DsrA RNA is a small [87-nucleotide] regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translationand turnover of specific mRNAs . Two targets of DsrA regulationare RpoS, the stationary-phase and stress response sigma factor[^s], and H-NS, a histone-like nucleoid protein and global transcription repressor . Genes regulated globally by RpoS and H-NS includestress response proteins and virulence factors for pathogenicE . coli . Here, by using transcription profiling via DNA arrays,we have identified genes induced by DsrA . Steady-state levelsof mRNAs from many genes increased with DsrA overproduction,including multiple acid resistance genes of E . coli . Quantitativeprimer extension analysis verified the induction of individualacid resistance genes in the hdeAB, gadAX, and gadBC operons. E . coli K-12 strains, as well as pathogenic E . coli O157:H7, exhibited compromised acid resistance in dsrA mutants . Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant . Thus, DsrA RNA plays a regulatory role in acid resistance . Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in eitherevent, our results suggest that DsrA RNA may enhance the virulenceof pathogenic E . coli.

 
Regulation by RNA, termed riboregulation, plays a substantialrole in modulating gene expression in bacteria [reviewed inreferences 14, 17, 26, 33, 42, 43, and 44] . In addition to theirclassically studied role in plasmid maintenance [reviewed inreference 42], Escherichia coli small RNAs act to change theconformation of target mRNAs [DsrA and RprA], block mRNA translationby occlusion of Shine-Dalgarno sequences [MicF, OxyS, Spf, andRyhB], degrade target mRNAs [DsrA, Oop, and RyhB], and titratespecific protein factors [OxyS and CsrB RNAs, 6S RNA], sometimesin combination.

Several E . coli small RNAs coordinate stress responses or virulence factors [reviewed in reference 14] . A principal advantage ofsmall RNAs as regulators is that they are not translated andtherefore cost less energy to produce than do proteins . Also, many bacterial small RNAs are relatively stable and can persist to target transcripts with high specificity by antisense interactions [reviewed in references 17 and 43] . Some small RNAs are degradedtogether with their target mRNAs [22].

One such RNA, DsrA RNA, is a small [87 nucleotides], multifunctional genetic regulator of E . coli . DsrA RNA modulates the levels of two global transcription regulators, RpoS [^s, the productof the rpoS gene] and H-NS [a nucleoid protein and transcriptionsilencer in bacteria, produced from the hns gene] . DsrA actsby sequence-specific RNA-RNA interactions to enhance translationof rpoS RNA and to stabilize rpoS message [18-20, 35] . In additionto its role at rpoS, DsrA also binds hns mRNA by specific base-pairinginteractions and blocks H-NS translation as it sharply increaseshns mRNA turnover [18] . The first stem-loop region of DsrA melts out to contact rpoS mRNA, whereas the second stem-loop region of DsrA base pairs with hns mRNA . This conformational change within DsrA acts to switch the translation state of two different mRNA targets [reviewed in references 1 and 17] . Like that ofmany small, noncoding regulatory RNAs, direct DsrA activityon mRNAs requires Hfq, an Sm domain RNA-binding protein andputative RNA chaperone [reviewed in reference 43].

DsrA perturbation of H-NS and RpoS results in increased transcription of downstream genes repressed by H-NS or activated by RpoS [19, 35] . H-NS and RpoS also act in concert to permit the transcriptionof a number of stress response and virulence factor proteins[reviewed in references 3 and 15] . Many genes require additionalregulatory proteins, in conjunction with H-NS and RpoS, to tailorspecific responses to particular environmental stresses [reviewedin reference 15] . The downstream effect of DsrA is therefore predicted to be the induction of a pleiotropic stress response.

Despite the study of key components of the DsrA regulatory network, the phenotype of DsrA activity in the cell has remained elusive. Here we used a genomics approach to define the downstream effects of DsrA in E . coli . DNA array-based transcriptome analysis suggests that DsrA stimulates acid resistance, which is known to enhance the virulence of pathogenic E . coli strains [9 and referencestherein] . Both the hdeAB and glutamate-dependent [gad] acidresistance systems were induced, although the arginine-dependent [adi] genes were apparently not induced by DsrA . Furthermore, both nonvirulent [K-12] and pathogenic [O157:H7] strains of E . coli had compromised acid resistance in dsrA-null mutants. DsrA therefore plays a role in cellular acid resistance, an important feature for the survival of enteric bacteria in low-pH environments and for the virulence of pathogenic E . coli.

