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Isolation of Bacteria from Remote High Altitude Andean Lakes Able to

Isolation of Bacteria from Remote High Altitude Andean Lakes Able to
Isolation of Bacteria from Remote High Altitude Andean Lakes Able to

Isolation of Bacteria from Remote High Altitude Andean Lakes Able to Grow in the Presence of Antibiotics

Julián R. Dib*1, Annika Weiss*2, Anna Neumann*2, Omar Ordo?ez1, María C. Estévez*1 and Maria

E. Farías*1

*1PROIMI, Planta Piloto de Procesos Industriales Microbiológicos, Av. Belgrano y Pasaje Caseros -(4000) - Tucumán, Argentina. CONICET Consejo Nacional de Investigaciones Científicas y Técnicas, *2Technische Universit?t Berlin, Fakult?t III, Umweltmikrobiologie Franklinstr. 29, 10587 Berlin, Germany

Received: October 24, 2008; Accepted: November 14, 2008; Revised: December 2, 2008

Abstract: High altitude Andean lakes are placed in Puna desert over 4400 above sea level. Completely isolated, they are

exposed to extreme environmental factors like high levels of salinity, UV radiation and heavy metals and low

concentrations of phosphorus. Nevertheless, they are the habitat of enormous populations of three flamingo species that

migrate among these Lakes. Previous reports have determined that bacteria isolated from these environments present high

levels of resistance to antibiotics.

The aim of this work was to determine the diversity of antibiotic resistant bacteria in water from Andean Lakes and their

connection with flamingo enteric biota. Bacteria from water and birds faeces from high altitude Lakes: Laguna (L.)

Aparejos, L. Negra, L. Vilama and L. Azul (all are located between 4,200 and 4,600 m altitude) were isolated by plating

in five different Antibiotics (ampicillin, 100 g ml-1; chloramphenicol, 170 g ml-1; colistin , 20 g ml-1; erythromycin, 50

g ml-1and tetracycline 50 g ml-1). 56 bacteria were isolated and identified by 16 S rDNA sequencing. Antibiotic

resistance profiles of isolated bacteria were determined for 22 different antibiotics. All identified bacteria were able to

growth in multiple ATBs. Colistin, ceftazidime, ampicillin/sulbactam, cefotaxime, cefepime, cefalotin, ampicillin and

erythromycin were the most distributed resistances among the 56 tested bacteria

The current results demonstrated that antibiotic resistance was abundant and diverse in high altitude Lakes. Also the

present article indicates some useful patents regarding the isolation of bacteria able to grow in the present of antibiotics. Keywords: Andean lakes microbiology, antibiotics resistance in environmental bacteria, extreme environments, water birds.

#Dedicated to the memory of Dr. Sandra Caziani, a great ornithologist and woman. Her spirit keeps

on flying with the birds in the Puna sky.

INTRODUCTION

Extreme environments are interesting resources for microorganisms with exceptional phenotypic and genotypic characteristics. High Altitude Andean Lakes are sites with extreme conditions like high UV incidence, high arsenic concentrations, high salinity and oligotrophy, among others. Microbial communities living in such aquatic ecosystems are tolerant to large fluctuations in environmental factors in addition to steady-state extreme conditions [1-3]. These Lakes have demonstrated to be a source of microbial diversity and strategies that allow microorganisms to survive under severe conditions [4-6]. UV radiation is considered one of the most extreme conditions microorganisms living in these environments have to cope with [5]. In response to this, the cell repair mechanisms increases mutational events. Spontaneous resistance to antibiotics is known to emerge under mutagenic conditions; in fact, bacterial mutagenesis after UV stress has been observed with spontaneous resistance to rifampicin or nalidixic acid [4, 6].

*Address correspondence to this author at the PROIMI, Planta Piloto de Procesos Industriales Microbiológicos, Av. Belgrano y Pasaje Caseros -(4000) - Tucumán, Argentina. CONICET Consejo Nacional de Investi-gaciones Científicas y Técnicas; Tel: 54-381-4344888 # 24;

Fax:54-381-4344-887;E-mail:mefarias@https://www.doczj.com/doc/3616199884.html,.ar;

mefarias2001@https://www.doczj.com/doc/3616199884.html,.ar

*1Authors who contributed equally to this work.

Bacterial resistance is the ability of microorganisms to continue growing in the presence of a cytotoxic compound. Environmental microorganisms are successful creators of new biosynthetic pathways that generate new bioactive compounds (many of them cyto-toxic) while maintaining evolutionary successful ones that occurred millions of years ago [7]. Many of these natural products function as ATBs, or at least generate a molecular response in bacteria that is equivalent to resistance. Therefore, environmental bacteria are a reservoir of resistance to novel ATBs [8-15]. In previous studies at our laboratory we have demonstrated the presence and correlation of resistance to UV-B radiation, arsenic and ATBs (Azithromycin, Erythromycin, Clarithro-mycin, Roxithromycin, Streptomycin, Chloramphenicol, Gentamicin, Kanamycin, Tetracycline and Ampicillin) in two pristine Andean Lakes, L. Azul and L. Vilama, situated at 4,400 and 4,600 m asl, respectively, where ATB selective pressure is supposed to be absent [4-6]. Although these Lakes are quite distant from each other (700 km), the bacteria isolated from both Lakes showed similar ATB resistance patterns. As has been mentioned before, flamingoes are the predominant vertebrate fauna in these Lakes. They feed themselves by filtrating water from these Lakes and they migrate between them. Birds have been postulated as microbial dispersers [10, 11, 16].

1574-891X/09 $100.00+.00 ? 2009 Bentham Science Publishers Ltd.

