Get 20M+ Full-Text Papers For Less Than $1.50/day. Subscribe now for You or Your Team.

Learn More →

Molecular epidemiology of Panton-Valentine Leukocidin-positive community-acquired methicillin resistant Staphylococcus aureus isolates in pastoral communities of rural south western Uganda

Molecular epidemiology of Panton-Valentine Leukocidin-positive community-acquired methicillin... Background: The emergence of multidrug resistant Staphylococcus aureus strains, including methicillin resistant (MRSA), is a global concern. Treatment of bacterial infections in Uganda’s health care settings is largely empirical, rarely accompanied by laboratory confirmation. Here we show the burden, characteristics of MRSA and epidemiology of Panton-Valentine Leukocidin (PVL) positive strains in asymptomatic carriers in pastoral households of south-west Uganda. Methods: Nasal swabs from 253 participants were cultured following standard methodology. MRSA strains were identified by detection of the mecA gene and SCCmec typing, and PVL genes detected by PCR. Pulsed Field Gel Electrophoresis (PFGE) was done to evaluate possible transmission patterns. Spa typing of PVL positive isolates was done to study the epidemiology of virulent strains in this setting. Results: S. aureus was isolated in 29% (n=73) of theparticipants, of which48wereMRSAby mecA typing. PVL-encoding genes were found in 49.3% (n=36) of the73isolates, of which25werealso mecA positive. Among the PVL negative strains (n = 37), 62.2% (n = 23) carried the mecA gene. The most common SCCmec type was V, detected in 39 (18 PVL positive and 21 PVL negative) isolates. PFGE clustered 21/36 (58.3%) PVL positive isolates divided in four pulsotypes and 18/37 (48.6%) PVL negative isolates divided in eight pulsotypes. The most prevalent Spa types were t318 (26.5%, n = 9) and t645 (20.6%, n = 7); while other common Spa types were t11656 (n =3), t127 (n = 3) and t355 (n =3). Conclusion: The study shows a high prevalence of community acquired (CA)-MRSA, and PVL-positive isolates with two predominant spa types in rural Uganda, further complicating infection control strategies in these underprivileged communities. * Correspondence: basiimwe@chs.mak.ac.ug Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, Milano, Italy Universita Vita-Salute San Raffaele, Via Olgettina 58, Milano, Italy Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 2 of 7 Background In African countries, CA-MRSA has been reported in About a third of healthy individuals in the community a few studies in Mali [17], Nigeria [18], Algeria [19], are asymptomatically colonized with Staphylococcus Egypt [20] and Gabon [21]. In Uganda, a few studies aures (S. aureus) in the nostrils [1], a very important have been done at tertiary health care centers [22–25], finding considering the fact that nasal carriage of S. aureus and these have focused on HA-MRSA. Community studies has been associated with subsequent infection [2], and have focused on S. aureus carriage in raw milk and its carriers are an important source of spread of infection in products as well as in urban milk vendors [26, 27]. We set communities. A major concern is the world-wide emer- out to establish the prevalence and molecular epidemiology gence of methicillin resistant S. aureus (MRSA) in the of PVL-positive CA-MRSA isolates from pastoral commu- community [3]. In contrast with health care associated nities of rural south-western Uganda so as to inform public MRSA (HA-MRSA) infections, community associated health on how to develop effective strategies to prevent MRSA (CA-MRSA) infections can occur in healthy indi- spread MRSA in these and surrounding communities. viduals [4], suggesting that these strains have greater viru- lence. Skin and soft-tissue infections represent about 90% Methods of cases of CA-MRSA infection, mostly characterized by Study area abscesses or cellulitis with purulent drainage [3]. The study was carried out in rural pastoral households A predominant feature of CA-MRSA is the presence of Kiruhura district, South Western Uganda. The area is of the Panton-Valentine Leukocidin (PVL) genes that en- a rangeland, populated by agro-pastoralists and season- code a S. aureus exotoxin that induces lysis of mono- ally itinerant semi-nomads whose livelihoods mainly de- cytes and neutrophil granulocytes [5]. Additionally, there pend on consumption and trade of cattle and their is evidence that PVL-positive S. aureus strains susceptible products; and small subsistent crop farming. to methicillin (MSSA) may be reservoirs for the develop- ment of PVL-positive MRSA via the integration of the Study design and sampling staphylococcal cassette chromosome mec (SCCmec)ele- A cross-sectional study of S. aureus carriage in the area ments including the mecA gene conferring methicillin re- was conducted as part of a zoonotic diseases study sistance [6]. Another key feature of CA-MRSA is that the among pastoral communities of Kiruhura district, South strains mainly harbor SCCmec types IV and V [7–9], and Western Uganda, in 2013. A total of 196 homesteads a relationship between CA-MRSA, SCCmec type IV and V from two sub-counties (100 from Kanyaryeru and 96 and PVL has been confirmed in some studies [10, 11]. from Sanga) were recruited and sampled. The sub- While efforts have been made to map out the geo- counties were purposively selected by the zoonoses study graphical predominance and spread of different CA- because they bordered Lake Mburo National Park, with MRSA clones worldwide [3], little is known about its a porous human-animal interface. All participants did magnitude and genetic composition in developing coun- not have a history of recent hospitalization, and the only tries, especially in Africa. Molecular analytical tech- demographics characteristics recorded were sex and self- niques such as pulsed field gel electrophoresis (PFGE), reported age. In each household, only a nasal swab was multilocus sequence typing (MLST), spa and SCCmec taken for any participant and these were inoculated into typing have been used to show both the spread and evolu- Brain-Heart Infusion (BHI) broth in 15 ml propylene tion of MRSA. PFGE is still considered the gold standard tubes and stored on ice before same day transportation for typing MRSA isolates, and is one of the most discrim- to the Department of Microbiology at Mbarara University inative typing methods [12]. While MLST is an excellent of Science and Technology for culture. tool for investigating clonal evolution of MRSA, it is ra- ther expensive, labour intensive and time consuming. Se- Sample culture, isolation and identification quences from the polymorphic region of the S. aureus Initial bacterial culture and isolation was done according protein A (spa) gene have been used to develop a single- to methods described by Cheesbrough [28], with minor locus sequence typing technique for MRSA [13] with a modifications. Briefly, 50 μl of culture broth was inocu- discriminatory power between PFGE and MLST, and lated on 5% sheep blood agar medium and incubated for ability to investigate both molecular evolution and out- 18–20 h at 37 °C. Preliminary identification of the bac- break situations [14], while remaining simple. teria was carried out based on colony characteristics The resistance of S. aureus to methicillin is caused by such as shape, size, color and hemolysis patterns. Iso- the mecA gene, located on a mobile genetic element, the lates suspected of being S. aureus were shipped in BHI staphylococcal cassette chromosome mec (SCCmec) [7]. with 20% glycerol to the Emerging Bacterial Pathogens Currently, eleven main SCCmec types (I to XI) are Unit (EBPU) at San Raffaele Scientific Institute in Milan, known [15], and Zhang et al. developed a multiplex PCR Italy, for identification and molecular characterization. for the characterization of SCCmec type I to V [16]. At the EBPU, all isolates were sub-cultured on mannitol Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 3 of 7 salt agar (Oxoid Ltd, Hampshire, England), then on blood 65.8% (n=48) of the73isolates. Theproportionof agar (Becton Dickinson, Heidelberg, Germany), and sub- PVL positive isolates carrying the mecA gene was 25/36 jected to the tube coagulase test in rabbit plasma with (69.4%) (Table 1), while 23/37 (62.2%) of the isolates EDTA (Remel, KS, USA). MRSA strains were identified by without the PVL gene possessed the mecA (Table 2). detection of the mecA gene and SCCmec typing. Among the PVL positive isolates, 18 of the 25 mecA DNA extraction, SCCmec typing and PVL genes detection Bacterial DNA was prepared from isolated colonies sus- Table 1 PFGE profiles, Spa types, mecA, and SCCmec types of pended in 500 μl Triton X-100 lysis buffer with 1% Triton PVL positive