Unknown spa types, spa repeats, and relatedness of MRSA isolated from horses, dogs, cats, and their human handlers

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a public health threat and a major cause of hospital-acquired and community-acquired infections. This study aimed to investigate the genetic diversity of MRSA isolates from 2015 to 2017 and to characterize the major MRSA clones and anti-biogram trends in Palestine.Methodology: Isolates were obtained from 112 patients admitted to different hospitals of West Bank and East Jerusalem, originating from different clinical sources. Antibiotic susceptibility patterns, staphylococcal chromosomal cassette mec (SCCmec) typing, and Staphylococcus aureus protein A (spa) typing were determined. Also, a panel of toxin genes and virulence factors was studied, including: Panton-Valentine Leukocidin (PVL), ACME-arcA, Toxic Shock Syndrome Toxin-1 (TSST-1), and Exfoliative Toxin A (ETA).Results: Of the 112 confirmed MRSA isolates, 100% were resistant to all β-lactam antibiotics. Resistance rates to other non- β-lactam classes were as the following: 18.8% were resistant to trimethoprim-sulfamethoxazole, 23.2% were resistant to gentamicin, 34.8% to clindamycin, 39.3% to ciprofloxacin, and 63.4% to erythromycin. All MRSA isolates were susceptible to vancomycin (100%). Of all isolates, 32 isolates (28.6%) were multidrug- resistant (MDR). The majority of the isolates were identified as SCCmec type IV (86.6%). The molecular typing identified 29 spa types representing 12 MLST-clonal complexes (CC). The most prevalent spa types were: spa type t386 (CC1)/(12.5%), spa type t044 (CC80)/(10.7%), spa type t008 (CC8)/(10.7%), and spa type t223 (CC22)/(9.8%). PVL toxin gene was detected in (29.5%) of all isolates, while ACME-arcA gene was present in 18.8% of all isolates and 23.2% had the TSST-1 gene. The two most common spa types among the TSST-1positive isolates were the spa type t223 (CC22)/(Gaza clone) and the spa type t021 (CC30)/(South West Pacific clone). All isolates with the spa type t991 were ETA positive (5.4%). USA-300 clone (spa type t008, positive for PVL toxin gene and ACME-arcA genes) was found in nine isolates (8.0%).Conclusions: Our results provide insights into the epidemiology of MRSA strains in Palestine. We report a high diversity of MRSA strains among hospitals in Palestine, with frequent SCCmec type IV carriage. The four prominent clones detected were: t386-IV/ CC1, the European clone (t044/CC80), Gaza clone (t223/CC22), and the USA-300 clone (t008/CC8).

The goal of this study was to delineate the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in Taiwan. Ninety-six MRSA isolates were collected from the blood cultures of different patients during the period July to December of 2008. The spa typing, staphylococcal chromosomal cassette (SCCmec) typing, mec-associated direct repeat unit (dru) copy numbers, and toxin genes (sea, seb, sec, tst, lukS/F) of each isolate were determined. Thirty-eight, 28, 18, and 12 MRSA isolates were SCCmec type II, SCCmec type III, SCCmec type IV, and SCCmec type V, respectively. Most (31/38, 81.6%) of the SCCmec type II isolates were of spa t002 with four dru repeats. Some of them also carried the sec or tst toxin gene (67.7 and 80.6%, respectively). Of the 28 SCCmec type III MRSA isolates, 15 (53.6%) were of t037 with 14 dru repeats, and all also carried the sea gene. Of the 18 SCCmec type IV MRSA isolates, 13 (72.2%) were of t437 with nine dru repeats, and ten of them also had the seb gene. Among the SCCmec type V MRSA isolates, nine were type V(T). Five (55.6%) of them were of t437 with 11 dru repeats, and all contained the lukS/F gene. The clonal spreading of SCCmec MRSA strains with specific spa and dru types was found. Further longitudinal, multiple-site surveillance is required in order to define the MRSA evolution in Taiwan.

