Pathogenicity of bacterial isolates associated with high mortality in duckling in Behira province

 Pathogenicity of bacterial isolates associated with high mortality in duckling in Behira province

Rania, I. Elmeslemany¹   Ahlam, E. Yonis²

¹ Bacteriology,   ²Biotechnology

Reference Laboratory for Veterinary Quality Control on Poultry Production, Damanhour Branch, Animal Health Research Institute, Agricultural Research Center, Egypt

Abstract

During 2020–2022, a survey of 25 farms of ducklings of various breeds (Pekini and Muscovy), that ranged  from one day up to a month of  age suffering from various symptoms and  mortality rates varied from 10 to 35 % revealed isolation of Klebsiella pneumonae   36 % whereas the prevalence of E coli, Staph aureus and Salmonella Typhimurium were 32%, 28% and 16% respectively.  Following PCR confirmation of Klebsiella pneumonae, the presence of the its virulence genes were surveyed and  results showed that  uge (44%) and rmpA (22%), as well as an antimicrobial susceptibility profile,  which revealed 100% resistant to Ampicillin, Amoxicillin and Penicillin G  followed by77 % toward tetracycline, 22% toward streptomycin . Contrarily, they were extremely sensitive to ciprofloxacin and enrofloxacin, as well as 77%  to cefotaxime and 66%  to gentamycin. The surveillance of class 1 integron ( intl1) and  genes of antimicrobial resistance  demonstrated that (66%) of isolates  harbor Int1,whereas only 33% of the isolates involved blaSHV, they all had the tetA and blaTEM genes. Salmonella and E. coli isolates were serotyped, and it was discovered that the E. coli isolates belonged to six different O-serogroups, including O55:H7, O148:K25, O86: K61, O114:H21, O26:H11 and O127:H6 while   Salmonella isolates were recognized as S. Typhimurium. . In our investigation, invA gene is present in 100% of the S. Typhimurium isolates, while the mgtC gene is present in 75% of them.

Keywords

Virulence genes   , antimicrobial resistance, integron (class 1) .  Ducks

1. Introduction

              Bacterial diseases are the leading cause of mortality globally, and due to haphazard use of antibiotics, antimicrobial resistance has become an emerging threat (Bhattarai et al., 2021).  Klebsiella pneumonia infects both humans and animals worldwide, and these infections are associated with resistance to crucial antibiotics (Marques et al., 2019), it consider a major zoonotic   bacteria of the Enterobacteriaceae family (Wang et al., 2020). In poultry, K. pneumoniae is one of the respiratory pathogens causing high mortality in chicks and broilers (Hamza et al., 2016). K. pneumoniae exhibits variety of virulence elements such as capsules, mechanisms of iron removal,   endotoxins, binders, and antibiotic resistance, which have been found to be important in pathogenesis (Zhang et al., 2018). The establishment of K. pneumoniae strains carrying diverse resistance genes has significantly expanded over the previous few decades (Wu et al., 2019). Antibiotic resistance genes can be acquired by bacteria from mobile components, which aid in their distribution throughout various bacteria (Blair et al., 2015). Due to their presence on plasmids, pathogenicity islands and   transposons, integrons are potent mobile genetic components that can migrate between various bacteria. (Firoozeh et al., 2019).

               Class 1 integrons are particularly prevalent in gram-negative bacteria, like K pneumoniae, and are often documented. In integrons antimicrobial resistance genes were encoded by internal gene cassettes and have two conserved areas, including the 3′ and 5′ conserved segments (3′ CS and 5′ CS), in their structure (Lima et al., 2014). In K. pneumoniae the expression of ESBL is one way by which bacteria become cephalosporins and monobactams resistant while   expression of carbapenemase is the second pathway, that   aids in the development of resistance to all obtainable β -lactams (Riwu et al., 2020).

             One of the most serious infections affecting ducks, salmonella infection which has a huge impact on both public health and the economy (Yang et al., 2019). A wide variety of clinical symptoms in poultry, such as septicemic lesions, suppurative dermatitis, and suppurative arthritis, are linked with Staphylococcus aureus infection  (Elfeil, 2012). Pathogenicity of it   was investigated in an experiment by Amen et al. (2019) on 7-day-old chicks and resulted in mortality of 100%, 100% and 26.7% of chicks through subcutaneous injection, oral and intra nasal route.

The goal of this study was to look into the main bacterial infection linked to high duckling mortality rates as well as its virulence characteristics.

2. Materials and Methods

2.1. Ethical approval:

                  No experimentation on animals was done as part of this research.  Freshly dead and diseased duckling were euthanized then samples were collected in accordance with the regulations of Animal Health Research Institute and the General Organization for Veterinary Services.