isolates and 20 μl of 1 mg/ml lysostaphin, incubated at 37 °C for Isolate ID Pulsotype Spa type mecA SCCmec 1 h, followed by phenol-chloroform extraction. SCCmec T095Na A1 NT + V typing was done by multiplex PCR using primers and pro- T101Na A1 NT + IVc tocols for SCCmec types and subtypes I, II, III, IVa, IVb, T047Na A1 t127 + IVc IVc, IVd, and V and the mecA gene according to Zhang T070Na A1 t127 + IVc et al. [16]. The MRSA strain COL (SCCmec type I, mec gene complex B and ccr gene complex 1) was used as T056N A2 t318 - - positive control. Additionally, amplification of the T099Nd A3 t2393 + IVc Panton – Valentine Leucocidin (PVL)toxin genes, lukS- T149Na A4 t645 + IVc PV and lukF-PV, was performed using the S. aureus T101Ng A5 t318 + V strain ATCC49775 as positive control, and primers and T005Na B1 t318 + V protocols as described by Lina et al. [29]. T021Nb B1 t318 + V Genotyping of isolates T021Nc B1 t318 + V All isolates were subjected to molecular epidemiological T097Na B1 t318 + V analysis by PFGE after SmaI digestion using the CHEF T125Na B1 t318 + V Genomic DNA Plug kit (Bio-Rad) according to a stan- T141Nc B1 t318 + V dardized protocol [30]. PFGE was run using a CHEF T047Nd B1 t186 + V DRIII system (Bio-Rad). The InfoQuest FP (v5.1) soft- T156N B1 t186 + V ware (Bio-Rad Laboratories) was used to analyze PFGE profiles, according to interpretation criteria described by T019N B2 t186 + V Tenover et al. [12]. Clustering analysis was achieved T119Nb C1 t355 + V using Dice similarity coefficients and the unweighted T135Na C2 t729 - - pair group method with averages (UPGMA) at 1.5% T132Nb C2 t11656 - - optimization and 1.5% position tolerance. PVL-positive T139N C2 t11656 + V strains were further subjected to spa-typing according to T145N C2 t11656 + V Frenay et al. [13]; and spa types were assigned on the Ridom SpaServer (http://spaserver.ridom.de) curated by T022N C2 t645 - - the SeqNet.org initiative [31]. T041Na C2 t645 - - T099Nb C2 t645 + V Results T063N C3 t355 + V Socio-demographic characteristics and S. aureus carriage T053N D1 t1376 + IVc The study sampled 196 households, and nasal swabs T055N D1 t1376 + V were taken from 253 participants (172 males and 81 females) with average age of 14 years and a range of 6 to T039Na E1 t318 + IVc 48 years. Of the samples, 132 (52.2%) were Staphylococcus T041Nb E2 t645 + V spp, of which only 73 were S. aureus (carriage rate of 29%) T025N E3 t2393 + IVc and further characterized. The 73 isolates were obtained T030Nb F1 t064 + V from 48 (65.7%) males and 25 (34.3%) females. The aver- T158Nb G1 t127 - - age age of the 73 participants whose isolates were charac- T087 H1 t064 + V terized was 13, with a range of 6 to 45 years. T155Nd H2 t509 - - Prevalence of PVL and mecA genes T097Nb I1 t002 - - PVL-encoding genes were found in 49.3% (n = 36) of ID identification number of the isolate, + positive on PCR amplification, - negative the 73 S. aureus isolates, while mecA was detected in on PCR amplification, NT not typable Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 4 of 7 Table 2 PFGE profiles, mecA, and SCCmec types of PVL Molecular epidemiology of MRSA PVL positive and PVL negative isolates negative strains Isolate ID Pulsotype Spa type mecA SCCmec PFGE profiles of PVL positive and negative isolates were analyzed separately to understand any difference in T117Nb E1 t786 - - transmission within the two groups. Among the PVL T119Na E1 t786 - - positive isolates, 21/36 (58.3%) strains were found in T092Na E2 t186 - - four clusters (A1, B1, C2, D1, Table 1); while in PVL T058Nb E4 t7662 - - negative isolates, 18/37 (48.6%) were found in eight clus- T061Nd L1 t1236 + V ters (E1, L1, L3, F1, N1, H1, G1, Q1, Table 2). Some T061Nb L1 t1236 + V family members carried identical strains. In order to re- late the genotypes of the isolates in this study to global T059Na L2 Unknown + V epidemiology of S. aureus, we performed Spa typing of T059Nb L3 Unknown + V the PVL positive isolates. Only two of the 36 isolates T099Na L3 Unknown + V could not be typed by this technique, but they belonged T021Nd L4 Unknown + V to the same pulsotype (A1). In all, there were 13 Spa T032Nc F1 t064 - - types among the 34 typable PVL positive S. aureus iso- T131Nb F1 t064 - - lates. The most prevalent Spa types were t318 compris- ing of 26.5% (n = 9), of which seven isolates carried the T032Nb F1 T064 - - mecA gene; and t645 (20.6%, n = 7). The other common T061Nc N1 t2771 + V Spa types were and t11656 (8.8%, n = 3) and t127 (n = 3). T074Nc N1 t2771 + V The other Spa types, their frequencies and PFGE profiles T074Nb N1 t2771 + V can be seen in Table 1. T029Nb H1 t002 - - T101Nf H1 t002 + V Discussion T076N G1 t5739 + V Knowledge of the epidemiology of bacterial infections is T128Nd G1 t2680 + V very important for appropriate decision-making in the T018Nb G2 t616 + V treatment of arising infections. At a community level, it T092Nb P1 t4353 + V is also important to curb the spread of infection, in- T102Nb P2 t4353 - - cluding multidrug resistant strains. To our knowledge, T127Na Q1 Unknown - - this is the first investigation of Panton-Valentine Leukocidin-Positive CA-MRSA in asymptomatic semi- T137Ng Q1 t064 - - nomadic pastoralist communities in the East African T110Nb R1 t4523 - - region. The main findings of the study are a high preva- T111Na R2 t5187 + V lence of MRSA and PVL-positive isolates with a pre- T027Nb R3 t5187 + V dominant spa type (t318). T004Nb Unique t3772 + V This study isolated 132 Staphylococci isolates from the T033Nb Unique Unknown + V nares of 253 participants, of which 73 were coagulase positive S. aureus (carriage rate of 29%). The 73 isolates T005Nb Unique t951 + V of S. aureus in our study were obtained from 48 male T114Ng Unique Unknown - - and 25 female participants, with an average age of T151Nb Unique t5739 + V 13 years, with 56/73 (76. 7%) of the participants being T115Na Unique Unknown + IVa between 7 and 15 years old. This is similar to observa- T120N Unique t5187 + V tions elsewhere that CA-MRSA infections tend to occur T126N Unique NT + IVa in previously healthy younger patients [21, 32, 33]. Our finding is of public health importance because this T104Nb Unique Unknown - - school-going age group has potential of disseminating ID identification number of the isolate the strains far and wide in the communities. The car- riage rate in our study is more than double that in a study on milk men in and around Kampala city, Uganda, positive isolates were SCCmec type V, while the other where only 11 Staphylococci were isolated from 31 indi- seven were all SCCmec IVc. Among the PVL negative viduals, of which only 4/31 (13%) were S. aureus [26]. isolates, 21 of the 23 mecA positive isolates were Our finding, however, is in agreement with statistics SCCmec type V, while the other two were type IVa. from literature suggesting that between 25 to 35% of Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 5 of 7 healthy individuals are asymptomatically colonized with at the National Referral hospital in Mulago, Kampala, S. aureus in the nostrils [1, 21, 34, 35]. showed that SCCmec type V was the most predominant In the current study, the proportion of S. aureus iso- type [22], suggesting the presence of mixed CA-MRSA lates carrying the mecA gene, hence MRSA, was 48/73 and HA-MRSA genotypes in hospital settings in Uganda. (65.8%). This is generally high in comparison to commu- A further characterization of the 36 isolates carrying nity studies elsewhere. In the urban and peri-urban the PVL gene in this study was done. SCCmecIV and Kampala, the four isolates from milk men were all PVL are known to be molecular markers associated with MSSA. In a study of indigenous remote African Babongo the emergence of CA-MRSA worldwide [10]. The pro- pygmies living in Waka National Parc, Central Gabon, portion of strains carrying both the PVL and mecA all 34 S. aureus isolates were susceptible to Oxacillin/ genes was 25/36 (69.4%), while the mecA gene was de- Methicillin, and did not amplify for the mecA gene by tected in 23/37 (62.2%) of the isolates without the PVL PCR. The authors hypothesized that the result could be gene. While there was no significant difference in mecA due to limited use of antibiotics in that population. Stud- carriage, PFGE profiles for both PVL positive and nega- ies in North America and Australia, however, have tive isolates show that there were bigger clusters in PVL shown that native and indigenous populations have been positive strains (Table 1) compared to PVL negative associated with a high risk of colonization and infection strains (Table 2). In fact, 18/37 (48.6%) of the PVL with CA-MRSA which may be related to many of these negative isolates were in clusters (Table 1) compared