Characterization of Methicillin-resistant Staphylococcus aureus Strains and Profiling of Panton-Valentine Leukocidin Virulence Gene in Pyogenic Infections from Patients Attending a Tertiary Care Center in North India.

BackgroundMethicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen associated with nosocomial and community infections. Panton Valentine leukocidin (PVL) is considered one of the important virulence factors of S. aureus responsible for destruction of white blood cells, necrosis and apoptosis and as a marker of community acquired MRSA. This study was aimed to determine the prevalence of PVL genes among MRSA isolates and to check the reliability of PVL as marker of community acquired MRSA isolates from Western Nepal.MethodsA total of 400 strains of S. aureus were collected from clinical specimens and various units (Operation Theater, Intensive Care Units) of the hospital and 139 of these had been confirmed as MRSA by previous study. Multiplex PCR was used to detect mecA and PVL genes. Clinical data as well as antimicrobial susceptibility data was analyzed and compared among PVL positive and negative MRSA isolates.ResultsOut of 139 MRSA isolates, 79 (56.8 %) were PVL positive. The majority of the community acquired MRSA (90.4 %) were PVL positive (Positive predictive value: 94.9 % and negative predictive value: 86.6 %), while PVL was detected only in 4 (7.1 %) hospital associated MRSA strains. None of the MRSA isolates from hospital environment was found positive for the PVL genes. The majority of the PVL positive strains (75.5 %) were isolated from pus samples. Antibiotic resistance among PVL negative MRSA isolates was found higher as compared to PVL positive MRSA.ConclusionOur study showed high prevalence of PVL among community acquired MRSA isolates. Absence of PVL among MRSA isolates from hospital environment indicates its poor association with hospital acquired MRSA and therefore, PVL may be used a marker for community acquired MRSA. This is first study from Nepal, to test PVL among MRSA isolates from hospital environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1531-1) contains supplementary material, which is available to authorized users.

Aims: This study evaluated the influence of exposure to the hospital environment on methicillin-resistant Staphylococcus aureus (MRSA) carriage. The antibiograms of the MRSA isolates were examined. Materials and Methods: Nasal, throat, and web-space swabs were collected from 119 nursing students of the age group 18-23 years (exposed group) and 100 age-matched pharmacy students (nonexposed group). S. aureus was identified and antibiogram obtained as per Clinical and Laboratory Standards Institute (CLSI) guidelines. MRSA was detected by cefoxitin disc diffusion test and by growth on oxacillin screen agar as per CLSI guidelines. The presence of the mecA gene was confirmed by conventional polymerase chain reaction. Results: The MRSA carrier rates were 11.8% and 4% in the exposed and nonexposed groups, respectively. Association of exposure to the hospital environment with MRSA colonization was statistically significant. All MRSA isolates showed sensitivity to netilmicin, linezolid, tetracycline, vancomycin and teicoplanin. Among the exposed group, 71.4% MRSA isolates were resistant to ciprofloxacin, 64.3% to cotrimoxazole, 64.3% to erythromycin, 28.6% to gentamicin and 21.4% to clindamycin. Among the nonexposed group, 75% MRSA isolates were resistant to ciprofloxacin, 25% to cotrimoxazole, 25% to erythromycin, 25% to gentamicin and 25% to clindamycin. Conclusion: Exposure to the hospital environment was found to be a significant risk factor for MRSA carriage. Hospital-acquired MRSA (HA-MRSA) isolates showed greater resistance toward antimicrobials compared with community-acquired MRSA (CA-MRSA) isolates. This highlights the need for the appropriate institution of pharmacotherapy in cases of HA-MRSA and CA-MRSA infections and control of transmission by carriers.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial and community infections. These infections are becoming increasingly difficult to combat, because of emerging resistance to all classes of antibiotics. Panton-Valentine leukocidin (PVL) is an important virulence factor in MRSA and causes white blood cell destruction, necrosis, and accelerated apoptosis. The aim of this study was to determine the frequency of pvl-positive MRSA in a pediatric hospital, in Marrakech, Morocco. 53 isolates of MRSA were recovered in the hospital from December 2010 to May 2014, and confirmed with biochemical tests (coagulase, mannitol fermentation, and DNase). Then, polymerase chain reaction (PCR) was used to detect pvl. Among the 259 Staphylococcus aureus isolates collected from various clinical specimens, 53 were identified as MRSA; and the presence of the PVL gene was investigated in them using PCR analysis. Out of the 53 MRSA isolates, only 1 (1.89%) was positive for pvl This pvl-positive MRSA isolate was characterized as staphylococcal cassette chromosome mec IV (SCCmec IV), a type commonly associated with community-acquired MRSA infections. The study revealed a relatively low prevalence of PVL-positive MRSA among pediatric patients at the University Hospital Center CHU Mohamed VI in Marrakech, with only 1.89% of MRSA isolates testing positive for pvl. Despite this low prevalence, the presence of PVL-positive strains accentuates a potential risk for severe infections in vulnerable children. These findings underscore the imperative need for sustained surveillance and rigorous infection control measures to mitigate the spread of MRSA and other resistant pathogens.

