Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the invA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers.     

Contamination of Salmonella spp. in a pig finishing herd, from the arrival of the animals to the slaughterhouse

The aim of the present study was to investigate the contamination sources and the transmission of Salmonella within a pig finishing herd in Italy. Nine sets of samples were collected during the fattening period from cleaned and disinfected pens, animals at different ages (4 days after arrival, 90, 150, 170 and 240 days of age) and at slaughter. Salmonella was isolated from cleaned pens, individual faecal samples, the truck used to transport the pigs to the abattoir and after slaughter (cecal contents, mesenteric lymph nodes and carcasses). Several serovars were isolated: Salmonella typhimurium and Salmonella derby on farm; Salmonella bovismorbificans, Salmonella bredeney, Salmonella blockley, Salmonella hadar and Salmonella corvallis from the truck; S. derby, S. hadar, S. bredeney, S. bovismorbificans and Salmonella infantis at slaughter. Antibiotic resistance of the strains was tested and PFGE was carried out to investigate the on-farm epidemiology of Salmonella. The results showed that the environmental contamination may have represented a major source of infection for the pigs both on farm and during transport to the abattoir.     

动物源性沙门氏菌的耐药性分析及氟苯尼考类耐药基因的鉴定

为了解动物沙门氏菌的流行情况和药物敏感性及氟苯尼考耐药株的耐药基因分布,本试验对临床上疑似患沙门氏菌病的病料进行病原分离和细菌的多重PCR鉴定;采用K-B法测定分离株对23种抗菌药物的敏感性;选择氟苯尼考耐药菌株扩增floR、fexA、fexB、cfr和pexA基因。结果显示,共鉴定出61株沙门氏菌,其中肠炎沙门氏菌10株,鸡白痢沙门氏菌12株,鼠伤寒沙门氏菌39株。所有菌株对青霉素、红霉素、万古霉素耐药,90.16%对6种及6种以上抗菌药耐药。floR基因广泛存在于鼠伤寒沙门氏菌氟苯尼考耐药菌株中(8/12,66.67%),未发现其他耐药基因。研究结果表明鼠伤寒沙门氏菌是鹅源分离株中的优势血清型;floR基因主要介导沙门氏菌对氟苯尼考耐药性,但可能还存在其他机制。     

Biofilm-forming associated genotypic and phenotypic characteristics of Staphylococcus spp. isolated from animals and air

Biological monitoring is performed to detect and analyze microorganisms that have continuously made an effort to survive in the environment. Of such microorganisms, Staphylococcus spp. is considered a common cause of nosocomial and environmental infections., Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are required for the adhesion and biofilm formation of Staphylococci. Thirty-six and thirty-five Staphylococci isolated from animals and air, respectively, were analyzed. Biofilm formation and ten MSCRAMM genes were investigated using Congo red agar, tissue culture plate methods, and PCR. Airborne isolates were shown to have higher adherence and stronger biofilm formation than those from animals. The prevalence of MSCRAMM genes from air isolates was also higher than those from animals. Of the genes, eno was mainly associated with biofilm formation in both animals and airborne isolates (P < 0.05). Moreover, the rate of airborne isolates harboring the eno gene was higher than in animal isolates. These results indicated that analysis of MSCRAMM genes with a phenotypic assay might be a helpful bacterial control system for the environment.     

Molecular and virulence characteristics of multi-drug resistant Salmonella Enteritidis strains isolated from poultry

Forty-six Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strains were isolated from chicken meat, faeces, and eggshells collected from hatcheries throughout Korea. The strains were examined for the presence of antimicrobial resistance and virulence genes. All 46 isolates were resistant to at least one of 21 antibiotics used in this study, 30 (65.2%) were resistant to three or more antimicrobials, and a single remarkable isolate was resistant to 15 antimicrobials. The isolates were primarily resistant to penicillins, sulfisoxazole, streptomycin, tetracycline and quinolones.The high rate of resistance in S. Enteritidis strains, sometimes to multiple drugs, may complicate future options for treating human infections. Nineteen of the 21 penicillin resistant isolates carried the bla_TEM gene, while one strain, resistant both to penicillins and ceftriaxone, carried the bla_CTX-M gene. Thirty-seven of the 45 sulfisoxazole resistant isolates carried sul2, and 23/24 streptomycin resistant isolates carried both strA and strB. All 10 tetracycline resistant isolates carried the tet(A) gene. Most isolates harboured both SPI-1 and SPI-2-associated genes, and the spv operon, which are known to be associated with human infections. The presence of these genes suggests that these strains could give rise to public health problems if dispersed in the general human population.     

Molecular typing of Salmonella enterica serovar Enteritidis isolates from food-producing animals in Japan by multilocus variable-number tandem repeat analysis: evidence of clonal dissemination and replacement

Background

Salmonella enterica serovar Enteritidis is a zoonotic pathogen. Human infections are associated with contaminated eggs and egg products. In Japan, since 1989, the incidence of food-borne disease caused by S. Enteritidis has increased and a pandemic has occurred; however, little is known about changes that occurred before and after this pandemic event in the dominant lineage of isolates from food-producing animals. This study aimed to determine the S. Enteritidis lineages in Japan over the last few decades by using multilocus variable-number tandem repeat analysis (MLVA).

