Probiotic Properties of Lactic Acid Bacteria Isolated from Water-Buffalo Mozzarella Cheese

This study evaluated the probiotic properties (stability at different pH values and bile salt concentration, auto-aggregation and co-aggregation, survival in the presence of antibiotics and commercial drugs, study of β-galactosidase production, evaluation of the presence of genes encoding MapA and Mub adhesion proteins and EF-Tu elongation factor, and the presence of genes encoding virulence factor) of four LAB strains (Lactobacillus casei SJRP35, Leuconostoc citreum SJRP44, Lactobacillus delbrueckii subsp. bulgaricus SJRP57 and Leuconostoc mesenteroides subsp. mesenteroides SJRP58) which produced antimicrobial substances (antimicrobial peptides). The strains survived the simulated GIT modeled in MRS broth, whole and skim milk. In addition, auto-aggregation and the cell surface hydrophobicity of all strains were high, and various degrees of co-aggregation were observed with indicator strains. All strains presented low resistance to several antibiotics and survived in the presence of commercial drugs. Only the strain SJRP44 did not produce the β-galactosidase enzyme. Moreover, the strain SJRP57 did not show the presence of any genes encoding virulence factors; however, the strain SJRP35 presented vancomycin resistance and adhesion of collagen genes, the strain SJRP44 harbored the ornithine decarboxylase gene and the strain SJRP58 generated positive results for aggregation substance and histidine decarboxylase genes. In conclusion, the strain SJRP57 was considered the best candidate as probiotic cultures for further in vivo studies and functional food products development.     

In vitro assessment of safety and probiotic potential characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from water buffalo mozzarella cheese

The aim of this study was to evaluate the safety and probiotic potential characteristics of ten Lactobacillus spp. strains (Lactobacillus fermentum SJRP30, Lactobacillus casei SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus SJRP50, SJRP76, and SJRP149) that had previously been isolated from water buffalo mozzarella cheese. The safety of the strains was analyzed based on mucin degradation, hemolytic activity, resistance to antibiotics and the presence of genes encoding virulence factors. The in vitro tests concerning probiotic potential included survival under simulated gastrointestinal (GI) tract conditions, intestinal epithelial cell adhesion, the presence of genes encoding adhesion, aggregation and colonization factors, antimicrobial activity, and the production of the β-galactosidase enzyme. Although all strains presented resistance to several antibiotics, the resistance was limited to antibiotics to which the strains had intrinsic resistance. Furthermore, the strains presented a limited spread of genes encoding virulence factors and resistance to antibiotics, and none of the strains presented hemolytic or mucin degradation activity. The L. delbrueckii subsp. bulgaricus strains showed the lowest survival rate after exposure to simulated GI tract conditions, whereas all of the L. casei and L. fermentum strains showed good survivability. None of the tested lactobacilli strains presented bile salt hydrolase (BSH) activity, and only L. casei SJRP145 did not produce the β-galactosidase enzyme. The strains showed varied levels of adhesion to Caco-2 cells. None of the cell-free supernatants inhibited the growth of pathogenic target microorganisms. Overall, L. fermentum SJRP30 and L. casei SJRP145 and SJRP146 were revealed to be safe and to possess similar or superior probiotic characteristics compared to the reference strain L. rhamnosus GG (ATCC 53103).     

Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

Safety evaluation of two bacterial strains used in Asian probiotic products

Probiotics, known for their prophylactic and therapeutic properties, are routinely used by the medical community in various regions of the world. In some Asian countries, these products are controlled as pharmaceutical substances and must adhere to strict regulatory guidelines. However, outside of Europe where the European Food Safety Authority has recently adopted a Qualified Presumption of Safety approach for probiotics used in food and feed, current safety requirements do not necessitate screening for the presence of virulence and other risk factors, which may result in the inadvertent use of probiotic strains harboring harmful genes. A safety evaluation was conducted on Enterococcus faecium R0026 and Bacillus subtilis R0179 used in several commercial probiotic products marketed in Asia. Molecular techniques were used to verify the identity of each strain and antibiotic resistance profiles were determined towards clinically relevant antibiotics. Strains were subsequently screened for the presence of enterotoxins and virulence factors and were subjected to 28 days of repeated high-dose oral toxicity testing in rats. No risk factors or aberrant activities were identified using such a detailed approach. Thus, both microbes were deemed to pose low risk to the consumer and, therefore, safe for use as probiotics.     

