Molecular analysis of methicillin-resistant Staphylococcus aureus dissemination among healthcare professionals and/or HIV patients from a tertiary hospital

Abstract

INTRODUCTION:

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen in community settings. MRSA colonized individuals may contribute to its dissemination; the risk of MRSA infection is increased in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients, although the prevalence of colonization in this group is not well established. The present study addressed this issue by characterizing MRSA isolates from HIV/AIDS patients and their healthcare providers (HCPs) to determine whether transmission occurred between these two populations.

METHODS:

A total of 24 MRSA isolates from HIV-infected patients and five from HCPs were collected between August 2011 and May 2013. Susceptibility to currently available antimicrobials was determined. Epidemiological typing was carried out by pulsed-field gel electrophoresis, multilocus sequence typing, and Staphylococcus cassette chromosome (SCCmec) typing. The presence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and heterogeneous daptomycin-resistant Staphylococcus aureus (hDRSA) was confirmed by population analysis profile. Isolates characterized in this study were also compared to isolates from 2009 obtained from patients at the same hospital.

RESULTS:

A variety of lineages were found among patients, including ST5-SCCmecII and ST30-SCCmecIV. Two isolates were Panton-Valentine leukocidin-positive, and hVISA and hDRSA were detected. MRSA isolates from two HCPs were not related to those from HIV/AIDS patients, but clustered with archived MRSA from 2009 with no known relationship to the current study population.

CONCLUSIONS:

ST105-SCCmecII clones that colonized professionals in 2011 and 2012 were already circulating among patients in 2009, but there is no evidence that these clones spread to or between HIV/AIDS patients up to the 7^th day of their hospitalization.

Keywords:
Methicilin-resistant Staphylococcus aureus; HIV patients; Teicoplanin resistance; h-VISA; Daptomycin

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial and community infections¹. It is usually present in the environment as well as in the microbiota of the superior respiratory tract and skin. MRSA is transmitted by direct contact, and patient-to-patient transmission is largely via the hands of health care providers (HCPs). Undetected MRSA-colonized HCPs represent a major source of the bacterium in hospitals, since it can be transmitted from these individuals to high-risk patients, which can limit the success of other control measures. Indeed, a case in which MRSA transmission was reduced after carrier HCPs were identified and successfully decolonized has been described².

Individuals with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) are at increased risk for MRSA colonization and infection mainly due to their high antibiotic use¹ and high rates of hospital readmission. These patients can also harbor community-acquired MRSA, which often produces Panton-Valentine leukocidin (PVL)¹. However, the prevalence of MRSA colonization in this group is not well established.

We addressed this in the present study by investigating the rate of MRSA colonization in hospitalized AIDS patients and their HCPs. MRSA isolates were characterized genetically and phenotypically and compared to determine whether any transmission occurred between patients and/or HCPs. The isolates were also compared to isolate from 2009 that were collected from different patients at the same hospital.

METHODS

Sample collection

MRSA samples from two colonization sites [nares (N) and saliva (S)] were obtained from HIV/AIDS patients on days 1 and 7 of hospitalization and from HCPs with whom they had contact, in two specific units of a large Brazilian public hospital with 600 beds, of which 24 are occupied almost exclusively by HIV patients. From August 2011 to May 2013, 317 individuals with HIV/AIDS were hospitalized, and 266 agreed to participate in the study along with 73 HCPs; staffs that were on leave were not included. Samples were collected using swabs and stored in Stuart agar until bacterial isolation and identification. S. aureus was isolated on Mueller Hinton agar supplemented with 5% sheep blood and phenotypic identification was carried out using the Vitek system (BioMérieux, Marcy l'Etoile, France). Methicillin resistance was detected using the AST-P585 card (BioMérieux) and broth dilution. Once an HCP was identified as being colonized with MRSA, a decolonization protocol was carried out that included a chlorhexidine bath and application of 1ml silver sulfadiazine to the nares, which was repeated daily for 5 days. Additionally, 22 MRSA isolates from other patients from different wards at the same hospital collected between June and September 2009 that were archived by our group, and S. aureus strain N315 were included for determination of clonality. Isolates representing each pulsotype detected by pulsed-field gel electrophoresis (PFGE) were randomly selected for typing by multilocus sequence typing (MLST) and Staphylococcus Cassette Chromosome (SCCmec) typing. The study protocol was approved by the Research Ethics Committee of the Ribeirão Preto School of Nursing (no. 1304/2011).

