Marisa Accogli

Escherichia coli of human and avian origin: detection of clonal groups associated with fluoroquinolone and multidrug resistance in Italy

OBJECTIVES Poultry have been suggested as a reservoir for fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli (ExPEC). Our aim was to investigate whether genotypes associated with ciprofloxacin and multidrug resistance were shared among human and avian E. coli. METHODS We compared 277 human ExPEC isolates from urinary tract infection (UTI) and sepsis (142 susceptible and 135 ciprofloxacin resistant) and 101 avian isolates (68 susceptible and 33 ciprofloxacin resistant) by antimicrobial resistance phenotype, phylogenetic group and multilocus sequence type (ST). RESULTS Most ciprofloxacin-resistant isolates from both human and avian sources were multidrug resistant. Human and avian isolates strongly differed in phylogenetic group assignment (B2 and A predominated among human and avian isolates, respectively), but a shift towards group A associated with ciprofloxacin resistance was observed among human isolates (8/100, 8.0% versus 17/87, 19.5%, P =0.021 for UTI and 5/42, 11.9% versus 15/48, 31.3%, P = 0.028 for sepsis). Heterogeneity of ST clones was observed, with ST131 strongly predominant in human ciprofloxacin-resistant strains (58/135, 43.0%), but not in avian strains. However, two major ST clonal complexes (CCs; CC10 and CC23, both belonging to group A) associated with ciprofloxacin resistance and multiresistance were shared by human and avian isolates. CONCLUSIONS The major human and avian E. coli ST clones associated with multidrug resistance were identified. A subset of ST clones belonging to CC10 and CC23 poses a potential zoonotic risk.

Ciprofloxacin-resistant, CTX-M-15-producing Escherichia coli ST131 clone in extraintestinal infections in Italy

Quinolone and β-lactam resistance mechanisms and clonal relationships were characterized among Escherichia coli isolates resistant to ciprofloxacin and extended-spectrum cephalosporins associated with human extra-intestinal infections in Rome. The E. coli. ST131 clone was found to be prevalent. This clone invariably carried a specific pattern of substitutions in the topoisomerase genes and all isolates but one produced CTX-M-15. One ST131 isolate produced SHV-12. The new ST131 variant described here is of particular concern because it combines fluoroquinolone resistance and chromosomally encoded CTX-M-15.

First Report of Plasmid-Mediated Quinolone Resistance Determinant qnrS1 in an Escherichia coli Strain of Animal Origin in Italy

ABSTRACT A qnrS1-positive strain of Escherichia coli was detected among 73 poultry isolates showing ciprofloxacin MICs of ≥0.125 μg/ml. The qnrS1 gene was associated with a Tn3-like transposon, as previously described to occur in a Salmonella enterica serovar Infantis strain of animal origin, but the plasmid scaffold carrying this element resembled that of a plasmid previously identified in Salmonella enterica serovar Dublin. These elements suggest genetic exchanges among Salmonella and E. coli and a potential animal reservoir for the qnr genes.

IncI1 plasmids associated with the spread of CMY‐2, CTX‐M‐1 and SHV‐12 in Escherichia coli of animal and human origin

Fourteen plasmids carrying blaCTX -M-1, blaSHV -12 or blaCMY -2 genes from Escherichia coli of both avian and human origin were analysed. IncI1 plasmids were largely predominant. Plasmid mutilocus sequence typing and comparative analysis revealed that the blaCMY -2 -ST12-IncI1 plasmids from avian E. coli were identical to those previously found in Salmonella from humans, but different to those associated with human E. coli. The IncI1-ST3 plasmids carrying blaCTX -M-1 or blaSHV -12 were related to those previously identified in avian E. coli, but different to those identified in human E. coli. Overall, no plasmids shared by E. coli of both origin (human/avian) were identified; however, further investigations are needed.

Emergence of Escherichia coli ST131 sub-clone H30 producing VIM-1 and KPC-3 carbapenemases, Italy

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita, Rome, Italy; Department of Medical Biotechnologies, University of Siena, Siena, Italy; National Centre for Epidemiology, Surveillance and Health Promotion, Istituto Superiore di Sanita, Rome, Italy; Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; Clinical Microbiology and Virology Unit, Department of Laboratory Medicine, Careggi University Hospital, Florence, Italy

Carriage of Haemophilus influenzae in the oropharynx of young children and molecular epidemiology of the isolates after fifteen years of H. influenzae type b vaccination in Italy

