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Research article

Bacteriophage treatment of carbapenemase-producing Klebsiella pneumoniae in a multispecies biofilm: a potential biocontrol strategy for healthcare facilities

  • The p-traps of hospital handwashing sinks represent a potential reservoir for antimicrobial-resistant organisms of major public health concern, such as carbapenemase-producing KPC+ Klebsiella pneumoniae (CPKP). Bacteriophages have reemerged as potential biocontrol agents, particularly against biofilm-associated, drug-resistant microorganisms. The primary objective of our study was to formulate a phage cocktail capable of targeting a CPKP strain (CAV1016) at different stages of colonization within polymicrobial drinking water biofilms using a CDC biofilm reactor (CBR) p-trap model. A cocktail of four CAV1016 phages, all exhibiting depolymerase activity, were isolated from untreated wastewater using standard methods. Biofilms containing Pseudomonas aeruginosa, Micrococcus luteus, Stenotrophomonas maltophilia, Elizabethkingia anophelis, Cupriavidus metallidurans, and Methylobacterium fujisawaense were established in the CBR p-trap model for a period of 28 d. Subsequently, CAV1016 was inoculated into the p-trap model and monitored over a period of 21 d. Biofilms were treated for 2 h at either 25 °C or 37 °C with the phage cocktail (109 PFU/ml) at 7, 14, and 21 d post-inoculation. The effect of phage treatment on the viability of biofilm-associated CAV1016 was determined by plate count on m-Endo LES agar. Biofilm heterotrophic plate counts (HPC) were determined using R2A agar. Phage titers were determined by plaque assay. Phage treatment reduced biofilm-associated CAV1016 viability by 1 log10 CFU/cm2 (p < 0.05) at 7 and 14 d (37 °C) and 1.4 log10 and 1.6 log10 CFU/cm2 (p < 0.05) at 7 and 14 d, respectively (25 °C). No significant reduction was observed at 21 d post-inoculation. Phage treatment had no significant effect on the biofilm HPCs (p > 0.05) at any time point or temperature. Supplementation with a non-ionic surfactant appears to enhance phage association within biofilms. The results of this study suggest the potential of phages to control CPKP and other carbapenemase-producing organisms associated with microbial biofilms in the healthcare environment.

    Citation: Ariel J. Santiago, Maria L. Burgos-Garay, Leila Kartforosh, Mustafa Mazher, Rodney M. Donlan. Bacteriophage treatment of carbapenemase-producing Klebsiella pneumoniae in a multispecies biofilm: a potential biocontrol strategy for healthcare facilities[J]. AIMS Microbiology, 2020, 6(1): 43-63. doi: 10.3934/microbiol.2020003

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  • The p-traps of hospital handwashing sinks represent a potential reservoir for antimicrobial-resistant organisms of major public health concern, such as carbapenemase-producing KPC+ Klebsiella pneumoniae (CPKP). Bacteriophages have reemerged as potential biocontrol agents, particularly against biofilm-associated, drug-resistant microorganisms. The primary objective of our study was to formulate a phage cocktail capable of targeting a CPKP strain (CAV1016) at different stages of colonization within polymicrobial drinking water biofilms using a CDC biofilm reactor (CBR) p-trap model. A cocktail of four CAV1016 phages, all exhibiting depolymerase activity, were isolated from untreated wastewater using standard methods. Biofilms containing Pseudomonas aeruginosa, Micrococcus luteus, Stenotrophomonas maltophilia, Elizabethkingia anophelis, Cupriavidus metallidurans, and Methylobacterium fujisawaense were established in the CBR p-trap model for a period of 28 d. Subsequently, CAV1016 was inoculated into the p-trap model and monitored over a period of 21 d. Biofilms were treated for 2 h at either 25 °C or 37 °C with the phage cocktail (109 PFU/ml) at 7, 14, and 21 d post-inoculation. The effect of phage treatment on the viability of biofilm-associated CAV1016 was determined by plate count on m-Endo LES agar. Biofilm heterotrophic plate counts (HPC) were determined using R2A agar. Phage titers were determined by plaque assay. Phage treatment reduced biofilm-associated CAV1016 viability by 1 log10 CFU/cm2 (p < 0.05) at 7 and 14 d (37 °C) and 1.4 log10 and 1.6 log10 CFU/cm2 (p < 0.05) at 7 and 14 d, respectively (25 °C). No significant reduction was observed at 21 d post-inoculation. Phage treatment had no significant effect on the biofilm HPCs (p > 0.05) at any time point or temperature. Supplementation with a non-ionic surfactant appears to enhance phage association within biofilms. The results of this study suggest the potential of phages to control CPKP and other carbapenemase-producing organisms associated with microbial biofilms in the healthcare environment.



