3.1. Occurrence of Selected Microorganisms in Smoothies
Fruits and vegetables used for smoothie preparation have been repeatedly described as potential sources of many different microorganisms and, together with secondary microbial contamination, may pose a risk for consumers [
42,
43,
44,
45]. In the first part of this work, the monitoring of the prevalence of total fungi, yeasts, and aerobic bacteria (considering coliforms and enterococci) was performed.
Table 3 shows the quantitative representation of the mentioned microorganisms in the studied smoothie samples. Total aerobic bacteria, yeasts, and fungi were monitored for a general view of microorganism load in the smoothie samples. The number of total aerobic bacteria ranged from 2.9 to 7.3 log CFU/mL. The results correlate with the bacterial population level in fruits and vegetables, which ranged from 3 to 7 log CFU/g, according to the type of ingredient [
46,
47,
48]. The occurrence of yeasts and fungi was 1.8 log CFU/mL to higher than 3.0 log CFU/mL. One sample of green smoothie prepared from spinach, pear, and banana (GF1) did not contain any yeasts and fungi (
Table 3). The prevalence of yeasts and fungi in the smoothie samples was similar to other studies, where the numbers oscillated around 2.8 [
49] and 2.17 log CFU/mL [
50]. The highest occurrence of yeasts and fungi was detected in all samples obtained from Fresh Bar D (GD1—spinach, orange, carrot, banana; FD1—kiwi, orange, banana; FD2—strawberries, banana, apple) and Fresh Bar E (FE1—apple, cucumber, spinach; FE2—pear, beetroot, carrot, celery; FE3—grapefruit, orange, ginger). This could be due to the predominance of fruit smoothies. Fruits are more likely to be contaminated by molds and yeasts due to their composition, which is rich in sugar and other nutrients [
50,
51]. The origin of contamination could be via air, as both of the fresh bars were located inside shopping centers as open-space shops. It needs to be noted that air can play an important role in the transfer of spoilage microorganisms [
52,
53]. The second collected fruit smoothie from Fresh Bar F has also a higher occurrence of yeasts and fungi (FF1—strawberries, banana, orange) (
Table 3).
Coliform bacteria and enterococci could be considered indicator bacteria, pointing out the hygienic process of the smoothie preparation. The consumption of raw and thermally untreated foods contaminated with such bacteria may cause the spread of disease in humans [
54]. Coliform bacteria were detected in all tested smoothie drinks. Korir et al. (2016) registered total coliform bacteria in 38.7% of different fresh vegetables (414 samples) [
47]. The number of coliforms in the green smoothie samples ranged from 2.1 to 3.7 log CFU/mL. This difference could be caused by the limited number of samples as well as better manufacturing praxis and hygiene.
In one smoothie sample, the occurrence of
E. coli was observed. It was the green smoothie marked as GA1, which was composed of spinach, banana, melon, and lime. Previous research revealed that more diseases are connected to the consumption of contaminated leafy vegetables; therefore, green smoothies could carry a higher risk of containing potential bacterial pathogens such as
E. coli, as it was in this case [
55]. The sample marked as GA1 contained 150 CFU/mL of
E. coli. Consequently, the number of microorganisms was still at a satisfactory level following Commission Regulation (EC) No. 2073/2005, which was mentioned above (1000 CFU/mL) [
35].
Total enterococci were observed in all fresh bars examined. They were not detected in two samples of fruit smoothies. The first was collected from Fresh Bar C and was composed of banana, strawberries, and apple (FC2). The second was collected from Fresh Bar D, and the sample obtained kiwi, orange, and banana (FD1). The remaining eighteen smoothie samples (90%) were positive for the presence of enterococci. The results are slightly related to the study performed by Sun et al. (2021), where total enterococci were detected in 87.1% of vegetables, 100% of our green smoothies, and 59.8% of fruits (84.6% of our fruit smoothies) [
31]. Enterococci, a fecal-contaminated bacteria, can be mainly transferred to fruits and vegetables through irrigated water, organic fertilization of animal origin [
25,
56], or, additionally, in the case of smoothies, through low hygiene standards of personnel working with food or contact with surfaces [
57]. The difference between the green and fruit smoothies in the occurrence of total enterococci was insignificant as there was a disproportion of each type of smoothie, and the offer and demand of the fruit smoothies were greater than the green smoothies. The number of
Enterococcus spp. ranged from 1.1 to 2.9 log CFU/mL.
Many studies showed that the microbial contamination of ready-to-eat foods can cause health problems among consumers [
58,
59,
60,
61]. An example of this untreated food is the smoothie. It could be prepared at the time of selling with asymptomatic food handlers as potential carriers of microbes or with handlers with poor personal hygienic standards [
45,
62].
3.2. Occurrence of Antibiotic-Resistant Coliform Bacteria and Enterococci
A growing food safety concern is related to the role of food in human exposure to antimicrobial-resistant bacteria, including zoonotic, commensal, and environmental bacteria as resistance genes reservoirs [
63,
64,
65]. Commensal
Enterobacteriaceae in fresh fruit and vegetables may represent a source of transmissible antimicrobial resistance genes [
27,
28,
66]. According to this, one of the aims of this work was to study the occurrence of antibiotic-resistant coliform bacteria and enterococci in the studied smoothie samples (
Table 4 and
Table 5). Antibiotics were chosen as representatives of different antibiotic classes. The concentrations were applied according to the resistance thresholds given by EUCAST and CLSI [
36,
37].
