Prevalence and gene sequencing of extended spectrum β-lactamases producing Salmonella enterica serovar. Typhi from South-East Nigeria

Department of Pharmaceutical Microbiology, Dora Akunyili College of Pharmacy, Igbinedion University Okada, Edo State, Nigeria. Department of Pharmaceutics and Pharmaceutical Technology, Dora Akunyili College of Pharmacy, Igbinedion University Okada, Edo State, Nigeria. Department of Pediatrics, College of Medicine, Imo State University Teaching Hospital, Owerri, Imo State, Nigeria. Department of Applied Microbiology and Brewing, Faculty of Bioscience, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.


INTRODUCTION
Enteric fever is a systemic disease classified into typhoid and paratyphoid fever (Elumalai et al., 2014). Paratyphoid fever is a less severe type of enteric fever caused by Salmonella Paratyphi A, B and C (Cheesbrough, 2006 ;CLSI, 2011).
Typhoid fever being a systemic infection caused by Salmonella enterica serotype Typhi is a highly adapted human specific pathogen which possesses remarkable mechanism for persistence in host as reported by Onyenwe et al. (2011). It estimates for approximately 5.4 million cases worldwide per year (Elumalai et al., 2014). Typhoid fever is severe and caused by Gram negative bacterium S. enterica serovar Typhi (Elumalai et al., 2014). It can be transmitted via contaminated food, water and is mainly present in area with poor hygiene or low socioeconomic status (Mweu and English, 2008). According to the World Health Organization (WHO), they estimated that typhoid fever accounts for 222,000 deaths and 21 million infections annually on a global scale (WHO, 2015). Africa is classified under region with medium incidence rate per year (10-100/100,000 cases/year) centered on a 22 community-based incidence study with only three African countries included in the analysis (Crump et al., 2004). As of 2013, the annual mortality rate was reported at 2.8 per 100,000 person in Africa with 2.5 per 100,000 person reported for Nigeria (Health Grove, 2018). These numbers make typhoid fever an infectious disease worth investigating for better treatment plan and management.
The standard treatment for typhoid fever is the use of antibiotics which target the Salmonella bacteria (DrugBank, 2009). In the 1990s, there was the introduction of chloramphenicol which posed as the first line of treatment for typhoid fever. However, the development of resistance has led to multiple use of antibiotics including Co-trimaxazole, Ampicillin and third generation Fluoroquinolones and Cephalosporins. These antibiotics have contributed to reducing the mortality rate from 20 to 1-2%, according to Wain et al. (2015). Nevertheless, multidrug resistant (MDR) strains emerged with extended-spectrum β-lactamases (ESBLs) production affecting the effective treatment of Salmonella Typhi (Wain et al., 2015).
ESBLs are enzymes belonging to the β-lactamase group. They cause resistance to most β-lactam antibiotics including Cephalosporins, Penicillin, Monobactams and even early generation Cephalosphorins. ESBLs are divided into three main groups comprising TEM, SHV and CTX-M (Pitout et al., 2008). Previous studies indicated that the SHV family is derived from the Klebsiella species (Pitout et al., 2008), and are capable of hydrolysing first 3 generation Cephalosporins and Penicillin (D'Angelo et al., 2016). TEM are derived from Escherichia coli and have resistance against Penicillin, Ampicillin and firstgeneration Cephalosporin to extended spectrum Cephalosporins (D'Angelo et al., 2016). The CTX-M group is identified as the most prevalent type of ESBLs from Salmonella enterica (Pitout et al., 2008) and as their name suggest, they are the most powerful hydrolytic activity against CTX (D'Angelo et al., 2016). Also another Onyenwe et al. 193 study have however shown that majority of ESBLs in Salmonella are derived from TEM and SHV βlactamasefamily (Elumalai et al., 2014). Klebsiella pneumoniae, E. coli, and Klebsiella oxytoca are the most commonly studied ESBL-producing organism due to the ability of easily identifying the presence of β-lactamase (D'Angelo et al., 2016). Thus ESBLs confer resistance to these antibiotics and related oxyimino-beta lactams (Schneider, 2008). In typical circumstances, they are derived from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the amino acid configuration around the active site of these β-lactamases. However, there is infrequency when it comes to investigating in-vitro antibiotic combination therapy in ESBL S. Typhi infection. Thus, this study is aimed at investigating the prevalence and gene sequencing of ESBLs producing S. enterica serovar. Typhi from South-East Nigeria.