    Throughout this paper, by a topological dynamical system (X,T) (TDS for short) we mean a compact metric space (X,d) with a homeomorphism map T from X onto itself, where d refers to the metric on X. By a measure preserving system (MPS for short) we mean a quadruple (X,X,μ,T), where (X,X,μ) is a Borel probability space and T,T1:XX are both measurable and measure preserving, i.e. T1X=X=TX and μ(A)=μ(T1A) for each AX.

    Given a TDS (X,T), let M(X,T) be the set of all T-invariant Borel probability measures of X. In weak-topology, M(X,T) is a compact convex space. By Krylov-Bogolioubov Theorem M(X,T). For each μM(X,T), (X,BX,T,μ) can be viewed as a MPS, where BX is the Borel σ-algebra of X. Let Me(X,T) be the space of all ergodic measures of (X,T). Then Me(X,T) is the set of extreme points of M(X,T).

    Define

    E(T)={hμ(T):μMe(X,T)}

    where hμ(T) denotes the measure-theoretic entropy of the measure preserving system (X,BX,T,μ). By the variational principle of entropy supE(T)=htop(T), where htop(T) is the topological entropy of (X,T). The extreme case is that Me(X,T) consists of only one member, that is, (X,T) is uniquely ergodic. When (X,T) is uniquely ergodic, E(T)={htop(T)}.

    It is interesting to consider the case when E(T) is big. As a direct corollary of [7,Theorem 11], Katok showed that

    [0,htop(f))E(f) (1.1)

    for any C1+α diffeomorphism f on a two-dimensional surface, based on the fact that every ergodic measure of positive metric entropy is hyperbolic. Katok conjectured that (1.1) holds for any smooth system.

    Conjecture 1.1 (Katok). Let f be a Cr (r>1) diffeomorphism on a smooth compact manifold M, then (1.1) holds, i.e. for every a[0,htop(f)), there is μaMe(M,f) such that hμa(f)=a.

    We need to point out that Katok's conjecture implies that any positive entropy smooth system is not uniquely ergodic, though whether or not a smooth diffeomorphism of positive topological entropy can be uniquely ergodic is still in question (see [5] for Herman's example: positive entropy minimal C-smooth diffeomorphisms). In [13,14], Quas and Soo showed that if a topological dynamical system satisfies asymptotic entropy expansiveness, almost weak specification property and small boundary property, then it is universal, which implies the conclusion of Katok's conjeture. Recently, Burguet [2], Chandgotia and Meyerovitch [4], extended the result of Quas and Soo to request only the almost weak specification property.

    In this paper, we study intermediate entropy for affine transformations of nilmanifolds. Throughout this paper, by a nilmanifold G/Γ we mean that G is a connected, simply connected nilpotent Lie group, and Γ is a cocompact discrete subgroup of G. A homeomorphism τ of G/Γ is an affine transformation if there exist a Γ-invariant automorphism A of G and a fixed element g0G such that τ(gΓ)=g0A(g)Γ for each gG. Our main result is the following.

    Theorem 1.2. Let G/Γ be a nilmanifold and τ be an affine transformation of G/Γ. If (G/Γ,τ) has a periodic point, then E(τ)=[0,htop(τ)].

    Following Lind [11], we say that an affine transformation of a nilmanifold is quasi-hyperbolic if its associated matrix has no eigenvalue 1. As an application of Theorem 1.2, one has the following.

