Antimicrobial Resistance Patterns of bacterial Septicaemia infecting infants in Mbita Subcounty, Western region of Kenya

Background: Gram positive bacteria such Escherichia coli, Group B Streptococcus coagulase-negative staphylococci, Staphylococcus aureus , and Gram-negative bacteria such as Klebsiella and Pseudomonas species are listed as some of the bacteria etiologies for pediatric septicemia. These bacteria are rapidly becoming multi drug resistant to penicillin (or aminopenicillin), gentamicin, the pragmatic antibiotic treatment regimens. Further, the ever-increasing burden of bacteria septicemia infection due to extended-spectrum β-lactamase (ESBL) producing Gram negative bacteria cumulatively presents a major health concern in the management and treatment of bacterial septicemia. In this study we present data on the prevalence and type of antimicrobial resistant patterns among children with bacterial septicemia in Mbita Sub county Hospital, Western region of Kenya. Methods: Blood samples were obtained from 248 children whose parents/guardian consented. The bacterial isolation and characterization were done using the automated BACTEC 9240 system, conventional culture using morphology, Gram stain and biochemical identification. Further identification and resistant gene detection were determined using Polymerase Chain Reaction (PCR). Descriptive statistics were used to present data. Results: Eighty-four (33.9%) patients had septicemia where Staphylococcus epidermidis (28.6%), S. aureus (13.1%), Escherichia coli (13.1%) and single Salmonella Paratyphi B, Citrobacter freundii , Gemella morbillorum , Klebsiella pneumoniae , Lactococcus lactis cremoris , Pantoea spp, and Pseudomonas putida were implicated. The majority of gram-negative bacteria were resistant to penicillin (Ampicillins) 100%, 96.1% to tetracyclin, 84.6% to sulphonamides (Trimethoprim/sulfamethoxazole), 73.1% Aminoglycosides (Gentamicin) 73.1% and 19.2% to Quinolone (Ciprofloxacin). For gram positive bacteria majority 96.7% were resistant to sulphonamides (Trimethoprim/sulfamethoxazole) followed by tetracycline 76.7%, penicillin (Oxacilline) 73.3% and least resistant to Quinolone (Ciprofloxacin) 30%. Various antimicrobial resistant genes mecA, SulII, blaTEM, TetA aac (3) were identified. Conclusion. In this geographically defined region of Kenya, of the 33.9% children with septicemia, gram positive bacteria were the leading cause septicemia. High level resistance due to various resistant genes were seen all type of antibiotics by both Gram positive and negative bacteria. Rapid antibiotic resistant testing is encouraged for appropriate treatment and management of septicemia infection.


Characteristics of study patients
All the data was available for 248 out of recruited 281 children. The mean (± standard deviation -SD) age of the participants was 27.93 (±20.6) months with 30.6% of them aged between 1 to 12 months. The majority of the patients 50.8% were males, 48% from Rusinga locality and 91.9% HIV negative. The mean body temperature for the patients 38 O C (± 20.5) ranging between 37 to 40 O C. There were 58.9% patients with body temperatures above 37.6 O C. The mean WBC of the patients was 17720.9 Cells/ml (± 8929.1) Cells/ml ranging between 12075 to 22450 Cells/ml. about 25.4% of them had WBC above the normal levels of 10501 cells/ml. The mean respiratory rate (RR) of the patients was 30.6 (± 10.6) breaths/min ranging between 18 to 96 breaths/min with 71.4% having RR between 20 and 30 breaths/min. Co-infection/complications among the study patients included: malaria reported in 83 (33.5%) of the patients followed by respiratory illnesses 33 (13.3%), Hematologic diseases 31(12.5%), Gastrointestinal disorders 27(10.9%), malnutrition and meningitis in 9 (3.6%) each. There were 6 (2.4%) patients who had Nervous system diseases, 5(2.1%) with Ear nose and throat infections and 2 (0.8%) with HIV. There were 20 (8.1%) patients who reported no other co-infection.   Table 1 summarizes the drug susceptibility patterns for bacteria causing septicemia among study participants. Susceptibility testing showed that majority of gram-negative isolates were resistant to penicillin (Ampicillins) 100% followed by tetracycline 96.1%, sulphonamides (Trimethoprim/sulfamethoxazole) 84.6%, Aminoglycosides (Gentamicin) 73.1% while they were least resistant to Quinolone (Ciprofloxacin) 19.2%. For gram positive bacteria majority 96.7% were resistant to sulphonamides (Trimethoprim/sulfamethoxazole) followed by tetracycline 76.7%, penicillin (Oxacilline) 73.3% and least resistant to Quinolone (Ciprofloxacin) 30%.

