Typing of Staphylococcus aureus in order to determine the spread of drug resistant strains inside and outside hospital environment - Epidemiological Review

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ABSTRACT

Staphylococcus aureusLVRQHRIWKHPRVWLPSRUWDQWHWLRORJLFDOIDFWRUVRIERWKQRVRFRPLDODQGFRPPXQLW\DFTXLUHG

infections. Multidrug-resistant S. aureus LVIUHTXHQWO\LVRODWHGQRZDGD\V$QWLELRWLFVXVHGRQWKHKRVSLWDOZDUGH[HUW a selective pressure on the strains and favor resistant strains. Multidrug-resistant and highly virulent strains can spread QRWRQO\ZLWKLQWKHKRVSLWDOEXWDOVREHWZHHQKRVSLWDOV1XPHURXVVWXGLHVVKRZDSUHGRPLQDQFHRIRQHFORQHRQD VSHFLILFWHUULWRU\7KHVSUHDGRIVXFKGDQJHURXVFORQHVWRQHLJKERULQJFRXQWULHVDQGWKHHQWLUHFRQWLQHQWLVSRVVLEOH 7\SLQJPHWKRGVDUHYHU\XVHIXOLQLQIHFWLRQFRQWURODQGSUHYHQWLRQ0RGHUQPHWKRGVZKLFKDUHEDVHGRQVHTXHQF-ing are necessary in rationaliz7\SLQJPHWKRGVDUHYHU\XVHIXOLQLQIHFWLRQFRQWURODQGSUHYHQWLRQ0RGHUQPHWKRGVZKLFKDUHEDVHGRQVHTXHQF-ing of infection control programs. Typ7\SLQJPHWKRGVDUHYHU\XVHIXOLQLQIHFWLRQFRQWURODQGSUHYHQWLRQ0RGHUQPHWKRGVZKLFKDUHEDVHGRQVHTXHQF-ing of Staphylococcus aureus includes methods that allow to determine the spread of drug-resistant pathogens. ‘Gold standard’ is pulsed-field gel electrophoresis (PFGE), which relies on separating the DNA fragments after restriction cutting. MLST (Multi Locus Sequence 7\SLQJLVEDVHGRQDFRPSDULVRQRI³KRXVHNHHSLQJ´JHQHVHTXHQFHVFRQWUROOLQJWKHEDVLFFHOOIXQFWLRQV:LWKWKH 0/67PHWKRGLWLVSRVVLEOHWRGHPRQVWUDWHDEURDGLQWHUQDWLRQDOVSUHDGRIWKHVSHFLILFFORQHV+RZHYHUIRUHSLGH-PLRORJLFDOLQYHVWLJDWLRQV0/67VHHPVWREHWRRWLPHFRQVXPLQJDQGH[SHQVLYHWREHXVHGDVDEDVLFW\SLQJWRRO The complementary method is spa W\SLQJEDVHGRQWKHVHTXHQFLQJRIVKRUWUHSHWLWLYHVHTXHQFHVRIWKHSRO\PRU-SKLF;UHJLRQIURPWKHJHQHHQFRGLQJSURWHLQ$7KLVPHWKRGFDQEHXVHGIRUVWXG\LQJPROHFXODUHYROXWLRQRI

S. aureusDVZHOODVIRUWHVWLQJIRUKRVSLWDORXWEUHDNV,WLVIDVWHUDQGFKHDSHUWKDQ0/67,WLVDOVRQHFHVVDU\

WRVXEW\SHWKHHOHPHQWVUHVSRQVLEOHIRUPHWK\FLOOLQUHVLVWDQFH6&&mec), which allows to distinguish MRSA (Methicillin-resistant Staphylococcus aureusFORQHVZLWKDFRPPRQDQFHVWRUEXWGLIIHUHQWHSLGHPLRORJLFDO origin. All of those methods have their specific advantages and disadvantages and there is no single method HIILFLHQWDQGVXLWDEOHLQDQ\FDVH

.H\ZRUGV: Staphylococcus aureus, MRSA, typing, PFGE, MLST Staphylococcus aureus constitutes one of important

microorganism which forms the human commensal flora and is potentially infectious. S. aureus colonizes PDLQO\ZDUPDQGPRLVWUHJLRQRIPXFRXVPHPEUDQHV HVSHFLDOO\WKHQDVDOYHVWLEXOHZKHUHLWVUHFHSWRUVDUH located. From the literature data show that the coloniza-WLRQRIQDVDOYHVWLEXOHUHSRUWHGLQRISDWLHQWVDQG residents) is mainly associated with temporary coloni-]DWLRQRISKDU\Q[7KHSDVWKLVWRU\RIS. aureus colonization is an important risk factor of infection progress. Different strains produce diverse toxins and YLUXOHQFHDJHQWVZKLFKHQDEOHVWKHEDFWHULDWKHLQYDVLRQ and have a negative effect on the immunology system of the host (3). Many strains are resistant to numer-RXVDQWLELRWLFVZKLFKOLPLWVWKHWUHDWPHQWRSWLRQVDQG

result in spreading of the resistance genes on sensitive strains (3).

