Laëtitia Fabre

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections.

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.

Emergence of Extended-Spectrum-β-Lactamase (CTX-M-9)-Producing Multiresistant Strains of Salmonella enterica Serotype Virchow in Poultry and Humans in France

ABSTRACT During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum β-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The blaCTX-M-9 gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum β-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.

Variant Salmonella Genomic Island 1 Antibiotic Resistance Gene Cluster Containing a Novel 3′-N-Aminoglycoside Acetyltransferase Gene Cassette, aac(3)-Id, in Salmonella enterica Serovar Newport

ABSTRACT Salmonella genomic island 1 (SGI1) harbors an antibiotic resistance gene cluster and was previously identified in the multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, Paratyphi B, and Albany. This antibiotic resistance gene cluster is a complex class 1 integron and most often confers resistance to ampicillin (Ap), chloramphenicol (Cm)/florfenicol (Ff), streptomycin (Sm)/spectinomycin (Sp), sulfonamides (Su), and tetracycline (Tc) (ApCmFfSmSpSuTc profile). Recently, variant SGI1 antibiotic resistance gene clusters conferring different antibiotic resistance profiles have been identified in several S. enterica serovars and were classified as SGI1-A to -G. We identified a new variant SGI1 antibiotic resistance gene cluster in two multidrug-resistant S. enterica serovar Newport strains isolated from humans in France. In these strains, the Sm/Sp resistance gene cassette aadA2 inserted at the first attI1 site was replaced by two other aminoglycoside resistance gene cassettes. The first one contains a new resistance gene encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase that confers resistance to gentamicin (Gm) and sisomicin (Sc). This gene has been named aac(3)-Id. The second one harbors the Sm/Sp resistance gene aadA7. This gene cassette replacement in the SGI1 complex integron of serovar Newport strains constitutes a new variant SGI1 antibiotic resistance gene cluster named SGI1-H. The occurrence of SGI1 in different S. enterica serovars, now including serovar Newport, strengthens the hypothesis of horizontal transfer of SGI1.

Nosocomial Outbreak Caused by Salmonella enterica Serotype Livingstone Producing CTX-M-27 Extended-Spectrum β-Lactamase in a Neonatal Unit in Sousse, Tunisia

ABSTRACT In this study, we report an outbreak of Salmonella enterica serotype Livingstone resistant to extended-spectrum cephalosporins that occurred in a neonatal ward of the maternity department of Farhat Hached Hospital, Sousse, Tunisia, in 2002. A total of 16 isolates were recovered from 16 babies hospitalized in the ward during the period 1 to 16 July. All these babies developed diarrhea, and three of them developed septicemia. All the isolates demonstrated resistance to ceftriaxone and ceftazidime due to the production of an extended-spectrum β-lactamase (ESBL). The isolates were also resistant to aminoglycosides (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin) and sulfamethoxazole-trimethoprim. DNA profiles were determined by pulsed-field gel electrophoresis using the XbaI and SpeI endonucleases and by ribotyping with PstI digestion. They yielded the same patterns, showing that the outbreak was caused by a single clone. The ESBL was identified as CTX-M-27 by sequencing of PCR products and by isoelectric focusing. The ESBL resistance was transferred by a 40-kb conjugative plasmid. The mobile insertion sequence ISEcp1 was found to be located upstream of blaCTX-M-27 in the same position as that known for a blaCTX-M-14 sequence. A new gene named dfrA21, encoding resistance to trimethoprim and carried by a 90-kb plasmid, was characterized. The dfrA21 gene was inserted as a single resistance cassette in a class I integron. The babies were treated with colistin, and all except two recovered. The outbreak came to an end when appropriate actions were taken: patient isolation, hand washing, and disinfection of the ward.