Drug resistant genes among the multi drugs resistant bacterial etiological agents
The number of multi-drug resistant bacterial isolates included 4 Staphylococcus epidermidis, 4 S. aureus, 7 E. coli (4 Enterotoxigenic and 3 Enteropathogenic E. coli), 4 S. paratyphi (3 S. paratyphi A and 1 S. paratyphi B), 3 S. typhimurium and 3 P. aeruginosa S. aureus and S. epidermidis: Plasmids isolated from both S. epidermidis and S. aureus species harbored resistance genes, mecA and SulII, highlighting their role in dissemination of antibiotic resistance. High frequency of resistance was observed for Sulfamethoxazole-Trimethoprim (87.5%), Oxacilin (75.0%), Erythromycin (62.5%) and Amoxyclavulanic acid (50.0%). All the isolates were multiply resistant to between two to six antibiotics. One of the S. epidermidis did not carry any plasmid but showed phenotypic resistance to Oxacilin, Amoxyclavulanic acid, Sulfamethoxazole-Trimethoprim and Tetracycline (Table 2).

E. coli:
Plasmids isolated from the E. coli isolates studied harbored resistance genes, blaTEM, SulII, and TetA. High frequency of resistance was observed for Sulfamethoxazole-Trimethoprim (100%), Ampicillin (100%), Tetracycline (100%) and Gentamycin (85.7%). All the isolates were multiply resistant to between four and eight antibiotics. However, there was no apparent relationship between carriage of plasmids and antimicrobial resistance ( Table 2).
Salmonella spp: Plasmids isolated from the Salmonella isolates studied harbored resistance genes, blaTEM, SulII, aac (3) and TetA highlighting their role in transmission of resistance. High frequency of resistance was observed for Sulfamethoxazole-Trimethoprim (100%), Ampicillin (100%), Tetracycline (100%), Cefixime (100%), Gentamycin (100%) and Ceftriaxone (57.1%). All the isolates were multiply resistant to between to four to seven antibiotics. One of the Salmonella did not carry any plasmid but showed phenotypic resistance to Cefixime, Ampicillin, Sulfamethoxazole-Trimethoprim and Tetracycline. However, there was no apparent relationship between carriage of plasmids and antimicrobial resistance.

Discussions
Early diagnosis and early appropriate treatment are crucial in cases of bacterial blood infection. In severe sepsis, the case fatality increases for each hour the antibiotic treatment is delayed (Ferrer et al., 2014). Therefore, empirical antibiotic treatment has to be initiated before the results of blood cultures are available. However, as infections with resistant microbes is an escalating problem worldwide, it is increasingly challenging to maintain appropriate antibiotic regimens for initial empiric therapy (Nathan and Cars, 2014; WHO, 2014). Resistant pathogenic bacteria are found frequently worldwide (WHO, 2014). Studies have shown that most developing countries are home to a number of risk factors for the emergence and spread of antibiotic resistance. Misuse of antibiotics, over-the-counter and parallel market access, and counterfeit or poor-quality drugs, combined with substandard hygiene and living conditions, are the driving forces behind the emergence and spread of antibiotic resistance (Kelesidis et al., 2007). The potential for the development and rapid spread of new forms of resistance is highlighted by the recent worldwide proliferation of NDM-1-producing Enterobacteriaceae. The gene, which confers resistance to carbapenems, originated in India in 2009, and since 2010 NDM-1-producing Enterobacteriaceae have been reported in North America, Europe, and Asia (Kumarasamy et al., 2010). The WHO has recently heightened awareness of this pressing issue with calls for action to contain antibiotic resistance on a global scale (WHO, 2014). In Kenya, a regimen containing penicillin and gentamicin, plus metronidazole if an anaerobic infection is suspected, has been recommended for more than thirty years in sepsis with unknown focus and etiology (Lee et al., 2014). In recent years, however, increasing numbers of infections with methicillin-resistant S. aureus (MRSA), extended-spectrum beta-lactamase producing Enterobacteriaceae, and vancomycin resistant enterococci have been detected (Lee et al., 2014). Selection of inherently resistant microbes due to antibiotic use is also a challenge. Updated knowledge about the distribution of microbes in serious infection and their resistance against antimicrobial agents is needed to ensure appropriate empiric antimicrobial treatment regimens. It is also important to identify subgroups in which tailored regimens are required. This was the basis important aspect of this study

