Biocatalysis and Agricultural Biotechnology 22 (2019) 101401

Contents lists available at ScienceDirect

Biocatalysis and Agricultural Biotechnology

Persister cell development among , , Mycobacteriaceae and Staphylococcaceae biotypes: A review

Somanath Behera , Smaranika Pattnaik *

Laboratory of Medical , Dept. of Biotechnology & Bioinformatics, Sambalpur University, Odisha, India

1. Introduction Spectropho to metric as say) and more over 16S rDNA sequenc ings were adopted for the purpose of charac ter i za tion and identi fi ca tion. There 1.1 . Persisters was devel opment of Black coloured colonies on the Congo Red agar plates ( Fig. 1 ) indi cat ing the presence of biofilm sub stan tiated by Treatment of infec tious dis eases is often long and requires patients spectropho to metric (absorbance taken at 570 nm, λ) analy sis to take drugs even after they have seemingly recov ered. This is be- ( Kirmusauglu, 2017 ). This is to mention that clini cally rele vant multi cause of a phenom e non called persis tence, which al lows small frac- drug resis tant bacte ria with biofilm produc tion are the poten tial can - tions of the bacte r ial popu la tion to survive treatment despite being didates to produce persis ters ( Pang et al., 2019 ). Strong biofilm for- genet i cally sus cepti ble ( Martinecz and AbelzurWiesch, 2018 ). Stud ies mation is an impor tant prereq ui site for persis ter devel opment ( Singh have attrib uted this treatment failure to the presence of a small, tran- et al., 2017 ). siently multidrug -tolerant sub popu la tion of cells, called persis ter cells. Gentam icin (drug of choice) impreg nated discs (30mcg) had been Wood et al. (2013) had defined that bacte r ial cells may escape the ef- placed on the Nutri ent agar (NA) agar plates seeded with O/N fects of antibi otics without under go ing genetic change; these cells are (10 3 CFU/ml) cultures of BMS2 and incu bated at 37 °C for a period of known as persis ters. Fisher et al. (2017) had elabo rated that, persis ter 14 h. After incu ba tion period, it was observed that there was growth cells are sub popu la tion of transiently antibi otic -tolerant bacte r ial cells of a small popu la tion of colonies on the vicinity of zone of inhi bi tion that are often slow -growing or growth- arrested and are able to resume ( Fig. 2 ). From this obser va tion it was inferred that a sub popu la tion of growth after a lethal stress. These special cells survive antibi otic treat- persis ters arose in low lev els after antibi otic treatment and devel oped ments by reduc ing their metab o lism; i.e., rather than by mounting an as sec ondary popu la tion maintain ing bi- phasic growth curve pattern active response to the antibi otic stress, such as acti vat ing efflux as sug gested by Orman et al. (2016) . The killing of Gentam icin could pumps and rather than under go ing muta tion, they survive antibi otics have fol lowed a biphasic pattern — rapid death of the bulk of the pop- by becom ing dormant ( Kim and Wood, 2016 ). u la tion fol lowed by a dimin ish ing killing rate of a more tol erant sub - And more over, after the initial rapid elimi na tion of bacte ria, the popu la tion ( Keren et al., 2011 ). Hence, the bacte r ial popu la tion devel - rate of elimi na tion slows down by fol lowing a biphasic killing pattern oped in vicinity of zones of inhi bi tion was con sid ered as the persis - ( Lewis, 2010 ). Hence, persis ter cells have been identi fied in every ma- ters. jor pathogen, con tribute to the antibi otic tol erance observed in This small number of pheno typic variants, defined as ‘persis ters ’, biofilms,and are respon si ble for the recal ci trant nature of chronic in- are refrac tory to antibi otics and survive treatment. The mecha nisms fections ( Defraine et al., 2018 ). behind this phenom e non remain largely unknown in spite of recent A modi fied method of TD (Tol erance detec tion) test ( Gefen et al., advances, in great part because of the diffi culty in isolat ing the very 2016 ) for easy detec tion of bacte r ial tol erance and persis tence in clin- small fraction of the popu la tion that is in this state at any given time ical isolates us ing con ventional ‘Disc diffu sion ’ method ( Bauer et al., ( Cañas -Duarte et al., 2014 ). Moreover, the proce dure for persis ter cell 1966 ) was carried out in the labo ratory of Medical Micro bi ol ogy, isola tion relies on acti va tion of the SOS response to gether with strin - Dept. of Biotechnol ogy and Bioinfor mat ics, Sambalpur Uni versity, In- gent response ( Dorr et al., 2010 ). dia. Briefly,the strain BMS2 was availed from dept. of Micro bi ol ogy, With this prelim i nary obser va tion on the devel opment of persis ters Veer Suren dra Sai Insti tute of Science and Research, Burla, India. The of BMS2, this review work was initi ated with an aim to under stand ing appro pri ate diag nos tic bacte ri o log ical tools ( Pattnaik, 2017 ) like the biol ogy of persis ters of vari ous biotype groups (Enter obac te ri - colony morphol ogy, Bio typing tests, Biofilm as say (Congo Red and aceae, Pseudomonadaceae, Staphylo coc caceae and Mycobac teri aceae)

* Corresponding author. Department of Biotechnology & Bioinformatics, Sambalpur University, Jyoti Vihar, Burla, 768019, Odisha, India. E- mail addresses: som7071@ gmail. com (S. Behera), smaranika2010@ suniv. ac. in (S. Pattnaik). https://doi.org/10.1016/j.bcab.2019.101401 Received 3 April 2019; Received in revised form 6 October 2019; Accepted 25 October 2019 Available online 4 November 2019 1878-8181/© 2019 Published by Elsevier Ltd. S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401

which are noto rious and causal organ isms of chronic persis tent infec - tions.

1.2 . Phenotype of persisters

Persis ter cells con sti tute a sub popu la tion of dormant cells within a micro bial popu la tion which are genet i cally identi cal but pheno typ i - cally differ ent from regu lar cells ( Willenborg et al., 2014 ). Persis tence is linked to pre- existing hetero gene ity in bacte r ial popu la tions be- cause pheno typic switching occurred between normally growing cells and persis ter cells having reduced growth rates. When a popu la tion of genet i cally identi cal bacte r ial cells is exposed to a suffi ciently strong antibi otic treatment, most of the popu la tion is killed. The death of the major ity of the popu la tion takes place as a fast expo nen tial decay charac ter ized by a sin gle para me ter, the killing rate. After a few hours, the initial purely expo nen tial decay of the killing curve changes to a more complex, slowly decreas ing function. By the time the antibi otic is removed, a small fraction of the cells still survives. These cells have not genet i cally acquired antibi otic resis tance: They re grow anew popu la tion that is as sensi tive to the antibi otic ( Balaban et Fig. 1. Ap pearance of Black colonies on the Congo Red agar plates in dicate al., 2004 ). It was sug gested that they are transiently refrac tory to de velop ment of biofilm. The merged colonies of BMS2 have produced a thick killing, without having acquired resis tance through genetic modi fi ca - biofilm. The Red colour of Congo Red agar is clearly vis ible in the plate where tion ( Keren et al., 2004 ). It might be expected that non- genetic inher i - the colonies have not grown. (For in terpre ta tion of the refer ences to colour in tance would be involved in at least some as pects of the persis ter cell this figure leg end, the reader is referred to the web version of this arti cle.) phenom e non ( Day, 2016 ). This “bet- hedging strat egy” is said to be a survival strat egy under fluctu at ing envi ron ment con ditions ( Petchiappan and Chatterji, 2017 ). Hence, a bacte r ial popu la tion fol - lows more than one way to make persis ters with estab lish ment of plu- rality ( Allison et al., 2011 ; Muthuramalingam et al., 2016 ) and the con cept was well estab lished by mathe mat i cal deriva tions made by Ogle et al. (2017) . The Pheno typic plural ity is embod ied in the biofilm para digm and geno typic plural ity is embod ied in the con cepts of the supra- genome and the dis tributed genome hypoth e sis ( Ehrlich et al., 2005 ). Inter est - ingly, the dormant cells in one of these stud ies appear to be narrower than normally growing cells, sug gest ing that sta tionary - phase cel lu lar dwarfing ( Orunno et al., 2011 ) are antibi otic -resistant pheno types ( Miyaue et al., 2018 ). As an adaptive trait, persis tence is hetero ge - Fig. 2. The growth of a sub popu la tion of cells in the vicin ity of zone of in hibi - neous and emerges via multi ple mecha nisms ( Cabral et al., 2018 ). Al- tion formed around a Gentam icin impreg nated disc tested against a strain of Staphy lococ cus au reus (BMS 2). though persis tence mecha nisms are con served in terms of the gene function and pathways involved among differ ent bacte r ial species, they may vary in gene homol ogy and rela tive impor tance of a given pathway ( Cui et al., 2016 ). Further, there are differ ences in the mech-

Fig. 3. A model showing a picto rial view of heteroge nous resus ci ta tion of persis ters after Antibi otic removal. The persis ters being in side a biofilm survive and at - tain three types.

