Prevalence and characterization of extended-spectrum beta-lactamases-producing Salmonella enterica isolates in Saragossa, Spain (2001-2008)

We analyzed the prevalence of resistance to extended-spectrum cephalosporins (ESCs) among clinical strains of Salmonella enterica collected by the Laboratory of Clinical Microbiology in the University Clinical Hospital Lozano Blesa in the region of Aragón (Spain), for which very few epidemiological information exists. A total of 2,092 strains of S. enterica were identified in stool samples from patients with gastroenteritis. Five isolates showed an extended-spectrum beta-lactamase (ESBL) phenotype: four isolates of S. enterica serotype Virchow harbored the ESBL-encoding bla(CTX-M-9) gene and an isolate of serotype Enteritidis carried a bla(CTX-M-1) gene, which, to the best of our knowledge, is described here for the first time in this serotype of S. enterica. The five ESC-resistant isolates were also resistant to spectinomycin, streptomycin, kanamycin, sulfonamides, tetracycline, and trimethoprim as well as to nalidixic acid. The ESBL isolate of serotype Enteritidis, however, remained susceptible to kanamycin and nalidixic acid. A class 1 integron of 1.5?kb was detected for the four serotype Virchow isolates with the gene cassette dfrA16-aadA2. The bla(CTX-M-9) gene was carried by an ～300-kb IncHI2 conjugative plasmid in the case of the S. enterica serotype Virchow isolates. The bla(CTX-M-1) gene was carried by an ～100-kb IncI1-N conjugative plasmid for the serotype Enteritidis ESC-resistant isolate. All the four ESC-resistant strains of S. enterica serotype Virchow clustered together in a XbaI pulsed-field gel electrophoresis, which also revealed a strong similarity between them and some pulsotypes of S. enterica serotype Virchow from France.     

Repeated occurrence of diverse extended-spectrum beta-lactamases in minor serotypes of food-borne Salmonella enterica subsp. enterica

Screening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum beta-lactamase (ESBL)-producing human isolates. Salmonella enterica serotype Brandenburg harbored a multiresistant SHV-5 gene-carrying plasmid. S. enterica serotype Blockley and S. enterica serotype Hadar harbored a TEM-52 gene-carrying plasmid. An S. enterica serotype Virchow strain producing plasmid-mediated CTX-M-32 was isolated twice from poultry end products. All ESBL plasmids were self-transferable and carried by clones currently common in Greece.     

Clonal emergence of extended-spectrum beta-lactamase (CTX-M-2)-producing Salmonella enterica serovar Virchow isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003)

Antibiotic treatment is not required in cases of Salmonella enterica gastroenteritis but is essential in cases of enteric fever or invasive salmonellosis or in immunocompromised patients. Although fluoroquinolones and extended-spectrum cephalosporins are the drugs of choice to treat invasive Salmonella, resistance to these antibiotics is increasing worldwide. During the period 2000 to 2003, 90 Salmonella enterica serovar Virchow poultry and poultry product isolates and 11 serovar Virchow human isolates were found to produce an extended-spectrum beta-lactamase, CTX-M-2, concomitantly with a TEM-1 beta-lactamase. The bla(CTX-M-2) gene was located on a large conjugative plasmid (>100 kb). Pulsed-field gel electrophoresis indicated a clonal relationship between the poultry and human isolates. All these isolates displayed additional resistance to trimethoprim-sulfamethoxazole and tetracycline as well as a reduced susceptibility to ciprofloxacin (MICs of between 0.5 and 1 mug/ml). CTX-M-2-producing Salmonella with a reduced susceptibility to fluoroquinolones constitutes a major concern, since such strains could disseminate on a large scale and jeopardize classical antibiotic therapy in immunocompromised patients.     