S. aureus
The three out of four S. aureus isolates causing septicemia had the mecA antimicrobial gene while one carried SulII antimicrobial gene. One of the four isolates were multi-drug resistant to five different drugs (Oxacillin, Amoxicillin/Clavulanate, Trimethoprim/sulfamethoxazole, Tetracycline and Erythromycin). Two isolates were resistant to four different drugs and one isolate to two different drugs. Our study further shows that 75% of the S. aureus were Methicillin resistance. The S. aureus with varying antibiotic profiles have been associated with sepsis in other settings. In Kilifi District Hospital on the Kenyan coast Talbert et al., (2010) reported that all the Staphylococcus aureus blood culture isolates were susceptible to methicillin. The S. aureus causing neonatal sepsis in Tikur Anbessa University Hospital, Ethiopia; showed high-level resistance to ampicillin, cefiriaxone, cephalothin, chloramphenicol, and gentamicin (Shitaye et al., 2010

Approximate sizes of the plasmids
Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol. 10, No.10, 2020 Lithuania was resistant to Methicillin (Bobelytė, 2017). Our study and other showing significantly high prevalence of multidrug resistant S. aureus is worrying given bacteraemias due S. aureus are difficult to treat and is associated with 29-63% mortality (Kaasch et al., 2014;Fortuin-de Smidt et al., 2015). The emergence of new CA-MRSA strains in the community has huge implications on patient treatment (David et al., 2010).

S. epidermidis
There were 3 out of 4 (75%) S. epidermidis that had antimicrobial gene, mecA and only one isolate did not carry any plasmid. All four S. epidermidis were resistant to 3 to 6 different antibiotics. The strain that had no plasmid containing antimicrobial gene, mecA showed phenotypic resistance to Oxacilin, Amoxyclavulanic acid, Sulfamethoxazole-Trimethoprim and Tetracycline. Studies continue to report the importance of Coagulasenegative staphylococci as among the leading cause of nosocomial sepsis, especially in neonates (Marchant et al., 2013;Becker et al., 2014). Coagulase-negative staphylococci sepsis most often originates from the infection of indwelling medical devices, such as in catheter-related bloodstream infections or central line-associated blood stream infections (Vassallo et al., 2015). Most prominent among Coagulase-negative staphylococci infections are those due to the skin commensal S. epidermidis (Vassallo et al., 2015). However, the bacterial factors contributing to the development of sepsis, in particular in CNS, are poorly understood. Most S. epidermidis blood infections are caused by methicillin-resistant strains, with methicillin resistance rates even exceeding those found among S. aureus (Qin et al., 2017).
Methicillin-resistant S. epidermidis isolates from patients are cross-resistant to all b-lactam antibiotics, even though some might be susceptible to certain b-lactam agents by in vitro testing (Raad et al., 1998). Therefore, this pattern of resistance is similar to what has already been established with methicillin-resistant S. aureus. The prevalence of resistance has increased rapidly over the last three decades and has been attributed to the selection effect of the increasing use of b-lactam antibiotics. Studies have demonstrated an increase in the prevalence of resistant S. epidermidis in hospitals when isolates from 1964 were compared with isolates from 1986 or resistance and plasmid profiles (Raad et al., 1998). This resistance was plasmid-mediated. Some investigators attributed resistance in S. epidermidis to the action of the mecA gene (Raad et al., 1998). However, Mempel et al., (1994) demonstrated that S. epidermidis isolates could be methicillin resistant and lack mecA transcription.

E. coli
The seven pathogenic E. coli species harbored three types of antimicrobial gene, SulII, blaTEM and TetA. One pathogenic ETEC was resistant to 8 different antibiotics, 3 ETEC were multi-drug resistant to 4 different antibiotics. One EPEC was resistant to 5 different antibiotics while the remaining 2 EPEC were resistant to four different antibiotics. Similar studies exist showing high level antibiotic resistance to E. coli species isolated from neonatal septicemia cases. Studies showed resistance to penicillin/ampicillin ranging from 55% among E. coli isolates in Georgia (Macharashvili et al., 2009) to 100% among E. coli isolates in Uganda (Mugalu et al., 2006). Resistance to gentamicin among E. coli ranged from 0% in Pakistan (Mir et al., 2011), 21.7% in South Africa (Dramowski et al., 2015), 67 in India (Jyothi et al., 2013). In Lithuania, sepsis-causing pathogens E. coli was characterized by the development of increasing antibiotic resistance for which initial empirical antibiotic therapy might fail (Bobelytė et al., 2017). Resistant to third generation cephalosporins ranged from 6% for E. coli isolates in Uganda (Mugalu et al., 2006) to 48% in India (Jyothi et al., 2013). Concerning the extended spectrum betalactamase production in Enterobacteriaceae; one reported extended spectrum beta-lactamase production in 65% of E. coli isolates (Jain et al., 2006)

