Salmonella Infections among Pediatric Population in Qatar: Phenotypic Resistance and Associated Genotypic Determinants

Abstract

Salmonella is a significant public health burden worldwide and being the most common bacterial diarrheal illness among infants and young children. In the last few years, Qatar reports a high incidence of salmonellosis outbreaks coupled with a significant increase of Multidrug-Resistant (MDR) among pediatric populations every year. This study aims to elucidate the molecular mechanisms underlying resistance to ceftriaxone, cefepime, amoxicillinclavulanate tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, and azithromycin among Salmonella isolated from the pediatric population. A total of 246 Salmonella isolates were collected from children under 18 years old admitted to the Pediatric Emergency Center (PEC), Hamad Medical Corporation (HMC) from Jan. 2018 to Dec 2019 with gastroenteritis. Isolates were tested for antibiotic susceptibility against nineteen relevant antibiotics using E-test. Resistance was confirmed using PCR-specific primers for 38 genes. Resistance was detected against 14 antibiotics, and 38.2% of isolates were resistant to at least one antibiotic. Overall, we reported 23.9%, resistance to tetracycline 21.1%, ampicillin 18.7%, AMC, and 13% sulfamethoxazole-trimethoprim. Further, 16.2% of the isolates were Multidrug-Resistant (MDR), with 4.1% being Extended-Spectrum Β Lactamase (ESBL) producers. 90% of ESBL producers harbored one of ^bla CTX-M-Group. Class 1 AMC resistant samples showed the highest resistance to different antibiotics. Our results indicate a high antimicrobial resistance pattern of Salmonella and the presence of Class (1) cassette that involves the transmission and expression of the resistance among AMC resistance isolates, which might lead to increased multi-drug resistance. This study provides evidence guidance to activate and implement the pillars of an antimicrobial stewardship program in human health to reduce MDR salmonellosis.

Keywords: Salmonella; MDR; salmonellosis; pediatric; Qatar

Abbreviations

MDR: Multi-Drug Resistant; NTS: Non-Typhoidal Salmonella; AMR: Antimicrobial Resistance; ESBL: Extended-Spectrum Β Lactamase; FDA: Food Drug Administartion

Introduction

Salmonella is one of the most common etiological agents of foodborne diarrheal illness and typhoid fever worldwide, leading to mortality in complicated cases [1]. Importantly, children less than 5-years old account for approximately a third of deaths attributed to foodborne diseases (WHO, 2015). There is an increasing concern regarding this pathogen due to the emergence and spread of antibiotic-resistance and potentially more pathogenic strains [2]. Drug-resistance among gastrointestinal pathogens preferentially cause illness in persons receiving antimicrobial drugs for any medical condition. Consequently, emerging resistance in foodborne pathogens may result in increased burdens of illness and outbreaks in settings where patients are treated with antimicrobial drugs [3]. Antimicrobial therapy is not indicated in uncomplicated cases of gastrointestinal illness in patients without underlying illness. However, therapy is needed, and maybe lifesaving in selected patients with the prolonged febrile course of suspected underlying invasive illness [3]. Non-Typhoidal Salmonella (NTS) can be invasive and cause paratyphoid fever, which requires immediate treatment with antibiotics. Extended-spectrum cephalosporins being used favorably to treat salmonellosis in children [4]. The treatment of the Enterobacteriaceae family, including Salmonella, has been increasingly complicated by the emergence of resistant strains to firstline antimicrobial agents [5,6].

The gram-negative bacterial genus Salmonella is divided into two species, Salmonella enterica, and Salmonella bongori. Only the Salmonella enterica subspecies enterica is of clinical relevance for humans [7], and is further classified into more than 2,600 serovars. Salmonella can be broadly categorized as typhoidal and Non- Typhoidal Salmonella (NTS). The typhoidal Salmonella includes serovar Typhi, and the closely related serovar Paratyphi (A-C) that cause enteric fever [8]. The much larger NTS group causes infectious diarrhea worldwide and primarily induces acute, self-limiting gastroenteritis [9]. NTS can also cause various other disorders, including bacteremia, meningitis, and osteomyelitis, particularly among immunocompromised patients.

