Variable Number Tandem Repeat Fingerprinting Research Articles

Staphylococcus aureus from Subclinical Cases of Mastitis in Dairy Cattle in Poland, What Are They Hiding? Antibiotic Resistance and Virulence Profile.

Bovine mastitis is a common disease worldwide, and staphylococci are one of the most important etiological factors of this disease. Staphylococcus aureus show adaptability to new conditions, by which monitoring their virulence and antibiotic resistance mechanisms is extremely important, as it can lead to the development of new therapies and prevention programs. In this study, we analyzed Staphylococcus aureus (n = 28) obtained from dairy cattle with subclinical mastitis in Poland. The sensitivity of the isolated strains to antibiotics were confirmed by the disc diffusion method. Additionally, minimum inhibitory concentration values were determined for vancomycin, cefoxitin and oxacillin. Genotyping was performed by two methods: PCR melting profile and MLVF-PCR (multiple-locus variable-number tandem-repeat fingerprinting). Furthermore, the presence of antibiotic resistance and virulence genes were checked using PCR reactions. The analyzed strains showed the greatest resistance to penicillin (57%), oxytetracycline (25%) and tetracycline (18%). Among the analyzed staphylococci, the presence of 9 of 15 selected virulence-related genes was confirmed, of which the icaD, clfB and sea genes were confirmed in all staphylococci. Biofilm was observed in the great majority of the analyzed bacteria (at least 70%). In the case of genotyping among the analyzed staphylococci (combined analysis of results from two methods), 14 patterns were distinguished, of which type 2 was the dominant one (n = 10). This study provides new data that highlights the importance of the dominance of biofilm over antibiotic resistance among the analyzed strains.

The Prevalence of Virulence Determinants and Antibiotic Resistance Patterns in Methicillin-Resistant Staphylococcus aureus in a Nursing Home in Poland.

Nursing homes (NH) contribute to the regional spread of methicillin-resistant Staphylococcus aureus (MRSA). Moreover, residents are vulnerable to the colonization and subsequent infection of MRSA etiology. We aimed at investigating the molecular and phenotypic characteristics of 21 MRSA collected from the residents and personnel in an NH (Lublin, Poland) during 2018. All MRSA were screened for 20 genes encoding virulence determinants (sea-see, eta, etb, tst, lukS-F-PV, eno, cna, ebpS, fib, bbp, fnbA, fnbB, icaADBC) and for resistance to 18 antimicrobials. To establish the relatedness and clonal complexes of MRSA in NH we applied multiple-locus variable-number tandem-repeat fingerprinting (MLVF), pulse field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. We identified four sequence types (ST) among two clonal complexes (CC): ST (CC22) known as EMRSA-15 as well as three novel STs—ST6295 (CC8), ST6293 (CC8) and ST6294. All tested MRSA were negative for sec, eta, etb, lukS-F-PV, bbp and ebpS genes. The most prevalent gene encoding toxin was sed (52.4%; n = 11/21), and adhesins were eno and fnbA (100%). Only 9.5% (n = 2/21) of MRSA were classified as multidrug-resistant. The emergence of novel MRSA with a unique virulence and the presence of epidemic clone EMRSA-15 creates challenges for controlling the spread of MRSA in NH.

Molecular typing and antimicrobial resistance profiling of 33 mastitis-related Staphylococcus aureus isolates from cows in the Comarca Lagunera region of Mexico

Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphylococcus aureus. Little is known about the S. aureus lineages causing mastitis in Mexican cattle. The aim of this study was to type S. aureus isolates causing mastitis in cows from the Comarca Lagunera region in Mexico in 2015–2016. Multi-locus variable number tandem repeat fingerprinting (MLVF) of 33 S. aureus isolates obtained from 210 milk samples revealed the MLVF clusters A (n = 1), B (n = 26), C (n = 5) and D (n = 1). Spa-typing showed that clusters A and B represent the spa-type t224, cluster C includes spa-types t3196 and t416, and cluster D represents spa-type t114. The different spa-types were mirrored by the masses of protein A bands as detected by Western blotting. Antimicrobial susceptibility testing showed that one isolate was susceptible to all antimicrobials tested, whereas all other strains were resistant only to benzylpenicillin. These findings show that only four S. aureus lineages, susceptible to most antimicrobials, were responsible for causing mastitis at the time of sampling. Lastly, many isolates carried the same small plasmid, designated pSAM1. The high prevalence of pSAM1 amongst the antimicrobial-susceptible isolates suggests an association with bovine colonization or mastitis rather than antimicrobial resistance.

