Bactericidal efficacy of molybdenum oxide nanoparticles against antimicrobial-resistant pathogens.

Multidrug-resistant bacteria pose a major threat to effective antibiotics and alternatives to fight multidrug-resistant pathogens are needed. We synthetized molybdenum oxide (MoO3) nanoparticles (NP) and determined their antibacterial activity against 39 isolates: (i) eight Staphylococcus aureus, including representatives of methicillin-resistant S. aureus epidemic clones; (ii) six enterococci, including vancomycin-resistant isolates; and (iii) 25 Gram-negative isolates (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae), including extended spectrum beta-lactamases and carbapenemases producers. All isolates showed a MoO3 NP MIC of 700-800 mg l-1. MoO3 NP produced a clear inhibition zone for S. aureus and all Gram-negative isolates at concentrations ≥25 mg ml-1 and ≥50 mg ml-1 for enterococci. When the NP solutions were adjusted to pH ~7, the biocidal activity was completely abolished. MoO3 NP create an acidic pH and show a universal antimicrobial activity against susceptible and resistant isolates belonging to the most relevant bacterial species responsible for hospital-acquired infections.

Frequent MRSA nasal colonization among hospitalized children and their parents in Angola and São Tomé and Príncipe.

BACKGROUND: The prevalence of nosocomial meticillin-resistant Staphylococcus aureus (MRSA) was previously estimated as 23% in a paediatric hospital in Luanda, Angola and 18% in a general hospital in São Tomé and Príncipe. AIM: To evaluate the prevalence of S. aureus/MRSA colonization among hospitalized children and their parents at two hospitals in Angola and São Tomé and Príncipe. METHODS: In 2017, 127 hospitalized children and 129 of their parents had nasal swabs for S. aureus/MRSA carriage in the two countries. The isolates were tested for the presence of the mecA and Panton-Valentine leukocidin (PVL) genes, and characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and SCCmec typing. FINDINGS: Twenty of 127 children (15.7%) and 13 of 129 parents (10.1%) were MRSA nasal carriers. Three lineages comprised 88% of the MRSA isolates: (i) PFGE A-ST5-SCCmec IVa (N=15; 45%), associated with spa type t105, recovered in Angola alone; (ii) PFGE N-ST8-IV/V (N=7; 21%), associated with spa types t008/t121, recovered in São Tomé and Príncipe alone; and (iii) PFGE B-ST88-IVa (N=7; 21%), associated with spa types t325/t786, present in both countries. Fifteen child/guardian pairs were colonized with identical MRSA (N=8) or meticillin-susceptible S. aureus (N=7) strains. PVL was detected in 25% of isolates, including two MRSA (ST30-V and ST8-IVa). CONCLUSION: Hospitalized children and their parents are important reservoirs of MRSA. Infection control measures should focus on parents in order to minimize the spread of MRSA to the community.

Antimicrobial activity of octenidine against multidrug-resistant Gram-negative pathogens.

Multidrug-resistant (MR) Gram-negative (GN) pathogens pose a major and growing threat for healthcare systems, as therapy of infections is often limited due to the lack of available systemic antibiotics. Well-tolerated antiseptics, such as octenidine dihydrochloride (OCT), may be a very useful tool in infection control to reduce the dissemination of MRGN. This study aimed to investigate the bactericidal activity of OCT against international epidemic clones of MRGN. A set of five different species (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, and Pseudomonas aeruginosa) was studied to prove OCT efficacy without organic load, under "clean conditions" (0.3 g/L albumin) and under "dirty conditions" (3 g/L albumin + 3 mL/L defibrinated sheep blood), according to an official test norm (EN13727). We used five clonally unrelated isolates per species, including a susceptible wild-type strain, and four MRGN isolates, corresponding to either the 3MRGN or 4MRGN definition of multidrug resistance. A contact time of 1 min was fully effective for all isolates by using different OCT concentrations (0.01% and 0.05%), with a bacterial reduction factor of >5 log10 systematically observed. Growth kinetics were determined with two different wild-type strains (A. baumannii and K. pneumoniae), proving a time-dependent efficacy of OCT. These results highlight that OCT may be extremely useful to eradicate emerging highly resistant Gram-negative pathogens associated with nosocomial infections.

Frequent occurrence of trimethoprim-sulfamethoxazole hetero-resistant Staphylococcus aureus isolates in different African countries.