Findings

MLVA was used to analyse 79 S. Enteritidis isolates collected from chickens (n = 63), cattle (n = 12), pigs (n = 2), and goats (n = 2) during 1975–2009. The S. Enteritidis isolates showed 14 different MLVA allele combinations, which were classified into two major clusters (A and C) and a minor cluster (B). All the 62 isolates in cluster A were isolated after 1988, whereas 13 of the 17 isolates belonging to cluster B and C were isolated before 1989.

Conclusions

The MLVA results showed that cluster C was predominant before 1989, and isolates in cluster A disseminated since 1989 and replaced the previous dominant clone, suggesting that isolates of cluster A originated from imported S. Enteritidis infection.     

Salmonella enterica isolates from faeces of domestic reptiles and a study of their antimicrobial in vitro sensitivity

From October 2001 to February 2002, the faecal samples of 305 reptiles (165 saurians, 99 ophidians and 41 chelonians) were bacteriologically examined to detect Salmonella enterica. S. enterica was isolated from 73 (23.93%) faecal samples including 44 (60.27%) samples collected from saurians, 15 (20.55%) from chelonians and 14 (19.18%) from ophidians; considering the number of samples taken for each reptile group, S. enterica was isolated from the 36.58% of chelonians, 26.66% of saurians and 14.14% of ophidians. The isolates were distributed among 38 serotypes. Sixty-nine (94.52%) isolates were resistant to erythromycin. About one-third of the isolates was resistant to sulfisoxazole (35.61%), gentamycin (32.88%), amoxycillin (31.51%) and ampicillin (27.40%). All but one of the isolates were sensitive to chloramphenicol. A high percentages of isolates were sensitive to enrofloxacin (84.93%), nitrofurantoin (80.82%), trimethoprim (76.71%) and tetracycline (75.34%).     

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.     

Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland

Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.     

Prevalence and antimicrobial susceptibility of Salmonella isolates in Pekin ducks from South Korea

An investigation was carried out to determine the prevalence and antibiotic resistance of Salmonella serotypes at South Korean duck farms. A total of 7119 samples collected from 72 duck farms in five provinces were examined from 2011 to 2012. The overall prevalence of Salmonella serotypes was 43.4% (69/159) in duck flocks from 65.2% (47/72) of the duck farms. Eighty-five strains were isolated from 69 duck flocks. Three serotypes of Salmonella enterica were identified such as S. Typhimurium (39/85), S. Enteritidis (44/85), and S. London (2/85). The prevalence of Salmonella infection decreased significantly in 3-week-old ducks compared to that in 1-week-old ducks (P < 0.05). All isolates except one were resistant to at least one antimicrobial and 27% of the isolates were resistant to 5–16 antimicrobials. Our findings provide baseline information on the degree of Salmonella infection and distribution of Salmonella serotypes in ducks and indicate that ducks should be considered an important source of foodborne pathogens.     

Phylogenetic background of attaching and effacing <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from animals

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae gamma2/theta type, group B1 the eae beta1, eae kappa, eae zeta, and eae epsilon types, group B2 the eae alpha1, eae alpha2 and eae iota types, while the group E possessed the eae gamma1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.     

The anatomical distribution and antimicrobial susceptibility of yeast species isolated from healthy dogs

The aim of this work was to identify the predominant yeast species present at different anatomical sites in healthy dogs and to determine their in vitro antimicrobial susceptibility using a broth microdilution assay. Samples were collected from the preputial, vaginal, oral and perianal mucosae and the isolates cultured were identified according to their morphological characteristics and biochemical profile. Malassezia pachydermatis was the most commonly isolated yeast, followed by Candida parapsilosis, Candida tropicalis, Candida albicans, Saccharomyces cerevisiae and Rhodotorula spp.Minimum inhibitory concentrations of the azole derivatives ketoconazole, itraconazole and fluconazole against Candida spp. were 0.03–16 μg/mL, 0.06 to >16 μg/mL and 0.5–64 μg/mL, respectively and Candida isolates were sensitive to caspofungin and amphotericin B. Although all isolates of M. pachydermatis were sensitive to itraconazole, fluconazole, ketoconazole and amphotericin B, they were found to be resistant to caspofungin. The study has highlighted that Candida spp., M. pachydermatis, S. cerevisiae and Rhodotorula spp. are part of the normal canine surface microbiota and some of these organisms exhibit in vitro resistance to commonly used antimicrobials.     