2 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401 a nisms of persis ter cell forma tion in Gram- negative and Gram- positive geneity in cell length, possi bly con tributed to cell -to -cell varia tion in bacte ria. It has been reported by Kim and Wood (2018) that persis ters host and antibi otic stress tol erance ( Vijay et al., 2017 ). have low lev els of antibi otic -corrupting proteins and that their resus - In this con text, Khlebodarova and Likhoshvai (2018) proposed a ci tation is hetero ge neous which was evi denced by (i) imme di ate divi - con cept of ‘popu la tion drift’ in the space of multi ple neutrally cou- sion ( Michael et al., 2010 ), (ii) imme di ate elon ga tion fol lowed by di- pled muta tions lead ing “neutrally coupled co- evolution ” (NCCE), as a vision ( Barrett et al., 2019 ), (iii) imme di ate elon ga tion but no divi sion result of which there is appear ance of cells capa ble of carry ing out a ( Bos et al., 2015 ), (iv) delayed elon ga tion/di vi sion ( Yu et al., 2019 ), sin gle cell cycle in two or more al terna tive ways due to coupled tran- and (v) no growth of cells which are defined as VBNC (Viable but scription - translation sys tem. But Chib et al. (2018) could imagine al - non- culturable forms, Ayrapetyan et al., 2015 ). The Persis ter cells typ- terna tive models in which cells under stress prefer en tially parti tion ically form long polynucleoid fila ments (elon gated forms) and reach functional compo nents to one daughter, thus enhanc ing survival rates. maxi mum SOS induc tion after removal of ofloxacin. Eventu ally, cell While, Gerdes and Semsey (2016) proposed that persis ters do have divi sion resumes, giv ing rise to a new popu la tion ( Goormaghtigh and higher TolC lev els than the drug- sensitive sub popu la tion. Van Melderen and De Bast, 2009 ). Figs. 4 and 5 are hypo thet i cal pic- to rial repre sen ta tion of puta tive morpho types and events of persis ter 1.3 . Genomics of persisters cells after removal of antibi otic sagainst Gram neg a tive and Gram posi - tive bacte ria respec tively. Lewis (2012) has reported that the persis ters are devel oped due to The induc tion of elon gated forms of a MDR (Multi Drug Resis tant) the muta tions in hip A or hip B gene present in the HipAB lo cus. But and biofilm produc ing Klebsiella pneumo niae strain (BMKl- 2) in pres- dis tinct from drug- resistant mutants with genetic modi fi ca tion, persis - ence of Amoxyllin was evi denced by an in vitro study, which was car- ters do not replicate in the presence of antibi otics. However, once the ried out in the labo ratory of Medical Micro bi ol ogy, Dept. of Biotech- drugs are removed, they resume growth and form a popu la tion nology and Bioinfor mat ics, Sambalpur Uni versity, India. BMKl- 2 (a equally sus cepti ble to the antibi otics as the parental popu la tion. Per- biosample isolated strain, taken from a patient with chronic urinary sisters accu mu late fewer antibi otics as a direct result of increased ef- tract infec tion) was sub jected to TD test (ex posed with Amoxyllin) as fluxrate with higher expres sion of efflux - associated genes partic u larly described above, and the test con centra tion tol erant cells were ob- high expres sion of tolC , which is crit ical to promote persis ter forma - served under micro scope (at 1000X). The cells were found to be both tion. The Persis ters combine active efflux and passive dormancy to elon gated and normal sized cells ( Fig. 6 ). This was inferred that My- survive antibi otic attack ( Pu et al., 2016 ). It is also reported that cobac ter ial cel lu lar varia tions in growth and divi sion increase hetero - oxyR, dnaK, sucB, relA, rpoS, clpB, mqsR and recA were prominent per- sister genes involved in persis tence to multi ple antibi otics ( Wu et al.,

Fig. 4. Three types of typ ical Rod shaped cells formed in side the biofilm on ex posure to an tibiotics. Type I Persis ters elon gate but not killed by an tibiotics (marked in Red). Type II Persis ters elon gate but killed by an tibiotics (Marked in Green); Type III Normal Cells do not elon gate but killed by the ac tion of an tibi- otics (Marked Blue). (For in terpre ta tion of the refer ences to colour in this figure leg end, the reader is referred to the web version of this arti cle.)

Fig. 5. Three types of typ ical spheri cal bac ter ial cells are formed in side biofilmon ex posure to an tibiotics. Type I Persis ters are not killed by an tibiotics (marked in Orange spheri cal cells); Type II Persis ters are killed by an tibiotics (Marked as fuzzy Blue cells), Type III Normal cells killed by an tibiotics (Marked as fuzzy pur- ple cells). (For in terpre ta tion of the refer ences to colour in this figure leg end, the reader is referred to the web version of this arti cle.)

3 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401

Fig. 6. Left panel showing a sec ondary ring of colonies around the well impreg nated with Amoxyllin.the vicin ity of zones are resus ci tated with Persis ters of a test strain of Klebsiella pneumo niae (BMKl - 2). The Right panel is showing the Gram stained elon gated cells (in dicated by arrows) and nor mal cells.

2015 ). Further, Transcrip tional analy sis revealed that genes involved Zhang, 2007 ). This is perti nent to mention that the Phosphate (Pho) in guanosine tetraphosphate syn thesis are upreg u lated in persis ters, regu lon is a global regu la tory mecha nism involved in bacte r ial Pi (in - which represses transcrip tion and DNA replica tion and leads to organic Phosphate) manage ment that was first charac ter ized in Es- ofloxacin tol erance. Lactate dehy dro ge nase and sev eral ATP- binding cherichia coli ( Santos -Beneit, 2015 ). cassette transporters were upreg u lated in persis ters to adapt to anaer- o bic metab o lism ( Matsumoto et al., 2018 ). The 16S rRNA promoter 1.5 . Metabolomics of persisters rrnB P1 was proposed to be a persis ter reporter and an indi ca tor of toxin acti va tion regu lated by ppGpp ( Shan et al., 2017 ). Fig. 7 is a hy- Maisonneuve and Gerdes (2014) have defined the persis ters as pothet i cal model showing the co lo cali sa tion of hipBA promot ers for dormant cells having a low metabolic activ ity. This is perti nent to persis ter forma tion in Escherichia coli, Shigella sonnie and Klebsiella mention that antibi otics are target oriented mole cules. The mole c u lar pneumo niae , which are members of Enter obac te ri aceae biotype group. targets of a bacte r ial cell may be proteins, Enzymes, or Nucleic acids which are syn thesized in actively growing cells. Hence, the antibi otics 1.4 . Proteomics of persistes can not make their way to corrupt the essen tial or active targets of bacte r ial cells of dormant Persis ters, which have entered a state of In addi tion, envi ron men tal con ditions (nu trient avail ability, oxy - ‘freezing ’. As a result of which, there is no further target mole cule gen con centra tion, cell density, and sub -MIC antibi otic lev els) regu - syn thesis and no antibi otic target ing events among persis ters, which late expres sion of biofilmad hesins and the type of biofilm matrix pro- can avail the drug tol erance phenom e non and increased the number duced, which influ ences the rate at which cells in the biofilm enter of bacte ria with a long lag time ( Vulin et al., 2018 ). In addi tion, once the persis ter state and, accord ingly, the antibi otic tol erance of the cell divi sion begins, the growth rate is that of expo nen tial cells. Criti - biofilm ( Waters et al., 2016 ). Besides, PhoU protein is required for cally, the greater the ribo some con tent, the faster the persis ter cells persis ter forma tion in bacte ria ( Zhang and Li, 2010 ). PhoU, whose ex- resus ci tate. Therefore, there is acquain tance of ‘Coher ent diver si ty ’ pression is regu lated by envi ron men tal changes like nutri ent avail abil- which is evolved due to differ en tial physi cal as so ci a tion of parental ity and age of culture, is a global neg a tive regu la tor beyond its role in DNA strands. One of the parental strands of DNA can be physi cally as - phosphate metab o lism and facil itates persis ter forma tion by the sup- so ci ated with proteins appro pri ate for a survival strat egy whilst the pression of many impor tant cel lu lar metabolic processes ( Li and other strand can be physi cally as so ci ated with proteins appro pri ate for

Fig. 7. Co lo cal isa tion of hip genes of Escherichia coli, Shigella son nie and Klebsiella pneumo niae . Schematic showing the - 35 - 10 boxes of hipBA promot ers as bind - ing sites respec tively. ATG are the tran scription start sites.