Susceptibility profiling

The minimum inhibitory concentration (MIC) was determined for oxacilin, vancomycin, teicoplanin, daptomycin, tigecyclin, linezolid, and quinupristin-dalfopristin by broth dilution, according to Clinical and Laboratory Standards Institute (CLSI) guidelines³. MIC₅₀ and MIC₉₀ were calculated and CLSI breakpoints were adopted for classification except in the case of tigecycline, for which the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendation was followed⁴.

Heterogeneous vancomycin-intermediate Staphylococcus aureus screening

Heterogeneous vancomycin-intermediate Staphylococcus aureus screening (hVISA) screening was performed as previously described⁵. If a countable number (one to 30) of colonies was observed within 48h of incubation at 37°C on brain-heart infusion agar containing 4μg/ml vancomycin, the strain was designated as a possible hVISA⁵. S. aureus Mu3 and Mu50 were used as hVISA and VISA control strains, respectively, and were kindly provided by Keiichi Hiramatsu and Teruyo Ito (Juntendo University, Tokyo, Japan).

Population analysis profile

The population analysis profile (PAP)^{5) (6} to vancomycin or daptomycin was determined for samples that were positive for the hVISA screening or exhibited resistance to daptomycin after 48h of incubation^{6) (7) (8}. PAP to daptomycin was used to identify heterogeneous daptomycin-resistant Staphylococcus aureus (hDRSA) strains⁸.

Panton-Valentine leukocidin gene and hemolysis analysis

The PVL gene lukSF was amplified by PCR as previously described⁹. Mueller Hinton agar supplemented with 5% sheep blood was used to assess hemolytic activity.

Molecular characterization

SCCmec type was determined by multiplex-PCR as previously described¹⁰ using the following reference strains: S. aureus RN4220 (SCCmec negative), 10442 (SCCmecI), N315 (SCCmecII), 85/2082 (SCCmecIII), 4744 (SCCmecIVa), and WIS (SCCmecV).

PFGE was carried out after DNA digestion with SmaI¹¹. Data were analyzed with Bionumerics v.7.1 software (Applied Maths NV, Belgium)¹² using the unweighted pair-group method with arithmetic mean based on Dice coefficients, where optimization and tolerance were set to 0.5% and 1.25%, respectively. A similarity coefficient of 80% was selected to describe patterns representative of each pulsotype, which were further characterized by MSLT¹³. Sequence types were identified using the MLST database¹⁴. To limit redundancy, duplicate isolates from the same patient with identical SCCmec and pulsotype were considered as the same strain and included only once in the analysis.

RESULTS

Staphylococcus aureus was cultured from 101 (38%) tested individuals, and resistance to oxacillin was observed in 15 participants (5.6% of all participants or 14.8% of those colonized by S. aureus). A total of 13/15 HIV patients were found to be colonized by MRSA on the day of hospital admission, and only five of these remained colonized on day 7. Additionally, two patients were found to be colonized only on day 7 of hospitalization. Only 3/73 (4.1%) of HCPs (P1, P2, and P3) were colonized by MRSA during the study. P1 was colonized on three different dates, despite having undergone decolonization procedures¹⁵. Therefore, a total of 29 MRSA isolates were characterized: 24 from patients and five from HCPs (Figure 1).

FIGURE 1:
Similarity dendrogram. Similarity was calculated with Bionumerics v.6.5 software using the Dice coefficient with 0.5% optimization and 1.25% tolerance. ST: sequence typing; SCCmec: Staphylococcus cassette chromosome; DPC: daptomycin; LNZ: linezolide; TCP: teicoplanin; TGC: tigecycline; OXA: oxacillin; VAN: vancomycin; N: nares; S: saliva; HCPs: healthcare providers. Isolates collected in this study are indicated in bold to differentiate them from those obtained in 2009, which were used only for clonality analysis.*resistance after 48h.