Molecular Diversity of Staphylococcus aureus Colonizing the Upper Respiratory Tract of Residents and Staff in A Nursing Home.

Elderly people living in nursing homes are a high-risk population for Staphylococcus aureus infection. Multiple comorbidities, a weakened immune system, inadequate hygienic conditions, and crowding might increase the prevalence rates of this opportunistic pathogen. However, the epidemiological aspects, genetic diversity, and transmission of S. aureus in nursing homes are still poorly understood, especially in Poland. This study aimed to determine the genetic relatedness and prevalence of colonization of S. aureus isolated from the anterior nares and the throat of residents and staff in a nursing home located in Lublin, Poland. The study showed a high S. aureus prevalence rate among participants (46.1%), yet there was a low frequency of MRSA strains among residents (1.7%) and staff (0%). The multiple-locus variable-number tandem-repeat fingerprinting (MLVF) analysis demonstrated a high degree of genetic diversity of S. aureus strains colonizing the anterior nares and the throat of the participants. The occurrence of simultaneous colonization with more than one unique S. aureus strain in any one individual as well as the incidence of colonization with the same genetic variant of S. aureus in different individuals was observed. These findings suggest that inter-participant S. aureus transmission might contribute to the development of cross-infections.

Investigation of Streptococcus pyogenes virulence factors and typing by multiple locus variable number tandem repeat fingerprinting (MLVF) method

Streptococcus pyogenes is an important bacterial pathogen that colonizes the throat and skin of human beings and causes a wide variety of diseases ranging from mild infections like pharyngitis, tonsillitis and impetigo to severe invasive infections such streptococcal toxic shock syndrome, septicemia, and necrotizing fasciitis, and produces a wide variety of virulence factors. The aim of this study was to investigate the antibiotic resistance, virulence genes; [pyrogenic exotoxin genes (speA, C, G, H, I, J, K, L, M, smeZ and ssa), deoxyribonuclease genes (sdaB, spd3, sdc ve sdaD), protease genes (speB, spyCEP ve scpA) and inhibitor genes (mac and sic)] of S.pyogenes strains isolated from throat cultures of patients with symptomatic tonsillo-pharyngitis and typing by multiple locus variable number tandem repeat fingerprinting (MLVF) method. One hundred and fifty S.pyogenes isolates were identified by conventional methods and streptococcus group A latex kit (Biomerieux, France). Antibiotic susceptibility tests were performed by Kirby-Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute. DNA isolation was performed by using a commercial DNA isolation kit (Qiagen, Germany) in accordance with manufacturer's recommendations. The virulence genes were determined by multiplex PCR. MLVF method was performed with multiplex PCR using specific primers for repeated sequences within bacterial genome. All of the S.pyogenes isolates were susceptible to penicillin G, cefotaxime, ceftriaxone, chloramphenicol, clindamycin, erythromycin, levofloxacin, vancomycin and linezolid. Among streptococcal pyrogenic exotoxin genes the most frequent gene was smeZ (90.0%) followed by speG (88.0%), speC (58.7%), ssa (42.7%), speA (33.3%), speJ (24.0%), speK (18.7%), speH (14.0%), speI (13.3%), speL and speM (9.3%). Of the DNase genes, sdaB was detected in all strains (100%), spd3, sdc, sdaD genes were determined as 64.7%, 36.0%, 24.7% respectively. Protease genes (speB, spyCEP, scpA) and mac gene from the inhibitor genes were positive in all strains, and sic gene was positive in only 3 (2.0%) of the isolates. Thirty-two different patterns that contained two or more isolates were determined by MLVF analysis. Ninety one isolates were included in any of the 32 different patterns, while 59 isolates were defined as sporadic isolates. In conclusion, S.pyogenes isolates collected from throat cultures of patients with symptomatic tonsillo-pharyngitis in Konya/Turkey were susceptible to all antibiotics studied and have carried a very high rate of virulence factors. However the isolates were mostly clonally unrelated and sporadic. This study is the first report in Turkey, in which S.pyogenes isolates were typed by the MLVF method and a large number of virulence factors were investigated.

Multiple-locus variable-number tandem repeat fingerprinting as a method for rapid and cost-effective typing of animal-associated Staphylococcus aureus strains from lineages other than sequence type 398.