Test organism
A total of 150 isolates of S. enterica were randomly collected from the Routine Section of the Medical Microbiology Laboratory in four hospitals from Southeast part of Nigeria. The isolates were from patients diagnosed with enteric fever that visited the four hospitals. Thus, the codes were designated as O for isolates from Owerri, U for isolates from Umuahia, E for isolates from Enugu and A for isolates from Abakaliki, accordingly, between June and October, 2017. After investigation, 100 S. enterica serovars were identified and screened (25 each from unrelated patients from the four hospitals) by standard biochemical characterization, using microbact® identification kit-12E (oxoid-England). The antibiotic-sensitivity screening of the isolates (using McFarland standard of 0.5 ml dilution) were carried out by the multidisc agar diffusion method and E-test (Oxoid, India) as described by Onyenwe et al. (2012) on 20 ml molten Muller Hilton agar using antibiotics disc (oxoid, India), with a standard ATCC 14028 strain of S. enterica serovar Typhi. Members of the family Enterobacteriaceae that commonly express plasmid-encoded β-lactamases are TEM-1, TEM-2, and SHV-1 (Philippon et al., 2002;George et al., 2005).

Antimicrobial sensitivity testing
The screening for antimicrobial activity was carried out by the single disc agar diffusion method as described in Onyenwe et. al., (2011). The zones of growth inhibition were interpreted using CLSI breakpoint as standard zones depending on the antibiotics used (CLSI, 2011;CLSI, 2014).

Beta-lacatamase production test using Nitrocefin sticks
The nitrocefin™ sticks (oxoid, India) is a chromogenic  cephalosporin β-lacatamase indicator in a container (Murray et al., 2006). The Nitrocefin sticks were allowed to reach room temperature after removing it from the freezer. Then a well separated representative colony from the primary isolation medium was selected. The procedure was as described by Onyenwe et al. (2012). A positive reaction was shown by the development of a pink/red color. No color change was observed with organisms that do not produce beta-lactamase. The unused Nitrocefine stick was used as control.

Double disc synergy test (DDST) to detect ESBL's production
The test inoculum (0.5 McFarland turbidity equivalent) was spread onto Mueller-Hinton agar (MHA, Oxoid, India) using a sterile cotton swab. A disc of 20 μg Amoxycillin + 10 μg Clavunalic acid was placed on the surface of MHA; then discs of Ceftriaxone (30 μg), Ceftazidime (30 μg) and Cefotaxime (30 μg) (Oxoid, India) were kept around it in such a way that each disc was at distance ranging between 10 and 15 mm from the amoxil-clavulanic acid disc (centre to centre). The plate was incubated at 37°C overnight. The organisms were considered to be producing ESBL when the zone of inhibition around any of the broad-spectrum cephalosporin discs showed a clear-cut increase towards the amoxil-clavulanic acid disc (Onyenwe et al., 2011;Yah, 2010).

PCR amplification and agarose gel electrophoresis
The method was as described by Onyenwe et al. (2012). The purified DNA template in PCR Eppendorf tubes (2.0 μl DNA) was mixed with the Master Mix ready to load (HOT FIREPol ® DNA polymerase with 2.5 mM MgCI 2 , Solis Biodyne). The mixture of primers, DNA and the Master Mix were vortexed, to mix and then centrifuged before introducing it into the PCR machine (Eppendorf-Germany). The PCR products were then taken for electrophoresis, after amplification (CBS Scientific Company Inc.) on the agarose gel at 80 to 100 V and finally viewed on the UV light for visible amplified image of the genes. A DNA-marker of 100 base pairs was used.
The thermo cycling condition (Solis biodyne 5x FIREPol) Master Mix Ready to load used for ESBL Bla ctx-M was 30 to 35 cycles of 95°C for 30 s, 72°C for 1 min, 66.2 for 1 min, 72°C for 1 min, 95°C for 30 s (PCR timing 1.38-2.58 h). The Bla ctx-M universal primers used were Bla ctx-M -F(5'ATG TGC AGY ACC AGT AAR GTK ATG GC-3') and R(5'-TGG GTR AAR TAR GTS ACC AGA AYC AGC GG-3') where R in the sequence is purine, Y is pyrimidine, and S is G or C (this design is to occupy for the ambiguity of sequence variation among the CTX-M types) (Moubareck et al., 2005).