to groups being disadvantaged, due to their association 21/36 (58.3%) of PVL positive isolates, with the largest with low socio-economic status, crowded living condi- (B1) comprising of eight isolates (Table 1). These re- tions and frequent use of antibiotics [36, 37]. There have sults suggest that PVL positive isolates in the study been reports of absolute resistance to penicillin and high were more likely to be involved in chains of transmis- percentage resistance to other antibiotics in milk from sion compared to the PVL negative isolates. There has similar settings in central Uganda, hence risk of spread been recent evidence of familial spread of PVL carrying to humans through the food chain [27]. In our study MSSA strains in Israel [42] and MRSA strains in Italy community there is frequent usage, by farmers, of veteri- [43], while a previous study in Greece revealed that a nary antibiotics to treat nearly all ailments in their live- unique clone of PVL-positive MRSA had spread in both stock, due to poor outreach services by the veterinary the community and hospital settings, and was replacing department. It is therefore more likely that constant older clonal types [44]. In Central Gabon, Africa, PVL- contact with antibiotics for animal use, as well as encoding genes were detected in 55.9% of study iso- consumption of raw milk and its products without ob- lates, with authors concluding that the pygmies in that serving drug withdrawal periods, as is the culture in this study faced a risk of developing necrotizing infections, setting, are limiting future options for the management due to the virulence characteristic of the PVL. The of multidrug resistant microorganisms in both humans finding of a high proportion of isolates carrying both and animals in pastoral communities of Uganda. Among the PVL and mecA genes in our study may have the 48 isolates carrying the mecA gene, 39 (81.3%) were considerable implications on future strategies for infec- type V, the other being type IV or its subtypes (Tables 1 tion control in these underprivileged communities. and 2). Type IV and V SCCmec elements have strongly Amongthe PVLpositiveisolates, therewas apredomi- been associated with stains causing MRSA infections in nant circulating Spa type, t318, comprising of nine of the persons with no history of hospitalization, hence thought 34 (26.5%) typable isolates. This is the first time the Spa to be more related to CA-MRSA [38]. Furthermore, it type has been identified in a Ugandan setting. Moreover, has been shown that children may be at a higher risk of seven strains (77.8%) of this Spa type carried the mecA infection with SCCmec types IV and V, as well as PVL gene, pointing to the presence of a potentially virulent carrying strains, compared to adults [38, 39]. SCCmec methicillin resistant strain circulating in the community. type V, which comprised of 81.3% of the mecA positive This strain has been found to be pandemic, and mostly strains in our study, is known to be rare in Europe and PVL positive, also denoted ST30 by multilocus sequence the United States [33], and only recently seen in Greece typing. It has recently been mapped to have originated [40]. Because SCCmec type IV and V are known to be from Australia and disseminated to Brazil, United States, small and highly mobile elements, their dissemination in South Africa and Western Europe [3]. However, it is not a community population may be most commonly by related to the USA300, belonging to ST8 and leading cause transfers of strains from carriers to other individuals or of CA-MRSA in the USA. It has been revealed that PVL is from MRSA strains to Methicillin Sensitive Staphylococcus most frequent in pandemic CA-MRSA strains and certain aureus (MSSA strains), or even from coagulase-negative MSSA lineages, including ST30, appear to be a reservoir of staphylococci strain to an MSSA strain [41]. However, a CA-MRSA [6]. Surprisingly, ST30 has also been isolated recent study on patients with surgical site infections (SSI) from Babongo pygmies of Gabon in Africa, who are known Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 6 of 7 to have been separated from other humans over millennia Author details Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, ago [21]. In a study of MRSA in five African cities, only Via Olgettina 58, Milano, Italy. Universita Vita-Salute San Raffaele, Via one strain of this type was isolated from Antananarivo, 3 Olgettina 58, Milano, Italy. Department of Medical Microbiology, Makerere and none from Cassablanca (Moroco), Niamey (Niger), University College of Health Sciences, P. O. Box 7072, Kampala, Uganda. Dakar (Senegal) and Yaoundé (Cameroon). While SCCmec Received: 1 September 2016 Accepted: 14 December 2016 type V strains have been isolated from hospital settings in Uganda, none were Spa type t318 [22]. However, Spa type t645, the second most frequent type in our collection References (20.6%), also common in Western Europe and the Middle 1. Gorwitz RJ, Kruszon-Moran D, McAllister SK, McQuillan G, McDougal LK, East (http://spaserver.ridom.de), was found to be the most et al. Changes in the prevalence of nasal colonization with Staphylococcus aureus in the United States, 2001-2004. J Infect Dis. 2008;197:1226–34. frequent type isolated from SSI at Mulago National refer- 2. von Eiff C, Becker K, Machka K, Stammer H, Peters G, et al. Nasal carriage as a ral hospital [22], supporting the notion that there is a source of Staphylococcus aureus bacteremia. N Engl J Med. 2001;344:11–6. changing epidemiology reflected by community associated 3. DeLeo FR, Otto M, Kreiswirth BN, Chambers HF. Community-associated SCCmec genotypes being now more associated with hos- meticillin-resistant Staphylococcus aureus. Lancet. 2010;375:1557–68. 4. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, et al. pital infections as observed elsewhere [23, 45]. However, it Community-acquired methicillin-resistant Staphylococcus aureus in children is noteworthy that some spa types of PVL+ S. aureus in with no identified predisposing risk. JAMA. 1998;279:593–8. the present study (t186, t729 and t355) have been pre- 5. Loffler B, Hussain M, Grundmeier M, Bruck M, Holzinger D, et al. Staphylococcus aureus panton-valentine leukocidin is a very potent viously identified in studies from Africa, and they all cytotoxic factor for human neutrophils. PLoS Pathog. 2010;6:e1000715. share the MLST type ST88 [46]. 6. Rasigade JP, Laurent F, Lina G, Meugnier H, Bes M, et al. Global distribution and evolution of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus, 1981-2007. J Infect Dis. 2010;201:1589–97. Conclusion 7. Ito T, Okuma K, Ma XX, Yuzawa H, Hiramatsu K. Insights on antibiotic The main findings of this study are a high prevalence of resistance of Staphylococcus aureus from its whole genome: genomic CA-MRSA, and PVL-positive isolates with a predominant island SCC. Drug Resist Updat. 2003;6:41–52. 8. Ito T, Ma XX, Takeuchi F, Okuma K, Yuzawa H, et al. Novel type V spa type in a rural setting in Uganda. This has implication staphylococcal cassette chromosome mec driven by a novel cassette on future strategies for infection control in these under- chromosome recombinase, ccrC. Antimicrob Agents Chemother. 2004;48: privileged communities because of possibility of limited 2637–51. 9. Daum RS, Ito T, Hiramatsu K, Hussain F, Mongkolrattanothai K, et al. A novel treatment options. Antimicrobial stewardship programs methicillin-resistance cassette in community-acquired methicillin-resistant may be necessary in Uganda so as to create awareness and Staphylococcus aureus isolates of diverse genetic backgrounds. J Infect Dis. avert possible emergence multidrug resistant microor- 2002;186:1344–7. 10. Vandenesch F, Naimi T, Enright MC, Lina G, Nimmo GR, et al. Community- ganisms in the near future. acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence. Emerg Infect Dis. 2003;9:978–84. Acknowledgments 11. Shukla SK, Stemper ME, Ramaswamy SV, Conradt JM, Reich R, et al. The authors wish to thank the households that participated in the study. Molecular characteristics of nosocomial and Native American community- associated methicillin-resistant Staphylococcus aureus clones from rural Funding Wisconsin. J Clin Microbiol. 2004;42:3752–7. BBA was supported by a Marie Curie Fellowship (MARIE CURIE – COFUND) 12. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, et al. project No. 267264). Interpreting chromosomal DNA restriction patterns produced by pulsed- field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. Availability of data and materials 1995;33:2233–9. All data supporting the conclusions of this article are included within the article. 