Technological characterisation, antibiotic susceptibility and antimicrobial activity of wild-type Leuconostoc strains isolated from north Italian traditional cheeses

2013 ◽  
Vol 80 (4) ◽  
pp. 457-466 ◽  
Author(s):  
Stefano Morandi ◽  
Paola Cremonesi ◽  
Tiziana Silvetti ◽  
Milena Brasca
Keyword(s):  
Antimicrobial Activity ◽  
Antibiotic Susceptibility ◽  
Tetracycline Resistance ◽  
Raw Milk ◽  
Starter Cultures ◽  
Genetic Determinants ◽  
Tetracycline Resistance Genes ◽  
Reduction Activity ◽  
Genes Encoding ◽  
High Degree

Genotypic and technological properties, antibiotic susceptibility and antimicrobial activity of 35 Leuconostoc strains, isolated from different Italian raw milk cheeses, were investigated. RAPD-PCR was used to study genetic variability and to distinguish closely related strains. The results showed a high degree of heterogeneity among isolates. All the strains had weak acidifying activity and showed low proteolytic and lipolytic activities. Reduction activity, was generally low. All the Leuconostoc were susceptible to ampicillin, mupirocin, erythromycin, quinupristin/dalfopristin and tetracycline. Many strains were classified as resistant to oxacillin, ciprofloxacin and nitrofurantonin, while all isolates were found resistant to vancomycin. PCR-based detection did not identify any of the common genetic determinants for vancomycin (vanA, vanB, vanC1, vanC2, vanC3, vanD, vanE, vanG) or erythromycin (ermB and ermC). Tetracycline resistance genes were detected in 25 tetracycline susceptible strains, the most frequent one being tetM. One strain, belonging to Ln. pseudomesenteroides species, was positive for the presence of the int gene of the Tn916/Tn1545 trasposon family. This is the first time the conjugative transposon Tn916 has been detected inside the Leuconostoc species. All strains showed antimicrobial activity against Enterococcus faecalis and Ent. faecium. The presence of genes encoding amino-acid decarboxylases (hdc and tdc) was not detected. Some strains are interesting in view of their use in cheese production as starter and non starter cultures.

scholarly journals Detection of a Novel, and Likely Ancestral, Tn916-Like Element from a Human Saliva Metagenomic Library

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 548
Author(s):  
Liam J. Reynolds ◽  
Muna F. Anjum ◽  
Adam P. Roberts
Keyword(s):  
Resistance Genes ◽  
Metagenomic Library ◽  
Tetracycline Resistance ◽  
Human Saliva ◽  
Fusion Event ◽  
Tetracycline Resistance Genes ◽  
Antimicrobial Resistance Genes ◽  
Genes Encoding ◽  
Gene Fusion Event ◽  
Metagenomic Approach

Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916.

scholarly journals Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of Gram-Negative Bacteria

2002 ◽  
Vol 68 (4) ◽  
pp. 1786-1793 ◽  
Author(s):  
R. I. Aminov ◽  
J. C. Chee-Sanford ◽  
N. Garrigues ◽  
B. Teferedegne ◽  
I. J. Krapac ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Production Systems ◽  
Animal Feed ◽  
Antibiotic Resistance Gene ◽  
Tetracycline Resistance ◽  
Pcr Primers ◽  
Tetracycline Resistance Genes ◽  
Common Structure ◽  
Waste Lagoons ◽  
Genes Encoding

ABSTRACT Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H+ antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this tet subcluster unifies 11 genes encoding tet efflux pumps and includes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control tet templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded tet fingerprint of animal feed was found to be tetCDEHZ, while the corresponding fingerprint of total intestinal microbiota was tetBCGHYZ. Interestingly, the tet fingerprints in geographically distant waste lagoons were identical (tetBCEHYZ) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of tet genes in comparison with the fecal diversity could be detected, suggesting that the tet pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The tet efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of tet genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.