High rates of trimethoprim/sulfamethoxazole (SXT) resistance, a combination of two antifolate antibiotics trimethoprim (TMP) and sulfamethoxazole (SMZ), have been reported among Staphylococcus aureus isolates in Portuguese-speaking African countries. Our study aimed to evaluate the occurrence of TMP resistance markers in major SXTR methicillin-resistant S. aureus (MRSA) clones from these countries. We accessed also different fitness traits that could explain the success of these isolates over the Brazilian MRSA (the most successful SXTR MRSA clone worldwide but never identified in these countries). Minimum inhibitory concentrations for SXT, TMP and SMZ were determined, and genes encoding TMP resistance (dfrG, dfrA, dfrK and dfrB) were searched. Representatives of the Brazilian clone and of the major MRSA African clones were evaluated for their fitness by individual growth curves, competition assays, survival under desiccation, autolytic activity, resistance to oxidative stress, and also growth at high osmolarity and in acid and alkaline environments. Although all African isolates showed high-level resistance to TMP, the majority presented hetero-resistance to SXT. TMP resistance was linked to the presence of dfrG (78%), dfrA (19%) or both (3%) genes. Compared to the Brazilian clone, the African isolates showed higher growth rates and autolytic activity, and better survival to desiccation and alkaline conditions. Since isolates exhibiting SXT hetero-resistance are frequent in Africa, the implementation of standardized guidelines to detect this phenomenon is of major interest. The predominant MRSA clones in Portuguese-speaking African countries likely possess significant advantages over other clones, such as the Brazilian MRSA, that may explain their epidemiological success.

Methicillin-resistant Staphylococcus aureus among animals: current overview.

Currently, methicillin-resistant Staphylococcus aureus (MRSA) is a universal threat. After being well established in the healthcare setting, it has emerged in the community among people with no risk factors for MRSA acquisition, therefore imposing a new threat. The subsequent detection of MRSA colonizing or infecting animals as well as in food of animal origin was of major concern, revealing new reservoirs for MRSA. The major MRSA clonal lineages circulating in the different settings, i.e. in hospitals, in the community and among animals, are described here, differentiating between clones colonizing companion and food-chain animals. Particular attention is given to the widely spread livestock-associated MRSA clonal complex (CC) 398, which is mainly associated with professional exposure but may be of high pathogenicity. The recent detection of a mecA homologue, designated mecC, with a wide geographical distribution in Europe, and including a large diversity of hosts (food-chain, companion and wildlife animals and also detected in water samples) adds to the threat. Domestication as well as globalization of the livestock industry have intensified exchanges between human and animal bacteria. We report here several cases of transmission of MRSA between companion or food-chain animals and humans, as well as some MRSA clones of human origin that have adapted to new animal hosts eventually by losing useless virulence factors or acquiring new mobile genetic elements.

Contamination of public transports by Staphylococcus aureus and its carriage by biomedical students: point-prevalence, related risk factors and molecular characterization of methicillin-resistant strains.

OBJECTIVES: To analyse the contamination of public transports by Staphylococcus aureus and assess its carriage by biomedical students, focussing on the point-prevalence, related risk factors and molecular characterization of methicillin-resistant strains. STUDY DESIGN: Cross-sectional survey. METHODS: Methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolated from handrails of buses (n = 112) and trains (n = 79) circulating in Porto and from nasal swabs of local university students (n = 475) were quantified, characterized by molecular typing methods and related to possible risk factors. RESULTS: The MRSA prevalence in buses (16.1%) was not significantly different from trains (8.9%). There was also no identifiable association between the counts of MSSA and MRSA in buses and trains and the number of travellers in each sampling day, specific routes (including those passing by main hospitals) or other risk factors. Of the students, 37.1% carried S. aureus, and having a part-time job or smoking were found to be risk factors for carriage. EMRSA-15 (ST22-SCCmecIVh) was the prevalent MRSA clonal lineage, found not only in the buses (n = 14) and trains (n = 2) but also in the single MRSA-carrier among the students. The characteristics of the community-associated Southwest Pacific MRSA clone were found in a single ST30-IVa isolate, which may suggest a recent SCCmec acquisition by an MSSA background in the community. CONCLUSIONS: The spread of EMRSA-15, a common hospital-associated lineage, among different public transports and as a nasal coloniser is of concern and warrants adequate public health control measures.

Staphylococcus aureus in former Portuguese colonies from Africa and the Far East: missing data to help fill the world map.

