Molecular characterization of KPC-2-positive Klebsiella pneumoniae isolates from a neurosurgical centre in Argentina.

Carbapenem-resistant Enterobacteriaceae is a growing concern worldwide. Klebsiella pneumoniae is an important nosocomial pathogen with a high capacity for nosocomial spread. We described the occurrence of plasmid-encoded KPC-2-harbouring K. pneumoniae isolates recovered from a neurosurgical centre in Argentina. The blaKPC-2 gene was surrounded by ISkpn6 and ISkpn7.

Widespread dispersion of the resistance element tet(B)::ISCR2 in XDR Acinetobacter baumannii isolates.

Acinetobacter baumannii is a significant nosocomial pathogen often associated with extreme drug resistance (XDR). In Argentina, isolates of A. baumannii resistant to tetracyclines have accounted for more than 40% of drug-resistant isolates in some hospitals. We have previously reported the dispersion of the tet(B) resistance element associated with the ISCR2 transposase in epidemiologically unrelated A. baumannii isolates recovered from 1983 to 2011. This study extends this surveillance to 77 recent (2009-2013) XDR A. baumannii isolates with different levels of minocycline susceptibility. Isolates were examined by a pan-PCR assay, which showed six different amplification patterns, and specific PCRs were used for the confirmation of the the ΔISCR2-tet(B)-tet(R)-ISCR2 element. The tet(B) gene was present in 66 isolates and the ISCR2 element in 68 isolates; the tet(B) gene was associated with ISCR2 in all tet(B)-positive isolates. We conclude that this element is widespread in XDR A. baumannii isolates from Argentina and could be responsible for the emergence of tetracycline resistance in recent years.

Genetic Variability of AdeRS Two-Component System Associated with Tigecycline Resistance in XDR-Acinetobacter baumannii Isolates.

The emergence of tigecycline resistance has increased in the last years. Although tigecycline-resistant Acinetobacter baumannii isolates were described all over the world, few reports regarding the molecular basis of this resistance are available. It has been recognized that the overexpression of AdeABC efflux pump is related to the tigecycline-resistant phenotype. In 37 clinical A. baumannii isolates we first determined the tigecycline-resistant phenotype and then, within a selected group, we analyzed the sequence of the adeRS operon, which is involved in the expression of the AdeABC efflux pump. Nucleotide sequence analysis of adeR and adeS showed the presence of 5 and 16 alleles, respectively. These results expose a high genetic variability in both genes, the adeS gene being more susceptible to genetic variation. The presence of 2 AdeR and 2 AdeS new variants were reported. Two of the new AdeRS variants were present in the intermediate and the resistant tigecycline A. baumannii isolates, suggesting a putative role in the development of the observed phenotype. More studies need to be addressed to determine the role of the genetic variability observed in the adeRS operon.

Preferential carriage of class 2 integrons in Acinetobacter baumannii CC113 and novel singletons.

Our understanding of the distribution of integrons associated with multidrug resistance in Acinetobacter baumannii isolates around the world remains incomplete. The association between the class 1 and 2 integron A. baumannii-positive isolates (n = 60), recovered since 1982 from 11 Argentinean hospitals, and the circulating lineages, was investigated. While class 2 integrons were highly significantly associated with clonal lineage CC113B/CC79P (P = 0·009) and novel singletons (P = 0·001), class 1 integrons were found not to be associated with CC109B/CC1P or other lineages. The study reveals a differential distribution of class 2 integrons in lineages, and suggests that the prevalence of intI2 in Argentina is related to the emergence of novel singletons in recent years and to the abundance of CC113B/CC79P, which has been the local dominant lineage for several decades.

Molecular fingerprinting of Staphylococcus aureus isolated from patients with osteomyelitis in Argentina and clonal distribution of the cap5(8) genes and of other selected virulence genes.

