PRELIMINARY INVESTIGATION OF IN VITRO

HOI NGH! KHOA HOC C O N G N G H f SINH HOC T O A N Q U O C 2013

PRELIMINARY INVESTIGATION OF IN VITRO - INDUCED LEVOFLOXACIN RESISTANCE IN PSEUDOMONAS AERUGINOSA ATCC 9027

Dang Bao' Ngoc, Thai Van Chi, L e Tran Hong Ngoc, L e Thi Ly, N g u y e n T h l T h u Hoai International University, Vietnam National Udverstty, Ho Chi Minh City

SUMMARY

Soial exposure lo certain antibiotics at sub - MIC concentrations was beheved to induce its resistance. Ttiis study was designed lo iiirther investigate this phenomenon by using Pseudomonas aeruginosa ATCC 9027 and Levofloxacin (LEV) as study model. Pure strain of P. aeruginosa ATCC 9027 was continuously exposed to LEV with daily adjusted concenfrations lower than minimal inhibitory concentration (MIC). After 10 days of serial exposures, the LEV - MIC of this bacterium markedly increased from 0.25 to 8 mg/L (32 times) and remained at that level even when the organism was continuonsly cultured in antibiotic - fi^e medium for anoflier 10 days. This result mdicated diat this in vitro- induced LEV-resistance was iireversible. Impressively^this in Wiro-induced LEV-resistant P. aeruginosa also became cros^ resistant not only to other flouroquinolone antibiotics (Ciprofloxacin from 0.0625 to 2 mg/L, Moxifloxacia from 2 to 32 mg/L and Norfloxacin from I to 16 mg/L) but also to odier unrelated antibiotics (Chloramp^enicot from 32 to 1024 mg/L, Doxycycline from 1 to 512 mg/L, Teuacycline from 1 to 8 mg/L). Sequencing result of strains before and after LEV exposure oigyrA gene (topoisomerase IV subunit A) which is primarily involved in flouroquinolone resistance mechanism revealed only single mutation al position 58 (Aspartic acid changed into Glycine), This mutation was also repotted before in clinical fluoroquinolone- resistant P. aeruginosa strains indicating its important role in inducing Tesistance lo fiouroquinoloaes. Further mvestigation should be perfomied to see whether other gene mutation and regulation could also involve in this in vitro - induced resistance.

Keyw'.rds: Fluoroquinolones, minimal inhibitory concentration (MIC), antibiotic resistance mecimoisms, serial exposure, Pseudomonas aen^inosa, gyraseA •

INTRODUCTION

Pseudomonas aeruginosa is an aerobic GrarrvnegaSve bacterium which belongs to the class of Gamma protaobaderia and family Pseudomonadaceae (Lee et al. 2005). This bacterium can 5u^^^ve and persist under a bn^ad range of environmei;ital conditions. P. aeruginosa is particularly problematic for seriously ill patients. From 1992lo 1997, data from tha American National Nosocomial infections Surveillance System showed that P. aemginosa was responsible for 21 % oi pneumonias, 10% of urinary fract Infections, 3% of bloodstream Infections, and 13% of eye, ear, nose, and throat infecbons within ICUs in the United States (Richard ef al. 1999). A similar study conducted in Europe also showed that.

P. aenig/nosa was accountable for 3 0 % of pneumonias, 19% o f urinary t r a d infections, and 10% of bloodsfream infecbons (YonezawSL efa/, 1995).

Flouroquinolones (FQs) are bactericidal, rapidly acting antimicrobial dmgs with wide spectra. Among those, levofloxacin (LEV) has appeared as one of most effective ' f ' ! ? ^ ! ^ ^ f ! 1 ! l l ^ F ^ ^ ^ f^Qs against P. aemginosa. However, .the extensive arid iW't" '[|i!Vi.'.il'ili]'ii Ji;i; •'.:• 'uncontrolled usage FQs inciuding LEV has resulted in an

=doe«Hj==cdo«.«.ii==:=ic«»« increasing FQ resistance which could lead to treabnent f a i l t u r e j n P.^ _aeruginosa infecbon.. There are,several mechanisms leading to FQ resistance .among those mutab'on in DNA gyrase. the major target .of FQs. is widely accepted as most principal mechanism for high level FQ resistance (Figure 1) (Akasawa et al., 2 0 0 1 ; Hooper 1995; Lister el a l . 2009), DNA gyrase is an A2B2 type of tetra-enzymes with pairs of two different subunits A and B which are encoded by gyrA and gyfS genes respectively. It has been shown that there are hotspots, called the - quinolone resistance determining region (QRDR), for mutations withfn these genes (Piddock, 1999). These mutations were widely believed to play a major role in FQ resistance In P. aemginosa (Lee ef at., 2005; Kaneko e( at., 2000)."