Salmonella is emerging as a priority public health hazard in Qatar, being the most reported bacterial diarrheal illness among infants and young children, alarmingly associated with multiple incidences of salmonellosis outbreaks among this pediatric population each year. Salmonella is listed among the four commonly isolated Enterobacteriaceae from Hamad Medical Corporation (HMC, Personal communication). A previous study showed a high incidence of salmonellosis in Qatar, especially in children less than five years of age, with an overall annual incidence rate ranging between 12.3 and 30.3 per 100,000 population in 2004-2012 [10]. Nonetheless, this previous study does not characterize the molecular level of resistance to understand better the relatedness, typing of different isolates, and understanding their resistance mechanism. Besides, there are no data available after 2012 on salmonellosis incidences in the State of Qatar. The spread of resistant bacteria is linked to the misuse of antibiotics in human and animal populations and the consumption of contaminated food with resistant bacteria [11].

There are limited published data on antibiotic resistance profile and its associated genetic determinants in Qatar among Salmonella for the pediatric population. Therefore, a study is required to define and analyze the antimicrobial resistance and molecular epidemiology of Salmonella. Obtaining such information is crucial to tackle and mitigate the salmonellosis problem at the clinical, public health, and economic levels. The recurrent Salmonella outbreaks in Qatar and the increasing number of salmonellosis cases mandate rapid regulatory and monitoring reforms at the State level. This study aimed to profile the phenotypic resistance of Salmonella to the relevant antibiotics. Furthermore, to elucidate the molecular determinants underlying resistance to the most worldwide used antibiotics, including ceftriaxone, tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, and azithromycin.

This is the first study among Qatar’s pediatric population that demonstrates the correlation between genetic and phenotypic trends of antimicrobial resistance that would influence the development and implementation of stewardship programs to reduce pathogenic resistant bacteria’s burden on the community.

Materials and Methods

Clinical isolates

Ethical approval for this study was obtained from the Medical Research Centre, HMC, Doha, Qatar, protocol no. MRC-01-17- 198 and Qatar University approval # QU-IBC-2019/008. A total of 246 Salmonella isolates were collected between January 2018 and December 2019 from children (2-18 years of age) of different nationalities presented to the Pediatric Emergency Center (PEC), HMC, the primary provider of healthcare services in Qatar with symptoms mainly, fever and gastroenteritis. For each patient, demographic data such as age, nationality, and gender were reported.

Bacterial culture

Pathogens were isolated from human samples using the standard bacteriological procedure. Briefly, 1 g of the collected stool samples was diluted with 3ml of phosphate-buffered saline (PBS, pH 7.2; Sigma, St. Louis, MO, USA), and 500μl of this dilution was added to 5 ml of Selenite broth (Oxoid, Basingstoke, Hampshire, UK) for the enrichment and incubated at 37°C for 24-48 hr. The enriched samples were sub-cultured into MacConkey agar and incubated at 37°C for 24 hr. The non-lactose fermenter colonies tested negative with oxidase and produced hydrogen sulfide gas were sub-cultured into Hektoen Enteric Agar (Biolife-Italia). Suspected colonies (transparent green colonies with black centers) except S. paratyphi A, whose colonies appear without black centers, were identified by Matrix-Assisted laser Desorption/Ionization, MALDI-TOF, (Bruker Daltonik GmbH, Leipzig, Germany). On the other hand, blood culture samples were incubated on automated BACTEC FX blood culturing instrument (B.D., U.S.). Gram stain was performed on blood cultures flagged positive. Then, cultured isolates demonstrating Gram-negative bacteria were inoculated into MacConkey agar and blood agar. Suspected colonies were automatically identified using MALDI-TOF. Salmonella serology was performed using (Difco^TM Salmonella O Antiserum) and (Difco^TM Salmonella H Antiserum).