13. Frenay HM, Bunschoten AE, Schouls LM, van Leeuwen WJ, Vandenbroucke- Grauls CM, et al. Molecular typing of methicillin-resistant Staphylococcus Authors’ contributions aureus on the basis of protein A gene polymorphism. Eur J Clin Microbiol BBA collected samples and data, participated in performing all the laboratory Infect Dis. 1996;15:60–4. analyses and wrote the first draft of the manuscript; RB participated in analyzing 14. Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, et al. spa typing and interpreting PFGE data and critical revision of the manuscript; AT participated method for discriminating among Staphylococcus aureus isolates: in performing PFGE; DMC supervised the study and critically revised the implications for use of a single marker to detect genetic micro- and manuscript. All authors read and approved the final manuscript. macrovariation. J Clin Microbiol. 2004;42:792–9. 15. Shore AC, Coleman DC. Staphylococcal cassette chromosome mec: recent Competing interests advances and new insights. Int J Med Microbiol. 2013;303:350–9. The authors declare that they have no competing interests. 16. Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal Consent for publication cassette chromosome mec types I to V in methicillin-resistant Not applicable. Staphylococcus aureus. J Clin Microbiol. 2005;43:5026–33. Ethics approval and consent to participate 17. Ruimy R, Maiga A, Armand-Lefevre L, Maiga I, Diallo A, et al. The carriage The study protocol was approved by the Institutional Review Board of the population of Staphylococcus aureus from Mali is composed of a School of Biomedical Sciences, Makerere University College of Health Sciences; combination of pandemic clones and the divergent Panton-Valentine and by the Uganda National Council for Science and Technology. The study leukocidin-positive genotype ST152. J Bacteriol. 2008;190:3962–8. objective was explained in the local language and written informed consent 18. Ghebremedhin B, Olugbosi MO, Raji AM, Layer F, Bakare RA, et al. obtained from each participant before taking a sample. For participating minors Emergence of a community-associated methicillin-resistant Staphylococcus within recruited households, both assent from the minors as well as consent aureus strain with a unique resistance profile in Southwest Nigeria. J Clin from the parents were sought before a swab was taken. Microbiol. 2009;47:2975–80. Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 7 of 7 19. Ramdani-Bouguessa N, Bes M, Meugnier H, Forey F, Reverdy ME, et al. 40. Chini V, Petinaki E, Meugnier H, Foka A, Bes M, et al. Emergence of a new Detection of methicillin-resistant Staphylococcus aureus strains resistant to clone carrying Panton-Valentine leukocidin genes and staphylococcal multiple antibiotics and carrying the Panton-Valentine leukocidin genes in cassette chromosome mec type V among methicillin-resistant an Algiers hospital. Antimicrob Agents Chemother. 2006;50:1083–5. Staphylococcus aureus in Greece. Scand J Infect Dis. 2008;40:368–72. 20. Enany S, Yaoita E, Yoshida Y, Enany M, Yamamoto T. Molecular 41. Hallin M, Denis O, Deplano A, De Mendonca R, De Ryck R, et al. Genetic characterization of Panton-Valentine leukocidin-positive community- relatedness between methicillin-susceptible and methicillin-resistant acquired methicillin-resistant Staphylococcus aureus isolates in Egypt. Staphylococcus aureus: results of a national survey. J Antimicrob Microbiol Res. 2010;165:152–62. Chemother. 2007;59:465–72. 42. Adler A, Temper V, Block CS, Abramson N, Moses AE. Panton-Valentine 21. Schaumburg F, Kock R, Friedrich AW, Soulanoudjingar S, Ngoa UA, et al. leukocidin-producing Staphylococcus aureus. Emerg Infect Dis. 2006;12: Population structure of Staphylococcus aureus from remote African 1789–90. Babongo Pygmies. PLoS Negl Trop Dis. 2011;5:e1150. 43. Cocchi P, Taccetti G, Montagnani C, Campana S, Galli L, et al. Evidence of 22. Seni J, Bwanga F, Najjuka CF, Makobore P, Okee M, et al. Molecular transmission of a Panton-Valentine leukocidin-positive community-acquired characterization of Staphylococcus aureus from patients with surgical site methicillin-resistant Staphylococcus aureus clone: a family affair. Clin infections at Mulago Hospital in Kampala, Uganda. PLoS One. 2013;8:e66153. Microbiol Infect. 2013;19:1158–62. 23. Kateete DP, Namazzi S, Okee M, Okeng A, Baluku H, et al. High prevalence 44. Chini V, Petinaki E, Foka A, Paratiras S, Dimitracopoulos G, et al. Spread of of methicillin resistant Staphylococcus aureus in the surgical units of Staphylococcus aureus clinical isolates carrying Panton-Valentine leukocidin Mulago hospital in Kampala, Uganda. BMC Res Notes. 2011;4:326. genes during a 3-year period in Greece. Clin Microbiol Infect. 2006;12:29–34. 24. Ojulong J, Mwambu TP, Joloba M, Bwanga F, Kaddu-Mulindwa DH. Relative 45. Shittu AO, Okon K, Adesida S, Oyedara O, Witte W, et al. Antibiotic prevalence of methicilline resistant Staphylococcus aureus and its resistance and molecular epidemiology of Staphylococcus aureus in Nigeria. susceptibility pattern in Mulago Hospital, Kampala, Uganda. Tanzan J Health BMC Microbiol. 2011;11:92. Res. 2009;11:149–53. 46. Schaumburg F, Pauly M, Anoh E, Mossoun A, Wiersma L, et al. 25. Iramiot S, Bwanga F, Itabangi H, Nakaye M, Bashir M, et al. Prevalence and Staphylococcus aureus complex from animals and humans in three remote antibiotic susceptibility patterns of clinical isolates of Methicillin-resistant African regions. Clin Microbiol Infect. 2015;21(345):e341–8. staphylococcus aureus in a tertiary care Hospital in Western Uganda. British Microbiol Res J. 2014;4:1168–77. 26. Kateete DP, Kabugo U, Baluku H, Nyakarahuka L, Kyobe S, et al. Prevalence and antimicrobial susceptibility patterns of bacteria from milkmen and cows with clinical mastitis in and around Kampala, Uganda. PLoS One. 2013;8:e63413. 27. Kasozi KI, Tingiira JB, Vudriko P. High prevalence of subclinical mastitis and multidrug resistant staphylococcus aureus are a threat to dairy cattle production in Kiboga District (Uganda). Open J Vet Med. 2014;4:35–43. 28. Cheesbrough M. District Laboratory Practice in Tropical Countries, Part 2. 2nd ed. 2005. p. 62–70. 132-143, 157-234. 29. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO, et al. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis. 1999;29:1128–32. 30. Baldan R, Tassan Din C, Semeraro G, Costa C, Cichero P, et al. Severe community-onset infections in healthy individuals caused by community- acquired MRSA in an Italian teaching hospital, 2006-2008. J Hosp Infect. 2009;72:271–3. 31. Mellmann A, Friedrich AW, Rosenkotter N, Rothganger J, Karch H, et al. Automated DNA sequence-based early warning system for the detection of methicillin-resistant Staphylococcus aureus outbreaks. PLoS Med. 2006;3:e33. 32. Naimi TS, LeDell KH, Como-Sabetti K, Borchardt SM, Boxrud DJ, et al. Comparison of community- and health care-associated methicillin-resistant Staphylococcus aureus infection. JAMA. 2003;290:2976–84. 33. David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev. 2010;23:616–87. 34. Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis. 2005;5:751–62. 35. McNally LM, Jeena PM, Gajee K, Sturm AW, Tomkins AM, et al. Lack of association between the nasopharyngeal carriage of Streptococcus pneumoniae and Staphylococcus aureus in HIV-1-infected South African children. J Infect Dis. 2006;194:385–90. 36. Tong SY, McDonald MI, Holt DC, Currie BJ. Global implications of the Submit your next manuscript to BioMed Central emergence of community-associated methicillin-resistant Staphylococcus aureus in Indigenous populations. Clin Infect Dis. 2008;46:1871–8. and we will help you at every step: 37. Vlack S, Cox L, Peleg AY, Canuto C, Stewart C, et al. Carriage of methicillin- • We accept pre-submission inquiries resistant Staphylococcus aureus in a Queensland Indigenous community. Med J Aust. 2006;184:556–9. � Our selector tool helps you to find the most relevant journal 38. David MZ, Glikman D, Crawford SE, Peng J, King KJ, et al. What is � We provide round the clock customer support community-associated methicillin-resistant Staphylococcus aureus? J Infect � Convenient online submission Dis. 2008;197:1235–43. 39. David MZ, Crawford SE, Boyle-Vavra S, Hostetler MA, Kim DC, et al. � Thorough peer review Contrasting pediatric and adult methicillin-resistant Staphylococcus aureus � Inclusion in PubMed and all major indexing services isolates. Emerg Infect Dis. 2006;12:631–7. � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Infectious Diseases Springer Journals

Molecular epidemiology of Panton-Valentine Leukocidin-positive community-acquired methicillin resistant Staphylococcus aureus isolates in pastoral communities of rural south western Uganda

Loading next page...
