Changing dynamics of antibiotic resistant Escherichia in Caspian gulls shows the importance of longitudinal environmental studies.

This study is focused on Escherichia spp. isolates resistant to critically important antibiotics (cefotaxime, ciprofloxacin and colistin) among Caspian gull's (Larus cachinnans) chicks nesting in the Nove Mlyny Water Reservoir, Czech Republic. The prevalence of antimicrobial resistance (AMR) in bacteria within wild birds is commonly evaluated using a single sampling event, capturing only a brief and momentary snapshot at a particular location. Therefore, the Caspian gulls in our study were sampled in May 2018 (n = 72) and May 2019 (n = 45), and a water sample was taken from the reservoir (2019). We obtained 197 isolates identified as E. coli by MALDI-TOF MS. A total of 158 representative isolates were whole-genome sequenced, 17 isolates were then reclassified to Escherichia albertii. We observed a higher (86 %; 62/72) occurrence of ESBL/AmpC-producing Escherichia spp. among gulls in 2018 compared to 38 % (17/45) in 2019 (p < 0.00001). The decrease in prevalence was linked to clonal lineage of E. coli ST11893 predominating in 2018 which carried blaCMY-2 and which was not recovered from the gulls in 2019. Oppositely, several Escherichia STs were found in gulls from both years as well as in the water sample including STs commonly recognized as internationally high-risk lineages such as ST10, ST58, ST88, ST117, ST648 or ST744. Phylogenetic analysis of E. coli from EnteroBase from countries where these particular gulls wander revealed that some STs are commonly found in various sources including humans and a portion of them is even closely related (up to 100 SNPs) to our isolates. We demonstrated that the occurrence of AMR in Escherichia can vary greatly in time in synanthropic birds and we detected both, a temporary prevalent lineage and several persistent STs. The close relatedness of isolates from gulls and isolates from EnteroBase highlights the need to further evaluate the risk connected to wandering birds.

Detection of Plasmid-Mediated Resistance to Metronidazole in Clostridioides difficile from River Water.

Clostridioides difficile is one of the most important human pathogens. The identification of its possible sources is important for the understanding of C. difficile infection (CDI) epidemiology. A total of 16 water samples from wastewater and surface water in South Moravia in the Czech Republic and 82 samples of fish and gulls were collected between May and July 2019. C. difficile isolates were cultured by direct plating and after enrichment on chromogenic media. Susceptibility testing to eight antimicrobials was performed by Etest. C. difficile isolates were characterized by ribotyping, multilocus sequence typing, multilocus tandem repeats analysis, and toxin gene detection. Samples from fish and gulls were C. difficile negative; a total of 15 C. difficile isolates from 8 out of 16 water samples were cultured (6 out of 14 surface water samples yielded 6 isolates, and 2 out of 2 wastewater samples yielded 9 isolates). Direct plating was culture positive in 6 out of 16 samples (12 isolates), and enrichment culture was positive in an additional 2 out of 16 samples (3 isolates). Twelve different ribotyping profiles and 14 sequence types of clades 1, 4, and 5 were identified. Five isolates did not carry genes for toxins, and eight isolates carried genes for toxins A and B; the remaining two isolates (RT078) carried the genes for toxins A, B, and binary. All C. difficile isolates were susceptible to amoxicillin, moxifloxacin, tetracycline, and vancomycin and resistant to ciprofloxacin. A high level of erythromycin resistance (>256 mg/L) was detected in eight isolates. Clindamycin resistance was found in 14 isolates, 6 of which showed a high level of resistance (>256 mg/L) and carried ermB. Surprisingly, one isolate (RT010, ST15) showed resistance to metronidazole (12 mg/L) with the presence of the plasmid pCD-METRO. In conclusion, a diverse spectrum of C. difficile strains was found in wastewater and surface water. A recently discovered plasmid-bound resistance to metronidazole was detected in C. difficile from the surface water sample. IMPORTANCE The combination of direct plating and culture after enrichment was used in order to gain a spectrum of C. difficile ribotypes present in the water samples. Toxigenic C. difficile ribotypes detected in surface water and in wastewater treatment plants overlapped with those derived from patients with CDI and/or animals. Importantly, a recently discovered plasmid-mediated resistance to metronidazole, a drug used for the treatment of CDI, was detected in C. difficile from river water.

