Study of Antimicrobial Susceptibility And Biolm Formation of Cronobacter Sakazakii Isolates From Neonatal Sepsis In Southwest Iran

Background: The Cronobacter genus is a member family of the Enterobacteriaceae The isolates of C. sakazakii have been suggested to be responsible for fatal neonatal infections, which gives rise to sepsis, necrotizing enterocolitis, and meningitis, with a high mortality rates. The aim of the present study was to investigate the antimicrobial susceptibility and biolm formation of C. sakazakii isolates from neonatal sepsis in Southwestern Iran. Results: During the period of study, 734/1045 bacterial positive growth samples were collected from patients. Overall, from 734 bacterial positive growth samples 120 isolates were C. sakazakii based on culture, biochemical tests and PCR amplication. seventy-four (61%) neonates had primary sepsis and (33%) had late sepsis. Regarding birth bodyweight, (22%) neonate weighted below 1000 gr, 61 (50%) between 1500 and 2500, and (26%) more than 2500 gr. In case of C. sakazakii isolates, the highest resistance rates belonged to Ampicillin (70%), followed Amoxicillin (%59) and Ampicillin/sulbactam (83%). However, C. sakazakii had low levels resistance to cefepime and tetracycline. In total, of the 120 isolated bacteria, (70%) were biolm producers, of which, (37%) produced strong biolms, (15%) produced moderate biolms, (17%) were weak biolm producers and (28%) were not biolm producers. Conclusion: Taken together, the high rate of C. sakazakii in neonates was high in the NICU. Age, higher birthweight, and caesarian delivery were the most remarkable risk factors for C. sakazakii. The majority of C. sakazakii strains were hospital-associated, which is the indication of NICU admission patterns. Our ndings suggest that the active surveillance of neonates for C. sakazakii is required to be considered as a part of strategies to detect importation and prevent transmission of C. sakazakii within NICU. and the remaining CV was solubilized by incubating with 200 μL of 95% ethanol for 10 minutes. The optical density at 570 nm (OD570) of well was measured by the ELISA plate reader (μQuant; BioTek Instruments, Winooski, VT, USA), to evaluate the biolm formation capacity. S. epidermidis ATCC 35984 and TSB broth were used as positive and negative controls (ODc) for the biolm formation, respectively. The results were interpreted according to the criteria suggested by Zhang et al. capacity: an between birthweight quartiles and the increased risk of C. sakazakii colonization, contradicts previous results. Our study also identied caesarian section delivery as a risk factor for C. sakazakii. Multidrug-resistant and extensive drug-resistant C. sakazakii, as central nosocomial infections, were critical problems in NICU. In our country, Iran, antimicrobial agents are extensively been misapplied, and this misapplication has complicated the treatment of infections associated with MDR and XDR C. sakazakii. In our study, 70% of C. sakazakii were resistant to one or more antimicrobial agents and 53% and 44% were MDR and XDR, respectively. A high level of antimicrobial resistance among C. sakazakii isolates has been reported in previous studies from Iran (22, 23). The high-level β-lactam combination agents and penicillin resistance among C. sakazakii isolates have caused serious problems in healthcare settings worldwide (24). Similarly, ampicillin/sulbactam-resistant enterococci (83%) was detected with higher frequency than ampicillin resistance (70%) in our study. A higher frequency of aminoglycoside resistance was also identied in C. sakazakii isolates, an observation that supports previous reports (25). Moreover, our results are consistent with previous reports on the predominance of C. sakazakii with two or more antibiotic-resistant categories (26). chi-Square signicant MDR biolm = 6.708, df = 3, P = 0.082). Cronobacter varied abilities form biolms. Earlier studies have emphasized that C. sakazakii can attach to enteral feeding tubes within only 2 h of exposure. C. sakazakii also able bind to different surfaces viz latex, polycarbonate, and silicon. In biolms, C. sakazakii are protected by secreted extracellular polymeric substances that form a protective shield from desiccation tolerance and abiotic stresses. Another diculty related to the Cronobacter ability to form biolms is that it renders these microorganism more resistant to disinfection. The ability of Cronobacter species to attach to infant feeding equipment may make these surfaces reservoirs and sources of infection for the neonatal (30). It has been reported that there is a strong correlation between the presence of antibiotic resistance and the ability of an C. sakazakii isolate, in order to colonizes and persists in naonatal and produces biolm in vitro. According to our results, 73% of enterococci indicated biolm formation phenotype, which signies the vital role of biolm formation in NICU. Cronobacter infections reported previously mainly includ infants, particularly premature neonates, with clinical manifestation of sepsis. Taken together, the high rate of C. sakazakii in neonates was high in the NICU. Age (7–28 days), higher birthweight, and caesarian delivery the most remarkable risk factors for C. sakazakii. The majority of C. sakazakii strains were hospital-associated, which is the indication of NICU admission patterns. Our ndings suggest that the active surveillance of neonates for C. sakazakii is required to be as part of strategies detect C.