2.2. Sample Collection

           A total of 25 farms of ducklings of different breeds (Pekini and Muscovy), range from one day up to a month of age suffering from mortality rates varied from 10 to 35%   and different symptoms (depression, lethargy, body weight loss, inactivity, pale combs, reduced water and feed consumption, ruffled feathers,, diarrhea, omphalitis, lameness and   arthritis) during 2020-2022 from different localities in Behira province were   tested to detect the incremented bacterial isolates.

23. Bacterial Isolation and Identification

             The   internal organs (liver, heart, lung, kidney, spleen) and blood   were collected. The samples initially incubated in buffer peptone water, after that cultivated on different specific media. E coli isolation was performed on both Eosin methylene blue and MacConkey agar and identified by (IMVIC) (Quinn et al., 2002).  K. pneumoniae isolation was done on MacConkey and Blood agar and identified based on Gram’s staining, and biochemical (kumar Arya et al., 2020)then confirmed by PCR. (ISO 6579 2002)   was used for isolation of Salmonella. Staph aureus isolation   was performed on both Baird parker agar and Mannitol salt agar then subsequently recognized using biochemical tests (Quinn et al., 2002).

2.3. E. coli and Salmonella Serotyping:

           Salmonella serotyped according to (Grimont and Weill, 2007), whereas E. coli isolates were serotyped in accordance with Neter (1973)  in RLQP  Animal Health Research Institute using  antisera (Sifin diagnostics GmbH, Germany).

2.4. In-Vitro anti-microbial sensitivity test:

            The bacterial resistance profile was evaluated using the agar diffusion technique according to the Clinical Laboratory Standard Institute (Wayne, 2010). K.pneumoniae   isolates were subjected to antibiotic sensitivity test against nine commonly used antibiotics Aminoglycosides (gentamycin10 mg, streptomycin100 mg (high concentration), Cephalosporines (Cefotaxime 30 mg), Fluroquinolones (Ciprofloxacin 5 mg, Enrofloxacin 5 mg ),  Penicillines (ampicillin 10 mg, amoxicillin10 mg and Penicillin G 10 mg)Tetracyclines (tetracycline30 mg) .

2.5. Detection of Resistance Genes and Virulence-Associated Genes

I-DNA extraction

               DNA was extracted from samples using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH), according to manufacture method.

II-Amplification of PCR.

For K. pneumonia Table (1) illustrates the used primers (supplied from Metabion (Germany).

                A 25µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (RR310A) (Takara, Japan), 1 µl of each primer at a concentration of 20 pmol, 5.5 µl of water, and 5 µl of DNA template was used. The reaction was carried out in a thermal cycler by Applied biosystem 2720.

For Salmonella Table (2) illustrates   Salmonella typhimurium's target genes, cycling conditions, amplicon sizes, and primer sequences.

                A 25- µl reaction including 12.5 µl of Emerald Amp Max PCR Master Mix(RR310A)  (Takara, Japan), 1 µl of each primer at a concentration of 20 pmol, 4.5 µl of water, and 6 µl of DNA template was used. Thermal cycler T3 Biometra was used to carry out the reaction.

III-PCR Products analysis.

                      The PCR products were separated using 5V/cm gradient electrophoresis on a 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature. Each gel slot had 20 µl of the PCR products put in it for the gel analysis. To estimate the sizes of the fragments, a generuler 100 bp ladder (Fermentas, Thermo, Germany) was employed. A gel documentation system (Alpha Innotech, Biometra) took pictures of the gel, and computer software was used to analyse the information.

  Table (1): K. pneumonia target genes, Primers sequences, and cycling conditions.

Table (2 ): Salmonella typhimurium's target virulence  genes, cycling conditions, amplicon sizes, and primer sequences

3. Results

3.1. Serotypes and Bacterial Incidence

Overall, 36% of cases involved K. pneumonae, whereas 32%, 28%, and 16% of cases involved E coli, S. aureus, and S. Typhimurium, respectively. The majority of the O-serogroups that were identified by serotyping the E. coli isolates were O55:H7, O148:K25, O86:K61, O114:H21, O26:H11, and O127:H6, while Salmonella isolates identified as S. typhimurium

3.2. Screening of Genes Associated with Virulence

                  PCR was used to confirm K. pneumoniae isolates (fig. 1). Screening these isolates for presence of   virulence genes uge and rmpA revealed that   4/9   (44%) contain uge (fig.2) and 2/9(22%) contain rmpA (fig.3). All of the S. Typhimurium isolates in our study have the invA gene (100%) (Fig.4) while   75% of them have the mgtC gene (fig.4).