    Theorem 1.3. Let G/Γ be a nilmanifold and τ be an affine transformation of G/Γ. If τ is quasi-hyperbolic, then E(τ)=[0,htop(τ)].

    The paper is organized as follows. In Section 2, we introduce some notions. In Section 3, we prove Theorem 1.2 and Theorem 1.3.

    In this section, we recall some notions of entropy, nilmanifold and upper semicontinuity of entropy map.

    We summarize some basic concepts and useful properties related to topological entropy and measure-theoretic entropy here.

    Let (X,T) be a TDS. A cover of X is a family of subsets of X, whose union is X. A partition of X is a cover of X whose elements are pairwise disjoint. Given two covers U,V of X, set UV={UV:UU,VV} and TiU={TiU:UU} for iZ+. Denote by N(U) the minimal cardinality among all cardinalities of subcovers of U.

    Definition 2.1. Let (X,T) be a TDS and U be a finite open cover of X. The topological entropy of U is defined by

    htop(T,U)=limn+1nlogN(n1i=0TiU),

    where {logN(n1i=0TiU)}n=1 is a sub-additive sequence and hence htop(T,U) is well defined. The topological entropy of (X,T) is

    htop(T)=supUhtop(T,U),

    where supremum is taken over all finite open covers of X.

    A subset E of X is an (n,ϵ)-separated set with respect to T provided that for any distinct x,yE there is 0j<n such that d(Tjx,Tjy)ϵ. Let K be a compact subset of X. Let s(T)n(ϵ,K) be the largest cardinality of any subset E of K which is an (n,ϵ)-separated set. Then the Bowen's topological entropy of K with respect to T [1] is defined by

    hd(T,K)=limϵ0lim supnlogs(T)n(ϵ,K)n.

    Let Z be a non-empty subset of X. The Bowen's topological entropy of Z with respect to T is defined by

    hd(T,Z)=supKZK is compacthd(T,K).

    And the Bowen's topological entropy of a TDS (X,T) is defined by hd(T)=hd(T,X) which happens to coincide with htop(T).

    Next we define measure-theoretic entropy. Let (X,X,μ,T) be a MPS and PX be the set of finite measurable partitions of X. Suppose ξPX. The entropy of ξ is defined by

    hμ(T,ξ)=limn+1nHμ(n1i=0Tiξ),

    where Hμ(n1i=0Tiξ)=An1i=0Tiξμ(A)logμ(A) and {Hμ(n1i=0Tiξ)}n=1 is a sub-additive sequence. The entropy of (X,X,T,μ) is defined by

    hμ(T)=supξPXhμ(T,ξ).

    The basic relationship between topological entropy and measure-theoretic entropy is given by the variational principle [12].

    Theorem 2.2 (The variational principle). Let (X,T) be a TDS. Then

    htop(T)=sup{hμ(T):μM(X,T)}=sup{hμ(T):μMe(X,T)}.

    A factor map π:(X,T)(Y,S) between the TDS (X,T) and (Y,S) is a continuous onto map with πT=Sπ; we say that (Y,S) is a factor of (X,T) and that (X,T) is an extension of (Y,S). The systems are said to be conjugate if π is bijective. In [8], Ledrappier and Walters showed that if π:(X,T)(Y,S) is a factor map and νM(Y,S), then

    supμM(X,T)π(μ)=νhμ(T)=hν(S)+Yhd(T,π1(y))dν(y) (2.1)

    where π(μ)(B)=μ(π1(B)) for BBY.

    Let G be a compact metric group and τ:GG be a continuous surjective map. Let π:(X,T)(Y,S) be a factor map. We say that π is a (G,τ)-extension, if there exists a continuous map P:X×GX (we write P(x,g)=xg) such that:

    (1) π1(π(x))=xG for xX,

    (2) For any xX,g1,g2G, xg1=xg2 if and only if g1=g2,

    (3)T(xg)=T(x)τ(g) for xX and gG.

    The following is from [1,Theorem 19].

    Theorem 2.3. Let π:(X,T)(Y,S) be a factor map. If π is a (G,τ)-extension, then htop(T)=htop(S)+htop(τ).

