Nosocomial Outbreak Caused by Salmonella enterica
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微生物临床杂志 2005年第3期
Laboratoire de Microbiologie-Immunologie
Laboratoire d'Hygiene, Hpital Farhat Hached, Sousse, Tunisia
Centre National de Reference des Salmonella, Unite de Biodiversite des Bacteries Pathogenes Emergentes, Institut Pasteur, INSERM U 389, Paris, France
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 -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.
INTRODUCTION
Salmonellosis in humans is usually a self-limited gastroenteritis that does not warrant antimicrobial therapy. However, these infections can occasionally lead to life-threatening systemic infections that require effective chemotherapy. With the emergence of ampicillin-resistant Salmonella enterica serotype Typhimurium DT104 isolates, extended-spectrum cephalosporins (ESC) are currently the agents of choice for such chemotherapy, especially for children, for whom the use of fluroquinolones is not yet approved. The selective pressure created by the use of ESC has been described as one of the most important factors in the appearance of extended-spectrum -lactamase (ESBL)-producing bacterial isolates (46). Unlike other Enterobacteriaceae, Salmonella strains resistant to ESC due to production of ESBL have been described rarely, although there are an increasing number of reports of such strains throughout the world (26). ESBLs in Salmonella are derivatives of the plasmid-mediated class A -lactamases TEM, SHV, PER, and CTX-M (26).
In this paper, focusing on the microbiological aspects, we report for the first time the ESBL CTX-M-27 in the genus Salmonella. CTX-M-27-producing isolates of S. enterica serotype Livingstone were the cause of a nosocomial outbreak in the neonatal ward of Farhat Hached Hospital, Sousse, Tunisia, during the summer of 2002. Molecular characterization of ESBL-producing isolates was done by pulsed-field gel electrophoresis (PFGE) and ribotyping. The ESBL was characterized by isoelectric focusing (IEF) and molecular methods.
MATERIALS AND METHODS
Description of hospital outbreak. The outbreak occurred in the neonatal ward of the maternity department of Farhat Hached University Hospital, located in the city of Sousse, Tunisia. This ward is divided into three sections located in separate rooms: low-risk care, intermediate care, and the intensive care unit (ICU). The ICU admits preterm neonates or infants with other serious medical or surgical pathologies requiring intensive care. The age range of babies admitted to this unit is from birth to 2 months.
The outbreak period was defined as 1 through 21 July, 2002, that is, the dates from the first to the last patient for whom culture was positive for S. enterica serotype Livingstone. A case was defined as a patient with a stool and/or blood culture positive for S. enterica serotype Livingstone. The following information was extracted from the medical record for each case: age, sex, date of admission, diagnosis, instrumentation, medications, symptoms, results of cultures, and outcome. The characteristics of the 16 cases are listed in Table 1.
Bacterial strains and media. The 16 Salmonella isolates used in this study were recovered from stool specimens of the 16 babies. Cultures were performed using the following media from Bio-Rad (Marnes la Coquette, France): Salmonella-Shigella agar, Tryptocasein soy broth and agar, and Drigalski agar. All the isolates were identified with the API 20E system (BioMerieux, Marcy l'Etoile, France) and were serotyped on the basis of somatic O, phase 1 flagellar, and phase 2 flagellar antigens by agglutination tests with antisera (Bio-Rad and WHO Collaborative Centre for Reference and Research on Salmonella, Institut Pasteur, Paris, France) as specified by the White-Kauffman-Le Minor scheme (34).
Escherichia coli C1a (resistant to nalidixic acid) was used for conjugation experiments. E. coli DH10B was used for transformation experiments. E. coli ATCC 25922 was used as a susceptible control for the disk diffusion method and for MIC determinations. Six susceptible human isolates and one susceptible meat isolate of Salmonella serotype Livingstone were used as comparison strains for PFGE and ribotyping experiments. Five human isolates and the meat isolate were from Tunisia: isolates Tun-1, Tun-2, and Tun-3 were recovered from stool specimens at the Service d'Hygiene of the Farhat Hached University Hospital in 2001, 2002, and 2003, respectively; meat (poultry) isolate Tun-4 was collected at the Service d'Hygiene in 2002; isolates 01-8221 and 03-5725 were collected at the French National Reference Center for Salmonella (NRC-Salm) from stool specimens of two patients who acquired gastroenteritis in Tunisia in 2001 and 2003, respectively. Isolate 00-2311 from NRC-Salm was collected from a patient who acquired gastroenteritis in the French West Indies in 2000. Serotype Livingstone reference strain 336K was from the WHO Collaborative Centre for Reference and Research on Salmonella. Salmonella serotype Braenderup H9812 was used as a molecular size marker in the PFGE experiments.
Antimicrobial susceptibility. Antibiotic susceptibility was determined by the disk diffusion method on Mueller-Hinton (MH) agar (Bio-Rad) according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (41). The following antimicrobial agents (Bio-Rad) were tested: amoxicillin, amoxicillin-clavulanic acid, ticarcillin, ticarcillin-clavulanic acid, piperacillin, piperacillin-tazobactam, cephalothin, cefamandole, cefoperazone, cefoxitin, ceftriaxone, ceftazidime, cefepime, aztreonam, moxalactam, imipenem, streptomycin, spectinomycin, kanamycin, tobramycin, netilmicin, gentamicin, amikacin, isepamicin, nalidixic acid, ofloxacin, ciprofloxacin, sulfonamides, trimethoprim, chloramphenicol, and tetracycline.
MICs of the -lactams were determined by Etest (AB Biodisk, Solna, Sweden). ESBL phenotype was detected by using the ESBL detection Etest strips (AB Biodisk) and the double-disk synergy (DDS) method (16).
Preparation of crude extracts of -lactamase and IEF. Crude extracts of -lactamases were obtained by sonication. IEF was performed with a PhastSystem apparatus (Amersham-Pharmacia Biotech, Freiburg, Germany) as described previously (43).
PFGE. PFGE was carried out with a CHEF-DR III system (Bio-Rad) as described previously (43), except that the migration buffer contained 750 μM thiourea (38). The running conditions were 6 V/cm at 14°C for 18 h, with pulse times ramped from 2.2 to 63.8 s.
Ribotyping. Automated ribotyping was performed by using the RiboPrinter Microbial Characterization system (Qualicon; Wilmington, Del.) in accordance with the manufacturer's protocol (PstI digestion). Ribotype patterns were automatically compared by using Taxotron software (Institut Pasteur).
PCR amplification of -lactamase genes and class I integrons, and sequence analysis. Total DNA was extracted by using the InstaGene matrix kit (Bio-Rad) in accordance with the manufacturer's recommendations. PCR amplifications of blaTEM, blaSHV, blaCTX-M, and blaCMY were performed using the primers listed in Table 2. A CTX-M-9 group-specific PCR assay was carried out using primers CTX-M-9-F and CTX-M-9-R, amplifying an 856-bp fragment of blaCTX-M-9-like gene (Table 2). All amplifications were performed on 50-μl samples containing DNA (2.5 μl), primers (50 pmol each), deoxynucleoside triphosphate (200 μM), Taq DNA polymerase (1.25 U of Ampli Taq Gold; Roche Diagnostics) and its buffer, MgCl2 (2 mM), and dimethyl sulfoxide (10%). The cycling conditions included 10 min of denaturation at 94°C (1 cycle); 30 s of denaturation at 94°C, 30 s of annealing at 50°C (53°C for CTX-M consensus and 54°C for CTX-M-9 group), and 1 min of polymerization at 72°C (35 cycles); followed by 10 min of extension at 72°C. For amplification of class I integrons, primers 5'-CS and 3'-CS (21) were used as described previously (43).
