Antimicrobial Resistance, Serologic and Molecular Characterization of E. coli Isolated from Calves with Severe or Fatal Enteritis in Bavaria, Germany

Worldwide, enterotoxigenic Escherichia coli (ETEC) cause neonatal diarrhea and high mortality rates in newborn calves, leading to great economic losses. In Bavaria, Germany, no recent facts are available regarding the prevalence of virulence factors or antimicrobial resistance of ETEC in calves. Antimicrobial susceptibility of 8713 E. coli isolates obtained from 7358 samples of diseased or deceased diarrheic calves were investigated between 2015 to 2019. Considerably high rates of 84.2% multidrug-resistant and 15.8% extensively drug-resistant isolates were detected. The resistance situation of the first, second and third line antimicrobials for the treatment, here amoxicillin-clavulanate, enrofloxacin and trimethoprim-sulfamethoxazole, is currently acceptable with mean non-susceptibility rates of 28.1%, 37.9% and 50.0% over the investigated 5-year period. Furthermore, the ETEC serotypes O101:K28, O9:K35, O101:K30, O101:K32, O78:K80, O139:K82, O8:K87, O141:K85 and O147:K89, as well as the virulence factors F17, F41, F5, ST-I and stx1 were identified in a subset of samples collected in 2019 and 2020. The substantially high rates of multi- and extensively drug-resistant isolates underline the necessity of continuous monitoring regarding antimicrobial resistance to provide reliable prognoses and adjust recommendations for the treatment of bacterial infections in animals.


Introduction
Escherichia coli account to the major enteric and systemic pathogens of the Gramnegative rods within the family Enterobacteriaceae. Most of the E. coli colonizing the intestinal tract of animals and humans are commensal, but facultative pathogenic strains may cause intestinal disorder or even severe and life-threatening extraintestinal disease [1,2]. In calves, enterotoxigenic E. coli (ETEC) pose a leading cause of intestinal disease, especially within the first four days of life [3][4][5]. ETEC encode lipopolysaccharide structures (LPS) that may act as endotoxins, fimbrial adhesins and finally enterotoxins. The endotoxins within the blood stream cause fever, damage of endothelial cells and disseminated intravascular coagulation (DIC), that leads to acute shock and sudden death [1]. The serological LPS characterization in calves comprise the E. coli serogroups O8, O9 and O101, and respective serotypes O9:K35 and O101:K30, as these are known for endotoxin effect [6]. Further, the

Antimicrobial Susceptibility
Within the study period 8713 E. coli were isolated from 7358 diarrheic calves at the federal state veterinary laboratory in Bavaria, Germany (Table S1). This number matches an average count of 1740 isolates per year that is in accordance with previous years (data not shown). The results on antimicrobial susceptibility testing revealed mean non-susceptibility values of 28.1% for amoxicillin-clavulanate, 37.9% for enrofloxacin and 50% for trimethoprim-sulfamethoxazole (Figures 1 and 2, Table S1). The highest nonsusceptibility value of a substance within each antimicrobial class revealed 11.9% for tulathromycin (macrolides), 18.3% for colistin (polymixins), 61.9% for tetracycline (tetracyclines), 62.2% for spectinomycin (aminoglycosides), 69.7% for ampicillin (penicillins), 80.5% for cephalothin (cephalosporins) and 96.8 % for florfenicol (phenicols) (Figure 1). A 5-year tendency from 2015 to 2019, evaluated for amoxicillin-clavulanate, enrofloxacin and trimethoprim-sulfamethoxazole, revealed a statistically significant decrease of the nonsusceptibility rates for amoxicillin-clavulanate and enrofloxacin (p < 0.05) ( Figure 2, Table 1). Regarding trimethoprim-sulfamethoxazole a significant decrease was assessed from 51.9% to 47.8% between 2015 and 2017 regarding the non-susceptible E. coli isolates (p < 0.05). A subsequent increase was further revealed from 47.8% to 52.5% in the years 2017 to 2019 (p < 0.05) ( Figure 2, Table 1). Categorizing the 8713 isolates according to the ECDC expert proposal, 84.2% of the isolates (7336/8713) were multidrug-resistant, 15.7% (1368/8713) were extensively drug-resistant, eight isolates (0.1%) were pandrug-resistant and one isolate was susceptible to all antimicrobials tested. As we only tested antimicrobials licensed for the veterinary use, and none of the latest antimicrobials available on the market, we rededicated the eight presumably pandrug-resistant as extensively drug-resistant summing up to 1376 isolates in this specification ( Figure 3).