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of community (CA-MRSA) and health care-associated MRSA (HA-MRSA) infection (4). The strains of CA-MRSA as opposed to HA-MRSA VWUDLQVUHPDLQXVXDOO\UHVLVWDQWWRPDMRULW\RIDQWLELRWLFV H[FOXGLQJEHWDODFWDPV$FFRUGLQJWRMunckhof, the FULWHULRQRI&$056$GLDJQRVLVVKRXOGEHVHQVLWLYH-QHVVWRPRUHWKDQWZRGUXJVRIQRQEHWDODFWDPV CA-MRSA strains are usually more virulent and many of them can produce Panton-Valentine leukocidin (5).

From the perspective of hospital epidemiology and surveillance of infections among the most important are

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Monika Pobiega, Jadwiga Wójkowska-Mach, Piotr B Heczko

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VNLQDQGVRIWWLVVXHVLQIHFWLRQVEORRGVWUHDPLQIHFWLRQ and pneumonia. These infections may occur in hos-pitalized patients and residents in the long-term care IDFLOLWLHVDVWKHHQGHPLFLQIHFWLRQDQGLWPD\FRQWULEXWH to epidemic. It is estimated that S. aureus is the most IUHTXHQWO\LGHQWLILHGHWLRORJLFDJHQWRILQIHFWLRQV and accounts for 1% of all hospital-acquired infections (nosocomial infections). Approximately half of S.aureus infections in hospitalized patients are hospital-acquired LQIHFWLRQV,QWKH8QLWHG6WDWHVWKHLQFLGHQFHRI

S. aureus infections amounts to 9.13/1,000 admissions,

of which 43% are the cases associated with MRSA S. aureus is usually the most important pathogen REVHUYHGLQWKH,QWHQVLYH&DUH8QLWV,&8LQDGXOWV DVZHOODVLQQHZERUQV

Surveillance of MRSA which is connected with a decrease in frequency of infections of this etiology may have an indirect effect on prevalence of MRSA in the general population, i.e. on risk of ward personnel colo-QL]DWLRQDVWKH056$LVHTXDOO\IUHTXHQWO\REVHUYHGLQ FRPPXQLW\DFTXLUHGLQIHFWLRQV,WVKRXOGEHQRWHG that the significant risk factor of MRSA infection is colonization with this strain (3).

,WLVREVHUYHGWKDWPRUHFRXQWULHVDUHLPSOHPHQW-ing the routine MRSA screen,WLVREVHUYHGWKDWPRUHFRXQWULHVDUHLPSOHPHQW-ing of hospital inpatients (excluding daily admissions, dermatology and others) (13). Compulsory surveillance of methicillin-resistant isolates of S. aureus is also conducted, which as the pathogen of significance in the hospital epidemiology is VXEMHFWWRFRPSXOVRU\PRQLWRULQJZLWKLQWKHIUDPHVRI hospital-acquired infections control. Such surveillance is present in Polish hospitals and since many years in Finland, Norway, Sweden and The Netherlands (14).

$QWLELRWLFWUHDWPHQWDSSOLHGRQWKHZDUGH[HUWVSUHV-VXUHDQGFRQWULEXWHVWRVHOHFWLRQRIUHVLVWDQWVWUDLQV7KH strains which are multi-drug resistant and highly virulent, PD\FORQDO\VSUHDGLQVLGHKRVSLWDODQGEHWZHHQKRVSLWDOV On the territory of the given country, one specific clone is usually predominant. In the course of time, the clone may VSUHDGWRWKHQHLJKERXULQJFRXQWULHVDQGWKHZKROHFRQWL-nent. Due to the Multi Locus Seqence Typing (MLST), it LVSRVVLEOHWRGHPRQVWUDWHZLGHRIWHQLQWHUQDWLRQDOUDQJH RIVSHFLILFFORQHV,WPD\DOVREHHPSOR\HGWRH[DPLQH the molecular evolution of S. aureus (15). It is a reference PHWKRGIRUHVWDEOLVKLQJWKHEDVLFJHQHWLFVWUXFWXUHRIS.

