Valeria Bortolaia

Population Genetics of Vibrio cholerae from Nepal in 2010: Evidence on the Origin of the Haitian Outbreak

ABSTRACT Cholera continues to be an important cause of human infections, and outbreaks are often observed after natural disasters, such as the one following the 2010 earthquake in Haiti. Once the cholera outbreak was confirmed, rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. We used whole-genome sequence typing (WGST), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing to characterize 24 recent Vibrio cholerae isolates from Nepal and evaluate the suggested epidemiological link with the Haitian outbreak. The isolates were obtained from 30 July to 1 November 2010 from five different districts in Nepal. We compared the 24 genomes to 10 previously sequenced V. cholerae isolates, including 3 from the Haitian outbreak (began July 2010). Antimicrobial susceptibility and PFGE patterns were consistent with an epidemiological link between the isolates from Nepal and Haiti. WGST showed that all 24 V. cholerae isolates from Nepal belonged to a single monophyletic group that also contained isolates from Bangladesh and Haiti. The Nepalese isolates were divided into four closely related clusters. One cluster contained three Nepalese isolates and three Haitian isolates that were almost identical, with only 1- or 2-bp differences. Results in this study are consistent with Nepal as the origin of the Haitian outbreak. This highlights how rapidly infectious diseases might be transmitted globally through international travel and how public health officials need advanced molecular tools along with standard epidemiological analyses to quickly determine the sources of outbreaks. IMPORTANCE Cholera is one of the ancient classical diseases and particularly prone to cause major outbreaks following major natural disasters, such as earthquakes and hurricanes, where the normal separation between sewage and drinking water is destroyed. This was the case following the 2010 earthquake in Haiti. Rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. Sequencing the genomes of bacteria can give detailed information on whether isolates from different sites share a common origin. We used this technology to sequence isolates of Vibrio cholerae from Nepal, identify single-nucleotide polymorphisms (SNPs), and compare these high-resolution genotypes to the complete genome sequences of isolates from the Haiti outbreak. We provide support for the hypothesis that the isolates were brought to Haiti from Nepal. Cholera is one of the ancient classical diseases and particularly prone to cause major outbreaks following major natural disasters, such as earthquakes and hurricanes, where the normal separation between sewage and drinking water is destroyed. This was the case following the 2010 earthquake in Haiti. Rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. Sequencing the genomes of bacteria can give detailed information on whether isolates from different sites share a common origin. We used this technology to sequence isolates of Vibrio cholerae from Nepal, identify single-nucleotide polymorphisms (SNPs), and compare these high-resolution genotypes to the complete genome sequences of isolates from the Haiti outbreak. We provide support for the hypothesis that the isolates were brought to Haiti from Nepal.

High Diversity of Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Italian Broiler Flocks

ABSTRACT We characterized 67 Escherichia coli isolates with reduced susceptibility to cefotaxime or ceftiofur obtained from healthy broilers housed in five Italian farms. The blaCTX-M-1, blaCTX-M-32 and blaSHV-12 β-lactamase genes were identified on IncI1, IncN, or IncFIB plasmids. Considerable genetic diversity was detected among the extended-spectrum β-lactamase (ESBL)-producing isolates, and we identified indistinguishable strains in unrelated farms and indistinguishable plasmids in genetically unrelated strains. The detection of highly mobile plasmids suggests a potential animal reservoir for β-lactamase genes.

High diversity of plasmids harbouring blaCMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA

OBJECTIVES To determine the population structure and genetic relatedness of plasmids encoding CMY-2 β-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area. METHODS In total, 42 human and 73 companion animal isolates displaying an AmpC phenotype were isolated at a regional diagnostic reference laboratory in the upper Midwestern USA during 2009-11. Following PCR screening for transferable AmpC genes and plasmid transformation, blaCMY-2-positive plasmids were characterized by S1 nuclease PFGE, PCR-based replicon typing, antimicrobial susceptibility testing of transformants, conjugation experiments, plasmid multilocus sequence typing and restriction fragment length polymorphism. RESULTS blaCMY-2 occurred in 6 (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively. Usually plasmids carrying blaCMY-2 were conjugative (78%) and did not contain additional resistance genes (82%). The replicon types were IncI1 (52%), IncA/C (13%), IncFII (10%), IncI2 (5%), IncL/M (3%), IncB/O (2%) or non-typeable (15%). Related IncI1/ST12 plasmids were detected in one human and five canine isolates, while the remaining plasmids did not show similarity across host species. A novel epidemiological linkage of blaCMY-2 with IncL/M plasmids and a new CMY gene variant (blaCMY-108) were found in human isolates. CONCLUSIONS This study is one of the first One Health attempts to compare plasmids encoding CMY-2 β-lactamase among clinical isolates from humans and companion animals in the same region. The results indicate an unforeseen heterogeneity of plasmid backgrounds and suggest limited exchange between the two populations, in which blaCMY-2 occurred at very different frequencies and was harboured by distinct plasmid types.