Multidrug resistance in Salmonella enterica serotype Typhimurium from humans in France (1993 to 2003)

The aim of this study was to determine the distribution of the antimicrobial resistance phenotypes (R types), the phage types and XbaI-pulsed-field gel electrophoresis (PFGE) types, the genes coding for resistance to beta-lactams and to quinolones, and the class 1 integrons among a representative sample of Salmonella enterica serotype Typhimurium isolates collected from humans in 2002 through the French National Reference Center for Salmonella (NRC-Salm) network. The trends in the evolution of antimicrobial resistance of serotype Typhimurium were reviewed by using NRC-Salm data from 1993, 1997, 2000, and 2003. In 2002, 3,998 isolates of serotype Typhimurium were registered at the NRC-Salm among 11,775 serotyped S. enterica isolates (34%). The most common multiple antibiotic resistance pattern was resistance to amoxicillin, chloramphenicol, streptomycin and spectinomycin, sulfonamides, and tetracycline (ACSSpSuTe R type), with 156 isolates (48.8%). One isolate resistant to extended-spectrum cephalosporins due to the production of TEM-52 extended-spectrum beta-lactamase was detected (0.3%), and one multidrug-resistant isolate was highly resistant to ciprofloxacin (MIC > 32 mg/liter). We found that 57.2% of the isolates tested belonged to the DT104 clone. The main resistance pattern of DT104 isolates was R type ACSSpSuTe (83.2%). However, evolutionary changes have occurred within DT104, involving both loss (variants of Salmonella genomic island 1) and acquisition of genes for drug resistance to trimethoprim or to quinolones. PFGE profile X1 was the most prevalent (74.5%) among DT104 isolates, indicating the need to use a more discriminatory subtyping method for such isolates. Global data from the NRC-Salm suggested that DT104 was the main cause of multidrug resistance in serotype Typhimurium from humans from at least 1997 to 2003, with a roughly stable prevalence during this period.     

Emergence and outbreaks of CTX-M beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital

Sixty-two isolates of Enterobacteriaceae (35 Escherichia coli and 27 Klebsiella pneumoniae isolates) producing CTX-M-type beta-lactamases were collected between March 2000 and June 2003 in different wards of Charles Nicolle Hospital in Tunis (Tunisia). Sequencing identified the bla(CTX-M-15) determinant in 55 isolates and bla(CTX-M-16) in 7 isolates. The CTX-M-15-producing strains were isolated in several wards and consisted mainly of two successive clonal groups of E. coli and a major clonal group of K. pneumoniae. The second clonal group of E. coli belonged to phylogenetic group B2 and harbored more virulence factors than the first clonal group. Among the 22 transconjugants or electroporants obtained with selected E. coli and K. pneumoniae CTX-M-15-producing strains, a predominant plasmid restriction pattern was obtained with 17 isolates. The four CTX-M-16-producing strains of E. coli yielded the same pulsed-field gel electrophoresis (PFGE) pattern, while the three CTX-M-16-producing strains of K. pneumoniae yielded two different PFGE patterns. All of the CTX-M-16-producing isolates were recovered in the pediatric ward and had the same plasmid restriction pattern.     

Use of biochemical kinetic data to determine strain relatedness among Salmonella enterica subsp. enterica isolates

Classical biotyping characterizes strains by creating biotype profiles that consider only positive and negative results for a predefined set of biochemical tests. This method allows Salmonella subspecies to be distinguished but does not allow serotypes and phage types to be distinguished. The objective of this study was to determine the relatedness of isolates belonging to distinct Salmonella enterica subsp. enterica serotypes by using a refined biotyping process that considers the kinetics at which biochemical reactions take place. Using a Vitek GNI+ card for the identification of gram-negative organisms, we determined the biochemical kinetic reactions (28 biochemical tests) of 135 Salmonella enterica subsp. enterica strains of pig origin collected in Spain from 1997 to 2002 (59 Salmonella serotype Typhimurium strains, 25 Salmonella serotype Typhimurium monophasic variant strains, 25 Salmonella serotype Anatum strains, 12 Salmonella serotype Tilburg strains, 7 Salmonella serotype Virchow strains, 6 Salmonella serotype Choleraesuis strains, and 1 Salmonella enterica serotype 4,5,12:-:- strain). The results were expressed as the colorimetric and turbidimetric changes (in percent) and were used to enhance the classical biotype profile by adding kinetic categories. A hierarchical cluster analysis was performed by using the enhanced profiles and resulted in 14 clusters. Six major clusters grouped 94% of all isolates with a similarity of > or =95% within any given cluster, and eight clusters contained a single isolate. The six major clusters grouped not only serotypes of the same type but also phenotypic serotype variations into individual clusters. This suggests that metabolic kinetic reaction data from the biochemical tests commonly used for classic Salmonella enterica subsp. enterica biotyping can possibly be used to determine the relatedness between isolates in an easy and timely manner.     