The purified PCR fragments were sequenced on both strands by Genome Express (Meylan, France) using an ABI 100 DNA sequencer (Applied Biosystems, Foster City, Calif.).
The nucleotide sequences and the deduced protein sequences were analyzed with EditSeq and Megalign software (DNAstar, Madison, Wis.). The BLASTN program of the National Center for Biotechnology Information was used for database searches (1).
Resistance transfer determination. A resistance transfer experiment was carried out in liquid and on solid media (10). E. coli C1a, resistant to nalidixic acid, was used as the recipient strain. Transconjugants were selected on Drigalski agar (Bio-Rad) supplemented with ceftazidime (4 mg/liter) or kanamycin (20 mg/liter) and nalidixic acid (64 mg/liter). Plasmid DNA was transformed by using standard electroporation techniques with DH10B electrocompetent E. coli and a MicroPulser electroporation apparatus (Bio-Rad). Transformants were selected on MH agar containing ceftazidime (4 mg/liter) or kanamycin (20 mg/liter).
Plasmid analysis. Plasmid DNA was purified from bacterial cells by an alkaline lysis procedure using the QIAGEN (Courtaboeuf, France) plasmid minikit and was subjected to 0.8% agarose gel electrophoresis. Molecular sizes of plasmids were determined using Taxotron software by reference to plasmids of known sizes (RP4, 54 kb; pIP173, 126 kb) mixed with a supercoiled DNA ladder (Invitrogen, Groningen, The Netherlands).
Exploration of the upstream sequence of the blaCTX-M gene. The presence of ISEcp1 was investigated by a PCR assay using primers ISEcp1U1 and CTX-M-R (Table 2) as described previously (39). DNA sequencing of the PCR product with primer ISEcp1U1 and subsequent sequence comparison were performed as described above. DNA sequencing of the upstream region of blaCTX-M-27 was performed on plasmid DNA of transconjugant pC1a7-1 with primer M9W (located at bp 44 to 25 from the CTX-M-27 translational starting point) (Table 2).
Nucleotide sequence accession number. The coding sequence of dfrA21 has been deposited in the GenBank data library under accession no. AY552589.
RESULTS
Outbreak. Events began on 1 July 2002, with the isolation of S. enterica serotype Livingstone from a 2-month-old baby (index case) hospitalized for pneumopathy. The baby had been born in the maternity department and briefly hospitalized in the neonatal ward, but she had not received any treatment. She was rehospitalized 45 days later for pneumopathy and received antibiotic treatment (cefotaxime and gentamicin). After 10 days at the hospital (1 July), she presented with diarrhea, and S. enterica serotype Livingstone was recovered from a stool culture. The second affected baby appeared 1 day later. At 2 days after her admission to the ICU of the ward, the baby became seriously ill and septic. Bacteriological analysis of blood, stool, cerebrospinal fluid, and urine samples and the umbilicus catheter was carried out. S. enterica serotype Livingstone was isolated from stools, blood, and the umbilicus catheter.
Between 3 and 21 July 2002, there was a dramatic increase in the number of isolations of serotype Livingstone from babies hospitalized in the different sections of the ward. A total of 16 babies among 48 admitted to the ward in that period presented with loose green stools. S. enterica serotype Livingstone was isolated from the stools of all the babies and from the blood of three babies (Table 1). Babies were born by normal vaginal delivery or by caesarean section. The interval between admission to the ward and the onset of diarrhea ranged from 2 to 10 days, and the duration of illness ranged from 4 to 10 days. Among these babies, 11 were hospitalized in the ICU and were placed in an incubator.
The babies were treated with colistin, and all except two recovered. The death of these two babies was attributed to their underlying diseases. No recurrence of diarrhea due to S. enterica serotype Livingstone was found among the babies during the following days, and no other cases were noted after 21 July 2002. Finally, interrogation of the neonatal-ward staff did not reveal any recent diarrhea episodes; nevertheless, parents were not reached by the investigation.
Identification of Salmonella isolates. The 16 isolates were identified as S. enterica by biochemical characterization and as serotype Livingstone by serotyping (antigenic formula 6, 7: d: 1, w).
Antimicrobial susceptibility. All isolates had identical antibiotypes. By the disk diffusion method, all of them were resistant to amoxicillin, ticarcillin, piperacillin, cephalothin, ceftazidime, and ceftriaxone but remained susceptible in vitro to piperacillin-tazobactam, cefoxitin, and imipenem. The isolates were also resistant to aminoglycosides (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin), sulfonamides, and trimethoprim. Isolates were susceptible to nalidixic acid, ofloxacin, ciprofloxacin, chloramphenicol, and tetracycline. The DDS test was positive for all of the isolates (data not shown). Isolate 7 was selected as a representative strain for MIC determination. The ESBL detection Etest strips confirmed the production of an ESBL in isolate 7 (Table 3). The MICs of the -lactams determined by Etest for isolate 7 are shown in Table 3. Isolate 7 exhibited high levels of resistance to penicillins (ampicillin and ticarcillin MICs, >256 mg/liter) and broad-spectrum cephalosporins (ceftriaxone MIC, >256 mg/liter; ceftazidime MIC, 128 mg/liter).
Molecular typing. The clonal relatedness of ESBL-producing S. enterica serotype Livingstone isolates was assessed by PFGE using XbaI and SpeI (Fig. 1; also data not shown). Five other human isolates and one meat isolate collected during the period 2001 to 2003 in Tunisia or in France, from patients who acquired gastroenteritis during travel to Tunisia, were also tested. The XbaI and SpeI macrorestriction patterns obtained with the outbreak isolates were indistinguishable, except for those of isolates 4 and 6, which had an additional low-molecular-weight band (approximately 70 kb), possibly corresponding to a plasmid. The same restriction pattern was also observed for two isolates collected in France in 2001 and 2003 but acquired in Tunisia (01-8221 and 03-5725). The PFGE patterns of comparison strains isolated in Tunisia (Tun-1, Tun-2, Tun-3, and Tun-4) and in France (00-2311), and of reference strain 336K, differed by more than three bands from the patterns of outbreak isolates.
Another typing method (automated ribotyping using PstI) was performed on eight isolates (isolates 1, 2, 4, 7, 10, 12, 14, and 16) and seven comparison strains (Fig. 2). Outbreak isolates and comparison strains Tun-3, 01-8221, and 03-5725 displayed the same ribotype, which differed from those of the serotype Livingstone reference strain 336K and comparison strains Tun-1, Tun-2, and 00-2311.
Characterization of -lactamase genes and class I integrons. A representative isolate (isolate 7) was analyzed for the mechanism of antibiotic resistance. PCR analysis with the blaCTX-M consensus primers showed the presence of a 540-bp fragment in isolate 7, while no PCR product was observed with TEM, SHV, and CMY primers. The sequence from both strands of the PCR product from isolate 7 was 99% identical to the respective segments of CTX-M-16 (GenBank accession no. AY029068), CTX-M-9a (GenBank accession no. AF252621), and CTX-M-27 (GenBank accession no. AY156923).
A CTX-M-9 group-specific PCR assay performed on eight ESBL-producing S. enterica serotype Livingstone isolates gave the expected amplification product of 856 bp. DNA sequencing of the PCR product from isolate 7 and deduced amino acid sequence analysis revealed that the -lactamase was CTX-M-27, which differed from CTX-M-14 and CTX-M-16 by a single point mutation, Asp240Gly and Ala 231Val, respectively (Ambler numbering) (2). The sequence of isolate 7 was identical to that of blaCTX-M-27 except for two silent mutations located at positions 372 (with respect to the CTX-M translational starting point) (AG) and 570 (GA), as known for blaCTX-M-16.
Isoelectric focusing was compatible with the DNA analysis in showing a single -lactamase with an isoelectric point (pI) of 8.2 in isolate 7.
Isolate 7 contained a class I integron. A PCR product of approximately 700 bp was obtained by using primers 5'-CS and 3'-CS. The sequence from both strands of the PCR product was 98% identical to the entire sequence of the dfr13 gene cassette encoding resistance to trimethoprim (GenBank accession no. Z50802). The deduced amino acid sequence revealed 95% amino acid homology with dihydrofolate reductase type 13 and 84% identity with dihydrofolate reductase type 12 (GenBank accession no. AY126944). The new gene was named dfrA21. The translated polypeptides of dfrA21, dfr13, and dfr12 are shown in Fig. 3.
Resistance transfer and plasmid analysis. Conjugation studies were performed to determine whether ESBL resistance could be transferred from S. enterica serotype Livingstone isolate 7 to E. coli. Transconjugants were obtained on Drigalski agar containing nalidixic acid and ceftazidime. Resistance to aminoglycosides, sulfonamides, and trimethoprim was not cotransferred with ceftazidime resistance for three arbitrarily selected E. coli C1a transconjugants tested. Transconjugants manifested the ESBL phenotype as determined by a DDS test (data not shown). The plasmid contents of these three E. coli transconjugants were analyzed by agarose gel electrophoresis. A 40-kb plasmid was detected in all of the three transconjugants, and transconjugant pC1a7-1 was selected for further analysis. Transconjugant pC1a7-1 expressed only a -lactamase with a pI of 8.2. Interestingly, it exhibited lower resistance to ceftazidime (MIC, 32 versus 128 mg/liter) than the parental strain (Table 3). A CTX-M-9 group-specific PCR assay and subsequent sequencing performed on pC1a7-1 plasmid DNA confirmed the transfer of blaCTX-M-27 (data not shown). Despite multiple attempts in liquid or on solid media, resistance to kanamycin could not be transferred to E. coli by conjugation. Electroporation of plasmid DNA from S. enterica serotype Livingstone isolate 7 into E. coli DH10B successfully transferred kanamycin resistance. Aminoglycoside resistance patterns (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin) of three selected DH10B E. coli electroporants were similar to that of the parental strain, except for streptomycin resistance due to the genotype of DH10B. Resistance to sulfonamides and trimethoprim was cotransferred with kanamycin resistance but -lactam resistance was not. Analysis of plasmid contents of two selected DH10B E. coli electroporants, pDH-K1 and pDH-K2, revealed a single large plasmid (approximately 90 kb) in pDH-K2 and a large (90-kb) and a small (2-kb) plasmid in pDH-K1. PCR amplification of class I integrons using primers 5'-CS and 3'-CS was performed for DH10B electroporants pDH-K1 and pDH-K2. A PCR product of approximately 700 bp was obtained for both electroporants, as identified in the parental strain, which contained the dfr13 gene cassette encoding resistance to trimethoprim.
Plasmid DNA was purified from S. enterica serotype Livingstone isolate 7. Four plasmids, of 90, 40, 3, and 2 kb, were detected.
Genetic environment of blaCTX-M-27 in Salmonella isolate 7. The presence of ISEcp1, a mobile insertion sequence located upstream of several CTX-M genes, was investigated by a PCR assay using primers ISEcp1U1 and CTX-M-R. Isolate 7 and the E. coli transconjugant pC1a7-1 gave PCR products of about 900 bp (data not shown). Sequencing of the PCR product and plasmid DNA from pC1a7-1 revealed that the ISEcp1 element was located in the same position as that known in a CTX-M-14-producing E. coli isolate from China (9). Moreover, the 285-bp sequence upstream of blaCTX-M-27 was 100% identical to the corresponding sequence of blaCTX-M-14 (GenBank accession no. AF25262).
DISCUSSION
S. enterica serotype Livingstone was first isolated from human feces in 1951 (32) and since then has been rarely isolated from animals, animal food products, and patients with human salmonellosis (29). From 1999 to 2003, a dramatic increase in the number of isolations of this serotype in cases of human salmonellosis and from animal products has been observed in Tunisia. Indeed, it was the most frequently isolated Salmonella serotype in the country over the last 4-year period, particularly in cases of human salmonellosis. According to the Institut Pasteur de Tunis, during 2002, 21% of 720 Salmonella infections of humans in Tunisia were caused by S. enterica serotype Livingstone (unpublished data). In Tunisia, this serotype was also isolated from animals, especially from poultry (unpublished data). As there is no information concerning the distribution of fingerprints among serotype Livingstone strains in Tunisia, it would be very interesting to compare the outbreak clone, by PFGE or by other subtyping methods, with isolates collected at the Institut Pasteur de Tunis in order to determine if the outbreak clone was frequently found in the general population in Tunisia or if it is an emerging clone. Previous studies have recognized that among modern typing methods, ribotyping with restriction endonucleases PstI and SphI and PFGE with restriction endonuclease XbaI provide good discrimination among strains of various S. enterica serotypes, including Livingstone (22). In our study, we have used PFGE with restriction endonucleases XbaI and SpeI. Five unrelated susceptible human isolates and one susceptible meat isolate from Tunisia, collected between 2001 and 2003, have been included in molecular typing experiments. The outbreak PFGE pattern was indistinguishable from that of isolates 01-8221 and 03-5725, collected in France from two patients who acquired gastroenteritis in Tunisia in 2001 and 2003. These two comparison isolates exhibiting the same PFGE pattern as that of the outbreak clone did not harbor the plasmid carrying the blaCTX-M-27 gene. Three other human isolates and one meat isolate from Tunisia (isolated during 2001 to 2003) exhibited different PFGE patterns. These preliminary results indicate that the S. enterica serotype Livingstone outbreak clone has been also found outside the Farhat Hached University Hospital and is probably not an uncommon clone, but more isolates need to be tested in order to appreciate its prevalence in the general population of Tunisia. Liebana et al. (22) reported that 6 of 14 S. enterica serotype Livingstone strains were found to be untypeable by PFGE. Murase et al. (28) have recently reported, in a PFGE study of 50 Salmonella serotype Livingstone poultry isolates from Japan, that addition of 50 μM thiourea prevented degradation of the DNA of this serotype during electrophoresis. In our study, addition of 750 μM thiourea to Tris-borate-EDTA buffer (in both in the agarose gel and the electrophoresis buffer) was necessary in order to obtain XbaI fingerprints for all ESBL-producing isolates and for four of six comparison isolates from Tunisia (two others were typeable without thiourea). In this study, ribotyping was less discriminatory than PFGE; comparison strain Tun-3 exhibited the same ribotype profile as outbreak isolates, while PFGE patterns were different. This could be due to the use of a single restriction endonuclease. Double restriction with PstI and SphI would have been more interesting, as reported by Liebana et al. (22). However, the results of both approaches (PFGE and ribotyping) for the epidemiological evaluation of the isolates involved in this outbreak suggested that it was caused by the same clone.
