First Description of the Extended Spectrum-Beta-Lactamase Gene blaCTX-M-109 in Salmonella Grumpensis Strains Isolated from Neonatal Nosocomial Infections in Dakar, Senegal

Nosocomial infections are very common in African hospitals, particularly in neonatal units. These infections are most often caused by bacteria such as Escherichia coli, Klebsiella spp and Staphylococcus spp. Salmonella strains are rarely involved in nosocomial infections. Here, we report the first description of S. Grumpensis in neonatal infections in Senegal. Seventeen Salmonella strains were isolated from hospitalized infants’ stool samples. The following resistance phenotype was described in strains: AMXRTICRCFR FOXRCFXRCTXRCAZRIMPSATMRNARNORRCIPRTMRGMRTERSXTR. All isolates were susceptible to imipenem, 15 out of 17 produced an extended spectrum ß-lactamase (ESBL). blaOXA-1, blaSHV-1, blaTEM-1, blaCTX-M1 genes were detected in strains 8, 13, 5 and 8, respectively. blaCTX-M1 sequencing revealed the presence of blaCTX-M-109. Thirteen of the 17 Salmonella Grumpensis strains were analyzed by PFGE. These 13 isolates belonged to a single pulsotype and were genotypically identical. This is the first report of neonatal S. Grumpensis infections in Senegal, and the first report of blaCTX-M-109 in the genus Salmonella.


Introduction
The emergence and spread of antimicrobial resistance is a major public health problem. Multidrug-resistant bacterial infections due to prolonged hospitalization, antibiotic treatment and poor hygiene are an increasingly prevalent cause of morbidity and mortality [1]. The frequency of hospital-acquired neonatal infections unrelated to non-typhoidal Salmonella is increasing. Therefore, hospital-acquired infections are both an important cause of mortality and a major economic burden [2].
Salmonella strains usually cause mild gastroenteritis but can also lead to serious infection, especially in vulnerable populations (young children, the elderly and immunocompromised individuals) [3].
Animals, both domestic and wild, are the main reservoir of non-typhoid Salmonella (NTS). Human NTS infections are frequently due to the consumption of contaminated cooked or raw meat or animal products (poultry, burgers, eggs) [4], or human-to-human transmission. Human contamination via the fecal-oral route, resulting from inadequate hygiene, is also not uncommon. NTSs are predominant among multidrug resistant (MDR) bacteria. They are a major cause of food-borne infections and often cause collective or individual nosocomial outbreaks in developing countries, in particular in pediatric settings [5; 6].
The main aim of this work was to characterize both the resistance and molecular characteristics of Salmonella Grumpensis strains isolated during a nosocomial infection outbreak in a neonatal unit of a University teaching Hospital Center in Dakar, Senegal, in 2011.

Materials and Methods
Between March to May 2011, nosocomial infection occurred in the neonatal unit in the university teaching hospital (Abass Ndao Hospital in Dakar). The unit which has a capacity of 35-40 beds including a room with nine individual cribs as well as large cradles (each holding three or four babies) and an intensive care room for premature babies with five illuminated tables and an incubator. These tables can accommodate up to three babies. The medical staff in this unit during the study period consisted of one midwife, six assistant nurses, two nurses' aides, five temporary support staff and ten temporary doctors. Such suboptimal working conditions may be the cause of neonatal infection. Stool samples were collected from 16 infants with acute gastroenteritis hospitalized in the neonatal unit of the Abass Ndao Hospital in Dakar together with one sample from a bottle of antiseptic (eosin) used in care of the newborn babies.
All samples were sent to Hôpital d'Enfants Albert Royer laboratory for testing.

Ethics Statement
Consent could not obtained from the babies' mothers as they were illiterate but a physician explained that the samples would be analyzed and, if a pathogen was identified, further studies would be carried out. Mothers also requested that all data would be analyzed anonymously and they all agreed in principle.
• Water samples were taken from the various taps in the unit and unopened formula cans were sampled.
• Samples were taken of disinfectant solutions prepared on site.
Stool samples from 18 staff members were tested for Salmonella at IPD

Bacterial isolates
Clinical isolates were obtained by standard procedures from Salmonella Shigella (SS) agar plates (Becton Dickinson, diagnostics). Salmonella strains were serotyped with polyvalent antisera using an agglutination test (Bio-Rad) at the National Center for Enterobacteriaceae, Pasteur Institute according to the White-Kauffmann-Le Minor method [7].

Antimicrobial testing
Antibiotic susceptibility testing was performed by the disc diffusion method on Mueller Hinton agar according the standard recommendations of the French Society of Microbiology (CA-SFM, 2010) [8].

Sequencing
Sequencing was performed with the Big Dye terminator for the characterization of class1 integrons, and antibiotic resistance genes. DNA was sequenced in an automatic sequencer (ABI Prism 3100; Applied Genetic Biosystems) in both directions with the same PCR primers used for amplification of the target genes. Contig sequences were edited with Chromas Pro and compared in the BLAST program of the NCBI (http://www.ncbi.nlm.nih.gov/blast/BLAST).