Background: The main pathogen causing various nosocomial infections, especially in burn patients, is Staphylococcus aureus. An important virulence factor of this bacterium is leukocidin, which binds to white blood cells and leads to apoptosis and necrosis. Objectives: In the present research, we aimed at investigating the prevalence of the panton-valentine leukocidin (PVL) and LucED genes in methicillin-susceptible and resistant isolates of S. aureus isolated from burn patients in Kermanshah, Iran. Methods: Overall, 73 isolates of S. aureus were collected from burn wounds and identified by specific biochemical tests. After determining the susceptibility patterns of the isolates by the disc diffusion method, the frequencies of the PVL and LucED genes were assessed using specific primers and polymerase chain reactions. Results: The rates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) isolates were 58.9% (43 isolates) and 41.1% (30 isolates), respectively. In MRSA isolates, the highest antibiotic resistance was to penicillin (100%) and gentamicin (81.4%). Moreover, the frequencies of the LucED and PVL genes were 76.7% (56 isolates) and 27.4% (20 isolates), respectively, and the highest frequency of the LucED and PVL genes was observed in MRSA (81.4%) and MSSA (40%) isolates, respectively. Conclusions: Increased multidrug resistance pattern among S. aureus isolates with leukocidin production has led to treatment failure and severe infections. The development of infections can be prevented by the identification of isolates carrying the leukocidin gene, and early detection of MRSA isolates is feasible by using the PVL gene.

Background & objectives:The two major genotypic markers that distinguish community acquired (CA) from hospital acquired (HA) methicillin resistant Staphylococcus aureus (MRSA) isolates are the architecture of mobile genetic element (SCCmec type) and presence of panton valentine leukocidin (PVL) toxin. This study was conducted to determine the molecular characteristics of CA- and HA- MRSA and methicillin sensitive S. aureus (MSSA) isolates in Sikkim.Methods:A total of 150 clinical isolates of S. aureus isolated from various clinical specimens were subjected to duplex (mec-A and pvl gene) and multiplex (SCCmec typing) PCR.Results:Of the 150 isolates, 53 (35.33%) and 66 (44%) were positive for mec-A (MRSA) and pvl genes, respectively. Thirty eight (25.33%) met the definition of CA-MRSA and 15 (10%) of HA-MRSA and the remaining 63 (42%) and 34 (22.66%) as CA- and HA-MSSA, respectively. No significant difference was seen in the distribution of PVL toxin in MRSA and MSSA isolates, but it was significantly (P<0.001) high in overall MRSA isolates than in MSSA. The majority of the MRSA isolates showed a double amplification band of SCCmec type III plus V (54.71%), and only a fewer isolates were amplified by single DNA fragments of type I (1.88%), III (3.77%), IVa (1.88%) and V (11.32%). SCCmec types I, III, IVa, were found only in HA-MRSA isolates, whereas type V in both the CA- and HA-MRSA. AST pattern showed that 18.42 per cent (7/38) and 46.66 per cent (7/15) were multidrug resistant (MDR)-CA-MRSA and MDR-HA-MRSA, respectively.Interpretation & conclusions:The present results show that SCCmec type V MRSA has been on the rise, and genotypic markers such as pvl gene detection used for the differentiation of these clinically distinct isolates of MRSA may not be reliable.