Figure 2.
The mean value (bold) and the five-year trend on non-susceptible E. coli isolated fr calves revealed the highest proportion of isolates against trimethoprim-sulfamethoxazole, follow by enrofloxacin and amoxicillin-clavulanate. The trends regarding enrofloxacin and amoxicil clavulanate remain at a stable level and rather tend towards a decrease regarding the number non-susceptible isolates. The graph of non-susceptible isolates regarding trimethoprim-sulfame oxazole reveals a decrease, 2016-2017, followed by a steep increase of non-susceptible isolates 2019. The corresponding statistic parameters are presented in Table 1.   Table 1.
Antibiotics 2022, 11, x FOR PEER REVIEW 6 of 17 Figure 3. The classification of 8713 E. coli into extensively drug-resistant and multi drug-resistant isolates was carried out according to the expert proposal for standard definitions for acquired resistance. We categorized eight potential pandrug-resistant isolates in the category extensively drug resistant, as we only tested antimicrobials licensed for the veterinary use and did not include the latest antimicrobials available on the market.
The fimbrial antigen F5 agglutinated in 6.5% of the isolates (n = 7) in combination with the serotypes O101:K30, O101:K28 and O9:K35. The fimbrial antigen F4 agglutinated in 4.6% of the isolates (n = 5), and exclusively combined with the serotype O139:K82 (Tables 2  and S2). Table 2. The serologic and molecular characterization revealed 13 different serotypes known to be pathogenic for cattle and other species. Furthermore, four different genotypes were detected with five different coding sequences for fimbria and/or toxins in one or more isolates. Some of the isolates were untypeable/ seronegative and did not reveal any of the investigated virulence factors (green box).

Serotype
Additionally

Molecular Characterization
Within the molecular characterization, 14 PCR assays targeted genes for the expression of fimbria, adhesin, hemolysin and toxins. A positive result was obtained for 24 isolates and 35 single assays, respectively (Tables 2 and S2). The most frequently detected genes coded for the fimbria F17 (13.9%; 15/108), F41 (3.7%; 4/108) and F5. The latter was always detected in combination with the toxin gene coding for ST-I (6.5%; 7/108). Finally, the gene coding for stx1 was detected in two of 108 isolates (1.9%). Seven of 108 isolates (6.5%) carried more than one type of virulence-associated genes (Tables 2 and S2). The fimbrial antigens F4, F6, F18, O157, adhesin eaeA, hemolysin ehxA and the toxins LT, ST-II and stx2 were not detected in any isolate. The occurrence of F4 fimbria in the serotyping assays could not be confirmed in the PCR investigation (Tables 2 and S2). In all, 84 of 108 isolates were negative in all PCR assays (Tables 2 and S2).