 
Media, strains, and plasmids. Cells were grown in Luria-Bertani [LB] medium [28] . Where appropriate,antibiotics were used at the following concentrations: ampicillin,100 µg/ml; chloramphenicol, 25 µg/ml; tetracycline,10 µg/ml; kanamycin, 50 µg/ml; streptomycin, 50µg/ml . E . coli strain M182 is described elsewhere [8].E . coli O157:H7 strain 1957 is a clinical isolate from stoolor fecal material from 2001, provided by the Wadsworth CenterBacteriology Laboratory . Strain M182 dsrA::cat was made by P1transduction of the cat gene from C600 dsrA::cat, which was provided by Susan Gottesman [National Institutes of Health,Bethesda, Md.] . E . coli strain SM10 tra⁺ Kan^r [pir] [32] wasprovided by Kelynne Reed [Austin College, Sherman, Tex.].

Strain O157 dsrA::cat was constructed as follows . The dsrA::cat allele, along with ca . 900 bp of the flanking chromosomal DNA sequence, was amplified by PCR with primers W1748 [GCT CTA GAAAGA GAC AAC GAT AAC CTC G] and W1749 [GCT CTA GAG CGT AAT CCATTA CCT CCA G] and was cloned by blunt-end ligation into pir-dependent gene replacement vector pCVD442 [ori-R6K mob⁺ Amp^r sacB] [11]at a filled-in XbaI restriction site . The recombinant plasmidDNA was used to transform DH5 [pir] and plated on LB mediumcontaining chloramphenicol and ampicillin . The resulting plasmid,pCVD442 dsrA::cat, was screened by restriction analysis andthen used to transform E . coli SM10 . Separately, a streptomycin-resistantvariant of O157:H7 was selected by plating on LB medium containingstreptomycin . This O157 [Str^r] strain was mated with SM10 Kan^r/pCVD442dsrA::cat in liquid culture for 2 h at 37°C . E . coli O157 Str^r/pCVD-dsrA::cat was selected on plates [LB medium containing streptomycin and chloramphenicol] . Selected clones were checked for kanamycin sensitivity and ampicillin resistance by patching of coloniesonto plates . To prepare dsrA::cat chromosomal integrants, gene replacement was performed by growing cultures of O157 Str^r/pCVDdsrA::cat and plating on LB medium containing 5% sucrose and chloramphenicol. Most sucrose-resistant colonies were Amp^s, indicating loss of the pir-dependent gene replacement vector . The exchange of dsrA for the dsrA::cat chromosomal allele was verified by chloramphenicolresistance and by PCR with primers W1748 and W1749.

Plasmids pBRdsrA and pBRdsrA^*H were constructed by cloning the BamHI fragment of pDDS164 [34] or pDsrA^*H [19] into the BamHIsite within the Tet^r gene of pBR322 . Potential clones were selectedfor ampicillin resistance and then screened for insertionalinactivation of the Tet^r gene [Tet^s] on LB medium containingtetracycline . Clones were confirmed by restriction analysisand DNA sequencing.

Transcription profiling. Freshly streaked cells of strain M182 containing either pBR322or pBRdsrA were grown overnight in LB broth plus ampicillinat 37°C . Cells were then diluted 1:100 into 30 ml of LBbroth plus ampicillin for growth at 30°C to induce DsrA [35] . Cells were grown with vigorous shaking to an optical densityat 600 nm of 0.3 to 0.4 . Total cellular RNA was prepared andused to make labeled cDNA by reverse transcription in the presenceof [-³³P]dATP [NEN] . An oligonucleotide mixture complementaryto every E . coli mRNA 3' end [Sigma-Genosys] was used to primecDNA synthesis . The ³³P-labeled total cellular cDNA was usedto probe filter-based DNA arrays [Sigma-Genosys] as previouslydescribed [39] . Filters were then exposed to phosphorimagerscreens [Molecular Dynamics, Inc.] and scanned . Images werequantified with Arrayvision software [Imaging Research, St.Catharines, Ontario, Canada] . The experiment was performed twice.Hybridization signals were normalized to total genomic DNA standardspresent on each filter . Differential expression for each setof experiments was determined for individual genes by dividingthe signal from the DsrA-overproducing strain by that of theplasmid control . For images of arrays and spot data files, seethe supplemental material.