Susceptibility analysis confirmed β-lactam resistance in all putative MRSA isolates. Daptomycin resistance in isolate 80N was observed after a 48-h incubation; this was unexpected, since daptomycin is considered bactericidal. A population analysis of the isolate revealed a heterogeneous phenotype that included some daptomycin-resistant cells (Figure 2A).

One isolate (176N) was identified as hVISA, which was confirmed by PAP (Figure 2B). According to CLSI breakpoints³, this isolate exhibited an intermediate level of resistance to teicoplanin after 24h of incubation (MIC = 16μg/ml) and full resistance after 48h. However, it would be classified as resistant after 24h based on EUCAST breakpoints⁴.

FIGURE 2:
Population analysis profile to: A) daptomycin and B) vancomycin. CFU: colony forming units.

MIC₅₀ and MIC₉₀ (μg/ml) of all antimicrobials were determined for each strain, and were as follows: 128/>256 for oxacilin, 4/4 for linezolid, 0.5/1 for teicoplanin, 0.125/0.5 for tigecycline, 0.5/0.5 for quinupristin/dalfopristin and daptomycin, and 1/2 for vancomycin. MICs for each isolate are shown in Figure 1.

Only 2/29 isolates (6.9%) were PVL-positive (strains 199N and 273N), while 25 (86.2%) were fully hemolytic. Isolates from HIV patients showed considerable variation in terms of PFGE band patterns, which included eight pulsotypes (A-E, G, H, and J) in contrast to three pulsotypes (F, I, and K) among isolates from HCPs.

ST5, ST105, ST148, ST239, and ST30 were detected among isolates. Two of three HCPs were colonized by ST105-SCCmecII, a single locus variant (SLV) of the New York/Japan Clone, whereas one was colonized by ST5-SCCmecIV, the Pediatric Clone. The following lineages were observed in HIV patients: ST5-SCCmecIV, ST239-SCCmecIII, ST30-SCCmecIV (Southern Pacific clone), ST5-SCCmecV, ST105-SCCmecII, and ST148-SCCmecIV, all of which have been previously described^{16) (17}. Different SCCmec types were observed in isolates collected from Patient #7 on days 1 and 7 of hospitalization, although they were indistinguishable by PFGE (Figure 1).

Only Patients #1 and #12 were readmitted to this hospital during the period of study. Isolates from these patients from days 1 and 7 of hospitalization were indistinguishable. Although both isolates from Patient #1 collected at readmission were of the same lineage as that obtained at the first hospitalization (ST239-SCCmecIII), they were of a different pulsotype (G instead of H), and the isolates from days 1 and 7 of readmission were of different subtypes (G3 and G2). Isolates obtained from Patient #12 over a two-month period were indistinguishable and MRSA was detected only on day 1 of both hospitalizations.

The first P1 isolate (PAS 4N) was characterized by PFGE as pulsotype F. After decolonization¹⁵, this HCP remained colonized by the same MRSA clone (PAS 63N). A second decolonization procedure was carried out, and P1 was again found to be colonized, but this time by pulsotype K strain PAS 65N. Although the pulsotype had changed, all three isolates from P1 were of ST105-SCCmecII. There were no HIV patients colonized by the same type of MRSA as determined by PFGE of HCP samples examined during the course of this study.

When MRSA isolated in 2009 from different infection sites from patients in different wards of the same hospital was compared to those of HIV patients and HCPs in the current study, we observed that with the exception of pulsotype H associated with Brazilian Epidemic Clone (BEC), all other HIV patient isolates were unrelated to those obtained from patients in 2009 (< 80% similarity) (Figure 1). However, HCPs P1 (isolates PAS 4N and PAS 63N) and P3 (isolate PAS 31N) were colonized in 2011 by clones that were indistinguishable from those isolated from infections at this hospital in 2009.

