Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.     

Pulsed-field gel electrophoresis, plasmid profiles and phage types for the human isolates of Salmonella enterica serovar Enteritidis obtained over 13 years in Taiwan

AIMS: Plasmid profile, phage typing, and pulsed-field gel electrophoresis (PFGE) patterns of 124 Salmonella Enteritidis strains isolated in 1998-2002 in Taiwan were analysed and the results were compared with those of the 63 strains obtained in 1991-1997, so that molecular subtypes and epidemic strains for Salmonella Enteritidis over a 13-year period (1991-2002) could be elucidated. METHODS AND RESULTS: A total of 124 strains of Salmonella Enteritidis isolated from human in Taiwan between 1998 and 2002 were analysed by PFGE, plasmid analysis and phage typing. The results obtained were compared with those of the 63 strains obtained in 1991-1997, so that the clonal relationships for a total of 187 strains obtained over 13 years could be elucidated. For PFGE, restriction enzymes XbaI, SpeI and NotI were used for chromosomal DNA digestion. Results showed 28 PFGE pattern combinations for the 187 Salmonella strains. Of them, pattern X3S3N3 was the major subtype as 130 strains isolated from different locations during 1991-2002 showed this PFGE pattern. For all these 187 strains, the genetic similarity was higher than 80%. Plasmid analysis showed 17 distinct types, which consist of one to four plasmids and the predominant phage type of those strains was PT4 (71.6%) and PT6a (13.4%). The three methods identified different degrees of polymorphism in the following order: plasmid profile (18 types, D = 0.659) > PFGE (28 types, D = 0.512) > phage typing (13 types, D = 0.438). As PFGE patterns, phage type and plasmid profile were combined for subtyping, the 187 strains could be grouped into 46 subtypes and the discriminatory index was raised to 0.795. For these 46 subtypes, the predominant one was X3S3N3/P1/PT4, which contained 77 (41%) isolates. CONCLUSIONS: Most of the Salmonella Enteritidis strains from sporadic cases were with pattern X3S3N3. They were the prevalent and may be the epidemic strains found in Taiwan during 1991-2002. The present study suggested that the several variants were derived from a single clonal line and the genome for strains of Salmonella Enteritidis are highly conserved over a 13-year period (1991-2002). SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here are useful for epidemiolgical study of salmonellosis caused by Salmonella Enteritidis in Taiwan. Comparing the data of the present study with those obtained for strains from other countries, the major subtypes for Salmonella Enteritidis infection in the world can be elucidated.     

Prevalence of Salmonella enterica serovars and genovars from chicken carcasses in slaughterhouses in Spain

AIMS: To determine the prevalence of Salmonella enterica serovars in chicken carcasses in slaughterhouses in Spain and to examine genotypic relations among these serovars. METHODS AND RESULTS: A total of 336 chicken carcasses were collected from six slaughterhouses in Northwestern Spain. Salmonellae were isolated (ISO-6579-1993), serotyped, phage-typed, ribotyped and antibiotyped against 20 antibiotics. Salmonella strains were detected in 60 (17.9%) carcasses. Isolates belonged to nine different serotypes, with Salm. Enteritidis being the most common. Three strains (5%) were resistant to one antibiotic and 24 (40%) were multi-resistant (to more than one antibiotic). The most frequently encountered resistances were to sulphamides, fluoroquinolones and tetracycline. Ribotyping was able to differentiate isolates of the same serotype and phage type. CONCLUSIONS: The Salmonella serotypes and phage types detected are among those most frequently associated with human diseases in Spain. The large percentage of antimicrobial resistant strains is a matter for concern. A high genetic relationship between strains from different slaughterhouses was found. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides detailed information about Salmonella isolates from poultry in Spain. It emphasizes the importance of controlling this pathogen in poultry products, and suggests the need for more prudent use of antibiotics.     

Development of a real-time PCR assay for detection of gyrA mutations associated with reduced susceptibility to ciprofloxacin in Salmonella enterica serovar typhi and paratyphi A

A real-time PCR assay with the cycling probe method was used to detect mutations at codons 83 and 87 in the DNA gyrase A subunit encoded by gyrA in Salmonella enterica serovar Typhi and Paratyphi A clinical isolates. The susceptibility estimated from the results of the gyrA mutation assay was consistent with that identified by the culture method using an E-test. This assay allows rapid screening of S. enterica serovar Typhi and Paratyphi A with reduced susceptibility to ciprofloxacin.     

Phage types and pulsed-field gel electrophoresis patterns of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis isolated from humans and chickens

We analyzed 66 Salmonella Enteritidis isolates in 2002. Thirty isolates were obtained from human patients with diarrhea, and 36 were obtained from chickens. A total of ten phage types (PT) were identified in the human and chicken isolates. PT1 and PT21 were the predominant PTs in both the human (20% and 13%) and chicken (17% and 47%) isolates. Twelve pulsotypes were generated by PFGE and divided into two major groups. Most of the PFGE types were categorized into cluster group 1. Eighteen chicken isolates in cluster group 1 showed high-level genetic association (>95%) with 22 other human isolates. Additionally, six chicken isolates from cluster group 2 showed fairly high-level genetic association (>95%) with the other seven human isolates. The highest levels of genetic association in humans and chickens were seen with A5-PT21 (11 isolates), A2-PT1 (7 isolates), and B1-PT4 (6 isolates). The Pulsed-Field Gel Electrophoresis (PFGE) and phage typing provided conclusive evidence that human Salmonella infections are attributable to the consumption of contaminated chicken.     

