First Characterization and Emergence of SHV-60 in Raw Milk of a Healthy Cow in Japan

Abstract

ABSTRACT. During monitoring of raw milk samples from healthy cows for the presence of antibiotic resistant bacteria, one isolate of Klebsiella pneumoniae strain HUF-100 was found to be resistant to oxyimino-cephalosporins and aztreonam. It was found to carry a chromosomally-encoded extended-spectrum β-lactamase that has not been described previously, namely SHV-60. Thus, it must be expected that this strain will spread further among food-producing animals and thereby constitute a reservoir of this resistant strain and resistance gene that can transfer to and cause treatment problems for humans. The present study confirms the hypothesis that some of novel multiple antibiotic resistant zoonotic bacterial pathogens may initially emerge from food animals and reports, for the first time, this type of emergence in Japan.KEY WORDS: Klebsiella pneumoniae, milk, SHV β-lactamase.J. Vet. Med. Sci. 70(11): 1269-1272, 2008 Colonization of the udders of the healthy cows with multidrug-resistant zoonotic pathogens during lactation or dry periods is one of the most important threats affecting not only the dairy industry but also public health. Recently, sections of the population in developed nations have been misinformed by many sites on the internet and regard raw milk as more nutritious and better tasting than pasteurized milk [12]. Therefore, we conducted a study to monitor multidrug-resistant bacteria in the raw milk of California mastitis test negative healthy cows that had not received any antibiotics in their feed and that had not been treated with antibiotics for more than 3 months, factors not analyzed previously.Twenty milk samples were collected aseptically into sterile screw-capped bottles after discarding three streams of milk and were directly transported to the laboratory in an ice box. Twenty-five ml of individual milk samples was added to 225 ml of buffered peptone water and incubated at 37°C with agitation for 24 hr. Following incubation, the enriched samples were subcultured onto MacConkey agar plates and incubated for 24 hr at 35°C. An API-20E system (bioMérieux, Marcy l'Etoile, France) was used for biochemical identification of isolated strains. The characterized strains were tested for susceptibility to antimicrobials by disk diffusion and MIC methods using Muller-Hinton agar (Nissui Pharmaceutical Co., Tokyo, Japan) and MullerHinton broth (Oxoid), respectively, according to guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2005) [6]. A strain identified as Klebsiella pneumoniae, KPHUF-100, exhibited an uncommon phenotype, suggesting the presence of an extended-spectrum β-lactamase (ESBL) with a spectrum extended to oxyimino-cephalosporins and aztreonam. MIC determination in the presence of a fixed concentration of clavulanic acid (4 µg/ml; β-lactamase production was first confirmed by isoelectric focusing that was performed in a 111 Mini IEF Cell apparatus (Bio-Rad Laboratories) on polyacrylamide gel in the presence of ampholines in the pH range 3.5-10. Broadrange pI markers (Bio-Rad...

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