Salmonella serotype Livingstone has been isolated previously from poultry in other countries: Canada (11), Scotland (11, 35), Japan (28), England (11), and France (13). Between 1980 and 1985 in Scotland, poultry and poultry products were an important vehicle for human salmonellosis, since poultry meat accounts for >50% of all human Salmonella outbreaks where a food vehicle has been identified (35). The modern poultry industry had been developing in Tunisia during recent decades, providing a valuable inexpensive source of protein and currently representing the primary source of meat consumption by humans. Thus, despite the absence of epidemiological studies explaining the emergence and spread of this serotype in the country, we suppose that the emergence and spread of S. enterica serotype Livingstone might be related to poultry. Nevertheless, this opinion needs to be validated by further studies.
Commenting on the low incidence of serotype Livingstone infection in humans, Reilly et al. (35) suggested that this Salmonella serotype might have low virulence for humans. Like most nontyphoidal Salmonella serotypes, serotype Livingstone manifested itself only in individuals with predisposing factors such as immunocompromised status. In the present outbreak all patients had serious underlying diseases, which might have predisposed them to infection with the organism involved. Furthermore, they had been subjected to manipulations of the oropharynx by insertion of nasogastric tubes or by insertion of an umbilicus catheter. These manipulations might have provided opportunities for contamination of the babies by the hands of health personnel (17, 25).
Salmonella infections in hospitals are usually food-borne and have an explosive pattern, with an obvious food vector. In this outbreak, food as a source was excluded because milk was commercially prepared and other infants, hospitalized in the same sector of the ward at the same period, were fed with the same preparations but did not become infected with this strain. Therefore, it was concluded that horizontal transmission of the outbreak strain had occurred. Unfortunately, we were not able to trace the source of infections, because no further environmental investigation was implemented.
This outbreak happened in the summer, when the ward was understaffed and overcrowded. Overcrowding and understaffing are outbreak occurrence factors which are commonly observed in neonatal units throughout the world, particularly in developing countries, where limited personnel and facility resources contribute to the perpetuation of this problem. Understaffing in the neonatal unit reduces the likelihood that appropriate attention can be given to infection control practices, including hand hygiene, exacerbating the difficulty of implementing outbreak control measures (31). Finally, the outbreak came to an end when appropriate actions were taken: patient isolation, hand washing, and disinfection of the ward. No cases of S. enterica serotype Livingstone carriers or infection were reported thereafter.
For several years, the resistance of S. enterica to extended-spectrum cephalosporins had been increasingly reported through the world (26). A recent study reported that 1.6% of 431 Salmonella isolates from a broad network of sentinel hospitals distributed in Europe, the Americas, and the Western Pacific region were expressing an ESBL phenotype (46). ESC-resistant S. enterica strains have been reported previously in Tunisian hospitals. Salmonella serotype Wien, producing SHV-2 ESBL, caused a nosocomial outbreak in a neonatal care unit in Sfax in 1988 (14). In the same hospital in 2000, three ESC-resistant strains of Salmonella serotype Livingstone were found to produce cephalosporinase ACC-1 (36). One isolate also produced SHV-2. From 1997 to 1998, 31 isolates of ESC-resistant Salmonella serotype Mbandaka were isolated at University Hospital in Tunis (23). TEM-4 (ESBL), SHV 2a (ESBL), and ACC-1 (cephalosporinase) were identified in 30, 3, and 1 isolate, respectively. In 2001, two Salmonella serotype Wien isolates from the military hospital of Tunis were found to produce CMY-4 (cephalosporinase) and SHV-2a (4). One of these isolates also produced CTX-M-3 ESBL. This report describes the first identification of CTX-M-27 in the genus Salmonella and the second CTX-M-type ESBL-producing Salmonella isolate in Tunisia. CTX-M -lactamases, which preferentially hydrolyze cefotaxime, form a growing family that was characterized in the second half of the 1980s with the report of FEC-1 (24). Currently, the CTX-M family comprises at least 40 enzymes belonging to six groups (6). CTX-M -lactamases share extensive similarities with the chromosomal -lactamases of Kluyvera spp., enterobacteria that are rarely detected in medical microbiology, and it was suggested that the enzymes identified in Kluyvera ascorbata and Kluyvera georgiana were probably the progenitors of the CTX-M-2 and CTX-M-8 groups, respectively (15, 33). ISEcp1 or ISEcp1-like insertion sequences may be involved in the mobilization of blaCTX-M genes (8). Nine CTX-M -lactamases (CTX-M-2, CTX-M-3, CTX-M-4, CTX-M-5, CTX-M-6, CTX-M-7, CTX-M-9, CTX-M-14, and CTX-M-15) have been reported to be associated with different serotypes of S. enterica (6, 37, 45). In this report, the analysis of deduced amino acid sequences showed that blaCTX-M encoded a CTX-M-27 ESBL. This enzyme had been described recently in an E. coli isolate from France (7). It differed from CTX-M-14 by a single substitution, D240G, involved in the higher ceftazidime MIC. In the outbreak clone, the blaCTX-M-27 gene was located on a 40-kb conjugative plasmid which contained no additional resistance determinants. S. enterica serotype Livingstone isolate 7 and its E. coli transconjugant pC1a7-1 exhibited high levels of resistance to ampicillin (MICs, >256 mg/liter), ticarcillin (>256 mg/liter), and ceftriaxone (>256 mg/liter). They exhibited similar levels of resistance to amoxicillin-clavulanic acid (MIC, 8 mg/liter) and piperacillin-tazobactam (4 mg/liter); however, the MIC of ceftazidime for pC1a7-1 was twofold lower than that for isolate 7. This differential expression of ESC resistance between S. enterica and E. coli, which has been reported previously (5, 20, 27, 43, 45), may be due, in part, to the dissimilar genetic backgrounds of the E. coli recipient and Salmonella parental strains, resulting in lower -lactamase expression and activity than in the wild-type strain, or to differences between the E. coli and Salmonella envelopes, modifying antibiotic input into the cell.
Other resistance determinants (aminoglycoside, sulfonamides, and trimethoprim) were carried on a 90-kb nonconjugative but transferable plasmid. Trimethoprim resistance was encoded by a new dfrA21 gene inserted as a single resistance cassette in a class I integron.
The extended-spectrum cephalosporins were preferentially used as first-line drugs in the neonatal ward. Among 16 neonates, 10 had received cefotaxime and 2 had received ceftazidime. As previously reported (24, 30, 42), the emergence of highly resistant Salmonella in an environment characterized by a heavy use of antimicrobial agents may provide a setting that is conducive to nosocomial transmission. In our study, the emergence of the CTX-M-27 ESBL in the neonatal ward could be correlated with heavy use of extended-spectrum cephalosporins by the physicians. Efforts to promote the appropriate use of antimicrobial agents are essential for the control of multidrug resistance of bacterial pathogens (46). Rapid detection and prompt institution of control measures may limit outbreaks due to such an organism and prevent systemic infections, which are difficult to treat because of the few antibiotics available to which the organism is susceptible.
ACKNOWLEDGMENTS
We thank Ridha Ben Aissa, Institut Pasteur de Tunis, Tunis, Tunisia, for his collaboration.