The aim of this paper was to determine whether the ability to grow in the presence of ATBs is an extended characteristic of bacteria that inhabit these environments. Therefore, bacteria were isolated from water from four Lakes: L. Aparejos, L. Negra, L. Vilama and L. Azul (4,200, 4,400, 4,600 and 4,400 m asl respectively). Considering that flamingoes are the principal inhabitant of these environments and the fact that they only feed from water of these Lakes; flamingo feces were also included in the samples to broaden the search for bacterial resistance.

MATERIALS AND METHODS

Description of Sample Sites

General parameters of the environments studied are described in Table 1.

L. Negra, L. Aparejos and L. Azul are located in the Andes mountains in the Northwest of Argentina. They belong to the ‘Salar de la Laguna Verde’ in Catamarca province, Argentina (27o 34′ S; 658o 32′ W), a group of shallow lakes and salt flats Fig. (1). The area is practically unexplored mainly due to the lack of access roads. The distance among the three lakes is almost 40 km. Two of the highest mountains of the Andes are found in this area: Ojos del Salado (6,885 m) and Nevado Pissis (6,779 m). Rain fall is scarce and glaciers are found above 5,800 m. The water temperature was 5oC at the moment of sampling (1:30 PM) during a summer day in the southern hemisphere and maximum UV-B irradiance reached 10.78 W m-2 at 312 nm (half band with 300 to 325 nm).

L. Vilama is also located in the Argentine Puna desert but further north (22°35’S and 66°55’W)at 4,600 m altitude Fig. (1). L. Vilama is, inhabited with no access roads. The area is cold and arid and is exposed to strong winds. It endures large daily temperature fluctuations (- 40oC at night to -20oC during the day) and high solar radiation levels, especially during summer (8.94 W m-2 of UV-B at noon). It is an oligotrophic environment with no detectable phosphorus contents and consequently chlorophyll production is low (2.8 10-4 g L-1). It has hyper saline characteristics (117 ppm) with crystallizer ponds (i.e., ponds where sodium chloride precipitates) and high arsenic contents (11.8 mg L-1), the water is clear and average depth is only 20 cm Table 1. Gamma-proteo-bacteria, HGC bacteria and Firmicutes were the predominant phylogenetic affiliation of isolates from this lake and most of them, like Pseudomonas sp. and Brachybacterium sp.,presented high UV-B resistance [4].

The four Lakes are the habitat of three sympatric species of South American flamingoes: the Chilean Flamingo (Phoenicopterus chilensis), the Andean Flamingo (Phoeni-copterus andinus) and James’ Flamingo (Phoenicopterus jamesi) [17]. They feed on zooplankton, primarily composed of copepods and daphnias, and to minor extent algae, mainly diatoms and microbial community [18]. These flamingoes migrate only around these Lakes, which provide their only source of food [17, 18].

Sampling

Surface water samples were collected during early spring 2006 (September) in 10l acid-washed polyethylene bottles, after pre-rinsing the containers with lake water. Water samples were stored at 4oC until further processing in the laboratory (within approximately 24h after collection), which was located 800 km away from the sampling site. Fresh feces samples were taken from flamengoes and were retained axenic at 4oC until processing in the lab. Four samples were taken from each Lake (L. Vilama, L. Aparejos, L. Negra and L. Azul). Boyle in WO patent describes the cross-reaction and neutralization of multiple species by isolation of antibiotic [19].

Bacterial Isolation and Culture Media

Water and feces samples were plated R2A medium (0.5 g of yeast extract, 0.5 g of proteose peptone, 0.5 g of casamino

Table 1. Characteristics of Four Lakes in Argentina: Laguna Aparejos, L. Negra, L. Azul and L. Vilama

Lake L. Aparejos L. Negra L. Azul L. Vilama Geographic position Catamarca Province Catamarca Province Catamarca Province Jujuy Province

Global position 27o 34′S

68o 23′W

27o40¨S

68o 23′W

27o 38′S

68o 32′W

22o 35′S

66o 55′W

Depth (cm) 10 20 100 20

Altitude (m asl) 4,200 4,400 4,400

4,600 pH 6.5

6.8

7.5

7.1

Arsenic (mg L-1) 2.5

3

0.8

11.8

Phosphorus (mg L-1) ND** < 0,05 < 0.012 ND**

Salinity (ppm) 0,4 32 5 117

Chlorophyll ( g L-1) 5.6

10-4 3.0 10-4 1.9 10-4 2.8 10-4

Max UV-B registered (W m-2 in situ 280-312 nm) 9.8 10.8 10.78 8.94

ND: not determined.

** Below detection limit.

acids, 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of Na-pyruvate, 0.3 g of K 2HPO 4, 0.05 g of MgSO 4 x 7H 2O/liter; pH 7.2). Media were supplemented with five different ATBs: ampicillin (Amp), 100 g ml -1; chloramphenicol (Cmp), 170 g ml -1; colistin (Col), 20 g ml -1; erythromycin (Ery), 50 g ml -1and tetracycline (Tet) 50 g ml -1. Colonies were grouped into morphologically identical types; the homogeneity of different colony groups was corroborated by Rep-PCR (data not shown). Isolates were cultured in R2A broth at 20oC with shaking. Genomic DNA extraction, amplification and

sequencing of the partial 16S rDNA gene were carried out as described by Fernández Zenoff et al . [5] and resulting sequences were registered at GenBank Table 2. Chen et al . in US patent 7384778 showed the susceptibility of antimicrobial and the devices for the detection of pathogenic microorganisms [20].

Determination of Antibiotic Resistance

Isolates were tested by the disk diffusion method on Mueller-Hinton agar according to the recommendations of

A

B

C

Fig. (1). Locations of the high altitude Andean Lakes: Jujuy province with L. Vilama and Salar de la Laguna Verde, Catamarca province, with L. Negra, L. Aparejos and L. Azul (A ). View of Laguna Vilama (B ) and Salar de la Laguna Verde with L. Negra and L. Verde (C

).