Molecular typing by pulsed field gel electrophoresis (PFGE)
Isolates were typed by pulsed field gel electrophoresis. In brief, DNA from Salmonella Grumpensis isolates was prepared in agarose plugs [13]. Bacterial cells were embedded in low-melting-point agarose (Biorad, Marnes-la-coquette) and lysed with lysis buffer containing lysozyme and proteinase K. DNA was digested at 37°C with 10U/μl of the restriction endonuclease Xba I (Biorad, Marnes-la-coquette, France). Digested DNA from S. Braenderup H9812 was loaded every five lanes as the molecular marker, as recommended by Pulse Net [14]. The digests were run at 6V/cmat at 14°C for 16 hours in a 1% agarose gel in 0.5X Tris-Borate-EDTA buffer using the Genepath system (Biorad, Marnes-la-coquette, France). The gel was stained with ethidium bromide and photographed on an ultraviolet trans-illuminator (GelDoc, Biorad, France). The restriction endonuclease digests were compared visually and isolates with the same pattern of bands (same number and molecular weight) were considered to be the same strain.

Bacterial conjugation
Conjugation experiments were carried out with E. coli NalR as the recipient. The recipients were selected on Luria-Bertani (LB) medium containing streptomycin (25μg/ml), nalidixic acid (50μg/ml), trimethoprim (5μg/ml) and ampicillin (100μg/ml) and the donors were selected on LB containing only streptomycin and trimethoprim (BioMérieux, France). A total of 5 ml of fresh LB broth was inoculated with either recipient or donor bacteria and incubated for 24 h at 37°C. After overnight incubation, cultures were diluted at 1:50 (400μl in 20ml of LBB) and incubated at 37°C with strong agitation for 4 hours until they reached the logarithmic growth phase (OD 600nm = 0.6-0.8). Donor and recipient strains were then mixed at the following ratios 1:1; 1:2; 1:10 and incubated at 37°C for 3 hours. Transconjugants were selected on LB agar plates supplemented with streptomycin (25μg/ml), nalidixic acid (50μg/ml), trimethoprim (5μg/ml) and ampicillin (100μg/ml). PCR was performed on transconjugants to determine whether resistance genes had been transferred.

Results
Multiresistant bacteria were detected in environmental samples: Salmonella Grumpensis in a sample taken from a bottle of antiseptic (eosin) used for the care of newborns, and other Gram negative bacteria, including Salmonella Brandenburg with a wild-type resistance phenotype in a sample from a sink in the nurses' locker room. Stool samples from the 18 caregivers were all negative for Salmonella. All 17 isolates in this study were biochemically identified and serologically confirmed as Salmonella enterica serovar Grumpensis. Babies were empirically treated with syrup containing norfloxacin and metronidazole (10-20 mg/kg/day) or intravenous ampicillin and gentamicin (50-100 mg/kg/day). All the babies were cured.

Antibiotic susceptibility
Ten (10) out of 17 (58.8%) isolates showed the following antibiotic resistance pattern AMX R-TIC R CF R FOX R CFX R CTX R CAZ R IMP S ATM R NA R NOR R CIP R TM R GM R TE R SXT R , Fifteen isolates produced an extended spectrum ß-lactamase (ESBL). All isolates were susceptible to imipenem.
In conjugation experiments, recombinants acquired the same pattern of antibiotic resistance as strains of Salmonella Grumpensis. intI1 and resistance genes were transferred intact to recombinant strains, suggesting that there was a block transfer of resistance through conjugative plasmids.

Molecular typing
Thirteen of the 17 strains of Salmonella Grumpensis were analyzed by PFGE. These 13 (12 from stool samples + 1from antiseptic sample) isolates belong to a single pulsotype and are genotypically identical (Fig 1).

