Antibiotic susceptibility pattern

The susceptibility profile of the tested K. pneumoniae isolate demonstrated antibiotic resistance to ceftazidime, ceftriaxone, cefotaxime, cefepime, kanamycin, and gentamicin in addition to intermediate sensitivity to ofloxacin. Whereas it was sensitive to gatifloxacin, ciprofloxacin, amikacin, and meropenem. Recorded data showed that among the four tested classes of antimicrobial agents, the selected K. pneumoniae isolate was resistant to at least one antibiotic from three different antibiotic classes (cephalosporins, aminoglycosides, and carbapenems), thus it was considered multidrug-resistant (Table 2). In the meantime, the MIC of zinc sulfate was recorded at a value of 4 mg/ml.

Table 2 Antibiotic and antibiofilm susceptibility patternsAntibiofilm susceptibility profile of sole different antibiotics and in combination with zinc sulfate

Regarding the antibiofilm potentials of ceftazidime, ceftriaxone, cefotaxime, cefepime, and cefpirome, the recorded results indicated that there is a gap between the MBICs compared to the corresponding MICs on planktonic cells, where an increase in the MBICs was recorded in the order of 32-, 2.5-, 8-, 2-, and 16-folds, respectively. A similar pattern was detected in the case of gatifloxacin, ciprofloxacin, moxifloxacin, and ofloxacin where they showed higher MBICs by 8-, 8-, 2-, and 2-folds, respectively. Also, kanamycin, gentamicin, amikacin, ertapenem, and meropenem exhibited elevated MBICs by 4-, 4-, 2-, 2-, and 8-folds, respectively.

Results revealed that zinc sulfate at a concentration of 0.25 MIC (1 mg/ml) potentiates the antibiofilm effectiveness of ceftazidime, ceftriaxone, cefotaxime, cefepime, and cefpirome, where the MBIC decreased by a value of 16-, 8-, 4-, 4- and 8-folds, respectively. In the meantime, an enhancement in the antibiofilm potentials of kanamycin, gentamicin, amikacin, and ertapenem was recorded following combination with zinc sulfate in the order of 4-, 2-, 4- and 8-folds, respectively. On the other side, a reduction in the antibiofilm efficacies was observed following the combination between zinc sulfate and gatifloxacin, ciprofloxacin, moxifloxacin, ofloxacin, and meropenem by 4-, 4-, 2-, 2- and 4-folds, respectively. One dilution below the recorded MBIC of each of the tested antibiotics either in the presence or in the absence of zinc sulfate, where it confirms the biofilm formation (sub-MBIC), was further applied to examine the transcriptomic profile of biofilm formation genes as indicated in Table 2.

Effect of zinc sulfate on the transcriptomic profile of K. pneumoniae biofilm formation genes

In comparison to the control cells, the impact of various antibiotic concentrations (sub-MBIC) alone and in combination with zinc sulfate (0.25 MIC—1 mg/ml) against K. pneumoniae biofilm was assessed by comparing the expression profile of genes controlling biofilm formation using RT-PCR. Current findings demonstrated that zinc sulfate alone reduced the expression of biofilm-correlated genes luxS, mrkA, pgaA, wbbM, and wzm genes by 2.8-, 4.1-, 7.6-, 1.5- and 2.4-folds, respectively as compared to the control bacterial cells (Fig. 1).

Fig. 1

Gene expression profile of K. pneumoniae biofilm formation genes presented as fold change in the expression level post-exposure to zinc sulfate as compared to the control bacterial cells. BKP-122 biofilm forming K. pneumoniae clinical isolate, ZN zinc sulfate

Similarly, the examined biofilm-related genes were down-regulated following treatment with different classes of antibiotics. In this context, zinc sulfate enhanced the anti-biofilm efficacies of cephalosporins (e.g. cefotaxime, ceftriaxone, ceftazidime, cefpirome, and cefepime) via down-regulating the expression of biofilm-related genes in the order of 18-, 38-, 5-, 77- and 2-folds, respectively. Concerning the effect of zinc sulfate/cefotaxime combination on K. pneumoniae biofilm-associated genes, it was found that the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes were down-regulated by 16.5-, 21-, 18-, 7- and 35.4-folds as compared to that observed following exposure to cefotaxime alone, respectively. Also, zinc sulfate/ceftriaxone combination reduced the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 2.7-, 79.3-, 105-, 4.3-, and 1.7-folds, respectively. Moreover, the expression levels of the tested genes were decreased in the order of 9.9-, 6.9-, 6.1-, 5.9- and 9.3-folds following treatment with zinc sulfate and ceftazidime in combination, respectively. Analogously, zinc sulfate/cefpirome combination down-regulated the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 324.7-, 68.8-, 11.9-, 1.7- and 3.7-folds, respectively. Furthermore, the expression levels of luxS and wbbM genes were reduced by values of 4.3-, and 5.2-folds post-exposure to zinc sulfate/cefepime combination compared to that observed following treatment with cefepime alone, respectively (Fig. 2).

