Characterization of cultivable airborne bacteria and their antimicrobial resistance pattern in French milking parlour.

The main goal of this preliminary study was to quantify airborne particles and characterize the dominant cultivable bacterial species as well as some Gram-positive species, and their antibiotic resistance pattern, from environmental samples taken inside and outside of a dairy milking parlour. Sampling was performed over 2 days, in different seasons. The small viable particulate matter < 10 µm (bioaerosols) and cultivable bacteria reached their highest concentrations in the milking parlour. The majority of airborne bacteria in the milking parlour belonged to the genera Staphylococcus (41.9%) and Bacillus (20.9%). A total of 32 different bacterial species of Staphylococcus, Aerococcus, Bacillus, Pseudomonas, Serratia and Acinetobacter were identified. Many of these bacteria may be opportunistic pathogens, causing disease in humans or animals. We found low levels of acquired resistance to the antibiotics commonly used in human or animal infections caused by these opportunistic bacteria. More specifically, resistance to tetracyclines (13.4%), penicillin G (13.4%) and macrolides (7.5%) was identified in Staphylococcus sp. as was a methicillin-resistant S. hominis and resistance to spiramycin (n = 1), lincomycin (n = 1) and streptomycin (n = 2) in Aerococcus sp. An assessment of the occupational risk run by dairy farmers for contracting infections after long- or short-term exposure to micro-organisms requires further studies on the concentration of opportunistic pathogenic bacteria in dairy farm environments.

Pathogenic Escherichia coli in Dogs Reveals the Predominance of ST372 and the Human-Associated ST73 Extra-Intestinal Lineages.

Escherichia coli is a ubiquitous commensal and pathogen that has also been recognized as a multi-sectoral indicator of antimicrobial resistance (AMR). Given that latter focus, such as on resistances to extended-spectrum cephalosporins (ESC) and carbapenems, the reported population structure of E. coli is generally biased toward resistant isolates, with sequence type (ST)131 being widely reported in humans, and ST410 and ST648 being reported in animals. In this study, we characterized 618 non-duplicate E. coli isolates collected throughout France independently of their resistance phenotype. The B2 phylogroup was over-represented (79.6%) and positively associated with the presence of numerous virulence factors (VFs), including those defining the extra-intestinal pathogenic E. coli isolates (presence of ≥2 VFs: papA, sfaS, focG, afaD, iutA, and kpsMTII) and those more specifically related to uropathogenic E. coli (cnf1, hlyD). The major STs associated with clinical isolates from dogs were by far the dog-associated ST372 (20.7%) and ST73 (20.1%), a lineage that had commonly been considered until now as human-associated. Resistance to ESC was found in 33 isolates (5.3%), along with one carbapenemase-producing isolate, and was mostly restricted to non-B2 isolates. In conclusion, the presence of virulent E. coli lineages may be the issue, rather than the presence of ESC-resistant isolates, and the risk of transmission of such virulent isolates to humans needs to be further studied.

Variant O89 O-Antigen of E. coli Is Associated With Group 1 Capsule Loci and Multidrug Resistance.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli, major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli. A multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing strain - E. coli 26561 - represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd, flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) - 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli.

Absence of co-localization between pathovar-associated virulence factors and extended-spectrum ß-lactamase (blaCTX-M) genes on a single plasmid.

Extended-spectrum ß-lactamases (ESBLs) were reported in virulent food-borne Escherichia coli clones, and numerous genes encoding ESBLs and virulence factors (VFs) are plasmid-mediated. We investigated the plasmidic co-localization of ESBL genes and pathovar-associated VF genes isolated in 18 E. coli isolates from faecal samples of diseased cattle. From the rare ESBL-producing E. coli among the various pathovars, no plasmid co-localization was found between VF and blaCTX-M genes on a single plasmid. However, a link between replicon types and VFs was highlighted: EspP was associated with IncFIB and ToxB with IncB/O. Associations of IncF alleles to VF or CTX-M-types were also identified: CS31A was linked to the allele FIB38 and CTX-M-14 to IncFII2. Also, as illustrated here, IncFII and IncFIB were carried by two different plasmids in a single cell.