The aim of the present study was to determine the prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage among patients and healthcare workers in Angola (ANG), São Tomé and Príncipe (STP), Cape Verde (CV) and East Timor (ET), and to characterize the antimicrobial susceptibility, virulence content and population structure of all S. aureus. Despite the importance of MRSA as a major human pathogen, data from these former Portuguese colonies in Africa and Asia are scarce. A total of 2065 nasal swabs recovered between 2010-14 were included in the study. Antimicrobial susceptibility testing and molecular characterization of S. aureus showed: (i) a very high MRSA prevalence in ANG (61.6%), moderate in STP (25.5%), low in CV (5.6%) and null in ET; (ii) a high prevalence of Panton-Valentine leukocidin in STP (36.8%), ET (29.2%) and CV (28.3%) contrasting with ANG (7.9%); (iii) ST5-SCCmecIVa, ST8-IV/V and ST5-VI were the major MRSA clones in ANG (65.2%), STP (44.8%) and CV (50%), respectively; (iv) a high resistance to trimethoprim-sulfamethoxazole in ANG (66.5%) and STP (50.9%), to rifampin in ANG (77.3%), and to tetracycline in STP (26.3%) and ET (20.8%); (v) three major methicillin-susceptible S. aureus clones (ST15, ST508, ST152) were present in all four countries. Age <18 years (OR 2.03, 95% CI 1.24-3.31), previous surgery (OR 2.45, 95% CI 1.24-4.83), no smoking (OR 4.04, 95% CI 1.05-15.50), and longer hospitalization (OR 2.53, 95% CI 1.49-4.28) were risk factors for MRSA carriage. This study provided the first comprehensive overview on MRSA in former Portuguese colonies in Africa and Asia, missing data in the world map.

High prevalence of ST121 in community-associated methicillin-susceptible Staphylococcus aureus lineages responsible for skin and soft tissue infections in Portuguese children.

In order to evaluate the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Portugal, we analyzed a collection of 38 S. aureus isolates recovered from 30 children attending the pediatric emergency department of a central hospital in Lisbon due to skin and soft tissue infections. Molecular characterization identified seven clonal lineages among the 35 methicillin-susceptible S. aureus (MSSA) isolates, of which the major lineage PFGE A/t159/ST121 included 63% of the isolates. The three MRSA isolates belonged to the Pediatric clone PFGE D/t535/ST5-IV (n = 2) and to the European CA-MRSA clone PFGE G/t044/ST80-IVc (n = 1). All isolates harbored several virulence factors, namely, leukocidins. Panton-Valentine leukocidin (PVL) was produced by isolates from five MSSA lineages and by the ST80 MRSA. Of interest, this is the first reported isolation of CA-MRSA ST80 in Portugal.

Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI.

In order to obtain insights into the methicillin-resistant Staphylococcus aureus (MRSA) population structure in the Azores archipelago, 106 MRSA isolates were collected from patients attending an Azorean central hospital between January 2007 and February 2008. Antimicrobial resistance was determined for all isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec) typing and the presence of Panton-Valentine leukocidin (PVL). The majority of the isolates (87%, n = 92) belonged to the EMRSA-15 clone (ST22, SCCmec-IVh), followed by the Pediatric clone (ST5-VI/IVc) (11%, n = 12). The Berlin clone (ST45-IVa) and a new clone (spa type t1839, ST1339 and SCCmec V variant) were represented by single isolates. All of the isolates carried SCCmec types IV, V or VI and a non-multiresistant antibiotic profile, resembling the currently emerging community MRSA. Moreover, PVL was described for the first time to be associated with the Pediatric clone carrying SCCmec type VI. We provided the first description of the population structure of MRSA in the Azores islands, which seems to be shaped by genetic events occurring locally, as well as by the regular population exchange between the islands, continental Portugal, the United Kingdom and the United States.

Identification of the first vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolate from a hospital in Portugal.

A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) with intermediate resistance to vancomycin (minimal inhibitory concentration [MIC] of 4 mug/ml) was isolated in 2006 from a surgical wound of a patient hospitalized at the orthopedics ward of Hospital de São Marcos--Braga, in the town of Braga. A combination of molecular typing methods, including pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and staphylococcal chromosomal cassette mec typing, identified the vancomycin intermediate-resistant S. aureus VISA-BRAGA as a derivative of the epidemic MRSA (EMRSA)-15 clone, which has been isolated with increasing frequency from several Portuguese hospitals recently. Compared to another EMRSA-15 isolate with the same genetic background (including PFGE subtype) the VISA-BRAGA isolate exhibited relatively high oxacillin MIC, slow growth, loss of hemolytic activity, and increased resistance to vancomycin and to daptomycin although neither of these two antibiotics was used in therapy. The VISA-BRAGA isolate described here appears to represent the first S. aureus with decreased susceptibility to vancomycin identified in a Portuguese hospital.