Chromosomal Integration of the Extended-Spectrum β-Lactamase Gene blaCTX-M-15 in Salmonella enterica Serotype Concord Isolates from Internationally Adopted Children

ABSTRACT We report the emergence of Salmonella enterica isolates of serotype Concord (and its monophasic variant 6,7:l,v:-) producing the extended-spectrum β-lactamases (ESBLs) SHV-12 and CTX-M-15 in France and Norway between 2001 and 2006 (43 in France and 26 in Norway). The majority of these isolates were from adopted children from Ethiopia, most of whom were healthy carriers. Several symptomatic secondary cases were found in the adoptive families and health care facilities in France. Serotype Concord isolates collected before 2003 produced SHV-12 encoded on a 340-kb conjugative plasmid of replicon IncI1. Isolates collected after 2003 produced CTX-M-15. We detected two conjugative plasmids carrying blaCTX-M-15. One plasmid, approximately 300 kb in size, was positive for the IncHI2 replicon and the plasmid-mediated quinolone resistance gene qnrA1. The other plasmid, from one of the earliest CTX-M-15-producing isolates collected, was a fusion plasmid with IncY and IncA/C2 replicons and was 200 kb in size. However, we showed, using Southern hybridization of I-CeuI-digested chromosomal DNA and S1 nuclease analysis of plasmid DNA, that most isolates had a blaCTX-M-15 gene located on chromosomal DNA. Analysis of the flanking regions of the chromosomally located blaCTX-M-15 gene by cloning revealed an ISEcp1 truncated by an intact IS26 upstream from the blaCTX-M-15 gene and a truncated orf477 gene downstream from blaCTX-M-15. We found regions beyond the IS26 and the orf477 genes that were derived from IncA/C2 plasmids, suggesting the chromosomal integration of part of the blaCTX-M-15-carrying IncY and IncA/C2 fusion plasmid from early CTX-M-15-producing isolates.

Clonal Expansion and Microevolution of Quinolone-Resistant Salmonella enterica Serotype Typhi in Vietnam from 1996 to 2004

ABSTRACT Salmonella enterica serotype Typhi clinical isolates (n = 91) resistant to nalidixic acid (Nalr) were collected from sporadic cases and minor outbreaks throughout Vietnam between 1996 and 2004. These isolates were typed and compared by four methods: Vi phage typing, PstI ribotyping, XbaI and SpeI pulsed-field gel electrophoresis (PFGE), and single-nucleotide polymorphism (SNP) analysis. The results indicated that 65% of the isolates were not typeable by Vi phage typing. In contrast, the ribotyping and, with more accuracy, the SNP analysis methods indicated that all Nalr isolates belonged to a single clone (ribotype 3a, haplotype H58) that was found previously and that largely consisted of plasmid-encoded multidrug-resistant serotype Typhi isolates. PFGE demonstrated the occurrence of microevolution within this clone. We identified two major combined PFGE profiles: X1-S1 and X3-S6. X3-S6 predominated between 1996 and 2002 but was replaced by X1-S1 after 2002. Nevertheless, PFGE, with a Simpsons index of 0.78, was not considered an optimal discriminatory method for investigating typhoid fever outbreaks in Vietnam. The rate of quinolone resistance increased and the rate of multidrug resistance decreased during the study period. From 2002 to 2004, 80.6% of the isolates from South Vietnam were resistant only to Nal. The mechanism of Nal resistance in most of the isolates (94%) was a mutation in the quinolone resistance-determining chromosomal region of gyrA that led to the amino acid substitution Ser83Phe. No plasmid-located qnrA, qnrB, or qnrS was detected.