RESULTS
A total of 100 isolates of S. enterica serovar. Typhi were obtained out of the 150 screened from the four different hospitals from the Southeast region of Nigeria between June and October, 2017. The isolates were identified by cultural and biochemical characteristics using Microbact identification kit ® 2E, thereby identifying the isolate to be S. enterica serovar. Typhi. as shown in Table 1. Table 2 shows that only 2 (8%) isolates out of the twenty-five isolates recovered in GOPD unit from Federal Medical Center Owerri harboured plasmids, while 20 (80%) isolates produced the conventional β-lactamase. It was also observed that isolates recovered from a female child of 2 years in the CHOP unit produced the conventional β-lactamase enzyme but was resistant to only 4 (28.6%) antibiotics used in this study, while an isolate recovered in the EPU unit from a female child of 1 year old was resistant to the 4 (28.6%) of the antibiotics used but produced no conventional β-lactamase and harboured no plasmid.
From the results obtained from the S. enterica isolates (Table 3) from Federal Medical Centre (FMC) Umuahia, only 3 (12%) harboured plasmid, while 18 (72%) of the isolates recovered produced the conventional βlactamase enzyme. It was also observed that isolates recovered from 2 (8%) female children in the IPU produced β-lactamase, though no plasmids were found on them, they were resistant to only 5 (35.7) of the 14 antibiotic used. Table 2. Beta-lactamase production and plasmid profiling of isolates of Salmonella enterica from Federal Medical Center Owerri, in relation to gender distribution. Results from University of Nigeria Teaching Hospital (UNTH) Enugu (Table 4) showed that there were no plasmid recovered from the 25 isolates but 20 (80%) of the isolates produced the conventional β-lactamase enzyme. It was also observed from this study that an isolate recovered from a child of 10 years from CHOP unit produced no conventional β-lactamase, harboured no plasmid but was resistant to 9 (64.3%) of the antibiotics used for the study.

Org. code [S/N] Unit/Dept. Sex Age (years)
Results on the S. enterica recovered from Federal Medical Centre (FMC) Abakaliki (Table 5) showed that 4 (16%) of the isolates harboured plasmids, with one of the isolates habouring the plasmid coming from a female child of 6 years old in the NHIS unit. Analysis also showed that the isolates recovered from the female child of 6 years as described earlier produced the conventional β-lactamase and was resistant to 10 (71.4%) of the 14 antibiotics used in this study. Almost all the isolates recovered from this part of Southeast (Abakaliki) in this study, produced the conventional β-lactamase. This could be as a result of the fact that the isolates have been recovered from IPU and GOPD unit of the hospital. It was also observed that one isolate recovered from a female patient in the IPU unit harboured no plasmid, but produced the β-lactamase enzyme and was resistant to only one antibiotic used in this study (Table 5). The resistance by S. enterica on the number of antibiotics tested was found in FMC Abakaliki to be the highest in the region (South-East Nigeria).
From Table 6, analysis showed the hospital record of the age range of individuals (patients) from whom S. enterica isolates were collected in this study. A total of 9 (9%) of the patients as shown on the record sheet of the hospital had plasmid DNA harbored in the isolates recovered from them (both male and female). While 55 (55%) of the total isolates showed positive GryA (male and female), 14 (14%) of the isolates had ParC (male and female), and 13 (13%) positive Bla CTX-M type gene (male and female), and non harboured bla-SHV and bla-TEM, in the genomic DNA of the isolates S. enterica recovered from various age groups as shown in Table 6. Analysis revealed that the highest number of S. enterica isolates were recovered from patients between 18 and above, followed by the ages between 21 and 30 and the least ages between 0 and 10 years. Figure 1 shows the graphical representation of the genetic constituents of the isolates of S. enterica and their resistance factors as related to each of the 5 selected antibiotics screened. Out of the 29 isolates of S. enterica resistant to Ciprofloxacin, only 15 produced mutation in GyrA, 8 produced double mutation in ParC, 1 produced Bla CTX-M and three isolates harbored plasmids. Also, out of 22 isolates resistant to levofloxacin 9 produced mutation in GyrA genes, while ParC, Bla CTX-M , and plasmids were produced by only two (2) isolates each. Other resistance gene produced by each S. enterica isolates resistant to other antibiotics such as ceftriaxone (CRO), cefotaxime (CTX-M), and amoxiclavulanic acid (AMC) are as shown in Figure 1. Table 7 shows the result of the sequence alignment of the gene resulting from the PCR amplifications of the DNA complete genome of the isolate from the various hospitals (SO3, SO14, SA96, SU33, and SA98) as shown in Table 7. From  are as shown in Table 7.

