Increase in resistance to extended-spectrum cephalosporins in Salmonella isolated from retail chicken products in Japan.

Extended-spectrum β-lactamase (ESBL)-producing Salmonella are one of the most important public health problems in developed countries. ESBL-producing Salmonella strains have been isolated from humans in Asian countries neighboring Japan, along with strains harboring the plasmid-mediated extended-spectrum cephalosporin (ESC)-resistance gene, ampC (pAmpC). However, only a few studies have investigated the prevalence of ESC-resistant Salmonella in chicken products in Japan, which are the main vehicle of Salmonella transmission. The aim of this study was to investigate the prevalence of ESBL-producing, pAmpC-harboring, or carbapenem-resistant Salmonella in chicken products in Japan. In total, 355 out of 779 (45.6%) chicken product samples collected from 1996-2010 contained Salmonella, resulting in 378 distinct isolates. Of these isolates, 373 were tested for resistance to ESCs, cephamycins, or carbapenems. Isolates that showed resistance to one or more of these antimicrobials were then examined by PCR and DNA sequence analysis for the presence of the bla(CMY), bla(CTX-M), bla(TEM), and bla(SHV) resistance genes. Thirty-five resistant isolates were detected, including 26 isolates that contained pAmpC (bla(CMY-2)), and nine ESBL-producing isolates harboring bla(CTX-M) (n = 4, consisting of two bla(CTX-M-2) and two bla(CTX-M-15 genes)), bla(TEM) (n = 4, consisting of one bla(TEM-20) and three bla(TEM-52) genes), and bla(SHV) (n = 1, bla(SHV-12)). All pAmpC-harboring and ESBL-producing Salmonella isolates were obtained from samples collected after 2005, and the percentage of resistant isolates increased significantly from 0% in 2004 to 27.9% in 2010 (P for trend = 0.006). This increase was caused in part by an increase in the number of Salmonella enterica subsp. enterica serovar Infantis strains harboring an approximately 280-kb plasmid containing bla(CMY-2) in proximity to ISEcp1. The dissemination of ESC-resistant Salmonella containing plasmid-mediated bla(CMY-2) in chicken products indicates the need for the development of continuous monitoring strategies in the interests of public health

Large plasmid profiles of 70 <i>Salmonella</i> isolates.

<p>These isolates consisted of 25 isolates harboring <i>bla</i>_CMY-2 (23 <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis, one <i>S</i>. Manhattan, and one O-untypeable:r:1,5), nine isolates harboring other <i>bla</i> genes (three <i>S</i>. Infantis and six <i>S.</i> Manhattan), and 36 isolates susceptible to 11 antibiotics (18 <i>S</i>. Infantis and 18 <i>S.</i> Manhattan). The 70 isolates generated eight large plasmid profiles (LPPs). The <i>S.</i> Infantis isolates generated LPPs 1 (n = 41), 2 (n = 1), 3 (n = 1), and 4 (n = 1). All <i>S.</i> Infantis isolates harboring <i>bla</i> genes showed LPP 1, except for one <i>S</i>. Infantis isolate carrying <i>bla</i>_CTX-M-2 that was classified as LPP 2. All 18 susceptible <i>S.</i> Infantis isolates showed LPP 1, except for two isolates that were collected in 2009 and 2008 showing LPP 3 and LPP 4, respectively. One O-untypeable:r:1,5 isolate was classified as LPP 1. <i>S.</i> Manhattan isolates generated four different LPPs (LPP 5-LPP 8). LPPs 5, 6, 7, and 8 were found in one, 21, one, and two <i>S.</i> Manhattan isolate(s), respectively. <i>S.</i> Infantis, O-untypeable:r:1,5, and <i>S.</i> Manhattan are expressed as <i>S.</i> I, OUT, and <i>S.</i> M in the figure, respectively.</p

Pulsed-field gel electrophoresis (PFGE) and Southern hybridization images of <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis.

<p>Selected <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar (<i>S</i>.) Infantis isolates were selected to demonstrate the plasmid location of <i>bla</i>_CMY-2. (A) PFGE separation of S1 nuclease-digested genomic DNA from selected <i>S.</i> Infantis isolates, followed by Southern hybridization with a <i>bla</i>_CMY-2 probe. Lane 1, Lambda ladder marker; lane 2, isolate 1993; lane 3, isolate 2127; lane 4, isolate 2150; and lane 5, isolate 1737, which does not harbor <i>bla</i>_CMY-2. (B) <i>Bln</i>I-digested whole-genomic DNA from selected <i>S.</i> Infantis isolates, followed by Southern hybridization with a <i>bla</i>_CMY-2 probe. Lane 1, Lambda ladder marker; lane 2, isolate 1993; lane 3, isolate 2127; lane 4, isolate 2150; and lane 5, isolate 1737, which does not harbor <i>bla</i>_CMY-2.</p

Location of <i>bla</i>_CMY-2 in <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar (<i>S</i>.) Manhattan.

<p>(A) Pulsed-field gel electrophoresis (PFGE) separation of S1 nuclease- or <i>Bln</i>I-digested genomic DNA from <i>S.</i> Manhattan isolates, followed by Southern hybridization with a <i>bla</i>_CMY-2 probe. Lane 1, Lambda ladder marker; lanes 2 and 4, isolate 2179, which harbors <i>bla</i>_CMY-2; lanes 3 and 5, isolate 2129, which does not harbor <i>bla</i>_CMY-2. Lanes 2 and 3, S1 nuclease-digested genomic DNA; lanes 4 and 5, <i>Bln</i>I-digested genomic DNA. (B) Densitometric curves of PFGE separation with S1 nuclease- and <i>Bln</i>I-digested genomic DNA from <i>S.</i> Manhattan isolate 2179. The arrows show hybridization signals and corresponding positions of densitometric curves. Lambda ladder marker consisted of concatemers starting at 48.5 kb.</p

Pulsed-field gel electrophoresis profiles (PFPs) of 70 <i>Salmonella</i> isolates harboring <i>bla</i> and non-<i>bla</i> genes.

<p>The isolates consisted of 25 isolates harboring <i>bla</i>_CMY-2 (23 <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis, one <i>S</i>. Manhattan, and one O-untypeable:r:1,5), nine isolates harboring other <i>bla</i> genes (three <i>S</i>. Infantis and six <i>S.</i> Manhattan), and 36 isolates susceptible to 11 antibiotics (18 <i>S</i>. Infantis and 18 <i>S.</i> Manhattan). One <i>S</i>. Infantis isolate that harbored <i>bla</i>_CMY-2 could not be analyzed by PFGE because of damage that occurred during storage. The letters, names, and figures in parentheses on the right of the dendrogram are PFPs, resistance genes, and sampling year of isolates, respectively. The 44 <i>S</i>. Infantis isolates were subtyped as: PFP A (n = 1), PFP B (n = 3), PFP C (n = 12), PFP D (n = 1), PFP E (n = 1), PFP F (n = 1), PFP G (n = 1), PFP H (n = 6), PFP I (n = 3), PFP J (n = 1), PFP K (n = 1), PFP L (n = 6), PFP M (n = 1), PFP N (n = 3), PFP P (n = 2), and Q (n = 1). The 25 <i>S.</i> Manhattan isolates belonged to subtypes PFP R (n = 24) and PFP S (n = 1). One O-untypeable:r:1,5 isolate was PFP E. The scale indicates the percent similarity, as determined by the Dice coefficients.</p