Table 2.Phylogenetic Affiliations and ATB Resistance Profiles of Isolates from ATB Enriched Cultures from Water and Feces from L. Negra, L. Aparejos, L. Azul, and L. Vilama. W: Water without Antibiotics, WAmp: Water with Ampicilin, WCmp: Water with Chloramphenicol, WCol: Water with Colistin, WTet: Water with Tetracycline, WEry: Water with Erythromycin, F: Feces without Antibiotics, FCol: Feces with Colistin, FAmp: Feces with Ampicillin, FCmp: Feces with Chloramphenicol, FEry: Feces with Erythromycin, FTet: Feces with Tetracycline

Phylogenetic Affiliation

Closest Identified

Relative (Accession

Number)

% of

Identity

Source Color Resistance to

Antibiotics

Resistance to

No. of ATBs

Isolation Source

L. Negra

Actinobacteria

Ni2 Dietzia sp. AM711580 99 W Salmon/orange Ctx, Caz, Men, Fep

Cet, Col

6 WCol Firmicutes

Ni4 Bacillaceae AM711582 99 W, F White Ams, Ctx, Caz, Fep

Col 5 WAmp, FCol

FAmp

Ni6 Exiguobacterium sp.

AM711583 99 W Transparent

Ams, Ctx, Imp, Caz

Men, Taz, Fep, Cet

Col, Ery, Tet

11 W, WCol, WEry

WTet

Ni14 Bacillus pumillus

AM712182

99 W, F White Caz, Cet, Col 3 W, F

Ni17 Bacillus thuringiensis

AM712183100 W,

F Light

pink Ams, Ctx, Tms, Caz

Fep, Cef, Cmp, Col

8 WCol, F, FCol, F

Ni19 Bacillus thuringiensis

AM712184 98 W,

F Orange Ams, Tms, Imp, Caz

Men, Cip, Taz, Fep

Cef, Akn, Cmp, Col

12 WCmp,

F

Ni21 Bacillus thuringiensis

AM712185 100 F White Ams, Ctx, Tms,

Fep, Col

5 F

Alfa Proteobacteria

Ni3 Sphingomonas sp.

AM711581 99 W Yellow Cip, Cef, Col, Ery

Tet

5 W,

WAmp

WCol, WEry

WTet

Ni9 Sphingomonas sp.

AM711585 99 W Dark/intense

yellow

Men, Cip, Col 3 W, WCol

Ni13 Caulobacter sp.

AM712181

82 W, F yellow/beige Tms, Caz, Cip, Col 4 W, FEry, F Beta Proteobacteria

Ni12 Burkholderia cepacia

AM71158699 W Transparent/

yellow

Ams, Ctx, Men, Cef

Col, Tet

6 WTet

Gamma Proteobacteria

Ni7 Stenotrophomonas

maltophilia AM711584 99 W,

F Orange Ams, Ctx, Imp, Caz

Men, Taz, Fep, Cet

Col, Tet, Ery, Amp

12 W,

WAmp

WCol, WEry

WTet, FAmp, F

FCol, FEry

L. Aparejos

Phylogenetic Affiliation

Closest Identified

Relative (Accession

Number)

% of

Identity

Source Color Resistance to

Antibiotics

Resistance to

No. of ATBs

Isolation Source

Firmicutes

Api4 Bacillus cereus

AM711589 99 W, F Snow/white Ams, Ctx, Tms

Imp, Caz, Fep, Cef

Col, Amp

9 W,

WAmp

WCol, F

Api7 Bacillus cereus

AM711591 99 W,

F White Caz, Men, Cip, Col

Cmp, Amp

6 W, F, FAmp

FCmp, FCol

Api11 Bacillus cereus

AM711594 98 F Orange Ams,

Ctx,

Tms

Caz, Taz, Fep, Cef

Col

8 FCol

Api10 Staphylococcus sp.

AM711593

100 F Transparent Col 1 FCol

Api13 Brevibacterium sp.

AM711595 99 F

Yellow/orange

Tms, Caz, Fep, Cef

Col, Amp

6 FCol,

FAmp

Alfa Proteobacteria

Api5 Sphingomonas sp.

AM711590 99 W,

F Dark

yellow Ams, Ctx, Tms

Caz, Taz, Fep, Cef

Col

8 WCol,

F

Beta Proteobacteria

Api8 Burkholderia sp.

AM711592 99 W Yellow Gen, Men, Cet, Col

Amp

5 WAmp,

WCol

Gamma Proteobacteria

Api1 Pseudomonas

plecoglossicida

AM711587 99 W,

F Orange Ams, Men, Cef

Cmp, Col, Ery, Tet,

Amp

8 WAmp, WCol

WCmp, WEry

WTet , FCol

FAmp, FCmp

FEry, FTet

Api3 Pseudomonas

plecoglossicida

AM711588 99 W,

F Transparent/

light orange

Ams, Ctx, Tms

Men, Cet, Cmp, Col

Amp, Ery

9 FCol,

FAmp

Api17 Pseudomonas

plecoglossicida

AM711596 99

W

White Cmp,

Col 2 WCmp

L. Azul

Actinobacteria

A5 Nocardia sp. DQ112024 99 W Light Red Ery, Clm, Azm

Rox, Amp

5 W

A12 Dietzia sp. AM882683 98 F Camel Col 1 FCol

A1 Micrococcus sp.

