Phenotypic, antimicrobial susceptibility profile and virulence factors of Klebsiella pneumoniae isolated from buffalo and cow mastitic milk

Abstract

Studies on the prevalence and virulence genes of Klebsiella mastitis pathogens in a buffalo population are undocumented. Also, the association of rmpA kfu, uge, magA, Aerobactin, K1 and K2 virulent factors with K. pneumoniae buffalo, and cow mastitis is unreported. The virulence of K. pneumoniae was evaluated through both phenotypic and molecular assays. In vivo virulence was assessed by the Vero cell cytotoxicity, suckling mouse assay and mice lethality test. Antimicrobial susceptibility was tested by disk diffusion method. The 45 K. pneumoniae isolates from buffalo (n = 10/232) and cow (n = 35/293) milk were isolated (45/525; 8.6%) and screened via PCR for seven virulence genes encoding uridine diphosphate galactose 4 epimerase encoding gene responsible for capsule and smooth lipopolysaccharide synthesis (uge), siderophores (kfu and aerobactin), protectines or invasins (rmpA and magA), and the capsule and hypermucoviscosity (K1 and K2). The most common virulence genes were rmpA, kfu, uge, and magA (77.8% each). Aerobactin and K1 genes were found at medium rates of 66.7% each and K2 (55.6%). The Vero cell cytotoxicity and LD (50) in mice were found in 100% of isolates. A multidrug resistance pattern was observed for 40% of the antimicrobials. The distribution of virulence profiles indicate a role of rmpA, kfu, uge, magA, Aerobactin, and K1 and K2 in pathogenicity of K. pneumoniae in udder infections and invasiveness, and constitutes a threat for vulnerable animals, even more if they are in combination with antibiotic resistance.

Introduction

Escherichia coli, Enterobacter aerogenes, Klebsiella pneumonia, Serratia marcescens, and Streptococcus sp. are the major organisms found to cause environmental mastitis.¹ Among the Gram-negative bacterial causes of mastitis, Klebsiella spp. cause the most severe cases.^2–4

The search for the pathogenic mechanism of Klebsiella infections has identified a number of virulence factors in K. pneumoniae associated with primary pyogenic liver abscess which include capsular polysaccharides especially K1 or K2, lipopolysaccharides, hypermucoviscosity, adhesins, and iron acquisition systems.^5–11 However, very little is known about the presence and roles of virulence-associated genes products involved in the pathogenesis of K. pneumoniae associated with mastitis.

Subclinical diagnosis of Klebsiella infections commonly is through milk cultures performed at herd surveys. Several isolates that were identified as being K. pneumoniae based on phenotypic, failed to differentiate Raoultella spp., some E. cloacae isolates and a single Providencia isolate from Klebsiella spp.^13–15

Consequently, we evaluated Klebsiella by both phenotypic and molecular assays through the use of 16S rRNA for species identification and virulence genes for Klebsiella identification. Studies on the prevalence and correlated virulence genes of Klebsiella mastitis pathogens in a buffalo population are undocumented. Also, the association of the some virulence determinants: K1, K2, magA (mucoviscosity-associated gene specific to K1 capsule serotype), uge (uridine diphosphate galactose 4 epimerase encoding gene responsible for capsule and smooth lipopolysaccharide synthesis) and kfu (iron uptake system gene), rmpA gene (regulator of the mucoid phenotype), and aerobactin in buffalo and cow mastitis are unreported. Therefore, in the present study, we screened buffalo and cows affected by clinical or subclinical mastitis for Klebsiella species via microbiological, biochemical characteristics and molecular techniques. In addition, antimicrobial resistance of Klebsiella isolates was studied for their antimicrobial resistance pattern.