Characterization of SCCmec and spa types of methicillin-resistant Staphylococcus aureus isolates from health-care and community-acquired infections in Kerman, Iran

Assess the best approach to type methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcal protein A (spa) typing, multiple-locus variable number tandem repeat analysis (MLVA) or both. Discriminatory power of spa typing and MLVA was determined using 20,771 MRSA isolates. There were twice as many MLVA types (MTs) as spa types present in the collection. Among the top 70% of the isolates, 37 spa types and 139 MTs were found. MLVA diversity among the top-10 spa types was high (diversity index 0.96), while spa diversity among the top-10 MTs was much lower (diversity index 0.83). The probability that two MRSA isolates with the same spa type also had the same MT was low (Wallace's coefficient 0.27). By contrast, most MRSA isolates yielding the same MT also had the same spa type (Wallace's coefficient 0.90). MLVA is superior to spa typing and will suffice to characterize MRSA isolates for surveillance.

Methicillin-resistant Staphylococcus aureus in retail meat, Detroit, Michigan, USA.

Methicillin-Resistant Staphylococcus aureus in Retail Meat, Detroit, Michigan, USA

Emergence of SCCmec type III with variable antimicrobial resistance profiles and spa types among methicillin-resistant Staphylococcus aureus isolated from healthcare- and community-acquired infections in the west of Iran

Molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients with bacteremia based on MLST, SCCmec, spa, and agr locus types analysis.

The aim of this study was to better understand methicillin-resistant Staphylococcus aureus (MRSA) at the molecular level by investigating the genotypic characteristics and evolutionary patterns of MRSA clones in Shenyang, China. We analyzed the molecular epidemiology of 60 MRSA isolates in Shenyang, China, between 2002 and 2008, using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and S. aureus protein A (spa) typing. They were examined for their antimicrobial susceptibilities. Among the 60 isolates, ST239 was identified most frequently (34 isolates; 58%), followed by ST5 (20 isolates; 34%). Nine spa types were obtained and 4 PFGE strain families (A, B, C, and D) were resolved. Spa type t030, which corresponded to PFGE genotypes A1, A3, and A4, constituted 45% (27/60) of all isolates; spa type t037, which corresponded to PFGE type A2, accounted for 13% (8/60) of all isolates. These 2 spa genotypes belonged to ST239 and carried SCCmec type III. Isolates genotyped as spa type t002 comprised 27% (16/60) of the study set and included isolates typed as PFGE B1 and B2, ST5, and SCCmec II. Most of MRSA isolates belonging to ST239 were susceptible to trimethoprim-sulfamethoxazole. The minimum inhibitory concentration (MIC50) of vancomycin among MRSA isolates belonging to ST5 (2 mg/l) was higher than that for other isolates (1 mg/l). These data document 2 major epidemic MRSA clones in Shenyang, China: ST239-MRSA-SCCmec type III-t037/t030 and ST5-MRSA-SCCmec type II-t002.

BackgroundCombination therapy including an aminoglycoside antibiotic and a cell-wall active agent is considered the most suitable option to treat invasive infections with methicillin-resistant Staphylococcus aureus (MRSA). Dual drug therapy enhances the effectiveness of treatment and reduces the risk of resistance development. This study aims to elucidate the phenotypic and molecular resistance to aminoglycosides and methicillin, and the molecular epidemiologic characteristics of S. aureus in Ardabil northwest Iran.MethodsTotally, 118 S. aureus isolates collected from clinical specimens were investigated. Identification was performed using standard microbiological and molecular approaches. Aminoglycoside and methicillin resistance were evaluated using the disk diffusion assay, and the minimum inhibitory concentrations (MICs) of aminoglycosides were determined via the agar dilution method. The mecA gene encoding methicillin resistance and aminoglycoside modifying enzymes (AMEs) genes were detected using PCR. Molecular epidemiologic features of the isolates were determined using staphylococcal cassette chromosome mec (SCCmec) typing spa typing and ERIC-PCR assays.ResultsOf the isolates, 42.4% (n = 50) and 57.6% (n = 68) were identified as MRSA and MSSA, respectively. All MRSA isolates were mecA-positive. Among MRSA isolates, SCCmec type IVa (17; 34%) was predominant, followed by types IVc, V, III, II, and I. Resistance rates to gentamicin, kanamycin, tobramycin, and amikacin were 16.1%, 17.8%, 8.5%, and 8.5%, respectively. Overall, the aminoglycoside resistance and most non-aminoglycoside antibiotics were significantly higher in MRSA versus MSSA isolates. The prevalence of AME genes was as follows: aac(6’)-Ie-aph(2’’) (30; 76.9%), aph(2’’)-Ib (22; 56.4%), and ant(4’)-Ia (14; 35.9%). About 60% of aminoglycoside-resistant isolates harbored ≥ 2 AME genes. The t030 type was the most common spa type identified. The ERIC-PCR profiles categorized the isolates into 19 unique ERIC types.ConclusionsThis study reveals high aminoglycoside and methicillin resistance in S. aureus isolates from Ardabil hospitals. Predominant SCCmec type IVa and spa type t030 indicate specific molecular patterns. These findings highlight the need for continuous surveillance and targeted treatment strategies for MRSA infections.Clinical trial numberNot applicable.