Occurrence of virulence-associated genes in Streptococcus uberis and Streptococcus parauberis isolated from bovine mastitis.

Streptococcus uberis is one of the most important mastitis-causing pathogens. Although the pathogenesis and virulence factors required for the intramammary infection development are not yet well established, several putative virulence-associated genes have been described. This work aimed to investigate the presence of ten known and putative virulence-associated genes in S. uberis isolated from subclinical or clinical mastitis and its closely related species Streptococcus parauberis in 135 dairy farms in the Czech Republic. The PCR analysis detected that all the examined isolates possessed at least four virulence genes and most isolates carried eight out of ten virulence genes. All S. uberis isolates were positive for the oppF, gapC and sua genes. Among the most prevalent virulence-associated genes skc (98%) and pauA (97%) were also found. The hasA and hasB genes were always present together in 94% of the isolates. The genes cfu and lbp were detected in 6% and 2%, respectively. In the S. uberis isolates, 14 different virulence gene profiles were observed. The most frequent profile was hasA + hasB + sua + skc + pauA + gapC + oppF with variable hasC, observed in 86% of the tested isolates, occurring in 127 out of 135 farms. S. parauberis was identified very sporadically and, although it is closely related to S. uberis, only a rare occurrence of the examined virulence-associated genes was found.

Extended-spectrum beta-lactamase-producing Escherichia coli and antimicrobial resistance in municipal and hospital wastewaters in Czech Republic: Culture-based and metagenomic approaches.

Wastewaters serve as important hot spots for antimicrobial resistance and monitoring can be used to analyse the abundance and diversity of antimicrobial resistance genes at the level of large bacterial and human populations. In this study, whole genome sequencing of beta-lactamase-producing Escherichia coli and metagenomic analysis of whole-community DNA were used to characterize the occurrence of antimicrobial resistance in hospital, municipal and river waters in the city of Brno (Czech Republic). Cefotaxime-resistant E. coli were mainly extended-spectrum beta-lactamase (ESBL) producers (95.6%, n = 158), of which the majority carried blaCTX-M (98.7%; n = 151) and were detected in all water samples except the outflow from hospital wastewater treatment plant. A wide phylogenetic diversity was observed among the sequenced E. coli (n = 78) based on the detection of 40 sequence types and single nucleotide polymorphisms (average number 34,666 ± 15,710) between strains. The metagenomic analysis revealed a high occurrence of bacterial genera with potentially pathogenic members, including Pseudomonas, Escherichia, Klebsiella, Aeromonas, Enterobacter and Arcobacter (relative abundance >50%) in untreated hospital and municipal wastewaters and predominance of environmental bacteria in treated and river waters. Genes encoding resistance to aminoglycosides, beta-lactams, quinolones and macrolides were frequently detected, however blaCTX-M was not found in this dataset which may be affected by insufficient sequencing depth of the samples. The study pointed out municipal treated wastewater as a possible source of multi-drug resistant E. coli and antimicrobial resistance genes for surface waters. Moreover, the combination of two different approaches provided a more holistic view on antimicrobial resistance in water environments. The culture-based approach facilitated insight into the dynamics of ESBL-producing E. coli and the metagenomics shows abundance and diversity of bacteria and antimicrobial resistance genes vary across water sites.

Carbapenemase-Producing Gram-Negative Bacteria from American Crows in the United States.

Wild corvids were examined for the presence of carbapenemase-producing Gram-negative bacteria in the United States. A total of 13 isolates were detected among 590 fecal samples of American crow; 11 Providencia rettgeri isolates harboring blaIMP-27 on the chromosome as a class 2 integron gene cassette within the Tn7 transposon, 1 Klebsiella pneumoniae ST258 isolate carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and 1 Enterobacter bugandensis isolate with blaIMI-1 located within EcloIMEX-2.