4 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401 a growth strat egy, so al lowing divi sion to gen erate daughters with dif- mental changes and only weakly on the selec tive pressures of any ferent, coher ent pheno types ( Nana et al., 2018 ). Computer aided sim- given envi ron ment. u la tions predicted ( Bandyopadhyay et al., 2018 ) that cell lin eage cor- rela tions and the sensi tiv ity of growth to changes in toxin lev els coin - 1.6. 2 . Development of persister in Klebsiella pneumoniae cide in a crit ical regime. Further it was added that the dormant phe- (Enterobacteriaceae biotype) notype charac ter ized by down- regulation of energy produc ing (ATP The toxin -antitoxin (TA) sys tem has also been reported to be in- decrease) and biosynthetic factors ( Lewis, 2005 ). volved in the forma tion of drug- tolerant persis ter cells K. pneumo niae cells ( Yi- Qing et al., 2016 ). Li et al. (2018) had provided evi dence 1.6 . Biotypes and persiser development mechanism that persis ter forma tion is a growth phase- dependent and Type II per- sisters domi nate the persis ter sub popu la tion during the entire expo - The survival mecha nism adopted by multi drug resis tant Es- nential phase of Klebsiella pneumo niae . Carvalho et al. (2018) had cherichia ( Ramírez- Castillo et al., 2018 ; Odonkor and Addo, 2018 ), a given a plausi ble expla na tion on the effect of the switching dy namics member of Enter obac te ri aceae is Gram- negative, non– spore - forming, between sus cepti ble and persis ter cells on the capac ity of biofilms to facul tative anaerobe that ferment glu cose and other sug ars, reduce ni- grow, survive and recover from antibi otic shocks by test ing three trate to nitrite, and produce cata lase, do not produce oxi dase and are switching strategies: (1) con stant switches, (2) sub strate- dependent referred as Enter ics ( Donnenberg, 2015 ) was reviewed and infer ences switches and (3) antibi otic -dependent switches. Keaseya et al. (2019) were drawn. have empha sized about role of proteomic features engag ing persis ters to grow. They have explained that drug- specific changes to proteomes 1.6. 1 . Development of persister in strains of Escherichia coli included proteins for recep tor -mediated membrane transport and (Enterbacteriaceaebiotype) sugar utiliza tion, central metab o lism, and capsule produc tion, while Escherichia coli persis ters profile indi cated about downreg u lated mecha nisms common to both antibi otics included ele vated scav eng ing biosynthetic pathways, con sistent with their dormant nature, and over of reac tive oxy gen species and turnover of misfolded proteins. Resis - expres sion of toxin/an ti toxin (TA) modules ( Lewis, 2008 ; Kedzierska tance to combined antibi otics presented inte grated adjust ments to and Hayes, 2016 ). While, Van Melderen ( 2009 ) had sug gested TA protein lev els as well as unique drug- specific proteomic features, (Toxin and Anti toxin) loci are primar ily plasmid borne operons that which was demonstrated in a strain of Klebsiella pneumo niae sus cepti - promote plasmid inher i tance by the expres sion of a sta ble toxin, bility, which involved global remod el ing of the bacte r ial proteome to which is coun teracted by an unsta ble anti toxin. This TA sys tem en- counter the effects of antibi otics and sta bilize growth. ables cells to escape from antibi otics and the main cause of forma tion of persis ter cells. Therefore, in presence of antibac te r ial agents, bacte - 1.6. 3 . Development of persister in Salmonella typhi serovar typhi ria respond by acti vat ing intra cel lu lar ‘toxin ’ proteins, which arrest (Enterobacteriaceaebiotype) cell growth and induce forma tion of persis ter cells. When, antibac te r - Drescher et al. (2019) found persis ters and unsta ble SCVs as phe- ial agents are removed, persis ters can re- grow by syn thesiz ing ‘anti - notypic variants of Sal monella enter ica that were able to survive tox in’ proteins that sequester toxin proteins. A multi tude of differ ent ciprofloxacin expo sure. TacT toxin of Sal monella enter ica is known to protein - based anti toxin species are known to be actively degraded by induce a persis ter state in macrophages through the acetyla tion of a limited number of shared prote olytic pathways, strongly sug gest ing aminoacyl -tRNAs ( VanDrisse et al., 2017 ). Silva -Herzog et al., (2015) inter ac tion via compe ti tion between anti tox ins for degrada tion ma- had sug gested that of the six TA loci con served across Sal monella en- chinery ( Ogle et al., 2017 ). This view was well supported by Cho et terica sub species that cause sys temic infec tions in mammals and birds, al. (2017) that MazE anti toxin sequesters the activ ity of MazF toxin. exoge nous expres sion of vapC or sehA resulted in complete growth ar- Fenga et al. (2013) had also added that HipBA, a two- gene operon, is rest. In addi tion, ectopic expres sion of three RelE family member tox - one of many chromo so mally encoded toxin and anti toxin modules in ins enhanced tol erance to ciprofloxacin ( relE), hydro gen perox ide Escherichia coli and the HipA7 al lelic variant was the first vali dated ( relE- 3) or low pH ( relE- 5). Slattery et al., (2013) had revealed that high- persistence mutant. Further they have sug gested a sto chastic the shpAB lo cus ( Sal monella high persis tence) imparted a 3 - to 4 - model to gen erate bistabil ity and/or Mul tista bility ( Jayaraman, 2008 ) order- of - magnitude increase in survival after ampicillin expo sure of the HipBA sys tem, via the reci p ro cal coupling of free HipA to the throughout its growth phase and protected the popu la tion against ex- cel lu lar growth rate. Germaina et al. (2015) had defended that Sto- posure to multi ple antibi otics. Genetic charac ter i za tion revealed that chastic varia tion of [(p)ppGpp] in sin gle cells induced TA- encoded shpAB is a newly dis cov ered toxin -antitoxin (TA) module. The high- mRNases via a pathway involv ing polyphosphate and Lon protease. persistence pheno type was attrib uted to a nonsense muta tion in the 3′ Polyphos phate acti vated Lon degrade all known type II anti tox ins of end of the shpB gene encod ing an anti toxin protein. E. coli, thus the acti vated mRNases induced persis tence and multidrug tol erance. These dormant cells form sto chasti cally, but also in re- 1.6. 4 . Development of persister in Mycobacterium tuberculosis sponse to envi ron men tal cues, by vari ous pathways that are usu ally (Mycobacteriaceae biotype) con trolled by the sec ond messen ger (p)ppGpp ( Harms et al., 2016 ). The biol ogy of persis ters of Mycobac terium tu bercu lo sis causing la - The mecha nism of ciprofloxacin- induced persis ter forma tion was tent infec tion in host tis sues is largely unknown ( Velayati et al., 2016 ; found, in Escherichia coli, to be partially due to the expres sion of the Torrey et al., 2016 ). The Mycobac terium tu bercu lo sis phosphate - specific TisB toxin, which is involved in the decrease of ATP lev els and the transport (Pst) sys tem con trols gene expres sion in response to phos- stop of the proton motive force ( Mina and Marques, 2016 ). While, phate avail ability by inhibit ing the acti va tion of the SenX3 -RegX3 Renbarger et al. (2017) had reported that PhoU is a ‘Persis tence two- component sys tem under phosphate - rich con ditions, but the Switch'in volved in Persis ter forma tion and tol erance to multi ple an- mecha nism of commu ni ca tion between these sys tems is unknown tibiotics and stresses in Escherichia coli, an inter est ing note was given ( Namugenyi et al., 2017 ). But Wei et al. (2019) had proposed that the by Miyaue et al. (2018) about presence of a long -retention effect, or Mycobac terium tu bercu lo sis CRISPR- associated Cas1 involves persis - “memory effect ” ( Fig. 3 ) in the persis ter cell state of E. coli cells. tence and tol erance to anti- Tubercular drugs. Srinivas et al. (2018) Kussell et al. (2005) derived a mathe mat i cal model and proposed that had deter mined that the pre- existing persis ters are a hetero ge neous the opti mal rate of switching between normal and persis ter cells in Es- mix of 44 vapC30, mazF, and relA/spoT overex press ing cells. Deep et cherichia coli is found to depend strongly on the frequency of envi ron - al. (2018) presented a plausi ble model for VapC11- tRNA (toxin - target) inter ac tions and explained that in stress induced response, the

