Abstract
Introduction
Enterobacteriaceae are often reported as a typical bacterial population in raw milk from any mammalian origin. The frequent concern with bacteria, especially those related to this group of microorganisms, is their increasing resistance to antibiotics and the emergence of enzymes that degrade them. This study aimed to characterize isolates of Enterobacteriaceae from raw goat milk to expose associated safety problems and possible technological challenges.
Methods
Isolates from 21 raw goat milk samples purchased in the State of Rio de Janeiro, Brazil, were identified by mass spectrometry, after isolation on Violet Red Bile Glucose agar. The isolates were subjected to evaluation of proteolytic, lipolytic, hemolytic, and biofilm producing activities. Furthermore, resistance profiles and production capacity of enzymes that degrade antimicrobials were evaluated.
Results
Almost half of the 59 isolates (48%) belonged to the Enterobacter genus, with a significant prevalence of the Serratia (20%) and Klebsiella (11%) genera. The majority showed biofilm-producing activity (90%), while the activity of degradative enzymes was observed in approximately 20%. Few isolates were found with a profile of resistance to antimicrobials, with only one isolate of Klebsiella variicola being classified as multidrug-resistant. However, chromogenic culture media showed high production of extended-spectrum beta-lactamases and carbapenemases (54% and 46%, respectively), as a presumptive identification.
Conclusions
A considerable degree of virulence was observed in the Enterobacteriaceae isolates, as well as the potential for undesirable technological damage. The characterization and identification of the isolates contributes to the improvement of the risk monitoring process of goat’s milk.
Keywords: Enterobacteriaceae, proteolysis, beta-lactamases, carbapenem-resistant Enterobacteriaceae, lipolysis
Introduction
The Enterobacteriaceae family comprises several potentially pathogenic species, such as Escherichia coli and Salmonella spp., as well as opportunistic pathogens, such as Klebsiella pneumoniae. These bacterial species are increasingly problematic due to the possible acquisition of virulence and antimicrobial resistance genes. In the dairy industry, species of the Enterobacteriaceae family can enter the milk production chain and reach the human organism through the consumption of contaminated milk and dairy products. From a technological point of view, they can cause enzymatic degradation of proteins and lipids.1,2 Although there are several groups of microorganisms of importance in raw milk, such as Pseudomonas spp. and Acinetobacter spp., members of the Enterobacteriaceae family show considerable prevalence in dairy products, calling attention to the possible presence of pathogenic species in the product.1,3,4
Inappropriate selection and abuse of antibiotics offered to dairy cattle has led to the emergence of antimicrobial resistance, which is also associated with the virulence capacity.5 In addition, there is an increasing number of strains capable of producing enzymes that degrade antibiotics, a fact that characterizes a mechanism of resistance to antibiotics, such as extended-spectrum beta-lactamases (ESBL) and carbapenemases. Animal products, especially dairy and meat, are the main sources of ESBL-producing Enterobacteriaceae.6
Considering the substantial research carried out with bovine milk, goat milk is a modestly explored potential reservoir of these microorganisms having great implications in the food industry. This study aimed to characterize the virulence factors of Enterobacteriaceae isolated from raw goat milk, evaluate activities that produce thermoresistant degradative enzymes, and determine the antibiotic resistance profiles of the isolated bacteria.
Methods
Milk samples and bacterial isolation
Twenty-one samples of raw goat milk were used, acquired from eight producers in the state of Rio de Janeiro and transported under refrigeration to the Microbiology Laboratory of Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, in a short period of time. Sample collection was carried out in two periods (March and August 2018). Before starting the procedures, the packaging was disinfected. These samples are listed in a previous study carried out by our group, which also describes the isolation of the bacteria used in this work.4
Identification of the Enterobacteriaceae isolates
The isolates were identified using a MALDI-TOF mass spectrometer (Microflex LT, Bruker Daltonics, USA), according to Bizzini et al. (2010).7 As described by Ramos & Nascimento (2019),4 222 isolates were identified from raw goat milk samples, being mainly composed of Pseudomonas spp., members of the Enterobacteriaceae family and Acinetobacter spp. The isolates belonging to the Enterobacteriaceae family have not been characterized until the present work.