In veterinary medicine, Staphylococcus aureus is associated with a range of mild to severe infections. The high density of livestock in intensive farming systems increases the risk of disease spread and hampers its control and measures of prevention, making S. aureus one of the most important animal pathogens. Multiple-locus variable-number tandem repeat fingerprinting (MLVF) has been successfully applied to the characterization of livestock-associated meticillin-resistant Staphylococcus aureus (MRSA) ST398 but not to the characterization of a wide range of other animal isolates. The objective of the current study was to examine the effectiveness of MLVF for studying S. aureus strains isolated from households, farms and exotic animals in three regions of Poland. MLVF, random amplification of polymorphic DNA (RAPD), spa typing and diagnostic microarrays were compared to determine the most suitable combination of methods for veterinary purposes. MLVF generated results consistent with host and geographic origins, reflecting population structures with a high concordance to spa typing results. MLVF has been proven to be a rapid, highly discriminatory and cost-effective method suitable for molecular typing in veterinary settings.

A Snapshot on MRSA Epidemiology in a Neonatal Intensive Care Unit Network, Palermo, Italy.

Objectives: We performed a 1-year prospective surveillance study on MRSA colonization within the five NICUs of the metropolitan area of Palermo, Italy. The purpose of the study was to assess epidemiology of MRSA in NICU from a network perspective.Methods: Transfer of patients between NICUs during 2014 was traced based on the annual hospital discharge records. In the period February 2014–January 2015, in the NICU B, at the University teaching hospital, nasal swabs from all infants were collected weekly, whereas in the other four NICUs (A, C, D, E) at 4 week-intervals of time. MRSA isolates were submitted to antibiotic susceptibility testing, SCCmec typing, PCR to detect lukS-PV and lukF-PV (lukS/F-PV) genes and the gene encoding the toxic shock syndrome toxin (TSST-1), multilocus variable number tandem repeat fingerprinting (MLVF), and multilocus sequence typing (MLST).Results: In the period under study, 587 nasal swabs were obtained from NICU B, whereas 218, 180, 157, and 95 from NICUs A, C, D, and E, respectively. Two groups of NICUs at high prevalence and low prevalence of MRSA colonization were recognized. Overall, 113 isolates of MRSA were identified from 102 infants. Six MLVF types (A–F) were detected, with type C being subdivided into five subtypes. Five sequence types (STs) were found with ST22-IVa being the most frequent type in all NICUs. All the MRSA molecular subtypes, except for ST1-IVa, were identified in NICU B.Conclusions: Our findings support the need to approach surveillance and infection control in NICU in a network perspective, prioritizing referral healthcare facilities.

Methicillin-resistant Staphylococcus aureus nasal colonization in a level III neonatal intensive care unit: Incidence and risk factors

To describe epidemiologic features and identify risk factors for methicillin-resistant Staphylococcus aureus (MRSA) acquisition in a level III neonatal intensive care unit (NICU). A prospective, cohort study in a university-affiliated NICU with an infection control program including weekly nasal cultures of all neonates. Demographic, clinical, and microbiologic data were prospectively collected between June 2009 and June 2013. Molecular characterization of MRSA isolates was done by multilocus variable number tandem repeat fingerprinting, staphylococcal cassette chromosome mec typing, and on representative isolates by multilocus sequence typing and spa typing. Of 949 neonates, 217 (22.87%) had a culture growing MRSA, including 117 neonates testing positive at their first sampling. Of these latter infants, 96 (82.05%) were inborn and 59 (50.43%) had been transferred from the nursery. Length of stay and colonization pressure were strong independent predictors of MRSA acquisition. Among MRSA isolates, 7 sequence types were identified, with ST22-IVa, spa type t223, being the predominant strain. In an endemic area, early MRSA acquisition and high colonization pressure, likely related to an influx of colonized infants from a well-infant nursery, can support persistence of MRSA in NICUs. Surveillance, molecular tracking of strains, and reinforcement of infection control practices, involving well-infant nurseries in a comprehensive infection control program, could be helpful in containing MRSA transmission.

Genetic Diversity of Staphylococcus aureus in Buruli Ulcer

BackgroundBuruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment.MethodologyWe investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa-typing, and antibiotic susceptibility was tested.Principal FindingsNineteen (63%) of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26%) S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37%) patients were distributed over two different MLVF clusters. Wounds of three (16%) patients were colonized with isolates belonging to two different genotypes at the same time, and five (26%) patients were colonized with different S. aureus types over time. Five (17%) of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA).Conclusion/SignificanceThe present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene.

