Emergence of Gram-Negative Bacilli with Concomitant blaNDM-1- and blaOXA-48-Like Genes in Egypt

Multidrug-resistant Gram-negative organisms have emerged as a major threat to hospitalized patients, and are associated with serious morbidity and mortality. This study aimed to characterize carbapenem resistance genes among Gramnegative bacilli isolated from clinical samples from patients in the intensive care unit of Cairo University Hospital. A total of 211 samples were collected from patients showing clinical evidence of infection. Bacteria were isolated and identified by conventional microbiological methods. Acinetobacter baumannii isolates were furtherly characterized by polymerase chain reaction (PCR), using primers specific for blaOXA-51-like genes. The Kirby Bauer disc diffusion method was used to determine susceptibility patterns of isolates, and carbapenem resistance was further examined by a modified Hodge test. Positive isolates were tested for the presence of blaKPC, blaOXA-48, and blaNDM-like genes by PCR. NDM gene types were determined by direct sequencing. From the 211 samples, 229 Gram-negative bacilli were isolated. Fifty isolates (21.2%) were resistant to carbapenem. PCR analysis showed that none of the 50 isolates carried blaKPC-like genes, while 24 (48%) isolates carried blaOXA-48-like genes, 8 (16%) carried blaNDM-1, and five isolates (10%) carried both blaNDM-1 and blaOXA-48-like genes. These results indicate that continuous surveillance of these multidrug-resistant pathogens is urgently required. And that is very important is to activate the antimicrobial stewardship programs of which the most important is restriction of the big gun antibiotics like carbapenems, colistin, tigecyclin and vancomycin and restricting their prescription to privileged specialties.


Introduction
"Infections caused by Gram-negative bacilli (GNB) are of particular concern, especially in intensive care units (ICUs). These organisms are highly efficient at acquiring or upregulating genes that code for mechanisms of antibiotic resistance, especially in the presence of antibiotic selection pressure. Furthermore, they contain a variety of resistance pathways, and often contain multiple mechanisms targeting the same antibiotic [1]". "Gram-negative bacteria resistant to three or more antimicrobial classes, known as Multidrugresistant (MDR) Gram-negative organisms, have emerged as a major threat to hospitalized patients and have been associated with mortality rates ranging from 30-70% [2], [3]". "A range of Gram-negative organisms, including both non-fermenters (Acinetobacter baumannii and Pseudomonas aeruginosa) and fermenters (Enterobacteriaecae), are responsible for hospital-acquired infections. MDR organisms, including A. baumannii, P. aeruginosa, and extended-spectrum β-lactamase-producing or carbapenemase-producing Enterobacteriaceae, are increasingly being reported worldwide [1]".
"Patients in the ICU usually have serious co-morbid conditions or are compromised by invasive procedures, such as mechanical ventilation, surgery, and frequent use of vascular or urinary catheters. Infections in these patients are usually life threatening and may be caused by MDR organisms [6]". "Carbapenem antibiotics have traditionally been considered the last line of defense in treating infections caused by these bacteria but nowadays we have colistin (polymexin E) is last line of defense against MDR gram negative bacteria. [7]" "Recently Qureshi et al. reported Colistin-resistant A. baumannii almost exclusively among patients who had received colistin methansulfonate for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection which is very alarming data for the last backbone of treatment of MDR gram negative bacteria [8]". "Thus, the alarming spread of carbapenemases poses serious risk to these vulnerable patients, as only a few suboptimal therapeutic options remain available for treating such infections [9]". "Infections caused by carbapenem-resistant organisms have been associated with high mortality rates [5]"."Early detection and strict application of infection control measures have been reliable in reducing the likelihood of transmission in health care settings [10]". Therefore, the objective of this study was to characterize carbapenem resistance genes among GNB isolated from clinical samples from patients in one of the ICUs at Cairo University Hospital.

Study Design and Sample Collection
A prospective cross-sectional study was conducted over a period of 6 months (June to December 2013). Clinical samples were obtained from patients showing clinical evidence of infection at one surgical ICU of Cairo University Hospital. The research protocol was approved by the Research Ethics Committee of the Faculty of Medicine, Cairo University, Egypt.

