A novel gyrase inhibitor from toxin-antitoxin system expressed by Staphylococcus aureus.

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.

Assessment of prediction methods for protein structures determined by NMR in CASP14: Impact of AlphaFold2.

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Designing strategies for eradication of Helicobacter pylori based on prevalence patterns of infection and antibiotic resistance in a low-income, medically underserved community in the United States.

BACKGROUND: Regional variation in Helicobacter pylori resistance patterns is a significant contributing factor for the ineffectiveness of traditional treatments. To improve treatment outcomes, we sought to create an individualized, susceptibility-driven therapeutic approach among our patient population, which is one of the poorest in the nation. It is medically underserved, minority-predominant and has high incidence of H pylori infection. METHODS: We compiled various factors involved in the antibiotic resistance of H pylori from literature. We then created a predictive model to customize therapies based on analyzed data from 2,014 H pylori patients with respect to several of these factors. The predictions of the model were further tested with analysis of patient stool samples. RESULTS: A clear pattern of H pylori prevalence and antibiotic resistance was observed in our patients. We observed that majority of H pylori patients were women (62%) and over the age of 40 years (80%). 30% and 36% of the H pylori patients were African American and Hispanic, respectively. A median household income of less than $54,000, past H pylori infection, previous use of certain antibiotics for any infection decreased the chance of eradication. Results of the stool testing were consistent with model predictions (90% accuracy). CONCLUSION: This model demonstrates the predictive accuracy of H pylori infection and antibiotic resistance based on patient attributes and previous treatment history. It will be useful to formulate customized treatments with predicted outcomes to minimize failures. Our community attributes may contribute toward broad applicability of model for other similar communities.

Evolution of the genetic code; Evidence from serine codon use disparity in Escherichia coli.

Among the 20 amino acids, three of them-leucine (Leu), arginine (Arg), and serine (Ser)-are encoded by six different codons. In comparison, all of the other 17 amino acids are encoded by either 4, 3, 2, or 1 codon. Peculiarly, Ser is separated into two disparate Ser codon boxes, differing by at least two-base substitutions, in contrast to Leu and Arg, of which codons are mutually exchangeable by a single-base substitution. We propose that these two different Ser codons independently emerged during evolution. In this hypothesis, at the time of the origin of life there were only seven primordial amino acids: Valine (coded by GUX [X = U, C, A or G]), alanine (coded by GCX), aspartic acid (coded by GAY [Y = U or C]), glutamic acid (coded by GAZ [Z = A or G]), glycine (coded by GGX), Ser (coded by AGY), and Arg (coded by CGX and AGZ). All of these were derived from GGX for glycine by single-base substitutions. Later in evolution, another class of Ser codons, UCX, were derived from alanine codons, GCX, distinctly different from the other primordial Ser codon, AGY. From the analysis of the Escherichia coli genome, we find extensive disparities in the usage of these two Ser codons, as some genes use only AGY for Ser in their genes. In contrast, others use only UCX, pointing to distinct differences in their origins, consistent with our hypothesis.

Protein structure prediction assisted with sparse NMR data in CASP13.

CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and 15 N-1 H residual dipolar coupling data, typical of that obtained for 15 N,13 C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum.

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.

De novo protein design by citizen scientists.

Online citizen science projects such as GalaxyZoo1, Eyewire2 and Phylo3 have proven very successful for data collection, annotation and processing, but for the most part have harnessed human pattern-recognition skills rather than human creativity. An exception is the game EteRNA4, in which game players learn to build new RNA structures by exploring the discrete two-dimensional space of Watson-Crick base pairing possibilities. Building new proteins, however, is a more challenging task to present in a game, as both the representation and evaluation of a protein structure are intrinsically three-dimensional. We posed the challenge of de novo protein design in the online protein-folding game Foldit5. Players were presented with a fully extended peptide chain and challenged to craft a folded protein structure and an amino acid sequence encoding that structure. After many iterations of player design, analysis of the top-scoring solutions and subsequent game improvement, Foldit players can now-starting from an extended polypeptide chain-generate a diversity of protein structures and sequences that encode them in silico. One hundred forty-six Foldit player designs with sequences unrelated to naturally occurring proteins were encoded in synthetic genes; 56 were found to be expressed and soluble in Escherichia coli, and to adopt stable monomeric folded structures in solution. The diversity of these structures is unprecedented in de novo protein design, representing 20 different folds-including a new fold not observed in natural proteins. High-resolution structures were determined for four of the designs, and are nearly identical to the player models. This work makes explicit the considerable implicit knowledge that contributes to success in de novo protein design, and shows that citizen scientists can discover creative new solutions to outstanding scientific challenges such as the protein design problem.

