Cefotaxime-Resistant Salmonella enterica in Travelers Returning from Thailand to Finland

During 1993–2011, cefotaxime resistance among Salmonella enterica isolates from patients in Finland increased substantially. Most of these infections originated in Thailand; many were qnr positive and belonged to S. enterica serovar Typhimurium and S. enterica monophasic serovar 4,[5],12:i:-. Although cefotaxime-resistant salmonellae mainly originate in discrete geographic areas, they represent a global threat.

S almonella spp.are a common cause of foodborne ill- nesses globally, but illnesses caused by Salmonella infections vary from mild diarrhea (travelers' diarrhea) to severe generalized infections (1).Certain Salmonella serotypes are more commonly linked to human infections and for example, the monophasic 4, [5],12:i:-variant of S. enterica serovar Typhimurium has caused an increasing number of Salmonella infections in humans during the last decade (2).Antimicrobial agents, usually fluoroquinolones and extended-spectrum cephalosporins, are needed for the treatment of patients with invasive Salmonella infections (3).
The abundant use of antibiotics in human and veterinary medicine and in food production has led to antimicrobial drug resistance (4), and the numbers and proportions of extended-spectrum β-lactamase (ESBL)-and AmpC β-lactamase-producing strains of Enterobacteriaceae have increased worldwide (3,(5)(6)(7).Although reduced fluoroquinolone susceptibility among S. enterica isolates has increased since the late 1990s (8,9), Salmonella spp.have remained cephalosporin-susceptible. Coexistence of ESBL and plasmid-mediated quinolone resistance genes in Salmonella and in other Enterobacteriaceae genera have been reported and there are existing reports on extendedspectrum cephalosporin-resistant and ESBL-producing Salmonella isolates (3,4,10).
To date, Salmonella isolates that have acquired resistance determinants against fluoroquinolones and extendedspectrum cephalosporins have been reported only anecdotally in Finland.This study describes a systematic analysis of extended-spectrum cephalosporin-resistant Salmonella isolates in Finland during a 19-year period.

