Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry

doi:10.1016/j.vetmic.2010.03.019

Veterinary Microbiology

Volume 145, Issues 3–4, 26 October 2010, Pages 273-278

https://doi.org/10.1016/j.vetmic.2010.03.019 Get rights and content

Abstract

To gain more information on the genetic basis of the rapid increase in the number of isolates exhibiting non-wild type Minimum Inhibitory Concentrations (MICs) for cefotaxime observed since 2003, beta-lactamase genes of 22 Salmonella enterica and 22 Escherichia coli isolates from broilers in 2006 showing this phenotype were characterized by miniaturized micro-array, PCR and DNA-sequencing. Presence and size of plasmids were determined by S1-digest pulsed-field gel electrophoresis and further characterized by PCR-based replicon typing. Transfer of resistance plasmids was tested by conjugation and transformation experiments. To link resistance genes and plasmid type, Southern blot hybridization experiments were conducted.

In 42 isolates, five (bla_CTX-M-1, bla_CTX-M-2, bla_TEM-20, bla_TEM-52, bla_SHV-2) different extended spectrum beta-lactamase (ESBL)-genes and two (bla_ACC-1, bla_CMY-2) AmpC-genes were present. Three of the detected ESBL-genes (bla_CTX-M-1, bla_TEM-52 and bla_CTX-M-2) were located on similar types of plasmids (IncI1 and IncHI2/P) in both E. coli and Salmonella. Two other detected ESBL- and AmpC-genes bla_SHV-2 and bla_CMY-2 respectively (on IncK plasmids), were only found in E. coli, whereas the AmpC-gene bla_ACC-1 (on non-typable plasmids), and the ESBL-gene bla_TEM-20 (on IncI1 plasmids), were only detected in Salmonella. In two isolates, no ESBL- or AmpC-gene could be detected through these methods.

The increase in the number of E. coli and S. enterica isolates from the gastro-intestinal tract of broilers exhibiting non-wild type MICs for cefotaxime is mainly due to an increase in IncI1 plasmids containing bla_CTX-M-1. The reason for the successful spread of this plasmid type in these species is not yet understood.

Introduction

A rapid development of resistance to extended spectrum cephalosporins (ESC) has been observed in Enterobacteriaceae worldwide. In these organisms, resistance to this group of antibiotics is predominantly based on plasmid-mediated production of enzymes that inactivate these compounds by hydrolyzing their beta-lactam ring. The most frequently detected groups of these enzymes in bacteria of animal origin are extended spectrum beta-lactamases (ESBLs) and AmpC-type beta-lactamases. Both are important causes of treatment failures in humans when they are produced by pathogens.

ESBL as well as AmpC-producing bacteria are frequently present in the gastro-intestinal tract of animals (Carattoli, 2008, Li et al., 2007) and have been isolated from swine, cattle, turkey (Liebana et al., 2004), cats, dogs (Carattoli et al., 2005a), poultry (Hasman et al., 2005), wild animals (Costa et al., 2006) and horses (Vo et al., 2007). The gastro-intestinal tract of animals is seen as an important reservoir for bacteria that produce beta-lactamases, and a potential source for human pathogens to take up these resistance genes (Carattoli, 2008, Carattoli, 2009, Li et al., 2007). ESBL- and AmpC-genes are located on plasmids which enable them to spread very rapidly. Therefore plasmid characterization is an essential tool to understand the epidemiology of these genes.

Before 2003, isolates exhibiting non-wild type MICs (Schwarz et al., 2010) for ESC in Escherichia coli from broilers was observed only incidentally in the Netherlands. This changed after 2003 with the observation of an annual increase in non-wild type MICs for cefotaxime in commensal E. coli from Dutch broilers. In 2006, a similar increase was also observed in Salmonella enterica from various poultry sources (MARAN, 2009). The purpose of the present study was to characterize the beta-lactamase genes and the plasmids harboring these genes in E. coli and S. enterica from broilers.

