Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production

Staphylococcus aureus is an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureus strains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureus strains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureus is a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.     

Characterization of Staphylococcus aureus and Salmonella spp. strains isolated from bovine meat in Za?re.

A large majority (87.4%) of 190 Staphylococcus aureus isolates from fresh beef in Lubumbashi (Za?re) belonged to the human St. aureus ecovar; 81.2% of the phage-typed human strains were partially or solely lysed by phages of group III. Thirteen of the 52 tested strains (25.0%) were enterotoxin producers; nine of these (69.2%) were positive for staphylococcal enterotoxin A. Sixteen serotypes were identified among the 122 Salmonella isolates and nearly all these strains were susceptible to the 8 different antibiotics tested.     

Effects of milk on activity of antimicrobics against Staphylococcus aureus isolated from bovine udders

Addition of milk to Mueller-Hinton susceptibility test medium permitted measurement of milk effect on agar disc diffusion zone diameters obtained from Staphylococcus aureus herd isolates and stock strains. Milk reduced zone diameters for all antimicrobics tested when compared with zones obtained by standard methods. Antimicrobics most affected included novobiocin, streptomycin, gentamycin, tetracycline, and vancomycin. Growth curves of a reference strain of Staphylococcus aureus in Mueller-Hinton broth with and without milk indicated no effect on growth rate of the organism, suggesting the observed effect was due to the action of milk on the antimicrobics being tested. The described method offers a simple in vitro test for milk effects on antimicrobic activity.     

Antimicrobial drug susceptibility of Staphylococcus aureus strains isolated from bovine and ovine mammary glands

The activity of selected antimicrobial agents against Staphylococcus aureus was determined with the agar disk diffusion test to determine the diameter of the zone of inhibition and the E-test for determination of the minimal inhibitory concentration (MIC). The 92 S. aureus strains used in this study were isolated from bovine (n = 76) and ovine (n = 16) intramammary infections. Four antibiotics, which are frequently used in mastitis therapy were chosen: penicillin-G, ampicillin, kanamycin, and cephalexine. The fifth compound (oxacillin) was used to detect methicillin-resistant S. aureus, but no such strains could be found. According to the evaluation criteria, 65.2 (penicillin) to 93.5% (kanamycin, cephalexine) S. aureus strains were susceptible to the antibiotics tested. Ovine S. aureus strains reveal a lower resistance rate than bovine isolates. Comparison of the results of the two methods of susceptibility testing shows, with exception of penicillin and ampicillin, satisfactory agreement. Analyzing the results of the MIC endpoints and the zone diameter values, very major errors, according to the error rate bounded method of Metzler and DeHaan, occurred at an error rate of 3.3% for penicillin and 3.8% for ampicillin.     

Virulence factors and genetic variability of Staphylococcus aureus strains isolated from raw sheep's milk cheese

Contamination of dairy products with Staphylococcus aureus can be of animal or human origin. The host pathogen relationship is an important factor determining genetic polymorphism of the strains and their potential virulence. The aim of the present study was to carry out an extensive characterization of virulence factors and to study the genetic variability of S. aureus strains isolated from raw ewe's milk cheese. A total of 100 S. aureus strains isolated from cheese samples produced in 10 artisan cheese factories were analyzed for the presence of enterotoxins (sea-see) and enterotoxins-like genes (seh, sek, sel, sem, seo, sep), leukocidins, exfoliatins, haemolysins, toxic shock syndrome toxin 1 (TSST-1) and the accessory gene regulator alleles (agr). Strains were also typed using pulsed-field gel electrophoresis (PFGE). AMOVA analysis carried out on PFGE and PCR data showed that the major component explaining genetic distance between strains was the dairy of origin. Of the total isolates 81% had a pathogenicity profile ascribable to "animal" biovar while 16% could be related to "human" biovar. The biovar allowed to estimate the most likely origin of the contamination. Minimum inhibitory concentrations (MICs) of nine antimicrobial agents and the presence of the corresponding genes coding for antibiotic resistance was also investigated. 18 strains carrying blaZ gene showed resistance to ampicillin and penicillin and 6 strains carrying tetM gene were resistant to tetracycline. The presence of mecA gene and methicillin resistance, typical of strains of human origin, was never detected. The results obtained in the present study confirm that S. aureus contamination in artisan cheese production is mainly of animal origin.     