Screening of Carbapenem-Resistant Isolates
"Isolates were screened for carbapenem resistance using an imipenem and meropenem disk diffusion method. Isolates were categorized as sensitive, intermediate, or resistant according to CLSI guidelines (CLSI, 2012) [13]". Isolates were tested for carbapenemase production by modified Hodge test (MHT) as described in the CLSI guidelines. MHT-positive K. pneumoniae ATCC1705 and MHT-negative K. pneumoniae ATCC1706 were used as control strains for the assay.

Genetic Characterization of Carbapenem-Resistant Isolates
All clinical isolates found to be resistant to imipenem or meropenem and/or positive by modified Hodge test were examined for the presence of bla KPC , bla OXA-48 , and bla NDMlike genes by PCR. The PCR conditions and methodology were as described previously (14)(15)(16) (Table 1).

DNA Sequencing and Analysis
Any identified bla NDM genes were sequenced to determine the NDM variant of the enzyme using the primers listed in Table 1. PCR products were purified using a MinElute Gel Extraction Kit (Qiagen, Valencia, CA, USA). The purified PCR products were directly sequenced on both strands using a 310 Capillary Array Sequencer and Big Dye Terminator chemistry (Applied Biosystems, Foster City, CA, USA). NDM sequences were analyzed using the BioEdit Sequence Alignment Editor (Ibis Therapeutics, Carlsbad, CA, USA), and results were compared to a reference sequence (GenBank accession no. LC032101).

Statistical Analysis
All statistical analyses were performed using SPSS version 15 for Microsoft Windows (SPSS Inc., Chicago, IL, USA). Data were statistically described in terms of frequencies (number of cases) and percentages.
When studying carbapenem-resistant isolates in relation to the site of infection, we found that respiratory infections yielded the highest number of carbapenem-resistant GNB (24, 48%), followed by wound infections (13, 26%) ( Table  3). All 50 carbapenem-resistant isolates were MDR, with 100% resistance to ampicillin, β lactam/β-lactamase inhibitors, first, second, third, and fourth generation cephalosporins, and carbapenems. In addition, 49 (98%) isolates were resistant to trimethoprim-sulfamethoxazole, 41 (82%) were resistant to aminoglycosides, and 39 (78%) were resistant to fluoroquinolones. However, all isolates were sensitive to tigacycline, polymyxin B, and colistin. No specific patterns were detected in relation to the absence or presence of resistance genes.
PCR analysis revealed that none of the isolates carried bla KPC -like genes, while 24 of the 50 resistant isolates (48%) carried bla OXA-48 -like genes, eight (16%) isolates carried bla NDM , and five (10%) carried both bla NDM and bla OXA-48 -like genes ( Table 4). Sequencing of the bla NDM genes revealed that all isolates contained the bla NDM-1 variant.