5 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401 anti- toxin mole cules are degraded by cel lu lar proteases. The result ing such as the gen eral stress response or the SOS response, in con junc - free tox ins inhibit transla tion by degrad ing either mRNA or rRNA or tion with the sec ond messen ger (p)ppGpp that is al most al ways in- tRNA, thereby repro gram ming cells to reduce cell growth to facil itate volved in persis ter forma tion ( Harms et al., 2016 ). Granta et al. cell survival in the presence of antibi otic -or host induced stress. Un- (2012) had found that persis ters were surviv ing in the envi ron ment like members of the γ- and β- lin eages, which encode with drop in dis solved oxy gen (DO) sat u ration (20%) in the face of two function ally diver gent RSH homologs (RelA and SpoT), Mtb en- bacte ri ci dal antibi otics. Zhang et al. (2012) had also made a simi lar codes a sin gle bifunc tional RSH enzyme, RelMtb ( Dahl et al., 2003 ; state ment that Tuber cle bacilli (Mtb) reside in differ ent micro envi - Sala et al., 2014 ; Singh et al., 2010 ), which is con served in all My- ronmen tal con ditions that include high oxy gen (in cav ities) or low cobacterium species ( Dutta et al., 2019 ). Although Korch et al. (2015) oxy gen (in host macrophages or in granu lo matous lesions) con tent, had reported thatMy cobac terium tu bercu lo sis pos sesses three Rel TA nutri ent star vation, oxida tive stress, and acidic pH, all of which affect modules (Rel Mtb ): RelBE Mtb , RelFG Mtb and RelJK Mtb (Rv1246c - their metabolic sta tuses. Such varied con ditions con sti tuted the basis Rv1247c, Rv2865 -Rv2866, and Rv3357 -Rv3358, respec tively), which for produc ing hetero ge neous bacte r ial popu la tions, includ ing non- inhibit mycobac ter ial growth when the toxin genes: relE, relG, relK) replicating persis ters and growing bacte ria with differ ent capac i ties are expressed inde pen dently of the anti toxin genes: relB, relF, relJ but for persis ter forma tion. Kubistova et al. (2018) had inves tigated and sub stan tial evi dences regard ing these T/A modules for persis ter for- proposed that salt or oxida tive stress could play a role in the forma - mation is lack ing in liter a ture. Increased cell size and cell -to -cell vari- tion of S. aureus persis ters out side the host's intra cel lu lar inter face. a tion in cell length were as so ci ated with bacte ria in sputum and in- While, Portillo et al. (2019) sug gested that sigB and sigE were over- fected macrophages. A study provided by Shi and Zhang (2010) had expressed in early adapta tion to low oxy gen con centra tion inde pen - vali dated that PhoU is involved in persis tence not only in E. coli but dently of the carbon source. also in M. tu bercu lo sis . Zhang et al. (2012) had reviewed and men- tioned that there are 56 upreg u lated persis ter - specific genes, includ ing 1.8 . Methods for studying persisters and its development many toxin -antitoxin (TA) modules, univer sal stress protein (UspA), SigF, anti- sigma factor RsbW, and DnaE2, with 5 genes being common In past, Keren et al. (2004) had devel oped a gen eral method for to the differ ent persis ter models, includ ing Acr2 α- crystallin heat isolat ing persis ters through ly sis of regu lar cells by ampicillin and also shock protein, GntR transcrip tional regu la tor family protein (Rv1152), estab lished a gene expres sion profile of persis ters con taining toxin - PdhA pyruvate dehy dro ge nase compo nent, Rv3290c (Lat) encod ing antitoxin (TA) modules and other genes that could block impor tant an l- lysine -epsilon -aminotransferase, and Rv2517c encod ing a hypo - cel lu lar functions such as transla tion. But Orman et al., (2016) fo- theti cal protein. cussed on the metabolic activ i ties of natively gen erated Escherichia coli persis ters in presence of aminogly cosides. They had performed 1.6. 5 . Development of persister in aeruginosa LIVE/DEAD fluo rescent indi ca tor cell ly sis stain ing, metabolic amino- (Pseudomonadaceae biotype) gly coside poten ti a tion as say, and more over persis ter measure ments Survival mecha nism opted by cells of Pseudomonas aerugi nosa was as say. Canas and Duarte (2014) were able to develop a novel method observed to be as so ci ated with hip mutants. Mulcahy et al. (2010) had for persis ter isola tion, based on a combi na tion of al kaline and enzy - proposed that high- persister mutants (hip mutants) of Pseudomonas matic ly sis that targets the cell membrane. Their proto col was to iso- aerugi nosa produce ele vated lev els of drug tol erant cells. Wang and late persis ter cells of Type I and Type II by rapidly killing normally Wood (2011) sug gested that nutri tional envi ron ment deter mines the growing cells us ing a mixture of lytic so lu tions. Further, isola tion was role, that quorum sens ing plays in Pseudomonas aerugi nosa biofilm for- done of only Type I persis ters by lysing cells of normally growing cells mation. and type II persis ters. Rowe et al. (2016) took inter est and enumer - ated the persis ters by adding lethal con centra tion of a bacte ri ci dal an- 1.6. 6 . Development of persister in Staphylococcus aureus/epidermidis tibiotic to a popu la tion of cells. The bulk of the popu la tion died (Staphylococcaceae biotype) rapidly and the surviv ing persis ter fraction could be enumer ated at Staphy lococ cus gen era also resist action of antibi otics making some vari ous time inter vals by remov ing an aliquot, washing with 1% NaCl, popu la tion of persis ter cells. Yang et al. (2015) had inves tigated and and plating ser ial dilu tions for colony count ing. The surviv ing persis - con firmedthat there are at least three sub popu la tions within a S. epi- ter fraction emerged in a clear biphasic curve in response to the an- dermidis biofilm: normal cells, dormant cells, and TBK cells. Biofilms tibiotic. The time period where the persis ter fraction remains con stant comprise of more TBK cells and dormant cells than their log - was referred to as the “persis ter plateau”. Henry and Brynildsen planktonic coun terparts ( Fig. 5 ) in a test strain of Staphy lococ cus epi- (2016) had devel oped Persis ter - FACSeq, which was a method that dermidis . Shapiro et al. (2011) had demonstrated the presence of per- uses fluo rescence - activated cell sort ing, antibi otic tol erance as says sister cells at high con centra tions in sta tionary planktonic cultures and next gen era tion sequenc ing to inter ro gate persis ter physi ol ogy and in biofilms of S. epider midis RP62a. Persis ter cell numbers in S. and its hetero gene ity. As a proof- of - concept, they had used Persis ter - epider midis biofilmsare quite low at first but within 24 h they become FACSeq on a library of reporters to study gene expres sion dis trib u - high. The persis ter number increases as the cell density increases in tions in non- growing Escherichia coli, and found that persis tence to planktonic cultures and biofilms. The pre- exposure of bacte ria to salt ofloxacin was inversely corre lated with the capac ity of non- growing stress caused a 1 –2.5 order of magni tude increase in persis ter forma - cells to syn thesize protein. Grassi et al. (2017) had isolated Persis ter tion in the bacte r ial popu la tion after antibi otic expo sure, depend ing cells (PCs) of Pseudomonas aerugi nosa and Staphy lococ cus aureus at on the type and con centra tion of the antibi otic used ( Kubistova et al., high effi ciency through membrane - perturbing antimi cro bial peptides 2018 ). (AMPs) and two clini cally used peptide - based antibi otics, (col istin and dapto mycin). It was reported that expo sure of sta tionary - phase cul- 1.7 . Environment and cues for persister development tures to opti mized con centra tions of the uncou pling agent cyanide m- chlorophenylhydrazone (CCCP) was able to gen erate at high effi - Regu larly growing bacte ria differ en ti ate into persis ter cells sto - ciency PCs exhibit ing an antibi otic -tolerant pheno type to ward differ - chasti cally at a basal rate, but this pheno typic con version can also be ent classes of antibi otics, which were sorted by flow cytom e try. While induced by envi ron men tal cues indica tive of immi nent threats for the Windels et al. (2019) had presented a novel and highly effec tive per- bacte ria. Size and compo si tion of the persis ter sub popu la tion in bacte - sister isola tion method involv ing cephalexin, an antibi otic that in- rial commu ni ties are largely con trolled by stress signal ing pathways, duces exten sive fila menta tion of sus cepti ble cells. They could sepa rate

6 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401 the antibi otic -tolerant cells by size after a short cephalexin treatment, tides can steril ize a sta tionary culture of Pseudomonas aerugi nosa . In and des ignated the isolated cells as ‘genuine persis ters ’ us ing mi- addi tion, rifampin, an inhibitor of RNA polymerase, was sug gested be- crofluidic device. Choudhary et al. (2019) had explored the CRISPRi- cause it “kills” by prevent ing persis ter resus ci tation. Alumasa et al. based genetic repres sion for better under stand ing the effect of DNA (2017) had added that Ribo some Rescue Inhibitors can kill actively gyrase deple tion on Mtb physi ol ogy and response to anti- TB drugs. It growing and non replicat ing persis ter Mycobac terium tu bercu lo sis cells. was reported that, suppres sion of DNA gyrase drasti cally affects intra - While Chung and Ko (2019) had shown that combi na tion of col istin and extra cel lu lar growth of Mtb. Inter est ingly, gyrase deple tion in and amikacin antibi otics would be effec tive at eradi cat ing persis ter Mtb leads to acti va tion of RecA/LexA - mediated SOS response and cells. In addi tion, the killing effect on persis ter cells was differ ent with drug tol erance via induc tion of persis ter sub popu la tion. Chemi cal in- respect to the order of antibi otic treatment because, the mecha nism of hibi tion of RecA in gyrase - depleted bacte ria results in rever sion of persis ter cell forma tion by col istin is differ ent from that by other an- persis ter pheno type and better killing by antibi otics. tibiotics.