The molecular fingerprinting of a collection of 94 Staphylococcus aureus isolates from patients with osteomyelitis in Argentina was performed. Twenty-three SmaI pulsed-field gel electrophoresis (PFGE) types and 37 spa types were identified. The isolates were assigned to 23 sequence types (STs). The proportion of methicillin-resistant S. aureus (MRSA) isolates was significantly higher among cap5 S. aureus (35/61) compared with cap8 S. aureus (8/33) isolates (p = 0.0025). Twenty-four of the 94 isolates carried the lukS-PV/lukF-PV genes, which were significantly associated to cap5 [(23/38) compared with cap8 S. aureus isolates (1/32) (p = 0.0001)]. Forty of the 94 isolates carried genes of the egc locus (seg/sei). The distribution of seg/sei genes among isolates was related to certain clones. Isolates of the four agr types were found in the S. aureus collection. Whereas agr I isolates were evenly distributed among cap5 and cap8 S. aureus isolates (32/61 and 14/33, respectively), the agr II group was composed of 29 cap5 S. aureus isolates and agr III was composed of 16 cap8 S. aureus isolates. Two clones originally associated to animals (ST 188, 7 isolates and ST 1796, 5 isolates) were associated with chronic osteomyelitis and lack of capsular polysaccharide (CP) production. Loss of CP production remains the single factor among those investigated that is associated with chronic osteomyelitis.

Tn7::In2-8 dispersion in multidrug resistant isolates of Acinetobacter baumannii from Chile.

Acinetobacter baumannii is considered an important pathogen in our hospital environment having a well-known capacity to acquire different mechanisms of antibiotic resistance. Previous studies in our laboratory had exposed the high dispersion of class 2 integrons in this species. In the present study, we analyzed 7 multiresistant intI2 positive A. baumannii isolates, 6 of which were found to harbour the Tn7::In2-8 element. Our results demonstrate the unusually high distribution of Tn7::In2-8 among different A. baumannii clones from Chile, suggesting a particular behavior of these elements at geographical level.

[Serovars of Salmonella enterica subspecies enterica and its antimicrobial resistance in slaughterhouse pigs]. / Serovariedades de Salmonella enterica subespecie enterica en porcinos de faena y su resistencia a los antimicrobianos.

A study was carried out in order to determine the prevalence of Salmonella and its serovars among porcine slaughterhouses, to evaluate the antimicrobial resistance profiles and to know the presence of class 1 integrons as possible reservoir of resistance. From a total of 386 samples from four porcine slaughterhouses of Buenos Aires and Santa Fe Provinces (Argentina), 93 (24.1%) Salmonella enterica subspecies enterica strains were identified, 52 (55.9%) from cecal contents and 41 (44.1%) from ileocecal lymph nodes. Thirteen serovars of S. enterica were found, the most prevalent were: S. Schwarzengrund, S. Heidelberg, S. subspecie I 6,8:e,h:-, S. Derby and S. Bredeney. Fifteen antimicrobials by the agar dilution method were tested: amikacin, gentamicin, ciprofloxacin, cephalotin, cefotaxime, enrofloxacin, fosfomycin, polimixin-B, tetracycline, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, ampicillin, nitrofurantoin, and nalidixic acid. According to the CIM determination, 73% Salmonella enterica subspecies enterica strains were sensible to all the antimicrobials tested. Antimicrobial resistance was observed to tetracycline in 24 (25.8%) of 93 strains, to chloramphenicol in 22 (23.7%), to streptomycin in 22 (23.7%), to trimethoprim-sulfamethoxazole in 20 (21.5%), to ampicillin in 18 (19.4%), to nitrofurantoin in 3 (3.2%) and to nalidixic acid in 3 (3.2%). Some isolates of S. Typhimurium, S. Heidelberg, S. Derby, S. Orion showed multidrug resistance and carried the class 1 integrase gene. The highest percentage of resistance corresponded to the antimicrobials currently used in veterinary and porcine farms.