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Laboratoire d'Hygiene, Hpital Farhat Hached, Sousse, Tunisia
Centre National de Reference des Salmonella, Unite de Biodiversite des Bacteries Pathogenes Emergentes, Institut Pasteur, INSERM U 389, Paris, France
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 -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.
INTRODUCTION
Salmonellosis in humans is usually a self-limited gastroenteritis that does not warrant antimicrobial therapy. However, these infections can occasionally lead to life-threatening systemic infections that require effective chemotherapy. With the emergence of ampicillin-resistant Salmonella enterica serotype Typhimurium DT104 isolates, extended-spectrum cephalosporins (ESC) are currently the agents of choice for such chemotherapy, especially for children, for whom the use of fluroquinolones is not yet approved. The selective pressure created by the use of ESC has been described as one of the most important factors in the appearance of extended-spectrum -lactamase (ESBL)-producing bacterial isolates (46). Unlike other Enterobacteriaceae, Salmonella strains resistant to ESC due to production of ESBL have been described rarely, although there are an increasing number of reports of such strains throughout the world (26). ESBLs in Salmonella are derivatives of the plasmid-mediated class A -lactamases TEM, SHV, PER, and CTX-M (26).
In this paper, focusing on the microbiological aspects, we report for the first time the ESBL CTX-M-27 in the genus Salmonella. CTX-M-27-producing isolates of S. enterica serotype Livingstone were the cause of a nosocomial outbreak in the neonatal ward of Farhat Hached Hospital, Sousse, Tunisia, during the summer of 2002. Molecular characterization of ESBL-producing isolates was done by pulsed-field gel electrophoresis (PFGE) and ribotyping. The ESBL was characterized by isoelectric focusing (IEF) and molecular methods.
MATERIALS AND METHODS
Description of hospital outbreak. The outbreak occurred in the neonatal ward of the maternity department of Farhat Hached University Hospital, located in the city of Sousse, Tunisia. This ward is divided into three sections located in separate rooms: low-risk care, intermediate care, and the intensive care unit (ICU). The ICU admits preterm neonates or infants with other serious medical or surgical pathologies requiring intensive care. The age range of babies admitted to this unit is from birth to 2 months.
The outbreak period was defined as 1 through 21 July, 2002, that is, the dates from the first to the last patient for whom culture was positive for S. enterica serotype Livingstone. A case was defined as a patient with a stool and/or blood culture positive for S. enterica serotype Livingstone. The following information was extracted from the medical record for each case: age, sex, date of admission, diagnosis, instrumentation, medications, symptoms, results of cultures, and outcome. The characteristics of the 16 cases are listed in Table 1.
Bacterial strains and media. The 16 Salmonella isolates used in this study were recovered from stool specimens of the 16 babies. Cultures were performed using the following media from Bio-Rad (Marnes la Coquette, France): Salmonella-Shigella agar, Tryptocasein soy broth and agar, and Drigalski agar. All the isolates were identified with the API 20E system (BioMerieux, Marcy l'Etoile, France) and were serotyped on the basis of somatic O, phase 1 flagellar, and phase 2 flagellar antigens by agglutination tests with antisera (Bio-Rad and WHO Collaborative Centre for Reference and Research on Salmonella, Institut Pasteur, Paris, France) as specified by the White-Kauffman-Le Minor scheme (34).
Escherichia coli C1a (resistant to nalidixic acid) was used for conjugation experiments. E. coli DH10B was used for transformation experiments. E. coli ATCC 25922 was used as a susceptible control for the disk diffusion method and for MIC determinations. Six susceptible human isolates and one susceptible meat isolate of Salmonella serotype Livingstone were used as comparison strains for PFGE and ribotyping experiments. Five human isolates and the meat isolate were from Tunisia: isolates Tun-1, Tun-2, and Tun-3 were recovered from stool specimens at the Service d'Hygiene of the Farhat Hached University Hospital in 2001, 2002, and 2003, respectively; meat (poultry) isolate Tun-4 was collected at the Service d'Hygiene in 2002; isolates 01-8221 and 03-5725 were collected at the French National Reference Center for Salmonella (NRC-Salm) from stool specimens of two patients who acquired gastroenteritis in Tunisia in 2001 and 2003, respectively. Isolate 00-2311 from NRC-Salm was collected from a patient who acquired gastroenteritis in the French West Indies in 2000. Serotype Livingstone reference strain 336K was from the WHO Collaborative Centre for Reference and Research on Salmonella. Salmonella serotype Braenderup H9812 was used as a molecular size marker in the PFGE experiments.
Antimicrobial susceptibility. Antibiotic susceptibility was determined by the disk diffusion method on Mueller-Hinton (MH) agar (Bio-Rad) according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (41). The following antimicrobial agents (Bio-Rad) were tested: amoxicillin, amoxicillin-clavulanic acid, ticarcillin, ticarcillin-clavulanic acid, piperacillin, piperacillin-tazobactam, cephalothin, cefamandole, cefoperazone, cefoxitin, ceftriaxone, ceftazidime, cefepime, aztreonam, moxalactam, imipenem, streptomycin, spectinomycin, kanamycin, tobramycin, netilmicin, gentamicin, amikacin, isepamicin, nalidixic acid, ofloxacin, ciprofloxacin, sulfonamides, trimethoprim, chloramphenicol, and tetracycline.
MICs of the -lactams were determined by Etest (AB Biodisk, Solna, Sweden). ESBL phenotype was detected by using the ESBL detection Etest strips (AB Biodisk) and the double-disk synergy (DDS) method (16).
Preparation of crude extracts of -lactamase and IEF. Crude extracts of -lactamases were obtained by sonication. IEF was performed with a PhastSystem apparatus (Amersham-Pharmacia Biotech, Freiburg, Germany) as described previously (43).
PFGE. PFGE was carried out with a CHEF-DR III system (Bio-Rad) as described previously (43), except that the migration buffer contained 750 μM thiourea (38). The running conditions were 6 V/cm at 14°C for 18 h, with pulse times ramped from 2.2 to 63.8 s.
Ribotyping. Automated ribotyping was performed by using the RiboPrinter Microbial Characterization system (Qualicon; Wilmington, Del.) in accordance with the manufacturer's protocol (PstI digestion). Ribotype patterns were automatically compared by using Taxotron software (Institut Pasteur).
PCR amplification of -lactamase genes and class I integrons, and sequence analysis. Total DNA was extracted by using the InstaGene matrix kit (Bio-Rad) in accordance with the manufacturer's recommendations. PCR amplifications of blaTEM, blaSHV, blaCTX-M, and blaCMY were performed using the primers listed in Table 2. A CTX-M-9 group-specific PCR assay was carried out using primers CTX-M-9-F and CTX-M-9-R, amplifying an 856-bp fragment of blaCTX-M-9-like gene (Table 2). All amplifications were performed on 50-μl samples containing DNA (2.5 μl), primers (50 pmol each), deoxynucleoside triphosphate (200 μM), Taq DNA polymerase (1.25 U of Ampli Taq Gold; Roche Diagnostics) and its buffer, MgCl2 (2 mM), and dimethyl sulfoxide (10%). The cycling conditions included 10 min of denaturation at 94°C (1 cycle); 30 s of denaturation at 94°C, 30 s of annealing at 50°C (53°C for CTX-M consensus and 54°C for CTX-M-9 group), and 1 min of polymerization at 72°C (35 cycles); followed by 10 min of extension at 72°C. For amplification of class I integrons, primers 5'-CS and 3'-CS (21) were used as described previously (43).