1.9 . Biofilm formation ability and persistence 3. Conclusion

Biofilms are surface - attached groups of micro bial cells encased in From this compar a tive review report on the survival mecha nism an extra cel lu lar matrix that are signif i cantly less sus cepti ble to antimi - adopted by the members of Escherichia coli, Sal monella typhi and Kleb- crobial agents than non- adherent, planktonic cells ( Hall and Mah, siella pneumo niae (Enter obac te ri aceae, Bio type group), Pseudomonas 2017 ). Cells in biofilmcan produce persis ter cells which do not grow aerurug inosa (Pseudomonadaceae), Mycobac terium tu bercu lo sis (My- or die in the presence of antibi otics. Stress con ditions in the host in- cobac teri aceae) and Staphy lococ cus aureus (Staphylo coc caceae), it was duce persis ter cells and influ ence biofilm forma tion by Staphy lococ cus observed that all the members are fol lowing the global survival mech- epider midis ( Fraiha et al., 2019 ). Roberts and Stewart (2005) through a nism. But it is impor tant to note that all the members undergo a their mathe mat i cal model, had hypoth e sized that the persis ter state is ‘Freezing ’ effect ’ (FE) in the presence of antibi otics. The said FE is re- a hypo thet i cal, highly protected state adopted by a small fraction of ferred to as hanging of enzy matic sys tem required for the growth and the cells in a biofilm. Persis ters were as sumed to be gen erated at a multi pli ca tion of bacte r ial strains for a certain period of time. While fixed rate, inde pen dent of the presence of sub strate or antimi cro bial analysing the biochem i cal proper ties of the said bacte r ial strains (un - agent. Cells were as sumed to revert from the persis ter state when ex- der study), it is found that all the four bacte r ial strains are of differ ent posed to the growth sub strate and inca pable of growth. Staphy lococ cus biotypes. Consid ering IMVIC test, E. coli is ++- - biotype, Kl. pneumo - aureus biofilms are embed ded in an extra cel lu lar matrix composed of niae is -- ++ biotype, P. aerugi nosa is -- - +biotype and S. aureus is - self- produced extra cel lu lar polysac cha rides, DNA, and proteins or +++ biotype. Likewise, these bacte ria exe cute differ en tial biochem i - host -derived matri ces such as fibrin, prompting specu la tion that lim- cal approaches in the reduc tion of Nitrate to Nitrite reduc tion, Urea ited drug diffu sion into biofilms con tributes to tol erance ( Waters et hydrol ysis, Coag u lase, Oxi dase produc tion, Carbo hy drate fermen ta - al., 2016 ) and leads to poor antibi otic pene tra tion, slow growth, adap- tion etc. But all the four Bio types opt a master mecha nism for stalling tive stress responses ( George and Halami, 2019 ) that have a marked of their metabolic reac tions and in most cases are facil itated by influ ence on cel lu lar physi ol ogy, and con tribute to the occur rence of biofilm produc tion. Hence, from this snapshot of a research review persis ter cells ( Acker and Coenye, 2016 ). Chemi cal signal ing commu - work carried out for this commu ni ca tion, it was under stood that that nica tion, a phenom e non called quorum sens ing (QS) has also active AA metab o lism is a crit ical medi a tor of persis tence, and as one would role for persis ter forma tion inside a biofilm( Maisonneuve and Gerdes, expect, the strin gent response, a major metabolic regu la tory sys tem 2014 ). con trolled by ppGpp ( Amato et al., 2014 ). Mok et al. (2015) inves ti- gated the role of seven global transcrip tional regu la tors (ArcA, Cra, 2. Prevention and killing persisters cyclic AMP [cAMP] recep tor protein [CRP], DksA, FNR, Lrp, and RpoS) on persis ter metab o lism. Eradi ca tion of tol erant persis ters is a seri ous challenge. But stud ies The modu la tion of TCA cycle activ ity appears as a hallmark in per- are in progress to challenge the persis ters. Pearl et al. (2008) had rec- sister metab o lism. This regu la tion must be precisely con trolled to ommended lytic phages infect ing bacte r ial persis ters of E. coli that avoid ROS forma tion with poten tially destruc tive impli ca tions for per- could lyse them when growth is resumed, sug gest ing that phage ther- sister cells. Mul tiple lines of evi dence sug gest that the metab o lism of apy could be a promising strat egy to eradi cate persis ters. But, Kim et persis ters can be tuned to al ter their sus cepti bil ity to ward antibi otics al. (2011) had shown that 3 -[4 -(4 -methoxyphenyl) piperazin - 1 -yl] or to trigger programmed cell -death - like processes. In a number of piperidin- 4 -yl biphenyl- 4 -carboxylate (C10), screened out of a chemi - cases, this is achieved simply by supple ment ing selected carbo hy - cal library, selec tively kills bacte r ial persis ters that tol erate antibi otic drates ( Prax and Bertram, 2014 ). In addi tion, target inac tiv ity and/or treatment but does not affect normal antibi otic -sensitive cells. C10 led global cel lu lar dormancy comprise mecha nisms such as reduced cel lu - persis ters to antibi otic -induced cell death by causing rever sion of per- lar energy, halted DNA replica tion and blocked transla tion support ing sisters to antibi otic -sensitive cells. Antibi otic killing of E. coli, P. aerug- persis ter forma tion ( Wilmaerts et al., 2019 ). inosa and M. tu bercu lo sis in the presence of oxy gen compared with an oxy gen -limited envi ron ment had been sug gested by Granta et al. Acknowledgement (2012) , which could be more effec tive. A combi na tion of specifi cally persis ter - targeted approaches, such as catch ing them when active and This review and exper i men tal stud ies are part of a Ph. D thesis reg- sus cepti ble, either by stimu lat ing them to “wake up” or by inter mit - istered under Sambalpur Uni versity. tent drug dos ing, kill Mycobac ter ial persis ters ( Zhang et al., 2012 ). Certain sug ars were shown to induce a proton motive force, which in turn led to increased uptake of aminogly cosides and thereby bacte r ial Appendix A. Supplementary data killing ( Helaine and Kugelberg, 2014 ). Mit o mycin ( Kwan et al., 2015 ) is a pro- drug which is con verted into a reac tive compound forming Supple men tary data to this arti cle can be found online at https:// adducts with DNA upon enter ing the cell. Prolonged treatment with doi. org/10. 1016/ j. bcab.2019. 101401 . aminogly cosides that cause mistrans la tion lead ing to misfolded pep-