The aim of our study w a s to invesbgate the in vitro- induced FQ resistance mechanism by detecting mutation of gyrA in LEV-reslstanl P. aeruginosa strain which was produced through serially exposing P. aemginosa ATCC 9027 to LEV. This preliminary Investigation would provide more evidence on how FQ resistance emerged rapidly in - Gram-negative badena such as P. aemginosa.

Rgure 1 . Flouroquinolone resistance mechanisms. Ruoroquinolone resistance is mediated by (1) overexpression of RND (resislanca-nodulation-divtsion) efflux pumps extradtng the * u g molecules fnJm the periplasmic and cytoplasmic spaces and/or (ii) mutational changes within the target genes coding (br topoisomerase IV (parC, parE) and gyrase {gyrA, gyrB). QuinokHie resistance determining regkms (QRQR) are denoted on the target genes (modified from Uster el al. 2009).

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• « M i | 5 M p 3 I K !

TopoW C«A gyrase

• HQI NGHI KHOA HQC C O N G NGHE SINH HQC T O A N Q U O C 2013

MATERIALS AND METHODS Preparation o f P. aeruginosa s t a r t i n g s t r a i n

P. aemginosa ATCC 9027 was subsequently cultured In Mueller Hinton agar (HImedia, India) for three passages for limiting genetic variance if present

A n t i m i c r o b i a l susceptibility t e s t i n g

Minimal Inhibitory concentraBoo (MIC) of levofloxacin (LEV; Stada, VN) for P. aemginosa ATCC 9027 was '^®'®™'"®1|^^

micro-dilution method according tei the recommendations given by the European Committee o n ™ ™ r n ^ D i a Suscepbbility Testing guidelines (EiJCAST 2013) The MIC result vras recorded afler 24 hours of Incubabon at 37 C.

Two baderial sbains (starting and LEV-exposed P. aemginosa A T C C 9027 strain) were tested for antibiotic s u s c e ^ b i l i t y to 8 different antibiotics including ciprofloxacin, chloramphenicol, norfloxacin, doxycydine (Slada, VN), cefalexhn (Pharimexco. VN), moxifloxacin (Medisure. Pakistan), tebacycline and ampicillin (Sigma-Aldrich, USA). T h e MIC experiments were performed by using 96- well plates with the same principle as described above (EUCAST 2013).

Induction and selection o f LEV- resistant mutants

P. aemginosa ATCC 9027 sbain was senaiiy cultured In Mueller Hinton Broth containing LEV at concentration just belijw its daily measured MiC value. Serial exposures o f P. aemginosa ATCC 9027 l o LEV were perfonned unfil no M I C Increase was observed (after 10 days). The induced LEV-resistant P. aemginosa ATCC 9027 sbain was collected after 10 days of LEV exposure (LEV-exposed strain) and conbnuously cultured for another 10 days in antibiotic- free medium (LEV-revertant strain). Starting and LEV-exposed strains were sent to Nam Khoa Biolek Company (HCM, VN) for 16S rRNA sequendng to confirm their identities.

Amplification and DNA sequencing

The primers were consbuded based on the published data of Saima e( ah gyrA F (5'-GTG TGC TTT ATG-CCC A T G A G - S') and gyrA R (5'-GGT TTC CTT TTC CAG GTC-3') for quinolone resistance determining region (QRDR) of gyrA (Salma et al., 2013). The volume of each PCR experiment was a 25 pl mixture containing 10x Buffer, 10 m M mix o f deoxynucieotlde biphosphates dNTPs (Sigma-Aldrich, Singapore), I U o f DNA Taq polymerase (Sigma-Aldrich, Singapore). 5 pM of each primer and 100 ng of the DNA template. The PCR Ihemial profile was 35 cycles consisting o f several steps: initial heal acbvabon at 94° C for 5 minutes, denaturation at 94° C for 1 minute, annealing at 5° C for p r i m e r gyrA in 1 minute and elongation at 72° C for 1 min. An elongation step at 72° C for 5 minutes was added at the 35 cycle lo complete the PCR reaction. The PCR products were checked by using agarose gel electrophoresis for expected amplified fragments then were sent to Nam Khoa Bidek Company (HCM, VN) for DNA sequencing,

RESULTS AND DISCUSSION

Alteration o f LEV susceptibility In P. aeruginosa ATCC 9027 after LEV- e x p o s u r e

Changes In antibldic susceptibility profile of P. serug/nosa ATCC 9027 during 1 0 - d a y exposure process to LEV and 1 0 - day reverlant process In anfabiotic- free medium were presented in Figure 1 . 168 riWJA sequendng results for P.

aemginosa identity of starting and the exposed strains showed no contamination during the antltriotic exposure.

