Context: Pseudomonas aeruginosa is an obsessive invader in the hospitals worldwide. It has powerful antibiotic resistance mechanisms, one of such being metallo beta lactamase, which is taking away from our hands our last resort, carbapenem group of antibiotics, to fight them. Not only they possess this metallic gun, they are exceptionally good gun dealers also, as the genes producing this metallo beta lactamase resides on plasmids and hence, are easily and efficiently transferrable among other bacteria, Thus it becomes a present day concern to detect the presence of MBL positive Pseudomonas aeruginosa in every hospital to have a powerful and effective antibiotic regimen in the future also. Aims: To determine the presence and extent of MBL positive Pseudomonas aeruginosa in a Midnapore Medical College, West Midnapore district, West Bengal, India. Materials and Methods: A total of 630 samples were processed, out of which 112 isolates of Pseudomonas aeruginosa were found. Among these, 20, were found to be imipenem resistant and these were subjected to EDTA impregnated imipenem disc potentiation test. Discussion: Out of 112 P. aeruginosa, 8 isolates (7%) turned out to be MBL producers. Also, a high susceptibility pattern was found for aminoglycosides. Conclusion: Although we found a low prevalence of MBL producing P. aeruginosa, still it is worrisome. Constant vigilance and careful selection of antibiotics are necessary. Keywords: Hospital infections, metallo beta lactamase, pseudomonas aeruginosa
The new millennium ushered in with a high hope that carbapenem group of antibiotics would bring the ultimate threat to the β lactam resistant bacteria. As they retain their activity and stability to hydrolysis by almost all β lactamases, carbapenems have become the mainstay of therapy to treat infections by β lactam resistant gram negative bacilli, including the ones producing ESBLs. [1] Unfortunately, everyday, million dollars of pharmaceutical research are being undone by single stroke of genetic evolution in bacteria. Resistance to carbapenems are being reported in many bacteria, and Pseudomonas aeruginosa, notoriously known worldwide for causing nosocomial infections, is chief among them. To resist carbapenems, gram negative bacilli have started producing 2 types of enzymes: serine carbapenemases, and Metallo β lactamases (MBLs). [2] These enzymes can hydrolyse not only carbapenems but many β lactams as well. [3] The genes responsible for production of MBLs lie on a plasmid, and hence can be horizontally transferred easily, efficiently and rapidly. [4] It is imperative to keep a hawk-eyed watch on the developing resistance pattern of Pseudomonas aeruginosa, especially in a hospital environment as this can only keep the increasing burden of hospital acquired infections in check. Our main aim was to have an inkling that, how much of a peripheral Medical College of ours, Midnapore Medical College and Hospital, situated in the west Midnapore district of the West Bengal state in India, mainly catering to the rural population, has been invaded by this bug with a metallic gun in its pocket.
Six hundred and thirty samples were collected during one year period from January to December 2010, in the department of Microbiology. The samples processed were: urine (392), pus / wound swab (175), sputum(33), knee joint fluid(7), and blood culture(23). Samples were cultured on MacConkies agar and Sheep Blood agar. Identification was done by biochemical test. [5] A total of 112 isolates of Pseudomonas aeruginosa were found and the isolates were stored at -20 C. Standard strains of Pseudomonas aeruginosa ATCC 27853 was used as control. Antibiotic sensitivity was done on Mueller Hinton agar with commercially available Discs (Hi-media, Mumbai) by Kirby-Bauer method. [6] The routine antibiotics incorporated were Amikacin (30mg), Gentamicin (10mg), Tobramycin(10mg), Ceftazidime (30mg), Ceftriaxone (30mg), Cefepime (30mg), Cefoperazone(7.5mg), Ciprofloxacin (5mg), Gatifloxacin (5mg), Levofloxacin (10mg ), Piperacillin-Tazobctum (100/10mg),and Imipenem(10mg). There are various recommended techniques for phenotypic determination of MBL, like Double disc synergy test using Imipenem and EDTA or Ceftazidime and EDTA discs, EDTA impregnated Imipenem Discs, E-test strip and microdilution technique for determining MIC of Imipenem. [7],[8],[9] We took up the EDTA impregnated Imipenem disc potentiation test. We used zone enhancement with EDTA impregnated imipenem discs. Test organisms were inoculated on the MHA plates as recommended by CLSI. [6] A 0.5M EDTA solution was prepared by dissolving 186.1 gm of Disodium EDTA.2H 2 O in 1000 ml of distilled water and its pH was adjusted to 8.0. This solution was sterilised by autoclaving. Then two 10mg imipenem discs were placed on test organism inoculated MHA and 5ml EDTA was added to one imipenem disc. After 16 hours of incubation at 35 0 c, the zones of inhibition around both the discs were measured. The strains with enhanced zones around EDTA impregnated imipenem discs were identified as MBL producing Pseudomonas aeruginosa.