7 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401

References Mycobacterium tuberculosis entry into quiescence and reduces persistence . Sci. Adv. 1 – 13 2019; 5: eaav2104 . Ehrlich , G. D. , Hu, F.Z. , Shen , K. , Paul Stoodley , P. , Christopher , J., 2005 . Bacterial Acker , H. V., Coenye , T. , 2016 . The role of efflux and physiological adaptation in biofilm plurality as a general mechanism driving persistence in chronic infections . Clin. tolerance and resistance . J. Biol. Chem. 1 – 11 . https:// doi. org/ 10. 1074/ jbc. R115. Orthop. Relat. Res. 437 , 20 – 24 . 707257 . Fenga , J., Kesslerb , D. A. , Ben -Jacoba , E. , Levinea , H. , 2013 . Growth feedback as a basis Allison , K. R. , Mark , P. , Brynildsen , M. P. , Collins , J., 2011 . Heterogeneous bacterial for persister bistability . Proc. Natl. Acad. Sci. 1 – 6 . persisters and engineering approaches to eliminate them . Curr. Opin. Microbiol. 14 , Fisher , R. A. , Gollan , B. , Helaine , S. , 2017 . Persistent bacterial infections and persister 593 – 598 . https:// doi. org/ 10. 1016/ j. mib. 2011. 09. 002 . cells . Nat. Rev. Microbiol. 15 , 453 – 464 . https:// doi. org/ 10. 1038/ Nrmicro. 2017. 42 . Alumasa , J.N. , Manzanillo , P. S. , Nicholas , D. , Peterson , N. D. , Tricia Lundrigan , T. , Fraiha , R. O. , Pereira , A. P. R. , Brito , E. C. A. , Borges , C. L., Parente , A. F.L. , Perdomo , T. R. , Baughn , A. D. , Jeffery, S. , Cox , J.S. , Keiler , K. C. , 2017 . Ribosome Rescue inhibitors Maced , L.R. , Weber, S. S. , 2019 . Stress conditions in the host induce persister cells kill actively growing and nonreplicating persister Mycobacterium tuberculosis cells . and influence biofilm formation by Staphylococcus epidermidis RP62A . J. Braz. Soc. ACS Infect. Dis. 3 , 634 – 644 . https:// doi. org/ 10. 1021/ acsinfecdis. 7b00028 . Trop. Med. 52 , 1 – 6 e20180001: 2019 . https:// doi. org/ 10. 1590/ 0037 -8682 -0001 - Amato , S. M. , Fazen , C. H. , Henry , T. C. , Mok , W.W. K. , Orman , M. A. , Elizabeth , L., 2018 . Sandvik , E. L., Volzing , K. G. , Mark , P. , Brynildsen , M. P. , 2014 . The role of Gefen , O. , Chekol , B. , Strahilevitz , J., Nathalie , Q. , Balaban , N. Q. , 2016 . TDtest: easy metabolism in bacterial persistence . Front. Microbiol. 5 , 1 – 9 . detection of bacterial tolerance and persistence in clinical isolates by a modified Ayrapetyan , M. , Williams , T. C. , Baxter , R. , Oliver , J.D. , 2015 . Viable but disk -diffusion assay . Sci. Rep. 7 (41284 ) , 1 – 9 . https:// doi. org/ 10. 1038/ srep41284 . nonculturableand persister cells coexist stochastically and are induced by human George , G. , Halami , P. M. , 2019 . Presence of extracellular DNA & protein in biofilm serum . Infect. Immun. 83 ( 11 ) , 4194 – 4203 . https:// doi. org/ 10. 1128/ iai. 00404 -15 . formation by gentamicin -resistant Lactobacillus plantarum . Indian J. Med. Res. 149 , Balaban , N.Q. , Merrin , J., Chait , R. , Kowalik , L., Leibler , S. , 2004 . Bacterial persistence 257 – 262 . https:// doi. org/ 10. 4103/ ijmr. IJMR_ 2022_ 17 . as a phenotypic switch . Science 10 (5690 ), 1622 – 1625 305 . Gerdes , K. , Semsey , S. , 2016 . Pumping persisters . Nature 545 , 41 – 42 . Bandyopadhyay , A. , Wang , H. , Ray , J.C. J., 2018 . Lineage space and the propensity of Germaina , E. , Roghaniana , M. , Gerdesa , K. , Maisonneuvea , E. , 2015 . Stochastic bacterial cells to undergo growth transitions . PLoS Comput. Biol. 14 (8 ) , e1006380 . induction of persister cells by HipA through (p) ppGpp -mediated activation of mRNA https:// doi. org/ 10. 1371/ journal. pcbi. 1006380 . endonucleases . Proc. Natl. Acad. Sci. 112 (16 ), 5171 – 5176 . Barrett , T. C. , Mok , W.W. K. , Murawski , A. M. , Brynildsen , M. P. , 2019 . Enhanced Goormaghtigh , F., Melderen , L.V. , 2019 . Single -cell imaging and characterization of antibiotic resistance development from fluoroquinolone persisters after a single Escherichia coli persister cells to ofloxacin in exponential cultures . Sci. Adv. 5 , 1 – 14 exposure to antibiotic . Nat. Commun. 10 (1 ), 1 – 11 . https:// doi. org/ 10. 1038/ eaav9462 . s41467 -019 -09058 -4 . Granta , S. S. , Kaufmanna , B. B. , Chandd , N. S. , Haseleya , N. , Hunga , D. T. , 2012 . Bauer , A. W., Kirby , W.M. , Sherris , J.C. , Turck , 1966 . Antibiotic susceptibility testing by Eradication of bacterial persisters with antibiotic -generated hydroxyl radicals . Proc. a standardized single disk method . Am. J. Clin. Pathol. 45 ( 4 ), 493 – 496 . Natl. Acad. Sci. 109 (30 ) 12147 – 1215 . Bos , J., Zhang , Q. , Vyawahare , S. , Rogers , E. , Rosenberg , S. M. , Austin , R. H. , 2015 . Grassi , L., Di Luca , M. , Maisetta , G. , Rinaldi , A. C. , Esin , S. , Trampuz , A. , Batoni , G. , Emergence of antibiotic resistance from multinucleated bacterial filaments. Proc. 2017 . Generation of persister cells of and Staphylococcus Natl. Acad. Sci. 112 (1 ), 178 – 183 . https:// doi. org/ 10. 1073/ pnas. 1420702111 . aureus by chemical treatment and evaluation of their susceptibility to membrane - Cañas -Duarte , S. J., Restrepo , S. , Pedraza , J.M. , 2014 . Novel protocol for persister cells targeting agents . Front. Microbiol. 8 , 1917 . https:// doi. org/ 10. 3389/ fmicb. 2017. isolation . PLoS One 9 (2 ), e88660 . https:// doi. org/ 10. 1371/ journal. pone. 008866 . 01917 . Cabral , D. J., Wurster , J.I. , Belenky , P. , 2018 . Antibiotic persistence as a metabolic Hall , C. W., Mah , T. -F. , 2017 . Molecular mechanisms of biofilm- based antibiotic adaptation: stress, metabolism, the host, and new directions pharmaceuticals . 11 resistance and tolerance in pathogenic . FEMS (Fed. Eur. Microbiol. Soc.) (14 ), 11 – 19 . https:// doi. org/ 10. 3390/ ph11010014 . Microbiol. Rev. 41 , 276 – 301 fux010 . https:// doi. org/ 10. 1093/ femsre/ fux010 . Carvalho , G. , Balestrino , D. , Forestier , C. , Mathias , J.D. , 2018 . How do environment - Harms , A. , Maisonneuve , E. , Gerdes , K. , 2016 . Mechanisms of bacterial persistence dependent switching rates between susceptible and persister cells affect the during stress and antibiotic exposure . Science 16 (6318 ), 354 . dynamics of biofilms faced with antibiotics? npj . Biofilms Microbiome 6 . www. Helaine , S. , Kugelberg , E. , 2014 . Bacterial persisters: formation, eradication, and nature. com/ npjbiofilms. experimental systems . Trends Microbiol. xx , 1 –8 . https:// doi. org/ 10. 1016/ j. tim. Chib , S. , Das , S. , Venkatesan , S. , Seshasayee , A. S. N. , Thattai , M. , 2018 . Using stochastic 2014. 03. 008 . cell division and death to probe minimal units of cellular replication . New J. Phys. Henry , T. H. , Brynildsen , M. P. , 2016 . Development of Persister -FACSeq: a method to 035004 . https:// doi. org/ 10. 1088/ 1367 -2630/ aab197 . massively parallelize quantification of persister physiology and its heterogeneity . Cho , J., Carr , A. N. , Whitworth , L., Johnson , B. , Wilson , K. S. , 2017 . MazEF toxin - Sci. Rep. 6 ( 25100 ), 1 – 17 . https:// doi. org/ 10. 1038/ srep25100 . antitoxin proteins alter Escherichia coli cell morphology and infrastructure during Jayaraman , R. , 2008 . Bacterial persistence: some new insights into an old phenomenon . persister formation and regrowth . Microbiology 163 , 308 – 321 . https:// doi. org/ 10. J. Biosci. 33 , 795 – 805 . 1099/ mic. 0. 000436 . Keaseya , S. L., Suhb , M. -J. , Das , S. , Blancettd , C. D. , Zengd , X., Andressonc , T. , Sund , M. Choudhary , E. , Sharma , R. , Kumar , Y. , Agarwal , N. , 2019 . Conditional silencing by G. , Ulrich , R. G. , 2019 . Decreased antibiotic susceptibility driven by global CRISPRi reveals the role of DNA gyrase in formation ofDrug -tolerant persister remodeling of the Klebsiella pneumoniae proteome . Mol. Cell. Proteom. RA 118 , population in Mycobacterium tuberculosis . Front. Cell Infect. Microbiol. 9 , 70 . 000739 . https:// doi. org/ 10. 3389/ fcimb. 2019. 00070 . Kedzierska , B. , Hayes , F., 2016 . Emerging roles of toxin -antitoxin modules in bacterial Chung , E. S. , Ko , K. S. , 2019 . Eradication of persister cells of Acinetobacter baumannii pathogenesis . Molecules 21 ( 6 ). https:// doi. org/ 10. 3390/ molecules21060790 . through combination of colistin and amikacin antibiotics . J. Antimicrob. Keren , I., Shah , D. , Spoering , A. , Kaldalu , N. , Lewis , K. , 2004 . Specialized persister cells Chemother. 1 –7 . https:// doi. org/ 10. 1093/ jac/ dkz034 . and the mechanism of multidrug tolerance in Escherichia coli. J. Bacteriol. 186 (24 ), Cui , P. , Xu , T. , Zhang , W.H. , Zhang , Y. , 2016 . Molecular mechanisms of bacterial 8172 – 8180 . persistence and phenotypic antibiotic resistance . 38 ( 10 ) , 859 – 871 20 . Keren , I., Minami , S. , Rubin , E. , Lewis , K. , 2011 . Characterization and transcriptome Dahl , J.L. , Kraus , C. N. , Boshoff, H. I.M. , Doan , B. , Foley , K. , Avarbock , D. , Kaplan , G. , analysis of Mycobacterium . mBio 2 (3 ) , 3 – 12 . https:// doi. org/ 10. 1128/ mBio. 00100 - Mizrahi , V., Rubin , H. , Barry , III, C. E. , 2003 . The role of RelMtb -mediated 11 . adaptation to stationary phase in long -term persistence of Mycobacterium tuberculosis Khlebodarova , T. M. , Likhoshvai , V.A. , 2018 . Persister cells – a plausible outcome of in mice . Proc. Natl. Acad. Sci. 100 ( 17 ) , 10026 – 10031 . neutral coevolutionary drifts . Sci. Rep. 8 , 14309 . https:// doi. org/ 10. 1038/ s41598 - Day , T. , 2016 . Interpreting phenotypic antibiotic tolerance and persister cells as 018 -32637 -2 . evolution via epigenetic inheritance . Mol. Ecol. 25 ( 8 ), 1869 – 1882 . https:// doi. org/ Kim, J.S. , Wood , T. K. , 2016 . Persistent persister misperceptions . Front. Microbiol. 7 , 1 – 10. 1111/ mec. 13603 . 7 . https:// doi. org/ 10. 3389/ fmicb. 2016. 02134 . Deep , A. , Tiwari , P. , Agarwal , S. , Kaundal , S. , Saqib Kidwai , S. , Singh , R. , Krishan , G. , Kim, J.S. , Wood , T. K. , 2018 . Single Cell Observations Show Persister Cells Wake Based 2018 . Structural, functional and biological insights into therole of Mycobacterium on Ribosome ContentbioRxiv . 2018 . tuberculosis VapBC11toxin – antitoxin system: targeting a tRNase to tackle Kim, J.- S. , Heo, P. , Yang , T. -J. , Lee , K. -S. , Cho , D. -H. , Kim, B. T. , et al. , 2011 . Selective Mycobacterial adaptation . Nucleic Acids Res. 46 (21 ), 11639 – 11655 . https:// doi. killing of bacterial persisters by a single chemical compound without affecting org/ 10. 1093/ nar/ gky924 . normal antibiotic -sensitive cells . Antimicrob. Agents Chemother. 55 (11 ), 5380 – Defraine , V., Fauvart , M. , Jan Michiels , J., 2018 . Fighting bacterial persistence: current 5383 . https:// doi. org/ 10. 1128/ aac. 00708 -11 . and emerging anti -persisterstrategies and therapeutics . Drug Resist. Updates 38 , 12 – Kirmusauglu , S. , 2017 . MRSA and MSSA: the Mechanism of Methicillin Resistance and 26 . https:// doi. org/ 10. 1016/ j. drup. 2018. 03. 002 . the Influence of Methicillin Resistance on Biofilm Phenotype of Staphylococcus Donnenberg , M. S. , 2015 . Mandell, Douglas, and Bennett’s Principles and Practice of aureus. The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus , Infectious Diseases . eighth ed . vols. 1 – 18 . Dorr , T. , Vulić , M. , Lewis , K. , 2010 . Ciprofloxacin causes persister formation by inducing Korch , S. B. , Malhotra , V., Contreras , H. , Clark -Curtiss , J.E. , 2015 . The Mycobacterium the TisB toxin in Escherichia coli. PLoS Biol. 8 (2 ) , e1000317 . https:// doi. org/ 10. tuberculosis relBEtoxin: antitoxin genes are stress -responsive modules that regulate 1371/ journal. pbio. 1000317 . growth through translation inhibition . J. Microbiol. 53 (11 ), 783 – 795 . https:// doi. Drescher , S. P. M. , Gallo , S. W., Pedro Maria Abreu Ferreira , P. M. A. , Alexandre , C. , org/ 10. 1007/ s12275 -015 -5333 -8 . Ferreira , S. , Oliveira , S. D. de O. , 2019 . Salmonella enterica persistercells form Kubistova , L., Dvoracek , L., Tkadlec , Melter , J.O. , Licha , I., 2018 . Microbial Drug unstable small colony variants after in vitro exposure tociprofloxacin. Sci. Rep. 9 Resistance Mechanisms Environmental Stress Affects the Formation of (7232 ), 1 – 11 . https:// doi. org/ 10. 1038/ s41598 -019 -43631 -7 . Staphylococcus aureus Persisters Tolerant to Antibiotics , vol. 24 . p. 5 . Dutta , N. K. , Klinkenberg , L.G. , Vazquez , M. J., Segura -Carro , D. , Colmenarejo , G. , Kussell , E. , Kishony , R. , Balaban , N. Q. , 2005 . Stanislas Leibler, S., bacterial persistence . Ramon , F., Rodriguez -Miquel , B. , Mata -Cantero , L., Francisco , E. P. D. , Chuang , Y. M. , Model Surviv. Changing Environ. Genet. 169 , 1807 – 1814 . Rubin , H. , Lee , J.J. , Eoh , H. , Bader , J.S. , Perez -Herran , E. , Mendoza -Losana , A. , Kwan , B. W., Chowdhury , N. , Wood , T. K. , 2015 . Combating bacterial infections by Petros , C. , Karakousis , P. C. , 2019 . Inhibiting the stringent response blocks killing persister cells with mitomycin C . Environ. Microbiol. 17 (11 ), 4406 – 4414 .

