Molecular detection of extended spectrum β-lactamase in gram negative bacteria isolated from intensive care unit patients in Wasit province, Iraq

Intensive care units (ICU) are epicenters for the emergence of antibiotic-resistant Gram negative bacteria and multi-resistant Gram positive infections, largely due to the inappropriate use of antimicrobials. In this study, a total of 100 clinical specimens (urine, sputum and pus) were collected from patients admitted in the ICU. Results showed eighty two were positive growth culture. The findings of the disc diffusion method used to test the isolates for antibiotic susceptibility showed that Amikacin was the most effective antibiotic against klebsiella pneumonia and pseudomonas aeruginosa. Phenotypically survey of ESBLs for all isolates were ESBL producers, of these, 13(68.4%) isolates were determined as ESBLs-producers following confirmatory testing to be ESBLs producers. Whereas, genotypically no isolate had ESBLs producers CTX-M -type ESBL. The prevalence of TEM, SHV , and OXA -1 were as follow: bla SHV 3(23 %). While bla TEM and bla OXA -1genes were absent among all isolates. Phenotypically survey of ESBLs, for (20) isolates pseudomonas aeruginosa 20(100%) isolates were ESBLs producers of these, 12(60%) isolates were determined as ESBLs-producers following confirmatory testing to be ESBLs producers. While genotypically isolates were ESBL producers 1(8.3%) of the isolates were ESBLs producers CTX-M -type ESBL was the most prevalent CTX-M 2 was 1(8.3%) , CTX-M 1 and CTXM9 were absent among all isolates. The prevalence of TEM, SHV , and OXA -type1 were as follow: bla SHV 4(33%). While bla TEM and bla OXA -1 genes were absent among all isolates. Phenotypically survey of ESBLs, for (17) isolates E. coli 17(100%) isolates were ESBLs producers of these, 10(58.8%) isolates were determined as ESBLs-producers following confirmatory testing to be ESBLs producers. Whereas, Genotypically no isolate ESBLs producers CTX-M -type ESBL, prevalence of TEM, SHV, and OXA -1 were as follow: bla SHV 1(10%),blaOXA-1 1(10%) . While bla TEM gene was absent among all isolates.


Introduction
Intensive care units (ICUs) are important departmants at the hospital for nosocomial infections.Although an ICU has 5%-10% of the hospital beds, 25%-50% of the nosocomial infections originate from the ICU [1].Both ventilator-related pneumonia (VRP) and catheter-related urinary tract infections (CRUIs) are the most common infections in the ICU [2].Many risk factors are responsible for nosocomial infection in the ICU [3].Some of the risk factors are related to the patient, whereas the others are related to the external factors.It is known that improving the risk factors decreases the infection, mortality, morbidity, and cost [2,4].The environmental conditions affect the infection rate in the ICU [5].Hospital-acquired infections (HAI) have been recognized for over a century as a critical problem affecting the quality of healthcare, and they constitute a major source of adverse healthcare outcomes [6].The emergence of multidrugresistant bacteria (MDRB) has become a public health problem, creating a new burden on medical care.In addition to this, ICU patients have an increased risk of infection due to their underlying diseases or conditions, impaired immunity, and exposure to multiple invasive devices (mechanical ventilation, central venous catheters (CVC), and urinary tract catheters), the incidence of ICU-HAI is 5-10-times higher than HAI rates in general wards [7].Intensive care units (ICU) are epicenters for the emergence of antibiotic-resistant Gram negative bacteria and multi-resistant Gram positive infections, largely due to the inappropriate use of antimicrobials [8].The aim was presented to detect spread of bacterial isolates from patients in ICU in Kut City, Wasit Province, Iraq, and to characterize it at the level of molecular analyses of its Phylogenic and antimicrobial resistance genes.

Material and Methods
A cross-sectional study was done in the intensive care unit (ICU) of the Alzahraa, Alkarama hospitals from the 3rd October 2021 to 20th February 2022.A total of 100 clinical samples, including: urine, sputum and pus culture media such as mannitol salt agar, MacConkey agar, blood agar, and chocolate agar.The growth showed different bacterial colonies whose morphological and biochemical characteristics were tested.Then DNA was extracted; purity and concentration were confirmed with Nanodrop.The purity of gram negative bacteria (1.8-2), and the concentration was between 50-360 ng/µl.Table 1 Primers' sequence of gram-negative bacteria Production of ESBLs was initially phenotypically discovered using the disk diffusion method (screening test), and was then verified using the double disc synergy test (DDST) [9].Wheraes an isolate is considered tobe ESBL producer in the case when it shows resistance to one or more of the 3rd.Generation cephalosporins asfollows: cefotaxime (≤ 27mm); ceftazidime (≤ 22mm); ceftriaxone (≤ 25mm) and aztreonam (≤ 27mm).According to who adapted this protocol as a reliable and efficient tool for the identification via multiplex PCR assays of the most common encoding of the beta-lactamase genes for gram negative bacteria?Gel Electrophoresis and Documentation [10] and finally the data's statistical analysis has been carried out with the use of SAS (Statistical Analysis System -version 9.1) [11].