Toolkit for NMR Studies of Methyl-Labeled Proteins.

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics, and explore mechanistic insights of large biomolecules by solution NMR. Methyls are relatively insensitive to chemical exchange-induced depolarization and provide superior probes of supramolecular interactions and allostery in such systems. In this chapter, we describe our systematic approach and contributions in the areas of sample preparation, data collection, and data analysis that streamline the application of methyl labeling in solution NMR studies of large proteins. We focus our effort on the initial and often onerous task of methyl resonance assignment and here we detail our approaches to simplify the process. We produce new methyl labeling combinations using Escherichia coli auxotrophs, increase speed, sensitivity, and resolution of NOESY experiments by employing 3D SOFAST-NOESY, and assign methyl resonances from raw data with spectral simulation tools and(or) automatically with minimal expert supervision using the MAGIC algorithm.

Combining Evolutionary Covariance and NMR Data for Protein Structure Determination.

Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupled with advances in methods for sequence covariance analysis can provide reliable residue-residue contact information for a protein from sequence data alone. These "evolutionary couplings (ECs)" can be combined with sparse NMR data to determine accurate 3D protein structures. This hybrid "EC-NMR" method has been developed using NMR data for several soluble proteins and validated by comparison with corresponding reference structures determined by X-ray crystallography and/or conventional NMR methods. For small proteins, only backbone resonance assignments are utilized, while for larger proteins both backbone and some sidechain methyl resonance assignments are generally required. ECs can be combined with sparse NMR data obtained on deuterated, selectively protonated protein samples to provide structures that are more accurate and complete than those obtained using such sparse NMR data alone. EC-NMR also has significant potential for analysis of protein structures from solid-state NMR data and for studies of integral membrane proteins. The requirement that ECs are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.

Characterization of YjjJ toxin of Escherichia coli.

Reminiscent of eukaryotic apoptotic programmed cell death, bacteria also contain a large number of suicide genes, which are in general co-expressed with their cognate antitoxin genes. These systems called the toxin-antitoxin (TA) systems are associated with cellular dormancy, and play major roles in biofilm formation and persistent multidrug resistance of many human pathogens. In recent years, the study on TA system toxins has become a hot topic due to the health implications of these toxins by virtue of their role in bacterial pathogenicity. Here we report functional characterization of a hitherto uncharacterized Escherichia coli TA toxin, YjjJ. YjjJ exhibits several uncommon properties: (i) unlike the genes encoding most type II TA system toxins, the gene encoding YjjJ is present as a single gene and not in an operon, (ii) despite being a homolog of the well-characterized toxin HipA, YjjJ seems to have different cellular target(s), and (iii) HipB, the cognate antitoxin of HipA, also acts as an antitoxin for YjjJ. This forms a basis for an interesting next step in the study of TA systems with respect to cross-regulation between various TA systems and the evolutionary as well as clinical significance of these observations.

Designing of a single gene encoding four functional proteins.

In the genomes of some organisms such as bacteriophages and bacteria, a DNA sequence is able to encode two different proteins, indicating that genetic information is compacted in DNA twice denser than in usual DNA. In theory, a DNA sequence has a maximal capacity to produce six different mRNAs, however, it is an intriguing question how many of these mRNAs are able to synthesize functional proteins. Here, we design a DNA sequence encoding four collagen-like proteins, two, (Gly-Arg-Pro)n and (Gly-Ala-Pro)n, from a sense mRNA and the other two, also (Gly-Arg-Pro)n and (Gly-Ala-Pro)n from its antisense mRNA, all of which are expected to form triple-helical structures unique to collagens. Other designs such as the combination of (Gly-Arg-Pro)n, (Gly-Val-Pro)n, (Gly-Thr-Pro)n and (Gly-Arg-Pro)n are also possible. The proposed DNA sequence is considered to contain the most compact genetic information ever created.

The role of the loop 1 region in MazFbs mRNA interferase from Bacillus subtilis in recognition of the 3' end of the RNA substrate.

