View of Pattern of Multi Drug Resistance with Biofilm Formation among Klebsiellapneumoniaisolated from Fecal Samples of Diarrheal Iraqi Patients

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Pattern of Multi Drug Resistance with Biofilm Formation among Klebsiellapneumoniaisolated from Fecal Samples of Diarrheal Iraqi Patients

Walaa Farhan Obaid and Shaimaa Obaid Hasson*

Al-Qasim green university, veterinary medicine collage, microbiology department, Iraq

*Corresponding author: [email protected]


Klebsiella pneumonia(K. pneumonia ) is an opportunistic pathogen, Most of its cause of health care associated infections (HAIs) and possess high stages of antibiotic resistance. Thesemicro organisms are great recognized for his or her capacity to provide biofilms


The cause of this take a look at is to decide the sample of antibiotic resistance and the capacity to provide biofilms of K. pneumoniaefrom fecal samples of diarrheal sufferers in a few Hilla hospitals. Bacteria collected was isolated from patients with diarrheal cases in some Halli hospitals form the period ( September-October 2020). All of them were collected from both sex and differenced ages (<10- 60) years and it isolates were cultured and diagnosed by ordinary method as well as by the Vitek2 system also, Antibiotic sensitivity test(AST) were done using Vitek2 system and biofilm production detected by Congo-red agar method. From one hundred fecal samples, 75(75%) K. pneumoniae isolates were obtained from 25 male (33.3%) and 50 females (66.6%) . All bacteria isolates were resistant to Ampicillin at (100%) and more sensitive to Ertapenem, Imipenem, Piperacillin/Tazobactam,Amikacin,Tigecycline at(100%).50( 66.6%) MDR isolates were obtained from K. pneumoniae and 52 (69.3%) biofilms products isolates with 23 (30.6%) non-biofilms were produced. Significant difference in (p <0.05).from the current study, Most of theK. pneumonia bacteria isolates have proven resistant to a wide range of tested antibiotics and are biofilm-producing. This was a strong correlation between MDR ability and Biofilm formation in K pneumoniae.

Key words:

K. pneumonia, Multi drug resistance(MDR), Biofilm Production


The Klebsiella genus is one of the earliest members of the Enterobacteriaceae family, first reported in 1885 via way of means of the German microbiologist Edwin Klebs (1836- 1911) have been first-ever Klebsiellastrainwas a capsulated bacillus [1]. Bacteria is a gram- negative, lactose fermenting, non-sporulation,non-motile, oxidase negative with a striking capsule of polysaccharides[2]. It was present anywhere in nature,whichpeople colonize the skin, gastrointestinal tract, or pharynx, urine, sterile wound, alsoin lots ofelements of the biliary tract, nostrilalso mouth is probablytaken into consideration as ordinaryflora [3]. Also, it'sopportunistic pathogen related toeach community-receivedalso nosocomial infections, inflicting pneumonia, abscess, bacteremia, also urinary tract infections [2]. Recently,different microorganisms [ K.

pneumoniae like ] evolve multi- drug-resistant, It has attracted increasing interest globally as an infectious microorganism due to the most recent upward push within a wide range of excessive bacterial infections antibiotic resistance, The problem of preparing powerful therapies is also


developing. It is now the second most unusual location for the cause of gram-poor bacteremia and also the first-order pathogen in contamination a hospital receives, especially in immunocompromised patients[4]. Bacteria is generate special enzymes that deactivate particular also goal elements of antibiotic. Beta-lactam are commonly the focutilized one by of means of produced enzymes, even as a few goal different drug classes, as well as fluoroquinoloness, aminoglycosides, trimethoprim, also sulfamethoxazole. This enzymes together with Metalloo- beta-lactamases, oxacillinases, prolonged range beta-lactamases, also K. pneumonia carbapenemasess, alsoa not the same special enzyme. These enzymes stand coded on the K.