    Remark 2.4. (1) In the above situation, Bowen shows that

    hd(T,π1(y))=htop(τ) for any yY, (2.2)

    where d is the metric on X. This fact is proved in the proof of [1,Theorem 19]. In fact, (2.2) holds in the more general situation of actions of amenable groups. This fact is given explicitly as Lemma 6.12 in the paper [10].

    (2) If G is a Lie group, H and N are cocompact closed subgroups of G such that N is a normal subgroup of H, then G/N and G/H are compact metric spaces and H/N is a compact metric group. Given further g0G and an automorphism A of G preserving H and N, one has the affine maps T:G/NG/N given by T(gN)=g0A(g)N, and S:G/HG/H given by S(gH)=g0A(g)H, and the automorphism τ of H/N given by τ(hN)=A(h)N. Then there is a map π:G/NG/H given by π(gN)=gH for gG, and a map P:G/N×H/NG/N given by P(gN,hN)=ghN for g,hG. These maps satisfy the conditions (1)(3) in the definition of (H/N,τ)-extension for the factor map π:(G/N,T)(G/H,S). That is, (G/N,T) is an (H/N,τ)-extension of (G/H,S). Hence one has by (2.2) that

    hd(T,π1(y))=htop(τ) for any yG/H, (2.3)

    where d is the metric on G/N.

    Given a TDS (X,T), the entropy map of (X,T) is the map μhμ(T) which is defined on M(X,T) and has value in [0,]. For any invariant measure μ on X, there is a unique Borel probability measure ρ on M(X,T) with ρ(Me(X,T))=1 such that

    Me(X,T)Xf(x)dm(x)dρ(m)=Xf(x)dμ(x) for all fC(X).

    We write μ=Me(X,T)mdρ(m) and call it the ergodic decomposition of μ. The following is standard.

    Theorem 2.5. Let (X,T) be a TDS. If μM(X,T) and μ=Me(X,T)mdρ(m) is the ergodic decomposition of μ. Then

    hμ(T)=Me(X,T)hm(T)dρ(m).

    We say that the entropy map of (X,T) is upper semicontinuous if for μn,μM(X,T)

    limnμn=μ implies lim supnhμn(T)hμ(T).

    We say that a TDS (X,T) satisfies asymptotic entropy expansiveness if

    limδ0supxXhd(T,Γδ(x))=0.

    Here for each δ>0,

    Γδ(x):={yX:d(Tjx,Tjy)<δ for all j0}.

    The result of Misiurewicz [12,Corollary 4.1] gives a sufficient condition for upper semicontinuity of the entropy map.

    Theorem 2.6. Let (X,T) be a TDS. If (X,T) satisfies asymptotic entropy expansiveness. Then the entropy map of (X,T) is upper semicontinuous.

    The result of Buzzi [3] gives a sufficient condition for asymptotic entropy expansiveness.

    Theorem 2.7. Let f be a C diffeomorphism on a smooth compact manifold M, then (M,f) satisfies asymptotic entropy expansiveness. Especially, the entropy map of (M,f) is upper semicontinuous.

    In this section, we prove our main results. In the first subsection, we prove that Katok's conjecture holds for affine transformations of torus. In the second subsection, we show some properties of metrics on nilmanifolds. In the last subsection, we prove Theorem 1.2 and Theorem 1.3.

    We say that a topological dynamical system (Y,S) is universal if for every invertible non-atomic ergodic measure preserving system (X,X,μ,T) with measure-theoretic entropy strictly less than the topological entropy of S there exists νMe(Y,S) such that (X,X,μ,T) is isomorphic to (Y,BY,ν,S). In [14], Quas and Soo show that toral automorphisms are universal, which implies the conclusion of Katok's conjeture. By using Quas and Soo's result, we have the following.

    Theorem 3.1. Let mN, Tm=Rm/Zm and τ be an affine transformation of Tm. Then E(τ)=[0,htop(τ)].

    Proof. We think of Tm as a group. Then there exist an element bTm and a toral automorphism A of Tm such that

    τ(x)=A(x)+b for each xTm.