Qualitative assessment of biofilm production
The isolates were used to inoculate Congo red agar using a spread plate method, with incubation at 37°C for 24 h.8 The Congo red agar was formulated from 15 g/L of nutrient agar (Merck, Brazil), 37 g/L of sucrose (Pro Analysi, Brazil), and 0.8 g/L of Congo red (VETEC, Brazil). Biofilm-producing isolates presented with black-colored colonies, while non-producing isolates were colorless or reddish.
Screening for proteolytic, lipolytic, and hemolytic activity
For the evaluation of proteolytic activity, skim milk agar was used, prepared with 1% skim milk powder (Molico®, Nestlé Brasil Ltda, Brazil) and 2% agar (Merck). The inoculated plates were incubated at 28°C for up to 7 days, and proteolysis was observed by the presence of degradation halos.9
To evaluate lipolytic activity, the isolates were used to inoculate modified Spirit Blue agar10 formulated with 1% peptone (Himedia, USA), 0.05% yeast extract (BD, USA), 2% agar, and 970 mL of distilled water. After autoclaving and cooling to 55°C, the lipase reagent, containing 12 mL of olive oil (Borges, Brazil), 0.12 mL of Tween 80 (Proquimios, Brazil), and 17.9 mL of distilled water, were added. Plates were incubated at 28°C for up to 7 days and the presence of a halo around the bacterial growth was observed.
The hemolytic activity of the isolates was evaluated by the inoculation of blood agar containing 5% defibrinated sheep blood (Laborclin, Brazil), and subsequent incubation at 37°C for 24 h. Hemolytic zones, represented by halos, were observed.
Antibiotic resistance profiles
To determine the resistance profile, the disk diffusion method was used, following the methodology described by the Clinical and Laboratory Standards Institute.11 Twenty-two antibiotics belonging to 12 classes were used, as described in Table 1. Microorganisms were classified as multidrug-resistant when they were resistant to at least one antibiotic from three or more different categories.12
Table 1. Antibiotics used to evaluate resistance of microorganisms of Enterobacteriaceae family.
| Categories | Antibiotic | Code | |
|---|---|---|---|
| Carbapenems | Ertapenem 10 µg | ERT | |
| Meropenem 10 µg | MER | ||
| Imipenem 10 µg | IPM | ||
| Penicillins | Ampicillin 10 µg | AMP | |
| Non-extended spectrum cephalosporins | 1stgeneration | Cefazolin 30 µg | CFZ |
| 2nd generation | Cefuroxime 30 µg | CRX | |
| Cefoxitin 30 µg | CFO | ||
| Extended-spectrum cephalosporins | 3rd generation | Cefotaxime 30 µg | CTX |
| Ceftazidime 30 µg | CAZ | ||
| 4th generation | Cefepime 30 µg | CPM | |
| Monobactam | Aztreonam 30 µg | ATM | |
| Tetracyclines | Tetracycline 30 µg | TET | |
| Quinolones | Ciprofloxacin 5 µg | CIP | |
| Levofloxacin 5 µg | LVX | ||
| Nalidixic Acid 30 µg | NAL | ||
| Aminoglycosides | Gentamicin 10 µg | GEN | |
| Tobramycin 10 µg | TOB | ||
| Phenicols | Chloramphenicol 30 µg | CLO | |
| Beta-lactams + beta-lactamase inhibitors | Amoxicillin + clavulanic acid 20/10 µg | AMC | |
| Piperacillin + tazobactam 100/10 µg | PPT | ||
| Ampicillin + sulbactam 10/10 µg | ASB | ||
| Ticarcillin + clavulanic acid 75/10 µg | TAC | ||
Presumptive identification of ESBL-producing and KPC-producing phenotypes
To evaluate KPC- and ESBL-producing phenotypes, rapid tests were performed using the CHROMagar®KPC and CHROMagar®ESBL chromogenic culture media. After inoculation with the isolates, the media was incubated at 37°C for 18-24 h. The results were interpreted according to the manufacturer’s instructions. In these chromogenic media, E. coli appears as pink or reddish colonies, while Enterobacter spp., Klebsiella spp., and Raoultella spp. appear as metallic blue colonies with or without a reddish halo. For the other genera of the Enterobacteriaceae family, there was no growth pattern available, and for this reason, they were not subjected to these tests.
