2016‒2017年辽宁省沙门菌耐药菌谱与分子分型

目的 分析2016‒2017年辽宁省沙门菌分离株的耐药特性与脉冲场凝胶电泳（PFGE）分子分型特征,为沙门菌引起的食源性疾病暴发、防控及抗生素使用提供参考数据。方法 对分离的54株沙门菌进行血清分型和药物敏感试验。根据PulseNet沙门菌标准PFGE分型技术,选取全部菌株进行PFGE分子分型分析,应用BioNumerics软件对菌株条带进行分析,确定菌株间的特征及相关性。结果 54株沙门菌血清型居首位的是肠炎沙门菌,占46.30％;其次是鼠伤寒沙门菌,占24.07％;共分为10个血清型。对13种抗生素的耐药分析显示多重耐药菌株为36株,占66.7％,其中耐3～5种的13株（24.1%）,耐6～8种的13株（24.1%）,耐9～11种的10株（18.5%）。54株沙门菌经聚类分析获得36种带型,相似度区间为49.7%～100.0％。结论 辽宁省沙门菌分离株多重耐药状况比较严重,相同血清型其PFGE带型相似度相对较高,同时具有较显著的优势带型特点;而且发现同一PFGE型菌株的耐药谱相对比较接近。     

High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

弗氏志贺菌4c和2a型菌株的脉冲场凝胶电泳分型特征

目的：了解北京部分地区弗氏志贺菌4c型（F4c）和2a型（F2a）菌株的分子分型特征。方法：对2005年和2006年自北京部分地区腹泻监测分离的弗氏志贺菌菌株（4c型10株,2a型20株）进行生化鉴定和血清分型,用PCR检测侵袭性抗原基因ipaH,用改良Kirby-Bauer纸片法检测菌株的耐药谱,用脉冲场凝胶电泳（PFGE）对菌株进行分子分型。结果：10株血清型鉴定为F4c的菌株中,有7株间的PFGE图谱存在相当的差异,Dice系数为0.78～0.92,而F2a菌株与大部分F4c菌株间的距离较远（Dice系数小于0.8）;F4c和F2a菌株对14种抗生素的耐药谱略呈差异。结论：采用PFGE能够很好地辨别弗氏志贺菌4c型和2a型菌株;弗氏志贺菌4c型菌株具有易变性,可在流行过程中产生PFGE图谱的差异、血清亚型的转换、耐药谱的变化等。     

Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative staphylococci

Thirteen Staphylococcus aureus and S. epidermidis strains obtained from nose and hand of two employees and one patient of a medical ward as well as two S. hemolyticus strains were analysed according to their restriction fragment length patterns (RFLP) by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI and SstII. Species identification of the isolates was performed by a system which includes 20 biochemical reactions. Furthermore, the antibiotic resistance patterns of the strains were determined. While several isolates exhibited identical antibiotic susceptibilities and biochemical profiles, differences in the RFLP were obtained. In three cases, S. epidermidis strains colonizing the skin showed an identical restriction profile as isolates from the mucous membranes of the same person. We concluded that the analysis of staphylococcal strains by PFGE is an important epidemiological tool with high discrimination power.     

Antimicrobial susceptibility, phage types, and pulse types of Salmonella Typhimurium, in São Paulo, Brazil

A total of 283 Salmonella Typhimurium strains isolated from cases of human infections and non human sources, were examined for antimicrobial susceptibility and the incidence of resistance was 38% and multiple resistance (to three or more antimicrobials) was 15%. All 43 multidrug-resistant strains (MDR) and 13 susceptible ones were characterized by phage typing and pulsed- field gel electrophoresis (PFGE). The strains encompassed 14 definitive phage types (DT), three were untypable (UT), and 18 atypicals or reaction does not conform (RDNC), which belonged to 21 PFGE patterns, A1-A21. The predominant phage types were DT49, DT193, and RDNC and two strains belonging to DT 104 and 104b were also identified. The most common PFGE patterns were A1 and A8. Analysis by PFGE and phage typing demonstrated that the most of the MDR were multiclonal and association among multiresistance, phage typing, and PFGE patterns was not so significant.     