Fig. 2

Transcriptomic profile of K. pneumoniae biofilm-associated genes expressed as fold change in the gene expression level post-treatment with a: cefotaxime, b: ceftriaxone, c: ceftazidime, d: cefpirome, and e: cefepime alone and in combination with zinc sulfate

Regarding the transcriptomic modulatory potentials of zinc sulfate in combination with aminoglycosides (e.g. kanamycin, gentamicin, and amikacin), results revealed that zinc sulfate in combination with the tested aminoglycosides could reduce the expression of biofilm-elated genes by 40-, 2602- and 20-folds, respectively. The recorded data showed that zinc sulfate/kanamycin combination down-regulated the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes by 32.7-, 68.8-, 116.9-, 1.7- and 3.7-folds, respectively as compared to that with kanamycin alone. Moreover, zinc sulfate/gentamicin combination reduced the expression of luxS, mrkA, pgaA, wbbM, and wzm genes in the order of 3548.3-, 1214.2-, 785.5-, 7541.3- and 163.1-folds, respectively as compared to that with gentamicin alone. Additionally, zinc sulfate/amikacin combination exhibited down-regulation of the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes by values of 0.12-, 32.7-, 65.3-, 1.5- and 3.2-folds as compared to that with amikacin alone, respectively (Fig. 3).

Fig. 3

Expression pattern of K. pneumoniae biofilm-correlated genes presented as fold change following exposure to zinc sulfate in combination with a: kanamycin, b: gentamicin, and c: amikacin compared to sole treatment with antibiotics

On the other side, the tested fluoroquinolones (moxifloxacin, ofloxacin, ciprofloxacin, and gatifloxacin) in combination with zinc sulfate showed an antagonistic effect. That was apparent in that the tested antibiotics alone showed down-regulatory potentials on the biofilm-related genes, however, the combination of these antibiotics with zinc sulfate exhibited a reduction in this down-regulation by 2-, 2-, 15- and 14-folds, respectively. Recorded results demonstrated that zinc sulfate in combination with moxifloxacin antagonizes the antibiofilm potentials of moxifloxacin via reducing the down-regulation of the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes by 0.057–0.0025-, 0.00016-, 0.0038- and 0.08-folds compared to that observed in case of moxifloxacin, respectively. A comparable pattern was detected in the case of zinc sulfate/ofloxacin combination, where the expression levels of the biofilm-related genes were reduced in the order of -0.42-, 0.00009-, 0.01-, 1.71- and -0.0035-folds compared to ofloxacin, respectively. Moreover, the down-regulation of the tested genes was decreased following the combination between zinc sulfate and ciprofloxacin by values of 0.053-, 0.0086-, 0.00033-, 0.00044- and 0.0072-folds, respectively, compared to that recorded post ciprofloxacin treatment. In the same context, zinc sulfate antagonizes the efficacy of gatifloxacin by reducing the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 0.26-, 1.247-, 0.223-, 0.000001- and 0.0068-folds as compared to that observed in case of gatifloxacin alone, respectively (Fig. 4).

Fig. 4

Gene expression levels of K. pneumoniae biofilm-associated genes following treatment with sole a: moxifloxacin, b: ofloxacin, c: ciprofloxacin, d: gatifloxacin), e: ertapenem, and f: meropenem as well as in combination with zinc sulfate

Concerning the effect of zinc sulfate on carbapenems, the results revealed an enhancement in the efficacy of ertapenem via down-regulating the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes by 5.1-, 1.7-, 3.2-, 8.1- and 0.7-folds, respectively. On the other hand, it antagonizes the effectiveness of meropenem by up-regulating the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 1.2- 1.7-, 0.0003-, 0.009- and 1.9-folds, respectively (Fig. 4).

Effect of different treatments on the bacterial growth

Recorded data revealed that a non-significant difference in the growth pattern was detected at different time intervals following different treatments as compared to the control cells. Thus, the tested concentration of zinc sulphate as well as antibiotics either alone or in combination didn’t show considerable inhibitory effect on the growth of the tested isolate during the formation of the biofilm as compared to the control cells (Fig. 5).

Fig. 5

Bacterial growth curve of K. pneumoniae isolate following treatment with zinc sulphate and different antibiotics as well as zinc-antibiotic combinations at different time intervals compared to the control