Discussion
Antibiotic treatment is the fundamental therapy regarding serious or life-threatening bacterial infections in man and animals [28,29]. Records regarding antimicrobial susceptibility on single substances are collected in many countries all over the world [43]. Worldwide this is a critical topic in line with the One Health issue [44]. Monitoring on the application and more important efficacy of antimicrobials regarding bacterial infections of farm animals is possible on principle in industrial countries. However, it is costly and difficult to standardize [36]. Published data from Canada in 2018 revealed a 51.6% susceptibility rate of 489 E. coli against trimethoprim-sulfamethoxazole, which is in consensus with our data (50%) (Figures 1 and 2) [45]. Tetracycline was accounted to be effective in 36.8% and resembles our findings at 38.1% (Figure 1) [45]. Further, authors from the United States and Germany determined similar high resistance rates for tetracycline, with 71.1% and 70.9%. These data rather resemble the rate of 61.4% revealed in the present study ( Figure 1) [46,47].
The antimicrobial class of fluoroquinolones includes enrofloxacin which is one of the substances of choice for the treatment of diarrhea in young cattle [29,48]. In Germany, the usage of fluoroquinolones has risen from 2011 to 2013 in human and veterinary medicine. This trend needs close monitoring to preserve the efficacy of the agent [27]. Fluoroquinolones are assessed as highest priority clinically important antimicrobials and as one of the few options for the treatment of serious Salmonella and E. coli infections in children recommended by the World Health Organization (WHO) [49]. The legislation reacted and passed a law in 2017 including obligatory antimicrobial susceptibility testing in case of the application of fluoroquinolones or third or fourth generation cephalosporines in Germany [50]. In the present study, the investigated E. coli isolated revealed a resistance rate of 34.1% regarding enrofloxacin (Figure 1). This finding correlates with published results from South America in 2017, with 36.4% [51].
Antimicrobial substances or closely related compounds may likewise be licensed for the use in man and animals. The application in an organism does trigger the development of antimicrobial resistance in present bacteria [49]. Legal restrictions regarding the use of cephalosporines, especially from the third and fourth generation, aim at a high prioritization of critically important antimicrobials in human medicine [49]. This is again in accordance with the terms of One Health [27,44]. The use of cephalosporines for the therapy of E. coli diarrhea in calves is a malpractice, as the effective therapeutic concentration is not reached within the gut [29]. Nonetheless, cephalosporin is the fifth-most commonly prescribed antimicrobial in the case of diarrhea with 8% according to a recent survey in Europe [30]. Regarding the third generation cephalosporine ceftiofur, a susceptibility rate of 86.4% could be determined in a study from Canada between 1994 and 2013 [45]. Significantly, our findings revealed 76.8% (Figure 1). Compared to data from the USA collected within the years 1960 until 2002 and in 2007, the resistance rate was at 7.4% and 11%, whereas in the present study the resistance rate of ceftiofur revealed 20.4% (Figure 1) [46,52]. This result is concerning, and the use of ceftiofur must be scrutinized critically, if not avoided completely. The resistance rates of the first generation cephalosporine, cephalothin, were lower in a comparable study regarding data within the period of 1960 to 2002, with 20.1%, in contrast to our results with an average rate at 46.1% from 2015 to 2019 (Figure 1) [46]. Currently, the standard antimicrobial therapy of mastitis in cows includes penicillins as well as first and second generation cephalosporines in the EU. Traces of antibiotics may reach the calves through the feeding of antibiotic contaminated waste milk [36]. To predict a reliable trend regarding the prevalence of ESBL-producing E. coli, PCR and sequencing methods should be applied to investigate the existence of ESBL-encoding genes as these are probably more accurate than the phenotypic characterization [53]. A study from 2013 revealed high rates (32.8%, 196 of 598 samples) of ESBL-encoding E. coli on dairy and beef cattle farms in Bavaria [54].
Completely inconsistent data are publicly available regarding the resistant rates for E. coli isolates and the substance florfenicol within the phenicol group. A 78% share of resistant isolates was determined in a study from the USA in 2006, only a 28% share from Canada in 2018, and a share of 35% from Bavaria, Germany, in 2002 [45,52,55]. In the present study, a rather higher resistance rate of 60.6% was determined for florfenicol ( Figure 1). There was no information about ages of animals within the American and Canadian studies [45,52]. Since lower resistance rates were previously published in older animals for the substances ampicillin, tetracycline, streptomycin, sulfamethoxazole and chloramphenicol, this might accordingly apply for florfenicol [32]. This argument, however, still does not explain the diverse results of the Bavarian study from 2002 and the present study (Figure 1) [55].