AM403127 98 W Yellow Ery, Clm, Azm

Rox, Sm, Km, Gen

Amp, Cmp

9 W

Phylogenetic Affiliation

Closest Identified

Relative (Accession

Number)

% of

Identity

Source Color Resistance to Antibiotics Resistance

to No. of

ATBs

Isolation

Source

Firmicutes

A3 Staphylococcus

saprophyticus DQ112023 97 W Orange Ery, Clm, Azm

Rox, Sm, Gen, Amp

7 W

A4 Bacillus pumilus

DQ217665 99 W White Ery, Clm, Azm

Rox, Sm, Amp

6 W

A14 Bacillus pumilus

AM882685 98 F Cream Ctx,

Caz,

Fep 3 F

A16 Bacillus pumilus

AM882687

98 F Transparent Ctx, Caz, Fep, Col 3 FCol

A15 Bacillus amyloliquefaciens

AM882686

99 F Cream Gen 1 F

A13 Bacillus pumilus

AM882684

98 F Cream Caz, Fep, Col 2 FCol

A10 Paenibacillus polymyxa

AM882681

98 F Transparent Col 1 FCol

A8 Bacillus subtilis

AM882680

98 F White No ATB-resistance found 0 F

Beta Proteobacteria

A11 Variovorax paradoxus

AM882682

98 F Cream Ams, Caz, Cef, Tet 4 FTet Gamma Proteobacteria

A2 Acinetobacter johnsonii

AY963294 99 W White Ery, Clm, Azm

Rox, Sm, Km, Amp

7 W

L. Vilama Actinobacteria

V2 Rhodococcus eritropolis

AM236137 97 W White Ery, Clm, Azm

Rox, Sm, Amp

Ams

7 W

V5 Brachybacterium sp.

AM236138 97 W Yellow Ery, Clm, Azm

Rox, Km, Amp

Ams

7 W

V7 Micrococcus sp.

AM403126 98 W Yellow Ery, Clm, Azm

Rox, Sm, Km, Amp Tet, Cmp

9 W

Firmicutes

V3 Bacillus vallesmortis

AM235882 100 W Light orange Ery, Clm, Azm

Rox, Amp, Tet, Cmp

7 W

V8 Bacillus vallesmortis

AM235883 100 W Light orange Ery, Clm, Azm

Rox, Tet, Cmp

6 W

V31 Exiguobacterium sp.

AM882701

97 W Orange/pink No ATB-resistance found 0 W

Phylogenetic Affiliation

Closest Identified

Relative (Accession

Number)

% of

Identity

Source Color Resistance to

Antibiotics

Resistance to

No. of ATBs

Isolation Source

V29 Staphyloccocus xilosus

AM882700

98 W Orange Caz 1 W

V27 Bacillus pumilus

AM882697 97 F Cream No ATB-resistance

found

0 F

V30 Bacillus pumilus

AM882699 98 F Cream Caz,

Fep 2 F

V26 Bacillus pumilus

AM882696 98 F Cream Caz,

Col 1 FCol

V23 Bacillus megaterium

AM882693 98 F Cream No ATB-resistance

found

0 F

V25 Bacillus sp. AM882695 98 F Cream Caz, Ctx 2 F V20 Bacillus sp. AM882690 97 F Yellow Col FCol Alpha Proteobacteria

V28 Rhodopseudomonas sp.

AM882698 98 W Transparent Ams, Tms, Gen

Caz, Akn, Mem

Cip, Taz, Fep

9 W

V22 Chelatococcus

asaccharovorans

AM882692

97 F Pink Caz 1 F

V18 Sphingomonas

paucimobilis AM882688 97 F Yellow No ATB-resistance

found

0 F

Beta Proteobacteria

V21 Janthinobacterium sp.

AM882691

98 F White Ctx 1 F Gamma Proteobacteria

V1 Pseudomonas sp.

AM403128 98 W Cream Ery, Clm, Azm

Rox, Sm, Amp

Ams, Cmp

8 W

V4 Enterobacter sp.

AM403125 98 W Cream Ery, Clm, Azm

Rox, Sm, Km, Amp

Ams, Tet, Cmp

10 W

V24 Stenotrophomonas

maltophilia AM882694 97 F Cream

Ams, Ctx, Imp, Gen

Caz, Cef, Akn

Mem, Cip, Taz, Fep

Amp

11 FAmp

V19 Stenotrophomonas

maltophilia AM882689 97 F Yellow Ams, Imp, Gen

Caz, Cef, Akn

Mem, Cip, Fep, Ery

9 FEry

the National Committee for Clinical Laboratory Standards (NCCLS) for ATB susceptibility. The following ATBs were assayed: Azm (azithromycin), 50 g; Ery (erythromycin), 50 g; Clm (clarithromycin), 50 g; Rox (roxithromycin), 50 g; Sm(streptomycin), 20 g; Gen (gentamycin), 10 g; Km (kanamycin), 20 g; Tet (tetracycline), 20 g; Amp (ampicillin), 100 g, Imp (imipenem), 10 g; Caz (cefta-zidime), 30 g; Ams (ampicillin/sulbactam), 10/10 g; Ctx (cefotaxime), 30 g; Tms (trimethoprim/sulfa-methoxazole), 1.25/23.75 g; Mem (meropenem), 10 g; Cip

(ciprofloxacin), 5 g; Taz (piperacillin/tazobactam), 100/10 g; Fep (cefepime), 30 g; Cef (cefalotin), 30 g; Akn (amikacin), 30 g; Cmp (chloramphenicol), 30 g and Col (colistin), 10 g.

Olson and Ceri showed the use of plate for antibiotic against biofilm infection [21].