Section snippets

Bacterial isolates and susceptibility testing

In the Dutch surveillance program on antibiotic resistance in bacteria isolated from animals, more than 500 E. coli and 1000 S. enterica isolates are tested each year for susceptibility to a panel of antibiotics. The Minimum Inhibitory Concentrations (MICs) of these isolates were determined for ampicillin, cefotaxime, ceftazidime, gentamicin, tetracycline, sulphamethoxazole, trimethoprim, ciprofloxacin, nalidixic acid, florfenicol and chloramphenicol by the broth micro-dilution method,

Antibiotic resistance phenotypes

Of 153 E. coli isolates obtained from Dutch poultry in 2006, 22 had non-wild type MICs for cefotaxime. In these 22 isolates, additional non-wild type MICs were found for ceftazidime (100%), sulphamethoxazole (95%), trimethoprim (86%), streptomycin (59%), nalidixic acid (55%), ciprofloxacin (50%), tetracycline (50%), chloramphenicol (32%), gentamicin (18%) and neomycin (14%).

Of 359 Salmonella isolates obtained from Dutch poultry in 2006, 12 S. Paratyphi B variant (var.) Java, 5 S. Infantis, 2 S.

Discussion

The results obtained in this study show that non-wild type MICs for cefotaxime in E. coli and S. enterica from Dutch broilers, can mainly be explained by the presence of IncI1 plasmids carrying bla_CTX-M-1 in both species. Although a variety of ESBL-genes and plasmids are found in both bacterial species, similar ESBL-genes and plasmid-families are those that are predominantly present (Table 2). This is understandable as E. coli and S. enterica isolates were derived from the same source: the

Acknowledgements

This work was supported by the Ministry of Agriculture, Nature and Food Quality as part of project nr. WOT-01-002-003 “ESBLs in farm animals”.

We would like to thank Muriel Mafura and Muna Anjum for teaching the miniaturized micro-array technique, Alessandra Carattoli for kindly providing the control isolates for the PBRT method to our laboratory and giving advice on the PBRT-results, and the European Community Network of Excellence MED-VET-NET workpackage 21 (contract no. FOOD-CT-2004-506122)

References (23)

B.M. Barton et al.
A general method for detecting and sizing large plasmids
Anal. Biochem.
(1995)
M. Batchelor et al.
Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria
Int. J. Antimicrob. Agents
(2008)
A. Carattoli
Animal reservoirs for extended spectrum beta-lactamase producers
Clin. Microbiol. Infect.
(2008)
A. Carattoli et al.
Identification of plasmids by PCR-based replicon typing
J. Microbiol. Methods
(2005)
X.Z. Li et al.
Beta-Lactam resistance and beta-lactamases in bacteria of animal origin
Vet. Microbiol.
(2007)
D.L. Paterson
Resistance in Gram-negative bacteria: Enterobacteriaceae
Am. J. Infect. Control.
(2006)
S. Schwarz et al.
Editorial: assessing the antimicrobial susceptibility of bacteria obtained from animals
Vet. Microbiol.
(2010)
A.T. Vo et al.
Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses
Vet. Microbiol.
(2007)
A. Carattoli
Resistance plasmid families in Enterobacteriaceae
Antimicrob. Agents Chemother.
(2009)
A. Carattoli et al.
Extended-spectrum beta-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003
Antimicrob. Agents Chemother.
(2005)

D. Costa et al.

Detection of Escherichia coli harbouring extended-spectrum beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal

J. Antimicrob. Chemother.

(2006)

Cited by (176)