Background
The Cronobacter genus is a member family of the Enterobacteriaceae and includes seven species, namely C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, C. universalis, and C. condiment. The isolates of C. sakazakii have been suggested to be responsible for fatal neonatal infections, which gives rise to sepsis, necrotizing enterocolitis, and meningitis, with a high mortality rate. (1) In spite of highlighting neonatal infections caused by Cronobacter species, studies have indicated that these bacteria can induce illness in both infants and adults, particularly in newborns, the elderly, and those with weakened immune systems. (2) The outbreaks of Cronobacter infections have recently been reported in many countries. (3)(4)(5)(6) Due to the inappropriate and excessive use of antimicrobial agents, the emergence and spread of multidrug-resistant and extensive-drug resistant strains have globally become a matter of increasing concern for public health. (7) Current surveys have speculated that Cronobacter species is less resistance to widely used antibiotics in comparison to other newborn pathogens, though multiple investigations have found drug-resistant strains of Cronobacter species (8-10) Therefore, it is essential to explore the antibiotic resistance of Cronobacter species, particularly those that are recovered from different clinical samples, to categorize the patterns of resistance and to formulate an e cient strategy for inhibiting the potential spread of these strains. (11) In recent years, a serious threat to newborn safety is the attachment and bio lm formation of newborn pathogens because the high likelihood of potential cross-contamination may result in serious problems for newborn safety. It has lately been found that the strains of Cronobacter species are capable of bio lm formation on numerous kinds of materials, e.g. stainless steel, polyvinyl chloride, silicone, and polycarbonate. The elimination of the established bio lms are highly di cult in virtue of the tolerance to sanitizing agents, posing a potential health risk to human health because within bio lms, there are microorganisms that my cause a persistent release of bacteria to newborn (12). The aim of the present study was to investigate the antimicrobial susceptibility and bio lm formation of C. sakazakii isolates from neonatal sepsis in Southwestern Iran.

Ethic statement
The present cross sectional study was conducted after obtaining approval from the Ethics Committee (IR.AJUMS.REC.1400.108) of the Ahvaz Jundishapur University of Medical Sciences and their submission to a liated hospitals.

Study Design and Population
This study design was carried out cross-sectional study from March 2021 to March 2019. The study was conducted on all blood sample of neonatal sepsis sent to the microbiology lab of Imam Khomeini Hospital, Ahvaz, Iran. The hospital serves as a referral center for the public hospital in Ahvaz city. The study included 1045 preterm neonates of 60/min, signs of respiratory distress (marked chest in drawing), grunting, cyanosis, fever or hypothermia, and elevated Creactive protein (CRP). For these neonates, blood cultures were collected and analyzed. Neonates were excluded from the analysis if the diagnosis of sepsis was presumptive and blood cultures were negative.

Data and Sample Collection
A total number of 1045 blood cultures were collected from cases suspected to neonatal sepsis admitted to the neonatal ICU at Imam Khomeini Hospital. The number of samples was limited by the consent rate. Neonates were subjected to complete clinical examination (temperature, respiration, color, presence of lethargy, or any neurological troubles and change in feeding pattern). Blood cultures were collected to be tested for the presence of bacterial infections, including C. sakazakii. Samples had been collected for general screening purposes. Approximately 1-4 ml of blood was aseptically collected from the peripheral vein and inoculated directly into BacT/ALERT blood culture bottles (bioMérieux, Marcy l'Etoile, France), which was monitored using the BacT/ALERT 3D instrument (bioMérieux, Marcy l'Etoile, France). The bottles were incubated at 37C • for 7 days, and positive specimens were inoculated onto Brilliance C. sakazakii chromogenic agar (DFI, Oxoid, United Kingdom), MacConkey agar, and blood agar and incubated for 24 h at 36C • . Also, a sample was streaked onto trypticase soya agar, which was incubated at 25C • . (14) Suspected colonies were further con rmed using C. sakazakii speci c primer con rmation.