Phenotypic antibiotic susceptibility testing

The antibiotic susceptibility testing was conducted using Phoenix (the NMIC/ID-5 panel, B. D. Biosciences, Heidelberg, Germany) per the manufacturer’s recommendations. This panel includes 15 antibiotics, namely Amoxicillin/Clavulanic Acid (AMC), Ampicillin (AMP), Ceftriaxone (CRO), Aztreonam (ATM), Cefepime (FEP), Ceftazidime (CAZ), Meropenem (MEM), Imipenem (IPM), Ertapenem (ETP), Piperacillin/tazobactam (TZP), Tigecycline (TGC), Ciprofloxacin (CIP), Levofloxacin (LVX) and trimethoprim/ sulfamethoxazole (SXT). At the same time, E-test (Liofilchem, Germany) was performed for Azithromycin (AZM), Fosfomycin (FOS), Tetracycline (TET), and Chloramphenicol (C) as they were not included in the Phoenix automated panel. MICs were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute CLSI (CLSI, 2017). Escherichia coli ATCC 25922 strain was used as a quality control organism. Isolates were confirmed as ESBL producers via the Double Disc Synergy Test (DDST) as previously described by Eltai et al. [12] AmpC Β-lactamase production was measured using cefoxitin; a zone diameter of (≥18 mm) was considered as AmpC-positive (CLSI, 2017). All intermediate resistant isolates were considered susceptible.

Molecular characterization of antibiotic resistance

Whole- genomic DNA was extracted from Salmonella bacterial cultures using QIAamp^® UCP pathogen Minikit (QIAGEN, Germany). Extracted DNA was then used to run PCR for 32 genes using previously published primers [11-21]. The conditions used for reactions were as follows: PCR mixture was made in the volume of 20μl containing 0.5μM of each primer, 1μl of DNA, 1× master mix (Hot star Taq plus master mix (QIAGEN, Germany) and H₂O up to 20μl. The reaction was amplified in Biometra TAdvanced Thermal cycler (Analytik Jena, Germany) under the following conditions: initial denaturation at 95°C for 5 min.; followed by 30 cycles of 30 s at 95°C, 1 min at 44-63 °C and 90 s at 72°C and a final extension step of 5 min at 72°C.

The 32 genes that responsible for the resistance of the most prescribed antibiotics were amplified. These genes include tet A, B, C, D, E and G (Tetracycline resistant isolates), catA, cmlA and floR (Chloramphenicol-resistant isolates), sul1, sul2 and sul3 (Trimethoprim–sulfamethoxazole-resistant isolates), ESBL genes ^blaTEM, ^blaCTX-M groups and ^blaSHV (Third generation cephalosporines), mphA, mphB, ermA, ermB, ereA, mefA, and msrA (Azithromycin-resistant isolates) and ampC, CMY2 and Class 1 gene cassette (Amoxicillin-clavulanate resistant isolates).

The only clear bands were considered for the further fingerprinting scoring. The scores’ ‘1’ and ‘’’0’ were given for the presence and absence of genes ‘ ‘amplicon’s band, respectively. The data obtained by scoring the Antimicrobial Resistance (AMR) genes were subjected to cluster analysis. A similarity matrix was constructed using Jaccard’s coefficient, where the similarity values were used for cluster analysis. Sequential Agglomerative Hierarchical Non-overlapping (SAHN) clustering was performed using the Unweighted Pair Group Method with the Arithmetic Averages (UPGMA) method. Data were then analyzed using Past software version 3.26 [22] based on Hamming similarity index with 100 bootstraps.