DISCUSSION

In this study, a variety of MRSA pulsotypes with few subtypes were isolated from HIV patients and HCPs. We observed no correlation between pulsotype and reduced susceptibility to antimicrobials. We found one hVISA isolate that also exhibited intermediate resistance to teicoplanin. The vanA gene responsible for complete glycopeptide resistance in S. aureus was not detected in any samples (data not shown). Teicoplanin resistance after vancomycin use has been reported¹⁸; however, none of the patients in our study were treated with vancomycin or teicoplanin.

The mechanisms underlying daptomycin resistance are not well understood^{19) (20}, but it is known that decreased susceptibility to daptomycin and vancomycin can co-occur both in vivo²⁰ and in vitro²¹ after selection with either antimicrobial²¹. Although there is evidence for cross-resistance²¹, in this study, the hDRSA isolate remained susceptible to vancomycin. Since none of the patients had been treated with daptomycin, the basis for resistance is unclear, but is currently under study. Heterogeneous type resistance to daptomycin has been previously observed, and is in part attributed to dltA overexpression. The detection of teicoplanin-intermediate S. aureus, hVISA, and hDRSA is of great concern since these represent last-line antimicrobials for treating MRSA infection.

ST105 and ST148 are SLVs of ST5 and account for the majority of isolates typed in this study. ST30 is a prototype of CC30 and is associated with community-acquired infection. The isolate ST30-SCCmecIV was positive for PVL, which is consistent with MRSA strains found in the community²².

Although many subtypes isolated from patients can be considered as closely related according to Tenover's criteria¹², only four pairs of patient strains yielded indistinguishable band patterns by PFGE. Interestingly, the isolate from P3 and the first two isolates from P1 clustered with MRSA from different patients isolated 2 years prior-i.e., a clone related to ST5-SCCmecII that was circulating among patients in 2009. There was no evidence that these clones spread to the HIV patients studied in 2011. This clone is common in hospitals around the world, and has replaced BEC as the predominant MRSA clone in Brazil²³.

Although it would be prudent, there is currently no rule in Brazil requiring removal from the work environment of a MRSA-positive HCP. British guidelines^{24) (25} for the control of MRSA in healthcare facilities include screening of staff for this bacterium. HCPs with colonized or infected hand lesions are required to be off work while receiving clearance therapy since they are a source of dissemination to non-colonized patients^{24) (25}. Moreover, HCPs work in different wards of a hospital, which increases opportunities for dissemination. Security and hygienic practices should also be verified more frequently, since HCPs can be colonized on different dates even after undergoing decolonization, as we observed here.

In conclusion, HCPs were found to be colonized by ST105-SCCmecII in 2011 and 2012 and this strain had infected other patients in 2009. Although the same pulsotype was detected, we cannot conclude that these HCPs were in direct contact with patients and that dissemination occurred. Finally, there was no broad dissemination of a specific MRSA clone among HIV patients up to the 7t^h day of hospitalization.

ACKNOWLEDGMENTS

We thank Michael S. Gilmore and Juliana Sposto Avaca Crusca for critical review of the manuscript.