The global establishment of a highly-fluoroquinolone resistant Salmonella enterica serotype Kentucky ST198 strain

While the spread of Salmonella enterica serotype Kentucky resistant to ciprofloxacin across Africa and the Middle-East has been described recently, the presence of this strain in humans, food, various animal species (livestock, pets, and wildlife) and in environment is suspected in other countries of different continents. Here, we report results of an in-depth molecular epidemiological study on a global human and non-human collection of S. Kentucky (n = 70). We performed XbaI-pulsed field gel electrophoresis and multilocus sequence typing, assessed mutations in the quinolone resistance-determining regions, detected β-lactam resistance mechanisms, and screened the presence of the Salmonella genomic island 1 (SGI1). In this study, we highlight the rapid and extensive worldwide dissemination of the ciprofloxacin-resistant S. Kentucky ST198-X1-SGI1 strain since the mid-2000s in an increasingly large number of contaminated sources, including the environment. This strain has accumulated an increasing number of chromosomal and plasmid resistance determinants and has been identified in the Indian subcontinent, Southeast Asia and Europe since 2010. The second substitution at position 87 in GyrA (replacing the amino acid Asp) appeared helpful for epidemiological studies to track the origin of contamination. This global study provides evidence leading to the conclusion that high-level resistance to ciprofloxacin in S. Kentucky is a simple microbiological trait that facilitates the identification of the epidemic clone of interest, ST198-X1-SGI1. Taking this into account is essential in order to detect and monitor it easily and to take rapid measures in livestock to ensure control of this infection.

Ceftriaxone-resistant salmonella enterica serotype Newport, France.

The multidrug-resistant (MDR) Salmonella enterica serotype Newport strain that produces CMY-2 beta-lactamase (Newport MDR-AmpC) was the source of sporadic cases and outbreaks in humans in France during 2000-2005. Because this strain was not detected in food animals, it was most likely introduced into France through imported food products.

Multiple-Antibiotic Resistance in Salmonella enterica Serotype Paratyphi B Isolates Collected in France between 2000 and 2003 Is Due Mainly to Strains Harboring Salmonella Genomic Islands 1, 1-B, and 1-C

ABSTRACT This study was conducted to investigate the occurrence of multiple-antibiotic resistance among 261 clinical isolates of Salmonella enterica serotype Paratyphi B strains collected between 2000 and 2003 through the network of the French National Reference Center for Salmonella. The 47 multidrug-resistant (MDR) isolates identified (18%), were characterized on the basis of the presence of several resistance genes (blaTEM, blaPSE-1, blaCTX-M, floR, aadA2, qacEΔ1, and sul1), the presence of Salmonella genomic island 1 (SGI1) by PCR mapping and hybridization, and the clonality of these isolates by several molecular (ribotyping, IS200 profiling, and pulsed-field gel electrophoresis [PFGE]) and phage typing methods. The results of PCR and Southern blot experiments indicated that 39 (83%) of the 47 S. enterica serotype Paratyphi B biotype Java MDR isolates possessed the SGI1 cluster (MDR/SGI1). Among these 39 MDR/SGI1 isolates, only 3 contained variations in SGI1, SGI1-B (n = 1) and SGI1-C (n = 2). The 39 MDR/SGI1 isolates showed the same specific PstI-IS200 profile 1, which contained seven copies from 2.6 to 18 kb. Two PstI ribotypes were found in MDR/SGI1 isolates, RP1 (n = 38) and RP6 (n = 1). Ribotype RP1 was also found in two susceptible strains. Analysis by PFGE using XbaI revealed that all the MDR/SGI1 isolates were grouped in two related clusters, with a similarity percentage of 82%. Isolation of MDR/SGI1 isolates in France was observed mainly between the second quarter of 2001 and the end of 2002. The source of the contamination has not been identified to date. A single isolate possessing the extended-spectrum β-lactamase blaCTX-M-15 gene was also identified during the study.

Extended-Spectrum-β-Lactamase (TEM-52)-Producing Strains of Salmonella enterica of Various Serotypes Isolated in France

ABSTRACT From 2002 to 2003, four isolates of Salmonella enterica serotypes Typhimurium, Enteritidis, Blockley, and Panama, isolated in France from patients with gastroenteritis, were found to produce extended-spectrum β-lactamase TEM-52. The study showed the blaTEM-52 gene to be located in a Tn3-like structure and carried by 100- or 32-kb conjugative plasmids.