Two different extended-spectrum beta-lactamases (ESBLs) in one of the first ESBL-producing salmonella isolates in Poland

Two extended-spectrum beta-lactamase (ESBL)-producing salmonella isolates, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, were analyzed. Both isolates produced the CTX-M-3 ESBL; however, their bla(CTX-M-3) genes were located on different plasmids. The serovar Typhimurium isolate also expressed another ESBL, SHV-2a, and probably the two ESBL genes had been acquired independently by the strain.     

Characterization of Salmonella enterica serotype newport isolated from humans and food animals

Salmonella enterica serotype Newport isolates resistant to at least nine antimicrobials (including extended-spectrum cephalosporins), known as serotype Newport MDR-AmpC isolates, have been rapidly emerging as pathogens in both animals and humans throughout the United States. Resistance to extended-spectrum cephalosporins is associated with clinical failures, including death, in patients with systemic infections. In this study, 87 Salmonella serotype Newport strains were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing and examined for the presence of class 1 integrons and bla(CMY) genes. Thirty-five PFGE patterns were observed with XbaI, and three of these patterns were indistinguishable among isolates from humans and animals. Fifty-three (60%) Salmonella serotype Newport isolates were identified as serotype Newport MDR-AmpC, including 16 (53%) of 30 human isolates, 27 (93%) of 29 cattle isolates, 7 (70%) of 10 swine isolates, and 3 (30%) of 10 chicken isolates. However, 28 (32%) Salmonella serotype Newport isolates were susceptible to all 16 antimicrobials tested. The bla(CMY) gene was present in all serotype Newport MDR-AmpC isolates. Furthermore, the plasmid-mediated bla(CMY) gene was transferable via conjugation to an Escherichia coli strain. The transconjugant showed the MDR-AmpC resistance profile. Thirty-five (40%) of the isolates possessed class 1 integrons. Sequence analyses of the integrons showed that they contained aadA, which confers resistance to streptomycin, or aadA and dhfr, which confer resistance to trimethoprim-sulfamethoxazole. One integron from a swine isolate contained the sat-1 gene, which encodes resistance to streptothricin, an antimicrobial agent that has never been approved for use in the United States. In conclusion, Salmonella serotype Newport MDR-AmpC was commonly identified among Salmonella serotype Newport isolates recovered from humans and food animals. These findings support the possibility of transmission of this organism to humans through the food chain.     

Diversity of strains of Salmonella enterica serotype enteritidis from English poultry farms assessed by multiple genetic fingerprinting

Reliable and sufficiently discriminative methods are needed for differentiating individual strains of Salmonella enterica serotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism was PstI-SphI ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of XbaI-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins of Salmonella serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.     

Characterisation of the first CTX-M-10-producing isolate of Salmonella enterica serotype Virchow

Microbiological analysis of a urine sample from an outpatient with symptoms of urinary infection detected >10(5) CFU/mL urine of Salmonella enterica serotype Virchow with resistance to cefotaxime. Molecular analysis demonstrated the presence of the gene encoding CTX-M-10 beta-lactamase in this clinical isolate. This is the first report of this enzyme in Salmonella spp.     

Occurrence of extended-spectrum β-lactamase-producing Salmonella enterica in northern Spain with evidence of CTX-M-9 clonal spread among animals and humans

Among the 1233 Salmonella enterica isolates obtained in two Spanish hospitals, five isolates (0.4%) (serovars: Virchow, four; Livingstone, one) had the phenotype of an extended-spectrum β-lactamase (ESBL) producer. The genetic characterization of the ESBL of S. enterica Livingstone revealed a bla _SHV-2 gene. The bla _CTX-M-10 gene in a phage-related genetic environment was found in one S. enterica Virchow isolate, and the bla _CTX-M-9 gene within the In60 integron was found in the three remaining Virchow isolates. These three isolates presented indistinguishable or closely related pulsed-field gel electrophoresis patterns among themselves and also as compared with the two other bla _CTX-M-9-containing isolates previously obtained from animals. ESBL production is an emerging mechanism of resistance in S. enterica in the two studied hospitals.     