pneumonias plasmids[5]. Klebselliaihas a few virulence elementstogether withtablet polysaccharide, lipopolysaccharide, kind 1 also kindthree fimbriae, outsidetissue proteins, alsofactors for iron acquisitionsalsonitrogengas supply usage. K. pneumoniaeutilizedthose virulence elements for be alivealsoto a stay away from the impregnablemachineat some point ofcontaminationin addition to biofilm formation itself [6, 7]. K. pneumoniae can products a heavysheet of extracellular biofilm that helps the microscopicengagement to dwelling or non- dwelling surfaces, protective antibiotics penetration, alsolowering its effects [8]. The antibiotic surrender of developedmicroscopicbiofilm is 10–1,000 instances that of planktonic microorganisms, alsomicroorganism in biofilms can withstalso phagocytosis, making them very tough to eliminate [9]. K. pneumoniae alsoK. oxytoca, each commensal of the human gastrointestinal tract, every so oftenreason diarrhea in people. Some of those diarrhea genic traces encode the thermos table or thermo labile toxin [10]. However, the positionof thosepollutantswithinside the pathogenesis of Klebsiella–related diarrhea has now no longer been clarified. There have been no sequences detected on this isolate that has been homologous to genes of input pathogenic E. coli traces that code for thermolabile also thermostable pollutants, that are produced via way of means ofa fewK. pneumoniaetraces[11, 12]. Around the world, antimicrobial resistance in the latest healthcare representing a developing issue. Several pathogenic microorganismtraces are Multidrug-resistant (MDR), which arequite simplyturning into widespread, that forming an excessivechance to patients. Today, the K. pneumoniae was taken into consideration the maximumfamous species of microorganisms that generate health care problems [13]. Aims this study a look atturned into to pick outthe connectionbetween antibiotic resistance stylesalso the biofilm-formation ability of K. pneumoniae fecal isolates from diarrheal humans.

2 . MATERIALS AND METHODS 2.1.Collection of sample

One hundred stool specimens had beengathered in disposable, smooth screw-capped, commercially to be hadpacking containersutilized for this purpose. All the specimens had been processed right away or utilized Carry Blair shipping media if not on time for 1-2 hours after their seriesafter which cultured. The current examine diarrheal isolates of K. pneumoniae from diarrhea sufferers to eachintercourse at distinct age group from AL- Hilla Teaching, AL- Qasim General Hospital, also Al-Hashimiya General Hospital from September-October 2020.

2.2. Isolation of microorganism

Fecal samples had been cultured in enrichment media ( heart infusion broth) had been incubated at 37C for 24hrs. then the bacterial growth was cultured to automatically


5352 media on (Nutrient also MacConkey agar ) had been incubating at 37 C for 24hrs. The positive growth culture was counted according to count bacteria , with biochemical check, staining, also microscopic exam had been done . Bacterial isolates recognized via way of means of the automatic gadget VITEK 2 to attain the very last diagnostic. The identity with VITEK 2 consists of an ID-GN card for gram-poor microorganisms.

2.3. Biofilm Formation Assay

1- Congo Red agar Test . Brain Heart Infusion Broth, agar supplemented with 50gm/l sucrose also 8gm/l Congo red had been organized consistent with[14]. Then study the end result as following: if the microorganismshaped black colonies with a dry crystalline consistency that changed intoimplying it biofilm manufacturer isolates whilst if it shapedcrimson colonies that changed intoimplying the non-biofilm manufacturer isolates [15].

2- Tissue culture plate Test (TCP) : (additionally referred to as semi-quantitative microtiter plate check (biofilm assay) defined via way of means of[16]changed intomaximumextensivelyutilizedalsochanged intotaken into consideration as preferredcheck for detection of biofilm formation as follow:

1-Isolates from purified agar plates were added to TSB containing 1% glucose, then incubated at 37 °C for 18 hours, then diluted in a ratio of 1:100 with pure TSB.

2- sterile, polystyrene, 96 pcs. The posterior biological tissue wells are filled with 150 μl of purified culture medium and a fine soup is used as a function to check the binding of unknown substances. Each subdivision turned into three vaccinations. After incubation for 4 hours at 37


3. After growth, gentlytouch the plate to remove all thecontents.Wash thesprings 4times with phosphate saline (PBS pH 7.2) to remove airborne bacteria.

4.Biofilms formed by adherent ‘sessile’ organisms in plate were fixed by placing in oven at 37C° for 30min

5. Allsourcesare stained with 5-crystal violet (0.1% w/v). The dyeing process is alsodoneby washingthoroughly with water extractedfrom the water, which is kept drying.6-150μl acetone/ethanol (20:80, v/v)binding bond. It gives a bluemelting. OpticalQuantity (O.)630nm had been recorded also the effectshad been interpreted consistent with a table (1).

Table 1: Interpretation of biofilm formation Mathur et al (2006).