DISCUSSION
ESBLs are enzymes belonging to the β-lactamase group. They cause resistance to most β-lactam antibiotics including Cephalosporin, Penicillin, Monobactams and even early generation Cephalosporin and Ciprofloxacin. Analysis in this study revealed that the cause of resistant to these antibiotics was not only as a result of plasmids but also some plasmids mediating enzymes and chromosomally mediating enzymes. Enzymes such as βlactamase enzymes and extended spectrum β-lactamase enzymes were detected to have initiated some forms of resistant (either enzyme inhibition or resistant mutants) to these antibiotics especially the Cephalosporines and Floroquinolones which has been recommended as a replacement by most authorities for the treatment of typhoid fever. Many reports have been put forward by several authors such as Brusch et al. (2010) that Floroquinolones are highly effective against susceptible organism, yielding a better cure rate than Cephalosporins, but that resistance to first generation Floroqninolones is widespread in many parts of Asia (Bruschet al., 2010). Furthermore, they stated that in recent years, third generation Cephalosporins have been used in regions with high Floroquinolones resistance rates, particularly in South Asia and Vietnam (Brusch et al., 2010) but much has not been reported in this part of Nigeria, especially in this study. Furthermore, in this study, analysis showed that both the third generation cephalosporin and the fluoroquinolones are still maintaining their choice as a replacement for the treatment of phenotypically detected chloramphenicolresistant strain of S. enterica serovars of Typhi in this part of Nigeria.
Analysis from this study showed that 13 (13%) positive bla CTX-M type gene were being harboured in the chromosomal DNA of the isolates S. enterica recovered