The Colonisation of Calves in Czech Large-Scale Dairy Farms by Clonally-Related Clostridioides difficile of the Sequence Type 11 Represented by Ribotypes 033 and 126.

To investigate a possible Clostridioides difficile reservoir in the Czech Republic, we performed a study in 297 calves from 29 large-scale dairy farms. After enrichment, faecal samples were inoculated onto selective agar for C. difficile. From the 297 samples, 44 C. difficile isolates were cultured (prevalence of 14.8%, 10 farms). The Holstein breed and use of digestate were associated with C. difficile colonisation (p ˂ 0.05). C. difficile isolates belonged to the ribotype/sequence type: RT033/ST11 (n = 37), RT126/ST11 (n = 6) and RT046/ST35 (n = 1). A multiple-locus variable-number tandem-repeat analysis revealed four clonal complexes of RT033 isolates and one clonal complex of RT126 isolates. All isolates were sensitive to amoxicillin, metronidazole and vancomycin. Forty isolates were resistant to ciprofloxacin, twenty-one to clindamycin, seven to erythromycin, seven to tetracycline and six to moxifloxacin. Moxifloxacin resistant isolates revealed an amino-acid substitution Thr82Ile in the GyrA. In conclusion, the calves of Holstein breed from farms using digestate as a product of bio-gas plants are more likely to be colonised by clonally-related C. difficile of ST 11 represented by ribotypes 033 and 126. The identified resistance to moxifloxacin with a Thr82Ile substitution in the GyrA highlights the need for further monitoring by the "One health approach".

Genomic analysis of Escherichia coli strains isolated from diseased chicken in the Czech Republic.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) can cause various extraintestinal infections in poultry, resulting in massive economic losses in poultry industry. In addition, some avian E. coli strains may have zoonotic potential, making poultry a possible source of infection for humans. Due to its extreme genetic diversity, this pathotype remains poorly defined. This study aimed to investigate the diversity of colibacillosis-associated E. coli isolates from Central European countries with a focus on the Czech Republic. RESULTS: Of 95 clinical isolates subjected to preliminary characterization, 32 were selected for whole-genome sequencing. A multi resistant phenotype was detected in a majority of the sequenced strains with the predominant resistance to ß-lactams and quinolones being associated with TEM-type beta-lactamase genes and chromosomal gyrA mutations respectively. The phylogenetic analysis confirmed a great diversity of isolates, that were derived from nearly all phylogenetic groups, with predominace of B2, B1 and C phylogroups. Clusters of closely related isolates within ST23 (phylogroup C) and ST429 (phylogroup B2) indicated a possible local spread of these clones. Besides, the ST429 cluster carried blaCMY-2, - 59 genes for AmpC beta-lactamase and isolates of both clusters were generally well-equipped with virulence-associated genes, with considerable differences in distribution of certain virulence-associated genes between phylogenetically distant lineages. Other important and potentially zoonotic APEC STs were detected, incl. ST117, ST354 and ST95, showing several molecular features typical for human ExPEC. CONCLUSIONS: The results support the concept of local spread of virulent APEC clones, as well as of zoonotic potential of specific poultry-associated lineages, and highlight the need to investigate the possible source of these pathogenic strains.

Vancomycin-resistant enterococci with vanA gene in treated municipal wastewater and their association with human hospital strains.