2.4. Antibiotic testing by VITEK-2 Compact

Antibiotic trying out become accomplished with the automatic VITEK-2 compact gadget primarily based totally on MIC method dedication through the Mean of OD value at 630nm Adherence Biofilm formation

<0.120 non Non

0.120-0.240 Moderately Moderate

>0.240 Strong High


usage of also AST-N222 playing cards gram-negative. These playing cards contained the subsequent Antibiotics, Ampicillin, Piperacillini/ tazobactami, Cefazolini, Cefoxitini, Ceftazidimei, Ceftriaxonei, Cefepimei, Ertapenemi, Imipenem, Amikacini, Gentamicini, Ciprofloxacini, Levofloxacini, Tigecyclinei, Nitrofurantoini, Trimethoprim/sulfamethoxazolei.



Frequencies and percentages were used to explain the variables in this study.

Associations among the antibiotic sensitivity sample also the biofilm generating potential of K. pneumoniae had been examined through Chi-rectangular checks the usage of(SPSS VER.16). The effects had been offered as incidence ratios with a 95% self-belief interim. Statistically importance become set if p-value .

3.Resultsand Discussion

3.1.Identification of Klebsiella pneumoniae

From September-October 2020, 75(75%) K. pneumoniae isolates have been tested from 100 general fecal bacterial isolates also 25% isolates of different Enterobacteriaceae(10% showed Escherichia coli, 10% showed Raoultella ornithinolytica also 5% showed Citrobacter sedlakii) Figure (1) display those results.

Figure (1):Number also Percentage of Enterobactericeae isolates from Human Diarrhea These results, the percentage of K. pneumoniaeseparatesbecomes 75% of the overall fecal samples bacterial isolates. This end resultbecomes in line with [17] who located that approximately 65% of inpatients with diarrhea are precipitatedthroughK. pneumoniae,also this outcome different from [18]. How based 22% K. pneumoniaelinesprecipitated diarrheal in sufferers. It is one of themaximumnot unusual place for Gram-terrible pathogen located in human’s nasopharynx alsowithinside the intestinal tract [19]. Also due to the fact this microorganism have lipopolysaccharide represents a criticalalsocriticalissue in bacterial pathogenicity, especially K. pneumoniae, as it's milesone of the superficial compositions of



Enterobactericeae bacteria

K.pneumonia E.coli

R.ornithinolytica C.sedlakii


5354 microorganism that assist it to withstalso phagocytosis, alsoit's milescharacterizedthrough its cappotential to set off the supplement factor [19].Virulence of K. pneumoniae is related to the presence of capsule also piles, to the manufacturing of lipopolysaccharides also siderophores, to allantois utilization, also to iron uptake systems, efflux pumps, also kind VI secretion systems [20]. Also, it have the piles is taken into considerationessential to the virulence of Klebsiella, because it protects the bacterium from phagocytosis also forestalls the microorganismthrough bactericidal serum factors [19]. Also,amongst this outcomesmicroorganism isolates had beenaccumulated from speciallong-timesufferershowever themaximum isolates had beenon the age <10[21]. K. pneumoniae is locatedwith inside the intestinal floraof healthful individuals, howevergenerally in small numbers [22].

Enterobacteriaceae considered as the maximum place pathogens remoted from fecal samples in growing countries. Underlying situations including malnutrition, loss of secure water, insufficient sanitation also of diarrhea spreading in growing countries[23]. Because of that,K.

pneumoniae had beenisolated in excessive numbers from the small bowel of people with acute diarrhea [24]also malnourished youngsterswho'vepersistent diarrhea [25].

All the fecal samples had beencollectedfrom 25 males (33.3%) also 50 females (66.6%). Most of K. pneumoniaeobtained from sufferers with diarrheal instancesof variousa long time (table 2).

Table (2):Demographic characteristics of patients with K. pneumonia infections P

value X2

No .of K.

pneumonia (%)

No . of

K.pneumoniae (n=75)

Status Patient



16.66 40%


<10 Age group (Years)

0 0



10 20-30


15 30-40


10 40-50


10 50-60


46.8 33.3%

25 Male



50 Female

X2 : chi-square test, * significant difference (P<0.05)

3.2. Biofilm Formation Discovery

Among the 75 K.pneumoniae isolates tested, Appeared on Congo Red agar 52(69.3%) isolates as biofilm production also 23(30.6%) isolates that have been now no longer biofilm producers (Fig 2)suggests those results. This end result similar with[26].The ability to form biofilm appears differently from one isolation to another due to the fact there are numerous elements such as physical and chemical factors . From the characteristics of K. pneumoniae, the physical interaction among Plug, form of the floor in which biofilm suspends, temperature, pH, etc. [27].


