Kanamycin Aesculin Azide Research Articles

Occurrence, Phenotypic and Molecular Characteristics of Vancomycin-Resistant Enterococci Isolated from Retail Raw Milk in Egypt.

The aim of this study was to determine the occurrence, phenotypic and molecular characteristics of vancomycin-resistant enterococci (VRE), isolated from retail raw cow's milk. One hundred milk samples collected from retail shops in Egypt were examined for the occurrence of VRE by using kanamycin aesculin azide agar supplemented with 4 μg/mL vancomycin. PCR was conducted to determine enterococcal species and to screen the isolated strains for the presence of antibiotic resistance and virulence genes. All isolated strains were characterized by antimicrobial susceptibility testing for 12 antibiotics. From 24 samples (24%), we recovered 22 isolates (91.6%) classified as VRE (minimum inhibitory concentration ≥32) and 2 isolates (8.3%) classified as intermediate resistant to vancomycin (≤16). Enterococcus faecium (29.1%), Enterococcus faecalis (12.5%), Enterococcus casseliflavus (16.6%), and Enterococcus gallinarum (4.1%) were identified by using multiplex PCR. The genus Enterococcus was resistant to clindamycin (100%), linezolid (91.6%), teicoplanin (91.6%), erythromycin (87.5%), and tetracycline (29.1%). Co-resistance to vancomycin, teicoplanin, and linezolid was detected in 83.3% of isolates. Antibiotic resistance genes vanB, tet(M), tet(L), and erm(B) were identified in 29.1%, 16.6%, 8.3%, and 4.1% of isolates, respectively. Virulence genes gelE and esp were detected in 16.6% and 12.5% of isolates, respectively. In conclusion, the high occurrence of co-resistance to vancomycin, teicoplanin, and linezolid reported in this study is alarming. The high frequency of linezolid resistance prompts increased the attention of researchers to routinely perform linezolid susceptibility in food isolates. This study declares potential food safety risks from consumption and improper handling of raw milk regarding clinically important bacteria and promotes necessary legislation for forbidding the selling and consumption of retail raw milk.

Development of a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci

Enterococci show higher proteolytic activities than other lactic acid bacteria and thus have received considerable attention in scientific literature in recent years. Proteolytic enzymes of enterococci have warranted the use of some species as starter, adjuncts or protective cultures and as probiotics, while in some strains they have also been linked with virulence. Consequently, the isolation and identification of proteolytic enterococci is becoming of increasing interest and importance. However, current screening methods for proteolytic enterococci can be time consuming, requiring a two-step procedure which may take up to 96h. This study describes a method, utilising Kanamycin Skim Milk Aesculin Azide (KSMEA) agar, for the isolation of proteolytic enterococci in one-step, thereby significantly reducing screening time. KSMEA combines the selective properties of Kanamycin Aesculin Azide Agar (KAA) with skim milk powder for the detection of proteolytic enterococci. Enterococci produced colonies with a black halo on KSMEA which were accompanied by a zone of clearing in the media when enterococci were proteolytic. KSMEA medium retained the selectivity of KAA, while proteolytic enterococci were easily distinguished from non-proteolytic enterococci when two known strains were propagated on KSMEA. KSMEA also proved effective at isolating and detecting enterococci in raw milk, faeces and soil. Isolates recovered from the screen were confirmed as enterococci using genus-specific primers. Proteolytic enterococci were present in the raw milk sample only and were easily distinguishable from non-proteolytic enterococci and other microorganisms. Therefore, KSMEA provides a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci.

Changes in antimicrobial resistance among faecal enterococci isolated from growing broilers prophylactically medicated with three commercial antimicrobials

A controlled environment poultry house (1600 m 2) was divided into two separated compartments. Three hundred twenty chick containers (each containing 100 chicks) were randomly allocated to each compartment. Antimicrobial products were provided (via drinking water) to one broiler group in three different growth phases for prophylactic purposes: lincomycin associated with spectinomycin from day 1 to day 3, trimethoprim associated with sulfamethoxazole from day 19 to day 21, and tylosin from day 26 to day 28. Feed and faecal samples from both groups were obtained on days 0, 4, 9, 14, 18, 22, 25, 29 and 33. One sample of the drinking water and samples from the broiler house environment were obtained one day before bird placement. A maximum of 16 enterococci isolated from Kanamycin Aesculin Azide Agar were tested for resistance using the disk diffusion method. A high prevalence of resistance to drugs such as tetracycline, erythromycin and nitrofurantoin was detected in enterococcal isolates from medicated and unmedicated flocks. Resistance occurrence in the non-medicated group revealed a shift in resistance profiles with the gradual change of the initial strains, isolated from the day-old chicks’ cloacal swabs, by others displaying a different phenotype. Resistance phenotypes obtained from faecal strains were correlated with those isolated from the farm environment and feeds.