Seasonality of antimicrobial use in Dutch food-producing animals
2023, Preventive Veterinary Medicine
Due to globally increasing antimicrobial resistance (AMR), it is pivotal to understand factors contributing to antimicrobial use (AMU) to enable development and implementation of AMR-reducing interventions. Therefore, we explored seasonal variations of systemic AMU in food-producing animals in the Netherlands. Dutch surveillance data from January 2013 to December 2018 from cattle, pig, and broiler farms were used. AMU was expressed as the number of Defined Daily Dosages Animal per month (DDDA/animal-month) per farm by animal sector, antimicrobial line (first, second, and third), antimicrobial class, and farm type. Seasonality of AMU was analyzed using Generalized Additive Models (GAMs) with DDDA/animal-month as outcome variable, and year and month as independent variables. Year and month were modelled as smooth terms represented with penalized regression splines.Significant seasonality of AMU was found in the cattle and pig sectors, but not in broilers. Significant seasonality of AMU was found mainly for first-line antimicrobials. In the cattle sector, a significant increase during winter was found for the use of amphenicols (an increase of 23.8%) and long-acting macrolides (an increase of 3.4%). In the pig sector, seasonality of AMU was found for pleuromutilins (p < 0.001) with an increase of 20% in October-November. The seasonality of pleuromutilins was stronger in sows/piglets (an increase of 47%) than in fattening pigs (16% increase). Only in fattening pigs, the use of amphenicols showed a significant seasonality with an increase of 11% during winter (P < 0.001). AMU in cattle and pig sectors shows seasonal variations likely caused by seasonality of diseases. In broilers, no AMU seasonality was observed, possibly due to the controlled environment in Dutch farms. In the context of the one health concept, future studies are necessary to explore whether this seasonality is present in other populations and whether it has implications for antimicrobial resistance in humans through the food chain.
Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae
2021, Plasmid
IncI1 has become one of the most common plasmid families in contemporary Enterobacteriaceae from both human and animal sources. In clinical epidemiology, this plasmid type ranks first as the confirmed vehicle of transmission of extended spectrum beta-lactamase and plasmid AmpC genes in isolates from food-producing animals. In this review, we describe the epidemiology and evolution of IncI1 plasmids and closely related IncIγ plasmids. We highlight the emergence of epidemic plasmids circulating among different bacterial hosts in geographically distant countries, and we address the phylogeny of the IncI1 and IncIγ family based on plasmid Multilocus Sequence Typing.
Selection of ESBL-producing Escherichia coli in the gut of calves experimentally fed with milk containing antibiotic residues
2021, Veterinary Microbiology
In the bovine sector, the spread of Enterobacterales producing extended-spectrum and AmpC β-lactamases (ESBL/AmpC) mostly concerns veal calves, and the use of waste milk containing antibiotic residues has been recurrently incriminated. In this study, calves were experimentally fed with milk containing either 2,000 μg/L or 20,000 μg/L of the critically important antibiotic cefquinome. The total counts of enterobacterales and ESBL-producing E. coli were monitored using non-selective and selective media. Our data highlighted the important combination of two main factors (cefquinome exposure and initial ESBL colonization level) in the ESBL selection and amplification process in the gut of calves. Results also proved the dose-independent effect of cefquinome administration on the selection and amplification of ESBL-producing E. coli. Finally, the bla_CTX-M-1/IncI1 ST3 plasmid was systematically recovered after cefquinome exposure, highlighting its epidemic success. Altogether, this work is one of the rare experimental studies providing quantitative information on the impact of waste milk containing antimicrobials on the ESBL load in calves’ microbiota, and the first one using cefquinome. These data emphasise the need for global guidelines on the use of waste milk on dairy farms in order to decrease the antimicrobial resistance burden in this sector.
Conjugative IncHI2 plasmid harboring novel class 1 integron mediated dissemination of multidrug resistance genes in Salmonella Typhimurium
2021, Food Control
Salmonella enterica serovar Typhimurium is one of the most important serovars among nontyphoidal Salmonella. The inappropriate and over usage of antimicrobials in clinical and animal husbandry has increased the prevalence of antimicrobial-resistant Salmonella Typhimurium (S. Typhimurium). Diverse mobile genetic elements (MGEs) such as plasmids, insertion sequences and integrons also have a crucial role in the rapid dissemination of multidrug resistance (MDR) bacteria. This study aimed to screen and analyze the genetic environment of MGEs carried by S. Typhimurium, and to clarify the multidrug resistance dissemination mechanism in S. Typhimurium. The results showed that, out of 255 S. Typhimurium isolates, 62 isolates, were screened out for the presence of IncHI2 plasmid by PCR amplification, of which 61 isolates were carrying both IS26 and class 1 integron. All the 61 isolates were resistant to at least one antimicrobial and 58 were MDR. The correlation between plasmids and antimicrobial resistance components was determined by conjugation, antimicrobial susceptibility test, PCR-based replicon typing (PBRT) and S1-PFGE experiment. The results of PBRT revealed that 15 transconjugants harboring two kinds of plasmid incompatibility groups including HI2 and I1. Further, S1-PFGE validated the presence of HI2 plasmid in 13 and I1 plasmid in 2 transconjugants, which was about 200–300 kb and 80–100 kb in size, respectively. Whole genome sequencing revealed that no significant difference (P > 0.05) was found at the number of antimicrobial resistance gene categories among 15 donor isolates, and ST34 was the predominant sequence type. Phylogenetic analysis based on the complete plasmid sequences revealed 15 IncHI2 plasmids in this study clustered in two different clades and clustered together with other IncHI2 plasmids isolated from China, suggesting that the prevalence and dissemination of IncHI2 plasmids from diverse sources and regions. Four kinds of IS elements-class 1 integron arrangement patterns were identified in the 15 conjugative IncHI2-plasmids, of which three patterns were characterized to be complex IS26-class 1 integron arrangement and the other one was IS256-class 1 integron arrangement. Overall, these results suggested that IncHI2 was one of the major plasmid lineages presented in the MDR S. Typhimurium strains. Furthermore, the presence of complex IS elements-class 1 integrons and resistance genes co-located on IncHI2 plasmids contributed to the prevalence and rapid dissemination of multidrug resistance genes in Salmonella.
Plasmid profile analysis of Escherichia coli and Salmonella enterica isolated from pigs, pork and humans
2022, Epidemiology and Infection
Leafy green vegetables are a common source of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) foodborne illness outbreaks. Ruminant animals, primarily cattle, are the major reservoir of STEC O157. Epidemiological, traceback and field investigations were conducted to identify potential outbreak sources. Product and environmental samples were tested for STEC. A reoccurring strain of STEC O157 caused two multistate outbreaks linked to romaine lettuce in 2018 and 2019, resulting in 234 illnesses in 33 states. Over 80% of patients interviewed consumed romaine lettuce before illness. The romaine lettuce was sourced from two California growing regions: Santa Maria and Salinas Valley in 2018 and Salinas Valley in 2019. The outbreak strain was isolated from environmental samples collected at sites >90 miles apart across growing regions, as well as from romaine-containing products in 2019. Although the definitive route of romaine contamination was undetermined, use of a contaminated agricultural water reservoir in 2018 and contamination from cattle grazing on adjacent land in 2019 were suspected as possible factors. Preventing lettuce contamination from growth to consumption is imperative to preventing illness. These outbreaks highlight the need to further understand mechanisms of romaine contamination, including the role of environmental or animal reservoirs for STEC O157.
Evaluation of in-feed supplementation of formic acid and thymol as non-antibiotic growth promoters and assessing their effect on antimicrobial resistant E.coli isolated in Turkey
2024, Veterinary Research Communications