aureus SRSXODWLRQZKLFKLVGRPLQDWHGE\VHYHUDOODUJH

clone complexes and includes several hundred sequence types (ST) (16). MLST consists in comparing conservative VHTXHQFHVRIKRXVHNHHSLQJJHQHVZKLFKFRQWUROWKHEDVLF functions of each living cell. The first stage of sequenc-ing is amplification of seven genes (arcC, aroE, glpF,

gmk, pta, tpi, yqiL) with seven pairs of starters (16). The

REWDLQHGQXFOHRWLGHVHTXHQFHVDUHWKHQFRPSDUHGWRNQRZQ DOOHOVIRUHDFKORFXVE\XVLQJWKHSURJUDPPHDYDLODEOHDW 0/67GDWDEDVHKWWSZZZPOVWQHW)RUHDFKLVRODWH

seven digital allels profiles are generated which define particular ST type. Due to the fact there are many allels in HDFKDQDO\]HGORFLLWLVLPSRVVLEOHWKDWWZRLVRODWHVKDYH the same profile accidently. The isolates of the same profile PD\EHWKHQFODVVLILHGWRWKHVDPHFORQDOW\SH7KH SURILOHVREWDLQHGGXHWRWKH0/67PD\EHHDVLO\FRPSDUHG EHWZHHQODERUDWRULHV,QWKHFDVHRIStaphylococcus, ap-SOLFDWLRQRI0/67PD\EHWLPHFRQVXPLQJDQGUHTXLUHV WKHVHTXHQFLQJRIODUJHQXPEHURIQXFOHRWLGHV)RU the purpose of epidemiological investigation, MLST is IRXQGWREHODERXULQWHQVLYHDQGFRVWO\WREHHPSOR\HG DVDEDVLFWRRORIW\SLQJ6RIDURYHU67W\SHVIRU

S. aureus KDYHEHHQGHVFULEHG

Spa typing is recommended as the method

supple-menting MLST. It consists in sequencing of short sequence repeats (SSR) from polymorphic X region of the protein A gene (spa,QWKLVUHJLRQWKH numerous spontaneous mutations (including loss and JDLQVRIVHTXHQFHUHSHDWVPD\RFFXU7RGDWH DSSUR[LPDWHO\GLIIHUHQWUHSHDWVDQGDERYH

spa KDYHEHHQGHVFULEHG7KHGLYHUVHYDULDWLRQV

of SSR correlates with the pathogenicity and virulence RIEDFWHULD'LYHUVLW\RIVpa gene, which is mainly FRPSRVHGRIUHSHDWVLVWKHFRQVHTXHQFHRISRLQW PXWDWLRQ ERWK GHOHWLRQ DQG GXSOLFDWLRQ 7KH comparison of spa types within Europe and the world LV SRVVLEOH GXH WR WKH LQWHUQDWLRQDO GDWDEDVH 5LGRP 6WDSK7\SH5LGRP*PE+*HUPDQ\KWWSVSDVHUYHU ULGRPGH7KHSURJUDPPHZKLFKLVDYDLODEOHDWWKH DIRUHVDLGZHELVWHDXWRPDWLFDOO\LGHQWLILHVWKHQXPEHU of sequence repeats and assigns it to the appropriate

spa type. SpaW\SLQJPD\EHDSSOLHGIRUWKHSXUSRVHV

of the analysis of molecular evolution of S. aureus and investigation of hospital epidemic. In comparison to MLST, it is less time- consuming and costly. However, it VKRXOGEHQRWHGWKDWGLVFULPLQDWRU\SRZHURIspa typing is lower than for PFGE. The most frequently identified

spa

W\SHLVWRFFXUULQJZLWKWKHSUHYDOHQFHRIDS-proximately 11% in many different European countries (excluding Poland), as well as in the United States and $XVWUDOLD7KHVXFFHVVLYHW\SHVRIJOREDOUDQJHDUHW WDQGWZKLFKDUHLGHQWLILHGLQ3RODQG