 
/lp/springer-journals/molecular-epidemiology-of-panton-valentine-leukocidin-positive-vRFg9TgDFK

References (50)

Publisher
Springer Journals
Copyright
Copyright © 2017 by The Author(s).
Subject
Medicine & Public Health; Infectious Diseases; Parasitology; Medical Microbiology; Tropical Medicine; Internal Medicine
eISSN
1471-2334
DOI
10.1186/s12879-016-2124-8
pmid
28056833
Publisher site
See Article on Publisher Site

Abstract

Background: The emergence of multidrug resistant Staphylococcus aureus strains, including methicillin resistant (MRSA), is a global concern. Treatment of bacterial infections in Uganda’s health care settings is largely empirical, rarely accompanied by laboratory confirmation. Here we show the burden, characteristics of MRSA and epidemiology of Panton-Valentine Leukocidin (PVL) positive strains in asymptomatic carriers in pastoral households of south-west Uganda. Methods: Nasal swabs from 253 participants were cultured following standard methodology. MRSA strains were identified by detection of the mecA gene and SCCmec typing, and PVL genes detected by PCR. Pulsed Field Gel Electrophoresis (PFGE) was done to evaluate possible transmission patterns. Spa typing of PVL positive isolates was done to study the epidemiology of virulent strains in this setting. Results: S. aureus was isolated in 29% (n=73) of theparticipants, of which48wereMRSAby mecA typing. PVL-encoding genes were found in 49.3% (n=36) of the73isolates, of which25werealso mecA positive. Among the PVL negative strains (n = 37), 62.2% (n = 23) carried the mecA gene. The most common SCCmec type was V, detected in 39 (18 PVL positive and 21 PVL negative) isolates. PFGE clustered 21/36 (58.3%) PVL positive isolates divided in four pulsotypes and 18/37 (48.6%) PVL negative isolates divided in eight pulsotypes. The most prevalent Spa types were t318 (26.5%, n = 9) and t645 (20.6%, n = 7); while other common Spa types were t11656 (n =3), t127 (n = 3) and t355 (n =3). Conclusion: The study shows a high prevalence of community acquired (CA)-MRSA, and PVL-positive isolates with two predominant spa types in rural Uganda, further complicating infection control strategies in these underprivileged communities. * Correspondence: basiimwe@chs.mak.ac.ug Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, Milano, Italy Universita Vita-Salute San Raffaele, Via Olgettina 58, Milano, Italy Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 2 of 7 Background In African countries, CA-MRSA has been reported in About a third of healthy individuals in the community a few studies in Mali [17], Nigeria [18], Algeria [19], are asymptomatically colonized with Staphylococcus Egypt [20] and Gabon [21]. In Uganda, a few studies aures (S. aureus) in the nostrils [1], a very important have been done at tertiary health care centers [22–25], finding considering the fact that nasal carriage of S. aureus and these have focused on HA-MRSA. Community studies has been associated with subsequent infection [2], and have focused on S. aureus carriage in raw milk and its carriers are an important source of spread of infection in products as well as in urban milk vendors [26, 27]. We set communities. A major concern is the world-wide emer- out to establish the prevalence and molecular epidemiology gence of methicillin resistant S. aureus (MRSA) in the of PVL-positive CA-MRSA isolates from pastoral commu- community [3]. In contrast with health care associated nities of rural south-western Uganda so as to inform public MRSA (HA-MRSA) infections, community associated health on how to develop effective strategies to prevent MRSA (CA-MRSA) infections can occur in healthy indi- spread MRSA in these and surrounding communities. viduals [4], suggesting that these strains have greater viru- lence. Skin and soft-tissue infections represent about 90% Methods of cases of CA-MRSA infection, mostly characterized by Study area abscesses or cellulitis with purulent drainage [3]. The study was carried out in rural pastoral households A predominant feature of CA-MRSA is the presence of Kiruhura district, South Western Uganda. The area is of the Panton-Valentine Leukocidin (PVL) genes that en- a rangeland, populated by agro-pastoralists and season- code a S. aureus exotoxin that induces lysis of mono- ally itinerant semi-nomads whose livelihoods mainly de- cytes and neutrophil granulocytes [5]. Additionally, there pend on consumption and trade of cattle and their is evidence that PVL-positive S. aureus strains susceptible products; and small subsistent crop farming. to methicillin (MSSA) may be reservoirs for the develop- ment of PVL-positive MRSA via the integration of the Study design and sampling staphylococcal cassette chromosome mec (SCCmec)ele- A cross-sectional study of S. aureus carriage in the area ments including the mecA gene conferring methicillin re- was conducted as part of a zoonotic diseases study sistance [6]. Another key feature of CA-MRSA is that the among pastoral communities of Kiruhura district, South strains mainly harbor SCCmec types IV and V [7–9], and Western Uganda, in 2013. A total of 196 homesteads a relationship between CA-MRSA, SCCmec type IV and V from two sub-counties (100 from Kanyaryeru and 96 and PVL has been confirmed in some studies [10, 11]. from Sanga) were recruited and sampled. The sub- While efforts have been made to map out the geo- counties were purposively selected by the zoonoses study graphical predominance and spread of different CA- because they bordered Lake Mburo National Park, with MRSA clones worldwide [3], little is known about its a porous human-animal interface. All participants did magnitude and genetic composition in developing coun- not have a history of recent hospitalization, and the only tries, especially in Africa. Molecular analytical tech- demographics characteristics recorded were sex and self- niques such as pulsed field gel electrophoresis (PFGE), reported age. In each household, only a nasal swab was multilocus sequence typing (MLST), spa and SCCmec taken for any participant and these were inoculated into typing have been used to show both the spread and evolu- Brain-Heart Infusion (BHI) broth in 15 ml propylene tion of MRSA. PFGE is still considered the gold standard tubes and stored on ice before same day transportation for typing MRSA isolates, and is one of the most discrim- to the Department of Microbiology at Mbarara University inative typing methods [12]. While MLST is an excellent of Science and Technology for culture. tool for investigating clonal evolution of MRSA, it is ra- ther expensive, labour intensive and time consuming. Se- Sample culture, isolation and identification quences from the polymorphic region of the S. aureus Initial bacterial culture and isolation was done according protein A (spa) gene have been used to develop a single- to methods described by Cheesbrough [28], with minor locus sequence typing technique for MRSA [13] with a modifications. Briefly, 50 μl of culture broth was inocu- discriminatory power between PFGE and MLST, and lated on 5% sheep blood agar medium and incubated for ability to investigate both molecular evolution and out- 18–20 h at 37 °C. Preliminary identification of the bac- break situations [14], while remaining simple. teria was carried out based on colony characteristics The resistance of S. aureus to methicillin is caused by such as shape, size, color and hemolysis patterns. Iso- the mecA gene, located on a mobile genetic element, the lates suspected of being S. aureus were shipped in BHI staphylococcal cassette chromosome mec (SCCmec) [7]. with 20% glycerol to the Emerging Bacterial Pathogens Currently, eleven main SCCmec types (I to XI) are Unit (EBPU) at San Raffaele Scientific Institute in Milan, known [15], and Zhang et al. developed a multiplex PCR Italy, for identification and molecular characterization. for the characterization of SCCmec type I to V [16]. At the EBPU, all isolates were sub-cultured on mannitol Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 3 of 7 salt agar (Oxoid Ltd, Hampshire, England), then on blood 65.8% (n=48) of the73isolates. Theproportionof agar (Becton Dickinson, Heidelberg, Germany), and sub- PVL positive isolates carrying the mecA gene was 25/36 jected to the tube coagulase test in rabbit plasma with (69.4%) (Table 1), while 23/37 (62.2%) of the isolates EDTA (Remel, KS, USA). MRSA strains were identified by without the PVL gene possessed the mecA (Table 2). detection of the mecA gene and SCCmec typing. Among the PVL positive isolates, 18 of the 25 mecA DNA extraction, SCCmec typing and PVL genes detection Bacterial DNA was prepared from isolated colonies sus- Table 1 PFGE profiles, Spa types, mecA, and SCCmec types of pended