Serovariedades de Salmonella enterica subespecie enterica en porcinos de faena y su resistencia a los antimicrobianos / Serovars of Salmonella enterica subspecies enterica and its antimicrobial resistance in slaughterhouse pigs

Se realizó un estudio para determinar la prevalencia de Salmonella y sus serovariedades en cerdos de faena, para evaluar sus perfiles de resistencia a los antimicrobianos y para conocer la presencia de integrones de clase 1 como posibles reservorios de resistencia. A partir de un total de 386 muestras de porcinos provenientes de cuatro frigoríficos de las provincias de Buenos Aires y de Santa Fe (Argentina), se identificaron 93 (24,1%) cepas de Salmonella enterica subespecie enterica, 52 (55,9%) de contenido cecal y 41 (44,1%) de nódulo linfático ileocecal. Se hallaron 13 serovariedades de S. enterica, las más prevalentes fueron S. Schwarzengrund, S. Heidelberg, S. subespecie I 6,8:e,h:-, S. Derby y S. Bredeney. Se probaron 15 antimicrobianos por el método de dilución en agar: amikacina, gentamicina, ciprofloxacina, cefalotina, cefotaxima, enrofloxacina, fosfomicina, polimixina-B, tetraciclina, cloranfenicol, estreptomicina, trimetoprima-sulfametoxazol, ampicilina, nitrofurantoína y ácido nalidíxico. Según se estableció mediante la determinación de la CIM, el 73% de las cepas de S. enterica subespecie enterica fueron sensibles a todos los antimicrobianos probados. Se observó resistencia a tetraciclina en 24 (25,8%) de las 93 cepas, a cloranfenicol en 22 (23,7%), a estreptomicina en 22 (23,7%) a trimetoprima-sulfametoxazol en 20 (21,5%), a ampicilina en 18 (19,4%), a nitrofurantoína en 3 (3,2%) y a ácido nalidíxico en 3 (3,2%). Algunos aislamientos de S. Typhimurium, S. Heildelberg, S. Derby y S. Orion presentaron multirresistencia y portaban el gen de la integrasa clase 1. Los mayores porcentajes de resistencia correspondieron a los antimicrobianos habitualmente utilizados en veterinaria y en las explotaciones porcinas.

A study was carried out in order to determine the prevalence of Salmonella and its serovars among porcine slaughterhouses, to evaluate the antimicrobial resistance profiles and to know the presence of class 1 integrons as possible reservoir of resistance. From a total of 386 samples from four porcine slaughterhouses of Buenos Aires and Santa Fe Provinces (Argentina), 93 (24,1%) Salmonella enterica subspecies enterica strains were identified, 52 (55,9%) from cecal contents and 41 (44,1%) from ileocecal lymph nodes. Thirteen serovars of S. enterica were found, the most prevalent were: S. Schwarzengrund, S. Heidelberg, S. subspecie I 6,8:e,h:-, S. Derby and S. Bredeney. Fifteen antimicrobials by the agar dilution method were tested: amikacin, gentamicin, ciprofloxacin, cephalotin, cefotaxime, enrofloxacin, fosfomycin, polimixin-B, tetracycline, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, ampicillin, nitrofurantoin, and nalidixic acid. According to the CIM determination, 73% Salmonella enterica subspecies enterica strains were sensible to all the antimicrobials tested. Antimicrobial resistance was observed to tetracycline in 24 (25,8%) of 93 strains, to chloramphenicol in 22 (23,7%), to streptomycin in 22 (23,7%), to trimethoprim-sulfamethoxazole in 20 (21,5%), to ampicillin in 18 (19,4%), to nitrofurantoin in 3 (3,2%) and to nalidixic acid in 3 (3,2%). Some isolates of S. Typhimurium, S. Heidelberg, S. Derby, S. Orion showed multidrug resistance and carried the class 1 integrase gene. The highest percentage of resistance corresponded to the antimicrobials currently used in veterinary and porcine farms.

Relatedness and social organization of coypus in the Argentinean pampas.