Table 4 presents antibiotic-resistant coliforms, which were not detected in all fruit smoothies collected from Fresh Bar A (FA1—melon, apple, pear, mint; FA2—pineapple, orange, coconut milk) and in one green smoothie obtained from Fresh Bar B (GB1—avocado, mango, spinach, apple) (
Table 4).
Ampicillin-resistant coliform bacteria have been registered in most smoothies. This could be due to the intrinsic resistance of most coliform bacteria to this antibiotic [
67]. In green smoothie samples from FSE A (GA1—spinach, banana, melon, lime) and fruit smoothie from FSE E (FE2—pear, beetroot, carrot, celery), the occurrence of both tetracycline- and chloramphenicol-resistant coliforms were observed. Tetracycline resistance was registered only in three smoothies (15%) as it was also detected in green smoothies from FSE B (GB2—apple, orange, carrot, celery, ginger). Resistance to chloramphenicol was also detected in fruit smoothies from FSE B (FB2—orange, apple, blueberries, strawberries) and D (FD1—kiwi, orange, banana) and in all samples taken from FSE F (GF1—spinach, pear, banana; FF1—strawberries, banana, orange). Altogether, chloramphenicol-resistant coliform bacteria were detected in 30% of all smoothie samples. Although the use of chloramphenicol in clinical praxis in Slovakia has been delimited [
67], the appearance of resistance to this antibiotic could be the consequence of cross-resistance. Gentamicin resistance was detected in 45% of collected smoothie samples, specifically in one sample from Fresh Bar C (FC3—blueberries, raspberries, strawberries, pineapple, apple) and in all collected samples from FSE D–F. The worst situation in the case of the occurrence of antibiotic-resistant coliform bacteria was observed in smoothies from Fresh Bar F (GF1—spinach, pear, banana; FF1—strawberries, banana, orange), where was not observed only resistance to tetracycline. In both green and fruit smoothies, high numbers of ampicillin, ciprofloxacin-, gentamicin-, and chloramphenicol-resistant coliforms were detected.
Resistant enterococci occurred in nine types (45%) of smoothies from all twenty smoothie samples from the FSEs (
Table 5).
Antibiotic-resistant enterococci were not detected in 55% of all smoothie samples. Fresh Bar C (GC1—spinach, mango, banana, orange; FC1—strawberries, mint, lime, apple; FC2—banana, strawberries, apple, FC3—blueberries, raspberries, strawberries, pineapple, apple) did not contain smoothies with the presence of antibiotic-resistant enterococci. Both fruit smoothies collected from Fresh Bar B (FB1—strawberries, acai, chia seeds, orange, apple; FB2—orange, apple, blueberries, strawberries) also did not contain antibiotic-resistant enterococci. Similarly, to coliform bacteria, a high abundance of ampicillin-resistant enterococci was determined. Ciprofloxacin-resistant enterococci were observed only in one fruit smoothie sample from Fresh Bar F (FF1—strawberries, banana, orange).
In four samples (20%) collected from three of the six tested fresh bars (GA1—spinach, banana, melon, lime; FA1—melon, apple, pear, mint; GB2—apple, orange, carrot, celery, ginger; FE3—grapefruit, orange, ginger), vancomycin-resistant enterococci (VRE) were registered. These bacteria were first identified in the mid-1980s and spread rapidly. They are currently a major problem in many healthcare institutions worldwide [
68]. In 2017, the WHO published a list of bacteria for which new antibiotics are urgently needed. VRE belong to the second group of resistant bacteria with a high priority [
69]. Torre et al. (2010) found VRE in one-third of analyzed fresh fruit and vegetable, especially in curly endive and lettuce [
70]. According to the irregular presence of VRE in these fresh bars, it could be assumed that their occurrence is affected by one specific smoothie ingredient. For example, in the case of the GB2 smoothie sample, it could be due to using celery, as was presented in another study [
70]. On the other hand, there is also a possibility of transfer due to inappropriate hygiene and handling during the drink preparation [
71].
Antibiotic resistance among coliform bacteria was more prevalent compared to antibiotic resistance among enterococci. It could be due to the higher initial microbial load of coliforms in the studied smoothie samples.
3.3. Identification of Antibiotic-Resistant Coliform Bacteria
Individual resistant strains were chosen from all used agar plates supplemented randomly with antibiotics. From all smoothie samples, 102 isolates were successfully isolated and identified (
Figure 1). Isolates were transferred from diagnostic media with an antibiotic to Mueller–Hinton media and consequently identified by a MALDI–TOF biotyper.