A survey for serotyping, antibiotic resistance profiling and PFGE characterization of and the potential multiplication of restaurant Salmonella isolates

AIMS: The aims of this research were (1) to determine the occurrence of Salmonella in Irish restaurant kitchens and (2) to investigate the serovar, genotype, antibiotic resistance profile and survival/growth profile of the Salmonella under catering chilled storage and temperature abuse conditions. METHODS: Five sites/tools in each of 200 randomly selected restaurant kitchens were examined for the presence of presumptive Salmonella spp. by enrichment. Serotyping, antibiotic resistance studies and genotyping were performed using the Kauffmann-White, CLSI and PulseNet methods, respectively. Survival/growth was investigated in milk, meat and vegetable products. RESULTS: Presumptive isolates from 15 of the 200 restaurant kitchens were recovered and confirmed as Salmonella positive. Seven different serovars showing a variety of antibiotic resistance profiles were detected. PFGE profiles suggested that isolates from geographically adjacent restaurants were related. Salmonella survived in foods stored at typical catering refrigeration temperatures and increased by approximately 0.8 log(10) CFU ml(-1) per day in food products stored under conditions of thermal abuse (20 degrees C). CONCLUSIONS: Inadequate hygiene has resulted in contamination of restaurant kitchens with Salmonella, which may persist/multiply in cross-contaminated foods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the need for greater hygiene in restaurant kitchens coupled with rapid chilling of food not for immediate consumption and reheating before subsequent serving.     

Distribution and genotypic characterization of Salmonella serovars isolated from tropical seafood of Cochin, India

Aims:  To determine the distribution of Salmonella serovars in seafood and to examine the intraserovar genetic variations in Salmonella enterica subsp. enterica serovar Rissen and Salmonella Weltevreden by polymerase chain reaction (PCR)-ribotyping and enterobacterial repetitive intergenic consensus (ERIC)-PCR methods.
Method and Results:  A total of 417 seafood samples collected over 2003–2006 from fishing harbours and fish markets of Cochin (India) was studied for presence of Salmonella serovars. Seafood samples were analysed for the presence of Salmonella by Bacteriological Analytical Manual (BAM), U.S. Food & Drug Administration (USFDA) method. The study indicated that 23·2% of the seafood samples were positive for Salmonella and a total of 241 Salmonella isolates comprising of 27 different serovars were isolated from seafood. S. Weltevreden, Salmonella Rissen, Salmonella Typhimurium and Salmonella Derby were found to be the most predominant serovars in seafood. PCR-ribotypes and ERIC-PCR profiles showed multiple genotypic profiles for S. Rissen and S. Weltevreden in seafood and the level of discrimination indices obtained was at 0·974 for S. Rissen and 0·988 for S. Weltevreden, respectively.
Conclusion:  The study highlighted the major Salmonella serovars in the seafood of Cochin (India) and molecular fingerprinting pattern revealed genetic variation among S. Rissen and S. Weltevreden.
Significance and Impact of the Study:  Widespread occurrence of Salmonella contamination in seafood and multiple clones of S. Rissen and S. Weltevreden detected in seafood, thus, indicated the diverse routes of Salmonella contamination in seafood.     

Virulence and antibiotic susceptibility of Aeromonas spp. isolated from drinking water

Aeromonas isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, cytotoxins, phospholipase, DNase, hydrophobicity and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics of Aeromonas isolates was also examined. Majority of the isolates displayed hemolytic activity against sheep erythrocytes, while only 7 of the 23 Aeromonas strains displayed DNase activity and 4 of the 23 Aeromonas strains tested were regarded as positive for phospholipase production. Most of the isolates showed cytotoxic activities in culture filtrate dilutions at titer of 1/8 or lower. No general relation between the strain isolated and the ability to interact with epithelial cells could be established. Using the bacterial adherence to hydrocarbons method, most of the strains were classified as highly hydrophilic. All five Aeromonas jandaei strains isolates, 9 of the 12 Aeromonas sp strains and four of the five Aeromonas hydrophila were multidrug resistant. The most active antimicrobial was ciprofloxacin (susceptible in 100% of the isolates), and the least active antibiotic was ampicillin (resistance in 92% of the isolates). The majority of the isolates tested were not killed by chlorine at 1.2 mg/l. Whether the high tolerance to chlorine of Aeromonas isolates can be linked to greater virulence is not know.     