    Let μh be the Haar measure. Then hμh(τ)=htop(τ). Let μh=Me(Tm,τ)νdρ(ν) be the ergodic decomposition of μh. Then by Theorem 2.5, one has

    htop(τ)=Me(Tm,τ)hν(τ)dρ(ν).

    By variational principle, there exists μMe(Tm,τ) such that hμ(τ)=htop(τ). Now we assume that a[0,htop(τ)). We have two cases.

    Case 1. A is quasi-hyperbolic. In this case, there is qTm such that A(q)=qb. We let

    π(x)=xq for each xTm.

    Then π is a self homeomorphism of Tm and πτ=Aπ. That is, (Tm,τ) topologically conjugates to a torus automorphism. By Quas and Soo's result [14,Theorem 1], there exists μaMe(Tm,τ) such that hμa(τ)=a.

    Case 2. A is not quasi-hyperbolic. In this case, we put

    H={xTm:(Aid)mx=0}.

    Then H is a compact subgroup of Tm and Tm/H is a torus. We let Y=Tm/H and πY be the natural projection from Tm to Y. The induced map τY on Y is a quasi-hyperbolic affine transformation and the extension πY is distal. Therefore, htop(τY)=htop(τ) and by Case 1 there exists μYaMe(Y,τY) such that hμYa(τY)=a. There is μaMe(Tm,τ) such that πY(μa)=μYa. Since the extension πY is distal, one has hμa(τ)=hμYa(τY)=a (see [6,Theorem 4.4]).

    This ends the proof of Theorem 3.1.

    Let G be a group. For g,hG, we write [g,h]=ghg1h1 for the commutator of g and h and we write [B1,B2] for the subgroup spanned by {[b1,b2]:b1B1,b2B2}. The commutator subgroups Gj, j1, are defined inductively by setting G0=G1=G and Gj+1=[Gj,G]. Let s1 be an integer. We say that G is s-step nilpotent if Gs+1 is the trivial subgroup. Recall that an s-step nilmanifold is a manifold of the form G/Γ where G is a connected, simply connected s-step nilpotent Lie group, and Γ is a cocompact discrete subgroup of G.

    If G/Γ is an s-step nilmanifold, then for each j=1,,s, GjΓ and Gj are closed subgroups of G and GjΓ/Γ is a closed submanifold of G/Γ (see Subsection 2.11 in [9]).

    We fix an s-step nilmanifold of the form G/Γ and an affine transformation τ of G/Γ such that

    τ(gΓ)=g0A(g)Γ for each gG

    where g0G and A is a Γ-invariant automorphism of G. For each j1, we let

    Aj:Gj1Γ/GjΓGj1Γ/GjΓ:Aj(hGjΓ)=A(h)GjΓ for each hGj1

    and

    τj:G/GjΓG/GjΓ:τj(hGjΓ)=g0A(h)GjΓ for each hG.

    It is easy to see that {Aj}jN and {τj}jN are well defined since A(Gj)Gj for each j1.

    For each j1, define the map πj+1 from G/Gj+1Γ to G/GjΓ by

    πj+1(gGj+1Γ)=gGjΓ for each gG. (3.1)

    It is easy to see that πj+1 is continuous and onto, and satisfies πj+1τj+1=τjπj+1. Hence, for each j1, πj+1:G/Gj+1ΓG/GjΓ is a factor map. We let bj=htop(Aj) for each j1. Then we have the following.

    Lemma 3.2. For each j1 and yG/GjΓ, hdj+1(τj+1,π1j+1(y))=bj+1 where dj+1 is the metric on G/Gj+1Γ.

    Proof. In Remark 2.4 (2), we let N=Gj+1Γ and H=GjΓ. Then both N and H are cocompact subgroup of G. Moreover, N is a normal subgroup of H. Hence (G/N=G/Gj+1Γ,τj+1) is an (H/N=GjΓ/Gj+1Γ,Aj+1)-extension of (G/H=G/GjΓ,τj). By (2.3), one has

    hdj+1(τj+1,π1j+1(y))=htop(Aj+1)=bj+1 for every yG/GjΓ.

    This ends the proof of Lemma 3.2.

    The following result is immediately from Lemma 3.2, (2.1) and Theorem 2.7.