Detection and quantification of Enterococcus gilvus in cheese by real-time PCR

The objective of this work was to investigate the occurrence of Enterococcus gilvus in cheese. For this purpose, a real-time PCR protocol using phenylalanyl-tRNA synthase ( pheS) as a target gene was optimized to evaluate the presence and abundance of this microorganism in Italian artisan cheeses. The real-time assay unequivocally distinguished E. gilvus from 25 non-target LAB and non-LAB species, demonstrating its absolute specificity. The assay performed well not only with purified DNA but also with DNA extracted from cheese samples artificially contaminated with E. gilvus. The dynamic range of target determination of the method in the cheese matrix (from 10 7 to 10 4 cfu/ml, covering three orders of magnitude) was lower and the detection limit higher than in vitro conditions, but still high enough to obtain an excellent quantification accuracy in cheese. Twenty commercially available cheeses were analyzed by real-time PCR and approximately 40% of the cheese samples contained E. gilvus at levels ranging from 4.17±0.10 to 6.75±0.01 log cfu/g. Such levels represented 0.1–10% of the total enterococci counted on kanamycin aesculin azide agar (KAA) from the corresponding cheeses. The successful isolation of E. gilvus from cheeses containing high loads of this species, as detected by real-time PCR, provided definitive proof on both assay specificity and presence of this organism in cheeses. Despite the relatively low sensitivity in cheese (≥4 log cfu/g), the real-time PCR described here may, however, be useful to detect E. gilvus rapidly when present at (sub)dominant levels within the enterococcal cheese microflora. The assay may be helpful to detect and quantify E. gilvus strains from food, thus enabling a better understanding of technological role, ecological and safety aspects in cheeses and other fermented food products of this infrequent species.

PRESENCE OF ENTEROCOCCI IN RAW COW'S MILK AND “PUZZONE DI MOENA” CHEESE

ABSTRACT Raw cow's milk and cheese samples collected from factories producing “Puzzone di Moena” located in the alpine province of Trento (northern Italy), were analyzed for their enterococcal content. Presumptive enterococci (Gram positive, catalase negative, coccal shaped and positive for growth on kanamycin aesculin azide) were isolated, verified for their growth in presence of 6.5% NaCl, 40% bile salts and esculin hydrolysis, and analyzed by a genetic polyphasic approach consisting of intergenic spacer (ITS)-polymerase chain reaction (PCR) between the 16S and 23S rRNA genes, denaturing gradient gel electrophoresis (DGGE) of the V3 region of the 16S rRNA gene and a sodA gene-based multiplex PCR. Results showed the presence of Enterococcus faecalis as the main species found in milk and cheese. Enterococcus casseliflavus and Enterococcus durans were also detected in raw milk. In order to exclude clonal relatedness, the isolates were subjected to randomly amplified polymorphic DNA-PCR (RAPD-PCR). Winter cheese was characterized by a higher biodiversity than summer production, both in terms of Enterococcus species and number of strains. No strain was found positive for vancomycin resistance as determined by vanA- and vanB-specific PCR. Two strains were shown to be characterized by an anti-Listeria behavior due to bacteriocin-like inhibitory substances (BLIS) production. BLIS producer strains persisted during the ripening of cheese. PRACTICAL APPLICATIONS Most of the traditional cheeses are made from raw milk, whose lactic acid bacterial population plays a major role during ripening. Enterococci from milk may survive the adverse hostile environment in cheese during ripening and be potentially transferred to consumers. Enterococcus strains isolated from milk involved in cheese production, and from ripened “Puzzone di Moena” cheeses, were shown not to host van genes, thus, they should not be involved in the process of antimicrobial resistance dissemination among intestinal microflora. Furthermore, it was found that bacteriocinogenic enterococci dominated over nonbacteriocin-like inhibitory substances producer Enterococcus strains during the cheese process, confirming previous studies showing that bacteriocin production may influence bacterial complexes. From an application standpoint, these results are basic in the selection of nonstarter lactic acid bacteria to be used as adjunct cultures in traditional cheese productions.

Quantification of Enterococcus italicus in traditional Italian cheeses by fluorescence whole-cell hybridization

The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91±0.17 to 7.34±0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening.