Production of Antimicrobial Substances

Antimicrobial substance producing organisms were identified using a modified agar-well diffusion assay as described by Portrait et al . [22] with Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 25923 and Listeria monocytogenes as control strains. RESULTS

Isolation of ATB Resistant Bacteria from Water and Feces

Phylogenetic affiliation of isolates, ATB resistance and isolation source (Water (W) or Feces (F)) with different ATBs are summarized in Table 2. Distribution of antibiotic resistance among the isolates is represented in Fig. (2). Members belonging to major phylogenetic groups like Firmicutes, Actinobacteria, and Gamma, Beta and Alfa proteobacteria were isolated from water and feces in all the Lakes assayed. Firmicutes were predominant in all the environments and bacteria resistant to multiple antibiotics were quite common in both water and feces isolates. L. Negra presented a medium salinity level, high UV exposure and low arsenic contents Table 1. Twelve bacteria were isolated from this Lake as shown in Table 2. Firmicutes were predominant (6 isolates) and most of them belonged to the spore-forming genus Bacillus . The only non spore-

forming isolate was Exiguobacterium sp., which, together with Bacillus thuringensis Ni19, presented the widest scope of ATB resistance (resistance to 11 and 12 ATBs, respec-tively). Alpha proteobacteria presented 3 isolates, belonging to Sphingomonas sp. and Caulobacter sp. The ATB resistance profile for Sphingomonas sp. Ni3 and Ni9 differed (resistance to 5 and 3 ATBs, respectively), denoting that they were different strains. Beta and Gamma proteobacteria were represented only by Stenotrophomonas maltophilia with resistance to 12 ATBs. All isolates from feces were also present in water, whereas many isolates were exclusively isolated from water.

L. Aparejos presented a low salinity level, high UV exposure and low arsenic contents. Together with L. Negra it had the lowest degree of oligotrophy and both Lakes harbored the largest population of flamingoes Table 1. Ten isolates were found in L. Aparejos, which are described in Table 2. As in L. Negra, Sphingomonas sp and Burkolderia cepacia were representatives of Alpha and Beta proteobacteria with resistance to 8 and 6 ATBs, respectively. Gamma-proteobacteria were represented by Pseudomonas plecoglossicida strains, which presented different ATB resistance profiles (2, 8 and 9) and were distributed between water and feces. Firmicutes were represented by Bacillus cereus with different ATB resistance profiles and two non spore–forming genera, Staphylococcus sp. and Brevibac-terium sp., with resistance to 1 and 6 ATBs respectively. Beta proteobacteria were exclusively isolated from water and the non spore-forming Firmicutes were exclusively found in feces; the remaining bacteria were common in both environments.

L. Azul presented low salinity, high UV exposure, low arsenic contents and extreme oligotrophic conditions since

Fig. (2).

Number and distribution of resistances to antibiotics of isolated bacteria form L. Negra, L. Aparejos, L. Azul and L. Vilama.

this Lake is located on a stone basin and no sediments were detected Table 1. The 13 isolates from this Lake are listed in Table 2. Firmicutes were predominant (8 isolates) with one non spore-forming isolate identified as Staphylococcus saprophyticus,which presented resistance to 7 ATBs. Among the spore-forming group, two isolates did not present ATB resistance. Actinobacteria were represented by Micrococcus sp. and Nocardia sp. with resistance to 9 and 5 ATBs, respectively. Dietzia sp. presented no ATB resistance. Gamma and Beta proteobacteria were represented by Acinetobacter johnsonii and Variovorax paradoxus with resistance to 7 and 4 ATBs, respectively. A low degree of association was found between culturable bacteria from water and feces, because only one isolate was isolated from both feces and water: Bacillus pumillus Table 2.

L. Vilama presented all three extreme conditions simultaneously: high salinity, high UV exposure and high arsenic contents Table 1. 21 isolates were identified from both water and feces Table 2. Bacteria belonging to Firmicutes predominated in water and feces (10 isolates). However, apart from Bacillus vallesmortis, they showed little ATB resistance compared to other isolates; especially isolates from feces presented resistance to 1 or 2 ATBs or no resistance at all. Three isolates were grouped into Alpha proteobacterias, and those identified as Rhodopseudomonas sp. showed resistance to 9 ATBs. Isolates identified as Sphingomonas paucimobilis presented no ATB resistance, which highly contrasts with the resistance profile of the same genus isolated from L. Negra. Gamma proteobacterium isolates were represented by Pseudomonas sp. and Entero-bacter sp., exclusively isolated from water, with resistance to 8 and 10 ATBs, respectively, and two strains of Stenotro-phomonas maltophilia with different ATB resistance profiles (resistance to 11 and 9 ATBs), which were exclusively isolated from feces. Beta proteobacteria were represented by Janthinobacterium sp., isolated from feces, and they presented only resistance to one ATB. The three actino-bacteria Rhodococcus eritropolis, Brachybacterium sp. and Micrococcus sp., were only isolated from water. The first two strains presented resistance to 7 ATBs and Micrococcus sp. to 9 ATBs. The above mentioned bacteria were distri-buted in water or in feces; there were no common bacteria between both isolation sources.

The most widespread ATB resistances were Col (22 resistances of 18 tested), Caz (16 resistances of 18 tested), Mem (12 resistances of 18 tested), Cet (18 resistances of 18 tested) and Ams and Ctx (13 resistances of 18 tested). Most resistant bacteria were isolated from L. Aparejos and L. Negra. Exiguobacterium sp., Stenotrophomonas maltophila and Bacillus thuringiensis N17 and N19 presented resistance to a high number of ATBs (10, 10, 9 and 12, respectively). N17 and Stenotrophomonas maltophila seem to be widely distributed not only in water but also in most bird feces, and consequently, these high ATB resistant bacteria could be spread by birds and therefore require more thorough studies. Pseudomonas plecoglossicida, Bacillus cereus and Sphin-gomonas sp. showed resistance to the highest number of different ATBs in L. Aparejos. Distribution of ATB resistant bacteria in water and bird feces in this Lake was also widespread.