Primer extension analysis. RNA was extracted and primer extension was performed as previouslydescribed [5, 18] . Primer sequences are as follows: adiA + 74, CTT GAT GGA GAA ACT CGC TTT CAA C; adiY + 83, AGT TCT CGC TAAAGC AAA GCG ATA C; gadA + 48, CGT TAA CAG CTT CTG GTC CAT TTCG; gadB + 64, GTT CCG ACC TTA AAT CCG TTA CTT G; gadX + 89,GGT GAG AAT ATA TTT ATG TCT TGC; nfnB + 37, TAT CCA TAA AGACTC CAT GTG AAA G; W538dsrA, GAA ACT TGC TTA AGC AAG AAG C.The hns- and stpA-specific primers [48] and the hdeA-specificprimer [2] are as previously described . All DNA oligonucleotideswere purified via elution from polyacrylamide gels . The DNAoligonucleotides were end labeled with [-³²P]ATP [Perkin-Elmer/NEN]and T4 polynucleotide kinase [New England Biolabs] as previouslydescribed [28], extracted with 1 volume each of phenol and thenwith chloroform-isoamyl alcohol [24:1], and purified by TE-10spin column chromatography [Clontech].

Acid resistance assays. Cultures of strains M182 and O157 and their respective dsrAmutants and merodiploid strains were grown overnight at 37°Cand tested by dilution into LB medium at pH 2.0 as previouslydescribed [13], except that samples were taken each hour forplating for up to 6 h of acid treatment at 37°C . Cells werediluted in 10 mM Tris-HCl [pH 7.5]-1 mM magnesium chloride priorto plating . Percent survival is given as the titer of the CFUof acid-tested cells compared to that of a zero-time, untreatedcontrol sample.

 
Global mRNA analysis. To determine the downstream effects of DsrA, we induced DsrAexpression from a plasmid . Changes in the expression of totalcellular RNA from cultures with and without DsrA overproductionat 30°C were compared by using genomic DNA filter arrays.Whereas transcript levels from many genes increased or decreasedseveralfold [Table 1], a number of genes related to acid resistancewere strongly induced by DsrA overproduction [Table 1, top].The hdeAB operon, which encodes the H-NS-repressed acid resistanceproteins HdeA and HdeB [12, 46, 47], produced 10- to 26-foldhigher hdeA transcript levels and 12- to 43-fold higher hdeBtranscript levels when DsrA was overproduced [Table 1, lines1 and 2] . Other H-NS- and RpoS-regulated acid resistance operonelements were also induced . For example, the gadAX operon, whichencodes glutamate decarboxylase, GadA, and its positive regulator,GadX [40], was induced as much as threefold [Table 1, lines4 and 5] . Similarly, a gene that encodes a positive regulatorof arginine-dependent acid resistance, adiY, was induced ca. fivefold [Table 1, line 7] [38].

 
Primer extension analysis. To verify and quantitate induction of the specific genes mentionedabove, as well as to check for the overexpression of severalgenes expected to be induced that were not seen by array analysis,we measured the levels of key transcripts by gene-specific primerextension analysis . Cells containing plasmid vector were comparedto cells overproducing either DsrA or a mutant DsrA variantfrom a plasmid . Identical growth conditions were used for thearray and primer extension analyses . The mRNA levels of the acid resistance genes hdeA, gadA, gadX, gadB, adiY, and adiAwere analyzed [Fig . 1; quantitation in Table 1, column five;data for adiY and adiA not shown] . As a positive control, we determined the transcript levels of stpA . StpA is an H-NS paralog that is induced in hns mutants [48] and that registered a 1.2-to 2.3-fold increase in the array analysis [Table 1, line 18].As a negative control, levels of the unrelated nfnB nitroreductasemRNA were also checked [4].