Replacement of methicillin-resistant Staphylococcus aureus clones in Hungary over time: a 10-year surveillance study.

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Hungary has been increasing and is now close to 20% among invasive isolates of S. aureus. In order to understand the evolution of MRSA in Hungary, two collections of isolates were studied: 22 representatives of a collection of 238 MRSA isolates recovered between 1994 and 1998, and a collection of 299 MRSA isolates recovered between 2001 and 2004. The isolates were first characterised by pulsed-field gel electrophoresis (PFGE) and were distributed into 19 different PFGE patterns. Representatives of each pattern were further characterised by spa typing, multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. The Hungarian clone that was predominant in 1994-1998 (PFGE E, ST239-III) had almost disappeared in 2003-2004, being replaced by the Southern German clone (PFGE B, ST228-I) and the New York/Japan epidemic clone (PFGE A, ST5-II), which represented c. 85% of the 2001-2004 isolates. Thus, this study describes, for the first time, the co-dominance and extensive spread of the New York/Japan clone in a European country.

Unusually high prevalence of nosocomial Panton-Valentine leukocidin-positive Staphylococcus aureus isolates in Cape Verde Islands.

Characterization of nosocomial methicillin-susceptible Staphylococcus aureus isolates from Cape Verde showed that (i) Panton-Valentine leukocidin genes were present in 35% of the isolates and (ii) half of the collection had the same genetic background as methicillin-resistant pandemic clones. Introduction of the staphylococcal chromosome cassette mec (SCCmec) into virulent and epidemic isolates could pose serious threats to public health.

Molecular characterisation of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone in a Mexican hospital (1999-2003).

Methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 216), collected between January 1999 and May 2003 in a tertiary-care university hospital in Guadalajara, Mexico, were characterised by antibiotype, pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments, and hybridisation of ClaI digests with mecA- and Tn554-specific DNA probes. Representatives of the single clonal type found were analysed by spa typing, multilocus sequence typing and staphylococcal chromosomal cassette mec (SCCmec) typing, and were tested for the presence of 22 virulence determinants and agr type. A single PFGE pattern was identified, with minor variations over time, with spa type 2, sequence type 5, SCCmec type II, agr type 2 and the presence of the enterotoxin genes seg and sei, the gamma-haemolysin variant gene hlg-v and the leukocidin lukE-lukD genes. In addition, the isolates showed antimicrobial resistance to beta-lactams, macrolides, chloramphenicol and imipenem, and susceptibility to gentamicin, rifampicin, trimethoprim-sulphamethoxazole and vancomycin. Following its appearance in 1997, this clone spread within the hospital, and is now present in most of the hospital units and wards.

High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study.

Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

Comparison of genetic backgrounds of methicillin-resistant and -susceptible Staphylococcus aureus isolates from Portuguese hospitals and the community.

In order to understand the origins of the dominant methicillin-resistant Staphylococcus aureus (MRSA) clones in Portuguese hospitals, we compared the genetic backgrounds of nosocomial MRSA with methicillin-susceptible S. aureus (MSSA) isolates from the same hospitals (n=155) and from the community (n=157) where they were located. Pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, and agr type analysis revealed that the genetic backgrounds correspondent to the dominant MRSA clones in Portuguese hospitals during the last 15 years (Iberian ST 247, Brazilian ST 239, and EMRSA-15 ST 22) were scarcely or not found among the present MSSA collection. The four major MSSA clones encountered (A-ST 30, B-ST 34, C-ST 5, and H-ST 45) correspond, or are very similar, to the background of other international MRSA pandemic clones, i.e., EMRSA-16, New York/Japan, Pediatric, and Berlin clones. However, with the exception of the Pediatric clone, none of these MRSA clones has been detected in Portugal. Our findings suggest the three major MRSA clones identified in Portuguese hospitals have not originated from the introduction of SCCmec into dominant MSSA backgrounds present in the Portuguese nosocomial or community environment but were probably imported from abroad. In contrast, the MRSA Pediatric clone might have originated in our country by the acquisition of SCCmec type IV into MSSA clone C. Furthermore, we provide evidence that the introduction of SCCmec into sensitive clones is most likely a relatively infrequent event that seems to depend not exclusively on the presence of a successful MSSA lineage.

Delineation of the endemic and sporadic methicillin-resistant Staphylococcus aureus clones in a Czech hospital.