in 500 μl Triton X-100 lysis buffer with 1% Triton PVL positive isolates and 20 μl of 1 mg/ml lysostaphin, incubated at 37 °C for Isolate ID Pulsotype Spa type mecA SCCmec 1 h, followed by phenol-chloroform extraction. SCCmec T095Na A1 NT + V typing was done by multiplex PCR using primers and pro- T101Na A1 NT + IVc tocols for SCCmec types and subtypes I, II, III, IVa, IVb, T047Na A1 t127 + IVc IVc, IVd, and V and the mecA gene according to Zhang T070Na A1 t127 + IVc et al. [16]. The MRSA strain COL (SCCmec type I, mec gene complex B and ccr gene complex 1) was used as T056N A2 t318 - - positive control. Additionally, amplification of the T099Nd A3 t2393 + IVc Panton – Valentine Leucocidin (PVL)toxin genes, lukS- T149Na A4 t645 + IVc PV and lukF-PV, was performed using the S. aureus T101Ng A5 t318 + V strain ATCC49775 as positive control, and primers and T005Na B1 t318 + V protocols as described by Lina et al. [29]. T021Nb B1 t318 + V Genotyping of isolates T021Nc B1 t318 + V All isolates were subjected to molecular epidemiological T097Na B1 t318 + V analysis by PFGE after SmaI digestion using the CHEF T125Na B1 t318 + V Genomic DNA Plug kit (Bio-Rad) according to a stan- T141Nc B1 t318 + V dardized protocol [30]. PFGE was run using a CHEF T047Nd B1 t186 + V DRIII system (Bio-Rad). The InfoQuest FP (v5.1) soft- T156N B1 t186 + V ware (Bio-Rad Laboratories) was used to analyze PFGE profiles, according to interpretation criteria described by T019N B2 t186 + V Tenover et al. [12]. Clustering analysis was achieved T119Nb C1 t355 + V using Dice similarity coefficients and the unweighted T135Na C2 t729 - - pair group method with averages (UPGMA) at 1.5% T132Nb C2 t11656 - - optimization and 1.5% position tolerance. PVL-positive T139N C2 t11656 + V strains were further subjected to spa-typing according to T145N C2 t11656 + V Frenay et al. [13]; and spa types were assigned on the Ridom SpaServer (http://spaserver.ridom.de) curated by T022N C2 t645 - - the SeqNet.org initiative [31]. T041Na C2 t645 - - T099Nb C2 t645 + V Results T063N C3 t355 + V Socio-demographic characteristics and S. aureus carriage T053N D1 t1376 + IVc The study sampled 196 households, and nasal swabs T055N D1 t1376 + V were taken from 253 participants (172 males and 81 females) with average age of 14 years and a range of 6 to T039Na E1 t318 + IVc 48 years. Of the samples, 132 (52.2%) were Staphylococcus T041Nb E2 t645 + V spp, of which only 73 were S. aureus (carriage rate of 29%) T025N E3 t2393 + IVc and further characterized. The 73 isolates were obtained T030Nb F1 t064 + V from 48 (65.7%) males and 25 (34.3%) females. The aver- T158Nb G1 t127 - - age age of the 73 participants whose isolates were charac- T087 H1 t064 + V terized was 13, with a range of 6 to 45 years. T155Nd H2 t509 - - Prevalence of PVL and mecA genes T097Nb I1 t002 - - PVL-encoding genes were found in 49.3% (n = 36) of ID identification number of the isolate, + positive on PCR amplification, - negative the 73 S. aureus isolates, while mecA was detected in on PCR amplification, NT not typable Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 4 of 7 Table 2 PFGE profiles, mecA, and SCCmec types of PVL Molecular epidemiology of MRSA PVL positive and PVL negative isolates negative strains Isolate ID Pulsotype Spa type mecA SCCmec PFGE profiles of PVL positive and negative isolates were analyzed separately to understand any difference in T117Nb E1 t786 - - transmission within the two groups. Among the PVL T119Na E1 t786 - - positive isolates, 21/36 (58.3%) strains were found in T092Na E2 t186 - - four clusters (A1, B1, C2, D1, Table 1); while in PVL T058Nb E4 t7662 - - negative isolates, 18/37 (48.6%) were found in eight clus- T061Nd L1 t1236 + V ters (E1, L1, L3, F1, N1, H1, G1, Q1, Table 2). Some T061Nb L1 t1236 + V family members carried identical strains. In order to re- late the genotypes of the isolates in this study to global T059Na L2 Unknown + V epidemiology of S. aureus, we performed Spa typing of T059Nb L3 Unknown + V the PVL positive isolates. Only two of the 36 isolates T099Na L3 Unknown + V could not be typed by this technique, but they belonged T021Nd L4 Unknown + V to the same pulsotype (A1). In all, there were 13 Spa T032Nc F1 t064 - - types among the 34 typable PVL positive S. aureus iso- T131Nb F1 t064 - - lates. The most prevalent Spa types were t318 compris- ing of 26.5% (n = 9), of which seven isolates carried the T032Nb F1 T064 - - mecA gene; and t645 (20.6%, n = 7). The other common T061Nc N1 t2771 + V Spa types were and t11656 (8.8%, n = 3) and t127 (n = 3). T074Nc N1 t2771 + V The other Spa types, their frequencies and PFGE profiles T074Nb N1 t2771 + V can be seen in Table 1. T029Nb H1 t002 - - T101Nf H1 t002 + V Discussion T076N G1 t5739 + V Knowledge of the epidemiology of bacterial infections is T128Nd G1 t2680 + V very important for appropriate decision-making in the T018Nb G2 t616 + V treatment of arising infections. At a community level, it T092Nb P1 t4353 + V is also important to curb the spread of infection, in- T102Nb P2 t4353 - - cluding multidrug resistant strains. To our knowledge, T127Na Q1 Unknown - - this is the first investigation of Panton-Valentine Leukocidin-Positive CA-MRSA in asymptomatic semi- T137Ng Q1 t064 - - nomadic pastoralist communities in the East African T110Nb R1 t4523 - - region. The main findings of the study are a high preva- T111Na R2 t5187 + V lence of MRSA and PVL-positive isolates with a pre- T027Nb R3 t5187 + V dominant spa type (t318). T004Nb Unique t3772 + V This study isolated 132 Staphylococci isolates from the T033Nb Unique Unknown + V nares of 253 participants, of which 73 were coagulase positive S. aureus (carriage rate of 29%). The 73 isolates T005Nb Unique t951 + V of S. aureus in our study were obtained from 48 male T114Ng Unique Unknown - - and 25 female participants, with an average age of T151Nb Unique t5739 + V 13 years, with 56/73 (76. 7%) of the participants being T115Na Unique Unknown + IVa between 7 and 15 years old. This is similar to observa- T120N Unique t5187 + V tions elsewhere that CA-MRSA infections tend to occur T126N Unique NT + IVa in previously healthy younger patients [21, 32, 33]. Our finding is of public health importance because this T104Nb Unique Unknown - - school-going age group has potential of disseminating ID identification number of the isolate the strains far and wide in the communities. The car- riage rate in our study is more than double that in a study on milk men in and around Kampala city, Uganda, positive isolates were SCCmec type V, while the other where only 11 Staphylococci were isolated from 31 indi- seven were all SCCmec IVc. Among the PVL negative viduals, of which only 4/31 (13%) were S. aureus [26]. isolates, 21 of the 23 mecA positive isolates were Our finding, however, is in agreement with statistics SCCmec type V, while the other two were type IVa. from literature suggesting that between 25 to 35% of Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 5 of 7 healthy individuals are asymptomatically colonized with at the National Referral hospital in Mulago, Kampala, S. aureus in the nostrils [1, 21, 34, 35]. showed that SCCmec type V was the most predominant In the current study, the proportion of S. aureus iso- type [22], suggesting the presence of mixed CA-MRSA lates carrying the mecA gene, hence MRSA, was 48/73 and HA-MRSA genotypes in hospital settings in Uganda. (65.8%). This is generally high in comparison to commu- A further characterization of the 36 isolates carrying nity studies elsewhere. In the urban and peri-urban the PVL gene in this study was done. SCCmecIV and Kampala, the four isolates from milk men were all PVL are known to be molecular markers associated with MSSA. In a study of indigenous remote African Babongo the emergence of CA-MRSA worldwide [10]. The pro- pygmies living in Waka National Parc, Central Gabon, portion of strains carrying both the PVL and mecA all 34 S. aureus isolates were susceptible to Oxacillin/ genes was 25/36 (69.4%), while the mecA gene was de- Methicillin, and did not amplify for the mecA gene by tected in 23/37 (62.2%) of the isolates without the PVL PCR. The authors hypothesized that the result could be gene. While there was no significant difference in mecA due to limited use of antibiotics in that population. Stud- carriage, PFGE profiles for both PVL positive and nega- ies in North America and Australia, however, have tive isolates show that there were bigger clusters in PVL shown that native and indigenous populations have been positive strains (Table 1) compared to PVL negative associated with a high risk of colonization and infection strains (Table 2). In fact, 18/37 (48.6%) of the PVL with CA-MRSA