With a 9% share of the most frequently listed antimicrobials, aminoglycosides remain at the fourth top position for the treatment of diarrhea in calves [30]. As these are almost solely used in the therapy of enterococcal endocarditis and multidrug-resistant tuberculosis in humans, they account to the high priority, clinically important antimicrobials in human medicine [49]. An application in veterinary medicine should therefore be prudent and well considered. Gentamicin belongs to the aminoglycoside antimicrobial class and has a withdrawal time for meat of more than 200 days in Germany for cattle and the indication of gastrointestinal disease. As this is economically hardly acceptable, the application of gentamicin is quite limited [48]. However, resistance to gentamicin among E. coli isolated from animals has been increasing from 0% to 40% between 1970 and 2002 within the United States [46]. Another long-term investigation from Germany revealed a further decrease of resistance rates including data from 2010 until 2013, and 2016 until 2017, respectively [47]. In the present study, the resistance rate of E. coli against Gentamicin was at 14.1% (Figure 1). Likewise, spectinomycin is an aminoglycoside antibiotic as well, and frequently used in combination with lincomycin for oral application in the treatment of simultaneous infection of the respiratory and the gastrointestinal tract in calves. The meat withdrawal time of 21 days is acceptable for farmers and practitioners and may be an explanation for the frequent prescription [48]. Within the present study and correspondingly a resistance rate of 48.9% was revealed in calves ( Figure 1).
As stated by the WHO, the antimicrobial class of polymyxins accounts for the highest priority in critically important antimicrobials regarding the treatment of serious infections with Enterobacteriaceae and Pseudomonas aeruginosa in human medicine [49]. Despite rather frequent prescription of polymyxins in the treatment of diarrhea in animals, investigated E. coli isolates are still highly susceptible [30]. In the present study, the resistance rate against colistin revealed to be only 1.8% (Figure 1). Corresponding to this suggestion, another study revealed that only 3.8% of the isolates were resistant to colistin [47].
The aminopenicillin family, as well as the preparation amoxicillin-clavulanate, belong to the high priority critically important antimicrobials for the therapy of Listeria and Enterococcus spp. infections in humans according to the WHO [49]. For the aminopenicillin, ampicillin, an alarming resistance rate of 76.3% was determined in E. coli published in a most recent study from Germany [47]. Regrettably, a rate of 69.5% was determined in the present work as a similar result (Figure 1). Consequently, the recommendation on the usage of ampicillin for the treatment of calf diarrhea cannot further be continued. The amoxicillin-clavulanate susceptibility rate averaged at 57% in Germany in 2013 [27]. In the present study, the average susceptibility rate was 71.9%, and the resistance rate was 8.6% (Figure 1). Accordingly, a recently published study reported 7% of resistant E. coli isolates in Germany in 2018 [34]. Analogical to the report on the resistance monitoring study 2018 of the Federal Office of Consumer Protection and Food Safety, Germany, we determined decreasing non-susceptibility rates regarding the clinically important antimicrobial amoxicillin-clavulanate [34]. In conclusion, the resistance rates of E. coli against amoxicillin-clavulanate have decreased since 2013 and remained on a constant level within the years 2015 and 2019. This is a positive trend is beneficial for the One Health point of view [27].
Comparing data originating from other continents and collected over the last 60 years clearly reveals an increase of resistance regarding E. coli in nine out of the 12 tested drugs, namely gentamicin, cephalothin, ceftiofur, enrofloxacin, trimethoprim-sulfamethoxazole, ampicillin, amoxicillin-clavulanate, florfenicol and tetracycline [27,34,[45][46][47]51,52,55]. Out of the 12 tested drugs in the present study, eight substances are similarly suitable for the treatment of human patients, namely gentamicin, spectinomycin, cephalothin, ampicillin, tetracycline, amoxicillin-clavulanate, colistin and trimethoprim-sulfamethoxazole (Figure 1) [49]. The application of these in veterinary medicine should be prudent due to the One Health aspect.
