Clindamycin resistant emm 33 Streptococcus pyogenes emerged among invasive infections in Helsinki metropolitan area , Finland , 2012 to 2013

In 2012, blood, skin and soft tissue infections caused by clindamycin resistant Streptococcus pyogenes (group A streptococcus; GAS) appeared to be increasing in the Helsinki metropolitan area. We compared monthly percentages of clindamycin resistant isolates in the area between 2012 and 2013, with those in 2010 and 2011. Resistance frequency in terms of patient age was also studied. We reviewed the medical records of bacteraemic cases in 2012 and 2013 and linked the data to emm types. To inform on the emm distribution among GAS isolated from skin and soft tissue infections during the epidemic, GAS isolates of one month (March 2013) were emm typed. For GAS blood, skin, and soft tissue isolates taken together, the proportions of clindamycin resistant isolates were significantly higher in 2012 and 2013 (23% and 17%, respectively) compared with the two previous years (3%, p<0,001). The erythromycin resistance percentages were almost equal to clindamycin (22% and 17%) in 2012 and 2013, respectively. Clindamycin resistance was most frequent in GAS isolates of 40 to 60 year-old patients (148/417; 36%). Among clindamycin resistant isolates, 12 of 14 blood isolates from 2012 to 2013, and 11 of 13 skin and soft tissue isolates from March 2013, were emm33. Emm33 GAS bacteraemia was associated with clindamycin and erythromycin resistance (odds ratio (OR): 7.0; 95% confidence interval (CI): 1.9–25.3). Infection focus was mainly the skin; either cellulitis (7/12) or necrotising fasciitis (3/12). All emm33 GAS isolates harboured the ermTR resistance gene with constitutive macrolides, lincosamides and streptogramines B (MLSB) phenotype. Emm33 GAS was responsible for the higher proportion of clindamycin resistance in skin, soft tissue, and blood isolates locally in 2012 and 2013. Introduction Streptococcus pyogenes (group A streptococcus; GAS) causes pharyngitis, skin and soft tissue infections, and invasive septic diseases [1]. Certain GAS emm types have been associated with tissue-specific infections [2], antibiotic resistance [3], and local epidemics [3]. Erythromycin resistance has been linked to various emm types, such as 4, 6, 12, 75, and 77 [4-7]. Most of these emm types have been identified in throat isolates.

Depending on the erythromycin resistance mechanism, isolates may also be resistant to clindamycin, although relatively rarely [8].
There is limited information concerning clindamycin resistance in GAS isolates causing skin and soft tissue infections.In Finland, the annual percentages of erythromycin and clindamycin resistance was only 2 to 3% in 2012 when all GAS isolates (including throat isolates) were analysed together [9].The figures for all GAS isolates have been the same also in Helsinki metropolitan area [10].In February 2013, while making the annual local antibiotic resistance statistics of 2012, a high proportion of clindamycin resistance was noticed among blood, skin and soft tissue GAS isolates.
In this study, we investigated whether a specific emm type was behind this phenomenon, by examining the laboratory data of GAS isolates in 2012 and 2013 in the Helsinki metropolitan area.We used baseline data of years 2010 and 2011 for comparison.The invasive GAS cases of 2012 and 2013 were analysed in detail and linked to emm types (blood isolates) to characterise common denominators behind the increase in clindamycin resistance.To obtain more information on the emm distribution in GAS isolates from skin and soft tissue infections, emm typing was performed on a set of such GAS isolates obtained during March 2013 in the Helsinki metropolitan area.

Setting
In Finland, the Division of Clinical Microbiology at HUSLAB is a clinical diagnostic laboratory that serves the Helsinki metropolitan area of ca 1.5 million population.It receives from the local laboratories all blood cultures flagged positive for bacteria by the BacT/ ALERT3D system (bioMerieux, Marcy l'Etoile, France) for bacterial identification and resistance analyses.The GAS blood isolates are routinely stored and sent to the national reference laboratory at the National Institute for Health and Welfare for emm typing.HUSLAB does the final identification and resistance analyses also for all other GAS isolates e.g.throat, skin, and soft tissue isolates of the Helsinki metropolitan area and keeps records of the resistance data for statistical analyses, however the bacterial isolates are not routinely stored.For this study, GAS isolates of skin and soft tissue infections of March 2013 were collected specifically, and stored at -70 °C.This was done to characterise the emm type distribution of both clindamycin susceptible and resistant GAS isolates from skin and soft tissue infections (while the epidemic was still going on), and to verify if the distribution was the same in these isolates compared with the routinely stored blood isolates.We were able to gather 78% (45/58) of the total skin and soft tissue GAS isolates of the month.