Results
Fifty-nine isolates of the Enterobacteriaceae family obtained from raw goat milk were identified as belonging to the genera Escherichia (5%), Enterobacter (48%), Raoultella (5%), Hafnia (5%), Pantoea (8%), Klebsiella (11%), Serratia (14%), Leclercia (2%) and Moellerella (2%). Two isolates did not have sufficient precision in mass spectrometry to determine their species, thus determining only the Enterobacter genus. The distribution by species is described in Table 2.
Table 2. Identification and characterization of the Enterobacteriaceae isolates.
| Identification | Isolate code | Biofilm Production | Proteolytic activity | Lipolytic activity | Hemolytic activity |
|---|---|---|---|---|---|
| Enterobacter spp. (n=2) | 0409 | + | - | - | - |
| 1206 | + | - | - | - | |
| Enterobacter asburiae (n=13) | 0401 | + | - | - | - |
| 0402 | + | - | - | - | |
| 0403 | + | - | - | - | |
| 0406 | + | - | - | - | |
| 0413 | + | - | - | - | |
| 1207 | + | - | - | - | |
| 1209 | + | - | - | - | |
| 1211 | + | - | - | - | |
| 1213 | + | - | - | - | |
| 1215 | + | - | - | - | |
| 1406 | + | - | - | + | |
| 1429 | + | - | - | + | |
| 1430 | - | - | - | - | |
| Enterobacter cloacae (n=9) | 0408 | + | - | - | - |
| 0412 | + | - | - | - | |
| 0414 | + | - | - | - | |
| 0415 | + | - | - | - | |
| 0416 | + | - | - | - | |
| 0501 | + | - | - | - | |
| 1208 | + | - | - | - | |
| 1409 | + | - | - | + | |
| 1418 | + | - | - | + | |
| Enterobacter hormaechei (n=3) | 0410 | + | - | - | - |
| 0411 | + | - | - | - | |
| 2101 | + | + | + | + | |
| Enterobacter kobei (n=3) | 1408 | + | - | - | + |
| 1411 | + | - | - | - | |
| 1424 | - | - | - | + | |
| Escherichia coli (n=3) | 0404 | + | - | - | - |
| 0405 | + | - | - | - | |
| 1205 | - | - | - | - | |
| Hafnia alvei (n=2) | 0514 | - | - | - | + |
| 0515 | - | - | - | + | |
| Klebsiella oxytoca (n=1) | 1501 | + | - | - | + |
| Klebsiella pneumoniae (n=1) | 2015 | + | + | + | + |
| Klebsiella variicola (n=4) | 1402 | - | - | - | - |
| 1403 | - | - | - | + | |
| 1404 | + | + | + | + | |
| 1909 | + | + | + | + | |
| Leclercia adecarboxylata (n=1) | 0701 | - | - | - | - |
| Moellerella wisconsensis (n=1) | 0604 | + | - | - | - |
| Pantoea agglomerans (n=5) | 0503 | + | - | - | - |
| 0507 | + | - | - | - | |
| 0510 | + | - | - | + | |
| 0512 | + | - | - | + | |
| 0519 | + | - | - | + | |
| Raoultella ornithinolytica (n=3) | 0502 | + | - | - | - |
| 0517 | + | - | - | - | |
| 1412 | + | + | - | - | |
| Serratia liquefaciens (n=8) | 0504 | + | + | + | - |
| 0505 | + | + | + | - | |
| 0506 | + | + | - | - | |
| 0508 | + | + | + | - | |
| 0509 | + | + | + | - | |
| 0511 | + | + | + | - | |
| 0513 | + | - | + | - | |
| 0520 | + | - | + | - | |
| Total (n=59) | 53 (90%) | 14 (24%) | 10 (17%) | 16 (27%) | |
The first two digits of the isolate codes represent the goat’s milk sample from which the isolate was obtained.