    Lemma 3.3. For j1 and νjM(G/GjΓ,τj), there exists μM(G/Gj+1Γ,τj+1) such that hμ(τj+1)=hνj(τj)+bj+1.

    We have the following.

    Corollary 3.4. htop(τj)=ji=1bi for j1. Especially, htop(τ)=s+1i=1bi.

    Proof. We prove the corollary by induction on j. In the case j=1, it is obviously true. Now we assume that the corollary is valid for some jN. Then for j+1, let πj+1 be defined as in (3.1). Then by Ledrappier and Walters's result (2.1) and variational principle Theorem 2.2, we have

    htop(τj+1)=supμM(G/Gj+1Γ,τj+1)hμ(τj+1)supμM(G/GjΓ,τj)(hμ(τj)+G/GjΓhdj+1(τj+1,π1j+1(y))dμ(y))htop(τj)+supμM(G/GjΓ,τj)G/GjΓhdj+1(τj+1,π1j+1(y))dμ(y)=ji=1bi+bj+1=j+1i=1bi,

    where we used Lemma 3.2. On the other hand, by Lemma 3.3 there exists μM(G/Gj+1Γ,τj+1) such that hμ(τj+1)=j+1i=1bi. Therefore htop(τj+1)=j+1i=1bi. By induction, this ends the proof of Corollary 3.4.

    Remark 3.5. We remark that the topological entropy of (G/Γ,τ) is determined by the associated matrix of τ [1]. That is

    htop(τ)=hd(τ)=|λi|>1log|λi|

    where λ1,λ2,,λm are the eigenvalues of the associated matrix of τ.

    Lemma 3.6. For j1 and νjMe(G/GjΓ,τj), there is νj+1Me(G/Gj+1Γ,τj+1) such that hνj+1(τj+1)=hνj(τj)+bj+1.

    Proof. We fix νjMe(G/GjΓ,τj). Let πj+1 be defined as in (3.1). By Lemma 3.3, there exists νM(G/Gj+1Γ,τj+1) such that

    hν(τj+1)=supμM(G/Gj+1Γ,τj+1)πj+1(μ)=νjhμ(τj+1)=hνj(τj)+bj+1.

    We fix such ν and assume that the ergodic decomposition of ν is

    ν=Me(G/Gj+1Γ,τj+1)mdρ(m).

    Then by property of ergodic decomposition, one has

    ρ({mMe(G/Gj+1Γ,τj+1):πj+1(m)=νj})=1.

    Therefore, for ρ-a.e. mMe(G/Gj+1Γ,τj+1),

    hm(τj+1)hν(τj+1)=hνj(τj)+bj+1.

    Hence by Theorem 2.5, one has

    hνj(τj)+bj+1=hν(τj+1)=Me(G/Gj+1Γ,τj+1)hm(τj+1)dρ(m)hνj(τj)+bj+1.

    We notice that the equality holds only in the case hm(τj+1)=hνj(τj)+bj+1 for ρ-a.e. mMe(G/Gj+1Γ,τj+1). Therefore, there exists νj+1Me(G/Gj+1Γ,τj+1) such that

    hνj+1(τj+1)=hνj(τj)+bj+1 and πj+1(νj+1)=νj.

    This ends the proof of Lemma 3.6.

    Now we are ready to prove our main results.

    Proof of Theorem 1.2. Firstly we assume that (G/Γ,τ) has a fixed point pΓ. We fix a real number a[0,htop(τ)]. We are going to show that there exists μaMe(G/Γ,τ) such that hμa(τ)=a. By Corollary 3.4, we can find an i{1,2,,s,s+1} such that

    s+1j=i+1bjas+1j=ibj.

    Since pΓ is a fixed point of (G/Γ,τ), there exists γΓ such that g0A(p)=pγ. Therefore,

    τi(pGi1Γ/GiΓ)=pγGi1Γ/GiΓp[γ,Gi1]Gi1γΓ/GiΓpGi1Γ/GiΓ,

    where we used the fact [γ,Gi1]Gi1. That is, (pGi1Γ/GiΓ,τi) is a TDS. We let

    π(phGiΓ)=hGiΓ for each hGi1.

