Tiziana Di Maggio

Rapid Dissemination and Diversity of CTX-M Extended-Spectrum β-Lactamase Genes in Commensal Escherichia coli Isolates from Healthy Children from Low-Resource Settings in Latin America

ABSTRACT A survey carried out in 2005 among members of a healthy population of children living in Bolivia and Peru revealed that fecal carriage of Escherichia coli strains resistant to expanded-spectrum cephalosporins was remarkably increased compared to that observed in the same settings in 2002 (1.7% in 2005 versus 0.1% in 2002). In this work, we demonstrated that this phenomenon was mainly related to the dissemination of CTX-M-type extended-spectrum β-lactamase (ESBL) determinants among commensal E. coli strains. Of 50 ESBL-producing isolates collected in the 2005 survey, 44 harbored a CTX-M-type and 6 an SHV-type (SHV-2 or SHV-12) ESBL. Compared to 2002 results, an increased diversity of CTX-M-type ESBLs was also observed: members of the CTX-M-1 group (CTX-M-15) emerged in Bolivia (where only CTX-M-2 was observed in 2002), while members of the CTX-M-9 group (CTX-M-14 and CTX-M-24) emerged in Peru (where only CTX-M-15 and CTX-M-2 were observed in 2002). A new CTX-M-2 variant named CTX-M-56 was also detected. Molecular characterization of the CTX-M-producing isolates and gene transfer experiments suggested that different mechanisms could be involved in the spreading of different CTX-M group determinants and revealed that additional resistance determinants for non-β-lactam antibiotics were preferentially carried by plasmids encoding certain CTX-M variants (CTX-M-15 and variants of the CTX-M-2 group). Three CTX-M-15-encoding conjugative plasmids from Peruvian isolates carried the new fluoroquinolone resistance gene aac(6′)-Ib-cr. To our best knowledge, this is the first report of the detection of aac(6′)-Ib-cr in Latin America.

Increasing Resistance in Commensal Escherichia coli, Bolivia and Peru

To the Editor: The global increase of antimicrobial-drug resistance, including resistance to the new and most potent antimicrobial agents, is a major public health concern. In low-resource countries, where bacterial infections are still among the major causes of death, especially for children, it is of particular concern (1). ANTRES (Towards Controlling Antimicrobial Use and Resistance in Low-income Countries—An Intervention Study in Latin America) is a research project on antimicrobial-drug use and resistance in low-resource countries of Latin America (see www.unifi.it/infdis/antres/default.htm). In 2002, the baseline ANTRES study showed a high rate of fecal carriage of Escherichia coli with acquired resistance to several antimicrobial drugs, especially older drugs (e.g., ampicillin, trimethoprim-sulfamethoxazole, tetracycline, streptomycin, and chloramphenicol), in preschool children from 4 urban settings in Bolivia and Peru (2). We report the results of a second cross-sectional study, conducted in 2005, that evaluated the evolution of antimicrobial-drug resistance in the studied areas. We studied healthy children 6–72 months of age from each of 4 urban areas: 2 in Bolivia (Camiri, Santa Cruz Department; Villa Montes, Tarija Department) and 2 in Peru (Yurimaguas, Loreto Department; Moyobamba, San Martin Department). The study design, sampling and inclusion criteria, methods, and ethical issues were the same as those of the baseline study (2). The study was carried out over 4 months (September–December 2005), the same seasonal period as in the previous study. No significant differences in sex ratios were found among children enrolled from the different areas, whereas minor differences were found for age. No statistical differences were found between the 2002 baseline study and the 2005 study results in terms of numbers of children (3,193 vs. 3,174) and sex ratios (0.94 vs. 0.95) (Table). Statistical analyses were performed by using Stata 9.0 (Stata Corp., College Station, TX, USA). Logistic regression models were used to compare the antimicrobial-drug resistance rates in 2002 and 2005, considering the combined influences of age, sex, city, and country. Table Antimicrobial drug–resistance rates of Escherichia coli as part of commensal flora in children, Bolivia and Peru, 2002 and 2005* *Expanded Table available online at www.cdc.gov/EID/content/14/2/338-T.htm. Data from the 2005 survey confirmed high resistance rates for ampicillin, trimethoprim-sulfamethoxazole, tetracycline, streptomycin, and chloramphenicol. The differences in resistance rates observed between 2002 and 2005 for these drugs, although sometimes statistically significant, are probably of limited epidemiologic relevance due to the high rates of antimicrobial-drug resistance found in the E. coli population in both surveys. The most relevant finding of the 2005 survey was the remarkable increase since 2002 in the resistance rates to fluoroquinolones and expanded-spectrum cephalosporins (Table). Molecular analysis showed that the dramatic increase in rates of resistance to expanded spectrum cephalosporins was mostly the result of dissemination of CTX-M-type extended-spectrum β-lactamase determinants (3). Concerning the association between sex and resistance rates, the higher resistance rates observed for some agents and in some settings for boys in the baseline study were not confirmed, except in 1 case (kanamycin in Camiri, p = 0.04) (2). Analysis by age (not performed for amikacin due to low numbers of resistant isolates) confirmed the occurrence of higher resistance rates for the youngest age group and an overall decreasing trend by age for all agents, except ciprofloxacin and gentamicin. For these 2 agents, resistance rates increased, although not significantly (p = 0.95 and p = 0.55, respectively) (2). Although we did not specifically address factors potentially involved in this phenomenon, we will address them in future investigations. Increasing resistance to fluoroquinolones and expanded-spectrum cephalosporins among E. coli clinical isolates has been observed in several parts of the world and complicates the management of infections (4,5). Recently, intestinal colonization with fluoroquinolone-resistant or extended-spectrum β-lactamase–producing E. coli of nonhospitalized persons has been described as an emerging phenomenon (6–9). Although the exact clinical implications of this phenomenon are not clearly established, colonization by these resistant strains is a public health threat at the community and hospital levels (8,9). The reasons for the increased prevalence of fecal carriage of these resistant E. coli strains by children from the studied areas are not clear. Data collected about household use of antimicrobial drugs excluded previous use of fluoroquinolones and expanded-spectrum cephalosporins (C. Kristiansson et al., unpub. data). The increased prevalence of resistant E. coli strains in preschool children most likely reflects increased exposure within a contaminated household setting, in the food chain, or both (6,8,10). Our findings support the need to continue monitoring the evolution of resistance in commensal E. coli, to evaluate the effects of these important reservoirs of resistance genes distributed in the community, to investigate the epidemiologic relationship with clinical isolates, and to define the role of the food supply. Investigation into whether carriage of resistant strains in adults correlates with data on antimicrobial-drug use in hospitals and in the community would also be of interest.