Toxigenic status of Staphylococcus aureus isolated from bovine raw milk and Minas frescal cheese in Brazil

A group of 291 Staphylococcus aureus isolates from mastitic cow's milk (n = 125), bulk tank milk (n = 96), and Minas frescal cheese (n = 70) were screened for staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, selj, and sell) and for the tst-1 gene encoding staphylococcal toxic shock syndrome toxin 1 by PCR assay. A total of 109 (37.5%) of the isolates were positive for at least one of these 11 genes, and 23 distinct genotypes of toxin genes were observed. Of the S. aureus isolates bearing SE genes, 17 (13.6%) were from mastitic cow's milk, 41 (41.7%) were from bulk tank milk, and 51 (72.9%) were from Minas frescal cheese. The occurrence of exclusively more recently described SE genes (seg through sell) was considerably higher (87 of 109 PCR-positive strains) than that of classical SE genes (sea through see, 15 strains). The SE genes most commonly detected were seg and sei; they were found alone or in different combinations with other toxin genes, but in 60.8% of the cases they were codetected. No strain possessed see. The tst-1 gene was found in eight isolates but none from mastitic cow's milk. Macrorestriction analysis of chromosomal DNA from 89 S. aureus isolates positive for SE gene(s) was conducted with the enzyme SmaI. Fifty-five distinct pulsed-field gel electrophoresis patterns were found, demonstrating a lack of predominance of any specific clone. A second enzyme, Apa I, used for some isolates was less discriminating than Sma I. The high genotype diversity of potential toxigenic S. aureus strains found in this study, especially from Minas frescal cheese, suggests various sources of contamination. Efforts from the entire production chain are required to improve consumer safety.     

A coagulase-negative variant of Staphylococcus aureus from bovine mastitis milk

Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates of Staphylococcus aureus which gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex? test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically 'coagulase-negative variant of Staph. aureus'. The fact that coagulase-negative Staph. aureus variants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis 'coagulase-negative staphylococci (CNS)' in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such as coa is necessary in these cases.     

Characterization of Staphylococcus aureus isolated from powdered infant formula milk and infant rice cereal in China

Dry infant foods are not sterile and could be contaminated with various bacteria including certain pathogens. The aim of this study was to investigate the prevalence of Staphylococcus aureus in infant foods and to characterize these strains. A total of 367 infant food samples, including 143 samples of powdered infant formula milk (PIF) and 224 samples of infant rice cereal (IRC), were collected in the Shaanxi Province of China during the period of July to August 2010 and screened for S. aureus. All S. aureus isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and detection of genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin (PVL), and toxic shock syndrome toxin 1. Among all the samples examined, sixteen of 143 PIF samples (11.2%) and 14 of 224 IRC samples (6.3%) were positive for S. aureus. From these positive samples, 29 S. aureus strains were isolated from PIF and 25 from IRC. Of these S. aureus isolates, 83.3% were resistant to at least one antimicrobial, 35.2% to three or more antimicrobials. Resistance was most frequently observed to erythromycin (75.9%), followed by ciprofloxacin (51.9%) and trimethoprim/sulfamethoxazole (27.8%), while significantly fewer isolates were resistant to gentamicin (22.2%), tetracycline (18.5%), or cefoxitin (3.7%). In addition, 63.0% of isolates were positive for one or more toxin genes tested. The three most predominant toxin genes were pvl (40.7%), seg (38.9%), and sec (18.5%), followed by sea (7.4%), seb (7.4%), sed (5.6%), and see (5.6%). The ets, tsst-1, seh, sei, and sej genes were not detected. A total of 39 PFGE patterns were generated among 51 selected food isolates. Our findings indicate that PIF and IRC in the Shaanxi province were contaminated with S. aureus, and many S. aureus isolates harbored multiple toxin genes and exhibited multiple antimicrobial resistance. In addition, these S. aureus isolates were genetically diverse. The presence of S. aureus strains in these infant foods poses a potential threat to infant health.     

Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina

A total of 206 strains of Staphylococcus aureus isolated from bovine clinical and subclinical mastitis in Argentina during 1996 to 1998 were investigated for their in vitro susceptibility to several antimicrobial agents. Minimum inhibitory concentrations that inhibit 90% of the strains tested reported in micrograms per milliliters were: 1.5, 0.5, 0.75, 1.50, 0.75, 1.0 and 0.125 for penicillin, oxacillin, cephalothin, gentamicin, erythromycin, clindamycin and ampicillin-sulbactam, respectively. Resistance was detected in 83 (40.3%), 24 (11.6%), 16 (7.7%) and 7 (3.4%) S. aureus isolates for penicillin, erythromycin, pirlimycin and gentamicin, respectively. No resistance was detected for oxacillin, cephalothin and ampicillin-sulbactam. Results indicated that S. aureus isolates in Argentina exhibited high resistance to penicillin of all antimicrobial agents tested.     

Bovine lactoferrin receptors in Staphylococcus aureus isolated from bovine mastitis.

A total of 103 Staphylococcus aureus strains isolated from bovine mastitis were tested for bovine lactoferrin binding in a 125I-labeled protein binding assay. More than 85% of the strains demonstrated high to moderate and a few showed little or no binding. Bovine lactoferrin binding to S. aureus cells was high when grown on blood, nutrient, or proteose-peptone agar, but the binding capacity was low with cells grown on salt rich media, in skim milk, or in broth. The kinetics of 125I-labeled bovine lactoferrin binding required approximately 90 min for complete saturation with optimal interaction in the pH range 4.0 to 7.0. The lactoferrin-staphylococci interaction was specific with a high affinity (association constant, Ka 14 x 10(6) L/mol). Scatchard plot analysis estimated the number of binding sites per cell at 7200 on strain SA-340. Unlabeled bovine lactoferrin effectively displaced the binding of the labeled ligand to strain SA-340 in a dose-dependent manner. Bovine lactoferrin binding was inhibited or displaced by human lactoferrin. Various plasma, connective tissue, or mucosal secretory proteins tested did not inhibit lactoferrin-staphylococci interaction. Bovine lactoferrin binding components on SA-340 were resistant to glycolytic enzymes and moderately susceptible to proteolytic digestion. Two proteins with an estimated molecular weight of approximately 92 and 67 kDa were identified as bovine lactoferrin binding components of S. aureus strain SA-340.     

牛乳腺炎金黄色葡萄球菌肠毒素A基因的克隆及序列分析

根据Genbank上公布的金黄色葡萄球菌肠毒素A的全序列,利用生物学软件Primer 5.0和oligo 6.0设计了一对特异性引物来扩增靶序列片段,经克隆预测序,结果表明扩增片段长度为101 bp,锦州分离株与标准菌株的基因片段序列相似性为100%,与Genbank上公布的金黄色葡萄球菌菌株(EF520720.1)SEA基因相似性达到99.14%。高度相似性结果为进一步研究建立分子检测技术奠定了实验基础。     

Characterization of Staphylococcus aureus isolated from chronically infected dairy goats

A herd of 88 Alpine goats in Northern Italy was monitored for a complete lactation. Milk samples were taken from each udder half during 8 monthly visits. Goats (n = 28) with ≥2 consecutive positive tests for Staphylococcus aureus in the same udder half were identified as chronically infected, and all of those had ≥4 positive tests of the 8 samples. Goats with no infections in either udder half during any visit were considered healthy (n = 26). Linear mixed models were used to examine the relationship between chronic infection by S. aureus and SCC and production traits. The bacteria isolated from one sample from each infected goat were genotyped on the basis of polymorphism in several genes and evaluated for the presence of genes encoding for enterotoxins. The bacteria isolated from each animal were also subject to a test for β-lactamase production and to minimum inhibitory concentration tests for 11 antimicrobial agents. As expected, SCC (log₂) was significantly higher in infected goats than in healthy goats (7.55 vs. 5.50). Also, mean log SCC from infected udder halves (8.02) was greater than that in uninfected udder halves from the same goats (6.44). No significant differences were observed in milk yield or for fat and protein percentages between infected and healthy goats. No genetic variability was observed among the bacteria isolated, suggesting that all were from the same strain, although isolates did vary in susceptibility to various antimicrobial agents. All S. aureus isolates were negative for the β-lactamase production test. The most effective drugs when tested in vitro were benzylpenicillin, amoxicillin plus clavulanic acid, cloxacillin, and cephalosporins.     