Materials and Methods

Sample collection

The study took place in private dairy farms surrounding Cairo, Egypt. A total of 525 milk samples were aseptically collected from apparently healthy (buffalo, n = 112 and cows, n = 125), subclinical (buffalo, n = 23 and cows, n = 10), and clinical mastitic animals (buffalo, n = 97 and cows, n = 158). The animals had not been treated with an antibiotic for at least 30 days prior to collection. Udders and teats of the randomly selected, lactating animals were cleaned, dried, and disinfected with 70% ethanol before sample collection. Three streams of foremilk was expressed from each quarter and then discarded. During sample collection, 10–15 ml of milk from each quarter was manually expressed into separate sterile 25 ml universal tubes. After gently suspending each sample, the milk was poured into a separate, unused container. As previously indicated,¹⁶ a composite milk sample representing one udder was created; this sample was comprised of all four quarters in one collection vial. A subsample of 15 ml of milk, taken from the composite milk sample, was transferred to sterile universal bottles. The milk samples were quickly transported to the laboratory under chilled conditions and stored at 4°C until bacteriologically analyzed, and had its antimicrobial susceptibility, pathogenicity, and molecular typing determined.

Milk samples were processed using routine methods for the isolation and identification of mastitis pathogens.¹² Briefly, approximately 0.01 ml of each milk sample was swabbed onto trypticase soy agar with 5% sheep blood and 0.1% esculin. Growth was evaluated after 24 and 48 hours of incubation at 37°C. For samples from buffalo and cows with CM, a 1∶1 mixture with Todd-Hewitt broth was prepared, and after 4 hours of enrichment at 37°C, additional TSA-BE plates were streaked and incubated in the same manner as the rest of the milk samples. Coliform isolates were identified based on colony morphology, subcultured onto MacConkey and incubated overnight at 37°C. Pink-yellow, mucoid, lactose-positive colonies were considered to be Klebsiella spp. and identified to the species level based on motility and biochemistry (citrate utilization and indole production).¹² The isolates were classified phenotypically as mucoid or non-mucoid. Colonies were touched with a loop; the loop was then lifted vertically from the surface of the agar plate. Mucoid phenotype was defined as being present when a string-like growth was observed to attach to the loop as it was lifted from the plate.¹⁷

Antimicrobial susceptibility

Once they had been isolated and identified, pure cultures of Klebsiella were tested for an antibacterial susceptibility panel of 28 antibiotics belonging to 11 drug classes (Table 3) with the disc diffusion assay on Mueller-Hinton agar. Testing was performed according to the recommendation of the Clinical and Laboratory Standards Institute.¹⁸ Antimicrobials were selected for testing based on the licensing for mastitis treatment in cattle, use in human medicine and potential resistant-determinant phenotypes.^19,20

Table 3. Antimicrobial susceptibility patterns of nine Klebsiella isolates.

Antimicrobial agents	Susceptibility
	Susceptible		Intermediate		Resistance
	n	%	n	%	n	%
Ampicillin (10 μg)	0	0	0	0	9	100
Carbenicillin (100 μg)	9	100	0	0	0	0
Cephotaxime (30 μg)	9	100	0	0	0	0
Chloramphenicol (30 μg)	0	0	2	17.4	8	82.6
Colistin Sulphate (10 μg)	0	0	2	17.4	8	82.6
Erythromycin (15 μg)	0	0	2	21.7	8	87.3
Flumequine (30 μg)	9	100	0	0	0	0
Gentamicin (10 μg)	9	100	0	0	0	0
Kanamycin (30 μg)	9	100	0	0	0	0
Neomycin (30 μg)	8	86.9	2	13.0	0	0
Nitrofurantoin (300 μg)	9	91.3	1	8.7	0	0
Oxytetracycline (30 μg)	7	69.6	3	30.4	0	0
Penicillin G (10 IU)	0	0	3	21.7	7	78.3
Streptomycin (10 μg)	0	0	2	17.4	8	82.6
Sulphamethoxazole/Trimpethoprim (1.25 μg+23.75 μg)	6	60.9	2	21.7	2	17.4

Capsule presence by light microscopy

Capsule formation was assessed by India ink method. An unstained region around central bacterial cells indicated the presence of a capsule.¹⁷

Assessment of Congo red uptake

The method of Qadir et al.²¹ was used. Congo red (0.003%, w/v) was incorporated into nutrient agar before autoclaving. Plates streaked with test strains were incubated at 37°C for 18 hours. Colonies were examined for the presence (red, crb +ve) or absence (white, crb −ve) of Congo red binding.