Those results , among the fifty two (69.3%) K.pneumoniae biofilm maker show result by tissue culture plate 35(46.6%) separates as solid, 15(20%) separates as moderate2(2.6%) separates as weak biofilm makers(Table3). This consequence remained in line with Nirwati et al.,2019. it had been rumored that out of the 167 K. pneumoniae tested, forty five isolates (26.95%) were known as high or medium biofilms also fifty separates (29.94%) were weak biofilm makers. The ability to create biofilms was totally different for every isolate as a result of many factors typically have an effect on the capacitance appreciate the physical also property of K. pulmonary, the physical interface between machineries, the kind of external on that the biofilms adhere, temperature, pH, etc.[27].

Table 3 Biofilm manufacturing capability of K.pneumoniae isolates by Tissue Plate Culture.

3.3. Antibiotic susceptibility profiles of K. pneumoniae

Most of K.pneumoniae have been resistant to wide rang variety of examined antibiotics. All K.

pneumoniae strain more resistance against Ampicillin at( 100%) also touchy to Ertapenem, Imipenem Piperacillin/Tazobactam, Amikacin, Tigecycline at (100%). As well as this bacteria resistance to Cefazolin (86.6%), Ceftriaxone, Ceftazidime, Cefepime, Trimethoprim/Sulfamethoxazole(60%) resistant respectively(Table 4) indicates those consequences. This document is supported through the observation performed by [21]. Who found were more resistances to Ampicillin [100%]. As nicely as we consequence comparable with [28]from whereinexcessivetouchy to Piperacillin/Tazobactam, Imipenem also Amikacin.

Exposure to antibiotics is the maximumcrucialissue in antimicrobial resistance. Numerous factors including antibiotics used in hospital, in the community, or even in animal production,

Number(%) Characteristics

23(30.6%) Non-biofilm producer

35(46.6%) Strong biofilm producer

15(20%) Moderate biofilm producer

2(2.6%) Weak biofilm producer

75(100%) Total

(Figure 2) K.pneumoniae biofilm formation on Congo Red Agar


5356 agriculture, as well as the environment have led to the flourishing of antibiotic resistance.Given the reality that antibiotics may be sold unfastened without a prescription, also antibiotics are utilized excessively withinside the society environment, it's miles very in all likelihood that the heavy also extended use of antibiotics is the number one issue with inside the unfold of contamination also resistance to difficult-to-deal with antibiotics[29].Among theantibacterial agents are circulating transmissibleplasmids, which may also carrydeterminants of virulence.

The capsule is usuallyreferred to as a clear polysaccharideinside K. pneumoniae isolates. The phenotype of mucosal capsules is an important virulence factor for K. pneumonia. The plasmid gene that regulatesthe mucosalphenotype (rmpA) hasbeen found togiveK. pneumoniae[6].

Which reported that 100% of K. pneumoniae was highly resistant , has been shown to ampicillin, the third generation of cephalosporin and aminoglycosides.

(Table 4) Antibiotic sensitivity profile of studiedK. pneumoniae Classes Antibiotics Resistance

rate %

MIC Sensitive rate %



Ampicillin 100% >16 0 0



0 0 100% <= 4

Cefazolin 86.6% >32 13.3% <= 4

Ceftriaxone 60% >32 40% <= 1


Cefoxitin 33.3% >32 66.6% <= 4

Ceftazidime 60% 8 40% <= 1

Cefepime 60% 2 40% <= 1


Ertapenem 0 0 100% <= 0.5

Imipenem 0 0 100% <= 0.25


Amikacin 0 0 100% <= 2

Gentamicin 20% >8 80% <= 1


Ciprofloxacin 6.6% 0.5 93.3% 1

Levofloxacin 6.6% >4 93.3% 1

Glycylcycline Tigecycline 0 0 100% 1

Nitro furans Nitrofurantion 46.6% 128 6.6% 32

Sulfonamides Trimethoprim/


60% >160 40% <= 20 X2 585.79

P value 0*



: chi-square test, * significant difference (P<0.05)