BACKGROUND Haemophilus influenzae is an important pathogen able to cause a wide spectrum of diseases in children. Colonization of the upper respiratory tract is a risk factor for developing disease. This study aimed to investigate the oropharyngeal carriage rate of H. influenzae in young children in two Italian cities, 15 years after H. influenzae type b (Hib) vaccination was introduced. Antibiotic resistant traits and genotypes of the colonizing H. influenzae isolates were investigated. METHODS Oropharyngeal swabs were obtained from 717 healthy children aged <6 years (June 2012-July 2013). Potential risk factors for H. influenzae colonization were investigated. H. influenzae isolates from carriage were characterized by PCR capsular typing, ampicillin susceptibility testing, resistance-associated gene sequencing and multilocus sequence typing (MLST). For comparison purposes, 38 non-typeable H. influenzae (NTHi) isolates from invasive disease were genotyped by MLST. RESULTS The overall H. influenzae carriage rate was 14.1% (101/717). Age, study site, presence of young siblings, and complete Hib vaccination status were independently associated with colonization. Of 101 isolates, 98 were NTHi, 2 were type e and 1 was type f. The overall ampicillin resistance rate was 15.8% (16/101). Resistance was mediated by TEM-1 β-lactamase production in half of isolates (n=8) or modifications in penicillin-binding protein (PBP) 3 in the other half (n=8). Several substitutions were discovered in PBP3 including the Asn526Lys change. Seventy-six different STs were identified among 98 NTHi isolates from carriage, with only 4 STs (ST12, ST57, ST238, ST1238) encompassing ≥ 3 isolates. Comparison of carriage and disease isolates found that several STs were shared between the two sources, although none of the major disease-associated STs were observed in carriage isolates. CONCLUSIONS NTHi is the predominant serotype in carriage. The importance of monitoring both NTHi colonization rate and circulating genotypes should be emphasized in the era of the Hib conjugate vaccines.

Identification of Haemophilus influenzae Clones Associated with Invasive Disease a Decade after Introduction of H. influenzae Serotype b Vaccination in Italy

ABSTRACT The introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines has changed the epidemiology of invasive H. influenzae disease, with a shift in the predominant serotype from Hib to nonencapsulated H. influenzae (ncHi). The objective of this study was to identify the genotypes/clones associated with invasive H. influenzae disease in Italy. Eighty-seven H. influenzae strains isolated in the years 2009 to 2011 within the National Surveillance of Invasive Bacterial Disease program were analyzed. Strains were characterized by serotyping, antimicrobial susceptibility testing, and multilocus sequence typing (MLST). Genetic polymorphisms in the blaTEM gene promoter region as well as the occurrence of both adhesin genes (hmwA and hia) and the IgA1 protease-encoding gene (igaB) were also investigated. Of 87 strains, 67 were ncHi and 20 were encapsulated. Eleven strains were β-lactamase positive, harboring the blaTEM gene. Most blaTEM genes (10/11) were associated with a Pdel promoter region exhibiting a 135-bp deletion; the remaining strain possessed the Pa/Pb overlapping promoter. MLST analysis showed that encapsulated isolates were clonal, with each serotype sharing a few related sequence types (STs). Forty-six different STs were identified among the 67 ncHi strains. Despite this heterogeneity, a group of closely related STs (ST103, ST139, and ST145) encompassed almost 25% of all ncHi strains and 45.5% of the β-lactamase producers carrying the Pdel promoter. These major ST clones were found to be associated with the hmwA gene but not with the igaB gene. To conclude, although the heterogeneity of the ncHi population was confirmed, diffusion of major successful ST clones was documented.

Neonatal invasive Haemophilus influenzae disease and genotypic characterization of the associated strains in Italy

TO THE EDITOR—We read with interest the article by Collins and colleagues demonstrating that nontypeable Haemophilus influenzae (NTHi) is responsible for most invasive disease occurring in the perinatal period and that early-onset neonatal NTHi disease (strain isolation from blood or cerebrospinal fluid within 48 hours of birth) is strongly associated with premature birth [1]. Although it is well known that following the routine use of H. influenzae type b conjugate vaccines, most invasive H. influenzae disease is caused by NTHi. The value of the Collins’s study was to draw attention to neonatal invasive disease that, to date, has probably been underestimated [2–4]. According to the authors’ conclusions, a better understanding of the responsible NTHi strains through molecular characterization is needed [1]. In Italy, 13 invasiveH. influenzae disease cases were detected in infants aged ≤31 days through the National Surveillance of Invasive Bacterial Disease (1 January 2009–31 March 2015) (Figure 1, inset). Of 13 infants (all presenting with septicemia), 9 (69.2%) were born prematurely (<37 weeks of gestation) and 5 were male. Early-onset invasive infection occurred in 11 neonates (11/13, 84.6%); all but 2 were born prematurely (Figure 1, inset). At discharge, all neonates were alive, but 2 had long-term complications (1 had moderate neurological impairment and 1 had hypoacusis). Looking at maternal factors in early-onset infection, 3 mothers had fever, 3 had chorioamnionitis, and 1 experienced premature rupture of membranes. The 2 neonates who developed late-onset invasive disease (>48 hours after birth) were born at 37 weeks and >38 weeks. Of 13 H. influenzae strains, 10 were sent to the National Reference Laboratory (Istituto Superiore di Sanità, Rome, Italy) where they were characterized. Serotyping,

