Antibiotic-resistant Propionibacterium acnes on the skin of patients with moderate to severe acne in Stockholm

The objective was to study the prevalence and antibiotic susceptibility patterns of Propionibacterium acnes strains isolated from patients with moderate to severe acne in Stockholm, Sweden and to determine the diversity of pulsed-field gel electrophoresis types among resistant P. acnes strains. One hundred antibiotic-treated patients and 30 non-antibiotic-treated patients with moderate to severe acne participated in the investigation. Facial, neck and trunk skin samples were taken with the agar gel technique. The susceptibility of P. acnes strains to tetracycline, erythromycin, clindamycin and trimethoprim-sulfamethoxazole was determined by the agar dilution method. The genomic profiles of the resistant strains were determined by pulsed-field gel electrophoresis. In the group of patients treated with antibiotics, resistant P. acnes strains were recovered in 37%, while in the non-antibiotic group of patients the incidence of resistant strains was 13%. Thus antibiotic-resistant P. acnes strains were significantly more often isolated from antibiotic-treated patients with moderate to severe acne than from non-antibiotic-treated patients (odds ratio, 3.8; P=0.01). There was a genetic diversity among the P. acnes strains. Forty-four different patterns of SpeI DNA digests were detected and two predominant clones were found. P. acnes strains exhibited different antibiotic susceptibility patterns and identical genotypes or vice versa. A person can be colonized with different strains with varying degrees of antibiotic resistance. The risk of increased resistance of P. acnes must be considered when treating acne patients with antibiotics, and especially long-term therapy should be avoided.     

Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline

Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 microg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 microg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.     

Characteristics of macrolide-resistant Mycoplasma pneumoniae strains isolated from patients and induced with erythromycin in vitro

Some patients with Mycoplasma pneumoniae infection are clinically resistant to antibiotics such as erythromycin, clarithromycin, or clindamycin. We isolated M. pneumoniae from such patients and found that one of three isolates showed a point mutation in the 23S rRNA gene. Furthermore, 141 EM-sensitive clinical isolates of M. pneumoniae were cultured in broth medium containing 100 microg/ml of erythromycin (EM). Among 11 EM-resistant strains that grew in the medium, point mutations in the 23S rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM-resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.     

Identification of Propionibacterium acnes by polymerase chain reaction for amplification of 16S ribosomal RNA and lipase genes

Propionibacterium acnes belongs to the cutaneous flora and is present in sebaceous follicles. The fatty acids that are released from sebum triglycerides by the action of this bacterial lipase play an important role in the pathogenesis of acne vulgaris. P. acnes is also involved in postoperative disorders and opportunistic infections in immunosuppressed hosts. Recently, it has been proposed that P. acnes causes sarcoidosis. Therefore, rapid isolation and identification of P. acnes is important. This study evaluated the polymerase chain reaction (PCR) for the detection of the 16S rRNA and lipase genes of P. acnes. The PCR used to detect the 16S rRNA gene could amplify the gene of P. acnes, but not the genes of the other tested strains of P. avidum, P. granulosum, P. lymphophilum, P. jensenii, P. acidipropionici and P. thoenii. The PCR to detect the lipase gene of P. acnes, however, could amplify not only the gene of P. acnes but also that of P. avidum. The PCR product of this lipase gene was not found in the strains of the other species tested. Therefore, the organism that has both the 16S rRNA gene and lipase gene was identified as P. acnes, while the strain with the lipase gene but not the 16S rRNA gene of P. acnes was characterized as P. avidum. These findings were confirmed by the conventional biochemical tests including lipase activity. Furthermore, out of the seven clinical isolates from acne vulgaris, four were identified as P. acnes and three as P. avidum by the PCR method and biochemical tests. The combination of two PCR, one for the detection of the 16S rRNA and the other of lipase genes was shown to be an easier, faster and more accurate method to identify P. acnes and P. avidum than conventional methods.     

Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan

AIMS: To investigate the existing antimicrobial susceptibility and genetic characteristics of Lactococcus garvieae isolates from cultured Seriola in Japan. METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents for 170 isolates were determined using the agar dilution method. Seventy-five isolates (44.1%) were simultaneously resistant to erythromycin (EM) (MIC>or=2 microg ml-1), lincomycin (LCM) (MIC>or=128 microg ml-1) and oxytetracycline (OTC) (MIC>or=4 microg ml-1). Resistance to EM was grouped as intermediate- and high-level resistant by MIC values. All resistant isolates possessed ermB and tet(S) genes. The number of different bands between pulsed-field gel electrophoresis patterns of 25 isolates and two ATCC strains (isolated in 1974), determined using two enzymes (ApaI and SmaI), did not exceed 3. CONCLUSIONS: The present resistance pattern observed with ermB and tet(S) is similar to that observed in previous reports. Moreover, the genetic characteristics of L. garvieae isolates from a wide area in Japan in 2002 and ATCC strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that EM-, LCM- and OTC-resistant isolates have been present for 15 years and that L. garvieae strains with same origin have spread among Seriola spp. in Japan since 1974.     