The Study
During 1993-2011, 43,171 S. enterica isolates were sent to the National Salmonella Reference Centre of the National Institute for Health and Welfare (THL) in Finland.This Salmonella collection contains ≈85% (range 75.9%-91.1%) of all Salmonella isolates collected annually in Finland during the study period.All of these isolates were screened for cefotaxime susceptibility (11).A total of 225 cefotaxime-nonsusceptible S. enterica isolates were identified; 183 of these, collected during 2000-2011, were genotyped.The isolates were screened and serotyped in the Bacteriology Unit at THL.
We confirmed phenotypic ESBL using disk diffusion tests (11).Cefotaxime-nonsusceptible isolates were screened for the ESBL genes TEM, SHV, and CTX-M by PCR (7).CTX-M-positive Escherichia coli, SHVpositive Klebsiella pneumoniae, and TEM-positive E. coli were used as positive ESBL controls.Isolates having only a TEM determinant were further classified by pyrosequencing (12).
We also screened the cefotaxime-nonsusceptible isolates for AmpC production.PCR was used to amplify the AmpC b-lactamase genes CMY, FOX, DHA, ACC, MOX, and EBC by using previously described primers (13).The AmpC multiplex-PCR reaction (50 µL) consisted of 0.2 pmol/µL of each primer, 0.06 U/µL AmpliTaq Gold DNA polymerase, 5 µL AmpliTaq Gold buffer, 2 mM MgCl 2 , and 0.2 mM dNTP mix (Life Technologies Europe, Espoo, Finland).The PCR program consisted of an initial denaturation at 94°C for 10 minutes, then 38 cycles of DNA denaturation at 94°C for 30 seconds, primer annealing at 64°C for 30 seconds, and extension at 72°C for 1 minute.
We determined susceptibility to the antimicrobial drugs ciprofloxacin, nalidixic acid, and meropenem using the standard agar dilution method according to the Clinical Laboratory and Standards Institute guidelines (11).We screened isolates showing reduced fluoroquinolone susceptibility; specifically, to ciprofloxacin (MIC ≥0.125 µg/mL), that were susceptible or resistant on a low level to nalidixic acid (MIC ≤32 µg/mL) (9) for transferable plasmidmediated quinolone resistance determinants.We screened the qnrA, qnrB, qnrS, and aac(6′)-Ib-cr genes with a previously described method (14).
We performed the statistical analysis using a log-binomial model and year as an explanatory variable to assess the log-linear trend in time in the percentage/proportion of cefotaxime-nonsusceptible S. enterica isolates.A p value <0.05 was considered significant.Statistical analyses were performed by using IBM SPSS Statistics Version 21 (IBM Corporation, Armonk, NY, USA).
During 2000-2011, of the 183 cefotaxime-nonsusceptible isolates, 95 produced ESBL and 88 produced AmpC.The number and proportion of ESBL-and AmpC-positive isolates varied and the number of cefotaxime-nonsusceptible isolates increased (Figure 2).The number of AmpCpositive S. enterica isolates was highest in 2008, and the number of ESBL-positive isolates was highest in 2011.During 2000-2005, 10 ESBL-positive isolates were found; these isolates had been identified in samples collected from travelers from Finland returning from the Mediterranean area, Egypt, and European countries.Isolates positive for the SHV gene mainly originated from Egypt.From 2006 onwards, the main geographic origin of ESBL-positive isolates was Southeast Asia; 61% (52/85) of the ESBL isolates originated from Thailand.During the same time, the CTX-M determinant (72/85 isolates) became more common than SHV.Of the ESBL positive isolates, 44 of 95 belonged to S. enterica ser.Typhimurium or the monophasic 4, [5],12:i:-variant of this serovar; 38 of these originated from Thailand.
AmpC-positive isolates were found from 2003 onwards.During 2003-2004, the AmpC-positive isolates were found in travelers from Finland returning from Spain, India, Mexico, and Africa.From 2005 onwards, the AmpCpositive isolates also commonly originated from Thailand (61/83 isolates).The most common AmpC gene was CMY.Of the AmpC positive isolates, 21 of 88 belonged to S. enterica ser.Typhimurium or a monophasic 4, [5],12:i:-variant of S. enterica ser.Typhimurium serotypes; 8 of these originated from Thailand.

Conclusions
In this study, we described a significant increase (p<0.001) in cefotaxime nonsusceptibility among Salmonella isolates, collected from patients in Finland during 1993-2011.In Salmonella spp., cefotaxime nonsusceptibility is thought to be linked to AmpC-type β-lactamases, and production of ESBLs to be more rare (3).According to our results, ESBL and AmpC production (51.9% vs. 48.1%)were equally common among the cefotaxime-nonsusceptible Salmonella serovars.
During the study period, a change in the geographic origin of cefotaxime-nonsusceptible Salmonella isolates was observed: its predominance in Egypt and the Mediterranean area shifted to Thailand and other Southeast Asian countries.We previously reported that Salmonella isolates with the qnr phenotype are concentrated in Southeast Asia, mainly Thailand (9).In this study, 37 ESBL-positive and 10 AmpC-positive S. enterica isolates were also qnr positive and 40/47 isolates were from Southeast Asia.These results were in concordance with previous reports: ESBL-producing Enterobacteriaceae are commonly isolated from patients returning from Southeast Asia (15) and ESBL and plasmidmediated quinolone-resistance mechanisms are commonly found in the same plasmids in Enterobacteriaceae and Salmonella (4,6).
We conclude that cefotaxime-nonsusceptible Salmonella isolates are already a threat for travelers to Southeast Asia.Because of the mobile nature of the ESBL and AmpC genes, qnr resistance determinants, and increased travel, this is a worldwide threat, and makes the treatment for invasive Salmonella infections even more challenging.