The resistance of S. aureus to methicillin is the re-VXOWRIV\QWKHVLVRIWUDQVSHSWLGDVH3%3D7KLVSURWHLQ VOLJKWO\ELQGVWRȕODFWDPDQWLELRWLFV$VDFRQVHTXHQFH HYHQLQWKHSUHVHQFHRIWKHVHDQWLELRWLFVWKHFHOOZDOO V\QWKHVLVLVQRWGLVUXSWHG7KXVWKHEDFWHULDPD\VXU-vive (15). The mecA JHQHZKLFKHQFRGHV3%3DLV located inside mec operon with regulatory genes (15). Due to the fact that there are more strains of S. aureus which have mecAJHQHLWLVLQGLVSHQVLEOHWRVXEW\SH PRELOHHOHPHQWVUHVSRQVLEOHIRUWKHUHVLVWDQFHWRPHWKL-cillin. These elements are defined as staphylococcal chromosomal cassette mec (SCCmec ,W HQDEOHV WR differentiate MRSA clones having a common ancestor

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Typing of Staphylococcus aureus No 3

EXWGLIIHUHQWHSLGHPLRORJLFDORULJLQV7KHFKDUDF-teristic elements of SCCmec are:

1. presence of mec gene complex which is composed of resistance gene to methicillin mecA and regulatory genes (mecR1 and mecI) and insertion sequences (IS1272 and IS431),

 SUHVHQFHRIccr gene complex which is composed RIUHFRPELQDVHJHQHVWKHVHFDWDO\]HH[FLVLRQDQG integration of SCCmec HOHPHQWVWREDFWHULDOFKUR-PRVRPHDQGDVDFRQVHTXHQFHDUHUHVSRQVLEOHIRU PRELOLW\RI6&&mec and surrounding elements, 3. presence of characteristic repeated sequences and

LQYHUWHGFRPSOHPHQWDU\ VHTXHQFHV DW ERWK HQGV (three regions J – J1 located at the right site of the FDVVHWWH - ORFDWHG EHWZHHQ FRPSOH[HVmec and

ccr and J3, located at the left site of the cassette).

The regions may activate plasmids and transposons, FDUU\LQJWKHGHWHUPLQDQWVRIUHVLVWDQFHWRDQWLELRWLFV RUKHDY\PHWDOLRQV

DELOLW\WRLQWHJUDWHDW¶HQGRIRSHQUHDGLQJIUDPH25) For the first time, SCCmecHOHPHQWVZHUHGHVFULEHG DQGFKDUDFWHUL]HGLQ6RIDUDWRWDORIHOHYHQ different SCCmecW\SHVKDYHEHHQLGHQWLILHG7KH epidemiological studies confirm that MLST, spa typ-ing as well as SCCmecVKRXOGEHSHUIRUPHGWRFODVVLI\ WKHFORQHFRUUHFWO\7KUHHGLIIHUHQWDSSURDFKHV on SCCmec HOHPHQWV W\SLQJ PD\ EH GLIIHUHQWLDWHG PHWKRGVEDVHGRQSRO\PHUDVHFKDLQUHDFWLRQ3&5 and its type in real time (Real-time PCR) or methods RIUHVWULFWLRQGLJHVWLRQ+RZHYHUWKHPRVWYDOXDEOHLV WKHPHWKRGSURSRVHGE\Kondo et alEDVHGRQVL[ multiplex PCRs:

x M-PCR 1 amplifies ccr (1-4) together with mecA gene x 03&5DPSOLILHVWKHFODVVHVmec$%DQG& x M-PCR 3 amplifies ORF from region J1 SCCmec

of I and IV types

x M-PCR 4 amplifies ORF of region J1 from SCCmec of II, III and V types

x 03&5DQGDPSOLI\JHQHVORFDWHGLQUHJLRQV- and J3

However, this method is time-consuming and re-TXLUHVFRQGXFWLQJRIODUJHQXPEHURIUHDFWLRQV,QWKH majority of cases for the epidemiological purposes, 03&5DQGDUHVXIILFLHQW03&5DQGDUHXVHG IRUVXEW\SLQJWKHGLIIHUHQFHVLQWKHUHJLRQ-03&5 5 and 6 identify integrated copies of transposons and plasmids. So far, the methods of typing SCCmec of W\SHV9,,;DQG;,KDYHQRWEHHQGHYHORSHG

The analysis of genotype similarity of S. aureus may EHSHUIRUPHGE\XVLQJSXOVHGILHOGJHOHOHFWURSKRUHVLV (PFGE). In the case of S. aureus typing, it is considered WREHDJROGVWDQGDUG3)*(UHOLHVRQHOHFWURSKRUHWLF VHSDUDWLRQLQSXOVHGILHOGRI'1$IUDJPHQWVREWDLQHG IURPFXWWLQJEDFWHULDOJHQRPHE\XVLQJVHOHFWHGUHVWULF-tion enzyme (SmaI for S. aureus). The separated DNA

