Eleonora Riccobono

High Prevalence of qnr Genes in Commensal Enterobacteria from Healthy Children in Peru and Bolivia

ABSTRACT A remarkable prevalence of qnrB (54%) and, at a lower level, of qnrS (14%) was discovered in pools of commensal enterobacteria from 310 healthy children living in Peru and Bolivia, using a metagenomic approach. Analysis of randomly selected enterobacterial pools revealed that qnrB was mainly carried by Escherichia coli and qnrS by Klebsiella pneumoniae. Investigation of 11 qnrB-positive isolates and 9 qnrS-positive isolates revealed the presence of plasmid-borne qnrB19 (n = 8), qnrB2 (n = 2), qnrB10 (n = 1), and qnrS1 (n = 9) genes.

Characterization of Small ColE-Like Plasmids Mediating Widespread Dissemination of the qnrB19 Gene in Commensal Enterobacteria

ABSTRACT In this work, we have characterized two small ColE-like plasmids (pECY6-7, 2.7 kb in size, and pECC14-9, of 3.0 kb), encoding the QnrB19 quinolone resistance determinant, that were carried by several clonally unrelated quinolone-resistant commensal Escherichia coli strains isolated from healthy children living in different urban areas of Peru and Bolivia. The two plasmids are closely related to each other and carry the qnrB19 gene as the sole resistance determinant, located in a conserved genetic context between the plasmid RNAII sequence (which controls plasmid replication) and the plasmid Xer site (involved in plasmid dimer resolution). ISEcp1-like or other putative insertion sequences are not present in the qnrB19-flanking regions or elsewhere on the plasmids. Since we previously observed a high prevalence (54%) of qnrB genes in the metagenomes of commensal enterobacteria from the same population of healthy children, the presence of pECY6-7- and pECC14-9-like plasmids in those qnrB-positive metagenomes was investigated by PCR mapping. Both plasmids were found to be highly prevalent (67% and 16%, respectively) in the qnrB-positive metagenomes, suggesting that dissemination of these small plasmids played a major role in the widespread dissemination of qnrB genes observed in commensal enterobacteria from healthy children living in those areas.

Plasmid-mediated fluoroquinolone resistance determinants in Escherichia coli from community uncomplicated urinary tract infection in an area of high prevalence of quinolone resistance

In Italy fluoroquinolones (FQs) are extensively prescribed in empirical therapy of uncomplicated urinary tract infection (UTI) despite recommendations in national guidelines and widespread antibiotic resistance in community. To survey the dissemination of plasmid-mediated quinolone resistance in a peak area of FQs consumption, E. coli strains from 154 community and 41 local hospital patients were collected; low level ciprofloxacin resistance qnrA, qnrB, qnrS, and aac(6)′-Ib-cr genes were screened by PCR and patterns of transferable resistances were determined. Clinical ciprofloxacin resistance in hospital doubled community value, while overall rates of FQ resistance genes were similar (31.6% and 27.8%). Prevalence of aac(6′)-Ib-cr gene was 11% in outpatients (21%, inpatients) and risk of harbouring this variant was significantly associated with gentamicin resistance; linkage to ceftazidime resistance was significant (P = 0.001) and six out of eight strains produced CTX-M-15 and TEM-1 beta lactamases. In transconjugants, the unique pattern ampicillin/kanamycin-gentamicin/ ESBL + was associated with aac(6′)-Ib-cr gene presence and with an increase of ciprofloxacin MIC value. Data highlight the need to monitor the resistance risk factors in the local community to provide clinicians with well-grounded guidelines for UTI therapy.