The purified PCR fragments were sequenced on both strands by Genome Express (Meylan, France) using an ABI 100 DNA sequencer (Applied Biosystems, Foster City, Calif.).
The nucleotide sequences and the deduced protein sequences were analyzed with EditSeq and Megalign software (DNAstar, Madison, Wis.). The BLASTN program of the National Center for Biotechnology Information was used for database searches (1).
Resistance transfer determination. A resistance transfer experiment was carried out in liquid and on solid media (10). E. coli C1a, resistant to nalidixic acid, was used as the recipient strain. Transconjugants were selected on Drigalski agar (Bio-Rad) supplemented with ceftazidime (4 mg/liter) or kanamycin (20 mg/liter) and nalidixic acid (64 mg/liter). Plasmid DNA was transformed by using standard electroporation techniques with DH10B electrocompetent E. coli and a MicroPulser electroporation apparatus (Bio-Rad). Transformants were selected on MH agar containing ceftazidime (4 mg/liter) or kanamycin (20 mg/liter).
Plasmid analysis. Plasmid DNA was purified from bacterial cells by an alkaline lysis procedure using the QIAGEN (Courtaboeuf, France) plasmid minikit and was subjected to 0.8% agarose gel electrophoresis. Molecular sizes of plasmids were determined using Taxotron software by reference to plasmids of known sizes (RP4, 54 kb; pIP173, 126 kb) mixed with a supercoiled DNA ladder (Invitrogen, Groningen, The Netherlands).
Exploration of the upstream sequence of the blaCTX-M gene. The presence of ISEcp1 was investigated by a PCR assay using primers ISEcp1U1 and CTX-M-R (Table 2) as described previously (39). DNA sequencing of the PCR product with primer ISEcp1U1 and subsequent sequence comparison were performed as described above. DNA sequencing of the upstream region of blaCTX-M-27 was performed on plasmid DNA of transconjugant pC1a7-1 with primer M9W (located at bp 44 to 25 from the CTX-M-27 translational starting point) (Table 2).
Nucleotide sequence accession number. The coding sequence of dfrA21 has been deposited in the GenBank data library under accession no. AY552589.
RESULTS
Outbreak. Events began on 1 July 2002, with the isolation of S. enterica serotype Livingstone from a 2-month-old baby (index case) hospitalized for pneumopathy. The baby had been born in the maternity department and briefly hospitalized in the neonatal ward, but she had not received any treatment. She was rehospitalized 45 days later for pneumopathy and received antibiotic treatment (cefotaxime and gentamicin). After 10 days at the hospital (1 July), she presented with diarrhea, and S. enterica serotype Livingstone was recovered from a stool culture. The second affected baby appeared 1 day later. At 2 days after her admission to the ICU of the ward, the baby became seriously ill and septic. Bacteriological analysis of blood, stool, cerebrospinal fluid, and urine samples and the umbilicus catheter was carried out. S. enterica serotype Livingstone was isolated from stools, blood, and the umbilicus catheter.
Between 3 and 21 July 2002, there was a dramatic increase in the number of isolations of serotype Livingstone from babies hospitalized in the different sections of the ward. A total of 16 babies among 48 admitted to the ward in that period presented with loose green stools. S. enterica serotype Livingstone was isolated from the stools of all the babies and from the blood of three babies (Table 1). Babies were born by normal vaginal delivery or by caesarean section. The interval between admission to the ward and the onset of diarrhea ranged from 2 to 10 days, and the duration of illness ranged from 4 to 10 days. Among these babies, 11 were hospitalized in the ICU and were placed in an incubator.
The babies were treated with colistin, and all except two recovered. The death of these two babies was attributed to their underlying diseases. No recurrence of diarrhea due to S. enterica serotype Livingstone was found among the babies during the following days, and no other cases were noted after 21 July 2002. Finally, interrogation of the neonatal-ward staff did not reveal any recent diarrhea episodes; nevertheless, parents were not reached by the investigation.
Identification of Salmonella isolates. The 16 isolates were identified as S. enterica by biochemical characterization and as serotype Livingstone by serotyping (antigenic formula 6, 7: d: 1, w).
Antimicrobial susceptibility. All isolates had identical antibiotypes. By the disk diffusion method, all of them were resistant to amoxicillin, ticarcillin, piperacillin, cephalothin, ceftazidime, and ceftriaxone but remained susceptible in vitro to piperacillin-tazobactam, cefoxitin, and imipenem. The isolates were also resistant to aminoglycosides (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin), sulfonamides, and trimethoprim. Isolates were susceptible to nalidixic acid, ofloxacin, ciprofloxacin, chloramphenicol, and tetracycline. The DDS test was positive for all of the isolates (data not shown). Isolate 7 was selected as a representative strain for MIC determination. The ESBL detection Etest strips confirmed the production of an ESBL in isolate 7 (Table 3). The MICs of the -lactams determined by Etest for isolate 7 are shown in Table 3. Isolate 7 exhibited high levels of resistance to penicillins (ampicillin and ticarcillin MICs, >256 mg/liter) and broad-spectrum cephalosporins (ceftriaxone MIC, >256 mg/liter; ceftazidime MIC, 128 mg/liter).
Molecular typing. The clonal relatedness of ESBL-producing S. enterica serotype Livingstone isolates was assessed by PFGE using XbaI and SpeI (Fig. 1; also data not shown). Five other human isolates and one meat isolate collected during the period 2001 to 2003 in Tunisia or in France, from patients who acquired gastroenteritis during travel to Tunisia, were also tested. The XbaI and SpeI macrorestriction patterns obtained with the outbreak isolates were indistinguishable, except for those of isolates 4 and 6, which had an additional low-molecular-weight band (approximately 70 kb), possibly corresponding to a plasmid. The same restriction pattern was also observed for two isolates collected in France in 2001 and 2003 but acquired in Tunisia (01-8221 and 03-5725). The PFGE patterns of comparison strains isolated in Tunisia (Tun-1, Tun-2, Tun-3, and Tun-4) and in France (00-2311), and of reference strain 336K, differed by more than three bands from the patterns of outbreak isolates.
Another typing method (automated ribotyping using PstI) was performed on eight isolates (isolates 1, 2, 4, 7, 10, 12, 14, and 16) and seven comparison strains (Fig. 2). Outbreak isolates and comparison strains Tun-3, 01-8221, and 03-5725 displayed the same ribotype, which differed from those of the serotype Livingstone reference strain 336K and comparison strains Tun-1, Tun-2, and 00-2311.
Characterization of -lactamase genes and class I integrons. A representative isolate (isolate 7) was analyzed for the mechanism of antibiotic resistance. PCR analysis with the blaCTX-M consensus primers showed the presence of a 540-bp fragment in isolate 7, while no PCR product was observed with TEM, SHV, and CMY primers. The sequence from both strands of the PCR product from isolate 7 was 99% identical to the respective segments of CTX-M-16 (GenBank accession no. AY029068), CTX-M-9a (GenBank accession no. AF252621), and CTX-M-27 (GenBank accession no. AY156923).