which may be related to many of these negative isolates were in clusters (Table 1) compared to groups being disadvantaged, due to their association 21/36 (58.3%) of PVL positive isolates, with the largest with low socio-economic status, crowded living condi- (B1) comprising of eight isolates (Table 1). These re- tions and frequent use of antibiotics [36, 37]. There have sults suggest that PVL positive isolates in the study been reports of absolute resistance to penicillin and high were more likely to be involved in chains of transmis- percentage resistance to other antibiotics in milk from sion compared to the PVL negative isolates. There has similar settings in central Uganda, hence risk of spread been recent evidence of familial spread of PVL carrying to humans through the food chain [27]. In our study MSSA strains in Israel [42] and MRSA strains in Italy community there is frequent usage, by farmers, of veteri- [43], while a previous study in Greece revealed that a nary antibiotics to treat nearly all ailments in their live- unique clone of PVL-positive MRSA had spread in both stock, due to poor outreach services by the veterinary the community and hospital settings, and was replacing department. It is therefore more likely that constant older clonal types [44]. In Central Gabon, Africa, PVL- contact with antibiotics for animal use, as well as encoding genes were detected in 55.9% of study iso- consumption of raw milk and its products without ob- lates, with authors concluding that the pygmies in that serving drug withdrawal periods, as is the culture in this study faced a risk of developing necrotizing infections, setting, are limiting future options for the management due to the virulence characteristic of the PVL. The of multidrug resistant microorganisms in both humans finding of a high proportion of isolates carrying both and animals in pastoral communities of Uganda. Among the PVL and mecA genes in our study may have the 48 isolates carrying the mecA gene, 39 (81.3%) were considerable implications on future strategies for infec- type V, the other being type IV or its subtypes (Tables 1 tion control in these underprivileged communities. and 2). Type IV and V SCCmec elements have strongly Amongthe PVLpositiveisolates, therewas apredomi- been associated with stains causing MRSA infections in nant circulating Spa type, t318, comprising of nine of the persons with no history of hospitalization, hence thought 34 (26.5%) typable isolates. This is the first time the Spa to be more related to CA-MRSA [38]. Furthermore, it type has been identified in a Ugandan setting. Moreover, has been shown that children may be at a higher risk of seven strains (77.8%) of this Spa type carried the mecA infection with SCCmec types IV and V, as well as PVL gene, pointing to the presence of a potentially virulent carrying strains, compared to adults [38, 39]. SCCmec methicillin resistant strain circulating in the community. type V, which comprised of 81.3% of the mecA positive This strain has been found to be pandemic, and mostly strains in our study, is known to be rare in Europe and PVL positive, also denoted ST30 by multilocus sequence the United States [33], and only recently seen in Greece typing. It has recently been mapped to have originated [40]. Because SCCmec type IV and V are known to be from Australia and disseminated to Brazil, United States, small and highly mobile elements, their dissemination in South Africa and Western Europe [3]. However, it is not a community population may be most commonly by related to the USA300, belonging to ST8 and leading cause transfers of strains from carriers to other individuals or of CA-MRSA in the USA. It has been revealed that PVL is from MRSA strains to Methicillin Sensitive Staphylococcus most frequent in pandemic CA-MRSA strains and certain aureus (MSSA strains), or even from coagulase-negative MSSA lineages, including ST30, appear to be a reservoir of staphylococci strain to an MSSA strain [41]. However, a CA-MRSA [6]. Surprisingly, ST30 has also been isolated recent study on patients with surgical site infections (SSI) from Babongo pygmies of Gabon in Africa, who are known Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 6 of 7 to have been separated from other humans over millennia Author details Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, ago [21]. In a study of MRSA in five African cities, only Via Olgettina 58, Milano, Italy. Universita Vita-Salute San Raffaele, Via one strain of this type was isolated from Antananarivo, 3 Olgettina 58, Milano, Italy. Department of Medical Microbiology, Makerere and none from Cassablanca (Moroco), Niamey (Niger), University College of Health Sciences, P. O. Box 7072, Kampala, Uganda. Dakar (Senegal) and Yaoundé (Cameroon). While SCCmec Received: 1 September 2016 Accepted: 14 December 2016 type V strains have been isolated from hospital settings in Uganda, none were Spa type t318 [22]. However, Spa type t645, the second most frequent type in our collection References (20.6%), also common in Western Europe and the Middle 1. Gorwitz RJ, Kruszon-Moran D, McAllister SK, McQuillan G, McDougal LK, East (http://spaserver.ridom.de), was found to be the most et al. Changes in the prevalence of nasal colonization with Staphylococcus aureus in the United States, 2001-2004. J Infect Dis. 2008;197:1226–34. frequent type isolated from SSI at Mulago National refer- 2. von Eiff C, Becker K, Machka K, Stammer H, Peters G, et al. Nasal carriage as a ral hospital [22], supporting the notion that there is a source of Staphylococcus aureus bacteremia. N Engl J Med. 2001;344:11–6. changing epidemiology reflected by community associated 3. DeLeo FR, Otto M, Kreiswirth BN, Chambers HF. Community-associated SCCmec genotypes being now more associated with hos- meticillin-resistant Staphylococcus aureus. Lancet. 2010;375:1557–68. 4. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, et al. pital infections as observed elsewhere [23, 45]. However, it Community-acquired methicillin-resistant Staphylococcus aureus in children is noteworthy that some spa types of PVL+ S. aureus in with no identified predisposing risk. JAMA. 1998;279:593–8. the present study (t186, t729 and t355) have been pre- 5. Loffler B, Hussain M, Grundmeier M, Bruck M, Holzinger D, et al. Staphylococcus aureus panton-valentine leukocidin is a very potent viously identified in studies from Africa, and they all cytotoxic factor for human neutrophils. PLoS Pathog. 2010;6:e1000715. share the MLST type ST88 [46]. 6. Rasigade JP, Laurent F, Lina G, Meugnier H, Bes M, et al. Global distribution and evolution of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus, 1981-2007. J Infect Dis. 2010;201:1589–97. Conclusion 7. Ito T, Okuma K, Ma XX, Yuzawa H, Hiramatsu K. Insights on antibiotic The main findings of this study are a high prevalence of resistance of Staphylococcus aureus from its whole genome: genomic CA-MRSA, and PVL-positive isolates with a predominant island SCC. Drug Resist Updat. 2003;6:41–52. 8. Ito T, Ma XX, Takeuchi F, Okuma K, Yuzawa H, et al. Novel type V spa type in a rural setting in Uganda. This has implication staphylococcal cassette chromosome mec driven by a novel cassette on future strategies for infection control in these under- chromosome recombinase, ccrC. Antimicrob Agents Chemother. 2004;48: privileged communities because of possibility of limited 2637–51. 9. Daum RS, Ito T, Hiramatsu K, Hussain F, Mongkolrattanothai K, et al. A novel treatment options. Antimicrobial stewardship programs methicillin-resistance cassette in community-acquired methicillin-resistant may be necessary in Uganda so as to create awareness and Staphylococcus aureus isolates of diverse genetic backgrounds. J Infect Dis. avert possible emergence multidrug resistant microor- 2002;186:1344–7. 10. Vandenesch F, Naimi T, Enright MC, Lina G, Nimmo GR, et al. Community- ganisms in the near future. acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence. Emerg Infect Dis. 2003;9:978–84. Acknowledgments 11. Shukla SK, Stemper ME, Ramaswamy SV, Conradt JM, Reich R, et al. The authors wish to thank the households that participated in the study. Molecular characteristics of nosocomial and Native American community- associated methicillin-resistant Staphylococcus aureus clones from rural Funding Wisconsin. J Clin Microbiol. 2004;42:3752–7. BBA was supported by a Marie Curie Fellowship (MARIE CURIE – COFUND) 12. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, et al. project No. 267264). Interpreting chromosomal DNA restriction patterns produced by pulsed- field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. Availability of data and materials 1995;33:2233–9. All data supporting the conclusions of this article are included within the article. 