3.3. Antibiotic Susceptibility

                     In-vitro susceptibility testing of K. pneumoniae isolates   showed varying degrees of resistance to tested   antimicrobial table (3) as all isolates (100%) were resistant to Ampicillin, Amoxicillin and Penicillin G   followed by77 % toward tetracycline, 22% toward streptomycin . However, they were extremely sensitive to ciprofloxacin and enrofloxacin,   as well as77% to cefotaxime and 66% to Gentamycin.

Table (3) Percentage (%) Susceptibility Pattern of K. pneumonae

Note: AMP: ampicillin;  AMX: amoxicillin; T:tetracycline S: streptomycin; CIP: ciprofloxacin;   ENR: Enrofloxacin;  CTX: cefotaxime; G: Gentamycin

3.4. Detection of Antimicrobial Resistance Genes

           Screening these isolates for presence of Int1, tet A, blaTEM and blaSHV   revealed that 6/9(66%) of isolates   involve Int1 (fig.5) and table (4),   all the isolates harbor tetA (fig.7) and blaTEM genes (fig.6) while only 3/9 (33%) involve blaSHV (fig.8).  

Table (4) Association between the occurrence of integrons and AMR phenotype in K.pneumonae

4. Discussion

                  One of the challenges to duck breeding is the early death of ducklings. This investigation revealed   the presence of Klebsiella pneumonae   in 36 % of examined farms (confirmed by PCR fig. 1), compared to the results obtained by Banerjee et al. (2019) (14.85%) and Mondal et al. (2022)(16.75%).It reported with E. coli as a cause of   variable diseases that can lead to death rates of up to 20–30% (Khelfa and Morsy, 2015).

                      One of the many virulence components that contribute to K. pneumoniae's pathogenicity is its capsule, which guards it from phagocytosis and potentially fatal serum factors  (Hsu et al., 2011). Surveying of Uge gene and rmpA   revealed their presence in 44% and   22% respectively (Fig.2) and (fig 3),   this finding demonstrated that the rmp gene was less prevalent than the uge gene, which may be related to what was mentioned by Yu et al. (2006) the prevalence of rmpA has been shown to be less frequent in strains from bacteremic cases than that   from liver abscesses. These   genes are responsible for invasion, pathogenicity and colonization for K.  pneumoniae   (Aher et al., 2012) , they also play a significant role in synthesis of  capsule, resistance of  phagocytosis, liver abscess, and infection of  blood.

               Li et al. (2019) attributed   high mortality rates of Klebsiella pneumonae   infection to its   antibiotic resistance, making them challenging to treat and manage. In-vitro sensitivity  testing   of K. pneumoniae   isolates  showed varying degrees of susceptibility to tested  antibiotics as all isolates (100%) were resistant to Ampicillin, Amoxycillin and Penicillin G highlighting these antibacterials' poor therapeutic effects for treatment of ducks against K. pneumoniae, followed by77% to tetracycline which is lower than that recorded by  Safika et al. (2022)(95%) and higher than that recorded by Zaghloul et al. (2021) (34.1) and Permatasari et al. (2020)(35.72%),while his result for resistance toward  streptomycin ( 21.4%) similar to our result ( 22%) . However, they had a high sensitivity to ciprofloxacin and Enrofloxacin (100% for each) and 77% to   cefotaxime compared to the 64.3% reported by Nnachi et al. (2015), as well as 66% sensitivity to Gentamycin and the remaining 33% showed moderate sensitive.

                Multidrug resistance in bacteria has been mostly disseminated via class 1 integrons Cambray et al., 2010, which has been documented in Gram-negative bacteria with its role in the distribution and spread of antimicrobial resistance (Deng et al., 2015). Surveying K. pneumonae isolates for Int 1 reveal its presence in 67% (6/9) (Fig.5) and table .4   which revealed the spreading of antibiotic resistance among isolated K. pneumonae as result of a significant association between the existence of class 1 integrons and the emergence of MDR (Li et al., 2013). Furthermore, analysis of isolated strains for genes associated with antibiotic resistance found that 100, 100, and 33% K. pneumoniae possessed the tet A , bla TEM (ampicillin resistance), and blaSHV (responsible for penicllinase hydrolysis) genes respectively. Compared with that reported by Mondal et al. (2022), 8.3% and 37.5% of K. pneumoniae isolates from ducks were found to possess blaSHV and blaTEM, while 13.33% and 33.33% were found to be positive, according to A. Banerjee et al. 2019. The results illustrated that all  bla SHV genes are present in strains positive for the class 1 integron, suggesting that intl1 and blaSHV genes may be carried on the same plasmid. The same observation was reported by Jones et al. (2005), while a low rate of association between   integrons and ESBL genes was found by Machado et al. (2007).Also there was a co-existence of two different ESBL genes ( blaTEM and bla SHV)  in the same strain in 33.3% of isolates.   Resistance to tetracycline is governed by tet genes, which are involved in either active efflux of the drug, ribosomal protection or enzymatic drug modification (Giovanetti et al., 2003). In spite of presence of tetA gene in all k. pneumonae there is 77% resistant and the remnant 22% were moderate sensitive and this result in accordance with that recorded by Xu et al. (2021),who examined isolates carried the wild-type tet A gene and found 75.8% of these tet A bearing isolates exhibited a tigecycline ( one member of tetracycline) resistant  phenotype and attributed this to tet A  mutants are often located in different types of plasmids, and these plasmids have different promoter and regulatory sequences that may result in different expression levels of tet A, in addition to occurring mutation of tet A under selective pressure. The examples of these promoters reported by other author as Zhang et al. (2019)reported the coexistence of mcr-1 and the tet A variant on the same plasmid from a K. pneumoniae isolate in human gut, and Yao et al. (2020) also reported an IncFII plasmid co-harboring blaIMP and tet A variant in a clinical K. pneumoniae isolates.