Salmonella spp
Six out of seven Salmonella species studied were found carrying at least three plasmids of varying sizes. Antimicrobial gene, SulII, was detected in the six Salmonella isolates while aac (3), TetA and blaTEM genes were detected in five Salmonella isolates. One of the Salmonella did not carry any plasmid but showed phenotypic resistance to Cefixime, Ampicillin, Sulfamethoxazole-Trimethoprim and Tetracycline. There was one Salmonella paratyphi B that was multi-resistant to five different antibiotics, three Salmonella paratyphi A were multi-resistant to six different antibiotics. Further, there were two and one Salmonella typhimurium that were multi-resistant to five and six different antibiotics respectively. Generally high frequency of resistance was observed for Sulfamethoxazole-Trimethoprim (100%), Ampicillin (100%), Tetracycline (100%), Cefixime (100%), Gentamycin (100%) and Ceftriaxone (57.1%). Studies confirms that the invasive forms of Salmonella disease include enteric fevers (typhoid and paratyphoid fevers) and NTS bacteraemia and are important causes of morbidity and mortality in Asia and Africa (Smith et al., 2014). In India a study among pediatrics sepsis patients attending Majeedia Hospital of Hamdard University in New Delhi showed various resistance profile in Salmonella typhi to various antimicrobials as follows ampicillin (46.4%), amoxicillin (27.3%), amoxiclav (15.4%), cefuroxime (10%), cefotaxime (25%), cefoperazone (10.5%), netilmicin (10.5%), ciprofloxacin (6.3%), chloramphenicol (9.1%) and tetracycline (37%). Salmonella typhi did not show resistance to gentamicin and amikacin (Alam et al., 2011). On the other hand, in the same study Salmonella paratyphi A did not show resistance to antimicrobials tested (Alam et al., 2011 In Zanzibar, the majority of the S. Typhi isolates 6/7 (86%) were multidrug-resistant (i.e. resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol), but susceptible to cefotaxime (Onken et al., 2015).

Pseudomonas spp
The antimicrobial gene, blaTEM, was detected in five of the Pseudomonas plasmids while four of the plasmids carried SulII, TetA and aac (3) antimicrobial resistance genes. High frequency of resistance was observed for Tetracycline (100%), Ampicillin (100%), Cefixime (80%), Cetriaxone (80%), Amoxyclavulanic acid (80.0%), Gentamicin (60.0%) and Sulfamethoxazole-Trimethoprim (60%), All the isolates were multiply resistant to between to two to six antibiotics. These results  (2011) reported no resistance to antibiotics including gentamicin among Gram-negative bacteria including Pseudomonas and E. coli. In Vietnam 56% of the sepsis causing Pseudomonas isolates were resistant to carbapenem resistant (Le et al., 2016). In Turkey, Pseudomonas spp which was the major causative pathogens of sepsis in children was 40.5% resistant to imipenem (Teke et al., 2016). Evaluation of the trends in antimicrobial resistance of bloodstream infections at a general hospital in Mid-Norway, showed, Pseudomonas spp were 100% resistant to cefotaxim and 6.9% to Imipenem (Mehl et al., 2017).
In conclusion, in this geographically defined region of Kenya, of the 33.9% children with septicemia, gram positive bacteria were the leading cause septicemia. Specifically, S. epidermidis and S. aureus. However, several other Gram-negative bacteria were implicated such as E. coli, P. aeruginosa, S. typhimurium and S. hemolyticus. It should be noted that gram-negative bacteria have often been implicated in the pathogenesis of severe sepsis and septic shock than the Gram-positive counter parts (Alexandraki and Palacio, 2010). Compared to Gram positive, majority of Gram-negative bacteria were resistant to penicillin (Ampicillins) 100% tetracycline 96.1%, sulphonamides (Trimethoprim/sulfamethoxazole) 84.6%, Aminoglycosides (Gentamicin) 73.1% and least resistant to Quinolone (Ciprofloxacin) 19.2%. The Gram-positive bacteria were resistant to sulphonamides (Trimethoprim/sulfamethoxazole) 96.7%, tetracycline 76.7%, penicillin (Oxacilline) 73.3% and Quinolone (Ciprofloxacin) 30%. The following antimicrobial resistant genes mecA, SulII, blaTEM, TetA aac (3) were identified However, we did not find apparent relationship between carriage of plasmids and antimicrobial resistance.