A CTX-M-9 group-specific PCR assay performed on eight ESBL-producing S. enterica serotype Livingstone isolates gave the expected amplification product of 856 bp. DNA sequencing of the PCR product from isolate 7 and deduced amino acid sequence analysis revealed that the -lactamase was CTX-M-27, which differed from CTX-M-14 and CTX-M-16 by a single point mutation, Asp240Gly and Ala 231Val, respectively (Ambler numbering) (2). The sequence of isolate 7 was identical to that of blaCTX-M-27 except for two silent mutations located at positions 372 (with respect to the CTX-M translational starting point) (AG) and 570 (GA), as known for blaCTX-M-16.
Isoelectric focusing was compatible with the DNA analysis in showing a single -lactamase with an isoelectric point (pI) of 8.2 in isolate 7.
Isolate 7 contained a class I integron. A PCR product of approximately 700 bp was obtained by using primers 5'-CS and 3'-CS. The sequence from both strands of the PCR product was 98% identical to the entire sequence of the dfr13 gene cassette encoding resistance to trimethoprim (GenBank accession no. Z50802). The deduced amino acid sequence revealed 95% amino acid homology with dihydrofolate reductase type 13 and 84% identity with dihydrofolate reductase type 12 (GenBank accession no. AY126944). The new gene was named dfrA21. The translated polypeptides of dfrA21, dfr13, and dfr12 are shown in Fig. 3.
Resistance transfer and plasmid analysis. Conjugation studies were performed to determine whether ESBL resistance could be transferred from S. enterica serotype Livingstone isolate 7 to E. coli. Transconjugants were obtained on Drigalski agar containing nalidixic acid and ceftazidime. Resistance to aminoglycosides, sulfonamides, and trimethoprim was not cotransferred with ceftazidime resistance for three arbitrarily selected E. coli C1a transconjugants tested. Transconjugants manifested the ESBL phenotype as determined by a DDS test (data not shown). The plasmid contents of these three E. coli transconjugants were analyzed by agarose gel electrophoresis. A 40-kb plasmid was detected in all of the three transconjugants, and transconjugant pC1a7-1 was selected for further analysis. Transconjugant pC1a7-1 expressed only a -lactamase with a pI of 8.2. Interestingly, it exhibited lower resistance to ceftazidime (MIC, 32 versus 128 mg/liter) than the parental strain (Table 3). A CTX-M-9 group-specific PCR assay and subsequent sequencing performed on pC1a7-1 plasmid DNA confirmed the transfer of blaCTX-M-27 (data not shown). Despite multiple attempts in liquid or on solid media, resistance to kanamycin could not be transferred to E. coli by conjugation. Electroporation of plasmid DNA from S. enterica serotype Livingstone isolate 7 into E. coli DH10B successfully transferred kanamycin resistance. Aminoglycoside resistance patterns (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin) of three selected DH10B E. coli electroporants were similar to that of the parental strain, except for streptomycin resistance due to the genotype of DH10B. Resistance to sulfonamides and trimethoprim was cotransferred with kanamycin resistance but -lactam resistance was not. Analysis of plasmid contents of two selected DH10B E. coli electroporants, pDH-K1 and pDH-K2, revealed a single large plasmid (approximately 90 kb) in pDH-K2 and a large (90-kb) and a small (2-kb) plasmid in pDH-K1. PCR amplification of class I integrons using primers 5'-CS and 3'-CS was performed for DH10B electroporants pDH-K1 and pDH-K2. A PCR product of approximately 700 bp was obtained for both electroporants, as identified in the parental strain, which contained the dfr13 gene cassette encoding resistance to trimethoprim.
Plasmid DNA was purified from S. enterica serotype Livingstone isolate 7. Four plasmids, of 90, 40, 3, and 2 kb, were detected.
Genetic environment of blaCTX-M-27 in Salmonella isolate 7. The presence of ISEcp1, a mobile insertion sequence located upstream of several CTX-M genes, was investigated by a PCR assay using primers ISEcp1U1 and CTX-M-R. Isolate 7 and the E. coli transconjugant pC1a7-1 gave PCR products of about 900 bp (data not shown). Sequencing of the PCR product and plasmid DNA from pC1a7-1 revealed that the ISEcp1 element was located in the same position as that known in a CTX-M-14-producing E. coli isolate from China (9). Moreover, the 285-bp sequence upstream of blaCTX-M-27 was 100% identical to the corresponding sequence of blaCTX-M-14 (GenBank accession no. AF25262).
DISCUSSION
S. enterica serotype Livingstone was first isolated from human feces in 1951 (32) and since then has been rarely isolated from animals, animal food products, and patients with human salmonellosis (29). From 1999 to 2003, a dramatic increase in the number of isolations of this serotype in cases of human salmonellosis and from animal products has been observed in Tunisia. Indeed, it was the most frequently isolated Salmonella serotype in the country over the last 4-year period, particularly in cases of human salmonellosis. According to the Institut Pasteur de Tunis, during 2002, 21% of 720 Salmonella infections of humans in Tunisia were caused by S. enterica serotype Livingstone (unpublished data). In Tunisia, this serotype was also isolated from animals, especially from poultry (unpublished data). As there is no information concerning the distribution of fingerprints among serotype Livingstone strains in Tunisia, it would be very interesting to compare the outbreak clone, by PFGE or by other subtyping methods, with isolates collected at the Institut Pasteur de Tunis in order to determine if the outbreak clone was frequently found in the general population in Tunisia or if it is an emerging clone. Previous studies have recognized that among modern typing methods, ribotyping with restriction endonucleases PstI and SphI and PFGE with restriction endonuclease XbaI provide good discrimination among strains of various S. enterica serotypes, including Livingstone (22). In our study, we have used PFGE with restriction endonucleases XbaI and SpeI. Five unrelated susceptible human isolates and one susceptible meat isolate from Tunisia, collected between 2001 and 2003, have been included in molecular typing experiments. The outbreak PFGE pattern was indistinguishable from that of isolates 01-8221 and 03-5725, collected in France from two patients who acquired gastroenteritis in Tunisia in 2001 and 2003. These two comparison isolates exhibiting the same PFGE pattern as that of the outbreak clone did not harbor the plasmid carrying the blaCTX-M-27 gene. Three other human isolates and one meat isolate from Tunisia (isolated during 2001 to 2003) exhibited different PFGE patterns. These preliminary results indicate that the S. enterica serotype Livingstone outbreak clone has been also found outside the Farhat Hached University Hospital and is probably not an uncommon clone, but more isolates need to be tested in order to appreciate its prevalence in the general population of Tunisia. Liebana et al. (22) reported that 6 of 14 S. enterica serotype Livingstone strains were found to be untypeable by PFGE. Murase et al. (28) have recently reported, in a PFGE study of 50 Salmonella serotype Livingstone poultry isolates from Japan, that addition of 50 μM thiourea prevented degradation of the DNA of this serotype during electrophoresis. In our study, addition of 750 μM thiourea to Tris-borate-EDTA buffer (in both in the agarose gel and the electrophoresis buffer) was necessary in order to obtain XbaI fingerprints for all ESBL-producing isolates and for four of six comparison isolates from Tunisia (two others were typeable without thiourea). In this study, ribotyping was less discriminatory than PFGE; comparison strain Tun-3 exhibited the same ribotype profile as outbreak isolates, while PFGE patterns were different. This could be due to the use of a single restriction endonuclease. Double restriction with PstI and SphI would have been more interesting, as reported by Liebana et al. (22). However, the results of both approaches (PFGE and ribotyping) for the epidemiological evaluation of the isolates involved in this outbreak suggested that it was caused by the same clone.