Discussion
During the study period, this neonatal nosocomial infection did not cause any deaths. No other case of nosocomial Salmonella Grumpensis infection was reported elsewhere. Salmonellosis is rarely acquired in a hospital setting [15]. When it occurs, bacteria are usually transmitted by hand, care equipment and sometimes, antiseptic solutions [16]. Serotypes frequently involved in outbreaks in neonatology are S. Typhimurium, S. Mbao, S. Eimsbuettel, S. Heidelberg and S. Worthington [17]. Salmonella outbreaks due to S. Ordain, S. Tel-el-Kebir, S. Mbao, S. Niloese and S. Poona have been reported in hospitals in Dakar [18]. This is the first report of neonatal S. Grumpensis infection in Senegal although this bacterium has been reported to cause infection in humans both in this country and elsewhere [19]. One Salmonella Grumpensis isolate was isolated from a bottle of antiseptic used for patient care reflecting poor hospital hygiene. Consistent with previous reports in the literature, we did not find Salmonella in the formula. Another source of Salmonella infection could be human-to-human transmission; despite extensive investigation, the source of the infection was never established.
The PFGE profiles of 13 isolates (including the strain from the eosin bottle) suggested that they all originated from the same clone.
The emergence of antimicrobial resistance within Salmonella species has been reported worldwide [20] and the proportion of resistant Salmonella strains is increasing in developing countries. Salmonella is naturally susceptible to the antibiotics routinely used to treat gastroenteritis. However, some strains produce ESBL, conferring resistance to aminopenicillins, cephalosporins, tetracycline and quinolones [21]. ESBL characterization revealed that eight isolates possessed bla CTX-M-1 genes, which are a common feature of Enterobacteriaceae [22]. blaCTX-M-1 PCR product sequencing showed that strains carried CTX-M-109 gene. In Senegal, this is the first description of this gene in Salmonella even among Enterobacteriaceae. CTX-M-109 gene was firstly described on Shigella species in Asia [23]. CTX-M producing Enterobacteriaceae strains are currently described in Senegal with CTX-M-15 particularly common [24]. The CTX-M-15 gene has been detected in Europe [25,26], Asia [27], Africa [24,[28][29] and America [30]. CTX-M group 1 gene was detected in eight strains but the bla CTXM9 gene was not found in any of the strains. The absence of a CMY gene in our isolates suggests that the genetic determinants of resistance to beta-lactams were not located on the bacterial chromosome. Plasmids carrying the bla TEM and bla SHV genes, like those bearing the bla CTX-M genes, often host resistance genes to other antibiotics (aminoglycosides, tetracycline, sulfonamides, trimethoprim and quinolone), and therefore may confer co-resistance similar to what we found in strains of S. Grumpensis. Furthermore, consistent with previous reports in the literature, some of our strains had group 1 bla CTX-M genes and other resistance genes such as a betalactamase oxacillinase (bla OXA-1 ), which confers resistance to aminopenicillin [31]. Antibiotics are widely used in intensive care units, and this could explain the emergence of antimicrobial resistance in bacteria in the past ten years [32]. The resistance spectrum of Salmonella strains is determined by antibiotic use in animals, leading to selection pressure and the transfer of antibiotic resistance genes [33]. Strains that express CTX-M ß-lactamases are multidrug resistant. These strains have also plasmid-mediated genes conferring quinolone resistance [34]. The gyrA gene was detected in 8 strains and the gyrB gene in 14 strains. We found qnrB and qnrS genes among 13 and 10 isolates respectively but it was not determined whether they were qnrB1, qnrB4, or qnrS1.
Bacterial resistance to quinolones is typically mediated by changes in the target enzymes DNA gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) or by the modulation of drug entry and efflux. The role of plasmid genes (qnrA, qnrB, qnrS) encoding resistance to quinolones has been described in Salmonella [20]. The qnr gene originally emerged in Enterobacteriaceae [21]. The presence of plasmid genes (qnrB, qnrS) in 13 and 10 isolates explains the strong resistance of Salmonella to quinolones. The qnr genes are frequently described in Salmonella strains [35]. But in another study in Senegal, qnr genes were not detected. Only chromosomal genes such as gyr and par with mutations conferring resistance were described [36]. Overall, 65% of strains were resistant to tetracycline, probably because this antibiotic is so often used in animals [37]. However, the only tetracycline resistance gene found was the tetA gene; the tetB gene, which often confers resistance to tetracycline in Enterobacteriaceae was not found in isolates [38]. In our study, we identified only class 1 integrons among fourteen Salmonella isolates. Characterization of class 1 integrons revealed the presence of aadA1 gene cassette encoding resistance to streptomycin and spectinomycin.
Conjugation experiments showed that all resistance genes were found in recombinants; therefore, "block" transfer of resistance genes occurred in the recipient strain of E. coli.
In addition, class 1 integrons from strains of Salmonella were transferred to E. coli during conjugation experiments. This suggests a plasmid origin for the genetic determinants of multidrug resistance among the strains of S. Grumpensis that caused outbreaks of gastroenteritis in the neonatal unit of Dakar Hospital. This means that multidrug resistance could rapidly spread and lead to treatment failure in many cases.

Conclusion
This investigation detected strains of Salmonella Grumpensis that can cause nosocomial infections that could be particularly devastating in developing countries where the antibiotics used to treat them are expensive and difficult to come by.
Salmonella Grumpensis found in a bottle of antiseptic had a similar resistance profile to isolates from infected newborn babies. Pulsed-field gel electrophoresis of Salmonella Grumpensis strains found in the infants' stools and the bottle of antiseptic confirmed the clonal link. Information about this outbreak in an intensive care unit is limited as the source of the contamination could not be identified; mothers and nurses were tested to investigate human-to-human transmission. The cleaning of surfaces in neonatal units is essential to remove multi-resistant germs, a simple way of controlling infection together with regular use of alcohol-based disinfectant solutions and training for care-givers. Establishment of an Infection Control service with adequate human and financial resources and the involvement of all personnel (staff awareness) are highly recommended.