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans , Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.     

Genetic diversity and safety aspects of enterococci from slightly fermented sausages

AIMS: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively. CONCLUSIONS: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.     

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.     

Molecular and probiotic characterization of bacteriocin-producing Enterococcus faecium strains isolated from nonfermented animal foods

Aims:  The characterization of four novel bacteriocin-producing enterococcal strains, isolated from nonfermented animal foods, was carried out with a view to evaluate their potential application as probiotics in raw and processed foodstuffs.
Methods and Results:  16S rRNA sequencing and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis allowed the identification and intra-specific grouping of Enterococcus faecium strains, which inhibited the growth of four relevant food-borne pathogenic and spoilage species. Enterococcus faecium strains exhibited remarkable probiotic profiles, being able to survive to pH 3·0 and to the presence of bile salts, pancreatin and pepsin. Enterococcus faecium strains evaluated did not exhibit bile salt hydrolase or haemolytic activity, but showed good adhesion properties, also exhibiting sensitivity to clinically relevant antimicrobial agents.
Conclusions:  In our study, DNA sequencing of the 16S rRNA gene and RAPD-PCR analysis were equally discriminatory for typing E. faecium strains. This study also confirmed the potential tolerance and survival of E. faecium strains isolated from nonfermented animal foods to the gastrointestinal tract.
Significance and Impact of the Study:  This study represents the first report on potential probiotic E. faecium strains isolated from nonfermented meat and fish. Their moderate heat resistance opens the way to their potential use as probiotics in minimally processed foods subjected to moderate heat processing.     

Leuconostoc mesenteroides SJRP55: a potential probiotic strain isolated from Brazilian water buffalo mozzarella cheese

The probiotic potential of Leuconostoc mesenteroides subsp. mesenteroides SJRP55, isolated from water buffalo mozzarella cheese was evaluated. The microorganism presented resistance to stressful conditions that simulated the gastrointestinal tract, and to the best of our knowledge, Leuconostoc mesenteroides subsp. mesenteroides SJRP55 was the first of this species with the ability to deconjugate bile salts. Tolerance to NaCl was temperature dependent, as well the results obtained by aggregation capacity. The strain presented good adhesion properties, β–galactosidase activity, viability in fermented milk during storage, inactive against Streptococcus thermophilus and sensitive to most of the tested antibiotics. Some analgesic medications inhibited the growth of the strain. Leuconostoc mesenteroides subsp. mesenteroides SJRP55 exhibited in vitro probiotic potential, and it can be better characterized through future in vivo tests. This bacterium presents higher functional properties compared to other studied strains, and therefore, it is a potential candidate for the application as a probiotic strain, which could be used by industries in the manufacture of functional milk-based products.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Incidence of virulence factors and antibiotic resistance among Enterococci isolated from food

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.     

Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium

Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated (cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Identification and characterization of enterococci from bryndza cheese

AIMS: To identify enterococci isolated from sheep milk cheese--bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. METHODS AND RESULTS: Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. CONCLUSIONS: Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.     

Screening for potential probiotic from spontaneously fermented non-dairy foods based on in vitro probiotic and safety properties

The aim of this study was to screen potential probiotic lactic acid bacteria from Chinese spontaneously fermented non-dairy foods by evaluating their probiotic and safety properties. All lactic acid bacteria (LAB) strains were identified by 16S rRNA gene sequencing. The in vitro probiotic tests included survival under low pH and bile salts, cell surface hydrophobicity, auto-aggregation, co-aggregation, antibacterial activity, and adherence ability to cells. The safety properties were evaluated based on hemolytic activity and antibiotic resistance profile. The salt tolerance, growth in litmus milk, and acidification ability were examined on selected potential probiotic LAB strains to investigate their potential use in food fermentation. A total of 122 strains were isolated and identified at the species level by 16S rRNA gene sequencing and included 62 Lactobacillus plantarum, 40 Weissella cibaria, 12 Lactobacillus brevis, 6 Weissella confusa, and 2 Lactobacillus sakei strains. One W. cibaria and nine L. plantarum isolates were selected based on their tolerance to low pH and bile salts. The hydrophobicity, auto-aggregation, co-aggregation, and antagonistic activities of these isolates varied greatly. All of the 10 selected strains showed multiple antibiotic resistance phenotypes and no hemolytic activity. The highest adhesion capacity to SW480 cells was observed with L. plantarum SK1. The isolates L. plantarum SK1, CB9, and CB10 were the most similar strains to Lactobacillus rhamnosus GG and selected for their high salt tolerance and acidifying activity. The results revealed strain-specific probiotic properties were and potential probiotics that can be used in the food industry.     