Isolation of nuc mutant isolates of Staphylococcus aureus from bovine clinical mastitis

Isolates of Staphylococcus aureus with a mutation in the nuclease (nuc) gene were recovered from cases of bovine mastitis in Poland. Three S. aureus isolates from cows in one herd had a 42 base pair duplication in the nuc gene. These isolates belonged to sequence type 97 (ST97) and clonal complex 97 (CC97). They had a different spa type and multiple-locus variable-number tandem-repeat fingerprinting (MLVF) subtype than a S. aureus isolate without the nuc mutation from the same herd. Isolation of nuc mutant S. aureus strains from cases of bovine mastitis may confound diagnostic PCRs based on detection of the nuc gene.

Genotyping of Staphylococcus aureus strains isolated from healthy persistent carriers

The paper presents results on the relatedness of Staphylococcus aureus strains colonizing the upper respiratory tract isolated from healthy persistent carriers. Genotyping was carried out using two methods--multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and pulsed-field gel electrophoresis (PFGE). By comparison of the results obtained by both methods, good correlations between MLVF and PFGE genotyping of strains isolated from the asymptomatic carriers were observed. Further studies are needed to evaluate methods useful for genotyping of S. aureus strains circulating in the community.

An Improved MLVF Method and Its Comparison with Traditional MLVF, spa Typing, MLST/SCCmec and PFGE for the Typing of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen, causing considerable morbidity and mortality. During the last 20 years, a variety of genotyping methods have been introduced for screening the prevalence of MRSA. In this study, we developed and evaluated an improved approach capillary gel electrophoresis based multilocus variable-number tandem-repeat fingerprinting (CGE/MLVF) for rapid MRSA typing. A total of 42 well-characterized strains and 116 non-repetitive clinical MRSA isolates collected from six hospitals in northeast China between 2009 and 2010 were tested. The results obtained by CGE/MLVF against clinical isolates were compared with traditional MLVF, spa typing, Multilocus sequence typing/staphylococcal cassette chromosome mec (MLST/SCCmec) and pulse field gel electrophoresis (PFGE). The discriminatory power estimated by Simpson’s index of diversity was 0.855 (28 types), 0.855 (28 patterns), 0.623 (11 types), 0.517 (8 types) and 0.854 (28 patterns) for CGE/MLVF, traditional MLVF, spa typing, MLST/SCCmec and PFGE, respectively. All methods tested showed a satisfied concordance in clonal complex level calculated by adjusted Rand’s coefficient. CGE/MLVF showed better reproducibility and accuracy than traditional MLVF and PFGE methods. In addition, the CGE/MLVF has potential to produce portable results. In conclusion, CGE/MLVF is a rapid and easy to use MRSA typing method with lower cost, good reproducibility and high discriminatory power for monitoring the outbreak and clonal spread of MRSA isolates.

Changing molecular epidemiology and high rates of mupirocin resistance among meticillin-resistant Staphylococcus aureus in Singaporean hospitals.

A prospective cross-sectional study was performed to determine the continuing shift in the molecular epidemiology of meticillin-resistant Staphylococcus aureus (MRSA) in Singapore. In total, 666 MRSA isolates from screening cultures performed between 7 and 20 January 2013 were obtained from all seven public sector hospitals in Singapore and were subjected to molecular typing using multilocus variable-number tandem-repeat fingerprinting with confirmatory multilocus sequencing typing for clustered isolates. Isolates were also tested for the presence of the orfX-ACME insert and the high-level mupirocin resistance gene ileS-2. The major circulating clones in Singaporean hospitals were ST22 (63.2%), ST45 (18.9%) and ST239 (10.7%). The orfX-ACME insert was only found in ST239 isolates (31/71, 43.7%), but ileS-2 was found in 207 (31.1%) of the MRSA isolates, varying between 10.0% and 47.8% among the hospitals. In conclusion, the molecular epidemiology of MRSA in Singaporean hospitals has continued to change, with ST45 now replacing ST239 in addition to the ongoing replacement of the latter by ST22. Although a greater proportion of ST239 isolates carry the orfX-ACME insert, the actual clinical impact may be marginal as ST239 MRSA continues to decline. Finally, high-level mupirocin resistance rates are remarkably high in local healthcare-associated MRSA, with implications for MRSA decolonisation and infection prevention. Further surveillance is required to monitor the changing epidemiological trends.