Salmonella serotype Livingstone has been isolated previously from poultry in other countries: Canada (11), Scotland (11, 35), Japan (28), England (11), and France (13). Between 1980 and 1985 in Scotland, poultry and poultry products were an important vehicle for human salmonellosis, since poultry meat accounts for >50% of all human Salmonella outbreaks where a food vehicle has been identified (35). The modern poultry industry had been developing in Tunisia during recent decades, providing a valuable inexpensive source of protein and currently representing the primary source of meat consumption by humans. Thus, despite the absence of epidemiological studies explaining the emergence and spread of this serotype in the country, we suppose that the emergence and spread of S. enterica serotype Livingstone might be related to poultry. Nevertheless, this opinion needs to be validated by further studies.
Commenting on the low incidence of serotype Livingstone infection in humans, Reilly et al. (35) suggested that this Salmonella serotype might have low virulence for humans. Like most nontyphoidal Salmonella serotypes, serotype Livingstone manifested itself only in individuals with predisposing factors such as immunocompromised status. In the present outbreak all patients had serious underlying diseases, which might have predisposed them to infection with the organism involved. Furthermore, they had been subjected to manipulations of the oropharynx by insertion of nasogastric tubes or by insertion of an umbilicus catheter. These manipulations might have provided opportunities for contamination of the babies by the hands of health personnel (17, 25).
Salmonella infections in hospitals are usually food-borne and have an explosive pattern, with an obvious food vector. In this outbreak, food as a source was excluded because milk was commercially prepared and other infants, hospitalized in the same sector of the ward at the same period, were fed with the same preparations but did not become infected with this strain. Therefore, it was concluded that horizontal transmission of the outbreak strain had occurred. Unfortunately, we were not able to trace the source of infections, because no further environmental investigation was implemented.
This outbreak happened in the summer, when the ward was understaffed and overcrowded. Overcrowding and understaffing are outbreak occurrence factors which are commonly observed in neonatal units throughout the world, particularly in developing countries, where limited personnel and facility resources contribute to the perpetuation of this problem. Understaffing in the neonatal unit reduces the likelihood that appropriate attention can be given to infection control practices, including hand hygiene, exacerbating the difficulty of implementing outbreak control measures (31). Finally, the outbreak came to an end when appropriate actions were taken: patient isolation, hand washing, and disinfection of the ward. No cases of S. enterica serotype Livingstone carriers or infection were reported thereafter.
For several years, the resistance of S. enterica to extended-spectrum cephalosporins had been increasingly reported through the world (26). A recent study reported that 1.6% of 431 Salmonella isolates from a broad network of sentinel hospitals distributed in Europe, the Americas, and the Western Pacific region were expressing an ESBL phenotype (46). ESC-resistant S. enterica strains have been reported previously in Tunisian hospitals. Salmonella serotype Wien, producing SHV-2 ESBL, caused a nosocomial outbreak in a neonatal care unit in Sfax in 1988 (14). In the same hospital in 2000, three ESC-resistant strains of Salmonella serotype Livingstone were found to produce cephalosporinase ACC-1 (36). One isolate also produced SHV-2. From 1997 to 1998, 31 isolates of ESC-resistant Salmonella serotype Mbandaka were isolated at University Hospital in Tunis (23). TEM-4 (ESBL), SHV 2a (ESBL), and ACC-1 (cephalosporinase) were identified in 30, 3, and 1 isolate, respectively. In 2001, two Salmonella serotype Wien isolates from the military hospital of Tunis were found to produce CMY-4 (cephalosporinase) and SHV-2a (4). One of these isolates also produced CTX-M-3 ESBL. This report describes the first identification of CTX-M-27 in the genus Salmonella and the second CTX-M-type ESBL-producing Salmonella isolate in Tunisia. CTX-M -lactamases, which preferentially hydrolyze cefotaxime, form a growing family that was characterized in the second half of the 1980s with the report of FEC-1 (24). Currently, the CTX-M family comprises at least 40 enzymes belonging to six groups (6). CTX-M -lactamases share extensive similarities with the chromosomal -lactamases of Kluyvera spp., enterobacteria that are rarely detected in medical microbiology, and it was suggested that the enzymes identified in Kluyvera ascorbata and Kluyvera georgiana were probably the progenitors of the CTX-M-2 and CTX-M-8 groups, respectively (15, 33). ISEcp1 or ISEcp1-like insertion sequences may be involved in the mobilization of blaCTX-M genes (8). Nine CTX-M -lactamases (CTX-M-2, CTX-M-3, CTX-M-4, CTX-M-5, CTX-M-6, CTX-M-7, CTX-M-9, CTX-M-14, and CTX-M-15) have been reported to be associated with different serotypes of S. enterica (6, 37, 45). In this report, the analysis of deduced amino acid sequences showed that blaCTX-M encoded a CTX-M-27 ESBL. This enzyme had been described recently in an E. coli isolate from France (7). It differed from CTX-M-14 by a single substitution, D240G, involved in the higher ceftazidime MIC. In the outbreak clone, the blaCTX-M-27 gene was located on a 40-kb conjugative plasmid which contained no additional resistance determinants. S. enterica serotype Livingstone isolate 7 and its E. coli transconjugant pC1a7-1 exhibited high levels of resistance to ampicillin (MICs, >256 mg/liter), ticarcillin (>256 mg/liter), and ceftriaxone (>256 mg/liter). They exhibited similar levels of resistance to amoxicillin-clavulanic acid (MIC, 8 mg/liter) and piperacillin-tazobactam (4 mg/liter); however, the MIC of ceftazidime for pC1a7-1 was twofold lower than that for isolate 7. This differential expression of ESC resistance between S. enterica and E. coli, which has been reported previously (5, 20, 27, 43, 45), may be due, in part, to the dissimilar genetic backgrounds of the E. coli recipient and Salmonella parental strains, resulting in lower -lactamase expression and activity than in the wild-type strain, or to differences between the E. coli and Salmonella envelopes, modifying antibiotic input into the cell.
Other resistance determinants (aminoglycoside, sulfonamides, and trimethoprim) were carried on a 90-kb nonconjugative but transferable plasmid. Trimethoprim resistance was encoded by a new dfrA21 gene inserted as a single resistance cassette in a class I integron.
The extended-spectrum cephalosporins were preferentially used as first-line drugs in the neonatal ward. Among 16 neonates, 10 had received cefotaxime and 2 had received ceftazidime. As previously reported (24, 30, 42), the emergence of highly resistant Salmonella in an environment characterized by a heavy use of antimicrobial agents may provide a setting that is conducive to nosocomial transmission. In our study, the emergence of the CTX-M-27 ESBL in the neonatal ward could be correlated with heavy use of extended-spectrum cephalosporins by the physicians. Efforts to promote the appropriate use of antimicrobial agents are essential for the control of multidrug resistance of bacterial pathogens (46). Rapid detection and prompt institution of control measures may limit outbreaks due to such an organism and prevent systemic infections, which are difficult to treat because of the few antibiotics available to which the organism is susceptible.
ACKNOWLEDGMENTS
We thank Ridha Ben Aissa, Institut Pasteur de Tunis, Tunis, Tunisia, for his collaboration.
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