Vancomycin-resistant enterococci (VRE) are pathogens of increasing medical importance. In Brno, Czech Republic, we collected 37 samples from the effluent of a wastewater treatment plant (WWTP), 21 surface swabs from hospital settings, and 59 fecal samples from hospitalized patients and staff. Moreover, we collected 284 gull cloacal swabs from the colony situated 35km downstream the WWTP. Samples were cultured selectively. Enterococci were identified using MALDI-TOF MS, phenotypically tested for susceptibility to antibiotics, and by PCR for occurrence of resistance and virulence genes. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used to examine genotypic diversity. VRE carrying the vanA gene were found in 32 (86%, n=37) wastewater samples, from which we obtained 49 isolates: Enterococcus faecium (44) and Enterococcus gallinarum (2), Enterococcus casseliflavus (2), and Enterococcus raffinosus (1). From 33 (69%) of 48 inpatient stool samples, we obtained 39 vanA-carrying VRE, which belonged to E. faecium (33 isolates), Enterococcus faecalis (4), and Enterococcus raffinosus (2). Nearly one-third of the samples from hospital surfaces contained VRE with the vanA gene. VRE were not detected among gulls. Sixty-seven (84%, n=80) E. faecium isolates carried virulence genes hyl and/or esp. Virulence of E. faecalis was encoded by gelE, asa1, and cylA genes. A majority of the E. faecium isolates belonged to the clinically important sequence types ST17 (WWTP: 10 isolates; hospital: 4 isolates), ST18 (9;8), and ST78 (5;0). The remaining isolates belonged to ST555 (2;0), ST262 (1;6), ST273 (3;0), ST275 (1;0), ST549 (2;0), ST19 (0;1), ST323 (3;0), and ST884 (7;17). Clinically important enterococci carrying the vanA gene were almost continually detectable in the effluent of the WWTP, indicating insufficient removal of VRE during wastewater treatment and permanent shedding of these antibiotic resistant pathogens into the environment from this source. This represents a risk of their transmission to the environment.

Salmonella enterica resistant to antimicrobials in wastewater effluents and black-headed gulls in the Czech Republic, 2012.

We investigated the presence and epidemiological relatedness of Salmonella isolates from a wastewater treatment plant (WWTP) in Brno, Czech Republic and from nestlings of black-headed gulls (Chroicocephalus ridibundus) at the Nove Mlyny waterworks, situated 35 km downstream from the WWTP. During 2012, we collected 37 wastewater samples and 284 gull cloacal swabs. From wastewater samples, we obtained 89 Salmonella isolates belonging to 19 serotypes. At least one resistant strain was contained in 89% of those samples. Ten different serotypes of Salmonella were detected in 38 young gulls, among which 14 (37%) were resistant to antimicrobials. Wastewater isolates were mostly resistant to sulphonamides and tetracycline, gull isolates to tetracycline and ampicillin. We detected the occurrence of blaTEM-1,tet(A), tet(B), tet(G), sul1, sul2, sul3, floR and strA resistance genes. For the first time, we identified a class 1 integron with the dfrA12-orfF-aadA2 gene cassette in S. Infantis. Using pulsed-field gel electrophoresis, we confirmed the presence of identical clusters of S. Agona, S. Enteritidis PT8, S. Infantis and S. Senftenberg in wastewater and black-headed gulls, thus indicating the possibility of resistant Salmonella isolates spreading over long distances in the environment.

Characteristics of Quinolone Resistance in Escherichia coli Isolates from Humans, Animals, and the Environment in the Czech Republic.

Escherichia coli is a common commensal bacterial species of humans and animals that may become a troublesome pathogen causing serious diseases. The aim of this study was to characterize the quinolone resistance phenotypes and genotypes in E. coli isolates of different origin from one area of the Czech Republic. E. coli isolates were obtained from hospitalized patients and outpatients, chicken farms, retailed turkeys, rooks wintering in the area, and wastewaters. Susceptibility of the isolates grown on the MacConkey agar with ciprofloxacin (0.05 mg/L) to 23 antimicrobial agents was determined. The presence of plasmid-mediated quinolone resistance (PMQR) and ESBL genes was tested by PCR and sequencing. Specific mutations in gyrA, gyrB, parC, and parE were also examined. Multilocus sequence typing and pulsed-field gel electrophoresis were performed to assess the clonal relationship. In total, 1050 E. coli isolates were obtained, including 303 isolates from humans, 156 from chickens, 105 from turkeys, 114 from the rooks, and 372 from wastewater samples. PMQR genes were detected in 262 (25%) isolates. The highest occurrence was observed in isolates from retailed turkey (49% of the isolates were positive) and inpatients (32%). The qnrS1 gene was the most common PMQR determinant identified in 146 (56%) followed by aac(6')-Ib-cr in 77 (29%), qnrB19 in 41 (16%), and qnrB1 in 9 (3%) isolates. All isolates with high level of ciprofloxacin resistance (>32 mg/L) carried double or triple mutations in gyrA combined with single or double mutations in parC. The most frequently identified substitutions were Ser(83)Leu; Asp(87)Asn in GyrA, together with Ser(80)Ile, or Glu(84)Val in ParC. Majority of these isolates showed resistance to beta-lactams and multiresistance phenotype was found in 95% isolates. Forty-eight different sequence types among 144 isolates analyzed were found, including five major clones ST131 (26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). No isolates sharing 100% relatedness and originating from different areas were identified. In conclusion, our study identified PMQR genes in E. coli isolates in all areas studied, including highly virulent multiresistant clones such as ST131 producing CTX-M-15 beta-lactamases.