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.

Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015–1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5–30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.

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

Characterization of IncI1 Sequence Type 71 Epidemic Plasmid Lineage Responsible for the Recent Dissemination of CTX-M-65 Extended-Spectrum β-Lactamase in the Bolivian Chaco Region

ABSTRACT During the last decade, a significant diffusion of CTX-M-type extended-spectrum β-lactamases (ESBLs) was observed in commensal Escherichia coli from healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n = 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to β-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). The blaCTX-M-65 module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli, Cronobacter sakazakii, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella enterica) and was stably maintained without selective pressure in these species, with the exception of K. oxytoca and S. enterica. Fitness assays performed in E. coli recipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost.

Effect of high N-acetylcysteine concentrations on antibiotic activity against a large collection of respiratory pathogens

ABSTRACT The effect of high N-acetylcysteine (NAC) concentrations (10 and 50 mM) on antibiotic activity against 40 strains of respiratory pathogens was investigated. NAC compromised the activity of carbapenems (of mostly imipenem and, to lesser extents, meropenem and ertapenem) in a dose-dependent fashion. We demonstrated chemical instability of carbapenems in the presence of NAC. With other antibiotics, 10 mM NAC had no major effects, while 50 mM NAC sporadically decreased (ceftriaxone and aminoglycosides) or increased (penicillins) antibiotic activity.

Antimicrobial susceptibility and emerging resistance determinants (blaCTX-M, rmtB, fosA3) in clinical isolates from urinary tract infections in the Bolivian Chaco