2014—2015年大连市伤寒沙门菌耐药性及分子分型研究

目的 了解大连市伤寒沙门菌的药物敏感情况及同源性特点,为临床用药和疾病预防提供科学指导。方法 选择15种抗生素、运用微量肉汤稀释法对65株伤寒沙门菌进行抗生素敏感试验;采用脉冲场凝胶电泳（PFGE）法进行聚类分析。结果 65株伤寒沙门菌对阿奇霉素100.00%敏感,对红霉素100.00%耐药,对其他13种药物有不同程度的耐药率（1.54%~73.85%）。发现1株多重耐药菌株。65株菌共产生41种PFGE带型,其中8株菌表现为同一型别。结论 大连地区临床分离伤寒沙门菌耐药形势严峻;聚类分析结果表明其PFGE型别较多,而且其中存在着优势菌株。     

High-Level Genotypic Variation and Antibiotic Sensitivity among Escherichia coli O157 Strains Isolated from Two Scottish Beef Cattle Farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Phenotypic and genotypic characterization of sorbitol-negative or slow-fermenting (suspected O157) Escherichia coli isolated from milk samples in Lombardy region

AIMS: To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS: Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS: The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.     

Comparison of Pulsed-Field Gel Electrophoresis, Ribotyping, and Plasmid Profiling for Typing of Vibrio anguillarum Serovar O1

A total of 75 Vibrio anguillarum serogroup O1 strains were studied with respect to their plasmid contents, ribotypes, and pulsed-field gel electrophoresis (PFGE) patterns. Eight plasmid profiles and six ribotypes were demonstrated, and one profile was dominant by both typing methods. In contrast, PFGE had very high discriminatory power, demonstrating 35 profiles. On the basis of PFGE patterns, a similarity matrix and a dendrogram were constructed. The results indicated that Scandinavian strains and southern European isolates (with some exceptions) belong to two different clonal lineages. A few strains from the United States and United Kingdom deviated considerably from each other and from Scandinavian and southern European strains.     

Use of pulsed field gel electrophoresis as an epidemiological tool for analysis of sporadic associated strains of Salmonella typhi isolated in Taiwan

In order to characterize the subtypes of Salmonella typhi which cause sporadic disease in Taiwan, 55 isolates of Salm. typhi obtained from unrelated patients of sporadic cases during 1992-96 were subjected to chromosomal DNA digestion and pulsed field gel electrophoresis (PFGE). When DNAs of these 55 Salm. typhi strains were digested with XbaI, 41 PFGE patterns were observed. Strains sharing the same XbaI digestion pattern could not be further discriminated by PFGE analysis using SpeI and NotI as digestion enzymes. Thus, considerable genetic diversity exists among the Salm. typhi isolates. Although strains of the same patterns were mainly isolated during the same time, recirculation of certain infectious strains could be possible. When 12 antibiotics, i.e. ampicillin, trimethoprim/sulfamethoxazole, erythromycin, norfloxacin, tetracycline, sulphonamide, streptomycin, neomycin, chloramphenicol, kanamycin, cefoperazone and gentamycin were used to test the antibiotic susceptibility for these Salmonella isolates, only three antibiogram patterns were obtained and 49 of the 55 Salm. typhi isolates were found to belong to one pattern. Phage typing and plasmid profiles were also poor in discriminating these strains. Thus, PFGE alone may be used as a powerful tool for analysis of sporadic associated Salm. typhi strains.     

Identification of fecal input sites in spring water by selection and genotyping of multiresistant Escherichia coli

The localization of fecal input sites is important for water quality management. For this purpose, we have developed a new approach based on a three-step procedure, including a preparatory phase, the screening of multiresistant bacteria using selective agar plates, and a typing phase where selected Escherichia coli isolates are characterized by antibiotic resistance profiles and molecular fingerprinting techniques (pulsed-field gel electrophoresis [PFGE]). These two well-known source tracking methods were combined in order to reduce cost and effort. This approach was successfully applied under field conditions in a study area located in the north-western part of Switzerland. E. coli isolates from spring water and surface water samples collected in this area were screened with selective agar plates. In this way, 21 different groups, each consisting of strains with the same pattern of antibiotic resistance, were found. Of these, four groups were further analyzed using PFGE. Strains with identical PFGE profiles were detected repeatedly, demonstrating the suitability of this method for the localization of fecal input sites over an extended period of time. Identical PFGE patterns of strains detected in water from two different springs were also found in the stream flowing through the study area. These results demonstrated the applicability of the new approach for the examination of incidents of fecal contamination in drinking water. The advantages of the described approach over genotyping methods currently being used to identify sources of fecal contaminants are a reduction in time, costs, and the effort required. Identical isolates could be identified without the construction of large libraries.     

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.     

Genetic analysis of Streptococcus agalactiae strains isolated from neonates and their mothers

Streptococcus agalactiae or group B streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis in neonates. One of the major questions is whether the GBS strains able to cause neonatal invasive disease have peculiar genetic features. A collection of S. agalactiae strains, isolated from cervix, vagina and rectum of 10 mothers and from throat, ear and umbilicus of their newborns was genetically characterized by pulsed-field gel electrophoresis (PFGE). This study demonstrated that the strains isolated from each mother and her child were all genetically identical but that the strains from the 10 mother/child pairs mutually were genetically heterogeneous and 10 different PFGE patterns were found. Although it has been suggested that PFGE would be able to identify virulence traits to direct decisions in antibiotic management, the heterogeneous feature of GBS strains does not support broad application.     