Microbiological methods
At HUSLAB GAS isolates are routinely identified by colony morphology with betahaemolysis on sheep blood agar and Lancefield grouping with latex agglutination (Latex Reagent A, Oxoid Ltd, Basingstoke, Hants, England).The resistance for erythromycin and clindamycin is routinely determined using the doubledisc diffusion method on Mueller-Hinton agar with 5% defibrinated horse blood with 20 mg/L beta-NAD (MH-F broth).In this study, additional minimum inhibitory concentrations (MICs) were determined for the clindamycin resistant skin and soft tissue GAS isolates of March 2013 and blood isolates of 2012 to 2013 after twice sub-culturing on horse blood agar.MICs were determined by Etests (bioMerieux SA, Marcy l'Etoile, France) for azithromycin, clindamycin, doxycycline, erythromycin, levofloxacin, moxifloxacin, tetracycline, and vancomycin, on Mueller-Hinton (MH)-F broth using 0.5 McFarland inoculum and incubated for 18 ± 2h with 5% CO2 at 35 ± 1 °C.Telithromycin susceptibility was tested by disc-diffusion method in similar conditions as in MIC determinations.European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2013 breakpoints were used to determine whether the zone inhibitions or MICs were considered susceptible (S), intermediate (I) or resistant (R).

Emm typing and resistance gene analysis of the collected group A streptococcus isolates
At the National Institute for Health and Welfare, the 45 GAS isolates of March 2013 and 109 blood GAS isolates from 2012 to 2013 were emm typed according to the guidelines provided by Centres for Disease Control and Prevention (http://www2a.cdc.gov/ncidod/biotech/strepblast.asp) as previously described [11].
DNA of the erythromycin and clindamycin resistant GAS isolates (blood isolates of 2012-2013; n = 14 and isolates of March 2013; n = 13) was extracted by suspending the colonies in 100 µl of TE buffer and boiling for 15 min, followed by centrifugation at 13,000 rpm for 2 min.The presence of erm, including ermB and ermTR, and mefA genes was detected by multiplex-polymerase chain reaction (PCR) including primers for amplification of mefA, ermB and ermTR genes, as described previously [12].Positive controls were S. pyogenes A569 for mefA, Escherichia coli with plasmid pJIR229 for ermB, and S. pyogenes A200 for ermTR [13].

Susceptibility data analysis
The susceptibility analyses of clinical isolates were made using WHONET 5.6 software.We analysed the resistance figures of GAS isolates of the Helsinki metropolitan area between January 2012 and December 2013, and compared the data to the baseline years, namely 2010 and 2011.Blood, skin, and soft tissue isolates were analysed separately from throat isolates.One isolate per patient, the most resistant one, was included in the analysis (WHONET definition).Data were expressed as percentage of isolates resistant or intermediate for erythromycin together and as percentage of isolates resistant for clindamycin, according to the EUCAST 2013 standard (http://www.eucast.org).The reason for this was that the standard did not include a zone diameter breakpoint for intermediate for clindamycin, but for erythromycin it did.Inducible clindamycin resistance was detected by antagonism of clindamycin activity by erythromycin (the D phenomenon) and if not present the isolate was reported susceptible.In HUSLAB the detected antagonism was reported resistant for clindamycin.

Analysis of clinical data and statistics
Electronic medical records of patients with a GAS positive blood culture between January 2012 and December 2013 in Helsinki metropolitan area were reviewed to identify underlying conditions and any common exposure between the patients.Age, sex, C-reactive protein (CRP) value, and leucocyte count at the time of diagnosis were registered.Diagnosed diabetes was recorded.Alcohol abuse was defined as a known social or medical problem caused by alcohol noted in the medical records.Intravenous drug abuse was registered similarly when mentioned in the records.Suspected focus of infection was registered.Presence of a cutaneous infection was described as either cellulitis (infections of the skin and underlying tissues such as erysipelas and deeper non-necrotising soft-tissue infection) or necrotising fasciitis (progressive, destructive, subcutaneous streptococcal infection with necrosis observed either directly or under surgery).Need for surgical procedures, complications, and stay at an intensive care unit due to GAS bacteraemia was recorded.Mortality within seven days after GAS positive blood culture was recorded.Data were analysed and compared using Fisher's exact or Pearson chi-squared tests, or t-test, Mann-Whitney U-test or analysis of variance (ANOVA), when appropriate, using SPSS for Windows statistical package (SPSS Inc., Chicago, IL).Logistic regression analysis was used to identify the risk factors.A p-value < 0.05 was considered statistically significant.