The aims of this study were to evaluate the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in retail foods in Shaanxi, China and to investigate antimicrobial resistance and molecular characteristics of these strains. A total of 1979 retail food samples were randomly collected during 2008-2012 from supermarkets and farmers markets and screened for S. aureus, and then S. aureus isolates were further examined to determine whether they were MRSA. MRSA isolates were further characterized by antimicrobial susceptibility test, pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, and SCCmec typing, and were examined for genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin 1. Among all the samples examined, four (1.4%) raw milk samples, six (2.3%) chicken samples, one (0.6%) pork sample, three (0.6%) ready-to-eat food samples, and three (2.5%) dumpling samples were positive for MRSA. No MRSA isolates were recovered from infant foods. A total of 23 MRSA isolates were recovered from the 17 MRSA-positive samples. Antimicrobial susceptibility tests showed that, among these MRSA isolates, resistance was most frequently observed to penicillin, ampicillin, oxacillin, cefoxitin, and clindamycin (each 100%), followed by erythromycin (95.7%) and clarithromycin (87.0%). The commonly detected toxin genes were pvl, seg, seb, sed, followed by see, sec, and sei. Seven spa types (t189, t377, t437, t899, t10793, t5762, and a new spa type) and three SCCmec types (II, IVb, and V) were identified. More than half (52.2%) of the MRSA isolates belonged to ST9, followed by ST88, ST59, ST188, ST72, and ST630. Our findings indicate that MRSA in food could be from both animal and human origin. Although the prevalence is low, the presence of multidrug resistant and enterotoxigenic MRSA strains in foods poses a potential threat to consumers and emphasizes the need for better control of sources of contamination.