Cytotoxicity of Klebsiella species

The procedure of Raja et al.²² was adopted. Cell Free Culture Supernatants of the nine Klebsiella isolates were prepared. Each bacterial strain was grown in Brain Heart Infusion broth. All of the culture supernatants were adjusted to pH 7.3–7.5. One hundred microliters of 5×10⁴ tissue culture cells per milliliter in Eagle’s minimal essential medium containing kanamycin and 10% fetal calf serum was added to the wells of 96-well microtiter plates and incubated in air containing 5% CO₂ at 37° for 2 days. Aliquots of 25 μl of the cell-free culture supernatant of Klebsiella diluted in a two-fold series with Dulbecco’s phosphate-buffered saline (pH 7.4) were then added to 100 μl of the monolayer cultures in a well, and the microtiter plates were incubated at 37°C in CO₂ incubator for 48 hours. The results of the cytotoxicity assay was expressed as a reciprocal of the highest final dilution of culture supernatant that caused rounding of more than 90% of cultured cells in the wells.

Animal inoculation

This work was performed in accordance with the recommendations in the updated Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.²³ All procedures were approved by the Cairo University Ethical Committee in compliance with the United Kingdom (UK) Animals (scientific procedures) Act of 1986 and all required approvals were obtained prior to the experiments.

Mice lethality test

Female BALB/cByl 6-week-old mice were used for inoculation. K. pneumoniae inocula consisting of 10²–10⁶ mid-logarithmic growth phase colony-forming units were diluted in 100 μl normal saline and injected intraperitoneally.²⁴ Survival of the inoculated mice was recorded and the LD₅₀ was calculated using the SigmaPlot (version 7.0) program from SPSS Inc. (Chicago, IL, USA).

Suckling mouse assay

The K. pneumoniae strains to be tested for production of heat stable enterotoxin were screened by the suckling mouse assay according to Giannella.²⁵ Strains were grown in casamino acid yeast extract and 0–1 ml was injected intragastrically in mice 2–4 days old. After three hours the mice were killed by decapitation and the ratio from gut weight divided by body weight was determined. Values greater than 0–0.83 were considered positive; values less than 0–0.75 were considered negative; intermediate results were considered doubtful.

Molecular typing

Molecular methods were used for strain typing and to confirm the species identity of isolates that had been classified as Klebsiella spp. based on phenotypic characteristics. Crude DNA extracts from Klebsiella isolates were obtained by 10-minute boil preparation and used as templates for m-PCR.²⁶ Strain typing was performed using m-PCR. The primers, sequences, conditions, and predicted sizes of the amplified products are outlined in Table 1. New Klebsiella-like colonies were boiled in 400 μl of 1× Tris-EDTA buffer (pH 8.0) for 10 minutes and centrifuged at 14 000 rev/min for 10 minutes to remove denatured proteins and bacterial membranes. Klebsiella spp. was distinguished on the basis of the 16S rRNA revealed by PCR. Amplification was performed in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA). Electrophoresis of amplified products was carried out using 1.5% agarose gels, with 20 5-mm-wide wells, run in 0.5× Tris-borate-EDTA buffer for 1.5 hours in a horizontal electrophoresis system at ∼95 V. Gels were stained with ethidium bromide and visualized through UV transillumination with the Molecular Imager Gel Doc XR system and Quantity One software, version 4.4.1 (Bio-Rad, Hercules, CA, USA).

Table 1. Oligonucleotide primers sequences and size of the PCR-targeted products for the Klebsiella species.