Potential Pathogenicity and Host Range of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates from Healthy Poultry

ABSTRACT Thirty of 33 epidemiologically unrelated extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from healthy poultry lacked the virulence genes commonly associated with human-pathogenic strains. The main zoonotic risk is associated with the broad host range of avian E. coli belonging to sequence type complex 10 and of IncN and IncI1 plasmids carrying bla CTX-M or bla SHV.

Human health risks associated with antimicrobial-resistant enterococci and Staphylococcus aureus on poultry meat

Enterococci and staphylococci are frequent contaminants on poultry meat. Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus are also well-known aetiological agents of a wide variety of infections resulting in major healthcare costs. This review provides an overview of the human health risks associated with the occurrence of these opportunistic human pathogens on poultry meat with particular focus on the risk of food-borne transmission of antimicrobial resistance. In the absence of conclusive evidence of transmission, this risk was inferred using data from scientific articles and national reports on prevalence, bacterial load, antimicrobial resistance and clonal distribution of these three species on poultry meat. The risks associated with ingestion of antimicrobial-resistant enterococci of poultry origin comprise horizontal transfer of resistance genes and transmission of multidrug-resistant E. faecalis lineages such as sequence type ST16. Enterococcus faecium lineages occurring in poultry meat products are distantly related to those causing hospital-acquired infections but may act as donors of quinupristin/dalfopristin resistance and other resistance determinants of clinical interest to the human gut microbiota. Ingestion of poultry meat contaminated with S. aureus may lead to food poisoning. However, antimicrobial resistance in the toxin-producing strains does not have clinical implications because food poisoning is not managed by antimicrobial therapy. Recently methicillin-resistant S. aureus of livestock origin has been reported on poultry meat. In theory handling or ingestion of contaminated meat is a potential risk factor for colonization by methicillin-resistant S. aureus. However, this risk is presently regarded as negligible by public health authorities.

Characterization of Isolates of Salmonella enterica Serovar Stanley, a Serovar Endemic to Asia and Associated with Travel

ABSTRACT Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding bla CMY-2 gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad.

Distribution and possible transmission of ampicillin- and nalidixic acid-resistant Escherichia coli within the broiler industry.

This study was performed to determine the origin and transmission of beta-lactam- and (fluoro)quinolone-resistant Escherichia coli in healthy, untreated broiler flocks. We focused on the dynamics of bacteria resistant to critically important antimicrobials for public and veterinary health in view of the possible link between antimicrobial resistant bacteria in farm animals and humans. By processing faecal samples collected with the sock method in broiler parent and broiler flocks, E. coli resistant to ampicillin and nalidixic acid were frequently isolated, while resistance to ciprofloxacin was detected at a very low frequency, and resistance to cephalosporins was not detected. Similarly, resistance to ampicillin and nalidixic acid were the only phenotypes detected in a collection of clinical E. coli isolates associated with first-week-mortality in broiler parent chicks. Although antimicrobial resistant E. coli were genetically diverse by means of amplified fragment length polymorphism (AFLP) typing, indistinguishable isolates were present in different flocks, including isolates from broiler parent chicks, broiler parents and broilers. In the absence of apparent selective pressure, the genotypic heterogeneity that we describe is likely the consequence of multiple introductions of antimicrobial resistant bacteria into the production system. The confinement under which broilers are raised limits the possibilities of bacterial transmission among different flocks. Our findings are consistent with vertical transmission of ampicillin- and nalidixic acid-resistant E. coli through the broiler production system. The persistence of antimicrobial resistant E. coli in healthy, untreated chicken flocks emphasises the need of careful evaluation of therapeutic options at any level of the broiler production.

The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance

ABSTRACT The origin of carbapenem-hydrolyzing metallo-β-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacterium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemase production was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA β-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.