Antimicrobial resistance of Enterococcus species isolated from produce

The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.     

Probiotic Potential of Bacillus Strains Isolated from an Acidic Fermented Food Idli

Present study is intended to assess the probiotic properties of Bacillus spp. isolated from idli batter, a traditional fermented food of Southern India and Sri Lanka. A total of 32 isolates were screened for potential pathogenic behaviour through haemolysis assay, DNase activity and antibiotics sensitivity. Two of the isolates were found to be potentially safe and identified as Bacillus spp. These strains were characterized for in vitro probiotic attributes and antioxidant activity. Both the strains showed strong acid and bile tolerance, transit tolerance, lysozyme tolerance, cell surface hydrophobicity, auto-aggregation, co-aggregation, biofilm formation potential and adhesion to human colon adenocarcinoma (HT 29) cell line demonstrating potential probiotic ability. These strains also exhibited considerable cholesterol binding, thermostability, β-galactosidase production, proteolytic, amylolytic and lipolytic activity. Cell-free supernatant inhibited the biofilm formation by Pseudomonas aeruginosa (KT266804) to 90%. Intact cells showed significant DPPH (41%), hydroxyl (31%), radical scavenging activity and lipid peroxidation inhibition (20.38%), while cell-free extracts exhibited significant superoxide anion radical scavenging activity (16.25%). Results revealed that isolates could be potential probiotic candidate after further assessment of in vivo probiotic properties and safety evaluation and could be utilised as starter cultures in functional foods.

     

Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance

Antibiotic-resistant Gram-positive bacteria are responsible for morbidity and mortality in healthcare environments. Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and Streptococcus pneumoniae can all exhibit clinically relevant multidrug resistance phenotypes due to acquired resistance genes on mobile genetic elements. It is possible that clinically relevant multidrug-resistant Clostridium difficile strains will appear in the future, as the organism is adept at acquiring mobile genetic elements (plasmids and transposons). Conjugative transposons of the Tn916/Tn1545 family, which carry major antibiotic resistance determinants, are transmissible between these different bacteria by a conjugative mechanism during which the elements are excised by a staggered cut from donor cells, converted to a circular form, transferred by cell-cell contact and inserted into recipient cells by a site-specific recombinase. The ability of these conjugative transposons to acquire additional, clinically relevant antibiotic resistance genes importantly contributes to the emergence of multidrug resistance.     

Genome-wide identification of ampicillin resistance determinants in Enterococcus faecium

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism.     

Presence of vancomycin and ampicillin-resistant Enterococcus faecium of epidemic clonal complex-17 in wastewaters from the south coast of England

The occurrence and diversity of vancomycin-resistant enterococci (VRE) in wastewaters from the Brighton and Hove area of south-east England were investigated. VRE were recovered from 71% of raw urban wastewater samples, 22% of treated urban wastewater samples, 15% of hospital wastewater sample and 33% of farm wastewater samples. Two hundred and eighty-eight isolates were typed and identified and the minimum inhibitory concentrations (MICs) to six antibiotics were determined for selected VRE. Vancomycin-resistant Enterococcus faecium (VREF) strains with a vancomycin MIC of more than 32 μg ml⁻¹ were examined by polymerase chain reaction for the vanA , vanB and esp genes. Twenty-three VREF with the vanA or vanB gene were further analysed by multilocus sequence typing which revealed that a cluster of VREF from both hospital and urban wastewaters belonged to the high-risk, epidemic, clonal complex-17 (CC17). Vancomycin-resistant Enterococcus faecium belonging to the CC17 group contained the purK-1 allele, were resistant to ampicillin and frequently ciprofloxacin, and usually contained the esp gene. To the authors' knowledge, this is the first report of CC17 strains isolated from urban wastewaters in the UK, and indicates that certain clones carrying antibiotic resistance or virulence traits indicative of the hospital environment can be detected in the urban wastewater system.