Staphylococcus aureus is associated with several infections and emergence of antibiotic resistance is the major hindrance faced worldwide to combat them. Alternation in the Penicillin binding protein (PBP2A) results in the decreased binding of β –lactam antibiotics which lands up in Penicillinase resistance, thereby giving rise to Methicillin-Resistant Staphylococcus aureus (MRSA) organisms. Panton Valentine Leukocidin (PVL) is a cytotoxin, one of the β-pore forming toxins. It belongs to the family of synergohymenotropic toxins, which has the ability to form pores in the membrane by its synergistic actions. Our study intends to analyze the distribution of PVL genes among the Methicillin resistant Staphylococcus aureus isolates from our part of the country by performing Polymerase chain reaction. Phenotypic identification of Methicillin resistant staphylococcus aureus was carried out by Cefoxitin disk diffusion method. Panton-valentine Leukocidin gene among the MRSA isolated was characterized by Polymerase chain reaction. Out of all the MRSA samples, 24% (12/50) were positive for PVL gene at 433 bp by gradient polymerase chain reaction and 76% (38/50) were negative for PVL gene among the MRSA isolates. All the isolated MRSA were clinically classified as HA-MRSA and CA-MRSA. Among the CA-MRSA, 9 (33.3%) were found to be PVL positive whereas only 3 (13%) HA-MRSA were seen as PVL gene positive. The present study highlights the high prevalence of PVL gene from the community acquired MRSA isolates.

To the Editor: Methicillin-resistant Staphylococcus aureus (MRSA) strains account for >40% of all hospital-acquired S. aureus infections in Italy (1). Although cases of community-acquired MRSA (CA-MRSA) infections have been reported in recent years (2), these isolates have not been characterized for Panton-Valentine leukocidin (PVL) (3); therefore, the presence of isolates with the typical characteristics of CA-MRSA (4) in Italy remains unknown. At the beginning of April 2005, a 37-year-old woman was admitted to the University Hospital Policlinico in Rome because of fever, cough, and headache. Her medical history was unremarkable. She was a teacher in a school for foreign students in Rome, smoked 3 cigarettes per day for 15 years, and reported no recent travel abroad. Her 5-year-old daughter had influenzalike symptoms in the previous week. At hospital admission, her temperature was 39°C, heart rate was108 beats/min, respiratory rate was 32 breaths/min, and blood pressure was 105/70 mmHg. Arterial blood gas analysis showed mild hypoxemia and hypocapnia (PaO2 73 mm Hg and PaCO2 34 mm Hg on room air). Leukocyte count was 24,360 cells/μL (81% polymorphonuclear cells), and platelet count was 506,000/μL. Chest radiograph showed infiltrates in the right upper and lower lobes and left lower lobe. Empiric treatment with clarithromycin and ceftriaxone was started, but the patient's clinical conditions did not improve. Culture of sputum samples obtained at admission yielded growth of MRSA. Computed tomographic scan showed multiple lung cavitary lesions, indicating necrotizing pneumonia. On day 3 of admission, antimicrobial drug therapy was changed to linezolid (600 mg 2 times a day). Fever resolved, and the patient's condition rapidly improved. The patient was discharged after 14 days of linezolid treatment. At discharge, leukocyte count was 6,040 cell/μL (58% polymorphonuclear cells), and arterial blood gas analysis showed PaO2 of 88 mm Hg. The MRSA isolate from sputum was susceptible to all the non–β-lactam antimicrobial drugs tested, including erythromycin, clindamycin, ciprofloxacin, tetracycline, kanamycin, and fusidic acid. With established molecular methods, the isolate was found to harbor SCCmec type IV (5); lukS and lukF, the genes coding for the 2 subunits of the PVL toxin; and hlg, the γ-hemolysin gene (3). The genetic background of the isolate was determined by multilocus sequence typing (MLST) (6) and sequence typing of the tandem repeat region of protein A gene (spa typing) (7). Results showed that the isolate belonged to ST30 according to the MLST database (http://saureus.mlst.net), and spa typing, analyzed by the Ridom Staphtype software (http://www.ridom.de), indicated a novel spa type, to which type 755 was assigned. ST30, 1 of 6 clones more commonly associated with PVL-positive CA-MRSA (4), is designated also the southwest Pacific (SWP) clone, because of the area in which it circulates. Recently, the SWP clone has caused CA-MRSA infections in northern European countries (England, Scotland, the Netherlands, Sweden, and Latvia) (8,9). Molecular analysis suggests that the SWP clone has evolved from a methicillin-susceptible clone of S. aureus, termed phage type 80/81, that was pandemic in the 1950s and considered to be unusually virulent and transmissible (8). In fact, strains belonging to phage type 80/81 carry the PVL gene and appear to have subsequently acquired methicillin resistance through horizontal transfer of SCCmec type IV. The spa type of the Italian isolate comprises 7 nucleotide repeats, indicated by XJ4AKAOM in the alphabetical code. This repeat sequence differs from that of the classical SWP clone, indicated by XKAKAOMQ (8), by only 1 bp in the second repeat and loss of the last Q repeat. In spite of these differences, the spa type is in substantial agreement with the MLST result and indicates that the Italian isolate is either a descendent or a local variant of the SWP clone. The most common clone of CA-MRSA described in Europe is ST80, spa type 44. CA-MRSA belonging to ST80 tend to be more antimicrobial drug resistant than isolates belonging to other clones (4). Resistance to fusidic acid, typical of ST80, has been proposed as a marker for CA-MRSA in Europe (10). In light of our finding, we cannot rely on resistance to fusidic acid to screen for PVL-producing CA-MRSA in our country. To our knowledge, this is the first report from Italy of necrotizing pneumonia caused by PVL-positive CA-MRSA. The presentation was typically that of a severe pneumonia that occurred in a previously healthy, young adult with no risk factors for MRSA acquisition, as described in other cases (11). This is also the first report of a SWP clone isolate in southern Europe; if the strain is circulating in Italy or is occasionally imported from the SWP area, whether our patient acquired it through contact with a foreign contact remains unknown.