Data showed that the MIC of UEV had increased significantly from 0.25 mg/L (starting strain) to 8 mg/L (32 times, exposed sbain). P. aeruginosa ATCC 9027 reached a maximum MIC of 8 mg/L LEV at day 7 and remained stable in the following 3 days. Interestingly, this MIC increase observed in the bacterium seemed t o be irreversible as no M I C decrease was seen after 10 days of culturing the exposed strain in anbblotic-free medium (Figure 1)

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Anltblotic exposure

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Figurs 2. Susceptibility of P. aerug/nosa ATCC 9027 to levofloxacin during exposure experiment, P.aeruginosa ATCC 9027 was senafly exposed to LEV al sub-f^IC concentrations. LEV-MiC became stable from day 7. The LEV-exposed strain at day 10 was conbnuously cultured in anlibiotic-free medium for another 10 days, however no decrease m LEV- MIC was observed. Jhi exposure process was repeated in a separate experiment and same data were recorded.

Cross-resistance o f P. aeruginosa t o different antibiotics

HOI NGH! KHOA HOC C O N G N G H E SINH HOC T O A N Q U O C 2013

Eight- -different antibiotics-(Chloramphenicol,-Doxycycline,--Tetracycliner "Norfloxacin,-Ciprofloxacin, -Moxifloxacm.

Ampicillin and Cefalexin) were used lo investigate the a o s s - resistance of the LEV- exposed P. aemginosa ATCC 9027 (Table 1). Impressively, this LEV-exposed sbain has shown a very sb-ong cnass-resistance to olher antibiotics except for ampicillin and cefalexin to which P. aemginosa ATCC 9027 was initially highly resistant. In details, the sbaln expressed resistance t o other FQs including noriloxacln, ciprofloxacin and moxifloxacin witti an 16-folds Increase MIC of anbbiotics Mutant strain also showed a - t o 5 1 2 - f o l d increase MIC of chloramphenicol (from 32 to 1024 mg/L), doj^cycline (from 1 to 512 mg/L) and teb^acydine (from 1 to 8 mg/L). This result indicated that serial exposure of P. aemginosa to LEV has resulted in a cross-resistance not only to FQs but also to other unrelated antibiotics (tebacyclines and chloramphenicol).

The mechanisms of this cross resistance are being further investigated Table 1. Antibiotic cross-resistance of LEV-exposed P. aeruginosa ATCC 9027 strains

MiC (mgfl-)

Antibiotics Starting LEV-exposed P. aeruginosa ATCC 9027 Chk)ramphenkX)l

Doxycydine Tetracycline Norfloxacin Ciprofloxacin Moxifloxacin Ampicillin Ce^lexin

32 1 1 1 0.0625 2

>=1024

>=1024 1024 512 8 16 1 32

>=1024

>=1024 Amplitication and sequencing o f gyrA gene

287 bp QRDR of gyrA gene was successfully amplified by using published pp'mers. This gene showed a point mutation al codon 58 with amino acid substitution from aspartic t o glycine (GAC-^ GGC), Docking analysis showed that this mutation affected directly the binding of gyrase A to FQs (data not shown). So far many additional genes have recently been analyzed to lllusbate furiher FQ resistance mechanism, such as merR, nfxB, parC genes (Tenover 2006). These related genes will be further investigated in in-vitro induced FQ- resistant P. aeruginosa In the near fuhjre, CONCLUSION

The serial LEV exposure of P. aemginosa resultedjn the irreversible resistance of P. aemginosa io LEV. Furthermore, the serial exposure t o LEV could also lead to the cross- resistance of P. aemginosa to other antibiotics not only the-ones of the same group, FQs, but also to ottier unrelated ones such as tetracyclines and chloramphenicol. This result raised a caution on application of antibiotics under MIC values leading l o generation multi-antibiotic resistant pathogens. The preliminary data on the mechanism of the in dtro - induced LEV - resistance highlighted a point mutation on the active region of subunit A of the target enzyme gyrase. Molecular modeling and docking study indicaied that substitution from Asp-58 to Gly-58 sttongly affected the binding of FQ to gyrase. However, whether single mutation could result In the high increase of LEV • MIC is still a questloFi. Furthermore, the mechanisms leading l o cross resistance to other antibiotics in the in vitro- Induced LEV - resistant P. aemginosa should be further investigated.

REFERENCES

Akasaha, T., Tanaka, M., Yamaguchi, A,, Sato, K,(2001) .Type.ll Topoisomerase Mutations In Flvoroquinalone-Redstant Clinic^ - Strains of Pseudomonas aenigiriosa Isolated in 1998 and 1999: Role d Target Enzyme in Mechanism of Fluomquinolone Resistance.

Antimicrob Agents Chemother 45 (8); 2263-2263.

Hooper, D C (2001). Emerging mechanisms of fluoroquindone re^stance. Emerg infect Dis, 7(2): 337-341.