Escherichia coli Producing CTX-M-1, -2, and -9 Group β-Lactamases in Organic Chicken Egg Production

A zoonotic contribution to the spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli at the community level has been proposed repeatedly (3), but information on the origin and frequency of ESBLs within different animal reservoirs is still limited. To improve the current understanding of the evolution and epidemiology of ESBLs in poultry farming, we investigated the occurrence of ESBL-producing E. coli in flocks of chicken egg layers reared in Danish organic systems, where low flock density, restricted antimicrobial use, and access to outdoor areas are regulated at the European level (4). From June to August 2009, we visited four randomly selected healthy layer flocks (flocks A, B, C, and D), reared at independent farms with no history of antimicrobial and biocide use. E. coli bacteria with reduced susceptibility to cefotaxime were recovered from flocks B and C using sock samples enriched in MacConkey broth supplemented with cefotaxime (2 μg/ml) (1). PCR analysis (6, 7) followed by partial sequencing revealed the presence of blaTEM-1 (n = 7), blaCTX-M-1 group (compatible with blaCTX-M-1/61) (n = 9), blaCTX-M-2 group (compatible with blaCTX-M-2/20/44) (n = 3), and blaCTX-M-9 group (compatible with blaCTX-M-14/17) (n = 4) genes (http://www.ncbi.nlm.nih.gov/ and http://www.lahey.org/Studies/) in randomly selected colonies (Table ​(Table1).1). We revisited farms B and C after 10 and 11 weeks, respectively, aiming to quantify the prevalence of CTX-M-positive chickens and the concentration of CTX-M-producing E. coli in feces. The study population remained unchanged, since no chickens had entered the farms in the interval between the two sampling points. At both farms, 2 (3%) out of 60 cloacal swabs collected from individual animals and processed as described previously (1) were positive for CTX-M-producing E. coli. Of 10 fresh fecal droppings collected from the floor on each farm, 7 (farm B) and 3 (farm C) samples yielded CTX-M-producing E. coli at concentrations ranging between 102 and 103 CFU per gram of feces, which accounted for ≤0.03% of total E. coli. By XbaI pulsed-field gel electrophoresis (PFGE) analysis, 30 CTX-M producers obtained from different sample types (sock samples, swabs, and fecal droppings) displayed 16 epidemiologically unrelated band patterns according to Tenover et al. (8). Two isolates were untypeable. Twenty strains representing distinct PFGE profiles and sample types carried blaCTX-M on transferable IncI1 (n = 7), IncN (n = 3), and untypeable (n = 10) plasmids, often containing additional determinants conferring resistance to tetracycline, sulfonamides, and trimethoprim, as demonstrated by conjugation and PCR-based replicon typing (PBRT) (2, 5) (Table ​(Table1).1). Restriction fragment length polymorphism (RFLP) analysis using ClaI and BglII showed that farm-specific IncI1 and IncN plasmids were widespread among different E. coli lineages in association with specific blaCTX-M genes (Table ​(Table1).1). All PBRT-untypeable plasmids, which demonstrated poor quality images by RFLP analysis, were approximately 149 kb in size. TABLE 1. Genetic and phenotypic traits of CTX-M-producing Escherichia coli from organic layers To our knowledge, this is the first description of CTX-M-producing E. coli in the Danish chicken production. The factors leading to the origin and persistence of these resistance genes of high clinical relevance in organic poultry not exposed to antimicrobial agents remain unknown. Further research based on plasmid sequencing and gene characterization is needed to elucidate the nature of the nonantimicrobial resistance genes located on blaCTX-M gene-carrying plasmids and their possible role in favoring the spread and maintenance of such plasmids in the absence of antimicrobial selective pressure.

Host-Specific Patterns of Genetic Diversity among IncI1-Iγ and IncK Plasmids Encoding CMY-2 β-Lactamase in Escherichia coli Isolates from Humans, Poultry Meat, Poultry, and Dogs in Denmark

ABSTRACT CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli isolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producing E. coli in Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensal E. coli isolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selected bla CMY-2-harboring plasmids. MLST revealed high strain diversity, with few E. coli lineages occurring in multiple host species and sample types. bla CMY-2 was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-Iγ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-Iγ/sequence type 2 (ST2), IncI1-Iγ/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources. IMPORTANCE CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli. This β-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported in E. coli as a result of production of plasmid-encoded CMY-2 β-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance in E. coli varies significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.