Statistical analysis

Data were introduced into Microsoft Excel 2010 (Microsoft Corporation, New York, USA) to generate figures and run the initial analysis. The relation between the resistance, nationality, age, and gender grouping was calculated using the Pearson test (GraphPad Software, San Diego, California, USA). A probability value (P-value) less than 0.05 was considered statistically significant. Past software, version 1.91 was used to construct a hierarchical clustering dendrogram, and Jacquard’s coefficient was applied to generate the similarity values for the cluster analysis.

Results

Demography of the study population

In this study, we tested 246 Salmonella isolates from children between 2-18 years of age. Among these, 220 (89.4%) were isolated from stool and 26 (10.6%) from blood. The demographic profile of the studied population was summarized in Table 1. The male: female ratio was 1.4:1 and 194 (78.9%) were under five years of age. The Qatari patient comprises 37.4% (n=92) of this study population, while other nationalities 62.6%. The typhoidal Salmonella among Qatari patient was 30.4%/28 (23 Paratyphi B, 2 Paratyphi A and 3 Typhi) and 38.3%/59 (38 Paratyphi B, 5 Paratyphi A and 16 Typhi) among non-Qatari. On the other hand non-typhoidal Salmonella isolates include 64.6% n= 159 (64 Qatari and 95 non-Qatari) including Salmonella group B (n=61), Salmonella group C1 (n=5), Salmonella group C2 (n=9), Salmonella group D (n=29), Salmonella group E (n=7), Salmonella Choleraesuis (n=8), Salmonella Enteritidis (n=13), Salmonella Typhimorium (n=12) and Salmonella group O (n=13).

  

    
Author, year, country of    study and PEDro scores 
    
Mean age of participants    (y); sample size 
    
Study groups and dosage    of intervention and control 
    
Outcome measure(s) 
    
Results: Time points of    assessment; Mean (SD) (or) n/N 
  
  

    
Liebergall- Wischnitzer    et al. [28], 2005 

      Israel 

      PEDro: 5/10 
    
Age range: 20-65*

      (mean age not reported) 
      
Exp: n = 30 

        Con: n = 29 
    
Exp: PE 

      Con: PFMT 
      
PE: 12 weeks (45 min    session/ week). 
      
PFMT: 4 weeks (30-min    sessions/ week). 
      
PE: Contraction and    relaxation of pubococcygeal muscles, anals sphincter, and eyelids. Open and    close mouth and press each finger of hands to thumbs. 
      
PFMT: NR 
    
→Pad test: Urinary leakage in    grams
    
12 weeks 

      Exp: 5.4 (8.8) 

      Con: 9.5 (16.0) 
  
  

    
Liebergall-Wischnitzer et    al. [10], 2009 

      Israel 

      PEDro: 5/10 
    
Exp: 47.3 (8.4) 

      Con: 47.9 (9.3) 
      
EA: n = 91 

        SA: n = 86 
    
Exp: PE 

      Con: PFMT 
      
PE: 12 weeks (45 minutes    session/ week). 
      
PFMT: 4 weeks (30 minutes    session/ week). After 4 weeks, 2 more sessions 3 weeks apart. 
      
PE: Contraction and    relaxation of pubococcygeal muscles, anals sphincter, and eyelids. Open and    close mouth and press each finger of hands to thumbs. 
      
PFMT: Raising the vagina    and anus. 
    
→Pad test: Urinary leakage    (grams)

      →Self-reported leakage amount
    
Leakage (grams); 12 weeks 

      Exp: 3.0 (7.2) 

      Con: 2.8 (4.3) 
      
Leakage amount; 12 weeks 

        Exp: 74/91 

        Con: 39/86 
  
  

    
Liebergall-Wischnitzer et    al. [36], 2012 

      Israel 

      PEDro: 5/10 
    
Exp: 46.7 (8.0) 

      Con: 46.6 (8.9) 
      
Exp: n = 65 

        Con: n = 59 
    
Exp: PE 

      Con: PFMT 
      
PE: 12 weeks (45 minutes    session/ week). 
      
