may play an important role for the strength of this antifungal activity.

Influence of time, toothpaste and saliva in the retention of Streptococcus mutans and Streptococcus sanguinis on different toothbrushes

Objectives The intraoral transmission of cariogenic and periodontopathogenic species seems to be facilitated by contaminated toothbrushes and other oral hygiene devices. The aim of this investigation was to analyze the in vitro retention and survival rate of Streptococcus mutans and Streptococcus sanguinis on different toothbrushes. The impacts of human saliva and antimicrobial toothpaste on these parameters were further evaluated. Material and Methods Part I: Four toothbrushes (Colgate 360°, Curaprox CS5460 ultra soft, elmex InterX, Trisa Flexible Head3) were contaminated by S. mutans DSM 20523 or S. sanguinis DSM 20068 suspensions for three minutes. Bacteria were removed from the toothbrushes after either three minutes (T0) or 24 hours (T24) of dry storage and grown on Columbia blood agar plates for the quantification of colony-forming units (CFUs). Part II: The effects of saliva from a caries-active or a caries-inactive person and of toothpaste containing 0.12% chlorhexidine digluconate were also tested. Results Part I: After three minutes of dry storage, approximately one percent of the bacteria were still detectable on the toothbrushes. After 24 hours, S. sanguinis exhibited a more pronounced decrease in viable cell numbers compared with S. mutans but the differences were not significant (Kruskal-Wallis test, p>0.05). Part II: The addition of human saliva from a caries-active or caries-inactive person slightly increased the retention of both streptococcal species at T0. The use of toothpaste had no influence on the amount of viable streptococci at T0, but it reduced the microbial load after 24 hours of storage. There were only slight nonsignificant differences (p>0.05) between the four toothbrushes. Conclusions In vitro bacterial retention and survival of S. sanguinis and S. mutans on different toothbrushes occurred. Within the limitations of this study, the use of human saliva or an antimicrobial toothpaste did not lead to significant differences in the microbial load on toothbrushes.

Caries experience in 7-, 12-, and 15-year-old schoolchildren in the canton of Basel-Landschaft, Switzerland, from 1992 to 2011.

OBJECTIVES To investigate the changes in caries experience and prevalence among schoolchildren of the canton of Basel-Landschaft, Switzerland, over the course of 20 years. METHODS A random sample of either schoolchildren aged 7, 12, and 15 years (in 1992) or aged 12 and 15 years (in 1997) or their respective school classes (2001, 2006 and 2011) was selected so that approximately 10% of schoolchildren could be examined. The childrens dmft and DMFT scores were determined according to the WHO methodology and analyzed using cluster-adjusted ordinary multiple linear regression modeling. RESULTS For all age groups, the respective dmft/DMFT values decreased steadily from 1992 to 2006 but increased again in 2011. However, the observed differences were not statistically significant over the examination years from 2001 to 2011. Schoolchildren with a migrant background had approximately two- to threefold higher dmft/DMFT values. CONCLUSIONS In the years from 1992 to 2001, a steady decline in caries was observed in all age groups of schoolchildren examined in the canton of Basel-Landschaft. However, in the subsequent 10 years, this decline has leveled off. The mean dmft/DMFT values are comparable to those in other parts of Switzerland. Migrants are a caries-risk group; the mean dmft/DMFT values were higher in schoolchildren with a migrant background than in the comparable Swiss children.