In a published study from Canada in 2018, 48.7% of multidrug-resistant E. coli were isolated from ruminants [45]. Within another study from the USA covering the years 1950 until 2002, a significantly increasing trend in resistance was observed for ampicillin, sulfonamide and tetracycline antibiotics regarding more than 1700 E. coli isolates. Two of these strains were identified as pandrug-resistant and originated from cattle in 2001 [46]. Further, multidrug resistance in E. coli increased from 7.2% to 63% between 1950 and 2002. Finally, 59.1% of the strains recovered form cattle were classified as multidrug resistant in the USA [46]. In the present study, we detected an even higher rate of 84.2% regarding multidrug resistance, 15.7% extensively drug-resistance and 0.1% pandrugresistance ( Figure 3). Furthermore, there were no exclusively susceptible isolates found amongst 108 isolates recovered in 2019 and 2020 from diarrheic calves in Bavaria (Table S2). Comparably high levels of antimicrobial resistance were published regarding the countries Brazil and Uruguay. Calves aged up to 60 days revealed a multidrug-resistance rate in E. coli at 78.7%, and at 61.6%, respectively [51]. As published, these bacteria occurred frequently in herds with high levels of diarrhea symptoms and subsequent antimicrobial therapy, as equally described in the present study [31].
Besides antimicrobial resistance, the determination of virulence regarding infectious agents is crucial in diagnostics. The discrimination from commensal E. coli was determined investigating virulence factors and evaluating the pathogenicity of isolates. As published, the E. coli serotypes O139:K82, O8:K87 and O147:K89 are pathogenic in swine [6]. However, in the present study, a fair amount of such isolates, six out of 108, were isolated from cattle, respectively (Tables 2 and S2). In laboratory diagnostics, implication of these serotypes should therefore be considered. Three isolates were identified as the serotype O78:K80, which frequently causes septicemia in calves (Table 2) [5,7,56]. However, more than one third, 38%, of the E. coli in this study revealed to be entirely seronegative (Tables 3 and S2), as it was as well published previously [57]. Preferably and in accordance with the One Health approach, the screening of E. coli isolated from diseased animals should always be of interest to identify zoonotic and human pathogenic serotypes [25]. As a matter of fact, formula associated with severe human syndromes included the serotypes O26, O103, O111, O117, O128, O145 and O146 respectively [13,22,23,58]. In recent studies, the fimbrial adhesins F17, F41 and F5 were frequently and significantly correlated with diseased calves compared to healthy animals [4,9]. These findings clearly correspond to the results of the present study (Tables 2 and S2). Other selective fimbrial antigens, F4, F6 and F18, occur frequently in isolates from diarrheic piglets [1, 10,59]. As to be expected, we did not detect these amongst our strains isolated from calves (Table S2). Even five serologically F4 positive isolates were not confirmed within our molecular in-vestigation (Tables 2 and S2). We assume that none of these isolates carry the specific primer sites, or agglutination was non-specific [9]. However, working at a federal state laboratory, we do research cross species infections especially among farm animals [60]. Furthermore, we consider the One Health approach, here especially the idea from farm to fork, and therefore continuously consider possible correlations between food-borne human pathogens and isolates from farm animals [27,44].
As published, hemolysis in E. coli isolates from piglets is a reliable diagnostic marker for virulence and pathogenicity [61][62][63]. Within the present study, only few (3/108) isolates revealed a hemolytic phenotype that was not even confirmed within the molecular analysis (Table S2). We conclude that hemolysis is not a relevant marker for virulence of E. coli isolated from calves in the present study. This statement is in accordance with prior publications [64,65].
Regarding the present study, ST-I was found in similar prevalence at a rate of 6.5% (7/108) compared to published data (Tables 2 and S2) [4,66]. The enterotoxins LT and ST-II were not detected in the present study (Table S2) and this again resembles data of relevant previous studies [4,56]. Concluding published data, ETEC isolated from calves only produced ST-I, whereas ETEC isolated from pigs may encode varying combinations of the enterotoxins LT, ST-I and ST-II [11,67]. In the present study, the detection rate of stx1 was very low and stx2 as well as intimin were not detected at all among the diarrheic calves' isolates (Tables 2 and S2). This finding matches the results of previously published data to a high degree [9,51,68]. Obviously, the detection rate of Shiga toxins rose with the number of colonies isolated from each clinical sample, suggesting the selection of up to 35 colonies [69,70]. In the present investigation however, only up to three colonies were analyzed per clinical sample (Table S2). Other published results suggested a positive correlation between animal age and the amount of Shiga toxin, supporting our findings including animals of young age [69][70][71]. Targeted infection studies with STEC led to severe disease and bloody diarrhea in neonatal calves, but more recent studies disproved this observation revealing a still controversial discussion [4,[72][73][74].