IUDJPHQWVDUHYLVLEOHLQWKHIRUPRIEDQGVZKLFKIRUP WKHEDQGLQJSDWWHUQW\SLFDOIRUDJLYHQVWUDLQ,QWKH FDVHRIPDMRULW\RIEDFWHULDWKHVHSDUDWHGIUDJPHQWV VL]HUDQJHVIURP.EWR0E7KHREWDLQHGUHVWULFWLRQ SDWWHUQVPD\EHDQDO\]HGE\XVLQJWKHFULWHULDSURSRVHG E\ Tenover et al. to differentiate the clonal groups DQGZLWKWKHXVDJHRISURJUDPPHIRUDQDO\VLVRI strains relatedness (eg. GelCompar of Applied Maths, GeneProfiler and others). Additionally, in the case of LQVHUWRQGHOHWLRQRIODUJHPRELOHJHQHWLFHOHPHQWVWR EDFWHULDOJHQRPHWKHPRGLILFDWLRQVZLOOEHYLVLEOHLQ EDQGLQJSDWWHUQV)XUWKHUPRUHSODVPLG'1$RI VL]HDPRXQWLQJWR.EGRHVQRWGLVUXSWWKHHOHFWURSKR-retic separation and successive analyses due to the small VL]HRISODVPLGV3)*(LVHPSOR\HGLQWKHDQDO\VLV RIJHQRW\SHVLPLODULW\EHWZHHQLVRODWHVHVSHFLDOO\LQ epidemiological investigations. It is highly discriminat-ing, however it is also time-consuming and requires VSHFLDOHTXLSPHQW0RUHRYHUWKHSUREOHPDWLFFRXOG EHWKHODFNRIUHVWULFWLRQVLWHVLQSDUWLFXODUVWUDLQV$VD UHVXOWWKH\DUHQRWVXEMHFWWRW\SLQJXVLQJWKLVPHWKRG )XUWKHUPRUHWKHUHVXOWVREWDLQHGLQODERUDWRULHV RIWHQFDQQRWEHFRPSDUHG

Typing of microorganisms covers the methods ZKLFKHQDEOHWRUHSURGXFHWKHWUDQVPLVVLRQURXWHVRI SDWKRJHQVDVZHOODVFRPSDUHWKHPZLWKJOREDOVSUHDG-LQJRIHVSHFLDOO\YLUXOHQWVWUDLQV,WHQDEOHVWRGHWHUPLQH the infection of S. aureus etiology in diverse population RISDWLHQWVDQGFRQWULEXWHVWRLWVUHGXFLQJ,WVKRXOGEH noted that there is no one universal method of typing ZKLFKLVLGHDOLQHDFKFDVH$OORIWKHPKDYHERWKDG-vantages and disadZKLFKLVLGHDOLQHDFKFDVH$OORIWKHPKDYHERWKDG-vantages. The successive important LQIRUPDWLRQLVWKHIDFWWKDWWKHGLIIHUHQFHVLGHQWLILHGE\ XVLQJRQHPHWKRGGRQRWKDYHWREHFRQILUPHGZLWKWKH usage of another technique. Thus, in some situations it is recommended to apply several typing methods simulta-neously in order to achieve more detailed information.

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chromosome mec (SCCmec) classification and typing PHWKRGVDQRYHUYLHZ3RO-0LFURELRO .RQGR<,WR70D;;HWDO&RPELQDWLRQRIPXOWLSOH[

PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and PDMRUGLIHUHQFHVLQMXQN\DUGUHJLRQV$QWLPLFURE$JHQWV &KHPRWKHU

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ESCMID Study Group of Epidemiological Markers (ES-GEM). Overview of molecular typing methods for out-EUHDNGHWHFWLRQDQGHSLGHPLRORJLFDOVXUYHLOODQFH(XUR 6XUYHLOOSLL 'RVWĊSRQOLQHWWSZZZ HXURVXUYHLOODQFHRUJ9LHZ$UWLFOHDVS["$UWLFOH,G 6WUXHOHQV0-+DZNH\30)UHQFK*/HWDO/DERUDWRU\

tools and strategies for methicillin-resistant Staphylococcus

aureus screening, surveillance and typing: state of the art

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