Most strains belonged to the
Enterobacter genus (52 strains of all isolated bacteria), predominantly isolated from plates with the addition of gentamicin (50%) and chloramphenicol (25%). In the case of
Enterobacter spp., most strains were found in fruit smoothies (61%), and the rest of this gender was detected in green smoothies (39%). The
Enterobacter spp. genus is predominantly habituated in soil and water and is often isolated from fruit and vegetable samples [
72]. However, this microorganism may also be found in human feces or the respiratory tract of men.
Enterobacter spp. has been noticed as a cause of urinary tract infection in hospital environments. This identification showed strains of
E. cloacae,
E. asburiae, and
E. ludwigii.
Enterobacter spp. are also associated with bacteremia but more in comparison to
Klebsiella spp. [
73].
Klebsiella species (30 stains of all isolated bacteria) were found in green as well as in fruit smoothies. The majority of
Klebsiella spp. was identified as
K. pneumoniae (60%) and predominantly was isolated from gentamicin plates (50%) and ampicillin plates (44%). This bacterium could represent a higher risk for staff preparing smoothies. It can be inhaled in the form of a bioaerosol produced during preparation and cause bronchopneumonia or bronchitis [
74].
K. oxytoca formed the rest of the isolated bacteria and was isolated from gentamicin (58%) and ampicillin (42%). Other coliforms were identified, such as
E. coli,
Serratia liquefaciens and
Serratia odorifera,
Morganella morganii,
Citrobacter gillenii,
Cronobacter sakazakii,
Raoultella ornithinolytica,
Kluyvera intermedia, and
Lelliottia amnigena. All isolates identified as
E. coli were ampicillin resistant. Two isolates were identified as
Rahnella aquatilis, which is rarely reported as a human pathogen.
Rahnella spp. is mainly the cause of infections between immunocompromised patients [
75]. The mentioned bacterium has been found in freshwater, soil, and certain animals, such as snails and certain beetles [
76], so the possible source in smoothies can be leafy vegetables or fruits that do not require peeling (in our case strawberries).
3.4. Efficiency of Washing the Raw Materials Used in Smoothies
Smoothie drinks and other ready-to-eat products are particularly important because of the microbial load of ingredients used during preparation as they are consumed without any heat treatment and therefore the present microorganisms are not devitalized. Proper treatment of the raw material is an important step in smoothie preparation. Ingredients used to prepare most fruit smoothies are mainly cleaned by removing the surface skin/peel, which could also eliminate a significant proportion of microorganisms. On the other hand, the decontamination of vegetables is much more complicated because vegetables are contaminated mainly by soil deposits, containing a wide range of bacteria. The external tissue inhabits a high number of different bacteria in fresh produce plants. In addition to this fact, bacteria have been detected within plant tissue [
77,
78]. It is very complicated to eliminate such endophytic bacteria by washing as it stays inside the plant [
79]. According to this, another aim of this work was to show how the proper washing of smoothie components can contribute to a decrease in the final microbial count in smoothie drinks (
Table 6).
As a control, a smoothie from unwashed components was prepared. The second sample consisted of smoothie components washed under a tap with running drinking water. The ingredients in the third smoothie sample (WS) were treated with an aqueous solution of commercial Sanosil disinfectant. From the one-way ANOVA analysis, we could conclude that treatments had a different effect on the monitored microorganisms. The highest number of all detected microorganisms was detected in the control sample. Washing the raw materials under running drinking water led to a reduction of the microbial count by less than one logarithmic order. The results relate to Falomir et al. (2010), who found out that washing ingredients with drinking water had an insignificant effect on the population of coliform bacteria [
80]. They surmise different mechanisms of adherence for these bacteria on plant surfaces, which could be responsible for this insignificant effect of cleaning ingredients under running drinking water [
80]. Washing raw materials also did not lead to the detection of the number of coliform bacteria resistant to ciprofloxacin. On the other hand, treatment with Sanosil solution had the best effect in decreasing the bacterial count. The WS sample contained the lowest number of total aerobic and coliform bacteria compared to the control sample and water-treated sample. The effect on coliform bacteria could be due to silver ions obtained in biocides, as Gram-negative species appear to be more sensitive compared to Gram-positive bacteria. According to research conducted by Silvestry-Rodriguez et al. (2007), silver ions have the potential to bind to negatively charged peptidoglycans located at the cell wall [
81].
The biocide agent Sanosil had a weak effect on the number of yeasts and fungi compared to the control sample. In the case of ampicillin-resistant bacteria, they were constantly decreased by the addition of washing. The WS sample decreased most of the monitored microorganisms, except ciprofloxacin-resistant coliform bacteria. Our results correlated with other studies with the use of detergent to eliminate bacteria [
82,
83]. The Sanosil biocide agent was the most effective, but as other studies have shown, efficiency is most likely related to microbial gender or the number of bacterial quantities, concentration of hydrogen peroxide in the solution, time of exposure, and many other factors [
39,
82,
83,
84].
Although this experiment was mainly demonstrative and focused on washing and sanitation using biocide Sanosil, it is important that other factors also affect the microbial safety of smoothie drinks. To establish a definitive correlation between cleaning methods and microbial quality, more variants with different microorganisms, different vegetables, fruits, and disinfection agents would need to be analyzed.