High prevalence of Salmonella and IMP-4-producing Enterobacteriaceae in the silver gull on Five Islands, Australia.

OBJECTIVES: The objective of this study was to investigate the silver gull as an indicator of environmental contamination by salmonellae and carbapenemase-producing Enterobacteriaceae (CPE) in south-east Australia. METHODS: A total of 504 cloacal samples were collected from gull chicks at three nesting colonies in New South Wales, Australia [White Bay (n = 144), Five Islands (n = 200) and Montague Island (n = 160)] and were examined for salmonellae and CPE. Isolates were tested for carbapenemase genes and susceptibility to 14 antibiotics. Clonality was determined by PFGE and MLST. Genetic context and conjugative transfer of the carbapenemase gene were determined. RESULTS: A total of 120 CPE of 10 species, mainly Escherichia coli (n = 85), carrying the gene blaIMP-4, blaIMP-38 or blaIMP-26 were obtained from 80 (40%) gulls from Five Islands. Thirty percent of birds from this colony were colonized by salmonellae. Most isolates contained the gene within a class 1 integron showing a blaIMP-4-qacG-aacA4-catB3 array. The blaIMP gene was carried by conjugative plasmids of variable sizes (80-400 kb) and diverse replicons, including HI2-N (n = 30), HI2 (11), A/C (17), A/C-Y (2), L/M (5), I1 (1) and non-typeable (6). Despite the overall high genetic variability, common clones and plasmid types were shared by different birds and bacterial isolates, respectively. CONCLUSIONS: Our data demonstrate a large-scale transmission of carbapenemase-producing bacteria into wildlife, likely as a result of the feeding habits of the birds at a local waste depot. The isolates from gulls showed significant similarities with clinical isolates from Australia, suggesting the human origin of the isolates. The sources of CPE for gulls on Five Islands should be explored and proper measures applied to stop the transmission into the environment.

Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA).

A series of twenty-one salicylanilide N-alkylcarbamates was assessed for novel antibacterial characteristics against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum inhibitory concentration was determined by the broth dilution micro-method with subsequent subcultivation of aliquots to assess minimum bactericidal concentration. The bactericidal kinetics was established by time-kill assay. Ampicillin, ciprofloxacin and vancomycin were used as reference antibacterial drugs. All the tested compounds exhibited highly potent anti-MRSA activity (⩽ 0.008-4 µg/mL) comparable or up to 250× higher than that of vancomycin, the standard in the treatment of serious MRSA infections. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl butylcarbamate and 4-chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl ethylcarbamate were the most active compounds. In most cases, compounds provided reliable bacteriostatic activity, except for 4-chloro-2-(4-chlorophenylcarbamoyl)phenyl decylcarbamate exhibiting bactericidal effect at 8h (for clinical isolate of MRSA 63718) and at 24h (for clinical isolates of MRSA SA 630 and MRSA SA 3202) at 4× MIC. Structure-activity relationships are discussed.

Low rates of antimicrobial-resistant Enterobacteriaceae in wildlife in Taï National Park, Côte d'Ivoire, surrounded by villages with high prevalence of multiresistant ESBL-producing Escherichia coli in people and domestic animals.