Antimicrobial resistance pattern of Salmonella serotypes isolated from food items and personnel in Addis Ababa, Ethiopia

Antimicrobial susceptibility test of 98 isolates of Salmonella was assayed from September 2003 to February 2004 using the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).The result revealed that 32.7% of Salmonella isolates were resistant to one or more of the 24 antimicrobials tested. Generally resistance for 13 different antimicrobial drugs was recognized. The most common resistance was to streptomycin (24/32, 75%), ampicillin (19/32, 59.4%), tetracycline (15/32, 46.9%), spectinomycin (13/32, 40.6%) and sulfisoxazole (13/32, 40.6%). All the three Salmonella Kentucky isolates showed resistance to at least 8 antimicrobials. Out of the 12 Salmonella Braenderup isolates, 10 (83.3%) showed multidrug resistance to ampicillin, spectinomycin, streptomycin, sulfisoxazole, sulfamethoxazole/trimethoprim, amoxicillin/clavulanic acid and trimethoprim. Among the 8 S. Hadar isolates 7 (86.5%) showed antimicrobial resistance. All the 6 S. Dublin isolates were resistant to carbadox (100%). All the 6 S. Haifa isolates were resistant for at least ampicillin, streptomycin and tetracycline. Up to ten different antimicrobial resistances pattern was observed. Multiple antimicrobial drug resistance was observed in 23 Salmonella isolates (23.5%). The level of antimicrobial resistance was significantly higher for isolates from chicken carcass (18/29, 62.1%) and pork isolates (5/22, 22.7%) (p = 0.003). The findings of the present study ascertain that significant proportion Salmonella isolates have developed resistance for routinely prescribed antimicrobial drugs and poses considerable health hazards to the consumers unless prudent control measures are instituted.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul, Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Bartonellae in animals and vectors in New Caledonia

Bartonellae are gram-negative facultative intracellular alpha-proteobacteria from the family Bartonellaceae. The natural history of bartonellae consists of a reservoir/host, which is a vertebrate with chronic intravascular infection with sustained bacteremia, and a vector (usually an arthropod) that transfers the bacteria from the reservoir to a susceptible yet uninfected host. In order to reveal the sources and reservoirs of Bartonella infection in animals and vectors in New Caledonia, we collected the blood samples of 64 dogs, 8 cats, 30 bovines, 25 horses and 29 wild deer Cervus timorensis russa and 308 associated blood-sucking parasites (14 keds Hippobosca equina, 258 ticks (22 Rhipicephalus microplus, 235 Rhipicephalus sanguineus, and 1 Haemaphysalis longicornis), 12 fleas Ctenocephalides felis and 24 dog lice Trichodectes canis). We isolated ten strains of Bartonella: four Bartonella henselae from cats and six Bartonella chomelii from cattle. The strains were characterized by sequencing of five genes (16S, ITS, rpoB, gltA and ftsZ). The six strains isolated from cattle were close to the reference strain of B. chomelii and were, probably, imported from France with cattle of Limousin race. PCR showed that 35% of keds collected from deer and 31% of deer were infected by B. aff. schoenbuchensis; all other samples were negative. Our data confirmed that in New Caledonia, as in other regions of the world, cats are the major reservoirs of B. henselae. We also confirmed that Hippoboscidae flies may serve as the vectors of ruminant-associated bartonellae.     

Phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. in Malaysia

Mycobacteriosis due to mycobacteria is one of the most common bacterial diseases in ornamental fish. We describe here the phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. using ATCC Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae as references. A total of four isolates (M1, M2, M3, M4) were obtained from four out of 106 fish samples using selective agar, and identified to Mycobacterium genus using acid-fast staining and 16s rRNA gene-based genus specific polymerase chain reaction. DNA sequencing and NCBI-BLAST analysis further identified isolate M1 as M. marinum and isolates M2, M3, M4 as M. fortuitum. Morphological, physiological and biochemical tests were carried out for phenotypic characterizations. Universal M13 and wild-type phage M13 RAPD dendogram was generated to illustrate the genetic relationship of the isolates and reference strains.     

Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection

Salmonella enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis or Gallinarum can colonise liver and spleen in particular hosts while infections with serovars Infantis, Agona, Hadar, etc. are usually limited to gastrointestinal tract. Reasons for this behavior are unknown, although it has been shown that sodCI and spv genes exhibit a strict distribution between more and less virulent serovars and they influence Salmonella virulence. However to what extent the presence or absence of these genes is associated with the increased virulence of serovars which possess them has never been addressed experimentally. In this study we therefore first confirmed the exclusive association of spvB and sodCI genes with the former group of serovars. In the next step we removed these two genes from S. Enteritidis genome and compared the virulence of such a mutant with the virulence of S. Infantis, S. Agona and S. Hadar for chickens and highly sensitive Balb/C mice. Single strain infection showed that the deletion of these two genes from S. Enteritidis resulted in the reduction of its virulence for mice but not for chickens. Mixed infection further confirmed these observations and indicated that in mice but not in chickens the virulence of sodCI and spv mutant was reduced to the natural virulence of serovars Infantis, Agona and Hadar. Although sodCI and spv genes do not influence S. Enteritidis virulence for chickens directly, they may be of an indirect effect through the increased persistence of S. Enteritidis in mice and increased probability of the reintroduction of S. Enteritidis into poultry flocks.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.