Behavioural and trapping studies of the social organization of coypus have suggested the occurrence of kin groups and a polygynous mating system. We used 16 microsatellite markers to analyse parentage and relatedness relationships in two populations (Jáuregui and Villa Ruiz) in the Argentinean Pampas. At Jáuregui, a dominant male monopolized most paternities, leading to a high variance in reproductive success between males and a high level of polygyny. At Villa Ruiz, variance in reproductive success was low among resident males and males were the fathers of zero to four offspring each. For females, no significant differences were found. Two different social groups in each study site were used to assess genetic relatedness within and between groups. These groups were neighbouring at Jáuregui but not at Villa Ruiz. At Villa Ruiz, coypus were significantly more related within than between groups, suggesting that behavioural groups were also genetic ones, and adult females were more related within than between groups, as should be expected for kin groups. This relationship was not found at Jáuregui. Our results provide support to previous studies based on behavioural and trapping data, which indicate that coypus form social groups and have a polygynous mating system. However, we found differences in social organization between the two populations. This is the first study to determine parentage and/or relatedness in coypus.

[Influence of penicillin minimum inhibitory concentration in the synergy between penicillin and gentamicin in viridans-group streptococci]. / Influencia de la concentración inhibitoria mínima de penicilina en la acción sinérgica de su combinación con gentamicina frente a estreptococos del grupo viridans.

Penicillin resistance rates higher than 60% have been recorded in viridans group streptococci by some authors during the 90's and recently such resistance was associated with higher levels of mortality in bacteremia. The lowest minimum inhibitory concentration of penicillin for which synergy with aminoglycosides is not yet possible is still unknown. In order to try to dilucidate this puzzle, a study on the susceptibility to penicillin of 28 strains of viridans group streptococci isolated from significant samples in the Hospital de Pediatría "Prof. Dr. Juan P. Garrahan" was carried out. Seven mitis group isolates presenting different susceptibility patterns were selected for performing time-killing curves with penicillin, gentamicin, and penicillin plus gentamicin, using higher and lower penicillin concentrations than their minimal inhibitory concentrations. Synergy was not observed when the penicillin concentration was lower than the minimum inhibitory concentration, at least in these strains with minimum inhibitory concentrations of gentamicin > or = 16 microg/ml. When using penicillin in higher concentrations than the minimum inhibitory concentration, synergy was found in five of the seven strains. Aminoglycoside-modifying enzymes were found in the two other streptococci.

Influencia de la concentración inhibitoria mínima de penicilina en la acción sinérgica de su combinación con gentamicina frente a estreptococos del grupo viridans / Influence of penicillin minimal inhibitory concentration in the synergy between penicillin and gentamicin in viridans group streptococci

Los porcentajes de resistencia a penicilina entre los estreptococos del grupo viridans han llegado a niveles superiores al 60% en algunos estudios realizados en la década pasada, y en recientes trabajos se los encontró asociados a un mayor índice de mortalidad en las bacteriemias. Aún no se conoce cuál es el nivel de concentración inhibitoria mínima de penicilina para el cual resulta imposible lograr un efecto sinérgico con algún aminoglucósido. Con este propósito, se estudió la sensibilidad a penicilina de 28 cepas de estreptococos del grupo viridans aisladas de materiales clínicamente significativos en el Hospital de Pediatría "Prof. Dr. Juan P. Garrahan". Se seleccionaron siete aislamientos pertenecientes al grupo mitis con distintas características de sensibilidad, y con ellos se ensayó la curva de muerte frente a penicilina, gentamicina y penicilina más gentamicina, con concentraciones de penicilina por encima y por debajo de su concentración inhibitoria mínima. En ningún caso se observó sinergia cuando la concentración de penicilina fue inferior a la concentración inhibitoria mínima, al menos en este grupo particular de estreptococos que presentaron concentraciones inhibitorias mínimas de gentamicina ³ 16 µg/ml. Se encontró sinergia en cinco de las siete cepas cuando se trabajó con concentraciones de penicilina superiores a la concentración inhibitoria mínima. En las otras dos, se detectaron enzimas modificadoras de aminoglucósidos.