BACKGROUND Bolivia is among the lowest-resourced South American countries, with very few data available on antibiotic resistance in bacterial pathogens. The phenotypic and molecular characterization of bacterial isolates responsible for urinary tract infections (UTIs) in the Bolivian Chaco are reported here. METHODS All clinical isolates from UTIs collected in the Hospital Basico Villa Montes between June 2010 and January 2014 were analyzed (N=213). Characterization included susceptibility testing, extended-spectrum beta-lactamase (ESBL) detection, identification of relevant resistance determinants (e.g., CTX-M-type ESBLs, 16S rRNA methyltransferases, glutathione S-transferases), and genotyping of CTX-M producers. RESULTS Very high resistance rates were observed. Overall, the lowest susceptibility was observed for trimethoprim-sulphamethoxazole, tetracycline, nalidixic acid, amoxicillin-clavulanic acid, ciprofloxacin, and gentamicin. Of E. coli and K. pneumoniae, 11.6% were ESBL producers. Resistance to nitrofurantoin, amikacin, and fosfomycin remained low, and susceptibility to carbapenems was fully preserved. CTX-M-15 was the dominant CTX-M variant. Four E. coli ST131 (two being H30-Rx) were identified. Of note, isolates harbouring rmtB and fosA3 were detected. CONCLUSIONS Bolivia is not an exception to the very high resistance burden affecting many South American countries. Optimization of alternative approaches to monitor local antibiotic resistance trends in resource-limited settings is strongly encouraged to support the implementation of effective empiric treatment guidelines.

In vitro activity of N-acetylcysteine against Stenotrophomonas maltophilia and Burkholderia cepacia complex grown in planktonic phase and biofilm

Stenotrophomonas maltophilia and Burkholderia cepacia complex (Bcc) have been increasingly recognized as relevant pathogens in hospitalized, immunocompromised and cystic fibrosis (CF) patients. As a result of complex mechanisms, including biofilm formation and multidrug resistance phenotype, S. maltophilia and Bcc respiratory infections are often refractory to therapy, and have been associated with a worse outcome in CF patients. Here we demonstrate for the first time that N-acetylcysteine (NAC), a mucolytic agent with antioxidant and anti-inflammatory properties, may exhibit antimicrobial and antibiofilm activity against these pathogens. The antimicrobial and antibiofilm activity of high NAC concentrations, potentially achievable by topical administration, was tested against a collection of S. maltophilia (n = 19) and Bcc (n = 19) strains, including strains from CF patients with acquired resistance traits. Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) ranged from 16 to 32 mg/ml and from 32 to >32 mg/ml, respectively. Sub-MIC concentrations (i.e., 0.25 × MIC) slowed down the growth kinetics of most strains. In time-kill assays, 2-day-old biofilms were more affected than planktonic cultures, suggesting a specific antibiofilm activity of NAC against these pathogens. Indeed, a dose- and time-dependent antibiofilm activity of NAC against most of the S. maltophilia and Bcc strains tested was observed, with a sizable antibiofilm activity observed also at 0.5 and 1 × MIC NAC concentrations. Furthermore, at those concentrations, NAC was also shown to significantly inhibit biofilm formation with the great majority of tested strains.

In vitro synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii grown in planktonic phase and in biofilms

Abstract Objectives To investigate the potential synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii strains grown in planktonic phase or as biofilms. Methods Sixteen strains were investigated, including nine colistin-susceptible (MIC range 0.5–1 mg/L) and seven colistin-resistant (MIC range 16–256 mg/L) strains. Synergism of colistin in combination with N-acetylcysteine was investigated by chequerboard assays. The activity of colistin/N-acetylcysteine combinations was further evaluated by time–kill assays with planktonic cultures (three colistin-resistant strains and one colistin-susceptible strain) and by in vitro biofilm models (three colistin-resistant and three colistin-susceptible strains). Results Chequerboard assays revealed a relevant synergism of colistin/N-acetylcysteine combinations with all colistin-resistant strains, whereas no synergism was observed with colistin-susceptible strains. Time–kill assays showed a concentration-dependent potentiation of colistin activity by N-acetylcysteine against colistin-resistant strains, with eradication of the culture by combinations of N-acetylcysteine at 8000 mg/L plus colistin at 2 or 8 mg/L. A static effect during the first 8 h of incubation was demonstrated with the colistin-susceptible strain exposed to 0.25 × MIC colistin plus 8000 mg/L N-acetylcysteine. A remarkable antibiofilm synergistic activity of 8 mg/L colistin plus 8000 mg/L N-acetylcysteine was demonstrated with all colistin-resistant and colistin-susceptible strains. The effects were greater with colistin-resistant strains (marked reduction of viable biofilm cells was observed at sub-MIC colistin concentrations). Conclusions N-acetylcysteine, at concentrations achievable by topical administration, was shown to revert the colistin-resistant phenotype in A. baumannii, and to exert a relevant activity against biofilms of colistin-susceptible and colistin-resistant A. baumannii strains.