• ¹
  Cole J, Popovich K. Impact of Community-associated methicillin-resistant Staphylococcus aureus on HIV-infected patients. Curr HIV/AIDS Rep 2013; 10:244-253.
• ²
  Ben-David MD, Mermel LA, Parenteau S. Methicillin-resistant Staphylococcus aureus transmission: The possible importance of unrecognized health care worker carriage. Am J Infect Control 2008; 36:93-97.
• ³
  Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 23^th Informational Supplement. CLSI document M100-S23. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania. 2013.
• ⁴
  The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. 2013. Version 3.1. [Internet] Available from: http://www.eucast.org http://www.eucast.org
  » http://www.eucast.org» http://www.eucast.org
• ⁵
  Hiramatsu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S et al. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 1997; 350:1670-1673.
• ⁶
  Cui L, Isii T, Fukuda M, Ochiai T, Neoh H, Camargo ILBC et al. An rpoB mutation confers dual heteroresistance to daptomycin and vancomycin in Staphylococcus aureus Antimicrob Agents Chemother 2010; 54:5222-5233.
• ⁷
  Howden BP, Davies JK, Johnson PDR, Stinear TP, Grayson ML. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clin Microbiol Rev 2010; 23:99-139.
• ⁸
  Cafiso V, Bertuccio T, Purrello S, Campanile F, Mammina C, Sartor A et al. dltA overexpression: A strain-independent keystone of daptomycin resistance and vancomycin in Staphylococcus aureus Int J Antimicrob Agents 2014; 43: 26-31.
• ⁹
  Lina G, Piémont Y, Godail-Garnot F, Bes M, Peter MO, Gauduchon V et al. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 1999; 29:1128-1132.
• ¹⁰
  Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J et al. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother2007; 51:264-274.
• ¹¹
  Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33:2233-2239.
• ¹²
  McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol2003; 41:5113-5120.
• ¹³
  Enright MC, Day NPJ, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus J Clin Microbiol2000; 38:1008-1015.
• ¹⁴
  Multi locus sequence typing. [Internet] [cited 2015 May 20]. Available from: Available from: http://www.saureus.mlst.net/ 2015
  » http://www.saureus.mlst.net/
• ¹⁵
  Rossini FP, Rodrigues B, Ferreira MVF, Silva MFI, Resuto TJO, Silva MHA et al. Protocolo para descolonização de pacientes colonizados ou infectados por Staphylococcus spp multidroga por Staphylococcus spp multidroga resistentes (MDR). Revista qualidade HC 2009; 41-44.
• ¹⁶
  Shuenck RP, Nouér SA, Winter CO, Cavalcante FS, Scotti TD, Ferreira ALP et al. Polyclonal presence of non-multiresistant methicillin-resistant Staphylococcus aureus isolates carrying SCCmec IV in health care-associated infections in a hospital in Rio de Janeiro, Brazil. Diagn Microbiol Infect Dis 2009; 64:434-441.
• ¹⁷
  Mimica MJBerezin EN, Damaceno N, Carvalho RB. SCCmec type IV, PVL-negative, methicillin-resistant Staphylococcus aureus in cystic fibrosis patients from Brazil. Curr Microbiol 2011; 62:388-390.
• ¹⁸
  Mainardi JL, Shlaes DM, Acar JF, Goldstein FW. Decreased teicoplanin susceptibility of methicillin-resistant strains of Staphylococcus aureus J Infect Dis 1995; 171:1646-1650.
• ¹⁹
  Pogliano J, Pogliano N, Silverman JA. Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins. J Bacteriol 2012; 194:4494-4504.
• ²⁰
  Hayden MK, Rezai K, Hayes RA, Lolans K, Quinn JP, Weinstein RA. Development of daptomycin resistance in vivo in methicillin-resistant Staphylococcus aureus J Clin Microbiol2005; 43:5285-5287.
• ²¹
  Camargo ILBC, Neoh H, Cui L, Hiramatsu K. Serial daptomycin selection generates daptomycin-nonsusceptible Staphylococcus aureus strains with a heterogeneous vancomycin-intermediate phenotype. Antimicrob Agents Chemother2008; 52:4289-4299.
• ²²
  Otter JA, French GL. Community-associated meticillin-resistant Staphylococcus aureus strains as a cause of healthcare-associated infection. J Hosp Infect 2011; 79:189-193.
• ²³
  Dabul ANG, Camargo ILBC. Clonal complexes of Staphylococcus aureus: all mixed and together. FEMS Microbiol Lett 2014351:7-8.
• ²⁴
  Ayliffe GAJ, Casewell MW, Cookson BD, Cox RA. Revised Guidelines for the control of methicillin-resistant Staphylococcus aureus infection in hospitals. J Hosp Infect1998; 39:253-290.
• ²⁵
  Coia JE, Duckworth GJ, Edwards DI, Farrington M, Fry C, Humphreys H et al. Guidelines for the control and prevention of meticillin-resistant Staphylococcus aureus (MRSA) in healthcare facilities. J Hosp Infect2006; 63S:S1-S44.

• This work was supported by the São Paulo Research Foundation [Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2013/02152-2)] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Publication Dates

• Publication in this collection
  Jan-Feb 2016

History

• Received
  10 Sept 2015
• Accepted
  27 Jan 2016