MazFbs is an mRNA interferase from Bacillus subtilis specifically recognizing UACAU. The X-ray structure of its complex with an RNA substrate has been also solved. When its amino acid sequence is compared with that of MazFhw, an mRNA interferase from a highly halophilic archaeon, recognizing UUACUCA, the 9-residue loop-1 region is highly homologous except that the V16V17 sequence in MazFbs is replaced with TK in MazFhw. Thus, we examined the role of the VV sequence in RNA substrate recognition by replacing it with TK, GG, AA or LL. The substitution mutants thus constructed showed significant differences in cleavage specificity using MS2 phage RNA. The primer extension analysis of the cleavage sites revealed that the VV sequence plays an important role in the recognition of the 3'-end base of the RNA substrate.

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.

A simple and cost effective method to independently and stereo-specifically incorporate [(1)H,(13)C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [(1)H,(13)C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [(1)H,(13)C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-(13)C,2,3-(2)H2,(15)N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[(13)CH3](γ2) labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for (2)H-glycine to prevent scrambling.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain.

We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and ß-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,ß-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,ß-dihydroxy-α-methylvalerate to α-keto-ß-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Suppression of the toxicity of Bac7 (1-35), a bovine peptide antibiotic, and its production in E. coli.

Bac7 (1-35) is an Arg- and Pro-rich peptide antibiotic, produced in bovine cells to protect them from microbial infection. It has been demonstrated to inhibit the protein synthesis in E. coli, leading to cell death. Because of its toxicity, no cost effective methods have been developed for Bac7 production in Escherichia coli for its potential clinical use. Here, we found a method to suppress Bac7 (1-35) toxicity in E. coli to establish its high expression system, in which Bac7 (1-35) was fused to the C-terminal end of protein S, a major spore-coat protein from Myxococcus xanthus, using a linker containing a Factor Xa cleavage site. The resulting His6-PrS2-Bac7 (1-35) (PrS2 is consisted of two N-terminal half domains of protein S connected in tandem) was well expressed using the Single-Protein Production (SPP) system at low temperature and subsequently purified in a single step by using a Ni column. The combination of protein S fusion and its expression in the SPP system at low temperature appeared to suppress Bac7 (1-35) toxicity. Both the purified His6-PrS2-Bac7 (1-35) and His6-PrS2-Bac7 (1-35) treated by Factor Xa were proven to be a potent inhibitor for cell-free protein synthesis.

Replacement of all arginine residues with canavanine in MazF-bs mRNA interferase changes its specificity.

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.

Molecular analysis of antimicrobial resistance in gram-negative bacteria isolated from fish farms in Egypt.

As little is known about antimicrobial resistance genes in fish farms, this study was conducted to monitor the incidence and prevalence of a wide range of antimicrobial resistance genes in Gram-negative bacteria isolated from water samples taken from fish farms in the northern part of Egypt. Ninety-one out of two hundred seventy-four (33.2%) non-repetitive isolates of Gram-negative bacteria showed multidrug resistance phenotypes and harbored at least one antimicrobial resistance gene. PCR and DNA sequencing results showed that 72 (26.3%) isolates contain tetracycline resistance genes and 19 (6.9%) isolates were positive for class 1 integrons with 12 different gene cassettes. The beta-lactamase-encoding genes were identified in 14 (5.1%) isolates. The plasmid-mediated quinolone resistance genes, qnr and aac(6')-Ib-cr, were identified in 16 (5.8%) and 3 (1.1%) isolates, respectively. Finally, the florphenicol resistance gene, floR, was identified in four (1.5%) isolates. To the best of our knowledge, this is the first report for molecular characterization of antimicrobial resistance in Gram-negative bacteria isolated from fish farms in Africa.

Prevalence and molecular characterization of ampicillin-resistant Enterobacteriaceae isolated from traditional Egyptian Domiati cheese.