Kaneko, A., Sasaki, J., Shimadzu, M , Kanayama. A., Saika, T , Kobayashi, I (2000). Comparison of gyrA and parC mutation and resistance levds among Huoroqutidone-resistant isolates and latmratory-denved mutants of oral slreptococd, 45{6): 771-775 Lee J. K„ Lee, Y. S.. P a * , Y K.. Kim,B. S (2005), Alternative in tfie GyrA and GyrB subunits of topoisomerase II and the ParC and ParE subudt of the IV In the ciprolloxacin-resistant elided isolaVon of Pseudomonas aeruginosa, int J Antimicrob agents, 25(4): 290- 295.- - f^;_

Lister PD, Woiler DJ. Hanson ND (2009). Antibactenal-reslstant Pseudomonas aeruginosa: clinical impact and Comdex regulation trf chromosamally encoded rebalance mechanisms. Clin Microbiol Rev.; 22:582-610

Piddock, L. J. V. (1999), Mechanisms of fluoroqdnolone resistance, an update 1994-1998 Dmgs 58(S2) 11-18.

Richards, M. J , Edwards, J. R., Culver. D, H., Gaynes, R, P {^9SB). Nosocomial Infections in medical intensive care units In the United States. Crit Care Med, 27 (5): 887-892,

Salma. R.. Dabboussi, F., Kassaa. I.. Khudary, R.. Hamze, M (2013). GyrA and ParC mutations In quinolone-resistant clinical isolates of Pseudomonas aemginosa from Ninl Hospital in north Lebanon. Joumai of infection and Chemotherapy, 19(4). 77-81.

Tenover. F.C (2006). Mechanisms dAnbmkrobidRedstance in Bacteria. The American Joumai of Medidne. 119 (6A): S3-S10

Yonezawa. M.. M. Takahata.. N. Malsubara (1995). DNa gyrase gyrA mutations In quinolones-resistanl clinical isolates of P.aerugino^.

Antimicrob. Agents Chemother. 39 1970-1972

HOI NGH! KHOA HOC C O N G N G H f SINH HQC T O A N Q U O C 2013

NGHIEN CCrU BilOC DAU V& Sy KHANG LEVOFLOXACIN CAM O'NG COA PSEUDOMONAS AERUGINOSA ATCC 9027 TREN MO HJNH PHONG THI NGHIEM

D^ng Bdo Ngpc, Thki Van Chi, Li Tr^n Hong Ngpc, L§ Thl Ly, Nguyen Thj Thu Hokl Tnrbng 09/ hgc Q u ^ te, Trubng Bai hgc Qu6c Gia Tp. Hi Chi Minh

idtATkt

Vifc tifip xiic Uen UK riia vi kfauln v6i Uifing sinh * nAng d$ nho hon nAng dd lie chS lii fiiiSu (MIC) dir^c tin ring se t?o " J ^ ^ ibuAc Nghian cuu niy nhini (im bieu ro hon bi& hrgng trin bing cich sii dimg Pseudomonas a^vginosa ATCC 9027 v^

Levofloxacin (LEV) lim in6 hinh nghiSn cfiu. P. aeruginosa ATCC 9027 dugc t i ^ xiic lien tpc irra^ 10 ngay vdi LEV6 nong dg Ih^ihonMIC. Kit qui cho thiy, W C cua LEV nhanh chong tang cao dat gii tii &i djnh sau 7 ngay (tfr 0,25 ISn 8 mg/L). Dfing chu j , chiing sau qni binh tiep xac vbi LEV van giii nguyen khi nang khing khi dugc nu6i dy hSn tvc tr*n m6i tmong khang c6 khing sinh. Hon th^ chftng niy cung cd fli tro nen da khftng vi Vhang chio khSng chi v6i cic khing sinh nhom flouroquinolone (tang tii 16 d ^ 32 lin voi CtproBoxacin, Moxifloxacin vi Nmfloxacin) mi cfin v6i khing sinh fliupc nhfim khac (ting tii 8 den 512 lan voi Chloramphenicol, Doxycycline v i Tetracycline). Trinh tir trSn chiing P. aeruginosa ban..dau v i sau nep xuc LEV ciia gyrA (topoisomerase IV subunil A) cho thiy ro^ dfit b i ^ fliay th^ Aspartic acid thinh Glycine 6 vj tri 58. Kit qui niy dong tbum v6i nhijng nghiSn cfiu trade diy tren cac chung P. aeruginosa lim sing khing fluoroquinolane. Cic bien doi phan tfi khic dang tiep tgc ixtgc phan tich thfim.

Tir khda: Fluoroquinotones, nong dfi fie chi tii fliiiu (MIC), co chi khing lhu6c, tiip xuc lien tiip, Pseudomonas aeruginosa, gyrase A

' fanhor for coirespondence. Tel: 0 8. 372 44270 - 3233; Email: ntlhoai@hcmiu.edu.vn