Molecular epidemiology of methicillin-resistant Staphylococcus aureus from different population groups in Argentina.

OBJECTIVES: In Latin America, methicillin-resistantStaphylococcus aureus (MRSA) is a leading cause of nosocomial infections. Limited studies have addressed the molecular epidemiology of MRSA clones in Argentina, characterised by continuous human migratory movements. The aim of this study was to describe the MRSA epidemiology, including distinct patient populations from different regions of the country. METHODS: MRSA strains were collected in epidemiological studies conducted from 2009 to 2015 in three cities (Formosa, Córdoba and Tucumán) and involving four population groups: community adult patients; hospitalised adults; hospitalised children; and healthy children (nasal colonisation). Antimicrobial susceptibility testing, SCCmec and Panton-Valentine leukocidin (PVL) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed. RESULTS: A total of 120 MRSA isolates were recovered with an important population diversity in the groups studied; in community adult patients, MRSA isolates corresponded to ST5, ST267 and ST1619; from hospitalised adults they were ST97, ST5, ST72, ST125, ST200, ST647, ST747, ST935 and ST2941; from hospitalised children they were ST5, ST30, ST34, ST1163 and ST1619; and from colonised children they were ST5, ST125, ST34, ST100, ST1619, ST207 and ST1163. Results of SCCmec typing showed SCCmec I, SCCmec IIIA, SCCmec IV and SCCmec ND associated or not with PVL genes. CONCLUSIONS: MRSA genetic lineages have differing distribution in the three regions. The most prevalent was ST5 in colonisation, community and invasive settings. Here we describe ST34-SCCmec IV clone for the first time in the hospitalised paediatric population. These findings contribute to the understanding of epidemiological changes in recent years.

Virulence factors and clinical patterns of multiple-clone hypermucoviscous KPC-2 producing K. pneumoniae.

Carbapenemase-producing Klebsiella pneumoniae (CRKP) are increasingly reported worldwide being necessary the local epidemiological monitoring. Our aim was to characterize the hypermucoviscous CRKP isolates collected in our hospital during a 6 months period. Carriage of the carbapenemase genes (bla KPC, bla NDM, bla VIM and bla OXA-48), extended spectrum ß-lactamases (bla SHV-2, bla CTX-M) and the virulence genes (magA, k2A, rmpA, wabG, uge, allS, entB, ycfM, kpn, wcaG, fimH, mrkD, iutA, iroN, hly and cnf-1) were determined by multiplex-PCR. Genetic relationship among the isolates was performed by PFGE and MLST. A total of 35 isolates were recovered, being the urinary and respiratory tract the most common infection sites (34.2%). The bla KPC-2 gene was present in all the isolates, coexisting with bla CTX-M-2 (45.7%), bla SHV-2 (28.6%), and bla CTX-M-2/bla SHV-2 (14.3%). The capsular serotype K2 corresponded with 68.6% of the isolates. Virulence factors frequency were variable [adhesins (97.1%), siderophores (94.3%) and phagocytosis resistance (wabG 48.5%, uge 80% and ycfM 57.1%)]. A total of 10 STs were identified although 40% of them clustered on ST25-CC65, and 17% to ST17. The incidence of KPC-2-producing K. pneumoniae reported by the hospital was 0.290 per 1000 admissions. In summary we described an epidemic scenario of multidrug resistant hypermucoviscous KPC-2 producing ST25 K. pneumoniae in our institution.

TT virus infection: role of interferons, interleukin-28 and 29, cytokines and antiviral proteins.

In 1997 a novel virus in the serum of a patient with acute post-transfusion hepatitis of non A-G etiology was identified. This agent was designed TT virus (TTV). It produces persistent viremia and no disease, but the mechanism of its persistence is poorly understood. In the present study mRNA expression of antiviral proteins as MxA, 2' 5' OAS, anti-apopotic protein, cytokines IL- 28, IL- 29 and IFN are examined in a subject affected by B lymphoma and positive for TTV DNA and RNA in this cellular subset, and in BJAB and Dohh2 cell lines.