13. Frenay HM, Bunschoten AE, Schouls LM, van Leeuwen WJ, Vandenbroucke- Grauls CM, et al. Molecular typing of methicillin-resistant Staphylococcus Authors’ contributions aureus on the basis of protein A gene polymorphism. Eur J Clin Microbiol BBA collected samples and data, participated in performing all the laboratory Infect Dis. 1996;15:60–4. analyses and wrote the first draft of the manuscript; RB participated in analyzing 14. Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, et al. spa typing and interpreting PFGE data and critical revision of the manuscript; AT participated method for discriminating among Staphylococcus aureus isolates: in performing PFGE; DMC supervised the study and critically revised the implications for use of a single marker to detect genetic micro- and manuscript. All authors read and approved the final manuscript. macrovariation. J Clin Microbiol. 2004;42:792–9. 15. Shore AC, Coleman DC. Staphylococcal cassette chromosome mec: recent Competing interests advances and new insights. Int J Med Microbiol. 2013;303:350–9. The authors declare that they have no competing interests. 16. Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal Consent for publication cassette chromosome mec types I to V in methicillin-resistant Not applicable. Staphylococcus aureus. J Clin Microbiol. 2005;43:5026–33. Ethics approval and consent to participate 17. Ruimy R, Maiga A, Armand-Lefevre L, Maiga I, Diallo A, et al. The carriage The study protocol was approved by the Institutional Review Board of the population of Staphylococcus aureus from Mali is composed of a School of Biomedical Sciences, Makerere University College of Health Sciences; combination of pandemic clones and the divergent Panton-Valentine and by the Uganda National Council for Science and Technology. The study leukocidin-positive genotype ST152. J Bacteriol. 2008;190:3962–8. objective was explained in the local language and written informed consent 18. Ghebremedhin B, Olugbosi MO, Raji AM, Layer F, Bakare RA, et al. obtained from each participant before taking a sample. For participating minors Emergence of a community-associated methicillin-resistant Staphylococcus within recruited households, both assent from the minors as well as consent aureus strain with a unique resistance profile in Southwest Nigeria. J Clin from the parents were sought before a swab was taken. Microbiol. 2009;47:2975–80. Asiimwe et al. BMC Infectious Diseases (2017) 17:24 Page 7 of 7 19. Ramdani-Bouguessa N, Bes M, Meugnier H, Forey F, Reverdy ME, et al. 40. Chini V, Petinaki E, Meugnier H, Foka A, Bes M, et al. Emergence of a new Detection of methicillin-resistant Staphylococcus aureus strains resistant to clone carrying Panton-Valentine leukocidin genes and staphylococcal multiple antibiotics and carrying the Panton-Valentine leukocidin genes in cassette chromosome mec type V among methicillin-resistant an Algiers hospital. Antimicrob Agents Chemother. 2006;50:1083–5. Staphylococcus aureus in Greece. Scand J Infect Dis. 2008;40:368–72. 20. Enany S, Yaoita E, Yoshida Y, Enany M, Yamamoto T. Molecular 41. Hallin M, Denis O, Deplano A, De Mendonca R, De Ryck R, et al. Genetic characterization of Panton-Valentine leukocidin-positive community- relatedness between methicillin-susceptible and methicillin-resistant acquired methicillin-resistant Staphylococcus aureus isolates in Egypt. Staphylococcus aureus: results of a national survey. J Antimicrob Microbiol Res. 2010;165:152–62. Chemother. 2007;59:465–72. 42. Adler A, Temper V, Block CS, Abramson N, Moses AE. Panton-Valentine 21. Schaumburg F, Kock R, Friedrich AW, Soulanoudjingar S, Ngoa UA, et al. leukocidin-producing Staphylococcus aureus. Emerg Infect Dis. 2006;12: Population structure of Staphylococcus aureus from remote African 1789–90. Babongo Pygmies. PLoS Negl Trop Dis. 2011;5:e1150. 43. Cocchi P, Taccetti G, Montagnani C, Campana S, Galli L, et al. Evidence of 22. Seni J, Bwanga F, Najjuka CF, Makobore P, Okee M, et al. Molecular transmission of a Panton-Valentine leukocidin-positive community-acquired characterization of Staphylococcus aureus from patients with surgical site methicillin-resistant Staphylococcus aureus clone: a family affair. Clin infections at Mulago Hospital in Kampala, Uganda. PLoS One. 2013;8:e66153. Microbiol Infect. 2013;19:1158–62. 23. Kateete DP, Namazzi S, Okee M, Okeng A, Baluku H, et al. High prevalence 44. Chini V, Petinaki E, Foka A, Paratiras S, Dimitracopoulos G, et al. Spread of of methicillin resistant Staphylococcus aureus in the surgical units of Staphylococcus aureus clinical isolates carrying Panton-Valentine leukocidin Mulago hospital in Kampala, Uganda. BMC Res Notes. 2011;4:326. genes during a 3-year period in Greece. Clin Microbiol Infect. 2006;12:29–34. 24. Ojulong J, Mwambu TP, Joloba M, Bwanga F, Kaddu-Mulindwa DH. Relative 45. Shittu AO, Okon K, Adesida S, Oyedara O, Witte W, et al. Antibiotic prevalence of methicilline resistant Staphylococcus aureus and its resistance and molecular epidemiology of Staphylococcus aureus in Nigeria. susceptibility pattern in Mulago Hospital, Kampala, Uganda. Tanzan J Health BMC Microbiol. 2011;11:92. Res. 2009;11:149–53. 46. Schaumburg F, Pauly M, Anoh E, Mossoun A, Wiersma L, et al. 25. Iramiot S, Bwanga F, Itabangi H, Nakaye M, Bashir M, et al. Prevalence and Staphylococcus aureus complex from animals and humans in three remote antibiotic susceptibility patterns of clinical isolates of Methicillin-resistant African regions. Clin Microbiol Infect. 2015;21(345):e341–8. staphylococcus aureus in a tertiary care Hospital in Western Uganda. British Microbiol Res J. 2014;4:1168–77. 26. Kateete DP, Kabugo U, Baluku H, Nyakarahuka L, Kyobe S, et al. Prevalence and antimicrobial susceptibility patterns of bacteria from milkmen and cows with clinical mastitis in and around Kampala, Uganda. PLoS One. 2013;8:e63413. 27. Kasozi KI, Tingiira JB, Vudriko P. High prevalence of subclinical mastitis and multidrug resistant staphylococcus aureus are a threat to dairy cattle production in Kiboga District (Uganda). Open J Vet Med. 2014;4:35–43. 28. Cheesbrough M. District Laboratory Practice in Tropical Countries, Part 2. 2nd ed. 2005. p. 62–70. 132-143, 157-234. 29. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO, et al. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis. 1999;29:1128–32. 30. Baldan R, Tassan Din C, Semeraro G, Costa C, Cichero P, et al. Severe community-onset infections in healthy individuals caused by community- acquired MRSA in an Italian teaching hospital, 2006-2008. J Hosp Infect. 2009;72:271–3. 31. Mellmann A, Friedrich AW, Rosenkotter N, Rothganger J, Karch H, et al. Automated DNA sequence-based early warning system for the detection of methicillin-resistant Staphylococcus aureus outbreaks. PLoS Med. 2006;3:e33. 32. Naimi TS, LeDell KH, Como-Sabetti K, Borchardt SM, Boxrud DJ, et al. Comparison of community- and health care-associated methicillin-resistant Staphylococcus aureus infection. JAMA. 2003;290:2976–84. 33. David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev. 2010;23:616–87. 34. Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis. 2005;5:751–62. 35. McNally LM, Jeena PM, Gajee K, Sturm AW, Tomkins AM, et al. Lack of association between the nasopharyngeal carriage of Streptococcus pneumoniae and Staphylococcus aureus in HIV-1-infected South African children. J Infect Dis. 2006;194:385–90. 36. Tong SY, McDonald MI, Holt DC, Currie BJ. Global implications of the Submit your next manuscript to BioMed Central emergence of community-associated methicillin-resistant Staphylococcus aureus in Indigenous populations. Clin Infect Dis. 2008;46:1871–8. and we will help you at every step: 37. Vlack S, Cox L, Peleg AY, Canuto C, Stewart C, et al. Carriage of methicillin- • We accept pre-submission inquiries resistant Staphylococcus aureus in a Queensland Indigenous community. Med J Aust. 2006;184:556–9. � Our selector tool helps you to find the most relevant journal 38. David MZ, Glikman D, Crawford SE, Peng J, King KJ, et al. What is � We provide round the clock customer support community-associated methicillin-resistant Staphylococcus aureus? J Infect � Convenient online submission Dis. 2008;197:1235–43. 39. David MZ, Crawford SE, Boyle-Vavra S, Hostetler MA, Kim DC, et al. � Thorough peer review Contrasting pediatric and adult methicillin-resistant Staphylococcus aureus � Inclusion in PubMed and all major indexing services isolates. Emerg Infect Dis. 2006;12:631–7. � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

Journal

BMC Infectious DiseasesSpringer Journals

Published: Jan 5, 2017

There are no references for this article.