Antimicrobial resistance genes can be found in all ecosystems, including those where antibiotic selective pressure has never been exerted. We investigated resistance genes in a collection of faecal samples of wildlife (non-human primates, mice), people and domestic animals (dogs, cats) in Côte d'Ivoire; in the chimpanzee research area of Taï National Park (TNP) and adjacent villages. Single bacteria isolates were collected from antibiotic-containing agar plates and subjected to molecular analysis to detect Enterobacteriaceae isolates with plasmid-mediated genes of extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR). While the prevalence of ESBL-producing E. coli in the villages was 27% in people (n = 77) and 32% in dogs (n = 38), no ESBL-producer was found in wildlife of TNP (n = 75). PMQR genes, mainly represented by qnrS1, were also present in human- and dog-originating isolates from the villages (36% and 42% in people and dogs, respectively), but no qnrS has been found in the park. In TNP, different variants of qnrB were detected in Citrobacter freundii isolates originating non-human primates and mice. In conclusion, ESBL and PMQR genes frequently found in humans and domestic animals in the villages were rather exceptional in wildlife living in the protected area. Although people enter the park, the strict biosecurity levels they are obliged to follow probably impede transmission of bacteria between them and wildlife.

First record of vancomycin-resistant Enterococcus faecium in Canadian wildlife.

In this study, we focused on spreading of vancomycin-resistant enterococci (VRE) to the environment. We studied that weather crows in Canada may be carriers and potentially reservoirs of VRE with vanA gene. We have found one multi-resistant isolate of Enterococcus faecium sequence type (ST) 448 with vanA gene on Prince Edward Island. This study is the first report of VRE in Canadian wildlife.

Antimicrobial-resistant Enterobacteriaceae from humans and wildlife in Dzanga-Sangha Protected Area, Central African Republic.

Antimicrobial resistance is a worldwide concern of public health. Unfortunately, resistant bacteria are spreading to all ecosystems, including the strictly protected ones. We investigated antimicrobial resistance in gastrointestinal Enterobacteriaceae of wild mammals and people living within Dzangha-Sangha Protected Areas, Central African Republic, with an emphasis on extended-spectrum ß-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. We compare resistance genes found in microbiota of humans, gorillas habituated and unhabituated to humans and other wildlife. In gorillas, we additionally investigate the presence of ESBL resistant isolates after treatment by ceftiofur. We found a considerable prevalence of multiresistant Enterobacteriaceae isolates with ESBL and PMQR genes in humans (10% and 31%, respectively). Among wildlife the most significant findings were CTX-M-15-producing Klebsiella pneumoniae in a habituated gorilla and a multiresistant Escherichia coli isolate with gene qepA in an unhabituated gorilla. Other isolates from wildlife were mostly represented by qnrB-harboring Citrobacter spp. The relatedness of resistant E. coli was investigated in a PFGE-based dendrogram; isolates from gorillas showed less than 80% similarity to each other and less than 80% similarity to human isolates. No ESBL-producing isolates were found in animals treated by ceftiofur. Although we did not detect any bacterial clone common to wildlife and humans, we detected an intersection in the spectrum of resistance genes found in humans and gorillas, represented by blaCTX-M-15 and qepA.

New derivatives of salicylamides: Preparation and antimicrobial activity against various bacterial species.

Three series of salicylanilides, esters of N-phenylsalicylamides and 2-hydroxy-N-[1-(2-hydroxyphenylamino)-1-oxoalkan-2-yl]benzamides, in total thirty target compounds were synthesized and characterized. The compounds were evaluated against seven bacterial and three mycobacterial strains. The antimicrobial activities of some compounds were comparable or higher than the standards ampicillin, ciprofloxacin or isoniazid. Derivatives 3f demonstrated high biological activity against Staphylococcus aureus (⩽0.03µmol/L), Mycobacterium marinum (⩽0.40µmol/L) and Mycobacterium kansasii (1.58µmol/L), 3g shows activity against Clostridium perfringens (⩽0.03µmol/L) and Bacillus cereus (0.09µmol/L), 3h against Pasteurella multocida (⩽0.03µmol/L) and M. kansasii (⩽0.43µmol/L), 3i against methicillin-resistant S. aureus and B. cereus (⩽0.03µmol/L). The structure-activity relationships are discussed for all the compounds.