 
The data confirm the induction of several types of acid resistance genes by DsrA, and the lengths of all cDNAs are consistent with transcription starting at the major promoter of each gene . Thelevels of hdeA transcript were increased 26-fold, corresponding exactly to those determined by the array analysis [Fig . 1, panel5, and Table 1, line 1] . The levels of gadA mRNA were also increased,by a factor of 3.5, again providing correspondence between thetwo methods [Fig . 1, panel 2, and Table 1, line 4], and stpA mRNA was increased 3.1-fold [Fig . 1, panel 4] . A modest 2.6-foldincrease observed for gadX mRNA was again corroborative [Fig.1, panel 1, and Table 1, line 5] . Interestingly, we also founda 3.5-fold increase in gadB mRNA, whereas such induction hadbeen undetectable by array analysis [Fig . 1, panel 3, and Table 1, line 6] . We attribute the differences between the two methodsto the fact that oligonucleotide cDNA primers for the array analysis were optimized for cloning of the PCR products displayed on the arrays [25] and not necessarily for annealing to thespecific mRNAs . Conversely, although the adiY transcript was3.7- to 5.7-fold elevated in the array analysis [Table 1, line7], we were unable to detect either adiY or adiA transcriptsby primer extension analysis [data not shown] . Regardless, sincegadB and gadC form an operon and since gadC is required forglutamate decarboxylase-based acid resistance [9], we can concludethat both the hdeAB and glutamate-dependent acid resistance[gad] genes are induced by DsrA.

To confirm that changes in transcript levels resulted from DsrA function, we measured acid resistance gene transcripts after induction of a DsrA variant, DsrA^*H, which is compromised in its ability to regulate hns or rpoS [Fig . 1, all panels, laneM] [19] . Variant plasmid pBRdsrA^*H generated levels of DsrA^*H mutant RNA comparable to those generated by the wild-type DsrAplasmid [Fig . 1, panel 6, compare lanes D and M] . The plasmidsthat produce DsrA and DsrA^*H are isogenic except for five pointmutations within the dsrA gene . DsrA^*H is unable to base pairwith hns mRNA and cannot significantly activate the translationof rpoS mRNA, although DsrA^*H can pair with an altered hns allele [19] . These data indicate that DsrA and not other plasmid productsor sequences induced these acid resistance genes.

Acid resistance phenotype test. The pattern of gene induction described above suggests a rolefor DsrA in acid resistance . Consistent with these observations,in preliminary experiments E . coli K-12 strain M182, overexpressingDsrA from one of several plasmids, displayed increased acidtolerance at pH 3.8 by a factor of 12- to 5,000-fold relativeto that of control M182 cells [data not shown] . To confirm aphysiological role for DsrA in acid resistance, we comparedE . coli M182 to an otherwise isogenic dsrA-null mutant [M182dsrA::cat] for the ability to survive immersion in pH 2 medium.Growth and acid treatment were at 37°C . The K-12 strainswith dsrA deleted were killed more readily at pH 2 than wasthe wild type [Fig. 2A and B, compare filled and open circles].The downward trend indicates a 10²- to 10³-fold disadvantage in survival at low pH between 1 and 5 h for the dsrA mutant, supporting a physiological role for DsrA.

 
To verify that DsrA is responsible for the acid resistance seen,we complemented the dsrA-null strain with wild-type or mutant DsrA produced in trans from a plasmid [Fig . 2A, filled and opentriangles] . Wild-type DsrA overproduction in the dsrA-null mutantrendered M182 strongly acid resistant even up to 6 h of treatmentat pH 2 . The strain overproducing DsrA was up to 10⁶-fold moreacid resistant than the dsrA mutant.

Curiously, we saw a partial restoration of acid resistance inthe dsrA-null mutant when we overproduced the altered DsrA^*H variant in trans from a plasmid [Fig . 2A, compare open circlesand open triangles] . The DsrA^*H-producing plasmid restored alevel of acid resistance comparable to that of the wild-typestrain [Fig . 2A, cf . filled circles and open triangles] . However,wild-type DsrA was considerably more effective than DsrA^*H forpromoting acid resistance survival when overproduced [10³-folddifference at 6 h] [Fig . 2A, cf . open and filled triangles].As DsrA^*H cannot act directly on hns or rpoS RNA, an independent mechanism of acid resistance is implied, possibly via directDsrA base pairing with other RNAs, such as, for example, putativemRNA targets argR, ilvI, and rbsD [19] . Nevertheless, the strongand persistent acid resistance phenotype of the DsrA-overproducingstrain, coupled with compromised acid resistance in dsrA-nullmutants relative to the wild type, substantiates the physiologicalrelevance of DsrA in acid resistance.