Genetic basis of macrolide and lincosamide resistance in Brachyspira (Serpulina) hyodysenteriae

Macrolide antibiotic resistance is widespread among Brachyspira hyodysenteriae (formerly Serpulina hyodysenteriae) isolates. The genetic basis of macrolide and lincosamide resistance in B. hyodysenteriae was elucidated. Resistance to tylosin, erythromycin and clindamycin in B. hyodysenteriae was associated with an A-->T transversion mutation in the nucleotide position homologous with position 2058 of the Escherichia coli 23S rRNA gene. The nucleotide sequences of the peptidyl transferase region of the 23S rDNA from seven macrolide and lincosamide resistant and seven susceptible strains of Brachyspira spp. were determined. None of the susceptible strains were mutated whereas all the resistant strains had a mutation in position 2058. Susceptible strains became resistant in vitro after subculturing on agar containing 4 micrograms ml-1 of tylosin. Sequencing of these strains revealed an A-->G transition mutation in position 2058.     

Genotypic characterization of clarithromycin-resistant Helicobacter pylori strains

Helicobacterpylori (Hp) resistance to clarithromycin, one of the antibiotics most used to eradicate infection, is connected with the presence of a point mutation on the level of adenine at position 2143 or 2144 of 23S rRNA. AIM: The aim of the study is to evaluate of the presence of these mutation vs control clarithromycin resistant Hp strains present in North Sardinia; to verify the real association between the type of mutation and the resistance-level; to use easier molecular biology methods to quickly locate the resistance-associated mutations beginning with the bioptic material. The clarithromycin susceptibility of Hp isolates was tested by the E-test method (antibiotic assay). Genomic DNA of Hp strains was amplified using specific primers for the domain V. of ribosomic 23S rRNA and sequenced after the reaction with a primer within the fragment 23S. At the same time PCR-RFLP reliability was examined underlining the presence of these mutations with BsaI, BbsI, MboII restriction enzymes. Two mutations in 2143 (A- - G) and 2144 (A- - G) were found by domain V sequencing. The strains with mutation 2143 are characterized by a greater resistance level (MIC>64 g/ml) than those with mutation 2144 (MIC <64 g/ml). Restriction endonucleases BbsI and MboII recognise the site containing the mutation 2143 (A- - G), while BsaI recognise the mutation 2144 (A- - G). These methods might enable us to identify the presence of Hp directly from bioptic material and possible clarithromycin resistance and plan a suitable therapeutic strategy and consequently a better control of the infection.     

Phenotypic and genotypic analysis of clarithromycin-resistant Helicobacter pylori from Bogotá D.C., Colombia

Resistance of Helicobacter pylori to clarithromycin is the most common cause of treatment failure in patients with H. pylori infections. This study describes the MICs and the presence of 23S rRNA mutations of H. pylori isolates from Bogotá, D.C., Colombia. H. pylori were isolated from gastric biopsies from patients with functional dyspepsia. Clarithromycin susceptibility was investigated by agar dilution and strains were considered resistant if the MIC was ≥1 μg/ml. DNA sequences of the 23S rRNA gene of strains resistant and sensitive to clarithromycin were determined to identify specific point mutations. Clarithromycin resistance was present in 13.6% of patients by agar dilution. The A2143G, A2142G and A2142C mutations were found in 90.5, 7.1, and 2.4% of H. pylori strains with resistance genotype.The resistant phenotype was associated with 23S rRNA resistance genotype in 85.7% of isolates. The point mutations in 23S rRNA were well correlated with MICs values for clarithromycin.     

建立一种反向斑点杂交法检测肺炎支原体23S rRNAV区A2063G基因突变的方法

目的建立检测肺炎支原体（Mycoplasma pneumoniae,MP）23S rRNA V区2063基因型的反向斑点杂交方法,并与PCR产物直接测序法的结果进行比较分析。方法19例自临床咽拭子标本分离培养的MP,用自行设计的反向斑点杂交法检测23S rRNA V区2063基因型,同时对相应序列测序分析。抽提标准菌株FH和127份经PCR测定MP阳性的临床标本基因组DNA,用MP阴性的基因组DNA抽提物5份作阴性对照,用反向斑点杂交法检测23S rRNA V区2063基因型。结果19例分离培养的MP,经反向斑点杂交法检测15株有23S rRNA V区基因A2063G位点突变;4株为2063A,与测序结果一致（Kappa一致性检验,P〉0.05）。经反向斑点杂交法检测,127份MP阳性的基因组DNA抽提物中有122份标本为A2063G位点突变,标准菌株FH和2份DNA抽提物的2063位点碱基为A,还有3份抽提物标本的2063位点碱基既有A又有G,5份阴性对照均无显色。结论反向斑点杂交方法能快速、准确检测临床MP 23S rRNA V区2063基因型。     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Primary resistance of Helicobacter pylori to antimicrobial agents in Polish children