Of the 112 isolates of P. aeruginosa, 20(17.8%) were showed resistance to imipenem. Among these, 8(7%) were seen to be MBL producers. The control strain ATCC 27853 P. aeruginosa did not show any zone enhancement. Out of 112 P. aeruginosa isolates in our study, 7 (%) were resistant to Amikacin, 14 (%) to Gentamicin, 13 (%) to Tobramycin, 92 (%) to Ceftazidime, 95 (%) to Ceftriaxone, 84 (%) to Cefepime, 101 (%) to Cefoperazone, 112 (%) to Ciprofloxacin, 30 (%) to Gatifloxacin, 34 (%) to Levofloxacin, and 3 (%) to Piperacillin – Tazobactum [Table 1].
Costerton and Anwar called Pseudomonas aeruginosa the most abundant life form on earth. It has been isolated from environments as diverse as water, jet plane fuel and disinfectant solutions due to its ability to utilise many different organic compounds and survive in the apparent absence of nutrients. It will be a dangerous situation if such a pathogen is not killed by the commonly used antibiotics. But this pathogen, notorious for its disease causing capacity, has also invented multiple mechanisms of antibiotic resistance. One of the most dangerous among them is metallo beta lactamase. The genes responsible for production of MBLs are VIM and IMP. Most of these genes are found as gene cassettes in integrons. In most instances, the MBL genes are fused with aacA4 genes which codes for aminoglycoside resistance,further compromising both antibiotic regimens. [2] Other than Pseudomonas aeruginosa, the bacteria that enjoy the ownership of this enzyme are plenty. The members of this August club are E.coli, K.pneumoniae, Enterobacter sps., C.freundii, Proteus sps., Acinetobacter etc. However, the presence of this enzyme was first reported in Pseudomonas in 1991 from Japan. [10] In different studies worldwide, variable resistance (4 – 60%) to carbapenems has been mentioned. [11],[12] In our country, the reports vary between 10 – 30%. [13] In our study we found a low prevalence (7%) of MBL producing Pseudomonas aeruginosa. However, to keep it in check, regular detection of this bacteria and judicious use of antibiotics to which it is still susceptible is mandatory. Interestingly, we found a high susceptibility pattern to Amikacin, Gentamycin and Tobramycin, the three aminoglycosides reported to be still active against Pseudomonas aeruginosa.[14] This has to be taken into account that although aminoglycoside resistance is alarmingly on the increase worldwide, it varies geographically and depends on differences in prescription patterns practiced in that particular area and infection control norms in that locality. [15] The people admitted to this hospital come from a large number of villages, and are mainly of a low socio-economic status. They most often have not enough money to buy antibiotics and hence, are not exposed to random antibiotics sold over the counter, a practice widely used in our country. This may be one of the reasons, we presume, that this bacteria is still susceptible to aminoglycosides here. On the contrary, it is difficult to explain why this same bacteria is totally resistant to an anti-Pseudomonal quinolone, Ciprofloxacin. An epidemiological study involving many factors is what we plan to conduct in future, in conjunction with antibiotic resistance gene pattern studies. But presently, presence of MBL positive Pseudomonas aeruginosa in this new and peripheral Medical College is of concern enough to add a few more extra furrows on the forehead of local medical community.
Source of Support: None, Conflict of Interest: None
[Table 1] |
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