PFMT: Once a week for 4    weeks. After 4 weeks, 2 more sessions 3 weeks apart for a total of 6 sessions    over 12 weeks 
      
PE: Contraction and    relaxation of the eyelids; raising the upper lip to the nose as the nose is    lowered to the upper lip; contraction and relaxation of the levator any    muscles alone or in conjunction with a “sh” sound. 
      
PFMT: Raising the vagina    from the chair (levator any) in sitting. 10s rest between Contractions.    Each contraction performed 1–2 minutes apart. 
    
→Urinary leakage in grams

      →Self-reported leakage amount
    
Leakage (grams); 12 weeks 

      Exp: 2.9 (7.1) 

      Con: 2.4 (4.4) 
      
Leakage amount; 12 weeks 

        Exp: 31/65 

        Con: 28/59 
  
  

    
Liebergall-Wischnitzer et    al. [39], 2013 Israel 

      PEDro: 4/10 
    
Exp: 48.5 (7.6) 

      Con: 47.8 (8.6) 
      
Exp: n = 64 

        Con: n = 78 
    
Exp: PE 

      Con : PFMT 
      
PE: 12 weeks (45 min    session/ week). 
      
PFMT: 4 weeks (30 min    session/ week). After 4 weeks, 2 more sessions 3 weeks apart for a total of 6    sessions over 12 weeks. 
      
PE: Opening and closing    the mouth (and feeling the contraction in the face, PFMs and anus) and palms    (and feeling the contraction in the shoulders and PFMs). 
      
PFMT: Raising the vagina    from the chair (levator any) in sitting. 10s rest between contractions. Each    contraction performed 1–2 minutes apart. 
    
→Self-reported leakage amount
    
12 weeks 

      Exp: 49/64 

      Con: 55/79 
  
  

    
Huang et al. [38], 2014 

      USA 

      PEDro: 5/10 
    
Exp: 60.5 (8.4) 

      Con: 62.4 (8.3) 
      
Exp: n = 9 

        Con: n = 9 
    
Exp: Yoga (group-based) 

      Con: Wait-list 
      
Yoga: 6 weeks    (supervised, 90 minutes/ session, twice weekly) 
      
Con: Yoga classes at a    local yoga studio. 
      
Yoga: Hatha yoga program    consisting of 8 yoga postures. 
      
Con: $180 gift    certificate for yoga classes at a local yoga 

        Studio. 
    
→Number of incontinence episodes
    
6 weeks 

      Exp: 0.71 (0.8) 

      Con: 0.21 (1.1) 
  
  

    
Huang et al. [34], 2019 

      USA 

      PEDro: 8/10 
    
Exp: 65.5 (9.1) 

      Con: 66.6 (6.8) 
      
Exp: n = 28 

        Con: n = 28 
    
Exp: Yoga (group-based) 

      Con: Attention 
      
12 weeks (supervised, 90    minutes/ session, twice weekly) 
      
Yoga: Hatha yoga program    consisting of 15 yoga postures. 
      
Con: Non-specific    stretching and strengthening exercises. 
    
→Number of incontinence episodes
    
12 weeks (change scores) 

      Exp: 0.8 

      Con: 0.4 
  
  

    
Note: Con: Control group;    Exp: Experimental group; NR: Not Reported; PE: Paula Exercise; PFM: Pelvic    Floor Muscle; PFMT: Pelvic Floor Muscle Training. *Age reported as    mean and SD unless specified. 
  

Table 1: Demographic profile of the study population (n = 246) with Salmonella
among pediatric (2 to 18 years old) in the State of Qatar.