Fifty-three isolates with biofilm-producing activity were detected, corresponding to 90% of the isolates. The use of Congo red agar is based on increasing the production of exopolysaccharides, allowing the quantitative assessment of biofilm formation in Gram-negative microorganisms.13,14
Proteolytic activity was observed in 10 isolates (17% of the total), with emphasis on the Serratia genus, where only one isolate did not present this characteristic. All isolates that exhibited proteolytic activity were observed in the first 48 h of the 7-day incubation. Lipolytic activity showed a similar prevalence, with a predominance of the Serratia genus. Also, 27% of the isolates (16) showed hemolytic activity, with the genera Pantoea, Hafnia, and Klebsiella being the most representative.
Antimicrobial resistance of the Enterobacteriaceae isolates was evaluated using the disk diffusion test. It was observed that seven isolates (11.9%) showed resistance to one or two categories of antimicrobials, as shown in Table 3. Only one isolate (1.7%) of Klebsiella variicola showed resistance to five antimicrobials (AMC, CFZ, PPT, CRX, and NAL) from four different categories, and was classified as multidrug-resistant (MDR).12 One Enterobacter hormaechei isolate showed resistance to imipenem. The antibiotics with the highest rate of resistance were tetracycline, ceftazidime, and cefoxitin.
Table 3. Antimicrobial-resistant Enterobacteriaceae isolates from raw goat milk.
| Identification | Isolate code | Antimicrobial resistance determined by disk diffusion | MDR |
|---|---|---|---|
| Enterobacter hormaechei | 2101 | IPM | No |
| Escherichia coli | 1205 | CFO | No |
| Klebsiella oxytoca | 1501 | CFZ, TET | No |
| Klebsiella variicola | 1909 | CFZ, TET | No |
| 1403 | CFO, TET, CFZ, CRX | No | |
| 1404 | AMC, CFZ, PPT, CRX, NAL | Yes | |
| Pantoea agglomerans | 0507 | CFO, AMC, CFZ | No |
| Raoultella ornithinolytica | 1412 | AMP, TET | No |
MDR – multidrug resistant; IPM – imipenem; CFO – cefoxitin; CFZ – cefazolin; TET – tetracycline; CRX – cefuroxime; AMC – amoxicillin + clavulanic acid; PPT – piperacillin + tazobactam; NAL – nalidixic acid; AMP – ampicillin.
Regarding the presumptive identification of ESBL-producing resistance phenotypes, evaluated by Chromagar® ESBL chromogenic medium, 37 isolates (54%) presented with a positive result, which is, with the growth of colonies of a certain bacterial genus according to the one indicated by the manufacturer. The genera Serratia, Hafnia, Pantoea, Leclercia, and Moellerella were not evaluated for the production activity of ESBL and KPC, as there were no growth patterns available from the manufacturer of the medium for these groups. In identification of KPC-producing resistance phenotypes, 27 isolates (46%) showed a positive result.
Discussion
Enterobacteriaceae are usually identified in raw milk, whether bovine or caprine, as they are part of the product’s natural microbiota and may have increased concentrations as a result of how good milking practices occur, consequently also being used as a hygiene indicator.15 Thus, it is important to evaluate their presence in raw goat’s milk, identifying the species and associating virulence factors and eventual resistance to antimicrobials, considering that in many localities human consumption of raw milk still occurs.