High-resolution typing by MLVF unveils extensive heterogeneity of European livestock-associated methicillin-resistant Staphylococcus aureus isolates with the sequence type 398

Methicillin-resistant Staphylococcus aureus sequence type 398 (MRSA ST398) has emerged in livestock worldwide. In particular, areas in Europe with high densities of livestock farming are affected. Consequently, the incidence of human colonization and infection with ST398 is rapidly increasing. Distinguishing different ST398 isolates with standard typing tools is problematic. The objective of this study was to examine the discriminatory power of Multiple-Locus Variable number tandem repeat Fingerprinting (MLVF) on a highly diverse ST398 collection. Our data show that MLVF combined with spa-typing is an attractive approach for high-resolution typing of ST398 isolates and unveiling their relatedness.

Rapid and high-resolution distinction of community-acquired and nosocomial Staphylococcus aureus isolates with identical pulsed-field gel electrophoresis patterns and spa types

Methicillin-resistant Staphylococcus aureus (MRSA) represent a serious threat for public health worldwide. Of particular concern is the emergence of community-acquired MRSA, which is often difficult to distinguish from nosocomial MRSA due to a lack of suitable typing methods for early detection. For example, the USA300 pulsed-field gel electrophoresis (PFGE) pattern includes both the ‘classical’ community-acquired USA300 clone with spa type t008 and an epidemiologically unrelated nosocomial clone with spa type t024. Likewise, spa typing cannot distinguish the classic USA300 from nosocomial MRSA with the spa type t008. Since the fast and high-resolution distinction of these S. aureus types is important for infection prevention and surveillance, we investigated whether multiple-locus variable number tandem repeat fingerprinting (MLVF) can be applied to overcome these limitations. Indeed, MLVF correctly grouped 91 MRSA isolates belonging to the classic USA300 lineage, nosocomial MRSA isolates with the USA300 PFGE profile and spa type t024, and nosocomial MRSA isolates with spa type t008 into 3 distinct clusters. Importantly, several sub-clusters were also identified, reflecting epidemiological relationships between the respective isolates. We conclude that MLVF has the discriminatory power needed to rapidly distinguish very similar community-acquired and nosocomial MRSA isolates and that MLVF-based sub-clustering of isolates is highly useful for epidemiological investigations, outbreak prevention, and control.

Microfluidic-Chip-Based Multiple-Locus Variable-Number Tandem-Repeat Fingerprinting with New Primer Sets for Methicillin-Resistant Staphylococcus aureus

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

Characterization of methicillin-resistant Staphylococcus aureus displaying increased MICs of ceftaroline

To characterize the mechanisms responsible for elevated MICs of ceftaroline for methicillin-resistant Staphylococcus aureus (MRSA). During the 2008 Assessing Worldwide Antimicrobial Resistance Evaluation ('AWARE') surveillance programme, four S. aureus collected from separate patients in Athens, Greece, demonstrated ceftaroline MICs of 4 mg/L. These isolates were clonally related and one strain (13101) was selected for further characterization. Two strains (4981 and 4977) displaying ceftaroline MICs of 1 and 2 mg/L, respectively, were included for comparison. All strains originated from the same hospital. Penicillin-binding protein (PBP) affinities for ceftaroline and comparators were determined. Strains were typed by single-locus typing (i.e. spa typing), multilocus sequence typing ('MLST') and by multiple-locus variable-number tandem repeat fingerprinting (MLVF). The presence of Pantone-Valentine leucocidin and the staphylococcal cassette chromosome mec types was assessed. We also performed nucleotide sequencing of the mecA (encoding PBP2a) promoter and ribosomal binding site (rbs) regions and mecR1. Ceftaroline demonstrated the highest PBP2a affinity with strain 4981 (ST5-MRSA-II) (IC(50) 0.06 mg/L; MIC 1 mg/L). Strains 4977 and 13101 (both ST239-MRSA-III) showed indistinguishable MLVF profiles. Ceftaroline PBP2a binding affinity in strains 4977 (IC(50) 0.25 mg/L; MIC 2 mg/L) and 13101 (IC(50) 1 mg/L; MIC 4 mg/L) was 4- and 16-fold lower than 4981, respectively. Strain 4981 contains a wild-type PBP2a, while strains 4977 and 13101 have N(146)K and E(150)K alterations in the non-penicillin-binding domain. Additionally, 13101 has one substitution (H(351)N) in the transpeptidase domain. Alterations in the mecR1, mecA promoter or rbs regions were not observed. Increased ceftaroline MICs were associated with decreased PBP2a binding affinity and reflected alterations in PBP2a.