Primer	Function	Forward	Reverse	bp	cycling conditions	Reference
K1	On the basis of mouse lethality, capsular serotype is the most virulent when injected intraperitoneally	5′-GGTGCTCTTTACATCATTGC-3′	5′-GCAATGGCCATTTGCGTTAG-3′	1283	95°C for 5 minutesAmplification (35 cycles of) 94°C for 30 seconds, 58°C for 90 seconds, 72°C for 90 secondsFinal extension 72°C for 10 minutes	6
K2		5′-GACCCGATATTCATACTTGACAGAG-3′	5′-CCTGAAGTAAAATCGTAAATAGATGGC-3′	641		27
16S rRNA	Used for species identification	5′-ATTTGAAGAGGTTGCAAACGAT-3′	5′-TTCACTCTGAATTTTCTTGTGTTC-3′	130		28
rmpA	Extracapsular polysaccharide synthesis regulator (regulator of the mucoid phenotype A)	5′-ACTGGGCTACCTCTGCTTCA-3′	5′-CTTGCATGAGCCATCTTTCA-3′	536		29
uge	Related to sugar nucleotide epimerases (for uridine diphosphate galacturonate 4-epimerase)	5′-TCTTCACGCCTTCCTTCACT-3′	5′-GATCATCCGGTCTCCCTGTA-3′	534	94°C for 1 minutesAmplification (35 cycles of) 94°C for 1 minute, 55°C for 45 seconds, 72°C for 2 minutesFinal extension 72°C for 7 minutes	17
kfu	Responsible for an iron uptake system	5′-GAAGTGACGCTGTTTCTGGC-3′	5′-TTTCGTGTGGCCAGTGACTC-3′	797		30
magA	Causes hypermucoviscosity (mucoviscosity-associated gene A)	5′-GGTGCTCTTTACATCATTGC-3′	5′-GCAATGGCCATTTGCGTTAG-3	1280		27
Aerobactin	A hydroxamate-type siderophore iron-uptake system	5′-GCATAGGCGGATACGAACAT-3′	5′-CACAGGGCAATTGCTTACCT-3′	556		26

Results

Prevalence and antibiotic profile

The results revealed the prevalence for K. pneumoniae from cow was 11.9%, while in buffalo, it was significantly lower (4.3%) (Table 2).

Table 2. Prevalence of Klebsiella species in buffalo and cow raw milk.

Animals	n of examined samples	K. pneumoniae		Total
		n	%	n	%
Buffalo raw milk
Apparently healthy animal	112	6	5.4	6	5.4
Subclinical mastitic animal	23	…	…	…	…
Clinical mastitic animal	97	4	4.12	4	4.12
Total	232	10	4.3	10	4.3
Cow raw milk
Apparently healthy animal	125	12	9.6	12	9.6
Subclinical mastitic animal	10	…	…	…	…
Clinical mastitic animal	158	23	14.6	23	14.6
Total	293	35	11.9	35	11.9

The obtained results recorded in Table 3 releaved that, all strains of Klebsiella species were susceptible to carbenicillin, cephotaxime, flumequine, gentamicin and kanamycin (100%). Most strains were susceptible to neomycin (86.96 %) and nitrofurantoin (91.30 %). On the other hand, most K. pneumoniae were resistant to ampicillin (100%), chloramphennicol, colistin sulphate, erythromycin and streptomycin (88.9%), and penicillin G (77.8%).

Relationship between capsular serotype, Vero cell cytotoxicity, and lethality in mice

The results in the present study indicated that all Klebsiella isolates were capsulated. When the mastitic strains were examined, 100% (9/9) cytotoxicity and lethality were observed with strains of K1 and K2 serotypes.

Relationship between mucoid phenotype, serotypes, and infection

The mucoid phenotype was observed in 100% (9/9) of strains from mastitic animals (Table 4); 100% (9/9) of the isolates of serotypes K1 and K2 were found to be mucoid.

Table 4. Distribution of the virulence genes within the isolated K. pneumoniae strains.

Animals	Virulence genes
	K1	K2	rmpA	kfu	uge	magA	Aerobactin
Buffalo
	D	ND	ND	D	D	ND	D
	D	ND	ND	ND	ND	ND	D
	D	D	D	ND	ND	D	D
	D	D	D	D	D	D	D
Cow
	D	D	D	D	D	D	ND
	D	ND	D	D	D	D	ND
	ND	ND	D	D	D	D	D
	ND	D	ND	D	D	D	ND
	ND	D	D	D	D	D	D

Note: D, detected; ND, not detected.

Association between phenotypic evidence of mucoidity, and presence of rmpA gene

Phenotypic evidence of mucoidity was highly correlated with the presence of the rmpA gene. Of the nine mucoid isolates, 77.8% (7/9) were rmpA gene-positive, and 22.2% (2/9) were rmpA gene-negative (Table 4).