Discussion
"In recent decades, the emergence of extended-spectrum βlactamase-producing bacteria has led to increased use of carbapenems in clinical practice, which in turn has led to the emergence of isolates containing carbapenemases [4]". We screened GNB responsible for different clinical infections in patients admitted to one of the ICUs at Cairo University Hospital, and investigated carbapenemase production by these isolates. We identified 50 isolates with carbapenem resistance patterns among 229 isolated GNB (21.8%). Although P. aeruginosa and K. pneumoniae were the most commonly isolated species (75 and 62 respectively), A. baumannii isolates showed the highest incidence of carbapenem resistance (46%)." In agreement with these results, Fouad et al. identified 547 nosocomial infections in three ICUs from three different hospitals in Egypt between January 2011 and September 2012 and found that the majority of infections were caused by Klebsiella spp., whereas A. baumannii showed the highest levels of imipenem resistance (74%) [17]". "Our study also revealed that respiratory infections yielded the highest number of carbapenem-resistant organisms (48%). These results are in partial agreement with Maltezou et al., who investigated infections caused by carbapenem-resistant GNB in hospitalized children. They isolated 71 carbapenem-resistant pathogens causing infections in 65 children, of which 25 cases were diagnosed with pneumonia (35.2%) [18]".
Although KPC-producers are now being identified at an alarming rate across the USA, France, Israel, Greece, Colombia, and China, and outbreaks of KPC-producing bacteria have been reported in many European countries, South America, and India (19-21), we did not detect any bla KPC -like genes among the 50 isolates that were positive by phenotypic methods for carbapenemases production. "This is in agreement with findings reported by Shibl et al. [4]", who tested 60 K. pneumoniae isolates from Saudi Arabia (the majority of which were from patients in an ICU) and did not identify any bla KPC -like genes. Together, these results indicate that bla KPC -like genes are not the major source of carbapenemases in the Middle East.
"The carbapenem-hydrolyzing class D β-lactamase OXA-48 was first described in a K. pneumoniae strain isolated from Turkey in 2004, and is now endemic in Egypt (4). OXA-48 carbapenemases are also endemic in countries around the Mediterranean, and are rapidly spreading into other countries in Europe [22], [23]". "Although many reports have described patients becoming infected with strains carrying bla OXA-48 -like genes during travel to Egypt [24], [25]", few surveillance studies have focused on this gene as a cause of carbapenem resistance in Egypt. "In a recent 6-month surveillance study of carbapenem-resistant GNB isolated from a cancer hospital in Egypt, only three isolates harbored this gene [26]". Our study identified a higher number of isolates (24 isolates) ( Table 3) than previously reported, which may indicate the rapid spread of carbapenem resistance in Egypt through the dissemination of bla OXA-48 -like genes. "Although bla OXA-48 is rarely detected in Acinetobacter spp., Goncalves et al. detected this gene in A. baumannii isolated from fecal flora of nursing home residents in northern Portugal [27]". "Importantly, bla OXA-48 is associated with transposons Tn1999 and Tn1999.2, which enable rapid transmission among GNB [28]".
" bla NDM-1 was initially identified in K. pneumoniae and E. coli recovered from a Swedish patient who was previously hospitalized in India, and has rapidly disseminated to other Enterobacteriaceae in several countries [29]". "NDMproducers are of particular concern as they also harbor multiple plasmid and chromosome-encoded resistance genes, resulting in a MDR phenotype [4]". "Recently, cases of NDM-producing A. baumannii have been described in Egypt, China, and Israel [20], [29], [30]". "Similar to bla OXA-48 , the way in which bla NDM-1 has spread between GNB in Egypt is not yet clear, but some of the identified cases in Europe had a history of travelling to Egypt [30][31][32]". In this study, eight isolates were shown to harbor bla NDM-1 ( Table 3), five of which also contained a bla OXA-48 -like gene (four K. pneumoniae and one A. baumannii). "Although this pattern of combined resistance has been reported previously in Lebanon and Tunisia [3], [23]", the rates identified in the current study are alarmingly high: five isolates versus one K. pneumoniae isolate each in Lebanon and Tunisia. However, there is little published data discussing the effect of the coexistence of several carbapenemases in GNB, an issue that requires close and continuous monitoring in infected patients.
"The emergence of such resistant strains represents a significant threat, not only to our country, but globally, especially as the dissemination of resistance genes is hastened by high rates of immigration and tourism [4]". This study has documented the emergence of NDM-1-and OXA-48-positive GNB in Egypt. NDM and OXA-48 type carbapenemases are increasingly reported in our region, with the Middle East and North Africa now regarded as secondary reservoirs for these carbapenemases. Several alarming reports have described the introduction of OXA-48-and NDM-expressing GNB to some European countries by patients previously hospitalized in Egypt. Healthcare workers, especially in ICUs, need to be aware of the emergence of these MDR isolates, as they are a significant health concern. Enhanced surveillance and detection of these MDR pathogens is urgently required so that patients can be identified quickly and appropriate infection control measures can be instituted to stop further dissemination. Further studies are also needed to clarify the epidemiological features of carbapenemase-producing isolates in Egypt. Also what is very important is to activate the antimicrobial stewardship programs of which the most important is restriction of the big gun antibiotics like carbapenems, colistin, tigecyclin and vancomycin and restricting their prescription to privileged specialties like infectious disease, intensevists, and pulmologists which can authorize other specialties in patients their clinical situation necessitates these big gun antibiotics.

Conclusion
This study indicates that continuous surveillance of these multidrug-resistant pathogens is urgently required. And that is very important is to activate the antimicrobial stewardship programs of which the most important is restriction of the big gun antibiotics like carbapenems, colistin, tigecyclin and vancomycin and restricting their prescription to privileged specialties.