The genera found were similar to those of other studies on goat milk bacterial populations. In Kenya, Serratia (20% of the total), Klebsiella (11%), Enterobacter (7%), and E. coli (6%) were identified.16 In China, 34% of the total isolates were identified as belonging to the Enterobacter genus.17 Bacteria frequently found in milk can remain present in its derivatives and lead to unwanted characteristics in these products, as in goat cheese produced with raw milk containing K. oxytoca, E. cloacae, H. alvei and P. agglomerans, all of which are associated with the production of gas and the consequent generation of “early blowing” in cheese.18
Gram-negative bacteria, mainly members of the Enterobacteriaceae family, are associated with the formation of biofilms in the dairy industry.19 In addition to being a potential source of contamination, biofilms can also increase the corrosion rate of the industrial material, reduce heat transfer, and increase the friction factor of the flowing fluid. In the case of the dairy industry, biofilms are often reported in the processing lines, storage tanks, heat exchangers, and separation systems.20 E. cloacae and K. oxytoca isolates of animal origin are capable of producing biofilms with large amounts of cells in short periods of time.21
In the present study, 90% of the total isolates showed biofilm-forming activity. In the context of the milk production chain, the presence of biofilms indicates a serious problem in processing, since once installed in milking utensils or equipment, biofilms are very difficult to remove. In the composition of the biofilm, in addition to pathogens such as E. coli, K. pneumoniae and members of the Enterobacter cloacae complex identified in this study, several deteriorators can co-exist, such as Pseudomonas spp.22
The production of proteases by microorganisms on skim milk agar is identified by the hydrolysis of milk casein contained in the culture medium, as evidenced by a halo around the colonies.9 Lipase production on spirit blue agar is observed by the degradation of oil lipids in the medium.10 The presence of thermostable enzymes in dairy products is a technological problem, as they are not destroyed by subsequent heat treatments. They remain in the product until the end of the production chain and cause deterioration, resulting in losses to the sector.23
The genus Serratia is frequently associated with the degradation of dairy products by thermoresistant enzymes, such as the production of proteases by Serratia spp., which may be close to 100% of the tested isolates. Although Pseudomonas spp. represents the genus of psychrotrophic microorganisms most related to proteolytic activity in raw milk, Serratia spp. and especially Serratia liquefaciens have been reported in raw milk as producers of thermostable proteolytic enzymes.24,25 Serratia spp. presents a worrying additional factor in relation to Pseudomonas spp., being able to produce biofilm in a more intense way.25 In the present study, this also occurs in goat’s milk, where all isolates of S. liquefaciens presented themselves as producers of biofilm, and the species was the one with the highest reported rate of proteolytic activity. Regarding other genera of the Enterobacteriaceae family, such as Enterobacter, Klebsiella, Raoultella, and Hafnia, protease-producing activity is not usually detected.23
Hemolytic activity is widely used as a tool in the investigation of pathogenic microorganisms that can use hemoglobin as a source of iron, which is essential for their survival and the occurrence of infections. Pathogens that present hemolytic activity secrete hemolysins in order to obtain hemoglobin or the prosthetic heme group from the breakdown of erythrocytes.26 In general, strains that show hemolytic activity are more virulent when compared to strains of the same species without this activity.27 Thus, the evaluation of hemolysin production among Gram-negative bacteria is an indicator of virulence factors, where Klebsiella spp. is the most relevant genre related to this aspect.28
In products of animal origin, the inappropriate use of antibiotics to treat the animal has a relevant role in terms of public health, causing the bacteria present in the derived food to have a high prevalence of resistance genes. Even though these genes are mostly associated with non-pathogenic species, horizontal gene transfer can occur for species potentially dangerous to human health, such as several members of the Enterobacteriaceae family, increasing the prevalence of antimicrobial resistance in food. The likelihood of horizontal gene transfer is even greater if resistance genes are located in mobile genetic elements of the cell, such as plasmids.29