The aim of this study was to address the prevalence and the molecular characteristics of antibiotic-resistant enteric bacteria isolated from one of the most popular types of Egyptian cheese. A total of 215 ampicillin-resistant enterobacterial isolates were obtained from 80 samples of Domiati cheese, and they were screened by PCR for a large pool of antibiotic resistance markers, including extended-spectrum beta-lactamases (ESBLs), class 1 and class 2 integrons, and plasmid-mediated quinolone resistance genes. It was determined that the most frequent mechanism of ampicillin resistance was from a TEM-1-type beta-lactamase. As well, SHV beta-lactamases, including SHV-1, SHV-25, and SHV-26, showed a high prevalence, and two novel SHV beta-lactamases, SHV-110 and SHV-111, were identified. Type CTX-M-14, OXY-1, OXA-1, and CMY-4 beta-lactamases were also detected in a few isolates. In addition, a novel AmpC beta-lactamase was detected that was designated CMY-41. Sequencing results of class 1 integrons revealed that the uncommon aminoglycoside resistance gene cassette aadA22 was found for the first time in an Escherichia coli strain. The other class 1 integrons harbored various common gene cassettes, including aadA1, aadA1a, aadA2, aadA12, dfr5, dfr7, dfr12, and dfr15. The only isolate that carried a class 2 integron contained dfrA1, sat2, and aadA1. Plasmid-mediated quinolone resistance determinants qnrS and qnrB showed a low prevalence. This study provides meaningful data on high antimicrobial resistance contained in Domiati cheese samples and reports for the first time the presence of beta-lactamases, plasmid-mediated quinolone resistance, and integrons in isolates from food of Egyptian animal origin.

Genetic basis of multidrug resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves in Egypt.

Up to this date, nothing is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals in Africa. Therefore, this study was carried out to screen the incidence of multidrug-resistant (MDR) strains of Salmonella from neonatal calf diarrhea in Egypt and also to characterize the molecular basis of this resistance. Nine unique Salmonella isolates were obtained from 220 fecal samples, and six of these showed multidrug resistance phenotypes and harbored at least two antimicrobial resistance genes. Four were Salmonella enterica serovar Typhimurium and two were S. enterica serovar Enteritidis. Class 1 integrons were identified in all MDR Salmonella isolates. The identified gene cassettes within class 1 integrons were as follows; aminoglycoside adenyltransferase type A (aadA1, aadA2 and aadA5), which confer resistance to streptomycin and spectinomycin, and dihydrofolate reductase gene cassettes (dfrA1, dfrA15 and dfrA15), which confer resistance to trimethoprim. A class 2 integron containing dfrA1-sat2-aadA1 gene cassettes was identified in only one isolate of S. enterica serovar Enteritidis. The beta-lactamase-encoding gene, bla(TEM-1), was identified in five isolates and the extended-spectrum beta-lactamase-encoding genes, bla(CMY-2) and bla(SHV-12), were identified in S. enterica serovar Typhimurium. Furthermore, the plasmid-mediated quinolone resistance genes, qnrB, qnrS and aac(6')-Ib-cr, were also identified. To the best of our knowledge, this is the first report of qnrS in S. enterica serovar Enteritidis, qnrB in S. enterica serovar Typhimurium, and aac(6')-Ib-cr in Salmonella of animal origin. Also, this is the first report of the molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Africa.

Genetic analysis of antimicrobial resistance in Escherichia coli isolated from diarrheic neonatal calves.

This study was carried out to screen and analyze the genetic basis of antimicrobial resistance in Escherichia coli strains isolated from neonatal calf diarrhea in Egypt. A total of 182 isolates of E. coli recovered from 91 diarrheic neonatal calves were analyzed for antimicrobial susceptibilities, the presence of class 1 and class 2 integrons and antimicrobial resistance genes. Nineteen isolates (10.4%) showed multidrug resistance phenotypes and harbored at least three antimicrobial resistance genes. PCR screening detected class 1 integrons in 19 isolates (10.4%) and class 2 integrons in 2 isolates (1.1%). The identified antimicrobial resistance genes within class 1 integrons were dihydrofolate reductase types: dfrA1, dfrA12, dfrA15 and dfrA17, which confer resistance to trimethoprim; aminoglycoside adenyltransferase types: aadA1, aadA2, aadA5, aadA7 and aadA23, which confer resistance to streptomycin and spectinomycin; and aminoglycoside acetyltransferase gene, aac(3)-Id, which confers resistance to gentamicin and sisomicin. Furthermore, many beta-lactamases encoding genes, plasmid-mediated quinolone resistance genes and florfenicol resistance gene were identified in this study. To the best of our knowledge, this is the first report for molecular characterization of antimicrobial resistance in E. coli isolated from diarrheic neonatal calves in Africa.