Relationship between aerobactin production and type of infection

The presence of the rmpA gene and phenotypic evidence of aerobactin production were closely correlated. 57% of rmpA gene-positive isolates were aerobactin producers (4/7); aerobactin was produced by 100% of isolates that were rmpA gene-negative (2/2) (Table 4).

Molecular identification and virulotyping

The results observed in Fig. 1 show the positive amplification of the 130bp fragment of primer specific for the 16s rRNA gene in all of the examined K. pneumoniae (9/9; 100%). The isolated K. pneumoniae strains revealed that they were encoded by K1 (6/9; 66.7%), K2 (5/9; 55.6%), rmpA (7/9; 77.8%) genes (Fig. 2), uge (7/9; 77.8%), aerobactin (6/9; 66.7%), kfu (7/9; 77.8%), and magA (7/9; 77.8%) genes (Fig. 3).

Figure 1.

Agarose gel electrophoresis showing positive amplification of product 130 bp fragment of 16srRNA gene of Klebsiella pneumoniae performed with specific primer. Lane 1: 100–1500 bp DNA ladder; lane 2: negative control E. coli ATCC 25922; lane 3: positive control K. pneumoniae ATCC 13883; lanes 4–6, 8, and 9: K. pneumoniae from cow milk; lane 7: negative control S. Typhimurium 14028; lanes 10–13: K. pneumoniae from buffalo milk.

Figure 2.

Results of the m-PCR amplification of products 16s rRNA, rmpA, k2, and k1genes at 130, 536, 641, and 1283 bp in all of the nine tested K. pneumoniae (lanes 4–13). Lane 1: 100–1500 bp DNA ladder; lane 2: negative control E. coli ATCC 25922; lane 3: positive control K. pneumoniae ATCC 13883; lanes 4–6, 8, and 9: K. pneumoniae from cow milk; lane 7: negative control S. Typhimurium 14028; lanes 10–13: K. pneumoniae from buffalo milk.

Figure 3.

Results of the m-PCR amplification of products uge, aerobactin, kfu, and magA genes at 534, 556, 797, and 1280 bp in all of the nine tested Klebsiella pneumoniae. Lane 1: 100–1500 bp DNA ladder; lane 2: negative control E. coli ATCC 25922; lane 3: positive control K. pneumoniae ATCC 13883; lanes 4–6, 8, and 9: K. pneumoniae from cow milk; lane 7: negative control S. Typhimurium 14028; lanes 10–13: K. pneumoniae from buffalo milk.

Discussion

Mastitis is one of the most economically problematic diseases of dairy cattle, particularly for the backyard farmers worldwide.³¹ The prevalence of mastitis varies between countries and geographical regions due to national differences in legislation, veterinary, and laboratory services and farmers’ management practices. Usually, the highest prevalence of mastitis is found in countries with a poorly developed dairy sector and lack of udder hygiene. Klebsiella mastitis is a major concern in the USA.^4,24,32,33 By contrast, Klebsiella mastitis outbreaks are only occasionally reported in Europe,^34,35 although a high rate has been reported in a study from the Alps in France (92%).³⁶ Our low prevalence rates are closely related to the findings in Canada³⁷ and significantly lower from several other global surveys on the prevalence of Klebsiella intra-mammary infections in Europe, Asia and South America^38–41 which have reported values ranging between 33.5% and 45.0 % and in Africa^42–49 which have reported a prevalence of 15–16%. The absence of Klebsiella spp. isolates in cases of subclinical mastitis highlights the insignificant role of this enterobacteria in subclinical mastitis contrary to other reports.^42–46,49

The existence of an oro-fecal transmission cycle has been suggested for K. pneumoniae in dairy herds, with fecal shedding resulting in the contamination of feed and water and the subsequent re-ingestion of the organism, resulting in renewed fecal shedding.^15,50–52 Fecal shedding of Klebsiella spp. contributes to pathogen loads in the environment, including in milking machines, recycled manure, bedding material, alleyways, and holding pens.^{4,15,34,35,51,53–55} Studies have illustrated a correlation between bovine teat colonization by Klebsiella and the use of sawdust bedding.^56–58