Patients with community-acquired pneumonia (CAP) infected with methicillin-resistant Staphylococcus aureus (MRSA) strains carrying the Panton-Valentine leukocidin (PVL) gene have severe clinical presentation and poor clinical outcomes. Antibiotics that suppress toxin production have been suggested for the management of these patients. The objective of this study was to compare the severity of disease and clinical outcomes of patients with hospital-acquired pneumonia/ventilator-associated pneumonia (HAP/VAP) infected with MRSA carrying the PVL gene with those patients infected with MRSA strains that do not carry the PVL gene. This was a multicenter observational study of patients with HAP and VAP. MRSA isolates were subjected to genetic analysis to define the presence of the PVL gene, the USA type and the staphylococcal cassette chromosome mec type. Severity of disease was evaluated with the Acute Physiology and Chronic Health Evaluation II (APACHE II) score. The primary clinical outcome was mortality at hospital discharge. A total of 109 cases of MRSA HAP/VAP were evaluated. The incidence of PVL(+) MRSA was 27%. APACHE II score at diagnosis of HAP/VAP was 21 ± 8 for PVL(+) MRSA and 20 ± 6 for PVL(-) MRSA (P = .67). Mortality was 10% (3/29) for patients with PVL(+) MRSA versus 10% (8/80) for patients with PVL(-) MRSA (P > .99). In patients with HAP or VAP due to MRSA, severity of disease and clinical outcomes are not influenced by the presence of the PVL gene. Therapeutic strategies directed to block PVL exotoxin may not impact outcomes in these patients.

Methicillin-resistant Staphylococcus aureus (MRSA) expresses the Panton-Valentine leukocidin (PVL) virulence gene, which is associated with community and hospital-acquired severe MRSA infections. The objective of this study was to determine the prevalence and antibiotic susceptibility profile with a focus on the presence of the PVL gene among MRSA isolates in healthcare settings. A total of 1,207 clinical specimens and 304 hospital environment swabs were collected in a tertiary care hospital in Nepal, and investigated following basic microbiological techniques. S. aureus was confirmed with the coagulase test. An antibiotic susceptibility test (AST) was performed by the Kirby-Bauer method and screening for MRSA was carried out by the cefoxitin disc diffusion method guided by the Clinical and Laboratory Standards Institute (CLSI), 2020. DNA was extracted and used in a polymerase chain reaction (PCR) to detect mecA and PVL genes. Of the 1,511 samples, 45 (2.9%) S. aureus (23 clinical and 22 environmental) were isolated. Among them, 69.6% (16/23) and 27.3% (6/22) were MRSA in clinical and environmental isolates, respectively. Twelve (52.2%) clinical isolates and seven (31.8%) environmental isolates were multidrug resistant. The majority of isolates were susceptible to vancomycin and linezolid. The PVL gene was detected in 18.2% (n = 4/22) of the MRSA isolates, of which three were from clinical sources and one was from an environmental swab. The prevalence of MRSA, and PVL-producing S. aureus were higher in the hospital setting. Hence, immediate and urgent implementation of infection control and sanitation measures are needed in the hospital.