Predominance of the fimH30 Subclone Among Multidrug-Resistant Escherichia coli Strains Belonging to Sequence Type 131 in Italy

TO THE EDITOR—We read with interest the article by Johnson et al, which demonstrates that, in extraintestinal pathogenic Escherichia coli (ExPEC), fluoroquinolone (FQ) resistance is currently associated with the rapid expansion of a single dominant multidrug-resistant (MDR) strain that emerged within sequence type (ST) 131 [1]. Although the strong predominance of the ST131 clone has been well described in the literature since 2008 [2– 5], the peculiarity of Johnson et al’s study was to analyze historical and recent ST131 isolates at the sub-ST level, revealing that a specific fimH-based subclone (H30) is currently responsible for most FQ-resistant ExPEC infections, at least in the United States. This H30 subclone was demonstrated to possess a unique and conserved gyrA/parC allele combination conferring FQ resistance, and the authors rightly commented on these data in support of its strict clonality. We are particularly interested in this issue, since ST131 is also currently predominant among MDR ExPEC in Italy and because some ST131 strains we previously analyzed were found to carry the same pattern of gyrA/parC substitutions described by Johnson et al [1, 6]. No information is available on the fimH-based subclones circulating in European countries. In this study, 172 ExPEC strains isolated from cases of urinary tract infections and sepsis in Italy during April 2012– December 2012 were analyzed. Staff a the 3 enrolled hospitals were asked to collect all consecutive MDR E. coli strains and 1 ciprofloxacin-susceptible non-MDR E. coli strain for every 3 MDR strains collected. “MDR” was defined as resistance to at least 3 antimicrobial agents of different classes (ampicillin, third-generation cephalosporins, ciprofloxacin, gentamicin, and trimethoprim/sulfamethoxazole). Of 172 strains, 119 were MDR strains that were resistant to ciprofloxacin; 9 were MDR strains that were susceptible to ciprofloxacin; and 44 were non-MDR strains that were susceptible to ciprofloxacin. Antimicrobial susceptibility testing, phylogenetic typing, polymerase chain reaction screening for ST131 followed by confirmation (by mdh and gyrB gene sequencing), characterization of the extended-spectrum β-lactamase (ESBL) and/or AmpC genes, and fimH-based subtyping of the ST131 strains were performed as previously described [1, 7–9]. To investigate which fimH allele circulated in Italy before 2012, an additional 91 E. coli ST131 strains previously identified were also subtyped [7, 10]. Of these 91 strains, 28 (24 ciprofloxacinresistant strains and 4 ciprofloxacinsusceptible strains) were isolated in 2006, and 63 (58 ciprofloxacin-resistant strains and 4 ciprofloxacin-susceptible strains) were isolated in 2009. By phylogenetic typing, overall, the majority of strains isolated in 2012 (110/ 172 [64%]) fell into phylogenetic group B2, followed by groups D (30/172 [17.4%]),

Food Reservoir for Escherichia coli Causing Urinary Tract Infections

To the Editor: We read with interest the article by Vincent et al. that compared Escherichia coli isolates from 3 sources (human urinary tract infections [UTIs], retail meat, and restaurant/ready-to-eat foods) by multiple molecular typing methods (1). This study has to be considered in the context of the larger debate about the possible animal origin of E. coli isolates that cause extraintestinal infections in humans (2–5), and the same authors (Vincent et al.) have declared, in the introduction, that their efforts were directed toward investigating the hypothesis that retail chicken is the main reservoir for extraintestinal E. coli. We strongly appreciate the amount of the experimental data and some interesting findings, but we are not totally convinced of the authors’ conclusions, particularly the assumption that the study strongly supports the preliminary hypothesis. First, the observation that only a low proportion (73/844, 8.6%) of the E. coli isolates analyzed belonged to clonal groups (defined as >2 E. coli isolates that had indistinguishable multilocus variable number tandem repeats and enterobacterial repetitive intergenic consensus 2 patterns), including members from >1 source, suggests an overall high degree of genetic heterogeneity among isolates from different sources. Second, looking at the single isolates within clonal groups reported in, twelve (2.9%) of the 417 isolates from retail meat shared multilocus variable number tandem repeats, enterobacterial repetitive intergenic consensus 2, and multilocus sequence types with some human UTI isolates; however, only 1 isolate (strain EC01DT06–1737–01) was also found to be indistinguishable from a human isolate (strain MSHS 161) by pulsed-field gel electrophoresis , indicating that identical genotypes (between isolates from retail meat and human infections) were observed only once. Although we agree that the finding of a partial overlap between multilocus sequence types of isolates from retail meat and from human UTI isolates is noteworthy (especially recovery of an ST131 isolate of avian origin), the emphasis posed for the role of food transmission in the dissemination of the E. coli strains that cause community-acquired UTIs, in our opinion, does not seem strongly supported by the experimental data. Nevertheless, the topic is relevant, and we would highlight the importance of further research on this issue.