Helicobacter pylori resistance to antimicrobial agents is an important factor compromising the efficacy of treatment. Therefore the aims of our study were: to determine the prevalence of H. pylori resistance to clarithromycin, metronidazole, amoxycillin and tetracycline in children prior to eradication therapy, to compare different methods of susceptibility testing and to detect mutations responsible for clarithromycin resistance. During 1996-2000, 259 H. pylori strains were isolated from antral gastric biopsies. Susceptibility to antimicrobials was determined by the agar dilution method and the Etest. Mutations in the 23S rRNA gene associated with clarithromycin resistance were analysed by PCR-RFLP and direct sequencing. Overall, ninety-six strains (37%) were resistant to metronidazole, 50 strains (19.3%) were resistant to clarithromycin, and 20 strains (7.7%) were simultaneously resistant to both drugs. All cultured isolates were sensitive to amoxycillin and only one isolate (0.4%) was resistant to tetracycline. The agar dilution method and the Etest showed a perfect category correlation for clarithromycin and 4% discrepancies for metronidazole. Primary resistance to clarithromycin was mainly associated with an A2143G mutation in the 23S rRNA gene of H. pylori. The study highlights the high prevalence of H. pylori primary resistance to clarithromycin in Polish children, which implies a need for pretreatment susceptibility testing.     

Macrolide resistance and <Emphasis Type="Italic">In Vitro</Emphasis> selection of resistance to antibiotics in <Emphasis Type="Italic">Lactobacillus</Emphasis> isolates

Spreading of resistance to antibiotics is of great concern due to the increasing rate of isolation of multiresistant pathogens. Since commensal bacteria may transfer determinants of resistance to pathogens, studies on development of resistance should include also lactobacilli. Resistance to macrolides, penicillins and tetracycline was determined in 40 isolates of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus crispatus, and Lactobacillus casei isolated from faeces of apparently healthy volunteers. Frequency of mutation and changes in susceptibility after serial exposure to these antibiotics at concentrations of 4× and 8× MIC were evaluated in susceptible isolates. Acquired resistance was defined as an increment in MIC values of at least four times in respect to the pre-selection values. Resistance to macrolides and/or tetracycline was identified in 14 and 4 isolates, respectively. ermB gene and A2058G mutation in 23S rRNA were detected in macrolide resistant isolates. Frequencies of mutation of susceptible isolates (n=26) were lower for ampicillin and erythromycin than for tetracycline. Serial exposure to antibiotics led to selection of resistant mutants. However, acquired resistance was rather unstable and was lost after subcultures in antibiotic-free medium in most mutants. Resistance to erythromycin was associated to a A2058G mutation in 23S rRNA. In conclusion, results indicate that resistance to macrolides and tetracycline is present among intestinal lactobacilli. Decrease in susceptibility following serial exposure to antibiotics might occur in lactobacilli, in a strain- and antibiotic-dependent way. Since lactobacilli are often used as probiotics, their ability to acquire resistance should be evaluated for isolates candidate to be included in probiotics based products.     

铜绿假单胞菌氨基糖苷16SrRNA甲基化酶基因分析

目的调查215株湖州地区临床分离铜绿假单胞菌对氨基糖苷类抗生素的耐药性和16S rRNA甲基化酶基因分布情况。方法收集2011年1月至2012年12月湖州地区临床分离铜绿假单胞菌215株,琼脂稀释法测定5种氨基糖苷类抗菌药物（庆大霉素、阿米卡星、妥布霉素、伊帕米星、奈替米星）的MIC值;PCR检测armA、rmtA、rmtB、rmtC、rmtD和npmA六种氨基糖苷类16S rRN甲基化酶基因,序列分析明确基因型。测定产16S rRNA甲基化酶菌株对常见抗菌的敏感性,并检测碳青霉烯耐药株产碳青霉烯酶情况。结果铜绿假单胞菌对异帕米星敏感率最高为81.4%,对5种氨基糖苷类抗生素全部耐药的22株菌株中,17株检出armA基因;未发现其他16S rRNA甲基化酶基因阳性菌株。17株armA阳性菌株对碳青霉烯类抗生素耐药5株（耐药率为29.4%）,对头孢他啶、头孢吡肟、哌拉西林/他唑巴坦、环丙沙星耐药率均超过40%。5株碳青霉烯耐药菌株中检测到2株产VIM-2型金属碳青霉烯酶。结论铜绿假单胞菌对氨基糖苷类抗生素耐药率高,检测到16S rRNA甲基化酶基因armA。产16S rRNA甲基化酶铜绿假单胞菌耐药性强,部分菌株同时产金属碳青霉烯酶,给临床抗感染治疗及院内感染控制带来挑战。     