In this study, only one out of three E. coli isolates (1205) showed resistance to a single antibiotic, whereas isolates of E. coli are frequently reported in the literature, as characterized by multidrug resistance in dairy products.30,31 Susceptibility to antimicrobials of isolates obtained from raw goat milk were evaluated by Mahlangu et al. (2018), where several genera of Enterobacteriaceae were found. The rates of resistance to antimicrobials were low, as in the present study, and the only isolates that showed MDR phenotype were Serratia spp. and E. coli.16 In isolates of Enterobacteriaceae, especially Serratia spp., a higher rate of resistance to ampicillin (76%) and tetracycline (24%) has been reported.32
The only isolate classified as MDR in this study (1404) was K. variicola, which has been recognized as an emerging pathogen in humans and has been shown to be a potential reservoir of resistance genes, in addition to being considered to cause mastitis in dairy animals.33,34 The relevance of this species has been underestimated by inaccurate detection methods, which fail to differentiate between members of the Klebsiella pneumoniae complex, which comprises seven species.34 In addition, K. variicola isolates were the ones that showed resistance to the greatest variety of antimicrobials, as shown in Table 3.
Comparatively, in a study carried out on bovine milk, ESBL-producing microorganisms were identified as E. coli (76%), Citrobacter spp. (10%), Enterobacter cloacae (6%), and Klebsiella oxytoca (4%).6 Most of the isolates in our work presented as ESBL producers, which was not evidenced in the disk diffusion tests, where a much smaller number of isolates showed resistance to any of the beta-lactam antibiotics tested, suggesting that the chromogenic culture media employed in this study generated a high rate of false positive results. However, this result may be a consequence of the fact that antibiotics whose corresponding isolate has intrinsic resistance were disregarded.11 Another fact that cannot be discarded is that in disk diffusion tests, sensitivity problems (false-negative results) may occur, mainly with metallo-β-lactamase-producing enterobacterial isolates exhibiting weak carbapenemase activity.35
Contradictory results were also observed with the detection of the production of carbapenemases using Chromagar® KPC. In the present study, a very low correspondence rate was observed between isolates that presented resistance to carbapenem in the disk diffusion test (only 1 isolate of E. hormaechei) and those that presented positive results in the chromogenic culture medium (27 isolates). Although this discrepancy can be justified by the existence of diverse carbapenemases and, consequently, different levels of expression especially by Enterobacteriaceae,36,37 other studies carried out with this chromogenic medium in Brazil also report the high rate of false-positive results for the detection of carbapenem resistant microorganisms from clinical samples.38
These results indicate that for an accurate characterization of the production capacity of carbapenemases and extended spectrum β-lactamases by members of the Enterobacteriaceae family isolated from raw goat milk, the detection of genes that encode these enzymes by conventional PCR become essential.
Conclusions
Enterobacteriaceae isolated from raw goat milk were shown to be potentially harmful to consumers’ health, exhibiting a high rate of biofilm-producing activity and significant rates of resistance to antimicrobials. The prevalence of genera such as Enterobacter and Klebsiella reveals a risk to the consumer’s health, since in many localities there is still consumption of raw milk. In addition, technological problems resulting from these microorganisms are evidenced by the production of thermoresistant enzymes, resulting in economic losses to the producer and low quality of the final product. From the identification and characterization data obtained on the species that are members of the Enterobacteriaceae family in raw goat milk, data on prevalence, virulence and resistance to antimicrobials associated with the isolates contribute to the improvement of the product quality and safety monitoring process and generate data for risk analysis models.
Footnotes
Authors’ contributions statement: GLPAR conducted the experimental work; GLPAR and JSN. analyzed the data and wrote the manuscript. Both authors read and approved the final version of the manuscript.
Conflicts of interest: All authors – none to declare.
Funding: None to declare.
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