Leakage of food‐borne Enterococcus faecalis through temporary fillings in a simulated oral environment

To evaluate the hypothesis that food-borne viable Enterococcus faecalis cells could enter the root canal space via coronal leakage. In a simulated oral environment under mastication the capacity of a calcium sulphate-based temporary filling material (Cavit W) to prevent leakage of E. faecalis from a cheese through the endodontic access cavity into the pulp chamber was assessed. Standardized class I access cavities were prepared in human maxillary molars. These were filled with Cavit of either 2 or 4 mm thickness (n=16, each). Empty access cavities served as positive, teeth filled with a light-curing composite material acted as negative controls (n=8, each). A cheese containing viable E. faecalis cells was placed on the occlusal aspects of test and control teeth, which were subsequently subjected to 680 mastication loads per day for 1 week in a masticator device perfused with artificial saliva at 37 degrees C. Leakage of E. faecalis from the cheese into the pulp chamber was assessed by culture on a kanamycin aesculin azide agar and compared between groups using Fisher's exact test. All of the positive controls showed pure growth of E. faecalis. In addition, one of the negative control teeth leaked. The 4 mm application of Cavit prevented leakage of E. faecalis significantly better than the corresponding 2 mm application: 1 of 16 specimens compared with 6 of 16 specimens had leakage, respectively (P<0.05). The current results substantiate the suspicion that food-derived microbiota could enter the necrotic root canal system via microleakage.

Analysis of enterococci using portable testing equipment for developing countries – variance of Azide NutriDisk medium under variable time and temperature

This report compares the enterococci count on samples obtained with Azide NutriDisk (AND) (sterile, dehydrated culture medium) and Slanetz and Bartley (SB) medium when exposed to a variable in incubation time and temperature. Three experiments were performed to examine the recovery of enterococci on AND and SB media using membrane filtration with respect to: (a) incubation time; (b) incubation temperature; and (c) a combination of the two. Presumptive counts were observed at 37, 41, 46 and 47 degrees C and at 20, 24, 28 and 48 h. These were compared to AWWA standard method 9230 C (44 degrees C, 44 h). Samples were confirmed using Kanamycin Aesculin Azide (KAA) agar. Friedman's ANOVA and Students t-test analysis indicated higher enumeration of enterococci when grown on AND (p = 0.45) than SB (p = < 0.001) at all temperatures with a survival threshold at 47 degrees C. Significant results for AND medium were noted at 20 h (p = 0.021), 24 h (p = 0.278) and 28 h (p = 0.543). The study concluded that the accuracy of the AND medium at a greater time and temperature range provided flexibility in incubator technology making it an appropriate alternative to SB medium for monitoring drinking water using field testing kits in developing countries.

Microbiological and physico-chemical changes in Genestoso cheese, a Spanish acid curd variety, throughout ripening

Abstract The aim of this work was to study the microbiological and physico-chemical changes in four batches of Genestoso cheese (two ripened in autumn–winter and the other two in spring–summer) throughout manufacture and ripening. In general, the pH decreased more quickly in the cheeses manufactured in spring which, moreover showed values lower in aw and higher in salt/moisture content. The highest counts of aerobic mesophilic bacteria, presumptive lactococci and presumptive leuconostoc were reached in 2 day old cheeses. Lactobacilli and fungic flora became predominant with ripening. The counts of enterococci differed between the batches, but in general the maximum counts in kanamycin aesculin azide were obtained after ripening for 2 or 7 days. The counts on mannitol salt agar were very low and disappeared in the first days of ripening. Enterobacteriaceae were not detected after 15 days of ripening in the cheeses manufactured in spring nor in the 7 day old cheeses of an autumn batch.

Study of enterococci and micrococci isolated throughout manufacture and ripening of San Simón cheese

The aim of this study was the enumeration and identification of enterococci and Micrococcaceae throughout the manufacture and ripening of five batches of San Simón cheese. The highest counts on mannitol salt agar (MSA) were obtained after 1 week of ripening in the samples taken from the core of the cheese and after 2 weeks in those from the surface. The percentage of strains that belonged to the Micrococcaceae was only 18·89% of the isolates from the core portion of the cheese and 35% with respect to those isolated on the surface. The predominant species both in the core and on the surface of the cheese was Staphylococcus sciuri. Of the strains isolated in MSA from the milk, curd and core of the cheese, 38·89% were from the genus Enterococcus and 26·25% of those isolated in the superficial portion were Enterococcus faecalis in both cases. During ripening the percentage ofMicrococcaceae decreased, while the percentage of enterococci increased, a phenomenon which can be related to the ability of enterococci to adapt to the conditions present in cheese during ripening. The maximum counts were obtained on kanamycin aesculin azide (KAA) agar after 1 week of ripening, both in the core and on the surface of the cheese, and were fairly stable during the whole ripening. Enterococci isolated in KAA agar constituted 79·63% of the isolates from the milk, curd and core of the cheese (and 90% with respect to the isolates taken from the surface), with predominance in both portions of the species E. faecalis.