Novel genotypes in Helicobacter pylori involving domain V of the 23S rRNA gene

BACKGROUND: Helicobacter pylori is a pathogenic bacterium that infects a half of the human population. In Chile, between 55% and 79% of people are colonized by H. pylori. At present, therapeutic strategies to eradicate the bacterium depend on the knowledge of its resistance to antibiotics. The clarithromycin resistance in H. pylori is associated with point mutations in the 23S rRNA. This study analyzes 23S rRNA gene mutations and minimum inhibitory concentration (MIC) for clarithromycin in H. pylori isolates from patients of the metropolitan region of Chile. MATERIALS AND METHODS: H. pylori isolates from 50 dyspeptic patients with no history of clarithromycin exposure were tested for clarithromycin resistance by agar dilution method. Resistant strains were analyzed for mutations in the 23S rRNA gene by polymerase chain reaction-based restriction fragment length polymorphism and sequencing. RESULTS: Primary resistance was observed in 10 isolates (20%). A single mutation was detected in four of the 10 isolates and two or more mutations in the other six cases. The C2147G transversion and G1939A, T1942C, and A2142G transitions in the peptidyltransferase region of domain V were novel. CONCLUSIONS: The study shows: 1, novel variants of the H. pylori 23S rRNA gene; and 2, a high prevalence of H. pylori displaying primary clarithromycin resistance with low level of MIC in an urban area of the Metropolitan Region of Chile.     

MAMA-PCR assay for the detection of point mutations associated with high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains

SYNOPSIS: Twenty Campylobacter jejuni and 16 Campylobacter coli strains isolated from humans and food/animals, including 17 isolates resistant to erythromycin, were analyzed. A combined mismatch amplification mutation assay-PCR technique was developed to detect the mutations A 2074 C and A 2075 G in the 23S rRNA gene associated with erythromycin resistance. All high-level erythromycin-resistant strains examined by DNA sequencing carried the transition mutation A 2075 G, whereas no isolate carried the A 2074 C mutation. No mutations were found among the susceptible and low-level erythromycin-resistant strains.     

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of 'Pathogenic', 'Commensal' and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two 'putative virulence' genes (eMLST) that provides improved high resolution typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA(1) clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA(2) strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA(1) and IA(2) strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.     

Occurrence of high-level mupirocin resistant coagulase-negative staphylococci in hospitals and their characterization

Occurrence of high-level mupirocin resistant coagulase-negative staphylococci in 5 hospitals in the region of Gdansk was determined. The study was carried out on 192 staphylococcal strains isolated from patients and medical staff. The mupirocin resistant strains were detected by 5 microgram mupirocin disc. The minimal inhibitory concentration (MIC) for mupirocin was estimated by E-tests. The mupirocin resistant strains were characterised by antibiotic sensitivity, including MIC for glycopeptides and oxacillin. Biotypes of resistant strains were also determined. Eight high-level mupirocin resistant strains (4.7%) were found. Only one strain expressed low-level resistance. All but one of high-level mupirocin resistant strains were resistant to methicillin. Six of them belonged to the S. epidermidis species but differences in the biotypes and antibiotic resistance patterns of these strains suggest they did not have a common origin.     

Genetic diversity of root-nodulating bacteria isolated from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Anti Propionibacterium acnes activity of rhodomyrtone, an effective compound from Rhodomyrtus tomentosa (Aiton) Hassk. leaves

Propionibacterium acnes have been recognized as one of the main causative agents in pathogenesis of acne. Twenty one isolates of P. acnes isolated from acne lesions were screened for lipase and protease activity which are reported to be associated in acne and inflammation. Interestingly, all P. acnes isolates demonstrated lipase activity. Similarly, 90% of test P. acnes produced protease enzyme. Antibacterial activity of the ethanol extract of Rhodomyrtus tomentosa (Aiton) Hassk. leaves and rhodomyrtone, its principle compound were tested against P. acnes using broth macrodilution method. The MIC(90) values of the ethanol extract and rhodomyrtone were 32 and 0.5 μg/mL, respectively. The numbers of the bacterial cells were reduced at least 99% after treatment with the ethanol extract and rhodomyrtone within 72 and 24 h, respectively. Cytotoxicity test of the extract and rhodomyrtone was performed on human normal fibroblast. The IC(50) values of the ethanol extract and rhodomyrtone were 476 and more than 200 μg/mL, approximately 15 and 400 folds higher than the MIC(90) values indicating that both substances were very low cytotoxic which could be applied as topical therapeutic anti-acne agents.