    Then for each hGi1, one has

    πτi(phGiΓ)=p1g0A(p)A(h)GiΓ=γA(h)GiΓ=A(h)γ[γ,A(h)]GiΓ=A(h)GiΓ

    where we used the fact [γ,A(h)]Gi since hGi1. Therefore πτi(phGiΓ)=Aiτi(phGiΓ) for each hGi1. That is πτi=Aiπ. Hence,

    (pGi1Γ/GiΓ,τi) topologically conjugates to (Gi1Γ/GiΓ,Ai).

    Notice that (Gi1Γ/GiΓ,Ai) is a toral automarphism and htop(Ai)=bi. By Theorem 3.1, there exists νiMe(G/GiΓ,τi) such that hνi(τi)=as+1j=i+1bj. Combining this with Lemma 3.6, there exists an ergodic measure μa=νs+1Me(G/Gs+1Γ,τs+1)=Me(G/Γ,τ) such that

    hμa(τ)=hνs+1(τs+1)=hνi(τi)+s+1j=i+1bj=a.

    Thus μa is the ergodic measure as required.

    Now we assume that (G/Γ,τ) has a periodic point. By assumption, there exists mN such that (G/Γ,τm) has a fixed point. Since τm is an affine transformation of G/Γ, by argument above, there exists μMe(G/Γ,τm) such that hμ(τm)=ma. Put μa=1mm1j=0τj(μ). It is easy to see that μaMe(G/Γ,τ) and hμa(τ)=hμ(τm)m=a. Thus μa is the ergodic measure as required.

    This ends the proof of Theorem 1.2.

    Proposition 3.7. Let G be an s-step nilpotent Lie group and A be a quasi-hyperbolic automorphism of G. Then for gG, there exists pG such that gA(p)=p.

    Proof. We prove the proposition by induction on s. In the case s=1, it is obviously true. Now we assume that the Proposition is valid in the case s=k. Then in the case s=k+1, we fix gG. Notice that G/Gk+1 is a k-step nilpotent Lie group. There exists ˜pG such that gA(˜p)Gk+1=˜pGk+1. There exists ˉgGk+1 such that gA(˜p)ˉg=˜p. There exists pGk+1 such that ˉg1A(p)=p. In the end, we let p=˜pp. Then

    gA(p)=gA(˜p)A(p)=˜pˉg1ˉgp=˜pp=p.

    By induction, we end the proof of Proposition 3.7.

    Proof of Theorem 1.3. This comes immediately from Proposition 3.7 and Theorem 1.2.

    W. Huang was partially supported by NNSF of China (11731003, 12031019, 12090012). L. Xu was partially supported by NNSF of China (11801538, 11871188, 12031019) and the USTC Research Funds of the Double First-Class Initiative.


    Abbreviation ANOVA: analysis of variance; AR: antibiotic resistant; CBR: CDC biofilm reactor; CDC: Centers for Disease Control and Prevention; CRE: carbapenem-resistant Enterobacteriaceae; CPKP: carbapenemase-producing ; CV: crystal violet; KPC: carbapenemase; EPS: extracellular polymeric substance; HAIs: healthcare-associated infections; HPCs: heterotrophic plate counts; MOI: multiplicity of infection; PBS: phosphate buffered saline; PSB: phage storage buffer; TSA: trypticase soy agar; TSB: tryptic soy broth; USD: U. S. dollars; SAO: soft agar overlay; DI: deionized; SEM: standard error of the mean;
    Acknowledgments



    This work was supported in part by internal funds provided under the Centers for Disease Control and Prevention's (CDC) Innovation Fund. Authors would like to acknowledge staff from Snapfinger Creek Advanced Wastewater Plant (Dekalb County, GA) and RL Sutton Wastewater Reclamation Facility (Cobb County, GA) for collecting wastewater samples. The use of trade names and commercial sources is for identification only and does not imply endorsement by the Public Health Service or the U.S. Department of Health and Human Services. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. CDC.

    Conflicts of Interest



    All authors declare no conflicts of interest in this paper.

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