Comparison of agar-based media for primary isolation of glycopeptide-resistant enterococci

OBJECTIVE: To compare four vancomycin-containing agar media for the isolation of glycopeptide-resistant enterococci (GRE) from clinical fecal specimens: kanamycin---aesculin---azide (KAA) agar; bile---aesculin---polymixin (BAP) agar; aztreonam---amphotericin blood (CBAA) agar; and neomycin blood (CBN) agar. METHODS: Fecal specimens from 125 patients were inoculated onto each medium. Media were examined for enterococci after incubation for up to 48 h. Enterococci were identified to species level, and glycopeptide phenotypes were determined by measuring minimum inhibitory concentrations of vancomycin and teicoplanin. RESULTS: GRE were isolated from 44/125 samples. Enterococcus faecalis and Enterococcus faecium isolates, expressing glycopeptide resistance of the VanA or VanB phenotypes, were recovered from 27/33 (82%) specimens on BAP medium, 26/33 (79%) on KAA medium, and 21/33 (64%) on CBN and CBAA media. Enterococcus gallinarum and Enterococcus casseliflavus isolates expressing low-level glycopeptide resistance (VanC phenotype) were recovered from 14/15 (93%) specimens on CBAA medium, 7/15 (47%) on KAA and CBN media, and 6/15 (40%) on BAP medium. CONCLUSIONS: The media tested in this study, with the exception of CBN medium, detected at least 75% of patients colonized by GRE. Further development of BAP, CBAA and KAA media is warranted to improve growth and selectivity.

Evaluation of media for the enumeration of faecal streptococci from natural water samples

The Most Probable Number technique using Azide dextrose broth and Ethyl violet azide dextrose broth, and the membrane filtration technique with the following agars: mEnterococcus, KF, Pfizer selective enterococcus, Kanamycin aesculin azide, Bile-Aesculin, Thallous acetate (Barne's), and Mitis-Salivarius were compared on the basis of their accuracy, specificity, selectivity, precision, and relative recovery efficiency characteristics. The results obtained indicate that none of the above mentioned procedures shows an optimal selectivity. The Most Probable Number technique was a good procedure on the basis of its high recovery efficiency and good selectivity. However, mEnterococcus agar using the membrane filtration technique showed the best performance characteristics of the enumeration media tested, because this method possessed the best recovery efficiency, precision and accuracy, and also a good specificity.

Culture media for enterococci and group D-streptococci

Lancefield group D-streptococci are contaminants of various food commodities, especially those of animal origin. They encompass the new genus Enterococcus comprising 13 known species and some species of streptococci which have their habitat in the intestine of animals, e.g. Streptococcus bovis, suis and equinus. The serologically based grouping may no longer constitute the best definition for streptococci from the food chain. Food hygiene monitoring systems using enterococci as indicators need reliable methods for selective cultivation and identification of marker strains. Up to now more than 100 modifications of selective media have been described for isolating streptococci or enterococci from various specimens. The selection of a medium requires either experience or consultation. It depends on the kind of specimen, the method of cultivation (plate count or membrane filter) and whether or not the habitat is heavily contaminated with other organisms. The choice of media is made more difficult as commercial versions of the same culture medium may vary in recipe and/or performance from producer to producer. Therefore, reviewing the literature may help in the choice of medium and confirmation tests. The selectivity and productivity of some commonly used or cited media are reported here, partly based on our own experience: citrate azide tween carbonate agar (CATC), kanamycin aesculin azide agar (KAA) and M-enterococcus agar (ME) including earlier results with aesculin bile azide agar (ABA), and thallous acetate tetrazolium glucose agar (TITG). No medium was completely selective for all group D-streptococci or for all enterococci but some media were highly selective for a single Enterococcus species, e.g., for E. faecalis which serves as indicator of human pollution. Confirmatory tests must be carried out when experience in the evaluation procedure is limited. Selective media for enterococci should be used only after or while checking in parallel their selectivity and productivity against appropriate test organisms.