                 E coli was prevalent in 32% of cases, which highlighted its significant involvement as a cause of duckling death.  It can cause a wide range of issues, but the most serious sickness strikes them between the ages of 2 and 6 weeks, when fatality rates can approach 43% (Punnoose et al., 2021). Numerous authors connected E. coli infection with mortality in ducklings, such as Islam et al. (2004)(11%), Bariha et al. (2019)(55%), Roshdy et al. (2012)(30.8%) in infected ducks that were more than a week old and still alive, while it was (28.4%) in recently dead ducks and Khelfa and Morsy (2015)(up to 20-30%) of mortality due to E coli with K. pneumoniae. The detected E coli in this investigation belonged to a variety of serotypes including (EPEC) O55:H7, (isolated from three farms), O86: K61, O114:H21 and O127:H6 (Ørskov and Ørskov, 1992), (EHEC) O26:H11 (Mainil, 1999) and Shiga toxin-producing E. coli (STEC) O148:K25.

                The findings of this investigation revealed that Staph aureus was present in 28% of the samples that were tested, which is greater than the percentages noted by Eid et al. (2019)(12.2%) and Amen et al. (2019)(6.6) , drawing attention to its significance as a cause of duckling mortality.  Staph aureus was identified by Meyer et al. (2021) in the event of severe mortality outbreaks in layer flocks, as the predominant isolate from several organs, such as lungs, liver, bone marrow and spleen, in addition to its recovery from   64.1% diseased and dead chickens by Bakeet and Darwish (2014). Also an outbreak of omphalitis was reported by Mondal and Sahoo (2014) in the week-old ducklings with different clinical (swollen abdomen septicaemia, reduction in feed and water consumption) which resulted in gradual severely dehydrated carcass.

              The current study found that the isolation of salmonella was 16%, which is greater than the 9.3% reported in Zhao et al. (2017)'s study , but virtually in agreement with EL-GAOS et al. (2020)(18.5%), Abdelaziz et al. (2020)(14.1) and Lam et al., 2002 (18%). It is consistent with the findings of Martelli et al. (2016) and Eid et al. (2019) that S. Typhimurium is the most common serotype in ducks that all salmonella isolates in this experiment were serologically identified as being. According to report of  Punnoose et al. (2021), S. typhimurium is responsible for duckling disease and death, particularly during the age of   two weeks.  It consider one of the main reasons of duckling death in Pekin duck farm suffering 95% mortality   According to (Badr and Nasef, 2016). 

                  A pathogenic bacteria's ability to infect hosts is enabled by its virulence factors. Pathogenic Salmonella have several virulence components that enable them to invade the host, persist there, and eventually spread diseases (Marcus et al., 2000). One of the initial stages in the Salmonella spp. pathogenic cycle is intestinal epithelial cell invasion (Galan et al., 1992). Invasion of host epithelial cells requires the bacterial membrane protein encoded by the invA gene   (Darwin and Miller, 1999).  All of the S. Typhimurium isolates in our study have the invA gene (100%) Fig.4 , which is identical to that found by Hamed et al. (2023), EL-GAOS et al. (2020) and Abdelaziz et al. (2020),while  75% of them have the mgtC gene, which, according to Alix and Blanc‐Potard (2008), is essential for intra-macrophage survival and proliferation in magnesium-depleted media as well as for organisms that enter host cells.

Conclusion

This study highlights the prevalence of pathogenic bacteria linked to duckling mortality, as outlined in K. pneumonae, E coli, Staph aureus, and S. Typhimurium, together with their virulence factors and patterns of reactivity to antimicrobial agents.