All isolates were tested for production of antimicrobial substances against Gram positive and Gram negative pathogenic bacteria Table 3. Seven strains presented production of antimicrobial substances against E. coli ATCC 35218, Staphylococcus aureus ATCC 25923 and Listeria monocytogenes.

DISCUSSION

Singer et al. [14] proposed that l andscape ecology, which links the biotic and abiotic factors of an ecosystem, might help untangle the complexity of antibiotic resistance and improve the interpretation of ecological studies. As an extension of these ideas ATB resistance was studied in an environmental context. Our results have demonstrated that highly irradiated pristine environments are a rich source of bacteria able to grow in the presence of multiple ATBs. To our knowledge this work is without precedent, since this is the first time that ATB resistance has been assessed at such high altitude and in such extreme environments.

ATB Resistant Bacteria are a Widespread Phenomenon in High Altitude Lakes

Bacterial resistance to ATBs was found in the four high altitude environments studied: L. Negra, L. Azul, L. Aparejos and L. Vilama. These results support the preli-minary ideas presented in a previous publication by our

Table 3.Production of Antimicrobial Substances Against Three Pathogens: Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 25923 and Listeria monocytogenes

Lake Strain

E. coli ATCC 35218 S. aureus ATCC 25923 L. monocytogenes

L. Aparejos Burkholderia cepacia AM 711592 - + -

L. Vilama Stenotrophomonas maltophilia AM882694 - - +

L. Vilama Bacillus sp. AM882690 - +

- L. Azul Bacillus pumilus AM882687 - - +

L. Azul Bacillus amyloliquefaciens AM882686 - - +

L. Azul Paenibacillus polymyxa AM882681 + + +

L. Azul Bacillus subtilis AM882680 - + +

group, which postulated that there exists a correlation between ATB resistance and UV-B radiation in extreme environments like high altitude Lakes [4]. Under extreme UV stress conditions, bacteria are known to increase muta-tional events as a last resistance mechanism, called error-prone repair [23]. In many cases, spontaneous resistance to ATBs is known to emerge under such mutagenic conditions, as a consequence of mutagenesis modified potential target genes. Other authors established a possible connection between oxidative stress resistance and resistance to ATBs [24]. It is known that UV produces high oxidative stress and consequently, a highly irradiated environment is expected to select oxidative stress resistant bacteria. There could also be a relationship with ATB resistance found in other irradiated environments. The fact that ATB resistance is more common in irradiated environments than in non irradiated ones could support this idea

Special attention should be given to Stenotrophomonas maltophila, since this seems to be the most widest spread bacterium, even that it was detected in DGGE bands from water and feces of from L. Aparejos, L. Negra, L. Azul and L. Vilama, the four Lakes and it was isolated from water and feces of from L. Negra, L. Azul and L. Vilama. In all the cases it was the most resistant bacterium to multiples ATBs (unpublished data). This pathogen has been increasingly recognized as an important cause of related to nosocomial hospital infections.Infection occurs principally, but not exclusively, in debilitated and immune-suppressed indivi-duals patients. Management treatment of S. maltophilia associated infections is problematic because many strains of the bacterium manifest resistance to multiple antibiotics [25]. DGGE bands corresponding to Acinetobacter sp. were detected both in L. Aparejos and L. Vilama, (unpublished data) this genera was reported by our group as high UV resistant bacteria [4]. In this report these microorganisms were also isolated from water and feces from L. Negra, L. Azul and L. Vilama and presented multiples ATB resistance. CURRENT & FUTURE DEVELOPMENTS

The ubiquity of multiple ATB resistant bacteria in the environments assayed supports the idea that pathogenic bacteria resistant to multiple ATBs are not a phenomenon that is restricted to human-modified environments. Besides, it shows that pristine environments could be considered important reservoirs of multiple ATB resistance in bacteria like Staphylococcus sp., Aeromonas sp., Stenotrophomonas maltophila and a large group of enteric bacteria. These ATB resistant bacteria could be spread by birds whose migratory patterns are not well established yet. In conclusion, from an epidemiological point of view we propose that pristine, UV irradiated environments should receive more attention as reservoirs of multiple ATB resistance. This may become even more important in the context of global warming that could modify the behavior and migratory patterns of birds and bring them and their ATB resistant microbiota in close proximity to human communities. ACKNOWLEDGMENTS

To Juan J Barreiro and Jose Britos for driving us safely along the Andean roads and Gendarmería Nacional for their assistance in the mountainous area. This work was supported by PIE CONICET 6268-6096, Fundación Antorchas No. 14248-133 and PICT-Agencia Nacional de Promoción Científica y Tecnológica. Cristina Estevez received a ANYPCT fellowship, Julian R. Dib and Omar Ordo?ez a CONICET fellowship and Annika Weiss and Anna Neumann received financial support from DAAD. CONFLICT OF INTEREST

None

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控制软件说明书 PC端软件FTM 安装及应用 系统运行环境: 操作系统中英文Windows 98/2000/ NT/XP/WIN7/ Vista, 最低配置 CPU:奔腾133Mhz 内存:128MB 显示卡:标准VGA,256色显示模式以上 硬盘:典型安装 10M 串行通讯口:标准RS232通讯接口或其兼容型号。 其它设备:鼠标器 开始系统 系统运行前,确保下列连线正常: 1:运行本软件的计算机的RS232线已正确连接至控制器。 2:相关控制器的信号线,电源线已连接正确; 系统运行步骤: 1:打开控制器电源,控制电源指示灯将亮起。 绿色,代表处于开机运行状态;橙色代表待机状态。 2. 运行本软件 找到控制软件文件夹,点击FWM.exe运行。出现程序操作界面:

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(2)电桥的灵敏度

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2、放空阀全开:放空阀指示为0% 3、润滑油压正常 4、润滑油温正常 5、动力油压正常 6、逆止阀全关 7、存储器复位:按下存储器复位按钮,即可复位,若复位不成 需查看停机画面。 8、试验开关复位:按下试验开关按钮即可,试验开关按钮在风 机启动后,将自动消失,同时试验开关也自动复位。 当以上条件达到时,按下“允许机组启动”按钮,这时机组允许启动指示变为红色,PLC机柜里的“1KA”继电器将导通。机组允许启动信号传到高压柜,等待电机启动。开始进行高压合闸操作,主电机运转,主电机运转稳定后,屏幕上主电机运行指示变红。这时静叶释放按钮变红,按下静叶释放按钮后,静叶从14度开到22度,静叶释放成功指示变红。 应继续观察风机已平稳运行后,按下自动操作按钮,启机过程结束。 B、停机画面: 停机是指极有可能对风机产生巨大危害的下列条件成立时,PLC 会让电机停止运转: 1、风机轴位移过大

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威利普LEDESC控制系统操作说明书

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分别对以上两个进程集合,计算使用先来先服务(FCFS)、时间片轮转法(时间片q=1)、短进程优先(SPN)、最短剩余时间优先(SRT,时间片q=1)、响应比高者优先(HRRN)及多级反馈队列(MFQ,第1个队列的时间片为1,第i(i<1)个队列的时间片q=2(i-1))算法进行CPU调度,请给出各进程的完成时间、周转时间、带权周转时间,及所有进程的平均周转时间和平均带权周转时间。

惠斯通电桥实验报告.pdf

南昌大学物理实验报告 课程名称:惠斯通电桥 实验名称:惠斯通电桥 学院:眼视光学院专业班级:眼视光151班学生姓名:许春芸学号:6303615024 实验地点:210座位号:30 座实验时间:第8周星期6上午10点10开始

一、实验目的: 1.掌握电桥测电阻的原理和方法。 2.了解减小测电阻误差的一般方法。 二、实验原理: 惠斯通电桥的电路四个电阻 R1.R2.R3.Rx 连成一个四边形,每一条边称作电桥的一个臂,对角 A 和 C 加上电源 E,对角 B 和 D 之间连接检流计 G,所谓桥就是指 BD 这条对角线,它的作用就是将桥的两个端点的电势直接进行比较。当 B.D 两点电势相等时,检流计中无电流通过,电桥达到了平衡,这时有:R2/R1=Rx/R3,即*Rx=(R2/R1)R3。若 R1.R2.R3 均已知,则 Rx 可由上式求出。 电桥电路可以这样理解,电源 E.R2.Rx 是一个分压电路,Rx 上的电压为[Rx/(R1+R2)]·E,又 E 和 R1.R3 也是一个分压电路,R3 上的电压等于[R3/(R3+R1)]·E,现在用检流计来比较 Rx 和 R3 的电压,根据电流方向,可以发现哪一个电压更大些。当检流计指零时,说明两电压相等,也就得出*式。 三、实验仪器: 线式电桥板、电阻箱、滑线变阻器、检流计、箱式惠斯通电桥、待测电阻、低压直流电源。 四、实验内容和步骤: 1、标准电阻 Rn 选择开关选择“单桥”档; 2、工作方式开关选择“单桥”档; 3、电源选择开关选在有效量程里; 4、G 开关选择“G 内接”; 5、根据 Rx 的估计值,选好量成倍率,设置好 R1R2 值和 R3 值,将位值电阻 Rx 接入 Rx 接线端子(注意 Rx 端于上方短接片应接好); 6、打开仪器市电开关、面板指示灯亮; 7、建议选择毫伏表作为仪器检流计,释放“接入”键,量程置“2mV”挡,调节“调零”电位器,将数显表调零。调零后将量程转入 200mV 量程,按下“接入”按键,也可以选择微安表做检流计; 8、调节 R3 各盘电阻,粗平衡后,可以选择 20mV 或 2mV 挡,细调 R3,使电桥平衡;

控制软件操作说明书

创维液晶拼接控制系统 软件操作指南 【LCD-CONTROLLER12】 请在使用本产品前仔细阅读该用户指导书

温馨提示:: 温馨提示 ◆为了您和设备的安全,请您在使用设备前务必仔细阅读产品说明书。 ◆如果在使用过程中遇到疑问,请首先阅读本说明书。 正文中有设备操作的详细描述,请按书中介绍规范操作。 如仍有疑问,请联系我们,我们尽快给您满意的答复。 ◆本说明书如有版本变动,恕不另行通知,敬请见谅!

一、功能特点 二、技术参数 三、控制系统连接示意图 四、基本操作 五、故障排除 六、安全注意事项

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图2 2.选择键,进入下一界面如图3 图3 3.选中项,再按键,进入下一界面如图4

图4 4.选择键,进入下一界面如图5 图5 5.选中项,再选择键,进入下一界面如图6

图6 6.选择键,进入下一界面如图7 图8 7.选择键,软件安装完成 二软件操作 选择WINDOWS 下开始按钮,选择程序,选择Wall Control项, 点击Wall Control软件进入大屏幕控制系统软件主界面如图9所示,整个软件分为3个区,标题区,设置区,功能区

图9 1.1标题区 大屏幕控制系统软件(只有管理员才可设置此项目) 1.2设置区 1.2.1系统 高级功能:管理员登录。 产品选型:选择拼接盒型号。 定时系统:设置定时时间。 幕墙开机:开机 幕墙关机:关机 退出:退出软件系统。 1.2.2设置 串口设置:设置使用的串口参数。 矩阵设置:设置矩阵的相关参数。 幕墙设置:幕墙设置参数。 幕墙颜色:幕墙颜色设置。 标志设置:更改幕墙名称。 系统设置:控制软件系统设置。 1.2.3工具 虚拟键盘:虚拟键盘设置。 硬件注册:可以通过时钟IC注册处理器的使用权限。 1.2.4语言 中文选择:选择软件语言类型为中文。 English:选择软件语言类型为英语。