8 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401

https:// doi. org/ 10. 1111/ 1462 -2920. 12873 . Escherichia coli isolates in urinary tract infections from Aguascalientes, Mexico: Lewis , K. , 2005 . Persister cells and the riddle of biofilm survival . Biochemistry (Mosc.) cross -sectional studyAnn . Clin. Antimicrob. Microbiol. 17 (34 ), 1 – 13 . https:// doi. 70 (2 ), 267 – 274 . https:// doi. org/ 10. 1007/ s10541 -005 -0111 -6 . org/ 10. 1186/ s12941 -018 -0286 -5 . Lewis , K. , 2008 . Multidrug tolerance of biofilms and persister cells bacterial biofilms pp Renbarger , T. , Baker , J.M. , Matthew Sattley , W., 2017 . Slow and steady wins the race: 107 -131 part of the current topics in Microbiology and immunology book series (CT an examination of bacterial persistence . AIMS Microbiol. 3 (2 ), 171 –185 . https:// Microbiology , 322 . pp. 107 – 131 . https:// link. springer. com/ chapter/ 10. 1007/ 978 -3 - doi. org/ 10. 3934/ microbiol. 2017. 2. 171 . 540 -75418 -3 . Roberts , M. E. , Stewart , P. S. , 2005 . Modelling protection from antimicrobial agents in Lewis , K. , 2010 . Persister Cells and the Paradox of Chronic Infections Dormant persister biofilms through the formation of persister cells . Microbiology 151 , 75 – 80 . https:// cells are tolerant to antibiotics and are largely responsible for recalcitrance of doi. org/ 10. 1099/ mic. 0. 27385 -0 . chronic infections Microbe . 5 (10 ) , 429 – 437 . Rowe , S. E. , Conlon , B. P. , Keren , I., Lewis , K. , 2016 . Bacterial Persistence . p. 1333 . Lewis , K. , 2012 . Persister cells: molecular mechanisms related to antibiotic tolerance . Sala , A. , Bordes , P. , Genevaux , P. , 2014 . Multiple toxin -antitoxin systems in Handb. Exp. Pharmacol. 211 , 121 – 133 . https:// doi. org/ 10. 1007/ 978 -3 -642 -28951 - Mycobacterium tuberculosis . Toxins 6 , 1002 – 1020 . https:// doi. org/ 10. 3390/ 4_ 8 . toxins6031002 . Li, Y. , Zhang , Y. , 2007 . Phou is a persistence switch involved in persister formation and Santos -Beneit , F., 2015 . The Pho regulon: a huge regulatory network in bacteria . Front. tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob. Agents Microbiol. 6 , 402 . https:// doi. org/ 10. 3389/ fmicb. 2015. 00402 . Chemother. 51 (6 ) , 2092 – 2099 . Shan , Y. , Gandt , A. B. , Rowe , S. E. , Deisinger , J.P. , Conlon , B. P. , Lewis , K. , 2017 . ATP - Li, Y. , Zhang , L., Zhou , Y. , Zhang , Z. , Zhang , X., 2018 . Survival of bactericidal antibiotic dependent persister formation in Escherichia coli . 8 (1 ) e02267 -16 . treatment by tolerant persister cells of Klebsiella pneumoniae . J. Med. Microbiol. 67 Shapiro , J.A. , Nguyen , V.L. , Chamberlain , N. R. , 2011 . Evidence for persisters in (3 ), 273 – 281 . https:// doi. org/ 10. 1099/ jmm. 0. 000680 . Staphylococcus epidermidis RP62a planktonic cultures and biofilms. J. Med. Maisonneuve , E. , Gerdes , K. , 2014 . Molecular mechanisms underlying bacterial Microbiol. 60 , 950 – 960 . https:// doi. org/ 10. 1099/ jmm. 0. 026013 -0 . persisters . Cell 157 , 539 – 548 . https:// doi. org/ 10. 1016/ j. cell. 2014. 02. 050 . Shi , W., Zhang , Y. , 2010 . PhoY2 but not PhoY1 is the PhoU homologue involved in Martinecz , A. , AbelZurWiesch , P. , 2018 . Estimating treatment prolongation for persisters in Mycobacterium tuberculosis . J. Antimicrob. Chemother. 65 (6 ), 1237 – persistent infections . Pathog. Dis. 76 (6 ), 1 – 10 . https:// doi. org/ 10. 1093/ femspd/ 1242 . https:// doi. org/ 10. 1093/ jac/ dkq103 . fty065 . Silva -Herzog , E. , McDonald , E. M. , Crooks , A. L., Detweiler , C. S. , 2015 . Physiologic Matsumoto , S. , Kawai , Y. , Miyagawa , S. , Iwamoto , Y. , Okuda , S. , Sánchez -Gorostiaga , stresses reveal a Salmonella persister state and TA family toxins modulate tolerance A. , Vicente , M. , Tsuneda , S. , 2018 . Unique transcriptional profile of native persisters to these stresses . PLoS One 10 (12 ), 1 – 18 . https:// doi. org/ 10. 1371/ journal. pone. in Escherichia coli. J. Biosci. Bioeng. 125 ( 1 ), 15 – 22 . https:// doi. org/ 10. 1016/ j. 0141343 . jbiosc. 2017. 07. 015 . Singh , R. , Barry , C. E. , Boshoff, H. I.M. , 2010 . The three RelE homologs of Mycobacterium Michael , K. A. , Daniel , D. J., James , C. J., 2010 . How antibiotics kill bacteria: from targets tuberculosis have individual, drug -specific effects on bacterial antibiotic tolerance . J. to networks . Nat. Rev. Microbiol. 8 (6 ) , 423 – 435 . https:// doi. org/ 10. 1038/ Bacteriol. 192 (5 ), 1279 – 1291 . https:// doi. org/ 10. 1128/ JB. 01285 -09 . nrmicro2333 . Singh , S. , Singh , S. K. , Chowdhury , I., Singh , R. , 2017 . Understanding the mechanism of Mina , E. G. , Marques , C. N. H. , 2016 . Interaction of Staphylococcus aureus persister cells bacterial biofilms resistance to antimicrobial agents . Open Microbiol. J. 11 ( 1 ), 53 – with the host when in a persister state and following awakening . Sci. Rep. 6 , 31342 . 62 . https:// doi. org/ 10. 2174/ 1874285801711010053 . https:// doi. org/ 10. 1038/ srep31342 . Slattery , A. , Victorsen , A. H. , Brown , A. , Hillman , K. , Phillips , G. J., 2013 . Isolation of Miyaue , S. , Suzuki , E. , Komiyama , Y. , Kondo , Y. , Morikawa , M. , Maeda , S. , 2018 . highly persistent mutants of Salmonella enterica serovar typhimurium reveals a new Bacterial memory of persisters: bacterial persister cells can retain their phenotype toxin - antitoxin module . J. Bacteriol. 195 ( 4 ), 647 – 657 . https:// doi. org/ 10. 1128/ for days or weeks after withdrawal from colony – biofilm culture . Front. Microbiol. 9 , JB. 01397 -12 . 1396 . https:// doi. org/ 10. 3389/ fmicb. 2018. 01396 . Srinivas , V., Peterson , E. J.R. , Baliga , N. S. , 2018 . Characterization and elimination of Mok , W.W. K. , Orman , M. A. , Brynildsen , M. P. , 2015 . Impacts of global transcriptional stochastically generated persister subpopulation in mycobacteria . SSRN Electron. J. regulators on persister metabolism . Antimicrob. Agents Chemother. 59 , 2713 – 2719 . 1 , 33 . https:// doi. org/ 10. 2139/ ssrn. 3299435 . https:// doi. org/ 10. 1128/ AAC. 04908 -14 . Torrey , H. L., Keren , I., Via , L.E. , Lee , J.S. , Lewis , K. , 2016 . High persister mutants in Mulcahy , L.R. , Burns , J.L. , Lory , S. , Lewis , K. , 2010 . Emergence of Pseudomonas Mycobacterium tuberculosis . PLoS One 11 (5 ), 1 – 28 . https:// doi. org/ 10. 1371/ journal. aeruginosa strains producing high levels of persister cells in patients with cystic pone. 0155127 . fibrosis. J. Bacteriol. 192 (23 ), 6191 – 6199 . Van Melderen , L., De Bast , M. S. , 2009 . Bacterial toxin -Antitoxin systems: more than Muthuramalingam , M. , White , J.C. , Bourne , C. R. , 2016 . Toxin -antitoxin modules are Selfish entities? . PLoS Genet. 5 (3 ). https:// doi. org/ 10. 1371/ journal. pgen. 1000437 . pliable switches activated by multiple protease pathways . Toxins 8 (7 ) , 1 – 16 . Vandrisse , C. M. , Parks , A. R. , Escalante -Semerena , J.C. , 2017 . A toxin involved in https:// doi. org/ 10. 3390/ toxins8070214 . Salmonella persistence regulates its activity by acetylating its cognate antitoxin, a Namugenyi , S. B. , Aagesen , A. M. , Elliott , S. R. , Tischler , A. D. , 2017 . Mycobacterium modification reversed by CobBSirtuin deacetylase . mBio 8 (3 ), 1 –14 . https:// doi. tuberculosis PhoY proteins promote persister formation by mediating pst/ SenX3 - org/ 10. 1128/ mbio. 00708 -17 . RegX3 phosphate sensing . mBio 8 (4 ), 6 – 7 . https:// doi. org/ 10. 1128/ mbio. 00494 - Velayati , A. A. , Abeel , T. , Shea , T. , KonstantinovichZhavnerko , G. , Birren , B. , Cassell , G. 17 . H. , et al. , 2016 . Populations of latent Mycobacterium tuberculosis lack a cell wall: Nana , G. Y. , Ripoll , C. , Cabin -Flaman , A. , Gibouin , D. , Delaune , A. , Janniere , L., isolation, visualization, and whole -genome characterization . Int. J. Grancher , G. , Chagny , G. , Loutelier -Bourhis , C. , Lentzen , E. , Grysan , P. , Audinot , J.- Mycobacteriology 5 (1 ) , 66 – 73 . https:// doi. org/ 10. 1016/ j. ijmyco. 2015. 12. 001 . N. , Norris , V., 2018 . Division -based, growth rate diversity in bacteria . Front. Vijay , S. , Vinh , D. N. , Hai , H. T. , Ha , V.T. N. , Dung , V.T. M. , Dinh , T. D. , et al. , 2017 . Microbiol. 9 , 849 . https:// doi. org/ 10. 3389/ fmicb. 2018. 00849 . Influence of stress and antibiotic resistance on cell -length distribution in Odonkor , S. T. , Addo , K. K. , 2018 . Prevalence of multidrug -resistant Escherichia coli Mycobacterium tuberculosis clinical isolates . Front. Microbiol. 8 – 12 . https:// doi. org/ isolated from drinking water sources . Hindawi Int. J. Microbiol. Article ID 7204013, 10. 3389/ fmicb. 2017. 02296 . 7 pages . https:// doi. org/ 10. 1155/ 2018/ 7204013 . Vulin , C. , Leimer , N. , Huemer , M. , Ackermann , M. , Zinkernagel , A. S. , 2018 . Prolonged Ogle , C. T. , William , H. , Mather , W.H. , 2017 . Proteolytically Coordinated Activation of bacterial lag time results in small colony variants that represent a sub -population of Toxin -Antitoxin Modules bioRxiv , vols. 1 – 18 . persisters . Nat. Commun. 9 , 4074 . https:// doi. org/ 10. 1038/ s41467 -018 -06527 -0 . Orman , M. A. , Henry , T. C. , Decoste , C. J., Brynildsen , M. P. , Engineering, B. , 2016 . Wang , X., Wood , T. K. , 2011 . Toxin -antitoxin systems influence biofilm and persister cell Bacterial persistence , 1333 . pp. 83 – 100 609 . formation and the general stress response . Appl. Environ. Microbiol. 77 , 165577 – Orruño , M. , Parada , C. , Kaberdin , V.R. , Arana , I., 2017 . InTech Open 405 – 421 . http:// 165583 0099 -2240/ 11/$ 12. 00 . https:// doi. org/ 10. 1128/ AEM. 05068 -11 . dx. doi. org/ 10. 5772/ 67777 . Waters , E. M. , Rowe , S. E. , O’Gara , J.P. , Conlon , B. P. , 2016 . Convergence of Pang , Z. , Raudonis , R. , Glick , B. R. , Lin , T. J., Cheng , Z. , 2019 . Antibiotic resistance in Staphylococcus aureus persister and biofilm research: can biofilms be defined as Pseudomonas aeruginosa : mechanisms and alternative therapeutic strategies . communities of adherent persister cells? . PLoS Pathog. 12 (12 ). https:// doi. org/ 10. Biotechnol. Adv. 37 (1 ) , 177 – 192 . https:// doi. org/ 10. 1016/ j. biotechadv. 2018. 11. 1371/ journal. ppat. 1006012 . 013 . Yi -Qing , W., De -Xi , B. , Dong -Qing , W., Hong -Yu , O. , 2016 . Prediction of type II toxin - Pattnaik , S. , 2017 . Diagnostic Bacterial Genomics . Pub. Himalaya Publishers Ltd. , antitoxin loci in Klebsiella pneumoniae genome sequences . Interdiscip. Sci. Comput. Mumbai . Life Sci. 8 , 143 – 149 . Pearl , S. , Gabay , C. , Kishony , R. , Oppenheim , A. , Balaban , N. Q. , 2008 . Nongenetic Wei , J., Lu , N. , Li, Z. , Wu , X., Jiang , T. , Xu , L., et al. , 2019 . The Mycobacterium individuality in the host – phage interaction . PLoS Biol. 6 (5 ) , e120 . https:// doi. org/ tuberculosis CRISPR -associated Cas1 involves persistence and tolerance to anti - 10. 1371/ journal. pbio. 0060120 . tubercular drug . BioMed Res. Int. 1 – 9 . https:// doi. org/ 10. 1155/ 2019/ 7861695 . Petchiappan , A. , Chatterji , D. , 2017 . Antibiotic resistance: current perspectives . ACS Willenborg , J., Willms , D. , Bertram , R. , Goethe , R. , Valentin -Weigand , P. , 2014 . Omega 2 (10 ), 7400 – 7409 . https:// doi. org/ 10. 1021/ acsomega. 7b01368 . Characterization of multi -drug tolerant persister cells in Streptococcus suis . BMC Portillo , P. , García -Morales , L., Menéndez , M. C. , Anzola , J.M. , Rodríguez , J.G. , Helguera Microbiol. 14 , 120 2014 . http:// www. biomedcentral. com/ 1471 -2180/ 14/ 120 . Repetto , A. C. , et al. , 2019 . Hypoxia is not a main stress when Mycobacterium Wilmaerts , D. , Windels , E. M. , Verstraeten , N. , Michiels , J., 2019 . General Mechanisms tuberculosis is in a dormancy -like long -chain fatty acid environment . Front. Cell. Leading to Persister Formation and Awakening Trends in Genetics . Infect. Microbiol. 8 . https:// doi. org/ 10. 3389/ fcimb. 2018. 00449 . Windels , E. M. , Meriem , Z. B. , Zahir , T. , Verstrepen , K. J., Hersen , P. , BVden , B. , Michiels , Prax , M. , Bertram , R. , 2014 . Metabolic aspects of bacterial persisters . Front. Cell. Infect. J., 2019 . Isolation of Persisters Enabled by ß- Lactam -Induced Filamentation 1 Microbiol. 4 , 1 – 6 . https:// doi. org/ 10. 3389/ fcimb. 2014. 00148 . Reveals Their 2 Single -Cell Awakening Characteristics . bioRxiv . Pu , Y. , Zhao , Z. , Li, Y. , Zou , J., Ma , Q. , Zhao , Y. , Ke, Y. , 2016 . Enhanced efflux activity Wood , T. K. , Knabel , S. J., Kwan , B. W., 2013 . Bacterial persister cell formation and facilitates drug tolerance in dormant bacterial cells . Mol. Cell 62 , 284 – 294 . https:// dormancy . Appl. Environ. Microbiol. 79 ( 23 ) , 7116 – 7121 . https:// doi. org/ 10. 1128/ doi. org/ 10. 1016/ j. molcel. 2016. 03. 035 . aem. 02636 -13 . Ramirez -Castillo , F.Y. , Moreno -Flores , A. C. , Avelar -Gonzalez , F.J. , Marquez -Diaz , F., Wu , N. , He, L., Cui , P. , Wang , W., Yuan , Y. , Liu , S. , Xu , T. , Zhang , S. , Wu , J., Zhang , W., Harel , J., Guerrero -Barrera , A. L., 2018 . An evaluation of multidrug -resistant Zhang , Y. , 2015 . Ranking of persister genes in the same Escherichia coli genetic