Epidemiological analysis of Salmonella enterica ssp. enterica serovars Hadar, Brancaster and Enteritidis from humans and broiler chickens in Senegal using pulsed-field gel electrophoresis and antibiotic susceptibility

AIMS: Salmonella Hadar, Salmonella Brancaster and Salmonella Enteritidis are the main Salmonella enterica ssp. enterica serovars isolated from poultry in Senegal. Our objective was to analyse the pulsed-field gel electrophoresis (PFGE) and antibioresistance patterns of strains belonging to these serovars and to assess the significance of broiler-chicken meat as a source of human infection. METHODS AND RESULTS: A total of 142 Salmonella isolates were analysed: 79 were isolated from Senegalese patients with sporadic diarrhoea (11 S. Hadar, nine S. Brancaster and 59 S. Enteritidis) and 63 from poultry (30 S. Hadar, 17 S. Brancaster and 16 S. Enteritidis). The PFGE of XbaI- and SpeI-digested chromosomal DNA gave 20 distinct profiles for S. Hadar, nine for S. Brancaster and 22 for S. Enteritidis. Each serovar was characterized by a major pulsotype which was X3S1 in 42% of S. Hadar, X8S1 in 53.8% of S. Brancaster and X1S2 in 43% of S. Enteritidis isolates. Human and poultry isolates of Salmonella had common PFGE patterns. Antibiosensitivity tests showed multiresistance (more than two drugs) was encountered in 14.5% of S. Hadar and in 5% of S. Enteritidis isolates. Resistance to quinolones was considered to be of particular importance and 14.5% of S. Hadar isolates were found to be resistant to nalidixic acid. CONLCUSIONS: The sharing of similar PFGE profiles among isolates from humans and poultry provided indirect evidence of Salmonella transmission from contaminated broiler meat. But most of the Salmonella isolates remained drug sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Efforts are needed to eliminate Salmonella from poultry meat intended for human consumption. This study has also highlighted the importance of continuous surveillance to monitor antimicrobial resistance in bacteria associated with animals and humans.     

鸽肠道沙门菌分离鉴定、药敏试验及毒力基因、耐药基因检测

目的 对安徽蚌埠某鸽场2014年初至2014年底5次送检以腹泻、败血症为特征的病死鸽肝组织进行病原菌分离鉴定,并测定其致病性、毒力基因和耐药基因。方法 将5次送检的病死肉鸽肝脏组织在麦康凯琼脂平板上进行病原菌的分离鉴定、同源性分析,并测定其毒力基因和耐药基因。结果 从上述病死鸽肝脏中分离到5株革兰阴性杆菌。基因比对结果显示,5株分离菌均为肠道沙门菌。分离菌均含有磺胺类耐药基因sul1,均不含β-内酰胺类耐药基因blaCMY-2,2株含β-内酰胺类耐药基因blatem-1,4株含氨基糖苷类耐药基因aadA1,2株含喹诺酮类耐药基因qnr。实验组小鼠多数于接种后24 h内死亡。分离菌均含肠毒素基因stn,菌毛基因invJ,毒力岛基因mis、orf31.9和pipC,3株含毒力岛基因基因sipA和ssaB基因。结论 该鸽场5次送检的病死鸽死亡是由肠道沙门菌感染引起的。5株分离菌致病性均很强,均含多重耐药基因和多种毒力基因。     

健康猪直肠粪便中沙门菌I类整合子与耐药基因的检测

目的了解安徽省规模化猪场健康猪直肠粪便中沙门菌分离株多重耐药情况及其与I类整合子和耐药基因的携带关系。方法采用标准K-B纸片法对22株沙门菌分离株进行15种抗生素敏感试验;应用PCR技术对沙门菌分离株进行I类整合子及耐药基因检测。结果 22株沙门菌分离株中有20株(90.91%)对2种以上抗生素耐药,属于多重耐药株,羧氨苄青霉素-四环素-卡那霉素-氯霉素-氟苯尼考是主要多重耐药谱;22株沙门菌中有19株(86.4%)携带I类整合子,tetB、aph(3)-IIa和cmlA基因分别检出最高。结论沙门菌多重耐药性与整合子携带之间的关系密切,耐药表型测定结果与耐药基因检测结果基本一致,基因组DNA携带的耐药基因种类多于质粒。     

PCR typing of tetracycline resistance determinants (Tet A-E) in Salmonella enterica serotype Hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium

The distribution of tetracycline resistance determinants Tet A-E was studied by PCR in 40 tetracycline-resistant Salmonella enterica serotype Hadar (S. hadar) isolates collected from human patients in 1996 and 1997, as well as in the microbial community originating from activated sludges of hospital and urban wastewater treatment facilities. A fast DNA extraction and purification method from activated sludges was used to provide amplifiable DNA. The method is based on the direct lysis of bacteria improved by bead-beating followed by DNA purification on polyvinylpolypyrrolidone spin columns to remove PCR inhibitors. The purified DNAs from salmonellae and activated sludges were characterized for the presence of tetracycline determinants with specific primer pairs designed on the basis of published sequences. The Tet A determinant was present in all clinical isolates and DNAs extracted from the bacterial community of the selected activated sludges. The Tet C determinant was identified in only one of the 40 clinical isolates and in six of the seven environmental samples. No signal was detected for Tet B, D and E determinants. This study revealed a high and stable prevalence of the Tet A determinant in both salmonellae clinical isolates and the microbial community of activated sludges from hospital and urban wastewater treatment facilities over a 2-year period.