第4章思考题和习题解答

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惠斯通电桥实验报告.doc

云南农业大学 物 理 实 验 报 告 实验名称:惠斯通电桥测量电阻 一、实验目的 (1)了解惠斯通电桥的构造和测量原理。 (2)掌握用惠斯通电桥测电阻的方法。 (3)了解电桥灵敏度的概念及其对电桥测量准确度的影响。 二、实验仪器 滑线式电桥,箱式电桥,检流计,电阻箱,滑动电阻器,待测电阻,电源,开关,导线等。 三、实验原理: 1.惠斯通电桥的测量原理 如图1所示,由已知阻值的三个电阻R 0、R 1、R 2和一个待测电阻R x 组成一个四边形,每一条边称为电桥的一个臂,在对角A 、B 之间接入电源E ,对角C 、D 之间接入检流计G 。适当调节R 0、R 1、R 2的阻值,可以使检流计G 中无电流流过,即C 、D 两点的电势相等,电桥的这种状态称为平衡态。电桥的平衡条件为 1 002 x R R R KR R = = (1) 式中比例系数K 称为比率或倍率,通常将R1、R2称为比率臂,将R0称为比较臂。

2.电桥的灵敏度 式(1)是在电桥平衡的条件下推导出来的,而电桥是否达到真正的平衡状态,是由检流计指针是否有可察觉的偏转来判断的。检流计的灵敏度是有限的,当指针的偏转小于0.1格时,人眼就很难觉察出来。在电桥平衡时,设某一桥臂的电阻是R ,若我们把R 改变一个微小量ΔR ,电桥就会失去平衡,从而就会有电流流过检流计,如果此电流很小以至于我们未能察觉出检流计指针的偏转,我们就会误认为电桥仍然处于平衡状态。为了定量表示检流计的误差,我们引入电桥灵敏度的概念,它定义为 n S R R ?= ? (2) 式中,ΔR 为电桥平衡后电阻R 的微小改变量,Δn 为电阻R 变化后检流计偏离平衡位置的格数,所以S 表示电桥对桥臂电阻相对不平衡值ΔR /R 的反应能力。 3.滑线式惠斯通电桥 滑线式惠斯通电桥的构造如图2所示。A 、B 、C 是装有接线柱的厚铜片(其电阻可以忽略),A 、B 之间为一根长度为L 、截面积和电阻率都均匀的电阻丝。电阻丝上装有接线柱的滑键可沿电阻丝左右滑动,按下滑键任意触头,此时电阻丝被分成两段,设AD 段的长度为L 1、电阻为R 1,DB 的长度为L 2、电阻为R 2,因此当电桥处于平衡状态时,有 111 000221 x R L L R R R R R L L L = ==- (3) 式中,L 1的长度可以从电阻丝下面所附的米尺上读出,R 0用一个十进制转盘式电阻箱作为标准电阻使用。另外电源E 串联了一个滑线变阻器RE ,对电路起保护、调节作用。为了消除电阻丝不均匀带来的误差,可用交换R 0与R x 的位置重新测量的方法来解决。也就是在测定R x 之后,保持R 1、R 2不变(即D 点的位置不变),将R 0与R x 的位置对调,重新调 节R 0为0R ',使电桥达到平衡,则有 221 000 111 x R L L L R R R R R L L -'''= == (4) 所以 221 000 111 x R L L L R R R R R L L -'''= == (5) 由式(5)可知,Rx 与R1、R2(或L1、L2)无关,它仅取决于R0的准确度。可以证明

控制系统说明书 V1.0

目录 1,系统概述--------------------------------------------------------------------------------------------------1 1.1 系统简介---------------------------------------------------------------------------------------------2 1.2 系统主要组成---------------------------------------------------------------------------------------2 1.3 系统硬件简要连接图------------------------------------------------------------------------------3 1.4 实际连线图------------------------------------------------------------------------------------------3 2,系统软件使用软件简要说明-----------------------------------------------------------------------------5 2.1 介绍---------------------------------------------------------------------------------------------------5 2.2 操作步骤---------------------------------------------------------------------------------------------5 2.3 取景窗口---------------------------------------------------------------------------------------------7 2.4 flash/cel文件的播放--------------------------------------------------------------------------------7 注1:连接网络的相关设置修改--------------------------------------------------------------9 注2:本机IP的查询----------------------------------------------------------------------------9 注3:本机IP的修改----------------------------------------------------------------------------10 注4:控制器IP的修改-------------------------------------------------------------------------11 3,对应表制作与选择-----------------------------------------------------------------------------------------12 3.1 介绍---------------------------------------------------------------------------------------------------12 3.2 操作步骤---------------------------------------------------------------------------------------------12 4,说明-----------------------------------------------------------------------------------------------------------14 4.1 ONC1A------------------------------------------------------------------------------------------------14 4.2 ONC1B------------------------------------------------------------------------------------------------14 4.3 ONC1C------------------------------------------------------------------------------------------------15 4.4 ONC1D------------------------------------------------------------------------------------------------15 4.5 ONC1E------------------------------------------------------------------------------------------------16 4.6 ONC1F------------------------------------------------------------------------------------------------17 4.7 ONC1G------------------------------------------------------------------------------------------------17 4.8 ONC1F------------------------------------------------------------------------------------------------17 5,附件-----------------------------------------------------------------------------------------------------------19 5.1 数码按钮控制板说明--------------------------------------------------------------------------------19 5.2 象素点排列说明--------------------------------------------------------------------------------------19

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