Acid resistance in a pathogen. The ability to survive at a low pH in the stomach is consideredto be a factor that permits pathogenic bacteria to establishan infection [9 and references therein] . We theorized that theenterohemorrhagic pathogen E . coli O157:H7 might use DsrA toinduce acid resistance . Accordingly, we assayed E . coli O157:H7and its dsrA null mutant for acid resistance . An E . coli O157 clinical isolate displayed patterns of acid resistance differentfrom those of K-12 laboratory strain M182 [Fig . 2B, cf . circles and squares], with the pathogenic strain E . coli O157 being considerably more acid resistant than K-12 . Again we found a clear trend of compromised acid resistance in the dsrA::cat mutant [Fig . 2B, cf . filled and open squares], with a 10- to75-fold reduction in acid resistance of E . coli O157 in theabsence of DsrA.

 
DsrA global effects. The mRNA levels of many genes increase, while levels of severalmRNAs decrease in response to DsrA overproduction [Table 1].The affected genes are distributed around the chromosome andperform multiple functions [Table 1 and Fig . 3A] . Most notably, acid resistance genes are induced by DsrA overexpression [Table 1 and Fig . 1] . Apart from acid resistance, no single unifyingtheme emerged from our analysis of these data, although regulatedgenes include membrane proteins and sugar metabolism operons[Fig . 3A] . These array data, in combination with studies ofspecific genes, serve as a departure point for generation oftestable hypotheses . The role of condition-specific regulatoryfactors may predominate; for example, only two of the flagellarregulatory and synthesis genes were induced [Table 1, lines22 and 23], and not a multigene cascade . Thus, hypotheses generatedfrom these data could be individually tested under appropriateconditions and strains, as was done for acid resistance [Fig.2] . Not all genes were detected by arrays [e.g., stpA, Table1, line 18], possibly because of suboptimal annealing of cDNAprimers, mRNA secondary structures, or degradation at the 3'ends.

 
DsrA enhances acid resistance. In comparisons of wild-type and dsrA-null mutants, both E . coliK-12 and pathogenic O157 strains are compromised in the abilityto survive a low-pH challenge if dsrA is knocked out [Fig . 2]. Also, dsrA mutants were rendered strongly acid resistant by overproduction of DsrA in trans . Taken together, these results imply a physiological role for DsrA in acid resistance . Multipleacid resistance responses [gadAX, gadBC, and hdeAB operons] are depicted as part of a DsrA regulatory circuit [Fig . 3B]. DsrA presumably acts indirectly, via H-NS and RpoS . However,in some cases DsrA may act directly [Fig . 3B, gray dotted arrow], by RNA-RNA interactions with specific target mRNAs, as it does with hns and rpoS . One such example is the periplasmic ribose-bindingprotein operon [rbs] transcript, which was identified as a potentialDsrA target by virtue of complementarity [19], and which isdepressed about threefold when DsrA is induced [Table 1, line14].

The greater effectiveness of wild-type DsrA than DsrA^*H in complementingthe dsrA-null mutant for survival at low pH should be considered[Fig . 2A] . Clearly, DsrA^*H cannot induce hde or gad acid resistancegenes [Fig. 1] . It is feasible that either DsrA^*H or plasmid sequences titrate out a regulatory molecule such as Hfq or LeuO, respectively [16, 36] . LeuO and Hfq have both been shown tocomplement an hns mutant for repression of certain acid-induciblegenes [29, 30] . Another possibility is that an additional, H-NS-independentpathway is responsible for DsrA^*H partially complementing theDsrA null mutant . DsrA interacts with hns and rpoS mRNAs bydifferent base pairing via discrete portions of the DsrA molecule[17] . The hypothesis that DsrA might interact with another targetvia different nucleotides than those mutated in DsrA^*H is thereforea viable option . DsrA contains regions of antisense complementarityto at least another three genes, namely, argR, ilvI, and rbsD [19], although direct base pairing between DsrA and these mRNAshas not been demonstrated.