Small qnrB-harbouring ColE-like plasmids widespread in commensal enterobacteria from a remote Amazonas population not exposed to antibiotics

Dipartimento di Biotecnologie, Sezione di Microbiologia, Università di Siena, Siena, Italy; Dipartimento Area Critica Medico Chirurgica, Clinica Malattie Infettive, Università di Firenze, Florence, Italy; Hospital Apoyo Yurimaguas, Yurimaguas, Peru; Malattie Infettive e Tropicali, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy; Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru; Dipartimento di Emergenza, Urgenza e dei Servizi Diagnostici, U. O. Microbiologia e Virologia, Azienda Ospedaliera-Universitaria Senese, Siena, Italy

Methicillin-resistant Staphylococcus aureus in hospitalized patients from the Bolivian Chaco

OBJECTIVES Information is lacking on the methicillin-resistant Staphylococcus aureus (MRSA) clonal lineages circulating in Bolivia. We investigated the prevalence and molecular epidemiology of S. aureus colonization in hospitalized patients from the Bolivian Chaco, and compared their features with those of the few clinical isolates available from that setting. METHODS S. aureus nasal/inguinal colonization was investigated in 280 inpatients from eight hospitals in two point prevalence surveys (2012, n=90; 2013, n=190). Molecular characterization included genotyping (spa typing, multilocus sequence typing, and pulsed-field gel electrophoresis), detection of virulence genes, and SCCmec typing. RESULTS Forty-one inpatients (14.6%) were S. aureus nasal/inguinal carriers, of whom five were colonized by MRSA (1.8%). MRSA isolates mostly belonged to spa-type t701, harboured SCCmec IVc, and were negative for Panton-Valentine leukocidin (PVL) genes. However, a USA300-related isolate was also detected, which showed the characteristics of the USA300 Latin American variant (USA300-LV; i.e., ST8, spa-type t008, SCCmec IVc, presence of PVL genes, absence of arcA). Notably, all the available MRSA clinical isolates (n=5, collected during 2011-2013) were also identified as USA300-LV. CONCLUSIONS Overall, MRSA colonization in inpatients from the Bolivian Chaco was low. However, USA300-LV-related isolates were detected in colonization and infections, emphasizing the importance of implementing control measures to limit their further dissemination in this resource-limited area.

Citrobacter braakii carrying plasmid-borne mcr-1 colistin resistance gene from ready-to-eat food from a market in the Chaco region of Bolivia

Department of Medical Biotechnologies, University of Siena, Siena, Italy; Department of Surgery and Translational Medicine, University of Florence, Florence, Italy; Department of Experimental and Clinical Medicine, University of Florence, Careggi University Hospital, Florence, Italy; Hospital B asico Villa Montes, Villa Montes, Plurinational State of Bolivia; Infectious and Tropical Diseases Unit, Florence Careggi University Hospital, Florence, Italy; Clinical Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy

Quinolone Resistance in Absence of Selective Pressure: The Experience of a Very Remote Community in the Amazon Forest

Background Quinolones are potent broad-spectrum bactericidal agents increasingly employed also in resource-limited countries. Resistance to quinolones is an increasing problem, known to be strongly associated with quinolone exposure. We report on the emergence of quinolone resistance in a very remote community in the Amazon forest, where quinolones have never been used and quinolone resistance was absent in 2002. Methods The community exhibited a considerable level of geographical isolation, limited contact with the exterior and minimal antibiotic use (not including quinolones). In December 2009, fecal carriage of antibiotic resistant Escherichia coli was investigated in 120 of the 140 inhabitants, and in 48 animals reared in the community. All fluoroquinolone-resistant isolates were genotyped and characterized for the mechanisms of plasmid- and chromosomal-mediated quinolone resistance. Principal Findings Despite the characteristics of the community remained substantially unchanged during the period 2002–2009, carriage of quinolone-resistant E. coli was found to be common in 2009 both in humans (45% nalidixic acid, 14% ciprofloxacin) and animals (54% nalidixic acid, 23% ciprofloxacin). Ciprofloxacin-resistant isolates of human and animal origin showed multidrug resistance phenotypes, a high level of genetic heterogeneity, and a combination of GyrA (Ser83Leu and Asp87Asn) and ParC (Ser80Ile) substitutions commonly observed in fluoroquinolone-resistant clinical isolates of E. coli. Conclusions Remoteness and absence of antibiotic selective pressure did not protect the community from the remarkable emergence of quinolone resistance in E. coli. Introduction of the resistant strains from antibiotic-exposed settings is the most likely source, while persistence and dissemination in the absence of quinolone exposure is likely mostly related with poor sanitation. Interventions aimed at reducing the spreading of resistant isolates (by improving sanitation and water/food safety) are urgently needed to preserve the efficacy of quinolones in resource-limited countries, as control strategies based only on antibiotic restriction policies are unlikely to succeed in those settings.