Resistance data of clinical group A streptococcus isolates
When the resistance data were analysed without throat isolates, the proportions of skin, soft tissue, and blood GAS isolates obtained from Helsinki metropolitan area that were clindamycin resistant in 2012 and 2013 were respectively 23% (199/866) and 17% (153/899

Emm types, clindamycin resistance, and clinical data of invasive group A streptococcus cases in 2012 and 2013
A total of 109 GAS positive blood isolates were identified in the Helsinki metropolitan area between January 2012 and December 2013.Figure 3 shows the emm type distribution of these invasive isolates.
Of the 109 GAS positive blood isolates, 14 were clindamycin resistant and these included 12 emm33, one emm28, and one emm89.Figure 4 shows the time distribution of the invasive GAS isolates resistant or sensitive for clindamycin with respective resistance genes.None of the emm33 isolates were susceptible for clindamycin.During the baseline years 2010 and 2011, when clindamycin resistance among isolates was at a low level, no invasive emm33 GAS were isolated in the Helsinki metropolitan area.
Table 1 compares the clinical data of emm33 cases to the cases with another emm type.In logistic regression analysis clindamycin and erythromycin resistance, alcoholism, and intravenous drug abuse (Table 1) associated with emm33 GAS bacteraemia.Of the 12 emm33 cases, 10 had infection focus on the skin or soft tissue.Three emm33 cases underwent a surgical procedure due to complications of GAS infection.There were no re-infections or need for intensive care in emm33 cases.

Emm types and laboratory referral data of skin and soft tissue group A streptococcus isolates of March 2013
A total of 45 GAS isolates from skin and soft tissue infections were gathered in March 2013 and emm typed.Emm typing revealed two isolates being S. dysgalactiae subsp.equisimilis and these were discarded from the analysis.The remaining 43 isolates represented emm types shown in Figure 5.Of these, 13 showed clindamycin resistance and these included 11, which were emm33.None of the emm33 were susceptible for clindamycin.Ten emm33 isolates were from a skin lesion or abscess as shown by the laboratory referral data in Table 2.

Discussion
During 2012 and 2013 emm33 GAS caused a local epidemic of skin and soft tissue infections in the adult population in Helsinki metropolitan area, Finland.The outbreak was detected as a marked increase in the proportion of isolates resistant to erythromycin and clindamycin.In most cases the primary infection focus was the skin, but a few GAS emm33 infections were invasive and caused necrotising fasciitis.All emm33 isolates were resistant to both erythromycin and clindamycin.
There was an association of emm33 with alcohol and intravenous drug abuse, however the number of patients was very low so these results have to be interpreted with caution.Alcohol abuse was marked positive if mentioned in the patient records.Since alcoholism is not always evident and not even always actively asked about by the doctor in the hospital, this information is most probably partly lacking from our data.The same counts for intravenous drug abuse.An association with alcohol abuse has nevertheless been reported for certain other emm types, such as emm59 and emm1 [14,15].
Emm33 belongs to the emm superfamily group D, which includes emm types causing skin infections, such as impetigo [2,16].Emm33 has further been characterised as a member of the D4 emm-cluster, which is able to bind plasminogen [17].Plasminogen binding may contribute to the skin tissue tropism by possible break down of tissue barriers facilitating dissemination and prolonged bacterial persistence in the skin [18].There is limited data concerning infections caused by this emm type.It caused some of the severe GAS infections of intravenous drug users reported in a study in the United Kingdom (UK) in 2003 and 2004, but was