To the Editor: It has recently become apparent that livestock can constitute a new methicillin-resistant Staphylococcus aureus (MRSA) reservoir and be a source of a novel and rapidly emerging type of MRSA. These livestock-associated MRSA clones are nontypeable by use of pulsed-field gel electrophoresis with SmaI and belong to sequence type (ST) 398 (1). MRSA ST398 clones account for 20% of all MRSA in the Netherlands (2), but the emergence of such clones has been described worldwide (3). Although ST398 transmission has been reported primarily between animals, persons with occupational exposure to livestock are at higher risk for MRSA carriage than the general population. Even though MRSA ST398 usually causes colonization, several cases of infections of variable clinical relevance, varying from skin and soft tissue infections (4) to endocarditis (5) and pneumonia (6), have been described over the past few years. Most instances of ST398 human carriers have been identified among persons who work at pig farms (7). Data regarding MRSA colonization of dairy farmers are less exhaustive and, to our knowledge, only 1 instance of direct transmission between cattle and humans has been proven. MRSA isolates from cows with subclinical mastitis in 2007 in Hungary were indistinguishable from MRSA isolates from the tonsil swab of a farmer who worked with these animals (8). We report a case of MRSA ST398 invasive disease in a cattle farmer, as well as a case of MRSA ST398 necrotizing fasciitis. In early April 2008, a 52-year-old man was admitted to an intensive care unit in Manerbio, Italy, because of severe sepsis and a large ulcerative and suppurative lesion on the right side of his neck. His medical history was unremarkable. He was a worker at a dairy farm, was obese, and did not report any previous contact with the healthcare system. At the time of hospital admission, he was oriented and cooperative. His temperature was 38.4°C, heart rate was 125 beats per minute, and blood pressure was 165/75 mm Hg. Arterial blood gas analysis showed hypoxemia and mild hypocapnia (PaO2 53 mm Hg and PaCO2 33.8 mm Hg on room air). Leukocyte count was 21,280 cells/μL (81.9% polymorphonuclear cells), and platelet count was 310,000 cells/μL. After blood samples were collected and aggressive surgical debridement of affected tissue was performed, empirical treatment with intravenous teicoplanin and imipenem was started. On the basis of histologic appearance of the intraoperative material and computed tomography scan images, necrotizing fasciitis was diagnosed. Culture of blood and necrotic tissue yielded MRSA. On day 3 after admission, antimicrobial drug therapy was changed to teicoplanin and clindamycin and, on day 7, to linezolid. Fever resolved in 3 days and the patient’s condition progressively improved. The patient was discharged after 31 days of antimicrobial drug therapy. The MRSA isolate was susceptible to all the non–β-lactam antimicrobial drugs tested (excluding tetracycline), carried the staphylococcal cassette chromosome mec type V, and was negative for Panton-Valentine leukocidin (PVL) genes. Multilocus sequence typing and sequence typing of the tandem repeat region of protein A gene (spa typing) showed that the isolate belonged to ST398 and spa type 899, respectively. Some issues are of concern. Although the MRSA isolate was PVL negative, its virulence resembled that of PVL-positive strains. Furthermore, it was resistant to tetracycline, as we expected because oxytetracyclines are the antimicrobial drugs most frequently used in pig and cattle farming (3). The major limitation of our study was that data regarding MRSA colonization of the farm are missing, so cattle-to-human transmission cannot be proven. However, because our patient did not have any other potential risk factor, dairy cows were probably the source of the human infection. In countries where community-acquired MRSA is common, all patients with serious S. aureus infections should be treated for MRSA until antimicrobial susceptibilities are known. Our report suggests that even in countries where community-acquired MRSA is still rare, being a cattle farmer may be considered an indication for early treatment against MRSA. The expanding knowledge of this zoonotic potential may undermine existing nosocomial MRSA control programs. In countries where a search and destroy policy (9) is adopted, such as the Netherlands, pig and cattle farmers may warrant screening and isolation at the time of hospital admission. Nevertheless, the first MRSA ST398 nosocomial outbreak has already been described (10). It is difficult to prevent persons with constant exposure to MRSA in their work or home setting from becoming MRSA carriers. Revisiting policies for the use of antimicrobial drugs on livestock farms, as well as improving hygiene measures, may therefore be necessary in infection control programs. However, before final recommendations can be made, further investigation is needed to determine the prevalence of MRSA among livestock and their handlers.

There is limited data on methicillin-resistant Staphylococcus aureus (MRSA) carriage in dental clinics. 1300 specimens from patients, health personnel, and environmental surfaces of a dental clinic in Egypt were tested for MRSA. Antibiotic susceptibility, biofilm formation, Staphylococcal protein A (spa) typing, SCCmec typing, and PCR-based assays were used to detect mecA, mecC, vanA, Panton-Valentine Leukocidin toxin (PVL), and toxic shock syndrome toxin-1 (tst) genes. Among 34 mecA-positive MRSA isolates, five (14.7%) were PVL-positive, seventeen (50%) were tst-positive, ten (29.4%) were vanA-positive, while none harboured mecC. MRSA hand carriage rates in patients, nurses, and dentists were 9.8%, 6.6%, and 5%. The respective nasal colonization rates were 11.1%, 6.7%, and 9.7%. 1.3% of the environmental isolates were MRSA-positive. Strong and moderate biofilm-forming isolates represented 23.5% and 29.4% of MRSA isolates. 24 MRSA isolates (70.6%) were multi-resistant and 18 (52.9%) harboured SCCmec IV. Among eight spa types, t223 (26.5%), t267 (23.5%), and t14339 (23.5%) were predominant. We noted an alarming genetic relatedness between 7 (20.6%) MRSA isolates and the epidemic EMRSA-15 clone, as well as a combined occurrence of tst and PVL in 3 (8.8%) isolates. Results suggest high MRSA pathogenicity in dental wards highlighting the need for more efficient surveillance/infection control strategies.

spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 (n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) in sheep and goat bulk tank milk from Southern Italy