Specific Bifidobacterium strains isolated from elderly subjects inhibit growth of Staphylococcus aureus

Cell-free, pH-controlled supernatants of thirty-eight Bifidobacterium strains isolated from healthy elderly subjects were subjected to antimicrobial activity assay. Bioluminescent indicator strains Staphylococcus aureus RN4220, Escherichia coli K-12, and Salmonella enterica serovar Typhimurium ATCC 14028 were used as targets of antimicrobial activity. The effect of nutrient depletion on the inhibition was eliminated with spent-culture controls. Three out of thirty-eight Bifidobacterium strains were capable of inhibiting the growth of S. aureus. The inhibition was equal to 23.2+/-19.1% to 50.4+/-26.7% of the inhibition caused by 50 IU/ml nisin. Reuterin-producing positive strain Lactobacillus reuteri SD2112 was capable of 86.0+/-24.6% inhibition, but Bifidobacterium lactis Bb-12, a known probiotic strain, showed no inhibition. None of the strains was capable of inhibiting the growth of E. coli or S. enterica. The observed inhibition by bifidobacteria was related to hydrogen peroxide formation and possible production of heat-stable proteinaceous compounds. The results suggest that production of antimicrobial substances other than organic acids is not common among Bifidobacterium strains typical of elderly subjects. However, specific strains were identified which showed considerable inhibitory activity against S. aureus.     

Characterization of multiple forms of lactophorin isolated from bovine milk whey

A glycoprotein that reacted to the antisoluble glycoprotein of bovine milk fat globule membrane was purified from the proteose-peptone of whey and designated lactophorin. Lactophorin was separated into seven components. Lactophorin and the seven components were rich in aspartic acid, threonine, serine, glutamic acid, leucine, and lysine. The content of threonine, glycine, isoleucine, lysine, and arginine varied in each component. The ratio of fucose, mannose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid of lactophorin, which contains about 18% saccharide, were 1, 6.6, 10.3, 5.5, 9.7, and 11.6, respectively, while the respective ratio of the seven components were 1, 5 to 6, 7 to 9, 3 to 4, 6 to 8, and 4 to 12. Sialic acid content varied in each component. Protein-carbohydrate linkage was N- and o-glucoside linkage. Lactophorin consisted of seven polypeptides (I to VII) with apparent molecular weights 17,000 to 67,000. Bands I, II, VI, and VII were glycoprotein. Bands VI and VII were major and had antigenicity to anti-soluble glycoprotein, while bands I to V were minor polypeptides. Component 1 consisted of only one polypeptide (VII), whereas the components 2 to 7 contained two major (VI, VII, or both) and several minor polypeptides. The sedimentation pattern of each component was a single and almost symmetrical peak. Sedimentation coefficient was 3.79 to 5.64 S and also varied in lactophorin. The results indicate that lactophorin has multiple forms.     

The prevention and removal of biofilm formation of Staphylococcus aureus strains isolated from raw milk samples by citric acid treatments

In this study, the antibiofilm activity of citric acid treatment on Staphylococcus aureus strains isolated from raw milk samples was evaluated. For this purpose, the prevention and removal of biofilm formation of S. aureus strains by citric acid treatments (2% and 10%) for 20 min were investigated for comparison with peracetic acid treatment (0.3%) on both microtitration plate and stainless steel coupons. The results indicated that the prevention and removal of biofilm formation and the numbers of prevented or removed S. aureus strains using citric acid treatments were observed to be higher than those using peracetic acid treatment on both surfaces. The prevention and removal of biofilm formation were substantially higher when the concentration of citric acid treatment increased from 2% to 10% and the stainless coupons were used. The results show that citric acid can be used as an alternative disinfectant in controlling biofilm formation in the dairy industry.     