Penicillin resistance rates higher than 60% have been recorded in viridans group streptococci by some authors during the 90's and recently such resistance was associated with higher levels of mortality in bacteremia. The lowest minimal inhibitory concentration of penicillin for which synergy with aminoglycosides is not yet possible is still unknown. In order to try to dilucidate this puzzle, a study on the susceptibility to penicillin of 28 strains of viridans group streptococci isolated from significant samples in the Hospital de Pediatría "Prof. Dr. Juan P. Garrahan" was carried out. Seven mitis group isolates presenting different susceptibility patterns were selected for performing time-killing curves with penicillin, gentamicin, and penicillin plus gentamicin, using higher and lower penicillin concentrations than their minimal inhibitory concentrations. Synergy was not observed when the penicillin concentration was lower than the minimal inhibitory concentration, at least in these strains with minimal inhibitory concentrations of gentamicin ³ 16 µg/ml. When using penicillin in higher concentrations than the minimal inhibitory concentration, synergy was found in five of the seven strains. Aminoglycoside-modifying enzymes were found in the two other streptococci.

Survey of molecular determinants in Gram-positive cocci isolated from hospital settings in Argentina.

BACKGROUND: In order to study the resistance mechanisms to aminoglycosides, tetracyclines and erythromycin, we investigated the genetic determinants on 85 Streptococcus spp., Staphylococcus spp., and Enterococcus spp. isolates collected from 46 hospitals of Argentina over a two-year period. METHODOLOGY: The MICs to amikacin, gentamicin, kanamycin, and streptomycin, tetracycline and erythromycin were determined by the standard broth dilution method according to CLSI recommendations. Detection of resistance genes to the antibiotic tested was assessed by the PCR standard technique whereas the clonal relationships of each species was performed by PFGE. RESULTS: Major heterogeneity was detected in aminoglycoside and erythromycin resistances. Indeed, 37.6% of the isolates harbored the aac(6')-aph(2'') genes; 27% harbored the aph(3')-IIIa and ant(6)-Ia genes along with the aac(6')-aph(2'') gene; 7% carried the ant(4')-Ia gene; and 71% harbored one or more of the erm(A), erm(B), erm(TR), mef(A), mef(E) and msr(A) genes. The tetracycline resistance was determined by the tet(M) gene and was found in 23 isolates that were resistant to this antibiotic. Spreading of tet(M) by the Tn916-like transposon was not a frequent event since the integrase of this element was detected only in 3 Streptococcus spp. isolates. Instead, a 370 bp fragment was detected that corresponded to a region of the CW459-like element integrase in 10 of 11 methicillin-resistant Staphylococcus aureus and in 3 group G Streptococcus isolates, a finding that implies a novel mechanism for tetracycline resistance spreading. CONCLUSION: This study demonstrates the wide spreading of resistance mechanisms in our nosocomial cocci population and underscores the importance of continuous and efficient epidemiological surveillance.

Multicenter study on spreading of the tet(M) gene in tetracycline-resistant Streptococcus group G and C isolates in Argentina.

A prospective multicenter study on invasive infections caused by beta-hemolytic streptococci was performed over 6 months and involved 42 centers from 16 cities in Argentina. Among 33 isolates recovered, 9 group G Streptococcus isolates (39.1%) and 2 group C Streptococcus isolates (20%) exhibited resistance to tetracycline and harbored the tet(M) gene. Genealogical analysis revealed that tetracycline resistance has a polyclonal origin in Argentina.

Development of a plasmid vector for easy selection of strong promoters.

A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of beta-lactamase activity in liquid or solid media.

Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Iq from the integron of a natural multiresistance plasmid.

The nucleotide sequence of a newly identified amikacin resistance gene, aac(6')-Iq (551 bp), is reported. It has 68.4 and 94.4% homology with the aac(6')-Ia gene and the recently described aac(6')-Ip gene, respectively. Analysis of its flanking sequences indicated that it is in the first cassette of a class I integron and has an attC site (59-base element) 108 bp in length.