The aim of this study was to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected between September, 2001, and February, 2003, at the regional hospital of Nový Jicín, Czech Republic. The isolates were characterized by susceptibility tests, HindIII ribotyping, and pulsed-field gel electrophoresis. Representatives of each clonal type were analyzed by multilocus sequence typing and staphylococcal cassette chromosome mec (SCCmec) typing. The prevalence of the most important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aac6'-aph2", aph3', and ant4') resistance genes was evaluated as well. Our results document the existence of two international MRSA clones: (1) the Iberian clone (ST247:SCCmec IA:PFGE A:ribotype H2), endemic in the hospital and associated to a single multiresistant phenotype; and (2) clone EMRSA-15 (ST22:SCCmec IV:PFGE H-ribotype H7), appearing in the beginning of 2002 and associated with three phenotypes. These two clones could be distinguished by antibiogram, distribution of macrolide and aminoglycoside resistance genes (ermA, aac6'-aph2", ant4' versus ermC and msrA in a few isolates), production of beta-lactamase, and presence of enterotoxin A (in the Iberian clone).

Surveillance of methicillin-resistant Staphylococcus aureus in a pediatric hospital in Mexico City during a 7-year period (1997 to 2003): clonal evolution and impact of infection control.

Between 1997 and 2000 a single multidrug-susceptible methicillin-resistant Staphylococcus aureus clone, M (sequence type 30 [ST30]-staphylococcal cassette chromosome mec [SCCmec] type IV), was present in a pediatric hospital in Mexico City, Mexico. In 2001 the international multidrug-resistant New York-Japan clone (ST5-SCCmec type II) was introduced into the hospital, completely replacing clone M by 2002.

Update on the major clonal types of methicillin-resistant Staphylococcus aureus in the Czech Republic.

The purpose of our study was the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 21 hospitals in the Czech Republic in the period 2000-2002 and comparison with previous results from 1996-1997. Strains were analyzed by pulsed-field gel electrophoresis (PFGE) of SmaI digests and ribotyping of HindIII digests hybridized with a 16S-23S DNA probe. The prevalence of the most clinically important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aph3', ant4', and aac6'-aph2") resistance genes was evaluated as well. Selected isolates representative of each clonal type were analyzed by multilocus sequence typing and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. Our results document the displacement of the Brazilian clone (ST239, SCCmec type IIIA, PFGE type B, ribotype H1) by a new clone that we named "Czech clone" (ST239, SCCmec type IIIA, PFGE type F, ribotype H6) and the maintenance of the Iberian clone (ST247, SCCmec type IA, PFGE type A, ribotype H2) exclusively in one hospital in the Czech Republic. In addition, we found a correlation between the distribution of aminoglycoside resistance genes and MRSA clonal types.

Application of molecular typing methods to characterize nosocomial coagulase-negative staphylococci collected in a Greek hospital during a three-year period (1998-2000).

A total of 143 methicillin-resistant coagulase-negative staphylococci (MR-CNS) collected between 1998 and 2000 at the University Hospital of Patras, Greece, were characterized by antibiogram and genomic typing to define the clonal types endemic in this hospital and their evolution during the 3-year period. These isolates corresponded to 93 methicillin-resistant Staphylococcus epidermidis (MRSE) and 50 other MR-CNS, which were isolated from patients in different wards, exclusively from blood and catheter tips cultures. Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with mecA and murE DNA probes were performed. The application of these methodologies demonstrated the existence, persistence and spread of MRSE, MR-Staphylococcus haemolyticus, and MR-Staphylococcus hominis clones in this hospital, whereas the SmaI/murE hybridization pattern was shown to be a valuable tool for the MRSE identification.

Evolution of sporadic isolates of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and their similarities to isolates of community-acquired MRSA.

Forty-one methicillin-resistant Staphylococcus aureus (MRSA) hospital isolates that clearly differed from the six major pandemic clones of MRSA in pulsed-field gel electrophoresis type, mecA and Tn554 polymorphism, and epidemic behavior were selected from an international strain collection for more detailed characterization. SpaA typing, multilocus sequence typing, and SCCmec (staphylococcal cassette chromosome mec) typing demonstrated extensive diversity among these sporadic isolates both in genetic background and also in the structure of the associated SCCmec elements. Nevertheless, the isolates could be grouped into restricted clonal complexes by using the BURST (i.e., based upon related sequence types) program algorithm, which predicted that most sporadic MRSA isolates evolved from pandemic MRSA clones. Several of the sporadic MRSA resembled community-acquired MRSA isolates in properties that included a relatively limited multiresistance pattern, faster growth rates, diversity of genetic backgrounds, and a frequent association with SCCmec type IV.