Results and discussion
The current study was conducted on a total of one hundred clinical specimens (urine, sputum and pus) were collected from patients admitted in the ICU.Bacterial isolates that were obtained from the clinical specimens have been initially characterized based on the cultural morphology as well as the biochemical tests.Results of culture showed colonies on MacConkey agar pink color with precipitation of bile salt around colonies, the refers to Escherichia coli isolates while results of biochemical tests for Escherichia coli were had given positive test for catalase, indole, methyl red, but negative for oxidase, voges-proskauer, simmon citrate, and TSI test showed A/A with gas , without H2S.This results diagnostic for Escherichia coli .Similar results was recorded by [12][13][14].
Results of identification of klebsiella pneumoniae were showed pink lactose fermenter mucoid colonies on MacConkey agar.Positive test for urease, voges-proskauer, simmon citrate , but negative for oxidase, methyl red, motility test, indole and TSI test showed A/A with gas , without H2S .Further confirmation done using Vitek2.Similar results were recorded by [15,16].
Results of Acinetobacter baumannii showed nonmotile and give negative result to oxidase, indole, methel red, voges proskauer and avirable to urea.All isolates were positive to catalase , simmons citrate and triple-sugur-iron test was alkaline / no change, these results are identical with those obtained by [17,18].The results of Pseudomonas aeruginosa showed that all isolates had given negative result for indole, methyl red, voges proskauer and urease test also, citrate assimilation was positive, motility was positive and TSI alkaline / alkaline or Alkaline / no change with no production of H2S and gas and isolated from cetrimide agar colonies appeared mucoid, smooth in shape, fruity odour, fluorescent green, and creamy pigments these results agreed with what mentioned by [13,[18][19][20].
The results of Proteus mirabilis appear as bacilli, rapidly motile by flagella.Swarming phenomena on blood agar plate.
It is non-lactose fermenter so it gives a pale colony on macConky agar.Negative test for indole and voges-proskauer but positive test for urease, simmon , s citrate , H2S is produced ,nitrate reduction motility and methyl reed .Similar finding was recorded by [15,18].
The results of Serratia.marcescens, showed that isolate had given result for indole, urease test , citrate assimilation , motility and catalase positive .Methyl red, voges proskauer and oxidase negative and TSI alkaline / acidic or acidic / acidic with no production of H2S and gas.The results of Burkholderia cepacia complex showed that isolate had given a result at negative for indole.But positive for methyl red, voges proskauer, urease test, citrate assimilation, motility, catalase and oxidase, TSI alkaline / acidic with no production of H2S and gas.
The results of Pantoea spp showed that isolate had given result for indole methyl red, urease test, motility,and oxidase negative.Voges-proskauer citrate assimilation, catalase positive, TSI alkaline / acidic with no production of H2S and gas.The results Enerobacter cloacae indicated that isolate had given result for indole, methyl red,urease test , motility and oxidase negative.Voges-proskauer, citrate assimilation and catalase was positive.TSI alkaline / acidic with no production of H2S and gas.The results of staphylococcus aureus appeard that isolates had given result for indole was negative , methyl red was positive , voges-proskauer was positive , urease test was positive , citrate assimilation was positive, motility was negative and TSI acidic / acidic with no production of H2S and gas , catalase was positive , coagulase was positive and oxidase was positive this results are consistent with findings from other Iraqi studies [20,21].Also similar result was recorded by [21][22][23].On blood agar staphylococcus haemolyticus fermenters as yellow colonies.Further biochemical tests were necessary for identification of staphylococcus haemolyticus from other species, all isolates were positive to catalase test, but negative for coagulase and oxidase [24].The results of Enterococcus faeciu showed that isolate had given negative results for indole, urease test, citrate assimilation, motility, catalase and oxidase.But positive result for voges-proskauer.TSI acidic / acidic with no production of H2S and gas.
These results agree with study in Wasit [31,32] which found to be highly resistant to (ceftazidime, ceftriaxone, azitreonam and cefotaxime) (100%) respectively.The maximal pseudomonas aeruginosa sensitivity has been to (amikacin, trimethoprime and cefoxitin) (60%) respectively.In pseudomonas aeruginosa, intrinsic antibiotic resistance is mediated by a decrease of outer membrane permeability, the expression of MDR, XDR and PDR efflux pumps, as well as the synthesis of antimicrobial inactivating enzymes (Hall et al., 2018).In our study, it was observed that the percentages (, MDR) were (70%), XDR were (15%) and PDR were (15%), as shown in the figure (3_9).This increase in percentage leads to multiple drug resistance of isolates to antimicrobials because of the province the outer membrane's permeability barrier antibiotics out of the bacterial cells

Screening and confirmation of ESBLs' production for K. pneumoniae
Presumptive ESBLs-producing K. pneumoniae were detected in 19 clinical specimens , were initially screened for ESBLs and result 18(94.7%).Of these, 13(68.4%)isolates were determined as ESBLs-producers following confirmatory testing to be ESBLs producers by CLSI complex disk detection (CLSI-CDD) method ( modified double-disc synergy test).