Other acid resistance gene networks that are independent ofDsrA [e.g., those regulated by EvgA-YdeO-YhiE or GadW] undoubtedlycombine with these networks to impart acid resistance [23, 41].It is noteworthy that E . coli O157 possesses the same threeacid resistance systems found in K-12 [9] . Since E . coli O157contains >1.4 Mb of DNA absent from K-12 [reviewed in reference45], it is likely that the pathogen utilizes additional anddivergent regulation of gene expression that significantly enhancesacid resistance.

Concerted acid and osmotic shock stress responses. Surprisingly, while low temperature induces DsrA [24], low temperaturedoes not induce gadA in wild-type or dsrA-null strains [R.A.L.,unpublished data], suggesting specific integration of appropriateenvironmental signals in acid resistance regulation . Also, besidesits role in acid resistance, DsrA protects E . coli from osmoticshock . DsrA induces the proU hyperosmotic shock operon proVWXby more than threefold [Table 1, line 9] [19] and the hypo-osmoticshock gene ompF by 2- to 2.5-fold [Table 1, line 12] . Under hyperosmotic conditions, dsrA knockout mutants are compromised for survival [21] . Thus, osmotic and acid stress responses maybe integrated via DsrA [Fig . 3B] . Interestingly, both hyper-and hypo-osmotic shock conditions induce expression of gad acidresistance genes [10], consistent with interdependence of acidand osmotic shock protective mechanisms . A unifying theme isthat maintenance of membrane chemiosmotic potentials and controlof cell permeability would be common to these and other stressresponses.

DsrA as a virulence factor coordinator. The mRNA for the acid resistance and virulence factor regulatoryprotein GadX is induced by DsrA overexpression [Table 1, line5; Fig . 1, panel 1, and 3B], repressed by H-NS, and activated by RpoS [Fig . 3B] [40] . GadX [formerly YhiX] is an AraC-likeregulator produced from the gadX promoter, as well as cotranscriptionallyfrom the gadAX operon . GadX functions as a master activator[and autoactivator] for gadAX, gadBC, hdeAB, and hdeD, and other acid resistance-related genes [Fig . 3B] [23, 41] . GadX alsorepresses Per, a regulatory protein of enteropathogenic E . colithat is absent from enterohemorrhagic E . coli [EHEC] strainssuch as O157 . In these enteropathogenic E . coli strains, Peractivates virulence factors produced from a bacterial pathogenicityisland [LEE] [31 and references therein] . In EHEC strains, LEE virulence factors are induced by quorum sensing and a regulatory cascade that involves RpoS induction, as well as factors that antagonize H-NS silencing of LEE [7, 37] . Coordination of acidresistance and adherence phenotypes by DsrA modulation of RpoSand H-NS levels could benefit bacteria that pass from the low-pHenvironment of the stomach to sites of potential attachmentand effacement in the intestine.

 
We thank Jonathan Hibbs and Susan Gottesman for generously providing bacterial strains; Kelynne Reed for strains and advice; LoriConlan, Colin Coros, Coby Slagter-Jäger, and David Edgellfor comments on the manuscript; and M . Carl and J . Dansereaufor help with the manuscript and figures, respectively . We appreciatethe services of the Wadsworth Center Molecular Genetics andGenomics Core Facilities . Sarah Woodson provided laboratoryresources, as well as valuable comments on the manuscript.

This work was supported by NIH grants GM39422 and GM44844 toM.B . and GM46686 to Sarah Woodson.

 
* Corresponding author . Mailing address: Department of Biophysics, Jenkins Hall, Homewood Campus, The Johns Hopkins University, 3400 N . Charles St., Baltimore, MD 21218 . Phone: [410] 516-6536 . Fax: [410] 516-4118 . E-mail: ral@jhu.edu .

Supplemental material for this article may be found at http://jb.asm.org.

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What Is Bioremediation?, What Is Cell Biology?, What Is Growth Medium?, What Is Genetics?, What Is Yeast?, r, Bacteriology, a, Microbe, i, Microorganisms, n, Microbiology, c, Bacteria, a, Yeasts, a, Microbial, s, Microbial, e, Microbial, n, Pasteurella, n, Erythromycin, c, Bacillus, e, Campylobacter, n, Streptococci, c, S. cerevisiae, a, Bacillus, r, Clostridia, o, Meningococcus, c, Pasteurella, c, Haemophilus, e, Staphylococcus aureus, n, Salmonella, c, Antimicrobial, o, Eubacter, s, Yeasts, n, Haemophilus




 

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