Genotypic characterization of Enterobacter sakazakii isolates by PFGE, BOX-PCR and sequencing of the fliC gene

Aims:  Enterobacter sakazakii is an emerging food-borne pathogen that can cause rare but severe forms of neonatal meningitis, bacteraemia and necrotizing enterocolitis. A rapid typing method at the strain level is needed to determine the monoclonality or polyclonality of the isolates during outbreaks.
Methods and Results:  The BOX-PCR fingerprinting technique, which targets the repetitive BOX sequences, and sequencing of the flagellin gene, fliC , were evaluated against a panel of 27 Ent. sakazakii strains from clinical and environmental sources. The typeability and discriminatory power of the techniques were compared with those of pulsed-field gel electrophoresis (PFGE), the reference genotyping method. BOX-PCR results yielded 92% agreement with PFGE results, whereas fliC gene sequencing was poorly discriminative.
Conclusions:  In our study, BOX-PCR and PFGE were similarly discriminatory to type Ent. sakazakii strains. The weak variability of the Ent. sakazakii fliC gene was related to the absence of the variable central domain present in most fliC genes of Enterobacteriaceae.
Significance and Impact of the Study:  The BOX-PCR typing provides an accurate discrimination and a rapid answer to identify clonal isolates of Ent. sakazakii .     

Characteristics of the MRSA clone with reduced susceptibility to vancomycin

The MIC of vancomycin was determined for all S. aureus strains isolated during 1997 in one hospital. MIC values for most isolates were in the range of 0.5-2 mg/l. In 18 strains, MIC was = 6 mg/L. All these strains were MRSA. Recently described VISA strains possessed MIC values for vancomycin equal or higher than 8 mg/l and such strains were not detected in the investigated group. Although strains with MIC = 6 mg/l are not VISA, but they are candidate for reduced vancomycin susceptibility, e.g. during therapy in compromised patients. Analysis of DNA of these strains by pulsed-field gel electrophoresis (PFGE) revealed that 15 of them shared a significant similarity, allowing to place them in the same group. The comparison data of phage patterns as well as antibiotic resistance patterns strongly suggest that all these strains were derivatives of a single clone.     

Pulsed-field gel electrophoresis as an epidemiological tool for clonal identification of Aeromonas hydrophila

Pulsedfield gel electrophoresis (PFGE) was used to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections that occurred over approximately 1 month in a French hospital. Five isolates from patients and 10 isolates from the water supply were characterized by biotyping and antibiotic susceptibility patterns and compared with 10 epidemiologically unrelated strains isolated from patients and rivers, by PFGE of digests of chromosomal DNA. Five environmental and four clinical isolates belonged to the same biotype and antibiotic susceptibility pattern type. The endonucleases XbaI, SpeI and SwaI gave satisfactory profiles whereas DraI did not. The profiles were stable, reproducible and discriminatory. The 10 epidemiologically unrelated strains exhibited 10 different patterns after digestion with XbaI , the least expensive, suitable endonuclease. PFGE is a rapid and discriminatory technique for the typing of Aeromonas hydrophila where a common origin of infection is suspected.     

Use of antibiotic susceptibility patterns and pulsed-field gel electrophoresis to compare historic and contemporary isolates of multi-drug-resistant Salmonella enterica subsp. enterica serovar Newport

Recently, multi-drug-resistant (MDR) Salmonella enterica subspecies enterica serovar Newport reemerged as a public and animal health problem. The antibiotic resistance of 198 isolates and the pulsed-field gel electrophoresis patterns (PFGE) of 139 isolates were determined. Serovar Newport isolates collected between 1988 and 2001 were included in the study. One hundred seventy-eight isolates were collected from the San Joaquin valley in California and came from dairy cattle clinical samples, human clinical samples, bulk tank milk samples, fecal samples from preweaned calves, and waterways. Twenty clinical isolates from humans from various regions of the United States were also included in the study. Resistance to 18 antibiotics was determined using a disk diffusion assay. PFGE patterns were determined using a single enzyme (XbaI). The PFGE and antibiogram patterns were described using cluster analysis. Although the antibiotic resistance patterns of historic (1988 to 1995) and contemporary (1999 to 2001) isolates were similar, the contemporary isolates differed from the historic isolates by being resistant to cephalosporins and florfenicol and in their general sensitivity to kanamycin and neomycin. With few exceptions, the contemporary isolates clustered together and were clearly separated from the historic isolates. One PFGE-antibiogram cluster combination was predominant for the recent isolates, which were taken from human samples from all parts of the United States, as well as in the isolates from California, indicating a rapid dissemination of this phenotypic strain. The data are consistent with the hypothesis that the reemergence of MDR serovar Newport is not simply an acquisition of further antibiotic resistance genes by the historic isolates but reflects a different genetic lineage.     