Screening and confirmation of ESBLs' production for Proteus mirabilis
The five proteus mirabilis clinical isolates were investigated phenotypically (screen and confirmatory tests) for ESBL production, were initially screened for ESBLs and result 5(100%) The result of the confirmatory examination was 2 (40%).Resistance of proteus mirabilis to third generation cephalosporins and monobactams were as the following: cefotaxime: 80%, ceftazidime: 40%, ceftriaxone: 100%, and aztreonam: 40%.

Molecular detecting Antibiotic Resistance Genes in gram negative bacteria
The results observed that all the isolates of Klebsiella pneumoniae, E. coli, proteus mirabilis, Burkholderia cepacia and Klebsiella oxytoca ) were negative for the presence of bla CTX-M1, bla CTX-M2, and bla CTX-M9 genes.Molecular detection of CTX -M ESBLs for isolate Pseudomonas aeruginosa showed that 1 (8.3%) of the isolate was positive for at least one CTX-M ESBLs gene from 12(60%) , the frequency of CTX-M ESBLs gene presence among Pseudomonas aeruginosa was as follows: blaCTX-M1 ,1(8.3%);bla CTX-M2, 1(8.3%); bla CTX-M9 0(0%) ,there was a significant association between the presence of ESBL genes in Pseudomonas aeruginosa and site of infection ,our study consistent with this study with in Jimma, Ethiopia [33,34] containing this bla CTX-M2 in the (3%); (8.2%) respectively.The results was consistent with [35] demonstrated the frequencies of this gene blaCTX-M1 (10%) in Erbil.
Detection of CTX -M ESBLs for isolate Acinetobacter baumannii 1(50%) of the isolate was positive for at least one CTX-M ESBLs gene, the frequency of CTX-M ESBLs gene from 2(100%) presence among Acinetobacter baumannii was as follows: bla CTX-M1,0(0%); bla CTX-M2, 1(50%); bla CTX-M9 0(0%) There was a significant association between the presences of ESBL genes .Our study is similar to these studies in in Saudi Arabia [36] carry this gene bla CTX-M2 (81%) and another study by [37] carry this gene bla CTX-M2 (43%).The agarose gel of PCR products was shown in Figure (3), (4).Pseudomonas aeruginosa isolate at product size.CTXM 5 666 bp Beta-lactam antibiotics are being used less and less due to extensive usage, which has led to a rise in the global dissemination of ESBL enzymes.Therefore, the most effective way to preserve the effectiveness of beta-lactam antibiotics is to fully identify ESBL through trials and restrict the consumption of beta-lactam antibiotics [38].
As for the results of blaSHV 1(10%) for one E. coli , the results of our study are similar to those of these studies [43,45] in China's Sichuan-Chongqing Circle.who carry this gene blaSHV (12%) ; ( 18%) respectively , and agree with this in Wasit [46] whose result is 13(29%).With regard to the results blaSHV 1(50%) for proteus mirabilis.Our results are similar to those of these studies in Japan [47,48] in Poland who carry this gene blaSHV (50%) ; (58%) respectively.
The results of the gene blaOXA 1(10%) were for E. coli, our study of this gene is consistent with the results of these studies [49,50] in Germany that contain this gene blaOXA ( 7% ) ; (14.9%) respectively.Correspond to the study in Wasit [14] whose result is (17.8%).As for the result blaOXA 1(100%), it was for Burkholderia cepacia .Our study is similar to the results of these studies [51,52] in the United States who carry this gene blaOXA (93% ) ; (100%) respectively .As shown figure (5) .However, TEM gene any positive results in all isolation bacteria in this study did not find.

Conclusion
According to the findings of current study in ICU, The highs frequency of specimens collection from intensive care unit patients were isolated from sputum and the gram-negative bacteria were the most common than the positive bacteria.Results of antibiotic susceptibility test revealed that the most active compound against Klebsiella pneumoniae and Pseudomonas aeruginosa were Amikacin.Phenotypically and genotypically of ESBLs for klebsiella pneumoniae showed no isolate had ESBLs producers CTX-M-type ESBL.The most prevalence of blaSHV and no isolate have blaTEM and blaOXA-1 genes, Phenotype and genotype of pseudomonas aeruginosa ESBL producers were carrying CTX-M2 and CTX-M1 in their isolates, also prevalence of blaSHV.AmpCß-lactamase comprised (blaEBC), Phenotype and genotype of ESBLs for E.Coli showed no isolate had ESBLs producers CTX-M-type ESBL.Also carries blaSHV, blaOXA-1 and no isolate have blaTEM gene.

Table 2
Thermal cycling program for multiplex pool.1 CTX group

Table 3
Thermal cycling program for multiplex pool.2