Polyclonal non multiresistant methicillin resistant Staphylococcus aureus isolates from clinical cases of infection occurring in Palermo, Italy, during a one-year surveillance period

BackgroundThe evolving epidemiology of methicillin resistant Staphylococcus aureus (MRSA) is characterized by the emergence of infections caused by non multiresistant MRSA carrying staphylococcal chromosomal cassette (SCC)mec IV or V in the healthcare settings. A molecular epidemiological analysis of non multiresistant MRSA isolates from four acute general hospitals was performed in Palermo, Italy, during a one year period.MethodsFor the purpose of the study, MRSA isolates were defined as non multiresistant when they were susceptible to at least three classes of non β-lactam antibiotics. Seventy-five isolates were submitted to antimicrobial susceptibility testing, multilocus sequence typing (MLST) and polymerase chain reaction (PCR) for SCCmec, accessory gene regulator (agr) groups, arginine catabolic mobile element (ACME) and Panton Valentine leukocidin (PVL) toxin genes. For epidemiological typing, Multiple-Locus Variable-Number Tandem Repeat Fingerprinting (MLVF) was performed on all isolates and pulsed field gel electrophoresis (PFGE) on ST8 isolates.ResultsNon multiresistant MRSA isolates were isolated from all hospitals. Resistances to ciprofloxacin, macrolides and tetracycline were the most prevalent. MLST attributed 46 isolates with ST22, 13 with ST8, eight with ST1, three with ST50 and three with ST398. SCCmec type IV was found in all isolates. PVL was detected in one ST22 isolate. All isolates tested negative for the ACME element. MLVF identified 31 different patterns, some subtype clusters ranging in size between two and 22 isolates. The closely related PFGE patterns of the ST8 isolates differed from USA300.ConclusionsA polyclonal circulation of non multiresistant MRSA along with blurring of boundaries between healthcare associated (HA)-MRSA and community associated (CA)-MRSA appear to be occurring in our epidemiological setting. A better understanding of spread of MRSA with the support of molecular typing can provide invaluable information in the epidemiological, microbiological and clinical fields.

Comparison of Modified Multiple-locus Variable-number Tandem-repeat Fingerprinting with Pulsed-field Gel Electrophoresis for Typing Clinical Isolates of Staphylococcus aureus

BackgroundMultiple-locus variable-number tandem-repeat fingerprinting (MLVF) is based on multiplex PCR, utilizing variable number tandem repeat. Our goal was to compare the performance of MLVF in distinguishing clinical Staphylococcus aureus isolates with that of pulsed-field gel electrophoresis (PFGE), which has traditionally been the gold standard.MethodsSixty-three clinically significant S. aureus isolates were tested using both PFGE and MLVF. Multiplex PCR for MLVF was performed using PCR primers for clfA, clfB, sdrCDE, sspA, and spa. PFGE was performed with genomic DNA fragments generated by SmaI endonuclease digestion. Banding patterns of MLVF or PFGE were analyzed using InfoQuestFP software.ResultsThe hands-on time of our modified method was about 3 h, on average, for each of 18 isolates. PFGE (80% cutoff) or MLVF (75% cutoff) separated all of the 63 isolates into 13 and 12 types, respectively. Three types generated by PFGE were identical to those generated by MLVF. PFGE and MLVF yielded similar Simpson's diversity indices, indicating similar discriminatory power. The overall concordance between PFGE and MLVF was low, as represented by adjusted Rand indices (0.266-0.278). PFGE predicted MLVF type better than MLVF predicted PFGE type, as reflected by Wallace coefficients (PFGE cutoff 80% vs. MLVF cutoff 75%, 0.389 vs. 0.233). Analysis of the relationship between a pair of isolates showed 91.0% concordance between the PFGE (80% cutoff) and MLVF (75% cutoff).ConclusionsOur simple, low-cost, modified MLVF protocol can effectively discriminate between S. aureus clinical isolates. MLVF can replace PFGE for the hospital infection control of S. aureus.

Use of the multiple-locus variable-number tandem repeat fingerprinting method and microfluidic chips for rapid genotyping of Staphylococcus aureus isolates

The early detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and a rapid and accurate identification of sources and routes of transmission are important objectives in hospital settings. In this study we investigated the application potential of Multiple-Locus Variable number tandem repeat Fingerprinting (MLVF) combined with microfluidic technology for a rapid discrimination of MRSA lineages in outbreak settings.