The antimicrobial susceptibility of udder pathogens varies greatly between studies. The disc diffusion technique is the most widely used method for determining the susceptibility of animal pathogens, especially in clinical work when it is necessary to determine the correct treatment. Therefore any comparison with studies that use other methods of susceptibility testing is not acceptable.⁴⁰ The prolonged use of antibiotics in the treatment of mastitis and as growth promoters has led to the additional problem of emergence antibiotic resistant strains, hence the constant concern about the resistant strains entering the food chain.⁶³ Klebsiella’s economic impact may be more devastating as many cows die or end up being culled.⁴ Klebsiella is usually referred to as particularly aggressive and is prone to cause severe clinical mastitis, which responds poorly to treatment and as a consequence, infections tend to be severe and long lasting with a fatal outcome.^3,24,60,64 Antibiotic-resistant Klebsiella on fresh vegetables and in sawdust pose a potential health problem with respect to bovine mastitis.⁵⁷ One possible contributing factor is the documented overuse of antibiotics in both agriculture and medical practice.^65,66 A possible explanation for the resistance phenomenon could be that ampicillin, chloramphennicol, colistin sulphate, erythromycin and streptomycin, and penicillin G have been the class of antimicrobial most widely used for treatment of several infections for many years.

The environment can contain a large variety of K. pneumoniae strains with pathogenic potential,^{24,52,54,59,60} and mastitis cases within a dairy herd are usually caused by many different strains which confirms our recording for K1 and K2.^33,51,61,62 Klebsiella produces 77 serologically distinct types of capsules¹¹ and their degree of virulence may be related to the mannose content of the capsular polysaccharide.⁶⁷ Mucoid strains of K1 or K2 serotype were more virulent to mice than non-mucoid strains of the same serotype.¹⁷ Although one of the few consistencies is that organisms that produce either the K1 or K2 capsular serotypes are more often associated with human disease,^11,28 both capsular serotypes were observed to be associated with the K. pneumoniae strains causing mastitis in our investigation, an indication that the source of Klebsiella infection was human.^11,68,69

Capsular serotypes K1 and K2 that carry genes magA⁷⁰ and rmpA^71,72 make the bacteria more invasive and more resistant to phagocytosis.^73,74 A previous study has shown that the mucoid phenotype may be due to a gene designated rmpA^71,72 which frequently coexists with aerobactin production.¹⁷ The strong association found in this study between mucoid phenotype A (rmpA-positive isolates) and aerobactin production suggests that the two virulence characteristics might be genetically coupled on a large virulence plasmid.^{17,29,70,71,75} This paper also shows that magA, uge, kfu, rmpA, and Aerobactin rather than the K1 and K2 capsule per se are important virulence genes in invasive K. pneumoniae strains causing mastitis.

Conclusion

The isolation of K. pneumoniae from milk of apparently healthy animals supports the notion that the presence of Klebsiella on teat ends may lead to opportunistic intramammary infections.⁷⁶ Therefore, to improve uptake of Klebsiella mastitis, control measures must be implemented: (1) the application of molecular or DNA-based methods in mastitis diagnostics; (2) insight into socioeconomic factors that motivate farmers to control Klebsiella mastitis; (3) hygiene of alleyways and holding pens is an important component of Klebsiella control on dairy farms; (4) mastitis controls will need to be developed to meet the specific requirements of an individual country or segment of the dairy industry; (5) identification of the virulence genes in K. pneumoniae strains causing mastitis will be helpful in tracing the origins of this infectious disease and vaccine production; and (6) pre-milking preparation procedures is of utmost importance. Cows with soiled udders arrive in the milking parlor, they are likely to still have Klebsiella on teat ends after pre-milking udder preparation. Not even the best pre-dipping disinfectant can cope with excess organic matter on heavily soiled udders.

Disclaimer Statements

Contributors None.

Funding None.

Conflicts of interest The authors declare no financial or personal conflict of interest that could inappropriately influence or bias the development, writing, and submission of this research and manuscript.

Ethics approval Yes.