Salmonella enterica serovar virchow with CTX-M-like beta-lactamase in Spain

Four Salmonella enterica serovar Virchow strains resistant to broad-spectrum cephalosporins were isolated from patients with gastroenteritis in 1997 and 1998 in Murcia and Barcelona, Spain. The isolates expressed a beta-lactamase with a pI of about 8 and a positive PCR when specific primers for CTX-M-9 were used. These results suggest the presence of a CTX-M-9 beta-lactamase in these strains.     

Use of molecular subtyping in surveillance for Salmonella enterica serotype typhimurium

BACKGROUND: Because Salmonella enterica serotype typhimurium is the most common serotype isolated from persons with salmonellosis in the United States, it is difficult to detect unusual clusters or outbreaks. To determine whether molecular subtyping could be useful in public health surveillance for S. enterica serotype typhimurium, the Minnesota Department of Health initiated the routine use of pulsed-field gel electrophoresis (PFGE) of isolates. METHODS: Beginning in 1994, all S. enterica serotype typhimurium isolates submitted by clinical laboratories to the Department of Health were subtyped by PFGE. A standard questionnaire was used to interview patients about possible sources of infection. RESULTS: From 1994 through 1998, 998 cases of infection with S. enterica serotype typhimurium were reported to the Minnesota Department of Health (4.4 cases per 100,000 person-years). PFGE was performed on 958 of the isolates (96 percent), and 174 different patterns were identified. Sixteen outbreaks with a common source were identified, accounting for 154 cases. PFGE subtyping made it possible to confirm 10 outbreaks that involved small numbers of cases in institutional settings. Of six larger, community-based outbreaks, four would probably not have been recognized without PFGE subtyping. These four outbreaks accounted for 96 of the 154 culture-confirmed outbreak cases (62 percent). Fifty-six of 209 isolates tested for antimicrobial susceptibility (27 percent) were resistant to at least five antimicrobial agents. The multidrug-resistant isolates identified had unique PFGE patterns. CONCLUSIONS: Routine molecular subtyping of S. enterica serotype typhimurium by PFGE can improve the detection of outbreaks and aid in the identification of multidrug-resistant strains. Combining routine molecular subtyping with a method of rapid communication among public health authorities can improve surveillance for S. enterica serotype typhimurium infections.     

Profile of Salmonella enterica subsp. enterica (subspecies I) serotype 4,5,12:i:- strains causing food-borne infections in New York City

Strains of newly emerging Salmonella enterica subsp. enterica (subspecies I) serotype 4,5,12:i:- causing food-borne infections, including a large food poisoning outbreak (n = 86) characterized by persistent diarrhea (14% bloody), abdominal pain, fever, and headache, were examined. The organisms were found in the stool samples from the patients. The biochemical profile of the organisms is consistent with that of S. enterica subsp. I serotypes, except for decreased dulcitol (13%) and increased inositol (96%) utilization. Twenty-eight percent of the strains showed resistance to streptomycin, sulfonamides, or tetracycline only; all three antimicrobial agents; or these agents either alone or in combination with ampicillin, trimethoprim, and trimethoprim-sulfamethoxazole. None of the serotype 4,5,12:i:- strains showed resistance or decreased susceptibility to chloramphenicol or ciprofloxacin. On pulsed-field gel electrophoresis (PFGE), the strains showed 11 or 12 resolvable genomic fragments with 18 banding patterns and three PFGE profile (PFP) clusters (i.e., PFP/A, PFP/B, and PFP/C). Seventy-five percent of the isolates fingerprinted were closely related (zero to three band differences; similarity [Dice] coefficient, 86 to 100%); 63% of these were indistinguishable from each other (PFP/A(1)). PFP/A(1) was common to all strains from the outbreak and 11 hospital sources. Strains from six other hospitals shared clusters PFP/B and PFP/C. PFP/C(4), of the environmental isolate, was unrelated to PFP/A and PFP/B. Nine band differences (similarity coefficient, 61%) were noted between PFP/A(1) and PFP/E of the multidrug-resistant S. enterica subsp. enterica serotype Typhimurium definitive type 104 strains. Whether these emerging Salmonella strains represent a monophasic, Dul(-) variant of serotype Typhimurium or S. enterica subsp. enterica serotype Lagos or a distinct serotype of S. enterica subsp. I is not yet known. Some of the phenotypic and genotypic properties of the serotype 4,5,12:i:- strains are described here.     