Level of resistant gene produced by Salmonella enterica
KEY Figure 1. Graphical representation of the genetic constituents, the number of antibiotics and resistant pattern of isolates that produced plasmids or a type of mutation in a gene detected using PCR amplification, out of the total isolates resistant to each antibiotic. from various age groups as shown in the Table 2.
Though not all the isolates were β-lactamase positive, as only 80% of the isolates produced β-lactamases, while 36% of the isolates were positive to ESBLs. Also, it was discovered that only 28 isolates had both the conventional β-lactamases and the ESBL's (Tables 2 to  5). This trend of result could have been the reason why there was multiple resistances to these antibiotics used in this study. According to Yujuan and Ling (2006), Valverde et al. (2008) andYah (2010) in their studies show resistance to broad spectrum β-lactams is highly mediated by ESBL enzymes, which has been increasing the world health problems in clinical settings. According to Yah (2010), the plasmid-borne βlactamases are also competent enough to hydrolyze βlactam antibiotics. Also, according to Soge et al. (2005), the first CTX-M-type β-lactamases in Nigeria were identified as plasmid-encoded enzymes in clinical isolates from the Enterobacteriaceae, and all 30 isolates of Klebsiella pneumonia in their study produced at least one β-lactamase and 17 (57%) produced a CTX-M βlactamase (Soge et al., 2005). Reports have also shown that the resistance of gastroenteric Salmonella strains to these antimicrobial agents is in large part due to the production of ESBLs encoded on plasmids as well as on the chromosome, as reported by David and Frank (2000), Yujuan and Ling (2006), Yah (2010) and Onyenwe et al. (2012).
According to the CLSI (2011) standard used for the sensitivity screening in this study, few of the isolates of S. enterica were sensitive to Chloramphenicol, though no molecular analysis was carried out to confirm the gene or enzyme responsible for it. It is worthy to note that due to resistance and safety concerns, chloramphenicol should no longer be a first line agent for indication of S. enterica treatment in developing nation like Nigeria as seen in this epidemiological survey using phenotypic characterization. According to Hakanen et al. (2001), the emergence of antimicrobial resistance in any part of the world may have a global bearing and thus deserves universal attention.
In this study, at least 3 S. enterica serovars harboring plasmids were found to be resistant to some groups of antibiotics (SA81, SA83, and SA85 isolates) including 2 members of the Fluoroquinolone (Ciprofloxacin and Levofloxacin) without harboring any resistance gene or mutation in their gyrA and parC regions in the chromosomes, hence may be an indication of plasmidmediated quinolone resistance (PMQR), no characterization was done on the gene. According to Hopkins et al. (2005) and Haugum et al. (2006), such resistance was reported only in Klebsiella and E. coli. Earlier studies have observed that in Salmonellae, the relative frequency of different mutations in gyrA was dependent on the quinolone antibiotics used for selection (Levy and Manshall, 2004). According to Lindtedt et al. (2004) it was discovered that a geographically dependent distribution of GyrA mutation was at codons 83 and 87 in  (Hakanen et al., 2001), which is in line with the present study. Several clinical treatment failures after the administration of Ciprofloxacin and other Fluoroquinolone to patients with typhoid fever due to strains with decreased susceptibility to the Fluoroquinolone have also been reported by Threlfall and Ward (2001). From this study, the result of the sequence alignment of the gene resulting from the PCR amplifications of the DNA complete genome of the isolate from the various hospitals (SO3, SO14, SA96, SU33, and SA98) as shown in Table 7 Table 7. In this study, the Fast Minimum Evolution and Neighboring Joining Taxonomic tree of some strains sequenced including their relatedness was also found for isolates SO3, SO14, SU33, SA96, and SA98 (data not shown).
In this study, the mutations responsible for the Fluoroquinolone resistance in the gyrA and ParC genes of the S. enterica serovars were investigated. The sequences for the Quinolone Resistance Determining Region (QRDR) of the gyrA gene of the isolates which showed reduced susceptibility to some Fluoroquinolone were detected. There was single mutation at the Ser-83-Tyr, Ser-87-Gly and Ser-83-Phe, while some were found in Asp-87-Gly or Asp-86-Gly in ParC gene. The sequence analysis also revealed that some of the positions of the amino acids in the gyrA mutation were identified as Asp-87-Asn or at Ser-83-Tyr. The gene (from isolate SA98) sequence alignment revealed that the S. enterica characterized in this study had 99% similarity or identity to the typed gene of S. Typhimurium strain 580 GyrA gene, partial cds (conservative domains) and 94% identical to the American Typed Culture Centre (ATCC) strain known as S. enterica subsp. enterica serovar Typhimurium strain ATCC 307 found in the genebank, respectively. Also in this study, some NCTC13348 (National Centre for Typed Culture) strains chromosome were found to be 94% identical to the quinolone resistant gene of the isolate sequenced in this study. Another gene (isolate SO3 having point and double mutation at position 83 and 87 codon) sequenced was also observed to be 94% identical to a strain, S. enterica subsp. enterica serovar Typhimurium str. DT104, which is also similar to the gene of the clinical isolate reported by Hirose et al. (2002), which caused nosocomial infections in the United States with high level of Fluoroquinolone resistance as shown in this study, according to the Fast Minimum Evolution and Neighboring joining tree, taxonomic analysis (Zheng et al., 2000;Aleksandr et al., 2008).

Conclusion
Based on this study, it could be deduced that Chloramphenicol may no longer be a first line agent for any indication in both developed and developing nations like Nigeria. But, in low income countries like Nigeria, unfortunately, Chloramphenicol is still widely used mainly because it is cheap and readily available. The most serious adverse effect associated with its treatment is bone marrow toxicity. This may occur in two distinct forms bone marrow suppression, which is a direct toxic effect of the drug, usually reversible. Secondly, aplastic anaemia, which is idiosyncratic in nature (rare, unpredictable and unrelated to dose) are generally fatal according to Onyenwe et al. (2011). Arising from this study, Fluoroquinolones such as Levofloxacin should only Onyenwe et al. 201 be used in patients who have failed at least one prior therapy, and also reserved for use in seriously ill patients who may require immediate hospitalization as specified by Johnson (2004, 2009) and Soge et al. (2005).