A variant of the Southern German clone of methicillin-resistant Staphylococcus aureus is predominant in Croatia

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant public health threat, causing skin and soft tissue infections (SSTIs) and severe complications in children. Virulence factors such as Panton-Valentine leucocidin (PVL) enhance its pathogenicity. This study investigates the epidemiology, antibiotic resistance and PVL production of community-acquired MRSA (CA-MRSA) infections among children. A nationwide multicenter retrospective study was conducted on children under 18 years diagnosed with CA-MRSA infections in Spain between 2018 and 2020. Positive MRSA cultures were obtained from biologic samples within the first 48 hours of hospital or primary care contact and analyzed for antibiotic susceptibility and PVL production. Epidemiologic, clinical and microbiologic data were described and compared with previous colonization data. Statistical analyses, including multivariate logistic regression, were performed to identify factors associated with infection. A total of 252 children with CA-MRSA infections were included, with a median age of 5.5 years (interquartile ranges: 2-10). SSTIs accounted for 89.3% of cases, and 23.6% required hospitalization. Migrant status (52.5%) and prior SSTIs (39.1%) were significantly associated with MRSA infection ( P < 0.05). MRSA strains causing infection exhibited high resistance to ciprofloxacin (32.2%) and clindamycin (18.3%). No differences in antibiotic susceptibility were observed between strains causing infection and colonization. Reinfection occurred in 21% of cases, despite 45.3% undergoing decolonization. PVL genes were detected in 76.9% of MRSA strains, and were significantly more frequent in infection compared to colonization ( P < 0.001). Migrant status and PVL toxin production are significant risk factors for MRSA infections. The high prevalence of antibiotic resistance highlights the importance of tailoring empirical treatment strategies based on local resistance patterns.

The Panton-Valentine leukocidin (PVL) genes of methicillin-resistant Staphylococcus aureus (MRSA) have previously been associated with severe infections. Here, the impact of the PVL genes on severity of disease and clinical outcome of patients with hospital-acquired pneumonia (HAP) or ventilator-associated pneumonia (VAP) due to MRSA was investigated in a single center observational study in a hospital in China. HAP due to MRSA was diagnosed in 100 patients and 13 of the patients were PVL positive, while VAP was diagnosed in 5 patients and 2 were PVL positive. The PVL positive patient group showed a significantly higher Acute Physiology and Chronic Health Evaluation (APACHE) II score (14.3 ±7.8 vs. 10.1 ±4.7, P = 0.005) and significantly more patients with CRP levels >80 mg/L (8/15 vs. 12/90, P = 0.006) or WBC counts >15x109/L (7/15 vs. 12/90, P = 0.006), indicating that the severity of disease is affected by the presence of the PVL genes. The outcome of the study was defined by 30-day mortality. Four (27%) of the PVL positive patients and four (4%) of the PVL negative patients died within 30 days (P = 0.01, Fisher exact test). Kaplan-Meier survival curves were generated for the PVL positive and PVL negative patient groups, which differed significantly (P = 0.003). Among the patients that died, the mean interval between diagnosis and death was shorter for the PVL positive patients (9.3 ±5.6 vs. 40.8 ±6.6 days, P = 0.013). Further analysis within the HAP and VAP patient groups showed that the presence of PVL in MRSA impacted the severity of disease and clinical outcome of HAP, but for VAP the number of patients included in the study was too low. In conclusion, in this single center study in a Chinese hospital the presence of the PVL genes in MRSA impacted the severity of disease and clinical outcome in patients with HAP due to MRSA.

can produce similar airway cast formation in humans; severe respiratory distress refl ects extensive obstruction of the respiratory system.Healthcare providers should be aware of the possibility of bronchial casts when examining children with infl uenza (H1N1) infection accompanied by atelectasis.Steroids can be administered early in infection to avoid cast formation, and antiviral drug therapy and respiratory support can be used for infl uenza (H1N1)-infected children in whom airway casts have developed.

with Atypical Resistance Profi le, Spain

The molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from neonates in a neonatal intensive care unit (NICU) were investigated by multilocus sequence typing (MLST), staphylocoagulase (SC) genotyping, staphylococcal cassette chromosome mec (SCCmec) typing, accessory gene regulator (agr) typing, and the presence of Panton-Valentine leukocidin (PVL). Among the 44 S. aureus isolates from nares in neonates between March and June 2014 at hospital in Busan, 27 (61.4%) were MRSA and 17 (38.6%) were methicillin-susceptible S. aureus (MSSA). The most prevalent clone in MRSA isolates was ST72-SC type Vb-SCCmec IV-agr I (n=26) and the remaining one was ST89-SC type I-SCCmec II-agr II. In MSSA isolates, the prevalent clone was ST121-SC type Va-agr IV (n=13), followed by ST72-SC type Vb-agr I (n=2), ST8-SC type III-agr I (n=1) and ST15-SC type X-agr II (n=1). All isolates did not possess the PVL. The data showed that the neonates in NICU carried high prevalence of ST72 MRSA and remarkably different clones with SC diversity between MRSA and MSSA isolates.