View all citing articles on Scopus

View full text

Article preview

Veterinary Microbiology

Abstract

Introduction

Section snippets

Bacterial isolates and susceptibility testing

Antibiotic resistance phenotypes

Discussion

Acknowledgements

References (23)

A general method for detecting and sizing large plasmids

Anal. Biochem.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

Int. J. Antimicrob. Agents

Animal reservoirs for extended spectrum beta-lactamase producers

Clin. Microbiol. Infect.

Identification of plasmids by PCR-based replicon typing

J. Microbiol. Methods

Beta-Lactam resistance and beta-lactamases in bacteria of animal origin

Vet. Microbiol.

Resistance in Gram-negative bacteria: Enterobacteriaceae

Am. J. Infect. Control.

Editorial: assessing the antimicrobial susceptibility of bacteria obtained from animals

Vet. Microbiol.

Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses

Vet. Microbiol.

Resistance plasmid families in Enterobacteriaceae

Antimicrob. Agents Chemother.

Extended-spectrum beta-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003

Antimicrob. Agents Chemother.

Detection of Escherichia coli harbouring extended-spectrum beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal

J. Antimicrob. Chemother.

Cited by (176)

Seasonality of antimicrobial use in Dutch food-producing animals

Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae

Selection of ESBL-producing Escherichia coli in the gut of calves experimentally fed with milk containing antibiotic residues

Conjugative IncHI2 plasmid harboring novel class 1 integron mediated dissemination of multidrug resistance genes in Salmonella Typhimurium

Plasmid profile analysis of Escherichia coli and Salmonella enterica isolated from pigs, pork and humans

Evaluation of in-feed supplementation of formic acid and thymol as non-antibiotic growth promoters and assessing their effect on antimicrobial resistant E.coli isolated in Turkey