Salmonella enterica serotype Gambia with CTX-M-3 and armA resistance markers: nosocomial infections with a fatal outcome

We report two cases of bacteremia caused by the Salmonella enterica serotype Gambia in our children's hospital, with one fatal outcome. The isolates showed indistinguishable genotypes and infrequent resistance markers: CTX-M-3 extended-spectrum β-lactamase and armA methyltransferase. This is the first report of S. Gambia exhibiting CTX-M-3 and armA markers involved in serious infections.     

Antimicrobial resistance of Salmonella serotypes isolated from slaughter-age pigs and environmental samples

The aim of this study was to determine the antimicrobial resistance patterns of Salmonella strains isolated from slaughter-age pigs and environmental samples collected at modern swine raising facilities in Brazil. Seventeen isolates of six serotypes of Salmonella enterica subsp. enterica were isolated out of 1,026 collected samples: Salmonella Typhimurium (1), Salmonella Agona (5), Salmonella Sandiego (5), Salmonella Rissen (1), Salmonella Senftenberg (4), and Salmonella Javiana (1). Resistance patterns were determined to extended-spectrum penicillin (ampicillin), broad-spectrum cephalosporins (cefotaxime and ceftriaxone), aminoglycosides (streptomycin, neomycin, gentamicin, amikacin, and tobramycin), narrow-spectrum quinolone (nalidixic acid), broad-spectrum quinolone (ciprofloxacin and norfloxacin), tetracycline, trimethoprim, and chloramphenicol. Antimicrobial resistance patterns varied among serotypes, but isolates from a single serotype consistently showed the same resistance profile. All isolates were resistant to tetracycline, streptomycin, and nalidixic acid. One isolate, Salmonella Rissen, was also resistant to cefotaxime and tobramycin. All serotypes were susceptible to ceftriaxone, norfloxacin, ciprofloxacin, ampicillin, gentamicin, and chloramphenicol. The high resistance to tetracycline and streptomycin may be linked to their common use as therapeutic drugs on the tested farms. No relation was seen between nalidixic acid and fluoroquinolone resistance.     

Nosocomial outbreak of pediatric gastroenteritis caused by CTX-M-14-type extended-spectrum beta-lactamase-producing strains of Salmonella enterica serovar London

CTX-M-14-type extended-spectrum beta-lactamase was first detected in Salmonella enterica serovar London strains which were isolated from three hospitalized pediatric patients with gastroenteritis. The isolates had pulsed-field gel electrophoresis patterns identical to those of the previously isolated antimicrobial-susceptible strains from community-acquired gastroenteritis, suggesting the susceptible clone acquired the resistance.     

Occurrence of extended-spectrum beta-lactamases in members of the genus Shigella in the Republic of Korea

A nationwide survey was carried out in Korea to assess the prevalence of Shigella strains producing extended-spectrum beta-lactamases (ESBLs). From 1991 to 2002, 5,911 clinical strains were isolated and screened for resistance to extended-spectrum cephalosporins. Twenty of the Shigella isolates were ESBL positive, based on the synergistic effects between clavulanate and selected beta-lactams (ceftazidime and cefotaxime). Nucleotide sequence analysis of these isolates revealed that they harbored bla(TEM-19) (eight isolates), bla(TEM-15) (five isolates), bla(TEM-52) (six isolates), bla(TEM-17) (one isolate), bla(TEM-20) (one isolate), and bla(CTX-M-14) (three isolates). All the ESBL-encoding genes in this study were carried in conjugable plasmids. Thus, TEM-19, TEM-15, TEM-52, and CTX-M-14 beta-lactamases can be considered common Korean ESBL types in Shigella sonnei and are probably transmitted through interspecies spread between medical facilities and the community in Korea. This is the first report of the presence of TEM-17, TEM-19, and TEM-20 in Korea and in S. sonnei.     

Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period

Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum beta-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 beta-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.