Prevalence, virulence and antibiotic susceptibility of Listeria monocytogenes isolated from sheep

2020 ◽  
Vol 21 (2) ◽  
pp. 48-52
Author(s):  
Shaimaa Elbar ◽  
Rasha Elkenany ◽  
Mohamed Elhadidy ◽  
Gamal Younis
Keyword(s):  
Spinal Cord ◽  
Listeria Monocytogenes ◽  
Antibiotic Susceptibility ◽  
Tetracycline Resistance ◽  
Raw Milk ◽  
Intestinal Content ◽  
Isolation And Identification ◽  
Antibiotics Susceptibility ◽  
Resistance Rates ◽  
Hlya Gene

Objective: This study was undertaken to determine the prevalence, virulence, and antibiotics susceptibility of Listeria monocytogenes isolated from hindbrain, spinal cord, milk, and intestinal content collected from sheep in the Dakahlia Governorate, Egypt. Design: Observational study. Samples: We analyzed 472 samples, including 72 hindbrain/spinal cord samples from emergency-slaughtered sheep, 300 raw-milk samples from apparently healthy sheep, and 100 intestinal content samples from slaughtered sheep at three abattoirs. Procedures: Isolation and identification of L. monocytogenes were performed using conventional techniques. The biochemically identified isolates were confirmed by 16SrRNA gene sequencing and examined for virulence-associated genes (hlyA and iap) as well as for antimicrobial susceptibility. Results: In total, 16 (3.39%) out of 472 sheep samples [5.56% (4/72) in hindbrain/spinal cord, 4% (12/300) in milk, and 0% (0/100) in intestinal content samples] were found to be positive for L. monocytogenes. All the confirmed isolates were positive for the hlyA gene (100%); meanwhile, none of them exhibited the iap gene. Antibiotic susceptibility testing showed high resistance rates to amoxicillin, cefotaxime, erythromycin (50% each), and vancomycin (37.5%). Sulfamethoxazole–trimethoprim and tetracycline resistance rates were 25% and 12.5%, respectively. On the contrary, all isolates were susceptible to amikacin, ciprofloxacin, and norfloxacin. Interestingly, 37.5% (6/16) of L. monocytogenes isolates exhibited multidrug resistance (MDR). The multiple antibiotic resistances (MAR) index of isolates ranged from 0.1 to 0.6. Conclusion and clinical relevance: Our data highlights the importance of awareness of virulent strains of MDR L. monocytogenes of sheep samples and potentially samples from other domestic animals in Egypt.

Tetracycline Resistance Patterns of Lactobacillus buchneri Group Strains

2016 ◽  
Vol 79 (10) ◽  
pp. 1741-1747 ◽  
Author(s):  
MARLIES FEICHTINGER ◽  
SIGRID MAYRHOFER ◽  
WOLFGANG KNEIFEL ◽  
KONRAD J. DOMIG
Keyword(s):  
Food Safety ◽  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Fermented Milk ◽  
Feed Additives ◽  
Starter Cultures ◽  
European Food Safety Authority ◽  
Tetracycline Resistance Genes ◽  
Lactobacillus Buchneri ◽  
Broth Microdilution Method