DNA extraction
The boiling colonies of C. sakazakii strains grown overnight on nutrient agar (Merck, Germany) were suspended in microtubes containing 500 μL of Tris-HCl-EDTA buffer, then the microtubes were placed in thermoblock (Denville Scienti c, Metuchen, NJ, USA) for 5 min at 95°C, and centrifuged at 14 000 rpm for 10 min at 4°C. The supernatant was used as the DNA template in the PCR assays. UV absorbance ratios, A 280 /A 260 were used to evaluate DNA extract purity using a Nanodrop instrument (Thermo Scienti c, Waltham, MA, USA). method was used to extract genomic DNA from C. sakazakii isolates.
Con rmation of C. sakazakii -speci c PCR ampli cation primers For de nitive identi cation, all 43 suspected C. sakazakii isolates were exposed to C. sakazakii -speci c PCR ampli cation primers. The C. sakazakii (ATCC 51329) strain was used as a positive control and Staphylococcus aureus ATCC 25923 strain was the negative control.

Minimum inhibitory concentration (MIC)
Colistin MICs were measured by E-test strips (Lio lchem, Italy) and interpreted based on (CLSI 2020) guidelines. After measuring the MIC with E-test method, the P. aeruginosa isolates with a MIC Equal to and lower than 2 μg/ml were considered as "intermediate" and MIC Equal to and higher than 4 μg/ml were considered as "resistant". Escherichia coli ATCC 25,922 was used as quality control for antimicrobial susceptibility testing (15).
Bio lm formation in 96-well microtiter plate (MTT) the bio lm formation capacity of isolates was evaluated using the crystal violet staining method. First, these isolates were inoculated in Mueller-Hinton agar at 37°C overnight. Then, these isolates were adjusted to 0.5 McFarland (~1.5×10 8 CFU/ mL) with normal saline (0.85% NaCl). A 10-μL aliquot of each suspension was then diluted 1:200 in 190 μL of tryptic soy broth (TSB) containing 1% glucose in 96-well polystyrene microtiter plates. Following incubation at 37°C overnight, the plates were washed three times with PBS, xed by adding 200 μL of methanol into each well, and stained with 200 μL of 0.1% crystal violet (CV) for 20 minutes. The plates were again washed three times to remove excess stain, and the remaining CV was solubilized by incubating with 200 μL of 95% ethanol for 10 minutes. The optical density at 570 nm (OD570) of each well was measured by the ELISA plate reader (μQuant; BioTek Instruments, Winooski, VT, USA), to evaluate the bio lm formation capacity. S. epidermidis ATCC 35984 and TSB broth were used as positive and negative controls (ODc) for the bio lm formation, respectively. The results were interpreted according to the criteria suggested by Zhang et al. Briefly, the isolates were classified into the several groups about the biofilm formation capacity: OD570≤ODc=no biofilm producer; ODc<OD570≤2×ODc=weak biofilm producer; 2×ODc<OD570≤4×ODc=moderate biofilm producer; and 4×ODc<OD570=strong biofilm producer. All experiments were performed in triplicate (17).

Statistical Analysis
Statistical analyses were performed using the Statistical Package for Social Sciences, version 22 (SPSS Inc., Chicago, IL, United States). Data are represented as the mean ± SD for continuous variables and as percentages for categorical variables.

Antimicrobials resistant rates of C. sakazakii isolates from neonatal sepsis
The resistance rates of C. sakazakii isolates to antimicrobial agents are illustrated in Table 3. In case of C. sakazakii isolates, the highest resistance rates belonged to Ampicillin (n=85/120; 70%), followed Amoxicillin (n= 71/120; %59) and Ampicillin/sulbactam (n=100/120; 83%). However, C. sakazakii had low levels resistance to cefepime and tetracycline. These isolates were then evaluating for colistin resistance of the MICs by using the E-test. Only (n= 20/120; %16) isolates were con rmed as colistin-resistant.  The prevalence rate of MDR and XDR C. sakazakii isolates from neonatal sepsis was identi ed by reference and shown in the table 4. Out of total 120 C. sakazakii isolated, (n=65; 53%) bacterial strains were MDR, and (n=53 ;44%) strains were XDR. No PDR isolated was detected in this study.