Highly bactericidal Ag nanoparticle films obtained by cluster beam deposition

UNLABELLED The recent emergence of bacterial pathogens resistant to most or all available antibiotics is among the major global public health problems. As indirect transmission through contaminated surfaces is a main route of dissemination for most of such pathogens, the implementation of effective antimicrobial surfaces has been advocated as a promising approach for their containment, especially in the hospital settings. However, traditional wet synthesis methods of nanoparticle-based antimicrobial materials leave a number of key points open for metal surfaces: such as adhesion to the surface and nanoparticle coalescence. Here we demonstrate an alternative route, i.e. supersonic cluster beam deposition, to obtain antimicrobial Ag nanoparticle films deposited directly on surfaces. The synthesized films are simple to produce with controlled density and thickness, are stable over time, and are shown to be highly bactericidal against major Gram positive and Gram negative bacterial pathogens, including extensively drug-resistant strains. FROM THE CLINICAL EDITOR The use of silver nanoparticle in health care is getting more widespread. The authors here describe the technique of cluster beam deposition for spraying silver on surfaces used in health care sectors. This may open a new avenue for future anti-bacterial coatings.

Low prevalence of methicillin-resistant Staphylococcus aureus nasal carriage in urban and rural community settings in Bolivia and Peru☆

OBJECTIVE To investigate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage in rural and urban community settings of Bolivia and Peru. METHODS MRSA nasal carriage was investigated in 585 individuals living in rural and urban areas of Bolivia and Peru (one urban area, one small rural village, and two native communities, one of which was highly isolated). MRSA isolates were subjected to molecular analysis for the detection of virulence genes, characterization of the staphylococcal cassette chromosome mec (SCCmec), and genotyping (multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE)). RESULTS An overall very low prevalence of MRSA nasal carriage was observed (0.5%), with MRSA carriers being detected only in a small rural village of the Bolivian Chaco. The three MRSA isolates showed the characteristics of community-associated MRSA (being susceptible to all non-beta-lactam antibiotics and harboring the SCCmec type IV), were clonally related, and belonged to ST1649. CONCLUSIONS This study provides an insight into the epidemiology of MRSA in community settings of Bolivia and Peru. Reliable, time-saving, and low-cost methods should be implemented to encourage continued surveillance of MRSA dissemination in resource-limited countries.

Carriage of antibiotic-resistant Escherichia coli among healthy children and home-raised chickens: a household study in a resource-limited setting.

We have previously observed high rates of acquired antibiotic resistance in commensal Escherichia coli from healthy children living in urban areas of Bolivia and Peru, including resistance to tetracycline and quinolones, which are not routinely used in childhood. In this work we investigated acquired resistance in commensal E. coli from healthy children and home-raised chickens in 12 households from one of the previously surveyed urban area in Bolivia, to ascertain the possibility of human-animal exchange of resistant strains in similar settings. The resistance rates to ampicillin, tetracycline, chloramphenicol, streptomycin, and trimethoprim-sulphametoxazole were overall high (≥50%) and comparable between children and chickens, whereas those to quinolones were significantly higher in chickens (81% vs. 29% for nalidixic acid; 43% vs. 10% for ciprofloxacin). Molecular characterization of tetracycline- and quinolone-resistant isolates (n = 66) from children and chickens of three selected households revealed a remarkable clonal diversity and, in some cases, the presence of the same resistant strains among children or among chickens living in the same household, but not between children and chickens. Several resistance plasmids were characterized, but inter-clonal plasmid dissemination was not detected. Overall, the results from the present study suggested that cross-transmission between children and home-raised chickens could not represent a major spreading mechanism for resistant E. coli in households of resource-limited settings with high human-animal promiscuity.