ABSTRACT Lactobacilli are applied as starter cultures for controlled fermentation in the production of food and feed. Among other lactobacilli, members of the Lactobacillus buchneri group are used in fermented milk, wine, and silage. Most of the L. buchneri species used for the manufacturing of food or feed are already on the list for qualified presumption of safety status and are recommended as biological agents by the European Food Safety Authority. Consequently, new strains intended as food or feed additives do not require any additional safety check than confirming the absence of transferable antibiotic resistance determinants. Of these determinants, tetracycline resistance genes are especially predominant in lactobacilli. Within this study, a total of 128 strains belonging to the L. buchneri group (L. buchneri, L. diolivorans, L. farraginis, L. hilgardii, L. kefiri, L. kisonensis, L. otakiensis, L. parabuchneri, L. parafarraginis, L. parakefiri, L. rapi, L. senioris, and L. sunkii) were examined for their susceptibility to tetracycline. Tetracycline MICs were assessed by the broth microdilution method according to ISO 10932/IDF 223. Subsequently, the presence of tetracycline resistance genes was investigated by using PCR. In addition, selected strains were tested for a broader range of tetracycline resistance genes by using a microarray technique. Applying the tetracycline cutoff values defined by European Food Safety Authority for heterofermentative and obligately homofermentative lactobacilli, 96.9% of the strains would have been categorized as tetracycline resistant. However, none of the tested tetracycline resistance genes could be detected by PCR or microarray analysis. Furthermore, the MIC distribution of all strains was unimodal and at the high end of the tested tetracycline concentration range (4 to 256 μg/ml). Thus, these data suggest that tetracycline resistance in the L. buchneri group strains is intrinsic, which complies with the requirements defined in the qualified presumption of safety outline.

scholarly journals A Multibacteriocin Cheese Starter System, Comprising Nisin and Lacticin 3147 in Lactococcus lactis, in Combination with Plantaricin from Lactobacillus plantarum

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
S. Mills ◽  
C. Griffin ◽  
P. M. O'Connor ◽  
L. M. Serrano ◽  
W. C. Meijer ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Lactobacillus Plantarum ◽  
Class Ii ◽  
Recipient Strain ◽  
Starter Cultures ◽  
Class I ◽  
Genetic Determinants ◽  
Content Type ◽  
Bacteriocin Producer ◽  
Lacticin 3147

ABSTRACT Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-bacteriocin-producing starters of Lactococcus lactis capable of producing the class I bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of class I bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the class II bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the class II producer proved to be most effective for reducing bacterial load. As emergence of bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications. IMPORTANCE We generated a suite of single- and double-bacteriocin-producing starter cultures capable of generating the class I bacteriocin lacticin 3147 or nisin or both bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a class II bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three bacteriocin-producing strains, as the double producer is not inhibited by either of the class I bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.

scholarly journals Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities

2001 ◽  
Vol 67 (4) ◽  
pp. 1494-1502 ◽  
Author(s):  
J. C. Chee-Sanford ◽  
R. I. Aminov ◽  
I. J. Krapac ◽  
N. Garrigues-Jeanjean ◽  
R. I. Mackie
Keyword(s):  
Antibiotic Resistance ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Antibiotic Resistance Gene ◽  
Tetracycline Resistance ◽  
Direct Result ◽  
M Gene ◽  
Tetracycline Resistance Genes ◽  
Genes Encoding ◽  
Gradient Gel Electrophoresis ◽  
Typing Methods

ABSTRACT In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W),tet(M), tetB(P), tet(S),tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated thattet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.

scholarly journals Prevalence of Tetracycline Resistance Genes in Oral Bacteria

2003 ◽  
Vol 47 (3) ◽  
pp. 878-882 ◽  
Author(s):  
A. Villedieu ◽  
M. L. Diaz-Torres ◽  
N. Hunt ◽  
R. McNab ◽  
D. A. Spratt ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Oral Bacteria ◽  
Healthy Adults ◽  
Resistant Bacteria ◽  
First Report ◽  
Tetracycline Resistance Genes ◽  
Oral Microflora ◽  
Genes Encoding ◽  
Ribosomal Protection Protein

ABSTRACT Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common tet gene identified was tet(M), which was found in 79% of all the isolates. The second most common gene identified was tet(W), which was found in 21% of all the isolates, followed by tet(O) and tet(Q) (10.5 and 9.5% of the isolates, respectively) and then tet(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had tet(L) and 1% carried tet(A) and tet(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of tet(W) in oral bacteria and the first report to show that tet(O), tet(Q), tet(A), and tet(S) can be found in some oral species.

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