DNA carryover in milk samples from routine milk recording used for PCR-based diagnosis of bovine Staphylococcus aureus mastitis

Real-time PCR techniques are increasingly used to detect udder pathogens from milk samples collected non-aseptically at routine milk recording. The objectives of this study were (1) to estimate the statistical associations between cycle threshold (Ct) values for Staphylococcus aureus in non-aseptically collected composite samples taken at routine milk recording from cows milked consecutively with the same milking unit and milk meter; and (2) to formulate practical and plausible guidelines for understanding the diagnostic implications of PCR testing for Staph. aureus intramammary infection at routine milk recording. The study included 4 herds with conventional milking parlors and repeatedly low Ct-values for Staph. aureus (representing a high DNA load) in bulk tank milk. Composite milk samples were collected from all cows at all milking units during routine milk recording using the Tru-Test electronic milk meter (Tru-Test Group, Auckland, New Zealand) and analyzed using the PathoProof PCR (Thermo Fisher Scientific, Vantaa, Finland) assay. Milking clock times were retrieved at each milk meter to establish the milking order of the cows at each unit. A multinomial logistic regression was applied to estimate the association between Ct-values from cows milked consecutively with the same milking unit and milk meter. The following groups were selected based on Ct-values: (1) 0–31.3, (2) 31.4–33.9, (3) 34.0–37, (4) 37.1–39.9, and (5) 40 (negative result). The association between groups from cows milked consecutively with the same milking unit and milk meter was statistically significant. Approximately 60% of cows were in Ct group 5 if the antecedent cow was also in Ct group 5, but only 20% of cows were in Ct group 5 if the antecedent cow was in Ct group 1. The probability of cows being in Ct group 1 was not markedly influenced by the group of the antecedent cow. Statistical relationships in the intermediate range gave a plausible indication of a dose-response relationship. Carryover of bacterial DNA via the milking unit and milk meter is very likely to affect PCR results for Staph. aureus. Therefore, information about milking order must be considered in mastitis control efforts. We suggest a practical interpretation of PCR results: cows with a Ct-value <32 can be labeled “very likely to be infected with Staph. aureus,” but cows with Ct-values of >37 and 32–37 can be labeled “very likely to be negative for Staph. aureus” and “uncertain Staph. aureus status,” respectively.     

Short communication: Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S. aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S. aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc, sea, seb, sec, sed, and see genes. The positive isolates were nuc, 100% (17); sea, 35.29% (6); seb, 5.88% (1); sec, 5.88% (1); sed, 29.41% (5); and see, 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S. aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk.     

Antimicrobial spectrum activity of bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk

Bacteriocins have attracted great attention as potential alternatives to antibiotics and chemical food additives. In the present study, 243 Staphylococcus isolates from milk samples (n = 110) of goat and sheep herds located in Fars province, Iran, were screened for antimicrobial substance production. Twenty-eight isolates showed an antagonistic activity against the indicator strain Micrococcus luteus ATCC 4698. The susceptibility of all antimicrobial substances to proteolytic enzymes allowed us to consider them as bacteriocin-like inhibitory substances (BLIS). The term BLIS is applied to uncharacterized proteinaceous antimicrobials produced by gram-positive bacteria. Based on molecular identi?cation methods, the isolates belonged to the species Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus pseudintermedius, Staphylococcus aureus, and Staphylococcus agnetis. Pulsed-?eld gel electrophoresis revealed a high level of genotype diversity among the Staph. chromogenes isolates. All of the isolates harbored nukA or bsaA2 genes, suggesting that their BLIS were related to nukacin or Bsa. The antimicrobial compounds from test strains were not effective against gram-negative pathogens, including Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Klebsiella pneumonia as well as the indicator mold Aspergillus fumigatus. All the gram-positive targets, including Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis Ef37 (a tyramine-producer strain), Lactobacillus saerimneri 30a (a histamine-producer strain), and methicillin-resistant Staph. epidermidis, were inhibited by the Staph. chromogenes isolates. Staphylococcus haemolyticus 4S12 was able to inhibit the majority of gram-positive bacteria. Listeria monocytogenes strains were the only indicators sensitive to the antimicrobial agents produced by Staph. agnetis 4S97B. The other Staphylococcus strains were ineffective on all the organisms tested. Based on their inhibitory capacities, the BLIS produced by the Staph. chromogenes isolates seem to be interesting candidates for developing novel antimicrobial agents.