3.4. Relationship between Biofilm formation and Multi Drug Resistant

K.pneumoniae MDR isolates have beendiscovered in 50(66.6%) isolates also 25(33.3%) isolates have been non- MDR(Table 5) The become no tremendousaffiliation between K. pneumoniae MDR also biofilm manufacturingabilityprimarily based totallyat thestatisticallyevaluationthe use of chi- rectangular tests These antimicrobial-resistant micro organisms have turn out to be a globalhasslealsothere may benonetheless very confinedfactsconcerning biofilm generatingabilityalso antimicrobial resistance of K. pneumoniae. This observationconfirmed that


antibiotic resistance become the biggest among K. pneumoniaealsoit's far a biofilm manufacturer from a non- biofilm product. This end result has been said in numerousresearch. Astudiesby[30, 31]highlights for excessivedrug resistance K. pneumoniae the capacityto supply pulmonary biofilms is associated with antibiotics resistance profile. The generaloccurrence of MDR K.

pneumoniae isolates on thisobservesbecome 50 (66.6%). Some precedingresearchsupport this excessiveoccurrence of MDR K. pneumoniae[32].

(Table 5)Relationship between Multi Drug Resistant (MDR) with Biofilm Producing among K.


Resistance of Antibiotic classes

Biofilm producer

Positive Negative

≥ 3classes 39(84%) 11(16%)

<3 classes 13(52%) 12(48%)

Total 52 23

X2 5.29*

P value 0.021

The MDR pathway encountered by microbes constitutes a major task in infection, and therefore, it is essential to monitor as well as improve screening also with antibiotics through antibiotic stewardship programs.Several research have proven that remedy with a set of antibiotics can allowsave you new resistance from risingtraceswhereinremedyscrew-ups are typicallyobserved in those whoget hold of antibiotic remedysimplestas soon asthat stillcrucial for physicians also microbiologists collaborate to make it also emphasized strong pollution control [33]. K.

pneumoniae isolates that confirmed résistance to 3 or extraspecifictraining of antimicrobials have beencategorized as multidrug-resistant (MDR) K. pneumoniae[34].Biofilm formation is a be aliveapproach for micro organismalso fungi to conform to their dwelling environment, specificallywithinside theadverse environment. Under biofilm protection, microbial cells in biofilms grow to be tolerant alsoproof against antibiotics alsoimpregnable responses, which will increase the problems of the medicalremedy of biofilm contamination. Clinical also laboratory examinations confirmed a clean link among biofilm contamination also clinical foreign bodies or static devices. Clinical also experimental observations[35]. It confirmedthat each one biofilm generating isolates suppliedextra resistant stylesas compared to the non-biofilm producers, however, no matter this end result, the protection mechanisms in biofilms vary from the onesaccountable for traditional antibiotic resistance[36]. In biofilms, it's far assumed that the protectingmasking of the adhesive biomaterial results interrible antibiotic penetration, adaptive responses to stress, also the formation of everlasting cells a multilayered defense, which will increasethe issue of eradication, specificallywhilstblended with the resistant nature of the microorganism itself[37, 38]. It seems that antibiotics resistance as well as bacterial capacity to biofilmsformation . It performsa critical function in the international spread ofK. pneumoniae, and to date the clearlinkbetween these elements has not been diagnosed in detail either. This finding is supported by [39]This has been showingvia way of means ofnumerousresearch in a few cases, the antibiotic remedyisn't sufficient to remove biofilm-forming infections.

Alsoconsequently,presentlyavailablecontaminationcontrol antibiotics have grown to bealso the


5358 consequenceshave been evaluated crucialalsopressing protocols for a hitremedy from biofilm- associated infections[40]. Generally, an allergy to antibiotics is vital to test. Collect medical specimens previous to antibiotics controlis likewise a vital point. Many medical doctorsindividuals who prescribe antibiotics do now no longerabsolutelyapprehend if simplest their beside the point recipes may want to have an impacton theimprovement of bacterial resistance. Initial settings Antimicrobial remedyprimarily based totally on medical microbiology the end result will lessenchoicestress on microorganisms in instances of contamination in hospitals. Thus, it's far from the highsignificanceof each hospital antibiotic counseling or supervision application for all pharmacists alsomedical doctorsat themaximumcorrectfoundation microbiological data. In conjunction with this directive's consistentattempt in tracking hospitals also infection control, medical audits have to be achieved to combat the speedyimprovement of antibiotic-resistant pathogens[36].

4. Conclusion

Most of theK. pneumonia bacteria isolates have proven resistant to a wide range of tested antibiotics and are biofilm-producing. This was a strong correlation between MDR ability and Biofilm formation in K. pneumonia.


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