Limits of the Study
The antimicrobial susceptibility testing was carried out with a standard panel of antibiotics currently used in veterinary diagnostics in Germany. The results are therefore limited to substances only partially prescribed in human diagnostics and sometimes even in veterinary medicine regarding other countries of the world.
A thorough molecular investigation of single isolates is fairly time consuming and costly compared to the benefit that might be drawn from the results. In routine diagnostics, the molecular methods therefore can hardly be kept up.

Study Design and Bacterial Isolates
At the Bavarian Health and Food Safety Authority in Germany 7358 fecal samples of diseased or deceased calves with enteritis younger than six weeks of age were analyzed and included in the present study. Samples were collected between January 2015 and December 2019. Clinical symptoms ranged from low general condition, diarrhea, fever, sepsis and sudden death, respectively. A total of 8713 E. coli strains were isolated and confirmed through positive fluorescence on ECD agar (Merck Millipore, Burlington, MA, USA) and a positive Kovacs-Indole reaction (Merck Millipore, Burlington, MA, USA). All isolates were subject to antimicrobial resistance testing, further analysis and cryopreservation at the internal vaccine laboratory.

Phenotypic Analysis and Serotyping
We deeper investigated a subset of 108 E. coli isolated in 2019 and 2020 originating from 66 diarrheic calves. The isolates were subcultured on Gassner agar (Oxoid Deutschland GmbH, Wesel, Germany) to differentiate specific colony morphology. The expression of potential virulent F5 fimbria was investigated by subculturing the isolates on pH 7.5 stabile, "minimum of casein" (Minca) agar (Sifin Diagnostics GmbH, Berlin, Germany) as previously published [76]. Finally, potential hemolytic properties of isolates were interpreted as described with subcultures on Columbia Sheep Blood Agar (Sifin Diagnostics GmbH, Berlin, Germany) [77]. Growth incubation was carried out for 18 to 24 h at 37 • C at all times. Serotyping for specific O-antigens was carried out using two polyvalent and 14 monovalent agglutination sera in a hierarchical approach according to the manufacturer's instructions (Sifin Diagnostics GmbH Berlin, Germany) ( Table 3). If an isolate showed a positive agglutination reaction with a polyvalent serum, but none with any correspondent monovalent or several reactions with various correspondent monovalent sera, it was categorized as untypeable. If an isolate showed no positive agglutination with any serum, it was categorized as seronegative.

Molecular Investigation
The molecular characterization of the E. coli isolates in the present study aimed at surface antigens, toxins and virulence factors. In all, 14 different target genes were of interest. Amongst were seven fimbrial genes F4, F5, F6, F17, F18, F41 and the outer membrane protein O157:H-. Further, two virulence genes were included, here adhesin intimin (eaeA), and enterohemolysin (ehxA). Finally, PCR targets coding for five toxins were screened, including heat-labile toxin (LT), heat-stabile toxin I (ST-I) and II (ST-II), Shiga toxin 1 (stx1) and stx2 (Table 4). Primer sequences were adopted from published protocols [9,39,40]. All 14 qPCR assays were performed applying a singleplex high resolution melting method, using AccuMelt HRM SuperMix (Quantabio, Beverly MA, USA) in 20 µL volumes according to the manufacturer's instructions. DNA was extracted after thermolysis. The primers were added in a concentration of 0.2 µM each, and 3 µL of template DNA was used. Polymerase chain reaction assays were conducted on a Stratagene MX3000P device (Agilent Technologies, Waldbronn, Germany). The cycling protocol comprised an initial single denaturation step for 10 min at 95 • C, followed by 40 cycles of annealing and polymerization for 30 s at 60 • C and 10 s at 95 • C. After completing amplification, the melting curve analysis was performed. Specific melting temperatures were determined for each molecular target and all tested isolates. Reference strains were used as positive controls and kindly provided from Prof. R. Bauerfeind (Justus-Liebig-Universität, Gießen, Germany), and purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany) ( Table 4).

Statistical Analysis
All statistical analyses were performed using the free software R Studio version 1.2.5033 (RStudio, Inc., Boston, MA, USA). Resistance trends of three clinically relevant antimicrobials amoxicillin-clavulanate, enrofloxacin and trimethoprim-sulfamethoxazole were evaluated by calculating a logistic regression model. The respective year was set as a continuous variable. The resulting odds ratio (OR) > 1 indicated an increased resistance trend, whereat an OR < 1 indicated a decreased antimicrobial resistance. The Wald test was used to determine the statistical significance of the year-antimicrobial trend. A value of p < 0.05 was considered significant (Table 1).

Conclusions
We conclude that an extensive monitoring, characterization and the analysis of antimicrobial resistance regarding enteritis causing E. coli is crucial to determine the currently raging serotypes, virulent genotypes and most important, the resistance situation. It is then possible to calculate reliable tendencies and prognoses from data collected over long terms in routine diagnostics. This is an important premise for objective and professional treatment recommendations regarding humans and animals within the scope of One Health. A further goal should be a slowdown of the increasing antimicrobial resistance situation that constitutes a global public health threat.