Number of isolates
Emm not the most common emm type in that study [19].
Intravenous drug abuse has been shown to be a risk factor for severe disease caused by GAS [20].
GAS is able to cause very local and timely limited epidemics, as shown in intravenous drug users in the UK [19].Interestingly, in 2012 in our neighbouring country, Sweden, there was an increase of invasive emm1 GAS diseases occurring mostly in patients over 80 years of age [21].The invasive emm1 numbers have remained stable in Helsinki metropolitan area between 2010 and 2013 indicating that the Swedish epidemic is local or has not reached Finland yet.
The Finnish National Institute for Health and Welfare receives all invasive GAS isolates from Finland for genotyping and strain collection.Since 2007, the main genotyping method has been emm typing.In Finland, the first emm33 invasive GAS isolates were found only in 2012 and they all originated from the Helsinki metropolitan area.From June 2013 onwards sporadic emm33 cases have been found also in other hospital districts, however emm33 still remains an uncommon genotype in Finland (Pieter Smit, personal communication, April 2014).
Several mechanisms underlie the macrolide and lincosamide resistance.M phenotype isolates carry the mefA gene, which causes efflux of the antibiotic and confers resistance to many macrolides with preserved susceptibility to clindamycin and streptogramin B [22].Emm4 GAS with mefA has previously caused high erythromycin resistance locally in Finland [23].
Interestingly, in our study none of the tested isolates carried the mefA resistance gene.The resistance data of the Helsinki metropolitan area showed also that most of the isolates with decreased susceptibility to erythromycin were also clindamycin resistant (either with inducible or constitutive phenotype) suggesting that the M phenotype was not generally present in the GAS isolates during the years 2012 to 2013.
Ribosomal methylation of the target of the antibiotics (ermA, ermB or ermTR) prevents binding of the antibiotics by causing a conformational change in the 23S ribosome [24].The ermB isolates usually show constitutive MLS B (cMLS B ) resistance to macrolides, clindamycin, and streptogramin B, while the ermA and ermTR isolates may show macrolide-induced resistance to clindamycin [13].In our study all emm33 isolates shared the ermTR macrolide resistance gene with a cMLS B resistance phenotype.This resistance phenotype is more common among ermB but has in rare occasions also been shown by isolates carrying the ermTR gene [25].
All the emm33 isolates were also The emm33 isolates in our study were also tetracycline susceptible, while Kataja et al. showed that inducible ermTR GAS isolates in Finland in 1994 and 1995 were mostly tetracycline resistant [25].To fully understand the resistance mechanism underlying emm33, the isolates should be examined in more detail.The fact that emm33 isolates had the same resistance gene and similar antibiotic resistance patterns supports the idea that they belong to the same clone (Pieter Smit, personal communication, April 2014).
Clindamycin is an important drug in the primary healthcare, especially, as it is used for treating GAS infections in penicillin allergic patients.The skin and soft tissue infections of intravenous drug abusers and of diabetic patients are often polymicrobial with anaerobic bacteria and staphylococci present making clindamycin the drug of choice for empirical treatment.In invasive, septic GAS infections clindamycin is used in combination with penicillin for better outcome possibly diminishing the bacterial toxin production [28].Spreading of a skin-tropic emm type with clindamycin resistance is of concern considering the empirical antibiotic treatment of the abovementioned patient groups.An announcement, aimed at the primary care and hospital doctors of the city of Helsinki, was released in spring 2013 by HUSLAB together with the infectious disease specialists of the Helsinki city hospitals concerning the proportion of clindamycin resistant GAS figures.It guided the empirical therapy of adult skin infections recommending that clindamycin should not have been used as monotherapy.The infectious disease specialists of the whole Helsinki metropolitan area were also informed, and additional antimicrobial susceptibility testing was conducted for the clindamycin resistant GAS isolates to find alternative drugs for penicillinallergic patients.Surveillance of the situation is important because emm33 GAS may spread to children since it is a potential impetigo-causing emm type [2].
The study shows that for resistance statistics it is important to analyse skin and soft tissue GAS separately from the numerous throat GAS.Different emm types are typical for distinct anatomical locations and important resistance phenomena may be masked if isolates are analysed only together.Here we documented a single, local, epidemic of a previously rare emm33 GAS causing skin and soft tissue infections with also invasive cases.This emm type caused rapid changes in macrolide and clindamycin resistance locally in the adult population.These findings had an impact on the empirical treatment of skin and soft tissue infections of the area.

Figure 1
Figure 1 Percentage of skin, soft tissue, and blood group A streptococcus isolates showing reduced susceptibility to clindamycin and erythromycin in Helsinki metropolitan area, Finland, 2012-2013 (n = 1,765)

Figure 5
Figure 5 Emm type distribution of skin and soft tissue group A streptococcus isolates, Helsinki metropolitan area, Finland, March 2013 (n = 43)

Table 2
Laboratory referral data of the group A streptococcus isolates from skin and soft tissue that were clindamycin resistant, Helsinki metropolitan area, Finland, March 2013 (n = 13) a Constitutive ermTR.b Inducible ermB.c Constitutive ermB.