Rapid phenotypic characterization of Salmonella enterica strains by pyrolysis metastable atom bombardment mass spectrometry with multivariate statistical and artificial neural network pattern recognition

Pyrolysis mass spectrometry was investigated for rapid characterization of bacteria. Spectra of Salmonella were compared to their serovars, pulsed-field gel electrophoresis (PFGE) patterns, antibiotic resistance profiles, and MIC values. Pyrolysis mass spectra generated via metastable atom bombardment were analyzed by multivariate principal component-discriminant analysis and artificial neural networks (ANNs). Spectral patterns developed by discriminant analysis and tested with Leave-One-Out (LOO) cross-validation distinguished Salmonella strains by serovar (97% correct) and by PFGE groups (49%). An ANN model of the same PFGE groups was cross-validated, using the LOO rule, with 92% agreement. Using an ANN, thirty previously unseen spectra were correctly classified by serotype (97%) and at the PFGE level (67%). Attempts by ANN to model spectra grouped by resistance profile-but ignoring PFGE or serotype-failed (10% correct), but ANNs differentiating ten samples of the same serotype/PFGE class were more successful. To assess the information content of PyMS data serendipitously associated with or directly related to resistance character, the ten isolates were grouped into four, three, or two categories. The four categories corresponded to four resistance profiles. The four class and three class ANNs showed much improved but insufficient modeling power. The two-class ANN and a corresponding multivariate model maximized inferential power for a coarse antibiotic-resistance-related distinction. They each cross-validated by LOO at 90%. This is the first direct correlation of pyrolysis metastable atom bombardment mass spectrometry with immunological (e.g. serology) or molecular biology (e.g. PFGE) based techniques.     

Phenotypic and genetic characterization of vancomycin-resistant enterococci from hospitalized humans and from poultry in Korea

Vancomycin resistant enterococci (VRE) isolates from humans (23 isolates) and poultry (20 isolates) were characterized by antibiotic susceptibility, vancomycin resistance transferability, pulsed-field gel electrophoresis (PFGE), and structural analysis of Tn1546-like elements. VRE isolates from humans and poultry showed different resistance patterns, transferability, and transfer rate. In addition to these phenotypic differences between humans and poultry VRE, PFGE and the structure of Tn1546-like elements were also distinct. Most poultry isolates (16/20) were identical to the prototype vanA transposon, Tn1546, while most human isolates (21/23) had multiple integrations of insertion sequence. The transmission of VRE and vancomycin resistance determinant between humans and poultry could not be demonstrated in this study.     

Characterization of recurrent and sporadic Listeria monocytogenes isolates from raw milk and nondairy foods by pulsed-field gel electrophoresis, monocin typing, plasmid profiling, and cadmium and antibiotic resistance determination

Following previous surveys to assess the incidence of Listeria monocytogenes in raw milk and nondairy foods processed in Northern Ireland, isolates were characterized as recurrent or sporadic on the basis of multilocus enzyme electrophoresis (MEE) analysis and restriction fragment length polymorphism typing. In the present study, 45 representative recurrent and sporadic electrophoretic types (ETs) previously identified by MEE were subjected to pulsed-field gel electrophoresis (PFGE) of genomic DNA macrorestriction fragments, monocin typing, plasmid profiling, and an examination of resistance to cadmium and nine different antibiotics. Although PFGE proved to be capable of subdividing a number of recurrent and sporadic ETs, the grouping of strains arrived at by PFGE and MEE were in broad agreement, and previous conclusions regarding the designation of L. monocytogenes strains as recurrent or sporadic remained unaltered. It is considered that PFGE was able to detect minor genetic changes in recurrent ETs which occurred during the time period in which food surveys were carried out. Production of type E monocin (Types A to E were found among the 45 strains), plasmid carriage, and resistance to cadmium occurred more frequently in recurrent than in sporadic strains and may be important with regard to the ability of L. monocytogenes to persist in food and food-processing environments. Only 2 of 45 strains showed resistance to any of the nine antibiotics tested: two sporadic strains were resistant to tetracycline (MIC, 64 microg x ml(-1)).