9 S. Behera and S. Pattnaik Biocatalysis and Agricultural Biotechnology 22 (2019) 101401

background demonstrates varying importance of individual persister genes in org/ 10. 1038/ s41421 -019 -0080 -3 . tolerance to different antibiotics . Front. Microbiol. 6 , 1003 . https:// doi. org/ 10. Zhang, Y., Li, Y., 2010. Phou (Perf), A Persistence Switch Involved in Persister 3389/ fmicb. 2015. 01003 . Formation and Tolerance to Multiple Antibiotics and Stresses as a Drug Target for Yang , S. , Hay , I.D. , David , R. , Speir, C. M. , Cui , B. , FeifeiSu , F., Anton , Y. , Peleg , A. Y. , Persister Bacteria, Patent Publication No. US 2010/ 0210602 A1. Lithgow , T. , Deighton , M. A. , Qu , Y. , 2015 . Antibiotic regimen based on population Zhang , Y. , Yew , W.W. , Barerc , M. R. , 2012 . Targeting persisters for tuberculosis control analysis of residing persister cells eradicates Staphylococcus epidermidis biofilms. Sci. society for Microbiology. All rights reserved . Antimicrob. Agents Chemother. 2223 – Rep. 5 (18578 ), 1 – 11 . https:// doi. org/ 10. 1038/ srep18578 . 2230 . https:// doi. org/ 10. 1128/ AAC. 06288 -11 . Yu , J., Liu , Y. , Yin , H. , Chang , Z. , 2019 . Regrowth -delay body as a bacterial sub cellular structure marking multidrug -tolerant persisters . Cell Discovery 5 (1 ). https:// doi.

10