Immunological characterization of Toscana virus proteins.

The genome of Toscana virus (Bunyaviridae family, Phlebovirus genus) consists of three single stranded RNA segments (L, M, S), with negative polarity. The L and M segments contain a single ORF in viral complementary sense and the S segment contains two ORFs in "ambisense" orientation. The M segment codes for three proteins in 3'-5' genomic orientation: a 30 kDa non structural protein and two 65 kDa glycoproteins, GN, and GC. In this paper we report the expression in E. coli of the S segment ORFs and of three regions of the L ORF. The expressed proteins were used to produce monospecific polyclonal antibodies in mice. By using these antibodies the N and the NSs proteins were unequivocally assigned to the S viral-complementary and viral-sense ORFs, respectively, and the L protein to the L ORF. We have found that like N and L proteins, NSs protein is associated with the viral nucleocapsids in mature virions, suggesting its possible involvement in early events of viral replication. NSs protein was also found associated with cellular polysomes. In virus-infected cells the anti-L antibodies recognized proteins shorter than the full-length L protein, possibly products of L subgenomic segments. Interestingly these defective products were not found in mature virions, suggesting specific mechanisms in virion assembly.

Detection of Toscana virus-specific immunoglobulins G and M by an enzyme-linked immunosorbent assay based on recombinant viral nucleoprotein.

An enzyme-linked immunosorbent assay (ELISA) based on the recombinant Toscana virus nucleoprotein (rN) has been developed. Its sensitivity and specificity for the detection of virus-specific immunoglobulins G and M in human sera were similar to those of the ELISA that is based on an antigen extracted from infected mouse brain and that is routinely used for serodiagnosis.

Completion of molecular characterization of Toscana phlebovirus genome: nucleotide sequence, coding strategy of M genomic segment and its amino acid sequence comparison to other phleboviruses.

The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence determined. The M RNA segment is 4215 nucleotides in length, and it contains a single major open reading frame (ORF) in the viral-complementary sequence, between nucleotides 18 and 4034, which can encode for a polyprotein of 1339 amino acids (Mr 149 kDa). The viral segment is expressed via a unique mRNA containing 10-14 non-templated nucleotides at the 5' end and it is truncated at the 3' end by about 140 nucleotides in a purine-rich region. In M predicted amino acid sequences, several hydrophobic regions have been identified. They could function as a signal sequence or a transmembrane region for the different proteins. Comparison of the deduced amino acid sequence of M precursor product revealed 38, 36, and 25% identity and 58, 56, and 47% similarity with those of Rift Valley fever (RVF), Punta Toro (PT) and Unkuniemi (UUK) viruses, respectively. Residues conserved among the proteins are mainly located at the COOH-portion of the precursor, while the major divergence is in the NSm coding regions. Based on sequence comparison and similarity of hydropathic pattern of TOS M segment with other phleboviruses the N-termini of TOS GN and GC glycoproteins were placed at residues 297 and 936 of the precursor.

Organization of the M genomic segment of Toscana phlebovirus.

The nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open reading frame in the viral-complementary sequence, which can encode a polyprotein of 1339 amino acids. To map the TOS M segment product(s), different regions of the putative M polypeptide were expressed as glutathione S-transferase fusion proteins, which were purified and inoculated into mice to produce hyperimmune sera. By Western blot analysis, a protein of approximately 30 kDa and two glycoproteins, G1 and G2, with the same molecular mass (approximately 65 kDa) were identified in TOS virus-infected cells. The 30 kDa protein, which reacted with antibodies raised to the NH2-terminal, was found to be a non-structural protein (designated NSm). By immunoprecipitation analysis of TOS virus-infected cell lysates, both treated or untreated with tunicamycin, the relative positions of glycoproteins G1 and G2 were determined. The gene order, with respect to the genomic M RNA, was found to be 3' NSm-G1 -G2 5'