Discussion
C. sakazakii is recognized as an opportunistic pathogen important for neonatal health worldwide. Neonatal sepsis, a major causes of neonatal mortality, is described as blood infections and imposes a signi cant health burden in infants, i.e. very low birth-weight and preterm infants (18). For the rst time in the present study, C. sakazakii infection is reported in cases of neonatal sepsis in Ahvaz, Southwestern Iran.
Among our 734 blood cultures, 120 cases (16%) were positive for C. sakazakii, representing a higher rate than that reported in the United States, which was virtually 1 in 100,000 infants and increased to about 1 in 11,000 infants of less than 1,500 g birth weight (19). Our study was further investigated by Alsonosi and associates who genotyped 51 Cronobacter strains from clinical isolates collected during six years. Their result indicated that C. sakazakii (65%) is the major detected species (20). Evidence has also been demonstrated that the susceptibility of preterm infants to infections are greater than any other age. A reason for such observation can be the trans-placental passage of antibodies peaks during the third trimester. Thus, it can be deduced that the majority of preterm infants have considerably declined humoral immune responses (21).
The higher rate of colonization in our study likely display the patterns of admission into the NICU because almost all neonates were admitted within 24 hours of birth. A case of C. sakazakii was regarded as hospital-acquired if it was isolated from an outpatient or an inpatient within 48 h of hospitalization; therefore, it appears that all the C. sakazakii isolates have been transferred from the hospital. However, some studies have examined prematurity and intubation as factors related to S. aureus colonization in the NICU population, but few have assessed the effect of age on colonization risk. We observed that neonates aged 7-14 and 15-28 days remarkedly increased risk for C. sakazakii colonization compared to younger neonates, though we did not nd any study in this matter for comparison. In this study, we discovered an association between higher birthweight quartiles and the increased risk of C. sakazakii colonization, which contradicts previous results. Our study also identi ed caesarian section delivery as a risk factor for C. sakazakii. Multidrug-resistant and extensive drugresistant C. sakazakii, as central nosocomial infections, were critical problems in NICU. In our country, Iran, antimicrobial agents are extensively been misapplied, and this misapplication has complicated the treatment of infections associated with MDR and XDR C. sakazakii. In our study, 70% of C. sakazakii were resistant to one or more antimicrobial agents and 53% and 44% were MDR and XDR, respectively. A high level of antimicrobial resistance among C. sakazakii isolates has been reported in previous studies from Iran (22,23). The high-level β-lactam combination agents and penicillin resistance among C. sakazakii isolates have caused serious problems in healthcare settings worldwide (24). Similarly, ampicillin/sulbactam-resistant enterococci (83%) was detected with higher frequency than ampicillin resistance (70%) in our study. A higher frequency of aminoglycoside resistance was also identi ed in C. sakazakii isolates, an observation that supports previous reports (25). Moreover, our results are consistent with previous reports on the predominance of C. sakazakii with two or more antibiotic-resistant categories (26).
In the current study, 14% of C. sakazakii isolates were high-level colistin-resistant, which is in good agreement with some former surveys (27,28). Microbial cells within bio lms have been exhibited to be 10-1000 times more resistant to antibiotics than the planktonic cells (29). Based on the results obtained in this study, 66% of C. sakazakii strains showed bio lm formation with variable degrees, and islets containing bio lm had higher antibiotic resistance (Fig. 1). In general, the results of the Pearson and chi-Square test showed no signi cant difference between MDR and bio lm formation variables (χ2 = 6.708, df = 3, P = 0.082). Cronobacter species have varied abilities to form bio lms. Earlier studies have emphasized that C. sakazakii can attach to enteral feeding tubes within only 2 h of exposure. C. sakazakii also was able to bind to different surfaces viz latex, polycarbonate, and silicon. In bio lms, C. sakazakii are protected by secreted extracellular polymeric substances that form a protective shield from desiccation tolerance and abiotic stresses. Another di culty related to the Cronobacter ability to form bio lms is that it renders these microorganism more resistant to disinfection. The ability of Cronobacter species to attach to infant feeding equipment may make these surfaces reservoirs and sources of infection for the neonatal (30). It has been reported that there is a strong correlation between the presence of antibiotic resistance and the ability of an C. sakazakii isolate, in order to colonizes and persists in naonatal and produces bio lm in vitro. According to our results, 73% of enterococci indicated bio lm formation phenotype, which signi es the vital role of bio lm formation in NICU.
Cronobacter infections reported previously mainly includ infants, particularly premature neonates, with clinical manifestation of sepsis.
Taken together, the high rate of C. sakazakii in neonates was high in the NICU. Age (7-28 days), higher birthweight, and caesarian delivery were the most remarkable risk factors for C. sakazakii. The majority of C. sakazakii strains were hospital-associated, which is the indication of NICU admission patterns. Our ndings suggest that the active surveillance of neonates for C. sakazakii is required to be considered as a part of strategies to detect importation and prevent transmission of C. sakazakii within the NICU.
30-Ling N, Forsythe S, Wu Q, Ding Y, Zhang J, Zeng H. Insights into Cronobacter sakazakii bio lm formation and control strategies in the food industry.