Draft Genome Sequences of Enterohemorrhagic Escherichia coli Encoding Extended-Spectrum Beta-Lactamases.

Extended-spectrum beta-lactamases (ESBLs) have rarely been observed among Shiga toxigenic Escherichia coli (STEC), and, to our best knowledge, only three ESBL-positive isolates of the enterohemorrhagic E. coli (EHEC) subpathotype have been reported. Here, we present the first draft genome sequences of two ESBL-positive EHEC isolates belonging to serotypes O111:H8 and O151:H16.

Assessment of Adhesins as an Indicator of Pathovar-Associated Virulence Factors in Bovine Escherichia coli.

The CS31A, F17, and F5 adhesins are usually targeted by serology-based methods to detect pathogenic Escherichia coli associated with calf enteritis. However, the virulence traits of the selected isolates are still poorly described. Here, from a set of 349 diarrheagenic E. coli isolates from cattle, we demonstrated a 70.8% concordance rate (Cohen's kappa, 0.599) between serology- and PCR-based approaches for the detection of adhesins under field conditions. A 79% to 82.4% correspondence between the two methods was found for fimbrial adhesins, whereas major discrepancies (33%) were observed for CS31A-type antigens. Various F17A variants were found, such as F17Ac (20K) (50%), F17Aa (FY) (18.9%), F17Ab (8.1%), and F17Ad (111K) (5.4%), including a high proportion (17.6%) of new F17A internal combinations (F17Aab, F17Aac, and F17Abc) or untypeable variants. In addition, the highest proportion of pathovar-associated virulence factor (VF) genes was observed among E. coli isolates that produced F5/F41 adhesins. A specific link between the heat-stable toxins related to the enterotoxigenic E. coli (ETEC) pathovar and adhesins was identified. STa was significantly linked to F5/F41 and EAST1 to CS31A adhesins (P < 0.001), respectively, whereas NTEC was associated with F17 adhesin (P = 0.001). Clustering between phylogroups according to the adhesin types was also observed. Also, few Shiga toxin-producing E. coli (STEC) or enteropathogenic E. coli (EPEC) pathovars were identified. Finally, no statistically significant difference was observed in the occurrence of extended-spectrum beta lactamase (ESBL) production according to the adhesins expressed by the isolates (P = 0.09). Altogether, this study gives new insights into the relationship between adhesins, VF, and antimicrobial resistance in calf enteritis and supports the need for further standardization of methodologies for such approaches.

Phylogenetic grouping and virulence potential of extended-spectrum-ß-lactamase-producing Escherichia coli strains in cattle.

In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx(1) and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla(CTX-M) genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.

CTX-M-15 extended-spectrum ß-lactamase in a shiga toxin-producing Escherichia coli isolate of serotype O111:H8.

We report the discovery of a CTX-M-15-producing Escherichia coli (STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic Escherichia coli (EHEC) infections. In line with the recent CTX-M-15/O104:H4 E. coli outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the E. coli population, including STEC isolates.

Impact of deoxynivalenol on the intestinal microflora of pigs.

Deoxynivalenol (DON), a mycotoxin produced by some Fusarium species, is a frequent contaminant of cereal. In the present study, 24 weanling piglets received either control feed or feed naturally contaminated with DON (2.8 mg/kg) for four weeks. Consumption of contaminated feed significantly reduced the animal weight gain during the first week of the experiment, but had a moderate effect on cultivable bacteria in the pig intestine. By contrast, changes in the intestinal microflora were observed by Capillary Electrophoresis Single-Stranded Conformation Polymorphism (CE-SSCP) in DON-exposed animals, suggesting an impact of this toxin on the dynamics of intestinal bacteria communities.

Use of ATP bioluminescence to determine the bacterial sensitivity threshold to a bacteriocin.

A new ATP bioluminescence-based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic-nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin-luciferase. Nisin-induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quantification of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a AN(tot):AN(ext) ratio = 1. For an initial concentration of 1.4 x 10(7) bacteria/mL, the nisin effectiveness threshold is 3.4 +/- 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells.