Evaluation of Polymyxin NP Test as a Rapid Method for Detection of Polymyxins Resistance in Enterobacteriaceae ‎

The carbapenemase-producing Enterobacteria-ceae are among the most clinically significant MDR bacteria. Such a rise drove an increase in the usage of polymyxins because of limited effective alternative antibiotic treatment. So, this global concern of polymyxins and the mounting use of colistin explicates the addition of acquired colistin resistance to the resistant traits in Enterobacteriaceae [3].

We aimed to assess the diagnostic efficacy of the rapid polymyxin NP test using Polymyxin B for the diagnosis of colistin- and polymyxin resistant Enterobacteriaceae and determine the frequency of mcr-1 and mcr-2 genes among the colistin-resistant Enterobacteriaceae at ASUH.

In the present study, the age of the patients ranged from 9 days to 77 years. The number of female and male patients included were equal (21/42, 50%). Concerning the sample type, it was observed that majority of isolates were retrieved from blood cultures (12/42, 28.6%).

In a similar pattern to our study, research conducted in Croatia reported that their patients harboring colistin-resistant isolates were equally distributed between both sexes (50% each), and their ages ranged from 6 to 84 years. The predominant specimen were blood cultures (9/24), followed by urine samples (5/24) [21].

As for the antibiotic susceptibility testing, all the tested isolates recorded a 100% resistance rate against almost all β-lactams with exception of ceftazidime (97.6%) and imipenem (90.0%). These findings came in concordance with the internationally high level of antimicrobial resistance among all classes of antimicrobial, especially in Egypt which caused surging in colistin use as the last resort antimicrobial.

In Egypt, a one-year retrospective study showed that Gram-negative isolates displayed a high rate of resistance to all used antibiotic classes. This study reported that resistance towards the Beta-lactam group ranged from 70% to more than 98% against different antibiotic members of this group [22]. Such a high resistance was demonstrated by other researchers as well. In Croatia, the sixteen enterobacterial isolates were resistant to almost all antibiotics; β-lactams, ciprofloxacin, and gentamicin. Except for colistin, the MIC90 of all antibiotics exceeded 128 mg/L [21].

Regarding antimicrobial consumption among the 42 patients included in our study, we found that 30 (71.4%) patients received antibiotics. Meropenem rated the first where 10/30 (33.3%) of our patients received it.

Other researchers reported a high overall antibiotic consumption among different hospitals. They found that Cephalosporins were the most usually prescribed antibiotics, followed by quinolones, penicillins, and carbapenems. They reported high significant pearson’s correlation coefficient factors (r) between carbapenem consumption and its resistance rate of 0.271 and 0.427 for E. coli and Klebsiella pneumoniae respectively [23].

Also, other investigators demonstrated that the rate of antibiotic usage among their patients was very high ranging from 32.9% to 91.7% and 59% of them were receiving one or more antibiotics. Third-generation cephalosporins ranked first among the prescribed antibiotics and accounted for 28.7%. Penicillins with beta-lactamase inhibitors and metronidazole derivatives accounted for 19.7% and 15.2% respectively [24].

Unlike our study, other researches in China and Egypt proved that cephalosporins are the most received drug [23, 24]. Whereas in the current study, Meropenem was the most frequently used one. This backs to the high level of multidrug resistance that was detected in the last years and obliged the physicians to prescribe carbapenem as the last option before resorting to polymyxins.

For colistin MIC results, our results ranged between 0.5 to 32 ug/ml. 18 isolates out of 42 were resistant to colistin (42.9%). The majority of resistant isolates were Klebsiella isolates (83.3%) in comparison to E. coli (16.7 %).

Similarly, a study conducted in Thailand reported that colistin resistance was identified in 13 (35%) E. coli and 213 (76%) Klebsiella pneumonia isolates, with MIC ranging from 0.5 to 32 mg/L and 0.25 to >128 mg/L respectively [25].

In an Indian study, they found that among their 138 isolates, meropenem, colistin, and double resistance were detected in 110, 31, and 21 isolates, respectively. Noticeably, 90.5% (19/21) of the dual colistin-resistant and carbapenem-resistant isolates were Klebsiella pneumoniae [26].

For polymyxin MIC done for our tested isolates, the results ranged between 0.5 to 16 ug/ml. Fifteen isolates out of 42 were resistant to polymyxin (35.7%). The majority of the resistant isolates were Klebsiella isolates (86.7%) and E. coli was (13.3%).

A Chinese study reported a lower resistance among 504 carbapenem-resistant Enterobacteriaceae isolates. Only 19 isolates exhibited resistance to polymyxin B, with MICs ranging from 4 μg/mL to ≥256 μg/mL [27].

Other researchersfound that out of 110 evaluated isolates, 51 isolates were polymyxin-resistant (MIC ranging from 4 to > 64 μg/mL) and 59 were polymyxin-susceptible (MICs ranging from ≤ 0.125 to 2 μg/mL) [28].

In light of results stated worldwide, we found that they agreed to a great extent with those reported in our study and this affirms the fact of increasing resistance against colistin and polymyxin B which are considered the last hope for treatment of MDR organisms, this foreshadows an upcoming worldwide disaster.

As regards the polymyxin NP test, the overall sensitivity, specificity, PPV, and NPV versus the colistin MIC BMD were 77.8%, 91.7%, 87.5%, and 84.6% respectively. While, versus the polymyxin MIC BMD, the overall sensitivity, specificity, PPV, and NPV were 93.3%, 92.6%, 87.5%, and 96.2% respectively.

Our results are in concordance with those reported by many authors where their studies showed excellent sensitivity and specificity [14, 29-32]. Other authors from Greece found that the Rapid Polymyxin NP test yielded a high sensitivity (99%) and a lower specificity of 82%. Although their results were different from ours, yet, these findings still point out that the Rapid Polymyxin NP test can be used as a screening test for early diagnosis of colistin-resistant isolates [33]. Thus, this test can represent a reliable alternative to BMD, especially in resource-limited settings.

Although the sensitivity of the test obtained in our study was lower for colistin MIC compared with the polymyxin MIC, many worldwide studies stated that the Rapid Polymyxins NP test exhibited an outstanding performance for detecting resistant and susceptible Enterobacteriaceae for both polymyxin B and colistin. We proved in our study that this test is easy to perform and could be accomplished within 4 hours compared to the MIC by the BMD method, which consumes around 24 hours.

A Brazilian study asserted this fact and reported that the required time to obtain the result was 2 hours for the Rapid Polymyxins NP test versus 24 hours for the BMD. In addition, this test is more advantageous, being less burdensome. This fast identification of polymyxin-resistant bacteria might aid in precisely identifying the optimum therapy choices, and rapidly implementing contact precaution measures, thus halting further outbreaks with MDR-isolates [29].

In our study, we carried out conventional PCR for confirmation of the presence of polymyxins resistant genes (mcr-1, 2 genes). In correlation with Colistin MIC, we found that 17/18 colistin-resistant isolates had the mcr-1 gene (94.4%) and five (27.8%) isolates harbored the mcr-2 gene. Whilst, correlating it with polymyxin MIC revealed that all the 15 (100%) polymyxin-resistant isolates were positive for the mcr-1 gene and 5/15 (33.3%) positive for the mcr-2 gene. We observed that the mcr-1 gene was found in all of the 14 Rapid Polymyxins NP positive isolates, while, the mcr-2 gene was detected solely in five isolates.

Although in our study mcr-1 and mcr-2 genes were present in most of our isolates, this represented a hindrance to finding a statistical relationship between the types of genes and the results of the rapid polymyxin NP test.

Research conducted in Switzerland found that the Rapid Polymyxins NP displayed 100% sensitivity when performed using polymyxin-resistant isolates of environmental, animal, and human origin and mediated by the plasmid carrying genes mcr-1 or mcr-2 [16].

On the other hand, investigators from Brazil reported that Polymyxins NP gave false-positive results in two polymyxins-susceptible isolates of their 19 selected isolates (11 polymyxins-resistant and 8 polymyxins-susceptible) carrying the mcr-1 gene [29].

A study in Thailand detected the mcr-1 gene in 11/37 E. coli isolates and their colistin MIC range was 4–32 mg/L, while, they found the mcr-1 gene in 4/280 Klebsiella pneumoniae isolates with a colistin MIC range of 4–64 mg/L [34].

The world has been suffering for a long time from the era that we fear the most; the era of antimicrobial resistance, this dreadful social challenge. Nowadays, the globe is attempting to find a way out of this trouble. But, since we are unable to add new therapeutic agents to our inventory, so, the only possible solution we have is to try decreasing the rate of colonization and infections with resistant strains, thereby reducing mortality and morbidity. To do so, we need to apply preemptive measures before we are left handcuffed without any antimicrobial choices.

In the current study, the rapid Polymyxin NP test proved to be a reliable, easy, affordable, and speedy assay to perform. Moreover, it exhibited promising performance compared to our reference technique. Hence, we can deduce that this test can be implemented as a screening tool in the outbreak management and active surveillance for the presence of Polymyxins resistance. Therefore, combined with PCR, it can play a substantial role as a part of infection control measures to contain antimicrobial resistance and prevent the transfer of resistance genes among patients.

CONCLUSION

The rapid polymyxin NP test is an easy, quick, sensitive, and specific test that can reliably detect both polymyxin-resistant and susceptible Enterobacteriaceae isolates, whatever the molecular mechanism of resistance.  Even more, it is less sophisticated and can provide results at least 16 hours before the BMD method.

The performance of the polymyxin NP test in the current study was lower for colistin MIC in comparison with the polymyxin B MIC.

Therefore, we recommend using the polymyxin NP test for early detection of polymyxin-resistant Enterobacteriaceae, especially in low resource health care settings. Further studies on broad scales with large sample sizes are recommended to study the effect of each mcr gene separately on the sensitivity and specificity of this test.

ACKNOWLEDGMENT: None.‎

List of abbreviations: CPE: carbapenemase-producing Enterobacteriaceae, MDR: multidrug-resistant bacteria, BMD: broth microdilution, MIC: minimum inhibitory concentration, PCR: Polymerase chain reaction, E.coli: Escherichia coli, CSF: cerebrospinal fluid, AST: Antimicrobial susceptibility testing, CIM: Carbapenem Inactivation Method, DDDs: defined daily dosages,

Funding: None.

Conflict of interest:  None.

Ethical approval: All study procedures were as per ethical guidelines of the 1975 Declaration of Helsinki and approved by the ethical committee of Faculty of Medicine, Ain Shams University. (Ethical approval number: FWA 000017585).