  

    
SUI: Paula exercise compared to PFMT
  
  

    
Outcomes
    
Illustrative comparative risks* (95% CI)
    
Relative effect

      (95% CI)
    
No of Participants (studies)
    
Quality of the evidence

      (Grade)
  
  

    
Assumed risk
    
Corresponding risk
  
  

    
Amount of urine leak in grams
    

    
The mean pad test leaks in the intervention    groups was 0.47 higher (1.79 lower to 0.84 higher)
    

    
360 (3 studies) [10,11,28]
    
⊕⊝⊝⊝ very lowa,b
  
  

    
SUI: Paula exercise compared to PFMT
  
  

    
Outcomes
    
Illustrative comparative risks* (95% CI)
    
Relative effect

      (95% CI)
    
No of Participants

      (studies)
    
Quality of the evidence

      (Grade)
  
  

    
Assumed risk
    
Corresponding risk
  
  

    
Number of women reporting small leaks
    
Study population
    
RR 1.26

      (0.88 to 1.80)
    
443

      (3 studies) [10,11,29]
    
⊕⊝⊝⊝ 

      very lowb,c 
  
  

    
545 per 1000
    
692 per 1000

      (485 to 986)
  
  

    
Moderate
  
  

    
475 per 1000
    
603 per 1000

      (423 to 860)
  
  

    
SUI: Yoga compared to control
  
  

    
Outcomes
    
Illustrative comparative risks* (95% CI)
    
Relative effect

      (95% CI)
    
No of Participants

      (studies)
    
Quality of the evidence

      (Grade)
  
  

    
Assumed risk
    
Corresponding risk
  
  

    

    
Control
    
Intervention
    

    

    

  
  

    
Number of incontinence episodes
    

    
The mean frequency of incontinence episodes in    the intervention groups was 0.83 lower

      (1.64 to 0.02 lower)
    
 
    
74

      (2 studies) [34,38]
    
⊕⊕⊝⊝ 

      lowd
  
  

    
Note: CI:    Confidence interval; PFMT: Pelvic Floor Muscle Training; RR: Risk ratio; *The    basis for the assumed risk (e.g. the median control group risk across    studies) is provided in footnotes. The corresponding risk (and its 95%    confidence interval) is based on the assumed risk in the comparison group and    the relative effect of the intervention (and its 95% CI).

      Grade Working Group grades of evidence.

      High    quality: Further research is very unlikely to change our    confidence in the estimate of effect.

      Moderate    quality: Further research is likely to have an important impact on    our confidence in the estimate of effect and may change the estimate.

      Low    quality: Further research is very likely to have an important    impact on our confidence in the estimate of effect and is likely to change    the estimate.

      Very    low quality: We are very uncertain about the estimate.

      aLack    of concealed allocation in two studies (Libergall-Wischnitzer 2012 and    Libergall-Wischnitzer 2005), lack of assessor blinding in one study (Libergall-Wischnitzer    2005), >15% dropout in two studies (Libergall-Wischnitzer 2012 and    Libergall-Wischnitzer 2009), and lack of intention-to-treat analysis in three    studies (Libergall-Wischnitzer 2012, Libergall-Wischnitzer 2009, and    Libergall-Wischnitzer 2005).

      bWide    CI.

      cLack    of concealed allocation in two studies (Libergall-Wischnitzer 2012 and    Libergall-Wischnitzer 2013), lack of assessor blinding in one study (Libergall-Wischnitzer    2013) and >15% dropout, lack of intention-to-treat analysis in three    studies (Libergall-Wischnitzer 2012, Libergall-Wischnitzer 2013, and    Libergall-Wischnitzer 2009).

      dAllocation concealment, assessor blinding and    intention-to-treat analysis not done in one study (Huang 2014). 
  

Table 2: Phenotypic resistant profiles of Salmonella isolates from hospitalized
pediatric in Qatar (n=246).

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Citation: Al Hadidi S, Abdelrahman H, Al Thani A, Ibrahim E, Yassine HM, Doiphode S, et al. Salmonella Infections among Pediatric Population in Qatar: Phenotypic Resistance and Associated Genotypic Determinants. J Bacteriol Mycol. 2021; 8(3): 1170.

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